Foodborne Disease Handbook, Volume 1: Bacterial Pathogens

Foodborne Disease Handbook Second Edition, Revised and Expanded Volume 1: Bacterial Pathogens

edited by

Y. H. Hui Science TechnologySystem West Sacramento, California

Merle D. Pierson Virginia PolytechnicInstitute and State University Blacksburg, Virginia

J. Richard Gorham Uniformed Services University of the Health Sciences Bethesda, Maryland

m M A R C E L

D E K K E R

MARCEL DEKKER, INC.

NEWYORK BASEL

ISBN: 0-8247-0337-5 This book is printed on acid-free paper.

Headquarters Marcel Dekker, Inc. 270 Madison Avenue. New York, NY 10016 tel: 212-696-9000; fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Drkker AG Hutgasse 4. Postfach 812, CH4001 Basel, Switzerland tel: 4 1-6 1-261-8482; fax: 41 -6 1 -26 1-8896 World Wide Web http://www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the headquarters address above.

Copyright 0 2001 by Marcel Dekker, Inc. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means. electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): l 0 9 8 7 6 5 4 3 2 1

PRINTED IN THE UNITED STATES OF AMERICA

Introduction to the Handbook

The Foodboine Disease Handbook,Second Edition,Revised and Expanded, could not be appearing at a more auspicious time. Never before has the campaign for food safety been pursued so intensely on so many fronts in virtually every country around the world. This new edition reflects at least one of the many aspects of that intense and multifaceted campaign: namely, that research on food safety has been very productive in the years since the first edition appeared. The Hcmdbook is now presented in four volumes instead of the three of the 1994 edition. The four volumes are composed of 86 chapters, a 22% increase over the 67 chapters of the first edition. Much of the information in the first edition has been carried forward to this new edition because that information is still as reliable and pertinent as it was in 1994. This integration of the older data with the latest research findings gives the reader a secure scientific foundation on whichto base important decisions affecting the public's health. We are not so naive as to think that only scientific facts influence decisions affecting food safety. Political and economic factors and cotnpelling national interests may carry greater weight in the minds of decisionmakers than the scientific findings offered in this new edition. However, if persons in the higher levels of national governments and international agencies, such as the Codex Alinlentarius Commission, the World Trade Organization, the World Health Organization, and the Food and Agriculture Organization, who must bear the burden of decision-making need and are willing to entertain scientific findings, then the information in these four volumes will serve them well indeed. During the last decade of the previous century, we witnessed an unprecedentedly intense and varied program of research on food safety, as we have already noted. There are compelling forces driving these research efforts. The traditional food-associated pathogens, parasites, and toxins of forty years ago still continue to cause problems today, and newer or less well-known species and strains present extraordinary challenges to human health. These newer threats may be serious even for the immunocompetent, but for the immunocompromised they can be devastating. The relative numbers of the immunocompromised in the world population are increasing daily. We include here not just those affected by the humanimmunodeficiency virus (HIV), but also the elderly: the veryyoung; the recipients of radiation treatments, chemotherapy, and immunosuppressive drugs; paiii

Handbook iv

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Introduction

tients undergoing major invasive diagnostic or surgical procedures; and sufferers of debilitating diseases such as diabetes. To this daunting list of challenges must be added numerous instances of microbial resistance to antibiotics. Moreover, it is not yet clear how the great HACCP experiment will play out on the worldwide stage of food safety. Altruism and profit motivation have always made strange bedfellows in the food industry. It remains to be seen whether HACCP will succeed in wedding these two disparate tnotives into a unifying force for the benefit of all concerned-producers, manufacturers, retailers, and consumers. That HACCP shows great promise is thoroughly discussed in Volume 2, with an emphasis on sanitation in a public eating place. All the foregoing factors lend a sense of urgency to the task of rapidly identifying toxins, species, and strains of pathogens and parasites as etiologic agents, and of determining their roles in the epidemiology and epizootiology of disease outbreaks, which are described in detail throughout the Foodborne Disease Hmdbook. It is very fortunate for the consumer that there exists in the food industry a dedicated cadre of scientific specialists who scrutinize all aspects of food production and bring their expertise to bear on the potential hazards they know best. A good sampling of the kinds of work they do iscontained in these four new volumes of the Handbook. And the benefits of their research are obvious to the scientific specialist who wants to learn even more about food hazards, to the scientific generalist who is curious about everything and who will be delighted to find a good source of accurate, up-to-date information, and to consumers who care about what they eat. We are confident that these four volumes will provide competent, trustworthy, and timely information to inquiring readers, no matter what roles they may play in the global campaign to achieve food safety.

I

Y. H. Hui J. Richard Gorham Dnvid Kitts K. D. Murre11 Wai-Kit Nip Merle D. Piersorl Syed A. Sattnr R. A. Smith David G. Spoerke, Jr. Peggy S. Starzfield

Preface

The first volume of the Foodborne Disease Handbook, Second Edition, Revised m d Expanded, focuses on bacterial pathogens. Although great strides have been made in food sanitation in general and in the application of HACCP principles in particular, hardly a week goes by without a report in the media of some outbreak of foodborne bacterial disease. It is evident that there is a gap between the principles and facts recorded in the first volume of the Handbook and the application of these facts and principles to the protection of the public’s health. Outbreaks of foodborne bacterial disease do not happen spontaneously-the principle of cause-and-effect remains fully operational. Moreover, people do not usually get sick by consuming wholesome food. Further, those who produce, manufacture, retail, and serve food have no wish to make people sick. Thus, when a foodborne disease outbreak occurs, it suggests that some (probably preventable) circumstance or set of circumstances was allowed to occur that permitted the proliferation of bacteria or the production of their toxins. As HACCP principles emphasize, prevention is the key to averting foodborne bacterial disease outbreaks. The editors and contributors to this volume of the Foodborne Disease Harzdbook have worked diligently to ensure that readers representing all aspects of the food industry now have readily at hand all the facts and principles needed to prevent foodborne outbreaks of bacterial diseases.

Y. H. Hui Merle D. Piersol? J. Richard Gorhnm

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Contents

Introduction to the Harldbook Prefkce Contributors Contents of Other Volurzres

...

111

17

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I. Poison Centers 1. The Role of U.S. Poison Centers in Bacterial Exposures David G. Spoerke, Jr.

1

11. Bacterial Pathogens 2.

Bacterial Biota (Flora) in Foods James M. Jay

23

3. Aerornonas hydrophilu Carlos Abeyta, Jr., Sarrruel A. Palrmbo. arrd Gerard N. Stelma, Jr.

35

4. Update: Food Poisoning and Other Diseases Induced by Bacilluscereus Kathleen T. Rajkowski and James L. Smith

61

5. Brucella Shirley M. Halling and Edward J. Young

77

6. Campylobacter jejuni Don A. Franco a ~ Charles d E. Willinrns

83

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7.

Clostridium botulinum John W. Austh and Karen L. Dodds

107

8. Clostridium perfringens Dorothy M. Wrigley

139

9. Escherichia coli Mcrrguerite A. Neill, Phillip I. Tarr, David N. Tcrvlor, and Marcia Worf

169

10. Listeria nzonocytogenes Catherine W. Donnelly

213

11. Bacteriology of Salmonella Robin C. Anderson and Richcrrd L. Ziprin

247

12. Salmonellosis in Animals David J. Nisbet and Richard L. Ziprin

265

13. Human Salmonellosis: General Medical Aspects Richard L. Ziprin and Michael H. Hume

285

14. Shigella Anthony T. Maurelli and Keith A. Lmnpel

323

15. Staphylococcusaureus Scott E. Martin, Eric R. Mvers, and John J. Iandolo

345

16. Vibrio cholerae Charles A. Kaysner and June H. Wetherirlgton

383

17. Vibrio parahaenzolyticus Tuu-jyi Chai and John L. Pace

407

18. Vibrio vuln@cus Anders Dalsgaard, Lise H@i, DebiLinttous: and James D. Oliver

439

19. Yersinin Scott A. Minnich, Michael J. Smith, Steven D. Weagant, crnd Peter Feng

47 l

111. Disease Surveillance, Investigation, andIndicatorOrganisms 20.

Surveillance of Foodborne Disease Ewer1 C. D. Todd

515

Contents

ix

21. Investigating Foodborne Disease Dale L. Morse, Guthrie S. Birkhead, and Jack J. Guzewich

587

22. Indicator Organisms in Foods James M. Juy

645

Index

655

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Contributors

Carlos Abeyta, Jr.

U.S. Food and Drug Administration, Bothell, Washington

Robin C. Anderson Food Animal Protection Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, College Station, Texas John W. Austin Bureau of Microbial Hazards, Health Protection Branch, Health Canada, Ottawa, Ontario, Canada Guthrie S. Birkhead New York State Department of Health, Albany, New York Tuu-jyi Chai Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan, Republic of China Anders Dalsgaard Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark Karen L. Dodds Health Canada, Ottawa, Ontario, Canada Catherine W. Donnelly Department of Nutrition and Food Sciences, University of Vermont, Burlington, Vermont Peter Feng Division of Microbiological Studies, U.S. Food and Drug Administration, Washington, D.C. Don A. Franco

Animal Protein Producers Industry, Huntsville, Missouri

Jack J. Guzewich U.S. Food and Drug Administration, Washington, D.C. Shirley M. Halling U.S. Department of Agriculture, Ames, Iowa Lise HGi Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark Michael H. Hume Food Animal Protection Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, College Station, Texas JohnJ. Iandolo Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma xi

xii

James M. Jay

Contributors

University of Nevada Las Vegas, Las Vegas, Nevada

Charles A. Kaysner Seafood Products Research Center, U.S. Food and Drug Administration, Bothell, Washington Keith A. Lampel Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Washington, D.C. Debi Linkous

Burroughs Wellcome Research Fund, Raleigh, North Carolina

Scott E. Martin Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois Anthony T. Maurelli Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland Scott A. Minnich Department of Microbiology, University of Idaho, Moscow, Idaho Dale L. Morse Wadsworth Center, New York State Department of Health, Albany, New York Eric R. Myers Nalco Chemical Company, Naperville, Illinois Marguerite A. Neil1 Department of Medicine, Division of Infectious Disease, Brown University School of Medicine, Providence, Rhode Island David J. Nisbet Food and Feed Safety Research Unit, Food Animal Protection Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, College Station, Texas James D. Oliver University of North Carolina at Charlotte, Charlotte, North Carolina JohnL.Pace Biochenlistry Department, Advanced Medicine, Inc., South San Francisco, California Samuel A. Palumbo U.S. Department of Agriculture, Philadelphia, Pennsylvania KathleenT.Rajkowski Department of Food Safety, Agricultural Research Service, U.S. Department of Agriculture, Wyndmoor, Pennsylvania James L. Smith Microbial Food Safety Lab, Agricultural Research Service, U.S. Department of Agriculture, Wyndmoor, Pennsylvania Michael J. Smith Department of Microbiology, University of Idaho, Moscow, Idaho David G. Spoerke, Jr. Bristlecone Enterprises, Denver, Colorado Gerard N. Stelma, Jr. U.S. Environmental Protection Agency, Cincinnati, Ohio Phillip I. Tarr University of Washington and Children’s Hospital and Medical Center, Seattle, Washington David N. Taylor Walter Reed Army Institute of Research, Washington, D.C. Ewen C. D. Todd Health Protection Branch, Bureau of Microbial Hazards, Health Canada, Ottawa, Ontario, Canada Steven D. Weagant U.S. Food and Drug Administration, Bothell, Washington

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Contributors

Xiii

June H. Wetherington U S . Food and Drug Administration, Bothell, Washington Charles E. Williams Consultant, Arlington, Virginia Marcia Wolf Walter Reed Army Institute of Research, Washington, D.C. Dorothy M. Wrigley Mankato, Minnesota

Department of Biological Sciences, Minnesota State University,

Edward J. Young VA Medical Center and Baylor College of Medicine, Houston, Texas Richard L. Ziprin Food and Feed Safety Research Unit, Food Animal Protection Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, College Station, Texas

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Contents of Other Volumes

VOLUME 2: VIRUSES, PARASITES, PATHOGENS, AND HACCP I. PoisonCenters 1. The Role of Poison Centers in the United States David G. Spoerke, Jr.

11. Viruses 2. Hepatitis A and E Viruses Theresa L. Cron.leans, Michael 0. Fmol-ov, Omana V. Nainan, and Harold S. Margolis

3. Norwalk Virus and the Small Round Viruses Causing Foodborne Gastroenteritis Hazel Appleton 4. Rotavirus Syed A. Snttnr, V. S u s m Springthorpe, and Jason A. Tetro

5. Other Foodborne Viruses Syed A. Sattar and Jason A. Tetl-o 6. Detection of Human Enteric Viruses in Foods Lee-Ann Jaykus 7. Medical Management of Foodborne Viral Gastroenteritis and Hepatitis SuZarwe M. Mcrtsui and Rarnsey C. Cheung xv

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Contents of Other Volumes

8. Epidemiology of Foodborne Viral Infections Thornns M. Liithi

9. Environmental Considerations in Preventing the Foodborne Spread of Hepatitis A Syed A. Sattar and Sabah Bidawid

111. Parasites 10. Taeniasis and Cysticercosis Zbigniew S. Pawlowski and K. D. Murre11

11. Meatborne Helminth Infections: Trichinellosis William C.Campbell 12. Fish- and Invertebrate-Borne Helminths John H. Cross

13. Waterborne and Foodborne Protozoa Ronald Foyer

14. Medical Management Pm1 Prociv 15. Immunodiagnosis of Infections with Cestodes Bruno Gottstein

16. Immunodiagnosis: Nematodes H. Ray Gamble 17. Diagnosis of Toxoplasmosis Alan M. Johnson and J. P. Dubey 18. Seafood Parasites: Prevention, Inspection, and HACCP Ann M. Admm and Debra D. DeVlieger

IV. HACCP and the Foodservice Industries 19. Foodservice Operations: HACCP Principles 0. Peter S q d e r , Jr. 20. Foodservice Operations: HACCP Control Programs 0. Peter Snyder, Jr. Index

VOLUME 3: PLANT TOXICANTS I. PoisonCenters 1. U.S. Poison Centers for Exposures to Plant and Mushroom Toxins David G. Spoerke, Jr.

Contents of Other Volumes

11. Selected Plant Toxicants 2. Toxicology of Naturally Occurring Chemicals in Food Ross C. Beier and Herbert N. Nigg

3. Poisonous Higher Plants Doreen Grace Lang and R. A. Smith 4. Alkaloids R. A. Smith 5. Antinutritional Factors Related to Proteins and Amino Acids Iwin E. Liener 6. Glycosides Walter Majak and Miclznel H. Berm

7. Analytical Methodology for Plant Toxicants Alister David Muir

8. Medical Management and Plant Poisoning Robert H. Poppenga 9. Plant Toxicants and Livestock: Prevention and Management Michcrel H. R a l p h

111. Fungal Toxicants 10. Aspergillus Zojia Kozcrkiewicz 11. Claviceps and Related Fungi Gretchen A. Kuldau and Charles W. Bacon

12. Fusariunl Walter F. 0. Marmas 13. Penicillium Johr~I. Pitt 14. Foodborne Disease and Mycotoxin Epidemiology Sara Hale Herlly and F. Xcwier Bosch 15. Mycotoxicoses: The Effects of Interactions with Mycotoxins Heather A. Koshinsky, Adrieme Woytowich, and George G. Khachatourians

16. Analytical Methodology for Mycotoxins James K. Porter 17. Mycotoxin Analysis: Immunological Techniques Fur1 S. Chrr

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xviii

Volumes Contents of Other

18. Mushroom Biology: General Identification Features Dmid G. Spoerke, Jr. 19. Identification of Mushroom Poisoning (Mycetismus), Epidemiology, and Medical Management David G. Spoerke, Jr. 20. Fungi in Folk Medicine and Society Dnvid G. Syoerke, Jr.

VOLUME 4: SEAFOOD AND ENVIRONMENTAL TOXINS I. Poison Centers 1. Seafood and Environmental Toxicant Exposures: The Role of Poison Centers Dnvid G. Spoerke, Jr.

11. Seafood Toxins 2. Fish Toxins Bruce W. Halstencl 3. Other Poisonous Marine Animals Bruce W. Hulsteacl

4. Shellfish Chemical Poisoning Lyndon E. Llewellyn 5. Pathogens Transmitted by Seafood Russell P. Hemlig

6. Laboratory Methodology for Shellfish Toxins David Kitts 7. Ciguatera Fish Poisoning Yoshitsugi Hokema and Jocmne S. M. Yoshikuwa-Ebesu 8. Tetrodotoxin Joame S. M. Yoslzikablta-Ebesu,Yoslzitsugi Hokamn, nnd TUIIKIO Noguchi

9. Epidemiology of Seafood Poisoning Lorn E. Flerning, Dolores Kat:, Judy A. Bean, and Robertl! Hnnurlond

Contents of Other Volumes

10. The Medical Management of Seafood Poisoning Donm Glad Blythe, Eileen Hack, Giavarmi Washirzgtofl,and Lorn E. Fleming 11. The U.S. National Shellfish Sanitation Program Rebecca A. Reid a d Timothy D. Durmce

12. HACCP, Seafood, and the U.S. Food and Drug Administration Kim R. Yomg, Miguel Rodrigues Krrnzrst, arid George Per)? Hoskin 111. Environmental Toxins

13. Toxicology and Risk Assessment D o r ~ ~J.l lEcobichorz 14. Nutritional Toxicology David Kitts 15. Food Additives Lasclo P. Sornogyi

16. Analysis of Aquatic Contaminants Joe W. Kiceniuk 17. Agricultural Chemicals Debra L. Browning and Car1 K. Winter 18. Radioactivity in Food and Water Hank Kocol

19. Food Irradiation Hark Kocol 20. Drug Residues in Foods of Animal Origin Austin R. Long and Jose E. Roybal 21. Migratory Chemicals from Food Containers and Preparation Utensils Yvonne V. Y ~ r m 22. Food and Hard Foreign Objects: A Review J. Richard Gol-ham 23. Food, Filth, and Disease: A Review J. Richard Gorham 24. Food Filth and Analytical Methodology: A Synopsis J. Richard Gol-hnm

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1 The Role of U S . Poison Centers in Bacterial Exposures David G. Spoerke, Jr. Bristlecorle Enterprises, Denver, Colorado

I. Epidemiology A. B. C. D. E. F. G. H. I. J.

1

AAPCC 2 Who staffs a poison center'? 3 What types of calls are received? 4 How calls are handled 5 What references are used? 6 How poison centers are monitored for quality 6 Professional and public education programs 7 Related professional toxicology organizations 7 International affiliations 9 Toxicology and poison center Web sites 10

11. U.S. Poison Information Centers

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References 2 1

1.

EPIDEMIOLOGY

Epidemiological studies aid treatment facilities in determining risk factors, determining who becomes exposed, and establishing the probable outcomes with various treatments. A few organizations have attempted to gather such information and organize it into yearly reports. The American Association of Poison Control Centers and some federal agencies work toward obtaining epidemiological information, while the AAPCC has an active role in assisting with the treatment of exposures. Epidemiological studies assist government and industry in determining package safety, effective treatment measures, conditions of exposure, and frequency of exposure. Studies on bacterial exposures provide information on the type of people most commonly involved. (e.g., children, adults at home, outdoorsmen, industrial workers, or bluecollar workers). Studies can also tell us which bacterial species are most commonly involved. The symptoms first seen, the onset of symptoms, and the sequelae may also be determined and compared to current norms. l

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A.

AAPCC

1. What Are Poison Centers and the AAPCC? The group in the United States most concerned on a daily basis with potential poisonings due to household agents, industrial agents, and biologics (including plants and mushrooms) is the American Association of Poison Control Centers (AAPCC). This is an affiliation of local and regional centers that provides information concerning all aspects of poisoning and often refers patients to treatment centers. This group of affiliated centers is often supported by both government, private funds, and industrial sources. Poison centers were started in the late 1950s, with the first center thought to be in the Chicago area. The idea caught on quickly, and at the peak of the movement there were hundreds of centers throughout the United States. Unfortunately there were few or no standards regarding what might be called a poison center, the type of staff, hours of operation, or information resources. One center may have had a dedicated staff of doctors, pharmacists, and nurses trained specifically in handling poison cases; the next center may just have had a book on toxicology in the emergency room or hospital library. In 1993 the Health and Safety Code (Sec. 777.002) specified that a poison center must provide a 24-hour service for public and health care professionals and meet requirements established by the AAPCC. This action helped to standardize the activities and the staffs of the various poison centers. The federal government does not fund poison centers, even though for every dollar spent on poison centers there is a savings of $2 to $9 in unnecessary expenses (1,2). The federal agency responsible for the Poison Prevention Packaging Act is the U.S. Consumer Product Safety Commission (CPSC). The National Clearinghouse for Poison Control Centers initially collected data on poisonings, information on commercial product ingredients, and biological toxic agents. For several years the National Clearinghouse provided product and treatment information to the poison centers who handled day-to-day calls. At first, most poison centers were funded by the hospital in which they were located. As the centers grew in size and number of calls being handled, both city and state governments took on the responsibility of contributing funds. In recent years the local governments have found it very difficult to fund such operations and centers have had to look to private industry for additional funding. Government funding may take several forms, as a line item on a state’s budget, as a direct grant, or as moneys distributed on a percall basis. Some states with fewer residents may contract with a neighboring state to provide services to its residents. Some states are so populous that more than one center is funded by the state. Industrial funding also varies, sometimes as a grant, sometimes as payment for handling the company’s poison or drug information-related calls, sometimes as payment for collection of data regarding exposure to the company’s product. Every year the AAPCC issues a summary of all kinds of exposures. A few bacterial exposures are listed in this log, most of which have to do with food poisoning.

2. Regional Centers The number of listed centers has dropped significantly since its peak of more than 600. Many centers have been combined into regional organizations. These regional poison centers provide poison information and telephone management and consultation, collect pertinent data, and delivery professional and public education. Cooperation between regional poison centers and poison treatment facilities is crucial. The regional poison information center, in cooperation with local hospitals, should determine the treatment capabilities of

Poison Centers and Bacterial Exposure

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the treatment facilities of the region and identify and have a working relationship with their analytical toxicology laboratories, emergency departments, critical care wards, medical transportation systems, and extracorporeal elimination capabilities. This should be done for both adults and children. A “region” is usually determined by state authorities in conjunction with local health agencies and health care providers. Documentation of these state designations must be in writing unless a state chooses (in writing) not to designate any poison center or accepts a designation by other political or health jurisdictions. Regional poison information centers should serve a population base of greater than one million people and must receive at least 10,000 human exposure calls per year. The number of certified regional centers in the United States is now under 50. Certification as a regional center requires the following. 1. Maintenance of a 24 hours per day, 365 days per year service. 2. Providing service to both health care professionals and the public. 3. Having available at least one specialist in poison information in the center at all times. 4. Having a medical director or qualified designee, on call by telephone, at all times. 5. Service should be readily accessible by telephone from all areas within the region. 6. Comprehensive poison information resources and comprehensive toxicology information covering both general and specific aspects of acute and chronic poisoning should be available. 7. The center is required to have a list of on-call poison center specialty consultants. 8. Written operational guidelines, which provide a consistent approach to evaluation, follow-up, and management of toxic exposures should be obtained and maintained. These guidelines must be approved in writing by the medical director of the program. 9. There should be a staff of certified professionals manning the phones (at least one has to be a pharmacist or nurse with 2000 hours and 2000 cases of supervised experience). 10. There should be a 24-hour physician (Board Certified) consultation service. 11. The Regional Poison Center shall have an ongoing quality assurance program. 12. Other criteria, determined by the AAPCC, may be established with membership approval. 13. The regional poison information center must be an institutional member in good standing of the AAPCC. Many hospital emergency rooms still maintain a toxicology reference such as the POISINDEX@system to handle routine exposure cases, but rely on regional centers to handle most of the calls in their area.

B. Who Staffs a PoisonCenter? The staffing of poison centers varies considerably. The three professional groups most often involved are physicians, nurses, and pharmacists. Who answers the phones is somewhat dependent on the local labor pool, moneys available, and the types of calls being

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received. Others used have included students in medically related fields, toxicologists, and biologists. Persons responsible for answering the phones are either certified by the AAPCC or are in the process of obtaining certification. Passage of an extensive examination on toxicology is required for initial certification, with periodic recertification required. Regardless of who takes the initial call, a medical director and the other physician back-up are available. These physicians have specialized training or experience in toxicology and are able to provide in-depth consultations for health care professionals calling a center. 1. Medical Director A poison center medical director should be board certified in medical toxicology or in internal medicine, pediatrics, family medicine, or emergency medicine. The medical director should be able to demonstrate ongoing interest and expertise in toxicology as evidenced by publications, research, and meeting attendance. The medical director must have a medical staff appointment at a comprehensive poison treatment facility and must be involved in the management of poisoned patients. 2. Managing Director The managing director must be a registered nurse, pharmacist, or physician or hold a degree in a health science discipline. The individual should be certified by the American Board of Medical Toxicology (for physicians) or by the American Board of Applied Toxicology (for nonphysicians). They must be able to demonstrate ongoing interest and expertise in toxicology. 3. Specialistsin Poison Information These individuals must be registered nurses, pharmacists, or physicians or be currently certified by the AAPCC as a specialist in poison information. Specialists in poison information must complete a training program approved by the medical director and must be certified by the AAPCC as a specialist in poison information within two examination administrations of their initial eligibility. Specialists not currently certified by the Association must spend an annual average of no less than 16 hours per week in poison centerrelated activities. Specialists currently certified by the AAPCC must spend an annual average of no less than 8 hours per week. Other poison information providers must have sufficient background to understand and interpret standard poison information resources and to transmit that information understandably to both health professionals and the public. 4. Consultants In addition to physicians specializing in toxicology, most centers also have lists of experts in many other fields as well. Poison center specialty consultants should be qualified by training or experience to provide sophisticated toxicology or patient care information in their area(s) of expertise. An infectious disease consult would be appropriate for bacterial infections.

C.What

Types of Calls Are Received?

All types of calls are received by poison centers, most of which are handled immediately; others referred to more appropriate agencies. Which calls are referred depends on the center, its expertise, and the appropriateness of a referral. Below are lists of calls that generally fall into each group. Remember, there is considerable variation between poison

Poison Centers and Bacterial Exposure

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centers, and if there is any doubt, call the poison center and they will tell you if your case is more appropriately referred. Poison centers do best on calls regarding acute exposures. Complicated calls regarding exposure to several agents over a long period of time that produce nonspecific symptoms are often referred to other medical specialists, to the toxicologist associated with the center, or to an appropriate government agency. The poison center will often follow up on these cases to track the outcome and type of service given. Types of Culls Usually Accepted Drug identification Actual acute exposure to a drug or chemical Actual acute exposure to a biological agent (plants, mushrooms, various animals) Information regarding the toxic potential of an agent Possible food poisonings Types of Calls Often Referred Questions regarding treatment of a medical condition (not poisoning) Questions on common bacterial, viral, or parasitic infections General psychiatric questions Questions regarding proper disposal of household agents, such as batteries, bleach, insecticides Questions regarding use of insecticides (e.g., which insecticide to use, how to use it) unless related to a health issue, for example, a person allergic to pyrethrins wanting to know which product does not contain pryrethrins Records of all calls/cases handled by the center shall be kept in a form that is acceptable as a medical record. The regional poison information center should submit all its human exposure data to the Association’s National Data Collection System. The regional poison information center shall tabulate its experience for regional evaluation on at least an annual basis. It 1983 the AAPCC formed the Toxic Exposure Surveillance System (TESS) from the former National Data Collection System. Currently TESS contains nearly 16.2 million human poison exposure cases. Sixty-five poison centers, representing 181.3 million people, participate in the data collection. The information has various uses to both governmental agencies and industry, providing data for product reformulations, repackaging, recall, bans, injury potential, and epidemiology. The summation of each year’s surveillance is published in the Anlerican Jorlrnal sf Emergency Medicine in late summer or fall.

D. HowCallsAreHandled Most poison centers receive requests for information via the telephone. Calls come from both health care professionals and consumers. Only a few requests are received by mail or in person. These often involve medico-legal or complex cases. Most centers can be reached by a toll-free phone number in the areas they serve, as well as by a local number. Busy centers will have a single number that will ring on several lines. Calls are often direct referrals from the 911 system. In most cases poison center specialists are unable to determine the exact bacterial species, so it is difficult to give specific information. When the species is known (as in some food poisonings), specific information is available. Information about culture sensitivities, rates of infection, or potential sources is usually not available, and such calls are often referred to a regional epidemiologist or regional infectious disease agency.

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Poison information specialists listen to the caller, recording the history of the case on a standardized form developed by AAPCC. Basic information such as the agent involved, the amount of agent, time of ingestion, symptoms, previous treatment, and current condition are recorded, as well as patient information such as sex, age, phone number, who is with the patient, relevant medical history, and sometimes patient address. All information is considered a medical record, and is therefore confidential. The case is categorized (using various references) as (1) information only, no patient involved, (2) harmless and not requiring follow-up, (3) slightly toxic, no treatment necessary but a follow-up call is given, (4) potentially toxic, treatment given at home and followup given to case resolution, ( 5 ) potentially toxic, treatment may or may not be given at home, but it is necessary for the patient to be referred to a medical facility, or (6) emergency-an ambulance and/or paramedics are dispatched to the scene. Cases are usually followed until symptoms have resolved. In cases where the patient is referred to a health care facility, the receiving agency is notified. This history is relayed, toxic potential discussed, and suggestions for treatment given.

E. WhatReferencesAreUsed? References used also vary from center to center, but virtually all centers use a toxicology system called POISINDEX, which contains lists of products, their ingredients, and suggestions for treatment. The system is compiled using medical literature and medical specialists throughout the world. Only a few bacterial infections (such as food poisonings) are listed in this resource. After accessing the individual bacterial or food-poisoning entry in the database, the physician or poison information specialist is then referred to a treatment protocol that may be for a general class of agents. An unknown skin irritation or potential infection would deserve a consult with an infectious disease specialist. POISINDEX is available on microfiche, as a CD ROM, over a network, or on a mainframe. It is updated every 3 months. Various texts may also be used. It is very difficult to identify bacterial infections based on infortnation given over the phone, so often the assistance of an epidemiologist and infectious disease specialist is used. Some poison centers have more experience with certain types of poisonings than others, so often one center will consult another on an interesting case. These are often more complex cases or cases involving areas within both centers’ regions. A recent trend has been for various manufacturers not to provide product information to all centers via POISINDEX, but to contract with one poison center to provide for poison information services for the whole country. Product information is given to that center only, and cases throughout the country can only be handled by that one center.

F. How Poison Centers Are Monitored for Quality Most poison centers have a system of peer review in place. One person takes a call, another reviews it. Periodic spot review is done by supervisors and physician staff. General competence is assured by certification and re-certification via examination of physicians and poison information specialists.

Poison Centers and Bacterial Exposure

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G. ProfessionalandPublicEducationPrograms The regional poison information center is required to provide information to the health professionals throughout the region who care for poisoned patients on the management of poisoning. Public education progranx aimed at educating both children and adults about poisoning dangers and other necessary concepts related to poison control should be provided. In the past, several centers provided stickers or logos, such as Officer Ugh, Safety Sadie, and Mr. Yuck, that could be placed on or near potentially toxic substances. While the intent was to identify potentially toxic substances that children should keep away from, the practice has been much curtailed on the new assumption that in some cases the stickers actually attracted children to the products. In the spring of every year there is a poison prevention week during which national attention is focused on the problem of potentially toxic exposures. Many centers run special programs for the public, including lectures on prevention, potentially toxic agents in the home, potentially toxic biological agents, or general first aid methods. Although this week is an important time for poison centers, public and professional education is a yearround commitment. Physicians are involved in medical toxicology rounds, journal clubs, and lectures by specialty consultants. Health fairs, school programs, and various women’s clubs are used to educate the public. The extent of these activities is often determined by the amount of funding received from government, private organizations, and public donations.

H. RelatedProfessionalToxicologyOrganizations ACGIH American Conference of Governmental and Industrial Hygienists Address: Kemper Woods Center; Cincinnati, OH, 45240 Phone: 5 13-742-2020 FAX: 5 13-742-3355 ABAT American Board of Applied Toxicology Address: Truman Medical Center, West; 2301 Holmes St.: Kansas City, MO, 64 108 Phone: 8 16-556-3112 FAX: 8 16-881-6282 AACT American Association of Clinical Toxicologists Address: c/o Medical Toxicology Consultants; Four Columbia Drive; Suite 810: Tampa, FL, 33606 AAPCC American Association of Poison Control Centers Address: 3201 New Mexico Avenue NW: Washington, DC, 20016 Phone: 202-362-7217 FAX: 202-362-8377 ABEM American Board of Emergency Medicine Address: 300 Coolidge Road: East Lansing, MI, 48823 Phone: 5 17-332-4800 FAX: 5 17-333-2234 ACEP American College of Emergency Physicians (Toxicology Section) Address: P.O. Box 61991 1; Dallas, TX, 75261-991 1 Phone: 800-798- 1822 FAX: 214-580-2816

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HPS Hungarian Pharmacological Society Address: Centeral Research Insttitute for Chemistry; Hungarian Academy of Sciences; H-l525 Budapest; P.O. Box 17; Pusztaszeri ut 59-67, Hungary Phone: 36-1-135-21 12 ISOMT International Society of Occupational Medicine and Toxicology Address: USC School of Medicine; 222 Oceanview Ave., Suite 100; Los Angeles, CA, 90057 Phone: 213-365-4000 JSTS Japanese Society of Toxicological Sciences Address: Gakkai Center Building; 4-16, Yayoi 2-chome; Bunkyo-ku; Tokyo 113, Japan Phone: 3-3812-3093 FAX: 3-3812-3552 SOT Society of Toxicology Address: 1101 14th Street, Suite 1100; Washington, DC., 20005-5601 Phone: 202-37 1- 1393 FAX: 202-37 1-1090 e-mail: [email protected] SOTC Society of Toxicology of Canada Address: P.O. Box 517: Beaconsfield, Quebec; H9W 5V1, Canada Phone: 5 14-428-2676 FAX: 5 14-482-8648 STP Society of Toxicologic Pathologists Address: 875 Kings Highway, Suite 200; Woodbury, NJ, 08096-3172 Phone: 609-845-7220 FAX: 609-853-041 1 SSPT Swiss Society of Pharmacology and Toxicology Address: Peter Donatsch; Sandoz Phmna AG; Toxicologtie 88 1/130; CH4132 Muttenz, Switzerland Phone: 41-6 1-469-5371 FAX: 4 l -6 1-469-6565 WFCT World Federation of Associations of Clinical Toxicology Centers and Poison Control Centers Address: Centre Anti-Poisons; Hopital Edonard Herriot; 5 p1 d'Arsonva1; 69003 Lyon, France Phone: 33 72 54 80 22 FAX: 33 72 34 55 67 1.

International Affiliations

The AAPCC and its members attend various world conferences to learn of toxicology problems and new methods used by these agencies. An especially close relationship has formed between the American and Canadian poison center associations. Once a year the AAPCC and CAPCC hold a joint scientific meeting and invite speakers and other toxicology specialists from throughout the world to attend. Some international affiliated organizations are listed with the North American groups above.

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J. Toxicology and Poison Center Web Sites

Association of OccupationalandEnvironmentalClinics This group is dedicated to higher standards of patient-centered, multidisciplinary care emphasizing prevention and total health through information sharing, quality service, and collaborative research. Address: [email protected] FingerLakesRegionalPoisonCenter Address: [email protected] Medical/Clinical/Occupational Toxicology Professional Groups A list of primarily U.S. professional groups interested in toxicology, including a description of each group, their address, phone numbers, and contact names. Keyword: poison centers, toxicology Address: http://www.pitt.edu/-martint/pages/motoxorg.htr-t1 PoisonNet A mail list dedicated to sharing information, problem solving, and networking in the areas of poisoning, poison control centers, hazardous materials, and related topics. The list is intended for health care professionals, not the lay public. The moderators do not encourage responses to individual poisoning cases from the public. Keyword(sj: poisoning, poison control centers II. U.S. POISON INFORMATION CENTERS The Poison Control Center telephone numbers and addresses listed below are thought to be accurate as of the date of publication. Poison Control Center telephone numbers or addresses may change. The address and phone number of the Poison Control Center nearest you should be frequently checked. If the number listed does not reach the poison center, contact the nearest emergency service, such as 91 1 or local hospital emergency rooms. The author disclaims any liability resulting from or relating to any inaccuracies or changes in the phone numbers provided below. This information should NOT be used as a substitute for seeking professional medical diagnosis, treatment, and care. ("Indicates a Regional Center designated by the American Association of Poison Control Centers.)

ALABAMA Birmingham Regional Poison Control Center* Children's Hospital of Alabama 1600 Seventh Avenue, South Birmingham, AL 35233- 171 1 (800) 292-6678 (AL only) (205 933-4050

Tuscaloosrr Alabama Poison Control System, Inc. 408 A Paul Bryant Drive, East Tuscaloosa, AL 35401

(800) 462-0800 (AL only) (205) 345-0600

ALASKA Anchorage Anchorage Poison Center Providence Hospital P.O. Box 196603 3200 Providence Drive Anchorage, AK 995 19-6604 (800) 478-3193 (AK only)

Poison Centers and Bacterial Exposure FairbaA-s Fairbanks Poison Center Fairbanks Memorial Hospital 1650 Cowles St. Fairbanks, AK 99701 (907) 456-7 182

Los Angeles Los Angeles County University of Southern California Regional Poison Center" 1200 North State, Room 1107 Los Angeles, CA 90033 (800) 825-2722 (213) 222-3212

ARIZONA

Orange University of California Irvine Medical Center Regional Poison Center" 101 The City Drive, South Route 78 Orange, CA 92668-3298 (800) 544-4404 (CA only) (7 14) 634-5988

Phoenix Samaritan Regional Poison Center" Good Samaritan Medical Center l 130 East McDowell Road, Suite A-5 Phoenix, AZ 85006 (602) 253-3334 Tucson Arizona Poison and Drug Information Center" Arizona Health Sciences Center, Room 1156 1501 N. Campbell Ave. Tucson, AZ 85724 (800) 362-0101 (AZ Only) (602) 626-6016

Richnlond Chevron Emergency Information Center 15299 San Pablo Avenue P.O. Box 4054 Richmond, CA 94804-0054 (800) 457-2202 (510) 233-3737 or 3738 Sacmrnento Regional Poison Control Center" University of California at Davis Medical Center 23 15 Stockton Boulevard Rm HSF-124 Sacramento, CA 95817 (800) 342-3293 (northern CA only) (9 16) 734-3692

ARKANSAS Little Rock Arkansas Poison & Drug Information Center University of Arkansas College of Pharmacy 4301 West Markham, Slot 522 Little Rock, AR 77205 (800) 482-8948 (AR only) (501) 661-6161

CALIFORNIA

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Fresno Fresno Regional Poison Control Center:': Fresno Community Hospital & Medical Center 2823 Fresno Street Fresno, CA 93721 (800) 346-5922 (CA only) (209) 445-1222

Sal1 Diego San Diego Regional Poison Center" University of California at San Diego Medical Center 225 West Dickinson Street San Diego. CA 92013-8925 (800) 876-4766 (.CA only) (619) 543-6000

Sail Francisco San Francisco Bay Area Poison Center" San Francisco General Hospital 1001 Potrero Avenue Rm 1E86 San Francisco, CA 94122 (800) 523-2222 (315) 476-6600

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12 Sun Jose Regional Poison Center Santa Clara Valley Medical Center 751 South Bascom Avenue San Jose, CA 95128 (800) 662-9886, 9887 (CA only) (408) 299-5112, 5113, 5114

COLORADO Denver Rocky Mountain Poison Center* 1010 Yosemite Circle Denver. CO 80230 (800) 332-3073 (CO only) (303) 629-1 123

CONNECTICUT Furmington Connecticut Poison Control Center University of Connecticut Health Center 263 Farmington Avenue Farmington, CT 06030 (800) 343-2722 (CT only) (203) 679-3456

DELAWARE Wilmington Poison Information Center Medical Center of Delaware Wilmington Hospital 501 West 14th Street Wilmington, DE 19899 (302) 655-3389

DISTRICT OF COLUMBIA Wushingtorz National Capital Poison Center* Georgetown University Hospital 3800 Reservoir Road, North West Washington, DC 20007 (202) 625-3333

FLORIDA Jucksonville Florida Poison Information Center University Medical Center 655 West Eighth Street Jacksonville, FL 32209 (904) 549-4465 or 764-7667 Tduhussee Tallahassee Memorial Regional Medical Center 1300 Miccosukk Road Tallahassee, FL 32308 (904) 68 1-54 1 1

Tampa Tampa Poison Information Center* Tampa General Hospital Davis Islands P.O. Box 1289 Tampa, FL 33601 (800) 282-3171 (FL only) (813) 253-4444

GEORGIA Atluntu Georgia Regional Poison Control Center* Cerady Memorial Hospital 80 Butler Street South East Box 26066 Atlanta, GA 30335-3801 (800) 282-5846 (GA only) (404) 616-9000 Macon Regional Poison Control Center Medical Center of Central Georgia 777 Hemlock Street Macon, GA 3 1208 (912) 744-1 146, 1100or 1427 Sclvunnnlz Savannah Regional Poison Control Center Memorial Medical Center Inc. 4700 Waters Avenue Savannah, GA 31403 (912) 355-5228 or 356-5228

Poison Centers and Bacterial Exposure HAWAII Honolulu Kapiolani Women’s and Children’s Medical Center 1319 Punahou Street Honolulu, HI 96826 (800) 362-3585, 3586 (HI only) (808) 941-4411

IDAHO Boise Idaho Poison Center St. Alphonsus Regional Medical Center 1055 North Curtis Road Boise, ID 83706 (800) 632-8000 (ID only) (208) 378-2707

ILLINOIS Chicago Chicago and NE Illinois Regional Poison Control Center Rush Presbyterian-St. Luke’s Medical Center 1653 West Congress Parkway Chicago, IL 60612 (800) 942-5969 (Northeast IL only) (312) 942-5969

Normal Bromenn Hospital Poison Center Virginia at Franklin Normal, IL 61761 (309) 454-6666 Springfield Center and Southern Illinois Poison Resource Center St. John’s Hospital 800 East Carpenter Street Springfield, IL 62769 (800) 252-2022 (IL only) (217) 753-3330 Urbana National Animal Poison Control Center

13 University of Illinois Department of Veterinary Biosciences 2001 South Lincoln Avenue, 1220 VMBSB Urbana, IL 61801 (800) 548-2423 (subscribers only) (217) 333-2053

INDIANA Indianapolis Indiana Poison Center* Methodist Hospital 1701 North Senate Boulevard Indianapolis, IN 46202-1367 (800) 382-9097 (317) 929-2323

IOWA Des Moines Variety Club Drug and Poison Information Center Iowa Methodist Medical Center 1200 Pleasant Street Des Moines, IA 50309 (800) 362-2327 (5 15) 24 1-6254

Iowa City University of Iowa Hospitals and Clinics 200 Hawkins Drive Iowa City, IA 52246 (800) 272-6477 or (800) 362-2327 (IA only) (319) 356-2922 Sioux City St. Luke’s Poison Center St. Luke’s Regional Medical Center 2720 Stone Park Boulevard Sioux City, IA 51 104 (800) 352-2222 (IA, NE, SD) (712) 277-2222

KANSAS Kansas City Mid America Poison Center

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Kansas University Medical Center 39th and Rainbow Boulevard Room B-400 Kansas City, KS 66160-7231 (800) 332-6633 (KS only) (913) 588-6633 Topeka Stormont Vail Regional Medical Center Emergency Department 1500 West 10th Topeka, KS 66604 (913) 354-6100

Wichita Wesley Medical Center 550 North Hillside Avenue Wichita, KS 67214 (316) 688-2222

KENTUCKY Ft. Tllor?m Northern Kentucky Poison Information Center St. Luke Hospital 85 North Grand Avenue Ft. Thomas, KY 41075 (513) 872-51 11 Louislille Kentucky Poison Control Center of Kosair Children’s Hospital 3 15 East Broadway P.O. Box 35070 Louisville, KY 40232 (800) 722-5725 (KY only) (502) 589-8222

LOUISIANA Houma Terrebonne General Medical Center Drug and Poison Infommation Center 936 East Main Street Hourna. LA 70360 (504) 873-4069

Monroe Louisiana Drug and Poison Information Center

Northeast Louisiana University School of Pharmacy. Sugar Hall Monroe, LA 7 1209-6430 (800) 256-9823 (LA only) (3 18) 362-5393

MAINE Portland Maine Poison Control Center Maine Medical Center 22 Bramhall Stree t Portland. ME 04102 (800) 442-6305 (ME only) (207) 87 1-2950

MARYLAND Baltimore Maryland Poison Center* University of Maryland School of Pharmacy 20 North Pine Street Baltimore, MD 21201 (800) 492-2414 (MD only) (410) 528-7701

MASSACHUSETTS Bostoit Massachusetts Poison Control System* The Children’s Hospital 300 Longwood Avenue Boston, MA 031 15 (800) 682-9211 (MA only) (617) 232-2120 or 735-6607

MICHIGAN Ahian Bixby Hospital Poison Center Emma L. Bixby Hospital 8 1 Riverside 8 Avenue Adrian, MI 49331 (5 17) 263-2412

Poison Centers and Bacterial Exposure Detroit Poison Control Center Children's Hospital of Michigan 3901 Beaubien Boulevard Detroit, MI 48201 Outside metropolitan Detroit; (800) 4626642 (MI only) (3 13) 745-57 11 G r a d Rapids Blodgett Regional Poison Center 1840 Wealthy Street, South East Grand Rapids, MI 49506 Within MI: (SOO) 632-2727

Kalmmcoo Bronson Poison Information Center 252 East Love11 Street Kalamazoo, MI 49007 (800) 442-41 12 616 (MI only) (6 16) 34 1-6409

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Hattiesburg Forrest General Hospital 400 S. 28th Avenue Hattiesburg, MS 39402 (601) 288-3235

MISSOURI Kansas City Poison Control Center Children's Mercy Hospital 2401 Gillham Road Kansas City, MO 64108-9898 (8 16) 234-3000 or 234-3430 St. Louis Regional Poison Center* Cardinal Glennon Children's Hospital 1465 South Grand Boulevard St. Louis, MO 63104 (800) 392-9 111 (MO only) (800) 366-8888 (MO, west IL) (314) 772-5200

MINNESOTA MONTANA Minrleapolis Hennepin Regional Poison Center* 701 Park Avenue South Minneapolis, MN 554 15 (612) 347-3144 (612) 347-3141 (Petline) St. Paul Minnesota1 Regional Poison Center* St. Paul-Ramsey Medical Center 640 Jackson Street St. Paul, MN 55101 (800) 222-1222 (MN only) (612) 221-2113

MISSISSIPPI

Jackson University of Mississippi Medical Center 2500 North State Street Jackson, MS 39216 (601) 354-7660

Dernver Rocky Mountain Poison and Drug Center Denver, CO 80204 (800) 525-5042 (MT only)

NEBRASKA Omaha The Poison Center* Children's Memorial Hospital 8301 Dodge Street Omaha, NE 681 14 (800) 955-9119 (WY, NE) (402) 390-5400, 5555

NEVADA Las Vegas Humana Hospital-Sunrise* 3186 Maryland Parkway Las Vegas, NV 89109 (800) 446-6179 (NV only)

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Reno Washoe Medical Center 77 Pringle Way Reno, NV 89520 (702) 328-4144

NEW HAMPSHIRE Lebanon New Hampshire Poison Center Dartmouth-Hitchcock Medical Center 1 Medical Center Drive Lebanon, NH 03756 (800) 562-8236 (NH only) (603) 650-5000

NEW JERSEY Newark New Jersey Poison Information and Education Systems" 201 Lyons Avenue Newark, NJ 071 12 (800) 962-1253 (NJ only) (973) 923-0764 Plzillipsburg Warren Hospital Poison Control Center 185 Rosberg Street Phillipsburg, NJ 08865 (800) 962-1253 (908) 859-6768

NEW MEXICO Albuquerque New Mexico Poison and Drug Information Center* University of New Mexico Albuquerque, NM 87 131 (800) 432-6866 (NM only) (505) 843-251 1

NEW YORK Buffalo Western New York Poison Control Center Children's Hospital of Buffalo 219 Bryant Street Buffalo, NY 14222 (800) 888-7655 (NY only) (7 16) 878-7654 Mineola Long Island Regional Poison Control Center" Winthrop University Hospital 259 First Street Mineola, NY 1 1501 (516) 542-2323, 2324, 2325 New York City New York City Poison Control Center* 455 First Avenue, Room 123 New York, NY 10016 (212) 340-4494 (213) 764-7667 Nyack Hudson Valley Regional Poison Center Nyack Hospital 160 North Midland Avenue Nyack, NY 10920 (800) 336-6997 (NY only) (914) 353-1000 Rochester Finger Lakes Regional Poison Control Center University of Rochester Medical Center 601 Elmwood Avenue Rochester, NY 14642 (800) 333-0542 (NY only) (716) 275-5151 Syracuse Central New York Poison Control Center SUNY Health Science Center 750 E. Adams Street Syracuse, NY 13210 (800) 252-5655 (3 15)476-4766

NORTH CAROLINA Ashville Western North Carolina Poison Control Center

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Poison Centers and Bacterial Exposure Memorial Mission Hospital 509 Biltmore Avenue Ashville, NC 28801 (800) 542-4225 (NC only) (704) 255-4490 or 258-9907 Charlotte Carolinas Poison Center Carolinas Medical Center 100 Blythe Boulevard Charlotte, NC 28232-2861 (800) 848-6946 (704) 355-4000 Durham Duke Regional Poison Control Center P.O. Box 3007 Durham, NC 277 I O (800) 672-1697 (NC only) (919) 684-8111 Greensboro Triad Poison Center Moses H. Cone Memorial Hospital 1200 North Elm Street Greensboro, NC 2740 1- 1020 (800) 953-4001 (NC only) (919) 574-8105 Hickon Catawba Memorial Hospital Poison Control Center 810 Fairgrove Church Road, South East Hickory, NC 28602 (704) 322-6649

NORTH DAKOTA Fargo North Dakota Poison Center St. Luke’s Hospital 720 North 4th Street Fargo, ND 58 122 (800) 732-2200 (ND only) (701) 234-5575

OHIO Akron Akron Regional Poison Center

17 281 Locust Street Akron, OH 44308 (800) 362-9922 (OH only) (2 16) 379-8562 Canton Stark County Poison Control Center Timken Mercy Medical Center 1320 Tinlken Mercy Drive, North West Canton, OH 44667 (800) 722-8662 (OH only) (216) 489-1304 Cincinnati South West Ohio Regional Poison Control System and Cincinnati Drug and Poison Information Center* University of Cincinnati College of Medicine 231 Bethesda Avenue ML #l44 Cincinnati, OH 45267-0144 (800) 872-5 11 1(Southwest OH only) (513) 558-51 11 Cleveland Greater Cleveland Poison Control Center 2074 Abington Road Cleveland, OH 44106 (2 16)23 1-4455 Col~u~b~w Central Ohio Poison Center* 700 Children’s Drive Columbus. OH 43205 (800) 682-7625 (OH only) (614) 228-1323 Dayton West Ohio Regional Poison And Drug Information Center Children’s Medical Center One Children’s Plaza Dayton, OH 45404- 18 15 (800) 762-0727 (OH only) (5 13) 222-2227

Lorain County Poison Control Center Lorain Community Hospital 3700 Kolbe Road Lorain, OH 44053 (800) 821-8972 (OH only) (216) 282-2220

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Sandusky Firelands Community Hospital Poison Information Center 1101 Decatur Street Sandusky. OH 44870 (419) 626-7423

J

PENNSYLVANIA

Toledo Poison Information Center of Northwest Ohio Medical College of Ohio Hospital 3000 Arlington Avenue Toledo, OH 49614 (800) 589-3897 (OH only) (419) 381-3897

Hershey Central Pennsylvania Poison Center* Milton Hershey Medical Center Pennsylvania State University P.O. Box 850 Hershey, PA 17033 (800) 521-61 10 (717) 531-6111

Youngstown Mahoning Valley Poison Center St. Elizabeth Hospital Medical Center 1044 Belmont Avenue Youngstown, OH 44501 (800) 426-2348 (OH only) (2 16) 746-2222

Lrrncnster Poison Control Center St. Joseph Hospital and Health Care Center 250 College Avenue Lancaster, PA 17604 (717) 299-4546

Znnesville Bethesda Poison Control Center Bethesda Hospital 2951 Maple Ave Zanesville, OH 4370 1 (800) 686-4221 (OH only) (614) 454-4221

OKLAHOMA

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(800) 452-7165 (OR only) (503) 494-8968

Oklalzom City Oklahoma Poison Control Center Children's Memorial Hospital 940 Northeast 13th Street Oklahoma City, OK 73104 (800) 522-4611 (OK only) (405) 27 1-5454

OREGON Portland Oregon Poison Center Oregon Health Sciences University 3 181 South West Sam Jackson Park Road Portland, OR 97201

Philadelphia Philadelphia Poison Control Center" One Children's Center 34th and Civic Center Boulevard Philadelphia, PA 19104 (215) 386-2100 Pittsbtqh Pittsburgh Poison Center* One Children's Place 3705 Fifth Avenue at DeSoto Street Pittsburgh, PA 15213 (4 12) 68 1-6669 Willinrmport The Williamsport Hospital Poison Control Center 777 Rural Avenue Williamsport, PA 17701 (717) 321-2000

RHODE ISLAND Providence Rhode Island Poison Center* 593 Eddy Street Providence, RI 02903 (401) 444-5727

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Poison Centers and Bacterial Exposure SOUTH CAROLINA Chrrrlotte Carolinas Poison Center Carolinas Medical Center 1000 Blythe Boulevard Charlotte. NC 28232-2861 (800) 848-6946

Columbirt Palmetto Poison Center University of South Carolina College of Pharmacy Columbia, SC 29208 (800) 922-1 117 (SC only) (803) 765-7359

SOUTH DAKOTA Aberdeen Poison Control Center St. Luke's Midland Regional Medical Center 305 S. State Street Aberdeen, SD 57401 (800) 592-1889 (SD. MN, ND. WY) (605) 622-5678

Rapid City Rapid City Regional Poison Control Center 835 Faimont Boulevard P.O. Box 6000 Rapid City, SD 57709 (605) 341-3333

Sioux Falls McKennan Poison Center McKennan Hospital 800 East 21st Street P.O. Box 5045 Sioux Falls, SD 571 17-5045 (800) 952-0123 (SD only) (800) 843-0505 (IA, MN, NE) (605) 336-3894

TENNESSEE Knoxville Knoxville Poison Control Center

University of Tennessee Memorial Research Center and Hospital 1924 Alcoa Highway Knoxville, TN 37920 (6 15) 544-9400 hlerrrphis Southern Poison Center, Inc. Lebanheur Children's Medical Center 848 Adams Avenue Memphis, TN 38103-2821 (901) 528-6048

Nashville Middle Tennessee Regional Poison Center, Inc. 501 Oxford House 1161 21st Avenue South B-101VUII Nashville, TN 37232-4632 (800) 288-9999 (TN only) (615) 322-6435

TEXAS Conroe Montgomery County Poison Information Center Medical Center Hospital 504 Medical Center Blvd.

TX 77304 (409) 539-7700 Dallas North Central Texas Poison Center* Parkland Memorial Hospital 5201 Harry Hines Boulevard P.O. Box 35926 Dallas, TX 75235 (.goo) 441 -0040 (TX only) (214) 590-5000 El Pnso El Paso Poison Control Center Thomas General Hospital 48 15 Alameda Avenue El Paso, TX 79905 (915) 533-1244

Galveston Texas State Poison Control Center University of Texas Medical Branch 8th and Mechanic Street

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20 Galveston, TX 77550-2780 (800) 392-8548 (TX only) (7 13) 654-1701 (Houston) (409) 765-1420 (Galveston)

Lubbock Methodist Hospital Poison Control 3615 19th Street Lubbock, TX 7941 3 (806) 793-4366

WASHINGTON Seattle Washington Poison Center P.O. Box 5371 Seattle, WA 98105-0371 (800) 732-6985 (within WA) (206) 526-2121

WEST VIRGINIA UTAH Salt Lake City Utah Poison Control Center* Intermountain Regional Poison Control Center 410 Chipeta Way, Suite 230 Salt Lake City, UT 84108 (800) 456-7707 (UT only) (801) 581-2151

Charleston West Virginia Poison Center’# West Virginia University 3 110MacCorkle Avenue, South East Charleston, WV 25304 (304) 348-4211 (800) 632-3625 (WV only) Parkersburg St. Joseph’s Hospital Center 19th Street and Murdoch Avenue Parkersburg, WV 26101 (304) 424-4222

VERMONT WISCONSIN Burlington Vermont Poison Center Medical Center Hospital of Vermont 1 1 1 Colchester Avenue Burlington, VT 05401 (802) 658-3456

VIRGINIA Charlottesville Blue Ridge Poison Center* University of Virginia Health Sciences Center Box 67 Charlottesville, VA 22901 (800) 451-1428 (VA only) (804) 924-5543 Richmond Virginia Poison Center Virginia Commonwealth University MCV Station Box 522 Richmond, VA 23298-0522 (800) 552-6337 (VA only) (804) 786-9123

Madison Regional Poison Control Center University of Wisconsin Hospital 600 Highland Avenue Madison, W1 53792 (608) 262-3702 Milwaukee Poison Center of Eastern Wisconsin Children’s Hospital of Wisconsin 9000 West Wisconsin Avenue P.O. Box 1997 Milwaukee, W1 53201 (414) 266-2222

WYOMING Omaha The Poison Center* Children’s Memorial Hospital 8301 Dodge Street Omaha, NE 681 14 (800) 955-9119 (WY, NE) (402) 390-5400, 5555

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REFERENCES 1. Harrison, D. L., Draugalis, J. R., Slack, M. K.. and Langly, P. C. (1996). Cost effectiveness of Regional Poison Control Centers. Arch. Intern. Med. 156:2601-2608. 2. CPSC. CPSC Chairman Ann Brown Suggests Information Technology Studyto Support Work of Poison Centers. News Release #94-047, Tuesday March 15, 1994.

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2 Bacterial Biota (Flora) in Foods James M. Jay Uitirlersityoj-Nevada Las Vegas. Las Vegm, NeIlacIa

I. Introduction 23 A. Soils 24 B. Waters 24 C. Human and animal sources 11. CommonFoodborneGenera A. Gram positives B. Gram negatives 28

24 24

24

111. DetectionandEnumeration 28 A. Choice of incubationtimeandtemperature B. Choice of culture medium 28 C. Selective and differential media 39 D. Ratio of numbers 29 IV. PrevalenceinFoods 29 A. Ready-to-use vegetables B. Spices 30 C. Meat products 31 References

1.

28

29

32

INTRODUCTION

Because natural foods come from either a soil or water environment, they may beexpected to contain at least some of the bacteria that are common to these environments unless steps have beentaken to effect their destruction. In addition, some, especially human pathogens, enter the food supply from infected animals and human handlers. Fortunately, only a relatively small percentage of known soil bacteria can be found on foods of plant or animal origin. In the case of foods from fresh and ocean waters, a higher percentage of the bacterial biota from these environments may be associated with such foods due in large part to a less diverse biota in contrast to that of soils.

23

24

Jay

A. Soils The number of bacteria in the top layer of a rich farm soil is typically around one billion per gram. Numbers differ for soils under active cultivation or where cover crops have been plowed under in contrast to those that have not been disturbed for several years. In the former, the bacterial biota is dominated by zymogeneous types that are active degraders of simple plant constituents, and they consist of both gram-positive and gram-negative heterotrophic genera. These bacteria are typically on the surface of plants when they are plowed under, and they are the ones most likely to be found on plant-based foods. Foodborne pathogens such as the proteolytic strains of Clostridium botulinum and Bacillus cereus are soil bacteria. The bacterial biota of stable soils is dominated by the autochthonous or indigenous types that consist of humus feeders. This group is dominated by gram positives such as the mycobacteria and nocardioforms, and they are slow growers. Also, stable soils are populated by autotrophic bacteria that are antagonized by utilizable organic matter. While any or all of these types may appear transiently on plant products, they lack the capacity to adhere, and importantly, they are slow growers or are outcompeted by the zymogeneous biota noted above.

B. Waters The bacterial biota of aquatic-based foods is less diverse than that of soils because of the general lack of humus feeders and chemoautotrophic types in the water column. In the case of fresh waters, the nonphotosynthetic biota is quite similar to that of the surrounding soils due to rain run-off. The significant difference between the biota of fresh and sea waters is that the latter consists essentially only of gram-negative bacteria. Further, except for marine coastal zones, where numbers of nonphotosynthetic bacteria may be high, the biota of the open oceans is quite low. The salinity and lower temperatures of marine waters allow for the existence and growth of halophilic bacteria such as Photobacterium spp. Also, true psychrophilic bacteria are found along with larger populations of psychrotrophic types. Foodborne pathogens such as Vibrio cholerae and Vibrio paralzaemolyticus are found in marine and estuarine waters.

C. Human andAnimalSources Although soil and water may be viewed as the primary and original habitats of all bacteria, some species have lost the capacity to grow and multiply in either of these environments (e.g., whooping cough and syphilis agents). Others maintain the capacity to live in these environments while acquiring the capacity to live as parasites or conlmensuals on human or animal hosts (e.g., the two Vibrio spp. noted above). The largest number of foodborne pathogens are most often found in or on human and animal hosts. Shigella spp. are found only in humans, whereas Salmonella spp. are found in humans and other vertebrate animals.

II. COMMON FOODBORNEGENERA A.

Gram Positives

The gram-positive bacteria most often recovered from foods are summarized in Table 1. The 40 genera listed represent those that may be recovered from fresh, fermented, and

Biota

Bacterial Foods

(Flora) in

25

Table 1 The Most Common Genera of Gram-positive Bacteria Found in Fresh and Processed Foods ~

Group/genus Spore-formers Alicyclobacillus Aneurinibacillus Bacillus Brevibacillus Clostridium Paenibacillus Sporolactobacilhrs Lactics Carnobacterium Enterococcus Lactococcus Lactobacillus Luctospltaem Leuconostoc Oenococcus Pediococcus Streptococcus Tetragenococcus Vagococcro Weissella Corynefomx & related Arthrobacter Colvnebacteriurrr Bifidobncterium Brevibacterium Caseobacter Cellulonlorms Microbacterium Propionibacterium Miscellaneous groups Aerococclrs Brochothrix Deirlococcus Ensipe1othri.x Halobacterium Hdococcus Kocuria Listeria Micrococcus Mycobacterium Plnnococc1rs Rubrobacter Staphylococclts

Morphology

~~

~

~~~

~

Common sources/conunents

R R R R R R R

Canned fruits, juices Same as Bacillus Soils, air, utensils Same as Bacillus Soil, air, utensils Same as Bacillus Soil, chicken feed

R C C R C C C C C C C R

Meats, poultry, vegetation Feces, water, vegetation Raw milk Gastrointestinal tract, vegetation Rumen, anaerobic sludge Vegetation, sugar refineries Grapes, wines Vegetation Raw milk Pickling brines Water, fish, feces Processed meats, vegetation

R R R R R R R R

Soils (rare in foods) Decaying organic matter Raw milk, feces Certain cheeses Meat carcasses Vegetation Vegetation Vegetation, cheeses

C R C R R C C R C R C R C

Raw milk Processed meats Extremely radiation resistant Cattle, raw milk Salt water Salt water Same as Micrococcus Vegetation, zoonotic transmission Air, dust, utensils, handlers Raw milk of infected dairy herds Sea water, seafoods (a halophile) Extremely radiation resistant Nasals/Sl;in of handlers; hides

26

Jay

processed foods. In no single edible food product would one find all of the bacteria listed in Table 1. Fresh foods such as ground meats may be expected to contain 10-20 of the genera noted. The gram positives are listed under three groups based on morphological and physiological features. The endospore-forming bacteria are widespread in nature, and some of these may be found in any fresh food product. As soil organisms, their primary entry into foods is via dust and soil contamination of ingredients and utensils. The genus Alicyclobacillus is of significance as a cause of spoilage of canned fruits and juices. Foodborne pathogens are found in the genera Bacillus and Clostridium. The lactic acid bacteria are very common in nature on plants and plant products, especially fruits. They are responsible for a number of fermented food products including sauerkraut, pickles, and cheeses. Although common on vegetation, the enterococci are also common in the gastrointestinal tract of mammals. The presence and numbers of enterococci in human feces led to their use as indicators of fecal pollution of waters and also to their use as indicators of sanitary quality of some fresh foods. In general, the lactics are as a group the most beneficial of all bacteria to humans relative to the food supply. In addition to the fermented foods they produce, their production of bacteriocins and their overall antagonism of foodborne pathogens have led to their designation and use as “protective cultures” (1). On the other hand, some lactics have been incrinlinated in human infections, with the lactobacilli, pediococci, and enterococci being most often reported (2,3). Coryneform bacteria are a somewhat loosely defined group of organisms that are common in soils. The term “coryneform” refers to a microscopic picture of young cells that display a club or wedge shape and undergo postsnapping division that leads to palisade formations. The cells of older cultures of coryneforms shorten and appear as coccoids, and the genera do not produce spores. As originally defined, they consisted of four genera: Arthrobacter, Cellulonzor~as,Co~vnebacterirrm,and Microbacterirrm (4). The other coryneform and coryneform-like genera in Table 1 share microscopic features similar to the four genera noted. Some of the coryneform genera are involved in food fermentations (e.g., Brevibncterizm and Propiorzibcrcterium), and the genus Bijidobacterium is very important in the gastrointestinal tract of infants. Some bifido strains are used as food starter cultures (5). Because of their association more with human feces than with those of other animals, the bidifobacteria have been suggested as indicator organisms for human fecal pollution of waters (for review, see Ref. 6). The miscellaneous group in Table 1 consists of 13 genera of bacteria that are phylogenetically diverse. The genus Brochotlzrix is significant on processed meats, especially those stored under CO., atmospheres. More information on the gram-positive biota of meats can be obtained from Holzapfel (7). The deinococci and rubrobacteria are important only in irradiated foods because of their extreme radiation resistance. The micrococci are widespread in nature, and some species along with Kocuricr may be found in a number of food products. Significant foodborne pathogens are found among the staphylococci, and they are discussed further in a later chapter. Listeria nzonocytogerzes causes listeriosis in animals and humans and is closely related to Ensipelothri.~rhusioynthiae, which causes erysipeloid in animals. Mycobncterimz pcrratlrberc.clrlosisis of concern in pasteurized milk as the possible cause of Crohn’s disease.

Bacterial Biota (Flora) in Foods

27

Table 2 Most Common Gram-Negative Bacteria Found on Fresh and Processed Foods Group/genus

Morphology

Enterobacteriaceae Arizona Cedecea Citrobacter Edwardsiella Emir1 in Enterobacter Escherichicl Hajk ia Kluyvera Klebsiella Morganella Obsermbacterium Parltoea Proteus Providencin Salmonella Serratia Shigella Yersinia Miscellaneous Acetobacter Acirzetobacter Aerontorras Alcaligenes Alteromonrts Arcobacter Bacteroides Burkholderia Brucella Caityylobncter Chrorlzobacteritlill Deiilobacter Desu~otomaculurrl Flavobucteriunl Glucoizobucter Megasphaera Moraxella Pectinatus Photobacterium Plesiornonas Pseudonlonas Psycltrobncter Ralstonin Shewanella Vibrio Xnnthonronas Zymophilrrs ~~

~~

~~

C = Coccus; R = rod; S = spiral.

Common sources/comments

R R R R R R R R R R R R R R R R R R R

Feces, water Feces, water Vegetation, water, feces Feces, water Vegetation. feces, water Vegetation, water Feces, water Water, feces, meats Feces Vegetation, feces, water Feces, vegetation, water Beer wort Feces, spoiled meats Feces, water, fresh foods Similar to Proteus Poultry, feeds, other animals Vegetation, waters, feces Human feces Water, fresh meats, feces

R R R R R R R R R R R R R R R C R R R R R R R R S R R

Fresh apples, cider mills Water, vegetation, refrig. foods Water, feces. seafoods Water, vegetation, feces Marine waters. seafoods Hogs, cattle, sheep, vegetation Feces, polluted waters (anaerobic) Plant pathogens, vegetation Cows, sheep, hogs; raw milk Raw poultry, milk, bovines Vegetation, raw milk Extremely radiation resistant Canneries, canned foods Vegetation Grapes, honey bees, beerdwines Spoiled beer Soil, water, refrig. foods Spoiled beer Sea water, spoiled fish Water, feces Soil, water, refrig. fresh foods Meat, fish, poultry Tomato wilt disease Rancid butter, marine/fresh waters Waters, seafoods, feces Contain numerous plant pathogens In beer yeasts

28

Jay

B. Gram Negatives Members of the family Enterobacteriaceae constitute the largest phylogenetic group of foodborne gram-negative bacteria, and 19 genera are listed in Table 2. Any or all of these organisms may be found in animal feces under appropriate conditions, hence the common designation “enterics.” All of the salmonellae and shigellae are human pathogens, and some species/strains of Escherichia, Klebsiella, and Yersinia are human pathogens. The four coliform genera are among the enterics (Citrobacter,Enterobacter, Escherichia, and Klebsiella). The genera Citrobcrcter,Enterobacter, and Emjinia are well-known plant inhabitants. Common as food spoilage organisms are some Citrobacter, Hafizia, Puntoetr, Proteus, and Serrcrtia species since they contain psychrotrophic strains. The miscellaneous group in Table 2 includes some that cause human illness (Brucella, Campylobncter, and Vibrio) and others that are well-known plant pathogens (Pseudomonas, Ralstonia, and Xmthomonas). The bacteria that are most conspicuous on refrigerator-spoiled fresh foods such as ground meats are in the genera Acinetobacter, Alcaligenes, Alteronzonns, Moraxella, Psychrobncter, and Pseudornonas, with the latter genus being the single most important of the psychrotrophic spoilage bacteria. More information on the foodborne bacterial pathogens can be obtained from the ICMSF monograph (8) and from the specific chapters that follow in this volume.

111.

DETECTION ANDENUMERATION

On the surface, the detection and enumeration of bacteria in foods could be carried out in the same way as for nonfood products. However, the experiences of many over the years indicate that it is not that simple. Factors that set a food analysis apart from that of, say, a blood or spinal fluid analysis are discussed in the following sections.

A.

Choice of Incubation Time and Temperature

A typical fresh food product contains some bacteria that grow well between 4 and 7°C and some that can grow between 42 and 46°C. Those that grow at the lower temperatures generally require longer incubation times. When colony-forming units (CFU) are to be enumerated, the long incubation can lead to overgrowth of the slow growers by some of the fast growers and spreaders such as Bacillus and Proteus spp. All too often, individuals who are not experienced food microbiologists incubate plates at 37°C and expect results in 18-24 hours. With the exception of Canlpylobacter spp., all foodborne bacteria of significance grow well at 30-32”C, and incubation time should be extended to 48 hours.

B. Choice of Culture Medium Because of the high heterogeneity of the foodborne bacterial biota, electing the proper culture medium is not easy. Those without experience with foodborne bacteria tend to use media that are too rich or complex. Such media tend to allow for overgrowth of some bacteria during prolonged incubations. Before carrying out bacterial determinations on foods, it is strongly recommended that a standard reference method be used: the two recommended reference works are the FDA Bacteriological Amlytical Mar2ual (commonly referred to as BAM) (9) and the Compendium of Methods-for the Microbiological

Bacterial Foods Biota (Flora) in

29

Examination of Foods, referred to as the Compendium (10). Both works contain recommended culture methods for all bacteria of significance in foods in addition to some bioassay, molecular, and genetic methods of analysis for cells and/or their products.

C.SelectiveandDifferentialMedia The use of a selective plating medium such as Macconkey agar to recover gram-negative bacteria from, say, spinal fluid is distinctly different from its use to recover gram-negative organisms from a food product such as fresh ground beef. In the case of spinal fluid, the bacterial biota is neither as heterogeneous nor as large in quantity as for ground beef. Further, when low dilutions of meat homogenates are planted onto the surface of selective media, meat particles and constituents are known to exert a neutralizing effect on the selective agents. The small food particles can serve as microniches for the growth of some bacteria that would otherwise be inhibited by the selective agent.

D. Ratio of Numbers Recovering bacterial pathogens from foods is often like trying to find the proverbial needle in a haystack. When human stool cultures are examined for salmonellae where salmonellosis is suspected, typically these organisms are at levels of 10s-108 CFU/g of stool, although lower numbers are sometimes seen. In this case, the ratio of salmonellae to nonsalmonellae is such as to allow for direct enumeration and isolation using appropriate selective media. The ratio of salmonellae to the background bacterial biota of a product such as ground beef is such that direct enumeration and isolation are all but impossible since the usually low numbers of salmonellae are typically overwhelmed by the much larger background biota. Thus, nonselective enrichments are necessary, and they are widely used in the food microbiology laboratory. The foregoing deals with the determination of viable numbers of bacteria, variously referred to as aerobic plate count (APC) or standard plate count (SPC) of organisms per gram or CFU. Rapid nonculture methods have been developed for all significant foodborne pathogens, and the references noted above are excellent sources. Although these methods have proven to beinvaluable, there continues to be a valuable place for the classical culture methods. Nonculture methods typically detect both viable and nonviable cells, and when it is desirable to know if viable cells are present, a culture method is required. The impact of molecular and other nonculture detection methods on bacteriological assessments of foods has been reviewed and discussed by Feng (1 l).

IV. PREVALENCE IN FOODS A.

Ready-to-UseVegetables

In consideration of the high incidence and prevalence of bacteria in the soils and waters that basic food products grow in, it should not besurprising to find relatively high numbers on products that have not been subjected to bactericidal treatments. The prevalence of

30

Jay

Table 3 Log,, Mean APC/g of a Variety of Ready-to-Use Vegetable Products from Four Different Countries Product

N

Means

Location

Ref.

Salad mix Parsley. fresh Parsely, frozen Lettuce Salad mix Carrot sticks Celery Chopped lettuce Cole slaw Radishes Bean sprouts Lettuce (hydroponic)

2 11 14 24 3 15 4 4 1 1 2 34

7.85 6.67 5.26 7.82. 7.92 5.60 4.50 52 0 7 .OO 6.04 7.76 7.35

England Germany Germany Italy Italy United States United States United States United States United States United States United States

12 13 13 14 15 16 16 16 17 17 18 19

One sample/month over a 23-month period.

bacteria in a variety of ready-to-use vegetable products is presented in Table 3, and it can be seen that numbers between 1 and 10 million/g are common. Additional ready-to-use vegetables are presented in Table 4 where on day 0 the APC means/g were between 10,000 and 1 million. However, after these products were held for 4 days at 4"C, the APCs of most increased to > 1 milliodg (20). Typically, the bacterial biota of products of this type consists of organisms on the plant products during their growth (e.g., many of the lactic genera and gram-negative bacteria such as Envinin and Enterobacter). Also, a significant part of the biota would be expected to come from handlers, cutting and processing equipment, storage containers, and the air.

Spices B. Numbers of bacteria per gram of retail-store spices are known to be high, and some examples are presented in Table 5 of spices examined in Austria (21). Of the 10 products presented, rosemary had the lowest APC, with a mean of 4.83 log/g, and black pepper Table 4 Log,,, Aerobic Plate Counts/g on Fresh-Cut, Ready-to-Use Vegetables Stored at 4"Ca Vegetables Chopped lettuce Salad mix Cauliflower florets Sliced celery Cole slaw mix Carrot sticks Broccoli florets Green peppers

APC day 0

APC day 4

4.85 5.35 4.82 5.67 5.13 5.13 5.58 5.99

5.63 6.05 5.45 6.59 6.95 6.27 6.59 7.22

The products had a recommended shelf life of 7 days at 4°C. Source: Ref. 20. a

Bacterial Biota (Flora) in Foods

31

Table 5 Log,,, Numbers/g of Microorganisms Found in Some Spices in Vienna, Austria Allspice Caraway Chili powder China spice curry Ginger Nutmeg Black pepper Rosemary Thyme

6.89 6.04 5.91 7.42. 6.79 7.00 5.53 7.34 4.83 6.7 1

Source: Ref. 21

and China spice were the highest, with 7.34 and 7.42/g, respectively. Typically, products of this type contain large numbers of molds, especially black pepper. Among the bacteria, sporeformers are usually abundant as well as gram-positive cocci such as the micrococci. Gram-negative bacteria are either absent or quite low in numbers in products of this type if they have been properly dried and packaged. Some spices (e.g., rosemary and thyme) are also known to possess some antibacterial properties.

C. Meat Products The bacterial biota of fresh ground meats is reflective of carcass contamination during slaughtering, processing, and storage. Since such products are kept at refrigerator temperatures, it is not surprising to find large numbers of psychrotrophic bacteria. Mean log,, APCs/g of ground beef tested in 1914 and in 1994 are presented in Table 6. Retail store fresh ground beefmay be expected to contain >lo5 CFU/g. This is true for the data presented in Table 6 prior to 1994 where the USDA reported a mean of only 3.90 CFU/g for563 nationwide samples of fresh ground beef (29). This surprisingly low numTable 6 Examples of Log,, Aerobic Plate Count (APC) Numbers of Bacteria/g of Fresh Ground Beef Year

Number

APC mean

Ref.

1914 1936 1957 1964 1975

44 41 96 26 5 5 140 140 563

7.23 6.49 7.5 1 6.72 5.30J 7.95b 6.30 6.81 3.90

22 23 24 25 26 26 27 28 29

1976 1977 1994 ~~~~~

On day 0. After 5 days at 4.5"C.

32

Jay

ber could be a reflection of the downward trend for appearance of bacteria in such products due in part to more attention being paid to sanitation throughout the production cycle or in part to the methodology employed in gathering these data. For example, the mean of 3.90 is the result of APC determinations being made at 35°C for 24 hours. For fresh meat products, this number would be higher if incubation was between 25 and 32°C for at least 48 hours. It was shown by Rogers and McCleskey (24) that the APC of retail store ground beef was considerably higher when plates were incubated at 7°C for 7 days than when incubated for 48hours at 30°C. From Table 6 it may be noted that >2.5 log cycle increase occurred after a 5-day incubation at 4.5"C in the meats sampled by Goepfert and Kim (26). Regarding the history of our knowledge of bacteria in fresh ground meats, the earliest proposed APC standard for acceptable product was made by Marxer in 1903 (30), and the limit was 106/g. The numbers in Table 6 for samples tested in 1914 and 1936 are reflective of this general level. The only U.S. state ever to adopt bacteriological standards for ground beef was Oregon, and it allowed up to 5 X 106/g (see Ref. 31). The Oregon law was in effect in 1973-1977. The APC limit in the Oregon standard is a reflection of what was considered safe and achievable under good production practices during that time. With APCs of approximately 106/g, fresh ground beef was rarely the source of foodborne outbreaks in the United States prior to the late 1980s. Whether the increased incidence of foodborne outbreaks from ground meats are related to lower numbers of background organisms is of possible concern, and it has been addressed (32). Of the genera of bacteria listed in Tables l and 2, most investigators have found around 20 in beef, poultry, fish, and sausage products. Ayres (33) identified 19 genera from refrigerated beef and 30 genera from the surfaces of beef, sausage, fish, and chicken (34). In a study of pork sausage, Sulzbacher and McLean (35) identified 16 genera. In a Canadian study, 19 genera were identified from fresh ground beef (36). In a study of the number of genera in fresh and spoiled ground beef, nine genera were identified from 69 isolates from the beef when fresh, and following the frank refrigerator spoilage of samples from the same batch, only four genera were found, with 16 of the 19 isolates being pseudomonads (37).

REFERENCES 1. Holzapfel. W. H., Geisen, R., and Schillinger, U. (1995). Biological preservation of foods with reference to protective cultures, bacteriocins and food-grade enzymes.Znt. J. Food Microbiol.. 24:343-362. 2. Aguirre, M., and Collins. M. D., (1993). Lactic acid bacteriaand human clinical infection. J. Appl. Bacteriol., 75:95-107. 3. Jett, B. D., Huycke,M. M., and Gilmore. M.S . (1994). Virulenceof enterococci. Clin. Microbiol. Rev., 7:462-478. 4. Jones, D. (1975). A numerical taxonomic study of coryneform and related bacteria. J. Gen. Microbiol., 8752-96. 5. Hughes, D. B., and Hoover, D. G. (1991). Bifidobacteria: Their potential for use in American dairy products. Food Teclznol., 45(4):74, 76, 78-80. 82. 6. Jay, J. M. (2000). Modern Food Microbiology, 6th ed., Aspen Publishers, Gaithersburg,MD. 7. Holzapfel, W. H. (1998). The gram-positive bacteria associated with meat and meat products. (1998). In The Microbiology of Meat and Poultn (A. Davies and R. Board, eds.). Aspen Publishers, Gaithersburg, MD, 1998.

Bacterial Biofa (Flora) in Foods

33

8. ICMSF. (1996). Microorganisms in Foods 5. Microbiological specifications of Food Pnthogem, Aspen Publishers, Gaithersburg, MD. 9. (1995). FDA Bacteriological Analytical Manual, 8th ed. AOAC Int., Gaithersburg, MD. 10. Vanderzant, C., and Splittstoesser, D. F., eds. (1992).Cornpendiurn of Methods for the Microbiological Examination of Foods. American Public Health Association, Washington,DC. 11. Feng, P. (1997). Impact of molecular biology on the detection of foodborne pathogens.Mol. Biotechnol., 7:267-278. 12. Brocklehurst, T.F., Zaman-Wong, C.M., and Lund, B. M. (1987). A note on the microbiology of retail packs of prepared salad vegetables. J. Appl. Bacteriol., 63:409-415. 13. Kaferstein, F. K. (1976). The microflora of parsley. J. Milk Food Technol., 39:837-840. 14. Ercolani, G.L. (1976). Bacteriological quality assessment of fresh marketed lettuce and fennel. Appl. Environ. Microbiol., 31:847-852. (1976). 15. Vescovo, M., Orsi,C., Scolari, G., and Torriani, S . (1995). Inhibitory effectof selected lactic acid bacteria onmicroflora associated with ready-to-eat vegetables.Lett. Appl. Microbiol., 21: 121-125. 16. Garg, S . , Churey, J. J., and Splittstoesser, D. F. (1990). Effect of processing conditions on the microflora of fresh-cut vegetables. J. Food Prot., 53:701-703. 17. Rafil, F., Holland, M.A., Hill, W. E., and Cerniglia, C. E. (1995). Survivalof Shigellaflerneri on vegetables and detection by polymerase chain reaction. J. Food Prot., 58:727-732. 18. Jinneman, K. C., Trost, P. A., Hill, W. E., Weagant, S . D., Bryant, J. L., Kaysner, C. A., and Wekell, M. M.(1995). Comparisonof template preparation methods from foods for amplification of Escherichia coli 0157 Shiga-like toxins type I and I1 DNA by multiplex polymerase chain reaction. J. Food Prot., 58:722-726. 19. Riser, E. C., Grabowski, J., and Glenn, E. P. (1984). Microbiology of hydroponically-grown lettuce. J. Food Prot., 47:765-769. 20. Odumeru, J. A., Mitchell, S . J., Alves, D. M., Lynch,J. A., Yee, A. J., Wang, S . L., Styliadis, S., and Farber,J. M. (1997). Assessment of the microbiological qualityof ready-to-use vegetables for health-care food services. J. Food Prot., 60:954-960. 21. Kneifel, W., and Berger, E. (1994). Microbiologicalcriteria of random samples of spices and herbs retailed on the Austrian market. J. Food Prot., 57393-901. 22. Weinzirl, L., and Newton, E. B. (1914). Bacteriological analyses of hamburger steak with reference to sanitary standards. Am. J. Public Health, 4:413-416. 23. Elford, W. C. (1936). Bacterial limitations in ground fresh meat. Am. J. Public Health, 26: 1204-1206. 24. Rogers, E. R., and McCleskey, C. S . (1957). Bacteriological quality of ground beef in retail markets. Food Technol., 11:318-320. 25. Jay, J. M. (1964). Beef microbial quality determinedby extract-release volume (ERV).Food Technol.. 18(10):133-137. 26. Goepfert, J. M., and Kim, H. U. (1975). Behavior of selected foodborne pathogens in raw ground beef. J. Milk Food Technol., 38:449-452. 27. Westhoff, D., and Feldstein, F. (1976). Bacteriological analysisof ground beef. J. Milk Food Prot., 39:401-404. 28. Foster, J. F., Fowler. J. L., and Ladiges, W. C. (1977). A bacteriological surveyof raw ground beef. J. Food Prot., 40:790-794. 29. U.S. Department of Agriculture, Food Safety and Inspection Service. (1996). Nationwide federal plant raw ground beef microbiological survey,August 1993-March 1994, USDA, Washington, DC. und der Haltbarkeit des Fleisches 30. Marxer, A. (1903). Beitrag zur Frage des Bakteriengehaltes bei gewohnlicher Aufbewahrung. For-tsckr. Vet.-Hyg., 1:328. 31. Carl, K. E., (1975). Oregon’s experience with microbiological standards for meat. J. Milk Food Technol., 38:483-486. 32. Jay, J. M. (1997). Do background microorganisms play a role in the safety of fresh foods? Trends Food Sci. Teclmol., 8:421-424.

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33. Ayres, J. C. (1960). Temperature relationshipsand some other characteristicsof the microbial flora developing on refrigerated beef. Food Res.. 25:l-18. 34. Ayres, J. C. (1960). The relationship of organisms of the genus Psezdonzorzns to the spoilage of meat, poultry and eggs. J. Appl. Bacteriol. 23:471-486. 35. Sulzbacher, W. L., and McLean, R. A. (1951). The bacterialflora of fresh pork sausage.Food Techno].,5:7-8. 36. Lefebvre, N., Thibault, C., and Charbonneau, R. (1992). Improvementof shelf-life and wholesomeness of ground beef by irradiation. 1. Microbial aspects. Meat Sci., 32203-213. 37. Jay, J. M. (1967). Nature, characteristics, and proteolytic properties of beef spoilage bacteria at low and high temperatures. Appl. Microbial.. 15:943-944.

Aeromonas hydrophila Carlos Abeyta, Jr. U.S. Food and Drug Administration, Borhell, Wadzington

Samuel A. Palumbo U.S. Department of Agriculture, Philadelphia, Pennsylvania

Gerard N. Stelma, Jr. U.S. Emironmentrrl Protection Agewcy, Cincinnati, Ohio

I. Introduction 36 11. Classification and Characteristics 37

A. Classification B. Characteristics

37 40

111. Pathogenicity 40 A. Aeromonas infections B. Enterotoxins 41 C. Other virulence factors

40 43

IV. Control 44

A. Low temperature 44 44 B. Modified atmosphere C. pH and NaCl 45 D. Nitrite 45 E. Bacteriocins 46 46 F. Water activity 46 G. Chemical food additives H. Multiple barrier approach 47 I. Inactivation 47 V. Detection and Isolation 50 A. General considerations 50 B. Procedures 50 C. Sampling, enrichment, and isolation procedures References 52

50

Abeyta et al.

36

I.

INTRODUCTION

Aeromonas hydrophila translated simply means a "water-loving, gas-producing' ' bacterium. It was first reported by Zimmerman (1) and then Sandrelli (2), who isolated the organism and demonstrated its pathogenicity in frogs. Since that time, Aeromoncrs species have been isolated from other aquatic animals such as finfish, shellfish, crustaceans, and amphibians (3-5). This group of organisms is pathogenic to many aquatic species and causes hemorrhagic septicemia (red sore disease) in many fresh water pond cultured and wild native fish (6,7). It is well established that Aeronzonas is a component of the intestinal flora of healthy fish (8,9) and is widely distributed in nature. A. hydrophila is commonly found in watersheds (5,10,11). The prevalence and distribution of Aeronzonns spp. in fresh water habitats and the marine environment are well documented. It appears that Aeronzonas spp. may not be truly indigenous to the marine environment but may have a transient existence after entering salt water via rivers or sewage inputs. Aeromonas that are shed in sewage can multiply in sewage lines to significant numbers prior to discharge into receiving waters (12). Aeromonas is also present in terrestrial warm-blooded animals (13), including humans (14), and have been associated with three types of human illnesses; extraintestinal, wound, and gastrointestinal infections (14-20). Extraintestinal diseases are the most common, with a mortality rate as high as 61% in septicemic patients that are immunosuppressed. Wound infections involving Aeromonas usually are linked with injuries incurred during recreation or other activities in the aquatic environment (21-23). For gastrointestinal infections, a common source of A. hydrophila outbreaks is such water supplies as natural mineral springs, marine water environments, chlorinated and unchlorinated domestic supplies, and watersheds polluted by sewage effluents (24,25). There are statistical relationships between Aerolnonas species in water sources and health risks. However, there is no firm documentation of serious health effects after exposure to domestic waters containing Aeromonas (24). A serious health risk may be present for immunosuppressed patients with underlying malignancies after exposure to domestic and recreational waters containing Aeromonns species (14,26,27). The presence of Aeromonas in the food chain is well documented. This organism can be readily isolated from seafoods, foods of terrestrial animal origin such as meats, dairy products, poultry, and vegetables (28-34). Its presence in the food chain is of great concern because of its capability of growth at refrigerated temperatures (35). Seafood products are the most common food source of Aeromorzas. Both finfish and shellfish are known reservoirs of this bacterium. In 1986, A. hydrophila was the causative agent of an oysterborne outbreak in Florida (36). An attack rate of 100% (N = 7) was reported. A. hydrophila was isolated from the remaining uneaten oysters and from stool specimens from patients. In cases of foodborne bacterial illnesses in which shellfish are implicated, laboratories have included Aeronzonns in the general screening for causative microorganisms. Controversy concerning pathogenicity still remains. At present there are great difficulties assessing the regulatory significance of Aeromonas species in foods (37). From a clinical standpoint, the organism is no doubt of great concern to immunocompromised patients with underlying malignancies. Foods containing high levels of Aeromonns species destined to these individuals should be regarded hazardous. On the other hand, the role of foods containing high levels of Aeromonas spp. destined for healthy individuals is

Aeromonas hydrophila

37

uncertain. Although Aeromonas has been implicated in cases of gastrointestinal illness from foods, the exact mechanism by which Aerornonas species cause disease is not understood fully. Confusion concerning the assessment of pathogenicity remains unsolved. This was made evident in humanfeeding studies of virulent viable strains of Aeromonas species (high doses ranging from lo4 to lolo) involving 57 volunteers; only 2 developed mild diarrhea (38). One explanation for the failure of these strains to elicit diarrhea was presented. Kirov et al. observed the inability of Aerornonas species to adhere to the intestinal mucosa (39). This observation was noted in the shift of pilated environmental isolates toward nonpilated forms once the intestinal tract was infected. The nonpilated forms isolated from stools would be noninfective when used in challenge studies. The objectives of this chapter are to review the available information concerning this emerging recognizable pathogen. The reader is given information needed to assess further epidemiology, pathogenicity, management, detection, and isolation.

II. CLASSIFICATION AND CHARACTERISTICS A.

Classification

The genus Aeromonas was first proposed by Kluyver and Van Niel in 1936, as described by Popoff in Bergey’s Manual of Systematic Bacteriology (40). Various investigators divided the motile aeromonads into species including A. hydrophila, A. punctnda, A. formicans, A.liquefuciens, A. anaerogenes,A. proteolytica, and A. cnviae andA. sobria (41,42). The complexity of the problem was shown by Popoff et al., who found that A. hydrophila could be divided into distinct groups based on their divergence by DNA/DNA hybridization (43). The mesophilic A. hydroplzilngroup is collectively referred to as motile aeromonads. The genus Aeromonas consists of two well-separated groups of organisms: (a) the psychrophilic nonmotile aeromonads known as Aeromonas sahonicidn subspecies (pathogenic to fish but not to humans) and (b) the mesophilic motile aeromonads (the A. hydrophila group), which are divided into the species A. hydrophila, A. sobria, and A. caviae. The A. hydrophila group is associated with human illnesses. Most recent, a fourth motile Aeromonas spp., A. veronii, has been added to this list (44); it is distinguished from the other motile aeromonads by a positive ornithine decarboxylase test. Motile aeromonads belong to the family Vibrionaceae. Separation of the genus Aeromonas from related members of the family can be accomplished by biochemical reactions (Table 1). Aeromonas species are easily confused with group F Vibrios (V. fluvialis). Vibrios and motile aeromonads can be differentiated by their phenotypical reactions: salt requirement for growth and 0/129 sensitivity. Motile aeromonads are facultative anaerobic, gram-negative straight rods with rounded ends, measuring approximately 0.3- 1.O pm in diameter and 1.O-3.5 pm in length. They are motile by means of a single polar flagellum in liquid medium, and their metabolism is both respiratory and fermentative. They break down carbohydrates to acid or acid and gas (CO? and H?). Nitrate is reduced to nitrite, and oxidase and catalase are produced. Optimum growth temperatures range from 22 to 28°C. NaCl tolerance ranges from 0 to 4%, and tolerance to pH ranges from 5.2 to 9.8. They produce exoenzymes such as amylase, protease, phospholipase, and DNase and are resistant to vibriostatic agent 01129 (2,4-diamino-6,7-diisopropylpteridine). The mol% G + C of the DNA is 57-63 (Bd, T,,,).

Abeyfa et a/.

38

Table 1 Differentiation of the Genus Aeromonas from Other Genera of the Family Vibrionaceae Test Plesiowonas

Vibrio

Inhibition by 0/129 10 P8 150 P8 Na' requirement for growth Oxidase Gas from glucose Fermentation of inositol Fermentation of mannitol Ornithine decarboxylasea Growth on thiosulfate-citrate-bile salt-sucrose agar

Aeronzonns R

R S

R R

S

+ +

-

+ + +

-

+ + +

-

-

-

+ D +

-

-

R. Resistant: S . sensitive; D, differs among biotypes. verorlii is positive (+).

Table 2 Differential Phenotypic Characteristics salmorlicidu

of Motile Aeronlonas Species and Aeromonas b

Motile Characteristics

Aerorttonns spp.

1

,..I

.

. L

A. snlnlonicida

"

Motility Oxidase Ornithine decarboxylasea Arginine dihydrolase Indole production Starch, gelatin, DNA and RNA hydrolysis Citrate (Simmons') Citrate (Christensen's) ONPG Brown water-soluble pigment Growth without NaCl Fermentation of sucrose Fermentation of maltose, galactose, and trehalose Fermentation of cellobiose, lactose. and sorbitol Fermentation of glycerol, dulcitol, rhamnose, inositol, xylose, raffinose, and adonitol Rods in singles and pairs Coccobacilli in pairs, chains, and clumps Growth in nutrient broth at 37°C Aeronzonas varonii is ODC Differs among strains. c Aberrant strains occur. Source: Ref. 30.

i

+ + + + + db d

+ +

+ +"

+ -

+ + + -

d

+ + +c

d

-

-

-

+

-

-

+

+ -

+.

t

I

Aeromonas hydrophila 39

Abeyta et al.

40 Table 4 DifferentiationAmong the Aeromonas Izydrophia Group ~

~~

~~

~

Biochemical test“

A. hydrophila

Motility Esculin hydrolysis Growth in KCN L-Arginine utilization L-Lysine utilization L-Arabinose utilization Fermentation of salicin Fermentation of sucrose Fermentation of mannitol Breakdown of inositol Acetoin from glucose (Voges-Proskauer) Gas from glucose Indole production Oxidase P-Hemolysis H2S from cysteine

A. sobria

A. cavine

+

+ + + + + + + +

+b

+ + + + + + + + -

-

+

+

+ + -

+, Typically positive; -, typically negative. Source: Ref. 45. B. Characteristics The differential characteristics of motile aeromonads from nonmotile Aeromonns species are presented in Table 2. The common characteristic that distinguishes the A. hydrophila group (motile aeromonads) from A. snlnlonicicla subspecies of course is motility, but other similarities include lack of pigmentation, growth in nutrient broth at 37”C, colonial morphology, and monotrichous flagellation in liquid medium. In contrast, A. sdnzonicida does not grow at 37°C (optimum growth is at 22-25°C) and are nonmotile. Aeromonads can be easily grown in most routinely bacteriological selective and differential media (Table 3). On nonselective media such as trypticase soy agar, they are difficult to distinguish morphologically among members of the Enterobacteriaceae. The leading factors to consider in choosing the optimal culture medium is the type of sample matrix and the selective agent that will eliminate other competing organisms. Speciation of the motile aeromonads has been proposed by numerous investigators (Table 4). Biochemical reactions that are important for speciating among motile Aeromonns are based on esculin hydrolysis, growth in potassium cyanide (KCN) broth, salicin fermentation at 26”C, gas from glucose, and H2S from cysteine but not from thiosulfate.

111.

PATHOGENICITY

A. Aeromonas Infections Gastroenteritis is the most common foodborne illness attributed to the A. hydrophila group. About three quarters of Aerornorzns gastroenteritis cases are “choleralike,” characterized by watery stools and a mild fever; vomiting may also occur in children under 2 years of age. The other one quarter of cases are “dysenterylike,’ ’ characterized by blood and mucus

Aeromonas

41

in the stools (17). Aeromonns-associated diarrhea is normally mild and self-limiting (19); however, severe cases of both types of diarrhea have been observed (46-50). Aeromonads also have been implicated as the cause of localized wound infections, pneumonia, and such disseminating infections as bacteremia or septicemia and meningitis (16,18,19,5155). Although disseminating Aeromorlns infections may originate in infected wounds (16), the patient's gastrointestinal tract is considered to be the source of Aeromonns species in those infections (56). Therefore, disseminating infections should also be considered as potentially foodborne. One of several controversial issues regarding Aeromonas pathogenicity is the question of whether the aeromonads are all opportunistic pathogens, capable only of attacking hosts with impaired body defenses, or whether some of these organisms are sufficiently virulent to pose a threat to normal hosts as well. The fact that Aeromonas species are associated most frequently with diarrhea in the very young or in older adults (17,57-59) and the unusually high rate of isolation of aeromonads from patients with hematological malignancies (60) suggest that theyare opportunists. On the other hand, there is accumulating evidence that Aeromoms spp. also infect normal adults. George et al. identified Aeromonas isolates as the primary infectious agents responsible for gastroenteritis in a number of adult patients with no underlying disorders (20), and several literature reports describe severe cases of bothcholeralike (46,6 1) and dysenterylike (48) diarrhea in individuals who were otherwise healthy. Surprisingly, even bacteremia (51) and septicemia (53), which are usually associated with immunocompromised hosts (14,56), have been observed in immunocompetent young adults. It can be argued that immunocompetent individuals infected by aeromonads were affected by unrecognized predisposing conditions; however, it is quite likely that some highly virulent strains exist among the members of this diverse genus, which consists of at least 9 validated and/or proposed species (62) and 13 known hybridization groups that are extremely heterogeneous in biochemical structural and genetic properties (63). B. Enterotoxins The most controversial issue related to Aeromonns pathogenicity concerns the relative roles of various putative enterotoxins in causing diarrhea. There are reports of both heatstable (56"C, 10 min) and heat-labile cytotonic enterotoxins and cytotoxic enterotoxins, some related and others unrelated to cholera toxin. The controversy is not over the variety, which is not unusual for a diverse group of organisms, of enterotoxins reported, but over the fact that researchers who reported evidence for one of these toxins usually were unable to observe any of the others. The various putative enterotoxins produced by Aeromonas species are described below. Ljungh et al. reported partially purifying an enterotoxin with a molecular weight of 15 kD that was stable after treatment at 56°C for 10 minutes (64,65). This molecule, which was serologically unrelated to cholera toxin (CT), caused fluid accumulation in the permanently ligated rabbit ileal loop (RIL), rounding of Y-l cells without death, and stimulation of cyclic AMP (adenosine monophosphate) synthesis. Further evidence for a heat-stable cytotonic enterotoxin was provided by Chakraborty et al., who reported cloning the cytotonic enterotoxin gene into Escherichia coli (66). Culture filtrates of the clone caused elongation of Chinese hamster ovary (CHO) cells and fluid accumulation in the RIL after treatment at 56°C for 20 minutes. These activities also were unrelated to CT. Both of these groups reported that the P-hemolysin was inactive in the RIL.

42

Abeyfa et al.

Potomski et al. (67) reported using affinity chromatography to isolate an A. sobria toxin that cross-reacted with CT, caused rounding of Y-l cells and fluid accumulation in both RILs and infant mice after heating at 56°C for 20 minutes. All of these activities were reportedly neutralized by antiserum to CT. Schultz and McCardell (68) also reported rounding of Y-l cells and stimulation of cyclic AMP synthesis by Aeronzorzns culture filtrates that were heated at 56°C for 20 minutes. These activities were partially neutralized by antiserum to CT. They also reported that DNA from strains producing the cytotonic enterotoxin reacted with one or more synthetic oligonucleotide probes coding for CT. Chopra and Houston (69) reported purifying a cytotonic enterotoxin that caused fluid accumulation in the RIL, stimulated cyclic AMP synthesis, and caused elongation of C H 0 cells. The purified toxin had a molecular weight of 44 kDa, was free of hemolytic and cytotoxic activities, and was not cross-reactive with antiserum to CT. The biological activities of the purified toxin were heat labile at 56°C. The first evidence for a cytotoxic enterotoxin was provided by Cumberbatch et al. (70), who reported a correlation between cytotoxic and RIL activities. All of their cytotoxic isolates also were hemolytic. They found no evidence for a separate cytotonic activity in any of their isolates. Turnbull et al. (71) and Burke et al. (72) also observed a strong correlation between enterotoxin production and hemolytic activity. Asao et al. provided the first direct evidence for a cytotoxic enterotoxin (73). They purified a hemolysin from A. hydrophila strain AH-l to electro-phoretic homogeneity and observed that it was cytotoxic to Vero cells and had enterotoxic activity in both the RIL and suckling mouse assays. The hemolysin had a molecular weight of 50 kDa and was heat labile at 56°C for 5 minutes. Stelma et al. (74) showed that the Asao hemolysin was P-hemolysin and that antiserum to the purified hemolysin completely neutralized the RIL activities of filtrates of P-hemolytic Aeronzonas isolates, indicating that P-hemolysin alone can cause the changes in intestinal permeability associated with diarrhea. These researcher also used polyclonal antiserum to show serological cross-reactions between the hemolysin purified from the Japanese strain, AH- 1, and hemolysins produced by isolates from diverse geographic origins. Several additional studies provided evidence that the Aeromonns P-hemolysins are a family of molecules related to the "aerolysin" originally described by Bernheimer and Avigad (75). Rose et al. (76,77) purified a 52 kDa protein toxin that possessed hemolytic, cytotoxic, and enterotoxic activities as well as serological cross-reactivities to both CT and the Asao (AH-l) hemolysin. The biological activities of their toxin were neutralized by homologous antiserum and antiserum against the AH-l hemolysin but not by antiserum against cholera toxin. Asao et al. (78) demonstrated that the hemolysin produced by A. hydrophila CA1 1, a U.S. Gulf Coast isolate, was related immunologically to AH-l hemolysin but also possessed unique antigenic determinants. Millership et al. (79), Kozaki et al. (80), and Stelma et al. (81) provided evidence that hemolysins from A. sobt-in and A. veronii also possessed enterotoxic activities and were serologically related to AH-l hemolysin. Evidence from several surveys suggests that the cytotoxic enterotoxins are the most common Aerornorzas enterotoxins. Cumberbatch et al. found no cytotonic activity in the filtrates of 96 isolates (70). Likewise, Johnson and Lior found no cytotonic activity in the filtrates of 73 isolates (82), and Seidler et al. found cytotonic activity in the filtrates of only 20 of 330 isolates (6%) (83). Stelma et al. found evidence for cytotonic activity in one of 24 isolates (74), but this was later shown to be caused by sublethal doses of partially denatured cytotoxic enterotoxin after heating at 56°C (84).

Aeromonas hydrophila

43

The absence of significant enterotoxic activity in the P-hemolysin purified by Ljungh et al. (64) may be due to differences in the purification procedures. Ljungh et al. used a six-step procedure that recovered only 0.6% of the original hemolytic units (64). In contrast, Asao et al. used a two-step procedure that recovered 65% of the original hemolytic units and probably yielded a product closer to the native molecule in all of its properties (73). The failure of the E. coZi clones carrying the A. hydrophila hemolysin gene to cause fluid accumulation in the RIL (66) was later shown to be due to the inability of the E. coli to release the hemolysin from the cells (85). Although the heat-labile cytotoxic enterotoxins appear to be the mostcotnmon Aeromonas enterotoxins, the relative roles of these toxins and the cytotonic e,nterotoxins in Aerornonas diarrhea are not known. Determination of the relative roles of these toxins will require the development of better animal models and either the use of strains that produce only one toxin or the use of transposon mutagenesis to inactivate various toxins one at a time. It has been agreed generally that A. cctvirre isolates were noncytotoxigenic and were not enteric pathogens (17,86). However, Namdari and Bottone recently reported detecting a heat-stable cytotoxin in culture filtrates of isolates grown in double-strength trypticase soy broth (TSB) (87). They, like earlier investigators, did not detect enterotoxin in filtrates of A. caviae grown in single-strength TSB. Several recent studies have also linked A. caviae to diarrhea in very young children and individuals aged 50 years or more (8890).

C. OtherVirulenceFactors The negative results of the human feeding study performed by Morgan et al. provided evidence that strains producing enterotoxin active in the RIL are not always diarrheagenic and that multiple virulence factors are involved (38). The strains used in that study did not possess either adhesion factors for colonization of the intestine or ability to invade tissues. Several studies have provided evidence that some aeromonads possess adhesion factors that correlate with the possession of pili (39,91,92). The most interesting study was that of Kirov et al. (39), who observed that environmental enterotoxigenic isolates possessed nutnerous pili, which appeared to be lost once infection was established. This suggests that in future studies environmental isolates may be more appropriate for human feedings than clinical isolates. The relationship between virulence factors and ability of Aeronzor~zsstrains to cause disseminating infections has not been established. Lethality tests with both normal mice and mice immunosuppressed by x-irradiation have focused primarily on relating virulence to biotype or phenospecies (93,94). The results of those studies and one of invasiveness to mammalian cells (95) indicated that A. hydrophila and A. sobria were inherently more virulent than A. caviae, but with significant strain-to-strain variation within a species. One property linked to virulence is a surface array protein (S layer) commonly found in human isolates from extraintestinal infections. The role of the S layer in human and animal infections is not clear, but it appears to be substantially different from the role of the S layer of A. salmonicida in fishdisease (96,97). Other properties of motile aeromonads that have been linked to virulence in other bacterial species include the ability to invade mammalian cells (95,98), resistance to serum bactericidal effects (99,100), production of a siderophore capable of removing iron from transferrin (101,102), a mechanism for utilization of iron from heme compounds (102), and production of proteases (103). Loss of

et

44

Abeyta

al.

viability after growth in broth containing 0.5% glucose (suicide phenomenon) has been associated with both enteropathogenicity and virulence (104), but the significance of this phenomenon is not known. The relative significance of each of these putative virulence characteristics cannot be determined until appropriate animal models are developed, preferably models in which the animals are compromised in a way that mimics the condition of the susceptible human host. i

IV. CONTROL The presence, survival, and growth of bacteria in foods can be controlled by the applying the three Ks: keep them out, keep them from growing, and/or kill them. The A. hydrophila group occurs widely in the aquatic environment (105), and this undoubtedly represents the major source of these bacteria in foods. Their widespread occurrence in virtually all fresh and processed foods surveyed attests to their ubiquitous presence in foods (105). It would thus appear that it is difficult if not impossible to keep them out of foods. Based on this, control of their levels would then depend on extrinsic and intrinsic factors such as low temperature, modified atmosphere, pH/acid, salt and water activity, and the use of food preservatives to keep them from growing and on measures such as high temperatures (55°C and above), irradiation, and low pH, especially when adjusted with organic acids, to kill them.

A.

Low Temperature

Low-temperature holding (5°C) or refrigeration of fresh and processed foods has traditionally been relied on by the food industry to prevent the growth of foodborne pathogens. However, A. hydrophila, along with other foodborne pathogens, has been observed to grow at holding temperatures typically used for fresh and processed foods (106). It has long been observed that A. hydrophila can grow at 5°C or below (35,107,108). Palumbo et al. (32) determined that naturally occurring A. hydrophila could grow competitively in various retail foods of animal origin during storage for one week at 5°C. Callister and Agger (34) made a similar observation for fresh vegetables held at 5°C. Using ground pork inoculated with A. hydrophila, Palumbo (109) verified the low-temperature competitive growth of this bacterium in a food and determined that additional factors such as NaC1, vacuum packaging, and pH can also affect its growth. Since A. hvdrophila can readily and competitively grow in foods held at 5"C, factors other than low refrigeration assume greater importance in controlling its growth in foods.

B. Modified Atmosphere Members of the family Vibrionaceae are facultative anaerobes (42). Broth studies (1 10) and surveys of red meats stored under various atmospheres (30,3 1,35,111-113) have verified this. Based on broth studies, Palumbo et al. (1 10) determined that overall A. hydrophila grew as well anaerobically as it did aerobically. The observations from the red meat storage surveys indicated that the meats' microflora and/or storage temperature rather than the packaging atmosphere appears to control the numbers and types of the bacteria that develop and are detected. When conditions favor the development of Pseudomonas

.

.

I~

Aeromonas hydrophila

45

species or lactic acid bacteria, these bacteria will dominate the Aeromonas species (31,109,113).

C. pH andNaCl After temperature, pH and NaCl are the next most important food parameters controlling the growth of most bacteria in foods. As a typical gram-negative bacterium, A. hydrophila is fairly sensitive to acid or low pH (<5.5) (35,114) and high NaCl (>4.5%) (35); in addition, as shown in Table 5, the limiting pH and NaCl levels are temperature dependent. While the observations in Table 5 were generated in brain-heart infusion (BHI) broth, similar responses were seen during studies of the bacterium in ground pork in which pH and NaCl interacted to restrict the growth of A. hydrophila at lower levels than with either factor individually (109). Using a multifactorial approach, Palumbo et al. (110,115) studied the individual effects and interactions of temperatures, pH, NaC1, and sodium nitrite on the growth kinetics of the bacterium in BHI broth. In these two studies, all factors interacted to increase lag and generation times as pH decreased and the NaCl level increased. For most studies described, HC1 was the acidulant; however, Palumbo and Williams (1 14) used different acidulants and observed differences in the response of A. hydrophila based on the acidulant used, with acetic and lactic acids being the most restrictive and H2S04and HC1 being the least so. Overall, the order of effectiveness for the acids tested are (listed from most restrictive to the most permissive): acetic, lactic, tartaric, citric, H2S04,and HCl. Their activity appeared to be related to their pK,s. D. Nitrite Sodium nitrite in combination with NaCl acts as the curing agent in cured meat products. Its continued permitted use in cured meat products is based on its anticlostridial activity. While members of the A. hydrophila group generally cannot grow in cured meats, they can be isolated from these products (1 16,117). The growth-inhibiting mechanism is sodium nitrite combined with their brine content, pH (about 6.0), vacuum packaging, and low storage temperatures (1 10). These conditions provide an environment in which A.

Table 5 Influence of Temperature on pH and NaCl Limits for A. hydrophila K144 Grown Aerobically in Brain-Heart Infusion Broth Limits of Temperature (C)

28

4

PH G at 5.5 NG at 4.5 NG at 5.5

G. Growth: NG, no growth. Highest level tested. Source: Ref. 35.

NaCl (9%) G at 4 3 G at 3.5 NG at 4.5

46

Abeyfa et al.

Izydrophiln cannot grow readily or compete with the normal flora of lactobacilli, micrococci, and yeasts found in these products.

E. Bacteriocins Bacteriocins are polypeptide antimicrobial compounds produced by various strains of lactic acid bacteria (LAB). There is increasing interest today in the “natural” preservation of foods, and the use of lactic acid cultures and/or the bacteriocin(s) isolated from them have been studied for their effectiveness against spoilage and foodborne pathogens. Lewus et al. (1 18) reported on a study in culture broth, which investigated the activity of various bacteriocin-producing LAB against A. hydrophila. They observed that LAB isolated from meats as well as known LAB cultures would inhibit the bacterium. Santos et al. (1 19) observed that the bacteriocin(s) of a dairy starter culture inhibited A. hydrophila in both skim and ewe’s milk. However, in studies by Kalchayanand et al. (120), neither nisin (4000 activity units/mL) nor pediocin (4000 activity units/mL) was individually effective against the bacterium in broth, but when combined with a preliminary heating or freezing treatment (stresses to damage the cell membrane and cell wall), A. hydrophila became sensitive. At this time, bacteriocins would appear to have some potential uses against this bacterium, but conditions for their optimal activity and effectiveness need to be developed.

F. WaterActivity Water activity (a,) is ameasure of the amount of free water available for microbial growth. Since all microbes need free water for growth, any food substance that binds water can control microbial growth. Some common food substances that can bind water include various salts including NaCl (its activity is discussed above), sugars and carbohydrates, and amino acid and proteins. In perhaps the only study of its kind currently available, Santos et al. (121) studied the effect of a, (adjusted with NaC1, glycerol, and polyethylene glycol) on the growth of three strains (food isolates) of A. hyc/rophiln at 28, 10, and 33°C. The minimum a, for growth varied with strain, temperature, and type of humectant, with NaCl being the most inhibitory and glycerol being the least at comparable a,$s.

G. ChemicalFoodAdditives Various miscellaneous chemicals (many are GRAS [generally regarded as safe]) are added to foods, and in addition to their other functions, they can control the growth of foodborne pathogens, including A. lzydoyhilu. Some of these include essential oils, liquid smoke, food preservatives, and polyphosphates. Stecchini et al. (122) studied the effects of the essential oils of clove, coriander, nutmeg, and pepper in botha model system and noncured cooked pork and observed that the bacterium was inhibited. Liquid smoke prepared from several species of wood also inhibited A . h,dr-ophi/a (123). When the food preservatives methyl p-hydroxybenzoate and potassium sorbate were tested against four psychrotrophic foodborne bacteria, A . hydrophila was the most sensitive (134). When Venugapal et al. (125) testedthe food preservatives butylated hydroxyanisole, propylhydroxy parabenzoate, and sodium tripolyphosphate against A. hydrophila, they observed that protease secretion was more sensitive than growth. When Palumbo et al. (126) tested the food polyphosphates Sodaphos, Hexaphos, sodium pyrophosphate, and sodium tripolyphosphate against the bacterium individually in culture broth, they observed only small changes

4

I

Aeromonas hydrophila

47

in growth kinetics (lag and generation times). However, when combined with 3.5% NaCl, the number of viable A. hydrophila declined to an undetectable level. This effect was also noted when the combination was tested in ground pork.

H. MultipleBarrierApproach As is apparent from the above discussion on the influence of specific factors in controlling the growth of A. hydrophila, most factors (NaCl [expressed both as a percent and as a,], sodium nitrite, and pH), at the levels generally encountered in foods, individually cannot restrict the growth of the bacterium in foods. Research from this laboratory has indicated that multiple barriers (factors) or the multifactorial approach (two or more factors at less than maximum inhibitory levels) can successfully be used to inhibit foodborne pathogens such as Listeria ~~lonocytogenes (127), Shigella fiexl1eri (128), Yersinia enterocolitica (129), E. coli 0157:H7 (130), and A. h,,drOphih (110,115). From these studies, predictive models or equations have been developed to allow description of how changes in such culture (food) parameters as temperature, pH, NaCl level, and atmosphere can alter a bacterium's growth kinetics (e.g., lag and generation times). Often, small changes in a single parameter can bring about dramatic increases in lag and generation times and a decreased hazard frotn that particular pathogen. These equations have been incorporated into a user-friendly, Windows-based computer software program (Pathogen Modeling Program, version 5.1, available from the Microbial Food Safety Research Unit (Eastern Regional Research Center [ERRC], U. S. Department of Agriculture, 600 E. Mermaid Lane, Wyndmoor, PA 19038 or from the ERRC website (WWW.ARSERRC.GOV). I. Inactivation There are several food-processing operations that can inactivate bacteria and other microorganisms. These include heating (cooking), irradiation, sanitizing and disinfecting, and acidification. In addition, treatments such as the lactoperoxidase system have been shown to inactivate bacteria in foods. 1. Heating Heating is one of the primary treatments for the destruction of pathogenic and spoilage bacteria in foods. As withother bacteria, the thermal resistance of A. hydrophila is affected by factors such as growth temperature, age of the culture, and heating menstruum [buffer or different foods] (13 1,132). Nishikawa et al. (133) determined that A. hydrophila was more heat sensitive than Salmonella typhiwzuiurn and E. coli 0157:H7 when heated in either hamburger or egg yolk. Condon et al. (13 1) calculated a DS5"C= 0.17 minute and a Z = 5.1 1°C for a single strain when heated in buffer. Using both clinical and food strains, Palumbo et al. (132) determined DlsJcof 5.2 and 4.3 minutes, respectively, when heated in saline or raw milk and a Z = 6.2"C. These few studies suggest that this bacterium's thermal resistance is similar to that of other gram-negative bacteria found in foods and the bacterium should be inactivated by the heat treatments given many food products during their normal processing

2. Irradiation Irradiation along with heating represents a means of putting energy into a system (culture broth or food) to inactivate microorganisms. Though there are very few studies on the

Abeyta et al.

48

radiation resistance of A. hydrophila (134,135), it does appear to be relatively radiation sensitive and pasteurizing doses aimed at eliminating other foodborne pathogens such as SuZmoneZZa and E. coli 0157 :H7 should also eliminate A. hydrophila (136). 3. Chlorine and Other Sanitizers Cattabiani evaluated the influence of various food plant sanitizers (disinfectants), including chlorine, on four strains of A. hydrophila (137). The results of his study are presented in Table 6; in general, the bacterium appears to have susceptibilities similar to other gramnegative bacteria found in foods. Knochel studied the chlorine resistance of motile Aeromonas species using two different methods and observed that Aeromonas species were more susceptible to chlorine that other gram-negative bacteria such as E. coli, Klebsiella species, and Pseudomonas aeruginosa (138). As can be seen from the data in Table 6 , A. hydrophila is inactivated readily by chlorine at the levels used for the treatment of drinking water. However, the bacterium can be isolated from chlorinated water supplies (24,139,140), even when the test for E. coli is negative (24,140). The recovery of the organism from chlorinated water may be explained as posttreatment recontamination, the presence of unusually large numbers of A. hydrophila, or the presence of organic matter that can inactivate the added chlorine. An alternate explanation may be offered: A. hydrophila is known to be injured by sanitizer treatments (141) and, when selective media are used to isolate the bacterium after treatment, the bacterium is not recovered and thought to be absent. Thus, the presence of the

,

Table 6 Sensitivity of A. hydrophila to Disinfectants Time of exposure at 25°C (min) Concentration

5

10

+ + + +

-

-

3+/l3+/1-

1+/31+/3-

-

-

-

3+/1-

2+/2-

1

Compound Sodium hypochlorite

Quaternary ammonium compound

Iodoform

2-Chlorophenol

Glutaraldehyde

5 PPm 2.5 ppm 1.25 ppm 0.625 ppm 0.31 ppm 1:12,500 1:25,000 1:50,000 1:100,000 10 PPm 5 PPIn 1 PP" 0.2% 0.1% 0.05% 0.125% 0.0625% 0.03 % 1

-, Sensitive. reduction of four or more log cycles in viable count; Source: Ref. 137.

-

+ + 3+/1+ + + + -

3+/1-

+

+. resistant,

+

+ + 1+/3+ + -

+

Aeromonas hydrophila

49

bacterium in such foods as poultry carcasses and vegetables may represent contamination via the potable water supply. 4. Organic Acids In the study by Palumbo and Williams (1 14) cited above in the section on pH and NaC1, the influence of organic acids in combination with temperature and NaCl on the growth kinetics (lag and generation times) of A. hydrophila was investigated. They also observed that certain combinations were lethal to the bacterium, These findings are presented in Fig. 1. Again, the form of the acidulant was important, with acetic and lactic acids being the most toxic and the two inorganic acids being the least toxic. While these findings suggest that A. hydrophila should not be a problem in various fermented and pickled foods, actual experimental studies and food surveys gave mixed observations. Aytac and Ozbas (142) determined that A. hydrophila inoculated into yogurt mix decreased to undetectable during the fermentation; however, Knochel and Jeppesen (117) were able to isolate the bacterium from 10% of the mayonnaise-based salads, often at levels of >105/g.

5. Lactoperoxidase System The enzyme lactoperoxidase (LP) in the presence of thiocyanate and hydrogen peroxide can produce an active antimicrobial system in milk; this system is bactericidal for gramnegative bacteria including A, hydrophila. Santos et al. (143) observed that A. hydrophila decreased to undetected in broth, skim milk, and ewes’ milk and indicated that the LP system, when used in combination with low temperatures, could be useful in controlling the presence of this bacterium in fluid dairy products. Santos et al. (144) determined that the LP system could effectively reduce the levels of A. hydrophila during the manufacture of the Spanish fresh sheep’s cheese Villalon. It would appear that the LP system could provide an adjunct to good manufacturing practices in controlling A. hydrophila in dairy foods in general.

4.4 4.0

S

p

3.6

W

,$ 3.2

2

2.8

S 2.4

8 2.0

l4

1.6

1.2 0

50

100

150

200

250

300

Hours at 5°C Fig. 1 Effect of different acids (HC1, sulfuric, tartaric, citric, acetic, and lactic) on the decline in viable count of A. hydrophila in BH1 broth at pH 5.0 and 5°C (0.5% NaCl). Dashed line-lower limit of detection (log,, = 1.33).

Abeyta et al.

50

V.

DETECTIONANDISOLATION

A.

GeneralConsiderations

In handling of suspect samples, immediate analysis upon arrival in the laboratory is preferred. Motile aeromonads have the capability of proliferating at refrigeration temperatures. If samples are to be analyzed within a few days, store at refrigeration temperatures. Samples kept longer than one week should be stored at -20 or -72°C. An enrichment procedure is necessary when analyzing frozen foods and foods that contained injured aeromonads or low levels of motile aeromonads.

B. Procedures Procedures suggested for isolating and enumerating motile aeromonads from foods are found in the Bacteriological AnalyticalMarzual (BAM) (45). These procedures have been used in Food and Drug Administration laboratories for the analysis of various foods samples. Since the publication of these methods, other procedures have been developed and are recommended in conjunction with BAM methodology for isolation and enumeration of motile aeromonads. These procedures are discussed in the next section.

C. Sampling,Enrichment,andIsolationProcedures The following procedures are based on analysis of a 25 mL or 50 g analytical unit at 1:9 (sample/diluent) ratio. For samples containing less that 25 g, add enough diluent to maintain a 1:9 ratio. For samples requiring enrichment techniques, the following procedures should be followed. Aseptically weigh a 25 g sample into a sterile wide-mouthed, screw-cap jar (500 mL) or other appropriate container (i.e., stomacher bags). Add 225 mL of sterile trypticase soy broth with ampicillin (TSBA) and blend for 2 minutes Loosen jar cap about 1/4 turn and incubate 24 t 2 hours at 35 ? 2°C. After incubation of enrichment broth for 18-24 hours at35"C, transfer 3 mm loopfuls of inoculum onto Macconkey agar (MAC), peptone-beef extract-glycogen (PBG) agar, and Yersinia selective agar (YSA) base supplemented with cefsulodin and novobiocin as described in BAM to yield isolated colonies. A recent recommendation is to use starch ampicillin (SA) agar instead of PBG and YSA. For enumerating aeromonads in TSBA, inoculate a 3- or 5- tube most probable number (MPN) series containing TSBA from serial dilutions. Incubate broth tubes for 18-24 hours at 35°C. Follow procedures as described above for isolation of aeromonads. For samples not requiring an enrichment procedure, aseptically weigh 25 g of sample into an appropriate container and add 225 mL of sterile 0.1% peptone water. Make appropriate serial dilutions and surface plate 0.1 mL portions onto selective media, as described below, with a sterile bent glass rods to distribute the entire inoculum evenly over the surface of the media. Starch-ampicillin (SA) agar developed by Palumbo et al. (32) is recommended for direct surface plating of samples. Starch hydrolysis was selected since this enzyme activity is largely restricted to Aerornorzas and Vibrio species. The use of ampicillin effectively suppresses growth of coliforms and members of the family Enterobacteriaceae. SA has been used effectively for red meats, chicken, raw milk, seafoods, and fresh vegetables. Most recent, marine environmental samples including shellstock oysters, sediment, and marine waters were evaluated by SA direct plating for enumeration of motile aeromonads (see Table 7). Results indicated that direct plating with SA was

Aeromonas

51

Table 7 Evaluation of Starch-Ampicillin Agar in Recovering Motile Aeromonads from Shellfish-Growing Waters in Humbolt Bay, Eureka, California Media Sample lo)

MCA/TSBA ( MPN)a

SAA/TSBA (MPN)b

MCA & SAA/TSBA (CFU)' (MPN)'

SAA

Oysters Water Sediment

1.17'(60)' 2.17 (90) 3.69 (100)

1.40 (70) 2.23 (90) 3.76 (100)

1.47 (70) 2.34 (90) 4.11 (100)

0.15 (10) 0.91 (70) 3.37 (100)

(I2 =

Most probable number(MPN) determinations in tryptic soy brothwith ampicillin (TSBA) plated onto MacConkey agar (MCA). h Plated onto starch-ampicillin agar (SAA). c Combined MCA and SAA. Colony forming unit determinations in direct plating onto SAA. e Log,(, perg or mL. Percent positive of motile aerornonads detected.

ENRICHMENT OR DIRECT PLATING

c SELECTIVE AGARS .1 OXIDASE (

c

A . HYDROPHlL 5.

+) MEDIUM(AHM)

SALT TOLERANCE OR VlBRlOSTATlC 01129

c DIFFERENTIATION AND CONFIRMATION .1 CONVENTIONAL TEST OR RAPID DIAGNOSTIC TEST (i.e., API) Hemolysin Gas from glucose H,S from cysteine Esculin hydrolyis Acetoin from glucose KCN Arginine Arabinose Salicin Sucrose

Fig. 2 Schematic diagram for the isolation of motile aeromonads. (From Ref. 45.)

52

Abeyta et al.

effective with sediment samples, but not for shellstock oysters and marine water, in which the enrichment procedure showed an advantage over SA direct plating. Identification and confirmation of motile aeromonads can be accomplished by following the schematic diagram of Fig. 2. Aeromonas hydrophila medium (AHM) described by Kaper et al. (145) is useful for rapid presumptive identification and differentiation from coliforms and enterics. AHM is a single tube medium testing for fermentation of mannitol and inositol, ornithine decarboxylation, indole production, motility, and H,S production from sodium thiosulfate and cysteine. Studies by Abeyta et al. found this medium useful in identifying environmental marine isolates (1 1). The efficacy of the multitest screening AHM was determined. Of the 1396 strains positive for oxidase, 76% (1,065) gave typical reactions in AHM. Of the 1065 isolates, 95% were confirmed as motile aeromonads. Other rapid test systems such as API 20E, VITEK, and MICRO-ID are useful in rapid identification of typical motile aeromonads, however, these systems have their limitations. For example A. hydrophila and V.JEuvialisare related closely biochemically. Such additional tests as salt tolerance are recommended before a final identification can be reached.

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33. 34.

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37. 38.

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79. Millership, S. E., Barer, M. R., Mulla, R. J., and Maneck, S. (1992). Enterotoxic effects of Aeromonas sobria hemolysin in a rat jejunal perfusion system identified by a specific neutralization with a monoclonal antibody. J. Gen. Microbiol., 138:261-267. 80. Kozaki, S., Asao, T., Kamata, Y., and Sakaguchi, G. (1989). Characterization of Aeromonas sobria hemolysin by use of monoclonal antibodies against Aeromonas hydrophila hemolysins. J. Clin. Microbiol., 27:1782-1786. 81. Stelma, G. N.. Jr., Johnson, C. H., and Spaulding, P. L. (1988). Experimental evidence for enteropathogenicity in Aeromonns veronii. Can. J. Microbiol., 34:877-880. 82. Johnson, W. M., and Lior, H. (1981). Cytotoxicity and suckling mouse reactivity of Aeronzonas hydrophila isolated from human sources. Can. J. Microbiol., 27:1019-1027. 83. Seidler, R. J., Allen, D. A., Lockman, H.,Colwell, R. R., Joseph,S. W., and Daily. 0. P. (1980).Isolation enumeration and characterization of Aeronzonas from polluted waters encountered in diving operations. Appl. Environ. Microbiol., 39:lOlO-1018. Kaylor, L. O., and Johnson, C. H. ( 1986). 84. Bunning, V. K., Crawford, R. G., Stelma, G. N., Jr., Melanogenesis in murine B 16 cellsexposed to Aerontonas hydrophilu cytotoxic entertoxin. Can. J. Microbiol., 32:815-819. 85. Chakraborty, T., Huhle, B., Bergbauer, H., and Goebel, W. (1986). Cloning expression and mapping of the Aeronlonas hydrophila aerolysin gene determinant in Escherichiacoli K12. J. Bncteriol., 167:368-374. 86. Janda, J. M., Reitano, M., and Bottone, E. J. (1984). Biotyping of Aeromonas isolates as correlate to delineating a species-associated disease spectrum. J. Clin. Microbiol.. 19:4447. 87. Namdari, H.,and Bottone, E. J. (1990). Cytotoxin and enterotoxin production as factors delineating enteropathogenicity of Aerontonas caviae. J. Clin. Microbiol., 28: 1796-1798. 88. Kuijper, E. J., Zanen, H. C. and Peeters. M. F. (1987). Aerontorzas-associated diarrhea in the Netherlands. Ann. Intern. Med., 106:640-641. 89. Kuijper, E. J., and Peeters, M. F. (1991). Bacteriologicaland clinical aspects of Aeromonasassociated diarrhea in the Netherlands. Experientia, 47:432-434. 90. Namdari, H., and Bottone, E. J. (1990). Microbiologic and clinical evidence supporting the role of Aerornonns caviae as a pediatric enteric pathogen. J. Clin. Microbiol., 28:837-840. 91. Clark, R. B., Knoop, F. C., Padgitt, P. J., Hu, D. H., Wong, J. D., and Janda, J. M. (1989). Attachment of mesophilic aeromonads to cultured mammalian cells. Curr. Microbiol., 19: 97- 102. 92. Hokama, A., Honma, Y., and Nakasone, N. (1990). Pili of an Aerontonas hydrophila strain as a possible colonization factor. Microbiol. Intmz~nol.,34:901-915. 93. Brenden, R. A., and Huizinga, H. W. (1986). Susceptibilityof normal and X-irradiated animals to Aeromonas hydrophila infections. Curr. Microbiol., 13:129-132. 94. Janda, J. M., Clark, R. B., and Brenden, R. (1985). Virulence of Aeromonas species as assessed through mouse lethality studies. Curr. Microbiol., 12:163-168. 95. Watson, I. M., Robinson, J. O., Burke, V., and Gracey, M. (1985). Invasiveness ofrleromonas spp in relation to biotype virulence factors and clinical features. J. Clin. Microbiol., 22:4851. 96. Janda, J. M., Oshiro, L. S., Abbott, S. L., and Duffey, P. S. (1987). Virulence markers of mesophilic aeromonads: Association of the autoagglutination phenomenon with mouse pathogenicity and the presence of a peripheral cell-associated layer.Irlfect. Inmun., 55:30703077. 97. Kokka, R.P.. Vedros, N. A., and Janda, J.M. (1991). Characterization of classic and atypical serogroup 0 : l l Aerornonns: Evidence that the surface array protein is not directly involved in mouse pathogenicity. Microb. Patlzog., 10:71-79. 98. Pazzoglia, G., Sack, R. B., Bourgeois, A. L., Froehlich, J., and Eckstein, J. (1990). Diarrhea and intestinal invasiveness of Aerontonas strainsin the removable intestinal tie rabbit model. Ilfect. Immun., 58:1924-1931.

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99. Janda, J. M., Brenden, R., and Bottone, E. J. (1984). Differential susceptibility to human serum by Aeromonas spp. Curr. Microbiol.. 11:325-328. 100. Palumbo, S. A., Bencivengo, M. M., Del Corral, F., Williams, A. C., and Buchanan, R. L. (1989). Characterization of the Aeromonas hydrophila group isolated from retail foods of animal origin. J. Clin. Microbiol., 27:854-859. 101. Barghouthi, S., Young, R., Olson, M. 0. J., Arceneaux, J. E. L., Clem, L. W., and Byers, B. R. (1989). Amonobactin, anovel tryptophan- or phenylalamine-containingphenolate siderophore in Aeromonas hydrophila. J. Bacteriol., 171:1811-1816. 102. Massad, G., Arceneaux, J. E. L., and Byers, B. R. (1991). Acquisition of iron from host sources by mesophilic Aeromonas species. J. Gen. Microbiol., 137:237-241. and distribution of extracellu103. Leung, K.-Y.,and Stevenson, R. M. W. (1988). Characteristics lar proteases from Aeromonas hydrophila. J. Gen. Microbiol., 134:151- 160. 104. Namdari, H., and Bottone, E.J. (1988). Correlationof the suicide phenomenon in Aerolnonas species with virulence and enteropathogenicity. J. Clin. Microbiol., 262615-2619. 105. Palumbo, S . , Abeyta, C., Jr., and Stelma, G. N., Jr. (1992). Aeromonas hydrophila Group. In Comperlditun of Methods for the Microbiological Examinationof Foods, 3rd ed., APHA, Washington, D.C., pp. 497-515. 106. Palumbo, S. A. (1986). Is refrigeration enough to restrain foodborne pathogens? J. Food Prot., 49:1003-1009. 107. Eddy, B. P., and Kitchell, A. G. (1959). Cold-tolerant fermentative gram-negative organisms from meat and other sources. J. Appl. Bacteriol., 2257-63. and temperature characteristics 108. Rouf, M. A., andRigney, M.M. (1971). Growth temperatures of Aeronzonas. Appl. Microbiol., 22:503-506. 109. Palumbo, S . A. (1988). The growth of Aeromonns hydrophila K144 in ground pork at 5°C. Int. J. Food Microbiol., 7:41-48. 110. Palumbo, S. A., Williams, A. C., Buchanan, R. L., and Phillips, J. G. (1992). Model for the anaerobic growth of Aeromonas hydrophila K144. J. Food Prot. 55:260-265. 111. Gill, C. O., and Reichel, M. P. (1989). Growthof cold-tolerant pathogensYersinia enterocolitica, Aeromonns hydrophila and Listeria monocytogenes on high-pH beef packaged under vacuum or carbon dioxide. Food Microbiol., 6:223-230. 112. Lee, B. H., Simard, R. E., and Holley, R. A. (1985). Effectof temperature and storage duration on the microflora, physicochemical and sensory changesor vacuum- or nitrogen-packed pork. Meat Sci., 13:99-112. 113. Grau, F. H., Eustace, I. J.. and Bill, B. A. (1985). Microbial flora of lamb carcasses stored at 0°C in packs flushed with nitrogen or filled with carbon dioxide. J. Food Sci., 50:482485, 491. 114. Palumbo, S. A., and Williams, A. C. (1992). Growth of Aeronlonas hydrophila as affected by organic acids. J. Food Sci., 57:233-235. 115. Palumbo, S. A., Williams, A. C., Buchanan, R. L., and Phillips, J. G. (1991). Model for the aerobic growth of Aerontonas hydrophila K144. J. Food Prot., 54:429-435. 116. Gobat, P.-F., and Jemmi, T. (1993). Distribution of mesophilic Aeromonas species in raw and ready-to-eat fish and meat products in Switzerland. Znt. J. Food Microbiol., 20:117120. 117. Knochel, S., and Jeppesen, C. (1990). Distributionand characteristics of Aeromonas in food and drinking water in Denmark. Int. J. Food Microbiol., 10:317-322. 118. Lewus, C. B., Kaiser, A., and Montville, T. J. (1991). inhibitionof foodborne bacterial pathogens by bacteriocins fromlactic acid bacteria isolated from meat. Appl. Environ. Microbiol., 57:1683-1688. 119. Santos. J. A., Lopez-Diaz, T. M., Garcia-Fernandez,M. C., Garcia-Lopez, M.-L., and Otero, A. (1996). Effectof a lactic starter culture on the growth and protease activityof Aeromonas hydroihila. J. Appl. Bncteriol., 80:13-18. 120. Kalchayanand, N., Hanlin, M. B., and Ray, B. (1992). Sublethal injurymakes gram-negative

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from bottle waters and domestic water supplies in Saudi Arabia. J. Food Prof., 49:471476. Cattabiani, F., and Brindani, F. (1988). Valutazione del Danneggiamento di Tip0 Structurale da Disinfecttanti in Aeronzonas, Vibrio, e Plesiomonas. Arch. Vet. Ital., 39:245-253. Aytac, S. A., and Ozbas, Z. Y. (1994). Survey of the growth and survival of Yersinia enterocolitica and Aerontonas hydrophila in yogurt. Milchwissenschafi, 49:321-324. Santos, J. A., Gonzalez. C., Garcia-Lopez, M.-L., Garcia-Fernandez, M.-C., Otero, A. (1994). Antibacterial activity of the lactoperoxidase system against Aerorrronns hydrophila in broth, skim milk and ewes' milk. Lett. Appl. Micr-obiol., 19:161-164. Santos, J. A., Lopez-Diaz, T. M., Garcia-Fernandez, M. C., Garcia-Lopez, M. L., and Otero, A. (1995). Antibacterial effect of the lactoperoxidase system against Aeronzorzns hydrophila and psychrotrophs during the manufacturing of the Spanish sheep fresh cheese Villalon. Milchwissenschnj?, 50:690-692. Kaper, J. B., Lockman, H., and Colwell, R. R. (1981). Aer-ortzoitns hydrophila, ecology and toxigenicity of isolates from an estuary. J. Appl. Bacteriol.. 50:359-377. Joseph, S. W., Janda, M., and Carnahan, A. (1988). Isolation, enumeration and identification of Aeromonas sp. J. Food Safety, 9:23-25. McCoy, R. H., and Pilcher, K. S. (1974). Peptone beef extract glycogen agar, a selective and differential Aeronzonns medium. J. Fish. Res. Board Can.. 3 1:1153- 1155. Havelaar, A. H., During, M.. and Versteegh, J. F. M. (1987). Ampicillin-dextrin agar medium for the enumeration of Aero.omonns species in water by membrane filtration. J. Appl. Bncteriol.. 62:279-287. Von Graevenitz, A., and Bucher, C. (1983). Evaluation of differential and selective media for isolation of Aeronlonas and Plesiomonas sp. from human feces. J. Clin. Microbiol., 17: 16-21. Janda, J. M., Dixon, A., Raucher, B., Clark, R. B., and Bottone, E. J. (1984). Value of blood agar for primary isolation and clinical implication of simultaneous isolation of Aeromonas hydrophila and Aerornonas ctlvine from a patient with gastroenteritis. Clin. Microbiol., 20: 1221-1222. Schubert, R. H. W. (1977). Uber den Nachweis von Plesionlonns shigelloides Habs and Schubert, 1962, und ein Elektivmedium, den Inositol-Brillantgriin-Gallesalz-agar. Ernst-Rodenwaldt-Arch., 4:97- 103. Schubert, R. H. W. (1967). Das vorkommen der Aeromonaden in oberirdische Gewassern. Arch. Hyg., 1501688-708. Roland, F. P., Salt-starch-xylose-lysine deoxycholate agar. A single medium for the isolation of sodium and non-sodium dependent enteric gram negative bacilli. Med. Microbiol. Irnmunol., 1631241-249. Rogol, M., Sechter, I., Grinberg, L., and Gerichter, C. B. (1979). Pril-xylose-ampicillin agar, a new selective medium for the isolation of Aeronlonas hydrophila. J. Med. Microbiol., 12: 229-23 1. Shread, P., Donovan, F., and Lee, J. (1981). A survey of the incidence of Aeromonas in human feces. Soc. Gen. Microbiol. Quart., 3:184. Altorfer, R., Alwegg, M., Zollinger-Iten, J.. and von Graevenitz, A. (1985). Growth of Aeromonas spp. on cefsulodin-irgasan-novobiocin agar selective for Yersinia enterocolitica. J. Clirt. Microbiol., 22:478-480. Hoban, D., Forsyth, W., Gratton, G., and William, T. (1981). Detection of Aeromonas hq" drophila from diarrhea stools using Macconkey Tween 80 agar. Abst. Ann. Mtg. Ant. Soc. Microbiol. C4. Hoban, D. (1983). Survey of diarrheal illness associated with Aeronzonas hyd?-oplzilain Manitoba. Abst. Ann. Mtg. Am. Soc. Microbiol. C151. Shotts. E. B., Jr., and Rinder, R. (1973). Medium for the isolation of Aeronzonas hydrophila. Appl. Microbiol., 26:550-553.

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4 Update: Food Poisoning and Other Diseases induced by Bacillus cereus Kathleen T. Rajkowski and James L. Smith U.S. Department of Agriculture, Wyndnoor, Pennsylvania *

I. Introduction 61 11. Foodborne Bucillus cereus Outbreaks62

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IV. V. VI. VII. VIII.

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A. Guidelinesforidentifying B. cereus outbreaks63 B. B.cereus foodborneincidentsreported in 1992-199763 Characteristics of the Food Poisoning Toxins Produced by Bacillus cereus A. Emetic toxin 65 B. Diarrheic toxin(s) 66 Nongastrointestinal Disease Induced by Bacillus cereus Infection 67 Occurrence inFood 67 Growth and Survival 70 Inhibition and Inactivation7 1 Isolation, Identifications and Characterization 7 1 FutureResearchNeeds72 References 73

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INTRODUCTION

The endospores of the genus Bacillus are ubiquitous in the environment and are capable of surviving adverse conditions. To date, Norris et al. (1) provide the most comprehensive list of conditions and environments from which Bucillus spores can be isolated. Since the spores are air-, soil-, and/or waterborne, it is only natural that various Bacillus spp. are isolated from foods and areas where food is produced. Since its first isolation in 1948

* Mention of brand or firm name does not constitute an endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned. 61

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associated with a foodborne disease outbreak, Bacillus cereus is considered the most common pathogen in the genus. Toxins produced by the vegetative cells lead to gastrointestinal upset and/or vomiting. In the United States, B. cereus foodborne diseases are a relatively minor concern: however, they are still a major concern worldwide. Recent reviews on various aspects of B. cereus and its disease potential have been published by Drobniewski (2), Fermanian et al. (3), Granum ( 4 3 , Granum and Lund (6), and Schultz and Smith (7). This chapter will summarize the reports of the more recent B. cerem foodborne outbreaks, toxin characterization, nongastrointestinal diseases caused by B. cereus infection, and the psychrotropic characteristics of B. cereus strains.

II. FOODBORNEBACILLUSCEREUS OUTBREAKS Data from the surveillance for foodborne disease outbreaks in the United States indicate that B. cereus is not a major concern. For the 10-year period 1983-1992, there were a total of 1396 bacterial foodborne outbreaks, with only 37 of those outbreaks due to B. cereus. Also, during that time period, there were a total of 83,5 10 cases of bacterial foodborne diseases, with 694 cases occurring in the B. cer-eus outbreaks. Thus, B. cereus was the cause of 2.7% of the bacterial foodborne outbreaks and 0.83% of the outbreaks cases. Death does not appear to be a consequence of foodborne B. cereus outbreaks (8,9). While foodborne disease due to B. cereus is not commonly reported, it is probable that the disease is underreported because most cases are sporadic. The ingestion of contaminated Chinese food and fried rice was responsible for almost half (45.9%) of the B. cereus foodborne outbreaks in the United States. Most outbreaks (67.6%) occulred during the months of May to October (8,9). During the 10-year reporting period, 43.2% of the B. cereus outbreaks occurred in commercial food establishments (cafeterias, delicatessens, restaurants) and 21.6% occurred in homes. In 28 of the 37 outbreaks, the factors (or errors) contributing to B. cereus outbreaks were reported. In 27 of 28 outbreaks (96.4%), improper holding temperature of the food was a contributing factor. The use of contaminated equipment, inadequate cooking, poor personal hygiene, and obtaining food from an unsafe source were contributing errors in 17.9, 17.9, 14.3, and 3.6%, respectively, of foodborne B. cereus outbreaks (8,9). Wilson et al. (10) indicated that Bacillus food poisoning has increased in Northern Ireland. During the 1 9 8 0 ~only ~ one Bncillus-induced foodborne incident was reported; however, nine incidents occurred in 1991 and four in 1992. B. cereus was isolated in most incidents. Bacillus subtilis was isolated in four incidents, however, B. cereus also was isolated from the food in two of those incidents. The foods involved were generally rice and/or chicken and in 10 of 14 incidents, the food was eaten in or taken out from Chinese restaurants. Clinically, the symptoms in almost all of the cases involved in the incidents were predominately vomiting and nausea with an incubation period of 1-6 hours. Thus, it appears that the emetic toxin was responsible for the majority of incidents occurring in Northern Ireland (10). Most of the incidents occurred during the months of April to August. It is probable that inadequate temperature control of cooked rice was the food-processing error that contributed to most of the Bncillrrs-induced food poisoning in Northern Ireland. The mean number of foodborne outbreaks in Taiwan for the period 1987-1993 was 81 (range: 57-93); however, in 1994 the number of outbreaks reported totaled 102 (1 1). A bacterial agent was identified in 74 of the 1994 outbreaks. Eleven of the outbreaks

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were due to B. cereus (14.9%) exceeded only by Vibrio parahaemolyticus (56.7%) and Staphylococcus aureus (20.3%). Snln?onella spp. accounted for only 8.1% of the foodborne outbreaks of bacterial origin (l 1). Food poisoning incidents involving B. cereus are common in the Netherlands. For the four year period 1991-1994, there were 124 incidents with 789 cases of foodborne disease with a bacterial etiology. B. cereus was responsible for 40 of 124 (32.3%) incidents with 172 of 789 (21.7%) cases. Chinese-Indonesian food accounted for 17 of 40 (42%) of the B. cereus-induced food poisoning incidents (12). Salmonella spp. caused fewer incidents (31/124) than B. cereus; however, there were more cases (290/789) with Salmonelln.

A.

Guidelinesfor Identifying B. cereus Foodborne Outbreaks

Two or more persons must become ill for an incident to be called a foodborne disease outbreak (9). In order to confirm that an outbreak is due to B. cereus, organisms must be isolated from the stool of two or more ill persons and not from the stool of controls; alternatively, an outbreak can be confirmed by isolation of 2 10’ B. cereleMs organisms from epidemiologically implicated food (provided that the food specimen has been properly handled). The incubation period for illness induced by emetic toxin is 1-6 hours. Vomiting is the major symptom seen in patients; some patients have diarrhea, but fever is uncommon. The incubation period for persons ingesting diarrheal toxin is 6-24 hours, with patients having diarrhea and abdominal cramps. A few patients show vomiting; fever is uncommon (9).

B. B. cereus Foodborne Incidents Reported in 1992-1 997 1. Two individuals developed gastrointestinal symptoms after eating spaghetti with homemade pesto. Both emetic and diarrheic toxin were involved in this incident. A large batch of the food had been prepared 4 days before the incident and although stored refrigerated, the food had been permitted to remain at room temperature for lengthy periods during the 4-day period. The preparation of a large batch of food which was intermittently subjected to temperature abuse over a 4-day period contributed to the outbreak (13). The outbreak was interesting because one patient died of fulminant liver failure induced by emetic toxin. 2. B. cereus was present in vegetable pakora (4.4 X lo5 CFU/g) and fried rice (2.0 X lo7 CFU/g) in two foodborne incidents involving five people who had eaten in an Indian restaurant in Scotland. All isolates belonged to the same enterotoxigenic serotype. There were a number of processing errors: rice was prepared too far in advance and in greater quantities than needed for a given safe time period. The cooked rice was not properly cooled and was at improper holding temperatures for long periods. The presence of B. cereus in foods other than rice indicated cross-contamination, and a high coliform count in foods indicated handling by person(s) with poor personal hygiene (14).

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3. At a college sport dayin Thailand, 470 individuals were ill with vomiting, nausea, and abdominal pain; approximately half ofthe individuals reported diarrhea. The ingestion of cream-filled eclairs was significantly associated with illness. The mean incubation period was 3.2 hours, which suggested a preformed toxin in thefood. Initial laboratory investigation indicated presence of B. cereus in the eclairs. A few days later, a different laboratory reported the presence of enterotoxin-producing S. aureus. A dual etiology for the outbreak may have been possible; however, before the eclairs had been sent to second laboratory, they had been at room temperature for 12 hours. In addition, the phage types of S. nureus isolated from the cooks were not similar to the phage type isolated from eclairs. Therefore, it is probable that the outbreak was due to B. cereus emetic toxin. The failure to refrigerate the eclairs after preparation and during serving led to the outbreak (15). 4. A stew ingested by 142 competitors at a Norwegian Ski Championship was the source of B. cereus-induced diarrhea. B. cereus was present in the stew at 2 X los CFU/g. Theincubation time was 12-36 hours, and most patients recovered within 24 hours. The kitchen in which the food was prepared was contaminated profusely with B. cereus. The problem was eliminated by thorough cleansing and decontamination of the kitchen with hypochlorite (16). 5. An outbreak of diarrheic foodborne disease involved 139 individuals attending a university field day barbecue in South Carolina. B. cereus was isolated from barbecued pork at levels > lo5 CFU/g. At least half of the pork was cooked 2 days in advance of the barbecue and held unrefrigerated for 18 hours before boning. The boned barbecued pork was then stacked 20 cm deep in metal trays and refrigerated. On the day of the barbecue, the pork was heated; however, the heating unit was not working properly. A hot temperature was not maintained during transportation to the site and while serving the pork to attendees. Preparation of the pork barbecue too far in advance and failure to maintain safe storage and serving temperatures were the food-processing errors that led to the outbreak (17). 6. Emetic toxin from B. cereus was responsible for food poisoning in 14 individuals (12 children and 2 adults) from two child care centers located in Virginia. The ill individuals had eaten chicken fried rice at a catered luncheon prepared by a local restaurant. Leftover chicken fried rice contained B. cereus at > lo6 cfu/g. Improper cooling of the cooked rice and improper holding temperatures contributed to the outbreak (18). 7. A catered wedding reception in California was the site of an outbreak of B. cereus food poisoning due to diarrheic toxin. The outbreak involved 55 individuals, with Cornish hen as the suspect food. The birds contained B. cereus at a level of 1.4 X lo6 CFU/g. There were several errors committed by the caterer which played a role in the outbreak. He was licensed to serve only 29 restaurant customers, yet on this occasion he was serving over 300 customers at two different locations on the same day. It was probable that the Cornish hens were not properly thawed, resulting in undercooked birds. In addition, the cooked Cornish hens were transported and held in an unrefrigerated van for several hours. That the caterer lacked facilities for large-scale food preparation was the chief reason for this outbreak (19).

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Symptoms found in the above outbreaks included diarrhea without vomiting or vomiting that was sometimes followed by diarrhea. The diarrheal toxin does not appear to induce vomiting, but on occasion the emetic toxin appears to 'give both vomiting and diarrhea. It would appear that under some conditions, emetic toxin-producing strains may also produce a diarrheal-type toxin. In the outbreaks listed above, the error in food preparation appeared to be improper holding of the food at temperatures high enough to allow growth of B. cereus. Prompt cooling of the food with refrigerated storage would have prevented the outbreaks. An interesting outbreak of B. cereus intoxication occurred in Germany, which involved Rottweiler puppies; B. cereus-contaminated milk powder was the food agent. Puppies fed formula containing milk powder (in addition to receiving mother's milk) became ill with vomiting and diarrhea. Five puppies, whose mother was not producing milk, were fed exclusively on formula containing the contaminated milk powder and died. B. cereus was present at a level of 2.7-3.5 > lo4 CFU/g in the milk powder. The presence of the organism in the powdered milk and symptoms suffered by the puppies indicated that this outbreak was due to intoxication by B. cereus (20).

111.

CHARACTERISTICSOFTHE FOOD POISONING TOXINS PRODUCED BY Bacillus cereus

A. Emetic Toxin Emetic toxin is preformed in foods, i.e., B. cereus at levels of 105-108 CFU/g of food produce toxin, which causes vomiting when the food is eaten. Symptoms-vomiting, nausea and malaise-appear within 0.5 to 5 hours. Duration of illness is generally 6-24 hours (5,6). Foods implicated in B. cereus emetic food poisoning include fried and cooked rice, pasta, noodles, and pastry. Agata et al. (21) found a strong association of emetic toxin production with the H1 serovar phenotype of B. cereus. Isolates of B. cereus that produce enterotoxin activity or hydrolyzed starch did not produce emetic toxin. The structure of the emetic toxin has been elucidated, and the compound has been named cereulide. Cereulide is a 36-membered cyclic dodecadepsipeptide with the sequence of cycb(D-O-Leu-D-Ala-L-o-val-L-Val)3with the formula, C57H9601SN6 and a molecular weight of l 19l .6. Cereulide is a potassium ionophore closely related to valinomycin (22-24). Cereulide induces vomiting in Suncus murinus (house musk shrew) and rhesus monkeys (25,26). The emetic toxin is not antigenic, does not act in the rabbit or mouse ileal loop test, produces vacuolation in HEp-2 cells but is not cytotoxic, is stable for 90 minute to 121"C, is stable to pH 2.0 and pH 11, is not hemolytic, and does not induce vascular permeability in the skin of the rabbit (5,26). Cereulide induces vomiting when it stimulates the vagus nerve by binding to the 5-HT3receptor. Using the S. murinus emetic model, Agata et al. (25) found that vagotomy eliminated the emetic action of cereulide. The 5-HT3receptor antagonist, ondansetron, also abolished cereulide-induced vomiting in Suncus. In addition, cereulide is a mitochondrial poison. Using rat liver mitochondria, Mahler et al. (13) showed that emetic toxin inhibited electron transport and uncoupled oxidative phosphorylation.

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Nothing is known about the synthesis of cereulide. It is probable that the cyclic peptide is enzymatically synthesized from its component amino acids and is not a product of a gene.

B. Diarrheic Toxin(s) The toxin(s) responsible for the diarrheal syndrome are formed in the small intestine of the host. Unlike the emetic toxin, which is preformed in food, any preformed diarrheic toxin, due to its loss of activity at pH 3 and its sensitivity to trypsin, would be destroyed as it passes through the gastrointestinal system (27). The infectious dose ranges from lo5 to lo7 organisms per host. Symptoms include abdominal pain, watery diarrhea, and occasionally nausea, which generally appear within 8-16 hours after ingestion of contaminated food. Duration of illness is generally 12-24 hours (6). Foods that are frequently implicated in B. cereus diarrheic food poisoning include meat products, soups, vegetables, puddings, sauces, milk and milk products. Enterotoxin activity is decreased or lost if thetoxin is subjected to a pH of 3, trypsin, chymotrypsin, pepsin, or heating at 80°C for 10 minute (4,27). Baker and Griffiths (27) demonstrated that the immunological activity of the enterotoxin was approximately four times more stable when heated in milk as compared to broth. However, since diarrheic activity was not tested, it is not clear that the toxin still retained the ability to cause disease. Granum and Lund (28) studied 85 strains of B. cerem isolated from dairy products and found that 50 (58.8%) were enterotoxigenic, 13 (15.3%) were psychrophilic, and 6 (7. l %) produced toxin at 6°C. The ability to grow and produce enterotoxin at refrigerated temperatures indicates that refrigeration may not be enough to protect consumers from B. cereus food poisoning. Hemolysin BL (HBL), a tripartite entity consisting of three protein components, possesses hemolytic, cytotoxic, dermonecrotic, and vascular permeability activity (29,30). In addition, HBL induces fluid accumulation in rabbit ileal loops, thereby indicating that it is an enterotoxin. HBL consists of three protein components: L? (46 kDa), L, (38 kDa), and B (37 kDa), and maximal expression of all HBL activities requires all three protein entities (30). Heinrichs et al. (31) and Suwan et al. (32) have cloned the genes for HBL (hbZC, hblD, IzblA, hZaB), determined the nucleotide sequence of the genes, and deduced the amino acid sequences of the three protein components. Lund and Granum (33,34) characterized a nonhemolytic enterotoxin complex from strains of B. cereus responsible for outbreaks of diarrheal food poisoning. The nonhemolytic enterotoxin (NHE) consisted of three protein moieties: B (105 m a ) , L2 (54 kDa), and L, (39 m a ) , and all components were needed for maximum cytotoxicity. Apparently, NHE was not tested in the ileal loop assay. The sequencing of NHE is underway (34). Diarrhea-inducing strains of B. cereus may produce either HBL or NHE or both (34). It would appear that there are at least two tripartite enterotoxins, HBL and NHE, in B. cereus. The role of “enterotoxin T” as an agent of diarrhea in B. cereus foodborne illness is not clear. Enterotoxin T consists of a single protein that is cytotoxic, positive in the mouse ileal loop assay and possesses vascular permeability activity (35). Enterotoxin T is a product of the gene bceT, which has been cloned (35). Granum and Lund (34) state that there is no real evidence that enterotoxin T causes food poisoning. Two commercial kits are available for the detection of B. cereus enterotoxins: the Oxoid BCET-RPLA and Tecra ELISA kits. The Oxoid kit measures L? of HBL, whereas t

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Bacillus cereus

67

the other kit measures L: of NHE: thus, the kits do not assay the same enterotoxin (34). But it would appear that both kits are necessary to detect enterotoxin production in food poisoning due to B. cereus. Beecher and Wong (36) developed a simple and inexpensive method for detecting HBL-producing strains of B. cereus. Their assay is based on a distinctive discontinuous hemolytic pattern produced by HBL-producing strains on blood agar plates.

IV. NONGASTROINTESTINALDISEASEINDUCED Bacillus cereus INFECTION

BY

In addition to causing classical food poisoning symptoms affecting the gastrointestinal system, B. cereus may cause disease in other areas of the body. Nongastrointestinal diseases that may result from B. cereus infections include (a) local infections such as ocular and wound infections, (b) bacteremia and septicemia, (c) central nervous system infections, (d) respiratory tract infections, and (e) endocarditis (2). Details from a number of case reports (1995-1997) of B. cereus-induced nongastrointestinal diseases are given in Table 1. Drobniewski (2) has a table listing cases of B. cereus-induced ocular infections that occurred during the period 1951-1990; another table lists other nongastrointestinal B. cereus infections that occurred during 1965-1992 (2). A table of clinical features that appeared in cases of B. cereus infections occurring in patients with leukemia during 19711995 is presented by Akiyama et al. (41). The cases of nongastrointestinal B. cereus infections presented in Table 1 and in the tables of Drobniecwski (2) and Akiyama et al. (41) indicate that individuals immunocompromised either by illness or medication are at particular risk for disease induced by this organism. Using both in vitro and in vivo methods, Beecher et al. (45) demonstrated that the B. cereus HBL enterotoxin had retinal toxicity and may be involved in endophthalmitis (inflammation of the inner eye). B. cereus is one of the major infective agents responsible for blindness in humans (45). Antibody neutralization of HBL factor in cell-free crude concentrated and dialyzed culture filtrates eliminated approximately 50% of the in vitro retinal toxicity. Purified HBL wasless toxic than culture filtrates containing the same level of HBL. These results indicate that B. cereus ocular virulence may be multifactorial and requires other toxic moieties in addition to HBL (45). Fulminant liver failure ending in death was apparently induced by the B. cereus emetic toxin cereulide (13). Purified emetic toxin from the disease causing strain inhibited electron transport and uncoupled oxidative phosphorylation in rat liver mitochondria (13). Many liver failure syndromes have been linked to mitochondrial injury and disruption of mitochondrial activity (40). Thus, cereulide acting as a mitochondrial poison led to liver failure in the patient described by Mahler et al. (13). The apparent correlation of nongastrointestinal disease syndromes with B. cereus diarrheic and emetic toxins warrants further study.

V.

OCCURRENCE IN FOOD

Some food sources from which B. cereus has been isolated are summarized in Table 2. In a more recent report, Hatakka (58) examined the microbiological quality of airline hot

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Table 2 Survey of Food Products from Which Bacillus cereus was Isolated and % Incidence Food product

5% Incidence

Rice and rice products Boiled rice Spices Milk products Chicken and meat products Buffalo meat Ready-to-serve moist food Powdered infant formula Nonfat dry milk Skim milk powder Buesco Blanco cheese soft hard Various food samples from a Spanish retail market Seed samples (alfalfa, mung bean, wheat or seed mixtures) Poultry products Pork carcasses Beef carcasses Chicken carcasses Raw beef Pork Chicken Ham Sausage Uncooked hamburger Cooked hamburger Meat product additives Tempeh samples

28.5 100 30 100 80 35 83 75 100 10.3 69 63 14.7 cells 82.4 spores 67.3

6.9 7 11 0 7.9 4.4 7.2 3.8 14.9 45.5 23.7 39.1 16

Country

Ref.

India

47

India United States

48 49

Japan

50

Venezuela

51

Spain

52

United States

53

England Sweden

54 55

Japan

56

Netherlands

57

meals over a 3-year period and found that 3% of the meals contained pathogenic bacteria, of which B. cereus was the most common pathogen. Using the fatty acid profiles for the identification of 229 B. cereus from environmental sources (dairy farm to processing plant), Lin et al. (59) determined that B. cereus spores in raw milk were the major source of B. cereus in pasteurized milk based on the comparison of the profiles from environmental and pasteurized milk isolates. The report of teGiffel and Beumer (60) confirmed that the presence of psychrotrophic B. cereus spores in raw milk resulted in contaminated pasteurized milk products. The vegetative cells in raw milk are rapidly killed during pasteurization at 65OC, whereas the endospores of B. cereus are heat-resistant (61). Properly pasteurized dairy products, when stored at recommended refrigerated conditions (maximum 7°C) and used within the expiration date usually cause no problems for healthy adults. More information can be found in the bulletins of the International Dairy Federation devoted to B. cereus in milk and milk products (62,63).

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VI.GROWTH

AND SURVIVAL

The Agricultural Research Service of the U.S. Department of Agriculture has developed a growth model for B. cereus, which can be found on the Internet at http:// www.arserrc.gov/mfs. The Pathogen Modeling Program Version 5.1 forWindows can be entered through the home page. The growth characteristics of B. cereus were reviewed by Schultz and Smith (7). The incidence of B. cereus in raw and processed foods, especially when held at low temperatures, has received considerable attention. The research data indicate that few foods are free of the organism and that growth and toxin fortnation can occur at psychrotropic temperatures (28). Bergann (64), reporting on the growth temperature requirements of 50 B. cereus strains, showed that more than half of the strains grew at 10°C, six at 8"C, and one at 6°C. Garcia-Armesto and Sutherland (65), after characterizing both psychrotropic and nlesophilic Bacillus species isolated from milk, found one B. cereus isolate that was clearly psychrotropic (grew at 6.5"C but not at 40°C in 2 days). The cold adaptation response was demonstrated by Foegeding and Berry (66) after determining the growth characteristics of 27 B. cereus clinical and food isolates. This cold adaptation was also reported by Christiansson et al. (67). They found that some strains of B. cereus were acclimated to grow at 8"C, and when the relationship between cytotoxicity and growth of these isolates was compared, most became cytotoxic at approximately lo* c f d n i l . The dairy isolates that produced cytotoxicity in milk at 8°C under aerated conditions also gave positive vascular permeability reaction with rabbits, which is indicative of B. cereus diarrheal toxin (67). In the study by Granum et al. (28), they found of the 85 B. cereus strains isolated from dairy products, 6 were both psychrotropic and enterotoxigenic and exhibited good growth at 6°C. While B. cereus is commonly found in milk and other dairy products and milk is a suitable substrate for toxin production under aerated conditions even at low temperatures, the incidence of B. cereus food poisoning in milk is low probably due to insufficient aeration during normal storage (67). Van Netten et al. (68) found that B. cereus isolates involved in two foodborne outbreaks of diarrheal illness were able to grow and produce toxin at 4-7°C. Strains of B. cereus of the emetic type, which were isolated during an outbreak involving pasteurized milk, were able to grow at 4-7"C, but enterotoxin production was not detected using the Oxoid test kit for diarrheal toxin. It is quite probable that the emetic toxin can beproduced at low incubation temperatures, although they did not test for it (68). Fermanian et al. (69) reported that a foodborne and clinical isolate grew at 10°C and that both isolates were toxin positive using the Oxoid kit. The presence of psychrotropic B. cereus strains in dried infant foods is a concern. Rowan and Anderson (70) reported that of the B. cereus isolated from reconstituted milkbased infant fornlulas (MIF), 1,4, and 16 strains grew at 4, 6, and 8"C, respectively, after 15 days. They were tested for diarrhea enterotoxin production, and 9 of the 21 isolates produced enterotoxin. The authors recommended brief refrigeration (4°C) storage of the reconstituted MIF forthis product to remain safe. They also tested the effect of maltodextrin on the growth and synthesis of the diarrheal enterotoxin in MIF and found that supplementing with 20.1% maltodextrin supported both growth and diarrheal toxin production when incubated for 14 hours or more at 25°C (71). Jaquette and Beuchat (72) reported that psychrotrophic B. cereus can survive and grow in reconstituted infant rice cereal. They recommended immediate consumption or holding the food for <48 hours at 58°C (72).

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VII.INHIBITIONANDINACTIVATION Schultz and Smith (7) reviewed the inactivation and growth inhibition of B. cereus. The inhibition by the addition of 50 IU of nisin to skim milk resulted in a decrease in numbers for 9 of the 12 psychrotropic B. cereus strains tested by Dufrenne et al. (73). They also reported that 250 IU decreased the bacterial numbers of all strains tested. Beuchat et al. (74) studied the effect of nisin and temperature on the growth of and enterotoxin production by psychrotropic B. cereus in beef gravy. They reported that as little as 1 pg of nisin/ mL may be effective in inhibiting or retarding growth of B. cereus and production of the diarrheal enterotoxin by both the vegetative cells and spores in beef gravy at 8°C. Jaquette and Beuchat (75) showed a correlation between pH, nisin, and temperature on growth and survival of B. cereus. Using a concentration of as low as 1 pg of nisin/tnL the effect was more pronounced in inhibiting the growth of B. cereus as the temperature decreased from 15 to 8 or 5°C and at more acidic conditions (pH 5.53). Not only does the pH affect the growth of B. cereus, but there are reports that as the pH of sporulation media decreases there is a drop in sporulation accompanied by a decreased D-value of the produced spores (76). In addition as the media pH decreases from 7.0 to 4.0, a decrease in D-value was reported by Mazas et al. (77). Mahakarnchanakul and Beuchat (78) studied the effect of growth temperature and NaCl concentration on the thermotolerance of a psychrotropic and mesophilic B. cereus. After a downshift in growth temperature from 30 to 10°C followed by incubation for 3 to 9 days, an increase in thermotolerance was observed for the psychrotropic strain when heated in BHI broth containing 2 or 4% NaCl compared to the thermotolerance in the broth containing 0.5% NaC1. They attribute the thermotolerance to the production of proteins with molecular weights of 63, 40 and 29 kDa for the psychrotropic strain. Gmnum (4) also suggested that changes in tetnperature may influence the ability of B. cereus to grow in minimally processed foods during distribution and storage.

VIII.ISOLATION,IDENTIFICATION, AND CHARACTERIZATION In their 1992 review, van Netten and Kramer (79) discussed the use of media for the isolation and enumeration of B. cereus in foods, and an earlier review (80) discussed the use of various media for isolation of B. cereus occurring in foods and clinical specimens. The selective mannitol-egg yolk-polymyxin (MYP) agar is recommended in the methods for isolation of B. cereus (81,82) ; whereas, either MYP or KG agars are, suggested for use by Harmon et al. (83). Regardless of which selective agar is used for isolation, confirmational identification still relies on the biochemical profiles (81,82,83). A rapid Bacillus identification system is available utilizing the API system (Analytab Products, Plainview, New York) which can be used to confirm isolates of B. cereus from selective agars (84). Since the review by Schultz and Smith (7) there have been nofurther updated reports on the use of the 23 flagella (H) antigens for serological typing of the B. cereus strains involved in food poisoning, and this typing method is still an effective way to distinguish between strains (85). The production, assay, and/or pathological effects of the various toxins produced by B. cereus have been reviewed by Granum (4), Granum et al. (28), Granum and Lund (6), Schultz and Smith (7), Gilbert and Kramer (86), Turnbull (87j, Kramer and Gilbert

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(80), Shinagawa (85), and Jackson (88). Anderson et al. (89) developed a rapid bioassay for detection of B. cereus emetic toxin based on the loss of motility of boar spermatozoa upon 24 hours of exposure to extracts of emetic B. cereus strains or contaminated food. The detection limit for food was 3 g of rice containing 106-107cfu of emetic B. cereus per gram. Currently there is no commercial available kit for the detection of the emetic toxin (cereulide) (5). A reversed passive latex agglutination test (Oxoid BCET-RPLA) (6) and an enzyme-linked immunosorbent assay (ELISA) (TECRATM BDE vusual immunoassay) (90) are available to test for the diarrheal toxin. The differentiation and genetic studies used to identify B. cereus from other Bacillus species was reviewed by Schultz and Smith (7). A phylogenetic tree of the group 1 Bacillus was constructed by Ivanov (91) using the G + C content in DNA. They showed a statistically reliable correlation between the evolutionary distances of 16s rRNA sequences (Ei) and parameter Pi having a coefficient of correlation between Ei and Pi equaling 0.97 for representatives of group 1, which include B. nrzthracis, B. cereus, B. circulnns, B.jinnus, B. Zautus, and B. subtilis. Based on the 16s rDNA and the 23s rDNA, Lechner et al. (92) propose that the psychrotolerant species of the B. cereus group be considered a new species that can grow at 4-7°C but not at 43°C be called Bacillus weiherzstephnnensis sp. nov. Nilsson et al. (93) developed a RAPD-PCR method for large-scale typing of B. cereus. The procedure was highly discriminatory for B. cereus strains and was found to give reproducible classification of RAPD fingerprints for five reference strains. Francis et al. (94) reported on the development of a single PCR assay using a major cold shock protein to discriminate between psychrotropic and mesophilic B. cereus strains. Koo et al. (95) obtained a recombinant antibody by reverse transcription-PCR that had B. cereus spore-binding ability. A rapid PCR-based DNA test for the enterotoxic B. cereus was reported by Mantynen and Lindstrom (96). Lin et al. (97) differentiated the B. cereus group (cereus, mycoides, and tlzuringiensis) using Fourier transform infrared spectroscopy. The absorbance peaks between 1800 and 1500 cm” for B. cereus were different from the other Bacillus studied. Carlson et al. (98) also concluded that B. cereus and B. thuringiensis should be considered one species after analyzing the pulsed-field gel electrophoresis and the multilocus enzyme electrophoresis profiles. The profiles were indistinguishable. Helgason et al. (99) concluded after analyzing the multilocus enzyme electrophoresis profiles of 154 B. cereus/ B. thuringiemis isolates from soil samples that a high degree of recombination occurred. Reports have implicated B. thuringiensis in gastrointestinal disease (2). Both B. cereus and B. thuringiensis were isolated from a gastroenteritis outbreak (100). All B. cereus and B. thurir~giensisisolates showed cytotoxic effects characteristic of enterotoxin-producing B. cereus.

IX. FUTURERESEARCHNEEDS More research is needed in the following areas: 1. There is a need to determine if the tripartite enterotoxins, HBL and NHE, are the only diarrheal toxins produced by food poisoning strains of B. cereus or if there are other toxins such as “enterotoxin T,” which is a monoprotein moiety. 2. Studies are needed to determine the intestinal receptor site(s) of the tripartite enterotoxins and how these toxins induce diarrhea.

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3. How B. cereus synthesizes cereulide (emetic toxin) is unknown; it is probably produced enzymatically, but there is no information available at the present time. 4. Two studies (13,44) indicate that HBL and cereulide play a role in the induction of B. cereus nongastrointestinal diseases. More studies are needed to confirm these observations and the potential role of the toxins in pathogenesis of the nongastrointestinal diseases. 5. Ribsomal operon ribotyping is of interest and results of study in this area may prove to be a useful tool in differentiation of the various bacilli.

REFERENCES 1. Norris, J. R., Berleley, R. C. W., Logan, N. A., and O’Donnell, A. G. (1981). The genera Bacillus and Sporolactobacillus. In The ProkaQlotes, Vol. 2 (M. P. Starro, H. Stolp, H. G. Truper, A. Balows, and H. G. Schlegel, eds.), Springer-Verlag,New York, pp. 1711-1742. 2. Drobniewski, F. A. (1993). Clin. Microbiol. Rev., 6:324-338. 3. Fermanian, C., Fremy, J.-M., and Lahellec, C. (1993). J. Rap. Meth. Autont. Microbiol., 2: 83-134. 4. Granum, P. E. (1994). J. Appl. Bacteriol., 76:61S-66S. 5. Granum, P. E. (1997). Bacillus cereus. In Food Microbiology: Fundanzentals and Frontiers (M. P. Doyle, L. R. Beuchat and T. J. Montville, eds.), ASM Press.Washington, DC, pp. 327-336. 6. Granum, P. E., and Lund, T. (1997). FEMS Microbiol. Lett., 157:223-228. 7. Schultz, F. J., and Smith, J. L. (1994). Bacillus: Recent advances in Bacillus cereus food poisoning research. InFoodborne Disease Handbook: Diseases Caused by Bacteria, Vol. 1 (Y. H. Hui, J. R. Gorlkam, K. D. Murrell, and D. 0 Cliver, eds.), Marcel Dekker, Inc., New York, pp. 29-62. 8. Bean, N. H., Griffin, P. M., Goulding, J. S., and Ivey, C. B. (1990). MMWR, 39, #SS-l. 9. Bean, N. H., Goulding, J. S., Lao, C., and Angujio, F. J. (1996). MMWR, 45, #SS-6. 10. Wilson, I. G., Wilson, T. S., and Kramer, J. M. (1993). Lancet, 342:928. 11. Pan, T. M., Chiou, C. S., Hus, S . Y., Huang, H. C., Wang, T. K., Chiu, S. I., Yea, H. L., and Lee, C. L. (1994). J. Fornzos. Med. Assoc., 95:417-420. 12. Simone, E., Goosen, M., Noterman, S. H. W., and Bergdorff, M. W. (1997). J. Food Prot., 60~442-446. 13. Mahler, H., Pasi, A., Kramer, J. M., Schulte, P., Scoging, A. C., BCir, W., and Krihenbuhl, S. (1997). N. Eng. J. Med., 336: 1142-1 148. 14. Dickson, A., and Morgan, M. (1995).Scottish Centre Infect. Environ. Health Weekly Report 29(5):7. 15. Thaikruea, L., Pataraarechachai, J., Savanpunyalert, P., and Maluponjiragul, U.(1995). Sotheast, Asian J. Trop. nted. Public Health, 26:78-85. 16. Granum, P. E., Naestvold A., and Gundersby, K. N. (1997). FEMS Microbiol. Lett., 157: 223-228. 17. Luby, S., Jones, J., Dowda, H., Kramer J., and Horan, J. (1993). J. Infect. Dis., 167:14521455. 18. Centers for Disease Control and Prevention. (1993). MMWR, 43:177-178. 19. Slaten, D. D., Oropeza, R. I., and Werner, S. B. (1992). Pub. Health Repts., 107:477-480. 20. Gareis, M., and Walz, A. (1994). Tiertirztl. Untschau., 49:319-322. 21. Agata, N., Ohta M., and Mori, M. (1996). Curr. Microbiol., 33:67-69. 22. Agata, N., Mori, M.. Hota, M., Swan, S., Ohtani I., and Isobe, M. (1994). FEMS Microbiol. Lett., 121:31-34.

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23. Isobe, M., Ishikawa, T., Suwan, S., Agata N., and Ohta, M. (1995). Bioorg. Med. Clzem. Lett., 5:2855-2558. 24. Ryan, P. A., Macmillan J. D., and Zilinskas, B. A. (1997). J. Bncteriol., 179:2251-2556. 25. Agata, N., Ohta, M., Mori, M., and Isobe, M. (1995). FEMS Microbiol. Lett., 129:17-20. 26. Sinagawa, K., Konama, H., Sekita, H., and Sugii, S. (1995). FEMS Microbiol. Lett.. 130: 87-90. 27. Baker, J. M., and Griffiths, M. W. (1995). J. Food Prof., 58:443-445. 28. Granum, P. E., and Lund, T. (1997). FEMS Microbiol. Lett., 157:223-228. 29. Beecher, D. J., and Wong, A. C. L. (1994). Appl. Environ. Microbiol., 60: 1646-1651. 30. Beecher, D. J.. Schoeni, J. L., and Wong, A. C. L. (1995). Infect. Irrtnrun., 63:4423-4428. 31. Heinrichs, J. H., Beecher, D. J., Macmillan, J. D., and Zilinskas, B. A. (1993). J. Bucteriol., 175:6760-6766. 32. Suwan, S., Isobe, M., Ohtani, I., Agata, N., Mori, M., and Ohta, M. (1995). J. Clzem. Soc. Perkin Truns., 1:765-775. 33. Lund, T., and Granum, P. E. (1996). FEMS Microbiol. Lett., 141:151-156. 34. Lund, T., and Granum, P. E. (1997). Microbiology, 143:3329-3336. 35. Agata, N., Ohta, M., Arakawa, Y., and Mori, M. (1995). Microbiology, 141:983-988. 36. Beecher, D. J., and Wong, A. C. L. (1994). Appl. Environ. Microbiol., 60:1646-1651. 37. Sato, K., Ichiyama, S., Ohmura. M., Takaski, M., Agata, N., Ohta, M., and Nakashima, N. (1998). J. Infect., 36:247-248. 38. Berner, R., Heinen, F., Pelz, K., Van Velthoven, V., Sauer, M., and Rorintheberg, R. (1997). Neuropediutrics, 28:333-334. 39. Meredith, F. T., Fowler, V. G., Gautier, M., Corey G. R., and Reller, L. B. (1997). Scund. J. Zrgect. Dis., 29:528-529. I., Akiyama, N., Mitani, K., Hirai,H.. Yazaki, Y., and Machi40. Motoi, N., Ishida, T., Nakano, nami, R. (1997). Acta Neuropatllol.. 93:301-305. 41. Akiyama, N., Mitani, K., Tanaka, Y., Hanazono, Y., Motoi, N., Zarkovic, M., Tange, T., Hirai, H., and Yazaki, Y. (1997). Intern. Med., 36:221-226. 42. Miller, J. M., Hair, J. G., Herbert, M., Herbert, L., and Roberts, F. J., (1997). J. Clin. Microbiol., 35:504-507. 43. Marley, E. F., Saini, N. K., Venkatraman, C., and Orenstein, J. M. (1995). Soutlz. Med. J.. 88:969-972. 44. Strittmatter, M., Hamann, G., Sahin, U., Feiden, W., Kohl, K., and Schinlrigk, K. (1995). Newenarzt, 66:785-788. 45. Beecher, D. J., Pulidok, J. S., Barney N. P. and Wong, A. C. L. (1995). h$ect. Zmmun., 63: 632-639. 46. Schafer, D. F., and Sorrel M. F. (1997). N. Eng. J. Med., 336:1173-1174. 47. Kamat, A. S., Nerkard, D. P., and Nair, P. M. (1998). J. Food Sufeq, 10:31-41. 48. Bachhil, V. N., and Jaiswal, T. N. (1998). J. Food Sci. Techrrol., 25:371-372. 49. Harmon, S. M., and Kautter, D. A. (1987). J. Food Prof., 54:372-374. 50. Suzuki, A., Kawanishi, T., Takayama, S., Haruta, M., Simizu, Y., Ogiwara, H., and Jinbo, K. (1984). J. Food Hygienic Soc. Jpn, 25: 106-1 11. 51. Arispe, I., and Westhoff, D. (1984). J. Food Prot., 47:27-35. 52. Mosso, A., Garcia-arribas, L., Cuena, J. A., and De la Rosa, C. (1989). J. Food Prot., 52: 184- 188. 53. Harmon, S. M., Kautter, D. A.. and Solomon, H. M. (1987). J. Food Prot., 50:62-65. 54. Sooltan, J. R. A., Mead, G. C., and Norris, A. P.(1987). Food Microbiol., 4:347-351. 55. Ternstrom, A., and Molin, G. (1987). J. Food Prot. 50:141-146. 56. Konuma, H., Shinagawa, D., Tokumaru, M., Onoue Y., Konno, S., Fujino, N., Shigehisa, T., Kurata, H., Kurata, Y., Kuwabara, Y., and Lopes, C. A. M. (1988). J. Food Prot., 51: 324-326. 57. Samson, R. A., Van Kooij, J. A., and De Boer, E. (1987). J. Food Prot., 50:92-94.

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cereus 75

58. Hatakka. M. (1998). J. Food Prot., 61:1052-1056. 59. Lin, S., Schraft, H., Odumeru. J. A., and Griffiths, M. W. (1998). Int. J. Food Microbiol., 43:159-171. 60. teGiffel, M. C., and Beumer, R. R. (1998). Tijdschr. Diergeneeskd., 123528-632. 61. Holsinger, V. H., Rajkowski, K. T., and Stabel, J. R. (1997). Rev. Sci. Tech. Ofs Int. Epiz., 16:441-451. 62. (1992). Internrctional Dui?? Federatiow Bulletin No. 275, Brussels. 63. (1993). Inter~ationcrlDui?? Federation Bulletin No. 287, Brussels. 64. Bergann, T. (1989). Mlz. Vet.-Med., 4423-25. 65. Garcia-Armesto, M. R., and Sutherland, A. D. (1997). J. D a i p Res., 64261-270. 66. Foegeding, P. M., and Berry, E. D. (1997). J. Food Prot., 60: 1256-1358. 67. Christiansson, A., Naidu, A. S., Nilsson. I., Wadstrom, T., and Pettersson, H-E. (1989).Appl. Enl*irort.Alicrobiol., 55:2595-2600. 68. van Netten, P., van de Moosdijk, A., van Hoensel, P., Mossel, D. A. A., and Perales, I. (1990). J. Appl. Bucteriol., 69:73-79. 69. Fernlanian, C., Lapeyre, C., Fremy, J. M., and Claisse, M. (1997). J. Doily Res.. 64551 559. 70. Rowan, N. J., and Anderson, J. G. (1998). Lett. Appl. Microbiol., 26:161-165. 71. Rowan, N. J.. and Anderson, J. G. (1997). Appl. Environ. Microbiol., 63: 1182-1 184. 72. Jaquette, C. B., and Beuchat L. R. (1998). J. Food Prot., 61:1629-1635. 73. Dufrenne, J., Bijwaard, M., te Giffel, M., Beumer, R., Notermans, S. (~1995).hzt. FoodMicrobiol.. 27:175-183. 74. Beuchat, L. R., Claverno, M. R. S., and Jaquette, C. B. (1997). Appl. Emiron. Microbiol., 6311953-1958. 75. Jaquette. C. B., and Beuchat, L. R. (1998). J. Food Prot., 61563-570. and Martin, R. (.1997). Lett. Appl. Micro76. Mazas, M., Lbpez, M., Gonzilez, I., Bernardo, A., biol., 25:331-334. 77. Mazas, M., Lbpez, M., Gonzilez, I., Gonzilez, J.. Bernardo, A., and Martin, R. (1998). J. Food Safen,, 18:25-36. 78. Mahakarnchanakul. W., and Beuchat, L. R. (1999). J. Food Prot., 6257-64. 79. van Netten, P., and Kramer, J. M. (1992). Int. J. Food Microbiol., 17:85-99. 80. Kramer. J. M., and Gilbert, R.J. (1989). Bacillus c e r e ~ and s other Bacillus species. InFoodborne bacterial pathogens (M. P. Doyle, ed.), Marcel Dekker, Inc., New York, pp 21-70. 81. Rhodehamel E. J., and Harmon, S. M. (1998). Bacillus cereus. In FDA BrrcteriologicalAltalytical Manzlal, 8th ed., AOAC International, Gaithersburg, Maryland. 82. AOAC OfficialMethods of Analysis. (1998). Bacillus cerezis. In Foods-AOAC OfJicicrl Method 980.31, Vol. 1 (P. Cunniff, ed.), AOAC International, Gaithersburg, Maryland. 83. Harmon, S. M., Goepfert,J. M., and Bennett, R. W. (1991).Bacillus cereus. In Compendium of Methods for the Microbiological E.yamination of Food (C. Vanderzant and D. F. Splittstoesser, eds.),American Public Health Assoc.. Washington, DC. 84. Logan, N. A. and Berkeley, R. C. W. (1984). J. Gen Microbiol.. 130:1871-1 883. 85. Shinagawa, K. (1990). Int. J. Food Microbiol., 10:125-142. 86. Gilbert, R. J. and Kramer, J. M. (1984). Biochenz. Soc. Trans., 12:198-200. 87. Turnbull, P. C. B. (1986). Bacillzcs cereus toxins. In Plrnnnacology of Bacterid Toxins (F. Dorner and J. Drews, eds.), Pergamon Press, New York. 88. Jackson, S. G. (1991). J. Assoc. 08 Anal. Chenz., 74:704-706. 89. Andersson.M.A., Mikkola, R., Helin, J., Andersson, M. C., and Salkinoja-Salonen, M. (1998). Appl Emiron. Microbiol., 64:1338-1343. 90. Tan, A., Heaton, S., Farr, L. and Bates, J. (1997). J. Appl. Microbiol., 82677-682. 91. Ivanov, V. N. (1998). Mikrobiol Z., 60:75-82. 92. Lechner, S., Mayr, R., Francis, K. P.. Pruss, B. M., Kaplan. T., Weissner-Gunkel,E., Stewart, G. S., and Scherer, S. (1998). Irtt. J. Syst. Bacteriol., 48:1373-1382.

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93. Nilsson, J., Sevesson, B., Ekelund, K. and Christiansson, A. (1998). Lett. Appl. Microbiol., 271168-172. 94. Francis, K. P., Mayr, R., von Stetten, F., Stewart, G. S., Scherer, S. (1998). Appl. Emiron. Microbiol., 64:3525-3529. 95. Koo, K., Foegeding, P. M., and Swaisgood, H. E. (1998). Appl. Enl-irort. Microbiol., 64: 2490-2496. 96. Mantynen, V. and Lindstrom, K. (1998). Appl. Emiron. Microbiol., 64:1634-1639. 97. Lin, S. F., Schraft, H. and Griffiths, M. W. (1998). J. Food Prot., 61:921-923. 98. Carlson, C.R., Caugant. D. A. and Kolsto, A-B. (1994). Appl. Environ. Microbiol., 60:17191725. 99. Helgason, E., Caugant, D. A., Lecadet, M"., Chen, Y.. Mahillon, J., Lovgen, A., Hegna, I., Kvaloy, K. and KolstG, A-B. (1998). Cz4rr. Microbiol.. 37:80-87. 100. Jackson, S. G., Goodbrand,R. B., Ahmed, R. and Kasatiya, S . (1995). Lett. Appl. Microbiol., 21~103-105.

L

5 Brucella Shirley M. Halling U S . Depurtment of Agriculture, Ames, Iowa

Edward J. Young VA Medical Center and Baylor College of Medicine, Houston, Texas

I. Introduction 77 11. Etiological Agent 78

A. Taxonomy 78 B. Hardiness 79 111. Epidemiology and Foods Involved 79 A. Foodborne brucellosis 80 B. Travel 80 References 81

1.

INTRODUCTION

Brucellosis is a zoonosis caused by species of the bacterial genus Brucella. Humans are always an accidental host, commonly contracting the disease by direct contact with infected animals or by ingesting contaminated dairy products. Only three of the six species of Brucella are important human pathogens: B. abortus, B. melitensis, and B. suis; these three species, respectively, have a natural host preference for the following food animals: cattle, goats and sheep, and swine (1). Brucella canis, principally a pathogen for dogs, rarely causes human infection and is not associated with the consumption of food (2). The risk of contracting brucellosis is highest among farmers, ranchers, abattoir workers, and veterinarians. Clinical and research microbiologists handling Brucella are also at increased risk of infection. In contrast, human brucellosis linked to the consumption of such foods as goat’s milk cheese is not occupation related and often occurs without direct animal contact. Brucellosis exists worldwide and is prevalent especially in countries where programs to eradicate the disease in domestic animals are inadequate. In the United States, programs to control bovine brucellosis have resulted in a dramatic decline in human brucellosis caused by B. abortus (3). Likewise, B. melitensis has been eradicated from native goats 77

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and sheep since 1972. Brucella melitensis is the most prevalent species worldwide, but cases of human infection with this species in the United States generally are linked to importation of contaminated goat's milk cheese from Mexico (4,5). Eradication of B. szris has beenless successful; however, human infection with this species is primarily a problem within the meat-packing industry (6). The true incidence of human brucellosis in many countries is unknown owing in part to the lack of accurate reporting mechanisms. Brucellosis is enzootic throughout the Mediterranean basin and in countries of the Arabian peninsula. In Kuwait, for example, the incidence of human brucellosis reached epidetnic proportions in 1985 (68.9 cases per 100,000 population) (7). With the introduction of brucellosis control measures, the incidence of human infection fell to 20.1 cases per 100,000population by 1988. Unfortunately, efforts to control the disease were hampered by the war with Iraq, and the incidence of human disease no doubt will rise again. Brucellosis in animals can result in economic losses estimated in the millions of dollars annually. Animal production is affected by abortions and infertility. The impact of brucellosis in humans is lost labor, morbidity, and occasional mortality.

II. ETIOLOGICALAGENT Bmcellr species are small, nonmotile, non-spore-forming, gram-negative coccobacilli measuring 0.5-1.5 pm by 0.5-0.7 pm. They grow aerobically at 37OC, although many strains of B. abortus require an atmosphere of 5%-10% CO, for primary isolation (8). Brucellae can be grown on simple defined media containing only a few amino acids and vitamins (for a review of nutritional requirements see Ref. 9). The optimal pH for the growth of blucella in vitro is 6.6-7.4. Brucellae grow rather slowly, especially on primary isolation. Therefore, clinical laboratories should be alerted to maintain cultures for at least 28 days before declaring them sterile. The useof lysis-centrifugation techniques may shorten the time required for recovery of Brwcellcr from clinical specimens (10). Brucella colonies are nonpigmented and nonhemolytic on blood agar. They are approximately 23 mm in diameter, and they appear concave, moist, and glistening. Rough mutants arise spontaneously, especially in liquid medium supplemented with glucose. BrucelZa strains are always catalase positive, but oxidase activity varies. Urease activity is extremely variable, with B. c m i s and B. suis showing stronger reactions than B. abortus or B. melitensis. Proteolytic activity is weak, and gelatin, milk, and serum proteins are not digested. Growth of brucellae is inhibited by certain dyes, a technique that has been used to differentiate species and biovars.

A.

Taxonomy

The oxidative utilization of certain amino acids, urea cycle components, and carbohydrates has been used for taxonomic purposes, although this largely has been replaced by specific phage lysis patterns (1 ). 1 Six Brzrcellll species currently are recognized; however, genomic analysis reveals a high degree of genetic relatedness (12). The exact phylogenetic position of Brucella species remains unclear, however, genomic studies indicate genotypic similarities with the genera Agrobacteriunz and Rhizobium, which are parasites of plants (13).

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B. Hardiness The brucellae are vegetative bacteria, and, lacking spores, they are susceptible to inactivation by such environmental factors as extremes of temperature, drying, and ultraviolet irradiation. Nevertheless, brucellae can survive in moist soil, manure, animal carcasses, and some foods for prolonged periods of time. Pasteurization by the holder method or by the short-term, high-temperature method effectively kills Brllcella organisms. Commonly used disinfectants, used at appropriate concentrations, also effectively inactivate brucellae. Brucellae can be found in contaminated foods for variable periods of time (for a review, see Ref. 14). Factors affecting the survival of Brucella in food include the type of product, the moisture content, pH, age, and the presence of other microorganisms. Brucellae have been shown to remain viable in butter and ice cream for up to a month and in soft cheeses for several months. Although brucellae can survive for months after inoculation into sterile milk, they die within 10 days in naturally contaminated milk or in milk that has soured and become acidic. In contrast to dairy products, meat and meat products are less common sources of brucellosis. Nevertheless, raw meat, organs, and blood can contain brucellae and are potential vehicles for the transmission of brucellosis (15).

111.

EPIDEMIOLOGYANDFOODSINVOLVED

Brzrcelln nzelitensis was isolated firstin 1886 by David Bruce onthe island of Malta from splenic tissue of servicemen who had died from Malta or Mediterranean fever. The epidemiology of brucellosis, however, remained obscure until 1905, when Zammit, working with the Mediterranean Fever Commission, identified antibodies to Brucella in the serum of native goats. Subsequently, the bacteria were isolated from blood and milk of seemingly healthy animals, and ingestion of unpasteurized goat‘s milk was shown to be the source of human brucellosis. Although goats and sheep are the major reservoirs of B. nrelitensis, other food animals, especially camels, are suspected to be the source of brucellosis in some countries. In Oman, for example, serological surveys among animals in the south of the country where brucellosis is prevalent revealed that 8% of camels had antibodies to Brucella (16). Since camel’s milk traditionally is ingested raw and boiling is said to spoil its flavor, it seems likely that camels are an important source of human brucellosis. In 1897, Bernhard L. F. Bang, a Danish veterinarian and physician, first isolated B. abortus from cattle suffering from contagious abortion. It was not until 1918, however, that the relatedness of B. abortus and B. n~elitensiswas recognized by the American bacteriologist Alice Evans. For some time thereafter, the question of whether or not milk from infected cattle could transmit brucellosis to humans was debated. Eventually, cases of human brucellosis associated with the ingestion of raw cow’s milk were documented, and pasteurization was shown to protect the milk supply. Brucella suis was isolated first by J. E. Traum in 1914 from an aborted swine: however, initial studies misidentified it as a biovar of B. abortus. In fact, the first bacteriologically proven case of human brucellosis occurring in the United States was caused by B. suis, but it was not until the isolate was reexamined years later that its true identity was recognized (17). The transmission of B. suis to humans is mainly by direct contact with diseased animals, especially during slaughter and butchering. Transmission by contaminated aero-

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1

sols has been implicated in outbreaks of human brucellosis caused by B. suis in abattoirs (18). In various parts of the United States, feral swine are infected with B. suis, posing a threat to hunters who may be unaware of the disease (19,20). In addition, B. suis biovar 4 is prevalent among caribou and reindeer in the tundra regions of Alaska, Canada, and northern Siberia, and cases of brucellosis among native peoples of these regions have been traced to the ingestion of undercooked meat and bone marrow (21). With the exception of rare cases involving caribou, the role of Brucella-infected wildlife as a source of human infection is controversial. More than 40 species of wild animals have been shown to be susceptible to Brucella (22), but, with few exceptions, the role of wildlife and their ectoparasites in the epidemiology of brucellosis remains unknown. Herds of bison and elk in the Yellowstone National Park are known to be infected with B. abortus, and under experimental conditions the disease has been shown to spread from wild animals to cattle (23). However, proven incidences of such transmission occurring in nature are rare.

A.

FoodborneBrucellosis

Studies by the Mediterranean Fever Commission in 1906 showed that brucellosis could be transmitted to uninfected goats and monkeys by feeding them B. melitensis organisms or milk from infected animals. The role of unpasteurized milk as a source of brucellosis was demonstrated further when prohibition of goat's milk by the military in Malta resulted in a dramatic decline in brucellosis cases among servicemen. Later outbreaks of human brucellosis were traced to the consumption of unpasteurized milk from cows infected with B. abortus; however, this species appeared to be associated less commonly with food than B. melitensis (24). The reason for this is apparent when one considers the studies of Morales-Otero, who showed that B. abortus produced disease more often when inoculated into the abraded skin of volunteers than when it was ingested (25). These findings also are consistent with the observation that the infectivity of intestinal pathogens varies according to their resistance to the bactericidal action of gastric juice. According to this hierarchy, B. abortus is killed more rapidly than B. melitensis, which in turn is killed more rapidly than Salmorzella typhi and ShigellaJEexneri (26). It also has been suggested that patients using antacids for peptic ulcer disease may be at increased risk of oral infection with Brucella (27). The gastric barrier is only one of multiple factors that determine whether infection results from exposure to enteric pathogens (28). The relative virulence of the organism, the infecting inoculum, the immune status of the host, and the route of inoculation are but a few of the factors that determine whether a pathogen will invade and become established in the tissues.

!

B. Travel With the increase in tourism throughout the world, the field ofmedicine has become firmly established (29). The risk to travelers of such infections as diarrhea, malaria, hepatitis, typhoid fever, and sexually transmitted diseases is well recognized. The risk of contracting brucellosis is perhaps lower, but no less serious (30). This risk is especially high when tourists visit countries where brucellosis is endemic and when they ingest traditional foods

F.,

. .". -

..... "

a.._

Brucella

81

(31). In recent years, outbreaks of human brucellosis in the United States have been traced to the importation of dairy products from brucellosis-endemic areas (32,33).

REFERENCES 1. Meyer, M. E. (1990). Current concepts in the taxonomy of the genus Brucella. In Animal Brzlcellosis (K. Nielsen and J. R. Duncan, eds.). CRC Press, Boca Raton, Florida, pp. 1-17. 2. Carmichael, L. E. (1976). Canine brucellosis: An annotated review with selected cautionary comments. Theriogenology, 6: 105-1 16. the U.S. Ann. Sclavo, 3. Brown, G. (1977). The historyof the brucellosis eradication program in 19:20-34. 4. Young, E. J., and Suvannoparrat, U. (1975). Brucellosis outbreak attributed to ingestion of unpasteurized goat cheese. Arch. Intern. Med., 135:240-243. 5. Taylor, J. P., and Perdue, J. N. (1989). The changing epidemiology of human brucellosis in Texas, 1977-1986. Am. J. Epidentiol., 130:160-165. 6. Buchanan, T.M., Hendricks, S. L., Patton, C. M., and Feldman, R. A. (1974). Brucellosis in the United States, 1960-1972: An abattoir-associated disease.111. Epidemiology and evidence for acquired immunity. Medicine, 53:427-439. and Marafie, A. A.(1988). Thenature of brucello7. Mousa, A. R.M., Elhag,K. M., Khogali, M., sis in Kuwait: Study of 379 cases. Rev. Infect. Dis., 10:211-217. 8. Huddleson, I. F. (1921). The importance of an increased carbon dioxide tension in growing Bact. abortus (Bang). Cornel1 Vet., 11:210-215. 9. Gerhardt, P. (1958). The nutrition of brucellae. Bact. Rev., 22:81-97. 10. Kolman, S . , Maayan, M. C., Gotesman, G., Rozenszajn, L. A., Wolach, B.,and Lang, R. (1990). Comparison of the bactec and lysis concentration methods for recovery of Brucella species from clinical specimens. Eur. J. Clin. Microbiol. Infect. Dis., 10:647-648. Brucellosis: Clinicaland Labora11. Corbel, M.J. (1989). Microbiologyof the genus Brucella. In tory Aspects (E. J. Young and M. J. Corbel, eds.), CRC Press, Boca Raton, F L , pp. 53-72. 12. Corbel, M. J. (1988). International Committeeon Systematic Bacteriology Subcommittee on the Taxonomyof Brucella, Report of meeting, September5 , 1986, Manchester, England(editorial). Int. J. System. Bacteriol., 38:450-452. of 16s rRNA from 13. Dorsch, M., Moreno,E., and Stackebrandt, E. (1989). Nucleotide sequence Brzlcella abortus. Nucleic Acids Res., 17:1765. 14. International Dairy FederatiodFederation Internationale de Latiterie. (1 988). Behavior of pathogens in cheese. Document 122, Federation, Brussels, Belgium. 15. Sadler, W. W. (1960). Present evidence on the role of meat in the epidemiology of human brucellosis. Am. J. Public Health, 50504-514. 16. Ismaily, S. I. N., Harby, H. A. M., and Nicoletti, P. (1988). Prevalenceof Brucella antibodies in four animal species in the Sultanate of Oman. Troy. Animal Herrltlz Prod., 20:269-270. 17. Huddleson, I. F. (1943). Brucellosis in Man and Animals. Commonwealth Fund, New York. 18. Kaufman, A. F., Fox, M. D., Boyce, J. M., Anderson, D. C., Potter, M. E., and Mortone, W. J. (1980). Airborne spread of brucellosis. Ann. NY Acad. Sci., 353:105-114. 19. Becker, H.N., Belden, R. C., Breault, T., Burridge, M. J., Frankenberger, W.B., and Nicoletti, P. (1978). Brucellosis in feral swine in Florida. J. Am. Vet. Med. Assoc., 173:1 181-1 182. 20. Zygmont, S. M., Nettles, V. F., Shotts, E. B., Carmen, W. A., and Blackburn, B. 0. (1982). Brucellosis in wild swine: A serologic and bacteriologic survey in the southeastern United States and Hawaii. J. Am. Vet. Med. Assoc., 181:1285-1287. 21. Chan, J., Baxter, C., and Wennman, W. M. (1989). Brucellosis in an Inuit child probably related to caribou meat consumption. Scand. J. Infect. Dis., 21:337-338.

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22. Rementsova, M. M. (1987). Brucellosis in Wild Animals. Amerind, New Delhi, p. 41. 23. Davis, D. S. (1990). Role of wildlife in transmitting brucellosis. In Ad~lancesin Brucellosis Research (L. G. Adams, ed.), Texas A&M University Press. College Station, p. 371. 24. Nelson, C. B., and Giblin, M. (1950). Milk-borne brucellosis in Minnesota.Minnesota Med., 331981-982. 25. Morales-Otero, P. (1933). Further attempts at experimental infection of man with a bovine strain of Brucella abortus. J. Infect. Dis., 52:54-59. 26. Garrod, L. P. (1937). The susceptibilityof different bacteria to destruction in the stomach. J. Pathol. Bacteriol., 45:473-474. 27. Steffen, R. (1977). Antacids-A risk factor in travellers brucellosis? Scarzd. J. Irzfect. Dis., 9~311-312. 28. Antacids and brucellosis (1978). Br. Med. J., 1:739-741. and Schar, M. (1987). Health prob29. Steffen, R., Rickenbach. M., Wilhelm, U., Helminger, A., lems after travel to developing countries. J. Irzfect. Dis.. 156:84-91. 30. Young, E. J. (1989). Brucellosis in travelersand Brucella that travels. In Travel Medicine (R. Steffen, H. 0. Lobel, J. Haworth, and D. J. Bradley, eds.), Springer-Verlag, NewYork, pp. 375-377. of travelers to 31. Arnow, P. M., Smaron. M., and Ormiste, V. (1984). Brucellosis in a group Spain. JAMA, 25 1505-507. 32. Eckman, M. R. (1975). Brucellosis linked to Mexican cheese. JAMA, 232:636-637. 33. Thapar, M. K., Young, E. J. (1986). Urban outbreak of goat cheese brucellosis.Pediatr. btfect. Dis.. 5:640-643.

6 Campylobacter jejuni Don A. Franco Anirml Protein Producers Industry, Huntsville, Missotwi

Charles E. Williams Consultant, Arlington. Virginia

I.

Campylobacter Species 83 A. Isolation, classification, and zoonotic aspects 84 B. Morphology, growth, and comparison with other bacteria

11. EpidemiologyandEpizootiology

85

86

Animal A. reservoirs 86 B. Agricultural animal reservoirs 86 89 C. Other animal reservoirs D. Human reservoirs 89 E. Environmental reservoirs 90 111. Occurrence and Survival in Foods of Animal Origin IV. Modes ofTransmission 92 V. Pathogenicity-Clinical Manifestations

90

93

VI. Economic Impactand Other Costs 94 VII.PreventionandControl VIII.

Summary and Conclusions 99 References

1.

96

99

CAMPYLOBACTER SPECIES

Se.vera1 Cmnpylobncter species are capable of causing foodborne disease in humans, but C.jejurzi and C. coZi appear to be the most prominent of the clinical isolates throughout the world. In 1983, scientists from the U.S. Centers for Disease Control and Prevention (CDC) conducted an epidemiological case/control study on an American university campus where students had been stricken with enteritis caused by C.jejuni. They found that 43% of those who had contracted the disease missed classes, and some 15% were hospitalized 83

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(1). Poultry was identified as the most frequent vehicle of disease transmission. Even the football team was affected; the training diet had been changed to provide a higher ratio of poultry to red meat, and a number of the players became ill. Poor food handling was thought to be an important factor in the majority of the cases (2; R. V. Tauxe, personal communication, 1991). Although the high percentage of cases (70%) in which poultry was implicated is considered unusual-raw dairy products or contaminated water are more often the vehicles of transmission-the fact that young adults were victims of the disease is‘not. CumpyZobncter resembles other enteric pathogens in the rate at which it strikes infants and the elderly, but the organism is unique in that young adults between the ages of roughly 20 and 30 years, and males more than females, are infected at a high rate. The infection, in other words, is not simply opportunistic. The reasons for this pattern are still to be discovered, as are the solutions to such other mysteries as the sporadic nature of cases, the relatively low and invariant infectious dose (the number of organisms required to induce an infection), and the seasonal dropoff of cases from November to January. These and other curiosities can be regarded as academic, however, because the methods of preventing infection by Ccmpylobncter are the same as those for ScdrnoneZZa and other pathogens commonly found in raw foods of animal origin.

A.

Isolation, Classification, andZoonoticAspects

Cnmpylobacter, first identified as animal pathogens about 90 years ago, became recognized widely in the 1970s for causing human illness (3-5). Two veterinarians, McFadyean and Stockman, discovered the organisms in 1909 while investigating the cause of epizootic abortion in ewes (6). In 1913, the same researchers demonstrated that the organisms could be observed in infectious abortions, but World War I prevented dissemination of their work to the public and to health workers (6). For more than 40 years the organism was thought to be exclusively an animal pathogen. During his 1919 investigation of infectious abortions in U.S. cattle, Theobald Smith isolated (along with the “Bacillus of Bang”) a bacterium he described as a spirillum (7). After completing his study, Smith became acquainted with the work of McFadyean and Stockman and inferred that he and they had been studying the same bacteria. He subsequently confirmed his finding with Taylor, and Vibrio-fetuswas proposed as the name of the organism (7). In 1931, Jones et al. ascribed outbreaks of winter dysentery in calves to a bacterium they called Vibrio jejuni(8). Doyle in 1944 attributed swine dysentery to a similar organism (9). In 1946, Levy reported an outbreak of acute diarrhea in humans associated with milkborne organisms that resembled Vibrio jejuni (10). In 1947, Vinzent isolated Vibrio fetus from the blood of three pregnant women, two of whom aborted (1 1). Despite these findings, however, strains affecting humans were not studied extensively until 1957, when King distinguished two groups of organisms isolated from human blood cultures (12). One group corresponded closely to V. fetus,as it then was described; the other group King termed “related vibrios.” In 1963, Sebald and Viron found that these two groups of Vibrio species differed biochemically and serologically from the classical cholera and halophilic Vibrios since their DNA base-pair ratio (G + C mol%) differed from that of the other Vibrio species (13,14). On the basis of these findings, Sebald and Viron proposed that these species be

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removed from the genus Vibrio and that they be called Campylobacter, meaning curved rod (14). In 1972, Butzler et al. isolated Canzpylobacter jejuni from 5% of Belgian children with diarrhea (15). These findings were confirmed later by Skirrow (16). Workers since have reported similar findings, and in some laboratories Campylobacter isolations have outnumbered those of SnlmoneZla and Shigella combined (5,17). As recently as 35 years ago, pathogens were isolated from the stools of fewer than 25% of persons with acute diarrhea. Today, the causative agent is identified in as many as 65-85% of acute diarrhea cases (18). Cnnzpylobacterjejuni is one of the most frequently identified organisms. Methods developed over the years for isolating the organism include species identification, serotyping, biotyping, and phage typing and such molecular typing techniques as pulse-field gel electrophoresis, random amplified polymorphic DNA, and ribotyping. Serogroup I1 was the most common of human isolates in a recent Canadian study of enteritis cases (20). Since its first isolation from human diarrhea stools in 1971, C. jejuni has become recognized as one of the leading causes of gastroenteritis (2). Finch and Riley conservatively estimated that more than 16,000 people were infected by Campylobncter in 1982 (19). They also determined that diarrhea caused by Campylobacter was as common as Salmonella and more common than Shigella. Since most laboratories did not culture for the organisms or report it, the results are significant. Comparative estimates for the United States in 1982 show that C. jejuni infections occurred at a rate approximately equal to that for hepatitis B and at a rate 10 times that for influenza (21). Between 1980 and 1982, meat and poultry were the vehicles of 4 of 23 foodborne outbreaks of campylobacteriosis reported to the CDC-a rate second only to unpasteurized milk (22). Between 1988 and 1992, 2 of 29 foodborne outbreaks of campylobacteriosis reported to the CDC were attributable to meat as the vehicle, while milk was identified as the vehicle in 5 outbreaks (23). A 1984 case-control study by the Communicable Disease Control Section of the Seattle-King County Department of Public Health reported that chicken consumption was the predominant risk factor associated with C. jejuni enteritis; this association was supported by microbiological isolations (54).

B. Morphology,Growth,andComparisonwith Other Bacteria CnmpyZobncter species (25-28) are small, non-spore-forming, gram-negative bacteria with a characteristic curved, S-shaped, or spiral morphology. The cells may vary from 0.5 to 8 pm in width. The cells are highly motile, with a characteristic rapid, darting, corkscrew-like motility. They have a single unsheathed polar flagellum at one or both ends of the cell. Virtually all members of the genus are oxygen sensitive and can grow only under conditions of reduced oxygen tension that vary from almost anaerobic to microaerobic for the different species. An aerotolerant Campylobacter group has also been described (28). Ccmpylobacter species grow best in an atmosphere containing 5-10% oxygen and thus are considered microaerophilic. Although most will not grow under aerobic or anearobic conditions, C. jejuni will grow in candle jars, simplifying isolation. All Campylobacter species grow at 37°C; however, C. jejuni grows best at 42°C (29). Because C. jejuni is a very common pathogen in humans, many laboratories use incubation at 42°C for optimal isolation; however, not all species can be detected at this temperature.

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CcrrnpyZobacter species tend to multiply more slowly than the usual enteric flora. Thus, they cannot be isolated from fecal specimens without the use of selective techniques. The most common isolation methods currently involve the use of antibiotic-containing media. Three such media, Skirrow’s, Butzler’s, and Campy-BAP, or variations of them, are in wide use (39). Butzler’s medium and Campy-BAP contain cephalothin, which inhibits C. fetus and several other Campylobncter species, but they are best suited for isolating C. jejurl i . Cnrrrpylobncter species can be distinguished from other microorganisms by several standard criteria and frotn one another through biochemical testing (30). Visible colonies usually appear on the plating media within 24-48 hours. Occasionally, growth takes place after 72-96 hours of incubation. The serotypic diversity of C. jejuni is broad; the organism is similar in this respect to other bacteria with an ecological niche in the gastrointestinal tract of mammals. More than 60 different serotypes based on somatic (0)antigen and 50 different serotypes based on heat-labile antigens (capsular and flagellar) have been identified (31). Advances in molecular and DNA technology have shown that Cnmpylobactel. forms a distinct group of eubacteria with Helicobacter and Arcobncter (32,33). Campylobncter jejuni is sensitive to drying or freezing temperatures, characteristics that limit its transmission. The organism, however, survives in milk or water kept at 4°C for several weeks. The standard concentrations of chlorine for water disinfection and pasteurization effectively destroy the organism (3 1).

II. EPIDEMIOLOGYANDEPIZOOTIOLOGY Campylobacteriosis is a worldwide disease of animals that can be transmitted secondarily to humans. Although important aspects of the transmission of the organism remain unexplained, considerable progress has been made in the understanding of both the reservoirs and the prevalence of the infection.

A. AnimalReservoirs Zoonoses, diseases transmitted from animals to humans, are remarkable not only for their frequent occurrence but because they almost always are unsuspected and unrecognized. This is as true for Cnmpylobacter as for other zoonotic pathogens. CnmpyZobacterspecies are commonly found as commensals of the gastrointestinal tract of wild or domesticated cattle, sheep, swine, goats, dogs, cats, rodents, and all classes of poultry. Extensive reports in the scientific literature demonstrate that the reservoirs of organisms in the animals cause infection and disease in humans. Animals or animal products have been identified conclusively as sources of infection in several outbreaks, and many of the Cnmnpylobncter serotypes that cause disease in humans have been isolated from animals (34). B. AgriculturalAnimalReservoirs 1. Cattle C. jejuni appears to be a normal commensal of all classes of bovines. The prevailing evidence supports transmission from adult cattle to calves, but not between adults. It is

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possible at any given time for several different serotypes to be present in a herd (34). Carcasses may become contaminated with intestinal contents during slaughter. In one outbreak, 525 specimens were obtained from 100 slaughtered beef cattle and examined for the presence of C. jejurzi and C. coli by direct plating and enrichment techniques. Of the 100 animals, 50 were positive for C. jejmi and 1 was positive for C. coli. The distribution pattern of positively infected animals, in decreasing order, was steers (57%), bulls (40%), heifers (40%), and cows (22%). Of those animals infected, significantly higher isolation rates were obtained from gall bladders (34%), large intestines (36%), and small intestines (32%) than from liver (12%) or lymph nodes (1.4%) (35). Similar rates have been reported in subsequent studies (61). In Finland, fecal samples from 200 cattle showed an incidence of 5.5% for C. jejmi (36). Similarly, feces collected from 90 cattle in Sweden showed positive isolates from 17 (19%) (37). In a milking herd, the presence of the organism does not necessarily lead to condemnation of the milk because C. jejzjuni is destroyed by pasteurization. A community outbreak of gastroenteritis in Vermont was traced to consumption of unpasteurized milk produced at a commercial dairy. Two different testing schemes showed a C. jeju~ziisolate from a sick patient and an isolate from a diseased cow to be of the same serotype (38).

2. Swine Carcass contamination by Curzpylobactel- species appears more common in swine carcasses than in cattle, sheep, or goats. The danger of intestinal spillage is greater with the procedures used in dressing swine carcasses than with those used in dressing the carcasses of ruminants. A Canadian study of cooler-ready hog carcasses to evaluate the degree of contamination of the carcasses and the slaughter environment showed that the largest number of Cmpylobncter isolates, mostly C. coli, came from fecal samples-hence, from the animals themselves (39j. Isolates of C. jejmi from 65 animals in Zaire (29% of the total number examined) showed the highest isolation rates in swine-42 positives (44%) of 95 animals examined. In swine, the youngest animals, weanling pigs less than 2 months old, were positive more frequently than the older ones: there were 13 positives out of 19 weanlings examined and 25 positives out of 72 adult animals examined (40). Points have been identified in the slaughtering and dressing process where contamination by Cumpyllobncter- and other organisms may be limited only by strict observance of sanitary procedures (41). The postslaughter chilling process tends to suppress Ccunpylobncter. replication on the carcasses. This suppression apparently is not caused by low temperatures, but is due to the drying effect of the forced ventilation used in the abattoir cooling rooms. C~~vrzpylobacter species appear to be sensitive to drying (42). A Canadian study of 118 isolates from hogs showed 1 15 positive for Cumnpylobncter-coli serotype (43). Samples collected from 300 normal slaughtered hogs during epidemiological studies performed at six different slaughterhouses were examined for the presence of C. -fetus jejuni. Of these samples, 182, or 60.796, were positive (44).

3. Sheep The relationship between sheep and Ccmpylobctcter has been documented since as early as 1909, when the genus was implicated in ovine abortion. C. jejjmni is an intestinal commensal inmany sheep herds, and surveys have shown the organism to be a common isolate. However, C. jejuni has not been found in sheep as often as in other food animals.

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Isolations of Campylobacter species were made from 16 of 197 (8.1%) sheep fecal samples examined in Norway (45). Stomach contents and heart blood were collected from 10% of lambs from 25 farms for survey of Carnpylobucter fetus. C. fetus infection was found in 102 of the 762 lambs examined (46).

4. Poultry Campylobncter species frequently are present in the intestinal flora of commercially raised birds and such wild birds as pigeons, sea gulls, crows, and ravens. High percentages of C. jejuni isolations in pigeons, obtained in a Spanish study, suggest a need for further study of this possible source of human infection (50). Isolations have been made from poultry early in the growing process, but many flocks escape infection. A Canadian study has shown that C. jejuni could be isolated from all of 108 chicken samples (43). Reports vary on the incidence of positive isolates. While researching the association of Carnpylobacterjejuni with laying hens, Doyle reported peak rates of C. jejurzi isolations of about 25% in October and in late April to early May (47). A Dutch study of broiler chickens found the highest rate of Campylobacter and Sulmorzelln contamination from June to September and the lowest in March and a positive correlation between Canzpylobrrcter and Salmonella colonization within flocks of broiler chickens (51 ). Potential routes of entry of organisms into a flock include infection of newborn chicks from older birds, contamination of feed and water, and wildor game birds. Infection of poultry is often without clinical manifestations. Contamination or recontamination can affect bacterial loads on poultry either in the growing process or at the processing plant. The Contamination ofpoultry houses by carrier flocks has been shown to lead to contamination of subsequent generations of poultry if cleaning and disinfection between removal of market-ready birds and introduction of new flocks is ineffective. When the birds were brought to slaughter, contamination was spread to the processing equipment. Ineffective sanitation at the end of the day contributed to the contamination of birds the following day (48). During the slaughtering process, C. jejurzi from the intestinal contents of processed birds spreads to the carcasses. There also appears to be an inverse relationship between the amount of intestinal carriage and contamination. In one study, C. jejzrni was found to be widespread in each of 15 poultry plants evaluated, but on some days the organism was not isolated from any samples. Slaughtering of heavily contaminated flocks may result in a contamination rate of up to 100% for end products and seems to be unrelated to the type of slaughtering process (49). A total of 138 samples from two California chickenprocessing plants was collected from four groups of birds (7-12 weeks old) in 11 lots over six mornings. Isolation rates for all sites for three scald-water temperatures described were 66.7% (60"C), 75.8% and 78.3% (53"C), and 55.1% (49°C). C. jejuni was isolated from all chilling tank samples, 94.6% of feather picker water samples, and 60-100% of fecal samples. The results demonstrate that chilling water and feather picker water were the major sites for cross-contamination (52). A study of Cmzpylobacter contamination of turkey carcasses at various stages of processing showed reductions of contamination at scalding, but recontamination at defeathering. C.jejuni counts peaked during evisceration but declined after chilling (53). Harris et al. reported a case-control study in Seattle (King County), Washington, where almost 50% of poultry from the processing plant surveyed were positive for C. jejuni. The highest positive rate occurred during the period July-October (54). This finding

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is consistent with earlier studies in England, Belgium, the United States, and South Africa, which showed a summertime peak of Campylobacter infection (55,56).

C. OtherAnimalReservoirs Epidemiological studies of C. jejuni and other Cumpylobacter species in such pets as dogs and cats have led to several findings: (a) C. jejuni may be isolated from both healthy and diarrheal dogs, (b) isolation rates are higher in puppies and kittens than in adult dogs and cats, and (c) isolation rates are higher in kennel populations of both dogs and cats than in pets raised in households. C. jejuni and C. coli have been isolated from 45% of clinically normal cats-the high rate of isolation possibly being attributable to scavenging of contaminated food from a poultry-processing plant. Campylobacter species were isolated from 49% of 144 stray dogs, 39% of 38 clinically normal puppies, and 38% of 42 diarrhetic puppies seen in a veterinary practice (57). In one breeding kennel, C. jejuni was isolated from the feces of 4 of 47 healthy adult dogs, 7 of 71 healthy puppies, 10 of 31 puppies with blood-stained diarrhea, and from the intestines of a puppy that had died from hemorrhagic enteritis (58). Campylobacter counts in normal dogs and cats show a wide range; they are highest (49% dogs, 45% cats) in immature animals, particularly strays or those living in kennels, and lowest (1.6% of dogs and cats) in adult animals living in households (16). C. upsaliensis was isolated from the blood and fetoplacental material of a woman in Turkey who spontaneously aborted after an 18-weeks pregnancy. The woman had had contact with a household cat, which tests showed may have been the source of the infection (60). A 1996 study of dogs and cats in Sweden found a high proportion to be colonized with C. upsaliensis, also found in children. A significant proportion of cats were colonized with C. Iwlveticus, a diarrheal agent in humans (61). These and other studies have shown the risks of Campylobacter transmission to humans from pets, even asymptomatic pets, the need for more efficient procedures to detect campylobacters in pets, and the importance of taking preventive measures, such as hand-washing, after contact with pets (62,63).

D. HumanReservoirs Fecal-oral person-to-person infection has been reported for C. jejuni. As with other enteric pathogens, those in contact with excreta of infected people are at risk. Transmission from asymptomatic infected food handlers is uncommon (31). Interesting reports of spread by human-to-human contact appear in the published literature, implicating food handlers as sources of the organism. These findings support the exclusion of food handlers from the work environment when they are affected, as well as during the period of carriage of the organism (59). A temporary food handler was the most probable source of an outbreak of acute gastroenteritis due to Campylobacter jejuni that occurred at a military base in Israel. Stool cultures were taken from 17 clinically affected as well as 23 asymptomatic soldiers. In 6 of the 17 patients with enteritis (35%), Campylobacter jejuni serotype ii was isolated, while the stool cultures of all the asymptomatic soldiers were negative. The temporary food handler had suffered from acute gastroenteritis before the outbreak but had not reported the illness. He was found to harbor the same serotype as the affected patients (64).

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E.Environmental

Reservoirs

1. Water Outbreaks of waterborne campylobacteriosis in the United States have been traced to the drinking, during outdoor recreational activities, of contaminated spring water or other water not meant for drinking. A May 1983 outbreak in Florida consisting of 871 cases was found to have occurred after a community water system had been contaminated by wild birds (65). Domestic or wild animals and birds potentially are significant sources of Cnrnpylobacter organisms found in polluted water. Back country (i.e., thinly settled rural areas) surface waters can be an important source of C. jejzmi. Infection should be considered as a cause of diarrhea in those who recently have returned from wilderness areas (66). In nature, C. jejzlni has been isolated from stream and river water, from the effluent of poultry-processing plants, and even from seawater samples. Ccrnzpylobacter species have been isolated from 50.4% of 540 (riverine) water samples from the Southampton area in England (34). Survival studies in Norway of thermophilic Carnpylobncter species revealed that all organisms survived better at 4°C. Chlorination drastically reduced the survival of all three strains examined. All strains survived for 10-15 days in polluted river water at 4°C. Removal of interspecific competition by sterile filtration did not enhance survival significantly (67). In 1992, Norwegian researchers reported that thermotolerant campylobacters had been isolated from polluted surface waters most often in a temperature range of 2.18.0°C, least often at temperatures higher than 15"C, and more often in autumn than in other seasons (68). 2. Soil Under conditions favoring replication, fecal contamination of soil could be a source of infection. Isolations of Curnpylobacter species have been made from both mud and sewage sludge in the United States. A 1994 survey of livestock wastes in the United Kingdom found Cmrpylobucter in a high proportion of the wastes. Cnrnpylobacter spp. were isolated more frequently than other pathogens such as Sulmonelln. Though considering the relatively poorer survival of the Canlpylobncter to make them less of a water pollution hazard, the researchers acknowledged the need for a greater understanding of the survival and transport of Currzpylobncter. in soil (69). 111.

OCCURRENCE AND SURVIVAL IN FOODS OF ANIMAL ORIGIN

Transfer of C. jejzwi to carcasses and to other meat and poultry products has become a serious problem for the meat and poultry industries. The most frequently implicated meat is poultry, with a C. jejzmi recovery incidence of 50% in store-bought poultry meat. Red meat carcasses also have yielded this bacterium, although at lower incidence. Food surveillance results of samples collected from February 1982 to September 1983 by researchers of the Seattle-King County Department of Public Health showed C. jejuni cultures at the following levels from retail products: poultry, 192 of 862 samples (22.370);beef, 1 of 230 samples (0.4%); pork, 0 of 142 samples; and lamb, 0 of 37 samples (54,70).

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A Polish survey found Canzpylobncter species on 80.3% of chicken, 48.0% of duck, and 38.0% of goose carcasses but on only 3.0% of turkey, 2.9% of pork, and 0.9% of beef carcasses (71). Samples collected in a survey of Canadian abattoirs from 1983 to 1986 yielded isolates of thermophilic Ccmpylobacter from 16.9% of pork, 22.6% of beef, and 43.1% of veal carcasses. C. jejuni biotypes I and I1 were the most frequently isolated in beef and veal. Biotype I was the most frequently isolated in pork, but 41.1% of the isolates from pork were C. jejuni biotypes I and I1 (72). Of Campylobacter-positive Samples in a Trinidad study of pathogen prevalence in raw meats and seafoods, most were from chicken (73). C. jejuni was found in 78 of 94 (82.9%) chicken wing packages analyzed on the day of delivery to supermarkets and in 15.5% or 45 out of 290 packages obtained from the supermarket shelves a few days later (74). About 800 fresh and frozen meat and poultry samples were analyzed at different field service laboratories of the U.S. Department of Agriculture, Food Safety and Inspection Service. Isolation levels of C. jejuni from fresh tissues were more than five times as high (12.1%) as those from frozen tissues (2.3%) in the samples analyzed. The prevalence of C. jejuni in fresh tissue according to species was avian, 21.3%; bovine, 4.7%; porcine, 8.6%; and ovine, 20.0% (75). A total of 6169 meat samples was examined in 31 laboratories. C. jejurzi was isolated from 98 (1.6%) of the samples, showing a low level of red meat contamination (76). Surveys continue to show the frequent presence of C. jejuni on whole birds or parts obtained at retail outlets and the need for food handlers and homemakers to practice good hygiene when preparing meals to prevent cross-contamination (77). There is evidence that the levels of C. jejuni on broilers sold at retail are affected by seasonal factors and the carrier state of the broilers (78). Studies of the incidence of C. jejuni on retail red meats have shown its presence on these products to be considerably less than on poultry (33,101). Refrigerated storage appears to cause the number of Campylobncter-positive red meat carcasses to diminish, although the effect of chilling on the bacteria appears to depend on the age of the animals. An Australian study showing Cnmpylobacter to be more prevalent on carcasses of feedlot cattle than on those of range cattle also demonstrated that chilling adult cattle carcasses for 20-24 hours significantly reduces the incidence of Canlpylobacter. On the other hand, Canzpylobncter species were found on almost all calf carcasses examined after chilling (79). Most cases of milkborne Cnmpylobacter enteritis have been associated with the consumption of unpasteurized milk. Research workers in Wisconsin isolated C. jejurzi from 1 of 108 (0.9%) milk samples obtained from the bulk tanks of 9 Grade A dairy farms and from 50 of 78 (64%) cows producing Grade A milk (80). In the Netherlands, C. jejuni was isolated from 2 of 1200 milk samples from farm bulk tanks, but not from 600 samples milk cans or 750 samples from cows with clinical mastitis (81). C. jejuni is a rather unique enteric pathogen. It differs markedly from other food poisoning organisms in that it normally does not multiply in food in numbers sufficient to cause infection, as do Sabnorzella species or Staphylococcus aureus, which produce a toxin in food associated with disease. The factors affecting survival and growth of C. jejuni usually are strict microaerophilic conditions: an atmosphere of 5% oxygen, usually with about 8-10% carbon dioxide and 85% nitrogen; an optimum temperature of between 42 and 45°C; and a roughly neutral pH (6.5-7.5) (82). The organism survives better in foods at refrigeration temperature than at room temperatures.

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IV. MODES OF TRANSMISSION Transmission can be defined as the pathway by which a pathogen spreads from its source to the host. It may occur through one or more of four different routes: contact, common vehicle, airborne, and vectorborne (83). Cumpylobacter may be transmitted by direct contact with contaminated animals or animal carcasses, through the ingestion of contaminated food and water, person to person from excreters with active infections, and by perinatal and childhood transmission. The following aspects of transmission are discussed from the foodborne perspective. The majority of Cnmpylobacter infections appear to occur sporadically without a clear determination of the transmission mode. The vehicles incriminated as sources of infection include poultry intended for human consumption, uncooked or poorly cooked meat and poultry products, unpasteurized dairy products, and uncooked foods subjected to possible cross-contamination by meat and chicken. In a case-control study in an urban Swedish community, two diseased persons had blood specimens positive for C. jejuni. Of the diseased persons tested, 8 of 10 had immunoglobulin-M-specific indirect fluorescent antibody (FA) titers to C. jejurzi, a significant association of chicken consumption and Cumpylobucter enteritis (84). C.jejuni was found almost routinely in beef sold in food stores. The bacteria survived on the food at 4°C for one week and frozen at -20°C for 3 months (37). In a Lima, Peru, market, 80 chicken handlers were studied for C. jejuni. The same number of individuals from the same market who did not handle chicken were sampled as controls. Results showed 15 persons (19%) positive for C. jejuni among handlers and 1 person positive among the control group (85). Raw hamburger was implicated in an outbreak that occurred in the Netherlands (87). Raw clams, probably contaminated by sewage, were described as the vehicle in an outbreak in New Jersey (34). Milk is an important and frequent source in the transmission of CnmpyZobacter enteritis. A community outbreak of Carnpylobncter enteritis was associated with the consumption of untreated milk, apparently from two cows with Campylobncter mastitis (87). A New Zealand report implicated raw milk as the cause of gastrointestinal illness among children in two different camp sites. Cnmpylobncter jejuni was isolated in 50 of the 88 affected children (88). A drink prepared with raw milk was associated with an outbreak of C. jejuni enteritis involving more than 500 participants in a jogging rally in Switzerland, with an attack rate of over 75% (89). An outbreak of Cnmpvlobacter jejuni enteritis followed the consumption of unpasteurized milk at an attack rate of around 50%. There were cases in all age groups, with the highest number in the 1 to 10-year-old group (90). C. coli was isolated from a 9-year-old British boy with persistent diarrhea, whose family had consumed raw goat's milk from a local farm. C. jejuni and C. coZi were found in the feces of goats from the farm, and C. jejuni was identified in samples of bulk milk (91). A Swedish researcher has implicated C. jejuni in milk with mastitis, even though no cases of Cnrnpylobacter mastitis have been reported except those induced experimentally (92). In another study, some 2500 English schoolchildren between the ages of 2 and 7 became acutely ill after drinking free school raw milk (59). A case-control study conducted in Seattle and King County, Washington, revealed an association between infection by Campylobacter jejuni coli and the consumption of raw milk and, to a lesser extent, mushrooms. The subjects in the study were more likely to be infected by tetracycline-resistant than tetracycline-sensitive strains (54).

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It is important to remember that many zoonotic infections are acquired by contaminating food from diseased animal tissues or from infected animal feces. Under normal processing procedures, it is extremely difficult, if not impossible, to produce meat and poultry carcasses that are free of bacteria. The slaughter and processing environment has the potential to perpetuate contamination because of the close contact of the tissues in the slaughterhouse with animal and poultry feces. It is inevitable that some of these contaminating bacteria will be Campylobncter species (93). The same basic factors apply to raw or inadequately heat-treated milk contaminated by feces at the time of milking. Chronic consumption of raw milk contaminated with C. jejuni appears to stimulate the production of antibodies resistant to the organism in healthy individuals. Those without the immunity thus conferred are more likely to be susceptible to infection (94). Fortunately, most milk consumed in the United States is pasteurized and devoid of harmful pathogens. The cycle of actual or potential contamination does not always end at the processing establishments. Clean meat and poultry can be contaminated at retail outlets if hygiene at the facility is poor or if insufficient attention is paid to the sanitization and cleanliness of such operational equipment as knives, cutting blocks, and slicing machines.

V.

PATHOGENICITY-CLINICALMANIFESTATIONS

Campylobncfer can be found in almost every country and now is recognized as one of the most common causes of bacterial diarrhea. Acute enteritis is the most common presentation of C. jejurzi infection, with symptoms varying from one day to one week or longer. A prodromal phase with fever, headache, myalgia, and malaise usually occurs 12-24 hours (but may last for up to 48 hours) before the onset of intestinal symptoms. The most common symptoms with Campylobncter infection are diarrhea, malaise, fever, and abdominal pain. The diarrhetic pattern can vary from loose stools to profuse, bloody, slimy, and foulsmelling stools (3 1,95). In some patients, cramping abdominal pain, typically periumbilical, can be the predominant manifestation of illness (31). Vomiting may occur but is rarely a marked feature. The illness frequently is selflimiting to within 1-4 days and usually lasts no more than 10 days, although there may be occasional relapses (96). A recurrence of symptoms is experienced by 25% of patients, often characterized by abdominal pain and varying from a relatively mild gastroenteritis to an enterocolitis with bloody diarrhea and accompanying abdominal pain lasting for several weeks (95). As with other intestinal pathogens, the clinical picture of C. jejuni infection varies from symptomless excretion to severe disease. Although it is normally a self-limiting disease, such complications as cholecystitis, peritonitis, septicemia, and meningitis have been reported. The small intestine is thought to be the main site of infection, but the colon often is involved (97). In the mid- 1980s, two fatalities from C.jejuni infections were reported in the Denver metropolitan area. The first case was in a previously healthy 26-year-old woman who died following a 2-day diarrheal illness. The second case was in a 69-year-old diabetic woman who died 19 hours after developing a gastrointestinal tract illness one day following hospital discharge for an orthopedic procedure (98).

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Archer reviewed the role of foodborne enteric pathogens (including C. jejuni) in the development of three seronegative spondarthropathies jankylosing spondylitis, Reiter’s disease, and reactive arthritis) (99), following gastrointestinal infections with gram-negative enteric bacteria. A molecular explanation of the etiology of these complications has been explored ( 100). Another potential complication of Cmnpylobacter infection is acute inflammatory neuropathy. There is increasing recognition that recent C. jejuni infection is the most common precursor of Guillain-Barr6 syndrome (101). Clinical investigations of gastrointestinal cases during the last two decades have led to the identification and description of new types of Cnnzpylobcrcter and Cuwpylobacterlike organisms (CLO). Australian researchers have reported on the colonization by spiral bacteria of the stomach antrum of patients with peptic ulceration and gastritis. They were found in 100% of patients with duodenal ulcers, 80% of patients with gastric ulcers, and 95% of patients with “active chronic gastritis’’ (102). Another Australian study, of blood cultures at Alice Springs Hospital, showed that of 72 cases of bacteremia that were identified, C. jejuni was the causative agent (103). Fennel1 and coworkers from the University of Washington in Seattle described new CLOs associated with proctitis in homosexual men. The organisms were isolated from 28 of 181 symptomatic homosexual men compared with 7 of 77 asymptomatic homosexual men and 0 of 150 normal heterosexual men and women (104). A study of Campylobncter isolates collected over two decades from South African children with gastroenteritis found that, for those cultures that could be serotyped, the most common serotype (0:41) was one that included strains associated with Guillain-Barr6 syndrome cases. Others were obtained from kwashiorkor and severe, chronic diarrhea cases. It is suggested that this serotype may represent a particularly virulent strain (105).

VI.

ECONOMICIMPACTANDOTHERCOSTS

While the reporting of foodborne disease has increased, particularly in Europe, the effect of disease on national economies and health has not been documented thoroughly. Limited attempts to document such costs have been made, but extrapolations from one or two outbreaks to an entire nation have been unreliable (106). This is unfortunate, because the failure to document the true costs leads to underfunding of research in what is one of the most important and prevalent public health problems in the United States (107). The true incidence of foodborne diarrheal disease is nearly impossible to determine, and outbreaks may go unreported for a variety of reasons. First, because of the limited duration of many bacterially mediated foodborne infections, individuals are not inclined to report them. Second, affected persons who do seek a physician’s opinion because of the severity or duration of diarrhea normally are treated symptomatically and without diagnostic tests. Third, even when the episode is severe enough to require hospitalization, the appropriate diagnostic tests may not always be performed. Fourth, by the time the epidemiology is associated with a foodborne illness, the suspected food seldom is available for analysis. Fifth; the public considers diarrhea to be a self-limiting, unpleasant nuisance, but not life-threatening and with few negative long-term health consequences (108). Hauschild and Bryan estimated the number of cases of foodborne and waterborne illness in the United States to be 1.4-3.4 million per year (109). This estimate was based on data from follow-up surveys conducted by the CDC following disease outbreaks. The

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ratio of estimated cases to those reported initially was 25:l for all foodborne diseases. Todd estimated the number of foodborne disease cases in the United States to be 5 million per year (106). In a 1977 German study, Krug estimated the cost of salmonellosis among humans to be $266 for sickness and $30 million for deaths (1 13). Morbidity accounted for 30%, mortality lo%, and undetected cases 60% of the total costs in this assessment. Costs also were expressed as proportions of the social costs as follows: 42% loss of leisure 23% welfare losses 16% losses in consumption 12% treating costs 6% examination costs 1% other costs Since 1962, estimates have been made of losses in the food-service industry due to foodborne illness. Most of thedocumented illnesses have involved Salstzonelln contamination; total costs per case ranged from $315 (1962 dollars) to $4867 (198 1 dollars) (1 13). Todd some years ago estimated the cost of all foodborne disease in the United States to be $1-10 billion annually (1 13). Included in Todd’s estimate were such economic factors as direct medical costs, lost wages and productivity, and industry loss through embargo, voluntary destruction, and recall. Because Campylobacfer j e j m i is a recently recognized, important cause of foodborne disease in the United States and the rest of the world, its economic impact can be estimated by a comparison with Salmonella incidence and prevalence. The incidence of campylobacteriosis has been reported to be 2.5 times that of salmonellosis (54). Skirrow reported that the incidence of campylobacteriosis exceeds the combined total cases of salmonellosis plus those of shigellosis (16). Archer and Kvenberg reported on work done by Hauschild and Bryan, who devised a ratio for salmonellosis of 29.5: 1 cases to isolations, based on data from postoutbreak questionnaires (109). In 1983, it was estimated that 1,147,000 cases of salmonellosis, a reportable disease, occurred in the United States, An extrapolation for the prevalence of carnpylobacteriosis would approximate 2,867,000 cases for that year. Accurately assessing the incidence of foodborne disease has posed obvious problems. In the United States, there have been recent indications that the CDC estimate, dating from the 1980s, of approximately 9000 deaths annually, is an inflated number that has not been scientifically validated. A definitive determination of the true number will have to be made if real progress in assessing risk is to be made. In the United States, an acceptable reference baseline does not exist aside from the extrapolations made by CDC epidemiologists. The Clinton administration initiative to build a new early-warning system for foodborne disease outbreaks will yield better information on which to base future prevention strategy. Increasing the number of FoodNet surveillance sentinel sites and using DNA “fingerprinting” technology to identify the source of infectious agents will enhance the process (1 10). 1996 U.S. calculations of the economic impact of campylobacteriosis, based on an estimated total 2,500,000 cases, yielded a result of $1,169.8- 1,373.8 billion annually. This result is a function, among other variables, of an estimated 200-730 deaths. Assuming that the vehicle in 55-70% of human illness cases of campylobacteriosis in the United States is food, the number of foodborne deaths in the United States attributable to Campy-

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lobctcter ranges from 110 to 5 11 per year. The annual costs of foodborne campylobacteriosis are $0.6-1 .O billion (1 11). In the United States, estimating total diarrheal disease is a continuing challenge, especially with regard to such nonreportable conditions as Camnpylobncter enteritis. The question persists: How many people seek a physician's care for diarrhea or an associated symptom (e.g., vomiting, cramps, nausea)? The answer depends on many variables that will continue to elude the resources of professionals in the field of foodborne disease. The fact that no specific methodology exists for valuing economic loss of a given illness, and that there is also no way of comparing the seriousness of specific illness, adds to the complexity of making adequate resource allocations. Economic impacts and cost profiles of such specific foodborne diseases as campylobacteriosis and salmonellosis are needed to support policy decisions to attack specific diseases or study risk and co-risk factors. We must continue to maintain an interest in developing better methods of recognizing various forms and impacts of loss and suffering from foodborne illness, living with risk factors, and invasive treatments. In summary, we must be aware that a condition like campylobacteriosis is much more than an inconvenience. It is responsible for severe illness, complications, operations, and hospitalizations for previously well persons in all age groups. Casnpylobncter enteritis is also expensive, not only for those who are ill, but for society at large, because outbreaks affect the economy adversely. Because it is a preventable disease, the cost of public health programs for the prevention and control of campylobacteriosis must be measured against the unnecessary costs of medical treatment and productivity loss.

VII.PREVENTIONANDCONTROL Prevention and control of most zoonotic diseases is difficult at best but assumes greater importance and significance when the causative agent C. jejrnni is distributed so widely among animal reservoirs and the environment. The survival of Cnmpvlobncter on fruits and vegetables also has become a subject of increasing attention and concern (112). It should be recognized that even a comprehensive, conscientious approach might not result in complete elimination of the organism. Also, resources are not available to carry out an unlimited effort against a particular foodborne pathogen like Campylobacter without including a number of other such bacteria as Yersinia enterocolitica,Aeromonns, and Listeria monocytogenes.Thus, there can be no assurance that any raw meat and poultry will be completely free of Campylobacter. Control measures are facilitated by the fact that C. jejuni is not a hardy survivor outside the host and does not survive well in food because it is sensitive to (a) drying, (b) 21% oxygen, (c) storage at 25OC, (d) acidic conditions, and (e) heat (1 15). Preventive and control measures must include the use of existing resources to reduce the level of infection in animals and subsequently in humans. Achieving this goal will require the concerted effort of livestock producers, food processors, veterinarians, physicians, public health officials, regulatory agencies, food chains (distributors and retailers), consumers, and research scientists. The livestock farmer can (a) practice sound sanitation procedures around the farm, (b) maintain disease-free animals, (c) use only feeds tested and found free of Campylobacter, and (d) prevent contamination of animals or food before or during transfer from farm to market. Cleaning and disinfection and maintaining a sanitary environment are

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essential. Changes in rearing practices, including improvement of the quality of litter, can reduce the transmission of Cnmpylobncter through poultry flocks (1 16). Administration of defined competitive exclusion cultures to chicks, colonizing them with protective bacteria, has been demonstrated to reduce colonization with C. jejuni (1 17).The implementation of disease-control principles will cost money, but the benefits of healthier livestock and the potential for establishing Cnrnpylobacter-free farms has to be a meaningful long-term objective. The food processor and distributor can assure the wholesomeness of food products by making mandatory the use of the HACCP (hazard analysis critical control point) system in their food-protection programs. The HACCP system includes elements that deal with (22):

1. Animal production controls-the wholesomeness and safety of meat and poultry are based in part on the health of live animals, their feed, and the environment in which they are raised. 2. Sanitary slaughter and dressing-the sanitary conditions during transport, slaughtering, and dressing; the rate of carcass cooling; and the time-temperature conditions of storage and distribution all have an effect on the microbial load on carcasses. 3. Processed products-cross-contamination of raw meat is minimized by assuring the cleanliness of workers’ hands, cutting boards, knives, and saws and by maintaining the condition of other utensils and equipment. 4. Raw (eviscerated, ready-to-cook) poultry-use of microbiological guidelines periodically to evaluate equipment surfaces and processing practices by examination of freshly processed carcasses. Companies should educate employees to observe correct personal hygiene principles, establish effective rodent and insect controls, limit nonessential traffic in all foodprocessing areas, and employ technically competent managers and supervisors who are conscious of and committed to the principles of food hygiene. The consumer should be conscious of basic sanitation and health practices, especially hand-washing and the use of clean equipment and cutting boards; proper methods of storing, preparing, and serving food; and the potential microbial contaminants of raw meat and poultry. The veterinarian can (a) help farmers to maintain disease-free animals and clean environments in which to raise them, (b) seek laboratory confirmation of suspected cases of campylobacteriosis in animals and report positive cases, (c) conduct sanitation inspection of the slaughter environment and product inspection to ensure an acceptable level of compliance with established standards of hygiene, (d) assist in the education of food-processing management and employees of proper sanitation practices, and (e) assist in the education of the public regarding the basic principles of food hygiene and the attributes of personal cleanliness in the prevention and control of foodborne diseases. The physician can (a) seek laboratory confirmation of suspected cases of campylobacteriosis in humans, (b) report all infections of Cnmpylobncter species to the proper health authorities and assist in the epidemiological work, when appropriate, (c) inform patients of needed precautions to take against the spread of the organism, especially to family and close contacts, and (d) assist in the education of the general public on the prevention of foodborne disease. The public health official can (a) keep local physicians informed of suspected outbreaks of campylobacteriosis, (b) follow up reported cases and investigate outbreaks, (c) take special care to obtain proper specimens for laboratory examination, (d) impress upon food processors

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the need for proper sanitation control, (e) create public awareness of campylobacteriosis, and (Q promote campylobacteriosis prevention and control (1 18). The research scientist can (a) maintain a close liaison with clinicians and epidemiologists, (b) keep abreast of the latest information applicable to Ccmpylobncter species, (c) assist in the adoption of acceptable standardized methods for isolating CnmpyZobncter species, (d) keep current with new or improved techniques for their isolation, (e) handle all cultures of Cntnpylobcrcter species with the necessary precautions to prevent spread from the laboratory, and (f) serotype all isolations and share information with collaborators and other researchers. In addition to these cooperative efforts to control the organism, innovative technologies show promise in minimizing or eliminating the Cun?pylobucterproblem. One of these, food irradiation, has been studied intensively for its antimicrobial effects. A major study showed that a medium dose of 250 kilorads (krad), combined with a handling environment temperature of 1.6"C (34.9"F). resulted in a product essentially free of bacteria that could be kept safely under refrigeration for up to 20 days. Foods irradiated at doses not exceeding 100 krad have been shown to be wholesome and safe for human consumption, requiring no safety testing to market (1 19). While irradiation can be effective in enhancing the safety of fresh products, there are some disadvantages. Irradiation can affect metabolic food processes, making the food less resistant to spoilage by various fungal diseases. Also, changes in the flavor or texture of irradiated food may be unacceptable to some consumers ( 120). The preponderance of research indicates, however, that irradiated foods are safe, can extend a product's shelf life, and can reduce the number of food spoilage microorganisms (121). Significant research has been aimed at evaluating the use of acetic acid or lactic acid in chilled water in poultry-processing plants to help produce a Snlnzonelln- or Canzpylobacter-negativepoultry supply (R. W. Johnston, personal communication, 1985). A 0.5% lactic or acetic acid wash has been observed to reduce the number of indigenous Cnnzpylobucter 10-fold in chickens (122). Studies involving the use of lactic acid and other chemical solutions on chicken inoculated with various pathogens, including C.jejuni, have shown significant reductions in the populations of these bacteria (123,124). This response to acid media in the water presumably was due to the acid sensitivity of C. jejuni, the preponderant strain in poultry (125). Other studies have found that 1.5 and 2% sprays of lactic acid or other organic acids reduce the numbers of Cnnzpylobacter and other organisms that could be isolated from pork carcasses (126,127). Recent research also has focused on the effects of trisodium phosphate treatments on poultry carcasses, with promising results ( 128,129). There are both immediate and long-term solutions to the problem of Cn~npylobcrcter. Immediate solutions involve pasteurization or cooking of animal-derived foods before consuming them and preventing cross-contamination of cooked food by raw food or surfaces and other utensils that contact raw food (130). The control of milkborne campylobacteriosis is simple because C. jejuni cannot survive pasteurization or such other forms of heat treatment as ultra-heat treatment. Freezing foods will reduce substantially the initial Ccmpylobncter population, but some organisms will survive and may remain viable for months. Although C. jejuni does not survive well in foods,refrigeration tends to prolong survival. Long-term solutions include reducing the organism's presence in food animals, developing systems to inactivate the organism in food before it reaches the retail level, and making the general public aware of the importance of food hygiene (e.g., through educational programs in the public schools).

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VIII. SUMMARYANDCONCLUSIONS Cninpylobacter jejmi was isolated first from human diarrheal stools in 1971. Since its identification as a human enteric pathogen, the organism has been recognized increasingly as one of the mostimportant causes of diarrheal disease in the UnitedStates and throughout the world. The study cited at the beginning of this chapter of Campylobncter infection on 303 U S . college campuses ( 2 ) illustrates the public health significance of the organism and highlights the need for further research. The study showed an incidence of Cnrnpylobncteer- in stool cultures of 13.2%, which exceeds the normal expected rate. C. jejmi enteritis is primarily a zoonotic disease with apparently different modes of transmission in the industrialized and developing countries. In the developed countries, the organism is spread primarily through foods of animal origin: on the other hand, evidence for the less developed countries is that fecal contamination of food and water and contact with sick people or animals predominate as the modes of transmission. These contrasting patterns of transmission are explained by the different levels of environmental hygiene in the developed and less developed countries, which have corresponding implications for international control of the organism. Transmission of the organism is minimized when the microbial environment is inhospitable to it. C. jejuni dies off rapidly at ambient temperatures and atmospheres and grows poorly in food. Food that has been mishandled, of course, can become a medium. Although milk has been cited tnost frequently as a vehicle for Carrzpylobncter worldwide, increasing attention is being paid to meat and poultry products as major reservoirs and vehicles. The principles of animal husbandry will play a significant role in controlling this ubiquitous organism. Using Canzyylobacter-free parent animals and raising Canpylobncter-free animals for slaughter will help immeasurably to protect the consumer from foodborne infection. The strictest standards of livestock hygiene and management will be required. Hygienic slaughter and processing procedures will preclude cross-contamination, while adequate cooling and aeration will cause a decrease in the microbial load. In addition, thorough cooking of meat and poultry products followed by proper storage should help in maintaining food integrity with less contamination. Campylobacteriosis is a universal problem and a challenge to all who work in the food safety arena. The solutions for control and prevention are not simple. More research is needed and close national and international cooperation between the veterinary and medical professions is imperative if progress is to be achieved in the long-term control of Cmlyylobacteer.

REFERENCES Tauxe, R. V., Deming. M. S., and Blake, P. A. (1985). Cnmpylobacter jejuni infections on college campuses: A national survey. AJPH, 75:659-660. 2. Tauxe, R. V. (1 992). Foreword: Transmission of human bacterial pathogens through poultry. In Colonization Control of Human Bacterial Enteropthogens in Poult91 (L. C. Blankenship. ed.), Academic Press, New York. Pp. xv-xxxiii. 3. Butzler, J. P., and Skirrow, M. B. (1979). Curzzpylobacter enteritis. Clin. Gastroenterol., 8: 737. l.

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29. Kaplan, R. L. (1980). Campylobacter. In Mrrnunl of Clinical Microbiology, 3rd ed. (E. Lennette, A. Balows, W. J. Hausler, Jr., et al., eds.), American Society for Microbiology. Washington, DC, p. 235. 30. Hebert, G. A., Hollis, D. G., Weaver, R. E., et al. (1982). 30 years of campylobacters: Biochemical characteristics and a biotyping proposal for Cumpylobacter jejuni. J. Clin. Microbiol., 15:1065. 31. Blaser, M. J. (1985). Campylobacter species. In Principles and Practice of Znfectious Diseases. 2nd ed. (G. L. Mandell, R. G. Douglas, Jr., and J. E. Bennett, eds.). John Wiley and Sons, New York, pp. 1221-1222. 32. Vandamme, P., Falsen, E., Rossau, R., Hoste B., Segers, P.. Tytgat, R., Ley, J. de. (1991). Revision of Campylobacter,Helicobacter, and Wolirzella taxonomy: Emendation of generic descriptions and proposal of Arcobacter gen. nov. Znt. J. Syst. Bacteriol., 41(1):88103. 33. Slurrow, M. B. (1994). Diseases due to Campylobacter, Helicobacter, and related bacteria. J. Cony. Path., 111:113-149. 34. Blaser, M. J., Taylor, D. N., and Feldman, R. A. (1984). Epidemiology of Campylobacter infections. In Campylobacter Infection in Man and Animals (J. P. Butzler, ed.), CRC Press, Boca Raton, FL, pp. 143-161. 35. Garcia, M. M., Lior, H., Stewart, R. B., Ruckerbauer, G. M., and Skijarevski, A. (1 985). Isolation, characterisation, and serotyping of Canzpylobacterjejltni and Carnpylobacter coli from slaughter cattle. Appl. Environ. Microbiol. 43:977-980. 36. Hanninen, M. L., and Raevuori, M. (1981). Occurrence of Campylobacterfetus subsp. jejuni and Yersinia enterocolitica in swine carcasses and the slaughterhouse environment. J. Food Prot., 33:441-445. 37. Svedhem, A., Kaijser, B., and Sjogren, E. (1981). The occurrence of Campylobacter jejzrni in fresh food and survival under different conditions. J. Hyg., 87:421-425. 38. Vogt, R. L., Little, A. A., Patton, C. M., Barrett, T. J., and Orciari, L. A. (1984). Serotyping and serology studies of campylobacteriosis associated with consumption of raw milk. J. Clin. Microbiol., 20:998-1000. 39. Mafu, A. A., Higgins, R., Nadeau, M., and Couisineau, G. (1989). The incidence of Salmonella, Car.~~pylobacter, and Yersinia enterocoliticnin swine carcasses and the slaughterhouse environment. J. Food Prot. 52:642-645. 40. Van D a m e , L. R. (1983). Isolation of Campylobacter jejuni from animals in Zaire. In Canzpylobacter ZZ (A. D. Pearson et al., eds.), PHLS, London. 41. Borch, E., Nesbakken, T., and Christensen, H. (1996). Hazard identification in swine slaughter with respect to foodborne bacteria. Znt. J. Food Microbiol., 30(1/2):9-25. 42. Oosterom, J., Beckers, H. J., van Noorie Jansen, L. M., and van Schothorst, M. (1980). An outbreak of Calnpylobacter infection in a barrack, probably caused by raw hamburger. Ned. Tijdsclzr. Diergeneeskd., 124:1631-1634 (in Dutch). 43. Monroe, D. L., Prescott, J. F., and Penner, J. L. (.1983). Canzpylobacterjejuni and Campylobacter coli serotypes isolated from chickens, cattle, and pigs. J. Clin. Microbiol., 18377878. 44. Oosterom, J. (1983). Carnpylobacter jejuni in poultry processing and pig slaughtering. In Campylobacter ZZ (A. D. Pearson et al., eds.), PHLS, London. 45. Rosef, D., Gondrosen, B., Kapperud, G., Underdal, B. (1983). Isolation and characterization of Campylobacter jejuni and Cmnpylobacter coli from domestic and wild mammals in Norway. Appl. Environ. Microbiol., 46:855-859. 46. Gumbrell, R. C. (1980). Preliminary Report on the Results of the First Year of a Five-Year Survey on Lamb Perinatal Mortality. Animal Health Laboratory, Lincoln, New Zealand, pp. 94-99. 47. Doyle, M. P. (1984). Association of Campylobacter jejuni with laying hen and eggs. Appl. Environ. Microbiol., 47533-536.

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48. Genigeorgis, C.. Hassuneh, M., and Collins, P. (1986). Cnnrpylobncterj e j m i infection in poultry farms and its effect on poultry meat contamination during slaughtering. J. Food Prot., 49:895, 899-903. 49. Hartog. B. J., DeWild,G. J. A., and DeBoer, E. (1983). Poultry asa source of Crtnzpylobncter 34: 116-122. (in German). jujurzi. Arch. Lebelzsmittellr?~g., 50. Casanovas, L., de Simbn, M., Ferrer, M. D., ArquCs, J., and Monzbn, G. (1 995.) Intestinal carriage of campylobacters, salmonellas, yersinias, and listerias in pigeons in the city of Barcelona. J. Appl. Bmteriol., 78:ll-13. 51. Jocobs-Reitsman, W. F., Bolder, N. M., and Mulder, R. W. A. W. (1994.) Cecal carriage of Cantpylobncter and Salmonelllr in Dutch broiler flocksat slaughter: a one-year study. Poultry Sci.. 73(8):1260-1266. 52. Wempe, J. M.. Genigeorgis, C. A., Farver, T. B., and Yusufu, H. I. (1 983). Prevalence of Cnnzp$obacter jejuni in two California chicken processing plants. Appl. Environ. Microbiol., 45:355-359. 53. Acuff, G. R., Vanderzant. C.. Hanna, M. 0..Ehlers, J. G., Golan, F. A., and Gardner, F. A. (1986). Prevalence of Canzpylobncter j e j m i in turkey carcass processing and further processing of turkey products. J. Food Prot., 49:712-717. 54. Seattle-King County Department of Health. (1984). Surveillance of the flow of Salmollella and Cnnzpylobacter in a community. Communicable Disease Control Section, Seattle-King County Department of Public Health. Contract #223817041. Report to U.S. Food and Drug Administration. Seattle-King County Department of Public Health, Seattle, WA. 55. Blaser, M. J., Wells, J. G., Feldman, R. A., Pollard, R. A., and Allen, J. R. (1983). Cnrnpylobircter enteritis in the United States. A multicenter study. Ann. Zrrt. Med., 98:360. 56. Mauff, A. C.. and Chapman, S. R. (1981). Campylobacteriosis in Johannesburg. S. Afi. Med. J., 59:217. 57. Burce, D.. Zochowski, W., and Fleming, G. A. (1980). Cunzpylobucter infections in cats and dogs. Vet. Rec. 107:200-201. 58. Crotti, D., andBoscato, U. (1983).Enteritisinthedog:Possibleetiopathogenicrole of Cnrtzpvlobncter. Obiettivi Doc. Vet., 5:51-54 (in Italian). 59. Jones, A., and Harrop, C. (1981). A study of Cnwrp?lobncter enteritis. J. Znt. Med. Res.. 9: 40-43. 60. Gurgan, T., and Diker. K. S. (1994). Abortion associated with Crrnrpylobacter rpaliensis. J. Clin. Microbiol. 32( 12):3093-3094. 61. Sjogren, E., Falsen, E., Kaijser, B., and Lindblom, G.-B. (1996). Carnpylobcrcter species in faeces from healthy pets in Sweden isolated by filter technique. In Cnnrpylobacters, Helicobacters, o?rdRelated Organism (D. G. Newell. J. Ketley, and R. A. Feldman, eds.), Plenum Press, New York. Pp. 471-473. 62. Moreno, G. S., Griffiths, P. L., Connerton, I. F., and Park, R.W. A. (1993.) The occurrence of campylobacters in small domestic and laboratory animals. J. Appl. Bacteriol., 75:49-54. 63. Altekruse, S. F., Hunt, J. M.. Tollefson, L. K., and Madden, J. M. (1994). Food and animal sources of human Carnpylobcrcter. jejzcniinfection. J. Am. Vet. Med. Assn., 20457-61. 64. Cohen, D. I., Rouach. T. M., and Rojol, M. (1984). A Cmnpylobacter enteritis outbreak in a military base i n Israel. Zsr. J. Med. Sci., 20:216-218. 65. U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control. (1983). Water-Reluted Disease Outbreaks. Arlrmnl Summni?:. CDC, Atlanta. GA. 66. Taylor, D. N., McDermott, K.T., Little, J. R., Wells, J. G.,and Blaser, M. J. (1 983). Cnnzyylobrrcter enteritis from untreated water in the Rocky Mountains. Ann. Znt. hled.. 99:38-40. 67. Gondrosen, B. (1983). Survival of thernlophilic canlpylobacters in water. In Canlpylobncter ZZ (A. D. Pearson, et al., eds.), PHLS, London. 68. Brennhovd. O., Kapperud, G., and Langeland, G.(1992). Survey of thennotolerant Cnnzpylobcrcter spp. and Yersirtia spp. in three surface water sources in Norway. Znt. J. Food Microbiol., 15(3/4):327-338.

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69. Easton, J. (1 996). Fats and transportof campylobacters in soil arising from farming practices. In Camnpylobacters, Helicobncters, and Related Orgrrnisrm (D. G. Newell, J. Ketley, and R. A. Feldman. eds.), Plenum Press, New York. Pp. 461-465. 70. Kotula. A. W., and Stern, N.J. (1984). The importance of Cantpylobncterjejuni to the meat industry: A review. J. Anint. Sci., 58:1561-1566. 71. Kwatiek, K., Wojton, B., and Stern, N. J. (1990). Prevalence and distribution of Cnnzpylobacter spp. on poultry and selected red meat carcasses in Poland. J. Food Prot., 53:127130. 72. Larmnerding, A. M., Garcia, M. M.. Manu, E. D., Robinson, Y . ,Dorward, W. J., Truscott. R. B., and Tittiger, F. (1988). Prevalence of Salnzonellu and thermophilic Cmzpylobacter in fresh pork, beef, veal, and poultry in Canada. J. Food Prot., 51:47, 51. 73. Adesiuyn. A. A. (1993). Prevalence of Listeria spp., Cnrnpylobrrcter spp., Salrnortelln spp., Yersinia spp., and toxigenic Esclzerichin coli on meat and seafoods in Trinidad.Food Microbiol., 10(5):395-403. 73. Kinde, A., Genigeorgis. C. A., and Pappaioanou, M. (1 983). Prevalence of Cnnzpylobncter jejuni in chicken wings. Appl. Emiron. A/licrobiol., 45: 11 16-1118. 75. Stern, N. J.. Green, S. S., Thaker, N., Krout. D.J., and Chiu, J. (1 984). Recovery of Cnnzpylobacter jqjrrni from fresh and frozen meat and poultry collected at slaughter. J. Food Prof., 47:37"373. 76. Turnbull, P. C. B., and Rose, P. (1982). Canzpylobacter jejuni and Salmonella in raw red meats. A Public Health Laboratory Service Survey. J. Hyg., 8829-37. 77. Flynn, 0. M. J., Blair, I. S., and McDowell, D. A. (1994.) Prevalence of Cunlpylobrrcter species on fresh retail chicken wings in Northern Ireland. .I. Food Prot., 57(4):334-336. 78. Willis, W. L. and Murray, C. (1997.) Cnnyylobcrcter jejzjlani seasonal recovery observations of retail market broilers. Pozrltly Sci. 76(2):314-317. 79. Grau, F. H. (l 98s).Ccmpylobncter jejzrrzi and Cmtpylobacter hyointesti~rnlisin the intestinal tract and on the carcasses of calves and cattle. J. Food Prot., 5 1957-861. 80. Doyle, M. P., and Roman. D. J. (1982). Prevalence and survival of Carnpylobacter jqjuni in unpasteurized milk. Appl. EmVror1. Micr-obiol., 44: 1154-1 158. 81. de Boer, E.. Hartog, B. J., and Borst, G. H. A. (1984). Milk as a source of Canzpylobucter jejuni. Netlt. Milk Dairy J., 38:183-194. 82. World Health Organization (WHO). (1984). Report of the WHO consultation on veterinary public health aspects of prevention and control of Cniizpylobncter infections in Moscow. WHO, Geneva. 83. Emory University School of Medicine, Department of Community Health. (1985). Course notes: Control of enteric infections in Third World countries. Emory University, Atlanta, Georgia. 84. Norkrans, G., and Svedhem, A. (1982). Epidemiological aspects of Cnnrpylobacter jejuni enteritis. J. Hyg.. 89:163-170. 85. Grados, O., Bravo, N., Butzler, J. P., and Ventura, G. (1983). Cnntpvlobncter infection: An occupational disease risk in chicken handlers. In Cnrlzpylobacter ZZ (A. D. Pearson et al., eds.), PHLS, London. Cnmpylobacter fetus subspecies jejurli innormal 86. Oosterom, J. (1980).Thepresenceof slaughtered pigs. Ned. Tijdscht-. Diergeneeskd., 105:49-50 (in Dutch). 87. Hutchinson, D. N., Bolton, F. J., Hinchliffe, P. M., Dawkins, H., Horsley, S. D., Jessop, E. G., Robertshaw, P. A., and Counter, D. E. (1985). Evidence of udder excretion of Campylobncterjejzmi asthecause of milkborne Canzpylobacter outbreak. J. Hyg., 94205215. 88. Brieseman, M. A. Raw milk consumption as a probable cause of two outbreaks of Canzpylobacter infection. N. 2. Med. J., 97:411-413. 89. Stalder, H., Tsler, R., Stutz, W., Salfinger,M., Lauwers, S., and Vischer, W.(1983). Contribution to the epidemiology of Canzpylobucter jejrcni. From asymptomatic excretion by a cow

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and

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in the cowshed to overt diseasein over 500 persons. Sclzwei:. Med. Wochenschr., 113245249 (in German). Porter, I. A., and Reid, T. M. (1980). A milkborne outbreakof Campylobacter infection. J. Hyg., 841415-419. Hutchinson. D.N., Bolton, F. J., Jelley, W. C.N., Matthews, W. G., Telford,D. R., Couitner. D. E., Jesop, E. G., and Horsley, S. D. (1985). Campvlobacter enteritis associated with consumption of raw goat's milk. Lancet, 1, 8436:1037-1038. Sandstedt, K. (1982). Carrzpylobcrcter transmission via food, especially milk. Statens Vet. Anstalt, Uppsala, Sweden, pp. 225-226 (in Swedish). Andrewes. C. H., and Walton, J. R. (1977). Viral and Bacterial Zoonoses. Balliere Tindall, London, pp. 18-19. Blaser, M. J., Sazie, E., and Williams, L. P. (1987). Theinfluence of immunity on raw-milkassociated Carnpylobncter infection. JAMA, 257:43-46. Mandel, B. K., De Mol. P., and Butzler, J. P. (1984). Clinical aspects of Canzpylobacter infections in humans. In Campylobacter Iizfection in M m and Animals (J. P. Butzler, ed.), CRC Press, Boca Raton, FL. Pp. 22-30. Benenson. A. S. (1980). Control of Cor)tnz~nicableDiseases in Man, 13th ed. American Public Health Association, Washington, DC, pp. 112-113. Butzler, J. P. (1982). Campylobacter enteritis. Itlfection 10 (supp1.2):567-569 (in German). Smith, C.S., and Blaser, M. J. (1985). Fatalities associated with CampyZobacterjejzmi infections. JAMA. 253:2873-2875. Archer, D. L. (1985). Enteric microorganisms in rheumatoid diseases: Causative agents and possible mechanisms. J. Food Prot., 48:538-545. Smith, J. L., Palumbo, S. A., and Walls, I. (1993). Relationshipbetween foodborne bacterial pathogens and the reactive arthritides. J. Food Sci., 13 (3):209-236. Wesley, I. (1997). Cnmpylobacter and related microorganisms incattle. In Beef Safery Synzp o s i m Proceedings, December 3-4, 1997. American Meat Science Association/National Cattlemen's Association, Chicago. Pp. 30-41. Marshall, B. J., and Warren, J. R. (1984). Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet, l , 8390: 13l l - 1315. Morey, F. (1996). Five years of Canzpylobacter bacteremia in Central Australia. In Cnq$obacters, Helicobacters. and Related Organisms (Newell et al., eds.), Plenum Press, New York. Pp. 491-494. Fennell, C. L., et al. (1984). Characterizationof Campylobncter-like organisms isolated from homosexual men. J. Irzfect. Dis. 149(1):58-66. Lastovica, A. J. (1996). Serotype distributionof isolates of C.jejzsni subsp. jejuni, C. jejuni subsp. doylei, C. coli, and C. upsaliensis from paediatric enteritis patients. In Campylobacters, Helicobacters, alld Related Organism (D. G. Newell et al., eds.), Plenum Press. New York. Pp. 481-485. Todd, E. C. D. (1985). Economic loss from food borne disease outbreaks associated with foodservice establishments. J. Food Prot., 47:322-328. Levy, B. S., and McIntire, W. (1974). The economic impact of a foodborne salmonellosis outbreak. JAMA, 230:1281-1282. Archer, D. L. (1984). Diarrheal episodes and diarrheal disease: Acute disease with chronic implications. J. Food Prot., 47322-328. Archer, D. L., and Kvenberg, J. E. (1985). Incidenceand cost of foodborne diarrheal disease in the United States. J. Food Prot., 48:887-894. U.S. Food and Drug Administration, U S . Department of Agriculture, U.S. Environmental Protection Agency, Centers for Disease Control and Prevention. (1997). Food safety from farm to table: A national food safety initiative. Report to the President, May 1997. Buzby, J. C., Roberts, T., Lin, C.-T. J.. and MacDonald, J. (1996). Bacterial Foodborne

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Clostridium botulinum John W. Austin and Karen L. Dodds Health Canada, Ottawa, Ontario. Cunncla

I. Introduction

108

11. The Organism: Classification and Characteristics

111. Epidemiology

IV.

V. VI.

VII.

108

109

A. Occurrence of spores in the environment 109 B. Occurrence of spores in foods 111 C. Incidence of botulism in North America 113 D. Incidence of botulism in Europe 115 E. Incidence of botulism in Asia 115 F. Incidence of botulism in other areas 116 G. Infant botulism 117 Clinical Aspects 118 118 A. Symptoms 119 B. Treatment C. Diagnosis 119 120 D. Case history 120 E. Prevention Detection 121 Neurotoxin 121 A. Structure 122 B. Mode of action 122 C. Therapeutic uses 123 Control in Foods 123 A. Factors affecting growth and toxin production 123 B. Thermal Inactivation 126 C. Inactivation by irradiation 126 References 127

108

I.

Austin and Dodds

INTRODUCTION

The species Clostridiun~botsrlinzun comprises gram-positive, anaerobic, rod-shaped, spore-forming bacteria. They are distinguished from other bacteria by the production of the most potent biological toxin known, botulinum neurotoxin (BoNT). The lethal dose for humans is estimated to be in the range of 0.1-1 ng/kg (l). Strains of C. botulim~rn are classified into seven types, A through G, depending on the serological specificity of the neurotoxin produced. Foodborne botulism, the most common form of botulism worldwide, is caused by ingestion of food contaminated with preformed BoNT, usually type A, B, or E. Because of the severity and life-threatening nature of this disease, C. botulimm has been extensively studied. Indeed, botulism has been described as “an archetype of catastrophic illness” (2). Originally, botulism was associated with sausages in central Europe (3). Presently, a wide variety of foods have been implicated in botulism, including such unusual products as hazelnut-flavored yogurt (4), garlic in oil (5), commercial cheese products (6,7), vacuum-packaged clam chowder, and commercial bean dip (8). However, the largest problem worldwide remains foods preserved in the home, whether from home canning, home curing, or home fermentation. Human botulism is currently classified into four categories. Foodborne botulism is caused by ingestion of food contaminated with preformed BoNT. Wound botulism is very rare and is due to infection of a wound with spores of C. botulirzurn, which grow and produce toxin in situ (9,lO). Infant botulism, first recognized in 1976 (11,12) and now the most common form of botulism in the United States, is caused by ingestion of viable spores that germinate and colonize the intestinal tract of infants under one year of age and are responsible for the local production of toxin. The fourth category, unclassified, includes cases of unknown origin and adult cases which resemble infant botulism (13). Animal botulism, which will not be discussed further, is typically caused by types C and D.

II. THEORGANISM:CLASSIFICATIONAND CHARACTERISTICS C. botulinum was defined in 1953 as the species designation for all organisms known to produce BoNT (14), although recent studies have identified strains of Clostridiunz baratii ( 1S ) and Clostridium butyricum(1 6- 18) as capable of producing neurotoxin (19). Strains of C. botulirzum are separated into seven types, A through G, based on the serological specificity of the toxin produced. Although serologically different, the toxins are structurally similar and have the same biological effect. Based on physiological differences, the species is also divided into four groups (designated Groups I to IV) (19,20). The classification of strains of C. botulin~rminto four physiological groups also agrees with results of nucleic acid hybridization and 16s ribosomal RNA sequencing studies (21-23). Group I strains are proteolytic, produce spores of high heat resistance, and have a minimum growth temperature of 10°C. This group includes all type A strains, which are all proteolytic, and proteolytic strains of types B and F (both of these types may be proteolytic or nonproteolytic). Strains in Group I1 are nonproteolytic, produce spores of a low heat resistance, and are capable of growing at refrigeration temperatures. Included in Group I1 are all type E strains, which are all nonpro-

Clostridium botulinum

109

teolytic, and nonproteolytic strains of types B and F. Group I11 includes type C and D strains. This group is generally nonproteolytic and, since it is not involved in human botulism, little is known regarding its heat resistance. Group IV includes type G strains and is classified as a distinct species, C. nrgerztinerzse (24). The emphasis in this chapter will be on Groups I and I1 since they are involved in human illness.

111.

EPIDEMIOLOGY

A.

Occurrence of Spores in the Environment

Fundamental to a knowledge of the potential botulism hazard from any food is a knowledge of thepossibility of contarnination of the food with C. botulinum. This contamination depends largely on the distribution and incidence of C. botulirzum in the environment (25). The serious nature of botulism has led many investigators worldwide to conduct surveys to establish the presence or absence of C. botulimrn in different environments. Summaries of these surveys are presented here, but readers should refer to the original publications or other reviews (25,26) for details regarding sampling. Spores of C. botulinum are commonly present in soils and sediments, but their numbers and types vary, depending on the location (Table 1). C. botulinum is often described as a soil organism, ubiquitous in nature, but this does not reflect veryaccurately its distribution in the environment. As the survey data show, spores of C. botuZinurn are spread widely in the environment, but the factors governing the geographical distribution of spores and the different types are still not understood well. C. botulinum spores are distributed widely in North America, but the spore load varies considerably, as does the predominating type (Table 1) (25). Soils in eastern and central United States usually contain type B spores, usually proteolytic, while type A spores predominate in the west (27). Type E is found infrequently, and only in damp to wet locations. However, in the region around the Great Lakes, and particularly around Green Bay of Lake Michigan, high numbers of type E are found in shoreline and sediment samples (28). Type E is also found in the coastal areas of Washington and Alaska (29,30). Type B predominates in the terrestrial environments of Britain, Ireland, Iceland, Denmark, and Switzerland (Table 1) (25). It is associated with most botulistn outbreaks in Spain, Portugal, Italy, France, Belgium, Germany, Poland, Hungary, and the former Czechoslovakia and Yugoslavia (3l), indicating wide distribution of this type in the European environment. Type B also predominates in the aquatic environtnents of the United Kingdom. Most European type B strains are nonproteolytic. The predominant serotype from all other aquatic environments studied in Europe is E. High numbers of type E spores are found in Scandinavian waters, particularly in the Sound and Kattegat, between Denmark and Sweden. C. botulirzurn type E is also prevalent in samples from the Baltic sea (32,33). The highest overall spore concentrations detected in Europe were reported in the Netherlands, where a mixture of types was found. Type E spores also predominate in most parts of the former U.S.S.R., except for the central portion, where type B spores are found most frequently (Table 1) (34). In general, surveys of Asia report lower numbers, with the exceptions of a high incidence of type E spores around the Caspian Sea (35), and a very high incidence of types A and B in the Xinjiang district of China (36,37). Types A, B, E, and F are found in areas of China north of 30" latitude, while types C and D are more common in parts of China south of 30" latitude (36). The incidence of type E spores is also high in much of northern

Austin and Dodds

I10 Table 1 Soiland Sediment Surveys for C. botztlinzrnz

Location

9% of Sample samples MPN size (g) positive per

North America Eastern U.S., soil 10 Western U.S.. soil 10 1 Great Lakes, soil S Green Bay, sediment 5 Washington coast, sediment l Alaska coast, soil Europe 50 Britain, soil 2 Britain coast, sediment Ireland, sediment 50 10 Iceland, soil 10 Faroe Islands coast, sediment Greenland coast, sediment 10 Scandinavian coast, Skagenak, 6 Kattegat. sediment 5 Norway coast. soil Sweden coast, soil 3-6 Denmark coast. sediment 10 10 Denmark, soil 0.5 Netherlands, soil Poland. Baltic coast, soil 5 Finland freshwater 300 300 Baltic coast Baltic offshore 500 Czechoslovakia, sediment 12 Switzerland. soil Italy. Rome, soil 7.5 Asia Former United Soviet Socialist Republic, soil Northeastern Southeastern Central Northwestern Far East 2 Iran, Caspian Sea. sediment China, Sinkiang. soil 10 Taiwan, soil 10 Japan. Hokkaido, soil 5-10 Japan, Ishikawa, soil 40-50 Japan. Honshu. sediment 5 Southem Hemisphere Brazil, cultivated soil 5 Paraguay, soil 5 Argentina, soil

19 29 2 77 71 41

Type kga A

B

(%'C)h

C/D

Ref. 27 27 28 28 29 30

64 16 0 0 3 0

12 14 0 0 0 0

12 8 100 100 96 100

0 0 0 0 0 0

100 100 100 100 0 0

0 0 0 0 0 0

0 0 0 0 100 100

0 158 0 159 0 160 0 41 0 41 0 41

0

100 100 99 100 0 32 97

0 32 0 32 0 32 0 41 0 41 0 161 0 162

0

100 100 100 0 0 0

0 33 0 33 0 33 0 163 27 164 0 165

0 86 0 73 6 0 86 0 5 87 92 0 19 2 3 20 0 100 100 0 9 33-82

0 34 0 34 0 34 0 34 0 34 0 35 0 37 5 166 0 167 0 40 0 168

4 3 l 46

0 0 0 0 0 0

100 7 84 67 13 94 32

>780 7 410 110 15 2500 76

0 0 0 0 0 0 3

0 0 93 22 0

0 0 1 0 7 46 0

61 58 88 32 34 1

372 500 1020

0 0 0 0 48 28 2 86

0 0 0 0 83 l4

0 0 100 6 0

0 8 16 14 0 0

14 17 77 0 8 8

0 0 0

0 0 7

86 57 10 14 67

7 0 20

35 24 34

F

21 12 33 62 24 0 1280 0 250 1 660 0

6 4 18 3 1 37

2 13 14 4 14 S7 70 78 4 56

E

2 18

93 25000 3247 373430 4 16

0

29 14 0

0 0 0

7 71 5

43 45 44

Clostridium botulinum

111

Table 1 Continued

Location Kenya. soil South Africa, soil Bangladesh, sediment Thailand. Hua-Hin, sediment Indonesia, sediment Java coast, sediment New Zealand, sediment

5% of Sample samples MPN size (g) positive per kg' A 5 30 10 10 50 10 20

25 3 37 3 21 13 55

Type (%)" B

0 33 S9 1 0 100 0 19 0 0 3 0 0 5 0 14 0 33 0 40 0

MPN calculated using the Halvorson-Ziegler equation In(n/q).where n and q = the number o i nontoxic samples. h As percentage of the types identified. Source: Adapted from Ref. 26.

=

C/D 11 0 100 83 100 67 100

E

F Ref.

0 0 0 17 0 0 0

0 46 0 47 0 41 0 43, 0 169 0 170 0 171

the total numberof samples analyzed

Japan (38,39). In other areas of Japan, the distribution of types is different, and type C predominates (40). In the tropical regions of Asia, types C and D replace type E as the predominant type in aquatic environments (41,42). Fewer surveys have been carried out in the Southern Hemisphere (see Table 1). Type A spores predominate in Brazilian and Argentinian soils (43.44). In Paraguay, the prevalent spore type was F, followed by A and C (45). In Africa, few surveys have been done, but one following an outbreak in Kenya found type A and C spores in soil (46), whereas in South Africa only type B spores were found (47). In summary, type A spores predominate in soils in the western United States, China, Brazil and Argentina, and type B spores in the eastern United States, the United Kingdom, and much of continental Europe. However, most American type B strains are proteolytic, while most European strains are nonproteolytic. Type E is the predominant type in northern regions, and in most temperate aquatic regions and their surroundings. Types C and D are found more frequently in warmer environments. The reasons for this distribution pattern are not understood well. Type A appears to be favored by neutral to alkaline soil with low organic content: this is consistent with its virtual absence in the highly cultivated soils of the eastern United States and Europe. Type E is psychrotolerant, which undoubtedly plays a role in its prevalence in the North and in many aquatic environments. Sirnilarly, the higher optimum growth temperatures for types C and D are consistent with their presence in warmer environments. B. Occurrence of Sporesin Foods The sequence of events resulting in foodborne botulism begins when C. botulitlrm contaminates a food. This often occurs during growth or harvesting, especially if a product originates in an environment with a high incidence of spores. However, contamination can also occur during or after processing. The presence of C. botulir~zm,mostly type E, in fish is demonstrated readily (see Table 2), although the incidence is lower than in environmental surveys (48). The level of contamination of prepared fish in the United States appears to be higher than in Europe. Numbers vary in Asia, with a particularly high result noted in fish from the Caspian Sea (35).

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Table 2 Incidence of C. botulinnm Spores in Prepared Fish Product Whitefish chubs eviscerated fresh smoked Frozen flounder, vacuumpacked Frozen, packaged fish Filleted fish Smoked fish Dressed rockfish Fish and seafood Salmon Vacuum-packed fish Vacuum-packed fish Smoked herring Smoked salmon Smoked eel Salted carp Smoked carp Rainbow trout eviscerated intestines Fish Fish and seafood

Origin

Sample 7o positive MPN size (g) samples per kg

Types identified

Ref.

Great Lakes

Atlantic Canada New York City Pacific NW California California Alaska England Viking Bank Sweden Denmark Baltic Sea Caspian Sea Caspian Sea Finland Finland Indonesia Osaka

10 10 1.5

100 5-10 10 70 24-36

20 20 2 2

1-5 5 30

13 1 10
20 63 5 0 0 3

8

128 128 172

14 1 70


173 130 174 175 176 131 177 177 178 179 180 35 35

0 0 6 3

181 181 182 183


A, B, C, D, F C, D

MPN determined by authors. All lots examined were contaminated. Soz4rce: Ref. 48. h

The level of contamination of meats is generally low (see Table 3) (48). These products are less likely than fish to be contaminated with spores prior to slaughter of the animals since the farm environment is generally less contaminated than the aquatic environment. Studies indicate a very low incidence of C. botrrlirzurn spores in meats in North America, where the average most probable number (MPN) is approximately equal to 0.1 spore per kg, while the incidence in Europe, where the average MPN is approximately equal to 2.5 spores per kg, is somewhat higher. The types most often associated with meats are A or B. C. botulinum often is present on fruits and vegetables, particularly those in close contact with the soil (49). Types A or B usually are identified. Products in which contamination has been detected include asparagus, beans, cabbage, carrots, celery, corn, onions, potatoes, turnips, olives, apricots, cherries, peaches, and tomatoes (48). A product of particular concern due to the high number of spores found is cultivated mushrooms, in which up to 2.1 X 10’ type B spores per kg have been detected (50). Spores in honey and other infant foods pose a unique hazard because, in some infants, the spores are able to colonize the intestines, produce toxin, and cause infant botulism. Honey is the only food implicated in infant botulism. Surveys for the presence of C. botulinum spores in honey suggest that the botulinum spore level in random samples

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Table 3 Incidence of C. botulinunz Spores in Meat and Meat Products Product Raw meat Cured meat Vacuum-packed bacon Cured meat Liver sausage Raw pork Vacuum-packed bacon

Sample size (g)

% positive

Origin North America North America Canada Canada Canada United Kingdom United Kingdom

3 30 75 75 75 30 25-175’’


samples

2 0-14b 4-73’

MPN per kg 0.1 0.5 0.1 0.2 0.2 <0.1-5” 1.0-7”

Types identified Ref.

c A, B A or Ba A A A, B, C A, B

184 185 186 186 187 188 189

Not specifically typed. Variation found on different sampling occasions. Sowce: Ref. 48. a

of honey is in the order of 1-10 per kg (48). However, in honey samples associated with infant botulism, the level is approximately lo4spores per kg (51-53). While C. botulinum has been detected in such other infant foods as corn syrup (54) and rice cereal (52), exposure of infants to botulinum spores via these foods seems to be minimal because the levels are low and unlikely to increase during manufacture and storage. Other foods exanlined, including dairy products (55), vacuum-packed products (56), fresh-cut packaged salads (57), and convenience foods (58), show a very low incidence of C. botulinum spores.

C. Incidence of Botulism in NorthAmerica Unlike other foodborne diseases, botulism outbreaks are defined as incidents involving one or more botulism cases. Table 4 shows the data for countries that have reported relatively frequent outbreaks. The accuracy of these data depend uponhow thorough the reporting system records incidents of botulism. As with other foodborne diseases, unrecognized and misdiagnosed cases of botulism occur, as evidenced by a 1985 outbreak in Vancouver, Canada, in which the initial diagnoses for 28 patients included psychiatric illness, viral syndrome, laryngeal trauma, overexertion, and a variety of other maladies (5). Similarly, initial diagnoses for patients in a 1993 outbreak in Georgia included labyrinthitis, transient ischemic attack, allergic reaction, mild glaucoma, and astigmatism (6). As well, in large areas of the world, particularly where botulism occurs infrequently, epidemiological data are scarce. In Canada, most botulism outbreaks have occurred in northern native communities (3 1). The foods involved were mainly such traditional native dishes as raw and parboiled meats from sea mammals, such fermented meats as muktuk (meat, blubber and skin of the beluga whale), and fermented salmon eggs. Type E was implicated in the majority of incidences. Toxin production in these outbreaks was attributed to keeping meats at ambient temperature for some time, often to attain a desired decomposition (59). The problem with the so-called fermented products urraq (seal flippers in oil), muktuk (meat of the beluga with blubber), nlicerak (mostly seal or whale fat), and ogsuk (mostly seal oil) is that the level of fermentable carbohydrates is too low to ensure a sufficiently rapid pH reduction to prevent growth of C. botulinum. Commercial products have been implicated in only four incidents in Canada since 1971 (31,53) involving bottled marinated mushrooms im-

Dodds 114 Table 4

and

Austin

Recorded Outbreaks of Foodborne Botulism

Countly Poland China United Statesd Italy France Japan U.S.S.R. Iran Canada Spain West Germany Alaska East Germany Hungary Portugal Norway Czechoslovakia Argentina Yugoslavia Belgium Denmarkg

Period 1984-87 1958-83 197 1-90 1979-87 1978-89 1951-87 1958-64 1972-74 1971-91 1969-88 1983-88 1971-88 1984-89 1985-89 1970-89 1961-90 1979-84 1980-89 1984-89 1982-89 1984-89

No. of outbreaks

No. of casesa

Predominant typeh

1301 986 286

1791 (3) 4377 (13) 619 (15) 310 304 (2) 479 (23) 328 (29) 314 (11) 213 (15) 198 (6) 154 (4) 117 (6) 52. (8) 57 (2) 80 (0) 42 (7) 20 (0) 36 (36) 51 25 (4) 16 (12)

B

-e

175 97 95 -e

87 63 63 48 33 31 24 19 17 16 12 11 11

A

A B B E B E E B B E B B B B, E B A

B E

Predominant food type' Meats Vegetables Vegetables Vegetables Meats Fish Fish Fish Meatst Vegetables Meats Fish Meats Meats Meats Fish Meats Vegetables Meats Meats Meats

Fatality rate shown in parentheses. Of outbreaks with type identified. c Of outbreaks with the food vehicle identified (vegetables includes fruits). Includes Alaskan data (mostly traditional lnuit meat dishes). e Number of outbreaks unknown. ' Mostly traditional lnuit meat dishes. e Greenland data included. Sorrrce: Ref. 31.

ported from the United States, bottled garlic in oil, also imported from the United States but temperature-abused locally, in-house bottled wild mushrooms, and commercial pati. Bottled garlic in oil also caused an outbreak in 1989 in the United States (60) and, as a result of these two outbreaks, garlic in oil can now be sold in North America only if a second barrier (e.g., acidification) in addition to refrigeration is present. In Canada, fatality rates have decreased from 58% during the 1950s to 10% in the 1990s. In Alaska, the situation is similar to that in northern Canada (61). All of the Alaskan outbreaks involved native Alaskans; they had ingested raw or fermented native foods, and type E was usually implicated. In the continental United States, the situation is quite different (31). Home-preserved vegetables are the foods most often implicated, and most outbreaks are caused by type A. Type E accounts for less than 5% of the outbreaks in the continental United States. Commercial foods seldom have been involved, but five outbreaks were associated with food service establishments, involving 130 cases from 1977 to 1989 (62). Temperature abuse of either food ingredients or the final product was often the problem. For example, one outbreak was caused by preparing a potato salad using leftover baked potatoes that

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had been held for up to 5 days at ambient temperature wrapped in aluminum foil (63). Temperature abuse of a commercial, canned cheese sauce resulted in a botulism outbreak in Georgia in 1993, involving eight cases and one fatality (6). Fatality rates in the United States have shown a steady decline, from 34% during the 1950s to 7% inthe 1980s. Fatalities due to type E showed the greatest decline, primarily due to the general distribution of type E antiserum, which first became available commercially in 1964.

D. Incidence of Botulism in Europe By far the highest number of cases of any country in the world reported on an annual basis is in Poland (see Table 4) (31). This probably reflects both a high local incidence of botulism and a very thorough surveillance system. In Poland and several other European countries, including France, Germany, Hungary, Portugal, the former Czechoslovakia, and Belgium, the predominant type involved was B and the foods most often implicated were such home-preserved meats as ham, fermented sausages, and canned products. A significant number of implicated foods were of commercial origin in Poland (25%), the former East Germany (27%), and Belgium (38%). In France, where 12% of implicated foods were of commercial origin, the manufacturers were generally small, local establishments. In Italy and Spain, type B also predominates, but the most commonly implicated foods were home-preserved vegetables. In Italy, most of the vegetables had been preserved in oil. A notable exception to botulism caused by home-preserved vegetables in Italy is a recent outbreak involving eight people in southern Italy who had consumed commercial acidified dairy cream (mascarpone cheese) (7,64). All patients required artificial ventilation, and one died. In Spain, all of the incriminated vegetables had been canned. Scandinavian countries recorded fewer outbreaks, and these mainly were associated with fish and type E. Botulism outbreaks have been rare in the United Kingdom; however, a large outbreak in 1989 involved 27 cases with 1 death (4). Type B toxin had been produced in an underprocessed hazelnut puree, which was then used to flavor a yogurt produced by a local dairy. Other European countries, including Austria, Switzerland, Bulgaria, and Greece, either reported few or no botulism outbreaks (3 1). There is no recent summary on botulism outbreaks in the former U.S.S.R. because epidemiological workon botulism there was discontinued (31). During the period of 1958-64, types A, B, and E were implicated in approximately equal numbers of outbreaks, with a slight predominance of type B (see Table 4). The type B outbreaks occurred mostly in the western region of the former U.S.S.R. and usually involved meats, nearly always home-cured ham, very similar to the situation in central Europe. Incidents associated with fish occurred mainly in regions around the Black Sea, the Sea of Azov, the Caspian Sea, and Lake Baikal. Surprisingly, type A was involved in as many outbreaks from fish as type E. The news agency TASS recently reported an outbreak of type E botulism in Irkutsk involving 12 cases and 1 death that was caused by smoked and salted omul fish.

E. Incidence of Botulism in Asia Foodborne botulism has been reported from only a few countries in Asia (see Table 4) (31). Israel reported an outbreak in 1987 that affected six people with one fatality (65). The incriminated food was kapchunka (salted, uneviscerated whitefish) contaminated with type E toxin. It had been bought in New York City and transported to Israel unrefrigerated. Iran reported a high incidence of botulism. The majority (97%) of investigated outbreaks

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was associated with type E and with fish or fish products. However, fish fleshwas responsible for only 10% of the outbreaks; the other 90% were caused by fish eggs. These eggs (ashbal) are salt-cured for several months and then eaten without further treatment. China recorded almost 745 outbreaks, involving 2861 cases and 421 fatalities, from 1958 to 1989 (36). Most were associated with type A, followed by types B and E. The northwestern province of Xinjiang recorded the majority of outbreaks (-80%), which were usually type A. Typically, vegetable products were incriminated, particularly fermented bean curd (74% of outbreaks) and fermented bean sauce (12%). The few incidents reported from the northeastern region were all associated with type E, but fish were not identified as the causative food. The only outbreak of foodborne botulism caused by neurotoxigenic C. butyric~rnoccurred in Guanyun, Jiangsu province in China. An isolate producing type E BoNT and giving the lipase- and lecithinase-negative colonies typical of C. bu~ricurnwas isolated from the salted and fermented paste of soybeans and wax gourds implicated in the outbreak (18). The majority of Japanese outbreaks occurred in northern areas and were associated with type E and fish or fish products. Izushi was the food most often implicated. To prepare izushi, fleshy pieces of fish are soaked in water for a few days, and then packed tightly into a tub with cooked rice, vegetables, salt, vinegar and spices and left to ferment, often for 3 weeks or longer. Izushi is eaten without further cooking. Two outbreaks in Japan were associated with commercial food. In one, vacuumpackaged, stuffed lotus rhizome caused a type A outbreak and involved 36 cases with 11 deaths. The second such outbreak was caused by imported bottled caviar with 21 cases and 3 deaths and was due to type B toxin. Taiwan also has reported a few botulism outbreaks, including an unusual one involving commercially canned peanuts (66).

F. Incidence of Botulism in Other Areas Argentina is the only country in the Southern Hemisphere to report a substantial number of botulism outbreaks (Table 4) (3 1). These occurred mainly in the provinces of Mendoza and Buenos Aires and between 30" and 40" south latitude. Most outbreaks were associated with type A, and the implicated foods were usually vegetables. For the period 1980-1989, the reported mortality rate was high, 36%, likely due to the frequent involvement of type A (67). Mexico, Guatemala, Venezuela, Peru, Brazil, and Chile are the other Latin American countries that have reported outbreaks. Chad, Egypt, Kenya, Madagascar, and Rhodesia are the only African countries with reported outbreaks (3 1). The first cases of type E botulism in Egypt were reported in 1991 (68). The outbreak, which involved 91 hospitalized patients and 18 deaths, was associated with ungutted, salted, uncooked fish called faseikh. In Kenya, two outbreaks were caused by native foods, one by sour milk prepared in a gourd and one by consumption of raw termites, considered a delicacy by several tribes. The termites were consumed, either raw or fried, after being kept for 3 days in a closed polyethylene bag (69). One of the two outbreaks reported from Madagascar was unusual because type E toxin was associated with a meat product (70). About 60 people were involved with 30 deaths, and locally manufactured bologna was the vehicle. Since 1942, Australia has recorded only six outbreaks of foodborne botulism, five due to vegetables and one due to canned tuna; the last reported outbreak was in 1992 (71). One outbreak of type A botulism was recorded in New Zealand, caused by homebottled fermented mussels and watercress, a traditional Maori dish (72).

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G. InfantBotulism Since the first two cases of infant botulism were diagnosed in the United States (1 l), over 1000 cases of infant botulism have been reported around the world, predominantly (more than 90%) from the United States (see Table 5) (73), where the annual number of infant botulism cases now exceeds the number of foodborne and wound botulism cases combined (74). California, Utah, and Pennsylvania report more than half of the cases (74). This distinct geographic disparity probably reflects both regional variation in physician awareness and the worldwide distribution of C. botulinum spore types. The disease, which affects otherwise healthy children usually less than 1 year old, is characterized by constipation, generalized weakness, and various neurological disorders (75,76). While most patients require hospitalization, fatal cases are rare. After its recognition asa disease, it quickly became evident that infant botulism presented a new pathogenic role for C.botulinum. The nature of the foods most often associated with foodborne botulism usually preclude the involvement of infants. The etiology of infant botulism involves ingestion of clostridial spores, apparently most often from the general environment, and germination, multiplication, and toxigenesis in the infant’s intestines. The only food implicated thus far is honey, which, as discussed in the section on the occurrence of spores in food, occasionally carries C. botuZinum spores in large numbers. Originally limited to C. botulirzum types A and B, the toxin types and organisms associated with the disease now include types E and F and toxigenic C. b u ~ r i c u mand C. bnrutii (16,17,77,78). There are several lines of evidence suggesting that the greatest exposure of infants to spores is from the environment. Since C. botulinum spores are widely distributed in soil and dust, people are always at some risk of ingesting them. Spores adhering to the surface of fresh fruit and vegetables may be ingested and, in the case of infants, spores in dust or dirt adhering to such toys as rattles that infants commonly put in their mouths

Table 5 Worldwide ReportsofInfantBotulism of

Country Argentina Australia Canada Chile Czechoslovakia Denmark France Hungary Italy Japan Spain Switzerland Sweden Taiwan United Kingdom United States

Number of cases infant botulism

Number of fatalities

>50

6 0 0 0 0 0 0 0 1 0 0 0 0 0 0

6 6 1 1 1 1 1

6 15 2 1 1 1 5 876a

Number of cases from 1986 to 1996.

?

Toxin type

Ref. 190, 191 81, 192-194 53, 195-197 198 199 200 20 1 202 16, 203-205 206-2 13 214, 215 216 217 218 2 19-222 76, 223

I18

Dodds

Austin and

maybe ingested. However, it appears that healthy children and adults are completely resistant to this form of exposure to C. botr~lirlrm,whereas many infants are not (79). A strong association between worldwide distribution of spores types and the incidence and type associated with infant botulism is best demonstrated in the United States. Type A cases predominate in the West while type B cases predominate in the East, reflecting the known distribution of spore types in the soil. This also is evident in Argentina, where all cases have been type A, the prevalent type in the soil. As well, more direct studies have implicated dust and dirt in the environment as the source of spores causing infant botulism. In every instance where C. botulirwrl was isolated from a case in California, the environmental isolate had the same toxin type as the clinical isolate (79,80). In Australia, C. botdirzm was isolated from environmental samples associated with three of four cases, and in each case the toxin type was the same as that isolated from the patient (81). Since one of the earliest studies of infant botulism implicated honey as the source of C. bofulirturrz spores (12), honey continues tobethe only food implicated in infant botulism (76). Spores of C. botrdimm have been isolated repeatedly from leftover honey fed to afflicted infants, and the spores' toxin types consistently matched that of the isolates from the infants' stools ( 5 3 3 ) . The California Department of Health Services now distributes a pamphlet advising against giving babies honey during their first year. This is the position of Health Canada, the U.S. Centers for Disease Control, the American Academy of Pediatrics, and the Sioux Honey Association of the United States, the world's largest honey producer.

W . CLINICALASPECTS A. Symptoms Foodborne botulism may vary from a mild illness, which may be disregarded or misdiagnosed, to a serious disease, which may be fatal within 24 hours (20). The onset of symptoms typically occurs 12-36 hours after ingestion of toxin, with a range from a few hours to 14 days. In general, the earlier symptoms appear, the more serious the disease. The first symptoms are generally nausea and vomiting, followed by neurological signs and symptoms, including visual impairments (blurred or double vision, ptosis, fixed and dilated pupils), loss of normal mouth and throat functions (difficulty in speaking and swallowing: dry mouth, throat, and tongue: sore throat), general fatigue and lack of muscle coordination, and respiratory impairment. Other gastrointestinal symptoms may include abdominal pain, diarrhea, or constipation. Diarrhea occurs early in the course of the disease, whereas constipation persists in the advanced stages. Nausea and vomiting appear more often in cases associated with type B and E than with type A. Dysphagia and muscle weakness are more common in outbreaks of types A and B than of type E. Dry mouth, tongue, and throat are observed most frequently in type B cases. Type E botulism tends to have the most rapid onset of symptoms, while type A botulism tends to be the most severe, as measured by intubation rates of patients (82). Respiratory failure and airway obstruction are the main causes of death. Fatality rates in the first half of the century were about 50% or higher, but with the availability today of antisera and modern respiratory support systems, they have decreased to about 10%. Botulism is often confused with other illnesses, including other forms of foodborne poisoning, myasthenia gravis, and carbon monoxide poisoning, but most commonly with

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119

Guillain-Barr6 syndrome. The neurological signs of botulism appear first in the cranial nerve area (eyes, mouth, and throat) and then descend; in Guillain-Barr&,the syndrome progresses in an ascending fashion, beginning in the extremities. The initial symptoms of infant botulism are less clear-cut. The most common, and usually the earliest, symptom is constipation (73). Medical attention is generally requested several days to a week later. The infants usually show a generalized weakness and a weak cry. Other symptonls may include feeding difficulty and poor sucking, lethargy, lack of facial expression, irritability, and progressive “floppiness.” Respiratory arrests occur frequently but seldom are fatal.

B. Treatment Initially, treatment of foodborne botulism is directed toward removing or inactivating the toxin by (a) neutralizing circulating toxin with antiserum, (b) enema or treatment with cathartics to remove residual toxin from the bowel, and (c) in the absence of vomiting, gastric lavage or treatment with emetics (26). Treatment with antiserum is most effective in the early stages of the illness. The impact of antiserum is obvious from the Chinese data; prior to the availability of antisera in 1960, the death rate in China was approximately 50%, but it was only 8% in the nearly 4000 patients who received antitoxin. Subsequent treatment is mainly to counteract the paralysis of the respiratory muscles by artificial ventilation. Optimal treatment for infant botulism consists primarily of high-quality supportive care (73). Approximately 25% of all affected infants require mechanical ventilation, and many require gavage feeding. The use of equine antitoxin and antibiotics are not recommended and do not appear to affect the course or outcome of infant botulism. A trial study using human botulism immune globulin to treat infant botulism began in 1992 and was completed in 1997 (83,234). Treatment with botulism immune globulin reduced hospital stay, intensive care unit stay, ventilator time, and tube feeding time.

C.

Diagnosis

The initial diagnosis of foodborne botulism is based on the patient’s signs and symptoms and, perhaps, food history. Outbreaks in which more than one patient presents with typical symptoms and have a common food history are recognized quickly, even in areas where botulism is not frequent, such as the massive outbreak in Egypt (68). However, the difficulties in diagnosing botulism were highlighted by the 1985 outbreak in Vancouver, Canada, in which the initial diagnoses for 28 patients included psychiatric illness, viral syndrome, laryngeal trauma, overexertion, and a variety of other maladies (5). This outbreak was particularly difficult to diagnose due to the slow onset of rather mild symptoms and the geographic distances between patients. The clinical diagnosis should be confirmed by detecting toxin or viable C. botulimnz in a suspect food or clinical sample or by epidemiological association with a laboratory-confirmed case (36). Serum, feces, enema fluid, stomach contents, and autopsy sections of the small and large intestines and of the liver are suitable specimens for toxin detection. Except for serum, these specimens are also suitable for detecting viable C. botulinzm. Diagnosis of infant botulism is difficult due to the lack of specific symptoms and variation in severity (73,76). It also requires identification of C. b o r ~ d i r rtoxin ~ ~ ~in sel’un1 or toxin and/or spores in the patient’s stools. Patients’ stools usually contain moderate to

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high toxin levels (up to 10' mouse lethal doses per g) and up to lo* viable spores per g, while toxin is seldom detected in theserum (77). Lack of physician recognition and limited diagnostic abilities in some areas are thought to contribute to the low numbers of cases reported from some areas.

D. CaseHistory The following case history is presented as one of the most severe, but nonfatal, cases of botulism documented (85). Within approximately 10 hours of eating only two mouthfuls of the suspect food, a patient in the United Kingdom had blurred and double vision followed by nausea and vomiting, difficulty in swallowing and talking, dryness of the mouth, and arm weakness. He suffered a respiratory arrest on arrival at the hospital and was intubated. A progressive flaccid paralysis ensued. He was given polyvalent antitoxin, a tracheostomy was done, and total parenteral nutrition (TPN) was started. While the patient was conscious, he essentially was paralyzed totally and blind due to complete ptosis. He could respond to questions by clenching his fists. There was little change in his condition for 100 days. His ptosis did not resolve until 46 days after onset of symptoms. TPN was stopped on day 158, and respiratory support was withdrawn after 173 days. He was discharged after 237 days, able to walk with the aid of crutches.

E. Prevention The only effective means of preventing foodborne botulism is by preventing toxin production in foods. Immunization of high-risk populations with botulinal toxoids has been considered; it is not considered effective and, at present, only laboratory workers at risk normally are immunized. In most cases, the preservation of high-moisture foods is geared toward control of C. botzrlirzum,which usually involves inhibition rather than destruction. Such control generally also ensures control of other foodborne pathogens and of many spoilage microorganisms. The effects of different factors on the growth of C. botulinrm~ in foods are described in a separate section below. Control of C. botulinum in foods is generally achieved by one of the following methods. 1. Low-acid, shelf-stable canned foods are preserved by a full thermoprocess. 2. Shelf-stable, canned, cured meats are preserved by a combination of thermoprocessing and addition of salt and nitrite. 3. Canned acid foods are preserved by a pasteurizing thermoprocess and acidity. 4. Products such as dry-fermented sausages are preserved by reduced water activity (a,J and pH and added nitrite. 5. Packaged raw or cooked meats or fish and shellfish are preserved by refrigeration alone. 6. Many meat and fish products are preserved by a combination of added salt and refrigeration. 7. A nutnber of cured meat products are preserved by a combination of added salt and nitrite and refrigeration. 8. Vacuum-packaged smoked fish are preserved by a combination of thermoprocessing, added salt, smoking, and refrigeration. 9. A few perishable products such as processed cheese, caviar, pickled fish, and acidified meats are preserved by decreased a, and pH and refrigeration.

Clostridium botulinum

V.

121

DETECTION

It is not necessary to isolate C. botulinum in pure culture from foods or a clinical sample in order to demonstrate its presence. Usually, the sample is inoculated into a nonselective enrichment medium. If the neurotoxin is present in the culture after incubation, the toxinproducing organism must have been present originally. BoNTs continue to be recognized by their lethal action in mice and neutralization with specific antisera. The mouse bioassay is generally more sensitive and more specific than several in vitro tests that have been developed. The sample, or an extract prepared by homogenizing it in a slightly acidic buffer, is clarified by centrifugation and normally filter-sterilized. Trypsin treatment may be required to activate low levels of toxin from nonproteolytic strains. The prepared sample is injected intraperitoneally into mice with and without neutralization with specific antitoxin. Typical signs of botulism are ruffled fur, pinched waist, labored breathing, limb paresis, and general paralysis before death. Definitive results are obtained if mice injected with untreated sample die within 72 hours, whereas mice injected with neutralized sample survive. Several investigators have developed ELISA (enzyme-linked immunosorbent assay) protocols for detecting the neurotoxin that approach or meet the sensitivity of the mouse bioassay (86). A new procedure based on detection of the endoproteinase activity of BoNTs has recently been developed (87,88). Common enrichment media for detecting viable C. botulinum are cooked meat medium (CMM), CMM glucose, chopped meat glucose starch (CMGS) medium, and trypticase-peptone-glucose-yeast (TPGY) extract broth, to which trypsin maybe added (TPGYT). Trypsin is necessary to activate the toxin produced by Group I1 organisms, and also may inactivate such potential inhibitors of C. botulinurn as boticins in mixed cultures. While foods may beinoculated directly, the sediments of centrifuged samples are preferred because potential growth inhibitors are removed. At least two tubes of media are inoculated. To select for spores, one tube is heated at 75-80°C or 60"C, respectively, depending on whether the suspected type belongs to Group I or 11. Spores of group I1 also may be selected by holding samples in 50% alcohol for 1 hour before inoculation. The other tube is incubated without any treatment to allow development of vegetative C. botulinunl cells in case few or no spores are present. Adding lysozyme to the medium may increase recovery of heat-injured spores. C. botulinum is identified after incubation of the enrichment medium by toxin analysis of the supernatant fluid using the mouse bioassay as outlined above.

VI.

NEUROTOXIN

As stated in the introduction to this chapter, seven serologically different neurotoxins are produced by various strains of C. botulinun~belonging to the four groups (19,89). They are produced during growth and generally accumulate in the culture fluids toward the end of the growth phase, largely through cell lysis, but toxin also is released during the logarithmic phase before any significant lysis occurs (90). In general, the designation of the strain is that of the toxin it produces. Group I strains produce type A, B, and F neurotoxins or, rarely, combinations of subtypes AB,AF, BA,and BF, where the subscripted type is produced in smaller amounts (19,23). Group I1 strains produce types B, E, and F neurotoxins. Group I11 strains produce C l and D neurotoxins and C? toxin and exoenzyme C3. C2toxin and exoenzyme C3 are not neurotoxins, but are ADP-ribosylating toxins, which have actin

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or the small GTP-binding proteins of the Rho family as substrates (91). Type G neurotoxin is produced by Group IV strains, which are now classified as C. nrgenfirzense (24).

A.

Structure

The neurotoxins are all very similar proteins with a molecular weight of approximately 150 kDa (92). They are synthesized as a single-chain protein with relatively low toxicity, which is activated when it is ‘nicked” by a number of proteases, including proteases of Group I, into a dichain molecule held together by a disulfide bond (93). The two components of the nicked toxin, a light (L) and heavy (H) chain, have molecular weights of approximately 50 and 100 kDa, respectively. Individually, the two components are not toxic, but toxicity can be restored by reestablishing the disulfide bond. The neurotoxins are released by bacterial autolysis and exist as complexes with other, nontoxic, proteins; these complexes are referred to as “progenitor toxins.” Three forms of progenitor toxins have been identified: 12S,16S, and 19S, respectively designated M (medium), L (large), and LL (extra large) toxins (1,201. The molecular weights of the progenitor toxins range from 500 to 900 kDa. The M form is the most common natural form, found in foods and cultures, along with the L form, which is a complex of the 150 kDa neurotoxin with an atoxic component. In the L form, a hemagglutinin is also part of the complex. M and L forms dissociate under mild alkaline conditions into the S, or derivative toxin, which is the single-chain protein. The LL form is only known for type A and was the first form of the botulinum toxin to be purified and crystallized. It has not been found in cultures and is likely an artificial aggregate. L

B. Mode of Action The detailed mechanism of action of the various BoNTs has recently been fully elucidated (94-96). The neurotoxins cause flaccid paralysis by blocking the release of the neurotransmitter acetylcholine at neuromuscular junctions (97). The mode of action can be divided into three steps: binding. internalization, and intracellular action (98). The C-terminal half of the H chains are responsible for binding, while the N-terminal half contains the domain required for internalization. The Lchains possess catalytic activity, which results in inhibition of exocytosis when they are released into the cytosol. The neurotoxin binds to a type-specific receptor on the presynaptic membrane, mediated by the carboxy terminus of the H chain (99). It appears that the different BoNTs bind to different receptors (1 00). While BoNT/B was recently shown to bind to synaptogamin I1 complexed with gangliosides (101-104), the receptor(s) for the other BoNTs have not been identified. The neurotoxin is internalized into the neuron by an initial receptor-mediated endocytosis (99), followed by pH-dependent membrane penetration. The amino terminus of the H chain then creates channels in the membrane, which permit the 50 kDa L chain to enter the cytosol (105,106). After this step, the toxin can no longer be neutralized. Finally, the internalized L chain acts as a zinc-dependent endopeptidase, which specifically cleaves proteins involved in vesicle docking and membrane fusion. BoNT types B, D, F, and G selectively cleave vesicle-associated membrane protein (VAMP) (also referred to as synaptobrevin) (107-109), while BoNT types A and E cleave the synaptosome associated protein SNAP-25 (110- 112). BoNT type C l degrades syntaxin/HPC-l ( 1 13,114). The

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cleavage of theproteins involved in vesicle docking and membrane fusion results in inhibition of acetylcholine release from the neuron and paralysis.

C. TherapeuticUses The use of BoNT to treat patients with disorders of muscle function originated with the use by Dr. Alan B. Scott of the Smith-Kettlewell Eye Research Institute (San Francisco, CA), in the 1970s of small doses of toxin A in the treatment of strabismus (crossed eyes caused by an imbalance of eye muscles) (115). Toxin injected into the problematic muscles, usually muscles that are contracting too much, causes temporary local paralysis and often results in long-lasting muscle realignments. Results of the first clinical trial in 1979 and many thousands of cases since have demonstrated its effectiveness and safety, with a few limitations (1 16). Since then, many other muscle disorders have been treated successfully including blepharospasm (an involuntary, spasmodic, repetitive eyelid closure) (1 17), torticollis (an involuntary contraction of neck muscles producing abnormal turning of the head) (1 18), spasmodic dysphonia (a contraction of the vocal chords causing a strained, strangled voice) (1 1g), hemifacial spasm (involuntary spasms on one side of the face) (119j, and various dystonias (involuntary muscle contractions in diverse areas of the body) including cervical dystonia (120), oromandibular dystonia (121), and focal dystonias of the hand (119). More recently, BoNTs have been used to treat common nondystonic conditions including hyperfunctional facial lines (122j and chronic anal fissure (123).

VII. A.

CONTROL IN FOODS Factors Affecting Growth and Toxin Production

The main factors controlling growth of C. botulinum in foods are temperature, pH, water activity (a%),redox potential, added preservatives, and other microorganisms. The growth of types A, B, and E, of Groups I and 11, has been intensively studied due to their involvement in foodborne botulism. Traditionally, food microbiologists have established maximum and/or minimum limits for these parameters which would permit growth of C. botulin z m (Table 6). and these limits have often been used in the control of C. botulinum. However, these factors seldom function independently; usually they act in concert, often having synergistic or additive effects.

Table 6 Properties of Groups I and I1 C. botzdinztm Property Toxin types Minimum temperature for growth Optimum temperature for growth Minimum pH for growth Inhibitory [NaCI] Minimum a, for growth D,,,IyCof spores D,?,'c of spores Source: Ref. 26.

Group I A, B, F 10°C 3540°C

4.6 10% 0.94 25 min 0.1-0.2 min

Group TI

B. E, F 3.3"C 18-25°C 5.0 5% 0.97 <0.1 min <0.001 min

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1. Temperature Due to the importance of refrigeration as a barrier to growth, studies have focused on determining the minimum temperatures permitting growth. The established lower limits are 10°C for Group I (20) and 3.0"C for Group I1 (124). However, these limits apply to relatively few strains and depend on otherwise optimum growth conditions. Irrespective of the actual minimum growth temperature, production of toxin generally requires several weeks at the lower temperature limits. The optimum growth temperature is in the range of 35-40°C for Group I organisms and in the range of 25-30°C for Group 11. The upper temperature limits, respectively, for Group I and Group I1 organisms are approximately 45-50 and 40-45°C.

2. pH It is generally accepted that the minimum pH permitting growth of C. botulinum Group I is 4.6, and many regulations worldwide use this limit. For Group 11, the limit is about pH 5.0. Thus, many fruits and vegetables are sufficiently acidic to inhibit C. botulinum by their pH alone, while other products, such as marinated mushrooms, are preserved by added acidulants. Several factors influence the acid tolerance of C. botulinum, including strain, substrate, temperature, nature of the acidulant, the presence of preservatives, a,, and redox potential (125). The growth of such acid-tolerant microorganisms as yeasts and molds may raise the pH in their immediate vicinity to a level that permits growth of C. botulinum (126). C. botulinunz also can grow in some acidified foods if excessively slow pH equilibration occurs. While high concentrations of proteins in laboratory media appear to protect C.botuZinum and permit growth at pH levels below 4.6, this does not occur in foods preserved by acidity (26). Current regulations stipulating a minimal acidity of pH 4.6 for the control of C. botulinum therefore are valid. The upper pH limits for growth are in the range of pH 8-9 but are of no practical consequence. 3. Salt and Water Activity Salt (NaC1) is one of the most important factors controlling C. botulinum in foods. Its inhibitory effect is primarily due to the depression of a, and consequently to its concentrationin the aqueous phase, also called the brine concentration (% brine = % NaCl X loo/% H20 + % NaC1). Under otherwise optimal conditions, the growth-limiting brine concentrations are about 10% for strains of Group I and 5% for strains of Group I1 (125). These concentrations correspond well to the limiting a, of 0.94 for Group I and 0.97 for Group I1 in foods in which NaCl is the main a , depressant. The type of solute used to control a, may influence these limits. Generally, NaC1,KC1, glucose, and sucrose show similar patterns, while glycerol permits growth at lower a, by up to 0.03 units (127). Full or partial replacement of NaCl with other salts has been evaluated in many products, with a slight reduction in control if a mixture of salts was used (125). The limiting a, may be raised significantly by such other factors as increased acidity or preservatives. 4. Atmosphere, Redox Potential Modified-atmosphere packaging (MAP) is being increasingly used to extend the shelf life and improve the quality of foods. However, MAP of perishable foods has been a concern because the extension of shelf life and inhibition of spoilage microorganisms might promote growth of C. botulinurn. The safety of MAP of fish has been studied extensively due to the high incidence of type E spores in fish (26,30,128,129). Furthermore, various researchers have shown that C. botuliizum can grow and produce toxin in MAP fish and,

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depending on conditions, toxin may be present before the fish is considered spoiled (130134). While there is a common assumption that C. botulinurn cannot grow in foods that are exposed to 02,the Eh (oxidation reduction potential) of most such foods is usually low enough to permit its growth (125). For example, initial atmospheres containing 20% O2 did not delay toxin production by C. botulinum in inoculated pork as compared to samples packaged with 100% N2, and some toxic samples contained 15% residual O2 ( l 35). Most MAP products rely on CO2to inhibit spoilage and pathogenic microorganisms (136), but CO1 may actually stimulate C. botulinum (137). Initial levels of 15-30% CO? did not inhibit C. botulinum in inoculated pork; only 75% CO2 showed significant inhibition (138). The safety of different atmospheres with respect to C. botulinum should be carefully investigated before use. C. botulinum grows optimally at an Eh of -350 millivolts (mV), but growth initiation may occur in the Eh range of 30-250 mV. The presence of other inhibitory factors lowers this upper limit. Once growth is initiated, the Eh declines rapidly (125).

5. Preservatives While effects on color and flavor are important organoleptic considerations in adding nitrite and nitrate to cured products, the most important role of nitrite in cured foods is the inhibition of C. botulinum. Nitrite is the functional constituent. While nitrate has little or no antibacterial activity, it is partially converted to nitrite by endogenous or added microorganisms. The exact mechanism of botulinal inhibition by nitrite is not known yet. Its effectiveness is dependent upon complex interactions among pH, salt, heat treatment, time and temperature of storage, and composition of food (125). Nitrite is rapidly converted to forms undetectable as nitrite during processing and continues to be converted with time until a constant low level is reached. Losses of 48-93% during processing alone have been reported (139). The effectiveness of nitrite and the duration of its effect may depend on a balance between the rate of nitrite depletion and the death rate of spores. The rate of nitrite depletion is dependent on product formulation, pH, and time and temperature during processing and storage. While nitrite is more effective at low pH, it is also depleted faster at low pH. However, a significant contribution of nitrite to the inhibition of C. botulirzurn continues even when nitrite is no longer detectable (26). Nitrite reacts with many cellular constituents and appears to inhibit C. botulinum by more than one mechanism, one of which is probably its reaction with essential ironsulfur proteins to inhibit the phosphoroclastic system that supplies the cell with energy (140,141). The reactions of nitrite, or nitric oxide, with secondary amines in meats to produce nitrosamines, some of which are carcinogenic, has led to regulations limiting the amount of nitrite used. Other compounds that are active against C. botulinum include sorbates, parabens, nisin, phenolic antioxidants, polyphosphates, ascorbates, EDTA, metabisulfite, n-monoalkyl maleates and fumarates, and lactate salts (125). The use of natural or liquid smoke has a significant inhibitory effect against C. botulinuln in fish, but appears to be insignificant in meats. 6. Other Microorganisms Other microorganisms have a very significant role in the control of C. botulinum in foods (26,125). Acid-tolerant yeasts and molds may make the environment more favorable for

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growth of C. botulirzurtz (126,143). Other microorganisms may inhibit C. botzdirzzrrn either by changing the environment or by producing specific inhibitory substances, or both. Lactic acid bacteria, including Lactobacillus, Pediococcus, and Streptococcus, can inhibit growth of C. botzrlirzzrrn in meat products, largely by reducing the pH, but perhaps also by the production of bacteriocins (143-146). The use of lactic acid bacteria and a fermentable carbohydrate, the “Wisconsin process,” has been permitted for producing bacon with a decreased level of nitrite in the United States (147,148). The growth of other microorganisms also may protect the consumer by causing spoilage that would make a toxic product less likely to be consumed.

B. ThermalInactivation Thermal inactivation is the most common method of sterilizing foods. C. botzrlir~urnspores of Group I, which are very heat resistant, are the target organisms for thermal processes. D-values (the time required to inactivate 90% of the population at a given temperature) vary considerably among C. botzrlirmnz strains and depend on how the spores are produced and treated, the heating environment, and the recovery system (149-153). Spores of types A and B are the most heat resistant, having D,2IrC values in the range of 0.1-02 minute. These spores are of particular concern in the sterilization of canned low-acid foods, and the canning industry has adopted a D-value of 0.2 minute at 121°C as a standard for calculating thermal processes. For the most resistant strains, z-values (the temperature change necessary to bring about a 10-fold change in the D-value) are approximately 10°C, which has also been adopted as a standard. Actual z-values may vary by several degrees. Despite the variations in D- and z-values, the adoption of a 12-D process as the minimum thermoprocess applied to commercial canned low-acid foods by the canning industry has ensured the production of safe products (26). Although strains of Group I1 are considerably less heat resistant (DInusC < 0.1 min) than those of Group I, their survival in pasteurized, refrigerated products is of concern because of their ability to grow at refrigeration temperatures (154-156). D*. c values of type E in neutral phosphate buffer are generally in the range of 0.2- 1.0 minute. While the D-values often are higher in foods, the z-values are essentially the same. The pasteurization of such products as crabmeat and processed fish should achieve a 10-log reduction of type E strains. Various regulations and guidelines for the safe production, distribution, and sale of refrigerated foods of extended durability have been published. The Advisory Committee on the Microbiological Safety of Food Working Group on Vacuum Packing and Associated Processes has produced recommendations for vacuum packed and sous vide foods to control growth of C. botuliwnz in foods (157). These recommendations include maintenance of chill temperatures and one or more of the following: a heat treatment of 90°C for 10 minutes or equivalent lethality, a pH of 5 or less, a minimum salt level of 3.5% in the aqueous phase, an a,of 0.97 or less, or a combination of heat and preservative factors that can be shown to prevent growth and toxin production by psychrotrophic C. botulinum. American recommendations from the National Advisory Committee on Microbiological Criteria for Foods include inoculated pack studies with C. botulimrm to determine shelf life.

C.

Inactivation by Irradiation

C. botzrlinzrr~spores are probably the most radiation-resistant spores of public health concern. D-values (irradiation dose required to inactivate 90% of the population) of Group

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I strains at -50 to - 10°C are in the range of 2.0-4.5 kGy in neutral buffers and in foods (125). Spores of type E are only marginally more sensitive, having D-values in the range of1-2 kGy. The goal of radappertization is to reduce the number of viable spores of the most radiation-resistant C. botulimlrn by 12 log cycles. D-values are affected by any pretreatment of spores, the presence of 02,irradiation temperature, and irradiation and recovery environments. In general, spores are more sensitive in the presence of 0, or preservatives and at temperatures above 20°C.

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173. Thatcher, F. S., Erdman. I. E., and Pontefract, R. D. (.1967). Some laboratory and regulatory aspects of the control of Clostridium botulinum in processed foods. In Botulism 1966 (M. Ingram and T. A. Roberts, eds.), Chapman and Hall Ltd., London, pp. 511-521. 174. Hayes, S., Craig, J. M., and Pilcher, K. S. (1970). The detection of Clostridium botulinur~r type E in smoked fish products in the Pacific Northwest. Can. J. Microbiol., 16:207209. 175. Lindroth, S. E., and Genigeorgis, C. A. (1986). Probability of growth and toxin production by nonproteolytic Clostridiuw botulinun? in rockfish stored under modified atmospheres. Znt. J. Food Microbiol., 3:167-181. 176. Baker, D. A., Genigeorgis, C., and Garcia, G. (1990). Prevalence of Clostridium borulinurn in seafood and significance of multiple incubation temperatures for determination of its presence and type in fresh retail fish. J. Food Prot., 53:668-673. 177. Cann, D. C., Wilson, B. B., Shewan, J. M., and Hobbs, G. (1966). Incidence of Clostridiuln botulinum type E in fish products in the United Kingdom. Nature, 211:205-206. 178. Johanssen, A. (.1965). Clostridiunz botulinum type E in foods and the environment generally. J. Appl. Bacteriol., 28:90-94. 179. Nielsen, S. F., and Pedersen, H. 0. (1967). Studies on the occurrence and germination of Cl. botzdinzrm in smoked salmon. In Botulism 1966 (M. Ingram and T. A. Roberts, eds.), Chapman & Hall, London, pp. 66-72. 180. Abrahamsson, K. (1967). Occurrence of type E Cl. botulinum in smoked eel. In Botulism 1966 (M. Ingram and T. A. Roberts, eds.), Chapman and Hall, London, pp. 73-75. 181. Hielm, S., Hyytia, E., Ridell, J., and Korkeala, H. (1996). Detection of Clostridium botulinum in fish and environmental samples using polymerase chain reaction. Znt. J. Food Microbiol. 31:357-365. 182. Suhadi, F., Thayib, S. S., and Sumarsono, N. (1981). Distribution of Clostridizrm botulinum around fishing areas of the western part of Indonesian waters. Appl. Environ. Microbiol., 41:1468-1471. 183. Haq, I., and Sakaguchi, G. (1980). Prevalence of Clostridizrnz botulinum in fishes from markets in Os&a. Jpn. J. Med. Sci. Biol., 33:l-6. 184. Greenberg, R. A., Tompkin, R. B., Bladel. B. O., Kittaka, R. S., and Anellis, A. (1966). Incidence of mesophilic Clostridium spores in raw pork, beef, and chicken in processing plants in the United States and Canada. Appl. Microbiol., 14:789-793. 185. Abrahamsson, K., and Riemann, H. (197 1). Prevalence of Clostridium botzrlinwn in semipreserved meat products. Appl. Microbiol., 21543-544. 186. Hauschild, A. H. W., and Hilsheimer, R. (1980). Incidence of Clostridiunl botulinum in commercial bacon. J. Food Prot., 43564-565. 187. Hauschild, A. H. W., and Hilsheimer, R. (1983). Prevalence of Clostridiurn botulinum in commercial liver sausage. J. Food Prot., 46:242-244. 188. Roberts, T. A., and Smart, J. L. (1976). The occurrence and growth of Clostridium spp. in vacuum-packed bacon with particular reference to Cl. perj?ignens (welchii) and Cl. botulinum. J. Food Technol., 11:229-244. 189. Roberts, T. A.. and Smart, J. L. (1977). The occurrence of clostridia, particularly Clostridium botulinum, in bacon and pork. In Spore Research 1976 (A. N. Barker, J. Wolf, D. J. Ellar, G. J. Dring, and G. W. Gould, eds.), Vol. 2, Academic Press, New York, p. 91 1. 190. Lentini, E., Fernandez, R., Ciccarelli, A. S., and Gimenez, D. F. (1984). Botulismo en el lactante, una neuva enfernledad? Arch. Arg. Pediatr., 82:197-198. 191. de Centorbi, 0. P., Centorbi, H. J., Demo, N., Pujales, G., and Fernandez, R. (1998). Infant botulism during a one year period in San Luis, Argentina. Zent. Bl. Bakteriol., 287:61-66. 192. Shield, L. K., Wilkinson, R. G., and Ritchie, M. (1981). Infant botulism in Australia-a case report. Aust. Paediatr. J., 1759. 193. Ryan, P. J. (1987). Infant botulism-the first reported case from Queensland. Med. J. Arrst., 1461105-106.

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194. Thomas, D. G. (1993). Infant botulism: Areview in South Australia(1980-89). J. Paediatr. Child Health, 29:24-26. 195. Austin. J., Blanchfield, B., Proulx, J.-F., and Ashton, E. (1997). Botulism inCanada-summary for 1996. Caw Commun. Dis. Rept., 23:132. 196. Hauschild, A.. Gauvreau, L., and Black, W. A. (1987). Botulism in Canada-summary for 1986. Can. Dis. Week. Rept., 13:47. 197. McCurdy. D. M., Krishnan, C., and Hauschild, A. H. (1981). Infant botulism in Canada. Can. Med. Assoc. J., 125:741-743. 198. Erazo, R., Marambio, E., Cordero, J., Fielbaum, O., and Trivino, X. (1987). Botulismo infantil: comunicaion de un caso. Rev. Med. Chi., 115:344-348. 199. Neubauer, M., and Milacek, V. (1981). Infant botulismtype B in central Europe. Abl. Bakt. Hyg., I. Abt. Orig. A., 250:540-547. 200. Balslev, T., Ostergaard, E., Madsen, I. K., and Wandall, D. A. (1997). Infant botulism. The first culture-confirmed Danish case.Neuropediatrics, 28:287-288. 201. Paty, E., Valdes, L., Harpey, J. P., Dore, F., Hubert, P., Roy, C., and Caille, B. (1987). Un cas de botulisme chez un nourrisson de 11 mois. Arch. Fr. Pediatr., 44:129-130. 202. Trethon, A., Budai,J., Herendi, A., Szabo, V.,and Geczy, M. (1995). Csecsemokori botuliznlus. Owosi Hetilap, 136:1497-1499. 203. Aureli, P., Fenicia, L., Creti, R., Bertini, E., Vigevano, F., DiCapua, M., and Pirozzi, N. (1989). Botulismoinfantile in Italia Aspetti clinici e microbiologicidi 5 casi. Riv. Ital. Pediatr., 15:442-447. 204. Fenicia, L., Ferrini, A. M., Aureli, P., and Pocecco, M. (1993). A case of infant botulism associated with honey feeding in Italy. Eur. J. Epidemiol., 9:671-673. 205. Moli, 0.L., Quarto, M., Larosa, C. G., Germinario,C., Zimbalatti, F., and Barbuti, S. (1996). A case of infant botulism: Clinical and laboratory observations. Ital. J. Pedintr., 22:234237. 206. Noda, H., Sugita, K., Koike, A., Nasu, T., Takahashi, M., Shimizu, T., Ooi, K., and Sakaguchi, G. (1988). Infant botulism in Asia. ANI.J. Dis. Child., 142:125-126. 207. Oguma, K., Yokota, K., Hayashi, S., Takeshi, K., Kumagai, M., Itoh, N., Tachi, N., and Chiba, S. (1990). Infant botulism due to Clostridizm botulinurzl type C toxin. Lancet, 336: 1449-1450. 208. Nabeya, T., Yano, R., Saito, T., Inoue, H., Shinohara, N.,Yokoyama, T., Nagai, S., Nishibayashi. Y., and Sakaguchi. G. (1989). Infant botulism was confirmed in Ehime Prefecture. Karlsenshogaku Zasshi-J. Jpw. Assoc. Infect. Dis., 63:268-272. 309. Tabita, K., Sakaguchi, S., Kozaki, S., and Sakaguchi, G. (1991). Distinction between Clostridium botulinum type A strains associated with food-borne botulismand those with infant botulism in Japan in intraintestinal toxin production in infant miceand some other properties. FEMS Microbiol. Lett., 79:251-256. 210. Morikawa, Y., Shishida, N., Toshima, M., Yoshioka, Y., and Nukina, M. (1994). A case report of infant botulism without a history of honey ingestion. Karlsenshogaku Zusshi, 68: 259-262. and Nakamura, S. (1996). The 21 1. Kakinuma, H., Maruyama, H., Takahashi, H., Yamakawa, K., first case of type B infant botulism in Japan. Acta Paediatr. Jpn., 38:541-543. 212. Toyoguchi, S., Tsugu, H., Nariai, A.. Kaburagi, Y., Asahina, Y., Ambo, K., and Katou, K. (1991). Infant botulism with Down syndrome. Acta Paediatr. Jpn., 33:394-397. H., and Nakamura, 213. Yamakawa, K., Karasawa, T., Kakinuma, H., Maruyama, H., Takahashi, S. (1997). Emergence of Clostridiurn botulinum type B-like nontoxigenic organisms in a patient with type B infant botulism. J. Clin. Microbiol., 35:2163-2164. 214. Torres Tortosa, P., Martinez Villalta, E., Rodriguez Caamano, J.. Lorca Cano, C., Puche Mira, A., and Borrajo, E. (1986). Botulismo dellactante. Presentacion de un caso. An. Esp. Pediatr., 24:193-196. 21 5. Lizarraga Azparren, M.A., Lopez Fernandez,Y., Pilar Orive,J., Latorre Garcia,J., Hermana

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216. 217. 218. 219. 220. 221. 222. 223.

Austin and Dodds Tezanos, M. T., and Rivera Aranda, A. (1996). Botulismo infantil: a proposito de un caso. An. Esp. Pedintr., 44:399-401. Gautier, E., Gallusser, A., and Despland, P. A. (1989). Botulisme infantile. HeIv. Pnediat. Acta, 43:521-530. Jansson, L., Bjerre, I., Schalen, C., and Mardh. P. A. (1985). Forstafallet av infantil botulism i Sverige? Lnkartidningen, 82:955-956. Wang, C. C., Chu, M. L., Liou, W. Y., Twu, P. H., Lin, C. H., and Lee, C. L. (~1988). Infant botulism: Report of a case. J. Formosan Med. Assoc., 87:919-922. Turner, H. D., Brett, E. M., Gilbert, R. J., Ghosh, A. C., and Liebeschuetz, H. J. (1978). Infant botulism in England. Lancet, i: 1277-1278. Smith, G. E., Hinde, F., Westmoreland, D., Berry, P. R., and Gilbert, R. J. (1989). Infantile botulism. Arch. Dis. Child., 64:871-872. Gilbert, R., Brett, M., and Kramer, J. (1993). A case of infant botulism. CDR Weekly, 3: 129. Cochran. D. P., and Appleton, R.E. (1995). Infantbotulism-Is it that rare? Dev. Med. Child Neurol., 37:274-278. Breuer, T., and Maslanka, S. E. (1997). Laboratory-confirmed botulismin the United States. January 1,1996-December 3 1, 1996. In 1997 Meeting of the InteragencvBotulism Research Coordintcting Committee, Bethesda, MD., 12-14 November, 1997.

8 Clostridium perfringens Dorothy M. Wrigley Minnesota State UniversiQ, Mankcrto, Mirtnesotu

I. Introduction 140 11. General Characteristics

140

Morphology A. 140 Toxins B. 141 C. Growthandsurvival of vegetative cells D. Sporulation and enterotoxin production 144 E. Germination 145 F. Genetics 146 G. Antibiotic sensitivities 147

142

111. Diseases 147 Gastroenteritis A. B. Relateddiseases

137 148

IV. Epidemiology 148 Human-to-human A. spread 148 B. Animal-to-human spread 149 V. Isolation and Identification 149 Plating A.techniques 149 B. Biochemical/Physiological tests C. Other tests 152 153 Transport D. of specimens VI. Enterotoxin Pathogenicity

152

153

Feeding A. studies 153 B. Biochemistry and mode of action C. Production 154 Purification D. 154 Enterotoxin E. assays 155 F. DNAprobes of theenterotoxingene156

153

VII. Prevention 156 References 157

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1.

INTRODUCTION

Clostridiun~ yegringens type A ranks among the most common agents of foodborne gastroenteritis (1-5). Some estimations place it as the leading cause because most cases are unreported or undiagnosed due to the mild nature of the symptoms. C. pegrilzgens gastroenteritis was described first in the early 1900s but received little attention until 1953 when Hobbs et al. (6) described the disease and began classification of gastroenteritis-associated strains (7). The organism is considered by many to be ubiquitous and has been isolated from soil, plants, and a variety of animals that includes cattle, poultry, and humans. All these sites may serve as reservoirs for infection, although animal and human sources appear to bethemost common. Its nutritional requirements and temperature ranges for growth are such that it may only replicate in mammals or other warm, nutrient-rich sites. The foodborne gastroenteritis follows ingestion of high numbers of vegetative cells. The cells sporulate in the intestines. It is during sporulation that the enterotoxin is produced. This protein, which appears to be a spore-coat protein, is released into the lumen of the gut and triggers the onset of diarrhea. Resolution of the diarrhea usually requires no treatment and is complete in several hours (7,s).Confirmation of C. perfringens as the etiological agent often is difficult due to the short duration of the spores in fecal material and the sensitivity of the organism to cold storage. Culture of C. perfringens from fecal material needs to be supplemented with confirmation of enterotoxin production (9,10). More serious diseases are also associated with C. perfringeus. Enterotoxigenic C. perfringem type A has been linked with sudden infant death (SID) more than any other bacterial species (1 1- 13). C. peI$”ngens type A also causes wound infections and is one of several agents associated with gas gangrene and myonecrosis. These infections usually are not caused by foodborne bacteria. While C. pe~fr-irzgenstype A is the most common of the foodborne clostridia, type C also is afoodborne pathogen. The first recorded outbreak was described as a necrotizing gastroenteritis and followed consumption of contaminated rations in Germany (14,15). More recent outbreaks have been seen in Papua New Guinea, where the disease is known as pig-bel (16).

II. GENERALCHARACTERISTICS

A.

Morphology

C. perfiilzgerzs is a nonmotile, gram-positive, anaerobic bacillus that has the capability to form an endospore. Strains associated with foodborne gastroenteritis more readily form spores than those associated with myonecrosis and gas gangrene. Spores are oval and subterminal. The length of the bacillus varies greatly. In glucose-rich media, the rods are short; in starch-based media, the rods are long. Capsules may be present, especially on bacilli from tissue specimens (l 7). Colonies of C. perfringens are generally white. When cultured on blood agar, colonies are typically surrounded by double zones of hemolysis due to the hemolytic activity of the alpha (a)-and theta (0)-toxins. On egg yolk agar, a zone of opalescence due to atoxin activity surrounds each colony. On sulfite-containing agars, black colonies form due to sulfite reduction.

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Table 1 Toxins of Clostridim perfringens Toxin (C. pe?jhkgens Activity type) a-Toxin (A,B,C,D,E)a P-Toxin (B,C) y-Toxin (B,C) &Toxin (B,C) €-Toxin (B.D) q-Toxin (A) 0-Toxin (A,B,C,D,E) L-Toxin (E) K-Toxin (A,C,D,E) h-Toxin (B,D,E) p-Toxin (A,B,D) v-Toxin (A,B.C,D,E) Enterotoxin (A)h

Phospholipase C, lethal, necrotizing Lethal, necrotizing Lethal Lethal, hemolytic Lethal, necrotizing Lethal Hemolytic, lethal ADP-ribosyltransferase, necrotizing, lethal Collagenase Proteinase Hyaluronidase Deoxyribonuclease Complex with plasma membrane

'C. pelfringens types shown to secrete the toxin. Types C and D secrete an enterotoxin distinct from type A. Source: Adapted from Ref. 17.

B. Toxins As a species, C. ye~fringensproduces a wide variety of toxins, which are used for typing the species. The activities are listed in Table 1. Two of the toxins, epsilon (E) and iota (I), are activated by trypsin. Theta-toxin is oxygen labile. The proteinase, lambda (1)toxin, degrades casein but not collagen (18). The types of C. perfringens (A, B, C, D, and E) are based onthe secretion of a-,beta (p)-, gamma (y)-, E-, and t-toxins. The characteristics of these five major toxins are given in Table 2. Numerous other toxins and enzymes are produced. 1. Toxins of C.perfringens Type A The toxins of C. ye$ringens type A have been studied extensively. The enterotoxin associated with gastroenteritis is discussed in Section VI. Alpha-toxin (phospholipase C) has received the most attention and the gene has been cloned and sequenced (19-21). The recombinant phospholipase C sequence has a promoter similar to the sigrng5 of Esclw richict coli. The recombinant protein at 42,528 daltons is identical to native protein. The N-tenninus has a 77-amino-acid sequence similar to the phospholipase C of Bacillus cereus and a 64% homology with thephospholipase C of C. bifermmztnns (21). The recombi-

Table 2 Conlparison of Major Toxins of Clostridium perfrirlgens Toxin

Molecular weight (daltons) producing Types

PI

Ref.

~~~

a-Toxin P-Toxin €-Toxin t-Toxin (a binary toxin)

43,000 28,000 34,250 47,500 7 1,500

5.4 5.4-5.5 5.6 5.2 4.2

A,B,C,D,E B,C B ,D E

19-21 33.138 34.35 39-41

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nant a-toxin has activity distinct from that of 0-toxin and no hemolytic activity against horse. sheep, rat, or rabbit erythrocytes. Alpha-toxin preferentially attacks the phosphorylcholine and sphingomyelin in tnenlbranes (22j. Alpha-toxin has no apparent activity in human gastroenteritis. Gastroenteritis associated strains usually produce less a-toxin than wound-associated strains (23). Whether a toxin has any true pathogenic effect in other intestinal diseases has not been determined conclusively. The toxin can kill intestinal villi cells (34). Alpha-toxin plays an important role in C. pelfriizgens gas gangrene and myonecrosis (25,26). Since these infections are complex and may involve more than one bacterial species, it is difficult to associate in vivo effects with a single agent or toxin. The epitopes of a-toxin have been mapped (27). Theta-toxin, perfringolysin 0, may have a significant rolein wound infections caused by C. pelfiiugerzs type A. It is thio-activated, binds cholesterol. and fornls pores in susceptible cells (28). This cytolysitdhemolysin kills polymorphonuclear cells and inhibits chemotaxis (29,30). It may act by a colloid-osmotic mechanism (3 1) that separates it from other hemolysins of similar function but different mechanism (e.g., streptolysin 0 )(32j. The gene for 0 toxin codes for a 54,000 dalton protein (29,30). 2. Major Toxins from Other C. perfringens Types The damage caused by p-toxin may be a major component of the disease pig-bel, a necrotic enteritis of humans. Beta-toxin is elaborated during vegetative growth. Production is optimal if the pH of the medium is maintained at pH 7.5 (33) The toxin is inactivated by trypsin. A recombinant p-toxin was not toxic in mice and induced neutralizing antibodies against C. p e ~ f i i ~ g e ltype z s C-produced p-toxin (34). Epsilon- and delta-toxins are associated with gastrointestinal disease in livestock. Epsilon-toxin is the principal toxin of C. per$-irzgens type D, which is associated with enterotoxemias in ruminants. The mode of action of €-toxin is not known completely, but it increases intestinal permeability and enters the blood stream: from there, it binds to brain endothelial cells and increases blood pressure. It also binds to Henle's loops and convoluted tubules in the kidney (35). Epsilon-toxin is synthesized as a prototoxin and then cleaved (36); the h-toxin may be responsible for the cleavage (37). Delta (@-toxin, a lytic toxin of 42,000 daltons, is amphiphilic. Its lytic activity is inhibited by Gm2 gangliosides (38). As one of the major toxins produced by C. perfringens types B and C, it is associated with dysentery in lambs and enterotoxelnias for sheep, goats, calves, and pigs (17). Iota-toxin is produced only by C. perfringem type E. It shares homologies with other binary toxins with ADP-ribosylating activity similar to the binary toxin C2 produced by C. botrdirzurn (39-41). To date, no disease state has been linked with C. peIfiingem type E.

C. Growth and Survival

of Vegetative Cells

C. perj?ingens has been studied extensively for factors that affect growth, enzyme/toxin production, sporulation, and spore germination and has been the subject of a number of reviews (17.43). The following sections focus on those factors associated with growth and recovery of C. perfringem in foods. As with other foodborne pathogens, many factors interrelate to stimulate optimal growth. In addition, strain characteristics differ, especially as relates to sporulation. At optimal temperature, pH, and nutritional requirements, generation times usually are below 15 minutes. One of the shortest reported is for strain NCTC

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8328, which can double every 7.1 minutes at 41°C (43). Theshort generation times permit the bacteria to rapidly reach infective doses necessary for disease. 1. Nutritional Requirements A defined medium for C. per-ingem developed by Boyd et al. (44) contains 19 amino acids. Of these, 14 are essential for growth. The lysine requirement can be eliminated by the addition of high concentrations of pyridoxamine or pyridoxal. Addition of uracil and adenine accelerates growth. With these growth requirements, C. perfiingens does well in many foods, especially meat and poultry. Sterilized ground beef is an excellent growth medium and supports a-toxin production (45,46).

2. Temperature Reported optimal temperatures for C. yeqfiringeru replication range from 41 to 45°C. Most reported optima are between 43 and 45°C. Maximum temperatures for growth are reported at 50-51°C (17). Above 50°C, the death rate increases. Culture at high temperatures increases thermal resistance and decreases the death rate (43,47). Minimum temperatures for growth range from 14 to 18.5"C. Incubation of cells between 0 and 10°C results in rapid cell death (17,48). The rapid loss of viable cells at 1-4°C accounts for the difficulty in isolating organisms or obtaining accurate estimates of the number of organisms from many refrigerated foods. Cold shock, the transfer of cells from 37 to 4 ° C on logarithnlically growing cells kills 96% within 10 minutes. Cultures become less sensitive to cold shock as the population approaches stationary phase growth (48). Maximum and minimum temperatures and survival in cold and heat vary with pH and growth medium. Cultures in cooked meat medium survive longer at 4°C than cultures in thioglycollate medium (1 7). 3. Oxidation Reduction PotentiallOxygen Requirements C. yer:fringens cells do not initiate binary fission in an oxygenated environment. Culture media are routinely steamed prior to use to drive off dissolved oxygen. To maintain anaerobiasis during culture, the reducing agents thioglycollate, cysteine, and OxyraseR(Oxyrase Inc., Mansfield, OH) are often added to media. Low concentrations of agar in a liquid medium retard oxygen diffusion into the medium. Overlays of agar or paraffin mixtures also slow or prevent reoxygenation. Anaerobic chambers perfused with a variety of gas combinations support C. pe$-ingens growth. In spite of its sensitivity to oxygen, C. perfringens will grow over a wide range of oxidation reduction potentials (Eh). Growth between - 125 and +350 mV (millivolts) has been reported. Walden and Hentges examined growth of C. yetfringens from fecal isolates and determined that elimination of oxygen is more important than Eh (49). The organism lacks an electron transport system and is, therefore, insensitive to electron inhibitors such as sodium azide, At least one enzyme essential for growth is oxygen sensitive (50). 4. Water Activity Minimum water activity (alv)ranges from 0.97 to 0.93 depending on the solute used. When glucose is used to adjust a , ,the minimum a, for growth is 0.96 (51). Extremes in temperature and pH raise the tolerated a, (40). The rate of growth is also affected by the solute in the medium (52).

5. pH Optimal pH for growth is between 6.0 and 7.0. Below pH 5.5 growth is inhibited. Below pH 5 , the bacterial cells die in several days. Metabolizing cells dramatically alter the pH

144

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in culture. In poorly buffered, glucose-supplemented media, the bacteria produce acids and drop the pH from an initial pH 7 to below pH 5 in several hours. The low pH affects survival and diminishes toxin production (53,54). 6. Curing Salts Sodium nitrite and sodium chloride act together synergistically to inhibit C.yelfiingens growth. The sensitivity of C. pelfringens to this combination of curing salts helps to make cured meats a rare vehicle for C'. yegringem gastroenteritis (one case from 1978- 1982 in the United States) (17). Individually, 7 4 % sodium chloride and 300 pg/mL sodium nitrite inhibit growth. Sodium nitrite inhibitory concentrations vary depending on bacterial strain, preparation of the salt solution, pH, and number of bacteria (55). D. SporulationandEnterotoxinProduction 1. Sporulation and Its Relation to Gastroenteritis Only endospore-forming strains cause gastroenteritis. Endospores form during the first 6 hours of culture in a sporulation medium. Depending on nutrient supplements in the sporulation medium, vegetative growth may continue, but the replicating cells do not contribute to the number of spores (53). Free spores are released between 7 and 8 hours after culture initiation (56,571. Under anaerobic conditions, the maximum number of spores in DuncanStrong sporulation broth (58) or ground beef (59) is reached by 10-12 hours. Spore also will form under aerobic conditions, but the maximum number is reached in 24 hours (60). Although there is evidence that some vegetative cells produce low levels of enterotoxin (61-66) and that some strains sporulate well without enterotoxin production (67), the connection between spore formation and enterotoxin production is well established. Enterotoxin-negative spore-fonning strains are not associated with gastroenteritis. The enterotoxin appears to be a spore coat protein (68) and is detectable in sporulation cultures after 2 hours of culture (69). One spore-forming strain considered negative by biological assays was shown to produce about 1 ng/mL enterotoxin by a more sensitive enzyme immunoassay. One ng/mL may be below the concentration needed to cause gastroenteritis (64). The sporeformers associated with C. yelfiingens gastroenteritis are divided into heat-sensitive and heat-resistant strains. Most heat-resistant strains that sporulate well in Duncan-Strong medium produce high concentrations of enterotoxin. Heat-sensitive spore-fommers do not sporulate as well. Therefore, enterotoxin is not always detectable by reverse-phase passive latex agglutination assays (70). 2. Nutrient Requirements for Sporulation Detection of spore formation by C.ye?fringens is important for the diagnosis of C. perfiirzgens gastroenteritis. Many strains do not form spores well in culture. Several media have been developed that provide good sporulation from a number of strains (58,7174). No one medium supports spore formation in all strains. Duncan-Strong medium or modification of Duncan-Strong medium are the most widely used. The components for 100 mL of Duncan-Strong mediutn are protease peptone, 1.5 g; yeast extract, 0.4 g; soluble starch, 0.4 g; sodium thioglycollate, 0.1 g; and Na2HP04-7H20,1.O g (58). Ting and Fung (75) and Sacks and Thompson (76) have developed defined media for sporulation studies. One study indicated that mutants of C. perfkingens type A (NCTC 8798) sporulate well in the defined medium of Sacks and Thompson but not in Duncan-Strong broth (77).

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While many components affect sporulation, carbohydrate composition is one of the most important. Starch, dextrin, and raffinose are used as carbodenergy sources. Glucose or lactose added to Duncan-Strong medium inhibit sporogenesis (78). However, Decaudin and Tholozan (77) demonstrated the 11 tnMof glucose in D medium resulted in anincrease in the percentage of spore formed for two mutants of C. pelfringem NCTC 8798. Depression of thepH of themedium due to fermentation of sugars appears to cause the inhibition. Sacks et al.demonstrated that the methylxanthines caffeine and theophylline enhance sporulation (79). In addition, they suppress the growth rate of C. yerjkingens. Guanosine also enhances spore fortnation in some strains (54). Several foods support good sporulation of C.pelfringens. Kim et al. cotnpared sporulation in a sporulation broth, Noyes veal broth, pea puree, and tuna (80). The best sporulation occurred in the sporulation medium and veal broth, but the pea and tuna also supported sporulation. In turkey, chicken, and beef, sporulation occurs in 14-18 hours (60,81,82). 3. Other Environmental Requirements The optimal temperature for sporulation is 35-40°C. Kim et al. examined five strains of C. yerjkkgerzs for temperature effects (80). Two strains produced a few spores (2-37 spores/mL) at 5°C; all five strains produced spores (1-63 sporednll) at 22°C. At 37"C, the number of spores in the culture ranged from 5 1 to 12 X 10' spores/mL. Provision of exogenous, heat-resistant amylase permits sporulation at higher temperatures (83,84). The role of amylase in sporulation is still being studied. Although vegetative cells of C. yerfiingems produce several amylases, a significant increase in one amylase occurs during sporulation (85). Extracellular amylase produced by C. ye@ingens during sporulation is rapidly inactivated above 43°C (83). Acid conditions in sporulation media inhibit spore formation. The pH range for spore formation is pH 6.0-8.0 (17,56). Spore formation decreases as vegetative cells in culture ferment sugars to acids. However, a brief exposure of the cells to acid prior to inoculation into sporulation media may enhance sporulation (86). Sporulation requires a higher a, than vegetative cell growth. Kang et al. induced sporulation in Ellner's medium at different a,v adjusted with NaC1, glycerol, or sucrose (51). Spores were found in the glycerol adjusted medium at an a,v of 0.98. Few studies have examined the effect of oxidation/reduction potential and oxygen on sporulation. The Eh of a medium at -450 mV changed to -400 mV during sporulation. In ground beef, spores formed more rapidly under anaerobic conditions than aerobic conditions (60,82). 4. Regulation of Sporulation The sporulation gene map appears to be similar to mapped positions for sporulation genes of Bncillus subtilis (87). Like B. subtilis, sporulation gene expression appears regulated through the activity of sigma factors. Three promoters with similarity to sigK- and sigEdependent promoters have been described (88). Some antibiotics interfere with sporulation at distinct stages. The antibiotics netropsin and distamycin allow sporulation to initiate but block that process at stage 111 (89).

E. Germination Spores can be classified as either heat resistant or heat sensitive based on their ability to germinate following heating. Heat generally enhances the recovery of cells from spore

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preparations. Heat-resistant strains require heat activation at 75°C for 20 minutes before they germinate well. It should be noted that under those conditions heat-sensitive spores will not germinate (70). Heat (90), alcohol (91), and the chaotropic salt CaCI, (92) enhance gemination. The disruption of hydrophobic interactions in spore proteins is essential for spore activation and suggests that spore activation involves conformational changes in spore proteins. Addition of lysozyme to plating medium helps in recovely of heat-injured spores (93-95). During germination, two acid-soluble proteins in the spore rapidly degrade. The two proteins are of the a/b type. One has 59 anlino acids and the other 60 (96). Another feature of C. perfringens spore proteins is a high degree of glycosylation (97). Two spore lytic enzymes, an amidase and a muramidase, are located on the external coat of the spore (98100). The deduced amino acid sequence for the muramidase indicates that it is mature as formed. The amidase is synthesized as a prepro-enzyme. The proform is thought to be noncovalently attached to the coat and is activated during germination (98,101). Spore activation is affected by components in the medium. Heat-resistant spores germinate well in medium with KC1 and Tris buffer. Heat-sensitive spores germinate better in medium supplemented with alanine, inosine, CaCL, and Tris buffer (70). Some spores require lysozyme for activation. Spores may contaminate food and germinate in the food. One study examined the sporulation and outgrowth of C. pei$-ilzgens in ground beef. Spores germinated well. The resulting vegetative cells were readily killed by 60°C (102). F. Genetics 1. Genetics of C.perfringens Type A Toxins The enterotoxin produced by C. yerfr.ingens type A has only one serological type (103); cloning and hybridization studies have confirmed one, highly conserved gene (104-106). Van Damme-Jongsten et al. constructed four probes from the cloned gene (10). The probes detect the enterotoxin gene in many spore-forming isolates from food, fecal material, and soil. Probe hybridization with strains of known enterotoxin activity corresponded to enterotoxin production. Several isolates from food and fecal material associated with C. perfriizgens gastroenteritis did not hybridize with any of the probes, which implies that those isolates might not be responsible for the outbreaks. Expression is associated with sporulation. Enterotoxin-negative strains, transformed withthe enterotoxin gene, express the gene on sporulation (107). Three promoters lie upstream from the enterotoxin gene. P1 is similar to the sigK-dependent promoters, and P2 and P3 are similar to sigE-dependent pronloters active in the mother cell of sporulating B. subtilis (88). The phospholipase C or a-toxin gene (plc) has been cloned (19-21). The gene is expressed from a single promoter (22). Mutagenesis of the histidine-68 or -148 results in loss of activity (108). Gene expression is partially regulated through the virR/virS locus (109). Homologies with other bacterial phospholipase C genes are limited to the genus Bacillrrs. The gene for @toxin (PfoA) has also been cloned into E. coZi (29,30). Like the phospholipase C gene, &toxin expression is positively regulated through the virR/virS locus (1 09). Expression is also stimulated by the product of yfoR, which codes for aDNAbinding protein (1 10). A small (<2000 daltons) secreted product from %positive strains derepresses the 8 gene and allows some ‘%-negative” stains to produce the toxin (111).

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2.

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Plasmids

C. pelflingens has plasmids that code for bacteriocins (1 12.1 13) and antibiotic resistance. C. yer.fringens has attracted some attention as a potential host for cloned genes (1 14,115).

Recovery of lysozyme-treated cells is good. Of the cells converted to protoplasts by lysozyme treatment in an osmotic stabilizing medium, 5% are recoverable on regeneration medium supplemented with bovine serum albumin (116). Transformation of L-forms of C. pelfiirzgens has been moderately successful (1 15,117). Stable L-forms are produced in brain-heart infusion with 10% sucrose and 2 units of penicillin G per ml. Stable L-forms also accept shuttle plasmids designed to transform both E. coli and C. pelfiingerzs (118).

G.AntibioticSensitivities There is little interest in determining antibiotic sensitivity of food isolates or fecal isolates from cases of C. pe@ingens type A gastroenteritis due to the short-term nature, role of sporulation, enterotoxin release, and mildness of the disease. Antibiotic sensitivity of isolates from chickens and swine indicate an increase in resistance to antibiotics, particularly tetracycline and macrolides, which are common additives to animal feeds (1 19-121). Strains associated with gas gangrene and other wound infections are susceptible uniformly to benzylpenicillin, metronidazole, and rifampin (122,123). Susceptibility to other antibiotics varies. Traub cautioned that minimum inhibitory concentration (MIC) data is not an adequate predictor for good prognosis (124) due to inadequate drug accumulation in the wound. Protein inhibitors affect phospholipase C production, but benzylpenicillin does not (125). Resistance genes against tetracycline (126,127), erythromycin (128), and chloramphenicol (129,130) have been determined. Chloramphenicol resistance is conferred by two separate genes. One resistance gene, cutP, is located on a large plasmid and is carried on transposons (129). The second resistance gene, cntQ, appears to be located on the chromosome (1 30). The e m P and e m B genes for macrolide resistance have been cloned (128). Other resistance genes for the macrolides probably exist because only 5 of 40 resistant strains hybridized with e m P and e m B probes (128). Only one tetracycline resistance gene (tetp) has been identified (126,127).

111.

DISEASES

A.

Gastroenteritis

Hobbs et al. were the first to confirm C. perfringens as a foodborne pathogen by finding bacteria of the same serotype in both the contaminated food and fecal specimens from an outbreak of gastroenteritis (6). The symptoms associated with C. yetfiingerzs gastroenteritis are abdominal pain and diarrhea. Nausea is rare (6,8). Onset is usually within 18 hours of ingestion of large numbers of C. pelfringerzs type A vegetative cells. Recovery is usually within 12 hours of onset. The contaminated food is most often meat, poultry, and such associated material as gravy or stock ( 1,2). The food often is precooked the day before serving and rewarmed. During the time between cooking and serving, surviving spores or newly introduced organisms replicate to high numbers. In cases in which the suspect food is available for analysis, the major species isolated is C.yelfiilzgerzs.

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The actual incidence of C. perfringens foodborne gastroenteritis is not known. Todd estimated that most cases are not diagnosed and thus are not reported due to the mild nature of the disease (5). In the United States, outbreaks are common and most often are associated with mass catering (13 1- 133). The disease is not limited by age, sex, or other criteria. C. pelfii1?gens has also been associated with a chronic gastroenteritis in patients on extended antibiotic therapy (134,135). In this nonfoodborne gastroenteritis, the organism carries the ent gene on an episome rather than on the chromosome as found in the traditional foodborne cases (136).

B. RelatedDiseases C. pe$ringer?s type C is the etiological agent in a foodborne, hemorrhagic enteritis first described in Germany (14,15) and later in Papua New Guinea, where it is called pig-bel (16,137). The enteritis is accompanied by diarrhea and abdominal pain. The stools are bloody with mucus. Death due to the enteritis has been reported. The disease appears to be due to the activity of the p-toxin, which causes necrosis of the intestine. Enterotoxin has no noticeable role. Delta- and theta-toxins may also contribute to the pathology (138). The disease is sporadic and occurs in epidemics. A predisposing factor appears to be a low-protein diet (16). Epidemics of pig-bel occur when youths who normally consume low-protein foods partake in a feast of roast pig. The contaminated pig is the vehicle for the organism (16,137). C. perfrilzgens type A is isolated from a majority of anaerobic infections (139). Infections include cellulitis, myonecrosis, and gas gangrene. The source of the infecting organism usually cannot be determined, and introduction into the body usually follows trauma. However, non-trauma-associated C. pelfiingens infections also are seen in patients with malignancies (140). Several of the toxins, but not the enterotoxin, are responsible for bacterial spread and tissue destruction (25,141). Alpha- and theta-toxin cause the most damage. Bacterial toxins have been implicated in sudden infant death syndrome (SID). C. per-ingens was found in 45.5% of affected infants but only in 19.6% of healthy babies. Furthermore, the enterotoxin was detected in 34.4% of fecal extracts from SID cases but not from healthy babies. Alpha-toxin was also present in SID fecal extracts (17.5%); none was found in the control group. Although other bacterial toxins, from E. coli, Staphylococcus uureus, and Clostridium diifJicile, were found, C. pe$ringelzs had the highest incidence (1 1). In another study, greater than 80% of SID fecal specimens had C. perfEngerls type A. Less than 3% of control non-SID cases were positive (142). IV. EPIDEMIOLOGY

A.

Human-to-HumanSpread

Humans appear to be a common source for many C. perfringens outbreaks. The contaminant often is introduced into a food during preparation (2). Adult levels of C. perfringens in fecal material are established by 6 months of age (17). Of populations studied, 1-52% of the individuals possessed heat-resistant spores in their feces (17,143,144). Heat-resistant spores commonly are associated with diarrheal disease, especially in Great Britain (7). In the United States, outbreaks have been associated with both heat-resistant and heat-sensi-

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tive spores of C. perfringens. When heat-sensitive spores are included in incidence studies from human fecal material, close to 100% of adults humans are positive for C. perfringens type A (17,145). However, at least one report indicates that vegetarians may lack C. perfringens (146j. The high incidence of C. perfringens in normal flora compounds the problems associated with outbreak confirmation in that positive cultures will not necessarily indicate the cause of the diarrhea. Detection of enterotoxin from isolates is necessary for confirmation. Spore counts of greater than 10" per g of fecal material are also needed for disease confirmation (147- 149).

B. Animal-to-HumanSpread C. perfringens type A is a ubiquitous organism and has been isolated from soil, animals, spices and herbs, dehydrated foods, and human fecal material ( l 19,120,150-153). Organisms associated with soil and dried foods tend to be in spore form. Whether isolates from all potential sources of C. yerfriMgerzs cause diarrhea in humans is not known. Fish harbor bacterial flora containing C. p e $ - i n g e n stype A but have not been associated with C, per$-ingens gastroenteritis. The prevalence of C.per$-ingens on pork, beef, and poultry and the number of outbreaks associated with these foods indicate that animals are a potential source of enterotoxin-producing strains. Pets may harbor the organisms (1 54). Examination of pigs with diarrhea revealed that the enterotoxin gene was episomal rather than chromosomal as typical of foodborne outbreaks (155). Meats that require extra handling in preparation are more likely to be contaminated with C. pelfringerzs than foods that require little handling (59,156), which implies that humans are the most common reservoir for human disease. The foods most likely to be contaminated with C. perfringens are beef, pork, lamb, chicken, and turkey (150153,157). Stews and other casserole-style foods made with meat products also provide an excellent growth environment for C. perfringem. Meat products in the United States are more likely to be contaminated with heat-sensitive spores than meat products in Great Britain (59). Cured meats, though, are poor vehicles for C. pelfiingerzs. In many outbreaks suspected of being caused by C. perfringens, the food is not available for analysis or has been stored improperly for assessment of C. pe$rirzgens (17). One hospital outbreak in Japan implicated a crab salad as the vehicle (l%), but the origin of the contamination was not determined. The bacterial source for domestic animals may be their feeds. Many chicken feeds and soy products are contaminated with C. perfringem (159,160). Soy meals also support bacterial growth, and the addition of soy meals to meat products may aid bacterial replication (45,46,16lj.

V.

ISOLATIONANDIDENTIFICATION

A.

PlatingTechniques

1. Isolation Isolation of C. perfringens from mixed cultures is relatively easy based on several characteristics: the ability to grow at 46"C, resistance to specific antibiotics, the ability to reduce sulfite to sulfide, and anaerobic metabolism. The difficulty in isolation lies in the number of

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other bacteria that contaminate the specimens, especially fecal specimens. Media selection depends of whether the specimen is fecal or food. C. perjhkgens requires an anaerobic environment for growth. C. perj?ingens may be isolated if oxygen concentrations are low and sufficient bacterial load is present to reduce the oxygen and permit the anaerobe to grow. However, if the number of C. perfiingens is low, then it is necessary to establish anaerobiasis in order to enrich for the organism. If sulfite reduction is to be used to enhance colony visualization, anaerobiasis also must be established (149). A variety of gases can be used to establish an anaerobic environment. Nitrogen, nitrogen-hydrogen, or nitrogen-hydrogen-carbon dioxide mixtures all permit growth. Hydrogen-carbon dioxide-generating systems that deplete oxygen (e.g., the GasPak anaerobic system [BBL, Becton Dickinson Microbiology Systems, Cockeysville, MD]) work well (17). Alternatively, oxygen scavengers, such as OxyeraseTM,can be added tothe media. A typical isolation medium for suspected C. perjl-ingem in meat and poultry products contains, tryptone, sulfite, and cycloserine (TSC) (see Table 3) and is supplemented with egg yolk to permit detection of a-toxin-producing strains (149,162). A variation of this medium is made without egg yolk (163) and is preferred at times when weak a-toxin producers are suspected. While most commercial media support the growth of pure cultures of C. per-ingem, they do not necessarily do as well for the organism in mixed cultures. Isolation of C. pe1;fiingelzsfrom chicken intestines on four different media demonstrated that sulfite-polymyxin-sulfadiazine nledium (164), tryptose-sulfite-neotnycin medium, Clostrisel (BBL), and lecithin-lactose agar were inadequate for good isolation (165). Another medium that contains oleandomycin, polymyxin, and sulfadiazine (OPSO) (see Table 3) is good for the selection of C. perj?ingens, but OPSO also supports fecal enterococcal growth (149). McClung-Toabe agar with added egg yolk also has been used for isolation of C. perfringens from foods (Difco, Ann Arbor, MI). Antibiotic addition to media is helpful in selecting for C. yeqringens. Cycloserine is added at 400 pg/mL final concentration. In OSPS, oleandomycin phosphate (Pfizer) and polymyxin B sulfate are added to give final concentration of 0.5 yg/mL oleandomycin phosphate and 10 IU/mL polymyxin B sulfate. Neomycin can be used with blood agar by spreading 0.06 mL of a 1% (w/v) sulfate over the plate (149). Due to C. perfi*ingens’optimal growth temperatures, the effectiveness of isolation can be improved by culture at 46°C rather than 35-37°C. However, media composition

Table 3 Media for Isolation _____

Tryptose (Difco) Soytone (Difco) Yeast extract Sodium metabisulfite Ferric ammonium citrate Ag‘W Source: Ref. 139.

15 15 5 1 1 20

For TSC: Cycloserine Egg yolk (optional)

400.5 pg/mL 0.08 mL/mL

For OSPS: Oleandomycin phosphate Polymixin B sulfate

0.05 Ing/InL 10 IU/mL

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affects culture growth at high temperatures. Marshall et al. demonstrated that C. perfiingerzs has difficulty growing at 46°C when cultured on tryptone-sulfite-neomycin agar (166j. Fecal specimens with spores may needto be heated to activate the spores. To activate spores, the fecal specimen typically is heated for 10 minutes at 80"C, which activates heat-resistant spores. Heat-resistant spores can survive heating at 100°C for 60 minutes (17). Heat-sensitive spores do not require heat for germination and may be killed during the 80°C incubation used to activate heat-resistant spores (167). Heating the specimen is also useful for decreasing the number of other species in the specimen.

2. Enumeration Enumeration of vegetative cells and spores aids in confirmation of C. pel-j'ritzgensas the cause of an outbreak. In suspect foods, high numbers (> 10"g food) of C. petfrirzgem suggest it as the cause of gastroenteritis (168,169). High numbers of spores also should be found in fecal specimens collected within 24 hours of symptom onset. Colony enumeration is performed by either tube methods or plate methods. Tube methods have the advantage of not requiring special anaerobic incubation chambers. A disadvantage of tube methods is that egg yolk cannot be incorporated into the agar for detection of a-toxin production. Typically, 150 X 19 mm tubes are filled with a selective and differential medium (e.g., TSC) and inoculated with dilutions of the fecal specimen or suspect food. To preserve anaerobiasis, the inoculated medium is topped with sterile medium or paraffin (149). An opaque white bar inserted in the tube at the time of inoculation enhances visibility of the colonies (170). Ali and Fung developed a double-tube assay for C. per-j'rirzgerzsenumeration (171). This entails insertion of an empty, thin, sterile test tube into a larger screw cap tube that contains the melted, inoculated TSC agar. The advantage of this method is that isolated colonies can be selected from the agar medium after removal of the thin inner tube. For plate counts, sealed pour plates or anaerobic culture chambers are necessary. With sealed pour plates, 14 mL of medium are inoculated with the sample and poured into a deep petri dish. This is covered by 10 mL of freshly molten medium. Reducing agents in the medium help to maintain anaerobiasis (149). Traditional spread plates on the appropriate medium will support growth when placed in anaerobic chambers such as the GasPak system or anaerobic incubators with the appropriate gas mixture. Alternatively, addition of the supplement Oxyrase permits incubation without specialized equipment. Although C. perjringens is relatively aerotolerant for an anaerobe, some organisms may be lost during plating and during the time needed to establish anaerobic conditions. Comparison of five media for recovery of heat-resistant spores indicates that TSC agar or Shahidi-Ferguson perfringens agar are better than tryptone-sulfite agar, sulfitepolymyxin-sulfadiazine agar, and tryptone-sulfite-neomycin agar (172). Plating on an antibiotic-containing blood agar has been reported as helpful in the isolation of heat-sensitive, spore-forming C. pe$&gens. Sutton and Hobbs demonstrated a correlation between hemolytic activity and heat sensitivity (167). Low numbers of C. per-j'rirzgem in foods are difficult to detect. Enrichment cultures coupled to most probable number (MPN) determinations can be used but are considered inaccurate and may represent the activity of nonclostridial species (173,174). Selective agents in themedium and incubation at 46°C improve recovery and aidpresumptive identification (148). Erickson and Deibel developed a rapid perfringens medium (RPM) that contains litmus plus supplement nutrients, polymyxin B, and neomycin sulfate (175). Iron-

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milk medium, a mix of whole milk and iron powder, also has been used for MPN enumeration of presumptive C. pel@ingerzs contamination of foods (176,177). When meat samples have low numbers of C. perfiiqerw, the organism has been enriched by placing the meat in vacuum packaging and incubating at 43-45°C. The gas produced by the organisms swells the bag, and drippings from the bag can be cultured for isolation (149). All methods used to detect C. perfringens need to be supplemented with additional tests for species confirmation and for enterotoxin production. B. BiochemicaVPhysiological Tests TSC and other selective media permit the growth of other clostridial species; therefore, species identification is necessary. C. perfringem has the following characteristics for identification: nonmotile, reduces nitrate, liquefies gelatin, and ferments glucose. Presumptive identification can be made based on “stormy fermentation” in iron-milk medium. Another characteristic is the ability to ferment raffinose but not salicin. C. perjhhgerzs has all the enzymes of the Embden-Meyerhof pathway for glucose degradation. It lacks glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (50). Alpha-toxin production can aid in identification. Bacteria growing on egg yolk agar and producing a-toxin will be surrounded by a zone of opalescence (149). Addition of anti-a-toxin to the medium will prevent the opalescence. Some strains associated with foodborne illness are weak producers of a-toxin. An enzyme immunoassay to detect a toxin is sensitive to 16 ng/mL (178). Rapid tests and multiple test strips with a number of biochemical tests in one system have become popular in laboratories for anaerobic, wound isolate identifications (179182). With these systems, C. perfrilzgens is identified with great reliability. The Vitek ANPMcard (Vitek Systems, bioMerieux, Hazelwood, MO) was used to correctly identify eight of eight strains of C. perfringens. The Patho-Tec (General Diagnostics, Cincinnati, OH), API ZYMTM(API Systems SA, La Balme les Grottes, Montalieu, Vericieu, France), and RapID ANA I1 (Innovative Diagnostic Systems, Atlanta, GA) correctly identified seven of seven. An-Ident (Analytab Products, Plainview, NY) also correctly identified known strains of C. perfkingens (179).

C. Other Tests Polymerase chain reactions (PCRs) using primers specific for C. per-ingens permit sensitive detection of the organism in a variety of specimens and give typing results similar to more classical methods (183,184). When coupled with electrophoresis, PCRs with a combination of primers can be used to look for the a-toxin, P-toxin, etoxin, and enterotoxin simultaneously. Alpha-toxin genes are detectable from direct enrichments of intestinal contents (185). One PCR reaction had a lower detection limit of 5 X 10’ bacteria from fecal specimens (186). Bacteriocin typing also may behelpful in identification of bacterial isolates responsible for outbreaks. Watson et al. typed 31 1 strains by bacteriocin patterns (187). Of the enterotoxin-producing strains, 92% were typeable. Serological typing initially was used to demonstrate that C. perfiirzgens was the cause of foodborne diarrhea (168,188). It still provides one of the best ways to determine if the organism found in the suspected food is the same as that isolated from fecal material and thus the agent of disease. Unfortunately, a significant number of isolates are still

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untypeable. One study found 24% of the isolates could not be typed by a panel of 75 antisera (188). Phage typing has not been adapted for use with C. perj%?gerzs. Yan used hostmodified phage to develop a typing scheme for C. peffringens isolated from human fecal material and other clinical specimens (189). Nine phage groups were used to define 10 typeable groups of C. pe@-ingens. Of the isolated organisms, 39% were not typeable by this method. Food isolates were not tested, and no correlation with enterotoxin production was made. Gas chromatography profiles of the fatty acid products of anaerobes are helpful in identifying some Clostridium species, but not C. yerjringens, which produces both acetic and butyric acid. Since C. pelfringens dies rapidly in cold storage, food samples may not yield high enough numbers to implicate it in a foodborne outbreak. Furthermore, the presence of carbohydrates in food may inhibit toxin production (46,54,76). Thus, direct toxin detection from foods may be unreliable.

D. Transport of Specimens Culture of C. yeifringens should be performed as rapidly as possible. Storage of specimens at 4°C can result in a loss of organisms as some die rapidly at that temperature. If plating cannot be immediate, then the food sample can be mixed 1:l (wthol)with 20% glycerol and stored on dry ice or at - 60°C (163).

VI.

ENTEROTOXINPATHOGENICITY

A.

FeedingStudies

The association of C. pelfkiizgens with gastroenteritis was not confirmed until 1953 when it was observed that both the associated food and the patients stools had high number of C. pelfiirtger~s.Of human volunteers fed 5.1 X lo*to 3 X 1O9 viable cells, 16 or l 8 became sick within 48 hours (8). Fecal specimens from the volunteers had high concentrations of spores. There is good correlation between the ability to cause gastroenteritis and positive rabbit ileal loop tests for enterotoxin (190). Ingestion of enterotoxin does not appear to play a major role in the disease. Eight to 10 mg of ingested toxin causes diarrhea in healthy adults (191j. Effectiveness of the toxin alone to cause disease is enhanced if gastric acids are buffered. Monkeys fed sodium bicarbonate developed gastroenteritis when fed 5 mg (192). Enterotoxin injected into rats or mice accumulates in the liver and kidneys, preferentially binding and killing parenchymal cells (193,194).

B. BiochemistryandMode of Action The enterotoxin of C. peffringerzs appears to be unique among the many bacterial enterotoxins. The single, 35,317 dalton polypeptide with 3 19 amino acids is produced in large quantities during sporulation and is released as free enterotoxin into culture medium concurrent with the release of spores from vegetative cells. The enterotoxin’s function in sporulation is not yet known. Hybridization studies using gene probes for the enterotoxin and numerous isolates of C. pelfringens reveal that only a small percentage of isolates

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have the enterotoxin gene (10) and that the protein is probably not essential to sporulation because there are spore-positive, enterotoxin-negative strains. The native protein is active. However, trypsinization increases activity threefold (195). The toxin binds to a 50,000 dalton protein in brush border membranes but is not enzymatic in activity. It forms a 160,000 dalton complex in the membrane with two membrane proteins of 50,000 and 7,000 daltons (196). Binding and cytotoxic activity are on different fragments of the toxin. Horiguchi et al. isolated a fragment that inhibits enterotoxin binding but has no cytotoxic activity (197). Binding of the toxin can be inhibited by monoclonal antibodies (198,199). Once bound, the toxin inserts into the membrane and remains associated with the membrane (200,301). A portion of the toxin remains on the external surface of the membrane (202). The result of the toxin’s presence is an increased permeability of the cell membrane to small molecules (203,204). The toxin creates ion-permeable “gates” in asolectin bilayers. The gates which are dynamic and bi-directional permit passage of molecules under 200 daltons (205j. Nucleotides and amino acids move through the gates and lead to cell death (206210). Addition of Ca+: may stabilize and enlarge the hole to permit small polypeptide movement (211,212). Calcium ions may not be necessary for cytotoxicity, McClane and Wnek examined both binding and permeability changes and concluded that there were two phases of enterotoxin activity (209). The first phase occurs during the first 7.5-8 minutes, is Ca+’ independent, and includes binding, insertion, complex formation, ion permeability, and inhibition of macromolecular synthesis. The second phase is Ca+?dependent and is accompanied by morphological damage to the cells. Permeability continues to increase, and the injured cells eventually lyse. In the intestines, cell death leads to desquamation of the villi, loss of fluid, and diarrhea.

C. Production Enterotoxin production is associated with sporulation frequency. Strains with a low frequency of sporulation in vitro tend to produce low concentrations of enterotoxin. Carbohydrates have a profound effect on sporulation. Many monosaccharides inhibit sporulation, while starch enhances it (54). A number of xanthines, such as caffeine, also enhance sporulation (79). Enterotoxin has a positive effect on its own production and enhances sporulation. Enterotoxin added to cultures decreased the lag phase, enhanced sporulation, and stimulated macromolecular synthesis in preexponential phase cells (213). Many sporulation media have been developed to enhance sporulation (61,69,7 173,75). Duncan-Strong medium continues to be the most widely used (61). Sporulation frequency in Duncan-Strong ranges from less than 0.1% to 90% (214). D medium is a defined sporulation and is used extensively in studies on enterotoxin production (76). All sporulation media need a complex carbohydrate such as starch or dextrin or the complex sugar raffinose. D. Purification The purification protocol of McDonel and McClane is used widely (214j. Preparation of the enterotoxin begins with an inoculum of a 9-hour culture of C. yerfi-ingem grown in fluid thioglycollate medium transferred to 20 L of fresh Duncan-Strong medium cooled

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155

to 37°C. For best production, the Duncan-Strong culture should be incubated until 80% of the vegetative cells form endospores. The bacterial cells are collected in a cold potassium phosphate buffer (pH 6.8) and sonicated to release the enterotoxin. Supernatant is collected by repeated centrifugation at 4°C. Enterotoxin in the supernatant precipitates on adjustment to 40% saturated ammonium sulfate at pH 7. Purification is enhanced by a second precipitation at 15% saturation with ammonium sulfate. The second precipitate then is resuspended in a sucrose buffer, and final purification is achieved on a Sephacryl 200TM column (Pharmacia LKB Biotechnology, Uppsala, Sweden). The fractions eluted off the column should be assessed for enterotoxin activity. The enterotoxin routinely is found in the second protein peak (214). A similar method is reported by Heredia et al. (215), with Duncan-Strong medium supplemented with raffinose. Other purification methods were described by Granum and Whitaker (69), Reynolds et al. (216,217), Dasgupta and Pariza (218), and Sakaguchi et al. (219). Purity is assessed by polyacrylamide gel electrophoresis. Satellite bands occasionally are seen and appear to be due to an endogenous protease (220).

E. Enterotoxin Assays Three basic groups of assays are used to identify or quantify C. yerjYingens enterotoxin: biological, serological, and tissue culture. DNA probes can be used to detect the presence of the enterotoxin gene (10). 1. Biological Assays Biological assays are some of the least sensitive assays to detect enterotoxin but have been useful in studies on mechanisms. The earliest assays were ileal loop assays. Rabbit, chicken, sheep, monkey, mouse, and calf intestinal loops have been used (17,190). LDS,,, mouse diarrheal, and rabbit and guinea pig erythemic reactions also have been used. Excised brush border membranes from the small intestine of rabbits have been used to examine the binding of radiolabeled enterotoxin (196,209).

2. Serological Assays Serological assays to detect enterotoxin have a long history (208,210,221-225). The most traditional serological tests are immunoprecipitation reactions in gels. In these assays, antibodies against the enterotoxin bind to the enterotoxin, forming a complex that precipitates. The most sensitive precipitations are counterimmunoelectroprecipitations (CIEPs), in which antiserum and enterotoxin are electrophoresed in a gel, and electroimmunoprecipitation (also known as rocket immunoelectrophoresis), in which the antiserum is in a gel and the enterotoxin is electrophoresed (223,226). The advantage of rocket immunoprecipitations is that the enterotoxin can be quantified based on the rocket height, which is proportional to the concentration of the enterotoxin. Both assays require a thin gel of buffered agar and predetermined optimal concentration of anti-enterotoxin antiserum (210). Enzyme immunoassays (EIAs), originally called enzyme-linked immunosorbent assays, have been modified both to detect and to quantify enterotoxin (208,224). Detection of 5 ng/g fecal material is possible. The absorbance for the samples can be compared to absorbance for known enterotoxin standards for quantitation (227). Enterotoxin inoculated into fecal samples has been detected at 1 ng/mL using an EIA (228,229).

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A variation on the liquid EIA is the DOT-EIA performed on nitrocellulose paper instead of microtiter plates. Mehta et al. diluted enterotoxin containing fecal samples and transferred 2 pL to nitrocellulose (230). The blots were visualized by treating with rabbit anti-enterotoxin, then anti-rabbit Ig conjugated with horseradish peroxidase, followed by the chromogen 3,3'-diaminobenzedine tetrahydrochloride. Reverse passive hemagglutination reaction with anti-enterotoxin antibodies fixed to human erythrocytes also detects enterotoxin in fecal extract (9). Reverse passive latex agglutination has also been reported (222). Unipath (Hampshire, England) markets a kit for perfringens enterotoxin reverse-phase latex agglutination (OxoidTM PET-RPLA). Sensitivity is approximately 2 ng/mL enterotoxin, and it is comparable to an EIA test for enterotoxin in fecal specimens (231). Other serological reactions have proved useful: single and double diffusion in gels, radioimmunoassay, and fluorescent antibody staining. EIA, radioimmunoassay, and reverse-phase assays are the most sensitive. The assays are less sensitive when the enterotoxin is in a biological specimen (e.g., fecal material) (208). 3. Tissue Culture Assays Tissue culture assays have used a variety of cell types. One of the more sensitive cell types is the Vero cell line. McClane and McDonel monitored enterotoxin by Vero cell assays based on changes in cellular morphology, cell detachment, decreased viability, plating efficiency, and alteration in cellular biochemical activity (207). These assays rely on microscopic observation; other studies have used "Rb efflux or neutral dye uptake to monitor enterotoxin damage to Vero cells (209,210,232).

F. DNA Probes of the Enterotoxin Gene Confirmation of a C. perj?ingerzs isolate as the cause of a gastroenteritis is enhanced if the isolate produces enterotoxin. Confirmation has been difficult since many strains do not sporulate well in vitro and therefore do not produce detectable enterotoxin. The use of DNA probes to detect the enterotoxin gene appears to be a useful alternative. They are sensitive and avoid some of the problems associated with enterotoxin detection in biologic a1 materials. Van Damme-Jongsten et al. cloned and sequenced the enterotoxin gene (erzt) (104) and constructed j'P-labeled probes. The probes hybridized with DNA from C. perfi-irzgens isolated from food and fecal material collected during confirmed outbreaks. The probe also detected the gene in one outbreak-associated strain that did not produce enterotoxin in culture. Comparisons of Western immunoblotting for the enterotoxin and PCRs to detect the gene further indicate the inability of some erzt gene-positive isolates to sporulate and produce the enterotoxin in culture (233). PCR reactions on beef samples could detect less than 10 colony-forming units per gram (234). The enterotoxin probes have indicated that the enterotoxin may have a major importance only in human disease. Isolates from sheep with C. pe$+lgens-induced diarrhea did not have the enterotoxin gene (10,235).

VII.

PREVENTION

Prevention of C. yegringens gastroenteritis depends on the elimination of spores from food or halting contamination during food processing and handling. Proper cooling of prepared foods and sufficient reheating to kill vegetative cells also will aid in prevention.

Clostridium perfringens

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Heat killing of spores can be difficult (93). Heat-resistant spores can survive heating at 80°C for over 1 hour. Heat-resistant spores have Dgs values ranging from 17.6 to 64 minutes (70). Viable spores of C. ye~fringensNCTC 8798 were recovered after 10 minutes at 105°C. At 120"C, 0.01% of this strain survived for 20 seconds (172). Heat-sensitive spores have Dgsvalues between 1.3 and 2.8 minutes (70). Thus, elimination of contamination by good hygiene and high-quality food sources becomes the best means to prevent infection.

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222. Harmon, S. M., and Kautter, D. A. (1986). Evaluation of a reversed passive latex agglutination test kit for Clostridium pe$ringens enterotoxin. J. Food Prot.. 49523-536. 223. Naik, S.. and Duncan, C. L. (1997). Rapid detection and quantitation of Clostridium perfr-ingeru enterotoxin by counterimmunoelectrophoresis. Appl. Empiron.Microbiol., 34: 125128. 224. Narayan, K. G.. Genigeorgis, C., and Behymer, D. (1984). Use of enzyme-linked immunosorbent assay (ELISA) in the quantitation of Clostridizlnz pe$ringens type A enterotoxin and antienterotoxin antibodies. Int. J. Zoorz., 10:105-1 10. 225. Uenlura, T., Sakaguchi, G., and Riemann, H. P. (1973). In vitro production of Clostridizrnz petfiingerzs enterotoxin by reversed passive hemagglutination. Appl.Microbiol., 26:381385. 226. Holliday. M. G. (1985). Rapid identification of Clostridium perfringeitsby counter-immunoelectrophoresis. Med. Lab. Sci., 42:322-325. 227. Bartholomew. B. A., Stringer, M. F., Watson, G. N., and Gilbert, R. J. (1985). Development and application of an enzyme linked immunosorbent assay for Clostr-idiunz per-ji-irzgens type A enterotoxin. J. Clin. Pnthol., 38:222-228. 328. Olsvik, O., Granum, P. E., and Berdal, B. P. (1982). Detection of Clostridim pe$ringens type A enterotoxin by ELISA. Acta Pcuhol. Microbiol. Immmol. Scnrzd., Sect. B, 90:445447. 229. Jackson, S. G., Yip-Chuck, D. A., and Brodsky. M. H. (1986). Evaluation of the diagnostic application of an enzyme immunoassay for Clostridium pe$r-ingens type A enterotoxin. Appl. Environ. Microbiol., 52969-970. 230. Mehta. R., Narayan, K. G., and Notermans, S. (1989). DOT-enzyme linked immunosorbent assay for detection of Clostricliurnperfringens type A enterotoxin. Int. J. Food Microbiol., 9:45-50. 231. Berry, P. R., Stringer, M. F., and Uemura, T. (1986). Comparison of latex agglutination and ELISA for the detection of Clostridium perfrirlgens type A enterotoxin in faeces. Lett. Appl. Microbiol., 2: 101-102. 232. Mahony, D. E., Gilliatt, E.. Dawson, S., Stockdale, E., and Lee, S. H. S. (1989). Vero cell assay for rapid detection of Clostridium ye@-ingewsenterotoxin. Appl. Erwiron. Microbiol., 552141-2143. 233. Kokai-Kun, J. F., Songer, J. G., Czeczulin, J. R., Chen, F., and McClane, B. A. (1993). Comparison of Western immunoblots and gene detection assays for identification of potentially enterotoxigenic isolates of Clostridium perfringens.J . dirt. Microbiol., 322533-3539. 234. Baez, L. A., and Juneja. V. K. (1995). Detection of enterotoxigenic Clostridium pe$ringens in raw beef by polymerase chain reaction. J. Food Prot., 58:154-159. 235. Van Damme-Jongsten, M., Haagsma, J., and Notemans, S. (1990). Testing strains of Clostridiiun? pe$ringerzs type A isolated from diarrhoeic piglets for the presence of the enterotoxin gene. Vet. Rec., 126:19 l - 192.

9 Escherichia coli Marguerite A. Neil1 Brown Universiq School of Medicine. Providence, Rhode Islarld

Phillip 1. Tarr University of Washingtoil and Children's Hospital ami Medical Center, Seattle. Washingtoll

David N. Taylor and Marcia Wolf Walter Reed Army Institute of Research, Washington, D.C.

I. Introduction

170

11. Enteropathogenic E. coli

170

A. Historical aspects 170 B. Characteristics of EPEC 171 Clinical C. manifestations 172 D. Epidemiology 172 E. Isolation and identification 173 F. Pathogenicity 173

111. Enterotoxigenic E. coli

175

Historical aspects 175 B. Characteristics of the ETEC 176 C. Characteristics of disease D. Epidemiology 177 E. Pathogenicity 178 IV. Enteroinvasive E. coli 180 A.

A. B. C. D. E.

Historical aspects 180 Characteristics of disease Epidemiology 181 Identification 181 Pathogenicity 182

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180

V. E. coli 0157:H7 and Enterohemorrhagic E. coli

182

A. Historical aspects 182 B. Characteristics of E. coli 0157:H7 and EHEC C. Characteristics of disease 184 D. Epidemiology 186 E. Isolation and identification 186 F. Pathogenicity 187

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7 70 VI. Enteroaggregative

E. coli

A. Historical aspects 189 B. Characteristics of the EAEC C. Clinical manifestations 191 Epidemiology D. E. Pathogenicity 19 1

189 190 190

VIT. Role of Food andWaterinTransmissionofDiarrheagenic VIII. Isolation

from Foods

E. coli

191

194

References 196

1.

INTRODUCTION

Over 100 years have elapsed since the original description of Bacterium coli cornnrzme by a German pediatrician, Dr. Theodore Escherich, in 1885 (l). His useofthesuffix “commune” recognized that this gram-negative organism, today known as Escherichia coli, was widely distributed in the intestinal flora of humans and warm-blooded animals. E. coli is the predominant facultative anaerobe in the human bowel and helps to maintain normal physiological function of the intestine (2). It was precisely this widespread and apparent commensal relationship between bacterium and host that obscured for many years the role of E. coli in causing enteric disease. When serological characterization was able to incriminate certain strains in the etiology of diarrheal illness, the species was finally recognized to be heterogeneous, containing both pathogenic strains and harmless inhabitants of the gastrointestinal tract. Previously, the diamheagenic E. coli were divided into four major categories: (a) the enteropathogenic E. coli (EPEC), (bjthe enterotoxigenic E. coli (ETEC), (cj the enteroinvasive E. coli (EIEC), and (d) the enterohemorrhagic E. coli (EHEC). Within each category, strains share distinct, category-specific virulence traits that are often plasmid encoded, common epidemiological and clinical features, and a similar pathophysiological mechanism of action. A fifth category is constituted by the enteroaggregative E. coli (EAEC), a recently recognized diarrheagenic E. coli. This group is composed of strains that have been epidemiologically linked to diarrheal disease but lack properties typical of the first four groups and possess a particular adherence phenotype. Another category of potentially diarrheagenic E. coli is the diffusely adherent E. coli (DAEC); because epidemiological and pathogenic features of the DAEC are less well understood, this group will not be discussed here. The five major categories of the diarrheagenic E. coli are the subject of this review.

II. ENTEROPATHOGENIC E. COLI

A.

Historical Aspects

The power of epidemiology to lay the groundwork for the discovery of an etiological agent is elegantly illustrated in the story of how EPEC came to be recognized as causes of infantile diarrhea (3,4). Pediatric diarrhea in the late 1700s was characterized by Dr.

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Benjamin Rush as a severe diarrheal illness occurring mostly in urban areas during the summer months (5), and from these observations came the terms “cholera infantum” and ‘‘summer diarrhea.’ ’ Even as epidemiological studies developed the corollary observations that disease was most severe in those less than 2 years old and those not breast-fed, mortality from childhood diarrhea fell dramatically in the United States and Great Britain. Explosive outbreaks in hospital nurseries during the 1930s were accompanied by high mortality rates and focused attention on the outbreaks’ likely infectious, but as yet undiscovered, causative agent. The association linking the bacterium E. coli with infantile diarrhea was made by Dr. John Bray, a British physician, who used his nose as well as his intellect in his investigations. Dr. Bray recognized that certain strains of E. coli from the stool cultures of children with diarrhea had a peculiar seminal odor, and that this same odor had been ascribed to the patients themselves by a pediatrician, Dr. Thomas E. D. Beavan, at the Hillingdon Hospital in Uxbridge, England. The antiserum prepared by Bray using these strains as an immunogen in a pet rabbit named Snowy agglutinated strains of E. coli in the stools of 95% of 50 infants with diarrhea and almost none (4%) of control E. coli from healthy children (6). The subsequent development of the Kauffman-White serotyping system, based on bacterial somatic (0),flagellar (H), and capsular (K) antigens (7j, was instrumental in proving that the hospital nursery outbreaks were related to specific serotypes. Studies of experimental infection helped to strengthen the epidemiological and serological association of these E. coli strains with diarrhea and convince the remaining skeptics that at least some serotypes of this normal inhabitant of the gastrointestinal tract could cause diarrhea, albeit mechanism unknown. In 1955, Neter (8) captured the diarrheal potential of these strains in his proposal of the term “enteropathogenic” E. coli (EPEC) for those serotypes with definite epidemiological association with childhood diarrhea. The recognition and definitionof the enterotoxigenic and enteroinvasive E. coli led to a renewal of skepticism that EPEC strains were pathogens, because the EPEC strains lacked the diarrheal mechanisms of the ETEC and EIEC and had no alternate explanation for their capability to cause diarrhea. Studies in volunteers by Levine et al. (9) demonstrated that EPEC strains lacking toxin production or invasiveness could cause diarrhea in humans. EPEC are now accepted as a distinct group of diarrheagenic E. coli, and considerable advances have been made in our understanding of the molecular pathogenesis of diarrhea from these strains.

B.Characteristics

of EPEC

1. Nomenclature The definition of EPEC strains has evolved over the past 40 years. Initially EPEC were defined solely on the basis of possession of certain somatic 0 antigens. This serogroupbased definition changed to a serotype-based categorization when EPEC were associated with particular O:H types. EPEC were subsequently shown to produce a characteristic histopathological finding, the attaching-effacing (A/E) lesion, which is now recognized as a hallmark of both their identification and their diarrheagenic properties. Other strains of E. coli, particularly those that produce Shiga toxin (Stx), may also produce A/E lesions. The current working definition formulated at the Second International Symposium on EPEC is that EPEC strains produce A/E lesions and lack the ability to produce Stx (10). Most EPEC by this definition cluster within certain O:H serotypes, but serotype alone is no longer the basis for definition of EPEC.

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Occasionally the tern1 “enteropathogenic” E. coli is used in a more broad fashion to denote any strain of E. coli that causes diarrhea. Such usage, while perhaps philosophically appealing, dilutes rather than enhances our conceptual understanding of the diarrheagenic E. coli and should be discouraged.

2. Serogroups Fifteen 0 serogroups had been described previously as including members of the EPEC: 018ab, 018ac, 026, 044, 055, 086, 0111, 0 1 14, 0119, 0125, 0126. 0127, 0128ab, 0142, and 0158 (1 1-14). Serotyping using the H antigen has shown that strains of different serotypes within the same 0 serogroup do not have equal pathogenic potential or occurrence in disease. Some serogroups, such as 055, are so rarely found in healthy persons that detection of this serogroup in a child with diarrhea may make further serotyping unnecessary as the strain is highly likely to be pathogenic. Other serogroups, such as 086, can occur commonly in normal bowel flora so detection of an 0 8 6 strain in a child with diarrhea necessitates further serotyping to determine if it falls into the one serotype (086: H34) associated with diarrheal disease anlong the 21 known serotypes in serogroup 086. Serological differentiation alone is insufficient to fully characterize the pathogenicity of an individual isolate and additional testing is required for classification into one of the groups of the diarrheagenic E. coli. Based on the current definition of EPEC and including genotypic characterization, those serotypes that characteristically contain EPEC include 055:H6,05S:NM, 086:H34, 0 1 11:H2. 011 1:H12, 0 1 11:NM, 0 1 19:H6, 0 1 19:NM, 0125ac:H21, 0126:H27, 0126: NM, 0127:H6, 0127:NM. 0128:H2, 0128:H12, and 0142:H6 (15). C. Clinical Manifestations There are no major distinguishing features of the diarrhea due to EPEC infection that can be considered pathognomonic. Typically the illness is an acute watery diarrhea with lowgrade fever and vomiting occurring in young infants (9,16). Severe disease resulting in death or prolonged diarrhea ( B 14 days) is not uncommon in young children, especially those less than 6 months of age (16- 19). In a study of community-acquired gastroenteritis, hospitalized children with illness attributed to EPEC had diarrhea for an average of 4.6 days compared to 2.4 days for children with nonbacterial gastroenteritis (20). These findings on duration and severity support the observation that the diarrhea due to EPEC infection may be more severe than that due to the other common causes of pediatric diarrhea such as rotavirus. The lack of symptomatic infection in older children and adults coupled with the not infrequent finding of such persons asymptomatically excreting EPEC strains (21,22) has led to the concept that imnlunity is acquired. However, EPEC are not common causes of traveler’s diarrhea even in geographic areas with a high incidence of EPEC, which may suggest that immunity is developmentally regulated. D. Epidemiology EPEC have been reported as a cause of childhood diarrhea from around the world (17,20,22,23), predominantly in children less than 2 years of age. Case-control studies comparing fecal isolation of EPEC with disease status have shown the most significant correlation of EPEC isolation and diarrheal illness in infants younger than 6 months (19). Both colostrum and breast milk have been shown to have both specific and nonspecific

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factors that inhibit EPEC adherence to HEp-2 cells (24) and breast feeding is protective against EPEC infection. Bottle feeding has been noted as a significant risk factor for EPEC infection. Explosive outbreaks in the community and in hospital nurseries were typical of the epidemiology of EPEC in Europe and North America through the 1950s, and mortality ranged up to 50% (19). These have largely disappeared, and this has been ascribed to improvements in sanitation and hygiene. In the developed world the current epidemiology of EPEC includes their implication as a cause of day-care center and occasional nursery outbreaks and community-acquired sporadic infantile diarrhea. Child care settings in the United States have been recognized as sites for EPEC-associated outbreaks (25,26), and chronic diarrhea was a prominent feature in these outbreaks. A recent study suggests that the frequency of EPEC as a cause of childhood diarrhea in the United States may be considerably underestimated (27). Although rare, outbreaks of EPEC infection in adults have been noted (28,29). The largest burden of disease due to EPEC is in the developing world. In some studies, EPEC accounted for one third of all infant diarrhea and rivaled rotavirus for the most common cause of diarrhea in the first year of life. The physical environment (linen, toys, scales, carriages, bathing area, dust) around an infant with EPEC diarrhea has been shown to be extensively contaminated (30). The route of transmission is likely a fecaloral one from contaminated foods or formula, from the contaminated hands of caregivers, or from soiled fomites. Respiratory transmission has also been postulated (30,31).

E. IsolationandIdentification EPEC have the same colony morphology on culture media and similar biochemical reactions as other E. coli. The definitive identification of EPEC-associated enteric disease is not possible with the diagnostic tools available in the average clinical laboratory. Serogrouping and serotyping are relatively uncomplicated but labor-intensive techniques, which were previously used as the basis to identify EPEC, but some strains have been described that do not fall into one of the classic EPEC serogroups (32). The current approach to identifying EPEC is to determine the adherence characteristics of the bacterial isolate and the absence of Shiga toxin production. Such testing may be performed either phenotypically or genotypically, with the latter based on the use of DNA probes and/or PCR assays. The A/E phenotype is demonstrated using the fluorescent actin stain (FAS) (33,34); this correlates with possession of e m gene sequences found within the locus of enterocyte effacement (LEE) pathogenicity island. Localized adherence (LA) to HEp-2 cells has generally correlated with the presence of the EAF plasmid and hfpA sequences (see below). Typical EPEC strains possess eae sequences and the EAF plasmid but do not produce Shiga toxin. Strains that lack the EAF plasmid but that otherwise have the key properties of EPEC (A/E phenotype and absence of Shiga toxin) are considered atypical EPEC. The intricacies of these diagnostic considerations have been recently reviewed in detail (15). F. Pathogenicity Despite the description of EPEC as the first recognized class of diarrheagenic E. coli, until recently our understanding of the sequence of events by which EPEC cause enteric disease has remained in a fairly primitive state in comparison to our understanding of the

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mechanisms of disease involved i n ETEC and EIEC infection. Recent reviews superbly summarize the explosion of work in this area (1 5,35). A distinct histopathological picture associated with human EPEC infection was first described by Polotsky et al. (36) in which the bacteria were observed to be very closely adherent to the epithelial cells and the surrounding microvilli were destroyed. Moon et al. applied the ternl, “attaching and effacing’’ first used by Staley et al. in 1969 (37), to describe the epithelial cell loss and cytoplasmic degeneration seen in the intestines of newborn pigs exposed to EPEC (38). Knutton st al. showed that EPEC attachment and effacement were associated with the accunlulation of actin within cultured cells at the point of EPEC attachment (33). Several other proteins of the cellular cytoskeleton have also been shown to accumulate beneath attaching and effacing EPEC strains (39,40). Studies on the molecular basis for the A/E lesion have now localized the genetic sequences for this distinctive histopathology to a large 35 kb chromosomal region called the LEE (41 ). Within the LEE are genes encoding several secreted proteins (espA, B, D), which appear to be involved in signal transduction and for which actual binding of the bacterium to the epithelial cell surface is necessary for them to effect signaling changes. Also contained in the LEE is the ecle sequence (E. coli attaching and effacing), which encodes intinin (42). This approximately 94 kb protein is involved in the intitnate adherence of bacteria to the cell surface and has 50% similarity to the invasin protein of Yersirzin. Another characteristic determinant in EPEC strains is their ability to adhere in localized microcolonies to epithelial cells in vitro (43). This feature is distinct from the diffuse adherence (or even nonadherence) seen with strains of ETEC, EIEC, or uropathogenic E. coli. This localized pattern of adherence was noted to be associated first in EPEC strain 0127:H6 with a 60 MDa plasmid designated pMAR2 (44) and subsequently in other EPEC strains with plasmids in the 55 to 70 MDa size range (45). These plasmids, termed “EPEC adherence factor” (EAF) plasmids, were noted to have a high degrees of homology at the DNA level. The basis for the bacterium-to-bacterium adherence in the localized adherence pattern is the bundle-forming pilus (BFP) (46), which has some sequence similarity to the toxinco-regulated pilus of Vibrio choler-ne.Whether BFP mediates bacterial adherence to the epithelial cell is unknown. A DNA probe was developed from a region of pMAR2 which was necessary for the localized adherence phenotype (47), and the EAF DNA probe has been utilized extensively in recent epidemiological studies detecting and characterizing EPEC (48-50). The importance of the 55 MDa plasmid in influencing both adherence phenotype and diarrheagenic potential can be seen in the following lines of evidence: first, nonadherent nonpathogenic laboratory strains of E. coli acquire the localized adherence trait with transfer of the plasmid; second, loss of the plasmid was correlated with loss of the capacity to exhibit localized adherence by several EPEC strains (51); and third, volunteers fed the EAF plasmid-cured strains had a much lower frequency of diarrhea than those receiving the parental EPEC strains that retained the plasmid (52). Not all EPEC strains hybridize with the EAF probe, although they have been implicated as diarrheal pathogens from epidenliological and volunteer studies and cause similar histopathological findings as EAF-positive strains. However, they have been given the designation atypical EPEC to distinguish them from the more common EAF probe-positive typical EPEC (50). Earlier descriptive studies of EPEC infection had shown intracellular bacteria by electron microscopy, suggesting that theorganisms could be invasive. However, the inability of EPEC strains to cause kerato-conjunctivitis in the guinea pig eye (Sereny test) and their lack of the very high molecular weight (140 MDaj plasmid of SkigeZZn and EIEC,

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which is associated with invasiveness, was taken as evidence that EPEC are not invasive. Revisiting the earlier observation of the intracellular location of EPEC, several investigators have recently shown that epithelial cells of various lineages can be invaded by EPEC strains (53,54) and the EAF plasmid increases invasion. How these events at the cellular level relate to the pathogenesis of EPEC diarrhea is not yet understood. The genetic basis of EPEC virulence, reviewed by Donnenberg and Kaper ( 3 3 , suggests a possible three-stage model for infection and disease production. Bacterial adherence to nlicrovilli is accomplished by a specific protein; subsequently the BFP allows bacteria to adhere to each other, contributing to the localized microcolonies seen in adherence studies. Both plasmid and chromosomal genes are required for this step. Secreted proteins contribute to signal transduction, with phosphorylation of several proteins involved in ion transport. Elevation of intracellular calcium levels is accompanied by microvillus effacement. Alteration of the cytoskeleton follows as the organisms adhere intimately and characteristic cuplike pedestals form. The epithelial cell may then be invaded by some of the closely associated surface EPEC organisms (35).

111.

ENTEROTOXIGENIC E. COLI

A.

HistoricalAspects

After EPEC, the second major group of the diarrheagenic E. coli to be recognized as a distinct pathogenic entity were the ETEC. A seminal observation was made by De et al. in the recognition of the similarity of findings induced in ligated ileal loops inoculated with either V. choleme or some serogroups of E. coli (55). Following De’s discovery of cholera toxinin 1959 (56), Taylor and Bettelheim noted that chloroform treatment of culture fluids of E. coli did not destroy their capacity to induce fluid secretion in the intestine, thus suggesting that enterotoxin(s) were made by these strains (57). The 1960s and 1970s saw the discovery of the enterotoxins, the formulation of a working definition of ETEC, and recognition of the profound importance of ETEC as causative agents of diarrhea in various settings. Smith and Gyles reported that two enterotoxins produced by E. coli could be distinguished on the basis of differential heat lability, a distinction that is reflected in the nomenclature of heat-labile toxin (LT) and heat-stable toxin (ST) (58). Gorbach et al. laid the groundwork for the appreciation of ETEC as important causes of human diarrhea in the tropics (59), while the further characterization of these strains by Sack validated their enlarging role as diarrheal pathogens (60). As noted in the section on EPEC, this body of work defining a basis for diarrhea from ETEC infection had the effect of casting doubt on the true pathogenicity of the EPEC, for which no mechanism had been defined. ETEC subsequently were shown to account for considerable proportions of infantile diarrhea in the developing world (61-65) in contrast to their relative unimportance in pediatric diarrhea in the developed world. Traveler’s diarrhea, a disorder of many sobriquets (“turista,” “Delhi belly,” “Rome runs,” “Aztec two-step.” “back door sprint,” “Montezuma’s revenge,” etc.), was added to the roster of settings in which ETEC were important etiological agents (66,67). ETEC also have been shown to cause diarrhea in military troops going into areas of lower hygiene and sanitation (68,69), including during the recent conflict in the Persian Gulf (70). Although generally uncommon in the United States, an outbreak of ETEC in the summer of 1998 in Chicago caused illness in more than 4000 people (71).

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B. Characteristics of the ETEC 1. Nomenclature By definition, ETEC strains produce either LT, ST, or both. However, enterotoxin production is not the sole determinant of pathogenicity; it also requires the presence of certain attachment factors called CAFs that permit adherence of bacteria to the human small intestine (see below). Depending on their complement of these attachment factors, ETEC may be pathogens of animals or humans or, rarely, both. 2. Serogroups Over 75 0 serogroups have been detected on ETEC strains, although half of the serogroups occurred only once (72). The most common ETEC serogroups are 06, 08, 025, 027, 078, 0128, 0153, 0148, and 0159 (72) (Table l). 0 serotyping is useful to identify ETEC causing outbreaks, but in endemic settings it isnot useful to identify ETEC because of the large proportion of ETEC with rare 0 serogroups. Further, ETEC 0 serogroups may overlap with nontoxigenic E. coli that commonly cause urinary tract infections or meningitis. However, 0 serogroup determination is useful for characterization of ETEC in endemic settings once they have been identified by other means. Over 30 H serogroups have been detected in ETEC. The most common are H9, H12, H16, H21, and H45. The role of flagella in ETEC pathogenesis is not clear (73), although by analogy to cholera it might be expected to play a role. Table 1 shows the association of H serotypes commonly associated with other factors. Attachment factors known as colonization factor antigens (CFA) can be considered a third serogroup. CFA/I, CFA/II, and CFA/IV have been found to be the most common (72) (Table l), but in some studies they occur on less than 30% of the ETEC (74-76). The terminology is somewhat confusing, because CFA/II may contain CS3, CS 1 and CS3, or CS2 and CS3. CFA/IV may contain CS6, CS4 and CS6, or CS5 and CS6. CS 1, CS2, CS3, CS4, CS5, and CS6 are distinct structures on the surface of bacteria. Attempts to find another common CFA have not been fruitful, although a number of CFA have been studied such as CS17, PCF020, PCFO159, PCFO166, and longus. To date, longus seems the most likely to be a common CFA (77), but more data are needed to assess these CFAs. The groundwork for an ETEC vaccine based on CFA types will require the combined epidemiological and molecular characterization of these antigens and continued determination of their relative distribution among the ETEC serogroups important in human disease.

Table 1 Common ETEC Phenotypes Toxin

Serotype 06:H16 08:H9 08:H9 027:H7 078:H12 0128:H12 0148:H28 0153:H45 0169:H-

CFA LTST LTST LT ST ST or LTST LTST ST ST ST

CFA/II (CS1 and CS3 or CS2 and CS3) CFA/II (CS3 alone) CS 17 CFA/IV (CS6 alone) CFA/I CFA/I CFA/IV (CS6 alone) CFA/I CFA/IV (CS6 alone)

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ETEC are common in environments with poor sanitation, and the common 0 and H serogroups and CFAs are all widespread throughout the developing world. In developed countries where ETEC infections rarely occur and no immunity exists among inhabitants, food and waterborne outbreaks of ETEC occur. In developing countries where ETEC is endemic and immunity to a wide variety of serotypes is acquired in early childhood, outbreaks rarely occur. The wide variety of serotypes increases the complexity of efforts to identify common antigens to include in vaccines against ETEC and to exploit for diagnostic assays.

C.Characteristics

of Disease

A noninflammatory diarrhea accompanied by nausea, mild to moderate abdominal cramping, and little if any fever is the typical illness produced by ETEC infection. Blood usually is not present inthe stool, and fecal leukocytes are absent. The diarrhea often starts abruptly 1-3 days after ingestion of ETEC. Because the mechanism of diarrhea production is so similar to that in V. cholerae infection, in its most severe form ETEC infection resembles cholera with severe dehydrating dian-hea and attendant high mortality. The course is somewhat variable depending upon the setting in which infection occurred. For most individuals with traveler's diarrhea, the illness is mild and usually lasts less than a week, but 5-10% may last longer. Regardless of the length of illness, the greatest impact is on the traveler's schedule (70,78). Under certain circumstances, as in military campaigns, even a mild illness may have dire consequences if a large proportion of the population is affected and is unable to perform scheduled duties. Because children are more sensitive to dehydration, ETEC infection can be severe in children (14,79). Frequent episodes of diarrheal disease can contribute significantly to malnutrition (62,80). Unlike EPEC, ETEC infections occur in nonimmune adults (travelers) as well as children. Fecal shedding of ETEC, either from symptomatic or asymptomatic infections, allows for continued transmission in areas of poor sanitation.

D. Epidemiology ETEC are not recognized as causative agents of diarrhea in Europe and North America (8 1,82), although occasional ETEC outbreaks have occurred (7 1,83-86). In the developing world ETEC is a major cause of childhood diarrhea. The attack rate increases with weaning, probably on a multifactorial basis that includes the withdrawal of factors protective against diarrhea found in breast milk, greater exposure to contaminated foods and water, and the decline in nutritional status that often accompanies weaning. Children in the developing world may sustain multiple clinically significant ETEC infections in the first 5 years of life as well as numerous asymptomatic infections (63,64,87). Although the attack rate declines after age 5, isolation of ETEC from asymptomatic people continues frequently, implying the development of immunity. Volunteer studies have shown that the inoculum required to produce illness with ETEC strains is fairly high, 108-10Joorganisms (88-91), an inoculum easily obtainable in unrefrigerated foods or contaminated water. Diminished host defenses such as decreased gastric acidity may allow illness to develop after receiving a smaller inoculum. For travelers, the risk of developing a diarrheal illness climbs proportionately with the length of stay in the geographic locale, with a 50% risk of illness being reached by

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a stay of 14 days. Studies have noted that the tnajority (20-75%) of traveler’s diarrhea is due to ETEC (70,92,93). The risk of ETEC infection appears to be highest with travel to Latin America and Africa, followed by travel to Asia (93). In contrast to people in endemic areas, asymptomatic infections with ETEC are infrequent in travelers.

E. Pathogenicity 1. Enterotoxins Two distinct enterotoxins, LT and ST (94), are the defining feature of ETEC strains. They may occur together or individually, and both cause secretory diarrhea. The names derive from their differing stability to heat: LT is a heat-labile toxin (inactivated after heating at 60°C for 30 minutes) and ST is stable after heating. A third enterotoxin called EAST has been found in some but not all ETEC strains (95). Evidence suggests that EAST alone tnay have pathogenic potential (29,96); it therefore may be a cofactor in ETEC. More data is needed to clarify the role of EAST in diarrheal disease. LT is a family of enterotoxins of varying similarity to each other and to cholera toxin (CT) of V. cholerue. The LT-I group includes LT of human origin (LTh) and porcine origin (LTp). LTh and LTp are very closely related to cholera toxin; they have 80% homology of amino acid sequences, the satne mechanism of action, and are neutralized by antibody to cholera toxin (94,97). Genes for both LTh and LTp are plasmid encoded. The LT-I1 group seems not to contribute to diarrhea in humans. LT holotoxin consists of one catalytically active A subunit and five B subunits, the latter being responsible for binding to a particular intestinal cell surface molecule, ganglioside GM,. Following binding to the GM, receptor, the A subunit of LT is internalized and enzymatically cleaved into the A1 and A2 subunits. The A1 subunit causes ADPribosylation of a regulatory protein, causing irreversible activation of adenylate cyclase. The resultant intracellular accumulation of cyclic adenosine monophosphate (CAMP)activates a protein kinase, causing absorption of sodium and chloride and secretion of chloride. Additional mechanisms may play a role in causing diarrhea. The watery diarrhea that results contains significant amounts of electrolytes. The discovery that neither CT nor LT impaired the transport of sodium from the intestinal lumen across the epithelial cells via a glucose and amino acid coupled pathway formed the basis for oral rehydration therapy (ORT) for diarrhea. ST differs from LT in size, structure, antigenicity, and tnechanism of action. ST resists heat inactivation, retaining activity even after boiling for 30 minutes (58), probably due to its heavy internal cross-linking by disulfide bonds. Two distinct forms of ST have been described: STa (also known as STA,STI, and ST1) and STb (also known as STII, STB, and ST2). Both are low molecular weight proteins that neither elicit an antibody response after natural infection nor are neutralized by antibody to LT or CT. STa is important in hutnan infection, whereas STb has been detected primarily in porcine strains. In contrast to the mechanism of LT, STa produces its effects immediately but irreversibly through the stimulation of guanylate cyclase with subsequent cyclic guanosine monophosphate (cGMP) accumulation (94,98j. The lack of commercial assays has limited the diagnosis of ETEC, but a GM,-based enzyme itnmunoassay (GM, ELISA), gene detection by hybridization, and PCR are assays in use in research settings. The detection of LT historically has been in tissue culture assays using Y-l adrenal cells or Chinese hamster ovary (CHO) cells, bothofwhich

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exhibit characteristic morphological changes following exposure to the toxin. Knowledge of the binding specificity of LT led to the development of GM, ELISA (99). Contrary to the relative ease with which LT can be detected, ST has not been detectable by tissue culture using nonintestinal cells. A suckling mouse model has been used for the detection of STa (98). After overcoming the lack of antigenicity of native ST, several ELISA methods have been developed (100- 102), including one that utilizes a monoclonal antibody (103). Specific DNA probes and primers now permit detection of LT and ST by hybridization (64,65,104-109) or PCR (107,110-113). ETEC strains may produce either LT or ST alone or in combination, and the genes for expression of these toxins are plasmid mediated. In many ETEC strains, one plasmid may carry genes for LT, ST, and a CFA. The distribution of toxin profiles among ETEC varies somewhat according to geographic region. In Thailand, Chile, and Mexico, colonization with strains expressing ST and LTST were associated with diarrhea (32,64,65), but more recently in Egypt, colonization with strains expressing only ST were associated with diarrhea (63). ETEC expressing only LT (which frequently lack CFAs) are isolated as frequently from symptomatic as asymptomatic volunteers. 2. Colonization Factors As noted above, enterotoxin production is one of two critical virulence determinants for enteric disease caused by ETEC; the other is possession of colonization factor antigens (CFAs) that permit the organism to attach to enterocytes in the proximal small intestine. The tissue specificity of the enterotoxins and the inefficient release of toxins requires that the bacteria be delivered at close range and attachment to the epithelial cell surface is the means to achieve this end. There has been intense interest in CFAs because of their potential development as vaccine components. CFA are long structures of repeated protein subunits on the surface of bacteria that promote attachment to receptors on epithelial cells of the intestine (1 14,115). As such they are fimbriae (also called pili) that are specific to ETEC. Electron micrographs show them as hairlike appendages projecting outward from all over the bacterial surface. Their structure somewhat resembles the flagellar antigens, but the CFA structures are much thinner and assembly mechanisms are much simpler than flagella. The repeating protein subunits are arranged in either a rigid structure 6-7 nm in diameter or a wiry, flexible structure that is thinner (2-4nm in diameter). Each structure may have thousands of subunits and reach lengths equal to the length of the bacterium. The CFA proteins are produced in culture at 37°C but not 18°C. The DNA sequences of many of these CFAs have been determined. Several ETEC colonization factor antigens have been described (1 14.1 15). CFA/I, CFA/II, and CFA/IV are the common colonization factors (see Table 1 and above). The nomenclature is inconsistent-those discovered after CFA/IV are named CS or PCF, and both CFA/II and CFA/IV are heterogeneous groups comprised of three distinct antigens; CFA/II expresses CS3 alone or in combination with CS l or CS2. CFA/IV expresses CS6 alone, but may express CS6 in combination with CS4 or CS6. To date, a predominant CFA has been described for all the main O:H serotypes of ETEC (Table l), but many ETEC isolates have been identified that do not contain any of the known CFAs, especially ETEC expressing LT alone, implying that many other CFA types may exist. The delineation of the full complement of CFA types and an understanding of their distribution among ETEC strains will determine the polyvalency of future ETEC vaccines based on colonization factors.

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CFAs exhibit striking host specificity, accounting in part for the lack of pathogenicity of many human ETEC strains in animals and vice versa. CFAs are plasmid encoded, often on the same plasmid as ST and LT, and are often associated with certain serotypes (Table 1) (72). The association of CFAs with diarrhea has been demonstrated a number of ways. In a study in Egypt, ETEC with CFAs were isolated more frequently from children with diarrhea than from controls, but ETEC without CFAs were isolated from both groups of children (63). Volunteer studies have shown that those receiving isogenic mutants of an ETEC strain lacking CFA/I did not develop diarrhea, while volunteers challenged with the parent strain did (89). In another study, volunteers immunized with an oral ETEC strain lacking LT and ST but containing the genetic sequence for its CFA developed much milder diarrhea and at a much lower rate compared to unimmunized controls when both were challenged with the parent strain (90). The most direct demonstration of the importance of CFAs to illness was a demonstration of passive protection from diarrhea by antibody to CFA/I (91).

IV.ENTEROINVASIVE

A.

E. COLI

Historical Aspects

EIEC are pathogenic E. coli that derive their virulence from their ability to invade epithelial cells. Not surprisingly, they originally were mistaken for Shigella, with which they share many biochemical phenotypes. The first association of EIEC with human disease was by Ewing and Gravatti in 1946 (1 16). These investigators analyzed a large sample of presumptive Shigellae from Allied soldiers serving in the Mediterranean during World War I1 who had received medical attention for dysentery. While most of the isolates were confirmed to be Shigellcre, a few strains were not easy to speciate but were described as “Shigella-like” because of their biochemical characteristics. In 1947, several dozen British school children were stricken with acute gastroenteritis, becoming manifest 14-24 hours after a common midday meal (1 17). Symptoms included fever, diarrhea, and vomiting. Dysentery was not noted. A “paracolon bacillus” was isolated from the stool cultures of the affected children, but not from any of the leftover food from the incriminated lunch. When fed to two human volunteers, an illness with symptoms resembling those observed in the children ensued. Subsequent serological and bacteriological assays demonstrated that the Shigella-like organisms of Ewing and Gravatti and the paracolon bacillus of Hobbs et al. were, in fact, E. coli of serotype 0124 (1 18).

B. Characteristics of Disease EIEC have a predilection for the colon, where they presumably exert their pathogenic effect by invasion. However, it is unknown if microscopic abnormalities are present in the small bowel, which would not be unexpected in view of the prominent vomiting from which many of the patients suffer. Despite their colonic venue and the invasive propensity of EIEC, dysentery seems to occur in only a small minority of patients in outbreaks (117,119). EIEC infection often begins with nonbloody diarrhea, which can be voluminous (120) and which is accompanied by abdominal pain, fever, and vomiting. Stools then

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become scanty, as is typical in dysentery due to Shigella. Most patients with EIEC diarrhea recover within 2 days without specific therapy. However, fluoroquinolones (121) in adults and ceftibuten and trimethoprim-sulfamethoxazole in children (122) have been used as therapy. The incubation period in outbreaks has been between 2 and 48 hours, with a median of 18 hours (117,119).

C. Epidemiology The epidemiology of sporadic cases of EIEC diarrhea and of less dramatic outbreaks than the ones described by Hobbs et al. ( l 17) and Marier et al. (1 19) is unknown. No doubt such situations exist (123- 126), and asymptomatic carriage can occur (127). The difficulty in determining the epidemiology of EIEC in general populations is the difficulty in identifying this organism in most clinical laboratories without an outbreak to prompt a more thorough investigation. EIEC in epidemics probably are foodborne, but few definite suspected vehicles have been implicated on microbiological and epidemiological grounds, because few outbreaks have been thoroughly investigated. The vehicles that have been identified include imported (to the United States) French cheese (1 19), old tofu (128), and guacamole (129). EIEC are a cause of endemic dysentery, albeit less frequently than Shigella, in developing parts of the world (130,13 1). EIEC have their highest age-specific incidence in children 3-5 years old (120) and are occasional causes of traveler’s diarrhea (132- 135). Person-to-person transmission also has been reported to occur (136).

D. Identification Like Shigella, EIEC are lysine decarboxylase negative (137) and are nonmotile. Early studies of EIEC strains, detected by differential biochemical reactions, suggested that most do not ferment lactose. More recent studies of EIEC using DNA probes as the format for detection have shown that the majority of isolates were lactose positive (120). No single biochemical test can be relied upon to identify EIEC specifically, and hence either lactosepositive or lactose-negative colonies that are confirmed to be E. coli and not Salmonella or Shigellu are EIEC candidates. Such strains can be typed in a reference laboratory to determine if they are in the classic EIEC serogroups (028ac, 029, 0112ac, 0124, 0136, 0143, 0144, 0152, 0164, and 0167) (138,139). EIEC serogroups do not overlap with those in the EPEC and ETEC. However, serogrouping may be complicated by the close antigenic relatedness of the 0 antigens of EIEC and Shigella. It is quite probable that EIEC not infrequently are misidentified as Shigella, an attribution that would not be considered aberrant clinically because of the similar clinical manifestations of EIEC infection and shigellosis. Invasion of cultured epithelial cells or positivity in the Sereny test (140) are the gold standards for confirmation of pathogenicity but are not practical for clinical laboratories. By the time these tests can be performed, the patient from whom a suspect EIEC strain was isolated usually has recovered. Serogroup 0167 does appear to produce both heat-stable and heat-labile enterotoxins (139). Despite the similarity of EIEC to Shigella, these strains have not been shown to produce Shiga toxin (Stx) I. or I1 (141), and they do not hybridize with a DNA probe that detects the gene sequences for these toxins (142).

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Cloned segments of the large, 140 MDa plasmid of EIEC have been used as a diagnostic probe (130,143), and polymerase chain reaction (PCR) amplification of other itlvasion-related sequences also has been described (144). Additionally, enzyme immunoassays that detect EIEC-specific secreted products, such as invasion-associated antigens (145,146), can be used toidentify these pathogens. These techniques are particularly valuable when seeking EIEC that do not ferment lactose and which would be overlooked on a standard stool culture. E. Pathogenicity As described above, the cardinal virulence trait of EIEC is the ability to invade epithelial cells. This trait is correlated with the presence of a large (140 MDa) plasmid in EIEC that also is found in Shigellcr (147). Many restriction endonuclease sites in these plasmids are not conserved, but the large plasmids nevertheless have considerable DNA homology to each other (147). Plasmidless EIEC do not invade, but the large plasmid of S. Jlemeri restores invasiveness (148). Much research has focused on the genetic structure and gene products of the 140 MDa plasmid in S. Jlexneri and, by extension, to EIEC (149). At least five genes spanning a distance of 20 kilobase pairs (kbp) appear necessary for the invasive phenotype (150). The four invasion plasmid antigens IpaA, IpaB, IpaC, and IpaD are encoded on the 140 MDa plasmid and are highly immunogenic. Mutations in ipaB, i p C , and i p D inhibit invasion. Recently, a provocative paper proposed that absence of loci in EIEC that encode proteins that might attenuate bacterial virulence (i.e., cadA, which encodes cadaverine) contributes to the pathogenicity of EIEC and Shigella (151). In addition to the Ipa gene products, a distant plasmid locus, \irF, encodes a gene product that is a positive regulator of ipuA-D (152). Also, virR, a chromosomal gene, can act in trans to suppress Shigella invasion (153). Additional putative virulence factors of EIEC include an exoribonuclease (Rnase R) (154) and a plasmid-encoded enterotoxin (155).

V.

E. coli 0157:H7 AND ENTEROHEMORRHAGIC E. COL/

A.

Historical Aspects

The known diarrheagenic groups of E. coli added a new member in 1982 with the discovery of E. coli serotype 0157:H7. This pathogen was the causative agent of two outbreaks of severe bloody diarrhea in Oregon and Michigan in whichtransmission occurred through consumption of contaminated fast-food hamburgers (156). Serotype 0157:H7 was considered rare because at the time only one prior isolation had been made in the United States, that being in 1975 from a woman in California with bloody diarrhea. Only six isolates had been noted in Canada from 1978- 1982 among more than 2000 E. coli isolates screened for enterotoxin and cytotoxin production (157). The term hemorrhagic colitis was used to describe the illness associated with E. coli 0157:H7 infection, and the term enterohemorrhagic E. coli (EHEC) evolved to designate serotype 0157:H7 and other serotypes that produce a similar clinical picture (4,158). In the decade since its discovery, E. coli 0157: H7 has been isolated with such an appreciable frequency in outbreaks of bloody diarrhea and in sporadic community-acquired diarrhea in western Europe and North and South America that it is no longer considered a "rare" serotype.

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A commonly asked question is whether E. coli 0157:H7 is truly a new (or newly emergent) pathogen or whether it simply has been present but notrecognized as a causative agent of disease. The retrospective assessment of large strain collections for this pathogen showed that it was veryinfrequently found among strains submitted for serotyping through the 1970s in the United States, Canada, and the United Kingdom (156,157,159). Population-based studies over the past 2 decades have suggested that the incidence of the hemolytic uremic syndrome (HUS) has been increasing, and HUS is now a well-recognized complication of E. coli 0157:H7 infection (see below). Taken together, these data along with evolutionary genetic analysis of E. coli 0157:H7 (160) suggest that this pathogen is both a new and an increasingly frequent causative agent of human illness. B. Characteristics of E. coli 0157:H7 and EHEC 1. MicrobiologicalFeatures There are no pathognomonic morphological features that distinguish serotype 0157:H7 or other EHEC serotypes from normal bowel flora cultured on solid media. All EHEC ferment lactose and have other biochemical reactions typical of E. coli (161). One distinguishing feature of serotype 0157:H7 is its lack of rapid (<48 h) fermentation of sorbitol. This has formed the basis of the most common screening procedure for this organism because 93% of E. coli isolated from humans do ferment sorbitol ( l 61,163). However, recently sorbitol-fermenting isolates of E. coli 0157:H7 have been reported (163,164). Serotype 0157:H7 also does not possess glucuronidase activity and, hence, cannot hydrolyze 4-methylumbelliferone glucuronide (MUG) to a fluorogenic product; it is therefore MUG negative (165,166). However, MUG positive variants have been isolated (163,164,167). Serotype 0157:H7 has been shown to grow well in broth media within the usual laboratory temperature range of 30-42°C and it survives freezing in ground beef quite well (168). At temperatures above 44-45°C serotype 0157:H7 grows poorly and as these temperatures are often used for the detection of E. coli in food samples, such conditions probably will negatively impact on the recovery of this serotype from food (168). Antibiotic susceptibility patterns of E. coli 0157:H7 isolates both from outbreaks and from sporadic cases had indicated that they are only rarely resistant and, even then, to very few antibiotics (169). This has seemed curious given the purported bovine source of these strains with transmission to humans via the food chain; this lack of resistance in E. coli 0157:H7 does not parallel the observations that have been made with salmonella in which an increasing frequency of multiple drug resistance has been observed (170). However, a multiply resistant isolate of E. coli 0157:H7 caused a large outbreak in Missouri (171); the outbreak strain was resistant to sulfisoxazole, tetracycline, and streptomycin, Resistance to these same three antimicrobials appeared in 13 of 176 (7.4%) strains from Washington state between 1989 and 1991, but it had not been seen in 30 isolates obtained between 1984 and 1987 (172). Antimicrobial resistance has been noted inseveral recent studies (173-176), but the data are too limited to assess whether there is a secular trend toward increasing antimicrobial resistance among these strains. Early efforts to place E. coli serotype 0157:H7 into one of the previously known groups of diarrheagenic E. coZi were unsuccessful. This serotype was not one of the traditional EPEC serotypes. Production of LT or ST could not be demonstrated, thereby precluding membership in the ETEC. The organism was not invasive, nor did it possess the invasion-associated plasmid that would have classified it as EIEC (156).

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Eventually serotype 0157:H7 was shown to produce verotoxin, a cytotoxic activity demonstrable on cultured Vero (African green monkey kidney) cells (177,178). and very rapidly thereafter this “verotoxin” was shown to be a family of cytotoxins of close structural and antigenic similarity to the Shiga toxin of SIzigellrr dyserzteriae (157,179,180). Excellent reviews of the family of Shiga toxins have been published detailing their history, genetics, and immunology ( 181,182).

2. Nomenclature The nomenclature of this group of diarrheagenic E. coli reflects the concurrent pathways of investigators who discovered cytotoxin production by E. coli 0157:H7, each of which chose a different name. Because of the recognition that these cytotoxins are related to one another with similar structure, functional activity, and cellular receptor, they have been designated Shiga-like toxins (SLT) or, more recently, Shiga toxin (Stx). Others have used the term Verotoxin, because the toxin activity was first observed as a cytotoxic effect on Vero cells (178). The terms “Shiga toxin” (or the older “Shiga-like toxin”) and “Verotoxin” are considered synonymous at this level of designation, although specific toxins (e.g., Stx2 and VT-2) may not be identical. Viewpoints for each nomenclature have been published (183,184). The designation Stx is used herein. Many strains of E. coli, including some EPEC strains, have been shown to produce Stx in varying amounts, but as most ofthese strains do not possess the other characteristics of the EHEC, they are not considered a member of this group. Thus, Stx production is necessary but not sufficient for strains to be considered in the EHEC category, and EHEC are one subset of the group of Shiga toxin-producing E. coli (STEC). All EHEC are considered human pathogens, whereas some but not all STEC are causative agents of disease. The term “EHEC” designates those serotypes of E. coli that often produce a clinical illness of severe bloody diarrhea (‘ ‘hemorrhagic colitis”) and that possess certain virulence determinants (Stx production, possession of a 60 MDa plasmid, and eliciting attaching and effacing histopathological lesions in an animal model). Levine et al. (185) proposed that serotype 026:Hll be considered an EHEC strain; however, an analysis of virulence properties of a collection of 026:Hll strains has shown that this serogroup is heterogeneous (186). Other EHEC serotypes include 026:H32, 055:H7, 0 1 13:H21, and 0 1 17:H14 (15). Serotype 0157:H7, as the most wellcharacterized, is considered the “prototypic” EHEC strain. C. Characteristics of Disease As one would expect, diarrhea is the hallmark of symptomatic infection with E. coli 0157: H7. Typically illness begins with a short prodrome of mild nonbloody diarrhea, which progresses within 24 hours to grossly bloody diarrhea, often associated with severe abdominal pain and cramping (187). Because of the copious amounts of frankly bloody rectal discharge, cases frequently have been mistakenly diagnosed as having a noninfectious cause, such as mesenteric infarction, inflammatory bowel disease, Meckel’s diverticulum, intussusception, and anatomical gastrointestinal bleeding. Hospitalization rates have been high (>50%), making hemorrhagic colitis a high-profile illness. Although the preponderance of cases reported in sporadic case series and the majority of cases in outbreaks have had bloody dialrhea, this may be due to ascertainment bias in that patients who are more severely ill or who are part of an outbreak may be more

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likely to have stool cultures for serotype 0157:H7 performed. It is now evident that nonbloody diarrhea can be seen with E. coli 0157:H7 infection (188-190) as well as with other serotypes (188). Patients with nonbloody diarrhea have been noted to be less severely ill ( 190). Many other enteric pathogens, such as Snlmorzelln, Shigella, and Camyylobncter, can be carried in the gastrointestinal tract asymptomatically, and E. coli 0157:H7 is no exception. Because persons without diarrhea rarely have stool cultures performed, documentation for such asymptomatic carriage has come from outbreak investigations and evaluations of family members in households with E. coli 0157:H7 infection (188,19 1193). The true frequency with which asymptomatic carriage occurs is unknown, and its importance in transmission is unclear (194). The hemolytic uremic syndrome (HUS) consists of an acquired Coombs’ negative hemolytic anemia, thrombocytopenia, and acute renal failure. It wasfirst described in 1955 (195), and small series and case reports had noted that mostpatients had a preceding diarrheal illness. A wide variety of microbiological and toxic agents had been described as “precipitants” of HUS, and it was assumed that HUS was the result of a final common pathway of injury resulting from a plethora of infectious and toxic insults. A few sporadic cases of E. coli 0157:H7 infection were noted to develop HUS (196,197), and both prospective and retrospective studies of HUS patients suggested a high frequency of infection with E. coli 0157:H7 or other STEC (198-200). Further studies have borne out this association (201-203). In one study (202), 100% of HUS patients cultured appropriately within 3 days of onset of illness had serotype 0157:H7 isolated; the detection rate fell if patients were cultured more than 7 days after onset, indicating that recovery of this serotype is highly time dependent. The proportion of patients infected with E. coli 0157:H7 who developed HUS has varied considerably (6-90%) among reported series and outbreak investigations. A complication rate of 8% has been observed in a recent prospective study of sporadic cases (204); however, the presence of various risk factors in the affected population may substantially increase this rate. The extremes of age, antimicrobial use, antimotility agents, and toxin type of the infecting strain all have been suggested to increase the risk of development of HUS after E. coli 0157:H7 infection. A few cases of thrombotic thrombocytopenic purpura (TTP) also have been described following E. coli 0157:H7 infection (205-207). This disorder consists of the cardinal features of HUS (hemolytic anemia, thrombocytopenia, and renal failure) along with fever and altered mental status. Prior series of patients with TTP have not indicated that many of these patients had antecedent gastrointestinal illness. The patients reported (205-207) all had bloody diarrhea and the overall features of their illness resembled those of severe HUS. Our current conceptual understanding of human infection with E. coli 0157:H7 is that a spectrum of clinical manifestations may result ranging from asymptomatic carriage to nonbloody or bloody diarrhea. Severe and life-threatening microangiopathic complications such as HUS and TTP may follow enteric illness with appreciable but variable frequency and thereby preclude volunteer studies with this pathogen. A rising incidence of HUS has been noted (208,209), suggesting either an increasing rate of human infection with E. coli 0157:H7 or an increasing rate of progression to this complication. Because HUS is a highly morbid disease with a fatality rate of5% and a considerable risk of serious long-term morbidity such as hypertension, chronic renal failure, and disability (210), a rising frequency of infection and/or its complications should provoke concern.

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Several helpful reviews on E. coli 0157:H7 (211,212) and HUS (213,214) have been published recently. D. Epidemiology Persons of all ages, ranging from one to 80 years, have been reported with E. coli 0157: H7 infection. In one population-based study in the Pacific Northwest, the highest agerelated incidence of infection was in children 0-9 years old (215). Serotype 0157:H7 is not an opportunistic pathogen, and it has produced severe illness in healthy individuals as well as those with chronic diseases. E. coli 0157:H7 has now been isolated on every continent except Antarctica (reviewed in Ref. 216). In North America, Canada and the tier of states along the U.S.Canadian border have tended to report both the largest number of outbreaks as well as sporadic cases. A striking seasonal pattern has been observed for both sporadic and community-acquired cases of E. coli 0 1 57:H7 infection, with a summertime predominance (187,216). Interestingly, a similar seasonality had been described for cases of HUS even prior to the "discovery" of E. coli 0157:H7. Doubtless this peak of symptomatic infection during the months of June through September represents some form of increased exposure to the agent, but whether this is because of periodic increases in contamination in the food chain or because of increased rates of exposure to a risk factor such as barbecuing are unknown. The mean incubation period for E. coli 0157:H7 infection as derived from outbreak investigations has ranged from 3.1 to 8 days (216). The wide variation in attack rates among outbreaks in which a food vehicle was implicated in transmission is probably due to differences in inoculum size, host susceptibility, and the presence of risk factors such as decreased gastric acidity and recent antimicrobial use. Person-to-person transmission has been noted, suggesting that the infectious dose is quite low, similar to that for Shigella infection. Quantitative recovery of E. coli 0157:H7 from foods involved in outbreaks indicates that the infectious dose can be 5100 organisms (217). Recently, outbreaks on an unprecedented scale have occurred in the United States involving 501 cases and 3 deaths (218), in central Scotland with 501 cases and 20 deaths (219). and in Japan with more than 5000 cases (320). These dratnatic outbreaks have strained the capacity of public health systems to investigate and control them, and the low infectious dose has necessitated a more stringent conceptual approach to food safety. Several facets of changing food animal production, large-scale food processing, and changes in consumer preferences have apparently provided a niche for the emergence of E. coli 0157:H7 as a human pathogen (221). With E. coli 0157:H7, as with many other pathogens, the major burden of disease is sporadic, rather than outbreak-associated. A new view of this situation was provided through prospective surveillance of E. coli in Minnesota during which all isolates were routinely subtyped using PFGE (332). Considerable heterogeneity was observed with >70 subtypes per year, but subtyping pertnitted the detection of outbreaks and small case clusters not recognized epidemiologically. These data could suggest that sporadic disease represents many "mini-outbreaks." Further work will need to define efficient and effective prevention strategies for outbreaks of both large and srnall scale.

E. IsolationandIdentification Virtually all procedures described to date for the primary isolation of E. coli 0157:H7 have been based on its lack of sorbitol fermentation (163,223). Colorless colonies on

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sorbitol Macconkey (SMAC) agar with morphology and biochemical reactions (indole positive, oxidase negative) typical of E. coZi may be serogrouped by slide, tube, or latex agglutination or by PCR assay for the rfb locus encoding the 0157 antigen (224) to detect the 0157 antigen. Serotyping can be completed by looking for immobilization in H7 antisera-containing motility media (162,223); however, some strains may be nonmotile (H-). Other strains of serogroup 0157 exist in nature, and although some are even sorbitol negative, none are of the H7 serotype (225). False-positive identifications of E. coli 0157: H7 have been reported (225-227). The recent description of sorbitol-positive strains may prove problematic for detection methods based on the lack of sorbitol fermentation if such strains are shown to occur elsewhere. Several techniques have been usedto subtype E. coli 0157:H7; those with the largest body of published experience include plasmid profile, toxin type, phage type (228), isoenzyme type (229), and analysis by pulsed field gel electrophoresis (230). Other less commonly used methods are ribotyping (23 1), insertion sequence subtyping (232), and restriction frequent length polymorphism using Stx probes (233). Isoenzyme profiling has been used to determine the placement of E. coli on the evolutionary tree of diarrheagenic E. coli and shows that the 0157:H7 group is a widely disseminated pathogenic clone, which has evolved relatively recently from an EPEC progenitor 0 5 5 strain (160). The other methods have mainly been used to "DNA fingerprint" strains to determine the interstrain relatedness, often in outbreak investigations. A superb overview has carefully delineated the published experience with these and related DNA-amplification subtyping approaches (234). Other methods for detection have been directed at demonstrating the presence of Stx, either directly in fecal filtrate or in culture supernatant from either pure or mixed cultures. Stx can be detected by tissue culture assay (direct cytotoxicity to Hela or Vero cells), enzyme-linked immunoassay, latex agglutination, colony immunoblot, and PCR assay (reviewed in Ref. 234). Although toxin-based detection methods can be extremely sensitive, their major drawback is that they may detect strains of unclear epidemiological and clinical significance (i.e., STEC not causally implicated in disease). As discussed in Sec. V.B above, not all STEC are EHEC and hence testing for additional virulence determinants is necessary for such strains. E. coli 0157:H7 produces a hemolysin, enterohemolysin (Ehly), whose phenotypic expression is visualized on washed sheep red blood cell agar (235). Ehly is distinct from a-hemolysin; E. coli 0157:H7 and some related EHEC possess only ehly sequences. The genes for Ehly are encoded on the large 60 MDa plasmid present in E. coli 0157:H7. DNA sequence-based methods (colony blot, PCR assay) have been described (236) and are particularly useful because not all strains express the ehly phenotype. Enterohemolysin production appears to be an interesting but imperfect correlate to pathogenic potential of the EHEC group as ehly-producing, non-Stx-producing E. coli have been found (234).

F. Pathogenicity Tremendous strides have been made in characterizing the molecular mechanism of action of the Stx family (for reviews see Refs. 181, 182). Both Stxl and Stx2 have a subunit structure consisting of one catalytically active A subunit and five B subunits responsible for binding to a specific cell surface receptor, globotriaosylceramide (Gb,). Both subunits are encoded on bacteriophage, and serotype 0 157:H7 produces either Stxl or Stx2 alone or in combination. Stxl differs by only one amino acid from Shiga toxin; Stx2 shares about 58% homology with Stxl. Some strains contain two copies of Stx2 genes, each

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copy with slightly different DNA sequences, and these differences probably account for the lack of cross-neutralization observed with certain antisera and for differences in biological activity on target cell lines (237,238). The biological significance of these differences is unclear. A third toxin, designated Stx2e (also known as SLT-IIe or VT2e) is produced by STEC strains of porcine origin (239). Although Stx2e has considerable DNA sequence homology to Stx2 (240), there are sufficient differences, particularly in the B subunit, that Stx2e binds to a different surface receptor than Stx2 (241). Stx2e is cytotoxic for Vero but not Hela cells. The role in human disease played by Stx2e-producing strains is unknown at present. These toxins function as protein synthesis inhibitors. Their RNA N-glycosidase activity leads to cleavage of a particular adenine residue in the 28s ribosomal RNA of the 60s ribosomal subunit (181). Both Stxl and Stx2 are extraordinarily potent, with cytotoxicity demonstrable from picogram quantities of toxin. Their biological activity is demonstrable on those cell lines such as Hela and Vero cells that express surface receptors for toxin; cell lines without these receptors are resistant. The exact role of these toxins in producing diarrhea or microangiopathy is presently unclear. Although a variety of animal models have been described that have employed various modes of toxin delivery, none have all the features of human infection, and in particular none duplicate the pathology seen in HUS. However, a substantial body of evidence has shown that for the parent toxin, Shiga toxin, both enterotoxic and cytotoxic activities are possessed by the same molecule, and these properties have also been shown for other members of the Stx family (242). The relationship of these biological activities to the clinical manifestations of diarrhea and dysentery was demonstrated in primate inoculation studies using toxin negative mutants of Shigella dysentericre 1 (243). Bloody diarrhea, the characteristic feature of dysentery, developed in animals fed the parent strain, and milder nonbloody diarrhea occurred in animals given the isogenic tox- mutant, thus implicating Shiga toxin in the colonic hemorrhage component of shigellosis. The compendium of evidence from several different animal models using a variety of bacterial constructs suggests that Stx expression by diarrheagenic E. coZi strains is responsible for the hemorrhagic aspect of diarrhea produced by other virulence determinants in those strains

(is). The pathological changes in HUS include endothelial cell damage, particularly in the glomerular endothelium in thekidneys. It has been hypothesized that systemic toxemia results from toxin release from the gastrointestinal tract with resultant damage to target endothelium. Support for this hypothesis largely comes from the lack of other obvious explanations such as bacteremia or cellular invasion to provide a connection between infection within the bowel lumen and end-organ damage in renal endothelium. Neutralizing antibodies to Stxl have been demonstrated in convalescent sera from patients with HUS (198). However, circulating toxin has never been demonstrated in patients with HUS or TTP, although this may bedue to rapid binding and internalization by cell types expressing surface receptors for toxin. Injection of toxin into laboratory animals has shown rapid uptake into tissues that are not typically affected (e.g., muscle) in HUS. Shiga toxin has been shown to be directly cytotoxic to human umbilical vein endothelial cells (244,245) directly cultured for short periods. Endothelial cells grown to confluence were demonstrated to be relatively resistant to Stx compared to Vero cells, a difference that may be ascribable to low levels of Gb3 in the endothelial cell membrane (246). The levels of cytotoxicity that could be demonstrated on these confluent endothelial cells were not aug-

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mented by lipopolysaccharide; a modest increase in cytotoxicity was seen with the addition of recombinant human tumor necrosis factor (246). Thus, a large body of mainly indirect evidence suggests that toxin is important in the pathogenesis of HUS but the pathophysiological sequence of events leading to microangiopathy distal to the bowel remains to be elucidated. A current area of active investigation is in the role of host-derived cytokines in HUS. Several studies have noted elevated serum levels of IL6 in HUS patients (247249); HUS patients showed higher levels of IL-10 and IL-IRa in comparison to hemorrhagic colitis patients (249). The extent to which cytokines alone versus those in combination with the effects of Stx contribute to both intestinal and extraintestinal disease is not clear at present. As would be expected by analogy with the other diarrheagenic E. coli, adherence properties of E. coli 0157:H7 are important in facilitating colonization. E. coli 0157:H7 exhibits an A/E phenotype like classical EPEC strains, not surprising given its origin from an EPEC progenitor serotype 055:H7. The molecular basis for A/E in 0157 strains has been elucidated (250) with identification and sequencing of the eae homolog in E. coli 0157:H7. The intimino157 protein encoded by the eaeo157gene has its greatest sequence divergence from the EPEC intimin in the C-terminal end, which binds the epithelial cell surface receptor. This in turn correlates with different sites of intestinal colonization for these pathogens-EPEC mainly colonize the small bowel, whereas E. coli 0157:H7 colonizes the large bowel (250). The sequence for the entire LEE of E. coli 0157:H7 was recently determined and compared to that for EPEC (251). For completely shared genes, the rate of divergence was greatest for those involved with bacteria1:host interactions, while genetic sequences for genes involved in other functions were highly conserved. This type of polymorphism of gene sequences specifically involved in pathogen-host interactions has been noted as an evolutionary strategy of several pathogens such as Borrelia burgdolferi and Streptococcus pyogenes (25 1).

VI.

ENTEROAGGREGATIVE E. COL!

A.

HistoricalAspects

The enteroaggregative E. coli (EAEC) are a relatively newly described, and somewhat controversial, cause of diarrhea. Their discovery illustrates the observation by Pasteur that "chance favors the prepared mind." Twenty, years ago, a particular type of adherence to HEp-2 cells had been described for EPEC (43). Using this assay, investigators intending to characterize the adherence phenotype of E. coli isolates from persons with diarrhea noted that some isolates exhibited a distinctly different form of adherence to HEp-2 cells than the localized adherence observed with EPEC strains (252,253). EPEC adherence is visualized as localized clusters of bacteria on the HEp-2 cell surface, appearing as a microcolony. Clearly distinguishable from this pattern was a palisading of bacteria in a stacked brick configuration, both on the cell surface as well as on the glass cover slip to which the HEp-2 cells adhered (254). This appearance was formed by "aggregative adherence" (AA), and E. coli strains exhibiting this phenotype were called enteroadherentaggregative E. coli or, later, enteroaggregative E. coli (EAEC). Aggregative adherence is distinguishable from diffuse adherence (DA); in the latter, bacterial cells are scattered across the surface of the HEp-2 cell, and there is no palisading observed on the glass cover slip (15).

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Subsequent epidemiological studies have more consistently isolated EAEC from persons with diarrhea compared to asymptomatic controls. although the frequency has varied considerably by geographic location (see below). For the diffusely adherent E. coli, there have been conflicting observations comparing the frequency of DAEC isolation from symptomatic persons compared with asymptomatic controls (254), and the DAEC are not yet fully accepted as a definite and distinct category of diarrheagenic E. coli. Although the EAEC were initially thought to be a type of EPEC, further support for classifying EAEC as a category separate from EPEC includes the lack of concordance of serotypes between the two groups and that EAEC strains are negative when tested with the EAF probe. The EAEC have caused diarrhea in both sporadic and outbreak settings as well as in volunteer studies (255). Thus a body of evidence has developed that supports the consideration of EAEC as a member of the diarrheagenic E. coli in a category distinct from the EPEC, ETEC, EIEC, and EHEC.

B. Characteristics of the EAEC 1. Serogroups Serogroups determination is not a basis for assigning isolates to the EAEC category. However, those serotypes characteristic of the EAEC include 03:H2,015:H18,044:H18,086: NM. 077:H18, Olll:H21, 0127:H2, and 0 nontypab1e:HlO (15). As previously noted, these serotypes are not overlapping with those of the EPEC category.

2. Identification As with the other categories of the diarrheagenic E. coli, there are no phenotypic or morphological features that distinguish EAEC on primary enteric isolation. The gold standard for assignment to the EAEC category is the demonstration in the HEp-2 adherence assay of the characteristic AA pattern. Varied culture conditions have been employed for preparation of the bacterial inoculum as well as performance of the adherence assay itself; however, at least one comparative study has indicated that the methods as originally described (43j yielded the best discrimination among adherence phenotypes (256). A DNA probe has been developed from a 1.0 kb fragment of a large plasmid from an EAEC strain (257). Nearly 90% of strains identified as EAEC by HEp-2 cell adherence hybridized with this DNA probe when the isolates had been obtained from Chile or south India (257.258); however, considerably fewer strains from Brazil, also identified on the basis of HEp-2 cell adherence, hybridized with the DNA probe (259). At present, DNA probe positivity is a helpful adjunct in categorizing EAEC strains: however, probe-negative strains that exhibit the AA phenotype in HEp-2 adherence are still potentially classifiable as EAEC strains. Ongoing epidemiological studies should help to define differences in pathogenic potential between probe-positive and probe-negative EAEC.

C. Clinical Manifestations A watery, noninflammatory diarrhea appears to be the most common clinical manifestation seen in outbreaks and in volunteer studies. In the latter, diarrhea has also been noted to be very mucoid. Several studies have associated EAEC with persistent diarrhea (259,260). EAEC have also been reported as a cause of diarrhea in an HIV-infected patient in the United States (261). EAEC have been detected more frequently in the stools of HIV-infected

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children in Zaire with diarrhea (41%) in comparison to HIV-uninfected children with and without diarrhea (30% and B%,respectively) (262).

Epidemiology D. EAEC have been isolated as causative agents of diarrhea in both the developing as well as the developed world. Rates of isolation have varied among locations (15,263). but essentially the EAEC appear to be widely dispersed geographically. EAEC outbreaks have been described in both the developed and developing world (260,264,265), and these have involved both healthy and malnourished individuals. In studies of pediatric diarrhea in India and Brazil, the EAEC are prominent causative agents of acute illness (258,266). In Brazil, persistent diarrhea was more frequently due to EAEC than to other pathogens (259). The importance of this finding is that there is greater overall workability with resistant diarrhea compared to acute diarrhea. Recently, an interesting but extremely disturbing observation noted the association of EAEC infection with growth retardation (267), and this was noted even for asymptomatic infection. These data indicate a much larger attributable morbidity due to EAEC infection than that of previous assessments based on acute diarrheal morbidity alone.

E. Pathogenicity At present, there is only a very incomplete picture of EAEC pathogenicity. Most of the work to date has been in studies of the AA phenotype and its genetic basis. A fimbrial structure designated aggregative adherence fimbriae I (AAF/l) has been identified, which appears to mediate adherence to HEp-2 cells (268). Excess mucus production has been identified in animal models and volunteer studies, and a role for excess mucus in enveloping bacteria proximal to the mucosa hasbeen postulated (15). Some degree of mucosal destruction has been noted in autopsy specimens, and cytotoxic effects on intestinal explants and cell lines has been noted by some, but not all, EAEC strains. A homolog of heat-stable toxin has been found in EAEC strains and designated EAST1 (269). Only half of EAEC appear to have this secretory toxin, which is also possessed by some EHEC strains. Using this available evidence, Nataro and Kaper have proposed a preliminary three stage model of EAEC pathogenesis (15). Initially, adherence to intestinal epithelial cells is mediated by AAF or other factors; subsequently enhanced mucus production entraps bacteria close to the cell surface, thus allowing delivery of toxin with resulting mucosal destruction.

VII.

ROLE OF FOOD AND WATER IN TRANSMISSION OF DIARRHEAGENIC E. COLI

Information on the source of transmission for the recognized groups of E. coli that cause diarrheal disease is derived mainly from outbreak investigations and prospective household-based studies (270). The former are more often carried out in the developed world largely as a consequence of improved recognition of outbreaks due to a lower background incidence of diarrhea and due to greater public resources. Foods important in transmission

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in the developed world may be essentially irrelevant in less developed countries due to nonavailability, nonacceptability, or differences in food-preparation practices. Prospective studies of food and water in the households of patients with diarrhea have more often been carried out in the developing world, and their major limitation is that often the food vehicles consumed by the index patient are no longer available. Despite the biases inherent in this imperfect data base, a few points are worthy of comment. ETEC are primarily transmitted via contaminated food and water, and person-toperson transmission is rare. This is most likely due to the relatively large inoculum needed ( lo8) to produce illness (68). Ingestion of nonrefrigerated foods and fecally contaminated water can result in intake of an inoculum of this magnitude; this is the mode of acquisition in ETEC-associated traveler’s diarrhea (271). Items considered at high risk for ETEC contamination include raw vegetables, such as in salads, untreated water, and ice (272). These observations have led to the dictum “Peel it, boil it, cook it, or forget it” as the main preventive measure for traveler’s diarrhea. The environmental conditions conducive to enterotoxin production in foodstuffs are almost never achieved, and hence, ingestion of preformed toxin does not seem to be an important means of becoming ill (273). Attempts to determine the source of infection in sporadic cases of ETEC diarrhea have not been highly successful. In Bangkok, different ETEC strains were isolated from market foods compared to isolates from children (274), and ETEC of the same serogroup as the index patient were detected in only two foods and two water sources in combined studies of over 100 households (275-278). Given the difficulties of such studies, as discussed above, these results should not be taken to preclude foodborne transmission of sporadic ETEC infection in an endemic setting. When EPEC-associated outbreaks of diarrhea in adults have been described, both food and waterborne transmission have been noted (28,279). Volunteer studies in adults showed that the inoculum necessary to produce disease was high, 10’ organisms, which would be consistent with that ingestible through contaminated food or water. It is possible, however, that infants are susceptible to lower inocula. The high secondary spread in hospital nursery outbreaks after the admission of an index case with EPEC gastroenteritis was invariably seen among the infants rather than the mothers, nurses, and family contacts (30,280). This finding is not explained by clinically inapparent infection as adult contacts of cases of EPEC diarrhea have not been shown to be colonized with these strains. All these findings together suggest that adults are fairly resistant to EPEC infection, but even such resistance can be overcome if the inoculum is high enough as has occurred in a few food- and waterborne outbreaks. The exact means of transmission of EPEC diarrhea among very young children is unclear. Large numbers of EPEC organisms are shed in the stool of infected infants, and the environment around these children becomes heavily contaminated (30j. The breadth of environmental surfaces that harbor EPEC and the ability of these strains to survive on dust and bedding create ample opportunity for the hands of caregivers to become contaminated and in turn perpetuate cross-infection. Aerosols containing EPEC have been shown in the immediate vicinity of patients with EPEC diarrhea (31) as has respiratory tract colonization in affected infants. This has raised the possibility that EPEC may be transmitted via an airborne route among young infants with ingestion of the inoculum following inhalation. Because a facile means of identifying EIEC in the routine clinical microbiology laboratory does not exist, information on transmission is drawn almost exclusively from outbreaks. Almost all such outbreaks have been foodborne, although at least one water-

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borne outbreak in Hungary has been reported (281). Volunteer studies showed that an inoculum of 10' organisms was necessary to cause disease in adults (68), a finding with which foodborne transmission is consistent. Person-to-person transmission has also been seen (136), not surprising given the similarity of EIEC to Shigella. Humans may be the main reservoir of the organism as they are for Shigella; evidence supporting this supposition is the lack of isolation of EIEC from animals and the linkage of outbreaks with either an infected food handler or fecally contaminated water, the latter contaminating the food item (Brie and Camembert cheese) during production in one large outbreak (1 19). Foodborne transmission of E. coli 0157:H7 has been repeatedly demonstrated since the original discovery in 1982 in which contaminated undercooked hamburger meat was the vehicle (156). In an almost eerie replay of the past, this same scenario was repeated a decade later in a large multistate outbreak of E. coli 0157:H7 infections associated with undercooked hamburgers from one fast food restaurant (218). The majority of outbreaks with E. coli 0157:H7 with a known vehicle of transmission have been due to contaminated beef (mainly hamburger), although less than half of all outbreaks have a known source. Consumption of undercooked ground beef has been shown to be a risk factor for illness in case-control studies of sporadic cases (282-283). Unpasteurized milk has transmitted E. coli 0157:H7 infection in several outbreaks in both the United States and the United Kingdom. The fairly common association of illness with bovine products has led to consideration that control of colonization in bovines would prevent most human disease. Given what is now known about the broad host range of E. coli 0157:H7, which includes wild animals such as deer, this approach is probably too simplistic. Other foods that have transmitted illness include dried venison (284) and sausage (216). Studies of retail meats not associated with human disease showed that E. coli 0157: H7 was recoverable from 3.7% of beef, 1.5% of pork, 2.0% of lamb, and 1.5% of poultry samples (285). Isolation rates may vary by geographic region as E. coli 0157:H7 was not detected in meat samples from Newfoundland or Ontario, Canada (286,287), or Thailand (288). Non-0157 STEC are frequently detected in raw meats (17% in one study) (289). However, many of these non-0157 STEC are of uncertain pathogenic significance, and there is controversy regarding the interpretation of the presence of STEC in foods (290,29 1). An unsettling development has been the recent instances of transmission of E. coli 0157:H7 infection by fresh produce such as lettuce (292), radish sprouts (220), alfalfa sprouts (293), and fresh unpasteurized apple juice or cider (294,295). These foods are eaten without additional cooking, posing a problem because of the extremely low infectious dose for E. coli 0157:H7. Sprouts are a special problem because the sprouting process itself is an amplification step; bacteria in the seeds can readily multiply to levels of lo7 cfu/g within 48 hours of sprouting the seed. E. coli 0157:H7 has remarkable ability to tolerate very low pH (<4) (296), and this acid tolerance probably contributes to its low infectious dose and prolonged survival in acid foods such as dry sausage and cider (216,297). Waterborne transmission of E. coli 0157:H7 has been described (171,298-300). Again reemphasizing the low infectious dose, in an outbreak due to a contaminated municipal water supply in a small town in Wyoming, some visitors became ill after consuming only small amounts of water during a several-hour stay (300). An interesting study from Chile has suggested that waterborne transmission may be less important than transmission by foods for all groups of diarrheagenic E. coli (301). Despite the access to clean water and toilet facilities within the home by nearly 70% of households, rates of infection with diarrheagenic E. coli remained high and suggested that

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transmission may bemore often foodborne than waterborne. Lack of adequate refrigeration was observed as probably contributing to illness by providing opportunity for bacterial multiplication in foods during the sumtner months when temperatures are high.

VIII. ISOLATIONFROMFOODS It is not within the scope of this chapter to provide a compendium of isolation techniques for the diarrheagenic E. coli, and the reader is referred elsewhere for comprehensive reviews (286,302). Several overall points are worth emphasizing here, however. Foodstuffs are obviously not sterile, and many bacteria other than E. coli may be present, complicating efforts to recover E. coli from the food item. One way to circumvent this problem is the use of an enrichment procedure that selects for the target E. coli group while discouraging the growth of other organisms. Such is the basis of isolation techniques such as that in the FDA Bacteriological Analytical Manual, which uses incubation temperatures of 44-45"C, which permit growth of some pathogenic E. coli while minimizing growth of other bacteria. There are several problems with this approach: (a) not all groups of the diarrheagenic E. coli are detectable with this method, e.g., the EHEC strain, E. coli 0157:H7, does not grow well at 44°C; (b) the phenotypic expression of those biochemical reactions that characterize E. coli may be poor at elevated temperatures; and (c) significant plasmid loss may occur during growth at an elevated temperature, thereby obviating the additional characterization of an isolate for plasmid encoded functions, e.g., enterotoxin production. Some of these difficulties have been surmounted by using an initial growth phase at 35°C in brain heart infusion broth to nonselectively permit growth and resuscitate injured or stressed microorganisms (303) before utilizing enrichment media such as tryptone phosphate (TP) broth (304) at 44°C to select for pathogenic E. coli. This procedure still does not allow for isolation of E. coli 0157:H7. Of all the diarrheagenic E. coli, only EHEC serotype 0157:H7 has a distinctive biochemical phenotype, allowing this to be the basis for initial screening procedures. This serotype does not ferment sorbitol rapidly, within 24-48 hours, but using this feature to discriminate E. coli 0157:H7 from other bacteria depends upon the background flora of similar phenotype in the original sample (305). Doyle and Schoeni (285,306) developed a method for direct isolation of E. coli 0157:H7 from foods with a sensitivity of 1.5 organisms per g, but the procedure has several steps and is time, labor, and materials intensive. A direct plating method (307), while simpler to perform, is several logs less sensitive, able to detect only 10'-104 organisms per g. Another method combining hydrophobic grid membrane filtration and using a monoclonal antibody has been reported with a sensitivity of 10 organisms per g (308) and was fairly rapid (24 hours) for negative samples, with positive samples requiring a further 2-3 days for analysis. A reactive disc blot ELISA technique has also been reported with similar turnaround times for positive and negative samples (309). Improved selective media incorporating cefixime and tellurite into sorbitol Macconkey media (310) have been combined with immunomagnetic bead separation (31 1) to provide a method very nearly equivalent to DNA amplification in sensitivity, with the advantage of providing a bacterial isolate available for additional subtyping. Regardless of method, all the aforementioned isolation procedures ultimately yield one or more E. coli isolates, which must be further characterized for the attributes that classify them as members of the EPEC, ETEC, EIEC, or EHEC. For EPEC, this entails

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0 and H serotyping, characterizing the strain for the presence of the A/E phenotype or e m gene and the EAF plasmid. Possible ETEC may be assayed by several methods for LT and/or ST production, but verification that an enterotoxin-producing strain is pathogenic for humans requires demonstration that the strain expresses a CFA type specific for humans. Candidate EIEC isolates can be characterized by combining serotyping with biochemical characterization, determining the presence of theinvasion-associated plasmid, and testing for invasion of Hela or HEp-2 cells in tissue culture or the ability to cause keratoconjunctivitis in the guinea pig eye. The prototype EHEC strain E. coli 0157:H7 is identified by 0 and H serotyping and Stx production or genotype. Other STEC are identified by the serotyping of isolates shown to possess the Stx gene, determination of the presence of the eae sequence and possibly Ehly. EAEC strains are categorized as such by serotyping and demonstration of the AApattern in tissue culture. All the methods noted above are dependent on recovery of a viable isolate and its subsequent growth in pure culture for further characterization and/or subtyping. Another methodology that circumvents the problems of viable organism recovery and labor- and time-intensive characterization procedures is the polynlerase chain reaction (PCR) assay. This technique allows for the rapid amplification of target DNA sequences to detectable levels within 24 hours. Depending on the specificity of the primers used in the reaction, the amplified DNA sequence can be organism specific (i.e., a conserved DNA sequence found only in E. coli allowing differentiation of E. coli from other Enterobacteriaceae), strain-specific (e.g., permitting amplification of SLT sequences used to delineate EHEC), or, even through the use of purposefully “cross-reactive” primers, the assay can be genus-specific (e.g.. allowing detection of members of the genus Mycobncteriunz without regard to species). PCR assays have been developed that allow for the rapid detection of target DNA sequences in pure isolates, thereby bypassing lengthy procedures that depend on the functional expression of such sequences, e.g., enterotoxin or cytotoxin production and detection. Validation that a genotype is always correlated with the previously described phenotype that characterizes a group of diarrheagenic E. coli, however, is necessary before PCR-based assays can supplant prior methods of strain characterization. PCR-based assays do not have inconsequential obstacles to overcome before they can become routine in almost any setting (3 12). They are exquisitely sensitive to contamination, although newer techniques have been developed that can overcome this problem. Their application to clinical specimens, human tissues, and environmental samples has been slowed by various inhibitors present in those specimens. Quality control in the individual assay itself as well as in the PCR laboratory is a very significant issue in order to ensure the scientific validity of the result. All nucleic acid amplification assays are limited by the fact that their end result does not yield a viable organism, thereby limiting further characterization such as the subtyping that plays an increasing role in epidemiological investigations. The diarrheagenic E. coli have particularly lent themselves to nucleic acid-detection methods because they have been among the first pathogens whose genetic basis of virulence has been defined. As such there has been a virtual explosion of publications describing PCR assays for their detection in foods and clinical and environmental samples. These are too extensive to be reviewed here. Many of these, however, were developed using artificially “spiked” samples and do not have side-by-side comparisons with other techniques, particularly in prospective studies. Nucleic acid amplification nlethodology has proven to be a very helpful and reasonably reliable means to categorize the diarrheagenic E. coli when pure cultures have been

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available. Its routine use on clinical and environmental samples and foodstuffs will need the types of validation studies mentioned previously. Those investigations coupled with focused epidemiological studies should enlarge our understanding of the ecological niche of the diarrheagenic E. coli and provide a more solid understanding on which to base disease-prevention strategies. This may be the means by which one of the most important achievements of public health in this century, safer food (313,314), is taken forward into the next millennium.

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Neil1 et al. clinical features of infection. epidemiology and pathogenesis. Curr. Clin. Top. Ilfect. Dis., 151230-252. Cobeljic. M.. Miljkovic-Selitnovic. B.. Paunovic-Todosijevic, D., et al. (1996). Enteroaggregative Esclzerichirr coli associated with an outbreak of diatrhoea in a neonatal nursery ward. Epidenriol. Infect., 117:ll-16. Eslava, C., Villaseca, J., Morales, R., Navarro. A., and Cravioto A. (1993). Identification of a protein with toxigenic activity produced by enteroaggregative Escherichia coli (abstract B-l 05). In Abstracts of the 93rd Gelzeral Meetirzg of the Americait Socieh for Microbiology, American Society for Microbiology. Washington, D.C., p. 44. Bhan, M. K., Raj. P., Levine, M. M., et al. (1989). Enteroaggregative Eschet-iclziacoli associated with persistent diarrhea in a cohort of rural children in India. J. hfect. Dis., 159:10611064. Steiner. T. S., Lima, A. A., Nataro, J. P., and Gue~rant,R. L. (1998). Enteroaggregative Escherichia coli produce intestinal inflammation in growth impairment and cause interleukin8 release from intestinal epithilial cells. J. Infect. Dis., 177:88-96. Nataro, J. P., Deng. Y., Maneval. D. R.. et al. (1992). Aggregative adherence fimbriae I of enteroaggregative Eschericlzia coli mediate adherence to HEp-2 cells and hemagglutination of human elythrocytes. Infect. I m m n . , 602297-2304. Savarino, S. J., Fasano, A., Watson, J., et al. (1993). Enteroaggregative Escherichia coli: heat-stable enterotoxin 1 represents another subfamily of E. coli heat-stable toxin. Proc. Natl. Acad. Sci. USA, 90:3093-3097. Echeverria. P., Serichantalergs, O., Changchawalit, S., and Sethabutr. 0. (1992). Escherichia coli gastroenteritis. In Food urd Waterborne Irzfections (A. Tu. ed.), Marcel Dekker. New York, pp. 71-101. Wood, L. V., Ferguson, L. E., Hogin, P., Thurman, D., Morgan, D. R., DuPont, H. L., and Ericsson, C. D. (1983). Incidence of bacterial enteropathogens in foods from Mexico. Appl. Em+-on. Microbiol., 46:328-332. Blaser, M. J. (1986). Environmental interventions for the prevention of traveler's diarrhea. Rely. Irfect. Dis., 8(Suppl 2): 142- 150. Doyle, M. P. and Padhye. V. V. ( 1989). Escherichia coli. Tn Foodborne Bacterial Pathogens (M. P. Doyle, ed.). Marcel Dekker, New York, pp. 236-281. Black, R. E., Merson, M. H., Rowe, B., et al. (1981). Enterotoxigenic Escherichia coli diarrhea: acquired immunity and transmission in an endemic area. Bull. WHO. 59263268. Echeverria, P., Seriwatana, J., Leksomboon, U., Tirapat, C., Chaicumpa, W., and Rowe, B. (1984). Identification by DNA hybridization of enterotoxigenic Escherichia coli in homes of children with diarrhea. Lancet. 1:63-65. Echeverria, P., Seriwatana, J., Taylor, D. N., Tirapat, C., Chaicumpa. W., and Rowe. B. (l 985). Identification by DNA hybridization of enterotoxigenic Escherichia coli in a longitudinal study of villages in Thailand. J. Itlfect. Dis.. 151:124-130. Echeverria, P., Seriwatana, J., Taylor, D. N., Yanggratoke, S., and Tirapat, C. (1985). A comparative study of enterotoxigenic Escherichia coli, Shigella, Aeromoms, and Vibrio as etiologies of diarrhea in Northeastern Thailand. Am. J. Trop. Med. Hyg., 34547-554. Echeverria, P., Taylor, D. N.. Seriwatana, J., Leksomboon, U., Chaicumpa, W., Tirapat, C., and Rowe, B. (1987). Potential sources of enterotoxogenic Escherichia coli in homes of children with diarrhea in Thailand. Bull. WHO, 65207-215. Costin, I. D., Voiculescu, D., and Gorcea, V. (1964). An outbreak of food poisoning in adults associated with Esclzerichia coli serotype 86:B7:H34. Pathol. Microbiol.. 27:68-78. Hinton, N. A., and MacGregor, R. R. (1958). A study of infections due to pathogenic serogroups of Escherichia coli. Can. Med. Assoc. J., 79:359-364. Lanyai B., Szita. J.. Ringelhann B., and Kovach K. (1959). Awaterborne outbreak of enteritis associated with Escherichia coli serotype. Acta Sci. Hzmg., 6:77-84.

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282. Slutsker, L., Ries, A. A., Maloney, K., et al. (1998). A nationwide case-control study of Escherichia coli 0157:H7 infection in the United States. J. Infect. Dis., 177:962-966. 283. Parry, S. M., Salmon, R. I., Willshaw. G. A., et al. (1998). Risk factors for and prevention of sporadic infections with vero cytotoxin (shiga toxin) producing Escherichia coli 0157. Lcrizcet, 351:1019-1022. 284. Keene, W. E.. Sazie, E., Kok, J.. et al. (1997). An outbreak of Escherichia coli 0157:H7 infections traced to jerky made from deer meat. JAMA, 227:1229-1231. 285. Doyle. M. P., and Schoeni, J. L. (1987). Isolation of Esclzericlzirr coli 0157:H7 from retail fresh meats and poultry. App. Erzviron. Microbiol., 53:2394-2396. 286. Ratnam, S., and March, S. B. (1986). Sporadic occurrence of hemorrhagic colitis associated with Escherichia coli 0157:H7 in Newfoundland. Can. Med. Assoc. J.. 134:43-46. 287. Read, S. C., Gyles. C. L., Clarke, R. C.. et al. (1990). Prevalence of verocytotoxigenicEscherichia coli in ground beef, pork and children in southwestern Ontario. Epidemiol. hlfect., 105:ll-20. 288. Suthienkul, O., Brown, J. E., Seriwatana, J., et al. (1990). Shiga-like toxin-producing Escherichia coli in retail meats and cattle in Thailand. Appl. Emiron. Microbiol., 56: 1135-1 139. 289. Samadpour, M., Ongerth, J. E., Liston, J., et al. (1994). Occurrence of Shiga-like toxin-producing Escherichia coli in retail fresh seafood, beef. lamb, pork, and poultry from grocery stores in Seattle, Washington. Appl. Erwiron. Microbiol., 60: 1038-1040. 290. Ta-r, P. I., and Neill, M. A. (1996). Perspective: the problem of non-O157:H7 shiga toxin (verocytotoxin)-producing Eschericlzia coli. J. Infect. Dis., 174: 1136- 1139. 291. Paton, J. C., and Paton. A. W. (1998). Pathogenesis and diagnosis of shiga toxin-producing Esclrerichia coli infections. Clin. Microbiol. Rev., 11:450-479. 293,. Ackers, M. L., Mahon, B. E., Leahy. E., et al. (1998). An outbreak ofEscherichia coli 0157: H7 infections associated with leaf lettuce consumption. J. Infect. Dis., 177:1588-1593. 293. CDC. (1997). Outbreaks ofEsrhet-iclziacoli 0157:H7 infection associated with eatingalfalfa sprouts-Michigan and Virginia, June-July 1997. MMWR, 46:741-734. 294. CDC. (1996). Outbreak of Escher-ichict coli 0157:H7 infections associated with drinking unpasteurized commercial apple juice-British Columbia, California, Colorado, and Washington, October 1996. MMWR, 45:975. 295. CDC. (1997). Outbreaks of Escherichitr coli 0157:H7 infection and cryptosporidiosis associated with drinking unpasteurized apple cider-Connecticut and New York, October 1996. MMWR. 46:4-8. 296. Leyer, G. J., Wang, L. L., and Johnson, E. A. (1995). Acid adaptation of Escherichio coli 0157:H7 increases survival in acid foods. A$,. Em~iron.Microbiol., 61:3752-3755. 297. Zhao, T., Doyle. M. P., and Besser, R. E. (1993). Fate of enterohemorrhagic Eschericlzia coli 0157:H7 in apple cider with an without preservatives. Appl. Emit-on. Microbiol., 59: 2526-2530. 398. Keene, W. E., McAnulty, J. M.,Hoesly. F. C., et al. (1994). A swimming-associated outbreak of hemorrhagic colitis cause by Escherichia coli 0157:H7 and Shigella sonnei. N. Engl. J. Med.. 331:579-584. 299. Blake, P. et al. (1998). Escherichia coli 0157:H7 outbreak among visitors to a water park. C h . Infect. Dis.. 27: 1033. 300. Olsen, S. J., Miller. G.. Breuer, T.. et al. (1998). A waterborne outbreak of E. coli 0157:H7 infections: evidence for acquired immunity. Abstracts of the 36th anma1 westing,Infectious Disease Socieo of America, November 12-15. 1998. Denver, Abstract 783. 301. Levine, M. M., Ferreccio, C., Prado. V., et al. (1993). Epidemiologic studies of Escherichia coli diarrheal infections in a low socioeconomic level peri-urban community in Santiago, Chile. A m . J. Epidemiol., 138:849-869. 302. Meng, J., Feng. P., and Doyle, M. P. Pathogenic Escherichicr coli. In: Compendium of Methods for the Microbiological Examination of Foods. American Public Health Association, Washington, D.C., 3000 (in press).

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303. Hitchins, A. D., Feng, P., Watkins, W. D., Rippey, S. R., and Chandler, L. A. (1992). Escherichia coli and coliform bacteria.In FDA Bacteriological Analytical Manual, 7th ed. Association of Official Analytical Chemists, Arlington. VA, pp. 27-49. 304. Mehlman, I. J., and Romero, A. (1982). Enteropathogenic Escherichia coli: methods for recovery from foods. Food Technol., 36(3):73-79. 305. Tarr, P., Tran, N. T., and Wilson, R. A. (1999). Escherichia coli 0157:H7 in retail ground beef in Seattle: results of a one-year prospective study. J. Food Pr-ot. 62:133-139. 306. Padhye, N.V., and Doyle, M. P. (1991). Rapid procedure for detecting enterohemorrhagic Escherichia coli 0157:H7 in food. Appl. Environ. Microbiol., 572693-2698. 307. Szabo, R. A., Todd, E. C. D., and Jean, A. (1986). Method to isolate Escherichia coli 0157: H7 from food. J. Food Prot., 49:768-772. 308. Todd, E. C. D., Szabo, R. A., Peterkin, P., Sharpe, A.N., Parrington, L., Bundle, D., Gidney, M. A. J., and Perry, M. B. (1988). Rapid hydrophobic grid membrane filter-enzyme-labelled antibody procedure for identification and enumeration of Escherichia coli 0157 in foods. Appl. Environ. Microbiol., 54:2536-2540. 309. Okrend, A. J. G., Rose, B. E., and Matner, R. (1990). An improved screening method for the detection and isolation of Escherichia coli 0157:H7 from meat, incorporating the 3M Petri film test kit-HEC-for hemorrhagic Escherichia coli 0157:H7. J. Food Prot., 53:936940. 3 10. Zadik, P. M., Chapman, P. A., and Siddons, C. A. (1993). Use of tellurite for the selection of verocytotoxigenic Escherichia coli 0157. J. Med. Micr-obiol., 39:155-158. 311. Chapman, P. A., Wright, D. J.. and Siddons, C.A. (1994). A comparisonof inmunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli 0157 from bovine faeces. J. Med. Microbiol.. 40:424-427. 3 12. Persing, D. H. (1991). Polymerase chain reaction: trenches to benches. J. Clin. Microbiol., 29~1281-1285. 313. CDC. (1999). Ten great public health achievements-United States. 1900-1999. MMWR, 48:241-243. 314. CDC. (1999). Safer and healthier foods. MMWR, 48:905-913.

10 Listeria monocytogenes Catherine W. Donnelly University of Vermont, Burlington. Vermont

I. Introduction 213 A. History and characterization of Listeria B. Distribution 215

214

11. Listeriosis in Humans 216

111. IV.

V. VI.

VII.

1.

A. Characterization of the illness listeriosis 216 B. Mechanisms of defense against Listeria in normal, healthy hosts 216 C . Listeriosis in immunocompromised hosts 218 Pathogenesis of Listericr monocytogenes 219 Foodborne Transmission of Listeriavzonocytogenes 221 A. Major foodborne disease epidemics 221 B. Sporadic cases of listeriosis with foodborne etiology 224 C. Other reports of foodborne listeriosis: epidemic and sporadic 225 Sources of Listeriu in Foods and Food-Processing Environments 226 Detection of Listeria in Foods 229 A. Selective procedures 229 B. Detection of sublethally injured Listeria 231 Summary 234 References 235

INTRODUCTION

Listeria mor~ocytogenes,the causative agent of the disease listeriosis, was first discovered almost 100 years ago. Within the past 15 years this organism has emerged as a foodborne pathogen of major significance. Prior to 198l, Listeria was recognized primarily as an animal pathogen, and it was suspected that humans acquired listeriosis through direct contact with infected animals. We now know that consumption of foods contaminated with Listeria can cause both sporadic illness as well as foodborne disease epidemics. In order to prevent foodborne listeriosis, it isnecessary to understand the disease, susceptible persons, distribution of the organism within the environment, and behavior of the organism in foods. This chapter is designed to summarize current knowledge with respect to the 213

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foodborne role of Listeria, with special emphasis on improvements in isolation procedures designed to detect the presence of Listeria in foods.

A.

History andCharacterization of Listeria

Listeria mor1ocytogenes is asmall (1.0-2.0 p X 0.5 p), gram-positive, facultatively anaerobic, rod-shaped bacterium, which is widely distributed in nature. The organism can exist in an intracellular state within monocytes and neutrophils, and its name is derived from the fact that large numbers of tnonocytes are often found in the peripheral blood of monogastric animals that this organism infects (1). Listeria is recognized as a causative agent of the disease listeriosis, a zoonotic illness that affects animals and humans. Reports of organisms resembling Listeria first appeared in the scientific literature in 1891 when Hayem of France observed small gram-positive rod-shaped organisms in human tissue (1 ). This observation was repeated 2 years later (1893) by Henle, who was working in Germany. Hulphers, working in Sweden in 191l , reported the presence of gram-positive rod-shaped organisms in the livers of rabbits and designated the organism Bacillus hepatis. Complete characterization of the organism now recognized to be Lister-in rzlorlocytogelles was achieved by Murray et al. in 1926 (2). These investigators, working at Cambridge University, observed infection of an animal colony caused by an etiological agent that induced pronounced monocytosis and caused hepatic lesions. The name given to this agent was Bacterium nzorlocytogenes. Pirie (3), working in South Africa one year later, isolated a small gram-positive bacillus from the livers of gerbils, which he designated Listerella hepntoliticcr, in honor of Lord Lister (4). In 1940, the genus/species designation Listeria nlotzocytogerzes was proposed for this organism. A detailed historical chronology can be found in the classic review article on Listeria and listeric infection published by Gray and Killinger (1 ). Presently included within the genus Listeria are the species L. rnonocvtogenes, L. illnoem, L. seeliger-i,L. welslzimeri, L. ivnrzovii, and L. grclyi (Table 1). DNA base composition and DNA-DNA hybridization studies led Stuart and Welshimer ( 5 ) to propose a new genus M~wraycrto include M. g r q i and M. grayi subsp. murrrryi. However, more recent examination of the genomic relatedness of L. gmyi and L. rmrrayi based upon DNA/DNA hybridization and multilocus enzyme electrophoresis reveals that these organisms should be considered as members of the single species L. grnyi (6). Members of the

Table 1 Characterization of Listeria Species Characterlstic P-Hetnolysis CAMP S. ourells R. rqrri Fermentation of mannitol xylose rhamnose Extremely Extremely Pathogenic YesIn humans

L. monocyrogenes

L. i1mncwii

L. seeligeri

L, imocua

L. rr-elsllimeri

+ +

+

+

-

-

-

+

-

-

-

-

~

-

+

-

-

-

-

-

+

+

-

-

-

+-

+

No rare3 cases

rare1 case

+ t No

Listeria monocytogenes

215

genus Listeria can be easily differentiated on the basis of the following biochemical reactions: reduction of nitrates to nitrites, P-hemolysis; acid production from mannitol, Lrhamnose, and D-xylose; and the CAMP test (Table 1). L. nzorzocytogenes and L. irzrzocrrcr are very closely related, and recent studies have confirmed that within the 16s rRNA, only 2. of 1281 base pairs differ between the two species (73). Only one species within the genus Listeria is generally regarded as capable of causing illness in humans, this species being L. monocytogenes. Three reports of human infection caused by L. ilwnovii exist in the scientific literature (9,10), along with one report of a case of human illness caused by L. seeligeri (1 1,12). Thus, L. ilwzovii and L. seeligel-i are extremely rare causes of illness in humans, and the main species of concern in human health is L. vzonocytogenes. L. irzrzocua and L. welshirnel-i are not capable of causing illness. These nonpathogenic species are of interest from a food microbiology standpoint since their presence indicates the potential for the presence of thepathogenic L. morzocytogenes. Although 16 serotypes of L. monocvtogeizes have been identified, three serotypes (4b, 1/2a, 1/2b) are responsible for 96% of human infections in the United States (13). A similar survey of strains from cases of human listeriosis in Britain between 1967 and 1984 revealed 1/2, 3, and 4 as the predominant serogroups causing human infection (14). Serotyping has poor discriminating power and is therefore of limited value as a subtyping method when compared to more advanced methods of genetic analysis. Of all available phenotypic and genotypic typing methods, pulsed-field gel electrophoresis (PFGE) is the preferred method for subtyping of L. nzonocytogenes strains (15), offering a high degree of discrimination of Listeria strains as well as reproducibility.

B. Distribution L. nzonocytogenes is very widely distributed in nature. This organism can be readily isolated from soil, water, sewage, green plant material, decaying vegetation, and numerous species of birds and mammals, including humans (1). Although widely distributed within mammalian species, cattle, sheep, and goats are the domestic mammals most frequently afflicted by listeriosis (16,17). A close relationship between onset of listeriosis in ruminants and feeding of contaminated silage has long been recognized ( l 7-20). Palsson (2 l ) reported that in Iceland, the relationship between silage feeding and onset of listeriosis is so strong that the disease has been referred to as "votheysveiki," or silage sickness. Presence of Listeria in silage is strongly influenced by pH, and samples having a pH in the range of 5.0-6.0 or above are far more likely to be sources of L. monocytogerzes than silage where the pH is below 5.0 (19,22-24). The tnost common disease syndrome of listeriosis in ruminants is encephalitis, leading to observations of nervous system involvement in cattle and sheep. Afflicted animals have been observed as disoriented, causing them to circle endlessly in one direction or another depending upon the direction in which their head is drooped. For this reason, listeriosis in ruminants is often referred to as "circling disease'' (425). Previous investigations have identified sheep as a major reservoir of Listel-icr in nature. In one study alone, 88% of tested sheep were identified as carriers of some member of the genus Listeria (26). Gray (17) used serotyping techniques to demonstrate the relatedness of isolates obtained from listeric sheep and the oat silage that they consumed. More recent studies have used strain-specific ribotyping to support the link between on-farm sources of Listeria (silage) and subsequent contamination of dairy-processing environments (27). Infected animals displaying symptoms of listeric infection may

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excrete L. rnorzocvtogerzes in milk, blood, and feces. High excretion rates of L. monocytogenes in milk from asymptomatic cows and goats have frequently been reported (28).

II. LISTERIOSIS IN HUMANS

A. Characterization of the Illness Listeriosis Because of its ubiquity in the environment, humans frequently come into contact with L. rrlorlocytogenes.Mere exposure to this organism does not necessarily dictate that infection will result. L. rnonocytogerzes is frequently shed in the stools of healthy humans who otherwise show no signs of the illness, thus confirming the high exposure rate to this organism (29). It is estimated that 5% of healthy humans harbor L. Inonocytogenes in their gastrointestinal tract. The first case of human listeriosis was described in 1929 (30), and since that time listeriosis has been recognized as a rare but often fatal illness. In adults, the disease listeriosis is characterized by the onset of severe symptoms including meningitis, septicemia, primary bacteremia, endocarditis, nonmeningitic central nervous system infection conjunctivitis, and flu-like illness (fever, fatigue, malaise, nausea, cramps, vomiting, and diarrhea) (3 1). Gastrointestinal symptoms are observed in approximately one third of documented cases of listeriosis (32). Whether this is due to a concurrent illness that disturbs the gastric mucosa, allowing invasion of L. nzoIzocytogems, or it is a nonspecific response to systemic infection, or it is actually part of the disease spectrum of listeriosis is unknown (29). Humans shown to be at high risk for acquiring listeriosis include pregnant women, neonates, the elderly, organ transplant recipients, or those receiving immunosuppressive therapy. In the latter case, treatment of patients with corticosteroids or antimetabolites renders a suppressed immune system. Persons suffering from chronic disorders such as alcoholism, malignancy, diabetes, heart disease, or acquired immunodeficiency syndrome (AIDS) have also been shown to be at risk. Additional underlying factors that have been reported in association with listeriosis include sarcoidosis, chronic otitis, collagen-vascular disease, idiopathic thrombocytopenic purpura, asthma, ulcerative colitis, and aplastic anemia (3 1). Age has been shown to be a predisposing factor in listeriosis. An 11% casefatality rate is documented in persons age 40 or under; a 63% case-fatality rate is recorded for persons over age 60 (29). Age-related reasons for increased incidence of listeriosis may include a decline of the immune system as a function of age, increased prevalence of immune-suppressive disorders, and increased dependence on immune-suppressive medications (29). Although the above-listed conditions may predispose patients to acquiring listeriosis, it should be noted that persons showing no apparent immunocompromising conditions have been shown to acquire listeriosis. The most recent estimates indicate that on an annual basis, approximately 1092 cases and 248 deaths occur due to listeriosis in the United States (13). This illness, while rare, has a case-fatality rate ranging from 23 to 35% (29,33).

B. Mechanisms of Defense Against Listeria in Normal, Healthy Hosts Cell-mediated immunity (CMI) plays an important role in dictating the resistance or susceptibility of a human host to infection by L. monocytogenes (34-36). Listeriosis occurs

Listeria monocytogenes

217

most often in persons with impaired CMI. CM1 is dependent upon the activity of mononuclear phagocytes as early response, nonspecific effectors, and specific T cells as a secondary response to infection. Since the response of T cells to infection takes time, resistance expressed at an infection site is most likely T-cell independent and is mediated by resident macrophages, which limit early bacterial proliferation (34). Thereafter, the CM1 response becomes sufficiently developed to effect control and ultimately eradication of listeriosis (37). In early phase host defense, macrophages function as primative scavenger phagocytes; later, macrophages function as final effector cells in lymphokine-mediated cellular immunity and in antibody-dependent cell-mediated cytotoxicity (38). Specific response to infection results in an increase of macrophages at the site of infection due to either the proliferation of resident macrophages or circulating blood monocytes, which are attracted to the infection site by substances produced by T cells (39-42). Polymorphonuclear leukocytes (PMNs) also contribute to phagocytosis and killing of Listeriu (34), and in the acute stages of infections by facultative intracellular bacteria, infected tissues are infiltrated by PMNs (43). As infection progresses, the number of macrophages at the infected site increases. With recognition of a microbial (listerial) antigen, macrophages become "activated," resulting in enhanced antimicrobial activity against that specific antigen as well as against nonrelated pathogens (44,45). Activated macrophages contain more lysosomes, display increased oxidative metabolic activities, and are more phagocytic (42,46). The accumulation of macrophages at the site of infection results in lesions structured in granulomas that contain mostly lymphocytes and macrophages. Within granulomas, the cells are tightly packed and their closeness allows cell to cell interactions, enhancing the destruction of Listeria (34). Two other roles of macrophages involve the production of interleukin-. 1 (leukocyte-activating factor), which specifically alters T-cell function, and the processing and presentation of immunologically active molecules to the lymphocytes (45). In work with murine models, nonspecific host defense mechanisms act against L. monocytogerzes in the first 2 days following challenge with Listeria (47). Within 10 minutes of injecting mice with a sublethal dose of L. monocytogenes, the organisms are trapped by the liver and spleen. Resident tissue macrophages in the liver destroy most Listeria, but surviving bacteria multiply within liver and spleen macrophages and are not inactivated until development of acquired cellular resistance. The main function of Listeria-sensitized T cells is to attract, focus, and activate macrophages at infective sites (43) by liberation of macrophage inhibitory factor (MIF), which encourages circulating blood monocytes to localize at the infection site, and by liberation of macrophage activating factor (MAF), now called y-interferon, which activates macrophages to enhance their phagocytic activity (34,48,49). Activation of T lymphocytes leading to acquired cellular immunity involves three distinct stages (50). Stage one involves the priming of prekiller T cells and the generation of Listeria antigen-specific helper T cells. In the second stage, helper T cells are stimulated by Listeria antigens to release a soluble substance or lymphokine (interleukin-2), which is a lymphocyte-activating factor. This causes helper cells to grow and proliferate and stimulates cytotoxic types (48). In the final phase, interleukin-2 converts the prekiller T cell into a L. nlorrocytogenesdependent cytotoxic T lymphocyte, which rapidly destroys the organism. Cytotoxic suppressor cells appear to be more important in eradication of Listeria than helper T cells (43). T-cell-deficient nude (athymic) mice have been shown to limit listeriosis to a chronic infection by relying on resident macrophage populations (37).

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In summary, primary infection with L. nlonocytogenes in a healthy human causes colonization of the liver and spleen. The dissemination from the infection site occurs through the lymphatic pathway. There is a short period of bacterial growth, which is stopped by the arrival of activated macrophages from the blood and peritoneal cavity. Thymus-derived leukocytes enhance the macrophage action, which results in elimination of bacteria and establishment of a cell-mediated resistance to further infections.

C. Listeriosis in lmmunocompromisedHosts Alteration of T-cell or macrophage function due to immunosuppression would result in an impairment of cell-mediated immunity allowing a listeric infection to occur after the primary infection or after further contact with Listerin. Patients with cancer (51) or undergoing treatment with steroids or cytotoxic drugs (46,52), pregnant women or neonates (1,53,54), renal transplant recipients, and elderly or alcoholic patients (29,31) are well known to be the primary target for listeriosis. The risk of complications due to L. nzonocytogenes infection has long been recognized in CMI-compromised conditions such as Hodgkin’s lymphoma and other hematological malignancies (55). North (40) suppressed cell-mediated immunity of mice to L. monocytogems by administration of an antimitotic drug and induced a listeric infection. Golnazarian et al. (56) showed that the L. monocyfogenes infectious dose for mice immunocompromised by administration of hydrocortisone acetate was much lower than for normal resistant mice. Studying the regulation of macrophages in mice with combined immunodeficiency (lack of T- and B-cell function), Bancroft et al. (57) demonstrated that, although numbers of macrophages were almost normal after primary infection, mice developed chronically high loads of bacteria. The same state of chronic listeriosis was noted in an experiment done by Newborg and North (37). 1. Pregnancy and Listeriosis During pregnancy, selective factors of CM1 become depressed to prevent rejection of the fetus by the mother. However, depression of these selective factors may result in decreased maternal resistance to L. nlorlocytogenes infections and thereby increase the maternal or fetal risk to onset of listeriosis (58). Such selective factors include shifts in levels of hormones or serum factors that affect lytnphocyte or macrophage synthesis, activation, or function during pregnancy (54). Plasma levels of hydrocortisone increase during pregnancy to levels three to seven times higher than those found in nonpregnant humans (54). Corticosteroids are known to suppress both lymphokine activation and phagocytic activity of macrophages. Low levels of IgM and decreased activity of the classic complement pathway during the neonatal period also occurs and demonstrates the importance of opsonization in the immune response to Listeria (29j. In humans, listeriosis occurs most often during the third trimester of pregnancy. Three outcomes are normally followed: an asyptomatic maternal infection and a resulting infected infant, a severely ill mother who enters premature labor and delivers a stillborn or severely ill infant, or an unaffected fetus with death of the mother (54). In most perinatal cases of listeriosis, the mother is usually mildly affected, exhibiting flu-like symptoms, but neonatal morbidity and mortality are common. In early-onset neonatal listeriosis, transplacental infection results in a syndrome known as granulomatosis infantisepticum, a necrotic disease of the internal organs (59). Spontaneous abortion of the fetus or stillbirth of the neonate are most common, but if the fetus infected in utero is born alive, recovery is not likely ( 1,59). Late-onset listeriosis occurs several days after birth, and infants are generally full-term and healthy at birth. Late-onset listeriosis is more likely than early-

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onset listeriosis to present as meningitis, and case fatality rates are lower than for earlyonset infection. Of late-onset cases reported in Britain during 1967-1985, 93% of cases presented evidence of central nervous system infection (60). Buchdahl et al. (61) described several cases in which complications due to listeriosis arose during the course of pregnancy. In one case, a mother at 32 week’s gestation developed a flu-like illness and developed irregular uterine contractions. Spontaneous membrane rupture occurred with release of meconium-stained amniotic fluid. The infant, delivered by ceasarean section, was found to have blood and cerebrospinal fluid contaminated with L. nzorzocytogerzes. Although the infant survived, neurological handicap was evident. The mother recalled consumption of a soft-ripened French cheese 9 days prior to delivery. 2. Listeriosis in Renal Patients Listeriosis is well recognized as a complication of renal transplantation. Most patients become ill while they are receiving immunosuppressive therapy, which increases their susceptibility to listeriosis (62). Meningitis is recorded as the most common presentation of listeriosis in renal transplant patients, and the fatality rate for listeria1 meningitis in renal transplant recipients is 38%. However, pneumonia due to L. mor~ocytogerzeshas also been observed in renal patients, suggesting a possible respiratory route of transmission (62). In a study of healthy renal transplant recipients, fecal carriage of L. rnorzocytogenes in 8 of 37 patients was documented (62). In a review of 83 cases of listeriosis in renal patients, Stamm et al. (62) found that one third of patients had been treated for acute rejection. Nieman and Lorber (31) have reported that hemodialysis is not a predisposing factor for most patients in acquiring listeriosis. However, Mossey and Sondheimer (63) reported four cases of L. monocytogerzes bacteremia associated with long-term hemodialysis and transfusional iron overload. None of these patients were receiving immunosuppressive therapy. Many surveys document a higher incidence of listeriosis in the tnonths from July to October, and the same seasonal variation has been reported for renal transplant recipients (62). 3. Listeriosis and Patients with AIDS Patients with AIDS exhibit an impairment of T-cell-mediated immune response and would therefore be expected to be at high risk for listeriosis. In early studies of the incidence of listeriosis in AIDS patients, L. morlocytogenes was rarely implicated as an agent affecting persons with AIDS (47). Reasons given for this surprising finding included the fact that AIDS patients who displayed frequent gastrointestinal tract infections were given multiple courses of antibiotic therapy, thus decreasing exposure to Listericr (47,64). Five cases of listeriosis in Los Angeles county between January 1985 and March 1986 occurred in patients with AIDS. Prior or concurrent gastrointestinal illness was recorded in three of the patients, and four patients had no history of prior antibiotic administration. This and subsequent investigations have shown that while listeriosis is a rare infection in patients exhibiting HIV infection, persons with AIDS have a 300- to 1000-fold increased risk of acquiring listeriosis compared with the general population (29,65). Persons with AIDS are therefore advised to refrain from ingestion of food items associated with listeriosis (64).

111.

PATHOGENESIS OF LISTERIA MONOCYTOGENES

The production of sulfhydryl-activated hemolysin, listeriolysin 0 (a-listeriolysin), is associated with the pathogenic potential of L. monocytogenes (66-70). Listeriolysin 0 is simi-

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lar to streptolysin 0 and pneumolysin, and antigenic cross-reactivity with these hemolysins as well as with hemolysins produced by L. ivanovii and L. seeligerihas been demonstrated (69,71). The virulence of Listeria species has also been associated with the ability to survive and grow intracellularly (72,73). Gaillard et al. (67,68) studied the role of hemolysin (listeriolysin 0)in pathogenicity of L. morzocytogenes. Working with transposon Tn1545, these investigators inactivated a genetic determinant for hemolysin production and obtained nonhemolytic mutants from hemolysin-producing strains. The loss of hemolysin production was shown to be associated with loss of virulence in a mouse model. The spontaneous loss of the transposon restored hemolysin production and virulence to the revertants (67). Further studies by Gaillard et al. (68) found that L. monocytogerm and L. ivnnovii invaded a continuous gut epithelial cell line, whereas L. seeligeri, L. i~nocua,and L. welskimeridid not. A nonhemolytic mutant of L. monocytogenes invaded these gut enterocytes at the same rate as the hemolytic wild type (67). This finding, which has been corroborated by others in a fibroblast 3T6 continuous cell line (74), demonstrates that listeriolysin 0 is not involved in invasion. Studies have been done that identified an extracellular protein (p60) that may be involved in the process of attachment and invasion of L. mojzocytoge1zes (75). Both listeriolysin and p60 are produced by all virulent L. morzocytogenes strains and are involved in the virulence of L. nrorzocytogenes(74,75). Under heat-shock conditions, listeriolysin is synthesized, whereas production of p60 no longer occurs (66). Protein p60 is found as both a major secreted protein, as well as on the cell surface of all L. monocytogems isolates. In addition, this protein possesses murein hydrolase and is involved in cell division. Rough mutants of L. nzor1ocytogenes that lack p60 form long chains of cells, which fail to separate. These mutants also show reduced uptake by 3T6 fibroblast cells (75). Factors other than hemolysin have recently been defined as essential virulence factors for Listeria monocytogenes. Hof and Rocourt (12) found that a construct of a virulent L. nzonocytogems EGD with selective blockade of phospholipase C production became avirulent. Tilney and Portnoy (76) demonstrated that L. r~onocytogenesis capable of bypassing the humoral immune system by remaining in an intracellular state and spreading from cell to cell. Following phagocytosis by host macrophages and escape from the phagocytic vacuole, Listeria are coated with actin filaments, form a pseudopod, dissolve the phagocytic vacuolar membrane, presumably by use of hemolysin, and repeat the cycle. It was postulated from this and other studies (72-74) that once Listeria enters macrophages, listeriolysin 0 is needed to lyse phagosomes, thereby releasing Listeria into the cytoplasm so that it can multiply. Listeria that lack hemolysin fail to grow in vivo because of an inability to dissolve the endosomal membrane and failure to escape from the endosome into the cytoplasm (73,76). L. rnonocytogenes can enter host cells in two distinct ways: through active ingestion by phagocytic cells such as macrophages, or through the production of specific gene products that control ingestion by normally nonphagocytic cells (68,76). Studies conducted by Gaillard et al. (77) identified a surface protein of L. monocytogerles, intemalin, which mediates bacterial invasion of epithelial cells. Once internalized, the life cycle of L. monocytogenes within both phagocytic and nonphagocytic cells is similar. The majority of virulence genes that produce products associated with the intracellular life cycle of L. rnonocytogenes reside on a region of the chromosome known as the PrfA-dependent gene cluster (78). This region is comprised of the genes prfA, plcA, hly, mpl, actA, and plcB. The prfA gene product is a positive regulatory factor, a 27 kDa regulatory factor that controls all virulence genes of the virulence gene cluster. The plcA product is phosphati-

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dylinositol-specific phospholipase C (PI-PLC), which contributes to vacuole escape in cells such as bone maTow-derived macrophages. The plcB gene encodes for a phosphatidylcholine-specific phospholipase C (PC-PLC), which, together with metalloprotease, the mpl gene product, enables listeriolysin-o independent escape of L. rnorzocytogerzes from primary vacuoles in human epithelial cells. Metalloprotease (mpl) pennits bacterial movement from the cytosol to the host surfaces and the ensuing cell-to-cell spread (76). The act A gene locus is responsible for the accumulation of actin around Listeria in the cytosol. The lack of one of these determinants has been shown to interfere with the pathogenicity of L. morlocytogerzes (1 1). Cowart (79j showed that hemolysin activity is stimulated in iron-deprived medium; cytolytic activity of hemolysin is maximally expressed at pH 5.5. Therefore, it can be assumed that when L. nlonocytogenes are engulfed in phagosomes that do not contain iron and have a pH value around 5.5, hemolysin production is maximized, allowing destruction of internal membranes surrounding them. An additional mechanism of intracellular survival is dependent upon theability of L. molzocytogenes to resist killing by oxidizing agents produced by phagocytes. The production of superoxide disrnutase (SOD) and catalase by L. rnonocytogenes has been associated with intracellular survival (72,80). Bortolussi et al. (72) demonstrated that resistance of L. monocytogelzes to hydroxyl radical (OH) during the log phase of growth was due to the production of sufficient amounts of catalase to inactivate this product. Welch et al. (80) found that catalase-negative strains of L. monocvtogerzes possessed at least twofold greater SOD activities than catalase-positive strains. Not all strains of L. monocytogenes are pathogenic. Further, within L. rnotzocytogenes strains, there may be particular serotypes that possess enhanced virulence potential. A survey conducted by Pinner et al. (81) showed that foods containing L. molzocytogenes serotype 4b were four times as likely to contain strains identical to patient strains than were foods containing serotypes 1/2a or 1/2b. These and other observations suggest that serotype 4b strains may have an enhanced capacity to cause human disease (81,82). Comparative virulence of Listerin strains is typically assessed on the basis of mouse lethal dose (LD,O)following intravenous or intraperitoneal inoculation (83,84). Numerous investigators have attempted to develop in vitro assays to measure virulence potential. Farber and Speirs (85) found that culture filtrates of L. morzocytogenes and L. ivarzovii were cytotoxic to eight tested continuous cell lines, whereas cell filtrates from L. innocucr had no effect on these cell lines. Pine et al. (83) found that use of human continuous cell line Caco-2 stained with trypan blue could differentiate virulent from avirulent L. r7lonocytogenes.

IV. FOODBORNETRANSMISSIONOF LISTERIA MONOCYTOGENES A.

MajorFoodborneDiseaseEpidemics

Listeria nzorlocytogenes has emerged as a foodborne pathogen of major significance within the last decade. A number of foodborne disease outbreaks worldwide have been linked to consumption of food products contaminated with L. morzocytogerles. This information has been summarized and is presented in Table 2. In 1979, listeriosis was diagnosed in at least 23 hospitalized patients in the Boston area (86). The vehicle of infection in this outbreak was linked to hospital food, and patients who had consumed lettuce, carrots, and

2

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9

5 S:

a

h

=!

3

a

c e Tt

e

2 3

t! CS t N

.d

a

d-

m

\o

S

9 4

m

Y

W

.4

3

5 m m m 00

m m 3

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radishes were more likely to contract the illness. Isolates from 20 of 23 cases were identified as serotype 4b. Symptoms reported by the afflicted patients included bacteremia or meningitis. Fifty percent of the patients involved in this outbreak were immunosuppressed because of cancer, chemotherapy, or steroid treatment. Curiously, 60% of the afflicted patients had reported the use of antacids or the antiulcer medication cimetidine. Cimetidine is a histamine-2 antagonist that blocks the H? effects of histamine, thereby decreasing gastric acid secretion (87), whereas antacids neutralize gastric acid. Itwas found that patients who had consumed antacids or cimetidine were more likely to develop hospitalacquired infection caused by L. monocytogenes. It was hypothesized that gastric acid neutralization following use of antacids or cimetidine predisposed humans to acquiring listeriosis as a result of ingestion of this organism via a foodborne vector. This finding has been corroborated in later studies, where patients were more likely than controls to have used antacids, laxatives, or H,-blocking agents prior to onset of listeriosis (88). In 1981. an outbreak of listeriosis occurred in the Maritime Provinces of Canada (89). The vehicle of transmission was identified as commercially prepared coleslaw. Cabbages used to prepare coleslaw were traced to a sheep farm where an outbreak of listeriosis had killed several sheep. Use of manure from infected sheep was suspected as a factor in this outbreak. Thirty-four cases of listeriosis in pregnant women resulted in spontaneous abortions, stillbirths, or live birth of ill infants. Seven nonpregnant adults who showed no evidence of immunosuppression had symptoms of meningitis, aspiration pneumonia, and sepsis. The overall mortality rate for this outbreak was 41%. All patient isolates were identified as serotype 4b, and L. monocytogenes isolates from unopened packages of coleslaw were also identified as serotype 4b. In 1983,49 patients in Massachusetts were diagnosed with listeriosis (90). Epidemiological evidence pointed to a strong association between consumption of pasteurized whole and 2% milk and onset of the illness. Forty-two patients were characterized with underlying illnesses such as cancer or alcoholism, and several patients were undergoing corticosteroid therapy. Seven of the cases involved fetuses or infants. The overall casefatality rate in this outbreak was 29%. Of 49 isolates available for serotyping, 32 were identified as serotype 4b. Despite numerous attempts, the epidemic serotype of L. monocytogerzes responsible for this outbreak was never recovered from the incriminated milk. The consumption of soft ripened cheese accounted for two major illness outbreaks of listeriosis in the 1980s. The first documented link between cheese consumption and an outbreak of listeriosis was reported in Orange County, California, in 1985 (91). In this outbreak, consumption of Jalisco-brand Mexican-style cheese was linked with the onset of 142 cases of listeriosis. The outbreak occurred during the period January l-August 15, 1985. Ninety-three (65.5%) of the cases were recorded in pregnant women or their offspring, while 49 cases were reported in nonpregnant adults. In all, 48 deaths resulted from this outbreak. A majority of the afflicted individuals (62%) were pregnant Hispanic women. In this outbreak, the cheese was most likely manufactured from a combination of raw and pasteurized milk, and the cheese plant that manufactured the incriminated cheese was found to be contaminated with Listeria. The epidemic strain was identified as L. moizocJjtogenesserotype 4b, and this serotype was recovered from unopened packages of Queso Fresco and Cotija Mexican-style cheese. A second outbreak of foodborne listeriosis linked to cheese was reported by Bula and coworkers (92). This outbreak occurred in Vaud, Switzerland, and was linked to consumption of Vacherin Mont D'or cheese. A total of 122 cases occurring during the period 1983-1987 were reported. The normal endemic rate of listeriosis in Switzerland is 5-10

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cases per million persons. During the outbreak period, the rate of listeriosis rose to 50 cases per million persons. Sixteen cases were reported in 1983,24 cases in 1984, 13 cases in 1985, 28 cases in 1986, and 41 cases in 1987. A mortality rate of 28% was associated with these cases. Of clinical isolates available from the epidemic period, 111 of 120 (93%) were serotype 4b of two unique phage types, and 85% of these strains matched the epidemic phage types isolated from Vacherin Mont D'or cheese. Dalton et al. (93) reported on an outbreak of listeriosis linked to chocolate milk served at a picnic. Forty-five individuals developed illness due to L. morzocytogenes characterized by fever and gastroenteritis without progression to invasive disease. Further, none of the patients reported immune deficiency or chronic illness and only one patient was pregnant and delivered a healthy baby. The median dose of Listerin consumed by patients may have been as high as 2.9 X 10" CFU per person. Proctor et al. (94) used PFGE to link four additional sporadic cases of invasive listeriosis to recalled 1% low-fat chocolate milk responsible for the previously mentioned outbreak. At the time of this writing, two active multistate outbreak investigations involving L. nzo;?ocytogerzes were reported by the Centers for Disease Control and Prevention (95,96). The first investigation of an 1l-state outbreak involving hot dogs and deli meats identified over 50 illnesses occurring during the period August 1998-January 1999 due to a single strain of L. monocytogerzes. This outbreak prompted a major nationwide recall of incriminated product. A second multistate outbreak involved a different strain of L. nzonocytogenes (serotype 4b), which was identified in 11 cases involving four states. These cases of illness occurred during August 1998-December 1998.

B. Sporadic Cases of Listeriosis with Foodborne Etiology Unlike the epidemics described previously, human listeriosis occurs most often as a sporadic illness (29). Information on whether the majority of these sporadic cases result from foodborne transmission is being provided by ongoing and active disease surveillance. For instance, from September to June 1987, the CDC conducted a population-based active surveillance for L. nlonocytogenes infections (97). This surveillance involved 154 patients from six regions of the United States (New Jersey, Missouri, Oklahoma, Tennessee, Washington, and Los Angeles). From this data, it was estimated that approximately 1700 cases of listeriosis occurred in the United States in 1986, for an annual incidence rate of 7.1 cases per million persons. Epidemiological evidence suggested that consumption of contatninated foods accounted for 30 of the 154 cases (20%) of listeriosis reported. Two foodborne sources were epidemiologically linked with onset of illness, these being uncooked hot dogs and undercooked chicken. As a result of this active surveillance, a recall of turkey franks commenced after being linked with the death of a patient in Oklahoma (98). L. r7zorzocytoge;zes serotype 1/2a strains of identical isoenzyme types were isolated from the patient as well as unopened packages of turkey franks (98). Between November 1, 1988, and December of 3 1, 1990, the Centers for Disease Control (CDC) conducted a second major case-control study in order to identify dietary risk factors for sporadic listeriosis (33). The population base in this active surveillance was in excess of 18 million persons distributed within five geographic regions of the United States. Cases were enrolled from patients identified through active surveillance. Underlying patient conditions included pregnancy, steroid therapy, cancer, renal dialysis, diabetes, HIV infection, liver disease, and organ transplant recipients. Three hundred and

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one cases of listeriosis were confirmed in this study. Foods in the refrigerators of patients were examined for presence of Listeria. Sixty-four percent of refrigerators (79 out of 123 examined) yielded a L. nzonocytogenes isolate. Of 2229 foods examined, 11% were positive for L. monocytogenes (81). Serotypes 4b, 1/2a, and 1/2b accounted for 95% ofL. monocytogenes isolates recovered from foods. Of the L. nzonocytogenes-positive foods, 33% matched the patient isolates. Sixty-seven percent of dairy isolates matched patient strains, implicating dairy products as sources of L. monocytogenes. Specific dairy product sources included Mexican-style cheese, feta cheese, and commodity cheeses. Thirty-two percent of sporadic cases of listeriosis could be attributed to eating foods purchased from delicatessan counters, Mexican-style and feta cheeses, and undercooked chicken (33). Preliminary CDC data for 1991 suggested a decrease of 30-40% in the number of cases compared with 1989-90. The annualized sporadic incidence for listeriosis was found to be 7.4 cases per million population, with an overall case fatality rate of 23%. Serotypes 1/2a (23%) and 1/2b (36%) together accounted for 59% of the cases; serotype 4b was isolated from 37% of the patients (33). Tapper0 et al. (13) reported a 44% decrease in rates of invasive listeriosis and a 48% decrease in the numbers of deaths due to listeriosis in the United States from 1989 to 1993. There were 1092 cases of listeriosis reported in 1993, resulting in 248 deaths, for an overall annual incidence of 4.2 cases per million population. Case-fatality rates remained similar (25% in 1989 compared to 23% for 1993). The decreased incidence rate was attributed to food industry efforts, sustained prevention efforts, and continued active surveillance.

C. OtherReports of FoodborneListeriosis:Epidemic and Sporadic Consumption of pat6 was cited as a likely cause of an epidemic of human listeriosis in the United Kingdom and the Republic of Ireland between 1985 and mid-l989 (99,100). The endemic rate of listeriosis in this region for the period between 1967 and 1982 was fewer than 100 cases annually. In 1987, the number of cases increased to 258; in 1988, 291 cases were reported, and in 1989, 250 cases were reported. The number of cases reported in 1990 showed a dramatic decrease to 90 cases, indicating that the outbreak period had ended. Food histories obtained during patient interviews revealed a significant association between pat6 consumption and onset of infection by L. monocytogenes. Surveys of the incidence of L. morzocytogelzes in pat6 revealed that pat6 from a single plant was more likely to be contaminated at a rate (48% positive) higher than that of other producers (4% positive). Two subtypes of L. monocytogenes, serotype 4b phage type 6,7 and serotype 4bX, accounted for the majority (30-54%) of the human cases. These subtypes accounted for 96% of all isolates of L. monocvtogenes from the pat6 produced by a single manufacturer. Raw fish and shellfish were epidemiologically linked to an epidemic of perinatal listeriosis in Auckland, New Zealand (101). A point source foodborne outbreak was epidemiologically linked with consumption of shrimp (102). Most patients suffered mild illness, with the exception of one woman who suffered miscarriage as a result of infection. An additional case of sporadic listeriosis was linked to consumption of contaminated fish (103). In 1983, a cluster of cases of perinatal listeriosis was reported in nine Hispanic mother-neonate pairs in Houston, Texas (104). All patient isolates from this outbreak were

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identified as serotype lb. The cause of this outbreak was unknown. This case was suspected to be of foodborne etiology, but attempts to identify a common source of exposure were unsuccessful. In Vermont, a case of listeriosis in a 76-year-old female patient was reported (105j. The patient presented mild respiratory congestion and chronic renal failure secondary to pyelonephritis upon admission to the hospital. Raw milk from the patient’s farm was incriminated as the vehicle of infection in this case of sporadic listeriosis. Isoenzyme typing and ribosomal RNA typing demonstrated that the raw milk isolates from the farm were indistinguishable from the patient isolates, and all isolates were confirmed as L. nlonocytogerles serogroup 112a. Two common source outbreaks of listeriosis were identified in Denmark between 1989 and 1990, which were epidemiologically linked to consumption of Danish blue-mild and hard cheeses (106). Jellied pork tongue was linked to a 1992 outbreak of listeriosis reported in France. This outbreak involved 279 cases, which resulted in 63 deaths and 22 spontaneous abortions (107). One year later (1993), a second meat-related outbreak was reported in western France linked to consumption of pork “rillettes.” A single strain of Listeria from this outbreak was linked to 39 cases of illness (108). In 1995, an epidemic of listeriosis occurring during the period January through July was linked to consumption of Brie de Meaux cheese (109). Bibb et al. (1 10) developed a multilocus enzyme electrophoresis (MEE) system for L. rnonocyfogerzes,which has proved useful in linking foodborne and clinical isolates of L. rnonocytogems. Baloga and Harlander (1 11) found that patient and product isolates were not distinguishable by serotyping, ribotyping, or multilocus enzyme electrophoresis. DNA fingerprinting proved to be the only method capable of proving relatedness of pairs of patient-product isolates. As mentioned previously, although phage typing has been successfully applied in epidemiological investigations, not all strains can be typed by this method. Pulsed-field gel electrophoresis is accepted as the preferred method to confirm relatedness of patientlfood product isolates (15 ) .

V.

SOURCES OF LISTERIA IN FOODS AND FOOD-PROCESSING ENVIRONMENTS

A host of unique properties possessed by Lisferiu render this a difficult organism to control in foods. Listeria can grow over a wide range of temperatures (- 1.5 to 45-50°C) (1 12114) and pH ranges (4.3-9.6) (115,116), survives freezing (117,118), and is relatively resistant to heat (90,119-127). Minimal water activity levels for growth of L. nzor1ocytog e r m and L. imzocna range from 0.90 to 0.97 (128,129). Shahamat et al. (130) reported survival of L. morzocytogerlesfor 132 days at 4°C in trypticase soy broth containing 25.5% NaCl. Lister-io is a psychrotrophic pathogen, and growth at temperatures as low as -0.1 to -0.4”C in chicken broth and pasteurized milk (13 1) and at - 1.5”Cin vacuum-packaged meat (114) has been recorded. The ability of L. morlocyfogenes to resist the heating temperatures used during milk pasteurization continues to be debated in the scientific literature (90,119-127). Fleming et al. (90), in their studies of an outbreak of listeriosis in Boston, concluded that “intrinsic contamination of the milk and survival of some organisms despite adequate pasteurization is both consistent with the results of this investigation and biologically plausible.’’ Fleming et al. reached this conclusion based upon the fact that milk involved in the outbreak came

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from farms where outbreaks of listeriosis had occurred, there was no evidence of improper pasteurization or postpasteurization contamination of the processed milk, and evidence in the scientific literature demonstrated the ability of L. rnonocytogenes to survive pasteurization (119,123,132). Comprehensive studies conducted by the U.S.Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA) (121,122) and by Health and Welfare Canada (125) have shown that Listeria is unable to survive normal conditions of milk pasteurization. Knabel et al. (127) found that growing L. monocytogenes at 43°C prior to heat inactivation caused an increase in thermotolerance, but a study conducted by Farber et al. (125) demonstrated that even under worst-case scenario conditions, which included cultivation of L. monocytogenes populations at 43°C prior to inactivation, pasteurization would render a 4.5 to 6.2 D process. Lovett et al. (133) and Beckers et al. (134) estimate that extremely low levels of L. nzonocytogerzes (0.5- 1.O ListerialmL) exist in commercial bulk tank raw milk. Therefore, while populations of L. monocytogems have been shown to survive minimum pasteurization treatments of 7 1.1“C/ 16 S in various laboratory studies, survival under actual conditions of commercial milk pasteurization and processing is unlikely. Listericr contamination of processed dairy products is most likely a function of postpasteurization from the dairy plant environment, and numerous surveys (1 35-138) document presence of Listeria within the dairy plant environment. Sources of Listeria within the dairy plant environment include floors in coolers, freezers, and processing rooms, particularly entrances; cases and case washers; floor mats and foot baths; and the beds of paper fillers (135). Pritchard et al. (139), in a study of dairy-processing facilities, found that those processing plants having a farm contiguous to the processing facilities had a significantly higher incidence of Listeria contamination than farms without an onsite dairy farm. Arimi et al. (140) used ribotype analysis to demonstrate the link between on-farm sources of Listericr contamination (dairy cattle, raw milk, and silage) and subsequent contatnination of dairy-processing environments. Raw milk is a well-recognized source of Listeria, and for this and numerous other microbiological reasons, consumption of raw milk should be avoided (105,134,141). Studies by Ryser and Marth (142-144) examined the fate of L. monocytogenes during the manufacture of cheddar, Camembert, and brick cheese. Rapid growth of Listeria to populations of 5 X lo7 CFU/mL is observed in Camembert cheese, which has a pH that increases during ripening, thereby creating a favorable growth environment for Listeria (143). In contrast, Listeria populations show a marked decline in viable population levels during ripening of cheddar cheese. However, population levels do not decline to undetectable levels. Current U.S. regulations call for cheese made from raw or subpasteurized milk to be ripened at 1.7”C (35°F) for at least 60 days prior to sale. Ryser and Marth have shown that aging alone will not ensure the production of Listerin-free cheddar cheese (142). Madec et al. (145) used the “Bactocatch” process (tangential membrane filtration) to eliminate L. monocytogelzes from raw milk. Treatment of skim milk at 50°C in conjunction with the “Bactocatch” process resulted in a 99% decrease in Listeria populations. This technique may enable elimination of Listeria during cheddar cheese manufacture. Genigeorgis et al. (146) evaluated the ability of 24 types of market cheeses to support growth of L. tnonocytogerzes. Cheeses able to support growth of L. monocvtogenes included soft Hispanic-type cheeses, Ricotta, Teleme, Brie, Camembert, and cottage cheeses (pH range 4.9-7.7). Cheeses not supporting growth and that resulted in gradual death of L. rnorzocytogelzes included Cotija, cream, blue, Monterey jack, Swiss, cheddar, colby, string, provolone, Muenster, feta, and Kasseri (pH range 4.3-5.6). A correlation was ob-

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served between growth of Listeria in cheeses having a pH of greater than 5.5 and in cheeses manufactured without a starter culture. Ryser et al. (147) examined the fate of L. monocytogenes during manufacture and storage of cottage cheese. L. molzocytogenes survived in both creamed and uncreamed cottage cheese during 28 days of storage at refrigeration temperatures and was recovered in higher numbers from creamed (pH 5.32-5.45) versus uncreamed (pH 5.12-5.22) cottage cheese. Hicks and Lund (148) examined the ability of L. monocytogenes to survive in creamed cottage cheese when stored at 4, 8, or 12°C for 14 days. The organism survived, but failed to increase in numbers, during storage in product with a pH range of 5.064.53. Chen and Hotchkiss (149), however, found that L. monocytogenes grew in cottage cheese stored at 7°C for 16 days or 4°C for 63 days, but would not grow under these conditions when modified CO2 packaging conditions were used. Conner et al. (150) investigated the effects of temperature, NaC1, and pH on the growth of L. monocytogenes in cabbage. Results indicated that cabbage juice provided a good substrate for growth of Listeria. The organism was found to survive well at 5°C even in the presence of 5% NaC1, and the organism could grow and tolerate a pH of less than 5.6. This study, together with the findings of Schlech et al. (89), confirms that cabbage can serve as a vector of transmission of L. monocytogenes to humans and demonstrates the potential for L. monocyfogenes to persist and proliferate on vegetables and in brines used to ferment vegetables. Potatoes and radishes have been identified as sources of L. monocytogenes during retail food surveys (15 1). Additional studies have confirmed growth and survival of L. monocytogenes on asparagus, broccoli, cauliflower, corn, green beans, lettuce, and radishes (152,153). During initial studies to prove efficacy of the USDA testing procedure for recovery of L. monocytogenes in meat and poultry products, McClain and Lee (154) recovered L. monocytogenes from 40% of analyzed frozen ground beef samples and from 52% of sampled pork sausage. Subsequent surveys (155) confirmed the presence of L. monocytogenes in 14 of 49 (28.6%) samples of domestic cooked beef, roast beef, and cooked corned beef. Glass and Doyle (156) examined the ability of L. monocytogerzes to grow on a variety of processed meat and poultry products. Growth and survival correlated well with product pH. Summer sausage (pH 4.8-5.2) supported poor growth of Listeria; cooked roast beef (pH 5.8) supported only slight growth of L. monocytogenes. L. monocytogenes grew well on those products having a pH near or above 6.0 (ham, bologna, and bratwurst). L. monocytogenes populations grew extremely well on sliced chicken and turkey, increasing by lo3lo5 CFU/mL during 4 weeks of storage at 4.4"C. Raw poultry is a well-recognized source of L. monocytogenes, and numerous surveys have confirmed the presence of L. monocytogenes in retail poultry samples (157,158). Bailey et al. (159) recovered L. monocytogenes from 23% of sampled broiler carcasses, the most prevalent serotype isolated being 1/2b. Ready-to-eat poultry products have been implicated as vectors of transmission of listeriosis to humans. Cooked-chilled chicken and turkey frankfurters were confirmed as vehicles of Listeria infection in England and the United States, respectively, during 1988 and 1989 (98,160). Gilbert et al. (161) confirmed the presence of L. monocytogenes in 12% of precooked, ready-to-eat poultry products collected from London-area retail establishments between mid-November 1988 and midJanuary 1989. Survival of L. wonocytogenes on chicken breasts processed by moist and dry heating methods has been demonstrated (162). Wenger et al. (163) examined a production facility that manufactured turkey franks in order to determine sources and incidence of contamination. L. monocytogenes was

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isolated from only 2 of 41 environmental samples from the plant, which included a cooler room floor and a conveyer belt attached to a peeler. Yet L. monocytogenes was isolated from six of seven retail lots of product produced over a 37-day production period. Product samples taken at the production line post peeler were more likely (12 of 14 samples positive) to be contaminated than samples from other production locations (2 of 40 samples positive). Therefore, product contamination was found to occur at a single point during the peeling process prior to packaging of product. Seafood is recognized as a source of L. nzonocytogenes.Weagant et al. (164) documented presence of L. monocytogenes in frozen seafood samples, which included shrimp, crabmeat, lobster tail, fin fish, and surimi-based products. Farber et al. (165) isolated L. monocytogenes from ready-to-eat shrimp, crab, and smoked salmon, and further laboratory studies demonstrated growth at 4°C of L. molzocytogenes on cooked crabmeat, lobster, shrimp, and smoked salmon. Jemmi (166), upon examination of 377 samples of smoked and marinated fish, found L. nzonocytogenes in 47 samples. A survey in Newfoundland conducted by Dillon et al. ( 167) also revealed the presence of Listeria in smoked seafood products.

VI.

DETECTIONOF LISTERIA IN FOODS

A.

SelectiveProcedures

Most selective enrichment and isolation procedures developed to date for the detection of Listeria take advantage of the resistance of this organism to selective compounds, which suppress growth of background contaminants. Selective agents commonly used in selective enrichment/plating media include acriflavine, naladixic acid, lithium chloride, moxalactam, and phenylethanol (1 33,141,168,169). Detection of L. nzonocytogenes in food products or food-processing environments is accomplished by use of a variety of standard or rapid microbiological procedures. Among the most widely used are protocols devised by the U.S. Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) for the detection of Listeria in meats (154,169- 171) and FDA for the detection of Listerin in dairy products (133,172). The FDA procedure initially involves enrichment of 25 g or 25 mL of sample into 225 mL of enrichment broth, M52 (172), which consists of a trypticase soy broth/yeast extract base supplemented with: monopotassium phosphate (anhydrous), 1.35 g/L; disodium phosphate (anhydrous) 9.6 g/L; acriflavin HC1 10 mg/L; nalidixic acid sodium salt 40 mg/L; cycloheximide 50 mg/L; and pyruvic acid (sodium salt 10% w/v aqueous solution) 11.1 mL. Samples are enriched without selective agents for 4 hours at 30°C. Following addition of selective agents (acriflavin, nalidixic acid, and cycloheximide), samples are incubated for an additional 44 hours for a total incubation period of 48 hours at 30°C. This procedure is a modification of the original procedure, which called for sample enrichment for 1 and 7 days at 30°C (141). The original broth was modified by increasing its buffering capacity, thereby positioning this media to be used successfully in conjunction with DNA probe and other methods, which are more sensitive than conventional cultural procedures. After 24 and 48 hours, EB cultures are streaked onto OXA (173) and Listerin plating medium (LPM) agar (170) or LPM plus esculin/Fe3' agars, both of which have replaced the originally recommended modified McBride agar (MMA) as the selective isolation media. PALCAM agar (174) may beused in place of LPM agar. This substitution brings the method into closer alliance with methodology used outside the United States

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and decreases reliance on the Henry technique. OXA and PALCAM plates are incubated (with optional use of a C02-air atmosphere) at 35°C for 24-48 hours, with LPM plates incubated at 30°C for 24-48 hours. LPM plates can be viewed using Henry illutnination or alternatively, esculin and ferric iron salt may be added to LPM to eliminate the need for Henry illumination. With OXA and PALCAM, Listeria colonies develop a black halo. It is recommended that five or more typical colonies be picked from OXA and PALCAM or LPM and transferred to TSAYE for confirmation of purity and typical isolated colonies. The selection of five colonies ensures that multiple species of Listeria, if present, will be identified. TSAYE plates are incubated at 30°C for 24-48 hours (35°C incubation may be utilized if colonies are not being used for wet tnount motility confirmation). Purified isolated are subjected to a series of standard biochemical tests, with a total of 10-1 1 days being required to isolate and confirm the presence of Listeria in food satnples via the FDA procedure. This procedure was specifically developed to optimize Listeria detection in milk and dairy products. The USDA-FSIS selective enrichment protocol for isolation of Listeria from raw meat and poultry was developed by McClain and Lee (154). The USDA-FSIS procedure for isolation of Listeria, while originally designed to detect the presence of Listeria in meats, has been subsequently used with success for isolation of Listeria from dairy products and environmental samples. The USDA scheme differs from the FDA procedure primarily in the selective enrichment and plating media used, along with the size of the sample tested. The revised USDA procedure (171) differs from the original method in that (a) LEB I1 has been replaced by Fraser Broth (175) as the secondary enrichment medium, (b) LPM agar has been replaced by Modified Oxford agar (MOX), and (c) the regulatory sample size has been increased to 25 g. Fraser broth and Modified Oxford Agar will both blacken during incubation due to the ability of Listeria spp. and other contaminants to hydrolyze esculin, with colonies of Listeria exhibiting black halos on Modified Oxford agar following 24-48 hours of incubation. MOX is also more selective than LPM or Oxford agar, with staphylococci and streptococci both generally unable to grow on MOX. Inadequacies in the original (176) FSIS procedure related to the use of Fraser broth as the secondary enrichment broth have been reported. False-negative results due to reliance on Fraser broth darkening and a 24-hour secondary enrichment have been reported by a number of laboratories (177,178). Kornacki et al. (178) compared recovery of L. rnonocytogerzes from Fraser broth incubated for 26 versus 48 hours. L. morzocytogerles was isolated from 60 of 1088 meat product and environtnental swabs from meat and dairy plants. A false-negative rate of up to 6.7% was recorded, attributed to the failure of L. nzorzocytogenes to be detected in Fraser broth at 26, but not at 48 hours, and by the failure of Fraser broth to blacken. Further, when primary enrichments were streaked directly to selective enrichment media, failure to recover L. monocytogerzes in eight samples was recorded. As a result of this study, a 48-hour incubation of Fraser broth was recommended. Thus, all Fraser broth enrichment cultures should be streaked regardless of color following 24-26 hours of incubation. Once cultures have been streaked to MOX, Fraser broth cultures should be reincubated at 35°C for an additional 24-hour enrichment period. MOX plates streaked from 24- to 26-hour Fraser broth enrichment cultures should be examined for presence of colonies displaying typical Listeria morphology. If present, isolation should proceed. If absent, a second MOX plate should be streaked from the 48-hour enrichment culture.

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A detection method widely used in Europe is the Netherlands Government Food Inspection Service (NGFIS) method, developed by Van Netten et al. (174). These authors have reported increased sensitivity with this method over that of the USDA procedure when used to examine foods containing L. monocytogenes at levels of less than 10 CFU/g. Food samples are enriched in L-PALCAMY enrichment broth for 48 hours at 30°C. After 24 and 48 hours, 0.1 mL of L-PALCAMY enrichment broth is plated onto PALCAM agar. Plates are incubated at 30°C for 48 hours under microaerophilic conditions (5% oxygen, 7.5% carbon dioxide, 7.5% hydrogen, and 80% nitrogen). Numerous studies have been conducted to compare the efficacy of these and other widely used detection/isolation protocols. Hayes et al. (179) compared use of the USDAFSIS procedure with cold enrichment as a means of identifying L. monocytogerzes in suspect food samples. Both procedures were able to identify L. nzonocvtogenes in 28 of 51 positive samples. The USDA-FSIS procedure identified 2 l samples missed by cold enrichment while the cold enrichment procedure identified an additional 2 samples that the USDA-FSTS procedure missed. A comparison of three enrichment methods was also made by Hayes et al. (180) when examining foods obtained from the refrigerators of patients with active clinical cases of listeriosis. Two thousand two hundred and twenty-nine foods were examined in the study, 1 % 1 of whichwere positive for L. ~)~onocytogenes. A comparative evaluation of three microbiological procedures was conducted on 899 of the examined foods. The USDA-FSIS (176), FDA (133), and Netherlands Government Food Inspection Service (NGFIS) (172) methods were not statistically different in their ability to isolate Listeria from the 899 samples. The FDA procedure detected L. monocytogelzes in 65% of foods shown to bepositive, while theUSDA-FSIS and NGFIS procedures detected L. monocytogerzes in 74% of foods shown to be positive. Thus, none of these widely used conventional methods proved to be highly sensitive when used independently for analysis of Listeria contamination in foods. It was noted, however, that use of a combination of any two methods improved detectability from 65-74% for individual protocols to 8791% for combined protocols. B. Detection of SublethallyInjured Listeria Most conventional and rapid methods for detection of Listeria in food product and environmental samples use highly selective enrichment media to facilitate growth over competitive background flora. These highly selective enrichment procedures do not account for recovery of sublethally injured Listeria, which could exist within a variety of heated, frozen, and acidified foods, or heated, frozen, and sanitized areas within food-processing environments. It is well recognized that Listeria can be injured as a result of exposure to a variety of processing treatments, which include sublethal heating and freezing, drying, irradiation, or exposure to chemicals (sanitizers, preservatives, acids) ( 122,142,181- 193). Under ideal conditions in food systems, injury is reversible and injured Listeria can repair sublethal damage. Repair of heat-injured L. nzonocytogems has been shown to take place in whole and 2% milk stored at 4°C (194). Several investigators have attempted to improve the sensitivity of current detection systems by recognizing that Listeria may exist in an injured state in food products and food-processing environments. All current detection procedures, withthe exception of cold enrichment, involve selective enrichment and/or selective plating. Cold enrichment is not feasible for routine sample analysis because several months may be necessary to

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record positive results. By failing to consider recovery of injured Listeria, current methodologies underestimate the true incidence of Listerin. Several previous studies have reported on the ability of commonly used plating media to recover injured Listeria. Among the compounds examined, phenylethanol, acriflavin, polymixin-acriflavin, and sodium chloride were found to inhibit recovery of thermally stressed and nonstressed Listeria (186,195- 198). It should be noted that these compounds comprise media that are routinely used for detection of Listeria. When examined for ability to quantitatively recover thermally stressed Listeria on solid media, Listeria enrichment broth (LEB) agar, modified McBride's agar (MMA), lithium chloride phenylethanol moxalactam (LPM) agar, and FDA enrichment broth agar showed significantly impaired abilities to recover injured cells. Warburton et al. (199) recently examined the ability of the modified FDA and USDA methods for isolating stressed and low levels of L. rnonocytogenes in food and environmental samples. These authors found the modified FDA and USDA methods to be comparable in their abilities to isolate stressed and low-level populations of L. wonocytogenes. However, the authors failed to assess the degree of injury within bacterial populations following exposure to sublethal stress. The percent injury existing within a population of bacterial cells can profoundly affect results of comparisons of media performance. Thus, it is difficult to determine whether valid conclusions can be drawn from these studies. Busch and Donnelly (184) developed an enrichment medium capable of resuscitating heat-injured Listeria. This medium, Listeria repair broth (LRB), permits complete repair of injured Listeria within 5 hours at 37°C. LRB is composed of the following constituents (per liter of distilled water): trypticase soy broth, 30.0 g; glucose, 5.0 g: yeast extract, 6.0 g; magnesium sulfate, 4.94 g; ferrous sulfate, 0.3 g; pyruvic acid (sodium salt), 10.0 g; MOPS-free acid, 8.5 g; and MOPS-sodium salt, 13.7 g. In studies that compared the efficacy of LRB in promoting repair/enrichment of heat-injured Listeria with that of existing selective enrichment media, repair was not observed in FDA enrichtnent broth (133), phosphate-buffered Listeria enrichment broth (PEB; Gene-Trak Systems, Framingham, MA). or UVM enrichment broth (l 69). Final Listeria populations in selective enrichment media following a 24-hour incubation were l .7-9.1 X lo8CFU/mL, compared with populations in LRB, which consistently averaged 2.5-8.2 X 10" CFU/mL. Studies with LRB were extended to examine the potential for repair of freeezeinjured and sanitizer-injured L. rnonocytogenes (193,200). Although variation in the susceptibility of L. nzonocytogerm strains was recorded, in general, L. wzorzocytogerzes is not highly injured by freezing. Golden et al. (1 17) compared the extent of freeze injury in four strains of L. rnof1ocytogenessubjected to freezing at - 18°C for 7 and 14 days. Percent injury was found to vary between 72 and 80%. El-Kest and Marth (201) recorded 65% injury and 55% death after one day of storage in phosphate buffer at - 18°C. Sallam and Donnelly (193) examined the ability of four commonly used dairy plant sanitizers to induce injury in L. monocytogenes when exposed at sublethal concentrations. UVM broth failed to support the growth of sanitizer-injured cells, while LRB medium permitted recovery of tested strains. Flanders et al. (202) examined the efficacy of using a repair step to increase sensitivity of recovery of injured Listeria from environmental sponge samples obtained from dairy processing plant environments. The USDA-FSIS Listeria isolation protocol using UVM-modified Listeria enrichment broth was compared with a modified USDA-FSIS format, which utilized LRB as the primary enrichment medium. UVM and LRB broths were also used in conjunction with rapid methods. Of 80 sites positive by any method, UVM and LRB showed similar recovery rates (87.5% and 88.8%, respectively). However, cultural methods combined with either rapid method from each broth increased

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sensitivity of detection to 97.5-98.8% when data from UVM and LRB enrichment was combined (202). Flanders et al. (203) also examined incorporation of ceftazidime into LRB (LRBC) to increase recovery of Listeria in dairy plant environmental samples. LRB, UVM, and LRBC enrichment media were evaluated for their abilities to identify Listeriapositive environmental samples. While no single broth was able to detect all Listeriapositive sites, LRBC detected 67 out of 89 positive sites (75.3%), and LRB and UVM each detected 60 out of 90 positive sites (66.7%). Combining results from any two broths increased recovery percentages from 66.7-75.3% to 82.2-94.4%. The combination of LRBC and UVM detected 94.4% of positive samples, and combining LRBS and LRBC recovered 9 1.1% of positive samples. In yet another study, Pritchard et al. (204) compared performance of UVM, LRB, and LRBC for isolation of Listeria from the dairy plant environment. Of eighty positive samples identified, 54 samples came from UVM medium, 56 were from LRB, and 57 of the positive samples came from LRBC medium. A total of 26 samples (32.5% of positive samples) were identified by either LRB or LRBC but not by UVM media. The use of UVM combined with either LRB or LRBC was shown to substantially increase the number of positive samples identified. When results of UVM and LRB are combined, 65 of 80 (8 1.3%) positive samples were identified. The combined use of UVM and LRBC resulted in identification of 74 of 80 (92.5%) positive samples. Despite improved recoveries when combined use of media is employed, these results illustrate the severe limitations associated with the regulatory procedures currently used to assure absence of Listeria in foods and food-processing environments. Ryser et al. (205) evaluated the ability of UVM and LRB to recover different strainspecific ribotypes of L. nzonocvtogenes from meat and poultry products. Forty-five paired 25 g retail samples of ground beef, pork sausage, ground turkey, and chicken underwent primary enrichment in UVM and LRB (30°C/24 h) followed by secondary enrichment in Fraser broth (35"C/24 h) and plating on modified Oxford agar. A 3-hour nonselective enrichment period at 30°C was used with LRB (with tested food) to enable repair of injured Listeria prior to addition of selective agents. Of 180 meat and poultry products tested, LRB identified 73.8% (133/180) and UVM 69.4% (124/180). Although there was not a statistically significant difference in these results, combining UVM and LRB results increased overall Listeria recovery rates to 83.3%. These results demonstrate that use of LRB for repair/enrichtnent of samples in conjunction with the USDA/FSIS method has the potential to improve recovery of Listeria from meat and poultry products. In this study, Listeria isolates were genetically discriminated using the automated RiboprinterTM Microbial Characterization System. A total of 36 different Listeria strains comprising 16 L. morzocytogelzes (including four known clinical ribotypes), 12 L. innocucI, and 8 L. welshimeri ribotypes were identified from selected positive samples (15 samples of each product type, 2 UVM and 2 LRB isolates per sample). Twenty-six of 36 (13 L. monocytogenes) ribotypes were detected using both UVM and LRB; whereas 3 of 36 (1 L. nzorlocytogenes) and 7 of 36 (3 L. morzocytogenes)Listeria ribotypes were observed using only UVM or LRB, respectively. Ground beef, pork sausage, ground turkey, and chicken yielded 22 (8 L. 111onocytogei1es), 21 (12 L. monocytogenes), 20 (9 L. nzonocytogenes), and 19 (1 1 L. nzonocytogenes) different Listeria ribotypes, respectively, with some Listeria ribotypes confined to a particular product. More importantly, striking differences in both the number and distribution of Listeria ribotypes, including previously recognized clinical and nonclinical ribotypes of L. monocytogenes, were observed when 10 UVM and 10 LRB isolates from five samples of each product were ribotyped. When a third set of six samples per product type was examined from which two Listeria isolates were obtained using only

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one of the two primary enrichment media, UVM and LRB failed to identify L. rnonocytogerzes (both clinical and nonclinical ribotypes) in two and four samples, respectively. These findings illustrate the complex microbial ecology of Listerin in foods and the limitations of existing detection procedures to fully represent the total distribution of Listeria isolates in foods. Furthermore, two of the L. vlonocytogenes ribotypes missed using UVM were known clinical isolates of serotype 1/2a, both of which were responsible for sporadic and epidemic cases of human listeriosis in England and Scotland (14). Continuing work (206) on enrichment of dairy environmental samples in UVM and LRB has shown that combining these two primary enrichment media into a single tube of Fraser broth for secondary enrichment yields a significantly higher 0, < 0.05) percentage of Listeria-positive samples than when either LRB or UVM are used alone. These findings, combined with reports of L. inlzocua being able to outgrow L. rvonocytogems in UVM and Fraser broth (207,208), suggest that different genetic types of L. rno1zocytogerzes may vary somewhat in their nutritional requirements or in abilities to compete with other genetic types of Listeria. Refinement of existing systems should consider the nutritional needs associated with specific genetic types (unique riboprints) of Listeria strains distributed in foods. Roth and Donnelly (192) assessed the survival of acid-injured L. morzocytogenes in four different acidic food systems, and they examined the efficacy of two different enrichment media, LRB and UVM, to recover acid-injured Listerin from acidic foods. Populations of L. nzorzocytogenes F5069 serotype 4b were injured in lactic (pH 3.0) and acetic (pH 3.5j acids. Two levels of injury were produced and monitored-one population with 99.99% injury and the second with approximately 95% injury. Four acidic food systems were used as models. Foods studied were fresh apple cider (pH 3.3), plain nonfat yogurt (pH 4.2), fresh coleslaw (pH 4.4), and fresh salsa (pH 3.9). Acid-injured Listeria were added to each acidic food system and monitored by selective and nonselective plating methods. Simultaneously, samples were tested using LRB and UVM enrichment procedures followed by standard isolation/identification procedures. Additionally, the survival of healthy L. monocvtogenes was also monitored. These experiments were conducted at 4°C (storage temperature) and 30°C (abuse temperature). Results indicated that acid-injured Listeria failed to repair in the foods tested, but survive in the tested foods for over a week. Storage temperature was found to affect survival, with 4°C storage having bacteriostatic effects, whereas 30°C imparted bacteriocidal effects. Parameters involved in the survival of acid-injured Listeria included the degree to which the bacterial population was injured (percent injury), storage temperature, and the pH of the food. At time points where differences were detected, LRB proved to be superior, detecting 22 of 54 samples, compared to UVM, which detected only 3 of 54 positive samples.

VII.

SUMMARY

Despite reductions in disease incidence due to Listeria morzocytogelzes, this organism remains the leading cause of death due to a foodborne pathogen (207). Recent multistate outbreaks of illness and death highlight the need for renewed collaboration among industry, university, and governmental agencies to control this dangerous but interesting foodborne pathogen. Improvements in testing methods are also needed to ensure adequate

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sensitivity of detection of regulatory procedures used to identify and ultimately control Listeria.

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162. Harrison, M. A., and Carpenter,S. L. (1989). Survival of large populations of Listeria monocytogenes on chicken breasts processed using moist heat. J. Food Prot., 52:376-378. 163. Wenger, J. D., Swaminathan. B., Hayes, P. S., Green, S. S., Pratt, M., Pinner, R. W., Schuchat, A., and Broome, C. V. (1990).Listeria rnonocytogerlescontanination of turkey franks: Evaluation of a production facility. J. Food Prot., 53:1015-1019. 164. Weagant. S. D., Sado, P. N., Colburn, K. G., Torkelson, J. D., Stanley, F. A., b a n e , M. H.. Shields, S. C., and Thayer, C. F. (1988). The incidence of Listeria species in frozen seafood products. J. Food Prot., 51:655-657. 165. Farber, J. M. (1991). Listeria rnonocytogems in fish products. J. Food Prot.. 54:922-923. 166. Jemni, T.(1990). Actual knowledge ofListeria in meat and fish products. Mitt. Geb. Lebewsmittel. Hyg., 81:144-157. 167. Dillon, R., Patel, T., and Ratnum, S. (1992). Prevalence of Listeria in smoked fish. J. Food Prot.. 55~866-870. 168. Donnelly, C. W., and Baigent, G. J. (1986). Method for flow cytometric detection ofListeria nzorzocytogenes in nilk. Appl. Environ. Microbiol.. 52689-695. (1989). FSIS method for the isolation and identification of 169. McClain, D., and Lee, W. H. Listeria r~lonocytogerzesfrom processed meat and poultry products. Lab Comm. No. 57, Revised May 24, 1989. U.S. Dept. Agric., FSIS Microbiol. Div., Beltsville. MD. 170. Lee, W. H., and McLain. D. W. (1986). Improved Listeria rnonocytogenes selective agar. AppI. Erz\iron. Microbiol., 52:1215-1217. 171. Johnson. J. L. (1998). Isolation and identificationof Listeria nzorzoc?ltogenesfrom meat, poultry and egg products. In USDA-FSIS Microbiology Laborator? Guidebook, 3rd ed., Vol. 1. Washington, D.C., pp. 8-1-8-18. 172. Hitchins, A. D. (1995). Listeria monocytogenes. In Food and Drug Adrrzinistration Bacteriological Analytical Manual, 8th ed., AOACInternational,Gaithersburg.MD,pp.10.0110.13. 173. Curtis, G. D. W., Mitchell, R. G., King, A. F., and Griffen, E. J. (1989). A selective differential medium for the isolation of Listeria nzorzocytogenes. Lett. Appl. Microbiol., 8:95-98. 173. Netten, Van, P., Perales. I., Van de Moosdijk, A., Curtis, G. D. W., and Mossel, D. A. A. (1989). Liquid and solid selective differential media for the detection and enumeration of L. ntorzocytogenes and other Listeria spp. M. J. Food Microbiol., 8999-316. 175. Fraser, J. A., and Sperber, W.H. (1988). Rapid detection ofListeria spp. in food and environmental samples by esculin hydrolysis. J. Food Prot., 51:762-765. (1989). Method for the Isolution and Identification 176. Carnevale, R. A., and Johnston, R. W. oj*Listeria monocytogenes fr-om Meat ancl Poultry Products. United States Department of Agriculture Food Safety and Inspection Service, Laboratory Communication No. 57, Revised May 24, USDA-FSIS, Washington, DC. for the simultaneous 177. Bailey, J. S., and Cox, N. A. (1992). Universal preenrichment broth detection of Scrlnzonella and Listeria in foods. J. Food Prot., 55:256-259. 178. Kornacki, J. L., Evanson, D. J., Reid, W., Rowe, K., and Flowers, R. S. (1993). Evaluation of the USDA protocol for detection of Listeria rnonocytogerzes. J. Food Prot., 56:441-433. 179. Hayes, P. S., Graves, L. M., Ajello. G. W., waminathan. B., Weaver. R. E., Wenger, J. D., Schuchat,A.,Broome. C. V.,andthe Listeria studygroup.(1991).Comparison of cold enrichment and the U.S. Departmentof Agriculture methods for isolatingListeria monocytogenes from naturally contaminated foods. Appl. Erniron. Microbiol., 57:2109-2113. 180. Hayes, P. S., Graves, L. M., Swaminathan, B., Ajello, G. W.. Malcolm, G. B., Weaver, R. E., Ransom, R.. Deaver, K.. Plikaytis, B. D.. Schuchat, A., Wenger, J. D., Pinner, R. W., Broome, C. V.. and theListeria study group. Comparisonof three selective enrichment methods for the isolation of Listericr monocytogenes from naturally contaminated foods. J. Food Prot., 55:952-959. and Marth, E. H. (1990). Acid-injury of Listeria rnonocytogenes. J. Food Prot., 181. Ahamad. N., 53126-29.

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182. Beuchat, L. R., Brackett, R. E., Hao, D. Y.-Y., and Conner, D. E. (1986). Growth and thermal inactivation of Listeria ~nonocytogenesin cabbage and cabbage juice. Can. J. Microbiol., 32~791-795. 183. Bunduki, M. M.-C., Flanders, K. J., and Donnelly, C. W. (1994). Metabolic and structural sites of damage in heat- and sanitizer-injured populations of Listeria morrocytogenes. J. Food Prot., 581410-415. 184. Busch, S. V., and Donnelly, C. W. (1992). Development of a repair-enrichment broth for resuscitation of heat-injured Listeria monocytogenes and Listeria innocua. Appl. Environ. Microbiol., 58: 14-20. 185. Cassiday, P. K., Brackett. R. E., and Beuchat, L. R. (1989). Evaluation of ten selective direct plating media for enumeration of Listeria rttortocytogenes in ham and oysters. Food Microbiol., 55:113-125. 186. Crawford. R. G., Beliveau, C. M., Peeler, J. T.. Donnelly, C. W., and Bunning, V. K. (1989). Comparative recovery of uninjured and heat-injured Listeria nzortocytogenes cells from bovine nlilk. Appl. Environ. Microbiol., 55: 1490-1494. 187. Curtis, G. D. W., Nichols, W. W. and Falla, T. J. (1989). Selective agents for Listerin can inhibit their growth. Lett. Appl. Microbiol., 8:169-172. 188. Garayzabel, J. F., and Genigeorgis, C. (1990). Quantitative evaluation of three selective enrichment broths and agars used in recovering Listeria microorganisms. J. Food Prot., 53: 105-1 10. 189. Golden, D. A., Beuchat, L. R., and Brackett, R. E. (1988). Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured and freeze-injured Listeria nronocytogenes from foods. Appl. Environ. Microbiol., 54: 1451-1456. 190. Golden, D. A., Beuchat, L. R., and Brackett, R. E. (1988). Inactivation and injury of Listeria monocytogenes as affected by heating and freezing. Food Microbiol., 5:17-23. 191. McCarthy. S. A., Motes, M. L., and McPhearson, R. M. (1990). Recovery of heat-stressed Listeria nronocytogenes from experimentally and naturally contaminated shrimp. J. Food Prot., 53:22-25. 192. Roth, T. T. and Donnelly, C. W. (1995). Injury of Listeria rnonocytogenes by acetic and lactic acids: mechanisms of repair and sites of sublethal damage. IF” Ann. Mtg. Book of Abstracts 81D-1, p. 246. 193. Sallam, S. S. and Donnelly, C. W. (1992). Destruction, injury and repair of Listeria species exposed to sanitizing compounds. J. Food Prot., 55:771-776. 194. Meyer, D. H. and Donnelly. C. W. (1992). Effect of incubation temperature on repair of heat-injured Listeria in milk. J. Food Prot., 55:579-582. 195. Leasor, S. B., ‘Abbas, C. A., and Firstenberg-Eden, R. (1990). Evaluation ofUVMas a growth medium for Listeria morrocytogenes. Abstr. Ann. Mtg. Amer. Soc. Microbiol. P39: 284. 196. Smith, J. L., and Archer, D. L. (1988). Heat-induced injury in Listeria rnonocytogenes. J. Zndust. Microbiol., 3: 105-1 10. 197. Warburton, D. W., Farber, J. M., Armstrong, A., Caldeira, R., Hunt, T., Messier, S.. Plante, R., Tiwari N. P., and Vinet, J. (1991). A comparative study of the “FDA” and “USDA” methods for the detection of Listeria monocytogenes in foods. Znt. J. Food. Microbiol., 13: 105-1 18. 198. Warburton, D. W., Farber. J. M., Armstrong, A., Caldeira, R., Tiwari, N. P., Babiuk, T.. Lacasse P., and Read. R. (1991). A Canadian comparative study of modified versions of the “FDA” and “USDA” methods for the detection of Listeria rnonocytogenes. J. Food Prot., 669-676. 199. Warburton, D. W., Farber, J. M., Powell, C., Tiwari, N. P., Read, S., Plante, R., Babiuk, T., Laffey, P., Kauri. T. Mayers, P., Champagne, M.-J.. Hunt, T,. Lacasse. P., Viet. K., Smando, R., and Fran Coates. (1992). Comparison of methods for optimum detection of stressed and low levels of Listeria monocytogenes. Food Microbiol., 9: 127-145.

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200. Flanders, K. P. (1991). Injury. resuscitation and detection of Listeria spp. From frozen environments, M. S. thesis, University of Vermont, Burlington, VT. 201. El-Kest, S. E. and Marth. E. H. (1992). Freezing of Listeria mortocytogenes and other microorganisms: a review. J. Food Prot., 55:639-648. 202. Flanders, K. J., Pritchard, T. J., and Donnelly, C. W. (1995). Enhanced recovery of Listeria from dairy plant processing environments through combined use of repair enrichment and selective enrichment/detection procedures. J. Food Prof., 58:404-407. 203. Flanders, K. J.. Beliveau, C. M., Pritchard T. J., and Donnelly. C. W. (1994). Enhanced recovery of Listeria from dairy plant environments using modified selective-enrichment media. IFT Ann. Mtg. Technical Program: Book of Abstr. 59C, p. 166. 204. Pritchard, T. J.. Flanders, K. J., and Donelly. C. W. (1995). Comparison of the incidence of Listeria on equipment versus environmental sites within dairy processing plants. Znt. J. Food Microbiol., 26:375-384. 205. Ryser, E. T., Arimi. S. M., Bunduki M. M.-C., and Donnelly, C. W. (1996). Recovery of different Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products with two primary enrichment media. AppI. Emiron. Microbiol., 62: 1781- 1787. 206. Pritchard, T. J. and Donnelly. C. W. (1995). Combined secondary enrichment of UVM and LRB primary enrichment broths increases the sensitivity of Listericr detection. IFT Ann. Mtg. Book of Abstracts, 34-2, p. 96. 207. Curiale, M. S. and Lewus, C. (1994). Detection of Listeria monocytogenes in samples containing Listeria inrlocurr. J. Food Prof., 57: 1048-1051. 208. Petran, R. L., and Swanson, K. M. J. (1993). Simultaneous growth of Listerin mortocytogenes and Listeria inrzocua. J. Food Prof., 56:616-618. 209. United States General Accounting Office. May 1996. FoodSafety: hlforntntion on Foodborne Zlbzess. Report to Congressional Committees. B-270753, pp. 1-31.

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11 Bacteriology of Salmonella Robin C. Anderson and Richard L. Ziprin U.S. Departwwnt of Agriculture. College Stcrtioiz, Te.ucrs

247 247 11. History andNomenclature 111. IsolationandCultivation 2-58 IV. Serotyping 25 1 V. Biotyping 252 VI. PhageTyping 253 253 VII. MolecularTyping I. Introduction

VIII.

Conclusions References

I.

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INTRODUCTION

Salnlorlella are gram-negative bacteria belonging to the family Er1terobacteriaceae (1). These bacteria are widely distributed throughout various ecological habitats, having been isolated from soil, water, foods, andthe intestinal tracts of humans and animals. The propensity of all Salrmlzelln to cause systemic or enteric infections in humans and animals makes them important pathogens, and despite many hygienic improvements in food production, these remain among the most important causative agents of foodborne disease (2-4j. Manifestation of disease can be varied depending on the serotype and host. For instance, certain host-adapted serotypes cause disease almost exclusively in their specific hosts, whereas other serotypes exhibit a broad host range specificity. Details regarding pathogenicity and disease in humans and animals have been more appropriately presented elsewhere in this text (see Chapters 12 and 13).

II. HISTORYANDNOMENCLATURE The study of salmonellae began with Eberth's first recognition in 1880, and the subsequent isolation by Gaffky (see Ref. 5 ) , of the bacillus causing human typhoid fever (now known 247

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as Sdmonella (ser.) Qphi). Shortly thereafter, Salmon isolated the bacterium then thought, but later disproved, to be the etiological agent of hog cholera. The genus was later named SnlmoneZZn by Lignikres in 1900 in honor of D. E. Salmon (see Ref. 5). Other salmonellae were soon isolated, and it became common for each new isolate to be named by the medical and veterinary fields for the diseases they caused or the species of animal afflicted. For instance, names such as Salrnorzelln abortus ovis, Salrnonellll bovis rzrorbijicms, Scrlrnor~ellayullormr, and Sdrnonelln gallinarml were used by the veterinary profession to designate bacteria afflicting specific hosts (i.e., sheep, cattle, and poultry). Names such as Snlrnonella Qylzi, Sdrnonel/a vphosa, and Sulmorzella pcrrcroplzi were used by the human medical profession to designate bacteria associated with a specific disease. More recently, each new isolate was often named for the geographic location from which it was isolated (e.g., Salrno~zellayantrnzcr, Salr.zronellu saintpnul ). Historical accounts of some of the early isolations are available (6,7). The use of many of the above-mentioned names continues to this day, because it provides a practical and established nomenclature, although to do so incorrectly implies that each is a different species. Since 1987, all Snlnlonella within the genus were classified as a single species based on their close relatedness as determined by DNA hybridization (8). The species initially named Salmorrella cholemesuis by LeMinor (9) was later renamed Salmonella entericn in order to avoid confusion of the former with an already named serotype (8). Scdnzonelln erztericnis divided into six subspecies: erzterica, scrlavrae, nrizonae, dinri,-orzae, houtenae. and indica, with each ascribed formal taxonomic status by the WHO Committee (10,ll). A second species, S. bongori, has now been proposed by Reeves et al. (13), who, using multilocus enzyme electrophoresis, showed that S. erlterica ssp. bow gori is sufficiently different from the others to warrant such classification. Publication of this new genus and species combination in the Irzter~ratiorzalJounrnl qf Systerncttic Bacteriology (13) has made the name available for use in bacteriological nomenclature. Such publication, however, ascribes no taxonomic rank, and thus S. bongori has been slow to be accepted as a second species by some microbiologists. Contemporary nomenclature, which we shall use throughout the remainder of our text, now appropriately conserves traditional connotations associated with important disease-causing salmonellae simply by presenting the serotype in Roman rather than in italic typeface thus negating any implication of formal taxonomic status (10,14-15). For example, the serotype formerly written as SalnzonelZn choleraesuis is more appropriately written as S. enterica subspecies erztericcr serotype (ser.) Choleraesuis, or less cumbersomely, as Salrnonella (ser.) Choleraesuis, Sdn?onekr Choleraesuis, or Choleraesuis. This method applies only to serotypes within the subspecies entericn, of which greater than 99% of salmonellae isolated from humans belong; the remaining subspecies are not named but rather are designated by their antigenic formulas preceded by their subspecies designation ( 15).

111.

ISOLATION AND CULTIVATION

Salmonellae are mesophilic heterotrophs, needing only simple inorganic salts containing nitrogen, phosphorus, sulfur, a source of divalent cations, and an organic substrate for carbon and energy to sustain life (5,16). Wild-type salmonellae are fully capable of synthesizing all their biochemical machinery from the components of simple defined media corn-

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posed of ammonium sulfate, phosphate buffer, a source of magnesium, and sodium citrate or glucose (4). While salmonellae grow best at moderate temperatures (35-37"C), they can grow over a much wider temperature range, with growth occurring at temperatures as low as 4°C (17) and as high as 48°C (18). Growth is also best at moderate pH (6.57.5) in conditions of high water activity and low osmolarity (4). Extensive descriptions of the genus have been published (14,16,19), and these provide the ultimate criteria for identification of isolates. Scdrrrorzella are nonsporeforrning, oxidase-negative, faculative anaerobes. They do not produce indole, do not hydrolyze urea, and do not deaminate phenylalanine or trytophan. Most reduce nitrate to nitrite, and most ferment a variety of carbohydrates with concomitant production of acid. D-Glucose is fermented via the mixed acid pathway, thus yielding a positive methyl red reaction; acetyl methyl carbinol is not produced by Salmonella, and they therefore yield a negative Voges-Proskauer reaction. Most Salmonella are motile via means of peritricious flagella; however, Salmonella serotypes Gallinarum and Pullorum are notable exceptions (16). Other prominent characteristics of salmonellae are that most produce abundant hydrogen sulfide, most decarboxylate lysine, arginine, and ornithine, and, with few exceptions (notably those belonging to subspecies arizonae and diariconae),most do not ferment lactose. These latter characteristics have been the basis for the development of numerous selective and differential media for the culture and presumptive identification of salmonellae (20). Examples of such media include Rambach agar (RA) xylose lysine decarboxylase (XLD) agar, ScllnloneZln-Shigella (SS) agar, brilliant green agar (BGA), brilliant green sulfite (BGS) agar, Hektoen enteric (HE) agar, Macconkey's agar, lysine iron agar, triple sugar iron (TSI) agar, dulcitol lactose iron agar, dulcitol lactose sucrose agar, Kliger iron agar, Levine eosin-methyline blue (EMB), and desoxycholate citrate lactose agar (2023). Samples to be cultured for Sdrnonella often contain too few cells to allow detection via direct plating on the selective and differential agars. Thus, in order to increase the sensitivity for detection it is necessary to increase the number of cells via enrichment and perhaps even via a prior preenrichment (24). Preenrichrnent is a process in which the sample is first cultured in a nonselective growth medium such as buffered peptone water or tryptic soy broth with the intent of allowing the growth of any viable bacteria, whether SalmorleZZcr or not. Preenrichment often is useful in allowing injured cells to recover and increase in number and sometimes provides a way to dilute any potential growth suppressors (24). Because of the nonselective nature of preenrichment, it is often necessary to diminish the proportion of competing bacteria via enrichment. Enrichment, in contrast to preenrichment, is a process by which growth of a desired bacterial species is favored and (or) growth of undesired bacteria is inhibited (25). A very simple form of enrichment may be used for some organisms. For instance, Listeria monocytogenes can grow at 4°C while most other bacteria of medical importance cannot. Thus, the former can be enriched via growth at refrigeration temperatures (4). In the case of Salmorzelln, enrichment is usually achieved by culturing samples in media containing inhibitors to restrict the growth of undesired bacteria. Following the enrichment period, the enriched cultures are spread onto selective and differential agar plates, and then after appropriate incubation the plates are examined for the presence of colonies exhibiting morphologies typical of Salmonella (25,26). Confirmation of colony type is typically confirmed via biochemical tests and serological examination. Both of these confirmation procedures are relatively easy toperform and interpret. For instance, suspect colonies are inoculated into triple sugar iron agar, lysine iron agar,

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urease test broth, and indole nitrate medium. Additional tests such as the ability to ferment a variety of carbohydrates or to utilize citrate may be used to further distinguish serotypes of major importance such as Snlrnorlella Choleraesis, Schorzelln Enteritidis, Scrlrnonelln Paratyphi, and Snlrnorlellcr Typhi (14,16). Ultimately, the final determination relies on the ability of the organism to react immunologically against antisera raised against particular antigenic determinants expressed on the cell surface (see next section). Enrichment media commonly used to enrich Scdrnorzellcr include the tetrathionate broth medium of Muller (27), Muller-Kauffman tetrathionite brilliant green (28), Leifson’s selenite cystine broth (29), and the more recent but now widely preferred RappaportVassilidis broth (30-32). The latter medium uses both malachite green and magnesium chloride as selective agents and has been proven to be very useful and more sensitive than the Muller-Kauffmann tetrathionate brilliant green. An added advantage of the RappaportVassilidis medium is that it can be formulated both as a broth or a semisolid medium (33-38). Comparisons of various multistep isolation protocols describing the use various enrichment and plating media are available (20,39-46). Just recently, a new medium component, ferrioxamine-E, has been reported to substantially enhance the selectivity of enrichment media for recovery of salmonellae (47). Some Scrlrnonella do not exhibit the typical biochemical characteristics of the genus and these present special problems diagnostically because they are easily tnissed on comtnonly used differential plating media. For instance, about 1% of the many Salr~zorwlla serotypes submitted to the Centers for Disease Control (CDC) ferment lactose, and hydrogen sulfide production can be quite variable (4). Bismuth sulfite agar has been used to culture and detect biochemically atypical Snlnzordn, but its sensitivity to storage and minor variations in preparation make it difficult and unpopular to use (22). A selective and differential medium devised by Devenish et al. (22) has gained widespread acceptance by many laboratories because it allows for detection of many lactose-fermenting and non-lactose-fermenting Saltnorrelln. This medium contains brilliant green as a selective agent for Sulnlorzella, ferric ammonium citrate for hydrogen gas detecton, novobiocin as an antimicrobial agent active against coliforms and Proteus (but not Snlmorlellcr), and glucose (the only carbohydrate used by all Schorzellr) as the carbon and energy source. Unfortunately, Snlt.zlonellcr Typhi grows poorly on this novobiocinbrilliant green-glucose agar. Moreover, it is important to note that none of the selective and differential media presently available are 100% reliable and foolproof. Thus, when dealing with samples of critical importance, it is recommended that more than one plating media be used. When screening for Snlmorwllcl Typhi or Snlrrlorlellcr Paratyphi, it is imperative that bismuth sulfite agar or bismuth sulfite brilliant green agar be used (48). The processes of preenrichment, enrichment, isolation on selective differentiation agars, and biochemical confirmation normally take several days, with each step often requiring overnight incubation. Consequently, there is considerable interest in the development of more rapid methodologies, particularly for diagnostic purposes. Presently, a variety of conlbination media exist that simultaneously test for several phenotypic characteristics [e.g., o-nitrophenyl-P-D-galactopyranoside-urease-indole broth (49), L-pyrrolidonyl peptidase activity (50), and malonate-dulcite-lysine agar (5l)]. Moreover, rapid test strip systems such as API System S.A. (bioMerieux Vitek, Inc., Hazelwood, MO) (521, MICRO-ID (General Diagnostics, Morris Plains, NJ) (53-55), Minitek (BioQuest Division, Becton Dickinson and Co., Cockeysville, MD) (56), RapID onE (57), and automated systems (58,59)have also been developed. Numerous immunological (e.g., enzymelinked immunoassays, immunodiffusion, or immunomagnetic separation) and nucleic

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acid-based (e.g., PCR amplification, in situ hybridization) procedures for detecting Salmonella have now been developed and commercialized. We refrain from mentioning all of the numerous methods, but rather refer the reader to an excellent review (60) and a few comparative studies illustrating that some of these procedures are as sensitive or even more so than conventional culture methods (6 1-69). Nevertheless, bacteriological cultivation of specimens remains the most often used and most trusted detection method.

IV. SEROTYPING Following the initial isolations of numerous different salmonellae from a wide range of habitats, it soon became apparent that a method was needed to classify this "one great group" of organisms (see Ref. 25). Serological typing arose out of that necessity as a powerful tool for differentiating salmonellae and has proven extremely useful in the epidemiological tracking of various outbreaks. This procedure, which involves the grouping of bacteria based on immunological reactions (agglutinations) of antisera raised against specific antigens expressed on the bacterial surface, allows for the differentiation and classification of serotypes that otherwise may be indistinguishable by biochemical methods. Development of our modern-day scheme is credited primarily to the work of Kauffmann and White, although notable contributions were made by Schutze, Andrewes, Felix, Smith, and Reagh (see Ref. 25). Officially recognized by the World Health Organization, the Kauffmann-White diagnostic scheme involves the primary subdivision of the bacteria into serogroups and further delineation into serotypes according to the specific somatic (0)or flagellar (H) antigens expressed on their cell surface. There are currently more than 2300 Snlnlonella serotypes (lo), and by convention each is now denoted by an antigenic formula with the major 0 antigen listed first, then the phase 1 H antigen(s), and then the phase 2 H antigen(s). The phase 1 H antigens are designated by lowercase letters and the phase 2 H antigens by Arabic numerals or, in some cases, by components of the e or z series (25). For instance, Salmonella Choleraesuis is 6,7:c: 1,5. Many microbiologists still refer to some serogroups by their traditional letter designation, i.e., group C, for Salmonelkz Choleraesuis (25). Serological specificity of the 0 antigens, which are part of the cell wall lipopolysaccharide (LPS), is determined by the type and order of various carbohydrate moieties making up side chains and the chemical nature of terminal groups attached to a lipopolysaccharide core. Presently, 0 antigens are designated by Arabic numerals from 1 up to 67, although the series isnot continuous because some 0 antigens previously assigned to bacteria now known not to be Scrlnlorzella have been deleted from the scheme (25). A particular Salrzlo1zeIla isolate is grouped into one of 46 possible 0 serogroups depending on which major 0 antigen is expressed on the cell surface (1 1,25,70). These 46 serogroups can be arranged into 17 chemotypes based on the polysaccharide composition. Each chemotype contains lipopolysaccharide composed of the same five sugars, plus up to three additional sugars, although differences in cross-linking patterns between the sugars in various chemotypes accounts for the antigen variation. The Kauffmann-White typing system is capable of detecting minor differences in the polysaccharide constitution of the bacterial cell, and in the case that more than one 0 antigen is expressed, only one is considered major-the others are considered minor (25). The commercial availability of antisera raised against 0 antigens now makes serogrouping a task readily performed at practically any laboratory; however, it is important

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for those doing the tests to be aware of certain pitfalls. For instance, cross-agglutinations can occur between some serotypes that share certain minor 0 antigens (25). Moreover, the polysaccharide component of Snlnzonella lipopolysaccharide is variable and can be influenced upon acquisition of genetic elements (e.g., plasmids) (11,15,71). Phage conversion can also alter the sugar composition of the lipopolysaccharide core, thereby changing the serotype and virulence attributes of a particular salmonellae (72-76). Nnalue et al. (72,73) have shown that phage conversion by the lysogenic bacteriophage P14 increased the length of the 0-antigen polysaccharide chains and increased mouse virulence. Chart et al. (76) have shown that phage conversion can change the virulent Salmorzella Enteritidis phage type 4 into the less virulent phage type 7 and that the change involves an alteration in the lipopolysaccharide component of the cell envelope. Some typhoid strains of Salnzonella and some strains of Salnzonellu Dublin and Scrlmonella Paratyphi C possess a distinct capsular antigen, named Vi in reference to virulence, that renders the 0 antigen resistant to agglutination by the anti-0 antibody (77). Thus, an organism that expresses phenotypic characteristics of SaZnzonella may react negatively with polyvalent anti-0 antisera because of the masking effect of the Vi antigen. Under such circumstances, it is imperative that the organism be tested with antisera specific to the Vi antigen, and then, following a boiling procedure to remove the Vi capsular material (0 antigens, being lipopolysaccharide in nature, are heat-stable), the organism should be retested for the presence of the 0 antigen. Results from such testing will delineate whether or not the organism in question in indeed a Scdnzonelln. Further differentiation of bacteria into serotypes is much more complicated than serogrouping due to possible phase variations that may be encountered among many of the Salwzonella serotypes. These variations not only make interpretation of the serological tests difficult but make preparation of antisera specific enough to discriminate between the various H antigens laborious and wrought with possible pitfalls (25). Thus, serotyping is usually done at public health laboratories that possess the necessary materials, antisera, and trained personnel.

V.

BlOTYPlNG

While serotype is the primary characteristic by which salmonellae are classified, certain variations in phenotype within a given serotype may also be used to further differentiate one organism from another. Organisms expressing different phenotypes are considered a different biotype, and it can be that these differences can be associated with differences in virulence. For example, Salmorzelln Pullorum and Sahzonel/a Gallinarum are considered the same organism by some authors and are treated as such in Bergey 'S Mar1ual of Systematic Bacteriology (16), e.g., as Salnzorzellc~Gallinarum-Pullorum. Yet these can be differentiated based on biotype (78,79) and have historically been treated as different bacteria that cause different disease syndromes in chickens. Salmonella Pullorum possesses ornithine decarboxylase activity and produces gas from glucose, whereas Scrlmorzellu Gallinarum does not. Other differences also exist between these two bacteria, notably the propensity of Snln~orzellaPullorum to cause pullorum disease, which is a septicemia of embryos and chicks, whereas Salmonellrr Gallinarum causes "fowl typhoid," which affects adult chickens (80,81). Biotyping schemes have been been devised to help differentiate other Salnlorlella serotypes such as Salnzorzella Agona (82), Snlmonella Livingstone

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(83), Salmonella Montevideo (84), Salmonella Senftenburg (85) and Salmonella Typhimurium (86-88).

VI.

PHAGETYPING

Salrnonella serotypes can be differentiated according to their susceptibilities to lytic bacterial viruses (bacteriophage) via a process called phage typing. A phage-typing system for Vi antigen positive salmonellae was developed in 1938 by Craigie and Yen, and Felix and Callow later developed phage-typing systems for Sulmonellu Paratyphi B and for Salmonella Typhimurium (see Ref. 89). Phage-typing systems have since been developed for several other Salrlzorzella serotypes (90-99). Phage typing can be extremely useful for epidemiological investigation (89,99). By the mid-1950s there were 33 recognized Viphage types, and their usefulness as epidemiological tools was well established. Similarly, there were more than 30 distinguishable phage types of Salmonella Paratyphi B. Bacteriophages useful for typing are distributed by the World Health Organization via collaboration with the Central Public Health Laboratory at Colindale in the United Kingdom, and an international phage-typing system for tracking human Salmonella infections is underway in Europe ( 100). Phage typing has proven useful regarding distribution of salmonellae in the North American poultry industry. For instance, Poppe et al. (101) examined Canadian broiler flocks for Salmorzellc~Enteritidis and found the flocks infected with fifty different Salmonella serotypes. Of the nine flocks infected with Schzonella Enteritidis, phage type 8 was found in seven flocks while phage type 13a was found in two flocks. Scdmonella Enteritidis phage type 4 is of particular interest to the poultry industry because these organisms seem to be especially virulent for chicks and may findtheir way to newly hatched chicks through transovarian transmission (102). Unfortunately, Salmonella Enteritidis phage type 4 has become so prevalent in many areas that information obtained via phage typing is of limited use when trying to determine the source of this bacterium in examined samples (103). When dealing with phage typing, it is important to remember that phage conversions can occur naturally when a particular bacterium becomes infected with bacteriophage or acquires new plasmids. For instance, acquisition of incN incompatability group R-plasmids by Salmonella Enteriditis 4 results in a phage conversion of this bacterium to phage type 24 (104). Moreover, acquisition of lysogenic phage can alter mouse virulence (76).

VII.

MOLECULARTYPING

Numerous typing methods based on the analysis of genetic elements have now beendeveloped, and while commonly used to compliment traditional typing methods, these have not yet replaced serotyping as the method of choice for typing salmonellae (15). Deternlinations of the plasmid size and copy number within a particular serotype enables the construction of plasmid profiles that are useful for monitoring infection patterns and possible transmission routes (105,106). The sensitivity of the typing method may be increased by creating a “plasmid fingerprint’’ via cleavage of the plasmid with selected endonucleases ( l 07). Threlfall et al. (108) have observed nine plasmid profiles among a strain of Salmonella Enteritidis phage type 4. They found sufficient variation in plasmid profiles among 13 other phage types to conclude that plasmid profiles are ‘an effective adjunct

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to phage typing for the subdivision of Sdmonelln Enteritidis" (108). Similarly, Wachsmuth et al. (109) found 16 distinct plasmid profiles among two phage types of Sulmollellcr Enteritidis, Colindale 8, and 13a. Plasmid analysis has been used in the epidemiological investigation of an instance of Scrhzone~/ncontaminated chocolate (1 10). in tracing an outbreak of salmonellosis to contaminated marijuana (11l), and to confirm that a single strain was responsible for recurrent infection in an immunocompromised patient (1 12). Plasmids are not always useful as epidemiological tools since not all strains contain plasmids. In one study of sahnorzella Typhi isolates obtained during a typhoid outbreak in Chile and Pent, only 17 of 141 isolates contained plasmids, and the plasmid profiles were not useful for subdividing the major Vi-phage groups (99). Thirteen of17 isolates contained identical plasmids suggesting a common origin among the 13 isolates (99), but the absence of plasmids among the other 124 isolates is clear evidence that plasmids are not required for virulence by organisms in this particular serotype and that virulence by Sdnlomdln Typhi is determined by the chromosomal genes. It thus seems likely that by being highly adapted to human beings, Scrlmolwlln Typhi has retained virulence genes for humans but lost genes needed for a wider host range. Plasmid analysis has also proven useful in elucidating the role of plasmids in virulence of other Salmonella serotypes (113). Helmuth and colleagues (114) reported that 90% of 337 isolates originating from 29 different countries contained serotype-specific plasmids. Of the seven serotypes represented in this study, four (Typhimurium, Dublin, Enteritidis, and Choleraesuis) contained plasmids that were associated with a lower mouse virulence (approximately 1 million-fold) as interpreted from LDS,,(lethal dose 50%) values. Plasmid contents were found to be unrelated to virulence in the other three serotypes (Infantis, Panama, and Heidelberg) (1 14). It has been proposed that the virulence plasmids of Salrl?onella Typhimurium and Dublin plus large plasmids (54 and 76-kbp) contained by Snlnlorlella Abortusovis, Salrnorzelln Enteritidis, and Snlrtlordlcr Paratyphi C comprise a family of related virulence plasmids (1 15). These plasmids were absent in strains of the following serotypes: Agona. Bovismorbificans, Heidelberg, Infantis, Panama, Paratyphi A, Paratyphi B, Saintpaul, Seftenberg, and Typhi. Moreover, virulence plasmids of Srrlmorzella Choleraesuis, Scrlmonella Dublin, and Scrlsnorwlln Enteritidis and the cryptic plasmids of Sdnlorwllcr Copenhagen and Sdmorzella Sendai are thought to belong to the same incompatibility grouping (1 16,117). Sulmorzella Typhimurium also contains high molecular weight plasmids (8590 kbp),which play a role in extraintestinal dissemination and replication of the pathogen within the tissues of mice (118). Similar large plasmids have been found necessary for the virulence of Salmonella Gallinanlm-Pullorum strains. Sabnolzella Typhimurium cells cured of their plasmids, thereby rendered avirulent in the mouse virulence assay, regain virulence by reintroduction of the virulence plasmid from Scrlrnorlella GallinarumPullorum ( 119). Beninger et al. (120) have shown that plasmids of different size and endonuclease restriction patterns found in Snlrnorlellrr Enteritidis and Scdnlolwllcr Choleraesuis often share a common 4 kb Eco-R1 restriction fragment with the 80 kbp virulence plasmid of Scdnzonella Dublin, pSDL2. Plasmid pSDL2. is required for the development of a lethal systemic infection in the mouse virulence test, and portions of pSDL2 are homologous to a virulence plasmid, pIB 1, of Yensillin species (121). Mouse virulence studies combined with molecular genetics studies of 22 common serotypes have demonstrated the essential role of plasmids containing the 4 kbp Eco RI restriction fragment for development of systemic infection in mice (121). Naturally occurring strains of the serotypes most often

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isolated from nontyphoid human systemic salmonellosis, serotypes Choleraesuis, Dublin, and Enteritidis, typically carry the virulence plasmids. It is possible that the 4 kbp region of the virulence plasmid is partly responsible for the ability of these serotypes to cause human systemic infections. However, virulence plasmids will not confer mouse virulence on all Sulmorzelka serotypes, e.g., Salnrorzellu Typhi and Paratyphi A, and this fact suggests that virulence also requires chromosomal determinant(s) (122). We are now beginning to understand the mechanisms by which plasmids operate to confer virulence. In some cases, they encode for outer membrane proteins that are necessary for replication within the host tissues (123-125). Plasmids may also be responsible for a reduced host immune response (126). Molecular analysis of chromosomal DNA sequences can also be usedto differentiate Salmonellcl; however, depending on the sequence analyzed and the method employed, the discriminatory power of these methods may be lacking (103,127). Thus, analysis of chromosomal DNA sequences are often performed in conjunction with another typing technique. Some of the DNA regions targeted include portions of the 16s rRNA gene (128,129). thefEiC flagella gene (130), virulence genes (1 3 1,132), and insertion sequences (133,134). Protocols involving polymerase chain reaction (PCR) amplification of selected nucleotide sequences have been developed to increase the sensitivity of these molecular techniques, but at present these often employ preenrichment cultural procedures of varing duration. Fingerprinting protocols involving electrophoretic separation of DNA fragments produced via digestion of chromosomal DNA with endonucleases (referred to as restriction fragment length polymorphism, RFLP) and(or) via PCR amplification have proven useful as tools for differentiating various Sall~orzelluserotypes that are not particularly amendable to phage or plasmid typing ( 103,135- 137). Use of pulsed field gel electrophoresis has been used to resolve fragments generated from the genome, and this enhanced the power to discriminate strains within serotypes (138,139) or phage types (140,141).

VIII. CONCLUSIONS Sdr~~onella are important pathogens that cause disease in humans and animals. Ubiquitous in nature, a few serotypes exhibiting host specificity tend to infect only their respective hosts, while many others exhibit broad host range specificity. Techniques, including nucleic acid and immunologically based methods, have been developed to detect and/or isolate Sullnonella from various specimens, but bacteriological cultivation remains the most used method. Serotype, biotype, and phage type are all determinants of host-range and virulence. Individual isolates vary in virulence, biochemistry, susceptibility to bacteriophage, and plasmid content. As alluded to previously, acquisition of additional genetic elements such as lysogenic phage (temperate phage or prophage) and extrachromosomal elements (plasmids) by a particular bacterium can bring about changes in phage type, serotype, and virulence.

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81. Snoeyenbos G. H. (1972). Pullorun~disease. In Diseases of Poultq, 6th ed. (M. S. Hofstad, B. W. Calnek, C. F. Helmboldt, W. M. Reid, and H. W.Yoder Jr., eds.), Iowa State University Press, Ames, pp. 83-114. 82. Barker, R., and D. C. Old (1982). Differential typing of Sahnonella agona: type divergence in a new serotype. J. Hyg. Camb., 88:413-423. 83. Odongo, M. O., McLaren, I. M., Smith, J. E., and Wray, C. (1990). A biotyping scheme for Salmonella livingstone. Br. Vet. J., 146:75-79. 84. Old, D. C., Munro, D. S., Reilly, W. J., and Sharp, J. C. M. (1985). Biotype discrimination of Salr~onellamontevideo. Lett. Appl. Microbiol., 1:67-69. 85. Tuchili, L. M., McLaren, I. M., Smith, J. E., and Wray, C. (1991). Differentiation of Salmonella senftenberg into biogroups. Vet Rec., 129530-531. 86. Anderson, E. S., Ward, L. R., de Saxe, M. J., Old, D. C., Barker, R., and Duguid, J. P. (1978). Correlation of phage type, biotype and source in strains of Salmorzella typlzimuriunz. J. Hyg. Camb., 81:203-217. 87. Brandis, H. (1970). Die Lysotypic von Salmonellen der Enteritisgruppe mit besonderer Beriicksichtigung von S. typltinrurinm. Zentralbl. Brrberiol., 222232-244. 88. Duguid, J. P., Anderson, E. S., Alfredsson, G. A., Barker, R., and Old, D. C. (1975). A new biotyping scheme for Salmonella typlzimul-ium and its phylogenetic significance. J. Med. Micl-obiol., 8:149-166. 89. Anderson, E. S., and Williams, R. E. 0. (1956). Bacteriophage typing of enteric pathogens and staphylococci and its use in epidemiology. J. Clin. Patlzol., 9:94-127. 90. Jayasheela. M., Singh, G., Sharma, N. C., and Saxena, S. N. (1 987). A new scheme for phage typing Salmonellae bareilly and characterization of typing phages. J. Appl. Bacterid., 62: 429-432. 91. Laszlo, V. G., and Csorian, E. S. (1988). Subdivision of common salmonella serotypes: phage typing of S. virchow, S. mawhattan, S. tlzonlpson, S. oranienburg, and S. bareilly. Acta Microbial. Hung., 35:289-294. 92. Vieu, J. F., Jeanjean, S., Tournier, B., and et Klein, B. (1990). Application d’une sirie unique de bactiriphages i la lysotypic de Sah?lorzel/askrovar Dublin et de Salmonella sirovar Enteritidis. Mbd. Malad. Iitfect., 20:229-233. 93. Ward, L. R., de Sa, J. D. H., and Rowe, B. (1987). A phage-typing scheme for Salmonella enteritidis. Epidemiol. Itgect., 99:29 1-294. 94. Hicknlan-Brenner. F. W., Stubbs, A. D., and Fanner 111, J. J. (1991). Phage typing of Salmonella enteritidis in the United States. J. Clin. Microbiol., 29:2817-2823. 95. Lilleengen, K. (195 1). Typing of Salmonella gallinarum and Salmonella pullor~mby means of bacteriophage. Acta Path. Microbiol. Scand., 30: 194-202. 96. de Sa, J. D.H., Ward. L. R., and Rowe, B. (1980). A scheme for phage typing of Salntonella hadar. FEMS Microbiol. Lett., 9:175-177. 97. Gershman, M. (1977). A phage typing system for Salrnonella heidelberg. J. Food. Prot., 40: 43-44. 98. Laszlo, V. G., Csak, K., and Csorian, E. S. (1988). A phage typing system for Salntonella infantis. Acta Microbiol. Hung., 3555-69. 99. Maher, K. O., Morris Jr., J. G., Gotuzzo, E., Ferreccio, C., Ward, L. R., Benavente, L., Black, R. E., Rowe, B., and Levine. M. M. (1986). Molecular techniques in the study of Salmonella typlti in epidemiologic studies in endemic areas: comparison with Vi phage typing. Am. J. Trop. Med. Hyg., 35:831-835. 100. Killalea, D., Ward, L. R., Roberts, D., de Louvois, J., Sufi, F., Stuart. J. M., Wall, P. G., Susman, M., Schwieger, M., Sanderson, P. J., Fisher, I. S. T., Mead, P. S., Gill, 0. N., Bartlett, C. L. R., and Rowe, B. (1996): International epidemiological and microbiological study of outbreak of Salmonella agona infection from a ready to eat savoury snack-I: England and Wales and the United States. Br. Med. J., 313:1105-1107.

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101. Poppe, C., Irwin, R. J., Messier, S., Finley, G. G., and Oggel, J. (1991). The prevalence of Snln~onellr~ enteritidis and other Salntonella spp. among Canadian registered commercial chicken broiler flocks. Epidentiol. Infect., 107:201-211. 102. O’Brien, J. D. P. (1988). Salmonella enteritidis infection in broiler chickens. Vet. Rec., 122: 214. 103. Olsen, J. E., Brown, D. J., Skov, M. N., and Christensen, J. P. (1993). Bacterial typing methods suitable for epidemiological analysis. Applications in investigations of salmonellosis among livestock. Vet. Q., 15:125-135. 104. Frost, J. A., Ward, L. R., and Rowe, B. (1989). Acquisition of a drug resistance plasmid converts Salnlonella enteritidis phage type 4 to phage type 24. Epiderniol. Infect., 103:243238. 105. Threlfall, E. J., and Frost, J. A, (1990). The identification, typing and fingerprinting of Salmonella: laboratory aspects and epidemiological applications. J. Appl. Bacteriol., 685-1 6. 106. Platt, D. J., Brown, D. J., Old, D. C.. Barker, R. M., Munro, D. S.. and Taylor, J. (1987). Old and new techniques together resolve a problem of infection by Salntonella tyl,’l?imurium. Epidemiol. Iilfect., 99:137-142. 107. Platt, D. J., Chesharn, J. S.. Brown, D. J., Kraft, C. A., and Taggart, J. (1986). Restriction enzyme fingerprinting of enterobacterial plasmids: a simple strategy with wide application. J. Hyg. Carnb., 97:205-210. 108. Threlfall, E. J., Rowe. B., and Ward, L. R. (1989). Subdivision of Salmonella enteritidis phage types by plasmid profile typing. Epidemiol. Itlfect., 102:459-465. 109. Wachsmuth, I. K., Kiehlbauch, J. A., Bopp, C. A., Cameron, D. N., Strockbine. N. A., Wells, J. G., and Blake, P. A. (1991). The use of plasmid profiles and nucleic acid probes in epidemiologic investigations of foodborne. diarrheal diseases. Int. J. Food Microbiol., 1277-90. 110. Kapperud, G., Lassen, J., Dommarsnes, K., Kristiansen, B. E., Caugant, D. A., Ask, E., and Jahkola. M. (1989). Comparison of epidemiological marker methods for identification of Salrnonellu t?.lyhinzuriunrisolates from an outbreak caused by contaminated chocolate. J. Clin. Microbiol., 272019-2024. 111. Taylor, D. N., Wachsmuth, I. K., Shangkuan. Y.-H., Schmidt, E. V., Barrett, T. J., Schrader, J. S., Scherach. C. S., McGee, H. B., Feldtnan, R. A., and Brenner, D. J. (1982). Salmonellosis associated with marijuana: A multistate outbreak traced by plasmid fingerprinting. N. Engl. J. Med., 306:1249-1253. 112. Mayer, K. H., and Hanson, E. (1986). Recurrent Salrnonellct infection with a single strain in the acquired immunodeficiency syndrome. Confirnlation by plasmid fingerprinting. Dingn. Microbiol. Infect. Dis., 4:71-76. typhimurittm and other salmonellae. 113, Gulig, P. A. (1990). Virulence plasmids of Sul~~onella Microb. Pathog., 8:3-11. 114. Helmuth. R., Stephan, R., Bunge, C., Hoog, B., Steinbeck, A., and Bulling, E. (1985). Epidemiology of virulence-associated plasmids and outer membrane protein patterns within seven conmon Salmonella serotypes. Infect. Immun., 48: 175-1 82. 115. Popoff, M. Y., Miras, I.. Coynault, C., Lasselin, C., and Pardon, P. (1984). Molecular relationships between virulence plasmids of Salmonella serotypes typlzirnuriunz and dublin and large plasmids of other Salmonella serotypes. Anrl. Microbiol. (Paris), 135A: 389-398. 116. Ou. J. T., Baron, L. S., Dai. X., and Life, C. A. (1990). The virulence plasmids of Salmonelln serovars typlzirnurizun,choleraesuis, dublirt, and enteritidis, and the cryptic plasmids of Salmonellt serovars copenhaget1 and sendai belong to the same incompatibility group, but not those of Salmonella serovars hcrban, gallinarurn, give, irlfantis and pullorunt. Microb. Pathog., 8:lOl-107. 117. Ou, J. T., and Baron, L. S. (1991). Strain differences in expression of virulence by the 90 kilobase pair virulence plasmid of Salmonella serovar Typhimurium. Microb. Pathog., 10: 247-25 1.

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118. Caldwell, A. L., and Gulig, P. A. (1991). The Salmonella t y h i n r w i t m virulence plasmid encodes a positive regulator of a plasmid-encoded virulence gene. J. Bacteriol.. 173:71767185. 119. Barrow, P. A.,andLovell, M. A. (1989). Functionalhomologyofvirulenceplasmidsin Srrllnonella gnllinasrcnr, S. pullort~nz.and S. ~phinturircnr.Irlfect. Imrnun.. 57:3 136-3 141. 120. Beninger, P. R., Chikami, G., Tanabe, K., Roudier, C., Fierer, J., and Guiney, D. G. (1988). Physical and genetic mapping of the Sultnonelln dzdblin virulence plasmid pSDL2. Relationship to plasmids from other Salr~ronellastrains. J. Clin. h e s t . , 81:l341-1347. 121. Krause, M., Harwood, J., Fierer, J., and Guiney, D. (1991). Genetic analysis of homology between the virulence plasmidsof Salnzonelln dublin and Yersinia pseudotuberculosis. Illfect. Imnun., 59:1860-1863. 122. Roudier, C., Krause, M., Fierer, J., and Guiney, D. G. (1990).Correlation between the presence of sequences homologous to the vir- region of Strlmonelln dublin plasmid pSDL2 and the virulence of twenty-two Saltnonelln serotypes in mice. Zrfect. Zmrnm., 58:1180-1185. 123. Taira, S., Riikonen, P., Saarilahti, H., Sukupolvi, S., and Rhen, M. (1991). The mkaC virulence gene of the Scrllnonellrr serovar Typhimurium 96 kb plasmid encodes a transcriptional activator. Mol. Gen. Genet.. 228:381-384. 124. Taira, S.. Baumann, M., Riikonen. P., Sukupolvi, S., and Rhen. M. (1991). Amino-terminal sequence analysis offour plasmid-encoded virulence-associated proteins ofSalmonella 8phinruriunz. FEMS Microbiol. Lett., 77:319-323. 125. Valone, S. E., and Chikami, G. K. (1991). Characterization of three proteins expressed from thevirulenceregion of plasmidpSDL2in Sal~no~~ellc~ dzcblin. hfect. I r n m m . , 59:35113517. 126. Hoertt, B. E., Ou. J., Kopecko, D. J., Baron, L. S., and Warren, R. L. (1989).Novel virulence properties of theSnblrorzelln typlzimwium virulence-associated plasmid: immune suppression and stimulation of splenomegaly. Plmnid, 21 :48-58. 127. Threlfall, E. J., Powell, N. G.,and Rowe, B. (1994). Differentiation of salmonellas by molecular methods. PHLS Micr-obiol. Dig.. 11:199-202. 128. Esteban, E., Snipes. K.. Hird, D., Kasten, R., and Kinde, H. (1993). Use of ribotyping for characterization of Scrlnronella serotypes. J. Clin. Microbiol., 3 1233-237. of 129. Nastasi, A., and Mammina, C. (1995). Epidemiological evaluation by PCR ribotyping sporadic and outbreak-associated strains of Suhnonella entericn serotype Typhimurium. Res. Microbiol., 146:99-106. 130. Kilger, G., and Grimont, P. A. D. (1993). Differentiation of Salmonella phase I flagellar antigen types by restriction of the amplified p i c gene. J. Clin. Microbiol., 31 : l 108-1 110. 131. Rexach, L., Dilasser, F., and Fach, P. (19931. Polymerase chain reactionfor Snlrnonellrr virulence-associated plasmid genes detection: a new tool in salmonella epidemiology. Epidenziol. Infect., 112:33-43. 132. Rhan, K., De Gandis, S. A., and Clarke, R.C. (1992). Amplification of an invA gene sequence of Srrlnlorzellcr Dphirnuriunz by polymerase chain reaction as a specific method of detection of salmonella. Mol. Cell Probes, 6:271-279. 133. Soria, G., Barbe, J., and Gibert, I. (1994). Molecular fingerprinting of Salnronelln typphinzuriurn by IS200-typing as a tool for epidemiological and evolutionary studies.Il4icrobiologia, 10:57-68. 133. Stanley, J., and Saunders, N. (1996). DNA insertion sequences and the molecular epidemiology of Salmonella and Mycobncteriurn. J. Med. Microbiol.. 35236-25 1. 135. Tompkins. L. S., Troup. N., Labigne-Roussel, A., and Cohen. M. I. (1986). Cloned, random chromosomal sequences as probes to identify Snlrnonella species. J. Infect. Dis., 154:156162. 136. Lin. A. W., Usera, M. A., Barrett, T. J., and Goldsby, R. A. (1996). Application of random amplified polymorphic DNA analysisto differentiate strains ofSnlrnonellrr enteritidis. J. Clin. Microbiol., 34:870-876.

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137. Olsen, J. E., Skov, M. N., Threlfall. E. J., and Brown. D. J. (1994). Clonal lines of Salnronellu entericn serotype Enteritidis documented by TS200-, ribo-. pulsed-field gel electrophoresis and RFLP typing. J. Med. Microbiol., 40: 15-22. 138. Suzuki, Y., Ishihara, M.. Matsumoto, M., Arakawa, S., Saito, M., Ishikawa, N., and Yokochi, T. (1995).Molecular epidemiology ofSnlmo~~ellu ewteritidis. An outbreak and sporadic cases J. Zrzfect., 31211-217. studied by means of pulse-field gel electrophoresis. 139. Thong, K.-L., Cheong, Y.-M., Puthucheary, S., Koh, C.-L., and Pang. T. (1994). Epidenliologic analysis of sporadicSulnlonelln hphi isolates and those from outbreaks by pulsed-field gel electrophoresis. J. Clill. Microbiol., 32:1135-1141. 130. Powell, N.G., ThreIfall, E. J., Chart, H., and Rowe, B. (1994). Subdivlsion of Sal~no~ellrr enteritidis PT 4 by pulsed-field gel electrophoresis; potential for epidetniological surveillance. FEMS Microbiol. Lett., 119: 193-198. 141. Powell, N. G., Threlfall, E. J., Chart, H.. Schofield. S. L., and Rowe, B. (1995). Correlation of change in phage type with pulsed field profile and 16s rrn profile in Solmorzellu enteritidis phage type 4, 7 and 9A. Epidemiol. Infect., 114:403-411.

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12 Salmonellosis in Animals David J. Nisbet and Richard L. Ziprin U S . Department of Agricrrlture, College Stutiorr, Texas

I. Sulmonelln in Companion Animals 265 11. Salnzorudlu in Cattle and Dairy Cows 267 111. Subuonella in Poultry 269 IV. Salmonella in Swine 274 References 275

Salmonella bacteria can colonize or infect a wide variety of domestic animals, both companion and agricultural animals. When colonized animals are asymptomatic, they may become an insidious source of inoculum for foods and, ultimately, humans. Rodents, pests, and reptiles may act asreservoirs and vectors for the transmission of Salmonellct (1). Insect pests have also been incritninated as Salmonella carriers (2). Symptomatic salmonellosis in animals, as in humans, can be a relatively benign illness with only minor gastrointestinal symptoms, or it can bea severe and life-threatening illness, Each animal species responds differently to specific serotypes, and therefore the nature of salmonellosis varies with the species infected, the infecting serotype, and the general condition of the infected animal, i.e., age and overall health status.

1.

SALMONELLA IN COMPANIONANIMALS

Salmonellosis has been observed in common pets such as cats (3- lo>and dogs (1 1-17) and in less common companion animals such as the horse (18-30). Salmonellosis in cats, dogs, and horses is particularly instructive because the full range of disease variability is readily seen in these animals. The percentage of asymptomatic Salmonella carriers among cats has been variously estimated at between 1% and 18% (3,4,6). Serotypes found in cats include Salmonella Derby, Salnzonella Typhimurium, SulrnoneZla Anatum, Salmonella Enteritidis, and Salmonella Newport. While SaZmoneZZn-infected cats are usual asymptomatic caniers, disease symptoms of salmonellosis in cats can include conjunctivitis and bacteremia. Feline salmonellosis may also present as a chronic febrile illness resembling human enteric fever (typhoid fever). 265

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The incidence of asymptomatic carriage in dogs has been estimated at between 0.6 and 27.6% of randomly sampled animals. depending on the study population. Fifty-three serotypes were found in one study of 8000 dogs in Florida (1 3).In 1996 Alaskan sled dogs participating in the Iditarod Trail Sled Dog Race were sampled for the presence of salmonellae. Salnnonelln was isolated from 18 of 26 (69%)normal asymptomatic dogs prior to the race during routine prerace veterinary inspection. Of these isolates 13 were Saltnorzella Typhimurium or Snlrnorlelln Typhimurium (Copenhagen); other serotypes isolated were Sulmorzella Infantis and Salrnolzella Reading. Two dogs were infected with both Salmonella Typhimuriunl (Copenhagen) and S c h o l d l n Infantis. During the race 30 dogs with diarrhea were sampled, 19 (63%) of which were Snlmorzelln positive: additionally 23 nondiarrahetic dogs were sampled, of which 13 (57%) were Salnzonelln positive. In this study no difference in the incidence of Salmonella was found between the prerace dogs and dogs sampled during the race (17).However a surprisingly high incidence of Salmonella in these Alaskan sled dogs was found compared to the prevalence reported in other dogs. Salmonellosis is also common in racing greyhounds. A study performed in 1993 found Snlnlonella in 11% of asymptomatic greyhounds and 76% of diarrhetic greyhounds (15).In that study a high prevalence of Snlnzor?ella was also found in the dogs' food that consisted of raw meat obtained from rendering plants (16).The increased prevalence of Scrlnzonella found in these working dogs may be associated with not only feed but also the stresses associated with rigorous training. The serotypes commonly isolated from dogs include those commonly isolated from humans. S a Z ~ ~ ~ o ~Typhimurium ~elln was isolated from 40% of the Scrlr.llonell~~-positive dogs. Salmonellu Enteritidis, Sdnlonelln Infantis, and Snlmor~ellnParatyphi B have all been transmitted to humans through an association with illdogs. Interestingly, Salmonella are not usually found in dog biscuits, dog candy, flakes, or kibbled products because these are heat-processed pelleted or extruded foods and the heat and pressure are sufficient to kill the bacteria. Salmonellosis in symptomatic dogs is similar to the disease in other mammals-fever, malaise, diarrhea, or abortion following uterine infection. In severe cases, symptoms may include hemorrhagic gastroenteritis and ultimately death (5,l 1,12,14). Salmonella Ohio has been observed to survive in dried canine fecal matter (1 1.6% moisture content) for at least 3 months (14). Similarly, horses may be asymptomatic carriers of Sulrnomlln. or they may experience clinical disease with dialrhea lasting 5-10 days. Fever may reach extretnes in instances of necrotizing colitis (106"F),and the infection may become a fatal septicemia. Young animals, under 4 months of age, may face a mortality rate as high as 50%. Snlmonella Abortus-Equi is a cause of abortion in pregnant mares. When the foals of infected mares are live-born, they commonly die within a few hours. When they do survive they may develop a septic arthritis (18).Although often reported in Asia and Africa, until recently Salrnonelln Abortus-Equi was no longer isolated from Europe and the United States (31).However, a recent outbreak was reported in Croatia in a herd of 38 horses, of which 26 were pregnant mares. In this outbreak 21 mares aborted suddenly and Salmonelln Abortus-Equi was isolated as the single causative agent in each case (32). Studies of the prevalence of salmonellae in asymptomatic horses have suggested carriage rates in the 0.2-15% range (33,34),and Salnzonelln Agona, which in 1975 was the fifth most frequently isolated serotype from humans, is commonly found in horses (1 1,3536). However, the incidence of asymptomatic carriage may be much higher. While the 0.2-15% rate is based on fecal or cecal cultures, enrichment culture of mesenteric lymph nodes from healthy horses demonstrated that 35 of 50 horses examined had asymptomatic SalnlonelZa infections (37).Fifteen different serotypes were recovered from the

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35 infected horses. These serotypes included the common serotypes found in human infections: Sctl~rrorzellc~ Reading, Scrlmonellcr Cerro, and Scdmolzelln Munchen (37). This high incidence of recovery of Salrzlor~ellerfrom lymph nodes establishes the status of horses as true carriers. It is well known among veterinary clinicians that acute clinical equine salmonellosis tends to occur when the animals are stressed. Smith et al. (34) list shipping, antibiotics, anthelminthic drugs, surgery, and prednisone as stresses associated with equine salmonellosis. The mechanisms by which stress causes asymptomatic carriage to transform into a clinical illness are largely unknown. Antibiotic treatments may simply allow the development of a large intraluminal Schnonelln population as a consequence of disruption of the normal intestinal microflora. Stresses such as shipping and veterinary treatment with immunosuppressive doses of corticosteroids undoubtedly impact in a negative wayon imnlunological defense mechanisms (38). Hospitalized horses are also at risk of becoming infected with Snlmorzellcr. A case-control study involving nosocomial cases was performed during an 1 l-yearperiod at the Veterinary Medical Teaching Hospital, University of California, Davis. This study revealed that horses in which nasogastric tubes were passed had a 2.9-fold greater risk of having Snbuolzellc1 isolated, compared with horses that did not undergo this procedure. Horses treated with antibiotics parenterally had a 6.4-fold greater risk, and those treated with antibiotics orally and parenterally had a 40.4-fold greater risk of developing salmonellosis, compared to horses not receiving such treatment. Additionally, horses admitted because of colic were 4.2 times more likely to have Snlmorzellcr isolated as those admitted for other reasons. Breed, age, and type of surgery did not appear to be risk factors (39). Recently the Large Animal Veterinary Teaching Hospital at Colorado State University was temporarily closed due to possible nosocomial infections of animals with Sabnomdla Infantis. The original source of the organism responsible for the outbreak was never determined, however, possible sources included hospital patients, hospital personnel, pigs housed in the facility used for teaching and research, wildlife (rodents or birds), and feed used for large animal patients (40). During the past several years there have been several reports of Salmonella outbreaks in veterinary teaching hospitals (33-35). Horses in such facilities may be particularly susceptible to Snlmonellcr infections due to factors such as long-distance transportation, dietary changes, anesthesia, surgery, concurrent disease, and treatment with antimicrobials (36-38). It is unfortunate that the horse is an expensive animal and is generally not well suited to use as a laboratory animal, as it might otherwise be a very useful model for studying the effects of stress on resistance mechanisms.

II. SALMONELLA IN CATTLEANDDAIRYCOWS Salmonellosis wasfirst observed in cattle as early as 1902, but infection of cattle by Snlrnolzelln had been considered rare as recently as the early 1960s (41,42). At that time a 5-year study at Cornel1 University of 731 Scdmorlelln Typhimurium cultures isolated from animal sources revealed that only 2 isolates originated in cattle (42). According to Jensen and Mackey (42), one study of 7365 salmonellosis outbreaks revealed that only 39 of these involved cattle. In 1965, a study of salmonellosis on Michigan farms revealed 39 outbreaks during a 20-month period with a 23% mortality rate among calves (43). Bovine salmonellosis is now considered an economically important disease to producers and continues to increase as a public health problem (44-52). Snlrno?zellnin dairy cattle

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is also of particular importance due to both the economic losses in animal production and because both milk and meat from infected cattle can pose serious public health risks. Salmonella infections of cattle range in severity from asymptomatic carriage to fatal bacteremia. Young animals have high mortality rates, but salmonellosis can be fatal in adult animals as well (53). The most frequently identified serotypes recovered from cattle are Scrlnrorlella Typhimurium, Salmonella Dublin, and Salmonella Typhimurium (Copenhagen) (50,5 1,54-57). Additionally, Salrnorzellcr Enteritidis (58) and Sulrnorlelln Muenster have also been isolated in milk and traced to cows with subclinical mammary infections (59). Corrier et al. (60) collected fecal samples from a herd of 200 feeder-calves and determined the prevalence of Snlrnonella carriage and the serotypes of the isolates. They monitored the herd from assembly at an auction market through the shipping process and at the feed-lot. Salmonellae were not isolated from samples collected at the farm or the auction market, but Salmonella-positive samples were found at the feed-yard. After 30 days at the feed-lot approximately 8% of the calves were infected, and the authors suggested that stressors immediately prior to arrival at the feedlot or during feedlot confinement may be contributing factors to the increase in fecal excretion of Salmonella. Serotypes recovered included Sabtzouella Anatum, Salnzonella Cerro, Saltnonellcl Newbrunswick, Salmor1ella Typhimurium (Copenhagen), and Subnonella Reading. Saltnorzellu Dublin was not isolated during this study. Salnlorlella Dublin is of particular concern. It causes septicemia in calves and abortion in adult dairy cattle. The feces of infected animals that have survived their infection may become persistently contaminated with Salrnorzella (61). Asymptomatic carriers also shed Scrlmonella Dublin in their milk. Concentrations of the organism in raw milk may approach 100,000 cells/mL, and epidemics of human salmonellosis have been traced to consumption of contaminated unpasteurized milk and dairy products (62,63). Salnzonella Dublin has a reputation for being very virulent to humans. It tends to infect older persons and patients suffering from chronic debilitating illness. Bacteremia has been observed in 75% of infected individuals, and mortality rates as high as 35% have been observed (62). In one case, an otherwise healthy 27-year-old, having sustained a minor abdominal injury during a fight, suffered a splenic rupture due to salnlonellosis caused by Salmorlella Dublin. The patient had consumed raw milk 12 days before his injury and had not had any gastroenterological symptoms or other significant symptoms of salmonellosis prior to his injury (64). Because the Dublin serotype is of special concern to public health and because there is a need to identify asymptomatic carrier cattle, considerable effort has gone into the development of immunological methods for determining the carrier status of individual animals. Wray and Callow (65) have developed a fluorescent antibody method, while Smith and colleagues (66,67) have worked to develop an enzyme-linked immunoassay (ELISA). The principle of the test is based on the fact that infected cattle develop antibodies against salmonellae, and these antibodies, which are present in milk, will react with Salmonella Dublin antigens bound to the solid surface of the microtiter plates. The bound antibodies are detected with horseradish peroxidase conjugated goat anti-bovine antisera (66,67). ELISA has been used to measure specific IgG titers in cattle and has proven useful for assessing vaccine responses, infection rates, carrier status, and epidemiology of bovine salmonellosis (68). Cattle often persistently shed Salmolzella Dublin in their milk, suggesting the presence of chronic mastitis. It has recently been discovered that bovine mastitis is accompanied by changes in the serum concentration of an acute phase protein, haptoglobin, which

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Animals

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is known to increase during mastitis (69,70). To these authors’ knowledge, haptoglobin measurements have not been used to identify possible carrier cows. There have been several efforts to immunize dairy cattle against Dublin. Besides reducing morbidity in cattle, the goal is to protect human health by reducing the incidence of Dublin-infected animals and hence Dublin-infected dairy products (7 1-79). Reported results have been mixed and inconclusive, but there have been successes. For example, Segall(71) reported that a vaccine strain of S. Dublin protected against the Dublin serovar but not against challenge with Typhimurium.

111.

SALMONELLA IN POULTRY

There is an extremely extensive literature on avian salmonellosis, and much of this literature has been compiled and interpreted in veterinary texts (80,81). Pullorum disease and fowl typhoid are acute lethal diseases of chickens caused by Salmonella GallinarumPullorum. While these diseases are of considerable interest and economic importance to poultry producers, Salrnonella Gallinarum-Pullorum does not have human public health significance. Chickens may also become infected with many of the other Salmonella serotypes. Usually only young chicks become ill, and disease among young hatchlings can result in high death rates with concomitant significant economic loss. Mortality rates may vary from negligible to 80% in unusually severe outbreaks (82j. Turkey poults are exceptionally susceptible to salmonellosis, and mortality rates may become extreme. Chicks seem in general to be more resistant than turkey poults. Chicks rapidly develop resistance to system salmonellosis during the first few days of life, especially between day 3 and day 5. By the seventh day it is nearly impossible to cause acute systemic illness, even with massive doses of bacteria given by direct intraperitoneal injection. This acquisition of resistance is tied to maturation of the immune system and the onset of cell-mediated immunity (8386). Usually the presence of salmonellae in a chicken flock has no effect on production whatsoever and goes unnoticed. Salmonella is therefore of no particular concern to the individual farmer. There are, however, two public health issues related to asymptomatic carriage: contaminated broiler carcasses and Snlrzlonella-contaminated shell eggs. Both are significant sources of salmonellae and pose serious public health issues. Snlmonellu bacteria are present on the skin, feathers, and in the feces of a small proportion of broiler chickens at the time of slaughter, and conditions in the slaughterhouses tend to allow the spread of these bacteria among the carcasses so that eventually a high percentage of the carcasses are contaminated to some degree; estimates range upward from 21% (87-89). The USDA’s Food Safety and Inspection Service in the early 1990s conducted one study at a chicken-processing plant located in Puerto Rico. Eight hundred samples were collected during 20 consecutive 8-hour shifts at five sites along the processing line. Seventy-seven percent of the “postautomatic cut’’ samples were culture positive for salmonellae, and 58% of the “preevisceration” carcasses were found to be contaminated with salmonellae (90,9 1). There have been several studies of the contamination incidence at different points during broiler production. However, with the initiation of the new Hazard Analysis Critical Control Points (HACCP) system for quality control in poultry-processing plants (92,93), the incidence is likely to decrease dramatically. Chicks may initially become infected in the hatchery (94), at the broiler house from con-

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taminated feed (95j, and from the droppings of animal pests (95). However, in one study, contamination of broiler carcasses was shown to mainly occur during transport and processing (89). It is not possible to obtain an accurate estimate of the proportion of human salmonellosis cases attributable to poultry. A given case could be attributed to any of many possible sources in the home, including pets, but the patient may only associate the illness with food recently consumed. Tauxe (96) has discussed some of the difficulties that the CDC faces when trying to estimate the proportion of human salmonellosis cases attributable to poultry. Estimates vary from 7.5 to 37% depending upon the methodology used and the assumptions underlying the methodology. Fortunately, careful attention to common household hygiene and cooking procedures and attention to food production hygiene and cooked food handling are sufficient to deal with health threats due to contaminated broiler carcasses. Nonetheless, the high rate of Sch?orzelln contamination among broiler carcasses has resulted in some considerable anxiety among the consuming public (96-99). Proper cooking of poultry as well as other food products that may be contaminated will certainly ensure that Scrlnzor~ellainfection does not occur due to ingestion of the cooked product. Improper handling of the product prior to cooking can lead to contamination of surfaces and utensils used during food preparation, and infections can occur due to contact with these surfaces or items. A somewhat different situation exists with regard to Sc~lnzolzelZn-contan7inatedshell eggs. First, the Salrnolzellcr Enteritidis serotype that is commonly associated with shell eggs appears to be more virulent for humans than other serotypes. Second, ordinary foodpreparation methods for egg-containing recipes may not always be adequate to control the organism. Third, while one may safely assume that for practical purposes all meat has some microbial contanlination and must be thoroughly cooked, the same assumption does not apply to eggs. Often egg-containing foods such as hollandaise sauce, mayonnaise, and bernaise sauces are prepared with uncooked or only partially cooked egg yolk (96,100104), and some people like to eat “3-minute” eggs in which the yolk is not fully cooked. Fourth, as regards the occurrence of large nosocomial (institutional) outbreaks (105-107), the nature of institutional food service operations associated with hospitals and nursing homes provides opportunity for dissemination of Snlvzonellcr Enteritidis among large populations of debilitated individuals. Indeed, during the 4-year period of 1985-1 988 there were 30 deaths among 4976 cases of Salmolzella Enteritidis food poisoning, 27 of which were among hospital and nursing home patients. From 1975 through 1987 there were 115 outbreaks of foodborne disease in nursing homes that involved nearly 5000 patients. Fiftytwo percent of these outbreaks and 82% of the deaths were attributable to Salrnorwlln of various serotypes, but Sulnzonellct Enteritidis was responsible for 56% of the deaths due to salmonellosis (96,103,108,109). The largest nosocomial outbreak due to Snlnzordln Enteritidis was investigated by the New York City Department of Health and involved 404 of 965 patients at one hospital. The outbreak resulted in nine deaths. Investigation traced the outbreak to raw eggs. traced back the eggs to the producing farm, and traced the infection to the ovary of a hen on the producing farm ( l lOj. Snlnzolzellu Enteritidis and sometimes other serovars (1 1 1,112jwill infect the ovaries of laying hens ( 113-1 16). The role of ovarian transmission of SalnlorzelZcr Enteritidis to the egg and the role of such a process in the natural dissemination of the organism is now generally accepted. Snlmonellrr can also penetrate and enter the egg through an intact shell as well as through minor cracks in the eggshell, and therefore it is not certain that all instances of outbreaks due to Scrlmonelle~contaminated eggs are the result of transovarian infection. Also, there is some evidence that while Scrlnzorder Enteritidis phage type 8 can

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penetrate the eggshell, it cannot enter the egg contents (1 17), as the inner membrane structure of the egg acts as a mechanical barrier protecting the egg contents. Invasive strains of Salmonella Enteritidis such as phage type 4 have been identified as the most likely source of egg contamination due to transovarian transmission and eggshell penetration (118,119). The presence of Salmonella Enteritidis (phage type unreported) has been reported in environmental samples collected from broiler fmns in the United States (120), but Saln1onella Enteritidis phage type 4 had not been isolated in U.S. poultry flocks until recently. In 1996 this especially virulent strain was isolated from a layer flock in the United States ( 121). Egg yolks obtained from Salnzonellcr Enteritidis-infected chickens contain antibodies directed against serogroup D antigens, suggesting that it may be possible to detect infected chickens, and possibly the eggs from infected chickens, by serological means (1 1 1,122- 125). Efforts are being made to improve upon the specificity of serological detection methods by focusing upon bacterial antigens that are limited to a very few serotypes, gm flagellin isolated from a highly motile strain of phage type 8 Sdmozzella Enteritidis, for example (126). There have been several proposals to vaccinate chickens against salmonellae. Recently, a live avirulent Salmonello vaccine was shown to induce excellent protection against intestinal, visceral, reproductive tract, and egg colonization, invasion, and/or contamination by Salnlorzella. The duration of this protection was for 11 months at which time the experiment was terminated (127). One disadvantage of a vaccination program would be the interference of vaccine-induced antibodies in serological testing for pullorum disease and interference in the serological detection of Salrnonellu Enteritidisinfected birds. Not all outbreaks of SaZmolleZZa Enteritidis are traceable to the consumption of contaminated eggs. In one large outbreak traced to a fast food restaurant, it was determined that an employee who had been ill with gastroenteritis contaminated the restaurant food. This employee continued working to the end of his shift even after experiencing the initial symptoms of illness and was probably responsible for infecting several other employees as well as thepatrons served during that shift. The source of his illness was not determined (128). Another restaurant-related outbreak was traced to sauces prepared from eggs. Eighty-one patrons were infected, 24 cases were culture-proven, and l 1 patients required hospitalization. Grade A intact extra-large eggs were the source of infection, and the eggs were traced to a farm at which Sulnzonelln Enteritidis was found. The isolate had plasmid profiles and antimicrobial susceptibility patterns indistinguishable from the outbreak isolate (103). In another series of restaurant-related outbreaks, scrambled eggs prepared from a large batch of raw egg mixture was implicated. At one of the restaurants the scrambled eggs were deliberately undercooked and 1 13 patrons developed gastrointestinal illness lasting more than one day. Of these 113 victims, 17 required hospitalization and 2 suffered Salmonella sepsis (129). Although these outbreaks are dramatic, restaurant food is usually quite safe. It has been estimated that nearly 46 tnillion people eat at more than 138,000 fast food restaurants per day, and there are remarkably few incidents of salmonellosis attributable to these establishments (128). Contaminated turkey has also been implicated in outbreaks of human salmonellosis (96). Salmonella Reading, Snl?nonellrHeidelberg, and Salmonella Saint-Paul are the three serotypes most frequently isolated from the birds (54,55), and these serotypes account for approximately 42% of the turkey isolates. Because salmonellosis is economically important to the turkey producer and because the turkey production system in the United States is unusually well vertically integrated, the turkey industry has greater means for Salmonella control than other animal industries (130). Critical control points have been identified

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that cover all phases of turkey production, turkey feed production, and whole carcass processing (130). A 5-year epidemiological study of Salmonella infections in turkeys has demonstrated the importance of preventing environmental contamination of breeding premises and revealed the importance of contaminated feed as a source of Salmonella infection (131). Surveys of animal-rendering facilities have demonstrated the need for additional hygiene practices in the animal feed industry. One survey of 83 rendering facilities revealed that 27% of analyzed samples were Salmonella positive (132). It is possible to produce Salmonellr-free turkeys if the feed is Scrlmonella-free, and ordinary prudent hygiene and pest control practices are followed. In one 4- to 5-year study, 2.5 million Salmonella-free poults were produced from 7500 hens and 600 toms. The producer used pelleted feed that did not contain animal protein, a frequent source of Salmonella, and samples of the feed were cultured upon delivery to the farm in order to ensure that the feed did not contain Snlnzorda (133). (The advantage of pelleting in the preparation of pet food has been previously mentioned.) Because of the importance of animal feed as a source of salmonellae to both turkey and chicken producers, the Animal Protein Producers Industry (APPI) has established an educational program for their members, which is designed to help them reduce the Salmonella contamination of their products. This Salnzonella Education-Reduction plan is based on the premise that careful attention to simple hygiene practices can help the animal feed producer produce feed free from salmonellae. Another approach to animal feed sanitation is the use of feed additives with possible antibacterial/anti-Salmonella properties: typically these are proprietary formulations of formic or propionic acid (134- 140). Ideally we wish to eliminate Salmonella from live birds so that the organisms do not reach the processing plant and cannot be disseminated during processing. It is possible to prevent introduction of Salmonella into a flock, and it may be possible to produce Salmonella-free chickens through a combination of relatively simple procedures-provided that they are made mandatory. In Sweden, a mandatory Scdmonella control plan has been in place for more than a decade for farms with flock populations above 5000 birds. The essentials of the plan are simple. Feed is produced in very well-regulated sanitary mills that are operated with the intent of producing Salmonella-free feed. All imported feed is tested and rejected if found to be contaminated. When salmonellae are found at the farm, the premises are quarantined and the birds are sent to special slaughter facilities designed to deal with contaminated birds. When flocks are found in which more than 5% of the birds are contaminated, the birds are destroyed and there is an indemnification program for the farmer. All breeding birds are routinely screened for salmonellae, and all dead birds on a farm are tested for salmonellae. The Nurmi competitive exclusion concept is utilized; that is, procedures are followed that aid the rapid establishment of normal intestinal flora in young birds. Young chicks with a normal “adult” intestinal flora resist colonization by salmonellae (14 1- 147). Recently there has been considerable research interest in improving the efficacy of competitive exclusion. Cox et al. (148) have attempted to ensure the early colonization of chicks by applying competitive exclusion bacteria it2 ovo, though this proved largely ineffective. And there have been attempts to improve control by manipulating the competitive exclusion flora in various ways (149-151). More recently, research interest was focused on defining the bacteria responsible for colonization control, and some studies have pointed to lactobacilli as important contributors to colonization control (152- 154). Other studies have shown that competitive exclusion cultures developed from a single strain of bacteria or from mixtures of strains from the same genus have not provided consistent

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protection against Sdmonella challenge (155), and it has been suggested that efficacious defined competitive exclusion cultures will need tobe more complex and contain a mixture of several bacterial isolates from different genera (156,157). Undefined competitive exclusion cultures contain mixtures of several hundred different bacterial isolates and have been shown to be efficacious, but cannot be used in many countries because they have not been approved by regulatory agencies. There are public health issues that arise any time food animals are deliberately exposed to undefined cultures that may contain pathogens or potential pathogens. In the United States these types of product are considered undefined drugs, and the U.S. Food and Drug Administration (FDA) has not yet given them regulatory approval. At least one undefined competitive exclusion culture has been shown to be efficacious in commercial field trials (158). An obvious alternative to undefined competitive exclusion cultures would be the development of an efficacious defined competitive exclusion culture. Historically, mixed defined competitive exclusion cultures have been difficult to develop due to lack of criteria for isolation, identifying, or maintaining the cultures in vitro (156,157), and this difficulty has hindered the development of a commercial product. Continuous-flow culture systems were recently used for the first time to maintain normal mixed microflora from the cecae of adult chickens in steady-state continuous culture (159,160). Subsequently this technique was utilized to develop a competitive exclusion culture characterized to contain 29 different bacterial isolates that was shown to control Sdmonelln colonization in experimental studies (16 1) and commercial field trials (162). Additionally, in an FDA-approved, doubleblinded, clinical trial, chicks treated with the culture at day-of-age were cecal Sulmonellu free at end of grow-out (163). In early 1998 this competitive exclusion culture (trade name PREEMPT@')was approved by FDA for use in commercial poultry production in the United States. Another method that has been investigated for use in controlling Salrnonella colonization in poultry is the use of various sugars provided to chickens in either feed or water. When chicks are given feed containing lactose, arabinose, or galactose, adherence of SnlInonella Typhimurium to the chick cecal surface is reduced (164), and numerous studies have now demonstrated that dietary lactose can sometimes be an effective adjunct to competitive exclusion (165-169). The mechanism by which lactose operates in conjunction with cecal microflora to control colonization is largely unknown. Because the chicken lacks the enzyme lactase, most of the dietary lactose is passed through the intestinal tract and becomes available for fermentation by lactose fermenting bacteria in the chicken cecae. Also, it has been suggested that sugars may compete with Salmonella for binding sites on the intestinal mucosal surface (170). Competition for binding sites on intestinal mucosal surfaces is, in our opinion, probably not a realistic mechanism, and there is very good circumstantial evidence that control is accomplished by other mechanisms including bacteriostatic/bactericidal volatile fatty acids produced in situ by the intestinal flora. The mechanism by which bacteriostatic volatile fatty acids may reduce colonization by intestinal pathogens has been well studied in chickens, and this mechanism may also operate to reduce Salmonella colonization within the intestinal lumen of swine (171). There is one report in which the Salmonella incidence was compared between pig farms that raised swine on liquid whey (naturally high in lactose) and farms that used ordinary drinking water. Salmonella bacteria were found at a lower percentage in swine on farms using whey than those using water. On farms where the premises were known to be contaminated, whey treatments reduced the proportion of pigs found to be culture positive. There was a highly significant statistical reduction in theproportion of culture-positive pigs given

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whey compared to those given water (172). The use of lactose to control Salrnonelln in commercial poultry flocks has not been adapted by the industry, most likely due to production economics and inconsistent experimental results.

IV.

SALMONELLA IN SWINE

We have already noted that Salmorzelln erzterica Choleraesuis has been a known swine pathogen since the mid-1880s. Approximately 1000 of the 1400 Scdrnor~ellaisolates from swine Sulrnorzella serotyped between July 1989 and June 1990 by the National Veterinary Services Laboratories, Ames, Iowa, were Salrrzonelln Choleraesuis. The remaining isolates were distributed more or less randomly among the other serotypes (54). Of 277 Sc~ln~or~elle~ isolates obtained from swine in Kansas during the 1979-1 983 period, 66% were Salmonella Choleraesuis, 8.6% were Salrnorzelln Typhimurium, and none of the remaining 25 serotypes isolated comprised more than 4% of the total. Of the top 10 clinical serotypes recovered from swine in 1996 as reported by National Veterinary Services Laboratories (173) and the top 10 serotypes recovered from humans as reported by the Centers for Disease Control in 1994 (174), three serotypes-Salmonella Typhimurium, Sahnomllcr Heidelberg, and Snlr~lonellaAgona-appear on both lists. Salrnonellu Agona has not been recognized as a significant pathogen of swine despite its importance in the human population (175). The USDA, Animal Plant Health Inspection Agency, Veterinary Service analyzed 6655 samples from 152 grower/finisher farms in a 1995 study. The sample and herd prevalence rates were 6.2% and 38.2%, respectively. The 10 most common serotypes recovered were Derby (33.5%), Agona (13.0%), Typhimurium (Copenhagen) (lO.l%), Bradenburg (8.0%), Mbandaka (7.7%), Typhimurium (3.6%), Heidelberg (3.6%), Anatum (1.9%), Enteritidis PT13A (1.7%), and Worthington (1.7%). Among the 58 Salmonellapositive farms, 15.8% were positive for Snlmonellrr Derby, while 6.6% of the farms were positive for Snlnzomlln Agona (176). Ferris and Miller reported that from July 1996 to June 1997, Snlnzonelln Choleraesuis (Kuzendorf) was the most frequently isolated serotype from swine (177). Although it has been well known that Sdmonelln Choleraesuis is highly pathogenic for swine in that it invades the tissues and establishes a carrier state in surviving swine, it has only recently been demonstrated that other serotypes can invade and persist within the lymphatic system of apparently healthy swine (178). Salrnorzelln Heidelberg has been isolated from clinically healthy swine, and the organism has been found to be aninvasive pathogen capable of dissemination from the intestines to the tissues with the concomitant establishment of a prolonged carrier state (179). Similarly, experiments with both Salnzonelln Newport and Salrnorzellcl Typhimurium demonstrated that these serotypes were able to establish themselves within swine tissues where they persisted throughout a 28-week study (180,181). Salmonellcr can be recovered from the gut even when the gastrointestinal tract is not the portal of entry. It has been demonstrated that the organism can be recovered from the gastrointestinal tract 3 hours after respiratory exposure, indicating that the respiratory tract is a portal of entry for salmonellae (182). The full significance of this method of infection has yet to be determined. Carrier pigs infected with salmonellae pose a potential public health problem when meat from infected animals is sold (183-1 85), and therefore disease prevention at the farm level is important. As with chickens, the use of clean Scrlrnorlellu-free feed is an important component of colonization control at the farm (1 86).Slaughterhouse sanitation is also an important additional component of disease prevention (187), and preslaughter handling at the abattoir will also influence the Snhnorzellu isolation rate (188,189). Fortu-

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nately, human infection with Salmonella Choleraesuis is rare, as this serotype is especially virulent and there is ahigh case fatality rate among people infected with Salrnonellcl Choleraesuis. The lower incidence of human salmonellosis attributable to pork may be a result of the fact that the public seems to understand that pork needs to be well cooked or processed in order to prevent the transmission of other historically significant diseases associated with undercooked pork products, such as trichinosis. Besides the potential public health issue posed by carrier pigs at slaughter, the animals themselves are prone to develop overt salmonellosis when stressed, and it has been estimated that nationwide swine salmonellosis costs producers $100 million yearly (190). For this economic reason, there is considerable interest in developing intervention methods (such as competitive exclusion and vaccination) and evaluating animal husbandry practices (such as all in all out vs. continuous flow rearing systems) that may help limit the spread of the Salnzonellcr problem at the farm level. Recently competitive exclusion cultures have been developed and efficacy in Scdmonellrr control evaluated. Initial results suggest that this might be a helpful method that will aide in the reduction of salmonellae on the farm (191,192). Live attenuated Salmonella vaccines have also been efficacious in reducing mortality and morbidity associated with salmonellosis (189,193,194), however, carrier animals still exist within the population, suggesting that other strategies are needed.

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157. Stavric, S. (1992). Defined cultures and prospects. Int. J. Food Microbiol., 55:245-263. 158. Blankenship, L. C., Bailey, J. S., Cox, N. A., Stem, N. J., Brewer, R.,and Williams, 0. (1993). Two-step mucosal competitive exclusion flora treatment to diminish salmonellae in commercial broiler chickens. Poult. Sci., 73: 1667- 1672. 159. Nisbet, D. J., Corrier, D. E., and DeLoach, J. R. (~1993).Effect of mixed cecal microflora maintained in continuous culture and dietary lactose on Salmonella hphirnurium colonization in broiler chicks. Avian Dis., 37528-535. 160. Nisbet, D. J., Ricke, S. C., Scanlan, C. M., Hollister, A. G., and DeLoach, J. R. (1994). Inoculation of broiler chicks with a continuous-flow derived bacterial culture facilitates early cecal bacterial colonization and increases resistance to Salmonella iypkimurium. J. Food Prot., 57:12-15. 161. Corrier, D. E., Nisbet, D. J., Scanlan, C. M., Hollister, A. G., Caldwell, D. J., Thomas, L. A., Hargis, B. M., Tomkin, T., and DeLoach, J. R. (1995). Treatment of commercial broiler chickens with a characterized culture of cecal bacteria to reduce salmonellae colonization. Poult. Sci., 74:1093-1101. 163,. Corrier. D. E.. Nisbet, D. J., Scanlan, C. M.. Hollister, A. G., and DeLoach, J. R. (1995). Control of Salntonellu Qphinzrwizcnt colonization in broiler chicks with a continuous-flow characterized mixed culture of cecal bacteria. Poult. Sci.. 74:916-924. 163. Corrier, D. E., Nisbet, D. J., Byrd 11, J. A., Hargis. B. M., Keith, N. K., Peterson, M., and DeLoach. J. R. (1998). Dosage titration of a characterized competitive exclusion culture to establish Salmonella colonization resistance in broiler chickens during growout. J. Food Prot.. 61:796-801. 164. McHan, F., Shotts. E. B., and Brown. J. (1991). Effect of feeding selected carbohydrates on the in vivo attachment of Salntonella typlrintzwizrm in chick ceca. AvianDis., 35:328331. 165. Conier. D. E., Hinton Jr., A. H., Ziprin, R. L., Beier, R. C., and DeLoach, J. R. (1990). Effect of dietary lactose on cecal pH, bacteriostatic volatile fatty acids, and Salmonella typhimuritun colonization of broiler chicks. Alian Dis., 34:617-625. 166. Corrier, D. E., Hinton Jr., A. H., Kubena, L. F., Ziprin, R. L., and DeLoach, J. R. (1990). Decreased Salmonella colonization in turkey poults inoculated with anaerobic cecal microflora and provided dietary lactose. Poult. Sci., 70:1345-1350. 167. Ziprin, R. L., Corrier, D. E., Hinton Jr., A. H., Beier, R. C., Spates, G. E., DeLoach, J. R., and Elissalde, M. H. ( 1990). Intracloacal Salr~lonellafyphirmriunt infection of broiler chickens: reduction of colonization with anaerobic organisms and dietary lactose. Avian Dis.,34:749753. 168. Ziprin, R. L., Elissalde, M. H., Hinton Jr., A. H.. Beier, R. C., Spates, G. E., Corrier, D. E., Benoit, T. G., and DeLoach, J. R. (1991). Colonization control of lactose-fermenting salmonellu typhinturizm in young broiler chickens by use of dietary lactose. Am. J. Vet. Res., 52: 833-537. 169. Corrier, D. E.. Hinton, Jr., A. H.. Ziprin, R. L., Beier, R. C., and DeLoach, J. R. (1991). Effect of dietary lactose and anaerobic cultures of cecal flora on Salmonella colonization of broiler chicks. In Colonization Control of Hurnan Bacterial Enteropathogens (L. C. Blankenship, ed.), Academic Press, Inc., New York, pp. 299-308. 170. Oyofo, B. A., DeLoach, J. R.. Corrier, D. E., Norman, J. O., Ziprin, R. L., and Mollenhauer, H. H. (1989). Effect of carbohydrates on Salmonella iyphinturiunz colonization in broiler chicks. J. Avian Dis., 33:531-533. 171. Prohaszka, L., Jayarao, B. M., Fabian, A., and Kovacs, S. (1990). The role of intestinal volatile fatty acids in the Salnlonella shedding of pigs. Zentralbl Veterinarmed [B] 37:570574. 172. Van Schie, F. W., and Overgoor, G. H. A. (1987). An analysis of possible effects of different feed upon the excretion of Salmonella bacteria in clinically normal groups of fattening pigs. Vet. Q., 9:185-188.

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173. Ferris, K. E., and Miller,D. A. (1996).Salmonella serotypes from animals and related sources reported during July 1995-July 1996. In Proc. U.S. Aninz. Health Assoc. Ann. Mtg., Little Rock, AR, 1996, pp. 505-526. 174. CDC Annual Summary, Salmonella Surveillance, 1994. 175. Williams, J. L. P. (1980). Salmonellosis. In Bacterid, Rickettsial, m d Mycotic Diseases (J. H. Steele, ed.), CRC Press Inc., Boca Raton, FL, pp. 570-583. 176. Bush, E. (1995). The 1995 NAHMS Swine Grower/FinisherStudy. In Proc. Lisestock Cons. Inst., Kansas City, April 5-7, pp. 207-208. from animals and related sources 177. Ferris, K. E., and Miller, D. A. (1997). Salmonella serotypes reported during July 1996-June 1997. In Proc. of the IOP' United States Animal Health Association Annual Meeting, Louisville, KY, 1997, pp. 423-443. 178. Keteran, K., Brown, J.. and Shotts. E. B. (1982). Salrlrorda in the mesenteric lymph nodes of healthy sows and hogs. Am. J. Vet. Res., 43:706-707. 179. Reed, W. M.,Olander,H. J., andThacker,H. L. (1985).Studies on thepathogenesis of Salrnonella heidelberg infection in weanling pigs. Am. J. Vet. Res., 46:230@-2309. 180. Wood. R. L. (1989). Swine as a reservoir of Salmonella: persistent infection with Salmonella typhimurium and Salmonella newport to market age. In Proceedings 93rdAnn. Meet. of the U.S. Animal Health Assoc., Las Vegas, NV, 1989, pp. 513-516. 181. Wood, R. L., Rose, R., Coe, N. E., and Ferris, K. E. (1991). Experimental establishment of persistent infection in swine with a zoonotic strain of Salmonella newport. Am. J. Vet. Res.. 521813-819. 182. Fedorka-Cray, P. J., Kelley, L. C., Stabel, T. J., Gray, J. T., and Laufer, J. (1995). Alternate routes of invasion may affect the pathogenesis of Snhonelln fyphirnuriunl in swine. Infect. Irnn~un.,632658-2664. 183. Oosterom, J., Dekker, R., deWilde, G. J. A., van Kempen-de Troye, F., and Engels, G. B. (1985). Prevalence of Car~lpylobacterjejuni and Salmonella during pig slaughtering. Vet. Q., 7:31-34. 184. Oosterom, J. (1987).Epidemiological studies of Sal~nonellnand Canpdobacter jejuni.Vet. Q., 9~348-354. 185. Mafu, A. A., Higgins, R., Nadeau, M., and Cousineau, G. (1989). The incidence of Salmonella, Campylobacter, and Yersinia enterocolitica in swine carcasses and the slaughterhouse environment. J. Food Prot., 52:642-645. 186. Linton, A. H. (1979). Salmonellosis in pigs. Br. Vet. J . . 135:109-112. 187. Childers, A. B., Keahey, E. E., and Kotula, A. W. (1977). Reduction ofSalmonella and fecal contamination of pork during swine slaughter. J. Am. Vet. Med. Assoc., 171:1161- 1164. 188. Craven, J. A., and Hurst. D. B. (1982). The effect of time in lairage on the frequency of Salmonella infection in slaughtered pigs. J. Hyg. Camb., 88: 107-1 11. 189. Morgan, I. R., Krautil, F. L.. and Craven, J. A. (1987). Effect of time in lairage on caecal and carcass Srdmonella contamination of slaughter pigs. Epidemiol. Itlfect., 98:323-330. 190. Schwartz. K. J. (1990). Salmonellosis in midwestern swine. In Proc. 94'l Ann. Meet. sf the U.S. A n i n d Health Assoc., pp. 443-449. 191. Fedorka-Cray, P. J., Bailey, J. S., Stern, N. J., and Cox, N. A. (1997). Mucosal competitive exclusion to reduce Salmonella in swine. In PI-oc. 2nd I d . S y p . on Epidenz. m d Control of Sahnonella in Swine, Copenhagen, pp. 164-166. 192. Nisbet, D. J., Anderson, R. C., Buckley,S. A., Fedorka-Cray, P. J., and Stanker, L. H. (1997). Effect of competitive exclusion on Sulmonella shedding in swine. In PI-oc. 2nd Intl. Synp. on Epidenl. and Control of Snlnlonella in Swine, Copenhagen, pp. 176-177. 193. Curtis, I.R., Porter, S. B., and Munson, M. (1991). Nonrecombinant and recombinant avirulent Salmonella live vaccinesfor poultry. In Colonization Control of H~martBacterial Enteropathogens i n f'ozdtry (L. C. Blankenship, ed.), Academic Press, San Diego, pp. 169-198. 194. Kramer, T. T., Roof, M. B., and Matheson,R. R.(1992). Safety and efficacyof an attenuated strain of Salmonella cholemszsis for vaccination of swine. Am. J. Vet. Res.. 53:434-348.

13 Human Salmonellosis: General Medical Aspects Richard L. Ziprin and Michael H. Hume U S . Department of Agriculture, College Station, Texas

I. Salmonella Serovars of Significanceto Humans 286 A. Host adaptation 286 Nontyphoid B. serovars 286 11. Typhoid Fever

287

Key A. features 287 Incidence B. rate 287 of S. Typhi 287 C. Diagnostic characteristics D. Typhoid fever 288 E. Incidence and drug resistance 289 F. Connection betweentyphoid fever and carcinoma290 G. Treatment and antibiotic resistance 290 111. Nontyphoid Salmonellosis 291 Key A. B. C. D. E. F. G. H.

features 291 Enterocolitis/Gastroenteritis 291 Nontyphoid bacteremia/septicenlia/enteric fever 292 Bacteruria 294 Asymptomatic carriage/nosocomial outbreaks 294 Multidrug-resistant strains 294 Treatment 295 Arthritis 296

IV. Mouse Model V.

298

Vaccines 299

A. Vaccination to prevent typhoid fever 301 B. Gal E mutants: alive-attenuated vaccine 301 C. Genetically "engineered"typhoid fevervaccine strains D. Vi antigen vaccines 303 VI. Conclusion 304 References 304

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I. A.

SALMONELLA SEROVARS OF SIGNIFICANCE TO HUMANS HostAdaptation

There are three host adaptation patterns among the Salmolzella serovars: highly host adapted, somewhat host adapted (or intermediate), and non-host adapted (1) [more simply stated, host adapted and ubiquitous (2)]. Human host-adapted serovars tend to cause the most severe illness. In contrast, the highly host-adapted chicken pathogens Scrlrnor~ella Pullorum and Salmonella Gallinarum are not human pathogens. While host adaptation patterns are somewhat linked with the severity of human illness, non-host-adapted serovars (ubiquitous serovars) often do cause severe illness (3). The most highly host-adapted organism of interest in human medicine is Scrlmonella Typhi, the cause of human typhoid fever. Typhoid fever is sometimes referred to as enteric fever, but the term enteric fever can be used to describe typhoid-like illness caused by other serovars. There is no animal source of S. Typhi and no evidence that the S. Typhi host range extends to animals other than human beings. S. Typhi is adapted totally to humans. Therefore, S. Typhi is always contracted from water or food that has been contaminated in some manner by another human being (3-8). There are very rare exceptions to human-to-human transmission, which occur as laboratory accidents (9- 12).

B. Nontyphoid Serovars Other Snlmorzelln serovars are host-adapted animal pathogens and sources of zoonotic infections, i.e., etiological agents of diseases of animals that are secondarily transmitted to human beings (13). Srdnzorzella Choleraesuis is a pathogen of swine and also causes severe systemic infections in humans (3,14). S. Choleraesuis is especially virulent. There is a high case fatality rate among infected individuals. Mycotic aneurysms (infection and weakening of arterial walls) may occur as a result of S. Choleraesuis infection and are sometimes fatal (3,14). Fortunately, the actual incidence of human S. Choleraesuis infection is rare. This rarity of incidence in humans can be attributed to the fact that most people are quite aware that pork products must be well cooked or properly processed to prevent transmission of other diseases such as trichinosis. Salmonella Dublin may cause septicemia in cattle and be transmitted to human consumers of raw milk or other unpasteurized dairy products. As happens with many infectious agents, S. Dublin tends to infect older persons, the very young, inmune-compromised individuals, and those suffering from chronic debilitating disease. S. Dublin can be highly invasive and virulent, and infections result in a high case fatality rate (3,1517). S. Dublin causes mycotic aneurysms (17) and has infected a prosthetic vascular graft (16). There is at least one citation that Dublin sometimes produces Vi antigen, the antigen believed to cause virulence in S. Typhi (18). Snlrr~orzellnEnteritidis and Snlrr~onellaSenftenberg are somewhat host adapted to chickens and turkeys. respectively (3). Both are capable of causing severe human illness, including death. S. Enteritidis is of particular public health concern, because chicken eggs destined for human uses are sometimes contaminated by S. Enteritidis. If the Contaminated eggs are not properly cooked or if eggs are used in a food containing raw or undercooked egg, the human consumer will become infected. S. Enteritidis is one of the major problem areas in human bacterial diseases.

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S. Senftenberg is generally associated with turkeys and has been known to find its way from turkey and turkey products into human food. It is a relatively rare cause of human illness. Nevertheless, S. Senftenberg has caused a serious nosocomial outbreak at a major medical/hospital center (1). The likely cause of the outbreak was cross-contamination of kitchen equipment by turkey meat. S. Senftenberg has been transmitted from a mother’s breast milk to her baby (19). There has been a recent report of a lung abscess due to Senftenberg (20). This incident is notable in part because the infection did not respond to conventional antimicrobial therapy and is one of numerous examples of the growing threat of multi-drug-resistant salmonellae in our environment. Multidrug-resistant strains have changed antimicrobial treatment protocols for systemic salmonellosis, as an ever-expanding list of antimicrobials must be used (6). Most Sch?oneZlu serovars (except as noted above) are not host adapted. Instead, they are found in a wide array of animal products, fruits, vegetables, and processed foods. Although these serovars tend to be somewhat less virulent than the host-adapted serovars (i.e., they are less likely to cause enteric fever-like disease), they are responsible for the overwhelming number of incidents of human salmonellosis (90%0), primarily uncomplicated enterocolitis (3,643).

II. TYPHOIDFEVER A.

KeyFeatures 1, Etiological agent: Sulnzonelln Typhi 2. Source of infection: human carrier (food or water contaminated with feces from a carrier, symptomatic or asymptomatic) 3. Nature of the infection: systemic infection, bacteremia 4. Onset of illness: slow (incubation period of 7-21 days, thenan incremental increase in fever to 103-104°F occurring during the first 1-2 weeks of illness) 5. Mortality: 10% in untreated cases, less than 1% when treated 6. Treatment: antimicrobial therapy, traditionally with chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole; more recently gentamicin, fluoroquinolones and cephalosporins. Multidrug-resistant strains exist, especially in underdeveloped countries.

B. IncidenceRate S. Typhi causes true typhoid fever. Worldwide, typhoid fever is responsible for between 12 and 33 million incidents each year and a mortality of 500,000-600,000 persons (21,22). Fortunately, true typhoid fever is a relatively rare illness within developed countries and the United States in particular. C.DiagnosticCharacteristics

of S. Typhi

Bismuth sulfite agar, though it has a reputation for being tricky to use, is the culture media of choice for isolating S. Typhi (23). Once a putative SnZmoneZZn is isolated, an attempt should be made to determine its serogroup, as serogrouping will also confirm that the organism is salmonellae. S. Typhi is a serogroup D organism, which has two important serological differences from S. Enteritidis: S. Typhi has a ‘d” phase I flagella antigen, L

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whereas Enteritidis has the phase I antigenic formula “g,m.” S. Typhi produces a somatic antigen, the Vi antigen (3,4,24), which was discovered early in this century when it was realized that some Salmonella strains failed to agglutinate with specific antiserum. Vi antigen masked the somatic antigens, thus interfering with the agglutination reaction. The name Vi antigen was chosen to denote the fact that the antigen was present in virulent strains (25). There is a practical implication of Vi antigen’s presence on the surface of S. Typhi. Any time an isolate has the usual phenotypic characteristics of salmonellae yet fails to react with polyvalent anti-0 antiserum, the possibility that the isolate is S. Typhi must be considered. The suspect organism must be tested with antiserum directed against the Vi antigen, and additionally the culture should be boiled to remove the Vi antigen and unmask the somatic antigens. The organism should then be retested with anti-0 antiserum. There are strains of Vi antigen-negative S. Typhi that cause typhoid fever. Typically illness due to these Vi-negative strains is not as severe as that associated with Vi-positive strains (26,27).

D. Typhoid Fever Although all human ailments caused by salmonellae share common features, typhoid fever is somewhat different from nontyphoid salmonellosis. Onset of typhoid fever is much slower (3,543). There is usually a brief gastrointestinal disturbance during the first day or two after ingestion of S. Typhi followed by a prolonged incubation period prior to the onset of serious symptoms. These may last from 1 to 3 weeks. Typhoid fever is characterized by a remittent fever. The fever increases over a period of days by stepwise increments. Many patients will develop “rose spots,” a skin rash characteristic of typhoid fever (4-8.22). Left untreated, fever may last for 2 or more weeks and the illness may cause death. The patient may experience intestinal perforation and hemorrhage, coma, delirium, and seizures. But, even in the preantibiotic era, 90% of patients eventually recovered. The case fatality rate is less than l% among individuals who have received appropriate therapy. Survivors often become constant asymptomatic carriers of S. Typhi. The organism remains present in the gallbladder and intestines. These asymptomatic carriers are most likely to be unknowing vectors of the disease (3-8). This carrier state causes a substantial public health hazard if the carrier happens to be a food handler. Carrier status is a feature of salmonellosis that is not limited to those who have recovered from typhoid fever. Indeed any Snlmonellcr infection can produce varying degrees of a postinfection carrier state (1,343). Typhoid fever involves a general invasion of the body by S. Typhi. In contrast, most other SalnzonelZa serovars usually cause only anenterocolitis with diarrhea. Systemic infections by nontyphoid serovars are relatively infrequent, whereas bacteremia is a constant feature of typhoid fever. The gastrointestinal tract is the portal of entry in typhoid fever. Organisms spread from the gastrointestinal tract to other organs-the lymphatics, the blood, the spleen, the liver-and produce a generalized infection. Then, all the pathophysiological effects of the ScdnloneZZr endotoxin (lipopolysaccharide) become apparent. With the organism causing a systemic infection, all major organ systems and tissues may become infected by S . Typhi. There is splenic enlargement, ulceration of the intestinal walls (sometimes with perforation leading to fatality), infection of the gallbladder, the bones (osteomyelitis), and brain or meningeal involvement. None of these features is unique to typhoid fever, and each may occur after infection by almost any Salmor~ella serovar.

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Because typhoid fever is a very serious health hazard and hygienic conditions cannot be assured during combat situations, it was recognized early on by military authorities that troops needed to be protected from typhoid fever. Military leaders mandated that their troops be vaccinated. Military vaccination against typhoid fever was first accomplished in 1904 with British troops and civilian travelers headed to India and was introduced to U.S. troops in 1911 (28-31). Early vaccines were merely saline suspensions of the organism inactivated by heating at 55-56°C for 1 hour. During the past two decades there have been extensive efforts to develop high-tech vaccines (3) (see Sec. V).

E. Incidenceand Drug Resistance Modern sanitation, hygiene, and public health measures (monitoring food handlers) have dramatically reduced the incidence of typhoid fever within the United States. Typhoid was very common during the pioneer days in North America. During the decade 19851994, the Centers for Disease Control and Prevention (CDC) received reports of only 2445 domestic typhoid cases-200 cases per year on average. Fortunately, only 10 of these incidents resulted in death (32). The overwhelming number of cases were clearly acquired through recent international travel (72%). Among the more recent cases, 12% of the Typhi isolates were resistant to at least three of the previously most effective antimicrobials-ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. This increased antimicrobial resistance is very worrisome, since typhoid fever, unlike Snlmonella enterocolitis, is a systemic infection that must be effectively treated with antimicrobial agents. As a consequence of increased resistance to traditional treatment, the cephalosporins and fluoroquinolone antibiotics are sometimes required for the treatment of typhoid fever. Most typhoid cases in the United States and the economically developed world are linked to international travel, but there are domestic cases. Approximately 20% of 55 cases in the Chicago area were in patients with no history of foreign travel. Of 55 cases reported to the Chicago and Cook County Health Departments during a 7-year period, 3 1% of the isolates were resistant to both ampicillin and trimethoprim-sulfamethoxazole. Forty percent of the cases involved contact with either food handlers or health care workers (33). There are relatively few outbreaks (as opposed to cases reported) of typhoid fever in the United States each year: only six were reported between 1980 and 1989. In Canada, there were 43 small outbreaks involving 116 persons during the decade 1983-1992 (34). One outbreak in the United States involved 60 persons who attended both a family picnic and a Latin food festival. Of the 60 attendees at the family picnic, 40% became ill and 16 cases were confirmed by culture. The likely source of organisms was a potato salad prepared with "intensive handling" and without adequate temperature control by a recent immigrant from El Salvador who was an asymptomatic carrier. This individual had elevated Vi antibodies in her serum and S. Typhi in her stools, strongly suggesting a carrier state. All of this indicates that typhoid fever, while rare, remains a serious concern as a foodborne disease (35). Another incident is similarly instructive. An 8-year-old girl was infected on two separate occasions with S. Typhi. The infective source was her grandmother, who was a known carrier (36). In this instance the child fortunately responded well to treatment with trimethoprim-sulfamethoxazole and the grandmother was treated successfully with the fluoroquinolone ciprofloxacin. A take-home lesson from these cases is that typhoid fever differs from other incidents of salmonellosis in part by the fact that the source of the

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infection is another human who is a carrier. As there are no nonhuman reservoirs of S. Typhi, the source of infection is radically different from the nontyphoid salmonelloses. Typhoid fever is not a zoonotic disease. One incident illustrates the role of a human source in the dissemination of S. Typhi. In 1981 there was a large outbreak of typhoid fever among guests at a New York resort hotel. Forty-three guests were eventually determined to have culture-confirmed cases of typhoid fever, while another 24 guests were ill, probably with typhoid fever, though it was not culture-confirmed. Twenty-one guests were hospitalized, two with bowel perforations. The source of infection was determined to be a 208 liter vat of orange juice prepared by the kitchen staff, one of whom was an asymptomatic carrier (37).

F. ConnectionBetweenTyphoidFeverandCarcinoma Recently, there has been some interest in the relationship between chronic microbial infections and carcinoma. It is well established that untreated Helicobacter pjdori infections may lead to gastric carcinoma (38-42). There is a sitnilar suspicion concerning chronic carrier states with salmonellae, in particular S. Typhi. Both typhoid fever and carcinotna of the gallbladder are endemic in India. The gallbladder is of course the site colonized by S. Typhi after an acute episode of thedisease. A thousand bile specimens were collected from cases of carcinoma of the gallbladder, cholelithiasis (stones), and other forms of biliary pathology. Samples were cultured and both S. Typhi and Snlrr1oneZla Paratyphi were detected in a significantly higher number of patients with carcinoma of thegallbladder than among controls with cholelithiasis (43,44).

G. TreatmentandAntibioticResistance When S. Typhi is susceptible to chloramphenicol, this drug remains the treatment of choice. In the United States there is reluctance to use chloramphenicol because of a 1 in 30,000 chance that a rare bone marrow aplasia will occur. Therefore, other antimicrobials are used, specifically ampicillin, amoxicillin, trimethoprim-sulfamethoxazole, and ceftriaxone (22). Because untreated asymptomatic carriage of S. Typhi can continue for years, effective sewage systems and surveillance of food handlers are necessary for keeping the incidence of typhoid fever low. Fortunately, these measures are very effective and have reduced the incidence of typhoid fever in the United States to negligible levels. Chronic carriage after recovery from enteric fever is, however, very difficult to eliminate. Sometimes surgical removal of the gallbladder is required to rid the patient of focal infection that does not respond well to medical treatment. When the infecting organisms are resistant to antimicrobials of first choice, the fluoroquinolone ciprofloxacin and other quinolones are efficacious, shorten the acute phase, and eliminate the carrier phase (45).However, the fluoroquinolones are not recommended for use in children (22). Instead the cephalosporins ceftriaxone, cefotaxime, and cefoperazone may be used (22). Multidrug-resistant strains sometimes fail to respond adequately to antimicrobial treatment. There have been isolates containing large plasmids that confer resistance to chloramphenicol, trimethoprim-sulfamethoxazole, ampicillin, and gentamicin. During the past decade these multidrug-resistant (MDR) S. Typhi have been responsible for numerous outbreaks throughout the world (37,46-48). For these MDR strains, aztreonam, cefixime, ampicillin-sulbactam, and furazolidone may be effective (22).

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The worldwide occurrence and concomitant vast useland abuse of antimicrobials that typhoid fever necessitates create a selective pressure for the accumulation and transfer of resistance genes among the various serovars. Hence, indirectly, the worldwide prevalence of typhoid fever is potentially significant to us here in the United States. Slowly, but certainly, and for a variety of reasons, MDR salmonellae of all serotypes are becoming serious public health problems. Vaccination programs in endemic areas are essential to reduce the worldwide incidence and protect society at large (see Sec. V).

111.

NONTYPHOIDSALMONELLOSIS

A.

Key Features Etiological agent: all Snbnorzelln serovars except S . Typhi Onset: rapid, hours to a few days, dose dependent Symptoms: enterocolitis and diarrhea, sometimes systemic infections Source of infection: food, water, animals and animal products, human feces Mortality: possible, especially with more virulent serovars (severe disease more probable in very young, aged, debilitated, and immunologically compromised individuals) 6. Treatment: antimicrobial therapy with chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole, gentamicin, fluoroquinolones, and cephalosporins (there is an ever-increasing incidence of MDR strains throughout the world, in both developing and developed countries) 1. 2. 3. 4. 5.

B. Enterocolitis/Gastroenteritis Whereas the onset of typhoid fever is insidious, the onset of nontyphoid salmonellosis tends to be dramatic. Onset is rapid, occurring within 12 hours (perhaps even earlier) to a few days after consumption of contaminated food or beverage. The initial symptoms are a dramatic diarrhea, which is sometimes accompanied by abdominal pain, nausea, vomiting, headaches, chills, myalgia, and low-grade fever. The diarrhea is due to invasion of the intestinal mucosa and a concomitant inflammatory response that leads to a secretory diarrhea mediated by activation of cyclic adenosine monophosphate (49,50). Some strains of Salmonella might produce enterotoxins that are biochemically/immunologically related to cholera toxin, E. coli heat-labile toxin, and Shiga toxin (50-52). But evidence that Salmonelln produces enterotoxins is marginal. Work with a real host, the calf, has shown that enterotoxins have a limited role in the causation of diarrhea. Instead, the invasion associated type I11 export system encoded on Salmonella pathogenicity island 1 has been shown to mediate fluid accumulation and PMN influx (53). Virulent organisms may cause bacteremia or nontyphoid enteric fever, and high fever should be a warning that the organism has invaded from the intestines. When invasion occurs, virulent organisms may establish themselves at focal loci within various tissues and organs throughout the body. However, healthy adults usually endure only a self-limited, uncomfortable diarrhea that resolves without treatment in 2 days to a week. Except when invasive disease is suspected, gastrointestinal salmonellosis requires no treatment beyond fluid and electrolyte replacement (6). Drugs to treat the diarrhea generally are not indicated. If used, bismuth subsalicylate (Pepto-Bismol") is preferable to antimotility drugs such as loperamide, which may increase the odds that the infection will become a systemic one. [Bismuth salts are compo-

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nents of culture media selective for salmonellae, so this author questions the antimicrobial benefit to the use of bismuth in the treatment of Salmonella enterocolitis. However, we have not thoroughly researched this question. A MedlineTMsearch, 1985 to present, using salmonellosis and bismuth as keywords returned only one citation (54),which does support the use of bismuth for its antisalmonella activity.]

C.

NontyphoidBacteremia/Septicemia/Enteric Fever

In most cases of Scrlr~zonelluenterocolitis/diarrhea, the organisms remain confined to the intestines and the infection is self-limiting. Typically the infection is foodborne, though some cases occur by direct transmission from an asymptomatic carrier to another human. Interestingly, it is possible for individuals to acquire a mixed infection, that is to coincidentally suffer from both salmonellosis and campylobacteriosis. A nursing home outbreak involved 119 of 580 residents ill with either Salnzonella Heidelberg, Cmzpylobacter jejuni, or both. Both organisms were recovered from 27% of the ill residents (55). Regardless of the identity of the infecting serovar, the lines of demarcation between diarrhea, gastroenteritis, enterocolitis, and a wider systemic infection may be blurred. One leads to the other as bacteria exit the intestines and establish within the organs. The severity of the resulting illness is influenced by a wide array of factors. The infecting dose, the serovar causing infection, the host’s general health, and the antibiotic resistance patterns of the invading organism all contribute to the course of the infection. The literature contains many reports of fatal salmonellosis among both previously healthy adults and those with predisposing factors: infants, elderly, immunocompromised, and otherwise ill or debilitated individuals. Some recent examples are given (56-63). A case of S. Enteritidis enterocolitis in a healthy 66-year-old man with one predisposing factor, rheumatoid arthritis, has been reported (56). The patient was hospitalized for diarrhea and abdominal pain and had been receiving predisolone for rheumatoid arthritis. Perhaps this anti-inflamlnatory agent was a factor in reducing the individual’s resistance. The patient died of hemorrhagic necrotic enteritis localized to the ileum. S. Enteritidis infections are often contracted from consumption of undercooked and improperly stored egg or egg products. S . Enteritidis is, therefore, of commercial and economic interest to the poultry industry. It is among the two or three most common causes of human nontyphoid salmonellosis. In 1994, contaminated eggs were responsible for an outbreak at a hospital in Mexico City. Ninety-eight employees who ate at the hospital cafeteria became ill. Cultures from the kitchen staff were negative, so the kitchen help was not the source of the outbreak. The source was traced to an egg-covered meat meal (64). Green and Vinker (59) reported a high incidence of Sdnonella infections among patients who suffer from systemic lupus erythematosus. As with the incident given in the previous paragraph, anti-inflammatory drug treatment was a predisposing factor contributing to illness. In the case reported, the patient had recurrent episodes of salmonellosis with two serovars, S. Typhimurium and S . Enteritidis. The S. Enteritidis infection was ultimately fatal. Four other cases of S. Enteritidis have been described in which the patients suffered from predisposing conditions: neoplasms, splenectomy, and hemophilia (60). All recovered from their illness, so the outcome is by no means necessarily bleak. Unfortunately, there are incidents of endovascular infection of atherosclerotic arteries. These infections of arterial walls are very dangerous as they can lead to fatality when the weakened artery

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fails. Fortunately improved medical and surgical care can save these patients. A case of S. Enteritidis mycotic aneurysm in a 65-year-old man has been described (one of many similar incidents). The patient presented with a protracted fever, weight loss, and thigh pain. Blood cultures were negative as were serological results, but a CAT scan revealed the aneurysm. A surgical repair was accomplished and S. Enteritidis was recovered from the arterial wall and the surrounding fluid (61). Sickle cell disease is yet another predisposing factor, and salmonellosis in sickle cell patients is especially dangerous. It is believed that capillary occlusion due to the sickling devitalizes the gut, permitting invasion. Then, reduced liver and splenic function and other reticuloendothelial system abnormalities suppress clearance of the bacteria from the blood. Salmonella osteomyelitis is a frequent complicating factor in such individuals and septic arthritis (active infection in a joint/synovial fluid), when it occurs, has a poorer prognosis. Aggressive surgical intervention is necessary (65). Somewhat surprisingly, despite frequent bouts of bacterial infections, septic arthritis is rare among HIV-positive patients. When it occurs, it is possible to culture the offending organism from the synovial fluid (66,67) (see Sec. 1II.H). Salmonella infections may occur in infants and even premature infants (68). It is possible for a pregnant woman to be the source of infection for an infant. There is a report of a premature infant delivered by cesarean acquiring a fatal Enteritidis infection from the mother. The child died shortly after birth, and S. Enteritidis was recovered from blood cultures and swabs of the child and from the placenta. The author describing the above case argues that antibiotic treatment should be given to women to treat Salmonella infections occurring during pregnancy. This procedure reduces the risk of transplacental infection of the fetus (57). Reports of salmonellosis during pregnancy or in the immediate neonatal period are not unusual (69-75). There may be drastic outcomes for the fetus or infant (70,71). At least one salmonellosis outbreak has occurred in a maternity and prenatal intensive care unit (73). In another case, the mother had contracted S. Enteritidis gastroenteritis (enterocolitis) one week before delivery. Antimicrobial treatment of the infant was described (75). In a study of 74 consecutive cases of salmonellosis, S. Typhimurium was the most common strain causing septicemia. S. Typhimurium infections are more frequently fatal during the neonatal period and in the presence of an associated disease such as congenital heart disorders or underlying immunodeficiency disorders (76). Nontyphi salmonellosis is 20 times more frequent in the HIV-positive population than in the general population, and a Snlmonella infection in AIDS patients may remain remarkably persistent (63). Increased susceptibility to salmonellosis among HIV-positive individuals is possibly due to both immunodeficiency and a deficiency in gastric acid secretion (77). Gastric acid deficiency allows Snlrnonelln entry past the stomach to the intestines and ultimately into the body (78-80). Judged from the amount of television advertising and the number of antacids found on pharmacy shelves, it is obvious that huge amounts of antacids are consumed each year. Omeprazole (PrilosecB) is advertised on television and prescribed as part of the format treatment for ulcers due to H. pylori infection and for common gastric acid reflux disorders. Omeprazole, like other potent antacids, predisposes the individual to salmonellosis (among other infections) by reducing the protective barrier offered by gastric acid secretions. Two cases of severe S. Enteritidis infections have been reported to have occurred in patients 4-5 weeks after the onset of omeprazole therapy (80). The difficulty with antacid use is

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that salmonellae survive at pH 4, whereas they die off rapidly at pH 2-3. Thus, normal gastric acidity is one of the mechanisms protecting the intestines from colonization (81). Chronic use of antacids makes it possible for salmonellae to pass through the stomach and into the intestinal tract. Once there, replication to large numbers occurs and the organism causes diarrhea and may invade the rest of the body. Not all fatal or complicated cases of salmonellosis involve the common serovars S. Typhi, S. Enteritidis, S. Dublin, S. Choleraesuis, or S. Typhimurium. Indeed, each of the many thousands of other serovars may cause serious illness. As just one example, Saltnonelkcl Weltevreden, certainly not a common isolate, caused a fatal salmonellosis in an individual whose condition prior to infection was one of relative good health. The infection was acquired during travel to Indonesia (82). Snln~or~ella infections can occur at any site in the body. Typically, the portal of entry is oral and invasion occurs at the ileum from whence the organisms travel and infect other sites. Sometimes the infections can occur in very unusual locations. Twelve cases of Salmonellu osteomyelitis of the skull have been reported. Two cases reported by one author involved extradural S. Typhimurium infections. Treatment included antibiotic therapy and surgical removal of the affected area (83). D.

Bacteruria

Twenty-eight cases of Salmonella bacteruria (bladder infections’)have been reported. Sixteen cases involved S. Enteritidis and 5 involved S. Typhimurium. Twenty-one patients were symptomatic and 7 were asymptomatic. Of the symptomatic patients, 16 hadcystitis, 3 pyelonephritis, and 2 suffered renal abscesses. Sixteen of the patients were immunocompromised, while the remainder had urological abnormalities (84).

E. AsymptomaticCarriage/NosocomialOutbreaks Chronic carriage poses a significant public health threat, especially when such carriage occurs in food handlers or hospital employees. We have already mentioned a typhoid fever outbreak at a resort hotel where orange juice became contaminated by a worker (37). In Italy an outbreak of SnlmonelZa Hadar occurred at a construction site canteen. In this large outbreak there were 448 symptomatic cases and 22 asymptomatic excretors of S. Hadar. The index case was a cook who had acute symptoms for 5 days before the onset of the episode (85). A nosocomial S. Enteritidis phage type 4 outbreak occurred in 1995. No common or continuing food source was located, but seven asymptomatic excretors of S . Enteritidis were identified among the patient population. The outbreak was likely due to personto-person transmission within the hospital itself (86). In another instance of nosocomial salmonellosis, fecal screening revealed a high carriage rate among the catering staff. The economic cost was staggering at 33,000 Pounds (approximately $70,000) (87). F. Multidrug-ResistantStrains Multidrug-resistant S . Typhinlurium has been around since the 1980s (88,89), and MDR strains of S. Typhi have been part of the typhoid fever landscape for more than two decades. These are in no way unusual. By the 1990s, 19% of the Typhi isolates in Britain were resistant to chloramphenicol and 92% of the isolates were resistant to trimethoprim,

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ampicillin, and gentamicin as well (3,46,90-92). Multidrug-resistant Typhi has found its way to the United States and overall where these isolates are not unusual (93-97). More recently a strain of S. Typhimurium known as DT104 has emerged, primarily in England, and found its way to the United States. DT104 stands for definitive type 104, and the organism is MDR to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracyclines (98). The prevalence of S. Typhimurium isolates with the five-drug pattern of resistance increased from 0.6% in 1979-1 980 to 34% in 1996 (98-100). The organism is associated with beef and other animals, including house pets (101-105).

G. Treatment Appropriate treatment for both acute salmonella enterocolitis and asymptomatic carriage is still unclear. Conventional wisdom argues against treating the acute infection except in cases where the infection translocates outside the guts. Antibiotics are not needed for treating uncomplicated Salmonellu enterocolitis. Fluid and electrolyte replacement are essential, but antimicrobials can usually be avoided except in high-risk patients (3,6). Bacteremia and focal infections indicative of invasion of salmonellae from the intestinal tract require antimicrobial therapy. Focal infections may also require surgery to remove the infected tissue and, in the case of aneurysms, to prevent a calamity (3,6). Chronic asymptomatic carriage of salmonellae after recovery from acute symptoms of salmonellosis is often difficult to treat. There has always been some concern about the effectiveness of antimicrobials for reducing the time to clearance of the carrier state. The effectiveness of antimicrobials for this purpose is somewhat problematic (106-1 lo), and it is generally heldthat long-term carriage rate is unaffected by such treatment (106,111-113). For treating systemic infection, effective therapeutics should be able to enter the phagocytic cells and have direct access to the microorganisms at their site of multiplication. Salmonella spp. penetrate into host cells and reside and even multiply there. A cure is achieved only when these intracellular bacteria are eradicated. Clearing the organism from the gut is not sufficient. Quinolones are accumulated in host cells and act upon bacteria in this peculiar intracellular environment. Ciprofloxacin is particularly useful because high concentrations may be achieved at the proper site. Clinical experience shows that in most instances a rapid amelioration of the acute disease is achieved. Thus, for microbiological, pharmacological, and clinical reasons ciprofloxacin is considered appropriate for the treatment of systemic infections with Salmonella spp. (114). However, studies of ciprofloxacin use in acute diarrhea showed that it did not reduce pathogen carriage after the acute episode (1 15,116). Nonetheless, there is some evidence that newer quinolones can work effectively (1 17). Oral doses of perfloxacin appear to have cleared the organism from affected children (117). Fifteen children who were excluded from school or nurseries due to asymptomatic convalescent-phase nontyphoid Salmonella carriage were treated with perfloxacin, and all were culture negative within 10 days of treatment onset (118). Overall the fluoroquinolones appear to shorten the duration of treatment. Nonetheless, two potential pitfalls have to be kept in mind: the risk of selecting for quinoloneresistant mutants and the risk of enhancing persistent carriage (1 19). Yet another pitfall is that quinolones are generally contraindicated in children and pregnant women because they may injure developing bones (22). This problem is not insurmountable. Perfloxacin may be especially useful in children (120). Moulin et al. (120) studied 42 patients (21 girls and 21 boys) aged 1 month to 12 years (mean 3.3 years) who were admitted to an

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hospital for severe Salmonella infections. Some of these patients had recovered from an initial acute episode of enterocolitis, became asymptomatic carriers, and then suffered a relapse of acute symptoms. Twenty-five of the patients were given perfloxacin. Among perfloxacin-treated patients, diarrhea and fever disappeared within 2 days of treatment. None developed side effects following its administration. Perfloxacin may be an alternative choice for treating severe Salmonella infections in children. It cleared the acute infection and reduced by 50% the number of asymptomatic carriers. Another report of the use of perfloxacin concluded that it is effective at eliminating the carrier state (121). Similarly, there continue to be encouraging studies of various new forms of the quinolones (122). Hof and Kretschmar (1 14) wrote, ‘‘One prerequisite for an effective treatment of a Salmonella enteritis with antibiotics is a reliable direct antibacterial activity of the drug. In comparison to most of [sic] all other usual antibiotics quinolones, especially ciprofloxacin, fulfill these conditions, because its activity is high and resistance of Salmorzella against quinolones is extremely rare. Since quinolone resistance is never plasmid coded, there will be even in the future no obvious risk of spreading of resistant strains.” Unfortunately this statement is not quite correct. Bacteria do develop resistance to fluoroquinolones. The parent compound affects DNA gyrase, and it is possible to develop mutations in the gyrA (123) and gyrB (124) genes, which increase resistance to nalidixic acid and the quinolone antibiotics in general (125,126). The reality that resistance to the quinolones is both possible and has already occurred in nature is fundamentally a scary one. A six-drug multiresistant organism, e.g., DT104 with quinolone resistance, would be extraordinarily difficult to treat because there are few if any antimicrobials left to which the organism might respond. For this reason it is important to monitor resistance patterns in new isolates and to control the spread ofany strains containing resistance to all six antimicrobials. Unfortunately, the prediction that fluoroquinolone resistance may become a problem is well founded. In England, DT104 started out as a problem in cattle but then rapidly spread to other livestock, including pigs and chickens. The use of olaquinodox in pig farms (in the United Kingdom since 1982) and enrofloxacin in poultry led to the appearance of DT104 isolates resistant to ciprofloxacin (98). Fourteen percent of the five-drug-resistant isolates were resistant to a fluoroquinolone (ciprofloxacin) (127,128). Other examples of six-drug-resistant strains have been reported (129).

H. Arthritis When bacteria find their way to a hostile environment, e.g., the high oxidative environment within phagolysosomes, they produce stress response proteins. Salmonella normally synthesize more than 30 proteins within the phagolysosome of a macrophage in response to the intracellular environment conditions (1 30-132). These proteins are involved in postinfection reactive arthritis. Reactive arthritis is a documented sequel to Salrnorlella infection that results from an inappropriate immunological response to Salmonella proteins. Typically, the illness starts a few days after an episode of enterocolitis, usually within less than 3 weeks from the onset of the gastrointestinal disturbance, and it tends to afflict those patients in whom the diarrheal symptoms persisted longer than usual. Reactive arthritis tends to afflict patients with a particular genetic predisposition-the HLA-B27 tissue type. Patients who develop reactive arthritis typically have mononuclear cells present in their synovial fluid. These cells react with a proliferative response to particular 65 kDa heatshock proteins (environmental stress response proteins) and to the antigens present on heat-killed Salmonella (3).

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The underlying immunological mechanisms responsible for reactive arthritis are unknown. Theories have included such ideas as anti-Salmonella antibodies reacting directly with the tissues, bacterial antigen initiation of proliferative responses, altered T-cell responses, changes in cytokine production within the joint, and so on. There is to date no satisfactory explanation of the role of the HLA-B27 tissue type, although several theories have been propounded and explored. Reactive arthritis triggered by salmonellosis is occurring more often than in the past, but the long-term prognosis for patients is unclear (133). Postinfection immunopathological reactions to salmonellosis may occur at sites other than the joints, for example, uveitis (inflammation of the vascular middle coat of the eye), iritis (inflammation of the iris of the eye), and urethritis (inflammation of the urethra). Postinfection reactions may include Reiter's syndrome, which is a triad of urethritis, conjunctivitis (inflammation of the conjunctiva of the eye), and arthritis. Reiter's syndrome is probably caused by an abnormal immune reaction to infectious agents (134). A retrospective study of patients hospitalized for salmonellosis at one facility during a 15-year period (1970-1986) found 63 patients with salmonellosis-related arthritis-of these 88% were HLA-B27 positive (133). The relationship between salmonellosis and arthritis is evident from a study in which 919 individuals suffered enterocolitis after eating at a single restaurant. All were sent questionnaires, 321 of which were returned. Twenty-three respondents reported persistent joint symptoms. Joint symptoms were reported most frequently by persons who suffered a diarrhea of long duration. Of these 23 respondents, 66% reported the extra-articular symptoms of Reiter's syndrome (135). Although the relationship between salmonellosis, reactive arthritis, and Reiter's syndrome seems well established, efforts to compare the presence of anti-S&zonelZa antibodies in the serum of patients and controls have failed to show a difference in the prevalence ofanti-SulnzonelZa antibodies between the two groups (136,137). Nor is the mechanism by which salmonellosis causes reactive arthritis atall clear, especially with regard to the role of the HLA-B27 antigen. The humoral response, as just mentioned, does not seem to explain the immunopathogenesis, but bacteria-specific T cells have been consistently found in the synovial fluid of individuals with reactive arthritis ( l 38). These cells may function as antigen-presenting cells, and antigen presentation by T cells may modify the antibacterial immune response (138). There is considerable circumstantial evidence that nlicrobial antigens locally present within the inflamed joints have a key role in the etiology of reactive arthritis. Synovial fluid has been found to have a powerful microbicidal activity, which likely accounts for the general failure to recover viable bacteria from the synovial fluid of individuals with reactive arthritis (139). Indeed, it is not possible to recover viable bacteria, and PCR studies have shown that bacterial DNA is not present in the joints of reactive arthritis patients. These two observations suggest that the disorder is essentially one of autoimmune dysfunction (140). Notwithstanding the above, it has long been speculated that persistence of causative microbes or their structures within the body is part of the mechanism of reactive arthritis. It has been postulated that HLA-B27-positive individuals develop complications eliminating the microbe after initial infection. There is some experimental evidence for this. It has been shown that the expression of HLA-B27 in cultured U937 cells does not influence their uptake of S . Enteritidis, but it remarkably impairs the elimination of the organism: "The impaired elimination of ReA-triggering microbes by HLA-B27+ monocytes may explain the persistence of ReA-triggering microbes in susceptible HLA-B27 + individuals" (141). S. Enteritidis survives within mouse fibroblasts transfected with HLA-B27, suggesting as above that the bactericidal effect is impaired (142). Further, nitrous oxide

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production is reduced in cells transfected with the HLA-B27 gene. All of this suggests a persistence by intracellular salmonellae in the HLA-B27 individual that does not occur in others. However, experimental models are extremely artificial, and as mentioned above there is no evidence of bacterial persistence, at least in the joints, which is where the pathology occurs (140). Another interesting hypothesis concerning reactive arthritis suggests that the pattern of cytokines normally present in the joints is somehow disturbed. Mononuclear cells were recovered from synovial fluid and assayed for interferon-?, tumor necrosis factor, interleukin-10, and interleukin-4. The cells were then stimulated with bacteria, and it was observed that stimulated cells secreted low amounts of interferon-y and tumor necrosis factor-a, but high amounts of IL-10. Exogenous IL- 12 enhanced interferon-? and tumor necrosis factor secretion. It was suggested that IL-10 and IL-12 secretion is involved in the regulation of the cytokine pattern in patients with reactive arthritis (143). In brief, there are many scientifically “sexy’ ’ ideas about the pathophysiology of reactive arthritis. In reality, there is little confirmatory evidence to support anyof the numerous hypotheses. In a classic understatement, it has been written that “the association between arthritis-causing bacteria and HLA-B27 positive cells is a complex event” (144).

W. MOUSE MODEL From experience with AIDS patients and others with disturbed immune function, it is known that natural resistance to Scdmo~zellnrequires normal cell-mediated defenses (63,77,145). T-cell defects, tnacrophage defects, neutrophil defects, metabolic defects of immune regulation, cytokine physiology, and atypical arachidonic acid metabolism all increase the chances that an individual will contract a systemic Sdnzorwlla infection. These phenomena are best studied with the mouse model, though it has well-recognized limitations. The dogma that the mouse has to be the model for all types of Salmonella infections, including unfortunately typhoid fever, has overstayed its usefulness. It is a poor model for studying diarrhea, which is best studied in other animal models (53,146). Medline@and Biosis‘@data bases searched with the Dialog“’ System search terms (mouse or mice) and (salmonellosis or Snlmonella) located 5455 and 6030 citations, respectively, from the 1960s through July 1998. These manuscripts deal with an extraordinary array of subject matter related to salmonellosis in the mouse: susceptibility and resistance factors, bacterial virulence factors, immunity, and vaccine development. One reason the mouse model is used so extensively is that no animal other than humanbeings contracts true typhoid fever [one abstract indicates infection of swine with S. Typhi (147j1. While the mouse is relatively resistant to S. Typhi, it is highly susceptible to some strains of S. Typhimurium. When infected with S. Typhimurium, the mouse develops an illness quite similar to human typhoid fever, murine typhoid fever. (For an excellent description of the mouse typhoid fever model and a presentation showing a correlation between in vivo reactions to various S. Typhimurium strains and an in vitro model of invasiveness, see Ref. 148.) From this manuscript we note that only a few cells of virulent S. Typhimurium are sufficient to cause murine typhoid fever, whereas avirulent strains of S. Typhimurium do not become lethal until mouse challenge doses are as high as lo7 cells per mouse. Similarly, S. Typhi does not kill mice until the challenge dose is in the 106-107 range ( 149).

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Human experience, veterinary medical experience, and the mouse model show that the general events involved in host resistance to salmonellae and the pathogenesis of salmonellosis are extremely complex. Effectively reviewing and organizing this literature is a monumental task and beyond the scope of this manuscript. The extensive mouse model literature contains highly complex experiments. conflicting and confounding data, and is almost ''intellectually indigestible." Still, the information obtained from mouse experiments paves the way for treatment strategies, for an understanding of pathophysiological events, and (most importantly) for development of prophylactic or preventive methodologies-vaccine development. Using the mouse model, we have learned a great deal about survival of salmonellae in the gastric environment, growth and replication of salmonellae within the gut (150), adhesion of salmonellae to intestinal epithelial cells, and interactions between salmonellae and the intestinal mucosa. We have learned about intracellular transport of salmonellae across the epithelial lining (151), toxin production, and numerous virulence factors (131,132,152-161). There exist a smorgasbord of citations relevant to the above subject matter, mostly citations of murine experiments, but a few from human or nonmurine studies (50-52,7881,150.156,162-204). Other subjects investigated with the tnouse model include, but are certainly not limited to, transport of salmonellae from the gut throughout the body by way of the circulatory system, growth and replication within phagocytic cells, intracellular residence and replication within nonphagocytic cells, and safe sites where salmonellae hide from host defenses (27,177,178,205-238). The mouse model has shown that the general series of events involved in resistance to salmonellosis include acid inactivation of ingested organisms within the gastric environment and the hostile intracellular environment of phagosomes (239,240), presence of secretory immunoglobulins within the lumen and wall of the intestines (241-243), chemotaxis of phagocytic cells to the site of infection (188,244,245), translocation and intracellular killing of internalized salmonellae (206,225-227,246,2471, antigen processing (233,248), T-cell responses and cell-mediated immunity (168,223,249-264), and humoral immune responses (168,252-254,256259,261,265,266). Lessons learned from studies of murine salmonellosis have led directly to significant developments in typhoid fever and nontyphoid salmonellosis prophylaxis, i.e., modern vaccines.

V.

VACCINES

Perhaps the most useful application of the mouse model has been in the realm of vaccine development. Mouse, animal, and human clinical experience have revealed that live-attenuated vaccine strains of salmonellae usually provide better protection than either killed salmonellae or purified antigens (3,267,268). Many experiments have been conducted to determine the relative roles of humoral and cell-mediated responses in resistance to salmonellosis (see citations immediately above). The consensus opinion is that both forms of immunity are important and active immunity is best accomplished with a vaccine that stimulates both humoral and cell-mediated phenomena. This goal is best accomplished with a live-attenuated Snlnlonellcl vaccine strain. The primary advantage of live-attenuated vaccines is that the live organisms are transported from the gastrointestinal tract into the body, where their presence initiates a strong immune response. The secondary advantage of live-attenuated vaccines is that

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mechanical injection is not necessary-the individual merely swallows the vaccine. This advantage is obvious for humans, who find injections objectionable and who sometimes suffer adverse responses at the injection site. Oral dosage is also a labor-saving advantage in agricultural practice, where injection is a costly procedure compared to adding a substance to drinking water or to feed. Currently there are at least two vaccines against typhoid fever on the market in the undeveloped world. One is a live attenuated vaccine and the other a purified injectable form ofVi antigen. To understand these vaccines it is best to first look at the parallel situation in the animal and agricultural world. Once vaccines are discussed in that context, it will be easier to understand the status of typhoid fever vaccines. Also, it must be noted that in the developed world there are too few human salmonellosis cases to justify mass immunization of the population. Vaccination is primarily appropriate for agricultural use. In nations with inadequate hygiene and public health systems the situation is reversed. The primary need is for vaccines suitable for human use against typhoid fever and nontyphoid enteric fever. Salmonellosis is an economic problem for segments of the agricultural industry. Turkey producers sometimes suffer economic losses due to salmonellosis in newlyhatched turkeys (269,270). Pigs are susceptible to salmonellosis and often develop systemic salmonellosis affecting growth and weight gain, etc. Pigs are especially susceptible to the serovar Choleraesuis. Pigs are also potential sources of salmonellosis for the consumers of pork products. The reason that pigs are not a primary human source of salmonellosis is probably that most people understand pork must be well cooked. Dairy cows may be infected with S. Dublin, but routine pasteurization usually protects the general public from this particular serovar (3). The most important agricultural need for Snlmorzelln vaccines is in the chicken industry. Broiler chickens are common sources of salmonellae, which find their way to the human food supply. Chickens themselves are very susceptible to the SnZw1onelln Gallinarum and S. Pullorum. S . Enteritidis infections in laying hens provide an active source of S. Enteritidis-infected table eggs that are a direct health hazard to the human community. A live-avirulent vaccine that prevents SnlnzoneZla Enteritidis infection of laying hens has been described, and this subject has been thoroughly reviewed (271). The usefulness of live-avirulent (live-attenuated) vaccines is based on three properties of the vaccine strain: an ability to invade from the gastrointestinal tract, an ability to initiate an effective immune response, and an assured inability to uncontrollably replicate within the body-avirulence. To achieve these goals, microbial geneticists developed avirulent vaccine strains containing double deletion mutations. Deletion mutations are irreversible, and the presence of two mutations assures that virulence cannot be regained by reverse mutation. The specific mutations were carefully chosen to assure that they adversely affected the ability of the vaccine strain to repeatedly replicate in animal or human tissue. The mutations introduced two requirements for exogenous metabolites-p-aminobenzoic acid and dihydroxybenzoic acid (272,273). As these are not present in host tissues, viable cells will invade and replicate for a few rounds but will not survive long within the host (274-280). These ’ ‘aromatic-deficient” (AnroA) strains had the distinct advantage that molecular geneticists could introduce foreign antigens into the vaccine strain. The vaccine strain was then able to serve as a carrier of additional antigens. Hence it became possible to simultaneously immunize against salmonellosis and another agent (254,25631-286). Through the years there have been a number of studies of AnroA vaccines in chickens (166,287-292). Results have not always been consistent. Some results have been very

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positive. Findings have included development of solid humoral immunity (287), reduced fecal shedding (288), protection against gut colonization and organ invasion (289), and serotype-specific responses (290). Serotype specificity was confirmed by Methner et al. (291), but protection against fecal shedding was not confirmed (292). Investigations of the properties of AaroA double deletion strains have continued for nearly two decades since the original work was published. During this time a competing vaccine strain was developed (174,27 1,293-295). The basic concept is the same, but the deleted genes are different. Adenylate cyclase and cyclic AMP receptor protein genes are deleted-Acyu, Acrp. This particular avirulent strain appears to be more efficacious in chickens than the AaroA, although this issue of efficacy is not entirely clear, Assuming the correctness of the assertion, it is both obvious and rational that part of the art of creating genetically altered vaccine strains is maintenance of immunogenicity for the target host species. As is the case with AaroA strains, Acyn, Acrp tnutants can be constructed containing DNA coding for unrelated antigens (202,255,256,296-298).

A. Vaccination to Prevent Typhoid Fever Early efforts to develop an attenuated or avirulent vaccine strain against S. Typhi did not involve high-tech genetic manipulations. Instead, random mutations were introduced into salmonellae with general mutagens, e.g., nitrosoguanidine. Virulence, protective activity, and the stability of avirulence was assessed with mouse virulence assays. While avirulent strains were readily obtained, avirulent strains generally contained multiple mutations that adversely affected properties such as intestinal colonization, invasion, and immogenicity. Sometimes the mutations responsible for avirulence were spontaneously reversible, i.e., the avirulent strain could regain virulence. So for these reasons, random mutagenesis did not appear appropriate for vaccine development. Nonetheless, the first live-attenuated S. Typhi strain fully developed for human vaccination against typhoid fever, comtnercialized, licensed, and actually put into widespread use was obtained by nitrosoguanidine mutagenesis.

B. Gal E Mutants: A Live-Attenuated Vaccine Smooth strains of S. Typhi are virulent and highly immunogenic in man. Similarly, smooth strains of S. Typhimurium tend to be more virulent than rough strains in the mouse typhoid fever model. It has been possible to expose a virulent wild-type strain of S. Typhi, Ty2, to a general mutagen and recover bacteria with mutations at multiple genetic loci. These cells have unusual phenotypic properties that lend themselves well to the specific purposes of a typhoid fever live-attenuated vaccine. They are known as gal E mutants (149,299). The gal E mutants are avirulent, but become virulent and immunogenic in the presence of galactose. When exposed to galactose, gal E strains synthesize a cell envelope component, which they cannot make in the absence of exogenous galactose and which contributes to virulence. However, the gal E mutants also have a very limited ability to survive in the presence of galactose. In practice, an avirulent strain is given as a vaccine. In vivo, the organisms are exposed to galactose. They then synthesize a cell wall component that is highly immunogenic. Indeed, these cells would also be virulent. However, as the cells accumulate galactose in vivo, they are killed by bacteriolysis: "the properties of the gal E in vivo are dependent upon two tnechanisms acting in opposite direction: a virulence-

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and immunogenicity-increasing biosynthesis of cell wall lipopolysaccharide and a vimlence-lowering galactose induced bacteriolysis” (299). In 1975 Germanier and Furer (299) reported that they were able to introduce the galactose epimerase mutations into a wild-type strain, S. Typhi Ty2. The mutantwas named Ty2 l a, and its safety and efficacy as a potential vaccine strain were initially evaluated using the mouse model of typhoid fever and the “mouse protection test.’. Mice were immunized with 10‘ viable Ty2 la, and it was observed that the vaccine organisms were cleared from their livers and spleens within 3 days. In contrast, Ty2 was still present in mouse tissues 15 days after inoculation even though the initial dose contained only lo4 viable cells. Four weeks after this initial ilnmunization with Ty2la, the tnice were challenged with an i.v. injection containing 5 X lo7viable Ty2 cells. (Remember that while S. Typhi is not especially virulent in mice, very high doses will cause mortality.) The vaccinated mice survived this high challenge dose and an even higher subcutaneous challenge (149). Ultimately Ty’la became a successful human vaccine strain (300). Interestingly, though the above explanation for the effectiveness and avirulence of gal E is frequently cited in the literature, the explanation is neither complete nor accurate. It turns out that while Ty2la has a mutation in gnl E , and while the nature of the mutation appears to adequately explain the avirulence of this organism, gal E mutations may not be the reason for the strain’s attenuation (301). Other potential typhoid fever vaccines have been developed by genetically altering Ty2, Ty21a, and by transferring virulence genes from Ty2 to avirulent S. Typhimuriunl vaccine strains. In general, it is too early to know how successful these will be as substitutes for Ty2la. Further, there are several properties of Ty2la that have contributed to its success as a vaccine, and it is not totally clear that thevarious modern genetic constructs can do as well as the original gcd E mutant.

C. Genetically Engineered Typhoid Fever Vaccine Strains Two genes are involved in the production of Vi antigen, via A and vin B. Some serovars of Snlnzonella besides S . Typhi contain only one or the other of these genes but not both and hence are Vi negative. The S. Typhi vic~B gene from Ty2 has been cloned into an S. Typhimuriunl Acya Adelto crp, which is an attenuated mutant of S. Typhitnurium. This recombinant was able to produce the Vi capsular polysaccharide and was also able to colonize the small intestines of mice and to invade through the mesenteric lymph nodes. Mice treated with the recombinant developed secretory antibodies to the Vi antigen and delayed hypersensitivity reactions by footpad testing. Mice were completely protected against challenge doses of virulent S. Typhi Ty2 (302). Ty541 and Ty 543 are nroA deletion mutants derived from Vi-positive and Vinegative strains of S. Typhi. Thirty-seven adult human volunteers received inoculations with doses as high as 10“’ vaccine organisms, and these individuals developed strong cellular immune responses. However, the humoral responses were weak. Additional deletion constructs have been developed (300). Genes involved in the regulation of virulence have been disrupted (303-306). Alteration of transcriptional regulator (PhoP) and environmental sensor (PhoQ) genes inhibits appendage formation necessary for invasion of the intestinal epithelium (307). After deleting the Phop/PhoQ virulence genes from S. Typhi Ty2, the resultant strain was a live attenuated typhoid fever vaccine believed suitable for testing in a limited number of hu-

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mans. Volunteers did not develop bacteremia or side effects and did develop solid IgA responses to S. Typhi lipopolysaccharides. Volunteers also developed IgG against S. Typhi. Humoral immune responses were measured and compared with those of control vaccinees who received four oral doses of S. Typhi Ty21a. In the most sensitive assays, 9 of 11 volunteers and 5 of 8 Ty2la control vaccinees had evidence of IgG directed against S. Typhi antigens. Ty800. the above genetic construct, apparently is safe and highly immunogenic in humans (308,309). There are many parallels between the requirements for typhoid fever vaccines and cholera vaccines. Many of the same genetic tricks have been applied to the development of oral attenuated vaccines against typhoid fever and cholera. S. Typhi Ty2la and Vibrio choleme CVD 103-HgR have been licensed for human use. Many studies have been conducted concerning the best way to deliver these, to store them, and to evaluate if they can be co-administered. The Ty2la and CVD 103-HgR (V. cholerae) vaccines have been coadministered to humans, alone and in combination with antimalarial drugs, polio vaccine, and yellow fever vaccine. Antimalarial drugs reduced the efficacy of the cholera vaccine but notthe typhoid fever vaccine and both vaccines could safely be co-administered (310-313). There are tnany other genetic constructs of various S. Typhi and S. Typhimurium strains that have been reported as avirulent and immunogenic. But a host of factors must be considered before any of these can be placed in prelitninary human trials and move on to field trials in the undeveloped world. An excellent review of the general subject has been written (300).

D. ViAntigenVaccines Although the bulk of research effort and practical experience has involved the live attenuated vaccine strain TY21a, some forms of typhoid fever vaccines do not employ live attenuated strains. Rather, purified Vi antigen is utilized as the immunogen. The earliest efforts to use Vi antigen as a vaccine immunogen occurred in 1954, but the highly purifiedVi antigen used inthis early study did not protect human volunteers. It was later found that the antigen had become denatured during manufacture. After manufacturing methods were changed to produce a nondenatured Vi antigen, there was anactive review of all studies involving the newly licensed vaccine composed of the Vi capsular polysaccharide. The Vi polysaccharide vaccine became licensed outside the United States, and studies were conducted among populations from the undeveloped world. Twenty-five micrograms of the purified polysaccharide was given by intramuscular injection, and it was observed that the vaccine was well tolerated. An antibody response lasting 3 years occurred in about 90% of subjects including children as young as 2 years of age. The Vi polysaccharide vaccine compared well with other typhoid vaccines with regard to safety, immunogenicity, and efficacy (3 14). Vi polysaccharide is a well-standardized antigen that is effective in a single parenteral dose, is safer than whole-cell vaccine, and may be used in children 2 years of age or older. Safety and efficacy were evaluated in at-risk populations, and a commercial Vi antigen vaccine was licensed for use in Chile, the Congo, Ivory Coast, Korea, the Netherlands, Peru, Philippines, Togo, and the United Kingdom. The Vi antigen has been conjugated to tetanus toxoid, diptheria toxoid, and cholera toxin in an effort to both increase cell-mediated immunity and co-vaccine against other important diseases (300).

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Initial trials indicated that purified Vi antigen vaccines protected humans for about 17-21 months, at which time surveillance was stopped. Additional follow-up studies showed that anti-Vi antibodies could be recovered from vaccinated subjects 3 years after vaccination. Repeat vaccination caused some additional local and systemic side effects. In efforts to improve the Vi polysaccharide vaccine, preparations consisting of a conjugate of the Vi polysaccharide and either heat-labile E. coli toxin or Pseudomonas aeruginosa exoprotein A were examined. These conjugates elicited higher antibody levels than the native Vi polysaccharide (3 15). The primary difficulty with Vi polysaccharide vaccines is the fact that there are Vi-negative strains of S. Typhi that are virulent. Even allowing for this problem, the Vi polysaccharide vaccines are not as efficacious as Ty2 1 a. The Ty2la protective effect lasts longer and protects a higher percentage of participants. Considering the disadvantages and inconvenience of an injectable vaccine versus an oral dose (Vi polysaccharide vs. Ty2la), the apparent current edge is toward oral administration of the Ty2la (26).

VI.

CONCLUSION

Typhoid fever, nontyphoid enteric fever, and Salnzonella enterocolitis are extraordinarily important human diseases. Each is acquired by ingestion of salmonellae and stands as a foodborne illness. Although these various forms of salmonellosis are strikingly similar to each other, there are important distinctions with regard to transmission, source of infective material, nature of the disease, preventive measures, vaccination potential, treatment, and sequelae. It is all too easy to overlook these differences and think of salmonellosis as a single disease caused by identical bacteria, Sdmonella choeraesuis (Salnlonella erzterica). In actuality the nature of salmonellosis is determined by numerous host factors, the infecting serovar, and an array of virulence factors which may or may not be present in any given serovar. Treatment varies according to the specifics of the disorder and the antimicrobial resistance patterns of the offending Salmonella strain. Similarly, the consequences and sequelae of an episode depend on the nature of the offending Sulmonella strain and host factors. Salmonellosis remains an important and common human illness, even in developed countries with well-developed food and water hygiene measures.

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289. Cooper, G. L., Venables, L. M., Nicholas, R. A., Cullen, G. A., and Hormaeche, C. E. (1993). Further studies of the application of live Salmonella enteritidis aroA vaccines in chickens. Vet. Rec., 133:31-36. 290. Cooper, G. L., Venables. L. M., Woodward, M. J.. and Hormaeche, C. E. (1994). Vaccination of chickens with strain CVL30, a genetically defined Snlrnonelln enteritidis aroA live oral vaccine candidate. Infect. Inamm ., 624747-4754. 291. Methner, U., Barrow, P. A., Martin, G., and Meyer, H. (1997). Comparative study of the protective effect against Snlnzonelln colonisation in newly hatched SPF chickens using live, attenuated Salntonelln vaccine strains, wild-type Salrnonella strains or a competitive exclusion product. bzt. J. Food Microbiol., 35923-230. 292. Tan, S.. Gyles, C. L., and Wilkie, B. N. (1997). Evaluation of an nroA mutant Snlvnonella typhirtturium vaccine in chickens using modified semisolid Rappaport Vassiliadis medium to monitor faecal shedding. Vet. Microbiol., 54:247-254. 293. Curtiss. R. D. R.; and Kelly, S. M. (1987). Snlmomdla typhimuriurtt deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic. Infect. Intmun., 55:3035-3043. 294. Curtiss, R. D., Kelly, S. M., and Hassan, J. 0. (1993). Live oral avirulent Salmonella vaccines. Vet. Microbiol., 37:397-405. 295. Curtiss, R., and Hassan, J. 0.(1996). Nonrecombinant and recombinant avirulent Salmonella vaccines for poultry. Vet. Itnnzmol. bnmunopathol., 54:365-372. 296. Maskell, D. J., Sweeney, K. J., O’Callaghan, D., Hormaeche, C. E., Liew, F. Y., and Dougan, G.(1987). Salmonelln typhimurium aroA mutants as carriers of the Escherichia coli heatlabile enterotoxin B subunit to the murine secretory and systemic immune systems. Microb. Pathog., 2:211-221. 297. Chabalgoity, J. A., Khan, C. M., Nash, A. A., and Hormaeche, C. E. (1996). A Snlnzonelln tyyhinzuriunz IztrA live vaccine expressing multiple copies of a peptide comprising amino acids 8-23 of herpes simplex virus glycoprotein D as a genetic fusion to tetanus toxin fragment C protects mice from herpes simplex virus infection. Mol. Microbiol., 19:791-801. 298. McSorley, S. J., Xu, D., and Liew, F. Y. (1997). Vaccine efficacy of Salmonella strains expressing glycoprotein 63. Itlfect. In~nzun.,65: 171-178. 299. Germanier, R., and Furer, E. (1971). Immunity in experimental salmonellosis. 11. Basis for the avirulence and protective capacity of gal E mutants of Salmonella t?,yhinzuriunz. Irzfect. Imnun., 4:663-673. 300. Ivanoff. B., Levine, M. M.. and Lambert, P. H. (1994). Vaccination against typhoid fever: present status. Bull. WHO, 72:957-971. 301. Hone. D. M., Attridge, S. R., Forrest, B., Morona, R., Daniels, D.. LaBrooy, J. T., Bartholomeusz, R. C., Shearman, D. J., and Hackett, J. (1988). A gnlE via (Vi antigen-negative) mutant of Snlrnonella typhi Ty2 retains virulence in humans. Itfect. Inz~mn., 56: 1326-1333. 302. Cao, Y., Wen, 2.. and Lu, D. (1992). Construction of a recombinant oral vaccine against Salrnonelln typhi and Salnzonellr typhinturiunz. Infect. Imnzm., 60:2823-2827. 303. Miller, S. I., Kukral, A. M., and Mekalanos, J. J.(1989). A two-component regulatory system (plroP phoQ) controls Salmonella vphimuriunt virulence. Proc. Nntl. Acnd. Sci. USA, 86: 5054-5058. 304. Miller, S. I.. and Mekalanos, J. J. (1990). Constitutive expression of theyhop regulon attenuates Snlwtortella virulence and survival within macrophages. J. Bncteriol., 172:2485-2490. 305. Miller, S. I., Loomis, W. P,, Alpuche-Aranda, C., Behlau, I., and Hohmann, E. (1993). The PhoP virulence regulon and live oral Salrnonella vaccines. Vaccine, l 1: 122-125. 306. Johnston, C.. Pegues, D. A., Hueck, C. J., Lee, A., and Miller, S. I. (1996). Transcriptional activation of Salrnonella hphimuriunt invasion genes by a member of the phosphorylated response-regulator superfamily. Mol. Microbiol., 22:7 15-727. 307. Reed. K. A., Clark, M. A., Booth, T. A., Hueck, C. J., Miller, S. I., Hirst, B. H., and Jepson, M. A. ( 1998). Cell-contact-stimulated formation of filamentous appendages by Salnronelln

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typhinmriunt does notdepend on the type I11 secretion system encodedby Salnzonellu pathogenicity island 1. Infect. Zr~zmun.,66:2007-2017. Hohmann, E. L., Oletta, C. A., and Miller, S. I. (1996). Evaluationof a phoP/phoQ-deleted, uroA-deleted live oral Salmonella @phi vaccinestrain in human volunteers. Vaccine, 14: 1924. Hohmann, E. L., Oletta, C. A., Killeen, K. P., and Miller, S. I. (1996). phoP/phoQ-deleted Suhnonella typhi (Ty800) is a safe and immunogenic single-dose typhoid fever vaccine in volunteers. J. Illfect. Dis., 173:1408-1414. Cryz, S. J., Jr., Pasteris, O., Varallyay, S. J., and Furer. E. (1996). Factors influencing the stability of live oral attenuated bacterial vaccines. Dev. Biol. Stand., 87:277-281. Cryz, S. J., Jr., Que, J. U., Levine, M. M., Wiedermann, G., and Kollaritsch, H. (1995). Safety and immunogenicity of a live oral bivalent typhoid fever (Salmonella t p h i Ty2la)cholera (Vibrio cholerue CVD 103-HgR) vaccinein healthy adults. Infect. Ir?tmun.,63:13361339. Kollaritsch, H., Furer, E., Herzog, C., Wiedermann, G., Que, J. U., and Cryz, S. J., Jr. (1996). Randomized, double-blind placebo-controlled trial to evaluate the safety and immunogenicity of combined Salmonella typhi Ty2la and Vibrio cholerae CVD 103-HgR live oral vaccines. Infect. Inzntun., 64: 1454- 1457. Kollaritsch,H., Que, J. U., Kunz, C., Wiedermann, G., Herzog, C., and Cryz, S. J., Jr. (1997). Safety and immunogenicity of live oral cholera and typhoid vaccines administered alone or in combinationwith antimalarial drugs, oral polio vaccine, or yellow fever vaccine.J. Irzfect. Dis., 175:871-875. Plotkin, S. A., and Bouveret-Le Cam, N. (1995). A new typhoid vaccine composed of the Vi capsular polysaccharide. Arch. Zntern. Med.. 155:2293-2299. Klugman, K. P., Koornhof, H. J., Robbins, J. B., and LeCam, N. N. (1996). Immunogenicity, efficacy and serological correlateof protection of Snlmonelln typhi Vi capsular polysaccharide three years after immunization. Vaccine, 14:435-438.

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14 Shigella Anthony T. Maurelli Uniforr~ed SemkesUuiversity of the Health Sciences, Bethesda, Maryland

Keith A. Lampel U.S. Food and Drug Administration, Washington, D. C.

I. Introduction 323 A. B.

The bacteria 323 Clinical features of shigellosis

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11. Pathogenic Mechanisms of ShigeIZu spp. 325 A. Hallmarks of virulence 325 B. Laboratory assays for virulence 326 C. Virulence-associated genes and gene products

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111. Epidemiology 330 IV. Detection Methods for Shigella A. General considerations B. Bacteriological methods C. Rapid detection methods

332 332 333 333

V. Treatment and Prevention 335 VI. Conclusions 336 References 336

1.

A.

INTRODUCTION The Bacteria

Bacteria of the genus Shigella are the causative agents of shigellosis or bacillary dysentery. This malady was first described by Hippocrates to indicate an illness characterized by frequent passage of stools containing blood and mucus accompanied by painful abdominal cramps. As with many other bacterial diseases, bacillary dysentery has made its impact

The opinions or assertions contained herein are the private ones of ATM and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences.

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on world history from ancient to modern times. Epidemic and endemic shigellosis in military encampments has influenced the outcome of wars by affecting the performance of troops, particularly during protracted conflicts and sieges. For instance, a survey of U.S. troops stationed in Saudi Arabia in 1991 during Operation Desert Shield found that 57% had experienced diarrhea (1). Of the 432 stool samples collected from military personnel, 49.5% contained enteric pathogens. Shigella and enterotoxigenic Escherichia coli were the predominant bacteria identified (26% and 30%. respectively). In 1898, Shiga successfully isolated a bacterium from the stools of a patient with dysentery and demonstrated the presence of agglutinating antibodies in the serum of the infected patient (2). Thus, bacillary dysentery was conclusively demonstrated to be both clinically and etiologically distinct from amebic dysentery, the causative agent for which had been identified 40 years earlier. In 1950, Shiga’s bacillus, Bacillus dysenteriae, was renamed Shigella dysenteriae, and Shigella was adopted as the generic name for the group of dysentery-causing bacteria. The genus Shigella is composed of four species based on serological similarity and biochemical reactions: group A (S. dysenterine), group B (S. Jlewzeri), group C (S. boydii), and group D (S. somei).Each group is further subdivided into serotypes and subserotypes, with the exception of S. sonrzci, for which only one serotype exists. Shigella are small, nonmotile, nonencapsulated, gram-negative rods and are typically lysine decarboxylate negative and lactose nonfermenting (except for a slow reaction with S. sonnei). They are facultative anaerobes with relatively simple nutritional requirements and belong to the family Enterobacteriaceae, tribe Escherichieae. Because Shigella and E. coli share many biochemical characteristics, they can be very difficult to distinguish from each other on the basis of biochemical tests (see Ref. 3 for a complete description of biochemical characteristics). Studies of DNA relatedness have shown that the four species of Shigella are genetically very closely related to E. coli with DNA homology of greater than 80% (4). Electrophoretic analyses of multilocus enzyme polymorphisms in E. coli and Shigella confirm the close genetic relatedness of the organisms (5). Thus, the weight of taxonomic evidence would argue for placing Shigella in the same genus as E. coli. However, due to historical and clinical considerations, the distinction between Shigella and E. coli has been maintained. Certain strains of E. coli, the enteroinvasive E. coli (EIEC), cause bacillary dysentery that is clinically indistinguishable from the disease produced by Shigella (6). EIEC often possess somatic antigens that are biochemically similar to and immunologically crossreactive with various Shigella serotypes (7). They also share certain biochemical traits with Shigella that are distinctly different from the reactions of typical E. coli (8). EIEC strains also possess a plastnid associated with virulence, which is functionally similar to the plasmids carried by Shigella spp. (9). B. ClinicalFeatures of Shigellosis Bacil1G-y dysentery is probably the most readily communicable of the bacterial diarrheas. Ingestion of as few as 100 organisms results in clinical disease when virulent strains of S. JIemeri and S. dysenterine are fed to volunteers (10). This characteristic is in stark contrast to other enteric pathogens such as Salmonella and Vibrio cholerae, which require ingestion of as many as 10,000-100,000,000 organisms, respectively, to cause illness. The extremely low infectious dose for Shigella explains why the disease is so easily transmitted from person to person and why the secondary attack rate can be so high when an

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index case is introduced into a family or into a crowded or institutionalized population. The clinical presentation of shigellosis can range from a mild diarrhea to a severe dysenteric syndrome with blood, mucus, and pus in stools. Typical symptoms include fever, cramps, tenesmus, inflammation and ulceration of the intestinal mucosa, and diarrhea and/ or dysentery. Watery diarrhea is common and often precedes dysentery. Large numbers of Shigellcr can be found in the stool during the acute phase of the disease, and, in fact, diagnosis can best be made by isolation of the organism from the patient's stool. The watery diarrhea is probably the result of jejunal secretion that cannot be reabsorbed by the colon due to transport abnormalities accompanying bacterial invasion and destruction of the colonic mucosa (1 1,12). The incubation period for shigellosis ranges from 1 to 7 days, but the illness usually begins within 3 days. S. dysenteriae causes the most severe disease and S. somei, the mildest. S. flexneri and S. boydii infections can be either mild or severe. Although the disease may be severe, shigellosis is self-limiting. If left untreated, clinical illness persists for 1-2 weeks and the patient recovers. In normally healthy individuals, bacillary dysentery is not generally a life-threatening illness, but in malnourished children, the elderly, and immunocompromised individuals, the disease may be lethal. Shigellosis remains a major cause of childhood mortality in developing countries (13). Complications that have been associated with shigellosis include intestinal perforation, toxic megacolon, dehydration, sepsis, seizures, Reiter's syndrome, and hemolytic uremic syndrome (14). The latter two syndromes are receiving increasing research attention. Reiter's syndrome, a form of reactive arthritis, is a postinfection sequela to shigellosis, which is strongly associated with individuals of the HLA-B27 histocompatibility group (15,16). Infections caused by a variety of other gram-negative enteric pathogens can also lead to this type of sterile inflammatory polyarthropathy (17). Experimental evidence suggests a role for molecular mimicry in the pathogenesis of reactive arthritis in that S. jlexnei-i antigens share a common epitope with the HLA-B27 molecule (18). Hemolytic uremic syndrome (HUS) is a rare complication associated with infection by S. dysenteriae 1 (19). HUS is also associated with strains of E. coli 0157:H7 (see Chapter 9), which produce high levels of Shiga-like toxin (20). It has been suggested that Shiga toxin (and the Shiga-like toxins) may cause HUS by entering the bloodstream and damaging to vascular endothelial cells such as those in the kidney (20,21).

II. PATHOGENICMECHANISMS OF SHIGELLA SPP. A.

Hallmarks of Virulence

Shigella spp. represent the classic example of bacterial agents that cause disease by a limited invasive process in the intestine. Many of the symptoms of shigellosis can be directly attributed to the ability of Shigella to invade the epithelial cells of the intestine, multiply intracellularly, and spread from cell to cell. One of the key early studies on the pathogenicity of Shigella was the demonstration that the ability to invade epithelial cells is an essential determinant of virulence (22). Spontaneous colonial variants of S. fle.weri 2a that do not cause disease in monkeys are unable to invade epithelial cells in tissue culture. Through the use of gene transfer studies with E. coli K-12, the role of intracellular multiplication after invasion was demonstrated. Matings between E. coli K-l2 donors and S. Jlexneri 2a recipients were used to generate S. flemeri-E. coli recombinant hybrids. A transconjugant that had inherited the .x$-rho region of the E. coli K-12 chromosome re-

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tained the ability to invade epithelial cells but showed a reduced ability to multiply within these cells (23). This transconjugant was unable to cause a fatal infection in the opiated guinea pig model and failed to cause disease when fed to rhesus monkeys (24,25). Intercellular spread of Shigella through the epithelial layer of the colon is another hallmark of virulence. Mutants of Shigella have been isolated that are competent for invasion and multiplication but are unable to spread between cells. These mutants affirm the essential role of this phenotype in Shigella virulence. In summary, invasion, intracellular multiplication, and intercellular spread are the prerequisites for virulence in Shigella. The pathology of shigellosis described above correlates well with the model of Shigella pathogenicity encompassing these three traits of the organism. The fourth hallmark of Shigella virulence defines the characteristic pattern by which the pathogen regulates expression of its virulence genes. Virulence in Shigelln spp. is regulated by growth temperature. Virulent strains of Shigella are able to invade mammalian cells after growth at 37°C but are totally noninvasive when cultivated at 30°C. This alteration of the organism’s phenotype is reversible because shifting the growth temperature to 37°C enables the bacteria to reexpress its virulence properties (26). Temperature regulation of virulence gene expression is a characteristic that Shigella shares with other human pathogens, such as Scllrnonelln typlzimurirrm, Bordetella pertussis, Yersirzin spp., pathogenic classes of E. coli, and Listeria rnonocytogenes (see Ref. 27 for review). Gene regulation in response to ambient temperature appears to be a useful bacterial strategy. It permits the bacteria to economize energy that would be expended on the synthesis of virulence products when the bacteria are outside the host. The system also permits the bacteria to coordinately regulate expression of a variety of unlinked genes that are required for the full virulence phenotype. Temperature regulation in S. jIe.uneri 2a is at the level of gene transcription (28) and is mediated by a chromosomal gene, virR/hns, which encodes a repressor (29). Regulation of virulence gene expression is very complex and includes both negative and positive control loops. In addition to the repressor gene virR/lms, two other genes, virF and virB. encode positive activators. A more thorough treatment of virulence gene regulation in ShigelZa can be found in several recent review articles (30,31).

B. LaboratoryAssaysfor Virulence Slligello are pathogens of humans and monkeys, and there are no other known reservoirs or hosts for Shigella infection. However, because monkeys are expensive and difficult to obtain, their usefulness as a tnodel for most studies is limited. Fortunately, small laboratory animal models have been developed, which, despite certain drawbacks, have been useful for measuring some aspects of Shigella virulence. Starved, opiated guinea pigs can be infected orally with Shigella. This model has helped to demonstrate the invasive nature of Shigella infection (22). The ligated rabbit ileal loop has been used to measure fluid secretion and tissue invasion (22.32). Probably the most widely used small animal model is the Sireny test (33). In this assay, a heavy inoculum of bacteria is gently instilled onto the corneal surface of the animal’s eye. The ability of the bacteria to invade the corneal epithelium, replicate, and spread leads to the development of keratoconjunctivitis. Guinea pigs, mice, and rabbits have been used as experimental animals in the Skreny test (34). None of these animals are natural hosts for Shigella. and dysentery is not an endpoint for any of the assays. Consequently, these assays measure only certain virulence characteristics of the pathogen. An inexpensive, accessible animal model that closely mimics the

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disease seen in humans (i.e., dysentery) is still needed to allow a full understanding of the pathophysiology and immunology of bacillary dysentery. Studies of host-parasite interactions between ShigelZa and mammalian cells in tissue culture have contributed greatly to our knowledge of the process of invasion. In monolayers of mammalian cell lines, such as HeLa and Henle, which are sensitive to invasion by Shigella, the bacteria multiply inside the host cells (Fig. 1) (22,35,36). A variation of the tissue culture invasion assay known as the plaque assay measures the ability of the bacteria to invade single cells in a monolayer of confluent cells and subsequently to multiply intracellularly and spread to adjacent cells (37). Bacteria that express these virulence traits produce a visible plaque in the monolayer as a result of the cytopathic effect of the cycle of infection and killing of neighboring cells. Thus, the plaque assay in many ways resembles the process that is postulated to occur in the intestinal mucosa: bacterial invasion of individual cells, spread to adjacent cells, and death of infected cells leading to necrosis, microabscess formation, and ulceration.

C.Virulence-AssociatedGenesandGeneProducts Much progress has been made over the last 10 years in advancing our knowledge of the genes and gene products essential for ShigelZa pathogenicity. This section will describe some of these virulence genes and their role in disease. For a more complete treatment of this subject, the reader is referred to several excellent review articles (38-40).

Fig. 1 Monolayer of eukaryotic cells infected with Shigellu @.weti. A confluent monolayer of mouse L2 cells was infected with a virulent strain of S. Jlesneri and incubated at 37°C to allow for invasion. The internalized bacteria are visible as dark-staining rod-like structures within the cytoplasm of the mouse L2 cells. Pseudopods ("fireworks") extending out from the infected cells contain bacteria and are caused by the ability of Shigella to polymerize actin filaments withinthe cytoskeleton of the infected host cell.Several cells visible in this field are not invaded. (Photo kindly provided by Robin C. Sandlin.)

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Shigella virulence is amultigenic phenomenon and involves genes on both the chromosome and a large 220 kilobase pair (kb) virulence-associated plasmid. The role of certain chromosomal loci was established in the early studies of Formal and colleagues, who used conjugational crosses between E. coZi K-l2 donors and S. jkxrzeri 2a recipients to demonstrate the importance of the q ~ l - r h aregion for intracellular multiplication (23). Another chromosomal locus called kcpA (located about 12 minutes on the Shigella chromosomal map) is essential for producing keratoconjunctivitis in the guinea pig in the Sireny test (41). The loss of the ability to provoke a positive Sireny test after transfer of this region of the chromosome of E. coli K-l2 into S. fle.nzeri is due to the transfer of the onzpT locus from E. coli. onzpT codes for a protease, and the IcsA protein, which is required for a positive Sireny test (see below) is sensitive to this protease. Wild-type strains of SAigelZa spp. do not carry the ompT gene. Thus, the kcpA locus is not a gene but the absence of a gene, normally found in E. coli, which abolishes the activity of a Shigella virulence gene product (42). The genes controlling Shigella 0-antigen biosynthesis map near the his locus (about 45 minutes on the Shigella chromosome). Replacement of this region with the corresponding region from E. coli K-l2 leads to negative results in the Sireny test and plaque assay (43,44). Theimportance of the lipopolysaccharide for Shigella virulence is underlined by the fact that mutant strains representing the “rough” chemotypes from Ra to Re (no 0antigen but complete to incomplete cores) retain the ability to invade tissue culture cells but are negative in the SLreny test and the plaque assay (45,46). The molecular basis for this phenotype is described later in this section. Although the genetic information required for 0-antigen biosynthesis in S. jkxtzeri is entirely encoded on the chromosome, this is not the case for S. dyserzteriae and S. sonmi. A 9 kb plasmid found in S. dysenteriae encodes the galactose transferase gene which is essential for 0-antigen synthesis in this organism (47). Genes for synthesis of the form I 0-antigen in S. sorzrzei are contained on a 180 kb plasmid, which also carries the genes required for invasion (48). The genes comprising the form I antigen locus on the virulence plasmid of S. somei have been cloned, characterized by restriction mapping, and expressed in heterologous genetic backgrounds (49). The structural gene for Shiga toxin is located on the chromosome of S. dysenterine and maps to about 28 minutes (50). Shiga toxin and the Shiga-like toxins produced by S. flexner-i and S. sonrzei are potent cytotoxins, which inhibit mammalian protein synthesis by cleaving the N-glycosidic bond at adenine 4324 in 28s rRNA (51,52). Experiments with isogenic Tax+ and Tox- strains of S. dysenteriae in the monkey infection model suggest that Shiga toxin contributes to the vascular damage in the colonic mucosa seen in the infected animals (53). Strains of enterohemorrhagic E. coli (EHEC) produce a toxin that is immunologically and functionally very similar to Shiga toxin of S. dysenter-iue 1. Production of this toxin is likely responsible for the HUS often associated with infection by S. dysenteriae 1 and EHEC (51). All virulent strains of Shigelln spp. and EIEC carry a 180-220 kb plasmid that contains all of the essential genes for invasion and intracellular multiplication (8,54,55). These plasmids are functionally interchangeable and share extensive DNA homology, suggesting that they descended from a common ancestor (56). The presence of the 180-220 kb plasmid in Shigellu can be detected by plating the organisms on medium containing the planar dye, Congo red. Wild-type (plasmid-bearing) strains bind the dye and form red colonies on this medium. Strains that have lost the plasmid or suffered deletions in plasmidborne genes produce white colonies (57). Although the Congo red binding phenotype is tightly associated with the ability to invade mammalian cells and is a useful aid for de-

l

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tecting virulent strains of Shigella, the mechanism of Congo red binding and its relation to invasion is unclear. Growth of Shigella at 37°C results in a high frequency of virulence plasmid deletion or loss. Under culture conditions in the laboratory, plasmid loss is enhanced by virulence gene expression at 37 "C (58). Growth at 30°C, a temperature at which plasmid virulence genes are repressed, results in bacterial populations that experience little or no plasmid deletion or loss. As a practical consideration, these observations should be kept in mind when culturing samples to be tested for the presence of Shigella by assays that are based on detection of virulence plasmid genes. A 37 kb region of the virulence plasmid of S. Jemzer-i 2a contains all of the genes necessary to permit the bacteria to penetrate into cells in tissue culture (59). Several different genetic loci within this region that contribute to the invasion phenotype have been identified (Fig. 2). The ipn (invasion plasmid antigens) operon is composed of four genes, ipaBCDA, which encode highly immunogenic peptides that are recognized by convalescent sera from experimentally challenged monkeys as well as humans recovering from shigellosis (59-61). The first gene in the operon, ipnB, encodes the "contact hemolytic activity'' of Shigella, which is necessary for the bacteria to escape from the phagocytic vacuole after penetration into thehost cell (62). The ability of S. Je-xneri to induce apoptosis in infected macrophages is an additional property attributed to IpaB (63,64). The precise roles for ipaC and ipnD are not known, but they appear to be involved in the initial step of contact and uptake. Evidence initially suggested that i p A is not necessary

mrrCHI J

K LM E

D

C-

A

" 1332 33 24 9 29 ,..4OORFlO

spa15 47

Fig. 2 Genetic map of the 220 kb invasion plasmid of ShigeZZnje.ateri 2a. A Snll restriction map of the virulence plasmid is shown in the center. Sections of SdZ fragments B and P (upper map) and fragments P, H, and D (lower map) are expanded to illustrate the virulence loci encoded in these regions. The expanded regions are contiguous. Arrows indicate the direction of transcription of the genes. The genes for icsB and ipgD are separated by 3 14 bp. (Genbank accession numberL04309 and M86530.)

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for invasion (65). However, recent studies indicate that IpaA is required for efficient invasion by its ability to recruit vinculin and a-actinin and initiate the formation of focal adhesion-like structures (66). A multicomponent complex consisting of IpaB, IpaC, and probably IpaA is found in the extracellular medium of Shigella cultures and likely is a key element in the actual invasion step (67). The ipgC gene product acts as a chaperon to prevent IpaB from complexing with IpaC in the cytoplasm of the bacteria (67). The ipa genes are highly conserved among the Shigella spp. and EIEC, and these genes have been used successfully as specific DNA probes for the identification of these organisms (see Sec. IV). Although the Ipa proteins are found on the surface of the bacteria and are secreted into the extracellular medium (61,68), the DNA sequences of the genes encoding these proteins suggest the absence of a typical signal sequence for their export to the surface. Transport of these proteins to the bacterial cell surface and beyond is dependent on a Type I11 secretion mechanism encoded by accessory genes on the invasion plasmid (69). The mui (membrane expression of invasion plastnid antigens) and spa (surface presentation of invasion plasmid antigens) loci, located upstream of the ipa operon, are composed of at least 20 genes, which encode the transport machinery (68,70-73) (Fig. 2). At least one other virulence locus that lies outside the 37 kb region has been closely studied for its role in virulence. The virG or icsA (intracellular spread) locus encodes a gene that is essential for the ability of Shigella to move within the host cell's cytoplasm and to spread from cell to cell (74-76). The bacteria induce the polymerization of actin monomers within the cytoplasm and cause the formation of long tails or projections from the cell. These molecular alterations are associated with bacterial motility within the cytoplasm and the movement of the bacteria from the infected cell to a neighboring cell. Shigella that are mutated in the icsA gene are still invasive in tissue culture cells, but they fail to produce plaques in the plaque assay. In the experimental monkey model, an icsA mutant is drastically decreased in virulence (77). An unusual characteristic of the IcsA protein is that it is asymmetrically localized in the outer membrane of Shigella, that is, it is present only at one pole of the bacterium (78). Polymerization of actin by IcsA forms a tail leading from the pole and provides the force that propels the bacterium through the cytoplasm. Thus, unipolar expression of IcsA imparts directionality of movement to the bacterium. Although the mechanism of unipolar localization of IcsA is unknown, it is dependent on synthesis of a complete lipopolysaccharide (LPS). LPS mutants of S. JEemeri 2a express surface IcsA in a circumferential fashion, and while the protein is still capable of polymerizing actin, movement of the bacterium is restricted (79,80). As mentioned earlier, rough strains of Shigella are invasive but fail to generate a keratoconjunctivitis response in the Skreny test. The molecular basis for this attenuated phenotype relates to the requirement for a complete LPS containing a full length 0-antigen side chain in order for IcsA to correctly localize to one pole of the bacterium. Another gene involved in intracellular motility and spread has been described (81). icsB maps next to the ipn operon and plays a role in lysis of the projections that facilitate intercellular spread of the bacteria. The exact mechanism of actin on IcsB is not known.

111.

EPIDEMIOLOGY

In the developing world, diarrheal diseases kill at least 5 million children each year. Endemic bacillary dysentery accounts for about 10% of the diseases in these areas (82.83),

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with children under 5 years at the greatest risk, Inadequate and untreated water supplies, poor sanitation, and overcrowding all contribute to the spread of shigellosis both by human contact and contaminated food and water supplies. S. dvserzteriae 1 is the organism responsible for epidemic outbreaks of shigellosis and continues to cause large numbers of infections in Central America, Southeast Asia, and the Indian subcontinent. In developing countries, such as Ethiopia, S. JJemer-iis the dominant Shigellcr recovered from patients and represents 50-70% of all shigellae identified (84). The incidence of shigellosis in developed countries is low primarily due to the widespread availability of modern sanitary and wastewater-treatment facilities (85). Nevertheless, sporadic outbreaks of shigellosis occur in industrialized countries and are caused predominantly by S. sonmi. For instance, S. sorzrzei is suspected as the causative agent of an outbreak due to imported contaminated iceberg lettuce that affected a number of European countries, including the United Kingdom, Sweden, and Norway (86). Although shigellosis can occur throughout the entire year, outbreaks occur most frequently in the summertime (87). The Centers for Disease Control and Prevention (CDC) reported that of outbreaks due to Shigella spp. in the United States since 1964, S. sorznei was the most common shigellae identified (64.4%) followed by S. jkxrzer-i (30.9%j, S. boydii (3.2%), and S. dysenterine (1.5%) (88). From 1973 to 1987 there were 104 outbreaks of shigellosis in the United States, representing 4% of the total incidence of foodborne outbreaks. However, there were 14,399 cases of shigellosis, third in totalnumber after Salmonella and Stcrplzylococcus aweus (89). Shigella and six other pathogens accounted for 93% of the outbreaks and 94% of the total number of cases. Shigelln caused four deaths during this period. Shigellae can spread rapidly in crowded and unsanitary conditions such as in camps for migrant workers, summer camps, refugee camps, and at mass gatherings, such as music festivals (90,91). The disease can spread rapidly, as was shown at one 5-day outdoor music festival where more than 3000 women developed shigellosis (91). The primary reasons for the spread of shigellae in foods are poor personal hygiene of food handlers and improper holding temperatures of contaminated foods. These factors may contribute to the trend that foods prepared by commercial or institutional companies are implicated in increasing numbers of Shigella outbreaks. Although any food can become contaminated, several types of food have been frequently associated with Shigella foodborne outbreaks. Shigella has been isolated from bakery products, fruits and produce, chicken, hamburger, potato salad, and finfish (87,88). Contaminated lettuce has also been a common source of outbreaks of shigellosis (86,92,93). From the number of outbreaks caused by contaminated foods it is evident that Shigella species can survive in food and water under various environmental conditions. Several reports show that Shigella species can survive for 50 days at room temperature (94), 5-10 days in acidic foods, and 7-14 days in refrigerated, fermented milk (95). Other reports have shown that Shigella can survive from 3 weeks to 3 months in seafood, milk, cheese, cooking oil, eggs, and soda (96). Many cases of shigellosis result from consumption of food and water contaminated by fecal matter from an infected individual. People most frequently affected are those with poor personal hygiene, such as young children i n day-care centers, persons in custodial care institutions (e.g., nursing homes and mental institutions), and those in lower socioeconomic groups who live under crowded conditions, often with inadequate water supplies and poor sanitation. Youngsters who display mild symptoms may contribute sig-

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nificantly to the increases in infection among parents and relatives of children in preschool and day-care environments because children generally have less developed hygienic practices and shigellae have a low infectious dose. Transmission among children is commonly associated with person-to-person or fecal-oral routes with ingestion of contaminated foods or water. Dissemination of Shigella from human carriers by houseflies is another possible route of transmission (97). The gradual increase in the age of males infected with S. JEe.wwri in the United States in recent years may be a reflection of increased homosexual transmission and the effect of the acquired immunodeficiency syndrome (AIDS). From 1968 to 1984, the average age of infection increased frOlll’5 to 24 years (98). Patients with shigellosis and with AIDS or human immunodeficiency virus (HIV) infections may experience longer and more severe symptoms and require longer antibiotic therapy. Therefore, contaminated foods present a greater risk to those with HIV or AIDS than to the general public.

IV. DETECTION METHODS FOR SHIGELLA

A. GeneralConsiderations The presence of any pathogenic microorganism in foods is a primary concern for public health. The rapid detection of Shigella in foods is critical because of the low infectious dose and the potential for rapid dissemination of the disease. Because the spread of shigellae in foods is due mainly to poor personal hygiene of food handlers, identification of the contaminated source is essential to reduce the number of infected people. Usually an outbreak of shigellosis is initially demonstrated from the results of the clinical laboratory. Shigellae are present in stools from 10’-109 colonyforming units (cfu) per gram, depending on the stage of the disease; postconvalescent patients may shed from 10’-103 cfu/g. With relatively high numbers of shigellae present in clinical samples, the detection of this pathogen is fairly straightforward. Conversely, several factors affecting the sensitivity (the minimum number of target organism that must be present) and specificity (targeting the intended pathogen) of the analytical method used cancomplicate the analysis of contaminated foods for the detection of shigellae. First, the food may have been adulterated or abused (e.g., temperature or time) as to adversely affect detection. There are many scenarios in which the food sample may be unsuitable for analysis or severely compromised. For example, several days may have elapsed before an outbreak is reported, and in that time period contaminated food may have been disposed of or stored improperly, rendering the pathogen nonrecoverable (dead or injured cells may be present). Injured shigellae may not be resuscitated under standard laboratory conditions (99). Second, the composition of each food may impede rapid identification of the causative agent. Unlike clinical specimens where most testing is performed on one type of material (stool) that has a high number of shigellae present, each potentially contaminated food item presents a different problem. These factors range from the composition of the food (pH, fats, oils) to the indigenous microbial population present in each. Therefore, all methods designed to detect shigellae in foods must take into consideration the low numbers and physiological state of this pathogen and the composition and physical state of each food to be analyzed. The competing microflora of each contaminated food sample is another important factor to consider in detecting Shigellcr spp., whether by classical bacteriological methods or by more rapid means, such as DNA probes. Some foods have a relatively low bacterial

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background, whereas in others, the normal flora is quite high. For example, alfalfa sprouts have greater than lo8cfu/g. A high background can have an adverse affect on the recovery of shigellae from suspected foods. Shigella can easily be outgrown by other bacteria present in the food sample. DNA-based methods can overcome some of the disadvantages of bacteriological methods.

B. BacteriologicalMethods Bacteriological methods designed for the detection of shigellae from foods can take 710 days for a final result. This regimen includes an enrichment step followed by presumptive and confirmation analyses. The Food and Drug Administration Bacteriological Arralyticnl Manual (100) details a protocol that is followed by the agency and by some food manufacturers. One difficulty in enriching for Shigella is to exclude other enterics, particularly E. coli. The enrichment step calls for a food sample to be inoculated into Shigella broth with 0.5-3 pg/mL novobiocin. The culture is grown anaerobically at 42-44°C for 20 hours. After growth, the culture is streaked onto Macconkey agar plates and incubated for 20 hours at 35°C. For further confirmation, suspect colonies are inoculated for a battery of biochemical and serological tests. Alternative bacteriological methods used to detect shigellae in foods require culturing of suspect foods in broth and subsequent plating on various selective media. Samples are added to gram-negative broth and selenite cystine broth, grown overnight, and it is recommended to plate onto two to three different selective media for isolation. These include (a) low selective media, such as Macconkey or eosin-methylene blue agar plates, (b) intermediate selective media, e.g., xylose-lysine-desoxycholate or desoxycholate citrate agar plates, and (c) high selective media such as Shigella-Sal~no~zelZ~ or Hektoen agar. Highly selective medium may be too stringent for some shigellae, such as S. dysenteriae. Suspect colonies are further identified by growth on triple sugar iron agar slants. A typical reaction for Shigella spp. is alkaline slant, acid butt, and no gas. Biochemical and serological tests are used for final confirmation. The bacteriological methods described for detecting shigellae in foods are labor intensive and time consuming; 10 days may be necessary to obtain a result. These methods also lack the specificity or sensitivity needed to be routinely implemented by food microbiology laboratories.

C. RapidDetectionMethods Because the number of shigellosis outbreaks continues to rise, rapid methods for detection have become more critical. Clinical laboratories have been using several types of rapid methods to detect shigellae. These include DNA-based methods such as colony hybridization (using DNA probes directed against a specific plasmid-encoded virulence determinant), in vitro amplification systems [i.e., polymerase chain reaction (PCR)], enzymelinked immunosorbent assays (ELISA) using antibodies to a virulence marker antigen (101,102), and serotyping (103). 1. DNA-Based Methods Several types of DNA-based formats are available (for review, see Ref. 104). The first gene probes for the detection of Shigella were isolated DNA fragments of the virulence plasmid (105-109). These probes were employed in colony hybridization assays to dis-

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criminate between strains that were pathogenic (i.e., retained the invasion plasmid) and nonpathogenic strains (cured of the invasion plasmid). However, all of these assays were designed to detect Slzigella in clinical samples. Synthetic oligonucleotides directed against one specific virulence gene, such as one of the ipcr genes, have been used to detect shigellae i n clinical samples (1 10,111). From the nucleotide sequence of these genes, an oligonucleotide was synthesized that is directed against the ipnH gene and has been shown to be useful in detecting Shigella in clinical specimens. PCR can be used when the target bacteria is present in low numbers, and this technique also has been used quite successfully with clinical samples (112,113). In contrast to the development of techniques for clinical use, methods for detecting shigellae in foods have not received as much attention. As mentioned previously, clinical samples (stool) are rather consistent in composition, and relatively high numbers of shigellae are present in positive specimens. Conversely, food has several problems that must be addressed. Specifically, each food matrix, as well as the physical state of the shigellae within the food, poses a unique challenge. DNA probes can be used to detect, by colony hybridization, virulent strains of Shigella and enteroinvasive E. coli in foods. However, the application of DNA probes can be severely limited in the case of abused food. In instances where the suspected food may have been abused either by temperature or time, an enrichment step in broth culture may be required. During this step, shigellae may be outgrown by indigenous microflora and the required number of target organisms ( lo5 cfuj may not be sufficient for hybridization assays, thus resulting in a false negative result. In developing a DNA probe for detecting shigellae in foods, a specific region on the large invasion plasmid resident in S. Jlesrzeri was used as a probe (114). A 2.5 kb HirzdZII fragment was shown to be specific for invasive shigellae and E. coli (106,107). The nucleotide sequence of this fragment was determined and a synthetic oligonucleotide based on this DNA sequence was used as a probe in colony hybridization to identify these bacteria seeded in foods (115). Although this probe was shown to be specific for enteroinvasive bacteria, its efficacy was dependent on the food tested. In some instances, such as with alfalfa sprouts, the indigenous bacterial background completely outcompeted the added S. .fZemer.i(10' cells/g of food) and no hybridization was seen with labeled probe and S. je.weri. To overcome some of the background problems associated with foods such as alfalfa sprouts [ 1.4 X lo8cfu/g], the PCR was used. PCR primers were made that were directed to the virulence-associated gene in the 2.5 kb HilzdIII fragment (l 16). In less than one day, a sample can be analyzed and the presence of pathogenic shigellae or enteroinvasive E. coli can be determined. PCR does not suffer the same limitations as DNA probes. Low numbers are not necessarily a concern. However, unlike stool samples, which are easily processed for analysis, the food components may have an adverse affect on the chemistry of the PCR reaction. As discussed above, shigellae have been isolated from several different foods, and each food may have to be analyzed in a different manner. Also, PCR does not discriminate between live and dead cells, which may be a concern in pasteurized products. Plasmid stability is another major concern for detection of shigellae, particularly with S. sorzrwi, which loses its plasmid quite readily, even after isolation and storage. Furthermore, it has been observed that spontaneous loss of the virulence plasmid frequently occurs upon culture of shigellae in the laboratory. One study showed that S. JEexneri grown at 37°C gave rise to spontaneous mutants that are avirulent due to plasmid

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rearrangement or curing (58). Smith et al. (117) studied the effect of Congo Red binding by S. JEemeri when the bacteria are grown in selective media (liquid and solid). Although most selective agar medium used to isolate S. Jlexneri from foods do not adversely affect Congo red binding, some selective media, e.g., Sulmolzellu-S~zigelluagar, desoxycholate, and violet red bile agar, do produce more white colonies. Loss of the plasmid renders most DNA-probe and PCR assays ineffective. Using a genetic marker, such as ipaH, which is present in the chromosome and on the invasion plasmid, may not satisfy regulatory concerns of government agencies. Since ipaH is present in both locations, this probe will not discriminate between pathogenic and nonpathogenic strains. Methods that stabilize plasmid loss could be incorporated into previously discussed methods. One possible alternative is to use antibiotics in a preenrichment medium before either probe or PCR analysis to reduce the microbial flora of the food sample. Because most Shigella outbreaks are first identified by clinical isolates and antibiotic profiles of the causative agent are performed, this information can be passed on to investigators, who will determine the source of the outbreak. This approach has been shown to be effective even with foods with high background flora, such as alfalfa sprouts and ground beef (A. A. Molina and K. A. Lampel, personal observation). One of the major disadvantages of using DNAprobes is the use of radioactive material to label the probes. However, nonradioactive labeling methods are also available. An alkaline phosphatase-conjugated oligonucleotide probe detected Shigella spp. with a sensitivity of 100% and a specificity of 8 1% (118). However, these tests were done with purified isolates. Further analysis with food samples needs to be performed to evaluate its efficacy. 2. Latex Particle Agglutination Several latex agglutination tests are commercially available and are used for the detection of Shigella spp. and Salmorzelln, primarily in the analysis of clinical samples. The Bactigen S~lr~zo~zelln-SJ~igelln Latex Agglutination Slide Test (Wampole Laboratories, Cranbury, NJ) and a color-coded latex agglutination test (Wellcotne Diagnostic, Research Triangle Park, NC) were compared for specificity and sensitivity using clinical specimens. The specificity of these methods ranged from 93.6 to 99.2%, although the number of samples tested with the slide test method was quite low. The sensitivity of the color-coated method was 97.1% (119,120). Bhaduri and Turner-Jones (1 2 l ) reported a hydrophobic latex particle agglutination assay that differentiates invasion-plasmid-bearing strains from nonpathogenic, plasmidless isolates. This method is rapid but has been tested only with pure cultures. Efficacy in detecting foodborne shigellae is still pending.

3. ELISA Antibodies were raised against an EIEC "virulence marker antigen." In an ELISA test using purified isolates, antibody identified only pathogenic strains of Shigella and EIEC (1 01,103). Although large numbers of clinical isolates could be screened within 24 hours with an ELISA, the detection of Shigelln in foods has not been assessed. V. TREATMENTANDPREVENTION Treatment for shigellosis includes fluid-replacement therapy, either intravenously or orally, for mild dehydration and the use of antibiotics to prevent further dissemination of

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the illness and to decrease morbidity. The disease is usually self-limiting. Therefore, by the time a positive culture is confirmed, antibiotic therapy may not be needed and is not recommended. The use of antimicrobials on mild cases may contribute to the propagation of antibiotic-resistant ShigelZa strains. However, in some instances antibiotics may be prescribed to prevent further advance of the disease to dysentery (122). The increase in numbers of antibiotic-resistant strains of shigellae has complicated treatment for shigellosis worldwide. These pathogens are known to be resistant to sulfonamides, ampicillin, trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol, and streptomycin. In a recent study of Shigella isolates in the United States, 32% were found to be resistant to ampicillin (1 23). In a study in Japan from 1971 to 1979, 66% of isolated shigellae were resistant to more than one antibiotic, and 97.5% of these resistant strains were capable of transferring the antibiotic determinants (124). In the United States, trimethoprim-sulfanlethoxazole,ampicillin, and erythromycin are the most common antibiotics used in the treatment of shigellosis (123). Nalidixic acid has been used as the drug of choice to counter multiple drug-resistant shigellae in developing countries. However, nalidixic acid-resistant strains have now appeared in Bangladesh, Africa, and on a Navajo reservation in the United States (123). AIDS patients with shigellosis may be subjected to relapsing infections with bacteremia and could be treated with a quinoline class of drugs. No vaccine to prevent shigellosis has been developed, although much research has been directed toward this goal. Several important factors can lead to the prevention or reduction in the number of foodborne outbreaks caused by Shigella. These are (a) better personal hygiene, particularly in areas lacking adequate sanitation facilities, ( b ) proper treatment of water and sanitary disposal of wastewater, (c) proper handling and storage of foods, and (d) health education for food handlers.

VI.

CONCLUSIONS

With the enormous growth in our knowledge of the pathogenic mechanisms of SJzigelZa over the past 20 years, a molecular picture of this invasive pathogen is beginning to emerge. However, further studies on the mechanism of host cell recognition, invasion, intracellular multiplication, spread, and cell killing are needed. The knowledge gained from these studies should lead to refinement of specific methods for detection of Shigella in food as well as clinical samples. In addition, this research should help us improve therapies for shigellosis and, hopefully, lead to an effective vaccine against the disease. In the meantime, continued vigilance of the safety of the food and water supply can provide an effective barrier against the spread of shigellosis. Health education of food handlers and close attention to hygiene and sanitary conditions by day-care workers and care providers at institutions will also contribute to reducing the incidence of shigellosis in the United States.

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which controls the ability of Shigella jlemeri to evoke keratoconjunctivitis. Irlfect. Irmzun., 3:73-79. 42, Nakata. N., Tobe, T., Fukuda, I.. Suzuki, T., Komatsu, K., Yoshikawa, M., and Sasakawa, C. (1993). The absence of a surface protease, OmpT, deternines the intercellular spreading ability of Shigella: The relationship between the ompT and kcpA loci. Mol. Microbiol., 9: 459-468. 43% Folmal, S. B., Gemski, Jr., P., Baron, L. S., and LaBrec, E. H. (1970). Genetic transfer of Shigella flexrzeri antigens to Escherichiu coli K-12. Iilfect. Inmun., 1279-287. 44. Gemski, Jr., P., Sheahan, D. G., Washington, O., and Formal, S. B. (1972). Virulence of Shigella jlexneri hybrids expressing Escherichia coli somatic antigens. Itfect. Imnwn., 6: 104-111. 45 Okamura, N., Nagai, T., Nakaya, R., Kondo, S., Murakami. M., and Hisatsune, K. (1983). HeLa cell invasiveness and 0-antigen of Sl1igellaje.xneri as separate and prerequisite attributes of virulence to evoke keratoconjunctivitis in guinea pigs. Infect. Inzmun., 39:505-513. 46. Okada, N., Sasakawa, C., Tobe, T., Yamada, M., Nagai, S., Talukder, K. A., Komatsu, K., Kanegasaki, S., and Yoshikawa, M. (1991). Virulence-associated chromosomal loci of Shigella identified by random Tn5 mutagenesis. Mol. Microbiol., 5:187-195. 47. Watanabe, H., Nakamura, A., and Timnis, K. N. (1984). Small virulence plasmid ofShigella dyserzterine 1 strain W30864 encodes a 41,000-dalton protein involved in formation of specific lipopolysaccharide side chains of serotype 1 isolates. Irfect. Irnmun.. 46:55-63. 48. Kopecko, D. J., Washington, O., and Formal, S. B. (1980). Genetic and physical evidence for plasmid control of Slzigella sorrnei Form I cell surface antigen. Infect. Inzrmm., 292072 14. 49. Viret, J. F., Cryz, Jr., S. J., Lang, A. B., and Favre, D. (1993). Molecular cloning and characterization of the genetic determinants that express thecomplete Shigella serotype D (Shigella sonrzei) lipopolysaccharide in heterologous live attenuated vaccine strains. Mol. Microbiol.. 71239-252. 50. Sekizaki, T., Harayama, S., Brazil, G. M., and Timmis, K. N. (1987). Localization of stx, a determinant essential for high level production of Shiga toxin by Shigella dysenteriae serotype 1, near pyrF and generation of stx transposon mutants. Infect. Imnzun., 55:2208-2214. 51. O'Brien, A. D., Tesh, V. L., Donohue-Rolfe, A., Jackson, M. P., Olsnes, S., Sandvig, K., Lindberg, A. A., and Keusch, G. T. (1992). Shiga toxin: Biochemistry, genetics, mode of action, and role in pathogenesis. Curr. Top. Microbiol. Immurzol., 180:65-94. 52. Endo, Y., Tsurugi, K., Yutsudo, T., Takeda, Y., Ogasawara, T., and Igarashi, K. (1988). Site of action of a vero toxin (VT2) from Escherichia coli 0157:H7 and of Shiga toxin on eukaryotic ribosomes. RNA N-glycosidase activity of the toxins. Eur. J. Biochem., 171:45-50. 53. Fontaine, A., Arondel, J., and Sansonetti, P. J. (1988).Role of Shiga toxin in the pathogenesis of bacillary dysentely, studied by using a Tox- mutant of Shigella dysenteriae 1. Infect. Irnmun., 56:3099-3109. 54. Sansonetti, P. J., Kopecko, D. J.. and Formal, S. B. (1981). Shigella sonnei plasmids: Evidence that a large plasmid is necessary for virulence. Infect. Immun., 34:75-83. 55. Sansonetti, P. J., Kopecko, D. J., and Formal, S. B. (1982). Involvement of a plasmid in the invasive ability of Shigella jlexneri. Infect. Irmun., 35:852-860. 56. Sansonetti, P. J.. d'Hauteville. H., Ecobichon, C., and Pourcel, C. (1983). Molecular comparison of virulence plasmids in Shigella and enteroinvasive Escherichia coli. A m . Microbiol. (Inst. Pasteur), 134A:295-3 18. 57. Maurelli, A. T., Blackmon, B.. and Curtiss, R. 111. (1984). Loss of pigmentation in Shigella je.vneri 2a is correlated with loss of virulence and virulence-associated plasmid. Irzfect. h nrun., 43:397-401. 58. Schuch, R., and Maurelli, A. T. (1997). Virulence plasmid instability in Shigellaflexrzeri 2a is induced by virulence gene expression. I??fect.Inzrnzm., 65:3686-3692. 59. Maurelli, A. T., Baudry, B., d'Hauteville, H., Hale, T. L., and Sansonetti, P. J. (1985). Clon*

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Maurelli and Lampel ing of virulence plasmid DNA sequences involved in invasion of HeLa cells by Shigella jexrteri. Infect. Zmmun., 49:164-171. Buysse, J. M., Stover, C. K., Oaks, E. V., Venkatesan, M.,and Kopecko, D.J. (1987). Molecular cloning of invasion plasmid antigen(ipa) genes from Shigella jexneri: Analysis of ipa gene products and genetic mapping. J. Bacteriol., 169:2561-2569. Baudry, B., Maurelli, A. T., Clerc, P., Sadoff, J. C., and Sansonetti, P. J. (1987). Localization of plasmid loci necessary forSlzigellnje.xneri entry intoHeLa cells, and genetic organization of one locus encoding four immunogenic polypeptides. J. Gen. Microbiol., 133:3403-3414. High, N., Mounier,J., Prbvost, M. C., and Sansonetti, P. J. (1992). IpaB of ShigellaJle.meri causes entry into epithelial cells and escape from the phagocytic vacuole. Mol. Microbiol., 11:1991-1999. Zychlinsky, A., Kenny, B., Mtnard, R., Prevost, M. C., Holland, I. B., and Sansonetti, P. J. (1994). IpaB mediates macrophage apoptosis inducedby Sltigelln Jlexneri. Mol. Microbiol., 11:619-627. Chen, Y., Smith, M. R., Thirumalai, K., and Zychlinsky, A. (1996). A bacterial invasin induces macrophage apoptosis by binding directly to ICE. EMBO J.. 15:3853-3860. Sansonetti, P. J., Hale, T. L., Dammin, G. J., Kapfer, C., Collins, Jr., H. H., and Formal, S. B. (1983). Alterations in the pathogenicity of Escherichia coli K-12 after transfer of plasmid and chromosomal genes fromShigella je-weri. Irzfect. Irnmzu~.,39: 1392- 1402. Tran Van Nhieu, G., Ben-Ze’ev, A., and Sansonetti, P. J. (1997). Modulation of bacterial entry into epithelial cells by association between vinculin and the Shigella IpaA invasin. EMBO J., 16:2717-2729. Minard, R., Sansonetti, P.J., Parsot, C., and Vasselon, T. (1994). The IpaB and IpaC invasins of Shigellajexneri associate inthe extracellular mediumand are partitioned inthe cytoplasm by a specific chaperon. Cell, 76:829-839. Andrews, G. P., Hromockyj, A. E., Coker, C., and Maurelli, A. T. (1991). Two novel virulence loci, mxiA and mxiB, in Shigella je-weri 2a facilitate excretion of invasion plasnlid antigens. Infect. Immun., 59: 1997-2005. Hueck, C. J. (1998). Type I11 protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev., 62:379-433. Venkatesan, M. M., Buysse, J. M., and Oaks, E. V. (1992). Surface presentationof Shigella JEexneri invasion plasmid antigens requires the productsof the spa locus. J. Bacteriol., 174: 1990-200 1. Allaoui, A., Sansonetti, P. J., and Parsot, C. (1992). MxiJ, a lipoprotein involved in secretion of Shigella Ipa invasins, is homologousto YscJ, a secretion factorof the Yer-siniaYop proteins. J. Bacteriol., 174:7661-7669. Allaoui, A., Sansonetti,P. J., and Parsot, C. (1993). MxiD, an outer membrane protein necessary for the secretion of the ShigellaJlexneri Ipa invasins. Mol. Microbiol., 7:59-68. Sasakawa. C., Komatsu. K., Tobe, T., Suzuki, T., and Yoshikawa, M. (1993). Eight genes in region 5 that form an operon are essential for invasion of epithelial cells by Shigella Jlexneri 2a. J. Bacteriol., 175:2334-2346. Makino, S., Sasakawa, C., Kamata, K., Kurata, T., and Yoshikawa, M. (1 986). A genetic determinant required for continuous reinfection of adjacent cells on large plasmid in Shigella jexneri 2a. Cell, 46:55 1-555. Bernardini, M. L., Mounier, J.. d’Hauteville, H., Coquis-Rondon, M., and Sansonetti, P. J. (1989). Identification of icsA, a plasmid locus of Shigella j7esner.i that governs intra- and inter-cellular spread through interaction with F-actin. Proc. Nntl. Acad. Sci. USA, 86:38673871. Lett, M.-C., Sasakawa. C., Okada, N., Sakai, T., Makino, S., Yamada. M., Komatsu, K.,and Shigellaje.xneri: IdentificaYoshikawa, M. (1989). virG, a plasmid-coded virulence gene of tion of the VirG protein and determination of the complete coding sequence. J. Bacteriol., 171:353-359.

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77. Sansonetti, P. J., Arondel, J., Fontaine, A., d'Hauteville, H., and Bernardini, M. L. (1991). ompB (osmo-regulation) and icsA (cell-to-cell spread) mutants of Shigella jexneri: Vaccine candidates and probe to study pathogenesis of shigellosis. Vaccine, 9:416-422. 78. Goldberg, M. B., Barzu, O., Parsot, C., and Sansonetti, P. J. (1993). Unipolar localization and ATPase activity of IcsA, a SItigellaJlexrzeri protein involved in intracellular movement. J. Bocteriol., 175:2189-2196. 79. Sandlin. R. C., Lampel, K. A., Keasler, S. P., Goldberg, M. B., Stolzer, A. L., and Maurelli, A. T. (1995). Avirulence of rough mutants of Shigella Jlemeri: Requirement of 0-antigen for correct unipolar localization of IcsA in bacterial outer membrane. Infect. Immun., 63: 229-237. 80. Sandlin, R. C . , Goldberg, M. B., and Maurelli, A. T. (1996). Effect of 0 side chain length and composition on the virulence of Slzigella jlexneri. Mol. Microbiol., 22:63-73. 81. Allaoui, A., Mounier, J., Prkvost, M. C., Sansonetti, P. J., and Parsot, C. (1992). icsB: a Slzigella Jlemeri virulence gene necessary for the lysis of protrusions during intercellular spread. Mol. Microbiol., 6:1605-1616. 82. Rohde, J. E. (1984). Selective primary health care: Strategies for control of disease in the developing world. XV. Acute diarrhea. Rev. Irgect. Dis., 6:840-854. 83. Stoll, B. J., Glass, R. I., Hug, M. I., Khan, M. U., Banu, H., and Holt, J. (1982). Epidemiologic and clinical features of patients infected with Shigella who attended a diarrheal diseases hospital in Bangladesh. J. Infect. Dis., 146:177-183. 84. Kotloff, K. L., Winickoff, J. P., Ivanoff, B., Clemens, J. D., Swerdlow, D. L., Sansonetti, P. J., Adak, G. K., and Levine, M. M. (1999). Global burden of Shigella infections: implications for vaccine development and implementation of control strategies. Bull. World Health Orgccn., 77:65 1-666. 85. Blaser, M. J., Pollard, R. A., and Feldman, R. A. (1983). Shigella infections in the United States, 1974-1980. J. Infect. Dis., 147:771-775. 86. Kapperud, G., Rorvik, L. M,, Hasseltvedt. V., Hoiby, E. A., Iversen, B. G., Staveland, K., Johnsen, G., Leitao, J., Herikstad, H., Anderson, Y., Langeland, G., Gondrosen, B., and Lassen, J. (1995). Outbreak of Shigella sorznei infection traced to imported iceberg lettuce. J. Clin. Microbiol., 33:609-614. 87. Smith, J. L. (1987). Shigella as a foodborne pathogen. J. Food Prot., 50:788-801. 88. Black, R. E., Craun, G. F., and Blake, P. A. (1978). Epidemiology of common-source outbreaks of shigellosis in the United States, 1961-1975. Am. J. Epidemiol., 108:47-52. 89. Bean, N. H., and Griffin. P. M. (1990). Foodborne disease outbreaks in the U. S. 19731987: Pathogens, vehicles, and trends. J. Food Prot., 53:804-817. 90. Wharton, M., Spiegel, R. A., Horan, J. M., Tauxe, R. V., Wells, J. G., Barg, N., Herndon, J., Meriwether, R. A., MacCormack, J. N., and Levine, R. H. (1990). A large outbreak of antibiotic-resistant shigellosis at a mass gathering. J. Infect. Dis., 162:1324-1328. 91. Lee, L. A., Ostroff, S. M., McGee, H. B., Johnson, D. R., Downes, F. P., Cameron, D. N.. Bean. N. H., and Griffin, P.M.. (1991). An outbreak of shigellosis at an outdoor music festival. A m J. Epiderniol., 133:608-615. 92. Davis, H.. Taylor, J. P., Perdue, J. N., Stelma, Jr., G. N., Humphreys, Jr., J. M., Rowntree, R., 111, and Greene, K. D. (1988). A shigellosis outbreak traced to commercially distributed shredded lettuce. Ant. J. Epidemiol., 128: 1312-1321. 93. Martin. D. L., Gustafson, T. L., Pelosi, J. W., Suarez, L., and Pierce, G. V. (1986). Contaminated produce, a common source for two outbreaks of Shigella gastroenteritis. Am. J. Epidenziol., 124:299-305. 94. Taylor. B. C., and Nakamnura, M. (1964). Survival of shigellae in foods. J. Hygiene (Camb.), 621303-3 11. 95. Wilson, F. L., and Tanner, F. W. (1945). Behavior of pathogenic bacteria in fermented milks. Food Res., 10:122-134. 96. Smith, J. L.. and Buchanan, R. L. (1992). Shigella. In Coriiper?diumof Methods for the Micro-

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15 Staphylococcus aureus Scott E. Martin Universir;\.of Illinois at Urbarta-C~lanzpaign, Urbarla, Illinois

Eric R. Myers Nnlco Chemical Company, Naperville, Illinois

John J. landolo University of Oklahomcr Health Sciences Center, Oklahorna City,Oklahornu

I. Introduction 346 11. ExtraintestinalDiseases348 111. Characteristics 349 A. Water activity 349 B. Extracellular enzymes 349 C. Toxins-hemolysins and

others 351

IV. Enterotoxins 352

A. Structure 353 B. Properties 355 Production C. 355 D. Detection 358 V. SublethalInjury363 A. Nucleic acids 363 B. Oxygen toxicity 364 C. Oxygen and sublethal injury 366 VI. Detection and Enumeration 367 A. Detection methods 370 B. Confirmatory tests 370 VII. Food Poisoning 371 VIII. Phage Classification 371 IX. Summary 372 References 372

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1.

INTRODUCTION

The genus Stcrphylococcus is found in section 12, Vol. 2, of Bergey 'SMorzunl of Systematic Bacteriology, the gram-positive cocci (1). The section includes aerobic genera (MicrococC I I S , Plemococcus, and Deinococcrrs),facultatively anaerobic genera (Staphylococcus, Stomatococcus, Streptococcm, Lelrcortostoc, Pediococcus, Aerococcz~s,and Gernella), and anaerobic genera (Peptococcus,Peptostreptococcrls,Ruminococcus, Coprococcus, and Sarcirm). The family Micrococcaceae consists of four genera: Micrococcus, Stonzcrtococcus, Plc~r~ococc~rs, and Stcryhylococcrrs. Differential properties of the gram-positive cocci include arrangement of cells, strict aerobes, facultative anaerobes or microaerophiles, strict anaerobes, catalase reaction, cytochromes present, major fermentation products from carbohydrates anaerobically, peptidoglycan, teichoic acid i n cell wall, and major menaquinones. Classification of Stcqdlylococcus eweus is as follows: Kingdom: Procaryotae Division: Firmicutes Class: Firmibacteria Family: Micrococcaceae Genus: Stcryhylococcus Species: crurerrs There are currently 32 species of Stcphylococcus recognized. Several other species are currently under investigation. Table 1 lists the 32 species found in the genus. The name StnpllylococcLrs is derived from the Greek stclph?)lo (bunch of grapes) and coccus (a grain or berry), hence Stophylococcus = the grape-like coccus. Staphylococci are spherical cells, 0.5-1.5 pm in diameter. which can occur as single cells, in pairs, or as clusters. Cluster formation occurs mainly during growth on solid medium and results from cell division occurring in a multiplicity of planes, coupled with the tendency for daughter cells to remain in close proximity. The three-dimensional appearance is apparent with wet

Table 1 Species of Stnphylococcr4s

S. cmreus S. capitis S. haertzolyticras S. sacclrasolyticm S. soprophyticus S. cohtrii S. .~yloslls S. cnrnosus S. hyiczts S. scirlr-i S. grrllirrcrrwn S. pasteut-i S. eqrdorLrm S. .felis S. sclrleiferi S. mtscoe

S. epidermidis S. \tmrlet-i S. l l o ~ ~ i n i s S. nwicrrlaris S. cnseolyticrls S. h-loosii S. simllans S. intet-meclirls S. ckromoyerws S. lentus S. lngdenensis S. cnprcw S. crt-lettcre S. piscifermentnrrs S. delplzin i S. litulus

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347

mounts, however, stained cells usually give the appearance of irregular sheets of cells. Staphylococci are strongly gram-positive, nonmotile, and asporogenous; capsules may be present in young cultures but are generally absent in stationary phase cells. Colony pigmentation on a nonselective medium such as tryptic soy agar can range from creamwhite to bright orange. Most strains of staphylococci are considered to be Class I1 (potential hazards). Staphylococcus hyicus is a Class IV (potential danger, extreme hazard) pathogen (2). Staphylococcal species are aerobes or facultative anaerobes and have both respiratory and fermentative metabolism. Staphylococci obtain energy via glycolysis, the hexose monophosphate shunt, and the tricarboxylic acid cycle. They are catalase positive and can utilize a wide variety of carbohydrates. Staphylococcus cuueus is the most common species in the Staphylococcus genus. It requires the presence of amino acids and vitamins for aerobic growth and, in addition, uracil and a fermentable carbon source for anaerobic growth. While S. azlreus is capable of anaerobic growth, the best growth occurs under aerobic conditions. The optimum temperature for growth is 35"C, although growth occurs from 10 to 45°C. The pH range for growth is between 4.5 and 9.3, with the optimum between pH 7.0 and 7.5. As environmental conditions become more restrictive, so does the pH range for growth. The major component of the cell wall of S. nureus is peptidoglycan, which accounts for 50% of the cell weight and provides shape and stability. Peptidoglycan is a polysaccharide polymer conlposed of unbranched, P-linked (1-4) chains containing alternating subunits of N-acetylmuramic acid and N-acetylglucosamine, with about 60% of the N-acetylmuramic acid residues 0-acetylated. The high level of 0-acetylation makes the cell wall resistant to lysozyme attack. Pentapeptide side chains are linked to the muramic acid residue and are cross-linked by a pentaglycine bridge to L-lysine of one chain and Dalanine on the other chain. Complexed with the peptidoglycan are a group of phosphatecontaining polymers of teichoic acids. Teichoic acid (about 40% of the cell wall weight) may be covalently bound to the peptidoglycan or to the cell membrane. The backbone of cell wall teichoic acid is an alternating sequence of ribitolphosphate and is linked to Dalanine by a-or P-glycosidic linkage through N-acetylglucosamine. The main components of the capsule of S. aureus are N-acetylaminouronic acids and N-acetylfucosamine. Eleven capsular serotype groups have been identified, with the genes for capsule production located in a single operon. Encapsulated bacteria are resistant to phagocytosis. A few strains of S. a u r e u ~are encapsulated, and these tend to be more virulent in animals. The capsules may be more common when grown in vivo but are lost upon cultivation. Typical colonies are described as smooth, raised, glistening, circular, entire, and translucent on nonselective agar. Colony coloration ranges from gray or gray-white through yellow-orange to orange. Ber-gey'S Manual (1) states that the pigments are triterpenoid carotenoids or derivatives of them and are located in the cell membrane. Pigmentation is usually apparent after 18-24 hours of growth at 37°C but is more pronounced when the culture is held at room temperature for a further 24-48 hours. Pigment is not produced during anaerobic growth or in liquid culture. In general, S. nureus growth is repressed in the presence of competing microorganisms unless they outnumber the other organisms present (3). Although D values differ with strain and heating menstruum, a D value of less than three minutes at 60°C indicates that S. nureus is not considered particularly heat-resistant. Heat resistance is influenced by pH, a,, and culture age. Heating meat

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Martin et al.

products to an internal temperature of 77°C is usually sufficient to kill any S. aureus present. S. aureus are resistant to temperatures below freezing but are sensitive to acidic environments and a variety of chemical compounds and antibiotics.

II. EXTRAINTESTINAL DISEASES Staphylococci constitute a normal part of the microflora of the animal body, being found on skin surfaces and hair, and in the nose, mouth, and throat. Staphylococcus epidermidis colonizes virtually all epidermal surfaces, while S. aweus primarily colonizes mucous membranes. The organism colonizes infants within a few days of birth. Polled0 et al. (4) studied 300 food handlers for the presence of S. clureus. These authors found that 83 (27.6%) were carriers of coagulase-positive staphylococci and 118 (39.3%) were positive for coagulase-negative staphylococci in the nasal fossae. Interestingly, males were found to carry coagulase-positive staphylococci more frequently than females. Thirty-six individuals (12%) were found to carry enterotoxigenic staphylococci. This rate contrasts with the 40-50% rate reported by Bergdoll (5). Asymptomatic staphylococcal carriers can be divided into several types: persistent carriers, who harbor a specific type of S. aureleMs for prolonged periods of time; occasional carriers, who sporadically carry pathogenic staphylococci; intermittent carriers, who harbor one type for a certain period of time and then harbor a different type; and noncarriers, those who never, or only rarely, carry virulent S. aureus. Symptomatic carriers are those people suffering from overt staphylococcal disease (6). Humans are considered to be the principal reservoir of S. nureus in nature. Staphylococci may produce disease in almost every organ and tissue of the body. S. nureus is the most common cause of suppurating infections (such as boils and pimples) and is among the longest recognized of the pathogenic bacteria. One of the first reports linking disease to S. nureus was in 1871 by the German scientist Van Recklinhausen, who observed cocci, which he isolated from the kidneys of a patient who died following a fever. The staphylococci are considered to be opportunistic pathogens whose invasion is dependent upon compromises in the skin or mucous membranes. The most frequent lesion is a cutaneous abscess or boil, which is a localized focus of purulent infection partially or completely walled off from the surrounding tissues. The sites of abscess formation are frequently hair follicles and sweat glands. A carbuncle is a series of interconnected abscesses. The spread of infection within the body and major organs and tissues may occur by extension to contiguous tissues or by way of lymphatics and then blood. Metastatic lesions are produced in a variety of tissues. More serious infections can occur in susceptible individuals: newborns, persons suffering burns or trauma, and during chronic debilitating disorders (cancer, cystic fibrosis, etc.). Serious staphylococcal disease is frequently the result of: nosocomial (hospitalacquired) infections and is most frequently caused by coagulase-negative staphylococci. They enter the body through wounds produced by foreign bodies such as catheters, shunts, needles, or other medical appliances. An intact phagocytic system is essential for normal host defense; abnormalities of humoral immune responses may contribute to disease susceptibility. Staphylococcal diseases can be disseminated in two primary ways: expelled from the upper respiratory tract by sneezing or by the hands, skin, or clothing of an asymptomatic carrier. Staphylococcal disease is more frequent in hospital patients than in the nonhospital population because of the lowered resistance of hospitalized individuals. Penicillin- and methicillin-resistant strains of S. aureus are of increasing concern, with 60-

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90% of all strains resistant to penicillin. Resistant strains appear rapidly in some patients after initiation of antibiotic therapy. Other antibiotics to which some staphylococci strains are resistant include erythromycin, neomycin, streptomycin, gentamicin, tetracycline, and chloramphenicol. Both plasmid(s) and the chromosome have been found to confer resistance in S. auwus. Examples of serious diseases include bacteremia, endocarditis, osteomyelitis, and pneumonia. Other diseases caused by S. a z 4 1 - e include ~~ scalded skin syndrome, toxic shock syndrome, enterocolitis, and staphylococcal food poisoning.

111.

CHARACTERISTICS

S. aur’eus is a bacterial species of great concern to the food industry. Staphylococcal food poisoning is a leading cause of foodborne disease not only in the United States, but in many other countries as well. It consistently competes with salmonellosis as the most prevalent cause of food-related illness. It has been suggested that staphylococcal food poisoning is the leading cause of foodborne illness in the world. Staphylococcal food poisoning occurs as the result of the ingestion of a heat-stable, preformed enterotoxin produced by the organism during growth (see below). A. WaterActivity One reason that S. aureus is of concern to the food scientist is the ability of this microorganism to grow at an a,of 0.83, which is too low for the growth of many competing bacteria. Most strains of S. nureus are highly tolerant to the presence of solutes such as salts and sugars and can grow over an a, range of 0.83 to >0.99. S. aureus grows best at an a, of B0.99, and growth at low a, values depends on other growth conditions being optimal. The minimum a, for growth is higher under anaerobic conditions (0.90). S. nureus can grow well in 16% NaCl (a, 0.88) at 37°C and can grow in media containing up to 50% sucrose (a, 0.97). However, enterotoxin production drops with a decrease in a , . Amin et al. (7) found proline betaine to be a staphylococcal osmoprotectant and at least as effective as glycine betaine and more effective than L-proline.

B. ExtracellularEnzymes S. nureus produces a large number of extracellular enzymes and toxins. Iandolo (8) stated that S. aui-eus is capable of producing at least 34 different extracellular proteins, although no single strain is capable of producing all of the proteins (Table 2). It is thought that production of many of these extracellular proteins aids the organism in its ability to invade healthy tissue and to help escape clearance by immunological mechanisms. Several of these enzymes are of value in the identification of the bacterium. Bjorklind and Arvidson (9) found that most extracellular proteins are produced after the end of exponential growth.

1. Coagulase Coagulase is one of the most important diagnostic enzymes produced by S. nureus. Two forms of coagulase exist: one free and the other cell-bound. The two differ immunologically and have slightly different actions. Cell-free coagulase is proteinaceous in nature, and different antigenic types have been identified. There is a93-100% correlation between coagulase production and enterotoxin production. A coagulase-positive S. aureus has the

Martin et al.

350 Table 2 Extracellular Proteins and Toxins Coagulase Enterotoxins A-E Elastase Protein A Lipase Leukocidin Collagenase Phospholipase Hemolysins Fibronectin-binding protein A Collagen-binding protein Clumping factor

of Stnplylococczu ~lzu-ezds

Endopeptidase Metalloprotease Penicillinase Serine protease Nuclease Pyrogenic exotoxin Staphylokinase Acid phosphatase Alkaline phosphatase Elastin-binding protein Fibronectin-binding protein B Matrix adhesin factor

Fibrinolysin Toxic shock syndrome toxin Exfoliative toxins A and B Staphylococcal lysozyme Succinic oxidase factor Hyaluronate lyase Lysostaphin Thiol protease Fibrinogen-binding protein

ability to cause blood plasma to form a fibrin clot. This enzyme, produced by mostvirulent strains, has been suggested to facilitate staphylococcal pathogenicity by causing a fibrin barrier that appears to localize acute staphylococcal lesions. However, the importance of this barrier in infection is not entirely clear, because coagulase negative S. nurezu may also be pathogenic. Clotting of blood in vivo involves the interaction of a number of components. Very briefly, a clot is formed by the plasma protein fibrinogen, which is present in a soluble form in the plasma and which is transformed into an insoluble network of fibrous material called fibrin. Staphylococcal coagulase acts by converting (or by facilitating the conversion of) the fibrinogen to fibrin. One of the reasons for the wide use of coagulase as a positive indication for the identification of S. azweus is that the test is easy to perform and very reliable. Cells of the suspect organism are mixed with commercially available human or rabbit plasma (with added citrate, oxalate, or EDTA present to chelate calcium, required for in vivo clotting), using either the tube method, performed in a test rube, or the slide technique, performed on a microscope slide. The slide or test tube is incubated at 37°C and read at 1 and 3 hours. Previously, and degree of clotting, however slight, was considered positive. However, current procedures, as described in the Bacteriological Arlalyticul Mnmcrl (lo), require that only a firm and complete clot that stays in place when the test tube is inverted is considered positive. Results of the slide test, in which cell clumping is positive, correlate well with those of the test tube method. Weers-Pothoff et al. (11) compared seven coagulase tests of staphylococcal identification. In general, the commercial tests were more rapid and of similar sensitivity to the standard tube or slide tests. Selepak and Witebsky (12) found that for certain isolates at least two colonies and 24 hours of incubation may be required for detection of some strains. Shivram and Kelkar (13) found a good correlation between production of coagulase, deoxyribonuclease, and thermostable nuclease. Of 164 strains, only one coagulase-negative strain demonstrated deoxyribonuclease and thermostable nuclease activities. 2. Heat-Stable Nucleases Heat-stable nucleases are another group of diagnostically important extracellular enzymes produced by S. aureus. These heat-stable nucleases, which are released into the growth medium, have been shown to attack both ribonucleic acid and deoxyribonucleic acid. S.

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aurezls is also capable of producing extracellular nucleases, which are not heat stable. The existence of two species of nucleases (heat stable and heat sensitive) may cause confusion in species differentiation. Gudding (14) reported that S. uureus, S. intermedizjs, and S. lzyiczls subsp. hyicus all produced thermostable nucleases. As suggested by their name, the heat-stable nucleases are extremely heat resistant and able to withstand boiling for 30 minutes without any significant loss in activity. Several tests have been described to measure heat-stable nuclease activity. The toluidine blue-deoxyribonucleic acid agar method is the currently recommended procedure. Briefly, an agar block (upon which an isolated S. mreus colony has grown) is cut out or a sample of a S. nureus broth culture is transferred to a test tube, which is heated for 15 minutes in a boiling water bath. A microslide is prepared by placing toluidine blue-deoxyribonucleic acid agar on the surface and cutting a small well in the agar. The boiled sample is placed into the well, and the slide is incubated in a moist chamber for 4 hours at 35°C. A positive result is indicated by a bright pink zone of DNA hydrolysis. As with coagulase, there exists between a 95 and 100% correlation between heat-stable nuclease production and enterotoxigenicity. Ratner and Stratton ( 15) reported no discrepancies between a thermonuclease test and the tube coagulase test from 250 human blood cultures. However, Adekeye (16) suggests that thermonuclease tests may not be as useful for confirming S. nureus isolates of animal (nonhuman) origin. In contrast to other extracellular staphylococcal toxins, thermostable nuclease is produced throughout the growth cycle (17).

3. Other Enzymes S. nureus produces additional extracellular enzymes, which are of diagnostic value and may aid in pathogenicity. Staphylokmase causes clot dissolution while several lipases are of diagnostic importance due to their ability to clear opacity on egg yolk agar. Hyaluronidase, formed by most S. nureus strains, is an enzyme that may promote diffusion through connective tissue by depolymerization of hyaluronic acid. Other enzymes produced by S. clweus include penicillinase, proteases (collagenase, elastase, endopeptidase, metalloprotease, serine protease, and thiol protease), lipases (phospholipases and phosphatases-acid and alkaline), fibrinolysin (converts fibrin to soluble products), protein A (a component of the outer layer of the cell wall that impairs opsonization by serum complement and delays phagocytosis), and lytic enzymes (staphylococcal lysozyme and lysostaphin).

C. Toxins-Hemolysins and Others Hemolysins, also refell-ed to as staphylolysins, are produced by S. nl.rreus and have been named alpha (a),beta (P), gamma (y), and delta (6) types; a single strain may produce one or more of the hemolysins. These extracellular proteins differ immunologically and in their ability to lyse erythrocytes from differing animal species (Table 3). Gamma-hemolysin and the Panton-Valentine leukocidin (which is lytic for leukocytes) are composed of two components, one of which is shared between the two. All but the &hemolysins produce a clear zone of P-hemolysis that surrounds S. m r e u s colonies when grown on blood agar. The pyogenic staphylococci are almost always hemolytic, as are almost all coagulase-positive strains. Other toxins produced by S. aureus include exfoliatin, leukocidin, and pyrogenic exotoxin, which cause, respectively, scalded skin syndrome, lysis of white blood cells, and fever. Toxic shock syndrome toxin is involved in staphylococcal toxic shock syndrome.

Martin et al.

352 Table 3 Staphylococcal Hemolysins ~~~~

~

Staphylococcal hemolysin

~

MW

Susceptible RBCs

t-28,OOO

Rabbit

k33,OOO

Sheep Human Guinea pig Rabbit Human Sheep Rabbit Human

Y

29,000 (I) 26,000 (11)

6

*21,000

Other effects Dermal necrosis; cytotoxic for leukocytes, platelets, and fibroblasts Cytotoxic for macrophages, fibroblasts

Cytotoxic for leukocytes and lymphoblasts: lethal for guinea pigs Lytic for lysosomes, mitochondria

IV. ENTEROTOXINS The enterotoxins produced by some strains of S. aurez4s are known to be the emetic cause of food poisoning (gastroenteritis). S. nureus is the predominant causative agent of staphylococcal food poisoning. However, strains of S. intermedius and S. hyicus have been found that produce enterotoxin (6). Staphylococcal enterotoxin (SE) was first studied by Dack and coworkers in 1930 (18). The enterotoxins are heat-stable, hygroscopic, water-soluble proteins released (secreted) form the cell. As little as 1.0 pg of ingested SE can cause symptoms of gastroenteritis (19). Despite being classified as enterotoxins, the toxins do not elicit fluid accumulation in ligated ileal intestinal loops (20). Rather they are presumed to function by affecting emetic receptors in the abdominal viscera, which stimulate the emetic and diarrheal response (21). Recently, the enterotoxins have also been categorized as superantigens due to their ability to stimulate mitogenic activity and cytokine production for a wide array of T-lymphocyte haplotypes (22). They are members of a toxin superfamily that includes staphylococcal toxic shock syndrome toxin- l (TSST- 1) and the streptococcal pyrogenic exotoxins (23). The staphylococcal enterotoxins uniquely produce gastroenteritis (20), while all members of the superfamily suppress immunoglobulin secretion (24), enhance gram-negative endotoxic shock (25),and induce fever and hypotension (20,23). Five enterotoxin serotypes were initially characterized (A-E) with multiple minor subtypes of serotype C (C,, C?, and C,) (8,20,23j. Since then, TSST-1 was mistakenly identified as serotype F (8), consequently there is no enterotoxin F, and serotypes G and H have been reported (26,27). A newly identified serotype I has recently been found. In addition, a silent variant of the enterotoxin A gene, s e d , was identified and shown to have a defective promotor (28). The toxins are related by sequence and immunological homologies. Enterotoxins A, D, E, G, H, and I share over 50% sequence homology and are immunologically cross-reactive proteins, while enterotoxins B and C,-C; share extensive sequence homology and are also immunologically cross-reactive (8j. Enterotoxigenic strains of S. nureus can produce one or more serologically distinct types of toxins. The enterotoxins are composed of single polypeptide chains of about 27 kDathat characteristically have an internal disulfide bridge near the middle of the molecule. They are secreted proteins that possess a signal peptide recognized by the SecYEG secretory pathway

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(29,30). This contrasts with the enterotoxins of gram-negative organisms that are secreted by specific transport proteins that are part of the operon encoding the toxin. The SEs are considered exotoxins composed of protein with no nonprotein residues (31). Mature SEs are composed of a single polypeptide chain of 230-239 amino acid residues, range in molecular weight from 26,360 to 28,494, and have a disulfide loop near the middle of the molecule (Table 4). Amino acid analysis of the SEs show them to be rich in lysine, aspartate, and glutamic acid (6) (Table 5). In addition, it has been suggested that their net positive charge may be important in their emetic activity.

A.

Structure

The nucleotide sequence of the genes for SEA, SEB, SEC1,SEC3, SED, and SEE (sen, seb, sec,, sec3,sed, and see, respectively) (32-37), and the amino acid sequences of SEA, SEB, and SEC, (38-40) have been reported. The SEs belong to a larger family of enterotoxins called the pyrogenic toxins (PTs), which include pyrogenic exotoxins A and B, TSST-1, and streptococcal pyrogenic exotoxin A-C (SPE A-C) (41-47). The PTs share a number of biological properties, which include lymphocyte mitogenicity, immunosuppression, pyrogenicity, and enhanced host susceptibility to endotoxic shock (48-54). Comparisons between the PTs have revealed 43-85% nucleotide sequence identity (33). Significant nucleotide and amino acid homology between entCI,entB, and SPE A has been recognized, while entC, and entB are more similar to each other than to SPE A (36). Couch and Betley (33) reported that SEC? has 98% nucleotide sequence homology with SEC,, while SECl and SEB exhibit 68% amino acid homology. Both SEB and SECl have significant structural homology, specifically in the amino- and carboxy-terminal regions, with its antigenic determinants, a similar molecular weight, a single disulfide bond, and no free sulfhydryl groups (41,5 1,55,56). The amino-terminal region in SEB and SEC, have been shown to be the antigenic site most responsible for cross-reactions with serological antibodies (57), while a high degree of dissimilarity in the amino-terminal of SEA may explain why antiserum to SEA does not cross-react with SEB or SEC, (38). In addition, Table 4 Chemical and Physical Properties of Staphylococcal Enterotoxins

Amino acid residues (mature toxin) (enterotoxin gene) Molecular weight (mature toxin) (enterotoxin gene) Isoelectric point Nitrogen content (5%) Emetic dose (EDso,pg per monkey) Maximum absorption (nm) Extinction (E”,) Sedimentation coefficient (So,”), S ~

per os. 0.05 mg/kg by IV route (19). Source: Refs. 19, 40,65, 67.

239 266

239 266

238 266

27,078 28,494 27,500 29,700 3 1,400 30,5 11 8.6 6.8 8.6 16.2 16.1 16.5

27,53 1 30,608 7.0 16.0

27,438 26,360 26,425 29,358 8.15 7.4 7 .O -

-

5 277 14.3 3.04

5-10 277 12.1 2.90


20 278 10.8

233 257

5 277 14.0 2.78

239 266

5 277 12.1 3 .OO

228 258

230 257

10-20 277 12.5 2.60

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354

Table 5 Amino Acid Composition (g/100 g protein) of Staphylococcal Enterotoxins Amino acid

SEA

SEB

SECS

SED

SEE

Alanine Arginine Aspartic acid Cysteine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Tryptophan Valine Amide NH2

1.9 4.0 15.5 0.7 12.6 1.o 3.2 4.1 9.8 11.3 1.o 4.3 1.4 3.0 6.0 10.6 1.5 4.9 1.8

1.3 2.7 18.1 0.7 9.5 1.o 2.3 3.5 6.9 14.9 3.5 6.2 2.1 4.1 4.5 11.5 1.o 5.7 1.7

1.7 1.7 18.2 0.7 8.6 3.1 2.9 3.8 6.4 14.0 3.5 5.4 2.2 5.0 5.7 10.1 1.o 6.0 1.5

2.0 3.4 16.7 0.7 13.2 2.7 2.7 6.0 9.3 12.9 1.1 4.8 1.4 5.1 4.5 7.2 0.6 4.1 1.7

2.4 4.5 15.1 0.8 12.2 4.1 3.0 4.3 10.1 10.8 0.5 4.5 1.9 4.7 6.4 9.8 1.7 4.4 1.7

Source: Ref. 5.

Huang et al. (38) was able to demonstrate remarkable structural homology in SEA at the half-cysteine residue located at position 106 with SEB and SEC,. Huang et al. (58) hypothesized that this half-cysteine residue may be the emetic site responsible for gastroenteritis. Mature SEA has 82, 72, 74, and 34 amino acid residues in common with SEB, SEC,, SPEA, and TSST-1, respectively (37,39,59-61). Betley and Mekalanos (34) were able to hybridize DNA probes derived from cloned entA gene to other types of SE, which suggested that a high amount of sequence divergence may have occurred between SEC, and SEB in the carboxy-terminal region. The nucleotide sequence for SPE A shares remarkable homology with entB and entCl (55,56), while Bloomster-Hautamaa et al. (59) have reported TSST-1 to have similarities in biological activities but no amino acid sequence homologies with SPE A, entB, and entC,. Couch and Betley (33) also reported comparisons between the entC3, entCI, or entB genes, which suggested that an ancestral entC,-like gene was formed by recombination between the e n C l and entB genes. Couch et al. (35) reported that the entE gene has 84% nucleotide sequence homology with the entA gene. They also reported that SEE is more closely related to SEA than it is to SEB, SECI, or SPE A. In general, the percentage of amino acid sequence homology among the mature forms of the enterotoxins varies from 29% to 82%, with SEA and SEE being the most closely related (35). Bohach and Schlievert (36) have suggested that the homologies shared by all the PTs may be required for their common biological properties, while, in contrast, those regions in which the molecules diverge may determine their unique toxicities. In addition, Bohach and Schlievert (62) have observed homologies in other studies comparing reported PT amino acid sequences indicating that several carboxy-terminal and centrally located stretches of primary sequences are uniformly conserved throughout the PT family.

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For example, they were able to uncover additional evidence that carboxyl regions of staphylococcal and streptococcal pyrogenic toxins shared biological activities and cross-reactive epitopes. Most of the work has been done on identifying the amino acid composition, the erzt genes, biochemical similarities, serological similarities, and biological action, while work concerning molecular events mediating the effects of the SEs remains relatively unknown. Since the SEs exhibit remarkable similarities, Singh and Betley (63) have suggested that sequences required for specific tertiary structure and amino acid residues required for common biological function are conserved among the SEs.

B. Properties 1. Heat Resistance The heat resistance of SE is dependent upon the purity of the preparation, the type of toxin, the initial amount, the pH, the heating menstruum, and the method of detection (64). Enterotoxin B has been shown to retain activity for up to 16 hours at 60°C and pH 7.3 (65). Heating SEB for 30-40 minutes in a boiling water bath does not completely destroy its ability to induce vomiting in monkeys or kittens. Borja and Bergdoll (66) heated a preparation of SEC for 30 minutes at 60°C with no change in serological reactions, while Bergdoll (67) heated SEA at 80°C for 3 minutes or at 100°C for one minute with a loss of its capacity to react serologically. In general, the heat resistance of SEA, SEB, and SEC is SEC > SEB > SEA (68). However, it should be noted thatthermal inactivation is more rapid in simple buffers than in more complex systems such as in culture media or foods. For example, Denny et al. (69) has shown greater thermal stability of SEA in beef bouillon (pH 6.0) than in phosphate buffer (pH 7.2), while Fung et al. (70) reported that at 60"C, SEC in phosphate buffer was inactivated in 60 minutes compared to 180 minutes in a culture medium. The for SEB ranges from 9.9 to 11.4 minutes and a zvalue of 25.8 to 32°C. Tibana et al. (68) and Bennett and Berry (71) provide evidence suggesting that SEA, SEB, SEC, and SED may survive legal retort temperatures and times. 2. Irradiation Resistance The toxins appear to be resistant to gamma irradiation. Read and Bradshaw (72) demonstrated that more than 2.7 and 9.7 Mrad were necessary for a one decimal reduction of SEB in buffer and milk, respectively. Recently, Modi et al. (73) examined the effects of irradiation on SEA using a sensitive ELISA system. In gelatin phosphate buffer, SEA was completely inactivated by irradiation at 8.0 kGy. However, in a 15% mince slurry, 2737% of SEA remained at 8.0 kGy. Interestingly, the protective effect of the mince slurry was seen up to a concentration of 30%, while the protective effects decreased as the slurry was increased above 30%.

3. Enzyme Resistance The SEs are resistant to the action of proteolytic enzymes such as trypsin, chymotrypsin, rennin, and papain (pH > 2.0) (67). However, SE is susceptible to pepsin at pH values of <2.0. This ability to withstand attack by proteases supports their ability to remain active after ingestion or to remain active in certain foods for several years. C.

Production

The staphylococcal enterotoxins are produced in both nutritionally rich and nutritionally fastidious media. According to Smith et al. (74), the following factors influence both

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growth and toxin production: size of inoculum, oxygen concentration, temperature, pH, sodium chloride concentration, a,, mineral ions, and media composition. The production of the SEs are differentiated into two groups based on their production characteristics (6). The first group consists of SEB and the SECS, which are produced in large quantities and are dependent on the conditions of incubation or culture conditions. The second group consists of SEA, SED, and SEE, which are produced in smaller amounts; their production is closely related to the growth of the strain rather than by incubation conditions. In general, a number of factors present in food determine whether or not SE can be produced. Any food that presents favorable environmental conditions and is contaminated with an enterotoxigenic strain of S. aureus is capable of causing staphylococcal food poisoning. The factors affecting staphylococcal growth and enterotoxin production have been extensively reviewed and will only be briefly mentioned here. The reader is further referred to reviews on the growth of S. aureus and the production of SEs that go beyond the scope of this chapter (75-77). The level of contamination can be significant for SE production. Higher inoculum levels will generally result in higher production of SE. In addition, higher inoculum levels will be more tolerant to environmental stresses. The SEs have been shown to be produced over the temperature range of 10-45°C at which S. aurez~scan grow. In general, temperatures that support adequate growth also stimulate production of enterotoxin in foods. The pH range of SE production is slightly more restricted than that for growth and has been reported to be between pH 5.2-9.0. In the pH range at which S. nureus does not grow well, enterotoxin production is also poor. The optimum range for enterotoxin production is influenced by a,. For example, SEA is produced at slightly lower a, values (0.90) than SEB (>0.90). The production of SEs may be influenced by aerobic or anaerobic growth conditions (78): staphylococci are able to grow under both conditions. However, growth under anaerobic conditions is slower, and cell numbers often do not reach those obtained under aerobic conditions. Although enterotoxin sometimes has been shown to be produced under anaerobic conditions, these observations are variable. Currently there is no evidence to suggest that enterotoxin can be produced in vacuum-packed foods (6). The production of SEB has been reported at the beginning of the early stationary phase (79-81), while SEA production has been reported to occur during the exponential phase of growth (82,83). In addition, Otero et al. (17) recently reported on the production of SECl and SEC2 during both the exponential and early stationary phases of growth. Numerous studies have suggested that sugars or other energy sources (amino acids) can affect toxin production by S. aweus in different laboratory media or various foods (6,76,80,84-86). Miller and Fung (87) were able to demonstrate that the production of SEB in a defined medium was dependent on the energy source. For example, they determined the minimum requirements for SEB production in a medium containing arginine, cysteine, phenylalanine, six organic salts, four vitamins, and monosodium glutamate. They concluded that the biosynthesis of one or more amino acids may be the limiting factor for growth and SEB production. Wu and Bergdoll (88) found that SEB production by S. nureus S-6 was dependent on the amount utilized of each of 18 amino acids. In a review of nutritional components on SE production, Bergdoll (6) reported on the significance of including an enzymatic digest of casein in media for increased enterotoxin production. He also noted the requirement of S. cmreus for B vitamins (niacin, thiamine, etc.), which could be satisfied by adding yeast extract to the growth medium. Another factor that has been found to influence SE production is glucose. In addition, Nychas et al. (78) demonstrated that the formation of exoprotein per bacterial cell in static

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cultures was influenced not only by glucose but also by other energy metabolites (lactate and amino acids). Their results on the repressive effects of glucose on SE production were in agreement with those reported by Coleman et al. (89). Glucose has been reported to have repressive effects on SEA, SEB, and SEC (86). SE production is also repressed by glucose under aerobic or anaerobic conditions (80,90-92). Hallis et al. (93) has shown using batch cultures that no effect was seen with medium composition or other culture conditions on SEA production other than the repressive effect of glucose. However, they observed that this simple catabolite repression of promoter function by glucose could be overcome by addition of exogenous cyclic adenosine monophosphate (CAMP). The production of SEs in foods has been reported (94). Approximately 25% of the S. aureus strains randomly isolated from foods have been found to produce different types of SEs (95). Gourama et al. (96) reported that the growth of S. aureus was excellent in clam chowder, with cell counts greater than los cfu/g after 12 hours at 42°C. Production of SEA and SED began after 3 hours, while maximal levels of SEA and SED were 0.29 and 1.6 ng/g, respectively, after 12 hours. In addition, growth in brain heart infusion broth produced SEA and SED concentrations of 21.9 and 36.3 ng/mL, respectively, after 24 hours at 37°C. However, Daoud and Debevere (97) found the production of enterotoxin to be low despite good growth of S. aureus in heated vegetables. Enterotoxins B and C are highly expressed exoproteins and can be produced at levels over 100 pg/mL, while the other toxins are produced at low levels (usually only a few pg/mL) (20). In spite of this, the majority of food-poisoning outbreaks are caused by serotypes A and D. A major factor in food poisoning by all types of staphylococcal enterotoxins is their high intrinsic level of heat resistance, which allows the toxins to survive heat treatments that are lethal to the producing organism. Consequently, it is not unusual to find toxic food products in which no staphylococci can be recovered. Genetically, the enterotoxins are quite diverse. The enterotoxin A (SEA) gene (sea) is borne on a group of temperate bacteriophages, which recombine with the chromosome within the beta-toxin gene (hlb) inactivating it and converting the recipient to an HlbSEA+ phenotype. Some members of the bacteriophage group also carry the gene for staphylokinase (sak) and simultaneously convert the recipient to the Hlb- S&+ SEA' phenotype (98-100). The enterotoxin B (SEB) gene (seb),on the other hand, is a chromosomal gene that is part of a unique element originally thought to be a highly defective bacteriophage (101), but more recent evidence (102; R. P. Novick, personal communication) links the gene to a mobile pathogenicity island. Moreover, the elements for tst and seb occupy the same locus and are mutually exclusive. The genes for enterotoxin C (SEC), E (SEE), G (SEG), and H (SEH) have all been shown to be chromosomal (26), and although likely, it is not known whether they are part of larger genetic elements as are the genes for enterotoxins A and B. Enterotoxin D (SED) is encoded by a gene contained on a large staphylococcal plasmid (pIB485 is the prototype plasmid), which also carries the gene ( s e j ) for enterotoxin J (SEJ) (103), which is located immediately upstream of sed and transcribed in the opposite direction. None of the enterotoxins are essential genes required for the growth of S. nureus. Because their origin can be traced to mobile elements, they are generally considered as accessory genetic material. Except for SEA, the expression of the enterotoxin genes is positively regulated by the staphylococcal master exoprotein control circuit ngr (accessory gene regulator) (102). This expression modulator is activated in late logarithmic phase, and its function is specifically required for the transcription of extracellular protein genes. Therefore, activation of ogr in late logarithmic phase explains why the enterotoxins (except for SEA) are produced in the stationary phase

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of growth. SEA is produced in the logarithmic phase of growth, and transcription of the gene is not under the influence of the ngr control circuit (8).

Detection D. The detection of enterotoxins in foods has been problematic. Only animals that can undergo an emetic response to the toxins are used, and the most reliable of these is the monkey (20). This model of enterotoxin poisoning is costly, complex, and requires elaborate animal care facilities. However, the standard method of detection is immunological and relies on precipitin reactions observed in various gel diffusion assays. Although many of the toxins are immunologically cross-reactive, quantitative and qualitative Ouchterlony diffusion assay against purified standard toxins can usually be used to identify the specific serotype present. A water extract of the suspect food is used for this assay, but in actual practice there is seldom enough material remaining after an outbreak to actually carry out this assay, and a diagnosis of staphylococcal food poisoning is usually made retrospectively. A food suspected of causing staphylococcal food poisoning must be assayed both for the presence of viable S. uz1rez4sand the presence of SE. Because of the greater heat resistance of SE conlpared with S. clureus cells, it is possible that a food could be the source of a staphylococcal foodborne disease outbreak and yet noviable organisms would be present. To confirm an outbreak of staphylococcal food poisoning, it is necessary to show the presence of SE in the implicated food. Traditional procedures for SE detection are time consuming, laborious, and lack sensitivity. The first early successful tests for the presence of SE utilized emesis of monkeys, which is still used today. A suspect food sample homogenate (usually 50 mL) is intubated into the stomachs of young rhesus monkeys (Mncnca nzulcrttn). The animals are then observed continuously for 5 hours. A positive sample results in vomiting of the animal. Kittens (intraperitoneal or intravenous injection) have been used in place of monkeys for SE detection. Both anitnal tests suffer from several problems such as the expense and labor involved in maintaining an animal colony, varying sensitivities with different animals, and the presence of interfering substances within a sample. Current methods for detecting SE are sensitive but suffer due to the inefficiency of toxin recovery after extraction from foods, specifically in foods containing low toxin concentrations. In general, most foods implicated in staphylococcal food poisoning outbreaks contain low levels of enterotoxins (
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359

Dimensionsof Well 0.357 cm % p- 0.238 cm

["l

I

1 1

A

Template

p&

J.

"b

TL--

2.54 cm

0.31 75cm

*

2.54 cm

Two layers of waterproof tape

I

l I

v2.54

k"---- 7.62 cm , L * /

c'm

Fig. 1 Microslideassemblyandspecifications.

Traditional methods such as the microslide assay or gel immunodiffusion assay are used for detection of SE. The microslide assay is based on the formation of precipitate on an agar matrix and is the currently recommended procedure for detection of SE. This microslide gel diffusion test is used to visualize precipitation and is described in detail in the BncferiologictdAnalytical Manual (10). As shown in Fig. 1, a microslide is prepared with the aid of a template, such that five wells are formed in highly purified Noble agar. Antisera is added to the center well, and known reference enterotoxins are placed in wells 3 and 5 (Fig. 2). Prepared sample is added to wells 2 and 4. The slides are then incubated in a humidity chamber at room temperature for 3 days. As the antigen (enterotoxin) and

e I

I

1

I

I

2

1

3

I

3

4

Fig. 2 Enterotoxin analysis with the microslide. The center well contains antibody to SE. Wells 1 and 3 contain SE. Slide 1: buffer solution in wells 2 and 4. Slide 3: the test sample in wells 2 and 4 contain SE. Slide 3: the test sample in well 3 contains SE, while the test sample in well 4 has no SE. Slide 4: the test sample in well 2 contains less SE than the test sample in well 4.

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Martin et al.

antisera diffuse from the wells, a precipitate forms where the concentration of antigen and antibody are at optimal proportions. The slide is then read by holding it over a source of light and against a dark background. Lines of precipitation are identified through their coalescence with the reference line of precipitation (Fig. 2). As little as 0.1 pg/mL or less of SE can be detected using this method. However, this level of sensitivity is difficult to achieve (21), while others have reported a number of inadequacies in this technique. For example, Pereira et al. (105) discovered that low-level production of SED (ng/mL) by specific S. a~e14.sstrains in laboratory media, not detectable by gel diffusion methods, can produce sufficient SED (ng/g) in foods to cause food poisoning. Ewald and Christensen (106) reported that 51% of 47 S. mreus strains tested produced SE by the ELISA technique, while only 35% were detected by the microslide immunodiffusion technique. The use of PHA and RPHA was proposed some 30 years ago (107,108). PHA utilizes specific antibodies at a constant concentration while the sample to be tested is varied. Following incubation, antigen-treated sheep red blood cells (SRBC) are added. Hemagglutination (HA) occurs only when antibody is not bound to antigen. As an alternative, RPHA utilizes specific antibody attached directly to the SRBC. Hemagglutination should generally occur within a %hour incubation period. The sensitivity of these techniques falls within a detection limit of about 1 ng/mL. However, due to difficulties associated with coating SRBC and nonspecific agglutination caused by food components, these assays have not been popular. To overcome these problems, Igarashi et al. (109) developed the RPLA. This assay utilizes latex particles coated with highly purified specific antibodies. However, when a commercial RPLA kit was compared to a commercial ELISA kit, RPLA failed to detect the required 0.1 pg of SE/100 g of food (1 10). Fujikawa and Igarashi (1 11) later improved RPLA by using high-density latex particles, which achieved the required detection level. Other serological assays that show great promise are the tracer-labeled immunoassays, which include RIA, ELISA, and LIA. These tracer-labeled assays can be defined as a type of binding assay that uses the antibody-antigen reaction as the base and a radioisotope, enzyme reaction, or luminescent reaction as the marker. RIA, which provides both high sensitivity and the ability to determine the level of SE, was one of the first tracer-labeled immunoassays utilized for SE detection (1 12-1 14). However, RIA has not been widely used because of the risks and the costs of handling and disposing of radioisotopes. An alternative to RIA for the detection of SE, which shows great promise, is ELISA. ELISA is classified as either homogeneous, where the immunoreactants are dispersed in solution, or heterogeneous, where the immunoreactants are bound to a solid matrix. The heterogeneous assay is further subdivided into the sandwich ELISA (Figs. 3 and 4) and the direct ELISA (Figs. 5 and 6). The ELISA test relies on two valid assumptions: (a) that antibody or antigen can be attached to a solid-phase support and still retain activity, and (b) that either the antigen or the antibody can be attached to an enzyme and the complex retain both its immunological and its enzymatic activities. Many antigens, as well as antibodies, can be attached to a variety of solid objects, such as paper disks, polyvinyl, or polystyrene, and retain activity and specificity. A number of enzymes (peroxidase, P-galactosidase, etc.) similarly have been successfully attached to both antigens and antibodies. Competitive assays can then be performed to both quantify and determine the amount of antigen present. One test proposed for SE detection involves a double antibody sandwich ELISA. In this technique, a polystyrene tray is coated with antibodies against SE and the tray

Staphylococcus aureus

367

W

W = wash

Y

+ = antigen

0= substrate

= antibody

t9

),

= enzyme-linked Ab

c]= coloredproduct

Fig. 3 Double antibody (sandwich ELISA). Antigen is added to wells coated with specific antibody; unreacted antigen is removed by washing; secondary antibody conjugated to an enzyme is added; a second wash is performed and a chromatic substrate added; a colored product is produced.

washed to remove any antibodies not attached. A solution prepared from the suspect food, thought to contain SE, is then incubated with the antibody-coated polystyrene tray. SE remains attached to the immobilized antibody on the plastic tray, while washing removes unattached material. A conjugate (antibodies made against SE that have the enzyme peroxidase attached) is then incubated with the trays. Washing removes excess conjugate. Finally, Hz02,the peroxidase substrate, is added. The rate of H202degradation is dependent on the amount of enzyme-labeled antibody, and that, in turn, depends on the amount of antigen in the test sample. The enzyme reaction is determined spectrophotometrically following the addition of the color-changing reagent 5-amino-salicylic acid. The double antibody sandwich method was first usedby Saunders and Bartlett (1 15) for the detection of SEA in foods. Since then, the method has been adapted and optimized for all the SE (1 16). Freed et a1 (1 16) reported detection limits of 0.1 ng of SE/mL of

V

W = wash

+ = antigen

1

= antibody

0= substrate

/I = enzyme-linked Ab = colored product

Fig. 4 Indirect antibody (sandwich ELISA). Antigen is added to wells coated with a specific antibody; after washing, secondary antibody is added; a second wash is performed and antispecies antibody conjugated to an enzyme is added; after a final wash a chromatic substrate is added; a colored product is produced.

Martin et al.

362

a

0 W

V

W = wash

1

+ = antigen

= antibody

= substrate

0 = antigen-enzyme = coloredproduct

Fig. 5 Competitive antibody (direct ELISA). Antigen to be tested and standard antigen-enzyme are added simultaneously to wells coated with specific antibody; the wells are washed and a chromatic substrate added: a colored product is produced.

food extract, using polystyrene beads as solid support. Ocasio and Martin (1 04) were able to obtain detection limits of 0.5 ng of SEB/mL in a variety of food extracts. In addition, Gomez-Lucia et al. (1 17) reported detection limits of 0.625 ng of SEA/mL of milk and 1 ng of SEB/mL of milk, while Fey et al. (1 18) compared four ELISA detection methods and concluded that the double antibody sandwich method was the best detection system. A relatively new and alternative approach to the detection of SE in foods is LIA. The LIA system utilizes chemiluminescent labels such as diazoluminol (1 19), isoluminol and its alkalated derivatives, and acridium esters (120-122). However, the low (1%) luminescent efficiency (ratio of photons emitted to the number of excited molecules formed) has limited the use of LIA for detecting SE in foods (123). Since the introduction of LIA, a number of improvements have occurred. For example, an isoluminol derivative [N-(4aminobuty1)-N-ethylisoluminolhemisuccinamide; ABEI-H3], which produces a higher quantum yield when conjugated to proteins (124,125), was used by Lohneis et al. (126) to detect SE in foods. Their immunoluminometric assay (TLMA), which utilized an ABEI-

W

W = wash

+ = antigen

r

= antibody

0= substrate

0 = antigen-enzyme = colored product

Fig. 6 Sequential antibody (direct ELISA). Antigen is added to wells coated with specific antibody; after reaction is complete the wells are washed and a chromatic substrate is added; color formation is inversely proportional to the antigen concentration.

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363

H-IgG conjugate, was only able to detect 0.40 ng of SEB/mL, while the ELISA was able to detect 0.15 ng/mL. However, the ILMA utilized a 30-minute sample incubation period as compared to 90 minutes required for the ELISA. Another example is an alternative to chemiluminescent compounds as labels. This alternative utilizes horseradish peroxidase (HRPO) as a label, which acts as a catalyst or a cofactor in the oxidative reaction of luminol by H202.Horseradish peroxidase conjugated to antibodies are stable and have been used in a number of LIA assays (127) as well as ELBA assays. Like allofthe chemiluminescent labels, the luminol-HRPO-H20, reaction also displays a low quantunl yield (1%). This problem was resolved by using enhancers of the luminol-HRPO-H202 reaction. Enhancers such as substituted phenols (i.e., p-iodophenol) provided as much as a 2500-fold increase in light emission over the unenhanced reaction (128). Ocasio and Martin (129) were able to use this reaction to develop an enhanced chemiluminescent immunoassay (CLIA) for SEB in foods.

V.

SUBLETHALINJURY

The food industry often employs some form of processing or low-temperature storage to extend the shelf life of perishable foods (130). Food processing, which may consist of the use of chemical preservatives, drying, freezing. heating, radiation, or a combination of these methods, is designed to reduce or eliminate vegetative cells of pathogenic and spoilage organisms in food products. Although most microorganisms are destroyed, some sublethally injured or stressed cells may survive and thus may be able to recover and grow at a later time. These stressed cells are usually more susceptible to an adverse environment or secondary stresses to which noninjured cells are resistant. Secondary stresses are also encountered in the form of selective agents used in the enumeration medium. The phenomenon of sublethal injury and stressed-cell recovery has been examined in several articles (130,13 1). Conditions that injure staphylococcal cells include heating (132,133), freezing and freeze-drying (134), irradiation (135), reduced water activity (136). and exposure to different chemicals such as acids and salts (137,138). Several sites of damage have been observed in sublethally injured cells. The most commonly reported are damage to the cytoplasmic membrane with theresultant leakage of cytoplasmic constituents (132,136,139,140) and the degradation of ribosomes and ribonucleic acid (RNA) (141,142). Other sites of damage include protein denaturation and enzyme inactivation (137,143).

A.

NucleicAcids

The involvement of RNA in injury was first recognized when investigators found that mildly heated suspensions of S. aweus lost 260 nm absorbing material into the heating medium. Tandolo and Ordal (133) found that the 260 nm absorbing material that leaked from heat-injured S. m r e u s was RNA or RNA-derived nucleotides. Later studies demonstrated that this material was due to the degradation of ribosomal RNA in addition to cellular pool components (141,144). The degradation of ribosomes and ribosomal RNA in vivo as a result of thermal stress has been examined in S. mrezls (141,144,145). Sucrose gradient analysis of ribosomes from heated cells revealed that the 30s subunit was selectively destroyed. Polyacrylamide gel electrophoresis of ribosomal RNA extracted from heated cells demon-

364

Martin et al.

strated that the 16s RNA had been degraded, while the 23s RNA appeared normal (1 45,146). Rosenthal and Iandolo (141) confirmed that during heating, 16s RNA was almost completely degraded and 23s RNA was altered. The heating of bacterial cells in the presence of a phosphate ion produces conditions that are conducive to RNA degradation by the stimulation of ribonuclease activity (147) and by the disruption of ribosome stability due to the chelation of intracellular ME”. Consequently, this degradation of bacterial ribosomes was due to the combination of phosphate and heat, both of which destabilized the ribosome, resulting in a susceptibility to nucleolytic attack. Recovery from injury represents a period of cellular rebuilding, during which time injured organisms repair damage produced by the sublethal stress. Since ribosome damage and ribosomal RNA degradation are major lesions within most heat-stressed organisms, it follows that ribosomal RNA synthesis and ribosome assembly should be major events in recovery (130). Iandolo and Ordal(132) first reported the need for RNA synthesis during the repair period. Sogin and Ordal (144) confirtned that RNA synthesis was required for recovery. They further demonstrated that RNA synthesis preceded protein synthesis and occurred even when protein synthesis was completely inhibited. In a subsequent study, Rosenthal and Iandolo (141) found that only the 30s subunit and 16s RNA were degraded during heating. However, the 50s subunit, as well as the 30s subunit, were regenerated during repair in the absence of protein synthesis (145). Flowers and Martin (148) examined the process of ribosome assembly during the repair of sublethal heat injury in S. mrez4s. Cells recovering from sublethal injury were found to contain three ribonucleoprotein particles (49S, 26S, and 30s). Polyacrylamide gel electrophoresis showed that the 49s particle contained 23s RNA, the 36s particle contained both 23s RNA and 16s precursor and mature RNA, and the 30s particle contained 16s and precursor 16s RNA. Particles with similar sedimentation properties were found in unheated cells. B. OxygenToxicity Molecular oxygen is only slightly soluble in water, with only 9 mg/L being dissolved at 20°C and one atmosphere of pressure (1 30,149). Only 7 mg OJL are dissolved in nutrient broth at 30°C. However, molecular oxygen is seven to eight times more soluble in organic solvents than in water, such that it probably becomes concentrated in the lipophilic cell membranes (150). Molecular oxygen is paramagnetic due to two of its valence electrons being unpaired. Molecular oxygen in this form is in the lowest energy configuration with the unpaired electrons in parallel spin and is referred to as ground or triplet oxygen. Triplet oxygen may be energized to yield singlet oxygen, which has the two unpaired electrons in antiparallel spin. Two forms of singlet oxygen exist and are of unequal energies. The first, which has the lower energy of the two, occurs when the unpaired electrons of the ground state become paired in the same orbital and are of antiparallel spins. The second occurs when one electron undergoes a spin inversion, with the two unpaired electrons remaining in separate orbitals. Singlet oxygen is exceptionally reactive and therefore poses a threat to the integrity of all cellular components (150). While it is very short-lived in aqueous media, it may be most damaging to those hydrophobic lipid/protein regions, such as membranes, where it reacts with carbon-carbon double bonds in polyunsaturated fatty acids, and where singlet oxygen would not be quenched and would be long-lasting (149). Triplet oxygen contains two unpaired electrons of parallel spin. These parallel electron spins forbid the direct entry of paired electrons (149). Therefore, in order for a divalent

Staphylococcus

365

reduction to occur, one electronic spin would have to be inverted in order to avoid the placement of two parallel spins in one orbital. As a result, whenever energetically feasible, univalent pathways of the reduction of oxygen are favored over divalent pathways. The first product in the univalent reduction of molecular oxygen is the hydroperoxyl radical in the protonated form and the superoxide anion radical in the ionized form (Table 6). The superoxide anion radical CO<-) can act as a powerful reducing or oxidizing agent and can also initiate free radical chain reactions. The superoxide radical and H202 can interact to forrn the hydroxyl radical OH', the most potent oxidant known. Finally, singlet oxygen may be produced by the reaction of 02"with O H (150) or by the interaction of two H202molecules (151). Hydrogen peroxide is formed by the two-electron reduction of molecular oxygen or by the disnwtation of superoxide. It is a strong oxidant and therefore has pronounced bactericidal effects, as a function of time, exposure, and concentration, on spore-forming and non-spore-forming bacteria (152). Hydrogen peroxide is produced by most aerobically growing cells (bacterial, plant, and animal) by the action of superoxide dismutase (SOD) and by some of the flavin-linked enzymes. Hydrogen peroxide is the most stable or least reactive of the intermediates formed in the univalent pathway, and yet it remains a strong oxidant capable of causing irreversible damage to various cell components. Hydrogen peroxide is a bactericidal agent that has been used as a topical antiseptic for many years (153,154). The effectiveness of H202 varies with the concentration, temperature, duration of treatment, and the type and number of organisms (152,155). The modes of action of H202on bacterial cells are numerous. For example, H202 can inactivate enzymes by modifying amino acid residues (156,157). The degradative effects of Hz02on DNA have been demonstrated. Hydrogen peroxide degrades DNA by changing the primary and secondary structures. The relative magnitude of degradation rates (158-161) is as follows: base destruction single strand breaks > double strand breaks > cross-linking. The third product produced in the univalent reduction of O2is the hydroxyl radical. The hydroxyl radical can theoretically be produced in a number of ways. Beauchamp and Fridovich (162) showed that thehydroxyl radical could be generated in significant amounts by a reaction between Oz.- and H202in an aqueous system. The OH' radical is the most potent oxidant known and can cause numerous types of injury.

Table 6 The UnivalentReduction of Molecular Oxygen

0,

+ H+ + le- +

H02(hydroperoxyl radical) radical)

0,

+ 2H' + 2e- +

O2

+ 3H' + 3e- + H 2 0 + OH.

H20z (hydrogen peroxide) (hydroxyl radical)

O2

+ 4H' + 4e- + 2H20

+ Oy- + H+ (superoxide

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Martin et al.

C. OxygenandSublethalInjury Demonstration of the toxic effects of oxygen were the results of attempts to improve the media used in the isolation of S. mrez4s. These studies examined the effects of agents that decompose H202(catalase, peroxidase, or pyruvate) in various solid and liquid media (137,138,163,164). The technique of dual plating on tryptic soy agar (TSA) and TSA plus 7.0% NaCl (TSAS) to measure the degree of stress was developed by Iandolo and Ordal (1 32). Prior to heating, enumeration of S. C ~ Z I ~ ~MF-3 L I S 1 was similar on both media. After heating at 52°C for 20 minutes in 100 mM potassium phosphate buffer (pH 7.2), enumeration on TSAS dropped to 0.0015% of that found on TSA, indicating that >99% of the population was injured (137). When either catalase or pyruvate was added to TSAS, however, enumeration of the heat-injured cells increased 14,000-fold, to approximately 76% of that found on TSA. Catalase addition to various other staphylococcal selective agar media was also effective in increasing enumeration ( 138). Addition of catalase concurrent with the presence of phosphatidylcholine and beef extract in Vogel and Johnson agar resulted in a medium that gave equal or better enumeration than Baird-Parker agar (164). Baird-Parker agar is currently recommended by the Food and Drug Administration (10) for the enumeration of S. nurez4s. Brewer et al. (163) developed a modified most probable number procedure employing either catalase or pyruvate addition. This method frequently detected the presence of low numbers of S. aureus missed by the currently accepted procedure. The observation that catalase or pyruvate increased the enumeration of heat-stressed S. nurezrs suggested that both ofthese agents were acting through the degradation of H701. Bucker et al. (165) allowed heat-injured cells to recover both aerobically and anaerobically. Cells allowed to recover anaerobically showed no sensitivity to NaC1, suggesting that although catalase levels were depressed, little or no hydrogen peroxide was formed. The optimal pH for S. cu4reus MF-3 1 catalase was 5-6 (130,166). The enzyme was stable over the pH range of 4-9. The apparent isoelectric point was 5.3 +- 0.1 pH units. With respect to temperature stability, purified S. awezrs MF-3 1 catalase was stable at 52°C after 45 minutes of heating. The enzyme was inactivated at 60°C for 10 minutes. However, this was dependent upon the concentration of the enzyme and the presence of protectants. The apparent subunit molecular weight was 64,000 2 1,000 daltons, whereas the apparent native molecular weight was 235,000 +- 5,000 daltons. Amino acid analysis revealed that S. aweus MF-31 catalase was similar to amino acid analyses of catalase from other sources. The iron content of S. mrezrs MF-31 catalase was found tobe 0.098%. The enzyme was inhibited by millimolar concentrations of sodium cyanide, sodium azide, and hydroxylamine, indicating the presence of a heavy-metal catalyst. The effects of salt, pH, and salt concentration also influenced activity. The chloride anion inhibited catalase activity at low pH, but this inhibition was influenced by the cation. Sodium chloride was more inhibitory at low pH than either potassium chloride or magnesium chloride. Superoxide dismutase activity has been shown to be lowered following sublethal heating of S. nzueus MF-31 (52"C, phosphate buffer) (167). When cells were heated for 90 minutes, SOD levels dropped approximately 20%, during which time >99% of the cells were killed. The observed loss in activity was not great, but this decrease in concert with a decrease in catalase levels may be significant. S. uurezls cells were sublethally heated and the SOD levels assayed both during the injury and the recovery periods. There was a significant (-85%) drop in SOD activity at the start of the recovery period. This decrease closely approximated the decrease observed in catalase activity following a heat

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stress. The levels remained depressed for approxitnately 2.5 hours until the cells began to multiply. After 2.5 hours, the SOD levels increased concurrently with increases in cell numbers.

VI.

DETECTIONANDENUMERATION

There are four reasons why a food or any ingredient is examined to determine the presence of S. aureus (130): 1. To confirm the presence of S. aweus following a food-poisoning outbreak 2. To determine whether or not a food is a potential source of staphylococcal food poisoning 3. To demonstrate postprocessing contamination 4. As part of a routine quality-control program It has been observed that when staphylococcal cells are sublethally stressed, many are no longer able to grow on selective media. The reasons for this failure, and the methods of overcoming it, have been extensively studied in S. c u u m s . The choice of the method for the detection of S. aureus depends upon the purpose for conducting the test and the product involved. When food is suspected as the source of a staphylococcal food-poisoning outbreak, large numbers of S. nureus are frequently present and sensitive methods may not berequired. More sensitive methods are required to detect small populations of S. aweus, which may bepresent as the result of postprocessing contamination. In many cases, S. aureus is not the sole or even the predominant organism present in a sample. For this reason, selective inhibitory media are employed for isolation and enutneration. Selective media utilize a number of different toxic chemicals to achieve selectivity. Some of the ingredients used include sodium chloride, tellurite, lithium chloride, and various antibiotics. A number of media have been suggested for the isolation of S. aureus from food when more than 100 per gram may be present. Some of these include Staphylococcal Medium 110, Vogel-Johnson Agar, egg yolk-sodium azide agar, tellurite-polymyxin-egg yolk agar, and Baird-Parker Agar (Table 7). Composition of typical staphylococcal selective media is found in Table 8.

Table 7 Examples of Selective Media for Staphylococcus nureus Agar medium Staphylococcus Medium 110 Vogel-Johnson

Egg yolk-sodium azide Baird-Parker

Selective agent Sodium chloride Lithium chloride Potassium tellurite Glycine Lithium chloride Potassium tellurite Polymyxin B sulfate Lithium chloride Potassium tellurite

Diagnostic agent Mannitol Gelatin Mannitol Tellurite Phenol red Egg yolk Tellurite

Martin et al.

368 Table 8 Staphylococcal Selective Media Composition Component Peptone Yeast extract Beef extract LiCl Glycine Sodium pyruvate Potassium phosphate dibasic Mannitol Phenol red Phosphatidyl choline DNA Agar Tellurite ( 1%) Egg yolk (50%) Catalase

B-P(g/L) PCVJ(g/L) VJ(g/L) pH 7.0 10 1 5 5 12 10

pH 7.2

pH 7.2

10 5 5 5 10

10 5

5 10 -

5

-

1s 10 mL 53 mL

10 0.025 2 2 16 10 mL

10 0.025 -

16 20 mL

-

780 units

B-P = Baird-Parker agar; PCVJ = phosphatidyl choline-Vogel and Johnson agar; VJ = Vogel and Johnson agar.

Most selective media are suitable for the enumeration of nomlal or unstressed S. aureus. However, due to processing, preservation, or other adverse conditions, sublethal stress may occur, resulting in the increased sensitivity of S. aur-eus to the selective agents. Because injured cells exhibit an increased sensitivity to selective agents, S. nureus may go undetected in conventional selective enumeration procedures. Baird-Parker and Davenport (168) demonstrated that the recovery of heated or dried cells of S. aureus may be lost or its activity reduced by heating or drying and that blood, which contains catalase, or the addition of pyruvate, helped in the enumeration by destroying H202produced by recovering cells. It has been found that Baird-Parker (B-P) agar is most satisfactory in enumerating injured cells when compared with other staphylococcal selective media (169-171). The addition of catalase to tryptic soy agar plus 7% NaCl (TSAS) (Table 9) and other selective media can increase the enumeration of themlally stressed S. aureus cells, while the addition of heat-inactivated catalase had little effect on enumeration (130,137,138). The addition of catalase to other selective media has been found to increase the enumeration of both heat-stressed and unstressed cells (138). Carlsson et al. (172) suggested that when phosphate and glucose were autoclaved together in a culture medium at neutral or alkaline pH, products resulted that rapidly auto-oxidized, forming these reactive species. Attempts have been made to develop a staphylococcal-selective medium that gives an enumeration equal to that of B-P agar while overcoming difficulties in the use of BP agar (e.g., with milk products) and its expense as described by Martin (130). Andrews and Martin (164) modified Vogel and Johnson agar by the addition of 0.5% beef extract, 0.2% DNA, 0.2% phosphatidylcholine (lecithin), and 780 units of catalase spread on the agar surface prior to inoculation. They found this phosphatidylcholine-Vogel and Johnson

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Table 9 Catalase and Enumeration of Thermally Stressed S. uureus MF-3 1 Cells3 Unstressed cells

Stressed cells %

%

Medium

CFU/mL

B-P VJ VJ + cat' TSA TSA + cat' TS AS TSAS + catc MSA MSA + cat' S1 10 S1 10 + cat' TPEY TPEY + catc

3.9 3.5 4.2 3.4 3.9 3.1 3.5 3.0 3.4 2.2 2.5 4.2 4.3

x lo9 X

X

x X X

x X X

109 lo9 lo9 109 109 lo9 109 109 109

X X 109 X 10' X

109

enumeration' 100 90 108 87 100 79 90 77 87 56 64 108 110

CFU/mL 2.4 X 6.2 X 2.9 X 1.9 X 2.5 X 1.8 X 1.2 X 2.5 X 7.2 X 2.3 X 3.7 x 8.0 X 2.5 X

109 10' lo9 109 109 107 109 lo6 lo8 10' los los 109

enumerationh 100 26 121 79 104 0.8 50 0.1 30 0.1 15 33 104

CFU = Colony-forming units; B-P = Baird-Parker agar; VJ = Vogel and Johnson agar; TSA = Tryptic soy agar; TSAS= Tryptlc soy agar 7% NaCl; MSA = mannitol salt agar; S1 10 = staphylococcal 1 10 agar; TPEY = tellurite polymyxin egg-yolk agar. Cells were heated in 100 mM potassium phosphate buffer (pH 7.2) at 52°C for 15 minutes. h Percentage of enumeration was calculated by dividing CFU/mL on the various media by CFU/mL on B-P and nlultiplying by 100. c Catalase (cat) activity was about 780 units per plate.

+

agar (PCVJ) gave an equivalent enumeration of stressed S. nureus cells and staphylococci from naturally contaminated food samples to that found for B-P enumeration. Enumeration with this medium was easier than with B-P and allowed ascertaining the production of DNase. Idziak and Mossel (173) modified the B-P agar formulation by replacing the egg yolk with pig plasma (B-PP) and found that, based on selectivity, diagnostic characterization, and increased sensitivity, B-PP agar was superior to B-P agar. Lachia (174) described a simplified method for the enumeration of S. nureus from food. He replaced the egg yolk in B-P agar with Tween 80 (0.05% wthol) and MgC12 (0.1%). When these compounds were added to the egg yolk-free B-P agar, the recovery of stressed cells were comparable to recovery on complete B-P agar. Mentzer-Morgenstern and Katzenelson (175) developed a single-step staphylococcal selective medium identified as 4-S agar. This medium permitted the isolation and identification of staphylococci and was achieved in a single step. Coagulase-positive staphylococci form small, grey to dark grey colonies surrounded by a dense, white opacity. Unfortunately, heat-stressed cells were inhibited by this medium, and a 3-hour preincubation period in brain heart infusion was required to enumerate these injured cells. This medium is reported to be very selective for S. uureus. When low numbers of S. nureus are expected in a food sample, a most-probablenumber (MPN) procedure is generally employed. The MPN technique is considered more efficient in the enumeration of low numbers of organisms or when high levels of competing

Martin et al.

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organisms are present (176-178). An MPN value is an estimate of the population and not a precise enumeration of viable organisms. Microbiological counts are reported as “number of microorganisms per quantity of sample by MPN method.” Strict interpretation of the confidence limits for a MPN value of 2O/g, for example, asserts that the true population density lies between 7 and 89/g in 95% of all samples (179). Because of its selectivity, NaCl (10%) has been incorporated into tryptic soy broth (TSBS) in a MPN procedure. After 48 hours in TSBS, suspected tubes must be streaked onto B-P agar for an additional 48 hours for confirmation (180). The enumeration of injured S. c~ureuscells in TSBS has been shown to be greatly depressed (163,180). Brewer et al. (163) found that the addition of 1% pyruvate or catalase significantly increased the enumeration of stressed cells. Because of the requirement to add catalase after autoclaving, pyruvate is considered more desirable. Other staphylococcal MPN media have been examined (181,182).

A.

DetectionMethods

1. High Numbers When a sample is thought to contain 21000 S. c~ureuscells per gram, the most widely recommended enumeration medium is B-P agar (183). The samples are suspended in sterile diluent and 1.0 mL is spread-plated in triplicate on B-P agar; plates are dried, inverted, and incubated for 48 hours at 35°C. Typical S. uureus colonies are black to dark grey, circular, smooth, convex, moist, frequently with a light-colored margin, surrounded by an opaque zone of precipitation, and frequently with an outer clear zone. The colonies have a buttery to gummy consistency (184). Unfortunately, the zones are not always apparent, causing some S. aurezls cells to be missed. 2. Low Numbers The Association of Official Analytical Chemists (AOAC) procedure for the detection of low numbers of S. nureus is a MPN procedure that utilizes MPN tubes containing TSBS and 1% sodium pyruvate (185). In a collaborative study, this method was shown to significantly increase the enumeration of low, middle, and high levels of S. nureus from naturally contaminated products and is highly selective. The MPN tubes are inoculated from appropriate dilutions and are incubated at 35°C for 48 hours. The tubes are confirmed positive by streaking on B-P agar incubated at 35-37°C for 48 hours. Lancette et al. ( l 85) reported that the addition of 1% pyruvate to TSBS in a MPN procedure gave a significantly better enumeration of both artificially and naturally contaminated foods than did TSBS alone. They also found that the addition of pyruvate to TSBS increased the recovery of heat-stressed and nonstressed staphylococci. Their results are similar to those of Brewer et al. (163), with both catalase and pyruvate giving improved recovery. For confirmation, the positive MPN tubes should be used to inoculate B-P agar, followed by biochemical tests.

B. Confirmatory Tests Confirmatory tests for the positive identification of S. aureus include gram-positive cocci, production of P-hemolysis on blood agar, the coagulase test, the catalase test (production of 0, gas during the degradation of H202),anaerobic utilization of glucose and mannitol, and the production of thermostable nuclease. An additional test frequently performed is

Staphylococcus aureus

371

to determine the sensitivity of the suspect isolate to the enzyme lysostaphin. Lysostaphin specifically lyses cells of thegenus StnphyZococcus.Hawcroft and Geary (186) have found that restriction length polymorphism analysis of genes for ribosomal RNA (' 'ribotyping' ') is a useful tool for staphylococcal identification.

VII.

,

FOODPOISONING

Any food that provides S. uureus with the proper nutritional and environmental requirements is a potential source of foodborne illness. In the United States, frequently implicated foods include baked ham, pork, salads (meat, potato), pastries with cream or custard fillings, dried milk, and whey. In manyinstances, the source of contamination with S. nureus is from food handlers who have open lesions due to enterotoxin-producing S. aureus or who are asymptomatic carriers. Contaminated food-handling equipment and milk from cows with staphylococcal mastitis are also potential sources of the organism. Frequently, S. ~ u r e u sis introduced into the food as a result of post-processing contamination. The food is then improperly stored at temperatures, which allow S. nureus to grow and produce enterotoxin. One exception to postprocessing contamination can occur in the production of naturally fermented sausage. In this case, there may be sufficient growth of S. nureus for enterotoxin to be produced before it is inhibited by lactic acid, produced during sausage fermentation. The most common symptoms of staphylococcal food poisoning are nausea, vomiting, retching, abdominal cramps, and diarrhea. These symptoms may vary such that there may be vomiting but no diarrhea, or diarrhea but no vomiting. When severe cases occur, there may be, in addition to the symptoms described above, headache, muscle cramping, and prostration. Occasionally, low fever may be present in some victims. The mechanisms of action of SE are not entirely understood, but it is postulated that the toxin acts directly in the abdominal viscera. The onset of the symptoms, typical for food intoxication, generally occurs within 1-6 hours following consumption of the contaminated food. The average time for onset is 2-3 hours, but symptoms may develop in less than 1 or after 6 hours. The development of symptoms is determined both by the sensitivity of the victim and the amount of toxin consumed. Recovery is generally complete within 1-3 days in otherwise healthy individuals; however, the more severe the symptoms, the longer the recovery period. The mortality rate of staphylococcal food poisoning is extremely low. although death does occur, usually among the elderly or very young.

VIII.

PHAGECLASSIFICATION

Strains of S. aureus are routinely identified by bacteriophage typing. The first demonstration of a bacteriophage was in England by Twort in 1915; this phage had the ability to lyse cells of S. nureus. Since that time a set of typing bacteriophages has been compiled for the typing of S. cw-eus. Five groups, divided into host-range specificities, each containing a varying number of phage species, are differentiated by the response of S. nureus cells to the set of typing bacteriophages (Table 10). One drop of each standardized phage suspension (routine test dilution or RTD) is placed on an agar surface previously inoculated with a heavy inoculum of the staphylococcal strain to be tested. The pattern of the zones of lysis is recorded following overnight incubation at 30°C. The use of phage typing is a

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372 Table 10 StaphylococcalTypingPhageGroups Phages group Phage Group I Group I1 Group I11 Group IV Miscellaneous

group within 29,52,52A,79,80 3A,3B,3C,55,71 6,7,42E,47,53,54,75,77.83A,84.85 42D 8 1,94,95,96

valuable epidemiological tool that can be used in determining the source of an enterotoxin producing strain. Types of S. nureus found in lytic group I11 are most frequently implicated in foodborne disease outbreaks. Not all cultures are typable by this procedure, and the susceptibility patterns of circulating strains vary in time and locality.

IX. SUMMARY S. aureus is a gram-positive bacterium that is capable of causing serious infections in susceptible individuals and is a leading cause of bacterial illness. Some of the important characteristics of S. aureus include its ability to survive and grow at reduced water activity and its ability to produce a wide variety of extracellular enzymes and toxins. Some of the more important include coagulase, heat-stable nucleases, penicillinase, hemolysins, toxic shock syndrome toxin, and staphylococcal enterotoxins. Staphylococcal enterotoxins are produced during the growth of S. nureus in foods, and < l pg can cause staphylococcal food poisoning in humans. These enterotoxins are heat-resistant and are produced under a variety of growth conditions. They are detected using either animal models or by immunological techniques. Cells of S. aureus can be sublethally injured by a number of treatments (heating, freezing, etc.) and sublethally injured cells become susceptible to environmental challenges to which unstressed cells show no effect. Oxygen and its toxic reduction products are involved in the failure of many staphylococcal selective media to enumerate and/or detect injured cells. Baird-Parker agar and a most-probable-number procedure using 10% NaCl and 1% pyruvate are recommended for the enumeration of samples containing high and low numbers, respectively, of S. cw-eus. Staphylococcal food poisoning is among the leading causes of foodborne disease outbreaks in the world. Symptoms include nausea, vomiting, diarrhea, and prostration.

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toxins A and D by Staphylococcus allreus in salad bar ingredients and clam chowder. J. Food Prot., 54( 11):844. Daoud, S. M., and Debevere,J. M. (1984). Growth and thermonuclease production Staplzyby lococcus aureus in vegetables. In Microbial Association and Interactions irz Food (I. Kiss, T. Deak, and K. Incze, eds.), D. Reidel Pub. Co., Dordrecht, pp. 165- 170. Betley, M.J., and Mekalanos,J. J. (1985). Staphylococcal enterotoxin Ais encoded by phage. Science, 229: 185- 187. Smeltzer, M. S., Hart, M. E., and Iandolo, J. J. (1994). Theeffect of lysogeny on the genomic organization of Staphylococcus aureus. Gem, 138:5 1-57. Coleman, D. C., Arbuthnott. H. P.. Pomeroy, H. M., and Birkbeck, T. H. (1986). Cloning and expression in Escherichia coli and Staphylococczls nureus of the beta-lysin determinant from Staphylococcus aureus. Evidence that bacteriophage conversion of beta-lysin activity is caused by insertional inactivationof the beta-lysin determinant.Microbial Pathog., 1:549564. Johns Jr., M. B., and Khan, S. A. (1988). Staphylococcal enterotoxin B gene is associated with a discrete genetic element. J. Bacteriol. 171:4799-4806. Lindsay, J. A., Ruzin A., Ross, H. F., Kurepina, N., and Novick, R. P., (1998). The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus. Mol. Microbiol., 29:527-543. Recsei, P.. Kreiswirth, B., O’Reilly, M., Schlievert, P., Gruss, A., and Novick, R. P. (1986). Stclphylococcus aureus by agr. Mol. Gen. Regulation of exoproteingeneexpressionin Genet., 20258-61. Ocasio, W., and Martin, S. E. (1990).19. New methodsfor staphylococcal enterotoxin detection. Devel. Ind. Microbiol., 3 1: 175. Pereira, J. L., Salzberg, S. P., andBergdoll, M. S. (1991).Production of staphylococcal enterotoxin D in foods by low-enterotoxin producing staphylococci. Int. J. Food Microbiol., 14(1):19. Ewald, S., and Christensen,S. (1987). Detectionof enterotoxin production byStaph~lococcus aureus from aviation catering meals by the ELISA and the micro-slide immunodiffusiontest. Int. J. Food Microbiol., 5:87. Johnson, H. M., Hall, H. E., and Simon, M. (1967). Enterotoxin B: Serological assays in cultures by passive hemagglutination. Appl. Microbiol., 15(4):815. Mores, S. A., and Mah,R. A. (1967). Microtiter hemagglutination-inhibition assayfor staphylococcal enterotoxin B. Appl. Microbiol., 15:58. Igarashi, H., Shingaki, M., Fujikawa,H.. Ushioda, S. H., and Terayama, T. (1985).Detection of Staphylococcus enterotoxins in food poisoning outbreaks by reversed passive latex agglutination. Zerztralbl. Bakteriol. Mikrobiol. Hyg. Abt. 1. Suppl., 14255. Thorpe, G. H. G., Kricka, L. J., Moseley, S. B., and Whitehead, T. P. (1985). Phenols as enhancers of the chemiluminescent horseradish-peroxidase-luminol-hydrogenperoxide reaction: Application in luminescence monitored enzyme immunoassays. Clin. Chenz., 3 l(8): 1335. test for the detection of Fujikawa, H., and Igarashi, H. (1988). Rapid latex agglutination staphylococcalenterotoxins A to E thatuseshighdensitylatex particles. Appl. Environ. Microbiol., 54(10):2345. Dickie, N., and Akhtar,S. M. (1982). Improved radioimmunoassay of staphylococcal enterotoxin A. J. Assoc. 08 Anal. Chem., 65(1):180. Miller, B. A., Reiser, R. F., and Bergdoll, M. S. (1978). Detection of staphylococcal enterotoxins A, B, C. D, and E in food by radioimmunoassay using staphylococcalcells containing protein A as immunosorbent. Appl. Emiron. Microbiol., 36(3):421. Orth, D. S. (1977). Statistical analysis and quality control in radioimmunoassays for staphylococcal enterotoxins A, B, and C. Aypl. Envil-on. Microbiol., 34:710. Saunders, G. C.. andBartlett, M. L. (1977). Double-antibody solid-phase enzyme immunoas-

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135. Frey, H. E.. and Pollard, E. C. (1968). The action of ganlmay-ray-irradiated medium in bacteria: relation to the electron transport system. Rndiat. Res., 35:59. 136. Hurst,A.,Hendry, G. S., Hughes,A.,andPaley, B. (1976).Enumeration of sublethally injured staphylococci is some foods. Cm. J. Bacteriol., 22677. 137. Martin, S. E., Flowers. R. S., and Ordal,Z. J. (1976). Catalase: its effect on microbial enumeration. Appl. Erniron. Microbiol., 32731. 138. Flowers, R. S., Martin, S. E., Brewer, D. G., and OrdalZ. J. (1977). Catalase and enumeration of stressed Staplzylococcus uureus cells. Appl. Erzvirorz. Microbiol., 33:1112. 139. Allwood. M., and Russell, A. D. (1970j. Mechanisms of thermal injury in nonsporulating bacteria. Ah,. Appl. Microbiol., 1289. 140. Witter, L. D., and Ordal. Z. J. (1977). Stress effects and food microbiology. In Antibiotics and Antibiosis in Agriculture (M. Woodbine, ed.), Buttenvorths, Reading, MA, pp. 102112. 141. Rosenthal, L. J., and Iandolo, J. J. (1970). Thermally induced intracellular alteration of ribosomal ribonucleic acid. J. Bacteriol., 103:833. 142. Witter, L. D. (1981). Thermal injury and recoveryof selected microorganisms. J. Daily Sci., 64: 174. 143. Bluhm, L., and Ordal, Z. J. (1969). Effect of sublethal heat on the metabolic activity of Stuplzylococcus cmreus. J. Bucteriol., 97: 140. 144. Sogin, S. J., and Ordal, Z. J. (1967). Regeneration of ribosomes and ribosomal ribonucleic acid during repair of thermal injury in Stuphylococcus aureus. J. Bucteriol., 94:1082. 145. Rosenthal, L. J., Martin, S. E., Pariza, M. W., and Iandolo, J. J. (1972). Ribosomal synthesis in thermally shocked cells of Staphylococcus aureus. J. Bacteriol.. 109:243. 146. Miller, L. L., and Ordal. Z. J. (1972).Thermal injury and recovery of Bacillus subtilis. Appl. Microbiol., 24:878. 147. Chababurtty, K., and Burma, D. P. (1973).The purification and properties of a ribonuclease from Sublrorrella fyphinzurizan. J. Biol. Chern., 243:671. 148. Flowers, R. S., and Martin, S. E. (1980). Ribosome assembly during recoveryof heat-injured Stuplzylococcus aureus cells, J. Bacteriol., 141:645. 149. Fridovich, I. (1982) Superoxide dismutase in biology and medicine. InPathology of Oxygen (P. Autor, ed.), Academic Press, New York, p. 1. 150. Morris, J. G. (1976). Oxygen and the obligate anaerobe. J. Appl. Bucteriol., 40229. 151. Koppenol. W. H., and Butler. J. (1977). Mechanism of reactions involving singlet oxygen and the superoxide anion. FEBS Lett., 83: 1. 152. Wardle, M.. and Renninger, G. (1975). Bactericidal effects of hydrogen peroxide on spacecraft isolates. Appl. Microbiol., 30:710. 153. Edsall, G.. and Ley, H. L. (1965). The prevention of infection. In Bacterial and Mycotic hfections of M m (R. J. Dubos and J. Hirsh. eds.), Lippincott, Philadelphia, p. 913. 153. Grump, W. (1979). Disinfectants and antiseptics. In Emyclopediu of Chemical Technology (Kirk, R. E. and Othmer, D. F., eds.), John Wiley & Sons, New York, p. 807. 155. Amin, V. M., and Olson, N. F. (1968). Selective increase in hydrogen peroxide resistance of a coagulase-positive Staplzyloc.occus. J. Bacteriol., 95: 1604. 156. Kleppe, K. (-1966).The effect of hydrogen peroxide on glucose oxidase from Aspergillus niger. Biochemistry, 5: 139. 157. Kong, S., and Davison, A. J. (1981). The relative effectiveness of OH, H202,O?,and reducing free radicals in causing damage to biomembranes: A study of radiation damage to erythrocyte ghosts using free radical scavengers. Bioclzim. Biophys. Acta, 640:313. 158. Massie, H.. Samis, H., and Baird, M. (1972). The kinetics of degradation of DNA and DNA by H202.Biochint. Biophys. Acta, 272539. 159. Rhaese, H., Freese, J. E., and Meizer, M. (1968). Chemical analysis of DNA alterations. 11. Alteration and liberation of bases of deoxynucleotides induced by hydrogen peroxide and hydroxylamine. Biochinl. Biophvs. Actu, 115:491.

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160. Yamamoto, N. (1969). Damage, repair and recombination. 11. Effect of hydrogen peroxide on the bacteriophage genome. Virology, 38:457. 161. Uchida, Y., Shigematsu, H., and Yamfugi, K. (1965). The mode of action of hydrogen peroxide on deoxyribonucleic acid. Enzymologia, 29:369. 162. Beauchamp, C., and Fridovich, I. (1970). A mechanism for the production of ethylene from methional. J. Biol. Cllent., 245:4641. 163. Brewer, D. G., Martin, S. E., and Ordal, Z. J. (1977). Beneficial effect of catalase or pyruvate in a most-probable-number technique for the detection of Staphylococcus aurem Appl. Environ. Microbiol., 34:797. 164. Andrews, G.P., and Martin, S. E. (1978). Modified Vogel and Johnson agar for Staplzylococcus aweus. J. Food Prot., 41:530. 165. Bucker, E. R. Martin, S. E., Andrews, G. P., and Ordal, Z. J. (1979). Effect of hydrogen peroxide and sodium chloride on enumeration of thermally stressed cells of Staphplococcus aureus. J. Food Prot., 42(12):961. 166. Martin, S. E., and Barrier, W. A. (1990). Influence of salt, pH and temperature on Staphylococcus nureus MF-31 catalase. Food Microbiol., 7:121. 167. Bucker, E. R., and Martin, S. E. (1981). Superoxide dismutase activity in thermally stressed Staphylococcus culre'ells.Appl. Environ. Mici-obiol., 41 (2):449. 168. Baird-Parker, A. C., and Davenport, E. (1965). The effect of recovery medium on the isolation of Staphylococcus nzweus after heat treatment and after the storage of frozen or dried cells. J. Appl. Bacteriol., 28:390. 169. Collins-Thompson. D. L., Hurst, A., and Aris, B. (1974). Comparison of selective media for the enumeration of sublethally heated food-poisoning strains of Stnplzylococcztsaureus. Can. J. Microbiol., 20:1072. 170. Rayman, M. K., Deyovod, J. J., Purvis, U., Kusch, D., Lanier, J., Gilbert, R. J., Till, D. G., and Jarvis, G. A. (1978). ICMSF methods studies. X. An international comparative study of four media for the enumneration of Staphylococcus aureus in foods. Can. J. Microbiol., 24: 274. 171. Stiles, M. E., and Clark, P. C. (1974). The reliability of selective media for the enumeration of unheated and heated staphylococci. Can. J. Microbiol., 20:1735. 172. Carlsson, J., Nyberg, G., and Wrethen, J. (1978). Hydrogen peroxide and superoxide radical formation in anaerobic broth media exposed to atmospheric oxygen. Appl. EnvirorI. Micro biol., 36:223. 173. Idziak, E. S., and Mossel, D. A. A. (1980). Enumeration of vital and thermally stressed Stnplzylococcus aureus in foods using Baird-Parker pig plasma agar (BPP). J. Appl. Bacteriol. 48:lOl. 174. Lachia, R. V. (1984). Egg yolk-free Baird-Parker medium for the accelerated enumeration of foodborne Staphylococcus aureus. Appl. Emiron. Microbiol., 482370. 175. Mentzer-Morgenstern, L., and Katzenelson, E. (1982). A simple medium for isolation of coagulase-positive staphylococci in a single medium. J. Food Prot., 45218. 176. Baer, E. F., Gilden, M., Wienke. C., and Mellitz, M. (1971). Comparative efficiency of two enrichment and four plating media for isolation of Staphylococcus mreus. J. Assoc. 08 A m l . Cltenl., 54:736. 177. Giolitti, G., and Cantoni, C. (1966). A medium for the isolation of staphylococci from foods. J. Appl. Bacteriol., 29:395. 175. Patterson, J. T. (1973). Comparison of plating and the most probable number techniques for the isolation of staphylococci from foods. J. Appl. Bacteriol.. 36:273. 179. Oblinger, J. L., and Koburger, J. A. (1984). The most probable number technique. In Conzpend i m of Methods for the Microbiological Examination of Foods (M. L. Speck, ed.), American Public Health Association, Washington, DC. pp. 99-1 11. 180. Lancette, G. A. (1986). Current resuscitation methods for recovery of stressed Staphylococcus nureus cells from food. J. Food Prot., 49:477.

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181. Van Dorne, H., Baird, R. M., Hendriksz, D. T.. Van Der JSreck, D. M., and Pauwels. H. P. (198 1). Liquid modification of Baird Barker’s medium for the selective enumeration of Staphylococcus aweus. Antonie van Leeuwenhoek; J. Microbiol, Serol., 47:267. 182. Chopin, A., Malcom, S., Jarvis, G., Asperger, H., Beckers, H. J., Berbona, A. M., Cominazzini, C., Carini, S., Lodi, R., Hahn, G., Heechen, W., Jans. J. A., Jervis, D. I., Lanier, J. M., O’Connor, F. O., Rea, M., Rossi, J., Seligmann, R., Tesone, S., Waes, G., Macquot, G., and Pivnick. H. (1985). CMSF methods studies. XV. Comparison of four media and methods for enumerating Stnplzylococcus nureus in powdered milk. J. Food Prot., 4821. 183. (1980). OfJicialMethods of Annlysis of the A.O.A.C., 13th ed., Association of Official Analytical Chemists, Washington, DC, Sec. 46. 184. Lancette, G. A., and Lanier, J. (1987). Most probable number method for isolation and enumeration of Staphylococcus nureus in foods: collaborative study. J. Assoc. Ofs Anal. Chent., 70:35. 185. Lancette, G. A., Peeler, J. T.. and Lanier, J. M. (1986). Evaluation of an improved MPN medium for recovery of stressed and nonstressed Staphylococcus aureus.J. Assoc. Ofs Anal. Clzem., 69:44. 186. Hawcroft, D., and Geary, C. (1996). The use of nonradioactively labeled probe system in an electrophoretic ribotyping method for the differentiation of strains of coagulase-negative staphylococci. Electrophoresis, 1755-57.

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16 Vibrio cholerae Charles A. Kaysner and June H. Wetherington U.S. Food and Drug Aduzinistratiorz, Bothell, Washington

I. Introduction 384 11. ClassificationandCharacterization

385

Classification A. 385 B. Characterization 386 Serological C. classification 386 111. DistributionandEcology387 Distribution A. 387 Ecology 388 B. C. The viable but nonculturable JV.

state

389

Relationship to WaterandFood389

Water 389 A. B. Food 390 V. Pathogenicity39 1 A. Characteristics of disease 391 Mechanisms B. of pathogenicity 393 C. Adhesion and colonization 394 D. Genetic regulation 394 E. Virulence of non-01/0139 V. cholerae VI. BiologicalandPhysicalControls

of Growth395

Temperature A. 395 B. Moisture and pH 396 C. Antimicrobials, disinfectants, and preservatives 397 VII. Principles of Detection in Water and Food 397 General A. considerations 397 Isolation B. procedures 398 Identification C. 398 D. Genetic-based procedures VIII.

Control of V. cholerae References 401

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1.

INTRODUCTION

Vibrio cholerae is a species that contains both harmless aquatic strains as well as strains responsible for the gastrointestinal disease cholera in the form of epidemics and global pandemics. A disease called cholera was mentioned in ancient writings nearly 2500 years ago (57). Cholera was the term given to any type of gastrointestinal disorder, particularly those with resultant diarrhea. The disease is also called Asiatic cholera, since the organism was subsequently determined to beendemic in that region and responsible for vast epidemics in both the Ganges basin of India and in Bangladesh. In recent times the population of the Bengal region has notbeen free of the disease. This intestinal infection is transmitted primarily by contaminated, untreated drinking water as well as by contaminated food. Cholera is rapidly spread among individuals after drinking water and foods have become contaminated due to inadequate sewage disposal and poor sanitation. The lack of potable water sources has been associated with cholera epidemics occurring primarily in regions of underprivileged, low socioeconomic populations, Crowded living conditions aid the rapid spread of the disease. In addition, pandemic waves of cholera have occurred along trade routes and in association with pilgrimage and human migration. This life-threatening but easily preventable and treatable disease still causes an estimated 150,000 cases each year on several continents (70,90,91), and in 1997, a nearly 20% mortality rate occurred in several countries in Africa. The Italian F. Pacini first described the causative agent of cholera in 1854 (67). He discovered that the intestinal contents of cholera victims contained large numbers of a curved bacterium that he called Vibrio cholera. His discovery, however, was overshadowed by that of Robert Koch, who studied the disease in Egypt during the 1880s. Koch demonstrated that cholera was caused by a comma-shaped organism, which he called Konlrmbnzillen. For several decades the name Vibrio comnzn was used. Pacini's work was finally recognized and Vibrio clzolercre, Pacini 1854 was designated as the type species of the genus. We now know that V. cholerae is a Gram-negative, facultative anaerobe that is a common organism throughout the temperate climates of the world. Koch first proposed that a toxin produced by V. comnm was responsible for the disease. But it was not until 1959 that a toxin was demonstrated by two groups working independently (17,23). Ten years later, the toxin was purified (29), which allowed for further investigations of its structure and mode of action. It was further demonstrated that two important characteristics were necessary for a strain of V. cholerae to cause cholera. One was the ability to produce cholera toxin. The second was the somatic antigenic type, which was designated 01. Biotypes of the 0 1 serogroup, Classical and El Tor, were found to be responsible for the epidemics and pandemic waves of the disease. The El Tor biotype currently is the more prevalent biotype and is endemic in several areas of the world. Classical strains have been isolated infrequently in Bangladesh since the advent of the seventh pandemic, seemingly being replaced by the El Tor biotype. Seven pandemics have been recorded since the early nineteenth century (Table 1). The first six originated on the Indian subcontinent and ended about 1925. They are believed to have been caused by the Classical biotype. The seventh pandemic originated in Indonesia in 1961 and has endured for nearly 40 years. This pandemic, which was the first to be attributed to the El Tor biotype, spread throughout Southeast Asia, into Asia and the Middle East, and reached the African continent in 1970. Subsequently, in early 1991 cholera appeared on the South American continent, which had been free of the disease for over a century. The initial cases were reported in Peru, from which theepidemic rapidly

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Table 1 CholeraPandemics Dates

Pandemic I I1 I11 IV V VI VI1 VI11

1817-1823 1829-1851 1852- 1859 1863- 1879 1881-1896 1899-1923/5 1961 -presenth El 1992-presenth

Indian subcontinent3 Indian subcontinenta Indian subcontinenta Indian subcontinenta Indian subcontinenta Classical Indian subcontinent3 Classical Indonesia Indian subcontinent

Tor 0139 Bengal

From Ref. 67. From Refs. 40, 63.

spread to bordering countries and into Central America. Over one million cases of cholera in 20 South and Central American countries resulting in more than 10,000 deaths have occurred (84,91). Through 1998, cases have been reported on a continual basis in Peru, Central America (El Salvador, Guatemala, Nicaragua, Belize), China (Guangzhou Province), Sri Lanka, Africa (Uganda, Kenya, Mozambique, Somalia), and India (70,91). In the fall of 1992, another explosive epidemic originating in the Bengal region of India occurred that was determined to be caused by a previously unknown serogroup of V. cholerae. This new serogroup was designated as 0139 and the strain as Bengal (1,26). This epidemic was similar in all aspects to those caused by the 0 1 serogroup, afflicting thousands of individuals and causing many deaths. The Bengal epidemic spread through India and subsequently reached Bangladesh, Nepal, Burma, Pakistan, Thailand, China, Malaysia, and Saudi Arabia. Imported cases were also reported in the United Kingdom and the United States. The Bengal strain is reported to share many virulence features with that of theEl Tor biotype of 0 1 V. cholerae of the seventh pandemic. Due to the similarity of disease, 0139 Bengal is considered to be the second etiological agent of cholera. Since this epidemic rapidly spread to other countries within a 2-year period, it is considered by some to be the eighth cholera pandemic (40,63,82). Several recent reviews are recommended to the reader that cover in more depth the information summarized here and below ( l .40,63,87) and recent electronic-based information (3 1).

II. CLASSIFICATION AND CHARACTERIZATION A.

Classification

In 1965, Veron (3) first proposed the family Vibrionaceae to group organisms into genera that were primarily oxidase-positive and motile by means of a single polar flagellum. This grouping was to differentiate these organisms from the Enterobacteriaceae and the recognized human pathogens in that family. Veron’s grouping was not intended to imply any ancestral relationship. Oxidase activity is the important character in differentiation of the Vibrionaceae from the common enteric pathogens. The Vibrionaceae currently contains four genera: Vibrio, Aeromonns, Plesiomonns, and Photobcrcteriurn. Vibrio species are Gram-negative, nonsporeforming, straight or curved rods and are facultative anaerobes by both a respiratory and fermentative metabolism (4). All Vibrios utilize D-glucose as a

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sole source of carbon and energy, and most utilize ammonium salts as a sole nitrogen source. Several of these species cause disease in humans as well as in marine vertebrates and invertebrates, such as fish, eels, and mollusks. Of the 34 recognized Vibrio species, V. clzolerae is probably the most notable. Within the past two decades, a closely related species, V. nzi~~zicz~s, was identified (16). This species has phenotypic characteristics similar to V. cholerne. One notable difference is the fermentation of sucrose. V. rninzicus does not ferment sucrose, and this biochemical test can be used for differentiation. Tolerance to salt is similar between the two species. Of notable importance is that some strains of V. mirrliczrs have been reported to produce an enterotoxin identical to cholera toxin (78). Human illnesses have been reported from the consumption of raw or undercooked shellfish in which the causative agent has been identified as V. minzicus (74). Although these infections are considered to be rare, clinicians should be aware of an organism similar to the disease spectrum of V. cholerne. Growth of all vibrios is stimulated by sodium ions in concentrations of 5 to 700 M’. Tolerance of various levels of NaCl in laboratory medium is used as a basis for species identification. Vibrios can be divided into two groups based on this requirement: those not requiring the addition of NaCl to laboratory media (V. cholerne, V. nzinzicrts) and the remaining halophilic species that require media supplemented with NaC1.

B. Characterization V. cholerae tolerates moderately alkaline conditions, with growth in medium at pH 9. They do not tolerate acidic conditions and rapidly decline in numbers at pH 5 or less. Most V. cholerm strains are also sensitive to the vibriostatic agent 0/129 (2,4-diamino6,7-diisopropylpteridine), although there are recent reports of epidemic strains resistant to this vibriocidal agent, including 0139 Bengal strains (40,63). The sensitivity patterns, however, can generally be used to aid in the differentiation of species. The biochemical properties of the clinically important species of Vibrios are published in a number of reference manuals (4,24,45,56). These properties can help to differentiate V. cholerne from another common environmental and food-associated species in the family Vibrionaceae, Aeronzonas hvdrophiln. Table 2 presents a list of the minimal features considered necessary for the identification of V. choZerae.

C.SerologicalClassification In addition to the production of cholera toxin (CT), the somatic serogroup determination is an important property to identify epidemic V. cholerne. Currently, over 150 0 serogroups have been identified antigenically, the majority of environmental strains encountered belong in serogroups other than 0 1 and 0139. The 0 1 serogroup, as mentioned above, had until recently been exclusively linked with epidemic and pandemic cholera. Until the emergence of the epidemic 0139 serogroup, isolates identified as V. cholerne that would not agglutinate in 0 1 antiserum were reported as non-01 V. cholerae. The recent literature now refers to these as V. clzolerae non-01/0139 strains. The routine determination of serogroups, other than 0 1 and 0139, in the laboratory is impractical, however. The 0 1 serogroup can be subdivided into the Ogawa, the Inaba, and the very rare Hikojima serotypes. The serotype distinction has been used for epidemiological purposes. However, recent reports indicate that serotype conversion can occur, so this distinction

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Table 2 Minimal Number of Characteristics Needed to Identify V. cholerne Percent Reaction positive Gram-negative, asporogenous rod Production of oxidase Glucose, acid under a petrolatum seal Glucose, gas D-Mannitol, acid meso-Inositol. acid Hydrogen sulfide (black butt on TSIj L-Lysine decarboxylase L-Arginine dihydrolase L-Ornithine decarboxylase Growth in 1% tryptone broth1

100 100 100 0 99.8 0 0 100 0 98.9 99.1

No sodium chloride added.

may be of limited value, whereas being of the 0 1 serogroup is of importance. V. cholerne 0 1 can also be divided into two biotypes, Classical and El Tor, based on hemolytic ability. Classical strains are nonhemolytic, whereas El Tor strains produce a P-hemolysin detectable on sheep blood agar plates. The 0139 serogroup is also hemolytic, resembling El Tor strains. Several other laboratory tests can be used to differentiate the 0 1 biotypes besides hemolytic ability, including a Voges-Proskauer reaction, inhibition by polymyxin B, agglutination of chicken erythrocytes, and a phage typing system (4,24,45,56). Of these differentiation tests, determination of the P-hemolysin and tolerance or susceptibility to polymyxin B are the most practical.

111.

DISTRIBUTIONAND ECOLOGY

A.

Distribution

Environmental investigations have determined V. cholerne to be a common bacterium in the temperate environments of the world. The majority of the bacterial species encountered in this environment are species of Vibrio. V. cholerae is part of the normal, free-living microflora of aquatic environments. Coastal areas with brackish waters and estuarine regions are niches for many species, including strains of toxigenic 0 1 V. cholerae. Epidemic cholera strains are endemic in several regions including Australia and the U.S. Gulf Coast and are sporadically involved in illnesses in those regions. V. cholerne 0 1 strains are occasionally encountered in the environment of nonendemic areas, but they are normally nontoxigenic and considered to be nonpathogenic (40). The predominant strains of V. cholerne encountered in these environments are of the non-01/0139 serogroups and are generally nonpathogenic to humans, although human infections resulting from these strains are occasionally reported. During the warmer months of the year, environmental samples frequently contain high levels of these non-01 /0139 strains. In regions of endemic human infection, the epidemic strains also appear in a regular seasonal pattern. Environmental factors trigger the dormant pathogen to multiply, which results in outbreaks.

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B. Ecology Ecological studies of V. cholerne (41) and also V. nzi~nicus(13) identified the physical factors that enhanced their incidence and distribution in the environment. Water salinity is an important physical parameter in the ecology of these organisms. V. cholerae is more prevalent and found in higher numbers in areas where water has a general salinity range of 2-25 parts per thousand (ppt). The lower salinity range of 2-5 ppt in areas with significant fresh water inclusion favors V. cholerne and its cousin, V. mimicrrs. These two species are less prevalent or rarely recovered in water of salinity 30 ppt or greater found in the open ocean. Many estuarine areas fluctuate in salinity due to the amount of rainfall and to fresh water inclusion from streams or rivers and tidal action, which will influence the incidence of these species. V. cholerne is seldom, if ever, isolated from water of a temperature below 10°C but can be frequently isolated from water when the temperature ranges between 15 and 35°C. Thus, a seasonal occurrence is typical in estuarine areas, with frequent isolations during the warmer summer months. Toxigenic V. cholerae was recovered from sea salt solution preparations for up to 70 days stored at 25°C (58), indicating the influence of temperature on the incidence of the organism. During colder or winter months, recoveries of this species can occasionally be made from the top layer of sediment, where they may beinsulated from the lower temperatures and remain dormant until the next summer season. The effect of environmental temperature changes also correlates with the frequency of reports of infections. In regions where the water temperature is warmer year-round, the seasonal incidence is not as dramatic. The environmental presence of pathogenic vibrios does not distinctly correlate with the presence of human sewage, although nutrients may be contributed by sewage influx that may enhance survival of the organism. In regions where epidemics have occurred and there has been aninflux of untreated human waste into the aquatic environment, endemic 01, and now 0139, V. choleme strains are more frequently encountered. Extreme climatic events have resulted inan increase inthe number of cases of cholera in many regions, along with other human diseases (10). These events resulted in increased rainfall and hurricanes causing flooding and also droughts. Flooding affects the availability of drinking water by contamination of sources and droughts effect hygiene by limiting sewage disposal. These weather conditions are related to a phenomenon termed El Nifio, resulting in warmer-than-normal ocean water temperatures, which has caused many regions to experience unusual weather this past decade. As an example, Tanzania reported only 1.424 cholera cases and 35 deaths in 1996. After heavy rains and flooding during 1997, over 40,000 cases and 2,200 deaths were recorded. Dramatic increases in cases have also occurred in South America since 1991 with assistance from El Nifio. A relationship exists between phytoplankton and zooplankton in the water and V. cholerne. This species is chitinoclastic, and its ability to digest chitin may play a role in its persistence in the sediment. V. cholerae are able to colonize copepods and crustacea that have chitinous exoskeletons. It is suggested that attachment to planktonic forms enhances survival during adverse environmental conditions. Similarly, V. cholerae was found to colonize other aquatic biota, such as water hyacinths in Bangladesh, probably aiding its persistence in fresh water environments (40). Due to the ability of V. cholerne to attach to material suspended in thewater, animals that reside in estuaries can be expected to pick up these vibrios during feeding. No particular animal reservoir has beenidentified; however, bivalve mollusks and finfish may contain

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Table 3 Recorded Outbreaks of Cholera Year

Location

No. cases

Source

Ref.

Philippines Italy Portugal Portugal Louisiana Gilbert Islands Texas Singapore Thailand Thailand Maryland Colorado

330 278 2467 136 11 572 15 37 24 71 3 1

Raw shrimp Raw seafood Raw/Undercooked cockles Bottled water Boiled/Steamed shrimp Raw clams, sardines Cooked rice Seafood Uncooked pork Uncooked beef Frozen coconut milk Cooked blue crab

39 2 6 7 9 55 38 33 81 80 85 30

~~

1961 1973 1974 1974 1977 1978 1981 1982 1987 1988 1991 1998

the organism on the surface or in the intestinal contents. Thus, seafood harvested from coastal and in-shore areas in endemic regions may frequently contain toxigenic V. C h d erue, which may ultimately be consumed by humans. Seafood commodities implicated in cholera infections include several species of finfish, squid, crustacea (shrimp, crab, lobster), and mollusca (oysters, clams, cockles, mussels) (Table 3).

C. The Viable but Nonculturable State V. cholerue and several other Gram-negative bacteria have been reported to survive in a viable but nonculturable state (VBNC) (14). This dormant state is induced by extended exposure to saline water and nutrient deprivation. The bacterial cells retain metabolic function but are not culturable on routinely employed nonselective bacteriological media. During this phenomenon the cells exhibit the starvation response, become ovoid in shape and reduced in size, and are detectable by epifluorescent microscopy. Cells can be induced into the VBNC state in the laboratory by inoculating sea-salt formulations adjusted between 0.5 and 2.5% (w/v) with NaCl and then storing at 5°C. When environmental conditions such as temperature and presence of nutrients become favorable, cells transform into a normal size and become culturable. VBNC cells of V. cholerae were reported to induce fluid accumulation upon injection into ligated rabbit ileal loops, thus showing a retention of virulence characteristics. The VBNC state is a plausible explanation for the persistence of this pathogen in the environment during adverse conditions, such as the colder winter water temperatures in temperate regions, while remaining undetected by laboratory examination. This dormant state may explain, at least in part, why endemic cholera strains cannot be recovered from the environment for periods of time, only to reemerge and once again infect humans.

W. RELATIONSHIP TO WATERAND FOOD A.

Water

Water plays a critical role in the transmission of cholera. During the second pandemic. Snow determined the role of water in the spread of the disease in England in the mid-

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1800s (67). A central supply of public drinking water in London, the “Broadstreet Pump,” became infamous. The drinking water had been contaminated with the epidemic strain by sewage discharge into the Thames River. Since Snow’s discovery, numerous investigations into cholera outbreaks have consistently identified water as the source of the organism causing rapid, epidemic spread of disease (44,59). In countries with recorded epidemics, the lack of potable water sources was the fundamental reason for spread of the disease. Household water supplies, i.e., cisterns used for collection and storage, often become contaminated by family members when washing their hands. Bottled, contaminated spring water was responsible for a cholera outbreak of over 100 cases in Portugal (7). The contaminated, untreated public water system in Trujillo, Peru, the drinking of unboiled water, and drinking from a household water storage container in whichhands had been introduced were all highly associated with the spread of cholera in that country (83). In addition, ice made from Contaminated water was also incriminated in transmission during this 1991 epidemic (73). Interestingly, cargo ships may have been responsible for transmitting the Latin American epidemic cholera strain to another country’s coastal waters (18) via contaminated bilge water (53). In underdeveloped countries, residents are urged to boil water prior to drinking and before use for food preparation. Water is most often collected from untreated sources, mainly due to the lack of public water systems. Because of the lack of public sewage collection and treatment systems, human waste is disposed of in a manner that contributes to the contamination of drinking water sources. Beverages that were prepared from contaminated water and sold by street vendors were associated with epidemic cholera cases in Ecuador (89). During outbreaks and epidemics, health officials struggle to assist in providing potable drinking water; this usually requires educating the public to boil any water collected and sometimes involves providing tablets that chemically treat drinking supplies. In more developed countries, cholera outbreaks have been virtually eliminated by the establishment of treated public water distribution systems.

B. Food Contaminated food also plays a major role in thespread of cholera (21,44,59,69).A variety of foods have been implicated in outbreaks of cholera, and a few of these reports are summarized in Table 3. Often it is difficult to determine whether the food became contaminated by direct environmental contact with the organism, by an infected food handler, or by the water used in food preparation. Water contaminated by sewage used to prepare food for workers on a U.S. off-shore oil platform (38) and likewise during the preparation of rice in Peru (73) is incriminated i n the spread of the disease. Similarly. vegetables irrigated with untreated sewage were associated with the transmission of cholera during the South American epidemic (83). In many countries, this led to monitoring of vegetables imported from South America to prevent the possible spread of cholera outside of Latin America. During 1992- 1994, numerous samples including fruits, vegetables, and frozen seafood were examined for the presence of V. cholerae by several countries. Foods investigated during epidemics include cooked rice and legumes, millet gruel, vegetables, and fruit-usually foods that are prepared using water or are normally washed prior to consumption. Generally, nonacidic foods are most frequently vectors of the disease because V. clzolerne does not tolerate acidic conditions. Other food products that have been linked to outbreaks in several countries are potatoes, gelatin, chopped eggs, frozen coconut milk, and contaminated meat preparations (i.e. pork, beef, and seafood). Implicated seafood is

Vibrio cholerae

391

usually associated with harvesting from estuarine areas that have been contaminated by sewage during epidemics or from regions where the epidemic strains of V. clzolerae have become endemic. Products implicated include both raw and processed seafood such as raw marinated, salted, or dried fish, shrimp, crab, and the filter-feeding mollusks oysters, clams, mussels, and cockles. Ceviche is raw marinated fish, which was suspected of cholera transmission in South America, but this was not substantiated. Fish used in its preparation were harvested from coastal waters found to be contaminated with the epidemic strain. In Ecuador, eating raw seafood and cooked crab was highly associated with the spread of the epidemic (89). Food items obtained from street vendors were highly incriminated in many Latin American countries (84) and also in other countries during earlier epidetnics (51). In the United States, the major domestic food vehicle for cholera in the past 25 years has been crabmeat (88). Cooked blue crabs harvested and processed in Louisiana were recently responsible for an illness in Colorado (30). Inspection of the processor found that cooked crab was allowed to contact surfaces that were also used for raw crab. In addition to other poor sanitation conditions, whole crab were not completely submerged when boiled. Investigations into the U.S. outbreaks have most often found that the implicated food had been processed (cooked in the case of seafood) but had become contaminated after processing. In a 2-year study of illnesses associated with raw oyster consumption in the state of Florida, the reported disease agents in decreasing order of frequency were V. ynml?ael71olE,ticrrs,non-01 V. cholerne, V. ~~ulrziJcus, V. hollisne, V. mirniczfs, V. jhvialis, and 0 1 V. clzolerne (46). Similar findings were also reported for infections reported in one year in four Gulf Coast states (49). In general, consumption of molluscan shellfish accounts for the majority of gastroenteritis cases from seafood in the United States (74). Because of the increase in travel and the transport of food products between countries, the potential for transmission of V. choleme has increased. An example is the outbreak of cholera from coconut milk imported into the United States (85). Food served on airlines has likewise been implicated in cholera infections (1 1,SS). Travelers returning from countries with current epidemics or areas of endemic cholera have become infected from food consumed prior to departure or served on the airplane. In a few instances, travelers transported Contaminated products in their baggage to another country. U.S. illnesses reported to have been caused by the non-01/0139 V. cholerae and also V. rzlirnicus have predominantly arisen from the consumption ofraw oysters (60,7 1,74). These reports have for the most part been gastroenteritis cases, however, septicemia and peritonitis cases have also resulted. They differ in disease manifestation from toxigenic V. choler-ne in that the gastrointestinal illnesses are not as severe and 0 1 V. clzolerne have not been demonstrated in extraintestinal infections.

V.

PATHOGENICITY

A.

Characteristics of Disease

Cholera, recently reviewed in detail (40), starts with an incubation period from several hours to 5 days after ingestion of water or food contaminated with toxigenic V. cholerae. Time of onset is dependent in part on the inoculum level ingested. The infective dose of V. choleme is believed to be about one million cells. In volunteers, approximately 10” CFU in buffered saline were necessary to induce diarrhea in volunteers (50), while only

392 Wetherington

and

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lo6 CFU ingested in a sodium bicarbonate solution to neutralize stomach acid induced diarrhea. The ingestion of lo6CFU in food such as rice and fish resulted in a 100% attack rate in a separate volunteer feeding study. This suggests that the ingested food can buffer the gastric acid, enabling the organism to reach and colonize the intestinal tract. Once colonized in the small intestine, V. choleme multiplies rapidly and produces cholera toxin. One known predisposition to severe infections is hypochlorhydria, a low level of stomach acid. Typical cholera is characterized by the sudden onset of vomiting and painless diarrhea, with the characteristic “rice-water” stools caused by the presence of mucous, developing as the disease progresses. Suppressed renal function, thirst, leg and abdominal cramping, and collapse due to marked dehydration and the resultant electrolyte imbalance follows in the severe diarrhetic episodes. Vomiting is common and occurs a few hours after the onset of diarrhea. Profuse secretory diarrhea is the main symptom, with resulting life-threatening dehydration. The fluid loss is so dramatic that an infected person can die within hours. These severe cases, “cholera gravis,” are reported to afflict about 15% of those infected by the classical biotype and but only about 2% of those infected by the El Tor biotype during an epidemic. Mild cases and asymptomatic infections occur most often. Moderate cases requiring medical attention occur in about 15% of those infected with the classical strains and only in about 5% of those with El Tor infections. An infected person sheds millions of these organisms in their feces, thus the disease can spread rapidly where poor sanitation and poor hygienic practices occur. The spread of the disease by personto-person contact has not been substantiated. Immediate treatment by rapid infusion of intravenous fluids is usually successful for the advanced cases, for replacement of the vital fluidslost. Oral rehydration is normally used for the milder cases and has been described as one of the most important therapeutic interventions developed in the twentieth century. Antimicrobial therapy has also been effective in shortening the duration of infection and reducing the carrier state; tetracycline is the drug of choice. However, widespread use of antimicrobials is strongly discouraged due to development of resistant epidemic strains, which are appearing more frequently. Cholera initiates an immune response in humans, and evidence exists for substantial infection-derived immunity. Several vaccines have been developed, some of which show promise for prevention of future epidemics. An easily administered vaccine, delivered orally, is desired. Currently live, attenuated bacteria, killed whole-cell preparations, wholecell toxoids, and a Salmonella-based carrier of V. cholercre antigens are preparations being evaluated in clinical trials. Non-01 /0139V. cholet-ae strains also cause human diarrheal illness and other clinical manifestations (46,60,61). Gastroenteritis is the most common of the illnesses reported, however, these strains have also caused wound, ear, septic, and peritoneal infections. Raw oyster consumption is highly correlated with the gastrointestinal infections. The vast majority of non-01/0139 strains do not produce cholera toxin and are not associated with epidemics. Two foodborne outbreaks of gastroenteritis have been reported as caused by non-01/0139 V. cholerme (61), and there are numerous individual reports of illness implicating consumption of rawshellfish (46,74). There is a seasonal occurrence of these gastroenteritis cases, accompanying the increased presence of these strains in the environment during summer and fall months. Generally, non-01/0139 gastroenteritis is reported to be mild or moderate in severity, however, occasionally a severe cholera-like disease has been reported. Symptoms reported include diarrhea, abdominal cramps, fever, and less frequently nausea and vomiting. Severe dehydration has been reported in a few patients.

.

Vibrio cholerae

393

Duration of illness can be from one to 10 days, and hospitalization and rehydration have been required for severe cases. There is no distinguishing symptom that would differentiate these infections from other gastrointestinal infections. Non-0 1/O 139 strains have also been isolated from cases of septicemia and peritonitis (10,65,7 1,75). The source of these infections was not identified in many of these cases, although some patients reported a recent history of seafood consumption or an association with seawater. A 60% mortality rate is reported for these infections. Epidemiological evidence from some cases suggests that septicemia could be acquired by invasion through the intestinal tract, similar to infections of V. vulnificus, a highly invasive species commonly encountered in seafood. The 0 1 epidemic strains have not been demonstrated to be invasive, however. Several of these patients had underlying medical conditions, such as liver disorders, that probably made them more susceptible to an invasive infection by these rare strains of V. cholerue. Isolates from septicemic patients are reported to be heavily encapsulated, similar to patient strains of V. vuZniJcz~~.

B. Mechanisms of Pathogenicity 1. Cholera Toxin The major determinant of virulence is the ability of V. cholerae to produce cholera toxin (CT) or choleragen. Strains of 0 1 and 0139 producing CT are considered fully virulent and capable of causing epidemics. Cholera toxin is a heat-labile protein toxin composed of a single A and five B subunits with an approximate molecular weight of 85,000 daltons (40). The initial action of CT is binding of the B subunit to the receptor, ganglioside G,,, on the cell membrane of epithelial cells. This binding is enhanced by the enzyme neuraminidase produced by the organism. The A subunit, after proteolytic cleavage to two further subunits, stimulates adenylate cyclase, resulting in increased cellular levels of cyclic AMP. Increased CAMPconcentration leads to increased chloride ion secretion by intestinal crypt cells and decreased NaCl absorption by villus cells, resulting in electrolyte movement into the lumen. The resultant osmotic gradient causes a massive water flow to the lumen, which overwhelms the absorptive capacity of the intestine, resulting in diarrhea. The gene for CT, ct.x-, was sequenced some time ago, which has allowed for molecular based studies of its action. The heat-labile enterotoxin of Escherichia coli was subsequently found to have similar homology to CT and to be similar in mode of action. Additional mechanisms may also be involved in the secretory effects of CT. Prostaglandins and the enteric nervous system both respond to CT and may add to the secretion that occurs with this disease. 2. Other Toxins In addition to CT, V. cholerae 0 1 and 0139 strains produce other toxins (40). Zonal occludens toxin (ZOT) increases permeability of the small intestinal mucosa by decreasing tissue resistance of the intercellular tight junction (zonal occludens). It is hypothesized that ZOT causes diarrhea as a result of leakage of water and electrolytes into the lumen by hydrostatic pressure due to the increase in permeability. Sequences of the zot gene have subsequently been found in non-01 /0139 strains. Accessory cholera toxin (ACE) causes fluid accumulation in ligated rabbit ileal loops and is suspected of forming an ion channel after its insertion into the epithelial cell membrane. The genes encoding these two toxins, Zot and m e , are located immediately upstream of the ctx genes encoding CT in what has been termed a “virulence cassette.’’

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The hemolysin that differentiates the El Tor from classical strains is cytolytic to c? variety of erythrocytes and mammalian cells in culture, is lethal to mice, and has been demonstrated to cause fluid accumulation in the ligated rabbit ileal loop assay. This hemolysin/cytolysin is also produced by non-0 1/O 139 strains and may play anaccessory role in diaqhea produced by those strains. Other reported toxins produced by 0 1 strains include a “new cholera toxin,’‘ a sodium channel inhibitor, and a Shiga-like toxin. Volunteer feeding studies have shown that ctx gene-deleted 0 1 strains can still elicit mild to moderate diarrhea in some individuals, thus these other toxins may be responsible for the diarrheal cases from which CT-negative strains were recovered. Additionally, some strains of non-01/0139 V. cholerne produce a heat-stable toxin, referred to as Nag-ST, similar to that produced by the enterotoxigenic E. coli. Although all of these toxins may contribute in part to diarrhea caused by strains that do not produce CT, their role in human pathogenesis needs further study.

C. AdhesionandColonization V. cholerne must first colonize the small intestine to begin the disease process. The toxin co-regulated pilus (TCP) is the most characterized colonization factor of this organism. These pili consist of long filaments that are laterally associated in bundles. Expression of the pilus is correlated with the expression of CT, hence the name. Another potential colonization factor, accessory colonization factor, has been described that may be involved in colonization via motility and/or chemotaxis, but its exact nature is still under study. The polar flagellum of V. cholerae, besides being involved in motility, may serve as an adhesin and an important virulence factor. In response to chemotaxins, motile V. choZerne are drawn to the mucosal surface of the intestine. Nonmotile but fully toxigenic strains show a markedly diminished virulence in animal models, indicating that motility is an important factor in establishing disease. V. cholerne has been reported to produce at least four hemagglutinins that may be involved in cellular adherence in the gut. Additional adherence factors that have been described and are potentially involved include several outer metnbrane proteins (OMP), the lipopolysaccharide (LPS) of the 0 1 strains, a polysaccharide capsule of non-01/01 39 strains, and the presence of other pili. Some strains of 0139 V. choZer-aeproduce a polysaccharide capsule, which is not found in strains of the 01 serogroup. V. cholerne also produces a siderophore, vibriobactin, seemingly not necessary for secretory disease, but which may be involved in establishing bacteremia by some 01/0139 strains. D. GeneticRegulation Several systems for the regulation of virulence genes in V. cholerne have been identified. The most extensively characterized regulon is ToxR, which controls expression of CT, the TCP colonization factor, the accessory colonization factor, two OMPs, and three lipoproteins. The control of seventeen distinct genes by toxR has been reported, and this gene is called the “master switch,” regulating the “virulence cassette.” This was demonstrated during a study of volunteers fed a V. cholerne strain with mutated toxR that did not illicit diarrheal symptoms, although it contained other virulence genes. Recently, a genetic pathogenicity island, PAI, was identified in epidemic strains (42). This chromosomal PA1 is present in epidemic and pandemic strains but not in nonpathogenic strains of the organism, including some of the 0 1 serogroup. It was proposed

Vibrio cholerae

395

to call this the vibrio pathogenicity island (VPI) to differentiate it from those of other bacterial species. The VPI may represent the initial genetic factor required for the emergence of epidemic and pandemic strains. VPI is 39.5 kilobases in size and contains the genes important in establishing disease, including those having a direct role in disease or an indirect role in the transfer and mobility of the VPI. It has been hypothesized that transducing phages may transfer a PA1 and that horizontal transfer may occur resulting in the emergence of new pathogenic strains. The VPI was demonstrated in two clinical non-0 l /O 139 strains associated with outbreaks, suggesting acquisition of the virulence package.

E. Virulence of Non-01/0139 V. cholerae Diverse variability of virulence exists among the non-Ol/O139 strains encountered from clinical specimens and foods. When fed to volunteers, tnost strains failed to produce illness. Inoculum levels of clinical non-01/0139 strains greater than lob were necessary to induce diarrhea in volunteers (60), seemingly a greater number than required for the toxigenic 0 1 strains. No toxins unique to these serogroups have been described, although some produced by 0 1 strains and other species have been reported, for example, the hemolysin/cytolysin of the El Tor biotype and the thermostable direct hemolysin, TDH, of K pnrahaemulyticus. A heat-stable enterotoxin, Nag-ST, is produced by some strains and a cholera-like toxin by others, which results in a less severe diarrheal disease than true cholera. In addition, the genes encoding CT, ZOT, and ACE have been detected in some strains. Other possible virulence factors reported are a Shiga-like toxin, various cellassociated hemagglutinins, and a polysaccharide capsule that could facilitate production of septicemia in susceptible hosts. The production of multiple virulence factors by non01/0139 V. choler-ne appears to be necessary to elicit a diarrheal illness, although host susceptibility may play an important role. However, case reports indicate that these bacteria have also caused gastroenteritis in healthy individuals as well as people with preexisting diseases. Obviously, the virulence mechanisms of the occasional non-01/0139 strains of V. cholerae associated with human infections remain unidentified and need further study.

VI.

BIOLOGICALANDPHYSICALCONTROLS OF GROWTH

A. Temperature V. ckolercre is a mesophilic organism that thrives in the temperature range of 15-40°C, with optimum growth at 37°C. The ability of this species to grow at 43°C has allowed for a temperature-selective approach to isolation (20) and is also used for identification (24.45). V. cholerae grow rapidly at temperatures in the range of 30-43°C. They do not grow at temperatures less than 10°C, consequently, storage of food at recommended refrigeration temperatures (<4.4"C) is essential to prevent multiplication. V. choler-ae 0 1 strains rapidly increased by 3-6 logloin 24-32 hours in cooked foods, such as rice, lentils, eggs, and raw vegetables, even at 22°C (48). Thus, an ambient storage temperature can be expected to allow for rapid increase in levels of the organism. Generally, a slow decline in numbers is reported during extended low-temperature storage. However, it appears that a refrigerated food may retain viable cells at a level that may cause illness up to the end

and

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Wetherington

of the shelf life of the product, at which time the food would likely become unacceptable for consumption due to the natural bacterial and autolytic decomposition. Freezing generally causes a reduction in levels of vibrios during storage by3-4 loglounits or greater. After inoculation into shrimp, crab, and oyster homogenates that were stored at 7 and -20°C for 31 days, the numbers of viable V. cholerue 0 1 were reduced between 10"- and lOS-foldfrom initial levels of 10h-107/g(72). V. clzolerne 0 1 was also recovered after 36 days at -20°C in peeled shrimp, a 6-10g decline after surface inoculation (64). Survival of the organism probably depends more on the freeze-thaw cycle and the protection offered by the food than on the actual storage temperature. V. choleme are susceptible to heat much like other non-spore-forming bacteria, and mild heating of a food reduces their numbers quite rapidly. While growth can occur up to about 45"C, above that temperature the numbers start to decline. Boiling rapidly destroys vibrios, and an internal temperature of 60°C for several minutes appears to be sufficient to eliminate the organism (8). Boiling water was reported to reduce V. cholerue 0 1 inoculated onto the surface of shrimp by 6-log within 2 minutes (64). Likewise, a lo4to 1OS-foldreduction was reported for the boiling of oysters when the internal temperature reached 60°C. A pasteurization temperature of 572°C for 30 minutes for these oysters was recommended to reduce V. cholerne levels 4-5 logloCS). In rice and fish, a 10-minute treatment at 60°C also eliminated 104-105 V. choleme (35), and a 30-second immersion of fruits and vegetables in boiling water killed the organism (37). D values in minutes reported for V. cholerne and the suspending medium are summarized in Table 4 (34,37,77).

B. Moisture and pH Foods with a high moisture content allow the survival of V. choleme. As with most other Gram-negative bacteria, the a , value must be greater than 0.93. The surface humidity of a stored dried or semi-dried product is an important factor, as is the water content of food. Sodium is required for growth of both V. cholerne and V. mimicus and the range of salt tolerance reported is up to 5% NaCl w/v in laboratory medium (24,45). The use of salt Table 4 D Values (min) Reported for V. cholerne Suspending medium Minutes D49

DS4

D60 D66

D7 I

1.70 8.15 1.04

5.03 0.63 0.39 1.60 1.22 0.32 0.30 0.30

Temperature is "C. From Ref. 77. h From Ref. 37. c From Ref. 34.

1% w/v peptone? Crabmeat homogenate" 1% wlv peptone Crabmeat homogenate 1% w/v peptone Shrimp homogenateh Crabmeat homogenate Crayfish homogenate' Shrimp homogenate Crabmeat homogenate Crayfish homogenate

Vibrio

cholerae

397

for food preservation must take into account the organism's tolerance to salt to obtain the required water activity value to inhibit growth. Foods of neutral or near neutral pH allow for survival of the organism, whereas levels decline rapidly in those with a pH of 5 or lower. The optimum pH range for growth is 7.0-8.5, however, growth occurs in the range of 6.0-10. Foods high in protein contain nutrients necessary for growth of this species and thus must be considered highly perishable and handled as such. V. choZercre survived only one day at pH 5 but up to 3 weeks at pH 8 and 10 at room temperature in cooked eggplant inoculated at 2 X 104/g(27). No growth was observed in food with a pH lower than 5.5 (59). Thus, acidic sauces used for food preparation can restrict growth if in contact with the organism. Growth of V. choleme was inhibited in a tomato-based sauce (pH 5.0), but the organism multiplied rapidly in peanut sauce (pH. 6.0) (79). If acidic sauces are used, contact of the sauce with the organism is an important factor necessary for preservation.

C. Antimicrobials,Disinfectants,andPreservatives Most V. cholerne strains appear susceptible to several antimicrobials, including tetracycline, chloramphenicol, the aminoglycosides, the quinolones, and the newer cephalosporins. Several of these are currently recommended by the World Health Organization for the treatment of cholera (43). Tetracycline has been the antimicrobial of choice for the treatment (40) since it is relatively inexpensive and is readily available. In a study of nine species of vibrio (including V. cholerm) from over 200 clinical and environmental sources tested for antimicrobial susceptibilities, resistance to sulfamethoxazole, trimethoprim, and the penicillins was reported (32,43). Resistance to a broad spectrum of antimicrobials is being reported with increasing frequency in strains of V. clzolerae 0 1 and 0139 worldwide. Thus periodic monitoring of the antibiotic susceptibility of strains is prudent. Due to this increased resistance, widespread use for disease treatment is discouraged. The susceptibility of V. choleme to common germicides was recently reported (76). The El Tor strain responsible for the epidemic in Perudid not survive any ofthe treatments with chemical germicides (30-min exposure at 20°C). The germicides included gluteraldehyde, formaldehyde, hydrogen peroxide, sodium hypochlorite, phenol, cupric ascorbate, and peracetic acid prepared at concentrations recommended for decontamination of medical devices. These results were also obtained even in the presence of a protecting protein, 0.1% serum albumin. The effect of disinfectants used in wash water for sanitation purposes will depend on the concentration of protein and other compounds that will complex with the antibacterials to inactivate them. Chlorine (100 ppm), a quaternary ammonium compound (50 ppm), and iodine (25 ppm) produced 6 log,,, reductions of V. cholerae 0 1 strains suspended in phosphate buffered saline after exposure for 2 minutes (54). However, the addition of 50 ppm chlorine to the wash water used in oyster processing failed to reduce the numbers of V. choler-ne in the blower/washer within 5- l 0 minutes (62), likely indicating the effect of suspended protein and carbohydrates from the oysters.

VII. PRINCIPLES OF DETECTION IN WATERAND FOOD A. GeneralConsiderations Under optimum conditions, V. cholerae has a rapid generation time that can be used advantageously in the laboratory. In addition, its ability to grow rapidly at an alkaline pH and

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at a high temperature, to resist the inhibitory effects of bile salts and sodium tellurite, and to tolerate salt can be exploited. The optimal NaCl concentration for V. cholercle was reported as 2% (w/v) (58). Nonselective enrichment broth formulations were employed prior to the use of a selective agar to detect low levels of V. cholerna in water and food. Enrichments are used to resuscitate stressed cells that can result from exposure to low temperature and nutrient deprivation in water and certain food-processing steps (i.e., drying, heating. freezing). Nonselective enrichment broths formulated with a final pH between 7 and 9 have been used successfully because V. clzolerne tolerate alkaline conditions. A medium widely used for detection and enumeration in food or water is alkaline peptone water (APW) consisting of 1% (w/v) peptone and 1% NaCl with a pH adjustment to 8.48.6 (24,45). APW has been the choice of many laboratories including the U.S. Food and Drug Administration, due to its relative low cost and ease of preparation (24). Enrichments are incubated at 35-37°C for 16-18 hours. For certain types of samples it is recommended that the surface pellicle of the broth be streaked to a selective agar after 6-8 hours, due to the rapid generation time of the organism. V. clzolerne multiply more rapidly than other microflora that may be present and that might interfere with its isolation. For food that may have been frozen, dried, or subjected to heat, enrichment broths should be reincubated after the 6- to 8-hour streak for an overnight period to resuscitate stressed cells (24). An elevated temperature method (20) takes advantage of the inhibition of growth at this temperature of many of the naturally occurring bacteria present and is recommended for the analysis of shellfish samples. A duplicate enrichment of sample in APW incubated at 42°C in addition to one portion of sample enriched at 35°C is recommended (19,24). B. IsolationProcedures After the incubation period, the surface pellicle of the enrichment broth is streaked to a selective and/or differential agar medium. The most commonly used is thiosulfate-citratebile salts-sucrose agar (TCBS) (22,24,40,45). This selective and differential medium was designed originally for detection of V. choler-cx but has also been effective for V. nzimicrrs. TCBS will differentiate the sucrose-fermenting species such as V. choler-ne and V. nlginolyticus from the non-sucrose-fermenting species V. nzimicus, V. pnrnlzcrenlolrvticlls, and V. vulrziJicus. TCBS is the recommended selective agar to be used in conjunction with another selective/differential agar, such as Cellobiose Polymyxin Coliston agar (52),and its modification (24), for differentiation of vibrios that may normally be present in marine water and seafood.

C. Identification The determination of oxidase activity is of pritnary importance to distinguish presumptive V. ckolercre and V. rnimicus isolates. The laboratory procedures for biochemical identification are quite straightforward, and procedures for identification are readily available. These two species have been well characterized, and tables of distinguishing characteristics are published in several manuals (4,24,45,56). The traditional biochemical procedures can be time consuming for complete isolation, identification, and virulence assay. Several commercial diagnostic kits are available, such as the APT 20E (bioMerieux Vitek, Inc.) for enteric organisms (66), and have been used successfully for more rapid identification. If V. choleme are isolated, serological testing for either serotypes 0 1 or 0139 is of primary importance for indicating the presence of a potential pathogen. Antisera for 0 1

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and 0139 are available commercially. The serological determination for the other somatic groups is impractical, and they are reported as non-01 /0139V. cholerae. Isolates of 0 1 or 0139 serotypes should be tested for the production of cholera toxin, since nontoxigenic 0 1 strains are occasionally encountered. The mere presence of a toxigenic 0 1/0139 V. choler-ae in food or water constitutes a public health hazard. Cholera toxin production can be detected by several means including the effect of culture filtrates on Y-l mouse adrenal tumor cells or Chinese hamster ovary cells (CHO), the production of intestinal fluid accumulation in infant mice, or by commercially available immunological procedures, such as the Oxoid “VET-RPLA” reverse passive latex agglutination diagnostic kit (24). Several monoclonal antibody-based coagglutination procedures for the detection of V. cholerae 0 1 are available (40). One has recently been developed for the rapid diagnosis of cholera by serogroup 0139 (12). D. Genetic-BasedProcedures The advent of genetic-based identification methods for pathogenic organisms offers the advantages of speed and specificity. Genetic probes are used for the detection of specific genes by colony hybridization (36). Gene probes for sequences of the cholera toxin gene, ctx, of V. clzolerae have been developed and in one report could detect lo4 CFU/g V. choler-aein samples (40). Probes can be labeled with a radioisotope or a nonisotopic label, leading to a broader application of a hybridization procedure. The sensitivity of some of these hybridization procedures needs to be enhanced to detect lower levels of toxigenic V. cholercw which may be encountered. The polymerase chain reaction, PCR, also offers rapid detection of toxigenic strains of V. cholercw in food and water samples. By amplification of DNA fragments of ctx, the pathogen can be detected in approximately 24 hours (47). The application of this genetic based technique can detect low levels of toxigenic V. cholerne from a suspect sample after a 6-hour enrichment step. PCR amplification of the CT gene was also valuable in detecting the Latin American epidemic strain in contaminated samples during epidemiological investigations (28). Molecular subtyping techniques have shown their value in identifying outbreak strains of V. cholerae for epidemiological purposes. This is very evident in the results of subtyping epidemic strains of V. cholercle 0 1 from Latin America (86) and the 0139 Bengal strain in India (26) and in tracing the spread of the organism. The application of pulsed field gel electrophoresis (PFGE) has also proven to be a very discriminating way to measure the RFLP of V. cholerme. Multilocus enzyme electrophoresis (MEE) is auseful epidemic marker system and also a way to measure the relatedness of V. cholerne strains (86). These techniques identified two distinct strains of toxigenic V. cholerne 0 1 currently epidemic in Latin America (25). Standardization of a ribotyping scheme has been proposed for V. clzolercle so that results obtained from various laboratories can be compared during an epidemiological investigation (68).

VIII. CONTROL OF V. CHOLERA€ The control of cholera in underdeveloped countries is a formidable and costly task, but two measures can help to control the spread of disease or at least reduce the number of cases. The most important measure is the disinfection treatment of water used for drinking

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and food preparation. Once treated, its protection by utilization of a piping system or by ensuring limited access to its source will reduce the chance of contamination. However, this would be extremely costly in the poorer countries. Education of people in areas that are at risk to at least boil water prior to drinking or use for food preparation is more feasible. In addition, point source chlorination of home water sources can be effective. In endemic areas, even modest improvements in food and water sanitation have resulted in fewer cases of infection (59). Costs of community systems for potable water dispersal and for sewage disposal are most likely prohibitive in economically stressed countries. Disposal of human sewage such that it cannot contaminate drinking water sources is the second important measure. In the Western Hemisphere, an estimated 80,000 metric tons of human feces is produced daily, only a small fraction of which is treated (5). Cholera easily becomes endemic in areas where there is a lack of sewage treatment and inadequate disposal to the environment. This has been evident in Latin American countries since the 1991 epidemic. Safer food-handling practices, safer food sources, and, most importantly, personal hygiene can all play a role in reducing the transmission of cholera by family household members and also by food vendors. Adequate sanitation requires uncontaminated water for cleaning and hygiene, which relates back to the first important control measure. For regions where cholera is endemic, the development of an effective vaccine offers some hope in at least reducing the number of cases and the mortality due to this disease. Across the world, there are many studies of the development of vaccines and trials to determine effectiveness. Investigations have included development using both attenuated cells and recombinant live vaccines (40,87). Most of these studies have concentrated on prevention of and treatment of 0 1 V. cholerae infections. The persistence of the 0139 serogroup in the Bengal region of India has also initiated development of a vaccine that includes components of those strains. Oral vaccine administration offers the most efficient, least costly means of prevention and treatment. Recently, the investigation into genetically altering a variety of potato to carry a vaccine has been investigated (1 5). This technology, if successful, could provide a simple, inexpensive way to protect against the disease (15). Gene-splicing techniques allow for insertion of the vaccine into the potato, but other common foods most likely will also be investigated. A “booster” of the vaccine could be given with another meal of the potato. It has been cautioned, however, that vaccines might be counterproductive if they decrease the motivation to improve sanitation. Food processors must adhere to strict sanitation and good manufacturing practices to assure a safe product. Because many countries export food products, an outbreak can occur at a location far removed from the source. Many countries have adopted hazard analysis critical control point (HACCP) programs to assure safety of products. Likewise, many of these countries require imported food to be produced under these HACCP agreements to reduce the spread of V. choleme as well as other pathogens. Perishable food needs to be handled properly during production as well as when it reaches the consumer. Refrigeration is of great importance in preventing the growth of the organism. Raw and cooked food must be stored below 4.4”C before preparing and serving. Thoroughly cooking a food to destroy V. cholerae and other bacteria can prevent many infections. Once a food is cooked, prevention of cross-contamination by contact with raw product is extremely important, as isappropriate hand-washing between handling of raw and cooked product. Once cooked, food must be stored at temperatures below 4.4”C (40°F) or above 60°C (140’F) and protected from recontamination if it is not consumed

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immediately. Cooking of some products, such as shellfish and other seafood, would require a change of certain cultural customs, however, as many recipes use raw product.

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Vibrio parahaemolyticus Tuu-jyi Chai Natioml Trriwarz Ocean Urliversity, Keelurzg, Taiwarz, Repllblic of Chitla

John L. Pace Adlmced Medicine, Inc., South San Flancisco, Crrlifomia

I. Introduction

408

11. Distribution

409

A.Geographyandmicroflora of aquaticandmarineorganisms B. Seasonal and environmental relationship 410

409

111. Impact on SeafoodSafetyandFood-PoisoningOutbreaks

410

IV. Taxonomy, Identification, and Serological Classification 4

11

A. Taxonomy 41 1 B. Detection and identification C. Serological classification V. Pathogenicity

41 l 41 2

4 13

A. Clinical aspects B. Hemolysins 414 Other C. virulence factors

313

VI. BiologicalandPhysicalControls

417 of Growth 418

A. Sodiumchloriderequirementandeffectofwateractivity418 B. Effects pH of 419 C. Temperature and radiation sensitivity 419 D. Susceptibility antibacterial to agents 330 Biological E. factors 420 F. Physical factors 420 VII. CellularActivities421 VIII.

RoleandComposition of theCellEnvelope

IX. SurvivalinAdverseEnvironment Starvation A.423 Injury B. and survival 324 X. Conclusion 424 References 425

423

422

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1.

Chai and Pace

INTRODUCTION

Vibrio yct~c~~~nenlol~~ticz~s continues to be an important organism causing 50-7096 of all cases of diarrhea associated with the consumption of seafoods in the summer season in Japan, Taiwan, and other areas (1 1,16,38,55,118,129). The numbers of large reported outbreaks caused by this microorganism have declined, while increased incidences of traveler's diarrhea have occurred (171,198). This pathogen is widely distributed in all coastal and estuarine environments (9,27,32,38,48,81,105,177,180,182).It has been detected in most brackish water and, surprisingly, has been associated with fresh water as well (19,20,141,142). V. ye~~~~he~emol~ticlrs has been isolated frotn both free-living and aquaculture species. Clearly, any source of seafood may serve as a vehicle for the pathogen. The wide distribution and often large numbers of the bacterium isolated from crustaceans and molluscans during harvest seasons and the potential spread by insects warrant continued concern (16,44,45,48,55,81,173,177). Further evidence has been reported on the role for Kanagawa phenomenon-negative strains of the bacterium as a potential cause of disease (64,66,68,69,115). Subsequent to the report by Miyamoto et al. ( 113) on the significant relationship between the Kanagawa phenomenon (hemolysis produced by V. ycr~e~hcren~ol~~ticus colonies cultured on Wagatsuma agar) and disease, it has become apparent that strains phenotypically nonhemolytic may produce disease as well (64,66,68,69). These other strains often produce hemolysins immunologically (thermostable direct hemolysin-related hemolysin, TRH) or genetically related to the thermostable direct hemolysin (thermostable direct hemolysin, TDH), and some carry the genetic potential to produce both types of hemolysin (1 19,120). Regulation of virulence factor expression by host-specific environmental cues encountered by the bacterium suggests that expression of the TDH and the ability to adhere to intestinal epithelial cells may be co-regulated (63,126,127). Capsule expression is also induced under similar cultural conditions in medium containing bile, a secretion found in the host intestine (127). The requirement for both TDH and capsule production for pathogenesis have been demonstrated by studies with bacterial mutants (139a). Production of a capsule and its chemical composition are major virulence factors for a nutnber of bacteria that may allow the bacteria to adhere to intestinal cells (139a,179). But again, Kanagawa phenomenon-negative strains produce a capsule and also adhere to intestinal epithelial cells (64,127). One of the original enigmas surrounding V. paralzaenzolvticus is even more perplexing today. Specifically, how can one differentiate between pathogenic and nonpathogenic strains? This is of some importance, because it is likely that many environmental strains isolated from seafood are not pathogenic. Conversely, many strains previously believed to be nonpathogenic based on the Kanagawa phenomenon can and do cause disease. It is an important goal to establish a method for rapidly differentiating pathogenic isolates from nonpathogenic microflora in seafood. Several groups have utilized molecular biology approaches to reach this goal. Methods utilizing PCR or hybridization to detect hemolysin genes associated with disease have been developed (84,101,102). Normal microflora and inoculated bacteria have been detected from both seafood and stool samples. Perhaps a tool can be developed for the effective differentiation of pathogenic V. parcrl1crenzolyticzfs,and the safety of seafood could be improved. Without a reliable and practical methodology for differentiation of pathogenic from nonpathogenic strains, both types of organisms must cause equal concern and both be subject to inspection (43,46).

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II. DISTRIBUTION A.

Geography and Microflora of Aquatic and Marine Organisms

V. yara}laemolq,ticus has been isolated from diverse coastal regions (12,47,56,82,83,117). As expected, these sites include estuarine and brackish bodies of water, but they may also include fresh water sources (19,ZO). In some locales the predominance of isolated strains are nonhemolytic, while other reports, particularly from Japan, have detected a significant proportion of hemolytic and toxigenic strains (136,141). Survival of this moderately halophilic organism in fresh water appears to be associated with adherence to algae (19,96,98,100,141). This is in accord with studies demonstrating an association between levels of V. pnrahaemolyticus and estuarine algae (96,100). Seasonal distribution has been observed for V. parnhaenzolyticus, with higher levels isolated during warmer months and following algal blooms (141). Simulation of these conditions (15 ppt salinity, 25°C) resulted in the highest level of adherence for both Kanagawa phenomenon-positive and -negative strains to Navicula spy. (19). Some studies have suggested that cold temperatures may induce nonculturability of V. parahaemol~ticusand that temperature upshift may induce recovery (124a). While association of V. paral~aemolyticuswith chitin or copepods/algae in nature may enhance survivability and the pathogen is often isolated from sediment during colder months, it is unlikely that the bacterium is induced to become nonculturable at low temperature and resuscitated when warnled at room temperature (79,96). Effects of temperature shifts on the viable nonculturable status of this bacterium in laboratory microcosms are believed to be due to cold injury and simple growth of surviving cells upon incubation at a permissive temperature (96). In addition to association with algae and zooplankton, the pathogen is commonly isolated from a wide variety of crustacea, mollusks, and finfish (12,44,71,88,89,95,117). Levels of K palwh~e~zzolytic~~s detected from Crassostrea japonica in Japan were reported to be as high as 1 X lo3CFU/g, with up to 16% TDH positive (98). In fact, V. pcrrahaernolyticus is often the most commonly isolated vibrio from bivalve mollusks. Oysters may actually concentrate the bacterium with levels approximately 100-fold greater in the mollusk than overlying water as compared to a 10-fold greater level of fecal coliforms in oyster than in the water (38). Again, Kanagawa phenomenon-positive strains have been detected from oysters with DNA probes as well as with the Kanagawa test (38,103). This includes both natural and cultured oyster populations (103). Levels of V. yrrrakaenlolyticus can rise in oysters stored as shell stock or shucked meats (16,55,71). Significant increases were observed for the bacterium from both stored oysters (Crcrssostren virgirzica) and clams (Mercennria ccrrnpechierzsis)(16,7 1). Levels of the bacterium in natural mollusk populations can change rapidly due to the improvement of test methodology, environmental changes, and other factors. For example, in July 1991 Kanagawa phenomenon-positive strains were not isolated from the gastropod Clithorz retropictus from the Sada estuary of southern Japan, but by September of that year levels had reached 2.0 X 10’ CFU/g (99). During laboratory studies with C. retropictus, TDH-producing strains were maintained at greater levels than negative strains and suggested that TDH-producing strains may persist even in the presence of a preponderance of nonhemolytic V. parahuemolyticus (97). The survival of the bacterium in C. retropictus may be due to a lack of recognition of V. parahuernolyticus and clearance by

Chai and Pace

410

the gastropod's hemocytes, which probably explains why the bacterium is a normal constituent of the marine invertebrate. B. SeasonalandEnvironmentalRelationship V. ya~nllcrer,tol?~ticl~s produces a chitinase and may play a major role in the cycling of chitinous materials in the estuarine environment (82,83). The chitinous nature of crustaceans associated with V. ycrl.n}znemolrvticusmay be important in mediating the interaction of the bacterium with its intermediate host (82,83). The organism also has been isolated from samples collected from Asia, the Mediterranean, Europe, Africa, South America, and North American areas (76,81). Zooplankton have exoskeletons composed of chitin and often can-y high numbers of V. ycrl.crlzaenzol?,ticlls(8233). The bacterium disappears from the water column during colder months and then increases with warming and increased numbers of zooplankton (31,82). The interaction with zooplankton probably is beneficial to the bacterium and aids in the survival of this mesophilic organism under reduced temperature (82). Increased levels of the bacterium in seafoods and incidence of foodborne disease caused by V. ycrl-c~hnenrol?)ticlis in Califomia during 1998 were most likely due to elevated coastal water temperatures caused by the recent El Nifio climate event (J. M. Janda, California State Dept. Health services, personal communication). Effects of salinity on the distribution of V. yal.cl}lnemol~lticusmay be related to effects on the adhesion of the organism to chitinous surfaces.

111.

IMPACT ON SEAFOODSAFETYAND FOOD-POISONINGOUTBREAKS

Microflora of marine organisms consumed by humans and V. p~~e11zcrer~zoZ~ticus appear to besynonymous. Whether it is oysters, clams, crabs, or fish, upon microbiological examination invariably V. ynl.nhctenzoZvticus will be detected (1,45,186). In a study by Fang et al. (45), V.pn~e~haernol~ticus was detected from 45.7% of seafood samples collected from retail markets in Taiwan. The detection frequencies included approximately 68% of bivalve shellfish, 48% of crabs, 44% of shrimps, 40% of fish samples, 32% of nonbivalve mollusks, and 22% of fish fillets (45). In fresh seafood sold in Guadalajara, Mexico, V. yerrwl~crer~~ol~ticrrs levels were higher during warm months, and positive samples included 71% of fish, 44% of oysters, and 28% of shrimp (164). Many of these isolates may be nonpathogenic, but other studies have reported incidence of Kanagawa phenomenon-positive isolates as high as 2% from market seafoods (186). In most cases detection is from raw, underprocessed, or improperly handled seafoods. However, as with kirasu, the partially dried sardine fry product from which V. ynr-crhael?.2oZvticus-associated gastroenteritis was first identified, various seafood products may harbor the pathogen (107). Foods as diverse as kippered herring, salted roe, cold-smoked fish, and frozen precooked and peeled shrimp may contain the bacteria, although often at low levels (25,30,86). One recent outbreak involved the consumption of raw oysters and spanned the states of California, Oregon, and Washington as well as British Columbia with 209 culture-confirmed infections (48). V. ynl-c~hcrernolytic~z~s levels do appear to correlate with incidence of disease; when levels reach 1 X 10' cells/ 100 mL of water from which the seafood is harvested, incidence of disease is more likely to occur (95). Bacterial levels as high as 1 X lo5 to 1 X 107/g of seafood have been detected (18).

Vibrio parahaemolyticus

41 1

W. TAXONOMY,IDENTIFICATION,ANDSEROLOGICAL CLASSIFICATION A. Taxonomy The family Vibrionaceae contains four genera: Vibrio, Aeromonus, Photobacteriurn,and Plesiornonns (10). The genus Vibrio comprises 20 major species with phenotypic and genotypic similarities. To determine the taxonomy of V. paruhnemolyticus in relation to other Vibrio species, a variety of methods have been utilized (10,31). Guanine-cytosine (GC) base composition for this organism is 46%, and V. ynrnhnemo1~)ticusstrains exhibit near 90% DNA homology. Other species related to V. pnrcdlaemolyticusby DNA homology are V. alginolyticus (65%), V. harveyi (55%), V. cnrnpbellii (55%), V. natriegens (52%), and V. vulnificus (32%) (10). Most of these species can also be related by determining the immunological relationships of superoxide dismutase (SOD) and alkaline phosphatase (AP). Results for alkaline phosphatase of the 14 species possessing this enzyme were similar (10). Electrophoretic mobility of dehydrogenases could also be used to separate V. paruhnemolyticus, V. cholerae, and V. fIuvin1is (1 14).

B. DetectionandIdentification Methodologies to detect V. purahnemolyticus from seafoods include those recommended by the U.S. Food and Drug Administration (FDA) (43) and a variety of other culture, fluorescent, and molecular techniques (82,102,112,123,179). FDA recommends a most probable number (MPN) method utilizing a homogenate of the seafood in alkaline peptone water. From these enrichment tubes, surface growth is transferred to TCBS agar and characteristic colonies are subjected to further biochemical tests and microscopic examination following Gram staining (43). V. pnrahnemolyticrds produces round blue-green colonies on TCBS agar and may not grow on mCPC agar (43). Typical colonies are transferred to gelatin agar, gelatin salt (GS) agar, and trypticase soy agar (TSA) with 2% NaC1. V. ynrnhaer~~olyticz~s should grow only on plates containing sodium chloride and evidence of gelatinase should be present around the colony (43). An oxidase test is perfomled on the colony from the GS plate, and other assays are inoculated from the TSA plate (43). Previously, the method of isolating and enumerating V. parahnenlolyticus as approved by FDA (167) included the MPN method in glucose-salt-teepol broth (GSTB) followed by plating on thiosulfate-citrate-bile salt-sucrose (TCBS) agar with subsequent determination of various physiological and biochemical traits (167). Other proposed methods have included (a) membrane filter procedures on medium containing copper sulfate, sodium cholate, alkaline pH, and 3% sodium chloride, (b) direct plating on TCBS agar followed by further differentiation, or (c) the use of several other selective media (42,85,18l). Fluorescent microscopy utilizing conjugated antibodies reactive with outer membrane proteins from V. pnruhcremolyticus following enrichment and the useof a fluorogenic substrate for the trypsin-like protease activity of V.puruhaemolyticus following growth in arabinose-glucuronate medium have also been proposed (112,178). V. pnrul~aemolyticus can be differentiated from other species by several physiological and nutritional traits including swarming on solid complex media (-), presence of lateral flagella on solid media (+), pigment production (-), arginine dihydrolase (-), oxidase (+), reduction of nitrate to nitrite (+), gas from glucose ( -), acetoin or diacetyl

4 12

Chai and Pace

production (-), growth at 40°C (+), utilization of sucrose (-), cellobiose ( -), D-gluconate (+), y-aminobutyrate (-), putrescine (+), ethanol (-), L-leucine (-), valerate (-), and P-hydroxybutyrate (-) (43). For the identification of V. pnrahnemolyticus, the minimum assays required include observation of a motile gram-negative rod, production of acid from glucose but no gas, sensitivity to vibriostat (150 pg), positive growth at 3-8% NaCl and 42OC, positive lysine and ornithine, and negative 0-nitrophenyl-P-D-galactopyranoside(ONPG) (10,43). Reaction on TSI agar should be alkaline slant, acid butt, no gas, and no H2S (43). However, most V. pnmhaenzolyticus isolates are reported H2S positive (169). It may be easier to detect this reaction using media other than TSI (77,169). Also, some strains of V. p a r c h e molyticus are ornithine negative ( 10). Synthetic gene probes have been utilized in a colony hybridization test for V. pnrcrIzaernolyticus (101,123). A short probe representing part of the tdz gene gave definitive determination of Kanagawa phenomenon-positive V. pnrahaemolyticzls but also detected V. holZisae (123). A second probe comprised of a 26-nler from the tdh gene was able to detect V. pnmlznemolyticus from seafood (103). Yet another probe used for colony hybridization tests, a 0.76 kb Hind111 fragment of chromosomal DNA labeled pR72H fragment coupled to digoxigenin-l 1-dUTP, detected 122 of 124 strains assayed and was not reactive with other vibrios or other enteric and nonenteric bacteria (102). A primer set based on the sequence of pR72H was utilized for PCR detection of V. parahaemolyticus (102). In artificially inoculated oysters cultured for enrichment in tryptic soy broth containing 3% NaCl for 3 hours at 35OC, the PCR assay was sensitive enough to detect initial bacterial levels as low as 9.3 CFU/g (102).Another primer set based on the gyrB gene of V. yamhuemolyticus was capable of detecting as little as 1.5 CFU/g from artificially inoculated shrimp homogenate by PCR amplification following preenrichment. Finally, PCR utilizing a tdlz-specific primer pair could detect V. pnra}lnernolyticus directly from fish homogenates, but again preenrichment increased sensitivity to as low as 10 CFU (85).

C. SerologicalClassification Serologically, V. parahae~~zol~~ticus has been separated into 11 heat-stable 0 serotypes and 71 primarily heat-labile K serotypes (43). It is unnecessary to determine the serotype for the identification of this organism, but the protocol is detailed in the FDA Bnctel-iological Ancdytical Manual (43). In northern Taiwan a total of 42 serotypes were identified from 1610 rectal swabs of food-poisoning patients from 1983 through 1993 (129,130). The predominant serotype was K8, which accounted for 36.8% of the total serotypes determined. The following types in descending order were K15 (10.8%), K12 (8.7%), K56 (7.9%), K63 (4.7%), K4 (4.2%), K41 (3.8%), K7 (2.9%), K54 (2.8%), and K29 (2.5%). Most of the V. parahnemolyticus incriminated in food-poisoning cases is found to be a single serotype in an outbreak. However, multiserotypes also occur in a single outbreak. Among a total of 112 foodpoisoning outbreaks associated with V. parrrhnenzolyticus in the 10-year period (19831993), only 54 (48.2%) were found to be caused by a single serotype. The other outbreaks caused by multiserotypes include two serotypes, 26.0%; three serotypes, 14.2%; four serotypes, 5.3%; five serotypes, 2.7%; six serotypes, 0.9%; seven serotypes, 1.8%; and eight serotypes, 0.9% (130)

Vibrio parahaemolyticus

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Comparisons of Japanese and U.S. isolates by serological methods suggest that pathogenic strains can be grouped separately and that groupings could be confirmed by growth assays for temperature and salt concentration (169). There does not appear to be any specific serotype restricted to a geographic region (146,169). Lateral flagella of V. ynrnhnemolvticus have two antigenic determinants that are different from those of the polar flagellum (149). Agglutination with antisera to lateral flagella has been used for identification of V. parnhaemolyticus (146,148). Organisms that were different biochemically from V. pnrnhaenzolvticus did not agglutinate, while both clinical and environmental isolates of the organism did (148).

V.

PATHOGENICITY

V. par-alznernol~ticlIsis a human pathogen primarily producing gastroenteritis and secondarily producing wound infections (76,81). The major symptoms of V. pnrahaemolyticus food poisoning are diarrhea, abdominal cramps, and nausea (16,55,81,106). In Japan, where there have been deaths caused by this organism, heart arrhythmias also have been noted (156). Onset of symptoms following consumption of contaminated seafood ranges from 4 to 96 hours; the illness usually is self-limiting and persists for 3-4 days (16,81). A few studies have demonstrated enterotoxic activities, epithelial cell adherence, or rabbit ligated ileal loop responses to V. parahuemolyticus (26,29,139,151,156,157,168). No definitive virulence mechanism has been proven responsible for the clinical syndrome caused by V. par-ahnemolyticus (55,8 1).

A.

Clinical Aspects

Clinical disease associated with V. parahoemolyticus most often involves cases of gastroenteritis (28,111). Occasionally, the associated diarrhea may be severe and appear choleralike (1 10.172). An 8-year survey of ill travelers returning to Japan through the Osaka Airport quarantine station reported a 21.5% incidence of V.pnmhaernolyticus among identified enteropathogens, with 90.2% TDH positive, in comparison to the incidence of other pathogens including Salrnorzellr~ spp. (28.6%) and enterotoxigenic Escherichincoli (ETEC, 11.5%) (198). A subsequent 2.8-year study at this site determined that V.pnrahaernolyticus accounted for 15.6% of identifiable disease (170). Overall during the period of 1979-199s at the quarantine station, this pathogen accounted for 1959 isolations from the 9766 of 29,587 stool samples examined bacteriologically (171). Similar results were found for the general population in Taiwan where V. yarnhaenzolvticzls accounted for over 35% of foodborne disease outbreaks during the period 1986-1995 (130). As has been reported in several studies, V.parnhnemolyticus is often identified as the only enteropathogenic species isolated from stool samples of patients (106). Illness may result in septicemia following oral infection or wounds (2,39,57, 80,124,143). Less frequently, reactive arthritis, panophthalmitis, and pneumonia have been reported (153a, 159,199). One interesting case involved a postoperative wound infection where the pathogen was isolated from wound drainage as well as the stool (2). The patient had consumed steamed crabs one week before the surgery to treat intestinal obstruction caused by colon carcinoma. Other risk factors include reduced gastric acidity and liver disorders ( 182).

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Chai and Pace

A remarkable symptom associated with gastroenteritis is an abnormal electrocardiogram (17a,65,166). Cardiogenic shock may ensue and is the principal cause of death associated with V.pnmhaemolyticus infection (166). In animals the TDH toxin has been shown to induce heart abnormalities (65a, 156). Small quantities of toxininjected into mice caused heart stoppage within one minute as well as cessation of spontaneous beating by myocytes in culture (53). Deaths caused by V.pnruhnernolvticus often follow ingestion of seafood. From 1981 to 1988 in Florida, 71% of fatal infections due to V. ynmhaenlolyticus followed the consumption of any seafood, and 57% followed the consumption of raw oysters (91). This is consistent with earlier reports in which gastroenteritis followed consumption of raw, underprocessed, or cross-contaminated seafood (153).

B. Hemolysins A variety of potential virulence factors including various hemolysins, toxins, and other virulent cellular substances are listed in Table 1. The thermostable direct hemolysin (TDH) or Kanagawa hemolysin is well known and is expected to play a major role in the pathogenesis of V.pnr~huer~zolyticus (3,113,120,156). A number of culture conditions that affect production of the TDH have been identified (33,126). These include culture with bile acids, culture in iron-restricted media, medium content of D-trytophan, and of course sodium chloride content of the medium (33,113,126). It is significant but not unexpected that culture under iron restriction might enhance hemolysin levels (33). This type of response has been widely described for bacterial pathogens (127). In vivo it is essential for a bacterial pathogen to obtain iron under limiting conditions (183,184). Lysis of erythrocytes produces a major source of iron for the microorganism (54). The finding that bile acids enhance cell viability and production of the hemolysin is also important. Bile acids are found in the host intestine and may serve a role in pathogenesis by stimulating the bacteria to produce virulence factors including hemolysins (126,127). Obviously, due to the cytotoxic nature of the TDH, this might also be expected to result in fluid accumulation or diarrhea. Animal passage does increase hemolysin production by V. pnrnl~aernolyticus isolates (87). TDH-positive strains are present in the aquatic environment, and the environmental strains do produce hemolysin in vitro (48aj. Thecause for the poor recovery of Kanagawa phenomenon-positive strains from the environment remains a mystery. However, a recent report by Okuda and Nishibuchi ( l 25b) brings to light a potential source for the Kanagawapositive strains. They report that a one-base difference in the promoter regulating expression of the tdz locus in Kanagawa phenomenon-negative strains is responsible for lack of hemolysin production, but that by a single point mutation Kanagawa phenomenonpositive subclones could arise. Several related forms of TDH with genes varying by minor nucleotide changes have been identified (196). These hemolysins have different electrophoretic mobility but identical antigenicity, and only slight changes in ability to cause hemolysis, vascular permeability, and mouse lethality (196). A variety of related hemolysins are produced by both Kanagawa phenomenon-positive and -negative strains (69,115,187,197). This family of hemolysins is related to the TDH. The TDH-related hemolysin (TRH) is heat sensitive as compared to TDH and immunologically similar but not identical (69). The TRH proteins are highly similar, whether from environmental or clinical isolates (197). Some of these hemolysins are different by as few as two amino acid residues, and many strains produce more than one type of hemolysin (187). Lytic

" "

aemolyticus

Vibrio

4 15

Table 1 Potential Virulence Factors and Some of the Responsible Genes of V. parahaemolyticus ~

factors Virulence StrainsCharacteristics gene

and

to tdh2 ~~

Hemolysins TDH (thermostable Produces vascular permeabildirecthemolysinor ity, cell lysis, cardiotoxKanagawa hemoicity lysin) (tdhS) tdhl KP+, chromosomal 119 (97.2) (T4750) 97.0 WP1 tdh2(td?A) 74,119 100W+, (T4750) chromosomal WPI tdhX KP+, chromosomal trhX KP+, chromosomal Weak or negative he- KP- intermediate or negamolysin (TDH-retive hemolysins lated hemolysins) tdh3 KP-, chromosomal tdh4 KP-, plasmidborne tdh5 K€-, chromosomal tdM KP-, chromosomal NAG-tdlt NT, plasmidborne trhl( =t7-11) K€-, chromosomal tr122 W-, chromosomal LDH and other Lecithin-dependent, phospholipase, AJlysophospholipase Toxins Enterotoxin Induces morphological changes in C H 0 cells Shigatoxin Cytoxic Enzymes Muscinase Degrades muscosa/glycoprotein Superoxide dismutase Induced by 02,renders microorganism better able to survive in host Others Adhesins Hydrophobic, can cause erythrocyte agglutination Lipopolysaccharide Endotoxic Kp+. Kanagawa-positive; Q rdh V. clzolercte non-0 1.

~~

% similarity

- ,

~

Ref.

~

TH3766 TH3766

98.4 68.1

187 187

AQ3776 A43776 AQ3860 THO12 91 AQ4037 AT4

98.6 98.6 98.9 98.4 98.6 68.6 68.8

119 119 3 119 4 118b 90b 147,190

152 144 76,167 34

29,132 152

Kanagawa-negative; NT, not tested.

activities of TRH proteins are somewhat different from the spectrum of activity for the TDH (69). Hemolysins produced by other species including nonagglutinable V. cholerae (NAG-rTDH) and V. hollisne also are highly related in structure and activity to the TDH (68,195). TDH may bind to several neuraminidase-sensitive gangliosides on target cell surfaces (158). GTIganglioside is able to inhibit activity of the TDH and is not found on

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horse erythrocytes, against which the hemolysin is poorly active (158). G,, and to a lesser extent Gbl,gangliosides also inhibit TDH-dependent cessation of spontaneous beating by mouse and rat myocardial cells (53). ''51-labeled TDH does bind to horse erythrocytes but, in contrast to human erythrocytes, does not lyse the equine cells (40,194). These findings suggest that GTIganglioside is required for lysis but not binding to erythrocytes (40,53,158,194). Neutralizing monoclonal antibody against the amino terminus (residues 1-3 1) of the TDH prevents binding to erythrocytes, and binding of antibody is prevented by prior interaction of the hemolysin and the erythrocyte (163). Binding of other monoclonal antibodies suggests that the carboxy terminus (residues 99-139) is involved in postbinding processes. A mutant TDH with a substitution of glycine at position 62 is nonhemolytic but retains approximately 50% of its binding capacity (161). Substitutions of TDH Trp65 or Leu66 also caused large decreases in hemolytic activity of the mutant hemolysins, demonstrating the importance of this region of the protein on its function (4). Hemolytic activity of TDH is due to pore formation in the target cells (22,67). Pore formation is temperature dependent, while the second step of lysis is temperature independent (67). TDH forms a pore in the target cell approximately 2 nm in diameter, and the pore complex is observable by electron microscopy (67). Effect of the TDH includes induced cation leakage in the erythrocyte (73). The potassium-induced leakage was sensitive to Zn'+ (72). Calcium influx is increased, and the Ca'+-activated K+ channel is stimulated (52). Results of this process in addition to hemolysis include hyperpotassemia in the rat and might contribute to the heart irregularities in addition to direct action of the TDH on myocytes through membrane depolarization (155). TDH causes calcium influx in human intestinal cell lines as well, but the relationship of cation influx to toxicity is less clear (13 1,162). Findings with rabbit ileum mounted in Ussing chambers suggested that the TDH induces intestinal chloride secretion in a manner consistent with calcium as a second messenger (13 1). Phosphorylation of erythrocyte proteins stimulated by TDH has also been observed (193). Detection of TDH has been approached by a number of methods. Toxin has been detected from ileal loop fluids at concentrations of a few ng/mL by ELISA (70). Elek tests have also been employed and are on par with Wagatsuma agar for detecting TDHpositive strains (1 16,117). Monoclonal antibodies against TDH may improve specificity, although antibodies reactive with both TDH and TRH have been utilized to compare clinical and environmental isolates of V. ynralwenzolyticus for hemolysin production. Some kits comprised of anti-TDH polyclonal seralatex beads actually detect both TDH and TRH due to cross-reactivity (163,194). Monoclonal antibodies produced against TRH also may have reactivity with TDH (66). Studies with ELISA method and anti-TRH monoclonals suggest that TRH-producing strains are predominantly isolated from clinical sources (69a). Alkaline phosphatase-conjugated and other nucleotide probes against tdh and td1 have been utilized to detect these genes and have been used to detect their presence in other vibrios (123,188). Polymerase chain reaction (PCR) has also proven useful for detecting the tdlz gene ( 154). Utilizing a nonisotopic microtiter-based assay method, as few as seven copies of tdlz from the target chromosome of V. parahoemolyticus could be detected with 35 cycles of amplification. PCR can detect as few as 100 cells bearing either the tdh or the related trh genes (101,154). Based on a genetic study completed on this organism, at least 12 different hemolysins including both TDH and TDH-related hemolysins have been identified and characterized (Table 1). All of these hemolysins were produced from their corresponding genes

Vibrio parahaemolyticus

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located presumably on V. parnhnemolyticus chromosomes except for NAG-tdlz and tdh4, which are plasmid borne. These identified hemolysin genes include at least tdhl, tdhS, tdh2, fdhA, tdhX, trhX, fdh3,tdh4, tclh5, tdh/l, NAG-fdh, trhI(trh), and trh2 (3,4,119,187). Two additional nucleotide sequences Vm-tdh and Vh-tdh, not shown in Table 1, were identified from V. mimicus and V. hollisae, respectively (90b,l18b). Following further comparative examination, the tdhS and tdhA genes are believed to be identical to tdhl and tdh2, respectively (1 19). The similarity of these hemolysin genes in comparison to tdh2 (tdhA) ranged from 68 to 99%. Additional genetic and physiological information can aid in differentiation between the predominant Kanagawa-positive strains of clinical isolates and the greater numbers of Kanagawa-negative strains existing in the estuarine environment.

C.OtherVirulenceFactors In the last decade, several studies regarding Kanagawa-negative V. paruhnemolyticus capable of causing disease have been reported (3,63,64,66,187). These clinical strains produce a similar hemolysin TRH (TDH-related hemolysinj, and some other strains phenotypically negative for hemolysis were positive genotypically for TDH, TRH, or both (22,150). Phenotypically negative clinical isolates either did not transcribe the responsible thermostable direct hemolysin (tdlz) or thermostable direct related hemolysin (trh) genes or did not export the proteins (150). Another TDH-related hemolysin has been isolated from a clinical Kanagawa-negative strain (64,69). This protein, thermostable direct immunological related hemolysin (TDH/I), was similar to TDH except for seven amino acid changes that result in different hemolytic profiles and electrophoretic mobility. More than 100 pg of TDH/I was required to produce ileal loop swelling, but live cells producing this hemolysin did produce a positive ileal loop response (69). Additional DNA sequences near the tdh gene also are not homologous in Kanagawa-negative strains, raising the possibility that associated virulence genes may be absent from nonpathogenic isolates (23). Hemolysins similar to TDH also have been purified from non-01 (nonagglutinable) V. cholerne and V. hollisae (121,122,192,195). V. parahaernolyticus also produces several enzymes that have hemolytic activity (Table 1). Among these are phospholipase A, lysophospholipase, and glycerophosphoryl-choline diesterase (147,190). No correlation has been found between plasmid content and the pathogenicity of V. pnrahaemolyticus (167). Previously the frequency of isolating urea-hydrolyzing V. pnrahaemolyticus was low, but now it is increasing (89,125). Production of urease may give the organism a competitive advantage in the host where 20% of urea nitrogen is excreted into the intestine. Alternatively, the purified urease caused intestinal fluid accumulation and was toxic in the suckling mouse assay (24). The gene encoding the urease enzyme may be genetically linked to the trh gene (75). Most Kanagawa-positive V. pnrahnemolyticus produce ligated ileal loop swelling, and the loops exhibit inflammatory changes with infiltration of granulocytes (139). Destruction of the mucosa was apparent with necrosis, ulceration, and hemorrhage. Most Kanagawa-negative isolates do not produce loop swelling (81,147,168). No serotype correlates with ability to induce loop swelling. Kanagawa-positive V. parnhnemolyticus are able to grow in the loop, but negative strains decline (168). Effective ileal loop doses of Kanagawa-positive V. pnrahaernol-yticusranged from 2.6 X lo5 to 7.7 X lo6 cells, and 2 X 10' to 3 X lo7 cells were required to produce diarrhea in human volunteers (168). Kanagawa-positive strains caused bacteremia in suckling rabbits, and 50% of animals fed

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V. paruhaemolyticus exhibited positive liver and spleen cultures (24a). Immunofluorescence of V. paruhae~~~olyticus demonstrated invasiveness in rabbits (2l). Evidence of an enterotoxic factor separate from the TDH also have been presented (17,151). These agents caused morphological changes in Chinese hatnster ovary (CHO) cells, lethality and diarrhea in mice, and enhanced skin permeability reactions in rabbits (17,65,89). Cytotoxicity for cultured human epithelial cells of bacteria grown with bile is increased (127), and V. parcrhaernolyticus does produce a secreted metalloproteinase with gelatinase and weak hemolytic activity (104). The lipopolysaccharide of V. purahnemolyticus exhibited lethal toxicity for mice, stimulated macrophages, and activated limulus amebocyte lysate similar to that from Enterobacteriaceae (5,6,152). Adherence of V. parnhaernolyticzrs to cultured epithelial cells has been assayed by several groups (27,29,49). One study (27) reported that Kanagawa-positive strains adhered in greater numbers, while other workers found no difference (49). Both studies used microscopic observation, which may be inefficient for the determination of adherence, and different cell lines used might have affected results. More recently, Kanagawa-positive V. paruhaernolyticus readily adhered to rabbit intestinal epithelial cells, while Kanagawanegative strains did not (29). This adherence was inhibited by fetuin, D-mannose, and Dglucose but not by antisera to the TDH (29). Kanagawa-positive V. paratzaemolyticus adhered to fixed human small intestinal mucosa ( l 89). Affinity was greatest for the ileal lymphoid follicle epithelium and less for microvilli-bearing absorptive cells. Similar results were found with rabbit small intestinal mucosa (186b). Hemagglutination activity has been described for V. paralznenzolyticus (29). Growth in medium containing bile or bile acids increased in vitro adhesion to cultured human intestinal epithelial cells (127). Interestingly, invasion of epithelial cells was inhibitable by chemicals known to inhibit invasion by other enteric pathogens (2a). The organism may cause disease in such shellfish as crabs and shrimp (95,174). However, Kanagawa-positive V. pnrahnernolyticus did not cause disease in brownhead minnows (61), and lipopolysaccharide (LPS) did not kill the juvenile fish when either immersion treated or intraperitoneally injected (60). Sterile culture filtrates of both Kanagawa-positive and Kanagawa-negative V. pnrahaer~~olsticus were not cytotoxic for cultured Chinook salmon epithelial (CHSE) 214 or epidermal papilloma carp (EPC) cell lines (127a). However, all strains, regardless of source, were hemolytic on blood agar prepared with fish erythrocytes.

VI.

BIOLOGICALANDPHYSICALCONTROLS OF GROWTH

A.

Sodium Chloride Requirement and Effect of Water Activity

V. parcrhnemolyticus is a slightly halophilic bacterium. The optimum NaCl concentration for the organism ranges from 2 to 4%, and poor growth is exhibited in media below 0.5% NaCl(92). The bacterium is inactivated rapidly in distilled water (104a). Growth at levels of 10% NaCl is inhibited, which is a useful trait in differentiating V. parcrlzoenlolyticzw from V. alginolyticus (10). The optimum water activity (a,v)for V. ynlzrhnernolyticus is 0.992 in the presence of NaCl, and the minimum is 0.948 (14). Requirements for a, and growth temperature are related, and the minimum a, is dependent on the solute

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(1 53a, 153b). A predictive model for growth rate of the organism for both laboratory media and foods based on the effects of a, and temperature has been reported (1 loa).

B. Effects of pH V. ynrahaemol~~ticus can survive in medium held under a broad pH range (14,168,169). However, the bacterium, like many microorganisms, grows best under slightly alkaline conditions. Several physiological responses are known to be affected by pH (83,90,146). Survival may be reduced during multiple stresses including a combination of chilling and acidic pH (13,175,176). The organism does exhibit an acid-tolerance response (185). Bacteria cultured at pH 7.5 and then shifted to pH 5.8 followed by pH 5.0 were more resistant to challenge at pH 4.4 (185). The adapted bacteria were also more resistant to thermal challenge and low salinity (158). Numerous proteins (6.4-96.9 kDa) were induced, while production of several proteins (25.3-91.7 kDa) was inhibited by acid adaptation. Enteropathogenicity of acid-adapted V. yarahaemolyticus in the suckling mouse model was also enhanced (185).

C. TemperatureandRadiationSensitivity V. pardmemolyticus is cold sensitive, and the storage medium affects survival of this organism (172a). The lowest recorded temperature of growth was in laboratory medium at 5"C, but, in general, V. yarahaemolyticus numbers decline when held at 10°C or below (16). Many survivors, however, can be isolated from whole shrimp, shucked oysters, or fish homogenate stored at low temperature, or from oyster shell stock (32,51,108). The primary site of cold injury may be the cell membrane since MgClz aids in recovery and the sublethally injured cells are more sensitive to detergents. V. yard?aemolyticusis a mesophilic organism with a maximum growth temperature of 42-44°C. The highest growth temperatures correlate with altered cell-envelope fatty acid composition and enhanced heat resistance (16a). Several strains of the organism exhibited z-values of 5.5-12.4"C when heated at temperatures from 49 to 55°C (35). Survivors could be detected in shrimp homogenate heated at 60 and 80°C when the inoculum level was 2 X lo6cells/mL (176). Heat sensitivity may also be affected by the physiological status of the pathogen. Glucose-starved cells exhibited increased resistance to heat, osmotic shock, and hydrogen peroxide (93). The significance of this finding is that the estuarine environment where these bacteria reside is low in nutrients, and recognizable differences have been reported between bacteria cultured in estuarine water and laboratory medium (128). Thus temperature sensitivity of the microorganism may be dependent on method of preparation and somewhat different from that observed from heat-processed seafoods. Sublethally injured V. yarnkaer~zolyticusalso can be recovered on nonselective media or TCBS agar with the addition of magnesium chloride (61a). Studies with antibiotics suggest that DNA, RNA, and protein synthesis are required for recovery from sublethal heat injury (61a). The pathogen is highly radiosensitive, exhibiting a decimal reduction dose (Dlo)of 0.03-0.04 kGy when exposed to 6"Co (38a). This sensitivity was unaffected by growth phase.

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D. SusceptibilitytoAntibacterialAgents 1. Food Additives A variety of antibacterial food additives are effective inhibitors of V. parahaernolvticus including potassium sorbate, sodium benzoate, glycerol, monolaurin, monocaprin, sucrose esters of fatty acids, and propyl-p-hydroxy-benzoate (15,177). Treatment of crabmeat or flounder homogenate inhibited the bacterium, and cell numbers were lower than in untreated samples (135). These agents also increase the heat sensitivity of the organism (15).

2. Antibiotics Quinolones have proven to be among the most effective antibacterial agents against this pathogen (46a). Against V. purahcremolyticus, MIC9"s offleroxacin and pazufloxacin were both 0.2 g/mL (47a). However, a gene encoding a putative drug efflux protein has been cloned following identification of a CCCP-sensitive norfloxacin efflux system from V. pnrahnemolyticus (1 14a). This gene was identified by cloning V. pnrahaemolyticus chromosomal DNA in an AcrAB minus E. coli mutant (105a). Transformants exhibited elevated resistance to norfloxacin, ciprofloxacin, ethidium, kanamycin, and streptomycin (1 14a). V. puruhnerrzolyticus norM, a putative efflux pump, was partially homologous to ydhE from E. coli and H. injuenzae. ydhE was cloned, and transfornlants also were more drug resistant, although their resistance profile was not identical. These findings suggested that NorM and YdhE have slightly different substrate specificities (1 14a). This organism is also sensitive to ampicillin/sulbactam combination, as well as to many third-generation cephalosporins, aminoglycosides, chloramphenicol, and tetracycline (46a). V.parahaemolyticus is resistant to older p-lactams due to p-lactamase production, and polymyxin Bresistant strains have been isolated (46a,93). E. Biological Factors Various biological factors affect the survival of V. parahaernolyticus. Bacteriophages lytic for V. parnhaemolyticus have been isolated from shellfish and natural waters, and phage levels increase with water temperature and mesophilic Vibrio levels (7). Other Vibrio species may be hosts for phages that are able to infect V. paralznernolyticus, but phages for the organism isolated from commercial oyster liquors were unable to infect E. coli, Proteusvulgaris,Pseudomonas aeruginoscr, or Salmonella newport (7,127a). Greater numbers of phages capable of infecting Kanagawa-negative strains were isolated from oyster liquors, which may reflect the predominance of this cell type in the environment. Many phages showed intergroup lysis (127a). Pseudomonas species in oysters may have an inhibitory effect on V. parahaemolytiCZAS(50). Kanagawa-negative strains were inhibited to a greater degree and the inhibitory agent elaborated by the Pseudomonas was hydrogen peroxide (34). Bdellovibrio, which parasitized V. parnl~aemolyticus,also is present in the estuarine environment (1 11a). The bacterial parasite might play a role in the distributions of V. paruhuer~zolyticusin seafoods or the environment where the Bdellovibrio has a lower optimal growth temperature than the pathogen.

F. PhysicalFactors Various physical factors can control the growth of V. parahnemolyticus. In addition to heat, ultraviolet (UV) light, radiation, and high pressure can inhibit the growth of this

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organism (145). Three strains of V. ynmhaemolyticus tested were found unable to survive or grow at 200, 400, 600, 800, or 1000 atmospheres (atm) of pressure. The evidence strongly supports the neritic or estuarine origin and habitat for V. pnralzaemolyticus since the deep ocean isolate Pseudornonas bnthycetes was capable of survival and growth at these pressures (145).

VII.CELLULARACTIVITIES

V. pnruhnemolyticus produces a variety of enzymes that are important in the utilization of substrates available in the natural environment. The organism produces chitinase, which may contribute to carbon cycling and survival (186a). The chitinase of V. yarnhnernolyticus does not appear to be inducible in estuarine water as the specific activities produced by several Kanagawa-positive and -negative strains were similar when cells were cultured in rich medium or estuarine water (128). Chitin probably can be used as a nutrient source, and its addition to estuarine water resulted in greater than 2 log more cells as compared to chitinless water (128). Chitin also provides a surface for the adherence of V. yarahnernolyticus with twofold greater numbers of attached cells than free-swimming cells (128). Solid/liquid interfaces generate physical forces that result in nutrient concentration and are important for microorganisms surviving under nutrient-limiting conditions. Estuarine waters are low in phosphate, and it is not unexpected that bacteria native to this environment would have enhanced levels of enzymes capable of metabolizing phosphorus-containing compounds. V. parahaemolyticus cultured in estuarine waters produces three- to fivefold higher levels of alkaline phosphatase than that cultured in rich medium (128). The availability of alkaline phosphatase necessary for the transformation of phosphate is provided by compartmentalization in the periplasmic space. The organism also produces an acid phosphatase that is activated by CO” (137). Production of several other proteins is also regulated by low-phosphate conditions (109). Another periplasmic enzyme produced by V.parnhnemolyticus is the 5”nucleotidase (138). This enzyme is responsible for the conversion of 5”nucleotidase to nucleoside and inorganic phosphate that can be used to sustain growth of the bacterium. In V.yarnhnenzolyticus, ATP can be used as a sole source of carbon, primarily because of the nucleotide’s conversion by 5”nucleotidase (138). This enzyme activity plays a major role in the cycling of phosphorus in natural waters where the small percentage of attached microplankton bacteria are highly active. Amylase produced by V.ynrahnemolyticus is capable of hydrolyzing dextran, starch, and maltose (160). The enzyme is secreted, regulated by catabolite repression, and expressed to a greater degree during stationary growth (160). This enzyme may give V. pnrahaernolyticus the option of utilizing some large carbon polymers when other carbon sources are unavailable. Defective cyclic adenosine 3’,5’-monophosphate-bindingmutants produce amylase constitutively (41). A respiratory Na+ pump generates an electrochemical potential used to drive transport of nutrients (138,165). This type of electrochemical potential can drive ATP synthesis and polar flagellar rotation, while proton-motive force drives movement of lateral flagella (109a). Chemotaxis occurs in V. parnhnepolyticus, and chemotactic control of lateral and polar flagella are similar (140). The optimal function of the flagella appendages is different and related to environmental conditions (solid/liquid, viscosity).

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VIII. ROLE AND COMPOSITION OF THE CELL ENVELOPE V.pnrnhaemolvticus has a typical gram-negative bacterial cell envelope consisting of inner and outer membranes and a peptidoglycan layer (94,128). The cell envelope plays a vital role in resistance to inhibitory agents, growth, and interactions of the bacterium with elements in the surrounding environment. The outer membrane acts as a permeability barrier to some antibacterials, detergents, and many hydrophobic or large hydrophilic substances. As reported for other gram-negative bacteria, aqueous channels that allow such small hydrophilic compounds as nutrients and some food additives to enter are formed in the outer membrane by proteins. The cytoplasmic membrane, which is the innermost layer of the cell envelope, is surrounded by a thin but rigid layer of murein or peptidoglycan. The area between the cytoplasmic membrane and the outer membrane is the periplasmic space where hydrolytic enzymes and binding proteins are located. Lipopolysaccharides (LPSs) are comprised of lipid A, core oligosaccharides, and 0-antigen-specific side chain and are localized in the bacterial outer membrane. LPS from V. pnruhaen~olyticushas a higher phosphorus content, and its polysaccharide component is shorter than the LPS of Enterobacteriaceae (36,37,58,62). The fatty acids involved in LPS ester linkages are myristic, plasmic, 3-lauroxylauric, and some unsaturated fatty acids, while 3-hydroxy myristic acid is amide linked (134). Lipid A of LPS has endotoxic properties and structural conservation. V. yarnhaernolvticus previously has been reported not to contain 2-keto-3-deoxyoctonate (KDO). Work by Han and Chai has shown that this organism indeed had KDO that was phosphorylated (59). Furthermore, they reported that the LPS of this organism has been demonstrated to lack 0-specific side chain but to contain a large proportion of phosphorylated sugars (59). These results indicate that this organism of marine origin possesses LPS fundamentally different from those of such Enterobacteriaceae as E. coli and Snlmomlln. These unique LPS components could play a significant role in ecology and its survival. Phosphatidylethanolamine (80%) is the predominant phospholipid, with lesser amounts of phosphatidylglycerol(l2- 18%) and cardiolipin (1-5%) (125a, 128) (Table 2). Phospholipid composition is variable and affected by culture condition (128) (Table 2). The predominant fatty acid species of V. yarnhnenzolyticcs are 16:l (40.2%), 16:O (11.2%), and 18:l (38%). V. parahuemolyticus cell-envelope proteins range in size from 27 to 150 kDa (36,94,128). Several of the major proteins are peptidoglycan associated, similar to porins Table 2 Cell Envelope Composition (%) of KP+ and KP- V. parnhaellzolvticus

KP+ strains Cell composition Cell envelope protein Total phospholipid Total lipopolysaccharide Phosphotidylethanolarnine Phosphotidylglycerol Cardiolipin

PPBE broth 50.1 17.0 32.9 86.0 10.6 4.2

Estuarine water 45.2 46.6 8.2 66.2 25.7 11.18.2

W- strains PPBE broth

Estuarine water

53.6 18.1 28.8 80.0

61.5 19.2 19.4 62.4 26.6

14.8

5.2

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of other gram-negative bacteria (128). Growth conditions that alter cell-envelope protein profile are similar to those for other gram-negative bacteria (26a,127a,128). Studies from 14 strains of both K+ and K-, regardless of the Kanagawa phenomenon reaction, demonstrated that proteins in common were 26.9, 27.5, 40.3, 52.4, 53.6, 56.4, and 75.7 kDa when the cultures were grown in rich medium. K+ strains had a much more uniform protein profile and more proteins in common than K- strains. Protein expression is also growth-condition dependent (128). A new major 120 kDa protein is observed in the cell envelope of V.pnrnlznemolyficus when cultured in estuarine water (128). A 30 kDa protein in the outer membrane is a phosphate-regulated porin (108a). To determine the possible intrinsic features of V. pnruhaernolyticus grown in estuarine water, Pace and Chai characterized the cell-envelope composition when cultures were grown in filter-sterilized bay water as compared with those grown in rich medium-proteose peptone-beef extract (PPBE) broth (128). Results shown in Table 2 indicate that cellenvelope composition varied extensively between the cells grown in estuarine water and rich medium for both K+ and K- strains. The cell envelopes contained about 50% protein, 18% phospholipid, and about 30% LPS for both K+ and K- strains. However, when the cultures were grown in estuarine water, the protein content decreased in K+ but increased in K- strains. A remarkable feature is that the LPS content of estuarine water-grown cells drastically decreased in K+ but decreased less in K- strains. This may be one of the possible reasons why K+ strains might not be stable in natural waters andwhy less than 3% of V. yarnhnemolyficus isolated from environmental water and seafood is K+ (84,156). Another interesting point is that estuarine water-grown cells of both K+ and K- strains greatly increased in cardiolipin. The significance of this change is not known, although cardiolipin may stabilize the bacterial membrane through divalentmediated cross-linking. K + strains grown in estuarine water also showed rapid decline in cell numbers after reaching stationary phase, while K- strains maintained the same capacity to absorb light. This suggests that differences between K+ and K- isolates may play a role in the low incidence of K+ isolates from environmental samples.

IX. SURVIVAL IN ADVERSEENVIRONMENT A.

Starvation

Many studies have been conducted on nutrient-deprived bacteria, and the spore form of some gram-positive bacteria is well defined (90a). For such gram-negative bacteria as V. pnrcl}znemol?lticus, a dormant state has been described (135a). The fact that environmental bacteria exhibit this type of starvation response is expected, since many of them live in nutrient-deficient waters (76a). In sterile estuarine water, V. yarn~zaernolyticus’scolony-forming ability declines after 24 hours and only l-5% of cells are metabolically active (acridine orange direct count). Cells able to form colonies decline significantly within 4 days at 35°C (16). At 20°C. colony-forming units persist for much longer periods of time. In contrast, C. jejuni does not become nonculturable at 40”C, but colony formation declines rapidly when cells are nutrient deprived at 37°C (135b). V. yarahuemolyticr~sand other gram-negative bacteria have reduced cell size, express new proteins and enzymatic activity, and exhibit changes in cell membrane phospholipids and membrane function when they are deprived of various nutrients (128). Under phosphate limitation, V. pnl-nllcrel7lolyticlIs produces increased levels of alkaline phospha-

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tase and an outer-membrane protein believed to be a phosphate-specific protein (109,128). When grown in estuarine water, the organism produces a 120 kDa membrane protein of unknown function, increased phosphatidyl glycerol with decreased phosphatidylethanolamine, and reduced levels of RNA (128).

B. InjuryandSurvival During seafood processing or handling, V. pnrnhaernolyticus cells are subject to heat, drying, and rehydration, refrigeration, freezing and thawing, high osmotic environments, irradiation, low pH, preservation, or sanitizers. Damage to the surface cellular structure or cell envelope of gram-negative bacteria has long been known to occur when cells are placed in adverse environments. EDTA and other chelating agents can solubilize or breach the LPS, rendering the cells more permeable to a variety of antibacterial agents and other preservatives. The LPS molecules in the outer membrane of the cells are damaged by freezing, drying, and heating (133). Heat can alter the normal structure and function of the cytoplasmic membrane. Damage to the cytoplasmic membrane upon heating a marine psychrophile results in the abnormal leakage of intracellular materials (61a). Injury to V. pnrnhnernolytic~scaused by cold shock, freezing, and heat (61a,78) was expressed as an increased sensitivity to NaCl in the plating medium (16a). NaCl at 2 or 3% or phosphate-buffer saline is used in the standard method for isolating V. pnrnhaemolyticus from food (43). This salt concentration can recover both normal and injured cells, while only normal V. pnrc~l~ner~zolyticus will grow in the presence of 8% NaCl(l0). Ray et al. reported that the use of a broth containing glucose salt teepol recommended for V. pnrnhae~nolyticusgave decreased recoveries of injured cells stressed by cold at 4°C or freezing at -20°C (132,133). The viable but nonculturable state has been observed in E. coli, C. jejurzi, V. cholerne, S. enteritidis, V. vulnijicus, and other organisms (1 18a,135b,186b). Nutrient deprivation or starvation can induce this physiological state, which is characterized by reduced or no detectable colony formation but with high levels of metabolically active cells observable by microscopy (135a,144). It has been reported with V. vubzificzu that lowering of the incubation temperature to 5°C results in nonculturability even in nutrient-rich medium (1 18a). Reduction in colony-forming ability by shifting V. vulrzijicus to low temperature can be inhibited by prior nutrient deprivation. Nilsson et al. have proposed that this temperature effect results in the inability to isolate V. vubzijiclrs from the coastal waters during the colder months (1 18a). The mechanism of resuscitation is not clear because the resuscitation rate under the current methodology is too low to reach a reliable number. It is likely to be a system of repair or recovery after cell injury or starvation or both. Nevertheless, the impact of viable but nonculturable and injured cells of V. pnrc1hcremolyticrrs is a serious concern in the protection of public health against pathogenic strains. A reliable and effective method for enumeration of viable but nonculturable and injured cells needs to be developed, although much basic information about the mechanism and genetic studies in this area should be worked out first.

X.

CONCLUSION

The recent increase in seafood consumption and growing dependency on aquaculture/ mariculture have made seafood product quality an even more important factor in ensuring

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public health. The inspection and surveillance of these products are also complicated by the different standards found in international trade and the continuous emergence of new, unconventional seafood products. As one of the major bacteria causing seafoodborne poisoning, V. yarahaemolvticms will continue to be a threat to both consumer safety and the seafood industry. The ubiquity of V. pamlzaemolyticus is due to its rapid growth and short generation time, wide range of growth temperatures, nonnutrient requirements, and its diverse habitat. Both pathogenic and saprophytic, V. ynmhaemolyticus exhibits complex functions. Its interaction with a wide range of living organisms emphasizes its importance in the natural habitat and environmental ecology. Many aspects of this organism have yet to be studied or understood. Exactly how V. parahnemolyticus is responsible for foodborne illness is a puzzle to microbiologists and physicians; it is unknown whether infection or intoxication is the cause. Furthermore, scientists have been unable to explain why such high inoculation doses are necessary to induce illness in experimental cases. The existence of a viable but nonculturable state of this organism is another threat to the public health. Strict prevention of cross-contamination is still a tedious task to consumers and seafood processors. The lack of genetic information about this species, which may differ from that of such enteric bacteria as E. coli and Salmonella, also hatnpers the understanding of this species. A key to answering these questions may lie in V. yarahaemolyticus's unique cell-envelope composition and structure, which are different from that of Enterobacteriaceae. What is the role of phosphorylated KDO and sugars in LPS in both the organism and in disease function? Further studies in these directions will help provide basic and practical information about V. parahoemolyticus, thereby improving control over product quality and consumer safety.

REFERENCES Abdelnoor, A. M., and Roumani, B. M. (1980). Characterization of some Vibriopnrnhaemolyticus strains isolated from seafoods in Lebanon. Zentralbl Bakteriol., 170:502-507. 2. Ahsan, N., Conter, R. L., and Appelbaum, P. C. (1988). Postoperative infection associated with Vibrio pardzaemolyticusin a patient without exposure to seawater. J. Clin. Microbiol., 26~1214-1215. 2a Akeda, Y., Nagayama, K., Yamamoto, K., and Honda, T. (1997). Invasive phenotye of Vibrio parahaemolyticus. J. Infect. Dis., 176: 822-824. 3. Baba, K., Shirai, H., Terai, A., Takeda, Y., and Nishibuchi, M. (1991). Analysis of the tdlz gene cloned from a tdh gene- and trh gene-positive strain of Vibrio parahaemolyticus. Microbiol. Imnunol., 35:253-258. 4. Baba, K., Shirai. H., Terai, A. Kumagai, K., Takeda, Y., and Nishibuchi, M. (1991). Similarity of the tdh gene-bearing plasmids of Vibrio choleraenon-01 and Vibrioparalzaemolyticus. Microbiol. Pnthog., 10:61-70. 5. Bandehar, J. R., and Nerkar, D. P. (1987). Stimulation of macrophages by lipopolysaccharide of Vibrio yarahaenlolyticrrs. Mici-obiol. Immunol.,3 1~683-689. 6. Bandehar, J. R., and Nerkar, D. P. (1988). Biological activities of lipid A from Vibrio parahaemolyticus: Stimulation of murine peritoneal macrophages. Microbiol. Irnmunol.. 32:275282. 7. Baross, J. A., Liston, J., and Morita, R. Y. (1978). Incidence of Vibrio pnrahaelnolyticus bacteriophages and other Vibrio bacteriophages in marine samples. Apyl. Emiron. Microbiol.. 36:492-499. 1.

426 8. 9. 10.

11. 12. 13. 14. 15.

16. 16a.

17.

17a.

18. 19.

20.

21.

22. 23.

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189. 190.

191.

192.

193.

194. 195.

196.

197.

198.

199.

437 labeled oligonucleotide probes for detectionof the genes for thermostable direct hemolysin (TDH) andgenes for thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus. Can. J. Microbiol., 38:410-416. Yamamoto, T., and Yokota, T. (1989). Adherence targets of Vibrio parahaenlolyticus in human small intestines. Irzfect. Inzmun., 572410-2419. Yanagase, Y., Inoue, K.. Ozaki, M., Ochi,T., Amano, T., and Chayono, M. (1970). Hemolysins and related enzymesof Vibrio paralzaerttolyticus. I. Identificationand partial purification of enzymes. Biken. J., 13:77-92. Yeh,M., Honda, T.,and Miwatani, T. (1985). Production by non-01 Vibrio cholerae of hemolysin related to thermostable direct hemolysinof Vibrio parahaentolyticus. FEMS Microbiol. Lett., 29:197-200. Yeh, M., Honda, T., and Miwatani. T. (1986). Purification and characterization of a Vibrio hollisae hemolysin that relates to the thermostable direct hemolysin of Vibrio parahaentolytiC M S . Cm. J. Microbiol., 32632-636. Yoh, M., Tang, G. Q., Iida, T., Morinaga, N., Noda, M.,and Honda, T. (1996). Phosphorylation of a 25 kDa protein is induced by thermostable direct hemolysin of Vibrio parahaenzolyticus. Int. J. Biochem. Cell Biol., 28:1365-1369. Yoh, M., Morinaga, N., Noda, M.. and Honda, T. (1995). The bindingof Vibrio paruhaemolyticus ''sI-labeled thermostable direct hemolysin to erythrocytes. Toxicon, 33:65 1-657. Yoh, M., Honda, T., and Miwatani, T. (1986). Purification and partial characterization of a non-01 Vibrio clzolerae hemolysin that cross-reacts with thermostable direct hemolysin of Vibrio parahaenlolyticus. hzfect. Inrmun., 52:3 19-322. Yoh, M., Honda,T., Miwatani, T.and Nishibuchi, M. (1991). Characterization of thermostable direct hemolysins encodedby four representativetdh genes of Vibrio parahnenzolyticus. Microb. Pathog., 10:165-172. Yoh, M.,Miwatani, T., and Honda, T. (1992). Comparison of Vibrioparahaemolyticus hemolysin (Vp-TRH) produced by environmental and clinical isolates. FEMS Microbiol. Lett., 71~157-161. Yoshida, A., Noda,K., Omura, K.. Miyagi, K., Mori, H., Suzuki, N., Takai, S . , Matsumoto, Y., Hayashi, K., and Miyata, Y. (1992). Bacteriological study of traveller's diarrhoea. 4) Isolation of enteropathogenic bacteria from patientswith traveller's diarrhoea at Osaka Airport Quarantine Station during 1984-1991. Kansemhogaku Zusshi, 66:1422-1435. Yu, S . L., and Uy-Yu, 0. (1984). Vibrio paralzaemolyticus pneumonia. Ann. Intern. Med., 100:320.

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18 Vibrio vulnificus Anders Dalsgaard and Lise H0i The Royal Veterinor?)am! Agricultwal UrIiversity. Frederiksberg, Derznm-k

Debi Linkous Burroughs Wellcome Reserrrcll Fund, Raleigh, North Carolina

James D. Oliver UtzilQersityof North Carolirzrr at Charlotte, Charlotte, North Carolina

I. Introduction 440 11. Distribution 340 Geography A. and ecology B. Growth and survival in oysters 441

440

111. Taxonomy.Isolation,andIdentification442 A. Taxonomy 442 B.Isolationusingpreenrichmentbrothsandselectivemedia442 C. Identificationusingserologicalandmolecularmethods447 IV. Pathogenicity 448 Infections with V. Iwlmjiczls (biogroup 1) B. Biogroup 2 strains 448 Virulence C. factors 449 D.Are all strains of V. IwlrziJicus virulent'? Genetics E. 455 F. Infectious dose and susceptible population A.

V.BiologicalandPhysicalControls

of Growth456

Sodium A. requirement 456 Effects B. of pH 456 C. Temperature sensitivity 456 Irradiation D. 458 E. Food additives 458 F. Antibiotics 458 VI.StarvationandSurvival Starvation A.458 B. The viable but nonculturable VII. Conclusion 462 References

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1.

INTRODUCTION

Vibrio vulnijicus, a halophilic marine vibrio, is an opportunistic human pathogen that can cause severe wound infections and septicemia, with mortalities for the latter as high as 60% (1,2). The first case of V. vulrzificus infection was possibly reported in the fifth century B.C. by Hippocrates (3). The king of the island Thasos in the Aegean Sea had an acute infection that was characterized by a swollen foot with red and black skin lesions, rapidly progressive septicemia, and death on the second day. It is suggested that this infection was caused by V. wlnificus (3). Most V. ~?ulrzijicus infections have been reported from the United States, Japan, and Taiwan, although since 1979 a number of cases in Holland, Israel, Belgium, Germany, Sweden, and Denmark have been reported (4-9). In the United States, V. vulnijicus septicemia, nearly always associated with the consumption of raw oysters, is responsible for about 95% of all seafood-related deaths and is the leading cause of reported deaths from any foodborne disease in the state of Florida (10). Following contact with seawater or shell- and finfish, V. vulrzificus also causes wound infections, which often require surgical debridement of the infected tissue and/or amputation. The bacterium has less often been described as a cause of gastroenteritis, and its role as a primary cause of gastrointestinal disease remains to be determined (1 1). The biology and pathogenicity of V. vulnijicus has been reviewed ( 1,12,13). This chapter is limited to the foodborne illness (primary septicemia) caused by V. I'Ulll~fiCLlS.

II.

DISTRIBUTION

A.

GeographyandEcology

V. ~~rrlrzificus can be isolated from a wide variety of aquatic, mainly estuarine, ecosystems, with the occurrence of the organism favored by high temperatures 0 2 0 ° C ) and intermediate salinities (15-25%) (14). V. vulrzificus has been reported on the Atlantic, Pacific, and Gulf coasts of the United States (15- 19). In a comprehensive study of the entire East Coast of the United States, approximately 1% of the culturable vibrios were identified as V. vz~1~zijicus (20). In temperate areas, V. vzrl~~zficus is less abundant than in subtropical waters, but V. ~~ulr.~iJcus has been isolated from coastal waters or implicated in human infections during the summer months in Denmark, Sweden, Germany, Holland, and Belgium (5,7-9,21,22). V. vulniJcus was recently isolated from the Mediterranean for the first time despite a high salinity (35%) that does not favor growth of V. vublijicus (23). However, clinical cases do not appear to have been reported from this area despite the millions of tourists who swim in the Mediterranean each year and the large volumes of oysters that are consumed. It is likely that V. vulniJcus occurs only in very low concentrations in these waters because of the high salinity, and the concentration is apparently too low to cause human infections. Oysters, clams, mussels, fish, plankton, as well as water and sediment have all been described as reservoirs and vehicles for V. vulrlijicus (1,15,20,22,24-26). During the summer months in the U.S. Gulf Coast, DePaola et al. (26) found considerably higher (2-5 logs) densities of V. ~~z11~z~ficus in estuarine fish than in surrounding water, sediment, or nearby oysters and crustacea. The highest densities were found in the intestinal contents of certain bottom-feeding fish ( 108/100g), particularly those that consumed nlollusks and

Vibrio vulnificus

44 1

crustaceans (26). It was suggested that the presence of high densities of V. vuZniJicus in the intestines of common estuarine fish may have both ecological (growth and transport) and public health (food and wound infections) implications. V. vul~~ificus has also caused disease in eel farms in Japan, Spain, Norway, Sweden, and Denmark (27-30). V. vulnijicus has been isolated from waters with temperatures from 7 to 3 1°C and salinities between 1 and 35% (15,23,25,3 1). V. vulniJicus tolerates wide ranges of salinities and temperatures and is abundant in water with temperatures above 20°C and salinities between 15 and 25% (14,17,19,32). Additional factors (e.g., sunlight, pH, nutrient factors, presence of competing bacterial populations, and grazing) may also affect the distribution of V. v u l nijicus in the environment. Grazing by protozoa is one of the main biological processes that control bacterial density in marine environments, but at the present time it is not known to what extent grazing affects the ecology of V. vulnijicus (33). The presence of bacteriophages lytic to V. vulnijicus in estuarine waters have recently been reported in sediments, plankton, shellfish, andthe intestines of finfish from the Gulf of Mexico (34,35). However, the number of plaque-forming units (PFU) did not correlate with densities of V. vulr~ijicus-the lowest number of PFU was found in intestinal contents from fish where the number of V. vulnijicus was highest (34). Oliver et al. (18,20) investigated the distribution and ecology of V. vulnijicus along the East Coast of the United States and found no correlation between the prevalence of V. vu1rzijicus and fecal coliforms. Tamplin et al. (16) described a negative correlation between V. vulniJicus andfecal coliforms; V. vulniJicus was most frequently isolated from samples with fewer than 3 fecal coliforms per 100 d. An inverse correlation between fecal coliforms and counts of V. cholerne was described by Dalsgaard et al. (36).

B. GrowthandSurvival in Oysters As consumption of oysters containing V. vulniJicus may cause primary septicemia, a number of investigations mainly in the United States have been studying the occurrence and susceptibility of V. vhijicus in oysters. During commercial harvest, oysters are typically held on the deck of the harvest vessel without refrigeration or icing until the vessel docks (37). In the United States, regulations by the Interstate Shellfish Sanitation Conference (ISSC) allow up to 10 hours before refrigeration during the warmestmonths (water temperatures >29"C) for oysters not intended for wet storage or depuration (38). However, the time shellstock oysters remain outside refrigeration appears to vary between the Gulf Coast states. Also, a recent comprehensive study of clinical cases in the United States revealed that less than 40% of 72 cases traced to a single site were the result of oysters harvested in waters of >29"C (38). Radu et al. (39) found that V. vuZniJicus failed to multiply in oysters kept at 13°C or below for 30 hours, whereas the numbers of the bacterium were significantly higher when oysters were held at 18°C or higher (see also Sec. V.C). A similar time-dependent decrease in number of recoverable V. vulnijicus cells in oysters during cold storage was found by Cook and Ruple (40). This indicates that endogenous V. vulrlificus can multiply in unchilled shellstock oysters. Similar increases in numbers of V. vulnijicus were found in a later study when the number of V. vulnijicus in summer-harvested oysters held without refrigeration was followed over a 14-hour postharvest period (37). It is therefore clear , that a reduction of the time oyster shellstock remains outside refrigeration can decrease consumer exposure to high numbers of V. vul~ificus,but shellstock must be cooled immediately after harvest to eliminate postharvest growth of V. vulnijicus (37).

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111.

TAXONOMY,ISOLATION,ANDIDENTIFICATION

A. Taxonomy Like other members of the genus Vibrio (family Vibrionaceae), V. 1-~zrZrz(ficus is a gramnegative rod, aerobic and facultatively anaerobic, motile by means of a polar sheathed flagellum, and is oxidase and catalase positive (41). The taxonomy of V. 1~1niJicuswasfirst investigated by Baumann et al. (42). V. vr~lnificzrswas described as a group of gram-negative, fermentative marine organisms, which were assigned to group C-2. Hollis et al. (43) designated the same organism as “lactose-positive Vibrio” or “L+Vibrio” since the ability to ferment lactose was one characteristic that could distinguish this species from Vibrio yarcdmenzolyticus and Vibrio dgirzolyticus. Today it is known that lactose fermentation is negative in up to 25% of the V. vulr.ziJicusisolates (44). Strains within group C-2 were found to be genetically related based on DNA/DNA hybridization and were assigned as a new species designation Berzeckecr vulrzijiccr (vrrlrzificn = wound in Latin) (45). Similar studies performed on the L+Vibrio concluded that this group was a species separate from V. parnhcrenzol?)ticusand V. crlgi~zolyticrrs(46). In 1979, the transfer of Berzeckecr vuln(fica (synonym = L+Vibrio) to the genus Vibrio was proposed and its name became V. 1~1rziJiczrs (47). The name V. 1~1rziJiczrs was given official taxonomic status in 1980 (48). V. wlnificus formerly was often misidentified as V. ycrmlzaelnol?lticus (43). The species V. ~?ulrziJicrrs comprises two biogroups, which in the original definition differed phenotypically, serologically, and in host range (49). V. ~~ulrziJicus biogroup 1 is ubiquitous in estuarine environments and is an opportunistic human pathogen (1,5,3 1). Biogroup 2 is typically recovered from diseased eels butis also reported to cause wound infections in humans after handling eels (5,7). Taxonomic traits for V. vrrlrzificus are shown in Tables 1 and 2. The division into biogroups has recently been questioned, and a division into serovars hasbeen suggested (29). This chapter discusses mainly the originally described biogroup, 1, which is the major foodborne human pathogen (49). B. IsolationUsingPreenrichmentBrothsand Selective Media The choice of including a preenrichment step in isolation of V. ~~ulrzijicrrs depends on four factors: (a) the expected concentration of V. wlnijiczrs in the samples, (b) how precise the results should be, (c) the conditions of the cells, and (d) the level and composition of background flora. Any preenrichment step should improve the ratio of target to background flora before a selective plating step. Preenrichment procedures often give improved recovery of V. ~~ul~zijicrrs compared to plating on selective media, although the choice of procedure should always be dependent on the sample type (24,50-52). The isolation of pathogenic Vibrio spp. is usually accomplished by culture methods that start with preenrichment in alkaline peptone water (APW) (1% peptone, pH 8.6, with 1% NaC1) to recover sublethally injured organisms, followed by plating onto thiosulfatecitrate-bile salts-sucrose (TCBS) agar (53). Early studies of the environmental distribution of V. vulnificzrs, as well as clinical investigations, used this protocol, which was developed for other Vibrio spp. and not optimized for the isolation of V. ~~ulrziJicus (18). Various enrichment broths have been tested for their capability in isolation of V. wlnijicus, includ-

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443

ing APW with various salt concentrations, marine broth, salt-polymyxin B broth, Horie's broth, Monsur's broth, and glucose-salt-teepol broth (24,50,54,55). Overnight preenrichment in APW with 1% NaCl at 35-37°Cgenerally gives the best recovery of V. vuZniJicus, and this procedure is recommended in the Bacteriological Analytical Munual of the U.S. Food and Drug Administration (FDA) (56). The use of APW in combination with cellobiose-polymyxin B-colistin (CPC) agar and modified CPC (mCPC) agar has been reported to be effective in recovering V. vulrzijicus from oyster and water samples (19,57-61). Sun and Oliver (61) found 82% (with correct morphology) of over 1000 colonies probed with a hemolysin gene probe to be V. vulnificus. Figure 1 show a flow diagram for the isolation of v. vulniJicus. Overnight preenrichment in APW withpolymyxin B (20 U/mL; APWP) gave higher recovery rates than preenrichment in regular APW in combination with mCPC agar when analyzing samples of coastal water and sediment in Denmark (22). APWP and mCPC agar was subsequently used with success for isolation of V. vulnijkus from fresh and frozen seafood (3 1,62). Other studies have also reported that different sample types require different isolation strategies for V. vulnijicus (24,51). Arias et al. (50) reported that 3-hour preenrichment in APW with 3% NaCl followed by streaking onto CPC agar was optimal for recovering V. vulrzijicus from seawater and shellfish samples from the western Mediterranean coast and that this culture technique gave more positive results than detection by direct PCR (50). The high salt concentration in the preenrichment may favor isolation of V. vulnijicus cells adapted to the high salinity of the Mediterranean (around 35%). Arguments for using both polymyxin B and colistin in a V. vuln@cus-selective agar have not been provided (58,63). Colistin and polymyxin B are both fatty acyl decapeptide antibiotics with bactericidal activity against most gram-negative bacteria and are known by the name "polymyxins" (64). The chemical composition of colistin and polymyxin B differs only in a single amino acid, and their mode of action and microbiological activity are identical (64). Hoi et al. (65) examined a collection of V. vulniJcus strains for their sensitivity to colistin and recommended a new medium termed cellobiose colistin (CC) agar. CC agar gave a better V. vulrziJicus recovery than TCBS, CPC, and mCPC agar in laboratory studies with pure cultures andwith Danish water and sediment samples. The recovery rate on CC agar was significantly better than on mCPC agar (65). The confirmation rate of presumptive isolates from CC agar was as high as previously reported for mCPC (approximately 95%) when taking into consideration the typical colony morphology of V. wlnijicus on this medium (flat, yellow colonies -2 mm in diameter) (31,65). However, further research is needed to determine whether CC agar enhances the recovery of V. wlrzijicus from oysters compared to current methods. TCBS agar gave a very low plating efficiency (1%) of both clinical and environmental V. vulnificus strains and cannot be recommended for the isolation of V. vulnificus (65). This is in agreement with other reports of low recovery of V. vulnificus on TCBS (66,67). V. vulrzijicus canbe isolated from blood or other clinical samples by culture on several media including blood agar and other nonselective agars. The use of TCBS was recommended when patients present with a compatible diarrheal illness and a history of eating raw seafood (68). However, the recent findings of low recovery rates of clinical V. vulnijicus strains on TCBS agar suggest that this medium should not be used for direct plating of clinical specimens (65).

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Fig. 1 Flow diagram for the isolation of V. vztlnijiczo.

C. IdentificationUsingSerologicalandMolecular Met hods FDA is currently using an enzyme immunoassay (EIA) in an ELISA format to identify presumptive V. vu/rziJicussubcultured from mCPC agar (56). The assay uses a V. vu/tz(ficus-specific monoclonal antibody (mAb) directed against an intracellular epitope of V. vubzijicus (56,58). No cross-reactions with other Vibrio species or non-Vibrio species have been described, and the ELISA format reduces assay time and facilitates handling of large numbers of test samples (58). The cell line producing the V. vulntficus-specific mAb is available at the American Type Culture Collection (ATCC HB 10393). V. vulnijiclrs can be identified one step beyond primary isolation with antiflagellar (anti-H) antibody (69). The agglutination reaction is fast and reliable and has been optimized to include the use of mAbs. However, the anti-core H antibodies are not available commercially, so the technique includes purification of flagellar protein and immunization of rabbits, which is time-consuming (69). Molecular techniques, particularly specific oligonucleotide probes, constitute a very sensitive andspecific tool for detecting V. vulnijicus. An alkaline phosphatase-labeled oligonucleotide probe directed toward the cytolysin gene of V. vulnijicrls was constructed by Wright et al. (70,71) (probe sequence: GAGCTGTCACGGCAGTTGGAACCA). This probe, termed VVAP, demonstrated 100% specificity and sensitivity for clinical and environmental isolates of V. vzh@cus, and numerous investigators have shown that cytolysin is produced by all V. tJulniJicusstrains, including both biogroups, and is species-specific (15,7%74). The sequence of the cytolysin gene has also been used for constructing primers for PCR identification (75,76). Two studies have argued that the use of the cytolysin

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gene as a target region in PCR amplification or as a target for an oligonucleotide probe is not suitable for the detection of V. vulniJcus (77,781. A nonessential gene, such as the cytolysin gene, could theoretically be lost or rearranged without affecting the viability of the bacteria. Instead, it has been suggested that primerdprobes directed against rRNA genes should be used since rRNA molecules are essential constituents of all living organisms and are present in growing cells in very high numbers (77,78).

IV. PATHOGENICITY A.

Infections with V. vulnificus (Biogroup 1)

V. vulniJcus causes both foodborne and wound infections throughout the world, and in the United States it carries the highest death rate of any foodborne disease agent (79). There are approximately 50 foodborne cases per year in the United States that require hospitalization. This bacterium is highly invasive, causing fulminant primary septicemia in persons at risk for infection, with mortality rates of approximately 60% (1). Infection resulting in primary septicemia is associated with consumption of raw shellfish containing the bacteria, especially raw oysters, with symptoms typically developing within 24 hours of ingestion. Death may occur within hours of hospital admission. Individuals who are immunocompromised or who have elevated serum iron levels, typically a result of liver disease (such as cirrhosis or viral hepatitis), are at highest risk for infection by this organism (2). Infections most frequently occur in males [82% of the cases reviewed by Oliver (l)], whose average age exceeds 50 years. The most common symptoms in the primary septicemia form of infection include fever (94%), chills (86%), nausea (60%), and hypotension (systolic pressure < 85% mm; 43%). These values are similar to those reported by Hlady and Klontz (2) in a recent study of 333 patients with Vibrio infections associated with raw oyster consumption in Florida. Hlady and Klontz (2) also found that 94% of patients were hospitalized for up to 43 days (mean of >8 days). An unusual symptom is the development (in 69% of patients) of secondary lesions, typically of the extremities, which often require surgical debridement and/or result in amputation (1). In addition to the primary septicemia that follows ingestion, V. vulnificus is known to infect wounds of otherwise healthy individuals, although the majority of patients with serious wound infections have an underlying disease (1$0). These occur most often as a result of contamination of preexisting wounds with seawater or after contact with fish or shellfish. Wound infection symptoms include localized pain, edema, erythema, with possible severe necrosis of the surrounding tissue requiring surgical debridement or amputation (l). Mortality rates following wound infection are approximately 25% (1,SO). In a review of 11 patients infected with V. ~~ulrzijicus during an unusually warm summer in 1994 in Denmark, Dalsgaard et al. ( 5 ) reported that 4 developed bacteremia, one of whom died, and 9 developed skin lesions. These infections and additional wound infections in 1995 were reported when water temperatures were above 20°C ( 5 ) (Fig. 2).

B. Biogroup 2 Strains While V. vulr~ijic~~s is a pathogen for humans, Tison et al. (49) reported that certain strains isolated from locations in Japan were pathogenic for eels. Recently, biogroup 2 strains have caused major disease problems in Danish eel culture (29,30). This subset of V. vul-

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1996

Fig. 2 Mean water temperatures from 1993to 1996 at three popular recreational beaches inDenmark. The bar shows the time period when the human V. vulrz~ficusinfections occurred in1994 and 1995.

nijkus strains was termed biogroup 2, based on phenotypic differences from the human pathogens that comprise biogroup 1. Biogroup 2 strains have been shown to possess similar virulence factors as biogroup 1, including production of exoproteins, uptake of various iron sources via phenolate and hydroxamate siderophores, and both LPS and capsule expression (81). However, the lipopolysaccharides of biogroup 2 strains are homologous, unlike those of biogroup l, which are heterologous (see Sec. 1V.D). Other differences between these biogroups include the fact that biogroup 2 strains are virulent for eels in either the encapsulated or nonencapsulated form, and nonencapsulated biogroup 2 isolates are able to utilize transferrin-bound iron (81). Recently, Amaro and Biosca (82) reported a V. vu1nZiJicu.sstrain isolated from a human leg wound that wasdetermined phenotypically to be biogroup 2, suggesting that at least some strains within biogroup 2 may also be opportunistic pathogens for humans. Biogroup 2 strains have also been associated with two cases of wound infections in Denmark (A. Dalsgaard and L. H@i,unpublished). There have been no human cases of V. vul~ziJicz~s associated with the consumption of infected eels, and the risks of such an infection appear to be very low. C. Virulence Factors A variety of factors have been implicated as possible virulence determinants for V. vulrzificus, including an extracellular hemolysin/cytolysin, an elastolytic protease, the ability to acquire iron from transferrin, the presence of a polysaccharide capsule and an endotoxic lipopolysaccharide, and resistance to the bactericidal effects of sera (for a review of these putative virulence factors, see Refs. 12 and 13). In addition, V. vulnijicus strains demonstrate a variation between virulent and avirulent forms, with virulent forms being encapsulated, serum resistant, and possessing the ability to acquire iron from iron-saturated transferrin, while avirulent variants lack these characteristics (83).

1. Exoenzymes A large number of extracellular compounds are produced by this bacterium, including hemolysin, elastase, collagenase, DNase, lipase, phospholipase, mucinase, chondroitin sul-

450

Dalsgaard et al.

fatase, hyaluronidase, fibrinolysin, and albuminase (84). An elastolytic protease has been purified and shown to be toxic for mice regardless of the injection route. Recent studies have also implicated V. vulnijicus proteases in the production of bradykinin ( S ) , which is an inflammatory mediator known to increase vascular permeability, cause vasodilation, and induce both pain and contraction of smooth muscle. Such proteases may be important in the intravascular dissemination of this pathogen, allowing septicemia to develop. The hemolysin produced by V. ~~uZ~zijicus strains has been isolated, purified, and shown to be lethal to mice when administered intravenously at concentrations as low as 3 pg/kg. While the hemolysin has been shown to be produced in vivo, a lack of correlation between hemolysin production and virulence has been demonstrated (86).

2. iron Utilization Elevated serum iron levels appear to be a critical element in the pathogenesis of V. vulrzijicus infections, with successful infection apparently requiring an increase in transferrin saturation. Indeed, Wright et al. (87) directly correlated virulence with host iron availability (Fig. 3). V. vdrzijicus does not appear able to grow in normal human serum (Fig. 4), while iron injections into mice prior to the injection of bacterial cells significantly lowers the LD50(Table 3). V. ~ ~ u l ~ z ~ fsimultaneously icus produces both phenolate and hydroxamate siderophores, with the phenolate siderophore enabling virulent isolates to acquire iron from highly saturated transferrin (88,89). Similar results have been reported for other ironbinding proteins such as lactofen-in and ferritin (89). It appears that cleavage of these proteins is caused by an exoprotease, resulting in iron release to the siderophore(s). Confirming that iron acquisition is required for V. vulrzijicus virulence, Litwin et al. (88) showed that a mutagenized virulent strain that lost phenolate siderophore production exhibited reduced virulence.

0

24

48

72

Time of Vibrio injection (h) Fig. 3 Effectofelevated serum iron levels on V. 1~1rziJicusinfection after chloroform (CC14) treatment (produces transient liver damage). Inocula of V. vulrtijiczrs (lo3,lo5, loqcfu) were injected for inocula of 10' and lo5 at 0, 24, 48, and 72 h after the injection of CCl.,. Increased mortality cfu correlated directly with increased serum iron (SI) levels, which were monitored over the same time period. Inocula of lo9 cfu always produced 100% mortality. (Adapted from Ref. 87.)

Vibrio vulnificus

451

10%

0

107J

3E

106-

105-

104-

0 -"- HS

1034

-0-

HS+O.Ol mg/mL

102-

+"

HS+O.I mg/mL

O I '

0

I

1

2

4

-

1

6

1

8

Time (h) Fig. 4 Growth of V. vulrzijicus in human serum. Pooled human serum (HS) was inoculated with V. vz~lniJicuswith and without iron additions (ferric ammonium citrate). (Adapted from Ref. 87.)

3. Capsule The presence of a capsular polysaccharide (CPS) is the best studied virulence factor of V. vuZmj?cus and is essential to its ability to cause human infection. Kreger et al. (90) demonstrated that an "antiphagocytic surface antigen" allows virulent V. 1~uZr2iJicusstrains to resist phagocytosis by human polymorphonuclear leukocytes. This antigen was subsequently shown by electron microscopy and ruthenium red staining to be an acidic polysaccharide capsule.

Table 3 Effect of Iron on LD50Determinations" Number dead Log I O inoculum

PBS

Fe"

8 7

6 5 4 3 2

5 5 5 5 4 4 0

1

0 -1

LD,,

=

1

X

10'

V. vulrtificus was inoculated into groups of mice (n = 5 ) at levels varying from lo8 to 10" cfu, concurrently with either phosphatebuffered saline (PBS) or iron (ferric ammonium sulfate at 4 pg/ g body weight). Source: Adapted from Ref. 87.

452

Daisgaard et ai.

Studies of virulent and avirulent strains have found a correlation between virulence and colony opacity (Fig. 5). All virulent strains are of the encapsulated, or opaque, colony type, whereas nonencapsulated, or translucent, cells are avirulent (83), supporting the presence of capsule as an explanation for resistance to phagocytic activity. Interestingly, we have observed that encapsulated cells mutate at a very high rate (typically 10-2-10-3) to produce nonencapsulated cells, with the loss of capsule correlating with loss of virulence. Whether such mutations are common in natural communities or are a consequence of laboratory manipulation is not known at this time. Reversion of translucent cells to opaque cells has also been reported in some strains, but at a very low (
4. Endotoxin The symptoms of V. ~)zrZnijicz~s septicemia, as well as the inflammatory response observed in wound infections, are typical of the endotoxic activity of LPS tnolecules, suggesting this molecule may be a major virulence factor. Indeed, McPherson et al. (91) demonstrated that intravenous injections of V. ~)ulniJicus LPS (-400 pg/kg body weight) in mice caused mean arterial pressure to decrease within 10 minutes, with death occurring in 30-60 minutes. This was similar to the response seen when equivalent amounts of LPS from Salmonella typhimuri~mwere injected. We now understand that LPS exhibits its toxic effects as a result of an overproduction of tumor necrosis factor (TNF), which subsequently leads to an overstimulation of nitric oxide synthase. In fact, increased mortality in cirrhotic mice due to V. vzhijicus infection is dependent on an in vivo TNF response (92). Purified V. vulnijicus LPS causes severe hypotension and death within one hour when injected into rats (93) (Fig. 6a). However, when a nitric oxide synthase inhibitor is administered to rats along with LPS, the lethality of the endotoxin is reversed (Fig. 6b). These data suggest that the stimulation of

Fig. 5 Opaque and translucent colonies of V. wZmj5cus on heart infusion agar. (From Ref. 83.)

Vibrio vulnificus

453

LPS 300 250

-

200

-

I

150 -

100

-

50 0 7 -20 0

l"

20 4 0 6 0 8 0 100 120 140 Time (minutes)

LPS

Q)

r

"\

,

I

0 -20 0

,

,

,

,

20 4 0

,

+

L-NMMA

I . , 60

160

,

I . ,

,

,

,

0

8 0 100 1 2 0 1 4 0 16C

Time (minutes)

Fig. 6 (A) Effect of intravenous injection of V. vulrtiJicusendotoxin (1 mg/kg body weight) on heart rate in male rats (11 = 5). (B) Reversal of endotoxin effects on heart rate through the administration of N-monomethylarginine (NMMA). Endotoxin (1 mg/kg body weight) injected into male rats (n = 5) at L, with N " A (20 mg/kg body weight) infused 10 minutes later at N. (Adapted from Refs. 91. 93.)

TNF, followed by nitric oxide production, leads to fatal endotoxic shock in response to V. vulnijicus LPS. No significant differences in chemical (94), electrophoretic (95), or resistance to phagocytosis (96) have been found between the LPS of virulent and avirulent V. vulr2iJicus strains. Also, it has not been possible to demonstrate a correlation between LPS type and colony morphology. These data suggest that avirulent strains are unable to produce infection not because they lack endotoxin, but because they lack the antiphagocytic capsule required to initiate infection of the host. Indeed, a comparison of rough and smooth V. vdnijicus lipopolysaccharide phenotypes suggests that rough isolates may be superior inducers of TNF compared to their smooth counterparts.

454

Dalsgaard et al.

One of the more interesting aspects of the disease caused by V. vuZr~iJicusis the significant difference in the rates of primary septicemia between males and females [82% of the cases reviewed by Oliver (1 ) occurred in males]. In an attempt to understand this difference, Huet-Hudson and Oliver (unpublished) examined whether there exist any gender differences in susceptibility to endotoxin. Following up on our previous studies (91,93), crude LPS extractions were injected intravenously into male and female rats at a dosage that was fatal to the majority of males. To our surprise, whereas 82% of the male rats died as a result of the injections, only 2 1% of the females died. When the females were ovariectomized and their estrogen levels allowed to deplete, their fatality rate increased to 75%. Ovariectomized females which were subsequently given estrogen exhibited decreased fatality in response to LPS injection. Further, male rats given estrogen exhibited a significantly lower mortality rate than normal rats. Thus, it is clear that the host response to V. vulr~ijiczrsLPS is significantly different for male and female rats, and while the exact mechanism of this response difference is not understood, these studies suggest an explanation for why males who consume raw oysters constitute the major risk group for V. ~ ~ Z n i J cprimary z~s septicemia.

5. Phagocytosis Multiple capsular types have been shown to exist for V. vu1nijicus (97,98), and pathogenicity in mammals is highly correlated with the presence of the antiphagocytic capsule. V. V U Z I Z ~ C Z ~ Shas been shown to be resistant to phagocytosis in the absence of serum opsonins. However, a correlation between V. vz~lnijicusvirulence and resistance to the opsonizing effects of normal human serum has also been reported, contributing to the host’s susceptibility to infection. Thus, the capsular polysaccharide of V. ~wlrzijicusstrains has the potential to modulate both the classical and alternative antibody pathways, which likely plays a major role in serum resistance.

D. Are All Strains of V. vulnificus Virulent? While V. wlrzijicus alone is responsible for 95% of all seafood-related deaths in the United States (l), it is unclear why more people don’t develop this infection. Many people would be expected to be susceptible to this bacterium based on the number of individuals who consume raw oysters and/or are exposed to seawater and are also known to have one of the underlying conditions that predisposes them to infection (2). However, only about 20 deaths are recognized in the United States each year from this bacterium. A possible reason for this discrepancy may be that notall strains of V. vzrhzific~sare identical in their capacity to cause infection. In fact, Buchrieser et al. (99) examined 118 strains of V. ~~ulnijicus isolated from three oysters and reported that no two strains had the same DNA profile based on clamped homogeneous electric field (CHEF) gel electrophoresis. Despite this, Jackson et al. (100) reported isolation of only a single V. vz~Zu~ficz4s strain (based on pulsedfield gel electrophoresis) from human tissues associated with disease. Thus, while unidentified host factors may be important for the development of disease, these studies suggest that there are significant genetic differences between strains and that all strains of V. vulr~ijiczrsmay not beequally pathogenic. Also, unidentified host factors may be important for the development of disease (see Sec. 1V.E). Of the many possible virulence factors that have been studied for V. ~~zrZrziJicus, the two that clearly seem most important in initiating infection are LPS and capsule expression. Because multiple CPS and LPS types have been shown to exist for V. vulrzijicus, it

Vibrio

vulnificus

455

is possible that certain capsule or LPS types are required for human infection. However, using mouse macrophages, Linkous et al. (96) were unable to detect any differences in resistance to phagocytosis among the 10 LPS serotypes or the 5 CPS serotypes identified in Siebeling's laboratory at Louisiana State University (101). While it is clear that virulence in V. vuhzijcus requires capsule, and likely endotoxin, these two factors do not appear to be sufficient to cause infection. This is based on our observation (unpublished) that a strain used in the laboratory for over 10 years lost all virulence for mice, yet exhibited no change in either CPS or LPS serotype compared to another isolate of the same strain that retained virulence (LDso< 10 cfu). Warner and Oliver ( l 02) have recently described the presence of a PCR-amplified DNA fragment that is present in 100%of clinical isolates but in only 13% of environmental isolates. It is tempting to speculate that this fragment is a portion of a gene that is essential to the virulence of V. vltlrziJicrrs.Such a situation would be similar to that of the Kanagawa hetnolysin present in all clinical isolates, but few environmental isolates, of V. ynrc~hccemolyticus.

E. Genetics Few researchers have studied the genetics of V. vulrz$czls, although what is known appears to be quite unique. Using both the randomly amplified polymorphic DNA (RAPD) PCR method and ribotyping, H@iet al. (103) reported that the 79 biogroup 1 isolates of V. vulniJicus examined were genetically very heterogeneous. Similarly, Warner and Oliver (102) found that all 70 V. vulnificzrs strains they examined by RAPD PCR produced unique banding patterns. These observations agree with several other studies that have employed similar or other methods for examining the genetic make-up of this species, including ribotyping. arbitrarily primed PCR, clamped homogeneous electric field gel electrophoresis, pulsed fieldgel electrophoresis, and amplified fragment length polymorphism (5,39,33,99,104).These observations suggest that all V. vulr?iJicusbiogroup 1 isolates are genetically very heterogeneous and that genomic rearrangements tnay be common in this species. Genotypes of V. vzhificus biogroup 2 strains appear much more homogenous than biogroup 1 strains. However, a recent study by H@iet al. (29) demonstrated a high degree of heterogeneity among biogroup 2 isolates of V. vuh[ficzts recovered from diseased eels in Denmark. Interestingly, when cells of V. vrrZ~zificusare starved or induced into the viable but nonculturable (VBNC) state (see Sec. VI.B), a loss of PCRamplification signals occurs (105). Whether this loss is also due to genomic rearrangements occurring during entry into these states is not yet understood.

F. InfectiousDoseandSusceptiblePopulation In almost all V. vzhiJicLIs infections resulting from the ingestion of raw oysters, the patient had an underlying chronic disease (1). The most common (80%) of these is a liver- or blood-related disorder, with liver cirrhosis secondary to alcoholism or alcohol abuse being the most typical (1,106). These diseases typically result in elevated serum iron levels, and laboratory studies have shown that elevated serum iron plays a major role in infection with V. Iwlm@ls (87). Other risk factors include hematopoietic disorders, chronic renal disease, gastric disease, use of immunosuppressive agents, and diabetes. Further, while

Dalsgaard et al.

456

cases in children have been reported, infections tend to be in males [82% of those reviewed by Oliver (l)] whose average age exceeds 50 years. The infectious dose of V. vulniJcus is not known. Using a mouse model, however, some insight to this question may be available. Wright et al. (87) observed that when mice were treated to produce serum iron overload, the LD5"decreased from approximately lo6 to a single cell. In a variation of these studies, the administration of small amounts of CC1, to produce short-term liver necrosis was found to increase serum iron levels, with a direct correlation between serum iron levels and lethality observed. These data agree with epidemiological studies, indicating that liver damage, and sometimes immunocompromising diseases, are major underlying factors in the development of V. vulnificus infections. These studies further suggest that extremely low numbers of this pathogen may be sufficient to initiate potentially fatal infections.

V.

BIOLOGICAL AND PHYSICALCONTROLS OF GROWTH

In comparison with V. cholel-ue and V. pnr.nlznenloZ~lticlIs,relatively little research has been conducted about the biological or physical controls of growth of V. vulniJcus. Summarized here are some of the studies that have been conducted.

A.

SodiumRequirement

While V. ~?uZniJcusis an obligate halophile, this requirement appears to be fulfilled by approximately 0.5% NaCl, a level typically found in most bacteriological media. Optimal NaCl concentrations appear to be approximately l-2%. The bacterium does not appear to have a demonstrable requirement for other cations or anions greater than those present in most media; thus, cultivation of this pathogen is not normally difficult. Growth at NaCl levels of 6% is possible but does not generally occur at 8% (Table 1).

B. Effects of pH Optimum growth of V. vulnijicus is in the mid-7 to mid-8 pH range. As with other vibrios, V. vulrzijicus survives and grows much better in alkaline than acidic pHs. Hijarmbia et al. (107) examined the effect of low pH (5.0) on survival of V. vuZrziJicus and reported that this stress resulted in an immediate loss of culturability on TCBS agar but did not produce cellular lysis. Most of the cells that had lost culturability retained viability, however, even when no cells could be cultured on this selective medium.

C.TemperatureSensitivity Like other vibrios, V. vulnificus is quite heat sensitive, with exposure to temperatures above 45°C causing death (but see Sec. V1.A on starvation and its effects on temperature sensitivity). Cook and Ruple (40) reported an average decimal reduction time at 47°C of 78 seconds for the 52 strains they studied. In shellfish, heating to produce an internal temperature of at least 60°C for several minutes appears sufficient to eliminate the pathogenic vibrios, with Cook and Ruple (40) reporting V. vuZr$cus to be eliminated from oysters when heated for 10 minutes at 50°C. This treatment did not appear to impart a

Vibrio vulnificus

457

noticeable cooked appearance or taste to the oysters and was proposed as a strategy to improve the safety of raw oysters. Very few reports exist on the survival of V. vulnijicus in seafood at low temperatures. Cook and Ruple (40) reported that V. vulnificus cells naturally occurring in oysters underwent a time-dependent decrease in recovery when either shellstock oysters or shucked oyster meats were held at 4, 0, or - 1.9"C. Cook (108) later found that, after harvest, V. vulnificus failed to grow in shellstock oysters stored at 13°C or below. In oysters held at 18°C or higher for 12 or 30 hours, however, V. vulnijicus numbers increased (see also Sec. 1I.B). Little is known regarding the effects of freezing on V. vulnificus in oysters. Panagaya and Oliver (unpublished) inoculated a TnphoA-marked strain of V. v~lnificzrsinto oysters, which were then held at 5, - 15, or -85°C. Results (Fig. 7) indicated a rapid die-off of the bacterium during the first 24 hours at all of these temperatures, with a more gradual decrease in viability over the remaining 7-day period of the study. This decrease was similar to that exhibited by the natural microflora present in the oysters (data not shown). Cook and Ruple (40) similarly reported that the organism remains culturable from oysters frozen at -20°C for 12 weeks. The persistence of V. vulnificus in oysters following freezing and storage at -2O"C, with or without vacuum packaging, was found to be dependent on the length of frozen storage time for cells packaged without vacuum, with a decrease from -lo5 to 10' cfu/g (109). Vacuum-packaged samples showed significantly lower concentrations of V. vulrzificus over a 70-day study period as compared to normal-packaged samples. Little is known about the prevalence of V. vulnificus in frozen seafood. However, Dalsgaard and Hoi (62) analyzed frozen shrimp products imported from Southeast Asia and found that only 3 of 46 (7%) frozen raw products and none of 61 frozen cooked products contained V. vulnificus (62). They concluded that the absence of V. vubziJicus in frozen cooked shrimp products and the surprisingly low prevalence of V. vulnificus in frozen raw shrimp, taking into consideration the autochthonous nature of V. vulnificz~s in tropical waters, suggest that V. vulnificus in imported shrimp products does not constitute a hazard to public health.

-

106 105

104

103

l

0

2

4 6 Time (days)

8

Fig. 7 Survival of V. vulnijkus in frozen oysters. Oysters were inoculated with a TnphoA-marked strain of V. ~~Ir~iJjcus, then frozen at 5. - 15, or -85°C. Samples were removed, thawed, blended, and examined for the presence of V. vulniJicus.

Dalsgaard et al.

458

D. Irradiation The use of ionizing radiation for reducing levels of V. vulnijicus in shellstock oysters has been studied by Dixon (1 10). He found that a dose of 1 kGy resulted in a decrease in over 5 logs, with no mortality in the oysters. Higher doses (e.g., 1.5 kGy) resulted in a total loss of V. vuZniJicus but carried increased oyster mortalities.

E. FoodAdditives Of 10 GRAS (generally recognized as safe) compounds tested against V. 1wlnijicIds, we found only lactic acid and diacetyl to be inhibitory in vitro. A subsequent study found only diacetyl to have a significant effect on the cells when present in shellstock oysters (1 11). In response to a report that V. vuln~ficuscould be killed through the application of cocktail or Tabasco@sauces to raw oysters, Sun and Oliver (112) examined the sensitivity of this species to a commercial horseradish-based sauce and to Tabasco sauce. Results indicated that, while Tabasco sauce (but not cocktail sauce) was highly effective in reducing the number of V. vuhijicus cells present on the oyster meat surface, little reduction in numbers present within the oysters occurred with either sauce. F. Antibiotics V. vulnijicus is sensitive to most commonly employed antibiotics (1 13). Various antibiotics including tetracycline (1 14- 117) and third-generation cephalosporins (118) have been recommended for the treatment of serious V. vz~lnijicusinfections. Dalsgaard et al. ( 5 ) demonstrated that a wide range of antibiotics were effective when wound isolates were studied in vitro. However, in cases with serious wound infections surgical debridement is essential.

VI.

STARVATIONANDSURVIVAL

A.

Starvation

When placed into a nonnutrient environment such as artificial sea water (ASW), V. v z d nijicus demonstrates a remarkable starvation-survival response. Figure 8 shows cells of three different V. vulnijicus strains that have been held at room temperature in ASW for over 8 years. Following a decrease in culturability during the initial 100 days, the cells have remained amazingly constant in their viability throughout the 8-year period. The reason for this prolonged survival is the ability of the cells to produce starvation-induced stress proteins. Such proteins are known to aid in the survival of nutrient-depleted cells (1 19) and to provide “cross-protection” against unrelated stresses (1 20). In the case of V. vul~tijicus,our studies have revealed the production of at least 34 proteins during the initial 26 hours of carbon starvation (121). Chloramphenicol studies indicated that, of the 34 proteins, those induced during the first 4 hours of starvation were essential for survival in the face of starvation stress. The response of V. vuln$kus to a combination of starvation and osmotic stress results in significant cross-protection against a variety of unrelated stresses (D. Smith and J. D. Oliver, unpublished). When cells are subjected to an osmotic upshift (e.g., a shift from the 0.5% NaCl content of heart infusion broth to ASW), coupled with a nutrient

Vibrio vulnificus

459

* CVD713t (#l) lEa8I 1E+07

1EN6 J

E 3

e

C7184t (#3)

_e_

CVD713t (H)

1EN5 1E+04

yl

0

1E43

l1 E 4 1 0

E

lo00 M 2000

2 3000

1

Time (days) Fig. 8 Long term survival of V. vrrlnijkus. Washed cells were inoculated into artificial seawater (ASW) and maintained in the dark. Samples were periodically tested for culturability at intervals over a 3 100-day period.

downshift (e.g., from heart infusion broth to ASW), the cells rapidly become resistant to the normally lethal effects of a 45-47°C heat treatment (Fig. 9). Indeed, significant protection is observed within 10 minutes of these combined stresses. Interestingly, neither stress alone induces this cross-protection; both the osmotic upshift and nutrient downshift are required. We have also found that this double stress leads to protection against ionic challenge (2.75 M NaClj, to 10% ethanol, and to oxidative challenge (0.2 mM H2O2j.Of potential significance for pathogenesis, we have observed that starved V. vulniJcus cells become resistant to the bacteriostatic/bacteriocidal effects of human serum (Fig. 10). In a separate study, we exanlined the ability of starvation to protect against a physical stress, that of sonication. As seen in Fig. 11, cells of V. vulnificus that were nutrient depleted developed resistance to the lethal effects of sonication, with 48 hours of starvation providing maximum protection. When nutrient was returned to the cells (at 96 h), resistance to sonication was rapidly lost. B. The Viable but Nonculturable State As exemplified by the study shown in Fig. 2, a strong correlation between cases of V. vulnijicus and water temperature has consistently been observed by nunlerous investigators. This correlation also agrees with epidemiological studies on the infections caused by V. wlrzijcus in the United States, which revealed a distinct seasonality (38). Because of this seasonality and the inability to isolate V. vulr~ijkusfrom water or oysters when water temperatures are low, there has been considerable investigation of the apparent dieoffof this species during cold weather months. While it is possible that V. whz[jicus overwinters in certain environments [e.g., DePaola et al. (26j reported its isolation during the winter from sheepshead fish], this seasonality is now attributed by many investigators to a cold-induced “viable but nonculturable” (VBNC) state. In the VBNC state, cells remain viable but are no longer culturable on the routine media normally employed for

Dalsgaard et al.

460

0.01 O - 1

0.001

;

l

I

1

I

I

0

10

20

30

40

50

60

Heat Stress at 45°C

(min)

Fig. 9 Survival of V. wlnificrls cells against heat stress (45°C) following starvation times of 0 (0) minute, 10 (m) minute, 20 minute (0),30 minute (O), 45 minute (O),and l hour (+).

their isolation (Fig. 12). This phenomenon, which has now been demonstrated in at least 16 genera, has been the subject of several recent reviews (122- 124). When the temperature downshift that induces this state in V. r~ulrzijicr~s is reversed, the nonculturable cells "resuscitate" back to the metabolically active and fully culturable state (125). Thus, resuscitation is believed to account for the reappearance of cells of this species in seawater and oysters when water temperatures rise (126). Entrance into, and resuscitation from, the VBNC state by V. vulniJicrts in a natural estuarine environment have been demonstrated (127). Oliver and Bockian (128) have also reported that cells of V. vulnijicus injected into mice in the

10000 I

cp

>

-g

1000

U) +.)

c Q)

f

100

n

1

L

10

Starvation Time (h) Fig. 10 Survival of V. vzrlnijiczrs following l-hour incubation in complement-inactivated human plasma following starvation for various periods in nutrient-free artificial seawater.

Vibrio vulnificus

461

Starvation Time (h) Fig. 11 Survival of V. vzhiJicus cells against physical stress (sonication) following carbon starvation. Cells starved in ASW were removed at different times of starvation, sonicated, and plated to determine the percent survival compared to control cells of the same population that were not sonicated. At the pointindicatedbythearrow,nutrient was returnedto the starvedpopulationand samples again removed at intervals and sonicated.

fully nonculturable (VBNC) state were capable of producing lethal infections in mice, with culturable cells being subsequently recovered from the dead animals. This indicates that resuscitation of VBNC cells within the host is possible, although such a scenario does not appear to be common, as evidenced by the low number of cases that occur during the cold months. The ability of VBNC cells to resuscitate when the stress (cold temperature) is iitninated or if reappearance is due to regrowth of a few nondetectable culturable cells has been debated in several studies (122,127,129-132). Recently, Bloomfield et al. ( 133) sug-

-

I

Q)

0

103102-

10" 0

2

4

6

8

Time (days) cells of V. ~~ZniJicus when Fig. 12 The typical VBNC response exhibited by exponential phase grown in heart infusion broth at 22 or 37°C and subsequently inoculated into an artificial seawater microcosm held at 5°C. Colony-forming units (0)on heart infusion broth, direct viable counts (U), and direct cell counts ( 0 )are used to demonstrate the phenomenon.

462

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gested a new plausible model to account at least partially for the VBNC phenomenon (134). They hypothesized that VBNC cells are not “unculturable” but that “we are simply failing to provide appropriate conditions to support culture” (133). They proposed that “sudden transfer of cells to nutrient-rich agar at temperatures optimal for enzyme activity initiates an imbalance in metabolism, producing a near-instantaneous production of superoxide and free radicals. In the absence of phenotypic adaptation, the cells are not equipped to detoxify superoxide. As a result, a proportion or all of these cells die.” Whether entry into the VBNC state is an active process developed for survival in the environment has yet to be conclusively shown. Weichart et al. (135) reported that the great majority of cells of V. vzrhijicus exhibit significant degradation of DNA and RNA as they enter the VBNC state. They also stated that “the integrity of ribosomes and nucleic acids may be maintained in a small fraction of the population for much longer than would be anticipated based on the ability of the cells to grow and divide, and these cells may retain the ability to recover and infect a suitable host.” As is the case with starvation, however, it isknown that cold stress proteins are induced in V. vulIziJicuson temperature downshift (1 36) and these might lead to crossprotection against environmental stresses. In fact, Weichart and Kjelleberg (132) have reported that the final stress resistance of cold-induced VBNC cells to sonication and ethanol exposure was equal to that of starved cells.

VII. CONCLUSION The microbiological parameters in seafood quality assurance programs have traditionally included analysis for the important human pathogens Vibrio pnrnhcrenlol~ticzw and Vibrio cholerc~e.However, other foodborne pathogenic Vibrio spp. are associated with human disease. V. vu11zijic~1s causes both foodborne and wound infections throughout the world. Although the bacterium is highly invasive, causing fulminant primary septicemia with mortality rates of approximately 60% in persons at risk for infection, such infection is only associated with the consumption of raw shellfish, mainly oysters, containing the bacterium. Studies from the U.S. Gulf Coast show that although oysters may contain high numbers of V. vulrziJcus ( 10”-104/g) and millions of people are consuming oysters, only about 30-50 cases associated with the consumption of shellfish are registered annually. Septicemia has not been associated with the consumption of any frozen seafood products or fresh finfish. V. ~wl~ziJicz4s has in a few cases been isolated from patients with gastrointestinal disease, however, its role as a primary cause of gastrointestinal disease remains to be determined. Analysis for V. vulI~ificusshould therefore not be part of a standard seafood quality assurance program except for molluscan shellfish intended for raw consumption. The presence of V. vulnijicus is notan indication of fecal pollution because the bacterium is part of the normal marine aquatic flora in mainly estuarine ecosystems with the occurrence of the organism favored by high temperatures (>20°C) and intermediate salinities (1 5-25%). Environmental and clinical strains both show a high degree of heterogeneity in phenotypic and genotyping tests, with few differences being found in their pathogenic potential using experimental animals. However, the low number of reported cases compared with the persons exposed to high numbers of V. v u l ~ ~ ~ f ithrough cus consumption of oysters suggest that all strains of V. ~~r~lrzificus are not equally pathogenic or that not all individuals in the defined risk groups are equally susceptible.

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Virulence is clearly dependent on the presence of an antiphagocytic capsule and most likely the endotoxin. Successful infection also generally requires certain underlying host syndromes, which predispose to this pathogen. Infection of such high-risk individuals carries an extremely high fatality rate, however. Numerous questions remain concerning this organism, including the role of the viable but nonculturable state in the epidemiology of the infection. The response of this organism to environmental stress may also prove to be a factor in disease production. Finally, the genetic heterogeneity of isolates of V. vulnificus should be noted. How can such variation exist for a single species? What is the role, if any, of the gene rearrangements that appear to be commonplace in this bacterium? And possibly most important, what tnechanisms of control can be applied to oysters such that the highly fatal primary septicemias produced by V. vuZniJcus can be reduced or eliminated?

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Dalsgaard et al. induced by Vibrio vulnificus lipopolysaccharide in the rat by inhibition of nitric oxide synthase. Microbiol. Pathog., 13:391-397. Bahrani, K., and Oliver, J. D. (1990). Studies on the lipopolysaccharide of a virulent and an avirulent strain of Vibrio vulnijicus. Biochem. Cell. Biol., 68:547-55 1. Bahrani, K. F., and Oliver, J. D. (1991). Electrophoretic analysisof lipopolysaccharide isolated from opaque and translucent colony variantsof Vibrio vzrlnifcus using various extraction methods. Microbios., 66:83-93. Linkous, D. A., Simpson, L. M., and Oliver, J. D. (1997). Comparison of pathogenicity among Vibrio vulnijiczls strains based on capsular and LPS serotypes. 97th General Meeting of the American Society of Microbiology, Miami Beach, FL, abstract B-210. Hayat, U., Reddy, G.P., Bush, C.A., Johnson, J. A., Wright,A. C., and Morris, J. G. (1993). Capsular types of Vibrio vulnijicus: An analysis of strains from clinical and environmental sources. J. Infect. Dis., 168:758-762. Simonson, J. G., and Siebeling, R. J. (1993). Immunogenicity of Vibrio vzclnifcus capsular polysaccharides and polysaccharide-protein conjugates.Infect. Imntun., 612053-2058. Buchrieser, C., Gangar, V. V., Murphree.R. L., Tamplin, M. L., and Kaspar, C. W. (1995). Multiple Vibrio vzhijiczrsstrains in oystersas demonstrated by clamped homogenouselectric field gel electrophoresis. Appl. Environ. Microbiol., 61:1163-1168. Jackson, J. K., Murphree, R. L., and Tamplin, M. L. (1997). Evidence that mortality from Vibrio vukzijicus infection results from single strains among heterogeneous populations in shellfish. J. Clin. Microbiol., 35:2098-2101. Martin, S. J., and Siebeling, R. J. (1991). Identificationof Vibrio vulnijicus 0 serovars with antilipopolysaccharide monoclonal antibody.J. Clin. Microbiol., 29: 1684- 1688. Warner, J. M., and Oliver, J. D. (1999). Randomly amplified polymorphic DNA analysis of clinical and environmental isolatesof Vibrio vu111iJicusand other Vibrio species. Appl. EnviYon. Microbiol., 65:1141-1144. Hgi, L., Dalsgaard, A., Larsen, J. L., Warner, J. M., and Oliver, J. D. (1997). Comparison of ribotyping and randomly amplified polymorphic DNA PCR for characterizationof Vibrio vulnijicus. Appl. Environ. Microbiol.. 63: 1674-1678. Arias, C.R., Verdonck, L., Swings, J., Garay, E., and Aznar, R. (1997). Intraspecific differenand ribotyptiation of Vibrio vulnijicusbiotypes by amplified fragment length polymorphism ing. Appl. Environ. Microbiol., 63:2600-2606. Warner, J. M., and Oliver, J. D. (1998). Randomly amplified polymorphic DNA analysis of starved and viable but nonculturable Vibrio vulnijicus cells. Appl. Environ. Microbiol., 64: 3025-3028. Desenclos, J. A., Klontz, K. C . , Wolfe, L. E., and Hoecheri, S. (1991). The risk of Vibrio illness in the Floridaraw oyster eating population, 1981-1988.Ant. J. Epidentiol., 134290297. Hijarrubia, M. J., Lazaro,B., Sunen, E., and Fernandez-Astorga,A. (1998). Survivalof Vibrio vulnifcus under pH,salinity and temperature combinedstress. Food Microbiol, 13:193-199. Cook, D. W. (1994). Effect of time and temperature on multiplication of Vibrio vulnijicus in postharvest Gulf Coast shellstock oysters. Appl. Environ. Microbiol., 60:3483-3484. Parker, R. W., Maurer, E. M., Childers, A. B., and Lewis, D. H. (1994). Effect of frozen storage and vaccum-packaging on survival of Vibrio vulnijicus in Gulf Coast oysters (Crrtssostrea Iirginica). J. Food Prot., 57:604-606. Dixon, W. D. (1992). The effects of gamma radiation (6nCo) upon shellstock oysters in terms of shelf life and bacterial reduction, including Vibrio vulnjficus levels. M.S. thesis, Univ. Florida. Sun, Y . , and Oliver, J. D. (1994). Effects of GRAS compounds on natural Vibrio vulnijicus populations in oysters. J. Food Prot., 57:921-923. Sun, Y., and Oliver, J. D. (1995). Hot sauce: No elimination of Vibrio ~~ulnijicus in oysters. J. Food Prot., 58:441-442.

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113. Farmer 111, J. J., Hickman-Brenner, F. W., and Kelly, M. T. (1985). Vibrio. In Manual of Clinical Microbiology (E. H. Lenette, A. Balows, W. J. Hausler, and H. J. Shadomy, eds.), ASM Press, Washington, DC, pp. 282-301. 114. Hoge, C. W., Watsky, D., Peeler, R. N., Libonati, J. P., Israel, E., and Morris, J. G. (1989). Epidemiology and spectrum of Vibrio infections in a Chesapeake Bay community. J. Inject. Dis., 160:985-993. 115. Fang, F. C. (1992). Use of tetracycline for treatment of Vibrio vulnijicus infections [published erratum appears in Clin. Infect. Dis. 1993; 16(2):346]. Clin. Zrzfect. Dis., 15:1071-1072. 116. Morris, J. G., and Tenney, J. (1985). Antibiotic therapy for Vibrio vulnificusinfection. JAMA, 253~1121-1122. 117. Bowdre. J. H., Hull, J. H., and Cocchetto, D. M. (1983). Antibiotic efficacy against Vibrio vulnificus in the mouse: Superiority of tetracycline. J. Pharmtlcol. Exp. Therp., 225595598. 118. Chuang. Y. C., Yuan, C. Y., Liu. C. Y., Lan, C. K., and Huang, A. H. (1992). Vibrio awhzificus infection in Taiwan: Report of 28 cases and review of clinical manifestations and treatment. Clin. Infect. Dis., 15:271-276. 119. Nystrom, T., Olsen, R. M.. and Kjelleberg, S. (1992). Survival, stress resistance, and alternations in protein expression in the marine Vibrio sp. strain S14 during the starvation for different individual nutrients. Appl. Environ. Microbiol., 58:55-56. 120. Koga, T., and Kawata, T. (1986). Composition of major outer membrane proteins of Vibrio vulniJcus isolates: Effect of different growth media and iron deficiency. Microbiol. Zrnmmol., 30: 193-201. 121. Morton, D. S . , and Oliver, J. D. (1994). Induction of carbon starvation-induced proteins in Vibrio vulnificus. Appl. Environ. Microbiol.. 60:3653-3659. 122. Oliver, J. D. (1995). The viable but non-culturable state in the human pathogen Vibrio vulnificus. FEMS Microbiol. Lett., 133:203-208. 123. Oliver, J. D. (1993). Formation of viable but nonculturable cells. In Stunntion in Bacteria (S. Kjelleberg, ed.), Plenum Press, New York, pp. 239-272. 124. Oliver, J. D. (1999). Public health significance of viable but nonculturable bacteria. In Nonczrlturable Microorganisms in the Environment(R.R. Colwell. and D. J. Grimes, eds.), New York: Chapman and Hall Publ. 125. Whitesides, M. D., and Oliver, J. D. (1 997). Resucitation of Vibrio vulnificusfrom the viable but nonculturable state. Appl. Environ. Microbiol., 63:1002-1005. 126. Oliver, J. D. (1995). The viable but nonculturable state. In Proceedings of the 1994 Vibrio wlnificus workshop, U.S. Food and Drug Administration. Washington, DC, pp. 63-72. 127. Oliver, J. D., Hite. F., McDougald, D., Andon, N. L., and Simpson, L. M. (1996). Entry into, and resusitation from, the viable but nonculturable state by Vibrio vulnificus in an eustarine environment. Appl. Environ. Microbiol., 61:2624-2630. 128. Oliver, J. D., and Bockian, R. (1995). In vivo resuscitation, and virulence towards mice, of viable but nonculturable cells of Vibrio vulnificus. Appl. Environ. Microbiol.,61:2620-2623. 129. Lefkowitz, A., Fout, G. S . , Losonsky, G., Wasserman, S . S . , Israel, E., and Morris, J. G. (1992). A serosurvey of pathogens associated with shellfish: prevalence of antibodies to Vibrio species and Norwalk virus in the Chesapeake Bay region. Anz. J. Epidemiol.. 135:369380. 130. Firth, J. R., Diaper, J. P,, and Edwards, C. (1994). Survival and viability of Vibrio vulniJicus in seawater tnonitored by flow cytometry. Lett. Appl. Microbiol., 18:268-271. from 131. Nilsson. L., Oliver, J. D., and Kjelleberg, S . (1991). Resuscitation of Vibrio ~.~ulnificus the viable but nonculturable state. J. Bncteriol., 173:5054-5059. 132. Weichart, D., and Kjelleberg, S . (1996). Stress resistance and recovery potential of culturable and viable but nonculturable cells of Vibro vulnificus. Microbiology, 142:845-853. 133. Bloomfield, S . F., Stewart, S . A. B., Dodd, C. E. R., Booth, I. R..and Power, E. G. M. (1998). The viable but non-culturable phenomenon explained? Microbiology, 144:1-2.

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134. Dixon, B. (1998). Viable but nonculturable. ASM News,64:372-373. 135. Weichart, D., McDougald, D., Jacobs, D., and Kjelleberg, S. (1997). In situ analysis of nucleic acids in cold-induced nonculturable Vibrio vtrlniJicrts. Appl. Em7I'ron. Microbiol., 63: 2754-2758. 136. McGovern, V. P., and Oliver, J. D. (1995). Induction of cold-responsive proteins in Vibrio vulr$cus. J. Bncteriol., 177:4131-4333.

19 Yersinia Scott A. Minnich and Michael J. Smith Urliversiv of Idrrho, Moscou: Idaho

Steven D. Weagant U.S. Food m d Drug Administration, Bothell, Washington

Peter Feng U.S. Food a i d Drug Admirlistmtiorz, Wrrshington, D. C.

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11. Yersirzirr Species 473 A. Characteristics of the organisms B. Serotypes 473 Other C. classification systems

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IV.ReservoirandDistribution477 V.Growth,Isolation,andIdentification A. Growth and survival B. Isolation methods Identification C. methods

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VI. Pathogenicity 483 A. Endotoxin 483 B. Enterotoxin 484 C. Cellular invasion 485 Iron-regulated D. proteins 486 E. Plasmid-associated virulence 487 F. Pathogenicity testing 496 VII.PreventionandControl498 References

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INTRODUCTION

Based on DNA homology and biochemical profiles, there are 11 recognized species in the genus Yersinicr (1-4), but only 3 species, Y. pestis, K enterocolitica, and Y.yseudotuberculosis, are known to be pathogenic to humans. Of these, Y. pestis, best known for causing the great pandemics of black plague in medieval Europe, is considered to be a zoonotic organism and is transmitted by direct contact with infected animals or humans or by the bites of flea vectors from infected animals (5). Y. pestis today appears only sporadically as epizootic plague in a number of rodent species, but human infections still occur on occasion as a result of contact with diseased animals or flea vectors. The Y. pestis complete genomic DNA sequence has been completed and is accessible on the Internet (ftp://ftp.scrnger.rrc.u~/~ub/~~yy). The other 2 pathogenic species, Y. enterocoliticn and Y.pseudotuberculosis, are known as enteropathogenic Yersinia and primarily cause gastroenteritis-like illness, known as yersiniosis. Both species are recognized as foodborne pathogens, because infections are usually transmitted by the consumption of contaminated food and water. In the United States, there are estimated tobe only 3,000-20,000 cases of yersiniosis per year (6,7); hence, cotnpared to Snlrnonella or Ccrr7zpylobacter, Yersinin species are not frequent causes of foodborne illness. But still, 5 outbreaks of gastroenteritis caused by Y. enterocoliticn have been reported and were traced to the consumption of contaminated water, food, and a variety of dairy products (8,9). Elsewhere, sporadic foodborne infections by Y. enterocoliticcr continue to be a common problem in northern Europe and Scandinavia (10,l l), and incidences of food and waterborne illness caused by Y. enterocoliticn and Y. pseudotuberculosis are also fairly prevalent in Japan (12). Although unrelated to foods, Y. erzterocolitica have also been implicated worldwide in cases of bacterial sepsis and endotoxic shock resulting from transfusion of contaminated blood or blood products (13). Yersiuin species are ubiquitous and maybe isolated from many environmental sources. They are also isolated frequently from foods, especially from a wide variety of meats, but pork remains to be the only known reservoir for pathogenic Y. enterocoliticcr (14-16). Because of the organism's ability to proliferate in cold temperatures, microbiological procedures to isolate Y. enterocoliticn and Y. pselldotuberclrlosis from foods usually include a lengthy cold enrichment followed by alkali treatment before plating onto selective media (17). The identification of Yersinia species, especially the biotypes of Y. enterocoliticn, requires extensive biochemical tests followed by serology to determine the many serotypes that exist in the species. The presence of Yersirlier in foods is not always associated with disease, because most yersiniae are not pathogenic (10,123).It is essential, therefore, to test all Yersi~zia isolates for virulence factors. The pathogenic mechanism of Yersinin spp. has not been fully determined, but it includes the production of an enterotoxin, invasion of intestinal cells, and a host of plasmid-encoded virulence factors regulated by a complex system of intracellular and extracellular signals. In this chapter we will specifically review the two enteropathogenic Yersirzia species that are important in foodborne illness with respect to the organism, its characteristics, classification, disease, outbreaks, growth and isolation procedures as well as the extensive amount of information that has been published on its virulence factors and the regulation of these factors.

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A.

Characteristics of theOrganisms

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Yersinia are gram-negative rod-shaped bacteria that are facultatively anaerobic. Like other genera of Enterobacteriaceae, Yersinin are oxidase negative, ferment glucose, and are motile by peritrichous flagella (19). Yersinia species are distinguished from other members of this family by their inability to ferment lactose, by their coccobacillary shapes, and by their loss of motility at temperatures above 30°C. Yersinia species are negative for lysine decarboxylase and arginine dihydrolase, and most, except for Y. pestis and Y. ruckeri, hydrolyze urea. Of the two enteropathogenic species, Y. erzterocoliticnand Y. pseudotuberculosis, that are important in foodborne illness, Y. pseudotuberculosisspecies do not decarboxylate ornithine, and both species produce little or no gas from the fermentation of carbohydrates. These two species also display a temperature-sensitive motility phenotype, becoming nonmotile at 37°C. Y. pestis is nonmotile. Y. enterocoliticn can be further subgrouped biochemically into seven biotypes designated as lA, lB, 2, 3, 4, 5, and 6. Currently, only strains in the biotypes 1B,2,3,4, and 5 are known to be pathogenic. These pathogenic biotypes and Y. erzterocolitica biotype 6 (now reclassified as Y. rnolleretti or Y. bercovierii and Y. kristerzsenii do not hydrolyze esculin rapidly (within 24 h) or ferment salicin (20). However, Y. enterocolitica biotype 6 and Y. kristerzsenii are relatively rare in the environment and can be distinguished from the pathogenic biotypes by their inability to ferment sucrose; they are also positive for pyrazinamidase (21). One note of caution, however, is that although most pathogenic Y. enterocolitica are positive for sucrose fermentation, some fully virulent, atypical phenotypic variants that are unable to ferment sucrose have been isolated from clinical samples (22). Characteristically, isolates of Y. pseudotubercrrlosis are negative for fermentation of ornithine, sorbitol, and sucrose. Otherwise, the species is fairly homogeneous biochemically, except for minor variations in the production of acid from melibiose, raffinose, and salicin (20). Also, unlike Y. erzterocolitica, isolates of Y. pseudotzrberculosis, especially those that are invasive for tissue culture cells, are esculin-positive,

B. Serotypes Strains of Y. erzterocoliticn and related species can be grouped serologically according to heat-stable somatic antigens. Wauters et al. (23) originally devised a scheme of 30 serogroups based on 34 antigenic factors. This was later expanded to add 20 more serogroups (24); however, many Y. erzterocoliticn-like organisms were later reclassified into separate species (35). as a result, Aleksic and Bockemuhl (1) proposed simplifying the serological scheme of Y. eilterocolitica into 8 serogroups based on 20 somatic factors. Not all Y. erzterocolitica are pathogens; however, pathogenic strains are widely distributed among serogroups and have been identified in serogroups 0:1,2a,3; 0:2a,3; 0:3; 0:8; 0:9; 0:4,32; 0:5,27;0:12,25; 0:13a,13b; 0:19; 0:20;and 0:21 (26). Those that predominate in human illness belong to serogroups 0:3, 0:8, 0:9, and 0:5,27. Clonal analysis of Y. erzterocolitica isolates by multilocus enzyme electrophoresis showed that the serotypes tend to cluster into two groups (27), and the pathogenic serotypes in these groups appeared to be distributed according to geographical niches (27,28). For example, the 0 : 9 serotype was found only in northern Europe and the 0:8 serotype was most frequently, and almost

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exclusively, isolated in the United States (27). Serotypes 0:20, 0:21, 0:13a,b, 0:18,and 0:4,32, which tended to be more virulent than the other serotypes, were also considered to be “American” serotypes. Based on recent reports, however, these geographical boundaries for serotype distribution may no longer be valid. Y. elzterocoZitica serotype 0:9 has now been isolated in the United States (29), and isolations of serotype 0 : 8 have also been reported from Europe and Asia (30). In Japan, the first case of gastroenteritis caused by Y. erzterocolitica serotype 0 : 8 was reported in 1991, and raw pork was the suspect vehicle (31). Also, serotype 0:3, which is supposed to be commonly found worldwide, was not a frequent isolate in the United States. Now, however, there is a marked increase in incidences of 0 : 3 infections in the United States (29,32), and it is the predominant Yersinia serotype isolated from stools (3334). The current broader dissemination of Yersirzia serotypes worldwide maybe attributed in part to increases in international importation and exportation of meat products (31 3 ) . However, infringement into natural habitats may also be a factor, as wild rodents in Japan have been found to carry Y. erzterocolitica serotype 0 : 8 and are suspected to be a source of this pathogen in human infections (36). The Y. pseudotllberculosis species is subgrouped based on a heat-stable somatic antigen, and at present there are six serogroups, designated by Roman numerals I-VI. Serogroups I, 11,111, and IV also have subtypes, but they are not as easily determined, because the antiserum to the serogroup will often cross-react with the different subtype strains and vice versa. Each of the six serogroups are known to contain pathogenic strains (37).

C.OtherClassificationSystems Aside from the conventional biochemical and serological typing schemes, other techniques are also used for epidemiological typing and classification of Yers-sinin.Phage typing has been used to some extent (38), but the limited availability of serotype-specific phages has precluded reliable classification. Genetically, Yersinia is ahighly diverse group; therefore, methods comparing DNA homologies of Yersinin have been useful for epidemiological studies. Y. etlterocolitica DNA is only 3-1696 homologous to DNA from other genera in Enterobacteriaceae (39) and 40-6096 related to DNA from Y. pseudotuberculosis and Y. pestis (40). The latter two species are virtually identical, showing greater than 90% DNA homology overall (41). Within species, yersiniae are highly conserved genetically; isolates representing nine different serotypes of Y. erlterocolitica showed 80-100% DNA homology (39). Multilocus enzyme electrophoresis has also been used as a technique to subtype Yersinia strains. This technique looks at the electrophoretic mobility of a panel of up to 21 enzyme proteins. These patterns provide distinctive profiles that can group by species, by serotype, and by epidemiologically related groups within serotypes (42,43). Genetic techniques have been used extensively to compare the relatedness of plasmids in Yersinin species. Restriction endonuclease analyses of plasmid DNA (REAP) showed no relatedness between the plasmids of avirulent Y. r~ckeriand that of virulent Y. enterocoliticn (44). However, the virulence plasmids from Y. et1terocoZiticn,Y. pestis, and Y. pseuclotubercrrlosis shared several restriction fragments (45). Restriction analysis of plasmids may also be used to determine differences in serotypes, because virulence plasmids from Y. yseudotl,fberculosisand Y. enterocolitica showed serogroup-specific restriction fragmentation patterns (46,47). In a study of 123 strains of Y. enterocoliticcr,Fukushima et al. found

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a close correlation between REAP patterns and geographic distribution (48). REAP was also used to subtype 678 strains of Y. pseudotuberculosis resulting in 16 distinct REAP patterns that were used in epidemiological investigations. Kaneko et al. (49) found REAP useful in subtyping Y. pseudotuberculosis strains within each serotype. Restriction analysis of chromosomal DNA (REAC) may also be used for epidemiological typing or identification of pathogenic Yersinia (50). Kapperud et al. compared the use of REAC to REAP and conventional phenotypic tests for differentiating strains of Y. erzterocoliticn and found it to be an effective supplement to other tests (51). However, the complexity and the large numbers of fragments obtained from the digestion of genomic DNA makes interpretation difficult. Analysis of genomic DNA may be facilitated by using probes to select small subsets of restriction fragments for comparison. This technique, known as restriction fragment length polymorphism (RFLP), has been used in typing Yersinia. Using RFLP and ribosomal RNA probes (ribotyping), several serogroups that were indistinguishable by other means can be effectively identified (52,53). Ribotyping may be used to trace reservoirs and the routes of infection in epidemiological studies of foodborne Yersirlia infections (52,53). DNA amplification using the polymerase chain reaction (PCR) has also been a useful method to differentiate serotypes of pathogenic Y. enterocoliticn. Using primers directed to the St gene that encodes for the enterotoxin, different size DNA fragments were amplified from pathogenic European serotypes as compared to American serotypes of Y. enterocoliticn (54). PCR has also been used to confirm the identity of suspect Yersinia cultures by amplifying genes that are limited to potentially virulent Yersinicr strains. (These PCR tests are explained in greater detail in Sec. V.B.)

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DISEASE

A. ClinicalManifestations The most prevalent symptoms of Yersinin infections are abdominal pain and fever (10). However, other gastrointestinal disorders such as diarrhea, nausea, headache, and vomiting may also be associated with the illness (10,5547). The minimal infective dose of Yersinia for humans has not been determined (14). The incubation period is about 24-36 hours (57), but periods of up to 11 days have been reported (14). The illness usually lasts 1-3 days (57); however, in some cases it may persist for 5-14 days or longer (14). Yersiniosis can manifest as diarrhea and abdominal pain, resembling gastroenteritis caused by other enteric pathogens (58). But when Yersinin invades the lymph system, the symptoms of fever and abdominal pain may closely mimic characteristics of acute appendicitis (58). Incidences of appendectomies being performed on yersiniosis patients have occurred in past outbreaks (10,55,58). Persistent Yersinia infections may lead to secondary complications such as erythema nodosum, septicemia (see Sec. VI.A), Reiter's syndrome, and reactive arthritis (lo,%). The latter illness is an autoimmune disease of the joints, presumed to be caused by host immune responses to Yersinia antigens (59). Presence of Yersinin antigens has been detected in the synovial fluid cells of affected joints (60). Patients prone to developing reactive arthritis from yersiniosis also have histocompatibility antigen HLA-B27 (59). Proteins that cross-react with HLA-B27 have been found on Y. yseudotubercrrlosis, suggesting that the arthritic condition may be induced by the molecular mimicry of bacterial antigens with HLA-B27 (59,61). Graves' disease or hyperthyroidism may be another complication associated with yersiniosis. Persons af-

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flicted with this autoimmune thyroid disorder have high titers of antibodies to Y. enterocolitica (62). Presence of thyrotropin binding sites on Y. enterocoliticn have been identified (63), suggesting that Graves’ disease may be caused by the cross-reactivity of these sites with receptors of thyroid-stimulating hormones. 1. Mechanism of Infection The mechanism of infection by yersiniae has not been fully established. However, based on animal infection studies and the types of virulence factors encoded by the pathogenic Yersinin species, a hypothetical mode of infection may be formulated. The pathogen, typically ingested as contaminants in foods and dairy products, is transported to the small intestine, where it adheres to the epithelial cell linings, via perhaps an adhesin produced by the virulence plasmid (see Sec. V1.E). Invasive factors encoded by these organisms (see Sec. V1.C) enable the bacteria to penetrate epithelial cell barriers and disseminate into the lamina propia (64), where the various plasmid-encoded virulence factors allow yersiniae to resist the bactericidal effects of serum and the phagocytic activity of macrophages (see Sec. V1.E). The pathogen may then be transported to the lymph nodes, enter the blood stream, and spread to the rest of host (65). A few incidences of direct infection into the blood via transfusion of Yersinia-contaminated blood products have also been reported (see Sec. V1.A).

2. Patient Susceptibility Age and physical condition of the host can influence the severity of Yersinia infections (55). The population most susceptible are children and the elderly, but infants less than 1 year old are most seriously affected (14,55,57,58). In European countries, Y. enterocoliticn infections most frequently involve children 1-3 years of age, and likewise, Y. pseudotuber” culosis infections also tend to occur in young adults between the ages of 1 and 16 (56). Although the sex of the host has not been established as a factor, the number of males infected by Y. pseudotuberculosis tend to outnumber females (56). Iron is a required growth factor for Yersinia; hence, increases in free iron in the mammalian tissues can also increase the susceptibility of hosts to Yersinia infections (66,67). Patients in South Africa suffering from Bantu siderosis, caused by excess consumption of alcoholic beverages that contain a large amount of iron, have been shown to be very susceptible to Y. enterocoliticn infection (68). Most iron in mammalian tissue is complexed with iron-binding proteins resulting in low free iron levels. Hence, the absence of free iron provides an antimicrobial effect for the host (65). To overcome this barrier, pathogenic Yersinia produce iron-binding proteins to sequester iron from the host (see Sec. V1.D). B. FoodborneOutbreaks The foodborne nature of yersiniosis is well established, and numerous outbreaks have occurred worldwide. Two outbreaks in Quebec, Canada, in the mid 1970s affected 138 schoolchildren and were traced to the consumption of raw milk (69). Five outbreaks of Y. enterocoliticn serotype 0 : 3 in Japan involved over 1100 school children, and although never confirmed, foods were the suspected sources of infection (70). In the United States, an outbreak in New York State in 1976 affected 217 students, of which 38 were culture positive. Pasteurized chocolate milk was the implicated source, and Y. enterocoZitica serotype 0:8 was isolated from the milk (71). In 1980, an outbreak in Washington State, also

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caused by serotype 0:8, affected 87 people. The source of contamination was traced to unchlorinated spring water used in the packaging of tofu, a soybean-based product (9). More outbreaks occurred in 1983 in southeastern United States, and again, pasteurized milk was implicated. A total of 172 cases were identified, and the causative organism was Y. enterocolitica serotype 0:13a, 13b (72). The most recent Yersinia outbreak occurred in 1989 in Atlanta, and the contamination, which affected 14 infants in the same household, was traced to the caretaker, who was preparing pork chitterlings (73). The unusual aspect about this outbreak was that it was caused by serotype 0:3, which is rare in the United States. Furthermore, two different phage types of serotype 0:3, as well as serotype 0: 1,2,3 were also implicated (74). Since then, only sporadic cases of Yersinicr infections have been reported from several New England states (75). It is interesting to note that although the primary reservoir of pathogenic Y. enterocoliticcr is swine, many outbreaks have implicated milk or milk products. Several microbiological surveys of milk showed that Yersinia are present in raw milk from cows and goats (71,76-79), but only in one instance was a potentially pathogenic strain isolated from pasteurized milk (71). Since Yersinin will not survive pasteurization, contamination of milk by Y. enterocolitica is probably due to deficient pasteurization or a breakdown in postprocess sanitation (76,78). Isolates of Y. enterocoliticn are also frequently found in streams, lakes, springs, and wells (80-84); hence, unchlorinated surface water has also been implicated as a vehicle in yersiniosis. In 1972, a 75-year-old man hunting in the woods in upstate New York was afflicted with Y. erzterocoliticn septicemia after drinking from a stream. Analysis of water taken from the mountain stream identified Y. enterocolitica of the same serotype and biotype as those isolated from the patient (85). The man recovered after 67 days of hospitalization and antibiotic therapy. Water was also implicated but not confirmed as the source of a yersiniosis outbreak at a ski resort in Montana (86). In the United States, Y. pseudotuberculosis is less commonly found than Y. enterocolitica, and although it is frequently associated with animals (including swine, birds, rodents, and hare), Y. pseudotuberculosis has not been implicated in foodborne illness and has only rarely been isolated from soil, water, and foods (87). In Japan on the other hand, several foodborne and waterborne outbreaks have been linked to Y. pseudotuberculosis (12,88). In one case, children were infected after drinking water from a garden pond that was contaminated with feces from a stray cat that carried Y. pseudotuberculosis (88). In Europe, a study done at a hospital in Ireland (89) found that 28% of patients presenting with symptoms of appendicitis symptoms and 11% ofpatients presenting with nonspecific abdominal pain had serum titer to Y. pseudotubercuzosis compared to 1% of controls.

IV.RESERVOIRANDDISTRIBUTION Swine are generally recognized as the primary reservoir for pathogenic Yersinia around the world. Y. enterocolitica have been isolated from tongues, throats, cecal contents, and feces of pigs; however, the isolation rate can vary greatly. Interestingly, Neyt et al. (90) recently determined that the pYV plasmids of Y. enterocoliticn low-virulence serotypes (0:1,2,3; 0: 1,2; 0:3; 0:9; and 0:5,27) contain a class I1 transposon, Tn2502, conferring arsenite and arsenate resistance. As these strains are of worldwide distribution, it suggests a clonal origin. Arsenicals were used for chemotherapy in Europe prior to World War I1 to treat swine for Serpulirla hyodysenteriae (formerly Treponema hyodvsenteriae)infections.

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Thus, these authors speculate that such arsenical treatments, and consequent gain of resistance, may have contributed to the establishment, or maintenance, of Yersinia in pigs. One study of 50 slaughter pigs at an abattoir found that only 2 (4%) carried the pathogenic 0:5,27 serotype (91). Other reports, however, showed that as many as 25 and 58% of the pigs sampled in Belgium and Denmark, respectively, carried pathogenic serotypes of Y. enterocoliticn (92). A more recent survey of 3375 slaughter pigs showed that Y. enterocoliticn was present in 808 (23.9%) of pigs and of the 107 pathogenic isolates obtained, mostly serotype 0 : 5 and a few of serotype 0 : 3 were identified (93). Although it is evident that pathogenic Yersinia are prevalent in swine, not all studies seem to support the hypothesis that swine is the reservoir for human infections. A study from China conducted over a period of 11 years showed no correlation between the serotypes of animal isolates versus those that caused infections in humans; hence, it suggested that swine may not be the source of pathogenic Y. enterocolitica in humans (94). Contaminated pork or pork products account for a large portion of yersiniosis infections worldwide; however, this organism is fairly prevalent and can be isolated from a variety of other foods. A survey of various products in France showed Yersinin to be present in raw vegetables, milk, ice cream, cakes, and pork products (95). Of the 666 samples examined, an average of 33% were contaminated, but only one Y. enterocoliticn out of the 180 isolated was potentially pathogenic. A similar survey in Denmark found Yersinirr to be present in 40-80% of pork products, l-17% of dairy products, 43% of raw vegetables, 8-20% of soy products, 22% of seafoods, and 9% of salads. In that analysis, 71% of the isolates were Y. enterocolitica, ,but only one strain isolated from pork tongues was found to be potentially pathogenic (96). Aside from pork, other meat products such as poultry can also be contaminated with potentially pathogenic serotypes of Y. enterocoliticn (97). More recent surveys from other parts of the world are also consistent with above findings. Studies from New Zealand and Australia showed that Y. enterocolitica was present in 3.4% of the cooked processed meats and seafood (98), in 18% of beef, 10% of lamb, and 12% of pork samples examined, but none of the isolates were pathogenic (99). Similarly, a survey from Argentina of 450 samples of cold, ready-to-eat foods, such as ham, salami, and cheese, showed that only 1-2% were contaminated with Y. enterocolitica. Although all of these isolates were serotype 0:9, a documented serotype known to be pathogenic, none of them harbored virulence factors (100). These studies consistently show that Yersinia are prevalent in foods, however-and fortuitously-the presence of pathogenic serotypes in foods seems to be rare. Finally, Y. enterocolitica have been found in samples of raw milk, but not in pasteurized milk (99); hence, they are not very resistant to heating. But as the above surveys showed, Yersinin species are present in many cooked foods, and some past outbreaks have also implicated pasteurized milk, thus indicating that postprocessing contamination may bea common problem. Environmental analysis of dairy plants in Vermont showed that Y. erzterocoliticn was present in 10.5% of the plant sites surveyed ( 101). Y. enterocoliticcr are also ubiquitous in the environment and may be found in lakes, streams, soil, and vegetation. The organism has been isolated from the feces of dogs, cats, goats, cattle, chinchilla, mink, and primates (80,102).Wild animals such as deer frequently harbor Y. enterocolitica (85), and in Japan (103) and the United States (104) it has been isolated from rodents as well as from flies and other insects. In the estuarine environment, many birds such as waterfowl and sea gulls may be important carriers, as Yersinin has also been isolated from oysters, clams, and shrimp (84). Consistent with those findings, Y. enterocolitica has been found to survive well in stream water at temperatures of 6-

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16"C, suggesting that surface water may serve as a persistent vehicle for this pathogen between animals and humans (105). The distribution of Y. enterocolitica is worldwide, but it tends to present greater problems in cooler climates. Yersiniosis is endemic to northern Europe where sporadic infections are common, especially in the fall and winter months. The seasonal distribution of Yersinia in the estuarine environment was also evident in Washington State, where peak levels occurred during winter months (unpublished data). Y. pseudotuberculosis is also found in tnany environmental sources and animals, and, like Y. enterocolitica, its primary reservoir also appears to be swine. Y. pseudotuberculosis is rarely found in the United States, but it seems to be fairly common in Japan. Pathogenic strains of Y. yse~~dotz~berculosis has been isolated from the throats of healthy swine and from retail pork samples surveyed in Japan (106), as well as from 20.6% of the freshwater samples analyzed (107). In Italy, a large survey of over 30,000 samples showed that Y. pseu~otuberculosiswas isolated from few clinical samples and animals examined but was not found in any of the environmental samples or from the dairy and raw meat products examined (108).

V.

GROWTH,ISOLATION,ANDIDENTIFICATION

A.

Growth and Survival

Yersinia can grow at temperatures of 0-44°C. In a rich medium, maximum growth is at 32°C with a doubling time of 34 minutes. Generation time rises to 40 minutes when temperature is increased to 40°C or decreased to 28"C, and at room temperature it is about 1 hour (109). Further decreases in temperature to 10°C increases doubling time to 5 hours (1 lo), and at 1°C generation time is close to 40 hours (1 11). Y. erzterocolitica shows a remarkable tolerance to low temperatures, and this attribute has been used effectively in isolation methods to selectively enrich for Yersirzin (see Sec. V.B). The optimal pH for growth of Yersinia is 7.6-7.9, but the organism will grow in pH ranges of 4.6-9.0 (109). The nature of the acidulent can also have an effect on minimum pH for growth. Karapinar and Gonul (1 12) demonstrated that acetic acid is more effective than citric acid in inhibiting growth. Y. enterocolitica is a versatile organism that can survive long periods in cool, dilute environments such as well water (1 13). Y. erzterocolitica can tolerate up to 5% sodium chloride in its growth medium (114). Growth rates of Y. enterocoliticn in pork decreased as the levels of NaCl, KC1, and CaCl? increased. CaCll was found to be more inhibitory than equivalent levels of NaCl or KC1. Concentrations of 2.2% CaClz (w/w)were enough to prevent outgrowth of Y. ejzterocoliticn in ground pork (1 15). However, Yersirzia are not very heat resistant. High-temperature, short-time pasteurization conditions of 71.8"C for 18 seconds easily kills Y. enterocolitica ( l 16,117). When some bacteria are exposed to elevated sublethal temperatures, they respond by synthesizing a group of heat-shock or stress proteins. Shenoy and Murano (1 18) found that Y. enterocolitica, when exposed to 45°C for 60 minutes, produced heat-shock proteins and were more resistant to subsequent lethal heat treatment. Evaluation of survival of control cells and heat-shocked cells in heated ground pork revealed that D values at 60°C increased from 1.7 to 6.7 minutes, respectively. They also found that heat-shocked cells grew as well as control cells when held in ground pork at 4 or 25°C (119). Gamma-irradiation has also been investigated as a means of eliminating Y. enterocolitica in pork products. The strains investigated were

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found to be sensitive to gamma-irradiation, with a D value of 0.25 kGy at 0°C. When samples of cooked ham, salami, and raw pork were artificially contaminated with Y. enterocoliticn at 106 cfu/g, cooked ham and salami were decontaminated with doses of 3 and 4 kGy, respectively. Viable cells could not be eliminated at a dose of 6 kGy. The dose of 1 kGy at -40°C was efficient in eradicating low numbers of naturally occurring Y. enterocolitica (120). Yersinia can withstand freezing and survive for extended periods in frozen food, even after repeated freezing and thawing (1 16). Destruction of viable cells under freezingthawing and constant freezing conditions at -20°C was more rapid in distilled water than in milk. Presumably proteins and fats in the food matrix provide a protective effect for Yersirzia (1 16). Several studies have examined the survival and growth of Yersinin in foods. On cooked beef or pork, Y. enterocolitica can increase by 6 log within 10 days at 7°C. At 25"C, the growth rate is even more rapid, attaining similar increases within 24 hours. Growth is much slower on raw beef or pork at both temperatures (1 11). Yersinia seeded into boiled eggs and boiled fish grows rapidly at 4°C (121). Y. enterocoliticn inoculated into pasteurized liquid eggs also grew well at low temperatures, but it was inhibited by the addition of 5% salt (122). Intofu (soybean curd) and pasteurized whole milk, Yersinia grows well at temperature ranges of 3-25°C (1 lo), however, growth may be slowed by the presence of psychrophilic microflora (1 14). Yersinin can also proliferate in seafoods, but at slower rates. Yersinin seeded in shucked oysters at 0-2°C or 5-7°C showed slow increases in numbers over 14 days (123). In raw shrimp and cooked crab meat stored at 5"C, Yersinia grew rapidly during the first week but declined in number with additional weeks of storage (123). Oxygen-free vacuum packaging and saturated carbon dioxide (CO,) controlled-atmosphere packaging (CAP) have been used to extend the shelf life of refrigerated foods. Gill and Reichel found that Y. enterocoliticn could grow in saturated CO, CAP beef at 50°C (124). Hudson et al. found that Y. enterocoliticn could grow slowly (generation time of 13.9 h) in vacuumpackaged sliced roast beef held at 30°C and even more slowly (generation time of 77 h) when held under saturated CO2 CAP. When held at - 1.5"C growth was slowed with a generation time of 32.1 hours in vacuum packaging and did not multiply under CO, CAP (125). B. Isolation Methods Yersinia spp. have been isolated from clinical samples by direct plating on a variety of differential and selective agars used for enteric bacteria. These include Snlmonelln-Shigelln agar with (126) or without deoxycholate, bismuth sulfite agar, and Macconkey agar with (127) or without Tween 80. The use of these selective media at 37"C, however, is not recommended for Yersinin isolation since the organism grows slowly and may be outgrown by other enterics. Furthermore, the virulence plasmid of Yersinin, pYV, is unstable and may be spontaneously lost during overnight growth at 37°C. Cefsulodin-IrgasanNovobiocin (CIN) agar, an agar specifically designed for isolation of Yersinin spp. at 32°C is useful for isolation (128). Methods to isolate Yersinia from foods are problematic due to competition and overgrowth by food microflora. Direct plating is rarely successful unless Yersinia numbers are very high. Thus, isolation of Yersinin from foods necessitates the use of enrichment techniques. Many enrichment techniques take advantage of the tolerance of Yersinia to growth temperatures as low as 4°C. As the enrichment temperature is lowered, the rate

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of growth is decreased and the time of enrichment increases. Cold temperature enrichment of samples in phosphate buffered saline for 2-4 weeks at 4°C improved recovery (129). Better results were obtained by including sorbitol and bile salts (130) and peptone in the enrichment medium as well as shortening the enrichment period to 10 days by increasing incubation to 10°C (13 1). There are also methods to selectively enrich for virulent Yersinin strains in foods. Modified Rappaport broth (126) as well as Irgasan-tarcacillin-potassium chlorate broth (132) are effective for Y. enterocoliticn 0 : 3 and 0:9, but not as effective in the enrichment of other bio/serotypes. Toora et al. proposed the use of modified tryptic soy broth (MTSB) supplemented with Irgasan (133). Bhaduri et al. modified this procedure by enrichment in MTSB without Irgasan at 12°C for 24 hours with shaking followed by addition of Irgasan and further selective enrichment at 12°C for 24-48 hours (134). Pathogenic Yersinin species in foods may also be detected using genetic methods. One such method is DNA colony hybridization, where DNA fragments containing sequences encoding traits known to be present in the target bacteria are labeled with a marker. These fragments then can be used to detect the hotnologous sequence in situ in suspect colonies on agar plates. Excised DNA fragments from the conserved calciumdependent region of the pYV virulence plasmid (45) were radiolabeled and used in DNA colony hybridization analyses of Y. enterocoliticn seeded into foods (135-137). The probes effectively distinguished potentially virulent Yersinin from normal flora in foods without the need for enrichment if present in high numbers. Synthetic oligonucleotide sequences derived from known sequences of genes determining pathogenic traits have been radiolabeled and used as probes for DNA colony hybridization. Such probes specific for the virulence plasmid have been evaluated, including one directed to the plasmid region that encodes for cytotoxicity to HEp-2 cells (138,139) and another that is specific to the yopA gene of the plasmid that encodes for the temperature-inducible outermembrane protein YadA (see Section V1.E) (140). Analyses for Y. enterocolitica in several types of seeded foods showed the detection efficiencies of these probes to range from 33 to 100% (135,138,140). Variations in food matrices and differences in the concentrations of normal flora in the foods affected probe sensitivity. Several probes specific for the invasion genes on the Yersinia chromosome have also been developed, but the potential of these probes to identify Yersinin species in foods have not been tested. The invasion-specific probes and other assays used for testing virulence of Yersinin are discussed in Section VI. F. Recently, nonradioactively labeled DNA probes have been prepared by polymerase chain reaction to Yersinia virulence genes and tested to identify plasmid-bearing Yersinia. They were also used successfully to detect Yersinia from artificially contaminated foods (141). The polymerase chain reaction (PCR) has also had numerous applications to the detection of potentially virulent Y. enterocolitica and Y. pseudotuberculosis from foods. It was first used by Wren and Tabaqchali (142) to detect potentially virulent Yersirzia species by amplification of the virF transcriptional regulator gene for outermembrane proteins important to the virulence mechanism. Kwaga et al. (143) targeted primers to the ail (attachment and invasion locus) gene of Y. enterocoliticn in a PCR assay. Nakajima et al. (144) combined the Wren and Tabaqchali primers with those the inv gene specific to potentially virulent Y. pseudotuberculosis and with primers for ail specific for potentially virulent Y. enterocoliticn. These primers were mixed in a multiplex PCR to simultaneously detect and differentiate potentially virulent Yersinin species. To this multiplex PCR, Weynants et al. (145) added the twist of adding primers specific to the gene encoding the 0: 3 antigen, a serogroup often pathogenic for humans. This allowed not only speciation

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and potential virulence of suspected isolates, but also serotype identification in the same reaction. One technical hurdle to the use of PCR for detecting bacterial DNA in environmental samples was the inhibition of the reaction by sample matrix components. To detect the presence of potentially virulent Y. enterocoliticn in fecal samples, Ibrihim et al. (146) used chemical extraction of DNA with PCR targeting yst (heat-stable toxin gene) of Y. enterocolitica. Harnett et al. (147) combined extraction of DNA with multiplex PCR to detect the presence of Y. enterocoliticn in artificially contaminated fecal samples using primers for ail, yst,and virF. Rasnlussen et al. (148) devised a method for PCR detection of Y. enterocoliticn cells from naturally contaminated pig tonsils. Swabs of tonsils were cold enriched for 7-10 days before treatment via immunomagnetic separation (IMS) to selectively remove target cells of Y. enterocoliticn from the enrichment broth. After IMS, Yersinia were detected by PCR with primers to ail. By using this IMS-PCR method, 80% of pig tonsil samples were positive compared to 68% by cultural techniques. Bhaduri and Cottrell(l49) have expanded this approach by swabbing various artificially Contaminated foods using a short selective enrichment and centrifugation and treatment with proteinase K prior to multiplex PCR for Y. enterocoliticn virF and uil to detect target bacteria. Despite the promise of these exciting developments in molecular methods, PCRbased detection methods have an inherent disadvantage of not isolating the contaminating culture for confirmatory tests. For this reason, they have not been adopted in routine testing for Yersinia in foods. However, PCR-based methods may have a place as screening tests for focusing culture-based techniques on those samples likely to be contaminated. Both Y. pseudotuberculosis and Y. enterocolitica are fairly resistant to alkaline conditions; therefore, pretreating enrichment cultures for a few seconds in 0.5% KOH prior to streaking onto selective agars can reduce the numbers of competing microflora (17). The selective media used for clinical sample analysis are also applicable for isolation of Yersirzinfrom foods. Among the more effective are Macconkey and Bismuth sulfite agars, but CIN agar has also proved to be reliable. CIN may be the preferred isolation medium because it is more differential for Yersirzia species, however, it isalso slightly more inhibitory to some Y. pseudotuberculosis strains (150). Combining the use of CIN and MacConkey’s agar with incubation at 30°C for 24 hours was found more successful than either alone (151). Another selective agar medium designed specifically for isolating Yersirlia is virulent Yersinin (VYE) agar. This medium has antibiotic supplements as well as esculin and was developed to differentiate potentially virulent biotypes from environmental strains (152). Recently, Bhaduri and Cottrell have suggested differentiating potentially virulent strains on Congo red brain heart infusion agarose medium after enrichment (153).

C. Identification Methods Conventional methods for the identification of Yersirzin species require a battery of biochemical tests. These are reviewed in detail elsewhere (10,151). Several commercially available miniaturized biochemical test kits, as well as automated microbial identification systems, have also been suggested for identifying Yersinia and other enterics (154,155). Many of these systems have been evaluated and are generally good for presumptive identification of Yersinia to the genus level but are not as reliable for speciation or biotyping (154,156-159). Conventional biochemical tests can be used to supplement the kits for speciating Yersiniu isolates and must be used for complete biotyping of Y. enterocolitica isolates.

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VI.

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PATHOGENICITY

As stated in the introduction of this chapter, the Yersinia are not large contributors to foodborne disease relative to other enteric pathogens. However, the disease-associated yersiniae have contributed tremendously to our understanding of molecular pathogenicity. Studies on the pathogenic Yersinia spp. have provided a common thread that has led to the unraveling of a pattern of common strategies for virulence in not only mammalian pathogens but plant pathogens as well. Indeed, many of the basic principles in molecular pathogenesis were first established in the yersiniae. Hallmarks of these discoveries include the first association of plasmids with virulence, the role of iron in the infectious process, the isolation of the first bacterial proteins involved with the invasion of eukaryotic cells, elucidation of the type I11 protein secretion system, and the importance of temperature as a key environmental cue for virulence gene activation. How did the yersiniae become so important in dissection of such key host-parasite interactions? In part, this is attributable to the common physiology among the pathogenic yersiniae, findings in one Yersinia species commonly extrapolated to the others. More importantly, many of the phenotypes associated with specific virulence attributes are pronounced, lending themselves well to genetic analyses. For example, all pathogenic yersiniae are calcium dependent at 37"C, and suppressors of this phenotype (i.e., calcium independent) were shown to be nonvirulent (159). Thus, from the early days, researchers had easily scorable phenotypes with which to work. Finally, as recombinant DNA and molecular genetics methodologies were developed for analyses of Escherichia coli and Salmonella typhimurium, these techniques were easily applied to the related yersiniae. There are a number of recent and excellent reviews on Yersinia virulence and particularly type I11 secretion systems (for recent reviews, see Refs. 160-163). In fact, over the past 8 years there has been such an increase in information that a book may be the only means to cover every detail. That being said, this section will focus on the essence of our present understanding of Y. erzterocolitica virulence, emphasizing an historical perspective as to how the Yersinin have been used so successfully as a model system. The pathogenic mechanism of Yersinia is highly complex and not fully understood, however, it involves multiple factors encoded by both chromosomal and plasmid genes. These virulence factors include endotoxin, an enterotoxin, several invasion genes, ironregulated proteins, and a large number of plasmid-encoded factors that are important in the process of virulence. These factors as well as motility are under a highly complex system of genetic regulation affected by temperature, DNA structure, histone-like proteins and other as-yet unidentified parameters.

A.

Endotoxin

Yersinin endotoxin is not a major factor in foodborne illnesses, but it is significant in clinical infections. Between 1987 and 1991, Y. enterocolitica was implicated in seven of the eight fatalities due to transfusions of bacteria contaminated red blood cells in the United States (164), and similar incidences of bacteremia and endotoxic shock have been reported around the world (74,164,165). In most instances, the source of Yersinia contamination appeared to be asymptomatic blood donors (1 64,165), and the blood used for transfusion showed no gross evidence of contamination (166-168). Other bacterial species have also caused sepsis (170), but Y. enterocoliticn is most commonly implicated, and it

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is estimated that so far it has caused about 40 cases of transfusion-related infections worldwide, with a mortality rate of greater than 60% (171). The higher frequency of transfusion-mediated Yersinin endotoxemia as compared to other bacteria may be attributed to several characteristics of this organism. First, Y. enterocolitica has the ability to proliferate in cold temperatures (166,168,179). At blood storage temperature of 4OC, 1 cfu/mL of Y. enterocoliticn inoculated into blood will undergo a lag phase of 10-20 days, then rapidly proliferate to lo8 or lo9cfu/mL (166,168). During log-phase growth, endotoxins produced may reach concentrations of 240-600 ngl mL (166). Second, iron affects the in vitro growth response of Y. enterocolitica (172). Hemolysis of red blood cells during storage may provide a readily available source of exogenous iron to stimulate growth of yersiniae (169,173). Third, Y. enterocoliticn produces plasmid-encoded outer proteins, designated Yops (discussed below), that enable the organism to resist the bactericidal effects of human serum and phagocytosis by macrophages and polymorphonuclear leukocytes. These abilities promote the survival of this organism in stored blood (169,174,175). A number of solutions have been proposed to prevent transfusion sepsis by Y. enterocoliticn, however, none of these seems to be viable (170,171). Screening and eliminating donors with gastrointestinal symptoms is only partially effective, because a third of the donors implicated in Yeminicl transmission did not exhibit gastrointestinal illness. Since Yersinin undergoes a 3-week lag phase before exponential growth in blood, it was proposed that the existing storage time for blood cells be lowered to 3 weeks. This, however, would have a devastating effect on blood supplies worldwide. Similarly, to include antibiotics in blood units to control Y. enterocoliticct may result in greater problems due to side effects or allergic reactions to the drug (170,171). Screening blood units is another alternative, but it is labor intensive and logistically difficult. The presence of Y. enterocolitica in blood may be detected by microbiological plate counts or by the use of hematology stains such as acridine-orange, Wright, or Wright-Giemsa stains (164). The level of Yersinia endotoxins in blood inay be determined by the Linzulus amebocyte lysate assay (165,166). A polymerase chain reaction assay may also be potentially applicable for the specific identification of Y. enterocolitica contamination in blood (176). All these methods, however, lack sensitivity and will not beable to detect the low numbers of Y. enterocoliticn cells that may be present at the time the units of blood are collected. B.

Enterotoxin

Pathogenicity of Yersinin may also involve the production of a heat-stable enterotoxin (ST) that is detectable by intragastric injection of culture filtrates into suckling mouse or by the rabbit ileal loop assay (10,177.178). The production of ST has been observed mainly in isolates of Y. enterocolitica but not in Y. pestis or Y. pseudotuberculosis (179). The ST of Yersirzia closely resembles the heat-stable ST toxin of enterotoxigenic Escherichia coli (10,180) in terms of heat resistance and pH stability (181,182). The molecular weight of 9700 estimated for the ST of Y. enterocoliticct is slightly larger than that of E. coli (178), but the toxins are immunologically cross-reactive (180,183). Both toxins also share common mode of action in stimulating cyclic guanosine monophosphate levels in the suckling mouse (182,183). In spite of these similarities, however, the implication of Yersinia enterotoxin in foodborne diseases is not fully established and remains controversial. For example, the Yersirzia ST toxin is not produced in vivo (10,184) and not detected in vitro at temperatures above 30°C (178), hence cannot be produced in the mammalian host. Also, there

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wasno correlation between production of ST and pathogenicity in mice (185-187); not all pathogenic serotypes of Y. enterocolitica produced ST (177,178), and other Yersinia species that were generally regarded as non pathogenic were also found to produce ST (188,189). Therefore, these factors tended to suggest that ST wasnot involved in virulence. Although ST is not produced in the host, intoxication due to ingestion of prefomled ST in foods was raised as a possibility for public health concern (188,190). Heat and pH stability of ST (1 8 1,182), as well as findings that 32-100% of Y. enterocoliticn isolated from milk were toxigenic (188), tended to support this concern. Other studies, however, showed that Yersinia produced ST in media only at 25°C (187,188), but not at 4°C (190). Also, despite good bacterial cell growth, no ST was produced in foods, milk, or milk-like media at 4 or 22°C (188,190). The absence of ST production at these commonly used food storage temperatures, plus the fact that no cases of foodborne intoxications due to Yersinia ST have been reported, would dispute the likelihood that performed Yersinia ST is a factor in foodborne illness. The S toxin is encoded by the yst gene on the chromosome of Y. enterocoliticn. Unlike physiological studies, genetic analysis of yst tended to suggest that there is a correlation between the presence of vst gene and pathogenicity of Y. enterocolitica (191). For example, infection of rabbits with isogenic ,st+ and yst- mutants confirmed that only yst+ strains of Y. erzterocolitica were able to cause diarrhea in rabbits (179). Similarly, DNA hybridization studies of pathogenic and nonpathogenic strains showed that yst-homologous gene sequences were present only in pathogenic Y. erzterocolitica and a few isolates of Y. kristensenii (191). A PCR assay for the yst gene also confirmed that a yst gene-like product was amplified from Y. kristensenii (192). Since Y. kristensenii are generally regarded as nonpathogenic, the presence of yst genes in this species would seem to contradict the role of ST in virulence. But subsequent hybridization studies using a DNA probe specific for the internal regions of the yst gene showed that only pathogenic isolates of Y. enterocolitica reacted with the probe (191). Although Y. kristensenii do not appear to produce ST, this species was found to be pathogenic to iron-loaded mice; hence, it may be carrying some other virulence factors (193). Finally, although ST is not produced by Y. enterocolitica at temperatures above 3OoC, transfer of the yst gene into E. coli resulted in the production of active toxin at 37°C (179). This suggests that yst gene expression in Y. erzterocolitica may be thermoregulated, analogous to the expression of the invasion gene, inv, in Y. pseudotuberculosis (194). More recent studies seem to provide stronger evidence that ST is a virulence factor in Yersinia. Production of ST may be dependent on the age of cultures, as fresh isolates of pathogenic Y. erzterocolitica were found to produce ST, but in strains of older collections yst had become silent, resulting in no toxin production (191). The absence of yst gene expression would account for the lack of ST production by some pathogenic serotypes, observed in earlier studies. Finally, genetic analysis of yst regulation showed that, even though the gene was not expressed at 37"C, low pH and increased osmolarity conditions, similar to that in the lumen, can induce yst to express at 37°C (195); hence, it is possible for Yersinin to produce the ST toxin in the infected host.

C. CellularInvasion Pathogenicity of Yersinia is closely associated with the presence of a virulence plasmid. However, isogenic strains of Y. enterocolitica and Y. pseudotuberculosis, with or without

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the virulence plasmid, showed equal efficiency in invading tissue culture cells (196-198). Therefore, the ability of Yersirzia to invade mammalian cells is chromosomally encoded. An invasion gene, designated inv, was first identified on the chromosome of Y. pseudotuberculosis (199,200). Transfer of the i m gene into E. coli conferred the invasive phenotype to E. coli (200). Expression of the im?gene in Y. yseudotube~-culosis is thermoregulated (194). The gene encodes a 103 kDa outer membrane protein (invasin) that binds to integrin receptors on mammalian cells, which stimulates Yersinia uptake (301,202). A similar i m gene and another gene, ail (attachment invasion locus), that also encodes for cellular invasion were identified on the chromosome of Y. erzterocoliticn (1 34,135). Although the inttasin protein of Y. enterocoliticu is slightly smaller than that of Y. pseudotrrberculosis, the inv genes from these species are 73% identical at the DNA level, 77% identical at the protein level, and the proteins share similar antigenic epitopes (136-1 38). The nil gene encodes for a 17 kDa Ail protein that also promotes attachment and invasion of tissue cells (133,138). Interestingly, Ail confers varying degrees of invasiveness to various pathogenic Y. erzterocolitica serotypes (133). Analyses of Ail proteins showed slightly different amino acid sequences, which may account for the differences in invasiveness among these serotypes (133). The invasion phenotype encoded by inv enables invasion of several cell lines (130,139), while that encoded by nil exhibits tissue cell specificity (134,139). The ability of Yersinia species to invade mammalian tissue cells appears to be correlated with pathogenicity because many avirulent yersiniae are not invasive (5,127,139). Analyses of Y. elzterocolitica isolates showed that inv gene sequences were present in both pathogenic and nonpathogenic strains (96,139). However, further analysis showed that the i12v sequences of avirulent strains were nonfunctional and these isolates were noninvasive (140). In contrast, genetic studies showed that the presence of ail is closely correlated with pathogenicity. DNA probing of disease-causing Y. erzterocolitica strains showed that only pathogenic isolates that were tissue cell invasive carried the nil gene (139). The presence of nil, therefore, appears to be closely associated with the potential for virulence in Y. enterocolitica (134,139). While im? and ail are the primary invasion genes in Yeninin, there is evidence that the enteropathogenic serotypes also have a virulence plasmid-mediated pathway for adhesion and low-level entry into mammalian cells (133,141,142). Although the precise function of invasin and ail proteins in cellular invasion has not been determined, studies showed that mice orally challenged with imp mutants of Y. pseudotuberczdosis exhibited slower rates of infection (143). This is consistent with the hypothesis that these invasion gene products enable Yersiiliu to penetrate intestinal cell walls to allow colonization and that the contribution of the virulence plasmid to pathogenesis probably occurs after penetration of the cell membrane (127,128,144,145). D. Iron-RegulatedProteins Iron acquisition is an important factor in Yersinin pathogenicity. Isolates of Y. kristensenii (215) and low-virulence Y. enterocolitica of serotypes 0 : 3 and 0:9 are lethal in the mouse model only when allowed to infect iron-loaded mice (172). In contrast, highly pathogenic Y. pseudotut7erculosis, Y. pestis, and Y. enterocolitica produce a set of conserved chromosomally encoded proteins to synthesize and uptake the Yersirlin siderophore yersiniabactin. Yersiniabactin's iron-complexing property is due to a phenolic group and three fivemembered heterocyclic thiazole moieties that serve as iron ligands (216). Thegene cluster

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encoding this iron uptake system is genetically unstable. Its spontaneous loss, or rearrangement, has been associated with large chromosomal deletions (102 kb high-pathogenicity island), loss of pigmentation, and virulence attenuation in Y. pestis (reviewed in Ref. 217). In Yersinin enterocolitica biotype 1B strain 8081, these genes are clustered on a 45 kb high-pathogenicity island (HPI) inserted into a resident tRNA-Asn gene (218). Pelludat et al. (219) examined this HP1 in biotype 1B strain WA-314 and identified five iron protein genes (Irps) within a 13 kb stretch of this region. These identified genes encode Irpl and Irp2, encoding previously designated HMWPl and HMWP2 (high molecular weight proteins 1 and 2) (220,221). These two proteins are antigenically cross-reactive (222). The predicted amino acid sequence of Irp 1 (3161 amino acids specifying a protein of predicted mass of 384.6 kDa) shows the C-terminal region to be highly homologous to Irp2 (HMWP2), accounting for immunological cross-reactivity. HMWP2 is a nonribosoma1 peptide synthetase, and recent biochemical studies show it to have the capacity for thiazoline formation (heterocyclization) (223). More recently, Gehring et al. (216) reported the complete sequence of the yersiniabactin region of Y. pestis. Their biochemical studies on Y. pestis yersiniabactin synthesis suggest that it is assembled in a modular fashion by HMWPl and HMWP2. Intermediates are passed from the amino terminus of HMWP2 to the carboxyl terminus of HMWP1. Because the genes for yersiniabactin are conserved, it is presumed that Y. enterocoliticn and Y. pseudotuberculosiswill have similar pathways. In summary, the genes for sequestering iron from the host have been identified, and the biochemistry of yersiniabactin assembly should be known in detail in the near future.

E. Plasmid-AssociatedVirulence 1. Yersinia Virulence Plasmids All three Yersinia pathogenic species require 2.5 mM calcium ion at 37°C. Without calcium at elevated temperature, the cells can only proceed through approximately two generations followed by growth arrest. Calcium dependence is, however, an unstable phenotype. That is to say, spontaneous suppressors can be isolated at frequencies of 1 X lops.This phenomenon, discovered in the 1950s (159), is similar to mutation rates associated with loss of plasmids and prophage or later identified transposition-associated events. Indeed, in 1980 a large virulence plasmid of 40 megadaltons (70 kb) correlated with calciumdependent growth and virulence in each species (186,224,225). Loss of calcium dependence at 37°C was associated with the loss of the virulence plasmid. Production of V (protein) and W (lipoprotein) antigens have long been recognized as virulence-associated properties of Y. pestis and Y. pseudotuberculosis (226-228). However, when pathogenic Y. enterocoliticn were also found to produce immunologically identical antigens (229), it suggested that all disease causing Yersiniae shared common mechanisms of pathogenicity. Studies on virulence of Y. enterocolitica serotype 0:8 showed that the production of V and W antigens was closely associated with calcium-dependent growth at 37”C, which in turn wasdependent on thepresence of the plasmid (186). Genetic studies and mouse models (186,197,226,229-232) verified the correlation of this plasmid withthe production of virulence antigens and with pathogenicity of Y. erlterocolitica. This plasmid, originally known as Vwa due to its association with V and W antigens, is genotypically designated as pYV. The related plasmid of Y. pseudotuberculosis is commonly designated as pIB, but in subsequent discussion the virulence plasmid from different pathogenic Yersinia species will be collectively referred to as pYV.

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The pYV plasmids of Yersinia species are similar in function (230,231) and size (233). However, DNA hybridization studies showed that these plasmids are not identical. The pYV of Y. enterocolitica and Y. pestis are only 55% homologous in DNA sequences (233), and homologies ranging from 58 to 87% have been reported even within species (197). For example, among pathogenic Y. enterocolitica, pYV of serotypes 0 : 3 and 0:9 are 90% homologous, but they shared only 75% homology with pYV of serotype 0:8 (234). In spite of these differences, the extent of sequence homologies among pYV of Yersirzia would suggest a common origin for this plasmid. A key observation in the early 1980s involved the correlation of the virulence plasmid with additional specific proteins. Portnoy et al. (197,235) showed that the outermembrane proteins of pathogenic yersiniae changed with temperature, but only in pYV' cells. Furthermore, mutations in specific regions of the pYV plasmid resulted in virulence attenuation or loss, and these changes were reflected by specific changes in outermembrane protein profiles. In addition to encoding calcium dependency and V and W antigen production, the pYV was shown to be responsible for other virulence-related phenotypes. These included auto-agglutination, serum resistance, uptake of crystal violet and Congo red dyes, cytotoxicity for tissue culture cells, production of Yersinin outer membrane proteins (Yops), guinea pig conjunctivitis (Sereny reaction), and lethality for mice and other animals (10,55,235). Two key observations were made with regard to pYV-encoded proteins in the late 1980s. First, Heesemann's laboratory showed that the outer membrane proteins induced at 37°C were actually secreted into the extracellular environment under calcium-limiting conditions (236). This was unusual because, at the time, the general dogma was that grampositive organisms secrete extracellular proteins, whereas gram-negative organisms, with few exceptions (e.g., E. coli hemolysin), export proteins to the periplasnl or the outermembrane, not the extracellular milieu. Second, Michiels et al. (237) reported that these proteins were secreted in an unprocessed form, i.e., secretion did not require proteolytic cleavage of a signal sequence. Hence, the Yersir~inouter membrane proteins were unique in this respect and appeared to be sec-independent. This property was confirmed. Originally referred to as Yops (for Yersinicr outer membrane proteins), with these two observations the Yop moniker for this set of proteins was modified; Yop was retained but now refers to Yersiizia outer proteins as opposed to the original context of outer membrane proteins. The secretion of Yops can readily be displayed in the laboratory. Y. erzterocoliticn is grown to mid to late exponential phase in calcium-depleted tryptic soy or BHI broth (Le., supplemented with 20 mM sodium oxalate and 20 mM MgSO, or some other chelation agent such as EGTA) at 25-30°C. The cells are then shifted to 37OC, and after 24 hours strands of proteineous material (insoluble Yops) can be spooled from the culture or precipitated from culture supernates with solvent or salt. SDS-PAGE gels can then be used to resolve individual secreted proteins. By the early 1990s the focus of work was well defined. Significant data indicated secreted Yops used a unique export pathway, they comprised a set of proteins essential for virulence, and their synthesis was induced by temperature. Hence, the stage was set to define each of these aspects: What is the nature of the export pathway? What is the function of each individual Yop? and How is the system regulated at the genetic level? In the ensuing 8 years, immense progress has been made in each of these areas. In particular the elucidation of the Yop secretion pathway led to the realization that this is acommon mechanism employed by numerous gram-negative pathogens for protein export (both plant and animal pathogens). Surprisingly, it was also determined that the mechanism of Yop

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secretion is related to flagellum biosynthesis. These secretion mechanisms have now been termed the type 111 system. 2. Type Ill ProteinSecretion The type 111protein-secretion system defines a set of dedicated secretory ports, or channels, on the surface of a gram-negative organism. About 20 conserved proteins are required to form the protein export channel. For Yersinia, this set of conserved proteins is denoted as Ysc and Lcr proteins (for Yersinia secretion and low calcium response). In contrast, the secreted proteins using this export pathway can vary considerably. That is, one finds the secretion machinery to be highly conserved from Yersinia to Xanthomonns, but the virulence proteins secreted are directed toward specific attributes of the host. Conserving the secretion apparatus in this manner, yet varying secreted proteins, has undoubtedly played a significant role in conferring gram-negative bacteria the ability to parasitize a wide spectrum of hosts ranging from mammals to plants. Other gram-negative human and animal pathogens employing type 111 protein secretion as a component of virulence expression include Scdmonella spp., Shigella spp., enteropathogenic E. coli (EPEC), and Pseudomonas neruginosn. A number of additional pathogens are suspected to operate type I11 systems based on preliminary sequence data. Plant pathogens with characterized type I11 systems, comprising HRP proteins involved in the hypersensitivity response, include Pseudomonas syringne, Xnnthomonns cmnpestris, Erwinin anzylovorn, and Rnlstonia solanacenarum (for recent reviews, see Refs. 160-163). For Y. enterocolitica, it is estimated that there are about 9-12 type I11 portals per organism. This is equivalent to the number of flagella made at 530°C (the relation of type I11 secretion and flagellum biosynthesis is discussed below). Uniform secretion of Yops from all export sites occurs in vitro when calcium ion is limiting, but secretion is polar and contact dependent in vivo when the organism encounters a mammalian host cell, as discussed below. In hindsight, however, in vitro Yop secretion in limiting-calcium medium may be a fortuitous artifact that led to the elucidation of this remarkable system. The milestones in the dissection of this system follow. The first insight into the mechanism of Yop secretion was made serendipitously. Two independent laboratories working on the developmental regulation of flagellum biosynthesis in the dimorphic organism Caulobacter crescentus sequenced the flagellar gene JEbF (now designatedJEhA)(238,239). When this sequence was compared to the GenBank, the predicted amino acid sequence was strikingly similar to the predicted protein encoded by Y. pseudotuberculosis 1cr.D. The IcrD gene maps within the highly conserved region of all pYV plasmids. Because the predicted amino acid sequence similarity between FlbF and LcrD was approximately 7096, it suggested a common function. Yet why a flagellar gene in the unrelated innocuous aquatic bacterium C. crescentus was related to a gene essential for virulence in Yersinin was enigmatic. Using phoA fusions, it was shown that both C. crescentus FlbF and Y. enterocolitica LcrD proteins were localized to the inner membrane and showed similar membrane-spanning domains (238,240). Mutations in lcrD abolish Yop secretion (240) and JEbF mutants fail to assemble flagella (238,239). These observations were the first hint that flagellum biosynthesis and Yop export were somehow related. As more DNA sequences became available in both flagellar and Yop secretion genes, the similarity between the two systems became more evident. Other flagellar homologs of Yersinia type I11 secretory components are listed in Table 1. All of these genes have the common feature of being required in either flagellar protein or Yop export. Hence,

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490 Table 1 Yersinia Type I11 Secretion: Lcr/Ysc and Flagellar Homologous Proteins

1. 2. 3. 4.

5. 6. 7. 8. 9. 10.

Yop secretion

Flagellar assembly

LcrD YscN YscQ YscR Yscs YscT Ys c u YscD YscJ YSCL

FlhA FliI FliY Flip FliQ FliR FlhB FliG FliF FliH

Source: Ref. 163.

the bacterial flagellum, generally regarded solely as an organelle for motility, was now further appreciated in a new venue-its more subtle function as a dedicated secretory portal for flagellar-specificproteins. Flagellin subunits, like Yops, are secreted in an unprocessed (sec-independent) manner. Furthermore, extracellular flagellar components, such as the hook and flagellin subunits, are translocated through the core of the basal body (241). When one considers the amount of flagellin protein comprising the flagellar filaments, one can conclude that the flagellar basal body is an exquisite secretion apparatus as well as a fine motor. Likewise, the amount of Yops secreted under pemlissive conditions is striking (up to 20% if total cellular protein) (160), being visually evident in culture supernates. Until these parallels were recognized, secretion of Yops was generally assumed to be firstdirected to the outermembrane and then uniformly released across the surface upon calcium ion chelation. As the number of virulence proteins showing similarity to proteins required for flagellar protein secretion increased, this view changed. This semblance suggested that Yops may be secreted from a dedicated portal analogous to the basal body of the flagellum. This connection between Yop and flagellar protein export was experimentally reinforced by the following observations. Genetic evidence had previously determined that YopE was responsible for cytotoxicity of eukaryotic host cells (242). Similarly, YopH was known to inhibit phagocytosis by macrophages (243), and Guan and Dixon (244) determined that YopH was a protein tyrosine phosphatase (PTPase). However, pure preparations of Yops had no cytotoxic effect when added to tissue culture cells (245). Furthermore, the possible role of YopH as an extracellular PTPase was not understood. Rosqvist et al. (246) determined that cytotoxicity of purified YopE required direct microinjection into Hela cells. Further, they showed that YopE cytotoxicity imparted by Yeninin cells in tissue culture was dependent upon YopD. That is to say that yopD- mutants could secrete YopE, but such mutants were not cytotoxic to host cells. Hence, the logical implication drawn by these authors from these experiments was that, in vivo, the Yop proteins are delivered across the eukaryotic membrane into the target cell cytoplasm and that delivery required functional YopD. This point was elegantly proven by two separate approaches. In what will undoubtedly be viewed as a classic experiment, Rosqvist et al. (247), using confocal laser microscopy with fluorescent-tagged anti-YopE antibodies, showed that the Yops were vectorally secreted into the host cell from the side of the bacterium in direct '

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contact with the host cell membrane. Additionally, these experiments showed that Yops emanated from distinct sites on the surface of the organism as opposed to a uniform release. Similarly, Sory and Cornelis (248) and Sory et al. (249) showed this same effect employing YopE- and YopH-adenylcyclase reporter fusions. This strategy employed fusing the calmodulin-dependent adenylcyclase domain of Bordetelkr pertussis cyclolysin to the secretion competent N-terminus of YopE. Y. enterocolitica harboring these fusion genes were allowed to infect tissue culture cells, and cAMP levels were monitored in the cells and in the outside medium. Because the fusion protein adenylcylase activity is dependent upon calmodulin, which is only present within eukaryotic cells, increased cAMP levels reflected internalization of the hybrid protein. These authors were also able to show that adenylcylase activity of the fusion protein was dependent upon YopB and YopD. With these two important studies, Yops were importantly recognized as two categorical groups: host cell effector Yops (e.g., YopE and YopH) and Yops required to translocate these effectors across the eukaryotic membrane (e.g., YopD and YopB). In summary, between 1990 and 1995, it was shown that Yop secretion is related to secretion of flagellar proteins, that Yop secretory sites are distinct on the cell surface, and that Yops are directionally delivered, or injected, into the cytosol of target host cells. These type I11 secretion organelles have recently been visualized under the electron microscope in SaZmorzeZlu typhinzuriurrz.Termed "needle-like" structures, they are strikingly similar to flagellar basal bodies in form (250).

3. Yop Function Below is a brief summary of effector and translocation individual Yop functions. It should be noted that each Yop has a cognate chaperone, designated Syc. These are covered in detail in the recent review of Cornelis et al. (160). Yop Effectors YopE. This 23 kDa protein accumulates in the perinuclear region of target host cells. It has cytotoxic activity causing host cells to round up due to disruption of the cytoskeleton. It has sequence similarity to P. creruginosn Exoenzyme S and the SptP protein of Scdmorzelln (160). YopP/J. YopP (Y. enterocolitica)and YopJ (Y. pseudotuberculosis) induce apotosis of the host cell. The protein has a molecular weight of 30 kDa and appears to remain localized in the cytoplasm of the host cell (160). YopT. YopT is the most recently described effector Yop (251). Mutants deleted for other effector Yops but secreting functional YopT disrupt the cytoskeleton of target cells. Secretion requires an intact type I11 apparatus and the YopT cognate cytosolic chaperone, Syc. The molecular weight of the protein is 35.5 kDa. YopM. Mutations in YopMgreatly attenuate virulence in mouse studies. This protein shows significant predicted atnino acid similarity to the SIzigelZcr IpaH protein. Early studies showed that this protein-mediated thrombin binding and in vitro studies showed it inhibited thrombin-induced platelet activation (160). Although this suggested an extracellular function, more recent studies show that Y. enterocolitica YopM is secreted into host cells (252). More detailed studies on Y. pestis YopM have shown it is targeted to the host cell nucleus (253). YopH. YopH is the best characterized Yop, with biochemical analyses and crystal structure confirming its function as a protein tyrosine phosphatase (PTPase)

a.

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(244). It is localized to the cytoplasm of the host cell, where it has been shown to dephosphorylate p13OCas and FAK (focal adhesion kinase) by Persson et al. (254). This activity prevents signal transduction between integrin receptors and intracellular signaling transducers. In macrophages, this activity inhibits phagocytosis. YopO/YpkA. This Yop has a molecular weight of 84 kDa. Designated YopO in Y. erzterocolitica, but in Y. pseudotubercuZosis it was designated YpkA when the predicted amino acid sequence was shown to have high similarity to serine/ threonine kinases of eukaryotes (Yersirlia protein kinase A). Mutant strains lacking YopO function show greatly reduced virulence in mice. In the mouse model, a y o p 0 - mutant passes normally through early stages of infection, but it does not colonize the spleen. The target protein of YopO/YpkA has not been reported (160). b. Yop Translocation-Associcrted Proteins Less is known at this time about the proteins involved in contact delivery of the Yop effector proteins described above. A brief synopsis is given below in the order of the protein molecular weights.

LUG. The LcrG protein (ca. 11 kDa) forms dimers and contains a heparin-binding domain. Mutations in IcrG abolishes directed translocation of all Yop effectors even though lcrG- strains are deregulated for Yop expression. That is, mutations in ZcrG confer the calcium-blind phenotype (160). YopK/YopQ. This protein has a molecular weight of ca. 21 kDa, and preliminary analysis of the role of this protein implicates it in the control of Yop translocation by modulation of the YopB pore. YopD. As mentioned above, YopD mutants were essential in demonstrating that Yop effectors are secreted into the host target cell cytoplasm (246,249). This protein, (33.3 kDa) is hypothesized to form the entry pore in the host cell membrane in association with YopB (160). LcrV. This protein is described as the protective antigen against plague and one of the early Yeniuia proteins described. Mutations in LcrV diminish YopB and YopD export. It is hypothesized that LcrV forms an integral component of the secretiodinjection apparatus ( l 60). YopB. This protein (41.8 kDa) is also essential for delivery of Yop effector molecules into the host cell (249). It associates with YopD and demonstrates homology with the RTX family of a-hemolysins and leukotoxins (pore-forming toxins). As such it is implicated in disrupting the host cell membrane. Purified YopB can cause lysis of sheep erythrocytes and disrupt lipid bilayers. YopB also displays sequence similarity to IpaB or ShigeZZa JEexneri and PopB of Pseudomonos aerugirlosa ( 160). Two additional proteins are implicated in the control ofYop effector secretion. YopN (32.6 kDa) is secreted in large quantities at 37°C in calcium-depleted medium. It is also synthesized at 37°C in the presence of calcium, where it is localized to the outer membrane. YopN is the hypothetical "lid" to the secretion channel and interacts with TyeA. This latter protein, recently reported (255), has a molecular mass of 10.8kDa

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and maps immediately downstream of yopN. Mutations in both yopN and QeA, like the aforementioned ZcrG, give a calcium-blind phenotype. In summary, the pYV-encoded Yops are induced by host temperature (37°C). Upon host cell contact, the translocation Yops form a pore in the host cell membrane through which the effector Yops are passed into the cytoplasm of this target cell. The contactdependent signal during this interaction stimulating Yop transfer is not known. However, it is tempting to speculate that activation of host cell receptors stimulates a flushing of calcium ion from the eukaryotic cytoplasmic membrane uncapping the export channel. 4. Virulence Gene Regulation

a. Secretion-Driver? Feedback Circuit on Transcription The regulation of flagellar genes served as an additional paradigm for investigating the control of Yop synthesis and secretion. Transcription of both flagellar and yop regulons is enhanced once protein secretion initiates. In other words, both systems canbe viewed as "secretion-driven" expression systems. An understanding of this "feedback" system was elucidated first for the flagellar regulon. Until the early 1990s it was unknown why mutations in any flagellar basal body gene (flagellar class I1 gene) resulted in transcriptional repression of flagellin, chemotaxis, and motor genes, all of which are positioned below basal body genes in the regulatory hierarchy (class I11 flagellar genes) (241). Hughes et al. showed the mechanism explaining this phenomenon (256), and it has a direct parallel in Yop synthesis. During flagellum biosynthesis, S. typhirnurium expresses an anti-sigma factor, FlgM, that directly antagonizes the sigma factor for flagellin gene transcription, FliA (oF). This interaction holds FliA in check until basal body synthesis is complete and competent for secretion of flagellin. Once flagellar hook assembly is complete, the anti-sigma factor, FlgM, is secreted from the cell through the basal-body hook complex. This dilution of FlgM from the bacterial cytoplasm allows FliA to then activate the next tier (class 111)of structural genes (flagellin, chemotaxis, and motor genes). Mutations in basal body assembly prevent FlgM secretion and, therefore, prevent FliA from promoting class I11 gene transcription (256). The parallel to the FliA-FlgM interaction for Yersinia yop gene expression was readily recognized based on published genetic studies. In fact, Rimpilainen et al. (257) speculated that Y. pseudotuberculosis LcrQ might play the equivalent role of FlgM several years before it was proven based on this genetic evidence. What are these similarities in regulation? First, reporter fusions to a number of yop genes showed they are induced upon a temperature shift to 37°C. However, yop gene expression remains at a low level when calcium ion is present (258-261). Chelation of calcium ion promotes secretion of Yops, which in turn results in a dramatic increase in yop gene expression levels. Therefore, calcium, in a not-yet-understood manner, blocks secretion and gene expression. Mutations in Y. pseudotuberculosispYV-encoded 1crQ phenotypically express and secrete Yops even in the presence of calcium ion, implying its role as a negative regulator comparable to FlgM. Secretion of LcrQ was shown in 1996 and fit the predicted model nicely (262). Interestingly, these findings could not be initially reproduced in Y. enterocolitica, which secretes a homolog of LcrQ termed YscM. This was because the Y. erzterocolitica pYV plasmid contains two copies of yscM. Mutations in bothyscM genes phenotypically resemble 1crQ mutants (263). The target of LcrQ (YscM)-negative regulation has notbeen determined.

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b. Temperature arzd Gene Regulation Temperature has a profound effect on Yersirzicr spp. within the narrow range of 30-37°C. Shifting cells to 37°C includes the following changes in cell phenotypes; motility is lost, porin synthesis is repressed in pYV+ cells, calcium-dependent growth initiates, the vop regulon is induced, and the cells morphologically change from single cells to chains that undergo autoagglutination (264). The Y. er~terocoliticcryop genes are generally dispersed around the pYV plasmid organized as monocistronic units (reviewed in Ref. 265). Transcription of Y. ellterocoliticct vop genes is dependent upon the positive activator VirF and temperature (261). VirF is a DNA-binding protein with sequence similarity to the AraC class of helix-turn-helix proteins (266). DNA footprinting experiments have identified the consensus VirF-binding site, TTTTaGYcTtTat (Y indicates C or T), for yopE, yopH, virC, and the 1crGVsycD.v opBD operon (267). The Y. enterocoliticcr virF gene is induced by temperature, even when placed in E. coli (266.268). In contrast, the homolog of VirF in Y. pestis, LcrF, is constitutively expressed, but yop gene transcription still requires elevated temperature (37°C) (269). When Cornelis et al. (270) fused Y. erzterocoliticn virF to the inducible tcrc promoter, artificial VirF overproduction at 30°C did not induce yop gene expression. Hence, both the natural constitutive expression of LcrF in Y. pestis and artificial manipulation of virF in Y. enterocolitica show that temperature is an integral signal in yop gene induction in addition to the positive activator. Several lines of evidence have implicated temperature-dependent changes in DNA topology as a factor in vop induction. Cornelis et al. (271) isolated constitutive yop mutants by transposon mutagenesis. DNA sequence identified the insertionally inactivated gene, ymoA (for Yersirzin tnodulator of virulence), to encode a histone-like protein. This protein is highly similar to the regulator of E. coli hemolysin expression, M z n , which has been shown to affect DNA supercoiling (272). YmoA mutants are not only constitutive for yop expression, but are VirF-independent. Yet temperature still enhances yop transcription in y r ~ o A -mutants (271), suggesting that at 30°C the yop promoters are structurally constrained and that temperature somehow alleviates this constraint. Rohde et al. (268) determined that induction of yop genes and repression of flagellar genes are coincident with changes in DNA supercoiling upon a temperature upshift from 25 to 37°C. Moreover, low levels of the DNA gyrase inhibitor novobiocin were shown to induce supercoiling, yop gene expression, and repress flagellin gene transcription at 25°C. As such, pharmacologically inducing supercoiling atlow temperature elicited a high-temperature phenotype. These authors also isolated a class of novobiocin-resistant mutant (DNA gyrase) that was constitutive for yop expression, like the aforementioned VmoA mutants, and determined that these mutants were also nonmotile (268). These data suggested that the reciprocal regulation of the flagellar and yop type I11 regulons was coordinate. Examination of the temperature requirement in yop expression by both groups suggested temperature-induced changes in DNA topology. mediated by YmoA and DNA supercoiling, appear to be required in the phenotypic temperature phasing of virulence genes between 30 and 37°C. Another factor in thermoregulation also indicates DNA topology is critical. The pYV plasmid is enriched for regions of intrinsic DNA bending (268) (J. R. Rohde, X. Luan, H. Rohde, J. M. Fox, and S. A. Minnich, in press). Furthermore, these intrinsic DNA bends can influence DNA supercoiling. Most striking is the fact that these bends are maintained by temperature until 37OC, whereupon they ‘melt’ (J. R. Rohde, X. Luan, H. Rohde, J. M. Fox, and S. A. Minnich, unpublished). Based on these facts, Rohde et

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al. (268) proposed the following model to account for thermoregulation in Y. enterocolitica. At low temperature (<37"C) DNA intrinsic bends are present and may bepreferential binding sites for histone-like proteins with repressor activity. This implied plasmid architecture maintains yop genes in a repressed state. Upon a shift to 37"C, intrinsic DNA bends melt, histone-like protein(s) is dislodged, and the plasmid undergoes compensatory modulations in supercoiling allowing the virF and yop promoters to form competent transcription complexes. Such a model, incorporating the synergism between intrinsic bends, histone-like proteins, and DNA supercoiling, is consistent with the physiological expression data. This model also suggests application in the temperature-induction of virulence genes in other pathogenic genera where these same influences are factors. Indeed, temperature-sensitive DNA bends have been recently shown to be an integral component in the temperature induction of the Shigella type TI1 regulon (273). Taken together, these data suggest that the cellular "thermostat" regulating Y. enterocoliticn plasmid-encoded virulence genes is a temperature-induced change in DNA structure.

c. Motility and Virule~zce Both foodborne pathogens, Y. yseri~otr.rbercr~losis and Y. enterocoliticn, phenotypically lose motility when shifted to host temperature (37°C) (264). In contrast, Y. pestis is classed as nonmotile, and flagella production has not been reported. Therefore, motility appears unnecessary for virulence in all three pathogens. However, there are several intriguing aspects of the Yersinicr flagellar regulon that warrant discussion. First, motility is coordinately temperature-regulated in a reciprocal manner with theyop genes as discussed in theprevious section (268). Second, some of the flagellar basal body genes are transcribed at 37°C (274) (M.J. Smith, C. Kifer, and S.A. Minnich, unpublished) although the flagellin (filament protein) genes are repressed (275) (V. Kapatral, M.J. Smith, and S.A. Minnich, unpublished). Third, Y. yestis, though not motile, contains the full regimen of flagellar genes on its chromosome. Initially determined by Southern hybridization (M.J. Smith, H. Rohde, and S.A. Minnich, unpublished), this fact is readily confirmed when one BLAST-searches the just completed Y. pestis chromosome sequence for flagellar gene homologies. (These sequence data were produced by the Y. pestis Sequencing Group atthe Sanger Centre and can be obtained from ftp:// ftp.sanger.ac.uk/pub/yyy.)The Y. pestis flagellar genes are present and organized in the same manner as other enteric bacteria (unpublished). Northern blots using Y. pestis mRNA isolated from cultures grown at 37°C and the Y. enterocoliticn flgE (flagellar hook gene) as probe show the presence of hook operon transcripts in Y. pestis, indicating part of the flagellar regulon is transcribed (M.J. Smith, C. Kifer, and S.A. Minnich, unpublished). Fourth, Y. euterocolitica with Tn5-ZacZ insertions is several flagellar genes fail to secrete Yopsat 37"C, even though these strains harbor pYV (276). Two of these transposon insertions have been identified; one is within j g G (flagellar rod gene) and the other upstream of fliA (flagellar sigma factor). These data are in contradiction to similar studies in S. trvyhimurirrr~where the production of the virulence type I11 regulon was not affected in motility mutants (250). However, at a minimum, the above data suggest that some mutations in the Y. enterocoliticcr flagellar type I11 system may disrupt proper localization, assembly, or regulation of the Yop type 111 secretory system. This may also explain why they are coordinately regulated (268,275) (V. Kapatral, M.J. Smith, and S.A. Minnich, unpublished). How different Y. enterocoliticn is in this respect from S. tyhimuriurn remains to be determined. Recently it was shown that S. ~yphimurizmcontains two type I11 secretion systems localized in two different pathogenicity islands on the chromosome. Each system is required for virulence. Tn5-ZncZ mutations within one type I11 locus nega-

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tively affect expression of the other type I11 system (277). Thus, coordinate regulation of type I11 protein secretion during infection may be a necessity. Finally, one additional piece of evidence shows the potential of flagellar involvement in virulence. Virginia Miller’s laboratory recently reported that a mutation in a Y. erzterocolitica gene encoding a secreted phospholipase attenuates virulence in the mouse model (278). Further studies show this enzyme is required for “swarming” motility and is under the control of the flagellar gene regulon (279). Therefore, motility may be required for the early stages of infection for the proper expression of this enzyme, perhaps even for directing its secretion.

F. PathogenicityTesting Both pathogenic and nonpathogenic Yersinin species are ubiquitous; hence, the presence of the organism in a food is not sufficient to implicate that food as the source of Yersinia infection. Therefore, all potentially pathogenic species of Yersinia isolated from foods need to be tested for virulence or virulence-associated phenotypes. With a few exceptions, most assays for Yersirzia pathogenicity measure virulence or virulence-associated factors encoded by the pYV virulence plasmid. 1. Cellular Invasion The ability of Yersinia species to invade mammalian cells can be determined using tissue culture models. The invasion of tissue cells by yersiniae is highly efficient, beginning with a half-hour lag phase, followed by rapid uptake of bacteria for 1-2 hours (280). Invasive bacteria may be visualized by acridine orange staining of monolayers for intracellular bacteria (28 1) or detected by a quantitative assay, using gentamicin to eliminate extracellular bacteria followed by lysis and microbiological plate counts of intracellular bacteria in the tissue cells (203). The use of DNA probes may be an alternative means to determine invasiveness of Yersinia species. Probes specific for the inv genes of Y. enterocoliticn and Y. pseudotuberculosisand for the ail gene of Y. enterocolitica have been used to characterize invasion genes in Yersinia species (139,208). Comparative analyses, using two synthetic oligonucleotide probes specific for the inv and nil genes, showed good correlation between probe reactivity and the ability of Yersinia isolates to invade HeLa cells (282).

2. Calcium-DependentGrowth In vitro calcium-dependent growth at 37°C by pathogenic isolates may be determined using magnesium oxalate agar (186,283) or other low-calcium medium (284). Virulent strains are calcium dependent and will yield pinpoint colonies after overnight growth, while avirulent strains produce larger colonies (283). DNA probes, specific for the calcium-dependency region of the virulence plasmid, may also be used as indirect evidence for this phenotype in pathogenic Yersinia (135,139). 3. Congo Red Binding Virulent Y. enterocolitica colonies bind Congo red dye to produce a reddish pigmentation that is absent in colonies of avirulent strains. The ability to bind Congo red is thought to be due to plasmid-mediated uptake of hemin (285); therefore, the assay merely identifies colonies of Yersirzia that bear the virulence plasmid. The phenotypes of Congo red binding and calcium dependency can be measured simultaneously using specially formulated differential media (286,287).

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4. Crystal Violet Binding Binding of crystal violet at 37°C is another test that identifies the presence of the virulence plasmid in Yersinia colonies (288). Although the mechanism of crystal violet binding by colonies of plasmid-bearing Y. enterocoliticn has not been fully elucidated, it is believed to be due to a cell surface protein or components encoded by the virulence plasmid (288).

5. Pyrazinamidase Activity Absence of this enzyme activity appears to be a characteristic of pathogenic serotypes of Y. enterocolitica (289). Although a close correlation exists between a negative test and the presence of plasmids in the pathogenic strains, pyrazinamidase activity has not been linked directly to the virulence plasmid. Hence, the test is an indirect indicator that the plasmid may be present and that there is a potential for pathogenicity (289). 6. Autoagglutination Strains of Yersinia species that are virulent in mouse assays will autoagglutinate in tissue culture media (290) or in phosphate-buffered saline (285) at 37°C but not at 25°C. Avirulent strains do not exhibit this property (290). Autoagglutination is attributed to the YadA protein, and it is closely correlated to the presence of the virulence plasmid in the Yersinia species (291). 7. Serum Resistance Resistance of virulent Y. enterocoliticn strains to the bactericidal effects of normal serum is another temperature-dependent plasmid-mediated property that occurs only at 37°C (292,293). In the presence of 10% normal human serum, avirulent strains of Y. enterocolitica are neutralized within 1 hour at 37"C, while virulent strains show resistance and begin to multiply after 2 hours (293). This property is also correlated with the plasmid-encoded YadA protein in Y. enterocolitica (29 1,294,295).

8. AnimalModels Numerous animal models have been used to assess virulence of Yersinin species for humans. These models, discussed in detail elsewhere (lo), include kerato-conjunctivitis in guinea pigs (Sereny test), lethality to mice and gerbils, enterocolitis in rabbits, and others. Mouse models, however, appear to be the most responsive to Yersinia infections, and this response is dependent on the presence of the pYV plasmid. Mice infected with European serotypes of Y. erzterocolitica via oral or intraperitoneal inoculations will produce diarrhea, but infecting mice similarly with the more virulent American serotypes usually results in lethality (10,296). Treatment of mice with iron increases the susceptibility of mice to Yersinia infections and even by less virulent strains. In addition, isolates of Y. kristensenii inoculated parenterally into mice presensitized with iron, were found to be virulent (215). These strains, which are generally regarded as nonpathogenic, did not express virulenceassociated phenotypes, did not carry the pYV virulence plasmid, and were not invasive, suggesting therefore that Y. kristensenii may be causing lethality in iron-loaded mice via other virulence mechanism(s), perhaps a secreted toxin (215). Numerous studies have evaluated and compared the various pathogenicity assays and their association with lethality in animal models or disease in humans. Analyses for the physical presence of the 42-48 Mdal virulence plasmid is unreliable since other Yersiniu species may carry plasmids of similar sizes that are not involved in pathogenicity (297). Most in vitro assays were able to moderately predict the presence of virulence plasmids in Yersinin, but no single assay was consistently better than others nor entirely

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reliable in predicting pathogenicity of Yersinin species (135,283,297,298). In fact, several studies showed that many in vitro pathogenicity tests were only somewhat correlated with virulence of Yersirzia in animal models (297,299,300) and tnay be entirely unrelated to the occurrence of diarrheal disease in humans (301). Therefore, it is evident that in vitro pathogenicity test results should be carefully interpreted and that a combination of tests may need to be used in assessing the virulence of Yersirzin species (299,300).

VII.PREVENTIONANDCONTROL Principal sources of yersiniosis outbreaks have implicated processed or prepared foods. During the investigation of a food processor that had produced Y. enterocolitica-contaminated foods, the organism was found in floor drains, on cleaning tools, in wash areas, on floor contact surfaces and in condensates within the food-processing facility (unpublished data). Therefore, key factors to prevention and control of Yersirlia must focus on the use of uncontaminated raw ingredients, proper preparation of food, and the proper cleaning and sanitation of the processing environment. Properly constructed and maintained facilities and equipment are critical in the control of yersiniae. Ceiling, floors, and walls should be smooth, tightly sealed, and water repellent. Doors and windows should be tight fitting and equipped with insect screens. Plant drainage systems should be adequate and accessible for easy cleaning, and the pipes should be insulated and in good repair. Water has been implicated in past yersiniosis outbreaks (9,85,86,301); therefore, water used in food processing and ice making should be treated and tested to ensure that it is free of Yersinia contamination. During preparation and processing of foods, possible sources of contamination are conveyors, belts, aprons, gloves, tables, mixing bowls, strainers, storage tubs, buckets, knives, and other implements that come in direct contact with food. Yersirzia can adhere and survive in biofilms on stainless steel and other surfaces (302); hence, these items and equipment have to be easily disassembled for daily cleaning and sanitizing. Although Yersinia is not noted for airborne transmission, it can be spread by splashing water from unclean surfaces onto food contact surfaces during clean-up. Also, if processing by-products are taken to waste facilities or to a farm for use as anitnal feed, the containers used must be sanitized before returning them to the food-processing environment. Inadequate cleaning of milk containers used to deliver outdated milk from a dairy to a hog farm may have been the cause of the largest Y. erzterocolitica outbreak in the United States (72,303). Yersinia grows well in cold, moist environments and can survive freezing, but it is killed by pasteurization, chlorine, and other commercial sanitizers. Yersinin is more susceptible to chlorine than other enteric bacteria. A treatment with 0.25 ppm chlorine dioxide for 5 minutes can reduce Y. ellterocolitica by4-5 log cycles (304). Yersirzia attached to stainless steel and other surfaces are slightly more resistant to chlorination (302). Since cross-contamination can occur during handling of postprocessed products and Yersinia can grow in foods at temperatures as low as 0- 1"C (11l), refrigeration alone may not be sufficient to prevent the growth of yersiniae in foods. Therefore, any fresh refrigerated foods must be suspect after extended periods of refrigeration (305). For a more detailed review of the factors that can effect growth and survival of Yersinia in foods, see Sec. V.A. These factors should be kept in mind when attempting to evaluate a process for the potential survival and outgrowth of Yersinin.

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Finally, food handlers need to understand the importance of sanitation and beresponsible for safe handling of food products. Good employee hygiene, such as use of clean clothing and footwear, washing hands prior to work or after handling unclean surfaces or raw ingredients, are critical to producing a wholesome, safe product. Knowledgable supervision of well-planned quality control and sanitation programs plus a commitment by management to adhere to and enforce rules of food sanitation are also essential.

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20 Surveillance of Foodborne Disease Ewen C. D. Todd Health Canada, Ottawa, Ontario, Canada

I. Introduction 5 16 11. Objectives of aFoodborneDiseaseSurveillanceSystem

517

111. Definitions of Terms519 IV.SurveillanceProgramsinEurope521 A. Notifiable foodborne diseases 521 B. Foodborne disease 522 V. SurveillanceProgramsinAustralia, A. Australia 529 B. New Zealand 53 Oceania C. 532

VI.SurveillanceProgramsin

New Zealand, and Oceania529

1

Asia

532

A. China 533 Taiwan 533 B. C. Hong Kong 535 D. Korea 535 E. Japan 536 F. Viet Nam 537 Thailand G.538 H. Cambodia 538 I. New Guinea (Irian Jaya and Papua New Guinea)539 J. Indonesia 539 K. Malaysia 539 L. Singapore 540 M.India,Pakistan,Bangladesh, Sri Lanka, and theMaldiveIslands N. Nepal 543 VII.SurveillancePrograms

in theMiddleEast543

A. Israel 543 B. Jordan 544 C. Lebanon 544 D. Saudi Arabia 544 E. 545 Iran F. Yemen 545 G. Bahrain 545

541

Todd

516 VIII. Surveillance Programs in Africa 545 A. Cholera in Africa 545 B. EHEC infections in Malawi and the Central African Republic C. Aflatoxicosis in Mozambique and other countries 546 D.Toxoplasmosis and brucellosis in the Sudan and other countries E. Egypt 547 F. Algeria 547 G. Tanzania, Ethiopia, and Kenya 547 H. Zambia and Zimbabwe 548 I. Liberia and the C6te d'Ivoire 548 J. Nigeria 549 K. South Africa and KwaZulu-Natal 549 L. Madagascar 550 IX.Surveillance

546 547

Programs in the Caribbean and Central and South America 550

Surveillance of foodborne disease 550 B. Cholera in Peruand other Latin American countries 551 C. Mexico 552 D. Argentina 552 Brazil 552 E. F. Peru 553 G. Other countries 553 X. Surveillance Programs in Canada and the United States553 A. Canada 553 B. The United States 556 A.

XI.

Sentinel,Case-Control, and Other Specialized Epidemiological Studies560 A. Europe 560 B. The United States

XII. Estimates

561

of Numbers of Foodborne Disease Cases and Their Costs 563

A. Number of cases 563 B. Number of deaths C. Costs 564

563

XIII. Control of Foodborne Disease565 XIV. Conclusion 566 References 567

1.

INTRODUCTION

Every society in the world has some interest in food safety and disease control, illustrated by the fact that new disease agents have been identified each decade over the last 100 years. In the last decade there has been a gradual shift in food safety priorities in developed countries, from chemical issues with potential chronic effects and reduced life expectancy to microbial and parasitic hazards with acute effects and subsequent long-term sequelae for a portion of those with infections. This change has been driven mainly by well-publicized outbreaks with hundreds or even thousands of cases and deaths, and the apparent inability of public health authorities to prevent them. Raw foods of animal origin, fresh fruits and vegetables, including juices, contaminated by Cyclosyor~cnteyatensis, Escherichia coli

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0 157, hepatitis A virus, Listeria monocytogenes, and Salmonella are the current concerns, along with enteric viruses in shellfish and parasites in meat. If foodborne disease surveillance is done, it is initiated and may becompleted at the local level, with some information occasionally appearing in thepress, but in many countries data are sent to a national center where an annual or other type of report may be prepared. However, no country has yet determined an accurate measure of foodborne diseases within its borders, and data from different countries may show distinct differences, which may reflect as much the way that these are collected and interpreted as thegeographic location and food habits of thepeople. Thus, caution must be advised in making too critical a comparison of foodborne disease statistics from nation to nation. Since 1980, many European and a few other countries have participated in an early warning and routine reporting system, coordinated through the FAO/WHO Collaborating Centre for Research and Training in Food Hygiene in Berlin; by 1995 46 countries had participated in this program (1). This increasing commitment to the documentation and control of foodborne disease indicates an increasing awareness of its significance in terms of morbidity, mortality, economic loss, and effects on trade between countries and common markets. The following discussion on worldwide surveillance of foodborne disease is partly based on previous reviews (2-6).

II. OBJECTIVESOF A FOODBORNEDISEASE SURVEILLANCE SYSTEM The broad objective of such a system is to identify the causes of foodborne disease so that prevention and control programs can be introduced and, if necessary, strengthened. There are several elements in a well-designed surveillance system. 1. Early alert of illnesses or potential illness to prevent further spread of disease. Local outbreaks are usually over before they can becontained unless a contaminated food is distributed to more that one establishment. But the situation is different for large-scale outbreaks. Although these may be infrequent, prompt action is essential, and often a team of government and industry officials work intensively on the problem until it is resolved. Typically, they involve a commercial product produced in one country and distributed widely in that country or imported from abroad. Examples include Clostridium botulinum in cheese (7), Salmonella agona in a snack product (8,9), and Staphylococcus cuueus in lasagne (10). Other examples include shellfish that have ingested toxic marine phytoplankton or been contaminated with viruses and need to be quarantined rapidly and sales stopped. 2. Notification of enteric or other specific diseases that are often foodborne through physicians reporting to a central epidemiological agency and reports of laboratory isolations of enteric pathogens to a reference laboratory. These are useful in showing trends in the morbidity of a disease over long periods of time, and information tends to be relatively current. Isolates may be serotyped, phagetyped, or characterized by molecular or genetic profiles to help give more precise information on the distribution of the strains. However, only certain enteric diseases are monitored, and usually there is very little information on the origin of the illnesses or even if they are foodborne. Nor do these tell us howto design control measures.

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3. Investigation of incidents of foodborne illness and reporting of results on a regular basis. Complaints of possible illnesses of foodborne origin are followed up using epidemiological principles, and clinical specimens and food samples are sent to the laboratory for appropriate analysis. Effective investigation depends on adequate training of the government officials who may bepublic health or environmental health inspectors, sanitarians, veterinarians, or physicians. The source of a complaint is usually a person who has become ill or knows someone who appears to have a bout of food poisoning and links this with a specific event or food establishment. Sometimes the earliest indication of an outbreak is through news media, and any such report should be quickly followed up by the investigation team. Laboratories have to be familiar with specific and sensitive methods to detect known microbial pathogens or their toxins, as well as agents of animal, plant, or chemical origin. It is also important to know how and where the food became contaminated, if microorganisms or parasites survived any process, and if pathogens grew on the food. Such knowledge will help develop better control strategies. The reporting of disease incidents to the state/provincia1 or national level is usually relatively passive, and data on many outbreaks will not be included in national publications. Also, most single or sporadic cases will be ignored because of the difficulty in linking one case to a contaminated food. However, large outbreaks are relatively uncommon and typically foodborne disease is probably more frequent in small clusters of cases, e.g., family reunions or individuals. Published reports are often delayed several months or even years owing to the requirement of compilation of data at different administrative levels, especially as this is often considered a low priority in the healthcare field. These data, however, can be used to develop preventive measures including hazard analysis critical control point (HACCP) systems, educational programs, and risk assessments. 4. The use of sentinel and special epidemiological studies to determine a more realistic level of morbidity of a foodborne disease. Because of the limitations of the passive reporting system, sentinel studies are considered as one approach to discover the extent of a foodborne disease in a community, the information from which can be extrapolated to a region or country. These studies depend on resources to conduct interviews and analyze clinical and food specimens. These can be focused on follow-up cases with a laboratory-confirmed diagnosis, or they can be directed to a more active searching for cases with the use of case-control studies. Therefore, only limited projects can be done and will not cover all foodborne diseases. Only some countries have utilized this approach to obtain new data on foodborne disease because of the high cost and commitment of trained personnel. A flow diagram showing the communication links in a typical foodborne disease surveillance system program is illustrated in Figure 1. 5. Estimation of health and economic impacts and setting directions for control programs. A foodborne disease surveillance system can also be used toestimate the health and economic impacts of foodborne diseases including cost-benefit and risk-benefit studies, to anticipate problems and focus research into areas of high risk, to evaluate the effectiveness of current preventive and control practices, and to provide information upon which to base goals and priorities for food-safety programs.

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Foodborne Dlsease Sporadic or Single Case

Outbreak

LOCAL

Public IIealthlEnvironmentaI Health Unit Including hledical Officers or Health Suspect Source of Marine Food Associated with Outbreak

z

/

LocallPrlvate Hospital I Laboratory Laboratory

/

Suspect Source of Animals Assoclated with Outbreak

STATE/ PROVINCIAL/ REGIONAL Food Laboratory

Clinical Rererence Laboratory

+ FoIIow-u~Studies on Sporadic Cases

NATIONAL Report of Outbreaks

Notification of Enteric Diseases

Human Isolations of Enteric Pathogens

Health Deoartment

/-Y

Publicallon or Health Statistics/ Special Studies in Communicable Disease Reports

INTERNATIONAL

Early Warning System for Other Countries

Routlne Reporting System Under FAOIWHO

Fig. 1 Generalized foodborne disease surveillance system. Note that in this figure, only the main directions of information flow are shown. However, there should be regular communication among the various agencies and with the originators of reports. The public health or environmental health unit is usually the primary agency responsible for identifying and investigating outbreaks and sporadic cases and for reporting the results to higher authorities. In some countries, veterinarians or physicians play a major role in investigation of outbreaks. In others. it is the food inspectors or sanitarians.

111.

DEFINITIONSOF TERMS

Most countries with a reporting system for foodborne diseases use similar terms: the following are based on Ref. 3. In this text, some agents and diseases are used in their shortened forms. Most of these relate to Escherichia coli infections, e.g., HUS (hemorrhagic uremic syndrome), EAggEC (enteroaggregative E. coli), EIEC (enteroinvasive E. coli),

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EHEC (enterohemorrhagic E. coli), EPEC (enteropathogenic E. coli), ETEC (enterotoxigenic E. coli), STEC (Shiga-like toxin-producing E. coli), VTEC (verotoxigenic E. coli), LT (labile toxin), SLT (Shiga-like toxin), VT (verotoxin), SRSV (small round structured virus), DSP (diarrhetic shellfish poison), PSP (paralytic shellfish poison), and GBS (Guillain-Barri syndrome). Inciderit: An outbreak or single case of disease associated epidemiologically or by laboratory analysis with a food vehicle. Outbreak: Incident in which (a) two or more persons experience a similar illness after ingestion of a common food (b) epidemiological analysis of the event implicates the food as the source of the etiological agent. Outbreaks of colfinned etiology: Outbreaks for which laboratory evidence of a specific agent is obtained and specified criteria are met. Outbreaks of umonjinned etiology: Outbreaks for which epidemiological evidence implicates a food source but adequate laboratory confirmation is not obtained. Single or sporadic case: One case definitely associated with the consumption of a contaminated food and, as far as can be ascertained, unrelated to other cases with respect to consumption of the implicated food. This differs from the situation of laboratory isolations of pathogens that may or may not be presumed to be foodborne. Case (outbreak associated): A person who has become ill following ingestion of food that has an etiological agent laboratory confirmed. It is not necessary to have all the cases in an outbreak with laboratory confirmation as long as there is good epidemiological evidence to associate them with the outbreak. NotiJiabZe disease: A case of a specific disease that must be reported by physicians with or without laboratory confirmation to local health authorities. These authorities may decide if the case meets the case definition before officially reporting the case. Isokate: The laboratory isolation of a pathogen that comes from a specimen of a person who meets the case definition of the corresponding disease. This is also reportable to local health authorities, who will communicate with the state/provincial/national agency responsible for collating the data. Laboratory isolates are often sent to reference center laboratories for confirmation and/ or further characterization. Rate: The number of outbreaks, outbreak-associated cases, single cases, hospitalized cases, or deaths divided by the total in that category and then multiplied by 1,000, 10,000, or 100,000 so as to convert the majority of entries into whole numbers. For known etiological agents, most countries restrict reporting to bacteria; some also use the word chemical to mean every kind of toxicant, natural or man-made. Etiological agent: Microorganism, animal, plant, toxin, or chemical substance that is capable of producing signs and symptoms associated with illness. Microorganisms: Viruses or cells or products of cells of bacteria, yeasts, or rnicroscopic fungi. These either invade the intestinal mucosa and cause damage in various tissues in the body or produce enterotoxins in the intestinal tract, or while multiplying in foods, produce metabolic products (toxins) that are ingested with the foods.

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Mycotoxins: Toxic metabolites produced by certain molds. Parasites: Roundworms, flatworms, protozoa, or flukes that invade human tissue through the alimentary tract. Plant: Whole or part of vegetable material, which are toxic components of the flowers, seeds, stems, roots, or bulbs, including mushrooms. Arzimal: Whole or part ofan animal, including toxic livers of mammals, marine products rendered toxic due to action by bacteria (histamine in fish) or algae (toxins typically from dinoflagellates or diatoms that have been ingested by sea creatures, typically reef fishes and shellfish), and birds that have eaten seeds poisonous to humans. Chenzicals: Metals, certain salts, and substances produced by man-made or natural processes. Included in this category are metals leached into solution by highly acid foods, nitrites, cleaning solutions, pesticides, compounds rendered rancid or foul-tasting, and extraneous matter of a physical or chemical nature. Substances (toxins) produced by animals, plants, or microorganisms are not included in this category. IV. SURVEILLANCEPROGRAMS IN EUROPE The WHO/FAO Surveillance Programme for the Control of Foodborne Infections and Intoxications in Europe has contacts with 46 countries, including most of Europe, all of Turkey, Israel, and the former U.S.S.R. (1). Most of the European data presented are derived from this document. Notifiable diseases that are potentially foodborne, epidemiological studies, and foodborne disease outbreaks are discussed.

A.

NotifiableFoodborneDiseases

Since 1980, many European and a few other countries have participated in an early warning and routine reporting system, coordinated through the FAO/WHO Collaborating Centre for Research and Training in Food Hygiene in Berlin (1). These reports are standardized as much as possible so that data can be compared, but because of the different degrees of commitment to reporting in these countries, direct comparisons of data between countries are questionable. However, some general statements can be made. Most of the 46 countries submitted data on notifications of specific foodborne diseases with or without laboratory confirmation and outbreak summaries. Nine of these countries reported on categories listed as "foodborne disease" and three on "infectious enteritis'' or "acute gastroenteritis." Notifiable foodborne disease was reported for 1992 in Albania, Denmark, Norway, Scotland, Poland, England/Wales, and Austria (16.5, 22.9, 64.9, 74.7, 126, 142.4/ 100,000,respectively) (Table 1). Austria, Denmark, England/Wales, and Scotland showed increases, and the reverse was true for Albania, Norway, and Poland, For infectious enteritis, which presumably includes most foodborne infections and other intestinal illnesses, the number of reported cases was higher than for foodborne disease alone; in 1992 the rates per 100,000 were 1000 cases in Norway, 306 in Germany, and 638 in Turkey (1989 data, not available for later years) (Table 1). Salmonellosis incidence rates varied tremendously, with ranges from 9 in Luxembourg to 424 in the Czech Republic. More were reported from central Europe than other areas. The trend was down in a few countries, such as Albania, Finland, and Sweden (e.g., in the last two countries from 150 and 66 in

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Table 1 Incidence Rates of Foodborne Diseases and Acute Enteritis in Some European Countries,1985-1993 Country

Population (millions)

Disease

3.3 7.8 5.2 4.3 37.8 38.8 56.7 49.7 5.1 79.8 4.3 56.7

Foodborne Foodborne Foodborne Foodborne Foodborne Foodborne Foodborne Foodborne Foodborne Enteritis Enteritis Enteritis

Albania Austria Denmark Norway Poland Spain Turkey EnglandIWales Scotland Germany Norway Turkey

1985 19901989

19921991

80.2 16.5 45.9 17.9 39.6 111.2 19.2 61.5 9.1 17.8 17.3 29 34 31 23.5 30.5 60.7 87.4 93.9 102 97 116 7.3 NA 12.2 NA NA 37.7 104 103 38.6 61.6 NA 170.9 77.8 147.7 967 1057 1100 242 NA638NA NA

1993

115.8 18.4

142.4 22.9

90.2 95

74.7 NA

103 58.6 221.9 1000 1000

126 64.9 306.4

NA 128.4 NA NA 58.9 NA NA 137 64 248.4 NA NA

Source: Ref. 1. NA = Not available.

1990 to 56 and 54 in 1993, respectively). For most countries, however, there appeared to be a peak in 1992. Rates for campylobacteriosis were generally lower than for salmonellosis, e.g., in 1992, 13.1 (Iceland), 32.4 (Israel), 41.6 (Finland), and 51.8 (Sweden). However, in countries where campylobacteriosis data has been collected for some time, the reverse is the case. For instance, in England and Wales in 1991, there were 32,636 campylobacteriosis cases compared with 22,627 salmonellosis cases; and in Scotland in 1992, there were 4915 campylobacteriosis cases compared with 2992 salmonellosis cases. Travel abroad to resorts on the Mediterranean Sea and to Asian and African countries was cited as a source for some of these infections. Although most imported cases are sporadic, there have been some well-documented outbreaks in tourist resorts, such as Zermatt in 1963, Kos in 1983, Salou in 1989, and the Dominican Republic in 1997, caused by Salmonella typhi or paratyphi), as well as on cruise ships in the 1980s and 1990s, caused by enteric viruses (1 1).

B. Foodborne Disease 1. Etiology Twenty-one countries reported foodborne disease outbreaks between 1990 and 1991/92/ 93 ranging from 5 in Albania to 2818 in Poland for a total of 14,537. In 19.5% of these the agent was unknown. Only in Israel (57.5% in 1992) and the Netherlands (91.5% in 1991) did outbreaks of unknown etiology exceed those of known etiology. Outbreaks of bacterial origin accounted for 95% of those where the etiology was known. The main agents causing the outbreaks in these 21 countries were Salrnonella (84.5%), Staphylococcusaureus (3.5%), Clostridium perj?ingens (3.0%), mushrooms (1.3%), Trichinella (1.5%), Clostridium botulinum (1.1%), Bacillus cereus (1.O%), Carnpylobacter (0.7%), Shigella and chemicals (0.5% each), and E. coli and histamine (0.4% each). Details are shown for 17 of these countries in Table 2. The foods, reported from 22 countries, most frequently implicated were eggs or foods with eggs as aningredient, including mayonnaise (25.4%), meat and meat products (23.4%), confectionery, sweets, cakes, pastries, pud-

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0

2

0

-f

c4

--

r-

Cl

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dings, desserts, and ice cream (17.0%), fish and shellfish (4.7%), milk and milk products (4.6%), and poultry and poultry products (4.2%). The only pathogen to be reported by all European countries where outbreaks were documented was ScrZmorzeZla, ranging from 0.6% of outbreaks (5.4% of known outbreaks) in the Netherlands (1991) to 89.4% in Romania (1990-92). Salwonella erzteritidis infections continued to be a major problem in Europe, with many outbreaks linked to consumption of partially cooked eggs or products made with raw eggs. The most frequently implicated strain was phage type (PT) 4. In Albania, there were three salmonellosis outbreaks from food served in canteens; poor storage of meat was a major contributory factor. In Austria, the mass catering outbreaks were almost exclusively caused by S. enteritidis PT 4 (in ham and egg spread, sauces, shashlik, mayonnaise salad, chicken, minced turkey, slightly cured sausage, bakery product with eggs, cheese cake with raw eggs, tiramisu). In the Czech Republic, about 30% of salmonellosis outbreaks were associated with eggs or egg products, followed by sweets, meat, and meat products. In Finland, Salmonella caused outbreaks from contaminated marinated meat, poultry, mayonnaise made with raw eggs, chocolate nuts, pizza, and sprouts. In France, Schorzelln was responsible for 8387% of outbreaks, with eggs and egg products being the main foods involved. Salmonella was also the major pathogen in Germany, responsible for 88% of outbreaks, often from raw eggs used in cakes, puddings, ice cream, homemade mayonnaise, sauces, and salads. In Iceland, however, the seven salmonellosis outbreaks were all either meatborne or from sandwiches. In Israel, meat was also the main vehicle for salmonellosis. In Italy, many of the S. enteritidis outbreaks, often from home-made cakes containing raw egg, were traced back to infected flocks. In Malta, the majority of outbreaks were caused by S. berta, both from food-service establishments and from homes; a variety of foods, including macaroni, lasagne, mayonnaise, ice cream, and curried rice, were involved. In Portugal, the foods associated with S. euteritidis outbreaks were omelet, mayonnaise, cakes, and desserts. In Slovakia, many contaminated foods led to salmonellosis outbreaks (e.g., egg and egg products, mayonnaise, ice cream, milk and milk products, salads, confectionery, poultry, and meat and meat products). In Sweden, several outbreaks involved Salmonella serovars: an elderly couple ate avocados, which contained S. ha@& imported from Israel; 352 persons traveling on a ferry were ill from S. crgoncr infections on several occasions between July and November 199l ; 5 cases of S. rzewport were traced to consumption of contaminated kebabs in a restaurant; 10 persons eating scampi in a restaurant suffered from S. btyeltevrederzinfection: S. enteritidis PT 4 was the cause of several outbreaks, including 153 medical staff at a social event with a sauce made from raw eggs, children eating a pyramid cake, children at a day-care center serving a home-made cheese cake, and several outbreaks from eggs used in food without further heating. In Switzerland, salmonellosis was associated with tiramisu and chocolate mousse. In England and Wales, eggs were the main food item responsible for Sabnonellcr incidents (e.g., in sandwiches with mayonnaise and lightly cooked egg-based saucesj. S. dublirz in imported Irish soft cheese affected 42 persons, and over 540 people were infected with S. typlzirnuriurn DT 12 from consumption of joints of cooked meat. In Scotland, farming families drinking raw milk and inadequately pasteurized milk accounted for a number of cases in several outbreaks. Compared with Snlmonella (84.5% of all outbreaks), other agents were documented much less frequently. Nevertheless, outbreaks from S. mreus were reported from many countries and usually involved ham, poultry, milk, and milk products like cream and cheese, egg products, cakes, pastries, fish, and vegetable products. C.pe$rirzgens caused outbreaks from mainly meat and poultry products, but also from soups, fish, potatoes with

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cheese, legumes, and salads. C.botulirzum incidents were reported, but little detail was given. Most seemed to arise from home-prepared food. In Norway, fermented raw trout (rakfisk) produced in small operations was the most frequent product associated with botulism. In Spain, the disease occurred in 18 outbreaks between 1990 and 1992; in 11of these (17 cases) the food was home-canned, and in 7 (22 cases) commercial products were implicated. In Portugal, three incidents in 1990 and two in 1991 occurred through consumption of cured ham. Outbreaks caused by B. cereus were documented, mainly from rice, but also meat, poultry, pit&,sandwiches, mashed potatoes, cheese with spices, bakery products, and sweets. Most campylobacteriosis illnesses are sporadic cases of unknown origin, and relatively few outbreaks were documented (mostly from the United Kingdom). Campylobncter outbreaks were also infrequently reported; vehicles mentioned were poultry, meat sauce, pork, unpasteurized milk, and pasteurized milk in bottles pecked by birds. One large outbreak of 350 cases occurred in Malta from roast chicken served in a hotel. Only 12 E. coli 0157:H7 outbreaks were reported, and these were all from England/ Wales, even although cases and outbreaks are known to have occurred in other countries (12). However, unspecified E. coli outbreaks were recorded in Germany, the Netherlands, Romania, Spain, and Scotland, and sotne of these were probably VTEC (also known as STEC) in origin. Yersiniosis (336 cases in 1990, 283 in 1991, 229 in 1992) arose in Lithuania mainly from consumption of contaminated vegetable salads in large catering establishments. Trichinosis outbreaks were documented in Lithuania (146), Bulgaria (18), Spain (16), and France (l), and many cases were recorded in Romania (4705) and Poland (738). Outbreaks of mushroom poisonings were reported in Hungary (158) and Israel (1). In addition, a few incidents were related to scombroid poisoning, brucellosis, hepatitis A, listeriosis, shigellosis, taeniasis, and enteritis from Klebsiella, streptococci, Vibrio parahaernolyticus, and SRSV. The places where food was most likely to be contaminated or mishandled were farms (21.5% of the outbreaks), restaurants (9.3%), homes (5.6%), and schools (0.7%). Food leading to outbreaks, however, was more likely to be eaten in the home (35.9%), restaurants, hotels, cafes, residential homes and bars (1 1.S%), and catering establishments (6.4%). Overall, themain factors contributing to outbreaks were temperature misuse (44.2% of outbreaks), contaminated raw material (16.0%). inadequate handling (15.9%), and contamination by personnel or equipment (14.9%). More specific factors that were identified included inadequate refrigeration or cooling of foods (19.3% of the outbreaks), preparation of meals too far in advance (9.3%), inappropriate storage (2.8%), and preparation of too large quantities (1.4%). The places where contamination or mishandling leading to outbreaks most frequently occurred (reported for onlyfive countries) was the farm (21.5%), restaurants (9.3%), homes (5.6%j, and catering establishments (3.1%). The places where food was eaten that led to outbreaks (reported for seven countries) were homes (35.9%), foodservice establishments ( l 1.8%),catering operations (6.4%), medical care facilities (5.0%), canteens (4.3%), and schools/day-care facilities (4.2%). Factors contributing to outbreaks were listed for seven countries; the main ones were temperature misuse (44.2%). environmental or personnel contamination (19.1%), Contaminated rawmaterial (16.0%), and inadequate handling (1 5.9%). 2. Epidemiological Studies In Switzerland, a case-control study to identify determinants for sporadic infections by salmonellae showed that eating food containing raw or undercooked eggs, particularly desserts, and intake of antacids (for S. enteritidis infections only) and travel abroad were

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associated with disease (1 3). Flocks positive for S. enteritidis have been imported into Switzerland (14). In Norway, 5 years after an outbreak of S. ~pl~irnurium 0:4-12 was traced to chocolate bars, there were still elevated numbers of infections by this strain in the country (15). Although most salmonellosis infections were acquired abroad, this was not the case with this strain, for which 46% were hospitalized. The strain also seemed to be the cause of fatal salmonellosis in wild passerine birds (such as finches), suggesting that there was a link between human and avian S. ~phimuriunl0:4-12 infections. Risk factors were drinking untreated water, contact with wild birds or droppings, and eating snow, sand, or soil. In Denmark, there was a very gradual decrease in cases of campylobacteriosis and yersiniosis, but for salmonellosis there were two distinct peaks in 1989 and 1992. The increase in 1992 was mainly due to S. erlteritidis and S. ~phirmriz41~1 (a phage type found in pigs). The notifiable foodborne and waterborne disease cases increased steadily between 1984 (192) and 1992 (1 189) with a slight peak in 1988 and a profile similar to that for salmonellosis, indicating that many of the foodborne diseases may have been caused by Snlmonelln. In Gemany, Cmnpylobacter j e j m i cases exceeded those of Snlvzorzella, and campylobacteriosis was the second most frequently notifiable disease. A survey showed that 10.9% of 485 retail food samples surveyed contained the pathogen with the following distribution: turkey liver, 66%; poultry, 50%; milk and milk products, 2.9%; fish, 2.3%: meat products, 0.8%; shellfish and sausage, 0% (16). Deepfrozen, raw products were more frequently contaminated than fresh, raw products (23.8% vs. 12.4%). Risk factors identified in sporadic cases of E. coli 0 1 57 infection in Wales and bordering English counties from 1994 to 1996 were beefburgers prepared in a fast food chain, cooked sliced meats sold by caterers, and contact with a farm (17). In Scotland, after 13 years of experience with E. coli 0157:H7 infections, the epidemiology is becoming better understood (18). The outbreaks have been associated with ground meat, unpasteurized milk, unpasteurized milk farm cheese, contaminated pasteurized milk, vegetables fertilized with cow manure, drinking and recreational water supplies, and person-to-person spread. Livestock, particularly cattle, are considered to be major reservoirs of this group of organisms. Nevertheless, the relatively high human isolation rates have not been satisfactorily explained. Children with HUS in the Czech Republic had evidence of VTEC infection (verotoxin in stools, antibodies to E. coli LPS in sera, or isolation of VTEC strains) (19). Serotypes 026:Hll and 0157 were the most frequently isolated. However, no source of the infections was identified. In the Netherlands, Belgium, and Germany, 78% of HUS patients had evidence of VTEC infections (20). E. coli 0157 was the most frequent causative agent, and VT-2 was the most frequent toxin produced. Where family members had E. coli 0157 isolated, the strains were identical by subtyping analysis. An argument has been made that an increase in foodborne disease might be connected with climate change, in particular warmer summers in England (21 ). The monthly incidence of this type of disease was found to be significantly associated with the temperature of the same and previous month. Thus, animals prior to slaughter may be more infected and the risks of microbial contamination and growth may increase in the derived meat products with higher temperatures. It was predicted that an additional 179,000 cases may occur by 2050 as a result of climate change. 3. FoodborneDiseaseOutbreaks The largest outbreak of E. coli 0157:H7 occurred in Scotland in November and December 1996, with hundreds of cases and 20 deaths. Those ill had eaten cold cooked meats, meat

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sandwiches, and cooked steak in gravy prepared by the same butcher who was later fined a relatively small amount for food hygiene and safety breaches. Previous outbreaks from beef had already been documented (22), but as a result of this episode, numerous changes in food hygiene were initiated nationally (23). Powdered infant formula was responsible for 48 known cases of salmonellosis in infants under 7 months old from 14 regions in Spain from January to June 1994 (24). The implicated strain was a lactose-fermenting S. virchow. It took some time for the illnesses to be linked with the Sdmorzella in the milk powder and recalls to be made. In England, another outbreak affecting infant food was caused by S. senftenberg in baby food cereal (25). Five persons who were suffering from S. enteritidis PT4 infection in England had attended the same gym and had consumed a protein-based beverage composed of milk powder and a raw egg as a body-building drink (26). For these people, the choice of food to suit their lifestyle outweighed the risks of enteric illness. A large Cnrrzpylobacteroutbreak of 72 cases occurred in 1992 at an outdoor music festival on the grounds of a farm in England (27). Untreated milk sold in l-L plastic containers from a local farm was implicated. A caterer infected 67 people who had attended an international AIDS conference in Wales (28). The food handler who had prepared the most likely food (coronation chicken) had been ill with a mild gastrointestinal illness 2 days earlier. She boned the cooked chicken with her bare hands. It was later found she had a SRSV infection and had transmitted it to some of the delegates even although she was symptomless atthe titne. A scombroid outbreak with15 cases also occurred in Spain from a fresh tuna sold at a supermarket in June 1994 (29). Typical symptoms of facial flushing, headache, diarrhea, nausea, and abdominal pain followed about 45 minutes after a peppery taste was noticed in the tuna. Histamine up to 580 ppm was found in leftover tuna samples and >35 pg/L in four urine specimens from the cases. Although botulism from dairy products is considered rare, some recent outbreaks show that this does occur. In 1989, the largest outbreak in theUnited Kingdom took place after commercially prepared hazelnut yogurt containing contaminated nut paste was consutned (30). In Italy in August and September 1996, at least 8 persons and one death resulted after tiramisu made with mascarpone cheese was eaten (3 l). Most of those stricken were children. The cheese contained spores of C. botuZi~zum,but it is uncertain how the contamination took place. The widely exported product was recalled on a massive scale. Overall, in Italy there were 33 cases of botulism in 1995 and 58 in 1996. Typical foods were vegetables preserved at home in oil or salty water or in sauces. In 1998 two cases of botulism, one fatal, occurred in a family in southern Italy after they ate home-preserved mushrooms bottled in oil (32). In the United Kingdom the link between human Creutzfeldt-Jakob disease (CJD) and consumption of beef that originated from cows with bovine spongiform encephalitis (BSE) was considered close enough to warrant bans by other countries on importing British beef. This type of CJD is supposed to be caused by a variant form of prion (V-CJD) that is found in bovine organ tissues. V-CJD is characterized by early age of onset, more prolonged duration, predominantly psychiatric presentation, and distinct brain pathology. In the United Kingdom, 25 persons diagnosed with V-CJD have died. The economic loss in 1996 to the European Community was $2.8 billion in subsidies to the beef industry for herd slaughtering and lost business (33). 4. Foodborne Disease Reports in Specific Countries The following are examples of some European countries that have reported additional foodborne disease data.

528

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a. France. In France, the number of outbreaks rose from 594 in 1990 to 732 in 1992. Where the agent was identified, Saknzorzella caused 83-8796 of outbreaks. Eggs and egg products were associated with many outbreaks, particularly after they were contaminated with S. enteritidis (1). Meat and meat products, as well as mixed foods, were also important vehicles for causing Salmonellcl, C. pelfrifzgens, and S. nureus outbreaks. Most fish- and shellfishborne outbreaks were caused by histamine and diarrhetic shellfish poison, respectively. Outbreaks involving dairy products such as cheese and ice cream and muchhandled prepared foods most likely arose from S. aureus intoxication. Outbreaks most frequently occurred after people had eaten at homes, schools, restaurants, canteens, hospitals and homes for the aged, holiday resorts, prisons, and religious gatherings. The most frequent contributing factors were contaminated equipment, faults in processing, inadequate cooling, contaminated raw ingredients, preparation too far in advance, and contamination through personnel. Botulism has been reported in France for many years. Of the 108 cases seen in a Poitiers hospital between 1965 and 1990, 83% had consumed home-cured ham contaminated with C. bofulinurntoxin, mainly type B (34). Raw goat milk cheese contaminated with Salmonelka parntyphi B infections affected 273 persons throughout the country in 1993 (35). Contaminated pork tongues in aspic sold in delicatessens was responsible for a major listeriosis outbreak with 279 cases, 22 abortions, and 63 deaths in 1992 (36). Consumption of wildboar meat led to two family outbreaks of trichinosis in 1993 and 1995 in the Languedoc region (37). Horse meat has been implicated in trichinosis outbreaks in France. The latest occurred in 1998, when 128 persons who ate horse meat imported from the Federal Republic of Yugoslavia were infected (38). b. Poland. The number of outbreaks in Poland steadily decreased from 991 in 1988 to 640 in 1992 (1). Salmonellosis fell from 35,268 in 1988 to 24,573 in 1992, botulism from 357 to 165, other bacteria from 4232 to 2633, and mushroom poisoning from 489 to 284 over the same years. S. aureus intoxications, however, remained much the same (517-491). From 1988 to 1992, most illnesses were associated with contaminated cakes, sweets, and ice cream (39.4%), meat dishes, including raw minced meat and eggs, poultry, and venison (25.8%), ready-to-serve food, e.g., croquettes, pancakes, dumplings, mayonnaise, salads (9.3%), eggs and egg products (4.0%), and milk and milk products (2.5%). Dishes prepared with uncooked eggs are common especially for fillings, e.g., custards, cheese and egg crepes, and pastries. Most outbreaks occurred at homes, with the most frequent places of contamination being farms, homes, schools, canteens, and vacation resorts.

c. Former U.S.S.R. Countries. The rate of salmonellosis per 100,000 in former U.S.S.R. countries (some in Europe and some in Asia) in 1990 were 16.7 (Georgia), 25 (Belarus for 1991-1992), 28.0 (Azerbaijan), 28.9 (Tajikistan), 32.0 (Ukraine), 37.2 (Uzbekistan), 37.7 (Turkmenistan), 38.2 (Armenia), 42.1 (Kyrgystan), 5 1.7 (Kazakhstan), 70.4 (Russian Federation), and 83.1 (Moldova) (1). S. enteritidis was the most frequently isolated serovar in Belarus, Moldova, and the Russian Federation and was often associated with poultry and eggs. In Russia, up to 85% of foodborne salmonellosis was caused by S. enteritidis (39). In the Russian Federation, salmonellosis rates rose from 70.4 in 1990 to 80.1 in 1992. In Russia, only 400-500 cases of campylobacteriosis are reported each year, probably because of lack of laboratory media and suitable incubators. There were 369 cases of botulism on average each year from 1986 to 1991 (death rate, 7.9) compared

"

Surveillance of Foodborne Disease

529

with 661 cases between 1992 and 1994 (death rate, 8.1%); the increase was due to the changing economic conditions and an increase in home canning and curing of mushrooms, vegetables, fruits, and fish (39). In Belarus, the last outbreak from brucellosis was in 1969 with 27 cases and 3,689 infected animals. However, yersiniosis occurs every year with a rate of 7.0 per 100,000 in 1991. In Moldova, most salmonellosis cases occur during the warmer months when large quantities of food are prepared and poorly stored. Moldova is reported to be free from trichinosis, the last outbreaks being in 1982. In July 1998, Kazakhstan officials have temporarily banned shashlik kebabs in Almaty, the country's capital, after five people were hospitalized as a result of eating meat from animals infected with anthrax (40). No deaths were reported. Over 100 restaurants and cafes in the capital had to find alternative dishes to serve their customers for over a week.

V.

SURVEILLANCEPROGRAMS IN AUSTRALIAyNEW ZEALAND, AND OCEANIA

A.

Australia

1. Surveillance In Australia, some trends in notifications of enteric diseases are apparent in 1991-95 data. Notifications for Campvlobacter, Listeria, and Salmonella isolations are increasing, and those for Yersinia are decreasing (Table 3). In 1995 there were 10,933 cases of campylobacteriosis (91.6 per 100,000 population), up over the previous 4 years; high regional rates were recorded from South Australia and Northern Territory (41). In the same year there were 5,895 salmonellosis cases (32.7 per 100,000population), with more cases recorded in the warmer months. There were also 29 cases of brucellosis, 58 of listeriosis, 734 of shigellosis, 69 of typhoid, and 305 of yersiniosis, but no cases of botulism. A study of campylobacteriosis cases in Tasmania showed a decrease in notifications after 1992, which coincided with the introduction of an infection control program in commercial chicken farms (42). 2. FoodborneDiseaseOutbreaks A summary of foodborne disease annual reports for the years 1980-1995 was published by Crerar et al. (43) (Table 4). Although the types of agents responsible for illness are similar to those in other industrialized countries, with Salmonella being the predominant cause of morbidity and mortality, the figures are relatively low for the 15-year period, and many more outbreaks probably occurred. From the data available, S. enteritidis does

Table 3 Notifications of Selected Enteric Diseases in Australia, 1991-1995 Year

Campylobacteriosis

Salmonellosis

1991 1992 1993 1994 1995

515 8,672 9,135 450 8,311 10,117 10,933

44 5,440 4,6 14 534,73 l 5,283 5,895

Source: Ref. 41.

Shigellosis 902 567 894 708 724 306 734

Listeriosis

Yersiniosis

38 34 58

414

Todd

530 Table 4 Foodborne Disease Outbreaks in Australia, 1980-1995, by Etiological Agent ~~

Agent Salmonella C. pelfrirtgews S. aureus Ccrnlpylobucter B. cereus V. parallrreiilol~ticus L. monocytogenes E. coli 0111 C. botulimm SRSV Rotavirus Hepatitis A virus Toxoplasnrn Scombrotoxin Ciguatera Mushroom poison Total known Total unknown Total

No. of outbreaks

Percent of outbreaks

No. of cases

Percent of cases

No. of deaths

27 14 9 5 5 4 2 1 1 11 1 1 1 2 1 1 86 42 128

53 11 7 4 4 3 2 0.8 0.8 9 0.8 0.8 0.8 2 0.8 0.8 67

2,053 280 99 106 27 181 13 23 1 2.267 55 7 13 8 30 5 4,438 1.5 14 5,932

35 5 2 2 1 3 0.2 0.4 0.02 38 0.9 0.1 0.2 0.1 0.5 0.08 75

5 0 1 0 0 2. 0 1 0 0 0 0 1

33 100

2 100

0 0 0 6 0 6

Source: Ref. 43.

not seem to be a major problem in Australia. Vibrio parahuemolyticus infections were more common than in North America or Europe, but this probably reflects the fact that most of Australia's population are close to the sea and consume seafood regularly. Scombrotoxin and viral outbreaks were also associated with seafood. S. snbandaka, which caused at least 54 cases of illness in Victoria and South Australia in 1996, arose from consumption of one brand of peanut butter (44). Individuals infected with this rare serotype occurred in other states and territories, and about half of the cases were younger than 5 years; links to peanut butter were only established in three Western Australian cases. One case also occurred from the same brand of peanut butter in New Zealand. The product was recalled in both countries. Another salmonellosis outbreak in the same year affected 52 persons-patients, staff, and spouses-from sandwiches served in a Brisbane hospital (45): the source was not identified. An outbreak in Melbourne in 1997 affected more than 700 people, about 3% of whom developed arthritis and half of whom are expected to have long-term problems (46). Food poisoning from tropical fish containing ciguatoxin and related toxins originating from benthic dinoflagellates is regularly reported from Queensland and Northern Territory coasts originating from a variety of fish including barracuda, Spanish mackerel, kingfish, coral trout, grouper, and reef cod. However, underreporting is suspected, and the rate is probably higher than the documented 1.6 cases per 100,000 population. In one outbreak in 1995, a family of four who ate a coral trout and developed typical gastrointestinal and neurological symptoms were given intravenous mannitol within 18 hours of the poisoning (47). The mother was 11 weeks pregnant, and the resultant child was observed for 2 years and suffered no ill effects.

"_

'

Surveillance of Foodborne Disease

531

Although sporadic cases of HUS have been associated with E. coli 0 1 11 and other VTEC in Australia, the first'outbreak was in 1995 when mettwurst produced by a small manufacturer infected many people and caused HUS in 23 children with one death (48). The manufacturing process was criticized for its lack of control procedures (no starter culture but the use of "backslop" inoculation of the meat ingredients, no monitoring of pH or water activity, no pasteurization) (49). This outbreak stimulated considerable interest in VTEC infections and HUS. In a study of 55 cases of Queensland children with HUS, most of those preceded by diarrhea were under 5 years of age ( S % ) , had reduced or no urine output (71"m), experienced hypertension (3 1"m), and developed seizures (29%). Eighty-five percent required transfusion, 56% antihypertensive therapy, 56% peritoneal dialysis, and 2% hemodialysis, which lasted 3-29 days, and 10% ventilation (50). One child died. In two other HUS incidents in Adelaide, E. coli 048:H21 and Enterobncter cloacae OR:H9, respectively, seem to have been responsible (51,52);both strains produced SLT-I1 toxins. In February and March 1996, the first 0157 outbreak occurred in Australia when 6 persons were infected from food served in a delicatessen on the Gold Coast in southeast Queensland (53). A food handler was the index case and may have contracted the infection from her pet dog, which had bloody diarrhea the week before she developed symptoms.

B. New Zealand 1. Surveillance In 1995 in New Zealand, the rates for campylobacteriosis, salmonellosis, hepatitis A, shigellosis, listeriosis, and VTEC disease were 223.0, 40.4, 10.3, 5.6, 0.4, and 0.2 per 100,000, respectively (54). The estimated rates have also been determined for giardiasis (158.1), rotavirus infection (141.4), yersiniosis (87.4), and cryptosporidiosis (66.6). There are probably about 300,000 cases of foodborne illness each year. For a New Zealand population of about 3.6 million, this represents one illness per 12 persons each year. Surveillance of listeriosis was conducted in 1995. There were 15 cases compared with 1l in 1994 (55). Three of the cases were perinatal, and two of the infants died. Of the remaining 12 cases, 11 had underlying disease or were elderly. None of the cases occurred in clusters, and there were no links to food. Risk factors for campylobacteriosis were determined through a case-control study from June 1994 to February 1995 (56). The main factors were consumption of raw or undercooked foods (especially poultry and unpasteurized dairy products) and untreated drinking water, overseas travel, and contact with animals. Thorough cooking of chicken could significantly reduce the incidence of campylobacteriosis. 2. Foodborne Disease Outbreaks In New Zealand, illnesses have been documented from C. botulinurrz in home-bottled meat and watercress, C. pe~fiingensin meat, chicken, and bean dip, Salmonella in pork, Canzpylobacter in chicken livers, and B. cereus in rice (57). More recent outbreaks include three separate episodes of S. Qplzirmriunz phage type 35 associated with consumption of bakery products in Christchurch in 1993 (58), two incidents in 1994, with people ill after eating curry probably contaminated with Clostridizm perfringens, one with 31 persons at an Auckland wedding reception in March (59) and the other with 59 attendees at a fashion show in April (60). The two C. per@ingerzsoutbreaks probably involved the same supplier, although this is not stated in the reports, and in one of them the practice for years had

Todd

532

been for large pots of meat to be left at room temperature because they were too big for the cold room. Hepatitis A occurred in Wellington in 1996 from delicatessen food contaminated by the owner/operator, who was the index case (61). In 1998, 64 people attending a local Maori hui outside Auckland and eating lunch consisting of roast pork suffered from gastroenteritis 1-3 1 hours later (mean incubation period, 12 hours) (62). The pig was home-killed and not inspected before being roasted without a meat thermometer. It was cooled for 90 minutes before serving. C. perfringerzs was the agent responsible. Outbreaks following huis have occurred before in 1997 and 1998. Illnesses associated with traditional Maori foods, however, are rarely reported.

C. Oceania Relatively few South Pacific countries reported diseases like salmonellosis (7 of 22 countries with a total of 221 ill), hepatitis A (10 countries, 121 cases), shigellosis (1 1 countries, 271 cases); however, practically every country recorded diarrheal illnesses (a total of 1,352/100,000 population) (63). Also, fish poisoning was significant (a total of 4,707 cases), with most poisonings in Fiji (1,653) and French Polynesia (856). On a population basis, these were highest in the Solomon Islands (2,125/100,000) and Kiribati (1,152/ 100,000). Although the details of these poisonings are not given, most would probably be ciguatera (64), PSP (65), or scombroid (histamine) poisoning (66). In many Pacific Islands seaweed is served as a side dish at meals. In 1994, boiled seaweed (Grncilurin coronopifolin) served at a picnic in Hawaii affected 7 persons with a burning sensation in the mouth and throat, headache, and gastrointestinal symptoms (67). Extracts of the seaweed killed mice, and therefore the etiological agent was probably a heat-resistant toxin. Illnesses from seaweeds Gracilarin and Grncilariopsishad been previously reported from Guam, Japan, and California with deaths in two of the episodes.

VI.

SURVEILLANCEPROGRAMS IN ASIA

Relatively little in the way of surveillance of foodbome disease is carried out in Asian countries. Most information is gleaned from specific but limited investigations and studies. Some studies cover several countries, e.g., parasitic diseases are widespread arising from consumption of raw or undercooked freshwater fish, shellfish, snails, frogs, and tadpoles. For instance, echinostomiasis, caused by the intestinal trematode fluke Echinostornu that encysts in freshwater mollusks as a secondary host, is prevalent throughout eastern Asia (e.g., 3% in the Philippines, 11-65% in Taiwan, 5% in China, 1% in Indonesia, and up to 50% in some parts of Korea and northern Thailand) (68). The disease is caused by at least 16 species transmitted by snails, the primary host. The disease is most prevalent in remote rural locations among low wage earners and women of childbearing age. Risk factors are promiscuous defecation and the use of night soil for fertilization of fish ponds. In addition, the likelihood of fungal intoxication is greater in areas where grain is stored over long periods of time. For instance, the cost of premature death from primary liver cancer and disability and morbidity to liver cancer attributable to aflatoxins in maize was estimated to be $A32.0 million in Indonesia, $A36.8 million in the Philippines, and $A7.7 billion in Thailand (69). The same estimates for peanuts were $A96.5 million in Indonesia, $A2.2 million in the Philippines, and $A16.0 million for Thailand. The difference in costs

Surveillance of Foodborne Disease

533

is related to the amount of aflatoxin ingested and is greatest in Indonesia and least in Thailand.

A. China Foodborne disease outbreaks are documented annually in China, and data for the years 1993-1995 are presented in Table 5. Overall, there were 1,365 outbreaks, 33,979 cases, and 246 deaths in 1993, 1167 outbreaks, 37,018 cases, and 264 deaths in 1994, and 947 outbreaks, 23,556 cases, and 244 deaths in 1995 (70-72). The decrease in 1995 was because of fewer outbreaks of microbiological and natural toxin etiology. The most frequently occurring outbreaks (in order) were caused by natural toxins, organophosphorous compounds, Salmonella, nitrite, Vibrio parahnemolyticus, S. nureus, Proteus, pathogenic or toxigenic coli-bacillus (probably E. coli), and histamine. Botulism was a relatively infrequent disease (7-1 1 outbreaks and 41-161 cases); the death rate for cases was 6.314.6%. A few illnesses resulting from ingestion of fungal toxins (mycotoxins and gossypol) were documented and, like botulism, had a high death rate (1.3-5.2%). The natural toxins are not defined but probably include seafood toxins such as ciguatoxin, paralytic shellfish toxin, and tetrodotoxin (the last since balloonfish are specifically mentioned in a list of foods). One disease limited to China is deteriorated sugar cane poisoning caused by Arthrinium spp. (molds) that grow on stored sugar cane in the winter months (73). The disease is characterized by sudden onset of gastroenteritis followed by toxic encephalopathy and a delayed dystonia in severe cases. It particularly affects children who like to chew the canes. The mold produces the toxin, 3-nitroproprionic acid, when the cane becomes mildewed because of poor storage. Another unusual disease is caused by Burkholderin cocoverlenans (Pseudomonas cocovenenms subsp. ferinofermentans) in fermented corn flour, potato products, and stored Tremella mushrooms. Poisoning may arise from ingestion of bongkrekic acid produced by the Burkholderia, leading to high mortality rates. Tremella is grown on cottonseed shells in homes and by small industries. Many outbreaks of chemical origin resulted in acute illness and deaths, unlike industrialized countries. The vibrio- and histamine-associated illnesses were probably mainly marine in origin. However, the most frequently implicated foods were meat and mushrooms. More illnesses were recorded in urban communities than in rural environments. The specific responsible groups listed were caterers, other food-service operators, and street vendors. A very large outbreak of hepatitis A occurred in Shanghai in January and February 1988, with 292,301 cases and 32 deaths. The virus was transmitted through clams contanlinated with sewage water (74,75). Hepatitis E can be transmitted by fecally contaminated water or possibly food, and there are endemic areas including China. For instance, in the Xinjiang region more than 100,000 cases were reported between 1986 and 1988 (76). From information available up to 1989 there were 746 outbreaks and 2,866 cases attributed to botulism in China with a 14.7% fatality rate. Most of the outbreaks (71.8%) were from home-fermented bean or cereal products (77). As more commercial products become available to the population, it is expected that fewer home-made products will be consumed and there will be correspondingly fewer botulism outbreaks.

B. Taiwan From 1986 to 1995 the number of foodborne disease outbreaks reported in Taiwan ranged from 57 to 123 (78). The main etiological agents were V. parahaemolyticus (197 out-

534

9 M

U

6

Surveillance of Foodborne Disease

535

breaks), S. aureus, mainly enterotoxin A-producing (169 outbreaks), and B. cereus (104 outbreaks). Over this period there were 31 outbreaks of salmonellosis (1,038 cases) and 10 of botulism, mainly A and B toxin types (19 cases). The most frequent serovar was S. typhimurium, and S. virchow had caused a number of large outbreaks. In recent years S. enteritidis was emerging as an important pathogen. In 1995, there were 123 outbreaks and 4,950 cases, the highest numbers since before 1986. It would appear from this study and that of Lee et al. (79) that the characteristics of outbreaks in Taiwan are more similar to those in Japan than in Korea. One Shigella outbreak in that year affected 646 persons (80). The Vibrio outbreaks occurred in the warmer months (April to October), and some were associated with seafood in lunch boxes delivered to schools or in catered food (78), Vibrio vulniJicrrs incidents also occur every year, and of the 28 cases between 1985 and 1990, most had ingested seafood or had exposed abraded skin to seawater (81). In two unusual outbreaks (1992 and 1994) schoolchildren mistook tung nuts from the tung oil tree (Alezlrites) for chestnuts and ate them; within 2 hours they developed gastrointestinal symptoms, fatigue, and headache (82).

C.

HongKong

From the number of enteric pathogens isolated from stools of patients in a major hospital in Hong Kong(83), it would seem that thedistribution of these pathogens in the population is similar to that in Japan, except that the proportion caused by Shigellu is higher and that of Vibrio is lower (Snlnlonella 52.5%, Campylobcrcter 16.6%, Shigella 11.3%. Vibrio 5.3% and EPEC 4.4%). Hepatitis A and E viruses have been implicated in shellfish outbreaks (84). Episodes of Vibrio yaralzner~zol~ticus and ~ ? u l ~ ~ i Jhave c u s been documented, mainly through consumption of shellfish. Dipping shellfish into hot water (“hot-pot”), a frequent practice, is not sufficient to destroy these pathogens. Seafood toxins, paralytic shellfish poison and ciguatoxin are regularly reported as causing illness in Hong Kong.

D. Korea The characteristics of foodborne outbreaks in Korea and Japan between 1971 and 1990 were compared (79) (Tables 6-8). There were considerable differences in the morbidity (3.0% in Korea, 29.2% in Japan) and mortality rates (2.48% in Korea, 0.07% in Japan), as well as agents involved (Vibrio spp. important in both countries, but Salmorzella was more a cause of outbreaks in Korea than S. c w m s , and vice versa for Japan). Most incidents occurred in the workplace and the home in Korea, whereas it was more in restaurants and hotels in Japan. Seafood was often implicated in both countries, but food of animal origin was much more frequently associated with outbreaks in Korea. The differences Table 6 Foodborne Disease Outbreaks by Etiology in Korea and Japan

Korea Japan

3.O 29.2

2.4s 0.07

23.1 14.s

14.9 47.3 24.8

0.5 0.2

37.6

6.8 3.5

17.1 9.6

536

Todd

Table 7 Foodborne Disease Outbreaks by Place of Eating in Korea and Japan

Country Korea Japan

Home Restaurant Workplace HotelSchool (9%) (%) (%)

38.8 17.2

10.6 32.7

5.3 11.5

(5%)

(%)

Retail store m )

19.1 c. 3

2.5 c. 4

0.5 c. 3

Other Unknown (%)

(%)

11.9 c. 16

1.3 c. 15

Solrrce: Ref. 82.

may have been due to types of food eaten, food-handling customs, and the fact that Koreans typically do not seek help at hospitals. Norwalk-like viruses are a common cause of gastroenteritis in young children, but it is not known how much a role food and water playin their transmission (85). Toxoplasmosis occurred after three men ate raw boar viscera and pork at a farmhouse in 1994 and also after five soldiers consumed raw liver and uncooked meat from a domestic pig in 1995 (86j. Three experienced chorioretinitis (two permanently blind in one eye), and five had lymphadenopathy.

E. Japan In 1987,40.6% of isolates were Snlnlorzelln, 19.9% were Vibrio spp., 16.7% were Campylobncter, 12.4% were E. coli, and 5.6% were Shigella. By 1990, S. enteritidis had become the dominant Snl1nor?elln serovar, and by 1992 salmonellosis was the leading cause of foodborne disease in Japan. In 1997, 11,000 people in 499 incidents of foodborne disease suffered from salmonellosis, three times greater than in 1992 (87). In the first 3 months of 1998, the number of salmonellosis cases increased dramatically with 1,770 people ill, almost five times the average number for the same period during the last 3 years. The figure included a massive outbreak of 1,100 elementary school students in Tokyo and the nearby prefectures of Kanagawa and Iwate. The import of foreign-hatched chicks for both poultry farming and egg production was a possible entry route into Japan for the Salmonella. The rise in Salrnonella incidents has been a trend over the last 5 years. Foodborne disease statistics have been kept by Japan for many decades, and the number of foodborne illnesses occurring each year was highest in the 1950s and 1960s, with up to 2,000 outbreaks reported annually. Little change, however, has been noted since 1982 (in 1982, 923 outbreaks and 35,536 cases; in 1990, 926 outbreaks and 37,561 cases. Slightly fewer outbreaks occurred in 1987 and 1988 (840 and 724, respectively). However, the proportion caused by V.pnrahnemolyticus increased from 33.4% of bacterial Table 8 Foodborne Disease Outbreaks by Implicated Food Grain/

Country Korea Japan

Vegetable Animal Seafood products mushrooms Confectionery (96) 31.8 21.7

+

in Korea and Japan Multiple foods

Unknown Other

m’o)

(%l

(%)

(%b)

(5%)

(%)

25 .O 3.6

17.5 14.6

2.9 1.2

18.3 9.6

1.9 1

2.6 48.3

Surveillance of Foodborne Disease

537

outbreaks in 1982 to 53.2% in 1990. More outbreaks occurred in the summedfall months (June-October), with a peak in August/September. After Vibrio spp., SaImotdla and S . aurezds were the dominant etiological agents. Poisoning from natural toxins (mushrooms, PSP, DSP, tetrodotoxin) accounted for 13.5% and 13.6% ofthe reported outbreaks in 1982 and 1990, respectively. The most serious was pufferfish poisoning (from tetrodotoxin); in 1982 there were 26 episodes and 8 deaths, but only 1 death in 1990. In fact, the reduction in deaths has been consistent over the years, from 41 1 in 1949 to 1 in 1990. Restaurant/ hotels and homes were the places where most food was mishandled that led to outbreaks. Infant botulism occurred for the first time in 1984, with C. botulimm type A spores being found in the infant’s stool, honey fed to the infant, and soil and dust specimens (89). In 1991, 21 persons in an inn near Tokyo suffered from cholera and one died after they consumed contaminated imported Korean clams (90). There had been a cholera outbreak in Korea at that time. Since 1984, there have been episodes of EHEC affecting hundreds of nursery or school children. In the largest outbreak (3 19 cases) there were 2 deaths. For example, one in 1990 affected kindergarten children who suffered neurological problems (e.g., stupor, deep coma, convulsions, tremors, and incontinence) arising from the action of verotoxin (91). A far greater problem occurred in 1996 from May to October, when the same pathogen caused a series of 24 outbreaks, with over 9,000 cases and 12 deaths. Several molecular subtypes of the E. coli were isolated (92). In the largest outbreak, in July and August in Sakai City (Osaka), a total of 6,309 schoolchildren and 92 staff from 62 elementary schools were affected and a further 160 secondary infections developed, mainly in family members of the schoolchildren (93). The high number of severely affected children may be related to an unbalanced diet resulting in protein malnutrition (94). In Habikino City (Osaka) another outbreak affecting 98 persons in a home for the elderly and three other small outbreaks in the same region occurred. All of the strains from these five outbreaks had identical DNA patterns, and radish sprouts from one farm were consumed by those ill. However, no isolates could be obtained from samples of soil, water, or sprouts on the farm. It has been shown, however, that E. coli 0157 can penetrate into the inner tissues of radish sprouts and disinfection will not remove them (95). In 1998, 39 persons suffered from E. coli 0157 infection after they consumed sushi made with soy sauce-seasoned ikzlra in restaurants in five Japanese cities (96). The ikura was produced from roe of domestically caught salmon.

F. Viet Nam In Viet Nan1 it was estimated that 30-57% of students in university hostels between 1984 and 1988 suffered from diarrhea, mainly because of pathogens being in poorly prepared and stored food (97). Street-vended meals in Hanoi in a 1990/91 survey often contained E. coli and C. perfringens. Most of the 5,714 documented illnesses between 1983 and 1988 were caused by Salmonella, E. coli, and S. aureus, and include 156 deaths (fatality rate of 2.7%). Because anthrax-infected cattle may be used for meat, hundreds of persons develop bacteremia, and 3-7 die each year (97). Chemical residues in foods are not adequately controlled, and manyillnesses are thought to be due to chemical poisonings. Deliberate illegal additives in alcohol, candies, and sweet products have caused intoxications; one such adulterated liquor caused 14 deaths. Foodborne disease is the most widespread public health problem in Viet Nam and the second leading cause of illness and death (98), even though cases are highly underreported. Infected food handlers and pesticide residues

538

Todd

in foods were considered to be important risk factors for foodborne disease in Viet Nam, although no direct links were made between these and investigated foodborne illnesses.

G. Thailand Although there has been national foodborne disease reporting since 1970, the focus appears to be mainly on chemical poisonings, particularly from insecticides, although these accounted for only 0.33% of the reported 207,580 cases of food poisoning between 1981 and 2986 (99). More details on poisonings were given in a later study (100). Between 1981 and 1987, insecticides accounted for 27.4% of outbreaks and 58.4% of cases. Because of the widespread use of insecticides, some of these have accidentally Contaminated desserts, beverages, fruits, and other foods. Methomyl, which looks like sugar or flour and with little odor, was responsible for 15 of the 18 insecticide-related outbreaks. Since 1987 this chemical was sold blue-colored in an attempt to reduce these poisonings. In addition, an alcoholic beverage containing methanol affected 10 males during a party: 5 died and one had permanent visual impairment. Poisonous plants, such as mushrooms (Awzcrnitn and CMoropl~yZlumspp.), cassava (raw roots), and wild plant seeds (Jcrtroplm spp.), caused 58.9% of outbreaks and 34% of cases, and poisonous animals, 11.0% of outbreaks and 6.5% of cases. Among the poisonous animals group, there was one episode of PSP with 63 cases (1 death) following consumption of green mussels containing 465-714 mouse units PSP toxins/g. In addition, there were four outbreaks associated with meals made with horseshoe crab meat and three outbreaks resulting from ingestion of pufferfish, presumably containing tetrodotoxin, although no analysis was done. Only serious intoxications seemed to have been documented, but many others undoubtedly occurred, such as the 2,207 reported cases of mild mushroom poisons, some of which could have been of bacterial origin, but no follow-up was done. Shigellosis is an important cause of diarrheal disease in Thailand, especially in children under 5 years of age (101). Whereas most of these are spread from person to person, one waterborne outbreak was identified in a rural region. A case-control study showed an association between the 242 cases of ShigeZ1aJlesner.i and drinking unboiled piped water. The water came from a river and had been unchlorinated for 6 weeks prior to the outbreak because of a lack of disinfection chemicals. Food handlers may have become infected through this source. Salmonellae were present extensively in chicken flocks and environmental samples in a 1991-92 study (102). They were found in all 7 parent breeder flocks and 13 broiler flocks and in 13 of 15 layer flocks examined. Of the 21 serovars isolated, no one was dominant. The shells and contents of eggs from layers were contaminated (4% and 2%, respectively), with S. enteritidis found only on one egg shell. Chicken is an important protein source in southeast Asian countries, and Thailand exports birds to several of these. Therefore, poultry could be a major vehicle for transmission of ScrZmor~elluto infect a large number of people. If virulent serovars such as S. enteritidis become established in flocks, the situation could become worse. H. Cambodia Diarrheal disease is a problem for very young children, since children less than 5 years of age with diarrhea constituted 15%of all outpatient consultations and 17% of those hospitalized. In the 7 most populated provinces in 1993, there were 906 cases of cholera (72 deaths), 13,323 cases of dysentery (2 deaths), 2,242 cases of typhoid (2 deaths), and 33,939 persons with diarrhea (32 deaths) (103). In the whole country in 1996, there were

Surveillance of Foodborne Disease

539

762 cases of cholera with 20 deaths, all from one province, but none of the cases was confirmed. Ice and bottled water is generally unacceptable according to French standards (93.3% for bacterial and 50% for chemical analyses) (103). A survey of restaurants showed that many were not clean or had some evidence of improper personnel hygiene; 75% had foods unacceptable by bacteriological analysis.

1.

New Guinea (Irian Jaya and Papua New Guinea)

Pigs play an important economic and cultural role in the tribes that live in the central highlands of New Guinea. In the Indonesian part of the island (Irian Jaya), the prevalence of cysticercosis, resulting from ingestion of Taenia solium, is the highest in the world according to Muller et al. (104), with a rate of over 30%. Pork is often consumed insufficiently cooked to destroy these parasites. Pig-bel, a severe form of Clostridium pe$ringens enteritis, is associated with ritual feasts involving consumption of roast pork in Papua New Guinea (105). After the feasts, portions of the pork are taken away for later consumption at ambient temperatures that allow germination of the spores and production of enterotoxin. There is of recent interest in kuru because of its possible similarity with V-CJD in the United Kingdom (106). Kuru was a fatal disease contracted though ritual consumption of the brains of dead persons by the Fore tribe in Papua New Guinea. Although this practice was banned in 1959, there are still some kuru-affected people, and questions are being asked about the amount of brain that was eaten and the length of the incubation period. J.

Indonesia

In a 5-month period in 1994, 408 children were monitored for diarrheal disease in Jakarta (107). Of the 36% of children with diarrhea during that time, 19.6% had ETEC isolated from rectal swabs: most of these children were under 2 years of age. These are similar results to those found in a rural area in central Java: ETEC were isolated from 19% of diarrhetic stools from 340 ill children (108). The incidence of typhoid fever in 1989 was high in southern Sulawesi, an island in Indonesia (3.1 per 100,000 population and a 5.1% case-fatality rate) (109). To account for this, a hospital-based case-control study was conducted in the city of Ujung Pandang (1.3 million population) on the southwestern tip of Sulawesi. Those at most risk were single, unemployed, or students or recent graduates of a university, whose lifestyle did not allow many home-prepared meals. It was also found that those hospitalized with the fever tended to eat food from street stalls and were not likely to use soap when washing their hands. Since typhoid fever occurred most frequently during a period when there was little rain, the authors postulated that only stagnant water was available, and any S. qphi present from fecal contamination could survive in it. Street vendors and households generally did not refrigerate food. Those hospitalized were severely ill, most likely because the dose was high as a result of growth of the pathogen in the food. It is possible that those that were not referred to a hospital could have had lower doses arising from water or another source.

K. Malaysia The incidence of food poisoning in Malaysia was 9.62/100,000 in 1981, with the most frequent etiological agents being S. aweus, V. paruhnemolyticus, and Salmonellu (1 10). In 1983 at a school canteen, 48 students eating meehoon (fried rice noodles) developed

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S. aureus intoxication. The organism was isolated from the vomitus and nasal swabs of three food handlers (1 10). Although food poisonings are frequent in Malaysia, they are rarely investigated by acceptable epidemiological procedures (1 10). Even in the meehoon outbreak the source of the S. nureus was not identified, since only scanty growth of the organism was obtained from the positive food handlers. The sources of these were not determined, but the V. pnrahnemol~~ticus infections probably arose from consumption of contaminated shellfish. ETEC seem to be a major cause of childhood watery and bloody diarrhea (66% of cases) in Malaysia, based on a study of 107 children with acute gastroenteritis (1 11). Salmonella was present in 10% of cases. Pathogens have been found in domestic foods, including B. cereus and S. nureus; even if there was a final heating stage, heat-resistant toxins could be produced (1 12). Snbnorzella has also been isolated from raw market foods and ready-to-eat items (1 13).An outbreak of Vibrio choleme 0139 with 52 cases in 1993 from a contaminated street food, rojak, led to a study of growth of the vibrios in a variety of street foods (1 14). It was found that strain 0139 could grow in cendol and tofu but did not grow in rojak gravy or noodles. In 1988, 25 ethnic Chinese were ill in a northwestern Malaysian state after eating a Chinese noodle, loh see fun (1 15). Seventeen of these were children who were admitted to hospital with acute hepatic encephalopathy, 13 of whom died. High levels of aflatoxins were found in the blood, brain, kidney, lung, and other postmortem tissues. The mean incubation period was very short for acute aflatoxicosis (8 hours). The cases were from six towns in two districts and had obtained their noodles from food stalls that were supplied by the same family-run factory. The noodles were made from rice, corn flour, tapioca flour, and wheat starch. None of these ingredients was found to contain aflatoxins at the time of testing, but it was assumed that one of the ingredients for one batch of noodles had to be highly contaminated to have such an effect on the children.

L. Singapore The main bacterial pathogens isolated from 7334 patients with diarrhea in Singapore were Salmonella ( 10.1%), Canzpylobncter (1.2%), Shigella (1.1%), Vibrio pnrtrlzaemol~ticus (0.8%), and V. ckolerae 0 1 (0.2%) (1 16). Salmonellosis outbreaks were associated with a variety of foods, such as coconut rice (nasi lemak), mutton curry, and Malay pancake (roti jala). Snlmonellu enteritidis was the main serovar, replacing S. typhinzurium in 1994. In 1995, 188 inmates in a penal institution were infected with S. enteritidis from canned sliced pork luncheon meat served at a lunch on March 26 (1 17). Although the specific cause of this outbreak was not determined, in previous outbreaks at Singapore institutions there were lapses in food hygiene. Another outbreak of salmonellosis, this time caused by serovar weltevreclen in 1996, affected at least 116 workers at a shipyard (118). Various cut fruits and vegetables including watermelon, pineapple, papaya, and vegetables in oyster sauce eaten over a 2-day period were implicated. These were sold from food stalls whose owners used industrial water derived from sewage effluent. Vibrio vul~~ificus was first diagnosed in Singapore in 1985. Since this country is surrounded by tropical waters and has the highest per capita consumption of seafood in the world, the risks of V. vulm!jicus infection are high. It is, however, not a notifiable disease in Singapore, and the true incidence of the disease is not known. Three hospitalized cases examined by Lee et al. (1 19) revealed a history of eating seafood-raw fish, crabs, and steamed cockles. None

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of them had evidence of wound infections. One of them, a fishmonger, died, and another had a leg amputated.

M. India, Pakistan, Bangladesh, Sri Lanka, and the Maldive Islands 1. Epidemiology Cholera is widespread in the Indian subcontinent and seems to be increasing, especially since the emergence of V. cholerae 0139 and drug-resistant strains (120- 122). Contamination of water is a typical problem, often from inadequate sewerage systems, common to many parts of Asia. In 1991-92, in Dhaka, Bangladesh, 451 children with acute diarrhea were compared with 602 matched controls (123). ETEC and EPEC were the only two of six pathogenic groups of E. coli looked for to be significantly associated with diarrhea. ETEC infections tended to be in the wet warm month of August, and EPEC infections peaked in the dry months of February to May. Flies and water samples were reservoirs for Cmzpylobacter, ETEC, Shigella, and Salnzonella (124). However, Canzpylobacter was the pathogen found most frequently in flies (12- 13% in urbadperiurban slums, 4% in hospitals, 2.6% in upper middle class homes, and 2% in markets) and water samples (30% in hospitals, 28% in markets, 12.5% in a village, 5.5% in riverdcanals, 4% in urban/ periurban slums). Therefore, Campylobacter may be a more important cause of morbidity than is currently recognized. This is particularly important now that a link has been made between Campylobacter and Guillain-Barr6 syndrome (GBS) in Kerala State, India (125). From serum samples taken from patients, 26% showed high antibody titers to Campylobacter jejurzi and 38% of stool specimens of new GBS cases were positive for C. j e j u d coli. Cooks and waiters in hotels in Poona had fecally contaminated hands (73% and 44%, respectively) based on coliform counts, and plates, spoons, forks, and kitchen towels were similarly contaminated (126). Apart from coliforms, these contained Salmonella, Proteus, and Pseudomonas spp. Hand-washing practices had not seemed to have improved from a more recent study in rural Bangladesh, partly because soap was too expensive to buy and partly because of religious and cultural practices (127). Similarly, mothers’ hands in rural communities in West Bengal are typically fecally contaminated (40%) compared with those of children (17%) (128). E. coli was also found in leftover food and water (59%) and utensils (27-32%). The contamination was highest in the wet summer season. Breastfed children were less likely to have E. coli isolated from their hands than partially breastfed children. There were 721 outbreaks and 1199 sporadic cases in the twin cities of Hyderabad/ Secunderabad between 1984 and 1989 (129). The majority of outbreaks affected 2-10 persons and occurred between February and June. The main vehicles of transmission were “stale” food (36.5%), rice dishes (23.5%), sweets (12.6%), and curry (9.8%). “Stale” food is probably food that has been left over from a previous meal at room temperature for a lengthy period of time, usually overnight (130). Chicken, pork, goat, and fish were the most frequent components of curries and rice dishes and were most often eaten at parties or in homes. S. aureus and Bacillus spp. were the most likely causative agents since they are often present in these foods. S. aureus was implicated in one outbreak in Hyderabad in which more than 100 persons fell ill after eating a sweet porridge (13 1). Home-prepared foods in small communities also contained pathogens, such as S. aureus,

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C.pe@-irzgens,and B. cereus but not Sdmonella. As in India, sweet dishes are vehicles for S. u w e m intoxication in Pakistan (133). One such outbreak from khoa, a confectionery with concentrated buffalo milk, caused eight persons to be hospitalized in the early 1980s. More recently, some samples of khoa obtained from manufacturers in a large Pakistani city contained up to los S. nureuslg. Salmonella was also found in khoa and in cheesebased confectioneries (132). Pulses, ground meat dishes, and chickpeas sold at bus and train stations in the same city contained 104-107 C. pegrirzgenslg, when the holding temperature was not hot enough (38-46°C) (133). Home-prepared foods in small communities also contained pathogens, such as S. uzmus, C. yerfiingens, and B. cereus, but not Salmor2elln. The main hazard identified was holding foods for long periods of time (e.g., overnight) at ambient temperatures (134). In India and many other Asian countries, cysticercosis from Taertin soliurn is a major public health problem because of the widespread consumption of insufficiently cooked pork (135). 2. Foodborne Disease Outbreaks The incidence of salmonellosis has increased in India in recent years, and some outbreaks have been investigated. In 1995 33 persons were admitted to hospital in Maharashtra after consuming vegetarian food served at a party (136). Twenty-three developed high-grade fever 24 hours after eating, one died, and one had irreversible cerebral damage with neurological complications. S. pnratyphi was isolated from 12 stool samples. In the Maldive Islands, a small outbreak of five persons eating salad containing carrots and peas in a cream base was caused by two Salrnomlla serovars: S. stanley and S. ormienburg were both isolated from the stools and the food (137). These isolates were resistant to the same antibiotics. Water was a probable source of an outbreak of EAggEC (EAEC) infection in south India (138). In one village, 69 of 451 residents experienced diarrhea, mostly watery but 24% with blood. The most frequent pathogen isolated from ill but not well persons was EAggEC. Following a feast in a village in Tamil Nadu, 25 of the 48 villagers developed diarrhea, abdominal cramps, and fever. An investigation showed that Yersiniu errterocolitica serotype 3, biotype 4, was the causative agent, being transmitted through buttermilk (139). Although all leftover foods had been discarded, this pathogen was isolated from a patient’s stool and water used to dilute the buttermilk. In addition, high anti-Y. enterocolitica antibody titers were found in sera of two patients, and toxin, determined by suckling mouse bioassay, was found in inoculated buttermilk kept for 6 hours at 4°C and room temperature. Soy milk is being encouraged as a substitute for animal milk in India. Unfortunately, an outbreak involving this product affected 35 of 263 schoolchildren drinking this at a midday meal in Dehli (140). The 30-minute incubation period and mild symptoms were indicative of S. uut-ells or B. cereus intoxication, although only E. coli at > 105/mLwas found in the milk. In a Sri Lankan village the carcass of a freshly dead monkey was made into a curry, and nine persons who ate this were subsequently infected with S. enteritidis PT 8; one 12-year old boy died (141). The curry containing monkey entrails and meat was insufficiently cooked. Mycotoxins have been responsible for illnesses in 1974 from aflatoxin in maize (15.6 ppm) and in 1987 from deoxynivalenol and other trichothecenes in wheat (142-144). In Bombay, 132 persons became ill and 4 died after eating fish that wereharvested from algae-rich water, and an algal toxin was believed to be responsible for their symptoms (145). In Kerala State, 500 persons were hospitalized, 7 of whom died after consumption of Perna mussels in September 1997 (146). PSP toxin levels exceeded 10,000 mouse units/100 g. The subsequent temporary ban on shellfish sales affected the jobs of 1000 families. A similar outbreak had occurred in 1984. Illnesses

borne

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from Vibrio vzrln$cus have occurred from consumption of seafood in southern India based on the finding of up to 107/g in intestines of a variety of market fish (147).

N. Nepal In Nepal 30,000-40,000 people die from gastroenteritis each year. The annual incidence of diarrheal diseases is 3.1-3.3 episodes per child, and 25% of childhood deaths are associated with diarrhea (148). Cholera outbreaks occurred in 1991 (1,800 deaths), 1992 (1,049 deaths), and 1995/96. In the latter outbreak, 1,017 children with acute diarrhea were studied by Pokhrel and Kubo (149), who found that the inadequate water sanitation system in Nepal contributed to the spread of enteric diseases. A WHO evaluation of food-safety practices showed that food for home preparation is generally purchased from markets where the hygiene is poor or from street-vended foods where sanitation is equally bad and the operators have no sanitation training (148). Proper food-safety education was thought to be essential in reducing future enteric illnesses.

VII. SURVEILLANCEPROGRAMSINTHEMIDDLEEAST

A.

Israel

Salmonellosis cases rose from 3,469 in 1989 to 4,542 in 1992, in contrast to a decrease of foodborne and waterborne diseases (all agents including those of unknown origin) from 2377 in 1989 to 1304 in 1992 (1). Campylobacteriosis cases decreased slightly over this period (from 1869 in 1989 to 1439 in 1992). In 1992, there were 5 C. perfringens, 3 Salmonella, 2 Shigellcr, and 2 S. aure-eus outbreaks; the remaining 28 outbreaks were of other or unknown etiology. Cheese, beef, and vegetables were the foods most frequently associated with outbreaks, and mass catering, restaurants, food-processing establishments, and homes were where the contamination was likely to occur. Some of the population, particularly non-Jews and Jews of Afro-Asian origin, were more likely to suffer from bacterial food poisoning; e.g., non-Jews (110/10,000) were twice as likely to be hospitalized as Jews (52/10,000) for bacterial food poisoning. This was probably because of different socioeconomic conditions and traditional food-preparation habits. Many of the non-Jewish communities had primitive water systems, inadequate sanitation, and poor food hygiene (150). This was borne out by a study of 399 hospitalized Arab infants in the West Bank, where there are villages and refugee camps with poor sanitation systems (151). They showed that 44% were infected with ETEC, and dehydration occurred in 58.3% of them and 28.5% failed to thrive. Religious differences are also reflected in the incidence rate per 100,000of the Israeli Arab population for echinococcosis (7.0 for Muslims, 22.5 for Christians, and 45.9 for Druze) (152). Home-slaughter of sheep, hunting of wildpig, and keeping of dogs were factors in thetransmission of theEchirzococcz4s tapeworm within these groups. In 1992, 197 air force personnel presented themselves to the base clinic with pharyngitis (153). Group A Streptococcr4s type 8/25 was isolated from the patients and a food handler. Processed white cheese was the vehicle served at a lunch. The asymptomatic handler mixed the cheese with his hands 24 hours before the meal and placed it in the refrigerator overnight before he went on vacation. It was put on the table for 6 hours at room temperature (28°C) before it was served. There were some secondary cases. No cheese was available for testing. Since this pathogen was shown to be able to grow in

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cheese, it was assumed that the organism from the handler mixed in with the white cheese multiplied during the 6 hours on the table. From December 1994 to February 1995, a peanut-flavored savory kosher snack item imported from Israel containing S. ngona infected 27 people in England and Wales and 10 in the United States. Information relayed to Israel helped identify the cause of more than 2,200 phage type 15 S. clgona infections in that country during the same time period (8,9) (see also Sec. IV). B. Jordan The isolation rate of enteric pathogens from healthy Jordanese food handlers was 6% for Salmonella, 1.4% for Shigella, and between 0.4 and 4.9% for eight types of intestinal parasite (154). Although this would appear to be a high risk factor for foodborne illnesses in food-service operations, there was no evidence that foodborne disease in Jordan was directly caused through contamination of food by infected food handlers. Of more significance was the incidence of brucellosis, with 33.2 and 46.2 persons infected with Brucella melitensis per 100,000 population in 1986 and 1987, respectively (155). The source of the Brucella was sheep and goats (infection rates >lo%) and possibly cattle (infection rate 2%). Unpasteurized milk and raw-milk cheese were frequently identified as vehicles of infection. In 17 cases of diarrhea admitted to a hospital in the Jordan Valley, only rota virus, EIEC, and EPEC were detected; six other pathogens looked for were not found (156). Sabnonella is widespread in poultry farms, broiler, layer, and breeder flocks, with 70% of birds having evidence of infection by serological testing (157). The serotypes most frequently isolated were S. gallinarum, S. euteritidis, and S. typhinzzu-irm.The yield of eggplants irrigated with treated effluent was twice that under conventional fertilizer application. However, the wastewater used for drip irrigation was shown to contain high levels of fecal coliforms, e.g., in July, 1.8 X 105/100 mL before chlorination, 4.6 X 10' mL after chlorination, and 8.1 X lo3 mL at the irrigation site (158). The fecal coliform count was over 100 times higher in irrigated soil than in dry soil. All fruits and leaves were negative for Snlmorzella and Shigella and had very low coliform counts.

C. Lebanon Salmonella outbreaks have also been documented from Lebanon (159). Raw meat and poultry products, milk and milk products, vegetables, and foods with multiple ingredients have been vehicles of transmission.

D. SaudiArabia Diarrhea in British troops deployed to Saudi Arabia was partly caused by ETEC (160). This pathogen is probably also present in the local population. The rate of brucellosis is high (617 per 100,000), particularly among the local Bedouin population, which is dependent on the raising of sheep, goats, and camels (161). There were 1.3 incidents and 22.4 cases of foodborne disease per 100,000 persons between 1982 and 1985 in the eastern province of Saudi Arabia (162). S. aureus was isolated more frequently than Salmo12ella from implicated foods, such as milk, fermented milk, cheese, meat, chicken, vegetables, and rice. Workers of Indian or Southeast Asian origin were the groups most frequently affected by outbreaks through mishandling in their work camps. Insufficient cooking and improper storage of food were the main factors contributing to the incidents. In one partic-

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ular outbreak in 1985, 168 of 419 Filipino workers at a workers’ camp in Damman, Saudi Arabia, contracted salmonellosis (163). A rice/meat/vegetable dish was the suspected food. One food handler and 57 cases were positive for S. rninnesota. Only those served at one meal were infected, and the source of the Salmonella was thought to be raw meat. In one town in central Saudi Arabia, an outbreak of typhoid fever was attributed to a cake (164). The cake containing cream had been kept overnight and served to schoolgirls on a bus the next day. There was a total of 19 ill, both those eating the cake and secondary cases.

E. Iran For those persons seeking medical aid in Tehran after experiencing an apparent attack of food poisoning, the most common etiological agents identified were C. pe~fringens (66.5%), Salmonella (17.8%), S. nureus (6.9%), E. coli (5.5%), B. cereus (1.6%), and C. botuhzum (1.0%). Most of these people (96%) had eaten in restaurants or mass-catering establishments (165).

F. Yemen An epidemic in Hodeida, Yemen, of 149 cases of diphtheria affected mostly children under 5 years of age. Those who had been previously vaccinated against the disease with at least three doses were generally protected (166). There were associations between those ill and consumption of water from a wheeled cart (possibly from direct contact with the infected driver) or locally made yogurt. It has been previously shown that milk can be a vehicle in the spread of diphtheria.

G. Bahrain Several large foodborne disease outbreaks have occurred in Bahrain including salmonellosis from improper handling of food in a hotel. Every school has a cafeteria, but the quality of the food is often questioned as being unappetizing, unhygienic, and containing foreign objects (167). The food workers had no training, and there was a potential for contamination by workers’ utensils, cloths, sponges, etc., growth of pathogens in food left at ambient temperature, and no reheating of items. Education and a HACCP plan were suggested solutions.

VIII.SURVEILLANCEPROGRAMS A.

IN AFRICA

Cholera in Africa

Cholera outbreaks have continued to occur in Africa since the seventh pandemic began in 1970. In a 1984 outbreak in Mali, millet gruel was associated with the illness. Typically, this gruel had curdled goat milk added to it, but at the time of the outbreak there was little milk available because of a drought. Laboratory studies showed that V. cholerae survived less than 6 hours in gruel with curdled milk compared with >24 hours in gruel alone (168). Other foods implicated in African outbreaks are peanut sauces (Guinea), leftover crabs (Guinea-Bissau), and rice meals prepared by persons preparing cholera victims for burial (Guinea and Guinea-Bissau). Direct person-to-person transmission is proba-

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bly rare because of the low rate of infection by care workers. However, in areas of crowding cholera spreads rapidly, and refugees are an increasing concern in Africa as a result of civil wars and international conflicts. It has caused severe morbidity and mortality in refugee camps in Malawi, Somalia, Ethiopia, and the Sudan (169). For instance, in April 1997 there was a cholera outbreak affecting 90,000 Rwandan refugees in three temporary camps in the Democratic Republic of Congo, with a high death rate. Between March 30 and April 20, a total of 1521 deaths was recorded, with a crude mortality rate estimated to be 9.9/10,000/day (170). The refugees were severely malnourished, and access by relief workers to the camps was difficult. How much of the cholera was due to food and water is not known, but both of these can be major vehicles of enteric pathogens in such situations. A study of one outbreak in Mozambican refugees in Malawi in 1988 indicated that there was a common source, which could have been food or water, but no environmental Vibrio isolates were found. Heavy rains destroyed some latrines 15 days before the outbreak, probably contaminating the local water table (169). In another outbreak in Malawi, three factors were identified with cholera: drinking river water, placing hands into drinking water in storage containers, and eating cooked peas kept overnight (166). B. EHEC Infections in Malawi and the Central African Republic During 1992, in the Malawan Lisungwi refugee camp holding 60,000 Mozambicans, 772 cases of abdominal cramps and bloody diarrhea were documented (171). The case fatality rate was 4.7%. The major factor contributing to illness was consumption of cooked food from the market. Based on analysis of stool cultures and the presence of the VT1 gene in some of these, the authors concluded that most of the cases were caused by E. coZi 0157:H7 and some by Shigella dysenteriae type 1. In the town of Zemio in the Central African Republic in 1996, 108 cases of bloody diarrhea were documented, with several HUS cases and 4 deaths (172). Viral hemorrhagic fever was originally suspected, and stool samples were negative for enteric pathogens. However, EHEC was eventually diagnosed based on the presence of virulence factors for this group of pathogens through PCR analysis of stools (presence of genes for SLTl and ene in 80% of specimens). In later hospital examinations of patients, E. coli 0157:H7 was isolated from two fatal cases of bloody diauhea. In other patients non-0157 EHEC were suspected. A case-control study showed that consumption of locally made meat pies (kanda) was associated with bloody diarrhea. Kanda is made by soaking smoked zebu cow meat in water for several hours and then mixing it with cooked marrow squash, wrapped in a banana leaf and steamed. It is then displayed in markets or roadside stands for up to several days at ambient temperatures until it is sold. Contaminated zebu meat was subsequently suspected as the cause of the 1996 outbreak.

C. Aflatoxicosis in Mozambique and Other Countries A consequence of consumption of moldy food is hepatocellular carcinoma (HCC); levels of aflatoxin B I have been found as high as 1.5 mg/kg of food. There was a strong association with mutations of the p53 gene in HCC and dietary aflatoxin intake in an international study of patients in 14 countries (173). The incidence of HCC is higher in Mozambique than any other country. Moldy grain, which contains aflatoxins, is often consumed under drought conditions. In Kenya and the Sudan, aflatoxins were present in the sera of children

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suffering from kwashiorkor but were metabolized in a different way than in children with other forms of lnalnutrition or in normally nourished children (174). The aflatoxins, therefore, may bea contributory factor to kwashiorkor. This is supported by more recent studies of Hendrickse (175). Because of the high humidity in tropical Africa, maize and cassava are often contaminated with aflatoxins, which are also found in up to 40% samples of breast milk, occasionally in high concentrations. Kwashiorkor-affected children have had as much as 4 pg aflatoxindkg body weight. The aflatoxin-induced immunosuppression may explain why the human immunodeficiency virus (HIV) is spreading so rapidly and aggressively in African infants.

D. Toxoplasmosis and Brucellosis in the Sudan and Other Countries Exposure to Toxoplasma gondii is relatively common in Africa, with a 18.2-61% prevalence of antibodies in the population of eight countries (176). In the Sudan, both Toxoplusma and Brucellu infections are probably associated with consumption of raw liver and intestines (177).

Foodborne disease increased in Egypt between 1989 (25 outbreaks, 115 cases, 3 deaths) and 1991 (146 outbreaks, 551 cases, 24 deaths) (178,179). The highest-risk foods in 1985 and 1986 were white cheese, fermented cream, meat/chicken, floudbutter oil, cabbage/ rice, and potatoes, and many illnesses occurred in homes (179). Street-vended food has been shown to contain pathogens, and many foods awaiting sale were at temperatures favorable for microbial growth (15-44°C) (180). Occasionally, acute illnesses directly associated with a food are documented. Between 1983 and 1985 in Egypt, three outbreaks from white cheese and two from cream/fermented cream were caused by EPEC. In 1991, the first major botulism outbreak with 20 deaths arose from ingestion of locally made faseikh (uneviscerated fish) (181). In 1994, three children died and six others suffered from severe diarrhea caused by E. coli 0157:H7 after they ate hamburgers, koshari, and dairy products in Egypt. As a follow-up to this, a survey of 175 foods obtained from slaughterhouses, supermarkets, and farmers' homes was conducted for E. coli 0157. This pathogen was detected in 6% ofunpasteurized milk, 6% of fresh retail beef, 4% of boneless chicken, and 4% of lamb meat samples (182).

F. Algeria An outbreak of botulism in the eastern provinces of Setif and Constantine killed 17 people in July 1998 and made another 100 persons ill after they ate rotten poultry and kashir, a processed meat (183). In addition, during the first halfof 1998, 1400 persons were reported to be ill from foodborne disease of unreported origin.

G. Tanzania,Ethiopia,andKenya In Tanzania, children <18 months old with diarrhea were infected with Cumpylobacter (22%) and ETEC (18%) (184). In Ethiopia, a prospective study of the incidence of campylobacteriosis showed that Cnmpylobncter spp. were isolated from 13.8% of 434 hospital-

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ized diarrheal cases in children under 15 (185), compared to 6.7% with Giardia and 3.2% with Shigella. The rate is a little higher than those in some other African countries e.g., Nigeria (1 l%), Rwanda (9.3%), and Zaire (8.6%). The disease seems to peak in the warm, rainy season. The pathogen was isolated more frequently from children with persistent diarrhea and from malnourished children. Contact of infants with chickens and cats may have been predisposing factors. From 1991 to 1993, many enteric pathogens were found in the stools of 862 diarrhetic children in the coastal town of Malindi in Kenya, but not so much Campylobacter: ETEC, EPEC, EHEC (13.8%), Salmonella (7.3%), Shigellu (6.5%), Camplobacter (4.9%), Entumoeba (7.8%), Giardia (4.9%), and rotavirus (16.1%) (186). There were many cases with multiple infections. The number of patients correlated with the rainy season, and drinking water was contaminated with up to 10’ coliforms/nL in 72% of households. A large cholera and shigellosis outbreak occurred in Malindi and Mombassa in 1994, again in the wet season. Reasons for the peak in illnesses at this time are the lack of a sewage system, flooding with an overflowing of latrines, and contaminated drinking water. An experiment with the Masai people of Kenya showed that solar radiation of drinking water by exposing water in plastic bottles to full sunlight reduced diarrhea in children 5- 16 years of age and could be used in communities where there are no other means of making water potable (187).

H. ZambiaandZimbabwe Case-control studies of diarrheal diseases in Zambia identified ingestion of relish (cooked meat or vegetable dishes) prepared by street vendors as a risk factor following an epidemic of Shigella dyselzteriae type 1 (188). Another study of a Lusaka city market and stall food showed that raw ground meat, chicken, chicken intestines, and dried minnows were contaminated with salmonellae; that pasteurized milk contained 10’ S. aur-euslml and caterpillars had lo7 B. cereuslg; and that leftover beef stew, chicken, and rice contained large populations of C. pellfringens, S. cumus, and B. cereus, respectively (189). Homes in Lusaka where diarrheal patients lived were examined for risk factors (190). Leftover cooked foods were most likely to be contaminated, such as nshima (boiled and whipped maize meal) and maize meal porridge, with 10’ B. cereuslg. Nshima is used as a weaning food. In Zimbabwe, unfermented porridge (pH 6 ) allowed the growth of Snlmoaella, Shigella, ETEC, and EPEC. Fermented foods, such as traditionally made in Zimbabwe, are not good vehicles for transmitting pathogens since most pathogens died within a few hours in sour porridge and mahewu because of the low pH (<3) (191). However, L. monocytogenes survived in traditionally fermented milk made both from raw or pasteurized milk, but longer under refrigeration conditions (192).

1.

Liberiaand C6te d’lvoire

Because food and water are thought to be the main means of transmitting these types of diseases, a survey of hygiene in houses where diarrhea had occurred was carried out in Liberia (193). In two communities, between 40 and 88% of stored water samples contained >lo3 enterobacteria CFU/100 mL, and 19-32% of adult food samples had >los enterobacteria CFUl100 g. Infant food such as formula, baby cereal, and traditional “rice water’’ were even more contaminated (56% with >10’ CFU/100 g) because they had been stored at room temperature for up to 8 hours. In the urban slum community it was a common

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practice to prepare food in advance of a meal and store it at ambient temperatures for up to 24 hours. It was only occasionally reheated, partly because of lack of fuel. Also, women only had time to make food once a day because they worked far from their homes. Unfortunately, no foods were examined for pathogens. These observations indicate that food- and waterborne diseases can be easily spread in such communities and cause a high morbidity. In Abidjan, C6te d’Ivoire (Ivory Coast), drinking water sold in bags to school pupils had up to 10’ E. coli/lOO mL and no residual chlorine present. This had the potential to cause illness ( 194).

J.

Nigeria

When strains of E. coli isolated from children with diarrhea in southwest Nigeria were examined, ETEC, EPEC, EIEC, EAggEC, and VTEC were found (195). The type of foods eaten may determine if they will be infected: e.g., ogi, a fermented maize porridge used for weaning infants in Nigeria, has a low enough pH to prevent growth of Salrzzomlln and EPEC (196). A case-control study in Nigeria showed that diarrhea in households was less related to poor food hygiene practices and more to improper disposal of feces (197). Human outbreaks of brucellosis have occurred in the past, and the disease is still endemic in Nigeria with a 3.1% seropositive rate in cattle (198). Of equal concern is the finding that Mycobacterium tuberculosis is present in slaughtered pigs (in lungs and lymph node), M. bovis in cattle, and M. avian in several animals (199), since human tuberculosis has a fairly high prevalence in the country. Because of limited laboratory procedures, it is not known how much tuberculosis comes from animal sources, but this study suggests that it could be one source of the disease. A 64-year-old male ate fried, prefrozen edible land snails collected locally in 1980. He developed diarrhea and abdominal cramps. His stool yielded a heavy growth of Aeromonas hydrophila, and thawed land snails had 1 X lo9 A. hydrophilalg (200). A 27-year-old male who ate with him did not suffer the same symptoms. A. hydrophila, Salmonella,or Shigella were isolated from about 40% of land snails in eastern Nigeria, indicating that this food source is a potential source of pathogens.

K.South

Africa and KwaZulu-Natal

In South Africa there are an estimated 12 million people without adequate potable water supplies and 21 million without safe sanitation systems (201). In 1995 and 1996, a Shigella dysenteriae type 1 in KwaZulu-Natal epidemic was responsible for thousands of cases and many hundreds of deaths. The impact of diarrhea for these two countries was estimated at 24 million cases in South Africa and 5.4 million (and an additional 76,000 of Shigella dysenterine type 1) in KwaZulu-Natal each year. For both countries, there would be a total of 54,000 deaths, and lost productivity was over 26 million days annually. The healthcare system would be burdened each year with over 5 million visits and 6.23 million hospitalization days. The total costs would be 3,375 million Rand (380 R/household) in South Africa and 905 million Rand (430 R for diarrhea and 67 R for dysentery/household) in KwaZulu-Natal. In South Africa, these figures represent 15% of the annual health budget spent on treating diarrheal disease, which costs 1% of the South African GDP (gross domestic product). These are the direct costs. The true costs may be 2.4 times higher. If these figures are extrapolated to all developing countries, the economic impact of diarrheal disease spread through contaminated water and food is enormous.

Todd

550 L. Madagascar

Shark is frequently caught and eaten in Madagascar, but one incident of ciguatera poisoning affected over 500 people who ate a shark in Novetnber 1993 on the east coast of Madagascar (202,203). The shark, 2 meters long and weighing 100-200 kg, was caught in a net and had been sold to six wholesalers for distribution to several villages. About 200 persons were hospitalized and 98 died, many of whom went into a deep coma preceding death. This high mortality rate is not typical of ciguatera. Some shark remains were available for analysis, and two new potent heat-stable lipid-soluble toxins, carchatoxin A and B, were isolated that were the likely cause of the severe illnesses. It is possible that this was similar to another incident of shark poisoning with cardiovascular symptoms, called selachian (pertaining to sharks and rays) ciguatera on Reunion Island, close to Madagascar (204).

IX. SURVEILLANCEPROGRAMS IN THECARIBBEAN AND CENTRAL AND SOUTH AMERICA A.

Surveillance of FoodborneDisease

All Central American, South American, and Caribbean countries have some form of notifiable disease system, with diarrheal diseases being one of the main causes of death in young children; e.g., the mortality rate per 100,000 for diarrheal diseases in children less than 5 years of age in 1994 was 9.8 in Nicaragua, 0.18 in Cuba, and 0.13 in Trinidad and Tobago (205). The causes of these are not generally known, but amebic dysentery, trichinosis, giardiasis, shigellosis, brucellosis, typhoid fever, E. coli, and hepatitis infections are all documented from Latin America and the Caribbean. There was little evidence, however, to link these with specific foods. In 1996, however, a more systematic approach to surveillance and reporting of foodborne disease has been attempted by the PanAmerican Institute for Food Protection and Zoonoses and the Pan American Health Organization (INPPAZ/PAHO). Information to date is limited, although many countries are contributing to the surveillance program. Between 1995 and 1997 there was a total of 2,236 foodborne outbreaks and 68,868 cases reported (205). The range of outbreaks for each of the 19 contributing countries was from <10 (Barbados, Jamaica, Panama) through several hundred (Chile and Mexico) to over 1,000 (Cuba). Reported deaths were highest in Mexico (74), Peru (58), Cuba, and Ecuador (both 12). For the 1,140 outbreaks of known etiology, the agents were bacterial (49%), chemical (47%), parasites, and viruses (2% each). Specific agents are shown by country in Table 9. There were 193 S. m r e u s outbreaks (4978 cases), 192 Salmonella outbreaks (10,971 cases), 417 ciguatera outbreaks (1,660 cases), 46 E. coli outbreaks ( 1591 cases), 22 C.pe$ringens outbreaks (1,053 cases), and 16 Shigella outbreaks (5577 cases). The foods implicated in outbreaks of bacterial origin were eggs/ mayonnaise (21%), dairy (21%), beef (19%), water (9%), fish (7%), and poultry (6%). The places where food was eaten leading to illness were identified in 2,236 outbreaks, with homes (42%), eating places (22%), restaurants, schools, and street vending (7% each) being the most frequent. In the Caribbean, the most cases from outbreaks were reported from Cuba (37,141), Bahamas (5,459), Trinidad and Tobago (414), and Antigua and Barbuda (180). In many of the Caribbean countries, ciguatera poisonings were listed as a major cause of the illnesses: Anguilla, Cayman Islands, British Virgin Islands (loo%), Montsenat (75%), Antigua and Barbuda (64%), and Belize (32%). Outbreaks associated

Surveillance Diseaseof Foodborne

551

Table 9 Bacterial Agents Causing Foodborne Outbreaks in Countries of Latin America and the Caribbean, 1995- 1997 Coulltly

SPP.

Argentina Bahamas Barbados Brazil Chile Costa Rica Cuba Dominican Rep. Ecuador El Salvador Guatemala Jamaica Mexico Nicaragua Panama Paraguay Peru Uruguay Venezuela Total

Snhonella SPP.

14 1 1 68 36 31 1 3 2

Staphylococcus

E. coli

Clostridium peq5ringen.s

Other bacteria

1

6 13 48 60 6 2 9

13

4 10 8

26

1 1

11 2 1 3 7

3

17

9 1 9 2 195

4

4

2

36

1 -

38 -

193

60

33

L-

72

Total 15 7 1 86 108 8 128 8 4 18 9 0 27 2 6 13 1 19 77 542

Source: Ref. J. Estupiiiin. personal communication.

with street vendors were reported from Chile and Mexico. Although street-vended food is frequently suspected as a source of foodborne outbreaks, there is rarely microbiological proof found during investigations. However, this type of food in the Dominican Republic had counts as high as 10" aerobic colony counts/g (206).

B. Cholera in Peru and Other Latin American Countries Cholera wasfirst documented inPeruin 1991 with a total of 600,000 cases (207). It rapidly spread to other countries and in 1994 caused 112,611 cases and 1,229 deaths, mainly in Peru, Brazil, El Salvador, Nicaragua, Honduras, Guatemala, Mexico, Bolivia, Ecuador, and Colombia (208). The total number of cases and deaths from 1991 to 1994 were 1,061,188 and 9,989, respectively. The source of the cholera was probably contaminated food. In 1991, V. cholerae 0 1 was found throughout Peru in water, sewage, finfish (skin and intestines), mollusks, and plankton with counts up to 1Os/1O0 mL (209). The disease was spread partly through consumption of street-vended foods and beverages containing ice (210). Undercooked or raw seafood may also have been implicated because these have been associated with cholera in the past (21 1). Shellfish may be contaminated with not only local sewage but also wastewater pumped from ships in harbor (212). Seafood (crab, shrimp, ceviche) was implicated in several incidents among U.S. travelers to Ecuador and Mexico in 1992 (211,213). From this information it is assumed that local persons also contracted cholera from seafood. In addition, 75 passengers on a flight to

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Los Angeles from Argentina with a stopover in Lima, Peru, were infected with V. cholerae (213,2 14). The economic loss in Peru in 1991 from lost markets and tourism, absence from work, medical care and lives lost due to the cholera epidemic was estimated at $495.3 billion (215). In El Salvador risk factors identified were eating cold or raw seafood and drinking water outside of the home (216). However, knowledge about how to prevent cholera, using soap, and eating rice were all protective.

C.

Mexico

In Mexico, colonization of infants by heat-labile ETEC increased 400-500% during the rainy season of those that received oat gruel. However, the risk of symptomatic infection was reduced by herbal tea and LT-ETEC-specific antibodies in the mothers’ breast milk. Also, the mothers’ level of education and the numbers of previous symptomatic infections affected the chances of illness (217). Salrnonelln may also be an important agent in Mexico, since one study showed that even with a method of limited sensitivity, 4.5% of chocolate samples in Guadalajara tested positive (218). Raw milk is allowed in Mexico and can be bought from street vendors. Therefore, there is a risk of enteric infections including listeriosis (219). In the northwest of the country seven crew of a fishing boat suffered from ciguatera poisoning after eating grouper in 1993 and two died after consuming a pufferfish (Sphaeroides sp.), probably containing tetrodotoxin, in 1995 (220).

D. Argentina In Argentina, S. enteritidis (22 1) and E. coli 0157:H7 (222) have been responsible for foodborne illnesses. Between 1986 and April 1990, 35 outbreaks of S. enteritidis affected 3,500 persons, largely through consumption of insufficiently cooked poultry and eggs used in mayonnaise. In Provincia de Buenos Aires, 23% of the outbreaks were caused by S. enteritidis, 44% by other bacteria, 27% by chemicals, and 6% were of unknown origin. In Argentina, 25-38% of children with bloody diarrhea are associated with VTEC infection, and 80.4% of children with bloody diarrhea eat meat 3 days a week (one third of them eat it undercooked) (223). Of 681 E. coli strains from 50 samples of raw meat, 19.4% produced SLT-I and SLT-11, and 31.5% of bovine stools carried VTEC. Of the bovine isolates, 22% and 44% were positive for eae and ehly genes, respectively. Only one 0157 strain was isolated. Therefore, non-O157:H7 VTEC need to be looked for in children with bloody diarrhea. Other diarrheagenic E. coli infections in young children have also been documented (224); these include ETEC, EPEC, and EAggEc. E. Brazil White cheese was implicated inan outbreak where both Sulrnouell~~ and S. aureus (106/g) were isolated (225). It had been previously reported that in 30 S. nureus outbreaks from two regions of Brazil in 1988-90, cream-filled cakes, white cheese, sausage, and milk were the foods implicated (226). In 1993, 21 1 children in a S50 Paulo state school suffered from S. elzteritidis infection as a result of eating a mayonnaise prepared with fresh eggs (227). Other enteric infections may derive from vegetables since high numbers of Aeronzorzas, including cnviae and hydrophila, were isolated from some samples of watercress, lettuce, and escarole in S50 Paulo (228).

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F. Peru Samples of vegetables from small markets in a Peruvian periurban slum, where cryptosporidiosis and cyclosporosis are endemic, contained Cryptosporidiump a ~ v u mand Cyclospora (14.5% and 1.8%, respectively) (229). Washing did not remove the oocysts completely. Therefore, these vegetables could be vehicles that allow these parasitic diseases to be endemic in the area. However, in a remote mountain community in Peru there were few opportunities for foodborne disease. Most foods were thoroughly cooked and eaten within a few hours, with few leftovers (230). At a Peruvian army base, 279 cases of diarrhea caused by Vibrio parahaemolyticus in recruits were traced to common meals served at a dining facility (23 l). No specific food item could be identified as the vehicle.

G.OtherCountries In Chile, 36 EHEC strains, including 13 0157, 5 026, 3 0 1 11, and 11 nontypable, were isolated from children with HUS between 1988 and 1996 (232). This study indicates that in Chile HUS is probably caused by several different EHEC strains. In Nicaragua, diarrhea in infants occurs in the first 12 months of life; the mortality rate was 2.55/1000 in 1994. In one study of children up to 2 years of age, ETEC was found in 38% of children with diarrhea, compared with 19% of asymptomatic controls (233). Since natural immunity develops with age, first exposures are the most severe, and vaccines are being developed to be given early in infancy. In Bolivia, the population of La Paz is exposed to the sewage-contaminated La Paz River. Water samples included ETEC, EPEC, EIEC, and Salmonella (234). Aeromorzas cavine were found in vegetables irrigated with river water. These same pathogens were found in children with diarrhea in La Paz and Sucre. This work indicates that contaminated water can cause infections through direct consumption or through food in contact with the water. In Costa Rica, a study of 640 samples of eight different vegetables (cabbage, tomato, lettuce, cucumber, carrot, radish, and coriander leaf and root) used for raw consumption were analyzed for parasites and fecal coliforms (235). Cysts of Giardia, Cryptosporidiunz, and Entamoeba were found in all eight vegetables; the E. coli counts in lettuce and coriander leaves were highest in the dry season (106/g). Botulism has been reported from Argentina, Brazil, Chile, Peru, and Mexico (236,237). Some babies in the Dominican Republic that were being treated for enteric infections came from homes where formula and other foods were prepared in a hazardous manner (238). Pathogens were found in these foods as well as in the kitchen environment.

X.

SURVEILLANCE PROGRAMS IN CANADAANDTHE UNITED STATES

A. Canada 1. Notifiable Foodborne Diseases and Other Studies Laboratory isolation data show that campylobacteriosis increased from 9653 cases in 1989 to 13,669 in 1993 and went back down to 10,499 in 1995 (239) (Table 10). The recent decreases may be a result of more analysis being done in private laboratories and not

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Table 10 Cases of Enteric Diseases in Canada, 1989-1995

Disease

1989

1990

1991

1992

Campylobacteriosis 9,653 Salmonellosis 8,984 8,742 8,762 Escherichia coli 0157:H7 enteritis 2,432

9,081

9,786

7,666 7,265

1,585

1,565

1,521

~

~~

1993

1994

Mean no./ 100,000 1995 1995 in

~

13,669 11,767 8,057 7,138 7,441 1,212

1,014

10,499

35 24

1,277

4

Source: Ref. 239.

being reported, rather than fewer cases occurring. Salmonellosis showed a slight decrease in the last few years (8,762 in 1989 to 7,138 in 1995), whereas E. coli 0157:H7 enteritis rose to a peak of 2,432 in 1989 after its recognition as a pathogen in 1982 and has since stabilized to over 1,000 cases each year (1,277 in 1995). Summer peaks are apparent for all three pathogens. A study of 80 farm families in Ontario, Canada, found that many individuals experienced mild or subclinical VTEC infection at an early age, which may give immunity to later exposures (240). VT1 antibodies were found in 41% of persons tested. E. coZi 0157: H7 and eight other serotypes were isolated from 21 persons on 16 different farms. Four of these serotypes were isolated from cattle living on the same farms. 2. Foodborne DiseaseSummaryReports Foodborne disease reporting in Canada was initiated in 1973 by the Health Protection Branch based on the approach used by the U.S. Centers for Disease Control and Prevention (CDC). There are, however, differences between the systems (e.g., definitions of microbiological, plant, animal, and chemical etiologies, and a more active pursuit of the information from the provinces). In addition, consumer complaint reports involving foodbome illness from the Health Protection Branch are tabulated along with the provincial data. Each year information is specifically requested by letter from contact persons (epidemiologists, food inspectors, environmental health officers, or laboratory staff) i n 10 provinces and two territories, with follow-up telephone calls to ensure some form of documentation is received. The reports are published as annual summaries (241-243) or 10-year summaries (244). Numbers of outbreaks and cases were similar from 1990 (1,044 and 6.027, respectively) to 1992 (1,069, 6,322, respectively) but dropped in 1993 (790, and 4,475) (243, Table 11j. Most of this decrease was for chemical and unknown etiologies as a result of some jurisdictions not submitting data. For microbial etiology there were fewer incidents caused by S. aure-eus,mold, and yeast and more by viruses. Foods most frequently associated with illnesses were meat, poultry, marine foods, and bakery products. Three or more incidents in 1992 and 1993 were caused by one agent in one type of food. These were B. cereus in Chinese foods (7 in 1992, 3 in 1993), Cumnpvlobacter in raw milk (4 in 1992) and chicken or turkey (4 in 1992, 5 in 1993), E. coli 0157:H7 in ground beef (1 1 in 1992 and 8 in 1993), Salmonella in poultry (10 in 1992, 8 in 1993), and S. uzueus in fish (4 in 1992) and poultry (3 in 1992 and 1993). Two episodes of salmonellosis were linked with consumption of eggs and egg products in 1993. Most mishandling tended to occur at food-service establishments, with the larger outbreaks in institutions.

Surveillance of Foodborne Disease 555

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556

3. FoodborneDiseaseOutbreaks In British Columbia in 1995, an outbreak of 61 cases of S. newport infection resulted from consumption of alfalfa sprouts from a single lot of seeds imported to British Columbia and Oregon (245). The same year in Ontario, an E. coli 0157:H7 outbreak occurred in a correctional institute, with 15 inmates and staff ill (246). This was attributed to inadequately cooked hamburgers and poor food-handling techniques. In Ontario in 1996, roast turkey served at a church supper was implicated in an outbreak of E. coZi 0157:H7 with 36 cases (14 confirmed and 4 seriously ill) (247). Three of the six ill food preparers contained 0157 in their stools. The turkeys appeared to be properly cooked and may have been contaminated during cooling on the kitchen counter. Other foods implicated in VTEC infections in Ontario between 1990 and 1995 were ground beef, chicken, beef, unpasteurized milk, pork, goat, seafood, eggs, and apple cider (248). Turkey, chicken, and eggs are not foods traditionally associated with VTEC infections. More recent E. coli 0 1 57 outbreaks were associated with salami (27 cases in April 1998) and salads (22 cases and 1 death in May 1998). Three separate incidents of ciguatera poisoning occurred from September 1996 to January 1997. Barracuda and doctorfish purchased in Ontario or Quebec were implicated, and leftover fish in two of the episodes were confirmed by tnouse bioassay. This increase in illness may reflect a growing market for tropical fish in temperate regions. In the summer of 1997, at least 42 persons developed Vibrio yurahnen?olyticus gastroenteritis after eating raw oysters in the Vancouver area, and at least an additional 160 others in California, Oregon, and Washington were also confirmed with the pathogen (249). Since such outbreaks are rare, it was deduced that the organism grew to large enough numbers in the Pacific coastal waters to cause the infections because of the atypical higher water temperatures (1-5°C above those in 1996).

B. The UnitedStates 1. Notifiable Diseases Notifiable diseases of enteric origin include salmonellosis, shigellosis and E. coli 0157: H7 disease (250). The numbers for the first two fluctuated from 1990 to 1996 with no clear trend noticeable; in 1996 there were 45,471 and 25,978 cases of salmonellosis and shigellosis, respectively (Table 12). These are considerable underestimates, as there are probably 46,000-231,000 foodborne cases of S. ~~phimuriurn DT104 alone annually (251). For hemorrhagic colitis and other conditions caused by E. coli 0157 infections, the trend is up from 1,420 in 1994 (when the disease was first made notifiable) to 2,139 in 1995 and 2741 in 1996. However, on a population basis fewer were reported per 100,000 in 1995 in the United States (1.01) than in Canada (5.2).

Table 12 Cases of Notifiable Enteric Diseases in the United States, 1992-1996 1992

disease

Enteric Salmonellosis 34,520 Shigellosis 14,926 Escherichiu coli 0157:H7 enteritis reported Not reported Not

19931995 17

1994

36.9 19.5 11

~

Source: Ref. 250.

1996

37,522 18,783

4 1,222 19,330

45,47 1 25,978

1,420

2,139

2,741

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557

2. Foodborne Disease Summary Reports In the United States individual states have been reporting outbreaks of foodborne illness since 1938, but the CDC foodborne disease outbreak surveillance system began in 1961, with reports published in 1966 and data being computerized since 1973 (252). New outbreak information can be added retrospectively to this data base. Local sanitarians investigate outbreaks, obtain laboratory analyses of clinical specimens and food samples, and send reports to the state. The state, in turn, screens these reports and forwards what it considers valid to CDC. Some investigations by CDC and other federal agencies are also included, but only limited material is received from the U.S. Food and Drug Administration (FDA) to be compiled with the state data. Outbreak information may be sent in a nan-ative style, but it is usually summarized on a one-page form. Publication of multiyear data are published in special issues of Morbidity and Mortalih) Weekly Report (253,254). These CDC summaries have been criticized for having incomplete reports of outbreaks and not always being of high quality (255-257). This is one reason for the establishment of the FoodNet program (see Sec. XI). Foodborne disease appeared to decline from 1990 (532 outbreaks, 19,883 cases) to 1992 (407 outbreaks, 11,015 cases), both for known and unknown etiology (258) (Table 13). There were 15, 10, and 8 deaths in 1990, 1991, and 1992, respectively. The agent causing most outbreaks was Salmonella, but even for this pathogen the number of outbreaks in 1992 (80) was much less than in 1990 (132). Part of this decline is attributable to a more restrictive definition of an outbreak introduced in 1992. Other pathogens responsible for foodborne illnesses were the traditional ones: S. aweus, C. pe$ringens, Shigella, B. cereus, and hepatitis A. Few E. coli 0157 outbreaks were documented during this time period. Both ciguatera and scombroid poisonings represented the majority of the chetnical illnesses. Outbreaks occurred at all times of the year with no particular peak, except perhaps for May and June. Restaurants, cafeterias, and delicatessens were the places where food associated with outbreaks was most frequently eaten. Foods most implicated were fish (scotnbrotoxin) and multiple vehicles (Salmorzella). Factors contributing to outbreaks were mainly improper holding temperatures, inadequate cooking, Contaminated equipment, and poor personal hygiene. Foodborne outbreaks in prisons were assessed in the United States from 1974 to 1991, with 88 outbreaks and 14,307 cases (259). The three main pathogens implicated were Sdmonella including S. enteritidis (37% of outbreaks of known etiology), C. perfringens (34%), and S. nureus (22%). About 50% of the outbreaks had no agent identified. Beef, poultry, meat and fish salads, and Mexican foods were the main foods implicated. Improper food storage and inadequate coolung were the main factors contributing to outbreaks. Because the HIV seroprevalence in prisons is higher than average (18.9% in 1989), the risk of serious infections is also high in prisoners from any source, including food. 3. Foodborne Disease Outbreaks As in other parts of the world, the epidemiology of foodborne disease in the United States is changing with new pathogens in foods not usually associated with illness (260). One new scenario is the result of low levels of contamination in food products that are widely distributed. One example of this occurred in 1994, when 593 cases of Salmonella erzteritidis were identified in Minnesota after a nationally distributed brand of ice cream was eaten (261). Pasteurized ice cream mix had been transported in a tanker previously used to carry nonpasteurized liquid egg. If it is assumed that the whole load of ice cream was contaminated at the same level as the sample taken (0.093 S. enteritidis PT 8/g), a possible

558

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Surveillance of Foodborne Disease

559

224,000 persons in the United States could have been ill. S. enteritidis in eggs and products in which eggs are used has been a major problem in the United States, with many outbreaks documented. However, S. ~phirnurizmDT104 is a new virulent strain that is affecting many people. Two raw milk Mexican-style cheese outbreaks were caused by this pathogen in California (262). In the same state, brucellosis is much less likely to be an occupational disease arising from handling infected animals or working in laboratories than through consumption of contaminated raw milk and cheese, with Hispanic populations most at risk (263). Fruits and vegetables are being increasingly implicated in outbreaks. In 1995, 62 cases of salmonellosis were associated with unpasteurized orange juice (264). Four Sahnoneh serovars were found in juice samples, unwashed fruit surfaces, and amphibians in addition to the processing facility. In 1993, consumers of apple juice in Massachusetts contracted cryptosporidiosis, where there was evidence that the cysts came from cattle manure contaminating the apples (265). Another apple juice outbreak occurred in 1996 in New York when 3 l confirmed and suspected cases were documented (266). In the same year, two juice outbreaks were caused by E. coli 0157:H7: one in Connecticut (66 persons and 1 death) and one in California, Colorado, and Washington as well as British Columbia (61 cases and 1 death) (266,267). This pathogen has also been responsible for outbreaks associated with lettuce, salad items, alfalfa sprouts, salami, and venison (268,269). The strongest evidence that Qclospora may be foodborne came in 1996, when raspberries (probably from Guatemala) caused severe diarrhea in more than 1400 persons in 15 states and Ontario. A similar outbreak occurred in 1997 in over 500 persons from fresh raspberries, also probably from Guatemala (270). In the same year, basil in salads (271) and mesclun lettuce served on a cruise ship (270) infected 308 and 224 cases with Cyclospora, respectively. Because of the lack of suitable methodology to isolate the cysts from food and its probable low infectious dose, no laboratory confirmation of the parasite in these products has yet been made. ETEC, more typically associated with developing countries, has caused a few outbreaks in the United States. After consuming buffet meals in a Minnesota restaurant in April and May 1991, 17 confirmed and 105 probable cases of ETEC were diagnosed (272). In 1993, airline passengers from North Carolina to Rhode Island ate a garden salad (47.1 1) and guests at a mountain lodge in New Hampshire ate tabouleh (78.1 l), with carrots being the only common element; ETEC strain 06:NM was the pathogenic agent in both outbreaks (273). The carrots could not, however, be traced to a single source. In 1994, there were up to 645 cases of ETEC among 1240 attendees following a banquet in Milwaukee (274). Pan-fried potatoes were the only food associated with the illness. These were cooked with cheese and spices and left at room temperature for extended periods of time. It is uncertain what the source of the ETEC was, but the contamination had to be extensive and considerable growth would have had to occur to allow so many people to become infected, since volunteers require 106-10'o cells to induce diarrhea (274). Other foods involved with ETEC were cornbread dressing in Louisiana and scallops on a cruise ship in the Caribbean (274). Listeriosis may be decreasing through industry, regulatory, and educational efforts (275). There were an estimated 1965 cases and 481 deaths in 1989 compared with 1092 cases and 248 deaths in 1993. However, these were hospitalized cases of invasive listeriosis. There also may be milder infections. In 1989, complaints of illness by two pregnant women led to the discovery that 10 of 36 persons who attended a party in New York developed mild listeriosis after eating snack foods (cooked shrimp was the most likely vehicle) (276); no one was hospitalized. In 1994, Listeria monocytogenes serotype 1/2b was isolated from 45 persons who were ill typically with diarrhea, fatigue, or fever after

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560

drinking chocolate milk at a picnic in Illinois (277). Only 4 persons were hospitalized. About lo9CFU of the same strain/mL were found in unopened milk cartons, and the dose consumed could have been about 10" CFU. In 1995, 15 of 26 persons attending a social function in Minnesota contracted cryptosporidiosis after eating chicken salad (278). It is probable that the host had contaminated her hands through changing a diaper from an asymptomatic child in her licensed daycare home, even although she washed her hands before preparing the salad. This type of transmission can be expected when the infectious dose is low (ID50= 132 organisms). Botulism has resulted from many different foods in the United States. For instance, a commercial canned cheese used as a cheese sauce on stuffed potatoes in Georgia in 1993 caused eight cases of type A botulism (7). The investigation indicated that the cheese was probably contaminated with spores after the can was opened, possibly from the potatoes. A more recent incident involved a cactus product. Three members of the Native American Church in Arizona were diagnosed with botulism after they and 10 others consumed peyote buttons, a cactus product with hallucinogenic properties that has been legalized for sacramental use in traditional American Indian religious ceremonies (279). The ceremonial tea the patients had drunk was made from buttons of the dried, alkaline-ground peyote cactus, which had been covered with water and stored in a closed jar for 2 months under refrigeration. The peyote was found to contain C. botulinum type B toxin. The prolonged nontraditional storage of unsterilized peyote likely produced an anaerobic and alkaline environment that favored the growth and production of the toxin.

XI. SENTINEL,CASE-CONTROL, ANDOTHER SPECIALIZED EPIDEMIOLOGICAL STUDIES A.

Europe

Because of their expense and considerable time commitment, limited numbers of sentinel or case-control studies have been carried out, and these only in a few countries. In Belgium, sentinel general practitioners have been established to improve the knowledge of infectious diseases derived from the national notification system. They also determine the extent of nonnotifiable diseases and other health problems observed in general practice (280). Similar programs exist in England/Wales organized by the Royal College of General Practitioners (281) and other European countries, e.g., Italy (282) and the Netherlands. In this last-named country, 42 sentinel general practitioners conducted studies on acute gastroenteritis from 1987 to 1991 and found that 1,500 cases per 100,000 persons occur each year, with campylobacteriosis and salmonellosis accounting for 15% and 5% of these cases, respectively (283,284). Because the Dutch sentinel study only measured foodborne disease as related to those who visited general practitioners and might have missed many cases, a study in one community was carried out in 1991. Over a 4-month period almost 2000 persons were questioned weekly about gastrointestinal complaints, consultations, medication, and time off work, and the results were sent to the National Institute of Public Health and Environmental Protection. In case of complaint, a fecal sample was to be sent immediately. The specimens were examined only for the presence of Salmonella and Campylobacter. It was found that 15,000/ 100,000 population experienced gastroenteritis, and in 10% of those interviewed it occurred more than once. This is equivalent to 2.5 million episodes of illness each year in the Netherlands. Only 10% (250,000) of these actually visited physicians; a similar number was derived from the general practitioner sentinel study (225,000). From laboratory results it was determined that at least 50% of

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these gastroenteritis cases were related to consumption of food or water, indicating that about 1,000,000 cases of food- or waterborne disease occur in the Netherlands each year (284). The proportion of S. enteritidis to all Salmonella cases increased from 2.4% in 1985 to 34.4% in 1991, with approximately 600 cases/100,000 population (285). Despite the fact that infected poultry breeding stocks are eradicated when they are discovered, the incidence rate is still going up. Five hundred general practitioners in France have been operating a sentinel system with a computer network for the surveillance of communicable diseases. In 1991 this system showed an annual incidence of 4,400 cases of acute diarrhea/100,000 population (286). Stool cultures were available for only 6% of these cases, with 21% containing Salmonella, 17% EPEC, 8% rotavirus, 4% Campylobacter, 2% Shigella, and 1% Yersinia. From these studies, it was estimated that there were 2.5 million cases of acute diarrhea annually-morethan the estimated 2 million influenza-like syndromes each year in France. VTEC infections have only recently been diagnosed in Italy (287). To confirm the significance of VTEC in Italy, a 3-year prospective study was set up with 49 children with hemolytic uremic syndrome and 39 matched controls (282); 75.5% of the HUS patients and 2.5% of the controls had serological or toxigenic evidence of VTEC infection. Special epidemiological studies are not only useful for generating numbers of cases but also for determining the most likely vehicle of transmission of a pathogen. For instance, a case-control study in Glasgow, Scotland, indicated that salmonellosis victims were 4.2 times more likely to have eaten poultry, particularly roasted poultry, than other foods, including boiled poultry and red meat (288). Salm-Net was established to identify trends in salmonellosis in Western Europe. Between 1993 and 1995 there was a fall of 6.8% in all isolates in seven contributing countries, with S. enteritidis and S. typhimurium representing about 75% of these. However, the trend for these two serovars was different; S. enteritidis decreased each quarter by a total of 16.4%, and S. typhimurium rose each quarter during the same three years by 15.2% (289). The decrease in S. enteritidis may represent efforts to limit infection in flocks and use unheated eggs less frequently in food items. It is also noted that many of the S. typhimurium strains are multidrug-resistant, and this leads to an emerging problem in enteric infections. Salm-Net also wants to type strains beyond their serological designation and is requesting phage typing for the most common serotypes. Another reason for the creation of Salm-Net is the rapid identification of an outbreak or contaminated product. A successful example of this was a kosher snack item imported from Israel into Europe and North America. A case-control study showed a strong association between infection with S. agona phage type 15 and consumption of a peanut-flavored savory snack imported from Israel. The combined testing of food from the United Kingdom, the United States, and Canada, where the product was imported, showed that the contaminated snacks were manufactured on at least seven separate dates during a 5-month period from October 1994 to February 1995. Ten cases were documented in the United States, 27 in the United Kingdom, and none in Canada or other European countries contacted through Salm-Net. However, as a result of the Salm-Net study, over 2,000 cases were recognized in Israel (see also Sec. VII). Voluntary recalls were carried out to avoid further illnesses.

B. TheUnitedStates Since it is known that an active surveillance system will identify more cases of a specific pathogen, e.g., S. enteritidis and Vibrio vu1niJicu.sin the United States, the use of sentinel

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counties can dramatically improve the reporting of certain foodborne illnesses and indicate a more accurate figure of the morbidity. In the United States campylobacteriosis, E. coli 0157:H7 infection, hepatitis, listeriosis, salmonellosis, and shigellosis have all been monitored by some form of sentinel study. In 1980-81, a 15-month nationwide hospital-based study showed that Campylobncter infections were more likely to occur in the summer and in an age range of 10-29 years (290). These results were essentially confirmed in another project, also in 1980-81, in which cases of C. jejuni infection and control persons were interviewed in King County, Washington, but the analysis failed to determine the origin of the pathogen (291). However, the organistn was isolated more frequently than Salmonella and Shigella combined. In the same county 2 years later, Harris et al. (292) studied food-consumption habits and found that consumption of chicken, particularly if it was raw or rare, was a risk factor for campylobacteriosis. Another case-control study at a university in Georgia indicated that students eating chicken, particularly raw or undercooked, or having contact with cats or kittens were more likely to have campylobacteriosis than other students (293). In fact, most sporadic cases seem to be associated with improper handling of poultry, since one drop of raw chicken juice (drippings) contains enough Canzpylobncter to cause an infection if ingested. A small study in England/Wales indicated that the number of isolates of Camnpvlobacter may considerably underestimate the extent of the disease, and the annual incidence could be as much as 1.1%of the population (28 1,294). In July 1995, the U.S. Department of Agriculture (USDA), FDA, and CDC collaborated on a project called the Foodborne Diseases Active Surveillance Network, or FoodNet, to collect more precise information on the incidence of foodborne disease in the United States (295). This project has links with state and local health departments at seven selected sites across the country (others are to be added). The primary purpose of the case-control studies is to identify patient behavior, food-preparation practices, and foodconsumption preferences, which are strongly associated with illness due to one of the specific bacterial pathogens just prior to becoming ill. Preliminary data indicate that 1.4 diarrheal episodes occur per person each year; 8% of these seek medical care. The isolation rate for Campylobacter, E. coli 0157:H7, Listerin, Salmonella, Shigella, Vibrio, and Yersinin combined was 50.4 cases per 100,000 persons in 1997. However, the isolation rate varied by pathogen: Cnlnpylobncter (49.4%), Salmonella (27.4%), Shigella (15.7%), E. coli 0157:H7 (4.2%), Yersinin (1.7%), Listeria (l%), and Vibrio (0.6%). Consumption of pink hamburgers or pink ground beef and visiting a farm are risk factors for sporadic infection from E. coli 0157:H7. Considerable attention has also been given to the surveillance of listeriosis because of its high morbidity and mortality risk to immunocompromised persons. A major study was conducted in 1988-90 in eight areas in four states (296); the incidence rate was 7.4 cases/million, and 23% of the infected cases died. Most of the cases were immunocompromised or pregnant and were more likely to have eaten soft cheeses or delicatessentype food. For immunocompromised persons, eating undercooked chicken wasan increased risk. The same specific types of L. monocytogenes from patients were also isolated from refrigerators of the patients and, in particular, from ready-to-eat food, such as processed meats, raw vegetables, leftovers, and cheeses (297). From these data it was estimated that 1965 cases and 481 deaths due to listeriosis occurred in 1989 in the United States (275). A similar study for 1993 showed that there were 1092 cases and 248 deaths, a reduction of 44% in cases and 48% in deaths. It was suggested that the combination of industry clean-up of plants, government regulations, and educational strategies caused this decrease. These estimates, however, are only for medically identified (normally hospital-

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ized) cases. Mild cases of listeriosis have been documented (276). The impact of those with a mild syndrome is not known, nor whether the numbers have been reduced. Hepatitis is intensively monitored in the United States, and one study conducted in Pierce County, Washington, over a 6-month period in 1984 indicated that the passive reporting system identified 65% of the actual cases (298). The increase in reporting through the active system, however, was largely for hepatitis B and non-A non-B; numbers of cases of hepatitis A, the disease most likely to be transmitted through food, did not change substantially from the passive reporting system. Two special projects examined the changing resistance of foodborne pathogens to antimicrobials in the United States. Resistance of Salmonella to antimicrobials rose in a study of isolates from 48 counties over a 5-year period (1979-80 to 1984-85) from 16% to 24% (299). Antimicrobial resistance of Shigella was also examined, this time in 25 counties in 1985-86 (300). Apart from showing that many strains are resistant to one or more antimicrobials, the authors indicated that strains derived from cases who traveled in other countries had a different pattern of resistance than strains from domestic cases. Risk factors for acquiring Shigella abroad included contaminated food and water and homosexual contact. The situation has worsened even further in recent years, with the proportion of resistant strains including those for the relatively new pathogen, E. coli 0157, increasing.

XII. A.

ESTIMATESOFNUMBERS OF FOODBORNE DISEASE CASES AND THEIR COSTS Number of Cases

It is recognized that in no country is the surveillance system accurate enough to determine the true number of foodborne illnesses that occur each year, and extrapolations of existing data must be made to estimate this. These have been made for all gastroenteritis of foodborne origin with considerable variation in case numbers and costs. The CAST Report (301) states that there are probably 6.5-33 million cases and up to 9,000 deaths each year in the United States from foodborne disease. Estimates have also been made for diseases of specific etiology. For instance, estimates of the number of salmonellosis cases in the United States have been determined by Hauschild and Bryan (302), Chalker and Blaser (303), Bennett et al. (304), Roberts (305), and Todd (306). Although different methodologies were used to calculate these, the range is not extreme (790,000-3,690,000), with a median figure of 1,920,000. This figure represents approximately 50 times the number of human isolations and 800 times the number of reported foodborne cases caused by Salmonella. Other specific foodborne diseases are less well reported than salmonellosis, and fewer attempts have been made to estimate these. Bennett et al. (304) derived case numbers based on literature searches, existing surveillance systems, and staff opinion at CDC, but the methodology is not specifically described. These figures were used by Roberts (305) to determine costs. A recent estimate by Mead et al. (307) puts the number of total cases at 16 million, most of them of unknown origin.

B.Number

of Deaths

The number of deaths has been difficult to determine, because there is no satisfactory reporting system to build these on. Since deaths associated with foodborne disease most

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frequently occur in aged persons in hospitals or nursing homes, at least in developed countries, death certificates are not reliable, because the cause of death usually listed is that of the major underlying disease of the patient. Bennett et al. (304) and Roberts (305) used the same ratio of deaths to cases for both known and estimated cases (e.g., 0.1% of salmonellosis cases = 1,920), whereas Todd (306) limited this rate for known case numbers only and l / 100 of this for additional estimated cases on the assumption that most of the seriously ill patients and resultant deaths would end up being known to investigators (estimated salmonellosis deaths = 32). Todd (306) admits that these values may be too conservative, but the total deaths for bacterial foodborne disease (7,041) suggested by Bennett et al. (304) and Roberts (305), seem high. In other studies in Canada (307), in Scotland (309), and in England and Wales (310,31 l), no extrapolation from known deaths to estimated deaths was considered-it was assumed that all deaths would occur in the reported cases. The high number of deaths was also recently challenged by Wilson (312). In fact, CDC has recently revised its estimates of numbers of deaths to be 5,000 each year (307). Nevertheless, foodborne disease could be a contributory factor in the deaths of cancer and AIDS patients, as well as those who have liver disease or are immunocompromised. The number of these types of deaths is much larger than statistics would indicate, but the extent of these is not even guessed at (313). Therefore, more precise ways of estimating such deaths have to be developed before the value of lives lost can be properly incorporated into a study of the economic or social impact of foodborne disease.

c.

costs

The published estimated costs for all foodborne disease in the United States range from $7.7 billion (314,315) to $10.7 billion (306); those for bacteria ranged from $5.8 (305) to $8.6 (306); and those for seven major pathogens (Salmonella, Campylobacter, E. coli 0 157:H7, S. ourem, Listeria monocytogenes, Clostridium pegringens, Toxoplasma gondii) ranged from $6.6 to $37.1 billion (316). Costs determined using the human capital approach by Todd (306) and Buzby and Roberts (316) were relatively similar for salmonellosis ($4.0 billion and $0.9-3.6 billion, respectively) and for staphylococcal intoxication ($1.5 billion and 1.2 billion, respectively), but there were differences for other diseases [e.g., campylobacteriosis-$1.6 billion by Todd (306); $0.8-5.7 billion by Buzby and Roberts (3 16)]. One reason was that Buzby and Roberts (3 16) incorporated the costs of long-term sequellae (e.g., GBs) into the overall economic assessment of campylobacteriosis as well as for E. coZi 0157 hemorrhagic colitis (hemolytic uremic syndrome) and toxoplasmosis (toxoplasmic encephalitis). Each of these adds substantially to the overall cost of foodborne disease. An estimate for foodborne disease of chemical, animal, plant, and parasitic as well as microbiological origin in Canada was 2.2 million cases (3 17). Most of these were caused by microbial infections or intoxications (1.1 million) or were of unknown origin (1.0 million). The total cost was $1.3 billion, with salmonellosis, staphylococcal intoxication, E. coli 0157:H7 infections, and C.pelfringens enteritis being the most expensive diseases. In England/Wales, costs of salmonellosis were determined on the basis of questionnaires sent to Environmental Health Departments for information on 1,482 cases (3 18). Overall costs amounted to &996,339or E789/case, excluding the value of lives lost, lossesto manufacturers, and long-term sequelae to infections. If these data are extrapolated to the 23,000 reported cases in England/Wales, costs would be E6.8 million for the public

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sector (investigating health departments, hospital, and physician costs), g9.5 million for lost production through time off work, and E1.8 million for family-related expenses for a total of E18.1 million. On the assumption that only 1 in 38 cases are reported (31 l), there would be 874,000 cases costing E688 million. If the costs per case for salmonellosis calculated by Mauskoff and French (320) (mild illness $222; moderate illness $890; severe illness $6,700) had been substituted for the above estimates of Roberts ($700), Todd ($1,348), or Sockett and Roberts (E789) (305,306,318), the losses would probably be somewhat less but more precise. When outbreak reports are made for economic purposes, therefore, it is important to include the severity of illness for each case. Even if the above examples are limited in their accuracy of case numbers and costs, they do indicate the magnitude of the social and economic impact of foodborne disease and justify considerable efforts to develop effective control measures.

XIII. CONTROL OF FOODBORNEDISEASE Control of foodborne disease can be accomplished by reducing the number of pathogens, or the amount of their toxins, to below that required to cause illness. This can be achieved through heat processing, increasing the acidity, or decreasing the water activity of the foods. Growth of pathogens, either as survivors or through recontamination, in potentially hazardous food can be prevented by appropriate packaging and storage at hot or cold temperatures. It is currently impractical to eliminate pathogens from raw materials, particularly meat, or for food workers to prepare food under sterile conditions. The traditional forms of evaluating and improving control procedures are inspection of facilities and operations, microbiological testing, and education and training (320). Since foodborne disease is increasing-or at least not diminishing-throughout the world, it is clear that these approaches have been limited in their effect. In fact, Mossell (321) deplores the lack of political will to better control foodborne diseases of animal origin. There have been, however, a few specific successes. For instance, salmonellosis outbreaks associated with commercially processed precooked roast beef, well documented in the United States in the 1970s and early 1980s, are no longer significant because of regulations introduced by the USDA in 1977 (253). Also, trichinosis outbreaks have been gradually decreasing in developed countries because swine are being fed heat-treated garbage and are kept penned better with less access to wild animals, including rodents, that can transmit the parasite. Wild omnivores, such as bear, wild pig, and walrus, however, continue to be infected, and meat requires proper heat treatment (322,323). Even hemorrhagic colitis from E. coli 0157:H7 and other VTEC may be controllable through specific heat-processing regulations for meat patties (253) and a widespread educational campaign (324). In addition to raw foods of animal origin, there are, however, new problems with fruits and vegetables contaminated with Salmonella, EHEC, Shigella, Cryptosporidium pnwum, and Cyclospora cayetanensis. In some situations we cannot explain the source of the contamination. Irradiation might be considered for some types of produce (e.g., berries) since there is no other apparent effective control measure. To achieve some degree of success requires cooperation between the primary food producers, the food processors, the food-service industry, the public, and government (321). This has partly been accomplished through the implementation of the hazard analysis and critical control point method (HACCP). The HACCP system identifies hazards,

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assesses their severity and risks of illness, determines the critical control points in an operation, institutes measures to control the hazards, establishes criteria to ensure that control is achieved, monitors critical control points and records data, takes action whenever monitoring results show that the criteria are not being met, and verifies that the system is functioning as planned (325). One of therecommendations of the Richmond Committee (12), written in response to the S. enteritidis egg-associated outbreaks, was the use of HACCP. HACCP has also been recommended to control cholera in the Americas (326) and for the seafood industry in the United States (327). In addition to adopting HACCP, it is important to understand why foodborne disease occurs and develop simple operating procedures that will reduce the opportunities for contamination and microbial survival and growth. To this end, the education and training of food workers in all food operations are essential (328-330). Persons especially at risk (e.g., AIDS patients) should be counseled about specific foods that should be avoided, such as raw or semi-cooked products (331). When it is known that certain control procedures become cost-effective, there may be more enthusiasm to adopt them. Curtin and Krystynak (307) determined that the economic benefits would exceed costs for education, sanitation, food irradiation, and Sdmorzella-free feed for control of Salnlonella in poultry in Canada. Yule et al. (308) also stated that food irradiation of poultry was economic in Scotland for preventing salmonellosis, as did the USDA in its submission for approval of this process in the United States (332). Thus, a process like irradiation, with potential for eliminating pathogens from foods, could be effective in reducing the number of enteric foodborne diseases.

XIV.

CONCLUSION

Although the socioeconomic impact of foodborne diseases is very high, there are limited effective control measures to reduce them, even in industrialized countries. One reason that control is difficult to achieve is because surveillance is inadequate and the burden of foodborne disease is not fully understood by policy makers. Another reason is that a consistent and coordinated effort by industry and government is required. With increasing world trade and travel, improvement of surveillance on a worldwide basis is all the more important. In both the United States and the United Kingdom, a sentinel general practitioner study is underway to find out the nature and causes of gastroenteritis. Both epidemiological and laboratory components are being incorporated into these programs. These initiatives should stimulate other countries to conduct appropriate surveillance programs, so that the real burden of foodborne disease can be determined at various national levels. If it is now being recognized as a major concern in industrialized countries, how much more of a problem is it in countries where urban growth is faster than the public health infrastructure can support and in rural areas where drinking water is frequently contaminated? The extent of the problem is not known because surveillance programs have linlited financial resources, often depending on voluntary work by health officials (333). However, networks have recently been developed in Latin America (334,333, and developing nations in other parts of the world need to take similar steps. Cholera epidemics would be more controllable through a good surveillance system coupled with effective educational programs. This issue is becoming increasingly important now that immigrants from developing countries are becoming more frequent, international travel is commonplace, and trade barriers between blocks of countries are coming down. International organizations, such as WHO and FAO, need to take a lead role in accomplishing better surveillance for

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both industrialized and developing countries by building on the existing expertise and insisting on the required funding to accomplish this. Since risk assessments, used to prioritize programs and to be consistent with GATT, are dependent on good outbreak data, food surveys, and population demographics, all countries have to be involved. The following need to be considered to assist in control measures (336): l. The necessity to detect specific diseases of contemporary importance and identify the factors that contribute to the occurrence of these diseases. 2. The rational use of surveillance and research data on specific etiological agents for the development of food-safety programs. 3. The implementation of HACCP systems for the safe production of a food item. To be effective this requires a proper hazard analysis of foods in each process, and this in turn depends on surveillance data on the likelihood of etiological agents being present in foods in sufficient quantity to cause illness. 4. The condensing of information from surveillance reports, research on ecology of microorganisms, and experience with hazard analyses of foods into educational material for health professionals, food operators, homen~akers,and schoolchildren. In addition to these, agencies given responsibilities for monitoring and controlling foodborne diseases must be organized in such a way that they can be effective. They need upto-date information on specific pathogens, guidelines to follow when an outbreak occurs, and the ability to cooperate with other government agencies as well as industry. To this end, a single food-inspection agency within one department has been established in Canada and is being considered in the United Kingdom and the United States. All the European countries arenow aware of the importance of surveillance of foodborne diseases and have made at least some contribution to the WHO Surveillance Programme for the Control of Foodborne Infections and Intoxications in Europe (1,337). This commitment is expected to increase over the next few years, partly because of the establishment of a single market in the European Economic Community in 1993. This single market allows a food product imported by one country to be sent to any of the other member states without further inspection (338). Therefore, a unified approach to food safety is essential in such an arrangement. This would be equally important for Canada, the United States, and Mexico in the North American Free Trade Association. In conclusion, it is clear that foodborne disease will not decrease in the foreseeable future without a major commitment to improve surveillance and develop effective control measures from all countries and also from international organizations.

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287. Caprioli, A., Edefonti, A., Bacchini, M., Luzzi. I., Rosmini, F., Gianviti, A., Matteucci, M. C., and Pasquini, P. (1990). Isolation in Italy of a verotoxin-producing strain of Escherichia coli 0157:H7 from a child with haemolytic uraemic syndrome. Eur. J. Epidenziol., 6:102104. 288. Oboegbulem, S. I., and Jones, R. B. (1992). Case-control study of poultry meatborne Salmonella infections. In Proc. 3rd World Congr. Foodborne Infections and Into.xications, June 26-19, 1992, Berlin, Instit. Vet. Med., Berlin, p. 60. 289. Fisher, I. S. T. (1997). Salnlonella enteritidis and S. typlzirnurium in Western Europe for 1993- 1995: A surveillance report from Salm-Net. Eurosunyeillarzce, 2( 1):4-6. 290. Blaser, M. J., Wells, J. G., Feldman, R. A., Pollard, R. A., Allen, J. R., and the Collaborative Diarrheal Disease Study Group. (1983). Campylobacter enteritis in the United States. A multicenter study. Ann. Intenz. Med., 98:360-365. 291. Johnson, K. E., Nolan, C. M., and the Canzpylobacter Laboratory Surveillance Group. (1985). Community-wide surveillance of Campylobacterjejuni infection. Evaluation of a laboratorybased method. Diagn. Microbiol. ITEfect. Dis.. 3:389-396. 292. Harris, N. V., Weiss, N. S., and Nolan, C. M. (1986). The role of poultry and meats in the etiology of Campylobacter.jejuni/coli enteritis. Am. J. Public Health, 76:407-411. 293. Deming, M. S., Tauxe, R. V., Blake. P. A.. Dixon, S. E., Fowler, B. S., Jones, S., Lockamy, E. A., Patton, C. M., and Sikes, R. 0. (1987). Cavzpylobacter enteritis at a university: Transmission from eating chicken and from cats. A m J. Epidenziol., 126:526-534. 294. Kendal. E. J. C., and Tanner, E. I. (1982). Campylobacter enteritis in general practice. J. HjJg. C U W I ~881155-163. ., 295. USDA. (1998). Report to Congress. FoodNet: An active surveillance system for bacterial foodborne disease in the United States. (http://u,ww.fsis.usda.gov/OPHS/rpcong97/ test.Iztm ) . 296. Schuchat, A., Deaver, K. A., Wenger, J. D., Plikaytis, B. D., Mascola, L., Pinner, R. W., Reingold, A. L., Broome. C. V., andthe Listeria study group. (1992). Role of foods in sporadic listeriosis. 1. Case-control study of dietary risk factors. J. An?. Med. Assoc., 267: 204 1-2045. 297. Pinner, R. W., Schuchat, A., Swaminathan, B., Hayes, P. S., Deaver, K. A., Weaver, R. E., Plikaytis, B. D., Reeves, M., Broome, C. V., Wenger, J. D., and the Listeria study group. (1992). Role of foods in sporadic listeriosis. 2. Microbiologic and epidemiologic investigation. J. Am. Med. Assoc., 3672046-2050. 298. Alter, M. J., Mares, A., Hadler, S. C., and Maynard, J. E. (1987). The effect of underreporting on the apparent incidence and epidemiology of acute viral hepatitis. Awl. J. Epidemiol., 125: 133-139. 299. MacDonald, K. L., Cohen, M. L., Hargrett-Bean, N. T., Wells, J. G., Puhr, N. D., Collin, S. F., and Blake, P. A. (1987). Changes in antimicrobial resistance of Salmonella isolated from humans in the United States. J. Anz. Med. Assoc.. 258:1496-1499. 300. Tauxe, R. V., Puhr, N. D.. Wells, J. G., Hargrett-Bean, N., and Blake, P. A. (1990). Antimicrobial resistance of Shigella isolates in the USA: The importance of international travelers. J. Infect. Dis., 1621107-1 11 1. 301. CAST Task Force. (1994). Foodbonze Pathogens: Risks and Consequences (P. M. Foegeding, and T. Roberts, eds.), Report No. 122. Council for Agricultural Science and Technology, Ames, IA. 302. Hauschild, A. H. W., and Blyan, F. L. (1980). Estimate of cases of food- and waterborne illness in Canada and the United States. J. Food Prot., 43:435-440. 303. Chalker, R. B., and Blaser, M. J. (1988). A review of human salmonellosis: 111. Magnitude of Salnzonella infection in the United States. Rev. Infect. Dis., 1O:lll-124. 304. Bennett, J. V., Holmberg, S. D., Rogers, M. F., and Soloman, S. L. (1987). Infectious and parasitic diseases. In Closing the Gap: The Burden of Unnecessaiy Illness (R. W. Amler and H. B. Dull, eds.), Oxford Univ. Press, New York, pp. 102-1 14.

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21 Investigating Foodborne Disease Dale L. Morse and Guthrie S. Birkhead New York State Department of Health, Albany, New York

Jack J. Guzewich U.S. Food and Drug Administration, Washington, D.C.

I. Introduction 587 11. Historical Perspective 588 111. Definition and Types of Foodborne Outbreaks 589 IV. Goals of Foodborne Outbreak Investigations 595 V. Surveillance and Reporting 597 VI. Outbreak Investigations 598 A. B. C. D. E.

Steps and components of an investigation 598 Epidemiological components 599 Environmental components 6 12 Laboratory components 616 Synthesis of epidemiological, environmental, and laboratory components

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Appendices 620 References 640

1.

INTRODUCTION

Foodborne illness causes considerable morbidity and associated losses of work and functional activity. In the United States from 1988 to 1992, 2423 outbreaks of foodbome disease with 77,373 cases of illness were reported to the Centers for Disease Control (CDC) (1). However, there are wide discrepancies in reporting of outbreaks, and the number of reported cases represents only a fraction of the actual cases that occur. In addition, an undetermined but substantial number of nationally notifiable cases of salmonellosis, shigellosis, and other enteric diseases are also foodbome, as are a number of milder unreported sporadic illnesses. Worldwide, diarrheal illnesses are the leading cause of childhood death and the second leading cause of death in general, behind cardiovascular diseases (2). While foodborne illnesses are of paramount importance as a contributor to diarrheal illness in developing counties, they also significantly affect the developed ones. The World Health 587

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Organization (WHO) estimated that from 1986 to 1989 foodborne diseases were the second-largest cause of morbidity in Europe, only second in importance to respiratory infections (3). In the United States, the number of cases of food-borne-related illness have been estimated at 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths each year (4). In 1997 the U.S. Department of Agriculture estimated the U.S. medical costs and productivity losses for seven food pathogens to range between $6.5 and $34.9 billion annually (5). Improvements in our knowledge and control of foodborne disease are dependent on surveillance and investigation of individual cases and outbreaks as they occur. These investigations need to be thorough, timely and include collection of sufficient epidemiological, laboratory, and environmental data to identify causes and target points at which preventive action can be taken. This chapter summarizes methods and procedures that can be used for investigating foodborne disease outbreaks.

II. HISTORICALPERSPECTIVE I am poured out like water, and all my bones are out of joint: my heart is like wax: 22:14 it is melted in the midst of my bowels.-Psalms

As the above quote illustrates, gastrointestinal illness has been around for a long time. Furthermore, since biblical times, food, particularly meat, has been recognized as a potential source of illness (6). In Leviticus 11:39, Moses commanded the Israelites not to eat meat from animals afflicted with wasting diseases. Both the Egyptians and Phoenicians had codes regarding the use and consumption of meat products. Both the Old and New Testaments told of the need for hand washing after touching anything unclean, particularly before meals. Such early philosophers and writers as Hippocrates, Euripides, and Ovid described foodborne illnesses occurring following the consumption of poisonous plants, fungi, and herbs. Xenophon (434-354 B.c.) described a food poisoning outbreak among a Greek army of 10,000 after they reached the Black Sea (7): The soldiers camped in some mountain villages which they found well stocked with food and honey and observed a great number of beehives. All of the soldiers who ate of the honey became delirious and suffered vomiting and diarrhea. Those who had eaten much of the honey behaved like mad people. Some of these died.

Since then, we have learned that poison can appear in honey derived from certain nectarbearing plants. The distribution of poisonous honey is closely related to that of poisonous rhododendron species. Of interest, the toxin from honey is called andromedotoxin because it commonly is found in Andromedu, a genus of rhododendron (7). Numerous episodes of intentional poisoning of food were described in the Roman and Greek times and in the Middle Ages and led to the use of food tasters by persons in power, as a protective measure. The Middle Ages saw widespread adulteration of food during its preparation, but, prior to microbial theories, food-related illnesses were attributed to other causes. By the late 1700s, ergotism, the most common form of fungal poisoning, was known to be caused by a fungal growth on cereals, primarily rye. The scale and severity of illness was extreme as reflected in the 10 epidemics recorded in Russia from

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1832 to 1864, which affected thousands and had a mortality rate of 42% (8). In the early nineteenth century, investigators felt food poisoning was due to the presence of chemical poisons. This was later revised as being due to putrefactive alkaloids from putrid and decomposing food. In 1872, Francesco Selmi called these alkaloids ptomaines, thus leading to the phrase ptomaine food poisoning, which took several years to discredit (though the term lives on). Some of the first investigations linking bacteria to foodborne disease were conducted by Johne in 1884 and Gaertner in 1888, who isolated bacilli from ill persons and meat associated with illness (6). This led to rapid descriptions of bacterial food poisoning, some of which were described in the 1920 book Food Poisoning and Food Zn$ections, which listed food-poisoning outbreaks in Britain from 1878 to 1918 (9). Polio appears to be the first virus recognized to be transmitted through food and was described in 1914 (10). Knowledge of the role of microorganisms has led to large reductions, but not elimination, of foodborne disease. Unfortunately, while the means to prevent microbial contamination are known, they are not always applied. Also, while introduction of such practices as pasteurization of milk have reduced the risk of foodborne illness greatly, such mechanization sometimes resulted in illness on a larger scale. Examples were the 1982 multistage outbreak of yersiniosis caused by pasteurized milk (1 1) and the 1994 outbreak of Salmonella enteritidis-related illness affecting an estimated 224,000 persons, which was associated with ice cream (12). Worldwide, the move to a global economy has made it possible for contaminated food to reach people in distant lands quickly. Some examples include multiple U.S. outbreaks of staphylococcal food poisoning caused by mushrooms imported from China ( l 3), cases of cholera associated with imported frozen coconut milk from Asia (14), outbreaks of cyclosporiasis associated with raspberries from Guatemala (15,16), cases of Snlmonella agorza-related illness caused by consumption of a snack product in Israel (17), and cases of hepatitis A in schoolchildren associated with strawberries from Mexico (18). Drug-resistant food pathogens also have emerged as a threat to humans in association with antimicrobial use on farms (19). A multidrug-resistant strain of Sulmonelln serotype Qphill-2urium (DT104 or R-type ACSSuT) emerged first in the United Kingdom in 1984 and has now spread to the United States (20). Recent acquisition of trimethoprim and fluoroquinolone resistance has been linked to veterinary use of these antibiotics (21). In addition, such newly recognized pathogens as enterohemorrhagic Escherichia coli, first recognized in 1982 (22) and subsequently linked to outbreaks via contamination of ground beef, cider, and fresh produce (23-27), continue to emerge, making constant vigilance necessary. As if the acute manifestation caused by microbial foodborne agents weren't enough, there is now increasing awareness of chronic or long-term sequelae that can develop as exemplified by Creutzfeldt-Jakob disease, associated with consumption of British beef infected with beef spongiform encephalitis (28), and Guillain-Barr6 disease, associated with Cnmpylobncter infections (29).

111.

DEFINITION AND TYPES OF FOODBORNE OUTBREAKS

Clusters or outbreaks of illness often are recognized when several persons experience similar symptoms after attendance at a common event. If the number of cases exceeds the expected or baseline number or represents an increased incidence, it also can be consid-

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ered an epidemic. Although airborne, person-to-person contact, waterborne, and foodborne routes of transmission need to be considered in these situations, a foodborne etiology often is suspected and needs to be ruled out if food was served. This is especially true in situations in which gastrointestinal symptoms predominate. Keeping a high level of suspicion and a low threshold for classifying outbreaks as of potential or suspected foodborne origin increases sensitivity but decreases specificity of detecting outbreaks. As a result, the number of outbreaks in which food is suspected and investigated as a source is far greater than the actual number confirmed. In New York State, for example, only 20% of initial suspected foodborne outbreaks are eventually confirmed (30). The CDC and Council of State and Territorial Epidemiologists have defined a confirmed foodborne outbreak as an incident in which (a) two or more persons experience a similar illness after ingestion of a common food, and (b) epidemiological analysis implicates the food as the source of illness (1). In the past, exceptions to this included a single case of botulism or chemical poisoning (3 1). Clinical and laboratory criteria for diagnosis depend on the etiological agent (see Appendix A) (32-34). Criteria for confirming that a particular food item is responsible for illness include (33): 1. Showing a statistically significant association between illness and consumption of the food 2. Demonstration of a dose-response relationship between the amount of food consumed and the risk of illness 3. Isolation of the etiological agent for illness from the food 4. Hazard analysis demonstrating obvious contamination and time/temperature abuse of an epidemiologically incriminated food Foodborne illnesses can be classified further as to types of causative agent, types of vehicle, and types of environmental source. Some examples of classifications in use are listed in Tables 1, 2, and 3.

Table 1 Types of Causative Agents Ref. ~~

35

~~~

Bacteria and their toxins Viruses Protozoans Helminths (intestinal worms) Poisonous fungi Toxic chemicals Poisonous plants Toxic animal products 1 (CDC guidelines) Bacterial Chemical Parasitic Viral Unknown

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Table 2 Classificationa of Vehicles of Foodborne Diseases General category Meat

Subcategory Beef

Pork

Process Raw Cooked, thin cut Cooked, thick cut Cooked, sliced Ground, thin Ground, thick Cured Dried Other RawIDried Cooked, thin cut Cooked, thick cut Cooked, sliced Ground, raw Ground, cooked Cured

Other red meats (e.g., lamb, mutton, goat, venison, wild game)

Organ meats Meat mixtures

Poultry

Fermented 0ther Raw Cooked, thin cut Cooked, thick cut Ground Dried Cooked Baked BoiledISimmered

Chicken

Raw Salads containing meatlpoultry Pasta containing meat Meat-filled pastry Other Whole, cooked

Duck

Portions, cooked Fabricated, cooked Cooked, sliced Cooked

Goose

Cooked

Examples Steak tartare Steak, veal cutlet Roast beef, prime rib Some luncheon meats Hamburger, meatballs, tacos, beef burrito Gyro, meat loaf Corned beef Jerky Prosciutto, cappicolo Chop Roast pork Some luncheon meats Hot dog, sausage, meatballs Ham, some luncheon meats, bacon Genoa salami Raw meat (other than beef or pork) Lamb chop Roast lamb Sausage Jerky Liver, heart, kidney, liver p'2te Meat pies, casseroles Meat stew, meat soups, chili Kibbe (Lebanese) Ham salad Lasagna, pizza with sausage, ravioli Cornish pastry Broiler. stewed or roasted chicken Wings, breasts, legs Nuggets, rolls Some luncheon meats Duck, pressed duck, Peking duck Goose, goose-liver pgt6

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592 Table 2 Continued General category

Subcategory Turkey

Cornish hen Game birds Other poultry Organ meats Poultry mixtures

Seafoods

Fin fish

Process Whole, cooked Parts, cooked Fabricated, cooked Cooked, sliced Cooked Cooked Cooked Baked Boiled/Simmered Salads containing meat/poultry Other Raw Cooked Smoked Retorted Acidified Salads Liquid mixtures

Fish eggs Shellfish

Crustaceans

Other seafoods

Marine mammals

Salted Salted Fermented eggs Raw Cooked Smoked Acidified Raw Cooked Raw Raw, acidified Cooked

Soups containing seafoods Salads containing seafoods Raw Cooked Fermented

Examples Roasted or smoked turkey Drumsticks Rolls, roasts Some luncheon meats Cornish hen Pheasant, quail Liver. gizzards, hearts Poultry pies, casseroles Chicken stew, poultry soups ChickedTurkey salad

Sushi, sashimi Fried/Baked/Broiled fish Smoked white fish, mullet, lox Tuna, sardines, salmon Herring Tuna salad Fish soups, chowders, gumbo, stews Kapchunka Caviar Salmon “stink” eggs Oysters, clams, mussels Fried/Baked/Broiled clams, mussels Oysters Oysters Small shrimp Shrimp, prawns, lobster, crabs, crayfish Ceviche Scallops, squid, urchins, octopus, sea cucumbers Gumbos, chowders Surimi salad, tuna salad, salmon salad Blubber Seal, whale meat Muktuk

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Table 2 Continued ~~

General category Eggs

Subcategory Eggs

Process Fresh Fresh blended Baked Frozen foods Dried Toppings Fillings containing eggs Combined

Salads containing eggs Salad dressings containing eggs Sauces containing eggs Miscellaneous foods containing eggs

Dairy products

Milk

Butter Ice cream/Milk Cream filling Cheese

Yogurt Produce

Leafy vegetables

Raw Pasteurized Dried Formulated products containing milk Salted/churned Butter blends Milk-based Other Milk-based Made of raw milk Made of pasteurized milk Soft Hard Mold-types Spreads Fermented Frozen Raw Cooked Fermented Cut/mixed

Examples Fried, soft boiled Scrambled, batters, French toast Lasagna containing eggs, quiche Egg-containing ice cream Foods containing dried eggs Meringue Eclairs, custard-filled Pastry Custard, omelets, cream filling, egg nog, tiramisu Egg salad Low-acid mayonnaise, Caesar's dressing Bernaise sauce, hollandaise sauce Seafood stuffing, rice balls, egg noodles, egg-pasta shells, tortelloni Raw milk Pasteurized milk Dry milk Infant formula, diet beverages Butter Whipped butter Ice cream, ice milk Cream-filled pastry Cheese, raw types Cheese, pasteurized types Camembert, Brie Cheddar Blue Cheese spreads Yogurt Frozen yogurt Lettuce, cabbage, cole slaw Spinach, turnip greens Sauerkraut Green salad, relish tray, cole slaw, salsa

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_

_

_

~

General category

~

Subcategory Root vegetables

Process Raw Cooked

Vine-stock-attached vegetables

Canned Raw Cooked

Canned Sprouts

Raw

Fruits

Raw

Juice Cooked Cut/Mixed Dried Melons

Raw

Nuts

Dressings

Dried Cooked Dried seeds, roots, leaves, flowers Steamed/boiled Fried Cooked

Desserts

Baked

Pasta (without meat/ eggs)

Boiled

Spices Cereals, pasta

Rice

Salads containing pasta Fungi

Sauces, salad dressings

Other cereal products Mushrooms

Raw wild Domestic Cooked High acid Low acid

Examples Potatoes, carrots, garlic, onion Mashed/Fried/Boiled/ Baked potatoes, potat0 salad Potatoes, carrots, beets Tomatoes, cucumbers, pimentos Beans, chick peas, squash, corn, bean burrito, corn, tofu Green beans, asparagus, peas Bean sprouts, alfalfa sprouts Oranges, pears, apples, berries, grapes, cherries Apple cider, orange juice Cobbler, pastry containing fruit Fruit salad Raisins, apricots, prunes Watermelon, honeydew, cantaloupe Peanuts, coconut Boiled peanuts Pepper, parsley White rice Fried rice Turkey stuffing, seafood stuffing Cakes/Cookies with low water activity toppings or fillings Spaghetti, macaroni, linguini, fettucini Macaroni salad

Wild mushroom Mushroom salad Sauteed mushrooms Mayonnaise, mustard, piccata Gravy

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Table 2 Continued General category Confectionery, highsugar

Subcategory Candy, syrup

Process Raw Cooked/Sweetened Combined

Beverages (other than water and milk)

Soft drinks Alcohol Water base

Mixed dishesh or platesh with no particular dominant ingredient

Acidified/Carbonated Fermented Distilled With ice Heated Cooked Chinese foods

Mexican foods

Multiple foods’ Pizza Epidemiologically implicated Other‘ Foods implicated, but do not fit into any of the above categories Unknown; an implicated food was not identified

Examples Honey Chocolate candy, hard candy, syrup Nondairy cream topping, icing Cola, root beer Wine, beer Moonshine whiskey Iced tea Coffee/hot tea Casseroles Chop suey, Chinese food combination plates Beans-ground meatrice combination plate Pizza

Drop categories that are infrequently reported and combine and list as “others” category; add category for foods commonly cited as vehicles but not included in the listing; for food combinations (e.g., soups, casseroles) classify under category of most significant food (e.g., tuna under seafood, fin fish, cooked). h Multiple foods are seldom the vehicle unless the same significant ingredientis incorporated into each or it is contaminated by the same person or processedon the same equipment and/or receivedthe same time-temperature abuse, but attack rates sometimes show similar statistically significant rates. Some multiple foods contain items that are frequently served together (e.g., spaghetti and meat balls; beans and ground meat in chili). If investigations fail to determine which of the two was the responsible vehicle, both would be listed in this category. c Cite the foods in the footnotes that fall in the “Other” category. Source: Ref. 36.

W. GOALS OF FOODBORNE OUTBREAK INVESTIGATIONS Public health officials have been given the responsibility of investigating suspected foodborne outbreaks. The primary goals in conducting such an investigation are as follows (33,35,37,38): 1. To determine the cause(s) of the outbreak. The determination includes the identification of not only the etiological agent but also the vehicle and means of transmission. For example, an outbreak of gastroenteritis might be identified as being due to SaZrnoneZZa enteritidis (agent) occurring as a result of ingestion of a casserole containing eggs (vehicle) that had been undercooked (means). 2. To prevent further cases of illness. Once the responsible food has been identified, additional cases can be prevented by the removal or proper disposal of the implicated

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596 Table 3 Factors That Contribute to Foodborne Disease Incidentsa Category Contamination

Survival/Lack of inactivation

Proliferation/Amplification

(Code)

Factorsb Toxic substance part of tissue Poisonous substance intentionally added Poisonous or physical substance accidentally/incidentally added Addition of excessive quantities of ingredients that are toxic in these situations Toxic container or pipelines Raw product/ingredient contaminated by pathogens from animal or environment Ingestion of contaminated raw products Obtaining foods from polluted sources Cross-contamination from raw ingredient of animal origin Bare-hand contact by handler/ worker/preparer Handling by intestinal carrier Inadequate cleaning of processing/preparation equipment/utensils Storage in contaminated environment Other source of contamination' Insufficient time and/or temperature during cooking/heat processing Insufficient time and/or temperature during reheating Inadequate acidification Insufficient thawing (followed by insufficient cooking) Other process failuresc Allowing foods to remain at room or warm outdoor temperature for several hours Slow cooling Inadequate cold-holding temperature Preparing foods a half day or more before serving Prolonged cold storage for several weeks Insufficient time and/or temperature during hot holding

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Table 3 Continued ~

Category

(Code) (P7) (P8) (P9) (P10) (P1 1) (P12)

~~

Factors Insufficient acidification Insufficiently low water activity Inadequate thawing of frozen products Anaerobic packaging/Modified atmosphere Inadequate fermentation Other situations that promoted or allowed microbial growth or toxic production'

Consider tabulating these factors for each major etiologic agent. ofplace mishandling (e.g., food processing. food service, homes) and total incidents. Numbers may exceed 100% because multiple factors frequently occur in any one outbreak. c Cite the contributory factors in the footnotes that fall in the "Other" categories. Source: Ref. 36. a

food(s), through the appropriate isolation and treatment of ill persons (and food workers, if any), and by corrections in any contributing food-handling practices. For example, if a casserole was the vehicle, any leftovers could be destroyed and containers properly cleaned. Persons with salmonellosis could be followed medically and temporarily restricted from high-risk activities (e.g., food handling, day care, patient care) in which secondary transmission is possible. Circumstances leading to the undercooking of the casserole should be reviewed and appropriate cooking practices implemented. 3. To preventfutureoutbrmks. The circumstances of the individual outbreak should be reviewed further to see whether there is a common feature that requires more widespread action. If a newly recognized agent, vehicle, or preparation procedure was involved, steps may need to be taken to warn others of this hazard and to eliminate the problem. For example, the emergence of S. enteritidis as a contaminant in Grade A shell eggs has required further steps beyond an emphasis on the need for thorough cooking. These steps have included testing of flocks to remove contamination at the source and substitution of pasteurized eggs for whole shell eggs.

V. SURVEILLANCEANDREPORTING Epidemiologic surveillance is the ongoing and systematic collection, analysis, and interpretation of health data in the process of describing and monitoring a health event. This information is used for planning, implementing, and evaluating public health interventions and programs. Surveillance data are used both to determine the need for public health action and to assess the effectiveness of programs (39).

The public health significance of a health event under surveillance is dependent on its magnitude (number of cases, incidence, and prevalence), severity (mortality rate, casefatality rate), and preventability. As discussed in Refs. 1, 2, and 6, a substantial number of foodborne outbreaks causing high morbidity rates almost always are preventable. While

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case-fatality rates are usually low, there are noted exceptions depending on the agent (e.g., botulism) and population affected (e.g., elderly, young, immunocompromised, malnourished). These factors make foodborne disease health events worthy of ongoing surveillance. Foodborne disease reporting began in the United States in the early 1900s in response to concerns about typhoid fever and infant diarrhea. Summaries of outbreaks of gastrointestinal illness attributable to milk began to be published by the Public Health Service in 1923, with summaries of outbreaks caused by other foods added in 1938. These reports served as the basis for a wide range of public health actions throughout this century (1). Today, all states investigate foodborne outbreaks and 39 have mandatory reporting (40). These reports are submitted to state health agencies by physicians, laboratories, health-care facilities, and local health units and then are sent to the CDC using a standard format. Although states have adopted the CDC’s standard case definitions, there are wide discrepancies in the level of surveillance and reporting. For example, from 1988 to 1992, New York State and Washington State reported 32% (774 of 2423) of U.S. foodborne illness outbreaks despite these areas having less than 10% of the population (1). Foodborne illness outbreaks are classified further as to etiological agent, vehicle, site, month of onset, and contributing factors. Despite limitations in quality, quantity, timeliness, and standardization of data, these reports have provided a wealth of information for control efforts. For outbreaks with known etiology, bacterial pathogens (66%) and chemical agents (26%) have been the largest contributors. However, while viral outbreaks contributed only 5%, studies of outbreaks with unknown etiology have shown us that viruses are often the cause (41). The extent to which viruses may contribute to foodborne illness was recently demonstrated by a CDC laboratory study of isolates from 90 outbreaks of nonbacterial gastroenteritis from 33 state health departments. Polymerase chain reaction (PCR) testing showed that 96% were due to “Norwalk-like viruses” (42). For the 5 1 outbreaks where the source of transmission was sought and reported, 47% were food related, 20% person-to-person, 6% waterborne, and 27% undetermined. While surveillance data have been invaluable for control at the local level, they have also shown such national trends as the large number of clam-associated Norwalk-like (43), egg-associated S. enteritidis (44) and hamburger-, cider-, and produce-associated E. coli 0157:H7 (2327) outbreaks in the 1980s, and 1990s.

VI.

OUTBREAK INVESTIGATIONS

A.

Steps and Components of an Investigation

Once a suspected outbreak of foodborne illness has been reported, a series of steps are taken. While described differently by different authors, these steps usually include certain basic elements, including: Receipt of initial data Verification of the diagnosis Determination of whether an outbreak has occurred Search for additional data and cases Description of cases in terms of time, place, and person Formulation of hypotheses

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Further analytical epidemiological, environmental, and laboratory studies Synthesis of findings with conclusions and recommendations Control measures Written reports These steps include epidemiological components to implicate a vehicle of transmission, laboratory components to identify the causative agent of infection, and environmental components to determine the circumstances that led to contamination. These components often must proceed simultaneously, but ina coordinated fashion, because information from one component may be critical to another. These components ideally involve personnel with specialized training in the areas of epidemiology, laboratory methods, and sanitary food preparation. In practice, the epidemiological and environmental investigations often are conducted by several people working closely together, but they are sometimes carried out by the same person, often a food sanitarian with epidemiological training. The laboratory components, by the nature of the expertise required, must be carried out by an experienced laboratory, often based in a public health department.

B. EpidemiologicalComponents 1. Introduction The epidemiological components provide the foundation for the entire investigation. Without epidemiological evidence linking ill persons with a possible vehicle of transmission, laboratory and environmental data often are not conclusive. There are both a science and an art to the investigation of foodborne illness outbreaks. For instance, logistical difficulties often are encountered in trying to interview and obtain valid information from ill and well individuals. The success of the investigation often will depend on how innovative the investigators are in developing field expedients to address these difficulties. At the same time, scientific rigor in the conduct of the epidemiological, laboratory, and environmental investigations is necessary to ensure that the correct vehicle of infection is identified and that spurious results can be minimized. An epidemiological investigation includes an initial assessment, planning, collection of data, and descriptive and analytic studies as described below.

2. Initial Assessment Initially, foodborne outbreaks often are brought to attention by a phone call from an ill individual or from a health-care provider who suspects that an outbreak has occurred. (Appendix B contains a sample of a New York State form used to record initial complaint data.) Less frequently, outbreaks are suspected from the review of sporadic case surveillance data gathered at public health departments or from other sources. The recent improvements in molecular diagnostic methods, such as pulsed field gel electrophoresis (PFGE), has led to anincreasing proportion of outbreaks being identified first in thelaboratory. The initial step in any investigation is to verify the diagnosis or to confirm that illness has occurred. This is followed by an assessment of whether there is an outbreak and, if so, its potential magnitude and importance. This initial assessment leads to a decision on whether to mount a further investigation and how vigorous that investigation should be. The factors to consider include the likelihood that a true cluster of cases of the same illness is present. This may be evident immediately in a large, common-source foodborne

600

et

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outbreak, or it may be more subtle in community outbreaks due to person-to-person transmission. In the latter case, the baseline rate of disease must be known and statistical techniques may need to be applied to determine whether there has been a significant increase over the baseline (45). If the symptoms of the initial case reports vary in their description and time and place of onset or several different agents have been identified, an etiologically related cluster may not have occurred. Additional factors to consider in the decision to initiate a full investigation include: The number of people possibly affected The severity of the suspected disease How recently the illnesses occurred The potential for continued disease transmission The potential to investigate a new agent, vehicle, or setting of infection The information obtained from the persons initially reported as ill is often important in determining the direction the investigation should take. The clinical symptoms will suggest the type of agent involved, and initial laboratory specimens already may have identified the agent. Initial cases also may provide important clues to identify a common source, the groups or cohorts of people exposed, and areas to target. At this initial stage, it is important to employ open-ended questions so as not to limit unnecessarily the focus of the investigation. Later in the investigation, the information gathered can be streamlined to that deemed important based on initial interviews. In the early stages of an outbreak investigation, it may be useful to survey the major providers of acute or emergency medical care serving the likely area where ill persons are located. Providers include emergency departments, physicians or physician groups, and laboratories. Surveillance for additional outbreak-related cases should be maintained at these sites throughout the investigation. In addition, persons named in initial case interviews should be contacted. In general, information gathered from initial cases should be placed in a “line list” format with ample space for adding new categories and comments. (Appendix C is a sample line list form used by the New York State Department of Health.) Later in the full investigation, information on both ill and well persons can continue to be recorded on a line list if the number of persons is fewer than 50 and they can be interviewed individually; otherwise, individual questionnaires can be handed or mailed to respondents. Data analysis for more than 50 respondents is handled most efficiently by computerization. 3. Planning the Investigation The epidemiological methods to be employed need to be considered early in the investigation. The cohort method is often used in foodborne outbreak investigations in which the whole group of exposed individuals is known; for example, all persons attending a wedding party or church supper at which a foodborne outbreak occurs constitute the cohort of persons at risk for illness. The investigation then proceeds by questioning all cohort members about illness and food consumption. In situations in which the cohort is too large, a random sample can be interviewed, in which case a modified cohort analysis still can be employed. In situations in which the cohort is not easily identifiable, for instance patrons at a busy restaurant, alternate methods such as a case-control study can be employed. The determination of which persons to interview usually will depend on the circum-

Foodborne

investigating

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stances of the outbreak and the characteristics of the group of ill or exposed individuals. For example, in an outbreak among attendees at a wedding luncheon, the list of attendees would allow the interview of the entire cohort with a standard questionnaire. In some situations, use of alternative data sources may be advisable. For instance, in an outbreak in a nursing home at which patients may not be able to provide reliable information, questionnaires may be completed by review of medical charts for symptom information and dietary records for food-consumption data. In other cases, it may be useful to focus the investigation on persons likely to be able to provide high-quality data. For example, employees in a nursing home or teachers in an elementary school may be better sources of information and certainly are easier to interview than patients or students. It also may be useful to question persons who are not typical members of the cohort or whose exposure to the outbreak setting was limited. Such persons may provide valuable clues to the outbreak vehicle. For example, in a foodborne outbreak of typhoid fever among guests at a resort hotel, several ill persons had eaten only one meal at the hotel (46). Interview of these sentinel persons served to focus the investigation on a specific time period and on certain food items that were the only ones eaten by those persons. 4.

Collection of Data

a. Types of Questiorulnires. The type of questionnaire used depends on the population and the method of administration. 1. Expanded line list. This method can be used if respondents will be questioned individually by an interviewer. 2. Written questionnaire, on-site administration. This method typically is used if a discrete cohort is available in one place (school class). Such questionnaires can be administered by interviewer or self-administered, depending on the size of the cohort and availability of interviewers. The makeup of the questionnaire obviously will differ depending on the situation (e.g., self-explanatory questions for self-administration). Even in the case of self-administered questionnaires, it often is useful to have someone available in person or by phone to answer questions. 3. Written questionnaire, mailed distribution. This method is used for larger groups that are not gathered together in one place (restaurant patrons). This type of questionnaire needs to be completely self-explanatory. A telephone number to call for questions is advisable. A second mailing to initial nonresponders, and possible phone follow-up may be necessary to assure an adequate response rate. A variation of distribution in an institutional setting is distribution via such intraoffice communication as inserting questionnaires into pay envelopes. 4. Telephone questionnaire. This method often is employed if time is a critical factor, as it often is in the midst of an outbreak and if the group of ill or exposed persons is widely scattered. In some situations, random digit dialing is used to identify controls for a case-control study. In this scenario, a list of four-digit random numbers is generated and appended to the three-digit telephone prefix of known cases to identify communitymatched controls. The success depends on the availability of interviewers and on the study population having telephones. A better response rate often is achieved if telephoning is done in the evening when respondents are at home or if work telephone numbers are available. 5. E-mailed questionnaires. Rapid advances in computerization have made it possi-

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ble to use e-mail to query certain populations. For example, we used such a method successfully to obtain information on a cohort of widely scattered corporate executives who had a common source exposure to hepatitis A at a training workshop. b. Questionnaire Spectjcs. disease outbreak includes:

Information to be gathered in a food- or waterborne

Demographic information Occupation or disease setting Symptoms and treatment 72-hour food history (may be longer for such agents as Listeria monocytogenes, E. coli 0157:H7, etc.) Additional risk factor information Medical history and laboratory findings Additional case reports A more detailed description of this information is presented below, and a sample collection form and Epi Info computer-generated questionnaire are included in Appendixes D and E. 1. Demographic information. This includes the name, address, phone number, age, race, and sex of ill and well individuals. 2. Occupation or disease setting. Foodborne outbreaks often are identified in such closed settings as school classrooms, places of employment, or among persons attending a common event. Initial information about the outbreak setting can guide the investigation and may have implications for control measures to prevent further cases regardless of the source. For instance, food workers and health-care or child day-care personnel with salmonellosis or other enterically transmitted diseases should refrain from working until infection has cleared. 3. Symptoms. Symptoms including diarrhea, fever, abdominal pain, nausea, vomiting, bloody stool, tenesmus (painful defecation), as well as the timing and sequence of the onset of these symptoms, are important pieces of information to collect. I11 persons’ suspicions about the causative food source sometimes can be helpful. Specific etiological agents often are suggested by the symptoms. For instance, sudden onset of vomiting with little diarrhea or fever suggests a staphylococcus toxin-mediated illness or other acute chemical intoxication. Fever with diarrhea may suggest such a bacterial pathogen as Salmonellcr, while bloody stools may suggest campylobacteriosis or E. Coli. 0154:H7. It is important to note that no combination of symptoms is necessarily pathognomonic, with the possible exception of the typical neurological symptoms of foodborne botulism, so that further laboratory studies to identify the pathogen always are warranted. 4. Food history. The standard practice in foodborne outbreaks is to take a detailed history of foods consumed by ill persons over the 72 hours preceding illness. This time period may be increased for agents such as Giardia, hepatitis A, or S. typhi, which have incubation periods longer than 72 hours. Using a calendar or asking the person to relate to their work schedule or other significant events may improve memory for foods consumed and their source. It is important to include water and other liquids consumed because these also can be vehicles for infection. Include “don’t know” responses. If possible, quantitate responses (number of glasses, number of servings). Do not forget items like gravies or sauces.

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5. Additional risk factor information. Factors that may relate to the risk of becoming ill include alcohol consumption, cigarette smoking, chronic medical diagnoses, medications such as antacids and steroids, or history of immunosuppression (e.g., HIV, cancer). 6. Medical history and laboratory findings. Medical history and physical findings may include fever, abdominal exam findings, and whether the stool was guaiac positive for occult blood. Laboratory test results may include Gram stain of stool, white blood cell count, and preliminary results of stool bacterial cultures. At this time, the questioner can request that stool samples be sent for bacterial culture on selective media, as well as samples of vomitus for toxin detection or stool for ova and parasite (O&P) examination and viral testing, if indicated. It is important to identify the sources of medical care for ill persons because they can coordinate the gathering of further information and provide medical follow-up. Medical providers, particularly hospital emergency departments, may be aware of other patients with similar symptoms. It may be important to inquire about this at the point of initial contact and again, as necessary, as the investigation proceeds. 7. Additional case reports. The persons interviewed are a good source of the names, addresses, and phone numbers of others who may have been exposed and affected. In particular, if an outbreak in a group is suggested, it is important to get the names and phone numbers of such key individuals as teachers, school principals, employment supervisors, or group leaders. c. Data Editing nlzd Computer Entry. Questionnaires collected from ill and well persons should be reviewed before entry into a computer. The purpose is to check for completeness of responses, to detect problems with completion or poor understanding of some questions by respondents, to make edits, and, in some cases, to code variables for computer entry. Computer entry can be done using a variety of software packages. Since keying errors can occur, double entry of data or limited verification of single-entry data should be undertaken to ensure valid data for analysis. Checking the data for outlying or inconsistent values (e.g., date of illness occurs before date of birth) is also an important mechanism to ensure valid data. 5. Descriptive Epidemiological Analyses Once questionnaire responses or line-listed data are available, the first step in the formal analysis of the outbreak is to conduct a descriptive analysis. This analysis seeks to describe the outbreak in terms of the time, place, persons, symptoms, and likely causative agent.

a. Case Dejinition. An important step in the analysis is to define a case of illness for the purposes of analysis. Case definitions can be flexible but should include a set of symptoms, usually including diarrhea, and a time frame for onset of these symptoms. Once a case definition has been defined, it should be applied uniformly for each person evaluated. Analyses can be done with case definitions modified to be more sensitive (e.g., any gastrointestinal symptom) or more specific (e.g., fever with diarrhea and abdominal pain). Measures of association with suspected food items (see below) often are stronger with a more specific case definition if the correct etiological food item has been identified. b. Symptoms. It is useful to make a frequency listing of all symptoms. Predominance of vomiting, diarrhea, fever, or bloody stools may suggest different etiological

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agents and serve as an indicator of the severity of the outbreak. Table 4 is an example of a symptom frequency table from an outbreak of Salmonella group B associated with consumption of coleslaw contaminated by a food worker during preparation. Note that diarrhea was part of the case definition and so is present in 100% of cases. Diarrhea and fever were much more common than vomiting, which is typical for a Salmonella outbreak. c. Time. The typical way to describe the time course of an epidemic is to use the datehime of onset information to draw a histogram of number of cases by time of onset (epidemic curve). Choice of time interval can be important. If in doubt, several intervals can be tried and the resultant epidemic curves examined. The pattern displayed by the epidemic curve can give an indication of whether transmission in the outbreak occurred from a point or a propagated source. The epidemic curve can point to a single case early in the outbreak who may have been the source of infection and can show later waves of cases indicating secondary transmission. Figure 1 shows an epidemic curve with a very rapid course over a period of 24 hours. This curve is froman outbreak of Salmonellcr group B associated with coleslaw that had been contaminated by an infected food worker during preparation. The sharp peak of case onsets indicates that a point source is involved and that the incubation period is very short, possibly indicating a large inoculum in the food vehicle. (Appendix A gives typical incubation periods for common foodborne pathogens.) Figure 2 shows the epidemic curve for S. eizteritidis illnesses in patrons of a restaurant. The occurrence of cases over several weeks indicates that this was a propagated outbreak. There was an ongoing source of infection in multiple ill food workers at the restaurant who continued to work although ill. Figure 3 shows the epidemic curve for culture-confirmed cases of typhoid fever in a point source associated with a resort hotel (46). The resemblance of Fig. 3 to Fig. 2 is only superficial; the typhoid case onsets are spread out in time because typhoid fever has an incubation period varying from 1 to 3 weeks, not because the outbreak was ongoing.

Table 4 Frequency of Symptoms Among I11 Persons, New York State Salmonella Outbreak #91-107 Symptom Diarrhea Loss of appetite Abdominal cramps Nausea Fever Weakness Perspiration Headache Muscle aches Chills Vomiting Dehydration Dizziness

Number of people affected

Percent affected

31 28 27 26 25 24 23 22 22 19 19 13 3

100 90 87 84 81 77 74 71 71 61 61 42 10

hvestigating Foodborne Disease

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Hour

01 OS 09 13 17 21 01 05 09 13 17 2101

Dare

20

Month July

21

22

OS 09 13 17 21 01 OS 09 13 17 21 01 05 09 13 23 24

Date of Onset

Fig. 1 Epidemic curve of Salmonella cases by date of onset, coleslaw-associated outbreak, New York, 1991.

Typically, diseases with longer incubation periods have less distinct peaks of onset over time. d. Place. Examining attack rates (number ill/group total) in various geographic subgroups of the outbreak cohort can help to focus the investigation into a productive area. Mapping cases (spot map) can also provide useful information.

s-

Fig. 2 Epidemic curve of Snlmorzelln cases by date of onset, restaurant associated outbreak, New York, 1991.

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11

10

-

-

m

9 -

‘ii

7 -

d

E

*5 4 -

3 2 1 -

Q , 1 1 l 2 1 3 1 1 l 5 l 6 1 7 I 8 ) 9 # ) 2 l 2 2 2 ~ 2 1 ~ 2 6 ~ ~I 229 3~ 4 S 6 7 8 9 1 0 1 1 1 2 1 3 1 1 151617

lune

Date of Onset

July

Fig. 3 Epidemiccurve of typhoid cases by date of onset,Hotel A, New York, 1991 (reports through August 21, 1989).

e. Person. The personal characteristics of cases (age, race, sex) and the attack rates within categories of these groups may provide additional clues to the source of the outbreak. For example, illnesses only among campers and not staff at a summer camp should help focus the search for a food source. 6. Analytical Epidemiological Studies Analytic epidemiological studies seek to determine a statistical association between certain food items and illness. The selection of the control or comparison group is a key step i n the analytic epidemiological analysis. In an investigation of illness among a cohort, the food-consumption histories (exposure) of all persons in attendance are collected and examined for association of eating certain food items with illness. Technically, this is a retrospective cohort study because the study is carried out and food-consumption (exposure) data are gathered after illness has occurred. In the analysis, relative risk (RR) estimates for illness by food consumed can be calculated directly. However, if the whole cohort of ill and nonill persons, or a random sample of them, cannot be interviewed, alternative approaches must be considered. If ill persons (cases) are known already, a control group must be selected to represent nonill persons. The key feature in the selection of members of the control group is that they be as much like the ill persons (cases) as possible, including having the opportunity or potential to have been exposed to possible vehicles of infection, but are not ill (47). Analysis of the risk of illness then is conducted in a case-control format.

a. Traditional Approach to Cohort Analysis. In the classical foodborne outbreak analysis described by Bryan (37) and others (331, questionnaire responses from ill and well persons are used to generate a food-specific attack rate table (see Appendix F). In a hypothetical example, 200 persons attended a wedding luncheon, 55 of whom (27.5%) became ill with diarrhea within 48 hours. Salmonella enteritidis was cultured from the

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Table 5 Food-Specific Attack Rate, Wedding Outbreak Example Number of persons eating food item Food item

I11

Well

Egg salad Tuna salad 70 Pasta salad

50 40 30

50

TotalI11

Percent I11

50 36 27

100 110 110

80

Number of persons not eating food item

5 15 25

Well

Total

Percent I11

Chi square

95 75 65

100 90 90

5 17 28

48.60 8.70 0.01

stool of several attendees. The food-specific attack rate for three food items is shown in Table 5. The percent of illness among persons eating and not eating each food item is calculated and compared. An implicated food item is identified by visual inspection of the data for foods with a high attack rate among consumers and a low attack rate among nonconsumers. The chi square test is used to determine the probability that the differences in attack rates by food consumption could have arisen from chance alone. This probability is expressed in a p-value. In the example, egg salad has the highest attack rate among consumers and the lowest attack rate among nonconsumers. The p-value corresponding to the chi square value of 48.6 is less than loR,making it highly unlikely that these results could have arisen by chance alone. A p-value of less than 0.05% traditionally is accepted as indicating that the result is significant statistically, although in fact it only means that the given results could have arisen by chance in less than 5% of cases. Several examples of disease outbreaks in which traditional food-specific attack rate table analyses were used can be found in Refs. 48-5 1. b. Relative Risk Approach to Cohort Analysis. The RR analysis is related to the food-specific attack rate method in that the same data used to construct a food-specific attack rate table are utilized. The calculation is visualized more easily if the data are arrayed in a 2 X 2 cross-tabular form (Table 6). The RR is calculated by dividing the rate (risk) of illness in food consumers by the rate of illness in persons not consuming the food. For example, in the interpretation of the RR of eating egg salad, persons consuming egg salad have a 10-fold increased risk of illness compared to those not eating egg salad (i.e., the RR of eating egg salad is 10).

Table 6 Format for Cohort Analysis, Wedding Outbreak Example Food

I11

Food item 1 b Ate

a

Did not eat Example: Egg salad Ate Did not eat

Well

Total a + b

C

d

c + d

50

50

100

5

95

100

Relative risk calculation

RR =

a/(a c/(c

~

+ b) + d)

RR = 50/(50 + 50) = 10.0 5/(5 + 95)

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608

In addition, the calculation of confidence intervals on the RR can be performed, providing a plausible range of values for the RR given statistical considerations (e.g., there is a 95% chance that the true increased risk associated with eating egg salad lies between 4.2 and 24 times that for persons who didn’t eat egg salad). The confidence intervals can be calculated by software packages such as Epi Info (52). A summary table for the three food items in the hypothetical outbreak is shown in Table 7. There are several desirable features of this analysis not available in the food-specific attack rate method. First, a measure of the strength of association (the relative risk) is calculated between a food and illness; for example, this might be stated as ‘‘consumers of egg salad were 10 times more likely to become ill than nonconsumers.” This provides a quantitative measure of risk that is not available in the traditional cohort analysis. The RR is a much more descriptive and understandable measure than the chi square, which says nothing about the strength of association but only the probability that this is a chance occurrence. The second advantage of the RR format is that 95% confidence intervals on the RR can be calculated, giving the investigator an idea of the range of plausible values for the risk, not just a point estimate. The RR plus confidence intervals also convey the role of probability in the results. A RR with a calculated 95% confidence interval that does not include the value 1.0 could have arisen by chance in less than 5% of cases, similar to the use of a p-value of less than 5% to indicate statistical significance. It is important to recognize that a food item could have an elevated RR of causing illness, but the confidence limits overlap 1 (or they-value isgreater than 0.05). This does not meanthat the food could not have caused the outbreak; rather, statistical significance at the somewhat arbitrary level of 5% was not achieved. Adding more respondents with similar exposure and disease characteristics eventually would result in statistical significance being achieved even though the RR may not change. Thus, the RR provides more information than the chi square value or p-value. A final advantage of the RR format is that stratified and multivariate analyses can be performed to examine the influence of several’factors on illness simultaneously or to control for other factors in examining the influence of one factor of interest. Several examples of disease outbreaks in which RR analyses were used can be found in Refs. 53-55. c. Cuse-Control Analysis. In a case-control analysis, the food-consumption and illness data are used to calculate an odds ratio that approximates the RR. In case-control studies, usually the whole cohort of potentially exposed persons has not been questioned and a group of nonill control subjects has been chosen in some manner to compare foodconsumption histories with ill cases. In the wedding luncheon example, suppose that there Table 7 Summary of Food-Specific Relative Risks, Wedding Outbreak Example ~~

item

Number of persons eating Number item food

food

Percent item Food

I11 Total Well

Egg salad Tuna salad Pasta salad

RR

=

50 40

50 70

30

80

I11

5100 110 110

Percent I11

Total Well

50 36

15

75

27

25

65

relative risk; C1 = confidence interval.

~~~

~

~~

of persons not eating

95

100 90 90

I11

RR

(95% CI)

5

10.0 2.2 1.O

(4.2-24.0) (1.3-3.7)

17 28

(0.6- 1.6)

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Table 8 Format for Case-Control Analysis, Wedding Outbreak Example Food

Case

Control

Odds ratio calculation

a

b d

OR = ad/bc

~~

Food item 1 Ate Did not eat Example: Egg salad Ate

C

Did not eat OR

=

2

16

odds ratio.

was insufficient time and personnel to interview all 200 attendees. A random sample of 25 ill cases and 25 well controls was selected for interview to facilitate a rapid analysis of the implicated food. Since the samples were drawn at random, the proportion of ill persons (cases) and well persons (controls) consuming egg salad was the same (91% and 34%, respectively) as among ill and well individuals in the cohort as a whole. Notice that since the number of controls chosen is arbitrary, there is no meaning to calculating the attack rate for each food item (percent of persons consuming a food item who became ill). Although the cross-tabular tables for each food item appear similar to those for the RR calculation, they are different qualitatively because the total in the nonill column is determined arbitrarily by the choice of the number of controls (Table 8). Because of this, the row totals and the percent of food consumers or nonconsumers who become ill (attack rate) are not calculated. Rather, the number of cases and controls (column totals) is fixed by the investigator. As with the RR, the odds ratio provides a measure of the strength of association between food consumption and illness. The interpretation of the odds ratio is similar to, but not the same as, the RR. In this case, ill cases were 20 times as likely as controls to have eaten egg salad. As with the RR, the confidence limits give a range of plausible values for the odds ratio. The calculation with confidence limits can be performed by the Epi Info software package (52). A summary table of the results for the case-control study for the three food items of the hypothetical wedding luncheon example is shown in Table 9. Note that an odds ratio of 1.0 ("even odds") for pasta salad indicates no association, positive or negative, with consumption of this food.

Table 9 Summary of Food-Specific Odds Ratios, Wedding Outbreak Example Number of persons eating food item Food item

Cases

Egg salad Tuna salad Pasta salad

23 18 14

OR = odds ratio; C1

=

Controls

9 12 11 14

confidence interval.

Number of persons not eating food item Cases

Controls

OR

(95% CI)

2 7 11

16 13

20.4 2.8 1.0

(3.4-203.50) (0.7-10.7) (0.3-3.5)

Morse et al.

610 Table 10 Summary Method Case-Control Data for Egg Salad Consumption, Wedding Outbreak Example Case ate

Number of pairs

Control ate

+ +

10 10 1 4

-

Several examples of disease outbreaks in which case-control analyses were used can be found in Refs. 56-58. d. Matched Case-Control Analysis. Matched case-control analysis is a variation on the case-control analysis in which controls are selected by characteristics that are the same as one or more cases. For instance, controls may be matched to cases by age (usually within an age range, e.g., 20-25 years, etc.), sex, or class (classroom, roommates, etc.). Matching often is done as a convenience in selecting controls, but it also may have the purpose of removing the influence of the matching variable (e.g., age) in the analysis. A caveat in matched analyses is that by matching, the investigator loses the ability to examine the role of the matching variable in causing illness since cases and controls will be identical in the distribution of the variable. When matching is done in the selection of controls, a matched analysis (McNemar test) should be used in the analysis. This calculation with confidence limits can be performed by the Epi Info computer package (52). In the wedding party example, assume that 25 ill persons were matched with a nonill spouse or other family member who also attended the party. Food-consumption histories then were used to determine the number of concordant case-control pairs (both ill case and matched control ate the suspect food item or both did not eat it) and the number of discordant casecontrol pairs (one ate but the other did not). Suppose that the questionnaires revealed that in 10 case-control pairs in which both ate egg salad, in 10 pairs the ill case had eaten egg salad but the matched control had not, in one pair the well control had eaten the egg salad but the ill case had not, and in 4 pairs neither case nor control had eaten the egg salad. These data can be summarized as in Table 10 or in tabular form as in Table 11. As can Table 11 Matched Case-Control Analysis, Wedding Outbreak Example Matched control Food Food item 1 Ate Did not eat Example: egg salad Ate Did not eat

Ate

Did not eat

a C

b d

10

10

1

4

Odds ratio (OR) calculation OR = b/c

OR

10 1

= -=

10.00

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61 1

be seen from the formula for the matched (McNemar) odds ratio in Table l 1, only the discordant pairs are important in determining the degree of association between case status and food consumption. This calculation of the matched (McNemar) odds ratio with confidence limits can be performed by the Epi Info computer package (52). The interpretation of the odds ratio is similar to that of the unmatched odds ratio. It is important when conducting a matched case-control analysis to analyze the data using the matched (McNemar) formula. It is inappropriate to analyze matched data in the standard case-control format. A summary of the matched analysis in the hypothetical wedding outbreak is shown in Table 12. Notice that case status had a negative association with eating pasta salad, that is, the odds ratio was less than 1, indicating a protective effect (statistically, not medically) of eating pasta salad. Examples of disease outbreaks in which matched (McNemar) analyses were used can be found in Refs. 59 and 60. e. Stratijied orMultivariateAnalysis. Stratified or multivariate analysis can be used with either RR or case-control investigations. Stratified analyses involve setting up multiple two-by-two tables between two variables, each table corresponding to a different level or stratum of a third variable, the influence of which the investigator is seeking to remove. In foodborne outbreaks, such an analysis often is done to determine which of two possibly implicated food items statistically associated with illness is the more important. In the wedding outbreak example, notice that although egg salad consistently appears to have the highest risk of disease, tuna salad also had measures of association (RR or odds ratio) severaIfold greater than 1, although statistical significance was not always achieved. It remains possible that tuna salad is responsible for some or all of the outbreak. This could be possible if both vehicles had been contaminated by a food worker, if the tuna was prepared in the same bowl or blender as the contaminated egg salad, or if egg salad was the only vehicle of transmission, but tuna salad was acting as a confounder. In this situation, egg salad eaters may have been more likely also to eat tuna salad, explaining an apparent association between tuna salad and illness. A stratified analysis is the technique to determine the influence of one variable, say egg salad, independent of another, the tuna salad, for instance. In the process, a RR for egg salad "adjusted" for the effect of tuna salad is calculated. The standard method for calculating adjusted measures of association in stratified Table 12 Summary of Food-Specific Odds Ratios from Matched Analysis, Wedding Outbreak Example Number of matched pairs Case: ~~

Food Egg salad Tuna salad Pasta salad

Ate

Ate

Did not eat

Did not eat

Did not eat

Ate

Did not eat

OR

(95% CI)

10 10 2

1 4 3

4 1 10

10.0 2.5 0.7

(1.3-78.1) (0.8-8.0) (0.1-4.0)

~~

Control: Ate 10 10 10

~~

OR = odds ratio; C1 = confidence interval.

Morse et al.

612 Table 13 Format for Stratified Cohort Analysis, Wedding Outbreak Example Food Conceptual example Table A: all ate food item 2 Ate food item 1 Did not eat Table B: none ate food item 2 Ate food item 1 Did not eat

I11

Well

Total

a

b d

a + b c + d

b d

a + b c + d

C

a C

Relative risk calculation

a(c RR (MH)

Food example Table A: all ate tuna salad Egg salad Ate Did not eat Table B: none ate tuna salad Egg salad Ate

47

25 2

25 45

50

25

25

50

3

50

53

Did not eat

=

+ d)

c-"c(a T+ b)

RR (MH) = 10.0 (95% C1 = 5.3-19.0)

RR = relative risk; MH = Mantel-Haenzel analysis; C1 = confidence interval, T = a

+ b + c + d.

analyses is the Mantel-Haenzel (MH) analysis (see Table 13). The analysis examines the association of food item 1 with illness, removing the influence of food item 2. A summary or adjusted RR (Mantel-Haenzel) is calculated. This calculation with confidence limits can be performed by the Epi Info computer package (52). Table 13 portrays the relationship between egg salad consumption and illness, first among tuna salad eaters and second among non-tuna eaters. Thus, the influence of tuna salad is removed. Each table (A and B) has an RR that can be calculated. These can be combined by the Mantel-Haenzel formula to a summary RR (10.0) showing the risk of egg salad independent of, or adjusting or controlling for, tuna salad consumption. It is important to use the summary RR only when the RRs from the individual tables are similar. (Different RRs in Tables A and B would indicate that the presence or absence of eating tuna salad is modifying the effect of egg salad. When tuna salad also is eaten, egg salad has a certain level of risk; when it is not eaten, there is a different risk for egg salad. Should this arise, simply report both RRs from the stratified tables and try to think of a medical explanation.) In this case, egg salad is associated with illness independent of tuna salad. But does tuna salad have any association with illness? This can be seen by constructing a table to look at the influence of tuna salad independent of egg salad (Table 14). In this example, tuna salad was found to have no association with illness.

C.

EnvironmentalComponents

1. Introduction An investigation of an alleged foodborne illness outbreak should include an environmental component, a food-preparation review, to determine if foods involved could have been

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613

Table 14 Format for Stratified Cohort Analysis of Tuna Salad, Wedding Outbreak Example Food Table A: all ate egg salad Tuna salad Ate Did not eat Table B: none ate egg salad Tuna salad Ate Did not eat

I11

Well

Total

25 25

25 25

50 50

2 3

45 50

47 53

RR (MH)

RR (MH) = 1.0 (95% C1 = 0.7-1.4)

RR = relative risk; MH = Mantel-Haenzel analysis.

contaminated or otherwise abused in a manner that would have led to the illness being investigated. Since a food-preparation review often is conducted by environmental health staff, it is important to differentiate between this activity and regulatory environmental health functions. A food-preparation review is a review of what happened prior to the consumption of a suspect food that could have resulted in that food making someone ill. (Appendix G contains an example of a food-preparation review form used by New York State.) A regulatory inspection focuses on present-time conditions by identifying all violations of a regulation regardless of the likelihood of those violations causing a food consumer to become ill. The hazard analysis critical control point (HACCP) food-preparation review system is an approach that industry and regulatory officials can use to identify and control food safety hazards before they endanger consumers (61). Three microbial etiology groups are the most important to consider when conducting a food-preparation review. Bacterial etiologies are reported most commonly in foodborne disease outbreaks in the United States, accounting for 79% of outbreaks and 90% of cases with known etiologies in the period 1988-1992 (1). Viral etiologies have been confirmed less frequently, but are likely to cause a significant proportion of foodborne morbidity (42,62). Parasites such as Cryptosporidiunz and Cyclospora have recently emerged as significant foodborne pathogens as well (15,16,27). The six factors necessary for a bacterial foodborne disease outbreak to occur are (34): Causative organism (etiological agent). Source and reservoir of the organism. Mode of dissemination of the organism from the source to a food. Food that has been contaminated must be capable of supporting the growth of the organism. 5 . Food that has been contaminated must remain in the temperature range suitable for proliferation of the organism long enough for the organism to multiply to sufficient numbers to cause illness or produce sufficient toxin to cause illness. 6. Quantity of contaminated food eaten must contain sufficient amounts of the organism or toxin to exceed susceptibility threshold of theperson who has eaten the food.

1. 2. 3. 4.

Viral, protozoal, helminthic, and chemical foodbome diseases require only factors 1, 2, 3, and 6. Diseases caused by plant toxicants would require only factors l , 2, and 6.

Morse et al.

614

A working knowledge of the microbial ecology of food is useful in fully understanding and applying these concepts in foodborne disease outbreaks of a bacterial etiology (37,63). 2.

Food-Preparation Review

CL Interview Manager and Food Workers. The first step in conducting a foodhazard analysis is to interview the food-preparation manager and all workers that were involved in the preparation and service of the suspect food or foods. The interviews should be conducted individually and in confidence to obtain as unbiased answers as possible. Ask each interviewee to describe, chronologically and in detail, his or her role in the food-preparation process. Ask them to identify sources of ingredients, quantities involved, equipment used, and where it was used. Record the date and time when each step took place. Identify steps in which contamination, microbial survival or destruction, or bacterial multiplication could have occurred. Inspect all workers’ hands for infected wounds and determine their food-consumption history for the suspect meal and 72 hours prior to it. Inquire about recent gastrointestinal symptonls or illness for each food worker. Followup questions should probe to provide insight based on initial responses.

b. Observe the Preprution Process. After you interview people who were involved in the preparation of the food, ask them to walk you through the process in the kitchen. Observe where the food was stored, prepared, and held prior to service. If possible, ask workers to prepare the same food items that are under investigation so that you can identify opportunities for contamination, measure container sizes, quantities of food, food temperatures at each step, ambient and refrigerator temperatures, and the time for each step. If water or foods containing water (e.g., ice or drinks) are suspect, inspect the water source to determine if it isprotected properly, if anyrequired treatment has been operating properly, and identify any possible cross-connections. As you watch the steps involved in food preparation, look for opportunities for cross-contamination from raw foods, soiled equipment or surfaces, workers’ hands, and so on. Consider at each step if bacteria, protozoa, or viruses of concern would survive or be destroyed and would have the opportunity (food, time, temperature) to multiply.

c. Obtain Food, Worker, Environmental S’ecimerzs. Obtain specimens of leftovers of all suspect foods. If leftovers have been discarded but can be recovered from the garbage, do so, as the findings still can be of some value. If foods from commercial sources are suspect, obtain unopened containers from the lot that is suspect and becareful to record all the information available as to container size, lot numbers, exact product description, inspection agency numbers, pull dates, wholesaler, and manufacturer. Refer this information along with epidemiological and laboratory findings to the appropriate state and federal agencies so that a traceback investigation can be initiated as soon as it becomes appropriate. Specimens of similar foods prepared on days other than the ones in question are of limited value; their findings can suggest that time/temperature abuse and/or contamination could have occurred, but they cannot be used to confirm that a foodborne etiology was involved. If agents such as Subnonellu, Shigella, or Cryytosporidium are suspected, request that all food workers provide stool specimens. Also obtain environmental swab specimens of food-contact surfaces that would have been in contact with suspect foods. Obtain swabs of infected wounds on workers’ hands and from exterior nares of noses if a staphylococcal etiology is suspected.

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615

d. Plot Titne/Tenzperature Graphs. Prepare timehemperatwe graphs for foods suspected in bacterial etiology outbreaks if foods that could have been affected adversely by timehemperatwe abuse are involved (see Fig. 4). On the vertical axis, mark temperatures between approximately 20 and 200"F,and on the horizontal axis mark time in onehour increments, with the firsthour being the time when the preparation process began and continuing to the time of service. Plot measured temperatures and times at cold holding, preparation, cooking, cooling, reheating, and holding for service. Connect the plotted points and interpret the graph by identifying those steps on the chart at which bacteria, protozoa, or viruses would survive or be destroyed and bacteria could multiply. Steps in which cooling foods are held between 70 and 120°F for more than 2 hours are of particular concern, as are steps when foods are held at temperatures between 45 and 140°F,when foods cooked for the first time do not reach an internal temperature of at least 140°F for most foods or 165°F for poultry, and when reheated foods do not reach an internal temperature of at least 165°F.

e. Diagram Process Steps and IdentiJj, Opportunities for Contamination, Sumivnl, Growth, and Destruction. Prepare a schematic flow chart ofthe preparation steps involved for the suspect foods, including all steps from raw ingredients to service to the consumer (see Fig. 5). Based on this flow chart and the timehemperatwe curves, identify steps at which agents of concern could have been introduced into the food and at which they would survive, be destroyed, or multiply, as appropriate.

f: IdentiJj, Possible Corztributing Factors. Likely contributing factors for agents of concern are steps at which microbial destruction should have occurred but did not, bacterial multiplication could have occurred, or possible contamination was not followed by a step that would have destroyed that contamination. Laboratory findings can help to confirm your interpretation of the food-preparation data by identifying an agent and, in some cases, the number of organisms or presence of toxin in leftover food. Worker specimens positive for the agent in stool or swab samples should be compared with interview data to verify if those workers could have contaminated food at the appropriate time. If

r

Cook Cooking and coolingperiod where destructionof vegetative bacteria and viruses canoccur

n

Reheakg and Holding

If temperatures are too low bacteria and viruses can survive

Fig. 4 Timehemperatwe graph of food preparation.

Morse et al.

616

0

D Sloragc

Q Cooling

lJ

Fig. 5 Diagram of 'ood preparation steps. (1) Opportunities for contaminettion from other raw foods. Opportunities for bacterial growth if temperatures are not cold enough. ( 2 ) Opportunities for contamination from workers, equipment and utensil surfaces, and raw ingredients. (3) Destruction of vegetative bacteria and viruses if time and temperature were adequate. (4)Opportunities for germination and growth of spores that would survive cooking and for growth of newly introduced vegetative bacteria. (5) Opportunities for introduction and contamination from raw foods. ( 6 )Opportunities for survival of bacteria, protozoa, and viruses if timekemperature is inadequate. (7) Opportunities for growth of bacteria if temperature is inadequate. (8) Opportunities for introduction of contamination from workers' hands or contaminated equipment.

a worker's specimen is positive for a bacterial agent such as SuZmomdZa or if a viral etiology is suspected, it will be necessary to determine from food consumption information, illness histories, and food-preparation interview data whether that worker was the source of the agent or a victim due to consumption of the contaminated food.

D. Laboratory Components 1. Introduction Laboratory identification and/or isolation of an etiological agent from human and food specimens are important components of a foodborne outbreak investigation. There are now a myriad of new diagnostic techniques and modified standard ones that can be used to identify bacterial, viral, and parasitic pathogens. Today's diagnostic armamentarium now ranges from direct observation of ova and parasites and specially stained smears, to standard culture techniques with modified media, to agent-specific serological, studies, to electron microscopy coupled with immunological techniques, to use of tissue cultures, molecular probes, PCR, and a number of DNA-fingerprinting methods such as pulsed field gel electrophoresis (PFGE). The increasing importance of DNA-fingerprinting methods, such as PFGE, was demonstrated in 1997 by Bender et al. (66), who subtyped 317 isolates of E. coli 0157:H7 in Minnesota over 2 years. They identified 143 distinct PFGE patterns, which was extremely useful in identifying real outbreaks and clusters that would have gone undetected and in ruling out outbreaks that were suspected by an increase of cases alone. More recently PFGE was used to identify a particular strain of Listeria mofzocytogenes associated with a multistate increase in listeriosis cases (67,68). Once the DNA pattern was identified, an epidemiological investigation showed an association with consumption of hot dogs (OR = 17.3, C1 2.4-160; y < O.Ol), which was confirmed by PFGE testing of isolates from the source product. While these techniques have made it possible to more successfully identify outbreak pathogens, specialized collection and analysis re-

617

Investigating Foodborne Disease Table 15 Collection of Patient and Food-Worker Specimens Agent

Specimen3

Amount

Media

Container

-

Sterile tube Sterile jar Sterile jar Sterile jar Sterile tube

~~~

Bacterial

Bacterial toxinb Virus Parasite

Blood serum Stool Urine vomitus Skinlnares

5-10 mL 10-25 n L 30 mL 10 mL Swab

Blood serum Stool Stool Blood serum Stool Blood serum

5-10 mL 10-25 mL 50 g 5mL 50 g 5 mL

Cary-Blair Cary-Blair

-

Buffered distilled water

-

Glycerol

-

10% formalin' ~

~~~

Sterile tube Sterile jar Sterile jar Sterile tube Sterile jar Sterile tube

~~~~

Refrigerate, do not freeze, all specimens listed here. h For example, to test stool for toxins from E. coli and C. botulincmt, serum for toxins from C. botulinum. Also 4% potassium dichlorate.

quirements make it essential to coordinate epidemiological and laboratory components closely. 2. Preparation for Specimen Collection Close attention to preliminary epidemiological and clinical data is essential to determine the likely diagnoses and the types of specimens to collect. The laboratory should be alerted in advance so that the collection methods, supplies, and transport are tailored to the suspected agent. Appropriate specimen-collection materials should be assembled, including report forms, sterilized containers, special media, ice packs, transport boxes, and blooddrawing equipment, as appropriate. It often is useful to give a unique number to all specimens from the same outbreak so that results can be collated more easily. Remember that you may only have one opportunity to obtain a specimen, so you should be prepared to do it right the first time. 3. Patient and Food Worker Specimens Specimens from humans should be obtained as early as possible because people are more likely to cooperate while ill and because agents usually are present only for a short time. However, collection of specimens after symptoms have ended still can be helpful for agents like Sulmonella, which are shed for a longer period. Patient specimens may have already been obtained under the orders of the patient's physician. If so, contact the examining laboratory and/or physician immediately to have any remaining specimens or isolates saved for further analysis. Attempts should be made to collect specimens before medication is given and to label, store, and ship them appropriately. Serum specimens should be collected during the acute illness and again 3-4 weeks later. It often is not necessary to collect specimens from everyone who is ill, but a complete set of specimens should be collected from at least 6-10 persons. Some general guidelines for human specimen collection are summarized in Table 15, with further elaboration given in Refs. 33 and 69. (An example of a clinical specimen collection report form used in New York State is included as Appendix H.)

Morse et al.

678

4. Food and Environmental Specimens If the source is unknown, specimens should be collected from all remaining foods that were consumed within 72 hours of illness. If only one meal is suspected, as many specimens of foods as possible should be obtained, along with water and beverage specimens. Samples should be collected from different locations in the food aseptically with sterile instruments and placed in sterile jars or plastic bags. Specimens should be labeled and transported under refrigeration to the laboratory. All product-identifying names, distributors, dates, and codes should be collected along with sales slips, invoices, and other shipping information available. Unopened containers of food also should be collected if they are from the same lot. Foods can be analyzed on a priority basis, with testing of suspected foods first. Other foods can be refrigerated and tested later if epidemiologically implicated. Some general guidelines for specimen collection are summarized in Table 16, with further details available in Refs. 33 and 69. (An example of a food sample collection report used in New York State is included as Appendix I.)

5. Laboratory Testing Laboratory testing should be performed only at laboratories capable of performing the desired tests. State or county laboratories familiar with these types of investigations should be the first choice unless otherwise indicated. Whenever positive results are found, the specimens and positive cultures should not be discarded in case confirmation or additional specialized testing is needed. Results should be phoned to investigators and followed by hard-copy results. 6. Steps to Maximize Laboratory Results Meaningless laboratory results can occur if specimens are not collected properly, not handled properly, not representative, not of sufficient quantity, not maintained at the proper Table 16 Collection of Food/Environmental Specimens Sample

Approximate quantity

Solid

Method of collection

Transport container

200 g

Cut or mix from several sites, aseptically

Sterile plastic bag or wide-mouth jar

Liquid Viscous Not viscous

200 mL 1-2 L

Mix or shake Filter and put filter pad in broth

Sterile bottle, jar, or plastic bag Sterile jar or sterile plastic bag

Frozen Small volume Large volume

Variable 200 g

Send intact Drill with a sterile, large bit from top to bottom, collect sterile hollow tube Follow method for appropriate physical state Streak suspect surfaces

Sterile jar or sterile plastic bag Sterile jar or sterile plastic bag

Dry

For chemical agents Environmental source

200Use g Up to 10 kg

Swab

Sterile jar or sterile plastic bag Glass jar; do not use plastic bag

Screw-top tube within buffered distilled water

Investigating Foodborne Disease

619

temperature, not accompanied by enough information, not delivered promptly, or if there is delay in analysis due to a failure to alert the lab in advance. To avoid these errors and maximize laboratory results, the following procedures should be routine: 1. Alert laboratory as soon as possible. 2. For each outbreak, submit specimens from patients, food workers, and suspected foods. 3. Include complete information. 4. Collect specimens in appropriate containers. 5. Deliver specimens immediately or store properly.

E.Synthesis of Epidemiological,Environmental, and Laboratory Components While the epidemiological, environmental, and laboratory components are described separately here, it should be clear that they need to be integrated closely at the beginning of, during the course of, and at the end of an investigation. Each component provides valuable information that, when synthesized, can lead to identification of the causative agent, vehicle, and circumstances that led to contamination. This synthesis should lead to a series of additional outbreak-specific control measures (general control measures usually are implemented during the early part of an investigation) and preventive actions to reduce the risk of future outbreaks. Crucial to this process is the writing of a final report with conclusions and recommendations. This report should document all components of the investigation. It should be written meticulously because it may serve as the basis for court cases and food industry changes in addition to its relevance as a local control document. For national reporting, the information also should be summarized and reported to the CDC using their standard form (see Appendix J).

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APPENDIX B

FORM USED TO RECORD INITIAL COMPLAINT DATA

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APPENDIX E SAMPLE FOODBORNE OUTBREAK QUESTIONNAIRE AGENCY NAME, DATE BACKGROUND INFORMATION This questionnaire is being distributed by the health department to help determine the cause of recent illnesses in the group. Please answer every question as completely as possible. If you have any questions, please ask the person collecting the questionnaires or call 999-9999. Your answers will be kept strictly confidential. Thank you for your assistance. IDENTIFYING INFORMATION Id Number l### Date of Interview <mm/dd/yy> . ." Last name Street {Address) (City) or town Tele{phone)

{First) name

&

initial

State CA > ZIP #ti## Work c h n g distance>

Person supplying {info)rmation (if not namedabove) GENERAL

&

DEMOGRAPHIC INFORMATION

Age I # #years or (if under age 2) age in {months) I # Date of Birth { N B ) <mm/dd/yy> Sex (H/F/Unk) FOOD CONSUMPTION HISTORY For the meal eaten at (restaurant name), which of the following items did you eat or drink? *** OR *** If you ate (meal) at (restaurant name) on (date and day of week) , which of the following items did you eat or drink? *** OR *** Which of the following items did you eat (purchase) from (store name) between (date) and (date)? Number of (CIRCLE ONE) Servings Not Sure Y N {a). (Food item name) {a) . # Y N Not Sure { b) (Food item name) {b) # Y N Not Sure {c). # {c) (etc.) Y N Not Sure I {e) {e}. (Beverage) Y N N o t Sure {f) Alcoholic beverage {f) # Y N Not Sure {g} # {g). Water

.

HEALTH OR ILLNESS INFORMATION Between (date) and (date), which of the following symptoms did you have? {Diarrhea) Y Maximum number of loose or watery {stools) in a 24-hour period #I

CIRCLE ONE N Not Sure



Investigating Foodborne Disease

631

Y N Not Sure (Nausea) Y N Not Sure (Vomiting) Y N Not Sure (D1ood)y stools Y N Not Sure Abdominal {Cramps) {Aching) of muscles or joints Y N Not sure Y N Not Sure (Sore throat) Y N Not sure {Headache) Y N Not Sure (Fever) If yes, please give highest (temp)erature measured

Which (symptom) occurred first? When did it first occur? Time ##:## ( A M ) or PM? < A >

Date <mm/dd/yy>

Did you see a (physician) for any of these symptoms? Waa a stool (culture) or rectal swab taken for culture? Were you (hospita1)ized for any ofthese symptoms? Were you given an {antibiotic) for these symptoms? If yes, please (specify) which one In the past year have you had anyother {medical} problem? If yes, please (describe) briefly Have you taken any (medications) in the past month? If yes, please {specify) which ones How many cigarettes do you {smoke} a day on average?

(Y/N) (Y/N) (Y/N) (Y/N)



(Y/N)



(Y/N)

#I

OTHER INFORMATION Have any friends, family members or other associates had symptoms similar to those mentioned above during the past (time interval)? {otherill} (Y/N) If yes, please give their names, the type of illness, and phone number or other information that might be helpful in contacting them for an interview.

Thank you for your assistance. If you have further questions or can provide more information about this problem, please call: Name Title Address Telephone number ************This Section For Investigatorts Use Only

***********C**

Does the illness information meet the (case) definition? CY>

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APPENDIX G FOOD-PREPARATION REVIEW FORM NEWYORKSTATEDEPARMNTOFHWTH Bureau of Community Sanitatbn and Food Protection

Food Preparation Review Complalnt Numbrr

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APPENDIX H CLINICAL SPECIMEN COLLECTION REPORT

Clinical Specimen

NE W YORK STATE DEPARTMENT OF HEALTH

Bureau 01 Communtty SanitationS Food Proledm

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Morse et al.

APPENDIX I FOOD SAMPLE COLLECTION REPORT NEW YORK STATE DEPARTMENTOF HEALTH

Bureau 01 CornmunHySanRaIlon and Food ProIedlon

Food Sample Collection Report

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Investigating Foodborne Disease

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APPENDIX J FORM APPROVE0

OMB NO 0920-0004

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INVESTIGATION OF A FOODBORNE OUTBREAK Thls form1s used to report foodborne dlsease outbreak investigations to CDC. Afoodbome outbreak is defined as the occurrence two of or more casesof a simllar lllness resultmg from the ingestlon of a common In food the United States Thls form has two parts Part 1 asks for the minmum data needed and Part 2 asks for additionalinformationForthisinvestlgatlontobecountedIntheCDCannualsummary.Part1mustbe completed. Weencourageyoutocompleteasmuch of Part 1 and Part 2 asyou can.

STATE USE ONLY

Part 1: Required Information

I i

Year

20-49 yrs.

6.Investigation Methods: (Check all that apply)

percent oftotal cases)

-%

0 lntervlewsofcasesonly investigatton 0 Casecontrol study 0 Cohort study (farm, manne estuary, etc 0 Food preparatlonrewew 0 Food product traceback

Male -%

2 50 yrs. -Yo

1-4yrs. -%

Month

5. Sex: (Estlmated

4. Approximate Percentage of Total Cases In Each Age Group: < l year -%

Day

Please send eprdemlc curve, rf avarlable

Female.

-%

5-1 9 yrs:-%

7. Implicated Food(s): (based on Reasons listedin Item 15on page 3)

0 Source lnvestlgabon )

0 EnvlronmentI foodsamplecultures

8. Etiology: (Name the bactena, virus, paraslte, or toxm Include specific detalls on toxm or organlsm. such as phage type,virulence factors, molecular tingerprinttng, antlblogram, metabolic profile. Cntena for confirmed etiologies are defined in MMWR 1996 /Vol. 45 / ss-5 / Appendlx B.) Etiology Serotype (if avail.) Other Characteristics

I

I

0 Etlology undetermined lsolatedlldentlfied 0 Could notbe determlned

0 Factoryorproductlonplant

from apply). (check that all 0 Patlent speumen(s) 0 Food speclmen(s) 0 Envlronment speamen(s) 0 Food worker speamen(s)

0 More than one ebology (Please llst In Comments)

9. Contributing Factors: (See list on page 2, check all that apply)

10. Agency reporting this outbreak:

0 Contnbuting factors unknown Contammation Factor OC3 OC2 DC1 OCIO

0 C11

Contact Person: OC4OC9 DC8 OC7 OC6 OC5

DC12 OC13 OC14

NAME 0 C15(descrfbefnComments) ON/A

Prollferatlon/AmplIficatlonFactor (bactenal outbreaks only)

0 P1 0 PI O

0 P2

OP3 OP4 OP5

0 P6

0 Pll

0 P12(descnbe m Comments) 0 N/A

OP7

Survlval Factor (mlcroblal outbreaks only): 0 S1 0 S2 0 S3 0 S4 0 S5 (descnbemComments)

0 P8

UP9

TITLE

PHONE NO. FAX NO E-MAIL

0 N/A

0 Yes 0 No Wasfood-worker implicated asthesourceofcontamlnatlon? If yes, please checkon/y one of followmg 0 laboratoryand epldemiologlc evldence 0 epldemlologlc evldence (w/o lab confirmatlon) 0 lab evldence(w/oeptdemlolcgic confirmation) 0 prior expenence makes thls the llkely source (please explam fn Comments)

Date of completion of this form: I

/ Day

" "

" "

Month

foodborne outbreak Comments:

Page 1 CDC 52 13 REV811999

Year

0 Initial Report 0 Updated Report 0 Final Report 0 Additional data suggests this is not a

.

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APPENDIX J Continued The following codes are to be usedto fill out Part 1 (question9) and Part 2 (question15). Contamination Factors:' C l - TOXICsubstance part of tissue(e.g., ciguatera) C2 Poisonous substance intentionally added (e.g , cyanide or phenolphthaleinadded to cause illness) C3 - Poisonous or physical substanceaccidentallylincidentallyadded (e g.. sanitizer or cleaning compound) C4 - Addition of excesswe quantities of Ingredients that are toxic under these sltuations (e.g , niacin polsonlng in bread) C5 - TOXICcontainer or pipelines(e.g , galvanized containers with acid food, copper pipe with carbonated beverages) C6 - Raw producffingredient contaminatedby pathogens from animalor environment (eg., Salmonella entend/fisIn egg, Norwalk in shellfish, E col/ In sprouts) C7 - Ingestion of contaminated raw products (e.g , raw shellfish, produce, eggs) C8 - Obtaining foods from polluted sources(e.g., shellfish) C9 - Cross-contamination from raw ingredientof animal origin (e.g., raw poultry on the cutting board) C10 - Bare-handed contactby handler/worker/preparer(e.g., with ready-to-eat food) C l 1 - Glove-handed contact by handledworkerlpreparer (e g., with ready-to-eat food) C12 - Handling by an infected person or carrier of pathogen (e.g, Staphylococcus, Salmonella, Norwalk agent) C13 Inadequate cleaningof processinglpreparatlon equipmenthtensils- leads to contamination of vehicle (e.g., cutting boards) C14 - Storage in contaminated environment leads to contamination of vehicle (e.g, store room, refrigerator) C l 5 - Other source of contammation @lease describe in Comments)

-

-

-

ProliferationlAmplification Factors:' P1 - Allowing foods to remain at room or warm outdoor temperature for several hours (e.g , during preparationor holding for service) P2 - Slow cooling (e.g., deep contamers or large roasts) P3 - Inadequate cold-holdlng temperatures(e.g., refrigerator inadequatelnotworking, iced holding inadequate) P4 - Preparing foods a half day or more before serving (e g, banquet preparation aday in advance) P5 - Prolonged cold storage for several weeks (e.g , permits slow growthof psychrophilic pathogens) P6 - Insufficient time andlor temperature during hot holding(e.g., malfunctioning equipment, too large a mass of food) P7 - lnsufficlent acldlficatlon (e.g., home canned foods) P8 Insufficiently low water activity (e.g., smokedkalted fish) P9 - Inadequate thawing of frozen products(e.g , room thawing) P10 - Anaerobic packaginglModified atmosphere(e.g , vacuum packed fish, salad in gas flushed bag) P1 1- Inadequate fermentation (eg., processed meat, cheese) P12 - Other sltuations that promote or allow microbial growth or toxic production (please descnhe in Comments)

-

Survival Factors:' S1 - Insufficient time and/or temperature during cookinglheat processing(e g., roasted meats/poultry, canned foods, pasteurization) S2 - Insufficient time and/or temperature during reheating(e.g.. sauces, roasts) S3 - Inadequate acidification (e.g , mayonnaise, tomatoes canned) S4 - Insufficient thawing, followed by insufficient cooking(e.g., frozen turkey) S5 - Other process failures that permit the agent to survive @/ease describe Comments) m Method of Preparation:2 (e.g., hard shell clams, sunny side up eggs) M1 - Foods eaten raw or lightly cooked M2 - Solid masses of potentially hazardous foods (e.g.. casseroles, lasagna, stuffing) M3 - Multiple foods (e g., smorgasbord, buffet) M4 Cooklserve foods (e.g., steak, fish fillet) M5 Natural toxicant (e.g , poisonous mushrooms, paralytic shellfish poisoning) M6 - Roasted meaffpoultry(e.g , roast beef, roast turkey) M7 - Salads prepared with oneor more cooked ingredients(e.g., macaroni, potato,tuna) M8 Liquid or semi-solid mixtures of potentially hazardous foods (e.g.. gravy, chill, sauce) M9 Chemical contamination (e.g., heavy metal, pesticide) M10 - Baked goods (eg., pies, eclairs) M1 1- Commercially processed foods(e.g., canned fruits and vegetables, ice cream) M12 - Sandwiches (e.g., hot dog, hamburger, Monte Cristo) M13 - Beverages (e.g., carbonated and non-carbonated, milk) M14 - Salads with raw ingredients (eg., green salad, fruit salad) M15 - Other, does notfit into above categories(please describe in Comments) M16 - Unknown, vehicle was not identified

-

-

' Frank L. Bryan, John J Guzewch, and Ewen C. D. Todd. Surveillance of Foodborne DlseaseIll.Summary and Presentationof Data on Vehicles and Contrtbutory Factors; TheirValue and Limitations. Journal of Food Protection, 60; 6:701-714, 1997 Weingold. S. E.,Guzewich JJ, andFudala JK. Use of foodborne disease data for HACCP risk assessment. Journal of Food Protection, 57; 9 820-830, 1994.

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APPENDIX J Continued Part 2: Additional lnformatior

I 11. Numbers of: Total cases for whom you have lnfonatlon avaliable

Cases wlth Outcome I Symptom

OUTCOME I SYMPTOM

much as

I

I

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I

I Deaih

Dosslblel

13. Duration of Acute illness Among Those Who Recovered:

(clrcle appropnate unlts) (Hours, days) - (Hours, days) __ (Hours, days) __

II

Healthcare Provlder Vlsit

I Hospltalization

I 0 Unknown

Vomiting

~

[ P l e a s e C o m D l e t e as

I 12. Incubation Period:

_

_

_

_

_

_

_

_

_

_

_

(circle appropnate unlts) Shortest __ (Hours, days) Shortest Longest - (Hours, days) Longest: Medlan ___ (Hours, days) Medlan 0 Unknown

_

Diarrhea

* Use the followmg terms, ~f appropriate,to descnbe othercommon charactenstlcs of cases.

Bloody stools

I Feverish

l

Abdomlnal cramps

l

I

anaphylaxis descendmg myalgla paralysls arthralgla bradycardia hemolytic throat bullous uremlc sore skln tachycardia (HUS) syndrome leslons thromobocytopenla hypotensionbradycardia reversal temperature ltchmg cough JaUndlCe coma wheezlng lethargy dlplopla paresthesla

flushlng sepbcemla headache

urticaria

I

\

14. If Cohort Investigation Conducted:

=

Event-specific Attack Rate

I

x l00 =

I # of exposed cases

#of exposed indlvid. for whom you have illness info.

15. Implicated Food(s): (Please provide known Information.)

I

Name of Food

Main Ingredients

I

Contammated Ingredient

I

(see below)

I 0 Food vehlcle couldnot be determined

I

Reason Suspected(choose all that apply) l Statlstlcai evldence from epldemlologlcai lnvestlgatlon 2 Laboratoryevldence (e g , ldentlficatlonof agent In food) 3 - Compellmg supportwe mfcrmatlon

-

~

~~

~

~

~~

~~

4 - Other data (e g , same phagetypefound on farm that supplled eggs)

-

5 Specific evldence iackmg but prlor experlence makes thls llkely source

~

17. Where was Food Eaten? (Check all that apply)

16. Where was Food Prepared? (Check allthat apply)

U Restaurant or deli 0 Day care center

0 Prrson, jail 0 Pnvate home

0 School 0 Church, temple, etc.

U Plcnlc U Far. festrval. other temporaryhoblle servlce

0 Camp

0 Contammatedfood imported IntoU S

0 Caterer 0 Grocery store

0 Commerual product, sewed wlthout further

U Hospital

0 (please Other descnbe)

preparation

0 Restaurant dell or 0 Day care center 0 School 0 Church, temple, etc 0 Camp Store U Grocery 0 Hosp~tal U Workplace cafeterta

0 Nurslng home 0 Prlson, jall 0 home Private 0 Plcnlc 0 Fair, festival. moblle or location 0(please descnbe) Other

0 Workplace cafetena 0 Nursmg home

19. Remarks: Briefly describe important aspects of the outbreak not covered above administration, economlc impact, etc) (e g., restaurant closure, product recall, ~mmunoglobulin

18. Other Available Info: 0 Unpubllshed agency report (please attach) 0 Epl-AId 0 Publicatton (please reference)

I

I

I

I

I

O Not avallable

State Health Departments: Please FAX this document to Biostatistics and Information Branch, DBMD, CDC, at (404) 639-2780. Page 3 CDC 52 13 REV 811999

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23. CDC. (1993). Multi-state outbreak of Escherichia coli 0157:H7 infections fromhamburgerswestern United States, 1992-1993. MMWR, 42:257-263. 24. CDC. (1997). Escherichia coli 0157:H7 infections associated with eating anationally distributed commercial brand of frozen ground beef patties and burgers-Colorado, 1997. MMWR, 461777-778 25. CDC. (1996). Outbreak of Escherichia coli 0157:H7 infections associated with drinking unpasteurized commercial apple juice-British Columbia, California, Colorado, and Washington, October 1996. MMWR, 45:975. 26. Tauxe, R., Kruse, H, Hedberg, C., Potter M., Madden, J., and Wachsmuth, K. (1997). Microbial hazards and emerging issues associated with produce. J. Food Prot., 60:1400-1408. 27. Altekruse, S . F., Cohen, M. L., and Swerdlow, D. L. (1997). Emerging foodborne diseases. Emerg. Infect. Dis., 3:285-293. 28. Pattison, S . J. (1998). The emergence of bovine spongiform encephalopathy and related diseases. Emerg. Irfect. Dis., 4:390-394. 29. Nachamkin I., Allos, B. M.and Ho T. (1998). Campylobacter species and Guillain-Barr6 syndrome. Clin. Microbiol. Rev., 11:555-567. 30. Bureau of Community Sanitation and Food Protection. (1980- 1997). New York State Annual Foodborne Reports (internal reports of the Bureau of Community Sanitation and Food Protection). New York State Department of Health, Albany. 31. Wharton, M., Chorba, T. L., Vogt, R. L., Morse, D. L., and Buehler, J. W. (1990). Case definitions for public health surveillance. MMWR, 39(RR- 13):1-43. 32. Bryan, F. L. (1982). Diseuses Transmitted by Foods, 2nd ed. Centers for Disease Control, Atlanta, pp. 1-101. 33. International Association of Milk, Food, and Environmental Sanitarians. (1999). Procedures to Investigate Foodborne Illness,5th ed. International Association of Milk, Food, and Environmental Sanitarians, Ames, IA, pp. 1-95. 34. International Association of Milk, Food and Environmental Sanitarians. (1996). Procedures to Investigate Waterborne Illness, 2nd ed. International Association of Milk, Food and Environmental Sanitarians, Ames, IA. 35. Tartakow, I. J., and Vorperian, J. H. (1981). Investigation of foodborne disease outbreaks.In Foodborne and Waterborne Diseases. AV1 Publishing, Westport, CT, pp. 239-258. 36. Bryan, F. L., Guzewich, J. J., and Todd, E. C. D. (1997). Surveillanceof foodborne diseases 111. Summary and presentation of data on vehicles and contributory factors: their value and limitations. J. Food Prot., 60:701-714. 37. Bryan, F. L. (1981). Guide for Investigating Foodborne Disease Outbreaks and Analyzing Surveillance Data. Centers for Disease Control, Atlanta, pp. 1-98. 38. Horowitz, M. A. (1977). Specific diagnosis of foodborne disease. Gastroenterology, 73:375381. 39. Klaucke, D. N., Buehler, J. W., Thacker, S. B., Parish, R. G., Trowbridge, F. L., and Berkelman, R. L., et al. (1988). Guidelines for evaluating surveillance systems. MMWR, 37(S-5): 1-18. 40. Chorba, T. L., Berkelman, R. L., Safford, S . K., Gibbs, N. P., and Hull, H. F. (1990). Mandatory reporting of infectious diseases by clinicians. MMWR, 39(RR-9):1-17. 41. Kaplan, J. E., Feldman, R., Campbell, D. S . , Lookabaugh, C., and Gary, G. W. (1982). The frequency of a Norwalk-like pattern of illness in outbreaks of acute gastroenteritis. Am. J. Public Health, 72:1329-1332. 42. Frankhauser, R.L., Noel, J. S., Monroe, S . S . , Ando, T., Glass, R.1. (1998). Molecular epidemiology of "Norwalk-like viruses'' in outbreaks of gastroenteritis in the United States. J. Zrtfect. Dis., 178:1571-1578. 43. Morse, D. L., Guzewich, J. J., Hanrahan, J. P., Stricof, R., Shayegani, M., Deibel, R., et al. (1986). Widespread outbreaks of clam- and oyster-associated gastroenteritis. N. Engl. J. Med., 314:678-681.

642 44.

45. 46.

47. 48.

49.

50. 51.

52. 53.

54. 55.

56.

57

58.

59.

60.

61.

62. 63.

Morse et al. St. Louis, M. E., Morse, D. L., Potter, M. E., DeMelfi, T. M., Guzewich, J. J., Tauxe, R. V.. et al. (1988). The emergence of Grade A shell eggs as a major source of Salmonella enteritidis infections. JAMA, 259:2103-2107. Centers for Disease Control. (1991). Guidelines for investigating clusters of health events. MMWR, 39(RR-II): 1-23. Birkhead G. S., Morse, D. L., Levine, W. C., Fudala, J. K., Kondracki, S. F., Chang, H. G., et al. (1993). Typhoid fever at a resort hotel in New York: A large outbreak with an unusual vehicle. J. Irzfect. Dis., 15:103-1 11. Schesselman, J. J. (1982). Case-Control Studies: Design, Conduct, Artalysis. Oxford University Press, New York, pp. 1-354. Seals, J. E.. Snyder, J. D., Edell, T. A., Hatheway, C. L., Johnson, C. J., Swanson, R. C., et al. (1981). Restaurant-associated type A botulism: Transmission by potato salad. Am. J. Epidenliol., 113:436-444. Engleberg, C. N.,Barrett, T. J., Fisher, H., Porter, B., Hurtado, E., and Hughes, J. M. (1983). Identification of a carrier using Vi enzyme-linked immunosorbent assay serology in an outbreak of typhoid fever on an Indian reservation. J. Clin. Microbiol., 18:1320-1322. Spitalny, K. C . , Okowitz, E. N., and Vogt, R. L. (1984). Salmonellosis outbreak at a Vermont hospital. South. J. Med., 77: 168-172. Vogt, R. L., Sours, H. E., Barrett, T., Feldman, R. A., Dickinson, R. J., and Witherell, L. (1982). Canzpylobacter enteritis associated with contaminated water. Ann. Intern. Med., 96: 292-296. CDC. (1997). Epi Info Version 6.04: A Word Processing, Database, and Statistics Program for Public Health. Centers for Disease Control and Prevention, Atlanta, pp. 1-593. Telzak, E. E., Budnick, L. D., Greenberg, M. S. Z., Blum, S., Shayegani, M., Benson, C. E., et al. (1990). A nosocomial outbreak of Salmonella enteritidis infection due to the consumption of raw eggs. N. Engl. J. Med., 323:394-397. Evans, M. R., Lane, W., Frost, J. A., and Nylen, G. (1998). A campylobacter outbreak associated with stir-fried food. Epidemiol. Infect., 121:275-279. Wharton, M., Spiegel, R. A., Horan, J. M., Tauxe, R. V., Wells, J. G., Barg, N., et al. (1987). A large outbreak of antibiotic-resistant shigellosis at a mass gathering. J. Infect. Dis., 162: 1324-1328. Reeve, G., Martin, D. L., Pappas, J., Thompson, R. E., and Greene. K. D. (1989). An outbreak of shigellosis associated with the consumption of raw oysters. N. Engl. J. Med., 321:224227. Mele, A., Rastelli, M. G., Gill, 0. N., DiBisceglie, D., Rosmini, F., Pardelli, G., et al. (1989). Recurrent epidemic hepatitis A associated with consumption of raw shellfish probably controlled through public health measures. Am. J. Epidemiol. 130540-546. Rushdy, A. A., Stuart, J. M., Ward, L. R., Bruce, J., Threlfall, E. J., Punia, P. and Bailey, J. R. (1998). National outbreak of Salmonella senfenberg associated with infant food. Epidemiol. Infect. 120:125-128. Fleming, D. W., Cochi, S. L., MacDonald, K. L., Brondum, J., Hayes, P. S., Plikaytis, B. D., et al. (1985). Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. N. Engl. J. Med., 312:404-407. Morse, D. L., Shayegani, M., and Gallo, R. J. (1984). Epidemiology of a camp Yersinia outbreak linked to a foodhandler. Am. J. Public Health, 74589-592. International Association of Milk, Food, and Environmental Sanitarians. (1991). Procedures to Implement the Hazard Analysis Critical Control Point System International Association of Milk, Food, and Environmental Sanitarians, Ames, IA, pp. 1-72. Cliver, D. 0. (1988). Virus transmission via foods. Food Technol., 42:241-248. International Commission on Microbiological Specifications for Foods. (1980). Microbial Ecology of Foods. Vol. 1, Factors Aflecting Life and Death of Microorganisnts. Academic Press. New York.

Investigating Foodborne Disease

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64. Barrett, T. J., Lior, H., Green, J. H., Khakhna, R., Wells, J. G., Bell, B. P., et al. (1994). Laboratory investigation of multistate food-borne outbreak of Escherichia coli 0157:H7 by using pulse-field gel electrophoresis and phage typing. J. Clirz. Microbiol., 32:3013-3017. 65. Gautom, R. K. (1997). Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli 0157:H7 and other gram-negative organisms in one day. J. Clin. Microbiol., 35: 2977-2980. 66. Bender, J. B., Hedberg, C. W., Besser, J. M., Boxrud, D. J., MacDonald, K. L., Osterholm, M. T. (1997). Surveillance for Escherichia coli 0157:H7 infections in Minnesota by molecular subtyping. N. Engl. J. Med., 337:388-394. 67. CDC. (1998). Multistate outbreak of listeriosis-United States, 1998. MMWR, 47:1085-1086. 68. CDC. (1999). Update: Multistate outbreak of listeriosis-United States, 1998-1999. MMWR, 4711117-1118. 69. Lew, J. F., LeBaron, C . W., Glass, R. I., Torok, T., Griffin, P. M., Wells, J. G., et al. (1990). Recommendations for collection of laboratory specimens associated with outbreaks of gastroenteritis. MMWR, 39(RR-14):1-13.

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22 Indicator Organisms in Foods James M. Jay University of Nevada Lus Vegas, Las Vegas, Nevada

I. Introduction 645 11. Indicators of Food Safety 645 A. Coliforms 645 B. Fecal coliforms 646 C. Escherichia coli 646

111. Rationale for the Use of Coliforms as Safety Indicators646 IV. Detection and Enumeration Methods 647 V. The Application of Forms to Food Safety 650 References 65 1

1.

INTRODUCTION

An indicator organism for foods is one that, if found in given products at specified levels, provides a warning that a safety or spoilage hazard may either exist or be imminent. Microbial and chemical indicators of food spoilage have been reviewed and discussed elsewhere (1-4) and are not discussed further here. This chapter is devoted to discussion of microbial indicators of food safety.

II. INDICATORSOFFOODSAFETY Among the microorganisms that have been suggested and investigated as food safety indicators are coliforms, fecal coliforms, Escherichia coli,enterococci, bifidobacteria, the family Enterobacteriaceae, coliphages, all gram-negative bacteria, and Bacteroides (for reviews and discussions, see Refs. 2 and 4). Since coliforms, fecal coliforms, and E. coli have emerged as the most useful safety indicators, the remainder of this chapter discusses this group.

A.

Coliforms

The coliforms are gram-negative, nonsporeforming rods that ferment lactose. They are represented by the genera Citrobacter, Enterobacter, Escherichia, and Klebsiella. Some 645

646

Jay

strains of Arizona hitzshawii and Hafiia alvei fit the general definition since they ferment lactose, but generally not within 48 hours. Also, some Pantoea ngglornernns strains ferment lactose within 48 hours. In general, at least 90% of coliforms of vertebrate animal origin are Escherichia that conform to E. coli type I strains. These Escherichia strains have the IMViC (indole, methyl red, Voges-Proskauer, citrate) pattern + + - -. Type I1 E. coli strains display the IMViC pattern - + - - and, along with other IMViC variations and non-Escherichia species, are found most often among insects, plants, and in soils. A large number of methods have been devised for the detection and enumeration of coliforms, and the reader is advised to consult Refs. 5-10 for approved isolation and enumeration methodologies.

B. FecalColiforms The group of fecal coliforms is defined by the production of acid and gas in EC broth that is incubated between 44 and 46"C, most often 44.5 or 45.5"C. For all practical purposes, a fecal coliform is E. coli type I, although some Citrobacter and Klebsiella species/strains grow and produce gas under these conditions. Some C. freundii strains have been shown to produce a heat-stable enterotoxin identical to that of some strains of E. coli (1 1). Some strains of C. fieundii from beef samples have been found to possess Shiga-like toxin genes ( 12).

C. Escherichiacoli As theprimary research organism of many biochemists, molecular biologists, and molecular geneticists, more is known about Escherichia coli than any other microbe. Although widely regarded as being a nonpathogen, strains that are pathogenic to humans and vertebrate animals have been recognized since 1945 (for review, see Ref. 13). These strains are refen-ed to as enteropathogenic E. coli (EC), and five distinct groups are recognized: enterohemorrhagic EC (EHEC), enteropathogenic EC (EPEC), enterotoxigenic EC (ETEC), enteroinvasive EC (EIEC), and enteroaggregative EC (EAggEC). The pathogenic EC strains are recovered by high-temperature incubation in EC broth (see Section ILB), although some if not all EHEC strains fail to grow in EC broth that contains the normal level of 0.15% bile salts but grow when the bile salt content is lowered to 0.1 12% (14). The EHEC strains can be detected by the use of sorbitol-Macconkey agar on which, after an 18-hour incubation at 35"C, sorbitol-negative colonies are selected and subjected to serological tests (9). The incidence of pathogenic EC strains in the feces of healthy adults and children ranges from about 2 to 15%. Among food handlers, 6.4% of 219 persons were found in one study to be carriers of pathogenic strains (15).

111.

RATIONALEFORTHE USE OF COLIFORMS AS SAFETY INDICATORS

In 1892, Schardinger was apparently the first person to suggest the use of E. coli as an index of fecal pollution and a safety indicator upon learning of the common association of this organism with human fecal matter (16). Three years later, T. Smith suggested that a test for E. coli could be used to assess the safety of drinking water (2). In 1901, the state of Massachusetts suggested that water and shellfish be examined for the presence/

Foods Indicator Organisms in

647

absence of E. coli, and in 1910 theAmerican Public Health Association used this organism to "score" fresh oysters (4). The term "coliform" came into use during the mid- 1920s when Enter-obacter ner-ogenes was shown to be biochemically so similar to E. coli. Because E. nerogenes is considerably less important than E. coli as a fecal indicator, a test for E. coli clearly is more indicative of fecal contamination. In a strict sense, the coliform index is a measure of fecal contamination. It emerged as a means of assessing the potability of drinking water and later was applied to food products. As indicators of fecal contamination, coliform tests are used best to assess the possibility or probability of intestinal pathogens. Although coliforms are used widely in shellfish sanitation, they do not always predict accurately the presence of some intestinal pathogens. For example, the correlation is poor between fecal coliforms and Vibrio cholerne in oysters (17) and between E. coli, Vibrio ynr~ihnerrlol?)ticus, and Yersillin erzterocolitica in fishand shellfish ( 18). It appears that coliforms cannot be used to predict scombroid poisoning organisms in fish (18). E. coli or coliforms alone were found in one study not to be predictive of the presence of Snlmotzellcl, V. pnrcll~uenlol~'ticUs, enteric viruses, or Staphylococcus nureus in shellfish from an estuary in Spain, but the simultaneous use of E. coli, enterococci, and coliphages were predictive (19). Coliform counts are of no sanitary significance for frozen blanched vegetables since many Enter-obactertypes occur on these products (20). Also, coliforms are not good safety indicators for Sulmonelln on poultry since they exist in a flock prior to slaughter and thus are unrelated to intestinal carriage (21). In general, coliforms in raw milk do not have the same meaning as they do in meats because human intestinal pathogens are not as common to the gastrointestinal tract of ruminants. While not directly related to safety, coliforms can be used for certain heatprocessed products as a measure of process adequacy and/or postprocess contamination.

IV. DETECTIONANDENUMERATIONMETHODS A large number of methods have been devised for detecting and enumerating coliforms, some of which are summarized in Table 1. The more classical of these are violet red bile agar (VRBA) plating and the MPN (most probable number) method. VRBA plating can be carried out with or without a resuscitation step. Without this step, VRBA is used alone with pour or surface plating. With a repair step for metabolically stressed coliforms, a basal layer of 8-1 0 mL of tryptic or trypticase soy agar is distributed evenly over the bottom of the plate and allowed to harden. About 2 hours later, diluted suspensions are added, followed by pouring with VRBA for total coliforms. The VRBA plating method can be adapted for E. coli by adding 4-methylumbelliferyl-beta-~-glucuronide (MUG) (100 parts per million [ppm]) to VRBA. Following incubation at 35°C (or 32°C for dairy products), VRBA-MUG plates are viewed under long-wave UV (ultraviolet) light. Suspect colonies may be confirmed by subculturing to brilliant green bile (BGB) broth and observing for gas production. The Petrifilm E. coli Count Plate (3M Co., St. Paul, MN) is another method that gives results comparable to direct VRBA plating. This dry-medium film method employs VRBA nutrients in addition to a substrate for glucuronidase. After incubation at 35°C (or 32°C for dairy products) for 24 hours, E. coli colonies are blue while those that are red with evidence of gas production are enumerated as coliforms. Another approved method for coliforms is the hydrophobic grid-membrane filter (HGMF) employing mFC (modified fecal coliform) agar without rosolic acid. The method

648 Table 1 Methods to Detect and Enumerate Coliforms in Foods ~~

Methods Plate-count methods VRBA plating Presumptive counts Confirmed results Anderson and Baird-Parker British roll tube Dry-medium film VRB medium EC count Broth cultures Presumptive MPN, LST broth Confirmed MPN, BGB broth EC broth, MPN Filter methods Standard membrane filter M-FC method M-7 h FC method Coli-Count Sampler HGMF-ELA ISO-GRID + SD-39 agar Glucuronate agar + MUG Polymerase chain reaction (PCR) Multiplex Immunomagnetic separation plus PCR BAX for screening Automated fluorescent Fluorogenic substrates LST + MUG X-GLUC plating MUGal mF method Defined substrate Presence-absence test Impedance Enzyme capture assay (ECA) Radiometric assays Nucleic acid probes DNA amplification (PCR) Glutamate decarboxylase Ethanol assay Immunomagnetic separation Coliphage assay Lux genes in phage Charon

Sensitivity

Primary use

-10lg -10lg -10lg 143

-10lg -10lg

Total coliforms Total coliforms E. coli type I E. coli Total or fecal coliforms E. coli



Total coliforms Total coliforms Fecal coliforms

-10lg

Total coliforms Fecal coliforms Fecal coliforms Fecal coliforms EHEC strains E. coli E. coli

1-10lg

EHEC strains


10lg

<10lg

1-6lg <3/g <3/25 g 1 cell 1l100 mL

-10lg -1O'lg


-

Stx genes EHEC EHEC

E. coli E. coli E. coli E. coli, coliforms Total coliforms E. coli Total coliforms EHEC strains E. coli E. coli Total coliforms EHEC E. coli E. coli

VRBA, Violet red bile agar; M-FC, Millipore filter count; HGMF-ELA, hydrophobic gridmembrane filter enzyme-labeled antibody; LST, lauryl sulfate tryptose; MUG, 4-methylumbelliferyl-beta-D-glucuronide;X-GLUC, 4-bromo-4-chloro-3-indolyl-beta-~-glucuronide; MUGal, 4-methylumbelliferyl-beta-D-galactoside; PCR, polymerase cham reaction.

649

Foods Indicator Organisms in

is carried out by filtering 1 mL or more of sample through a HGMF, transferring the membrane to a dry mFC agar plate, and incubating at 35°C for 18-24 hours. The dark blue or gray colonies are counted and coliform numbers are calculated. MPN procedures for total coliforms, fecal coliforms, and E. coli are summarized in Fig. 1. The classical MPN procedure employs lauryl sulfate tryptose (LST) broth in a 3- or 5-tube format with incubation at 35°C for up to 48 hours. The MPN presumptive test is completed following the reading of LST tubes for gas. The MPN confirmed test is carried out by transferring inocula from all gas-positive LST tubes to BGB tubes, which are incubated at 35°C for up to 48 hours. The MPN confirmed test is completed upon the reading of BGB tubes for the presence of gas. Numbers of coliforms are calculated from MPN tables by using the BGB tube results. Fecal coliforms can be determined from the same gas-positive LST tubes by inoculating EC broth and incubating at 44.5"C for 24

Serial dilutions of food homogenates

/

\

/

GAS-POSITIVETUBES

I'

Fecal

MPN presumptive test for total coliforms.

I

coliforms

l

\

\

+

Examine tubes under long-wave (365 nm) UV light.

+

Record fluorescentpositive tubes and calculate €. coli MPN from table.

From gas tubes, inoculate €C medium incubate at and 44.5OC for 24h.

From all gas+ tubes, inoculate BGB broth; incubate at35OC for

48h'

\dl

+

Inoculate LST MUG broth; incubate at35OC for 24-28 h.

Inoculate LST broth tubes; incubate at35OC for 40 h.

Record gas tubes and calculate MPN for fecal coliforms.

l

Record gas + BGB tubes and calculate MPN from table. MPN confirmed test for total coliforms.

Fig. 1 Summary of most probable number methods for total coliforms, fecal coliforms, and E. coli.

650

Jay

hours. Gas-positive EC tubes are recorded for MPN fecal coliform numbers. E. coli numbers can be determined by inoculating LST tubes that contain 50 ppm MUG. After incubating at 35°C for 24 or 48 hours, the LST-MUG tubes are viewed under long-wave UV light for fluorescence. and MPN E. coli is calculated from the appropriate MPN table. For more information on the detection and enumeration of coliforms, fecal coliforms, or E. coli, one of the standard reference works noted above should be consulted (9910). The EHEC strains are of special interest since, as members of an indicator group, they are also enteropathogens and cannot be recovered by some of the methods used for other E. coli strains. EHEC strains produce Shiga-like toxins (also designated verotoxins, verocytotoxins, or Stx) that are responsible for symptoms of hemorrhagic colitis, thrombotic thrombocytic purpura, and the hemolytic uremic syndrome. Although strain 0 157: H7 is the best known, there are many others that produce verotoxins. They are most commonly associated with dairy cattle, where their incidence in raw milk and some beef products has been found to be around 1.5-3.5%. However, they have been shown to be fairly common in untreated waters and on vegetables and fruits such as apples. Their true reservoir in nature is not known. More on the distribution of coliforms, especially pathogenic E. coli strains, can be obtained from the ICMSF monograph (22). A Petrifilm test kit for EHEC strain 0157:H7 has been developed that can detect 1 cell in 25 g of raw meat or 50 g of cooked meat in 26-28 hours. An enzyme-linked immunosorbant assay (ELISA) method has been presented for the rapid detection of 0 157: H7 strains in foods; the method consists of the enrichment of a 25-g sample in a selective medium for 16-18 hours at 37°C (23). The enrichment is applied to a sandwich ELISA with polyclonal antibodies specific for E. coli 0157 as capture antibody plus a monoclonal antibody specific for 0157:H7 and 026:Hll as detection antibody. The ELISA is completed within 3 hours, and the method was able to detect as few as 0.2-0.9 cell/g. A variation of this method employs a dipstick immunoassay with a 12-hour enrichment (24). Immunoassay results are obtained 4 hours later and the method is sensitive to 0.1-1.3 cells/g of inoculated beef. A PCR method for detecting EHEC strains has been reported that could detect as few as 1 colony-forming unit (CFU) in ground beef following a 6-hour broth enrichment at 42°C (25). This PCR method employed Shiga-like toxin genes I and 11. As noted in Sec. ILB, some C. fr-eundii strains isolated from beef samples have been shown to possess the Shiga-like gene 11, which could be detected by a PCR assay (12). The PCR method

Indicator Organisms in Foods

651

Table 2 Suggested/Recommended Limits for Coliforms and Esclzerichia coli as Components of Sampling Plans for Some Food Products Products ~

Group ~

Dried milk Pasteurized liquid, frozen, dried egg products Coated/Filled, dried, shelf-stable biscuits Precooked breaded fish Dried vegetables Further processed deboned poultiy products

~

~

iz

C

112

M

5 5 5 5 5 5

1 2 2 2 2 2

10 10 10 11 10: 10

10: 1o1 10’ 500 1o3 10’

~~~~~~~~~~

Coliforms Coliforms Coliforms E. coli E. coli E. coli

Source: Refs. 28 and 30.

of these organisms as sanitary indicators, as noted above. As previously noted, the historical use of the coliform index was for human fecal contamination. E. coli is known to exist in the gut of a number of animals where its presence does not relate to the normal occurrence of human intestinal pathogens, e.g., pigeons. The introduction of terms such as “surrogate” and “index” to be synonymous with indicators for specific products or conditions does not validate the widespread use of the coliform index as one of food safety regardless of product type. There is little doubt that foods are sometimes declared to be unsafe because of the improper use of the coliform index, but erring on the side of safety may be the better alternative. Whether coliforms, fecal coliforms, or E. coli, these indicators are used best as components of a total system of food sanitation. The best such system is the hazard analysis critical control point (HACCP) concept, which covers foods essentially from farm to home. Coliform numbers alone are much less meaningful than when they are applied as parts of such a system. When employed in an HACCP system, they are applied best in a sampling plan. Examples of this type of usage for suggested or recommended coliform or E. coli limits for six categories of foods are given in Table 2. For more information on sampling plans, see Ref. 28. A detailed explanation of the HACCP system can be found in Ref. 29.

REFERENCES 1. Jay, J. M. (1986). Microbial spoilage indicators and metabolites. In Foodborize Microorganisms and Their Toxiizs: Developing Methodology (M. D. Pierson and N. J. Stem, eds.), Marcel Dekker, New York. pp. 219-240. 2. Jay, J. M. (2000). Indicators of food microbial quality and safety. In Modern Food Microbiology, 6th ed. Aspen Publishers. Gaithersburg, MD, pp. 387-406. 3. Jay, J. M., and Shelef, L. A. (1991). The effect of psychrotrophic bacteria on refrigerated meats. In Biodeterioratioit and Biodegradation 8 (H. W. Rossmoore, ed.), Elsevier, London, pp. 147-149. 4. Mossel, D. A. A. (1978). Index and indicator organisms-a current assessment of their usefulness and significance. Food TecFzizol. Aust.. 3 0 2 12-2 19. 5. Association of Official Analytical Chemists (AOAC). (1997). OfJicial Methods of Analysis of the AOAC, 16th ed., Vol. 1, AOAC, Aslington, VA. 6. Association of Official Analytical Chemists (AOACj. ( 1995). Bacteriological Analytical Manual (BAM) 8th ed., AOAC, Arlington, VA.

652

I-.

day

I

7. Hartman, P. A., Deibel, F. H., and Sieverding, L. M. (1992). Enterococci. InCornpendium of Methods for the Microbiological E.uvnination of Foods (C. Vanderzant and D. F. Splittstoesser, eds.), American Public Health Association, Washington, DC, pp. 523-531. 8. Hartman, P. A., Petzel, J. P.,and Kaspar, C. W. (1986). New methods for indicator organisms. In Foodborne Microorganisms and Their Toxins: Developing Methodology (M. D. Pierson and N. J. Stern, eds.), Marcel Dekker, New York, pp. 175-217. 9. Hitchins, A. D., Hartman, P. A., and Todd, E. C. D. (1992). Coliforms-Escherichia coli and its toxins.In Compendium of Methods for the Microbiological Examination of Foods (C. Vanderzant and D. F. Splittstoesser, eds.),American Public Health Association, Washington, DC, pp. 325-369. 10. Standard Methods for the Esarnination of Water and Wastewater, 18th ed., American Public Health Association, Washington, DC, 1992. 11. Guarino, A., Giannella, R., and Thompson, M. R. (1989). Citrobacter freundii produces an 18-amino-acid heat-stable enterotoxin identical to the 18-amino-acid Escherichia coli heatstable enterotoxin (ST Ia). Infect. Immun., 57:649-652. 12. Schmidt, H., Montag, M., Bockemuhl, J., Heesemann, J., and Karch, H. (1993). Shiga-like toxin 11-relatedcytotoxins in Citrobacterfieundii strains from humans and beef sample, Infect. Inlntun. 61 :534-543. 13. Donnenberg, M. S., and Kaper, J. B. (1992). Enteropathogenic Escherichia coli. Iitfect. Inzntzm., 60:3953-3961. 14. Szabo, R. A., Todd E. C. D., and Jean. A. (1986). Method to isolate Escherichia coli 0157: H7 from food. J. Food Prot., 49:768-772. 15. Hall, H. E., and Hauser, G. H. (1966). Examination of feces from food handlers for salmonellae, shigellae. enteropathogenic Escherichia coli, and Clostridiwn pe$ringens. Appl. Microbiol., 14:928-933. 16. Schardinger, F. (1 892). Uber das Vorkommen Gahrung erregender Spaltpilze imTrinkwasser und ihre Bedeutung fur die hygienische Beurtheilung desselben. Wien. Klirt. Wachr., 5:403405, 421-423. 17. Colwell, R. R., Seidler, R. J., Kaper, J., Joseph, S. W., Garves, S., Lockman, H., Maneval, D., Bradford, E., Roberts, E.,Rammers, E., Hug, I., and Huq, A. (1981). Occurrence of Vibrio cholerae serotype 01in Maryland and Louisiana estuaries Appl. Environ. Microbiol.,41:555558. 18. Matches, J. R., and Abeyta, C. (1983). Indicatororganisms in fish and shellfish. (1983). Food Teclznol., 37(6):114-117. 19. Martinez-Manzanares, E., Molifiigo, M. A., Cornax, R., Egea, F., and Borrego, J. J. (1991). Relationship between classical indicatorsand several pathogenic microorganisms involved in shellfish-borne diseases. J. Food Prot., 54:711-717. 20. Splittstoesser, D. F. (1983). Indicator organisms on frozen vegetables. Food Technol., 37(6): 105-106. 21. Tompkin, R. B. (1983). Indicator organisms in meat and poultry products. Food Technol., 37(6):107-110. 22. ICMSF. (1996).Intestinally pathogenic Escherichia coli. In Microorganisms in Foods 5. Aspen Publishers, Gaithersburg, MD, pp. 126-140. 23. Padhye, N. V., and Doyle, M. P. (1991). Rapid procedure for detecting enterohemorrhagic Escherichia coli 0157:H7 in food. Appl. Environ. Microbiol., 67:2693-2698. 24. Kim, M. S., and Doyle, M. P. (1992). Dipstick immunoassay to detect enterohemorrhagic Escherichia coli 0157:H7 in retail ground beef. Appl. Environ. Microbiol., 58:1764-1767. 25. Gannon, V. P. J., King, R. K., Kim, J. Y., and Golsteyn Thomas, E. J. (1992). Rapid and sensitive method for detection of Shiga-like toxin-producingEscherichia coli in ground beef using the polymerase chain reaction. Appl. Environ. Microbiol., 58:3809-3815. 26. Hill, W. E. (1996). The polymerase chain reaction:Applications for the detection of foodborne pathogens. Crit. Rev. Food Sci. Nutr., 36:123-173.

Organisms

Indicator

Foods

653

27. Scheu, P. M., Berghof, K., and Stahl, U.(1998). Detection of pathogenic and spoilage microorganisms in food with the polymerase chain reaction. Food Microbiol., 15:13-31. 28. ICMSF. (1986). Microorganisms in Foods 2. Sampling for Microbiological Analysis: Principles and Specific Applications, 2d ed., University of Toronto Press. 29. ICMSF (1988). HACCP in Microbiological Safety and Quality, Blackwell Science, London. 30. Warburton, D. W., Weiss, K. F., Lachapelle, G., and Dragon, D. (1988). The microbiological quality of further processed deboned poultry products sold in Canada. Can. Inst. Food Sci. Technol., 21:84-89.

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Index

AAPCC (see American Association of Poison Control Centers) Accidents, laboratory, 286 Acinetobacter, 28 Acquired inmunodeficiency syndrome (AIDS), 216, 219, 293, 298, 332, 564, 566 Actin, 174 Adaptation to cold, 70 Additive, chemical food, 46, 420, 458 Adherence aggregative, 189, 191 assay of, 190, 418 diffuse, 189- 190 to epithelial cells, 413, 418 A/E (see Lesions, attaching-effacing) Aerococcus, 346 Aerornonas, 96, 385, 411 anoerogenes, 37 caviae, 37, 40, 43, 552-553 in chlorinated water, 48 formicans, 37 hydrophila, 35-59, 386, 549, 552 isolation of, 48, 50-52 lactoperoxidase effects on, 49 liquefaciens, 37 organic acid effects on, 49 pathogenicity of, 39-44 proteolytica, 37 punctata, 37 salmonicidcr, 37-38, 40, 43 sanitizer effects on, 48 sobria, 37, 40, 42, 43 veronii, 37-3811, 42 virulence of, 43, 44 Aflatoxicosis, 540

Aflatoxin, 533, 540, 542, 546-547 Agar, 29, 39, 50-51, 67, 140. 150-152, 187, 229, 230, 232-233, 249, 250, 287, 333, 347, 351, 366-367, 398,408, 411,416, 418,480, 482 Agricultural Research Service, 70 Agrobacterium, 78 Alcaligenes, 28 Aleurites, 535 Algae, 409, 542 Alkaloids, 589 Allocyclobacillus, 26 Alterornonas, 28 Amanita, 538 American Academy of Pediatrics, 118 American Association of Poison Control Centers (AAPCC), 2-4 American Board of Applied Toxicology, 4 American Board of Medical Toxicology, 4 American Journal of Emergency Medicine, 5 American Public Health Association, 647 American Type Culture Collection (ATCC), 447 Amino acids in enterotoxins, 354 Analysis of food, 28, 70, 113, 147, 398 Artdromeda, 588 Andromedotoxin, 588 Aneurysm, 293, 295 Animal husbandry, 99, 275 Animal Plant Health Inspection Service (APHIS), 274 Animal Protein Producers Industry (APPI), 272 Animal-rendering facilities, 272 Antacids, 223, 293-294, 525, 603 Anthrax, 529, 537

656 Antibiotics, 267, 287, 291, 295-296, 458, 484 aminoglycosides, 397, 420 ampicillin, 50, 287, 289-291, 294, 336 ampicillin-sulbactam, 290, 420 amoxicillin, 290 aztreonam, 290 benzylpenicillin, 147 cefixime, 290 cefoperazone, 290 cefotaxime, 290 cefsulodin, 480 ceftazidime, 233 ceftibutin, 181 ceftriaxone, 290 cephalosporins, 289-29 1, 420, 458 chloramphenicol, 147, 287, 289-291, 294-295, 336, 349, 397,420,458 ciprofloxacin, 289-290, 295-296, 420 colistin, 443 cycloserine, 150 distamycin, 145 enrofloxacin, 296 erythromycin, 147, 336, 349 ethidium, 420 fleroxacin, 420 fluoroquinolones, 181, 289, 291, 295-296, 5 89 furazolidone gentamicin, 290-291, 294, 349 kanamycin, 420 macrolide, 147 methicillin, 348 metronidazole, 147 nalidixic acid, 296, 336 neomycin, 150- 151, 349 netropsin, 145 norfloxacin, 420 novobiocin, 250, 333, 480, 494 olaquinodox, 296 oleandomycin, 150 pazufloxacin, 420 penicillin, 147, 348, 349, 397 perfloxacin, 295-296 polymyxin, 150, 151, 420, 443 quinolones, 295-296, 397, 420 resistance to, 45, 147, 183, 290-292, 296, 348, 349, 397,420, 542, 563, 589 rifampin, 147 streptomycin, 183, 295, 336, 349, 420 sulfadiazine, 150 sulfamethoxazole, 397

Index [Antibiotics] sulfisoxazole, 183 sulfonamides, 295, 336 susceptibility to, 147, 183 tetracycline, 147, 183, 294, 336, 349, 397, 420,458 trimethoprim, 397, 589 trimethoprim-sulfamethoxazole,181, 287, 289-291, 294, 336 Antibodies to Camnpylobacter, 93, 541 against Escherichia coli, 526, 552, 554 against salmonellae, 268, 271, 289, 297, 303 against shigellae, 324, 333 against Toxoplasma gortdii, 547 against vibrios, 41 1, 41 5, 447 anti-enterotoxin, 156 anti-0, 252 fluorescent, 92, 156 monoclonal, 154, 179 to yersiniae, 542 Antigens, 71, 86, 171, 187, 217, 251, 268, 271, 286, 296-297, 299, 302-304, 328, 333,422,447,451,475,492 colonization factor (CFA), 176-177, 179, 195 flagellar, 287 invasion plasmid, 182- 183 produced by yersiniae, 487 somatic, 288, 324, 473-474 Antimicrobials (see Antibiotics) Antisera, 251-252, 398-399, 418 anti-0, 252, 288 Antitoxin, 120 APC (see Plate count, aerobic) Appendicitis, 475, 477 Arcobacter, 86 Arizona hirdzawii, 646 Arsenic, 534 Arthrinium, 533 Arthritis postinfection reactive, 296-297, 325, 413, 475 rheumatoid, 292 and Salmonella infection, 296-298, 530 Arthobacter, 26 Ashbal, 116 Association of Occupational and Environmental Clinics, 10 Association of Official Analytical Chemists (AOAC), 370

Index Atmospheres, modified, 44, 124 Autoagglutination, 497 Bacillus, 26, 28, 146, 541 anthracis, 72 blindness caused by, 67 cereus, 24, 61-76, 141, 522-523, 525, 530-531, 534-535, 540, 542, 545, 548, 554-555, 557-558, 620 circulans, 72 cold tolerance of, 66 detection of, 66-67 disease caused by, 62, 68 dysenteriae, 324 endospores of, 61 jirn~us,72 in foods, 66, 69-70, 79 growth of, 70 hepatitis, 2 14 inactivation of, 71 infections caused by, 67 lautus, 72 liver failure caused by, 67 mycoides, 72 nongastrointestinal infections caused by, 67-68 subtilis, 61, 145-146 thuringiensis, 72 weihenstephanensis, 72 Bacteria autochthonous, 24, 457 autotrophic, 24 bifidobacteria, 26, 645 chemoautotrophic, 24 coliform, 250, 409, 441, 541, 544, 548, 553, 645-651 coryneform, 26 endospore-forming, 26, 61, 140, 144 fecal coliform, 645-646, 648-650 food-fermentation, 26, 108, 116, 533, 548 in fruit juice, 193, 559 gram-negative, 24, 27-31, 37, 78, 85, 170, 247, 324-325, 384-385, 389, 396, 412,422-424,442-443,473,488489, 645 gram-positive, 24, 25, 31, 108, 140, 214, 347, 372,488 halophilic, 24 heat effects on, 47, 396, 424, 456-457, 478-479 heterotrophic, 24, 248 humus feeding, 24

[Bacteria] inactivation of, 47, 71, 79-80, 86-87, 9091, 123-127, 596 intracellular, 174, 217, 220 isolation of, 63, 85, 90, 150, 177, 186, 193, 229, 248, 287,442-443,447, 480-482 lactic acid (LAB), 46 lactic acid effects on, 26, 98 mesophilic, 248 microaerophilic, 9 1 in milk, 647 nonsporeforming, 385, 396 pathogenic, 69, 71, 83, 90, 107, 140, 147, 175, 182-184, 187-189, 191, 213, 221,226, 247, 273-274, 286, 288, 291, 326-327, 332, 347, 385,407425,439-463,472-499, 533, 535, 539, 548, 553 protective, 97 psychrophilic, 24, 66 psychrotrophic, 24, 28, 31, 44, 69-71, 226 radiation effects on, 47, 98, 126-127, 419, 458,479-480 radiation-resistant, 26 sampling for, 50, 266, 268-269, 274, 651 shedding of, 216, 268, 288, 294, 301, 332, 392 in soil, 23, 24, 90, 109-1 11, 118, 477, 526 solar radiation effects on, 548 sporeforming, 3 1 sublethal injury to, 363-367 survival of, 596 typing systems, 63, 71-73, 86, 108, 141, 152-154, 171, 371-372 zymogenous, 24 Bacterial reproduction in the environment and in culture, 596 anaerobic, 150, 249, 333, 384-385, 560 differential media for, 29, 72, 15 1, 181, 249 enrichment media for, 121, 151- 152, 194, 229-230, 233, 249, 250, 333-334, 398,411,442-443,472,480 inhibition effects on, 47, 71, 125, 172173,249,420-421,479 isolation media, 287, 442-443, 447, 480482 NaCl effects on, 45, 47, 71, 123-124, 229, 386, 388, 397-398,418,424, 441,456, 479

658 [Bacterial reproduction in the environment and in culture] nitrite effects on, 45, 47, 125, 144 pH effects on, 45, 47, 66, 71, 78-79, 98, 123-125, 140, 143-145, 193, 215, 227-228, 234, 249, 293-294, 299, 331, 347, 386, 390, 392, 397, 419, 424, 456, 479, 482, 531, 548, 549, 560, 565 protective media for, 26 screening for, 183, 194 selective media for, 29, 78. 86, 151, 194, 249, 292, 333, 367,442-443, 472, 479,482 temperature effects on, 47, 90, 98, 123124, 140, 143, 146, 149, 151, 153, 157, 175, 226, 232, 234, 249, 326, 329, 331, 388, 395-396,409, 419. 424,440-441,456-457,461, 472, 478-480, 483, 494-495, 525, 532, 535, 539, 541, 543, 545-547, 549, 556, 565, 596, 615 Bacteriocins, 26, 46, 126, 147, 152 Bacteriological Analytical Manual (BAM), 28, 50, 194. 333, 350, 359, 412, 443 Bacteriophage, 252, 37 1-372, 420 Bacterium coli conmtune, 170 Bacterium nzonocytogenes, 214 Bacteroides, 645 Bactocatch process, 227 Bacturia, 294 BAM (see Bacteriological Analytical Manual) Bang, Bernhard L. F., 79 Bdellovibrio, 420 Beavan, Thomas E. D.. 171 Beneckea vulnijica, 442 Bergey ' S Manual of Systematic Bacteriology. 37, 252, 346-347 Beverages, 594 Biosis, 298 Biotyping of salmonellae, 252-253 Bismuth subsalicylate (see Pepto-Bismol) Blood and blood products, yersiniae in, 472, 476,483-484 Bongkrekic acid, 533 BoNT (see Neurotoxin, botulinum) Bordetella pertussis, 326, 491 Botulism, 108, 113-120, 527-529, 533, 535, 553, 590, 598, 602 animal, 108 detection of, 121

Index [Botulism] diagnosis of, 119 fatality rates of, 114-1 15 foodborne, 108, 115-116, 118, 547, 560 and Guillain-Barr6 syndrome, 119 inmunization against, 120 infant, 108, 112, 117, 537 misdiagnoses of, 113 prevention of, 120, 123-127 treatment of, 119 unclassified, 108 wound, 108, 117 Bray, John, 171 Brevibacterium, 26 Brochotlzrix, 26 Bruce, David, 79 Brucella, 28, 71-82, 547 abortus, 77-82 canis, 77-78 in domestic sheep and goats, 544 hardiness of, 79 melitensis. 77-80, 544 suis, 77-80 taxonomy of, 78 in wild mammals, 80 Brucellosis, 77-82, 525, 529, 544, 549-550, 559 and tourism, 80 Burkllolderia cocovenenans, 533 California Department of Health Services, 118 Campylobacter, 28, 185, 522-523, 529-531, 535-536, 540, 547-548, 554-555, 558, 560-562, 565 aerotolerant, 85 in bottled milk contaminated by birds, 525 classification of, 84 coli, 83, 87, 92, 147 fetus, 86-88 in foods, 91, 525 helveticus, 89 immunity to, 93 jejuni, 83-105, 292, 424, 526. 541, 562. 620 jejunilcoli, 541 mastitis caused by, 92 in pigeons, 88 reservoirs of, 86-90, 541, 562 in sea gulls, 88 thermophilic, 90-9 1 upsnliensis, 89

Index Campylobacteriosis, 85, 99, 292, 522, 525526, 528-529, 531, 543, 547, 553554, 560, 603 economic costs of, 94-96 and Guillain-Barr6 syndrome, 94, 541, 589 prevention and control of, 96-98, 531 related syndromes, 94 risk factors for, 562 waterborne, 90, 53 1, 541, 548 Canadian Association of Poison Control Centers (CAPCC), 8, 9 Carchatoxin, 550 Carriers birds, 478, 525 cats, 265-266, 562 cattle, 268, 526, 650 dogs, 265-266, 531 human, 287-288, 290, 294-296, 332, 348, 392 insect, 265, 408 poultry, 269, 526, 536, 538, 556 swine, 275. 286, 549 Caulobacter crescentus, 489 CDC (see Centers for Disease Control and Prevention) CDC Botulism Manual, 620 Cellulomonas, 26 Centers for Disease Control and Prevention (CDC), 83, 85, 95, 118, 224-225, 250. 270, 274, 289, 331, 554, 557, 562-564, 587, 590, 598, 619, 624n Central Public Health Laboratory, 253 Cereals, 594 Cereulide, 65-67, 72-73 CFU (see Colony-forming units) Cheese (see Milk and milk products) Chemotaxis, 421 Chemotypes, 25 1 Chicago Health Department, 289 Chickens, Salmonella-free, 272 C~zlorophyllum,538 Cholera, 176, 303, 383-401, 541, 543, 545546, 548, 566 Asiatic, 384 course of in humans, 391-392 foods associated with, 390-391, 589 hog, 248 and hypochlorhydria, 392 infantum, 171 from Korean clams, 537 pandemics of, 384-385 pathology of, 393-394 toxin, 386, 393-395

659 [Cholera] treatment of, 392, 397 vaccines against, 392, 400 Choleragen, 393 Chorioretinitis, 536 Cider (see Juice) Ciguatera (ciguatoxin), 530, 532, 535, 550, 552, 556-557, 622 selachian, 550 Cimetidine, 223 Circling disease, 2 15 Citrobacter, 28, 645-646 freundii, 646, 650 CJD (see Creutzfeldt-Jakob disease) Clithon retropictus, 409 Clostridium, 26 Clostridium argentinense, 109, 122 Clostridiunt baratii, 108, 117 Clostridium bifermentnns, 141 Clostridium botulinum, 24, 107-138, 517, 522-523, 530-531, 534, 537, 545, 555, 558, 560, 620 detection of, 121 environmental distribution, 109, 110 geographic distribution, 109. 110- 116, 118 neurotoxins of, 121-123, 258 physiological groups, 108-109, 123 spores of, 109-113, 117-118, 121, 126127, 527, 537 strain types, 108-109, 121, 127 in traditional Native American foods, 113-114 Clostridium butyricunz, 108, 116- 117 Clostridium diflcile, 148 Clostridium pelfringens, 139-168, 522-524, 528, 530-532, 537, 539, 542-543, 545, 548, 550-551, 555, 557-558, 564, 620 antibiotic resistance of, 147 cultural requirements of, 142-146, 149150 genetics of, 146-147 germination of, 145 isolation of, 149- 150 prevention of gastroenteritis caused by, 156-157 -spores and sporulation of, 140, 144-146, 148, 151, 154, 157, 539 toxic effects on animal hosts, 142 toxic effects on human hosts, 142 toxins of, 141-142, 152, 154, 175, 178179, 181, 188-189, 191

Index

660 [Clostridium perfringens] wound infections by, 140, 147, 413, 441, 448-449,452,458,462 Coagulase, 349-350 Coconut milk, 589 Coliform index, 650-65 1 Coliphages, 645, 647-648 Colitis, hemorrhagic, 182, 184, 556, 564565, 650 Colostrum, 172 Colony-forming units (CFU), 28, 63-64, 70, 224, 228,232, 332-334, 391-392, 409, 412,480,484, 548, 560, 650 Colonization, 272-274, 290, 294, 301-302, 348, 388, 552 Colorado State University, 267 Compendium of Methods for the Microbiological Examination of Foods, 28 Concentration, minimum inhibitory (MIC), 147 Congo Red, 496 Consumer Product Safety Commission (CPSC), 2 Contamination, 597 fecal, 647, 651 microbial, 589 postprocessing, 478 routes of, 48-49, 62, 79-80, 87-93, 99, 146, 148, 173, 177, 192-193, 227, 269-272, 289, 291, 300, 371, 384, 389-390.472,476, 525, 539, 541, 549, 553, 604 Cook County Health Department, 289 Coprococcus, 346 Corynebacterium, 26 Council of State and Territorial Epidemiologists, 590 Crassostrea japonica, 409 virginica, 409 Creutzfeldt-Jakob disease (CJD), 527. 589 variant (V-CJD), 527, 539 Crohn’s disease, 26 Cross-contamination, 63, 91-92, 98-99, 192, 270, 287, 391,400,414,425,498 Cryptosporidiosis, 531, 552-553, 559-560 Cryptosporidium, 553, 613-614, 624 pawum, 553, 565 Crystal violet, 497 Cyclospora, 559, 613, 624 cayetanensis, 5 16, 565 Cyclosporiasis, 589

Cyclosporosis, 553 Cysticercosis, 539, 542 Cytotoxicity, 217 Cytotoxin, 182, 184, 188, 191, 328 Deinococcus, 346 Deoxynivalenol, 542 Dialog System, 298 Diarrhea bloody, 93, 182, 184-185, 188, 531, 540. 542, 546, 552 caused by Aeromonas hydrophila, 4 1, 549 caused by Bacillus cereus, 66, 542, 554 caused by Camnpylobacter, 93-94, 292, 541, 544 caused by enteroaggregative Escherichia coli, 189-191, 549 caused by enterohemon-hagic Escherichia coli, 546 caused by enterotoxigenic Escherichia coli, 177-179, 539-541, 544, 553 caused by Escherichia coli 0157:H7, 546-547, 556 caused by Listeria monocytogenes, 559 caused by salmonellae, 266, 288, 291292, 294, 296, 298, 538, 541-542, 604 caused by shigellae, 325, 541, 546, 548, 56 1 caused by Stophylococcus, 37 1 caused by verotoxigenic Escherichia coli, 531, 549, 552 caused by Vibrio parahaemolyticus, 408, 417, 553 caused by Vibrio spp., 384, 392, 395, 538 childhood, 85, 177, 540-541, 543, 547550, 552-553, 587 community-acquired, 182 dehydrating, 177, 392 infantile, 170-171, 173, 175, 553, 598 nonbloody, 184- 185, 188 secretory, 178, 291, 392 summer, 171 traveler’s, 172, 175, 177-178, 181, 192, 408 watery, 178, 190, 325, 540, 542 Diphtheria, 545 Diseases costs of foodborne, 95-96, 294, 527, 532, 549, 552, 564-565, 588 control of foodborne, 565-566

Index [Diseases] diarrheal, 41, 177, 181-182, 190, 192, 291-292, 294, 296, 325, 384-401, 407-425,440-463, 532, 538-543, 546-549, 552-553 foodborne, 80, 94-95, 115, 148, 213, 221, 224, 247, 270, 286, 292, 304, 331, 384-401,407-425,440-463, 515567, 587-639 incidence rates of, 522 nongastrointestinal, 67-68, 73 noninfectious, 184 numbers of cases of, 563, 588 parasitic, 532, 539, 542, 548, 555, 558 reporting of, 97, 517, 562, 587 surveillance of foodborne (see Surveillance of foodborne disease) waterborne, 384-401, 477-478, 498, 526, 531, 533, 538-543, 548-551, 553, 56 1 Disinfectants (see Sanitizers) DNA fingerprinting, 226, 255, 616 DNA hybridization, 248, 251, 333, 481 DNA probes, 156, 173-174, 181, 190, 229, 332-335, 409, 481, 485 Dose, minimum infectious, 193, 292, 298, 559 for Bacillus cereus, 66 for Cryptosporidium, 560 for ETEC-caused symptoms, 177, 192 for Shigella, 324 for toxin of Clostridium perfringens, 153 for Vibrio spp., 391, 425, 455-456, 463 for yersiniae, 475 Dysentery, 188 amebic, 324, 550 bacillary, 323-336 endemic, 181 Eberth, 247 Echinococcosis, 543 Echinococcus, 543 Echinostoma, 532 Echinostomiasis, 532 Eels infected by Vibrio vulnificus, 448-449, 455 Eggs and egg products, 522, 524, 527-528, 536, 550, 552, 554, 556-557, 559, 561, 566, 593, 596-598 EIA (see Enzyme immunoassay) ELISA (see Enzyme immunoassay) El Niiio, 388, 410

661 Embden-Meyerhof pathway, 152 Encephalitis, bovine spongiform (BSE), 527, 589 Endospores, 61, 144 Endotoxin, 452-455,463,472,483-484 Entamoeba, 548, 553 Enteric fever (see Typhoid fever) Enteritis infectious, 521, 556 from Klebsiella, 525 Enterobacter, 28, 30, 645, 647 cloacae, 53 1 Enterobacteriaceae, 28, 50, 195, 247, 324, 385,422,473-474, 548, 645 Enterococci, 26, 645, 647 Enterocolitis, 287-289, 291-292, 295-297, 304, 349,497 Enterotoxins, 41-44, 64-67, 70-72, 140, 144, 146-149, 152-155, 175-176, 179, 192, 194-195, 291, 371, 386, 535, 539 amino acids in, 354 assays of, 155-156, 178, 358-363, 393394,484 detection of, 358-363 enzyme resistance of, 355 heat-labile, 393 heat-stable (ST), 349, 355, 372, 394-395, 484-485, 646 irradiation resistance of, 355 pathogenicity of, 178, 181-182, 288, 291, 393,483 produced by Staphylococcus, 352-363, 372 produced by Vibrio parahaemolyticus, 41 3 produced by yersiniae, 472, 484-485 purification of, 154- 155 Enzyme immunoassay (EIA, ELISA), 155156, 178-179, 194, 250, 268, 333, 335, 358-363,416 Enzymes, extracellular, 349, 449-450 Epidemic curve, 604-606 Epidemics (see Outbreaks) Epidemiology, 174 of botulism, 108-116, 528, 537 of brucellosis, 79, 80, 544 of disease caused by Campylobacter, 8690, 541 of disease caused by enteroaggregative Escherichia coli, 190- 191 of disease caused by enterohemorrhagic Escherichia coli, 186

662 [Epidemiology] of disease caused by enteroinvasive Escherichia coli, 181 of disease caused by enterotoxigenic Escherichia coli, 177-178, 531 of disease caused by Vibrio parnhaemolytiCuS, 407-425 of disease caused by Vibrio spp., 384401, 546 of disease caused by Vibrio vuh?i~$cus, 439-463, 540 of foodborne disease, 599-612 of growth retardation, 191 of listeriosis, 216, 528 phage typing in, 252, 37 1-372 plasmid analysis in, 254 ribotyping in, 475 risk factors, 77, 80, 149, 173, 185-186, 192, 216, 224, 266-268, 289, 325, 371. 413, 448, 455-456, 476, 526, 532, 546, 548, 552, 562-564, 603 of salmonellosis, 268, 272. 292 seasonal influences on, 88-89, 91, 186, 194, 219, 331, 387-388, 408-409, 440, 459, 479, 537, 541, 548, 562 of shigellosis, 330-332 Epi Info, 608-61 1 Ergotism, 588 Erwinin, 28. 30 amylovora, 489 Eqsipe1othri.u rhusiopathine, 26 Escherichia, 28, 645-646 Escherichia coli, 41, 48, 141, 148, 159-212, 304, 324, 326, 328, 333, 420, 422, 425, 486,488, 519, 522, 525, 53053 l , 533-534, 536-537, 541-542, 545, 549-551, 553, 558, 620, 645648 detection of, 179, 183, 186-1 87, 647-651 diarrheagenic, 159-212, 539-541, 546547, 549 diffusely adherent (DAEC), 170 enteroaggregative (EAEC. EAggEC), 170, 189-191, 520, 542, 549, 552, 646 enterohemorrhagic (EHEC), 170, 182189, 328, 520, 537, 546, 548, 565, 589, 646, 648, 650 enteroinvasive (EIEC), 170, 180- 182, 324, 330, 334-335, 520, 544, 549, 553, 646 enteropathogenic (EPEC), 170-175, 184, 489, 520, 535, 541, 544, 547-549, 552-553, 561, 646

Index [Escherichia coli] enterotoxigenic (ETEC), 170, 175-180, 324, 393, 413,484, 520, 539-541, 543-544, 547-549, 552-553. 559, 646 identification of, 181- 182 isolation of, 194 Shiga toxin-producing (STEC), 184, 187. 520, 525 uropathogenic, 174 verotoxigenic (VTEC), 520, 525-526, 531, 552, 554, 556, 561, 565 viable but nonculturable (VBNC) state of, 424 Escherichia coli 0157:H7, 47-48, 182-189, 325. 483, 516, 525-526, 547, 552554, 556-557, 562, 564-565, 602, 616, 650 antimicrobial resistance in, 563 isolation of, 194 reservoirs of, 193, 526, 537, 556 seasonality of disease caused by, 186 and thrombic thrombocytopenic purpura (TTP), 185, 188, 650 transmission routes of, 186, 193, 598 Escherich, Theodore, 170 Estrogen, 454 Euripides, 588 European Economic Community, 567 Evans, Alice, 79 Factor accessory colonization, 394 adherence, 394 certain attachment (CAF), 176 EPEC adherence (EAF), 174 lymphocyte-activating, 217 macrophage activating (MAF), 217 macrophage inhibitory (MIF), 21 7 positive regulatory, 220 virulence, 408, 449-454, 472, 476, 478, 483,485, 546 FAO (Food and Agriculture Organization), 517, 566 FAO/WHO Collaborating Centre for Research and Training in Food Hygiene, 5 17, 521 FDA (see Food and Drug Administration) Feeds, animal. 149 additives to, 272 contaminated, 272. pelleted, 272 Salmonella-free, 566

Index [Feeds, animal] sugars in, 273-274 whey in, 273 Fish and shellfish, 524, 526, 528-530. 532533, 535, 537-538, 540-542, 547548, 550-551, 556, 592, 646-647 Flies as reservoirs of Canlpylobacter, 54 1 of ETEC. 541 of Snlmonella, 541 of Shigella, 541 of yersiniae, 478 Fomites (see Vehicles of transmission) Food additives, 46, 420, 458 adulteration, 588 bacteria in, 23, 69, 79, 90, 94-95, 192, 413, 539, 647-648 ethnic, native and traditional. 113-1 14, 116, 391, 524-525, 527, 531-532, 536-537, 539-540, 542, 546-549 fermented, 548-549 handling, 84, 89, 91, 270, 289, 294, 33 1332, 371, 390,400,498-499, 527, 537, 538, 540-541, 543-545, 556, 646 home-preserved, 108, 114-1 15, 120, 525, 527-529, 53 1 indicator organisms in, 645-65 1 infant, 112-1 13, 527 intoxication, 540, 542, 564 low-acid canned, 120 poisoning, 588-589 poisoning, staphylococcal, 589 tasters, 588 vacuum canning of, 126 workers, 597, 602, 604, 611, 616, 618 Food and Drug Administration (FDA), 50, 227, 229, 232, 273, 333, 366, 398, 411, 443, 447, 557, 562 FoodNet, 95, 557, 562 Food Poisoning and Food Iilfections, 589 Food Safety and Inspection Service (FSIS), 9 1, 229-230 Fowl typhoid, 252, 269 Fruits, bacteria in, 96, 112, 117, 529, 559, 565, 594 FSIS (see Food Safety and Inspection Service) Gaaffky, 247 Gaertner, 589

663 Gastroenteritis from Aeromonas hydrophila, 40-41, 549 from Bacillus cereus, 62, 72, 542 from Campylobacter, 85, 89, 94, 541, 554 from Clostridium pe$ringens, 140, 144. 147, 151, 153, 156, 542-543, 545 community-acquired, 172 from enteropathogenic Yersinia, 472-499, 542, 561 from EPEC, 192, 547-549, 552, 561 in Europe, 560-561 necrotizing, 140, 292 nonbacterial, 172, 598 nonfoodborne,148 number of cases of, 563, 588 sentinel studies of, 566 surveillance of, 515-567 from Vibrio paralzaemolyticus, 410. 540, 556 from Vibrio spp., 391-393 from Vibrio lwln(ficus, 440-463, 540 GATT (General Agreement on Tariff and Trade), 567 Genzellu, 346 GenBank, 489 Germination of spores, 145-146, 539 Giardia, 548, 553, 602 Giardiasis, 53 1, 550 Gossypol, 533-534 Gracilarirr coronopifolia. 532 Gracilmiopsis. 532 Graves' disease, 475-476 HACCP (see Hazard Analysis Critical Control Points) Hnetnophilus influerzzae,420 Hajjia, 28 ctlvei. 646 Hand-washing, 89. 97, 400, 499, 539, 541 Hayem, G., 214 Hazard Analysis Critical Control Points (HACCP), 97, 269, 271, 400, 518, 545, 565-567, 613, 651 HCC (see Hepatocellular carcinoma) Health Canada, 118, 227 Health Protection Branch, 554 Health and Safety Code, 2 Helicobncter, 86 pylori, 290. 293 Hemolysin, 220-221, 488 produced by Vibrio pnrrrl1nemolyticus, 408-409, 413, 416-417 produced by Vibrio wlrliJicus,449-450

Index Henry illumination, 230 Hepatitis, 550, 562-563 A virus, 517. 525, 531-533, 535, 557558, 589, 600, 624 E virus, 533, 535 Hepatocellular carcinoma (HCC), 546 Hippocrates, 588 Histamine, 522, 528, 532-534 HIV (see Human immunodeficiency virus) Honey, 112-1 13, 117-1 18, 537. 588 Host( S ) accidental, 77 adaptations, 286-287 specificity of, 180 Houseflies as vectors of shigellae, 332 Human immunodeficiency virus (HIV), 190, 224, 293, 332, 547, 557, 603 Humidity, effects of, 87 HUS (see Syndrome, hemolytic uremic) Hyaluronidase, 35 1 Hygiene food, 93, 97-98, 157, 270, 287, 289, 304, 331, 400. 498-499, 527, 540, 543, 549 personal. 62-63, 97, 173, 175, 193, 288, 331,400, 499, 539, 557 in turkey production, 272 Hyperthyroidism, 475 Hypochlorhydria, 392. IAMFES Procedures to Investigate Foodborne Illbless, 624n IAMFES Procedures to Investigate Waterborne Illbless, 624n Iditarod Trail Sled Dog Race, 266 Immunity, 93, 177 acquired cellular, 2 17 cell-mediated (CMI), 216-218, 269, 299, 302-303 humoral, 299, 301-303 lymphokine-mediated cellular, 217 natural. 553 Immunodiffusion, 250 Immunomagnetic separation, 250, 648 Immunosuppression, 2 18-2 19, 603 Inactivation by irradiation. 48, 98, 419, 458, 479-480, 565-566 thermal, 47, 126, 175, 419. 457, 479 Incubation time and temperature, 28, 32, 45, 71, 85-86, 96, 194, 229-230, 287. 329, 333, 461

Infections bacterial, 6, 40, 62, 67, 88, 90, 92-94, 140, 175, 182, 185, 192, 222, 271, 290-291, 296, 384-401,440-463, 47 1-499 inapparent (asymptomatic), 88-89, 172, 177-179, 181, 185, 191-192,265266, 268-269, 274, 287-290, 292, 294-296, 348, 371,483, 527. 543544, 560 systemic, 295 wound, 140, 147, 413, 441, 448-449, 452, 458, 462 Inoculum (see Dose) Insect control, 97 Insects as food, 548 Interferon, 217, 298 Internalin, 220 International Dairy Federation, 69 International Journal of Systematic Bacteriology, 248 Interstate Shellfish Sanitation Conference (ISSC), 441 Intimin, 174, 189 Intoxication, food, 540, 564 Irradiation of food, 47-48, 98, 126-127, 458, 565-566 Jatropha, 538 Johne, 589 Juice, bacteria in apple, 193, 559 cider, 589, 598 orange, 559 Kanagawa phenomenon, 408-410, 412, 414, 416, 418, 420-421, 423 Klebsiella, 28, 48, 525, 645-646 Koch, R., 384 Kocuria, 26 Kommabazillen, 384 Kuru, 539 Kwashiorkor, 547 Lactobacilli, 26, 126, 272 Large Animal Veterinary Teaching Hospital, 267 Latex agglutination tests, 335, 399 Lesions attaching-effacing (A/E), 171, 173-174, 184, 189, 195 secondary, 448

Index

8,

Leuconostoc, 346 LigniCres, 248 Listerella hepatolitica, 214 Listeria, 213-215, 218-219, 223, 225-227, 229-230, 231-234, 529, 562 characterization of species of, 214 heat-injured, 232 host defenses against, 217 repair broth (LRB), 232-234 sublethally injured, 231-232 virulence of, 220, 252 Listeria grayi, 214 Listeria grayi nturrayi. 214 Listeria innoma, 214-215, 220-221, 226, 233-234 Listeria ilmovii, 214-215, 220-221 Listeria rnonocytogenes, 26, 47, 96, 213234, 249, 326, 517. 530, 555, 562, 564, 602, 616-617 acid-injured, 234 associated with cheese. 225-227, 561 associated with fish and shellfish, 225, 559 associated with meat and meat products, 233, 562 associated with milk, 227, 548, 552, 560 associated with pati, 225 associated with poultry products, 227-228, 233, 562 associated with raw vegetables, 562 causing encephalitis in ruminants, 215 causing spontaneous abortion, 21 8 causing stillbirth, 2 1 8 culture of, 230-231 pathogenesis of, 219-221 psychrophilic, 249 survival of in cottage cheese, 228 Listeria seeligeri, 2 14-2 15, 220 Listeria welshimeri, 214-215, 220, 233 Listeriolysin, 219-221 Listeriosis, 213, 215, 223-225, 525, 528529, 531, 559, 562-563, 617 associated with antacids, 223 associated with cimetidine, 223 associated with dairy products, 226. 552 associated diseases, 216 in central nervous system, 219 chronic, 217-218 early-onset, 218-219 environmental sources of, 215, 229, 29 1 epidemiology of, 216, 528 geographic distribution of, 215

665 [Listeriosis] in immunocompromised hosts, 218-219, 562 late-onset, 218 neonatal, 2 18 occurrence in domestic cattle, 215 occurrence in humans, 216, 218 occurrence related to age, 216 and pregnancy, 218-219, 223, 559, 562 and renal transplants, 21 9 risk factors for, 216, 218, 223, 528, 562 seasonal occurrence, 2 19 Longus. 176 Macacn rmlatta, 358 Macrophages, 217, 220, 298, 329, 456, 484, 490,492 Mannitol, 530 Mantel-Haenzel (MH) analysis, 6 12 Meat and meat products, 31-32, 522, 533, 543-544, 547, 550, 552, 554, 588589, 591 Bacillus cereus in, 64, 66, 69, 525 bacteria in, 31-32, 90, 112 Brucella in, 78-79 Carnpylobucter in, 92, 96-97, 99, 525 Clostridium in, 108, 115-116, 143, 146149, 152, 528, 531-532, 542 consumed raw, 547-548, 565 Escherichia coli in, 193, 527, 546, 556, 562 ground beef, 26, 31-32, 92, 193, 556, 562, 565, 589, 650 Listeria rnonocytogenes in, 224, 228 salmonellae in, 266, 268, 295, 524, 528, 548, 565 shigellae in, 548 yersiniae in, 472, 478 Media defined, for Clostridium perfringens, 143 differential, 29, 40, 250 enrichment, 29, 50-51, 121, 152, 231232, 250, 266, 333-334, 398, 411, 442-443,472,479-480 isolation, 39, 50-51, 71, 84, 150, 186187, 249, 287,442-443, 447, 480482 selective, 29, 48, 86, 150. 152, 250, 333, 442-443,472,479.482 sporulation, 144-145, 154 Mediterranean Fever Commission, 79 Medline, 298

666 Mercerzaria canlpechiensis. 409 Methods bacteriological, 333, 647 for classification (taxonomic), 37-40, 52, 108. 398 for culturing, 50, 230 for detection, 66-67, 231. 233-234, 367371, 397-398, 517, 647-650 elevated temperature, 398 endotoxin assay, 484 epifluorescent microscopy, 389 fluorescent antibody, 268, 41 1 hydrophobic grid-membrane filter (HGMF), 647-649 for isolation, 50, 52, 229, 287, 398, 442443, 447, 480-482 most probable number (MPN), 369-370. 41 1, 647-650 Petrifilm E. coli Count Plate, 647, 650 Microangiopathy, 188-1 89 Microbucterit~m,26 Micrococcaceae, 346 Micrococci, 26, 31 Micrococcus. 346 Microorganisms causing spoilage, 98, 120, 124 Milk and milk products, 49, 69-70, 79, 84, 91, 99, 193, 524, 528, 543-545, 547, 550, 552, 556, 589, 593, 598, 647 Bacillus cereus in, 65-66, 69-70, 81. 525 breast. 172-173, 177, 287, 541. 547, 552 Brucella in. 77-79, 544 Can1plobacter in, 92, 525-526, 531, 554 Clostr-iditmt in, 108, 527 Escherichia coli in, 193 Group A Streptococcus in, 543-544 Listeria nzorzocytogenes in, 223-227, 552, 560 Mycobacteriunt pn”tuberculosis in, 26 raw. 650 salmonellae in, 268-269, 286, 300, 524, 528, 542, 545, 559 shigellae in, 331 Stupllylococcus aureus or its toxins in, 528, 533, 542 yersiniae in, 472, 476-478, 542 Miller, V., 496 Molds, 125, 546, 554-555 Monosodium glutamate, 623 M0ra.t-ella. 28

Index Morbidiv m d Mortality Weekly Report, 557 Moses, 588 Murine typhoid fever. 298, 301 Mushroom(s), 522, 594 Cawlpylobacter in, 92 Clostridiwn in, 113-1 14, 527, 529 poisoning, 525, 528, 530, 533, 537-538, 623 staphylococci in, 589 Mycobacterium, 195 avian, 549 bovis, 549 paratuberculosis, 26 tubercrdosis, 549 Mycotoxins, 533-534, 542 National Advisory Committee on Microbiological Criteria for Foods, 126 National Clearinghouse for Poison Control Centers, 2 National Data Collection System, 5 National Institute of Public Health and Environmental Protection, 560 National Veterinary Services Laboratories, 274 Native American Church, 560 Netherlands Government Food Inspection Service (NGFIS), 231 Neurotoxin botulinum (BoNT), 108, 116, 121-123 mode of action of, 122 New Testament, 588 Nitrosamines, 125 North American Free Trade Agreement. 567 Norwalk-like virus, 536, 558, 598 Nucleases, heat-stable, 350-35 1 Nurnli competitive exclusion concept. 272273, 275 Old Testament, 588 Omeprazole (see Prilosec) Operation Desert Shield, 324 Opsonization, 218 Organisms, indicator. 26 Organizations, professional toxicology, 7-8 Osmolarity. 249 Outbreaks of foodborne disease, 36, 62-63, 113-116, 175, 177, 181, 192, 516567 annual number of cases, 588

Index [Outbreaks of foodborne disease] associated with fish/shellfish, 386, 407425, 440-463, 524, 528, 530, 532533, 535, 537-538, 540-542, 548551, 556 causative agents of, 590 caused by bacteria, 589, 598 caused by chemical agents, 598, 602, 622 caused by Clostridium Botulinum, 114116, 119-120, 547, 558, 560, 613 caused by Escherichia coli, 186, 55 1, 558 caused by Listeria monocytogertes, 21 6217, 222-225, 234, 559, 602 caused by parasites, 6 13 caused by salmonellae, 267, 270-27 1, 288-290,292-294, 538-539, 541, 558, 614, 616 caused by Salmonella Typhi, 254, 287, 289, 539, 602 caused by shigellae, 331, 541, 558, 561, 614 caused by Staphylococcus, 372, 558, 564 caused by Vibrio, 384, 387-388, 390, 392, 558 caused by viruses, 613 caused by yersiniae, 472, 542, 561 classification of, 590-595 contributing factors to, 61, 63, 173. 177, 186, 192, 324, 331, 371, 384, 388, 392,454-455,476, 525-526, 528, 531, 539, 543. 544-546, 548-549, 557. 596-597, 615-616 costs of, 195, 294, 552, 588 definition of, 589-590, 598 and dose-response relationship, 590 epidemiological aspects of, 599-612 environmental aspects of, 612-619, 646 fatality (mortality) rates associated with, 173, 185, 216, 223-224, 266-269, 275, 286-288, 325, 448, 462-463, 483, 533, 535, 539, 546, 550, 553, 564, 589 incubation period of, 62, 147, 153, 181, 186, 325, 371, 391, 413, 448, 475, 542, 604, 620-624 investigating, 587-639 laboratory aspects of, 616-619 prevention of, 697 reporting of, 97, 518. 587, 597-598 reports of, 63-65, 67-68, 70, 80, 85, 89, 92-95, 191 surveillance of, 515-567, 597-598

667 [Outbreaks of foodborne disease] symptoms associated with, 61-63, 66-67, 93-94, 96, 118-120, 140, 147, 151, 172, 177, 179-180, 184-185, 188, 216, 271, 286-288, 291, 325, 371, 413,448, 475-476, 484, 527, 535, 559, 603-604 types of, 589-590 viral, 598 Outbreaks of waterborne disease, 90, 94, 177, 192-193, 332, 384, 389-390, 407-425, 439-463, 477, 526, 531, 533, 538, 539-543, 548, 550-551, 553, 561, 602 Ovid, 588 Packaging controlled atmosphere (CAP), 480 vacuum, 480 Pacini, 384 Pan American Health Organization (PAHO), 550 Pan American Institute for Food Protection and Zoonoses (INPPAZ), 550 Pantoea, 28 agglomerans, 646 Parasites, 517, 532, 548, 553, 555, 558, 613 Pasteur, 189 Pasteurization, 48, 79, 87, 93, 98, 120, 226227, 300, 396,476-477,479,498, 524-526, 531, 548, 557, 589 Pathogenic processes, 37, 122, 142, 148, 154, 174-176, 178, 181-182, 184, 187-189, 191, 219-220, 274, 288, 291, 293-294, 297, 299, 325-327, 394,408,413,472,483 Pathogens drug-resistant, 589 foodborne, 24-26, 28, 44, 62, 69, 72, 7980, 85, 91, 108, 116, 120, 140, 147, 175, 177-178, 182, 189, 191, 221, 226, 247, 270, 286. 288, 292, 304, 331, 347,400,407-425,440-463, 47 1-499, 5 15-567 gram-negative, 37, 78, 85, 170, 247, 396, 412,422-424,442-443,473,488489, 645 gram-positive, 24-25, 108, 140, 214, 347, 488 population control measures for, 123- 126, 273, 275, 286, 300, 531, 566

Index [Pathogens] population reduction measures for, 86, 90-91, 98, 126, 127, 157, 227, 27 1-273, 289-290,400,420-421, 565 PCR (see Polymerase chain reaction) Pediococci, 26, 126 Pediococcus, 346 Pepto-Bismol, 291 Peptococcus, 346 Peptostreptococcus, 346 Pest control, 272 Peyote, 560 pH (see Bacteria, pH effects on) Phagocytosis, 217, 220, 451-452, 454-455, 484, 490. 492 Phenotypes, 252 Photobacterium, 24, 385, 41 1 Phytoplankton, 388, 409, 551 toxic marine, 5 17 Pig-bel, 140, 148, 539 Pilus, 174, 179 toxin co-regulated (TCP), 394 Plague, 472 Plannococcus, 346 Plants, poisonous, 588, 613 Plasmid(s), 147, 173, 178-179, 182, 184, 187, 190, 194-195, 253 adherence factor (EAF), 174 analysis, 254 cloned segment of, 182 cryptic, 254 fingerprint, 253 invasion, 334 profiles, 254, 271 resistance, 290, 296, 349 virulence, 254, 324, 328-329,472, 476, 480, 483,485, 497 Plate count, 648 aerobic (APC), 29-32 standard (SPC), 29 Plesionzorzas, 38, 385, 411 Pneumolysin, 220 POISINDEX, 2, 6 Poison arsenic, 534 diarrheic shellfish (DSP), 520, 528, 537 paralytic shellfish (PSP), 520, 532-533, 535, 537-538, 542, 623 Poison control or information centers, 1, 1020

Poisoning algal toxin, 542 bongkrekic acid, 533 cassava, 538 chemical, 590, 622 ciguatera (ciguatoxin), 530, 532-533, 535, 550, 552, 556-557, 622 food, 6, 62-63, 67, 118, 349, 356, 358, 367, 371, 543, 545, 564, 589 Jatropha seed, 538 methanol, 538 methomyl, 538 mushroom, 525, 528, 530, 537-538 nitrite, 533-534 by organochlorine compounds, 534 by organomercury compounds, 534 by organophosphorous compounds, 533534 by pesticide residues, 537-538 pufferfish, 537-538, 552 scromboid (scrombotoxin), 525, 527, 530, 532, 557, 622, 647 seaweed, 532 shellfish, 533, 538, 542, 623 sugarcane, 533 Poison Net, 10 Poison Prevention Packaging Act, 2 Polio, 589 Polymerase chain reaction (PCR), 72, 152, 155, 173, 178, 182, 187, 195, 250, 251, 255, 297, 333-335, 399,408, 412,416, 443, 447, 455, 475, 481482,485, 546, 598, 616,648, 650 Portal of entry, 274, 288, 294, 448 Poultry and poultry products, 32, 49, 62, 64, 69, 84-85, 88, 90, 92, 96-97, 99, 143, 147, 224-225, 228, 252-253, 271-272, 286, 296, 300, 524-526, 531, 536, 538, 541. 544, 547-548, 550, 552-553, 556, 560, 562, 566, 591, 647 ready-to-eat, 228 PREEMPT, 273 Probes DNA, 153, 173-174. 181, 190,409 EAF, 174 molecular, 6 16 Propionibncterilsrn, 26 Prilosec, 293 Proteins environmental stress, 296, 470

Index [Proteins] extracellular, 488 heat-shock, 296, 479 Proteus, 28, 250, 533-534, 541 vulgaris, 420 Protozoa, 44 l Pseudomonads, 32 Pseudomonas, 28, 44, 541 aeruginosa, 48. 304, 420, 489, 491-492 bathycetes, 42 1 cocovenenans ferinofennentarzs, 533 syringae, 489 Psychrobacter, 28 Ptomaines, 589 Public Health Service, 598 Pullorum disease, 252, 269, 271 Pyrazinamidase, 497 Quarantine, 272 Radappertization, 127 Ralstonia, 28 solanaceaarunt, 489 Raspberries, 589 Redox potential, 123-124, 143, 145 Regional Poison Center, 2 Reservoirs or sources of contamination animal, 36, 80, 86, 140, 149, 193, 291, 477-478 birds, wild, 88, 90, 477-478, 525 cattle, 77, 86-87, 99, 140, 215, 526, 650 domestic pets, 77, 89, 117-120, 149, 266 environmental, 36, 44, 61, 69, 90, 108, 118, 140, 149, 173, 177, 192, 215, 227, 229, 268, 270, 274, 384, 388, 390,440,472,477-479,498, 538, 539, 612-619, 646 feces, 266, 268. 270, 287. 291, 294, 371. 384, 388,477-478,526,532-533, 539. 548-549, 551. 559 feed, 272 flies, 541 food, 289, 291, 300, 371, 384, 440, 472, 476,498, 539 goats, 215, 541 humans as, 193, 271, 371, 532, 540 oysters and other seafoods, 440, 448, 454, 457-458, 462,478, 517, 535, 540, 543, 551, 554-556, 559, 647 poultry, 88, 99, 140, 149, 536, 538, 541, 544, 550, 552, 554, 556, 562

669 [Reservoirs or sources of contamination] reptiles, 265 rodents, 265, 474, 477-478, 565 sheep, 77, 87-88, 215, 223 swine, 77, 87, 149, 472, 477-478, 542, 549 water, 384, 440, 526, 533, 538-539, 540544, 548, 550-551, 553, 650 Rhizobium, 78 RiboprinterTM Microbial Characterization System, 233 Ribotypes and ribotype analysis, 227, 233234, 399,455,475 Richmond Committee, 566 Rodent control, 97 Rotavirus, 530-53 1, 544, 548, 561 Royal College of General Practitioners, 560 Ruminococcus, 346 Rush, Benjamin, 171 Salm-Net, 561 Salmon, D. E., 248 Salmonella (salmonellae), 24, 48, 63, 91, 95, 185, 247, 249-254, 265-269, 273274, 286-289, 291, 294-296, 304, 324, 33 1,413, 422, 425, 489, 49 1, 5 17, 522-526, 528-531, 533-545, 548, 550-555, 557-558, 560, 562, 565, 604, 614, 616-617, 620, 647 Abortus-Equi, 266 Abortusovis, 248, 254 Agona. 252, 254, 266, 274, 517, 524, 544, 561, 589 Anatum, 265, 268, 274 antigens of, 268 arizonae, 248, 249 bacteruria caused by, 294 berta, 524 bongori, 248 Bovismorbificans, 248, 254 Bradenburg, 274 Cerro, 267-268 chocolate contaminated by, 254, 552 Choleraesuis, 248, 250-251, 254-255, 274-275, 286, 294, 300, 304 Choleraesuis (Kuzendorf), 274 Copenhagan, 254 Derby, 265, 274 diarizonae, 248-249 DNA sequences of, 255, 297

670 [Salmonella (salmonellae)] Dublin, 252, 254-255, 268-269, 286, 294, 300. 524 Education-Reduction plan, 272 eggs contaminated by, 269-271, 286, 292, 527-528, 536, 552, 554, 557, 559, 561, 566 enterica, 248, 304 enterica bortgori, 248 entericn Choleraesuis. 248, 274 Enteritidis, 250, 252-255, 265, 268, 270271, 274, 286-288. 292, 294, 297. 300, 524-529, 535-536, 538, 540, 542, 544, 552, 557, 559. 561, 566, 589, 595, 597-598, 604, 606-607 feed contaminated by, 272 Gallinarum, 248, 249, 252, 286. 300, 544 Gallinarum-Pullorum, 252, 254, 269 Hadar, 294 haifa, 524 Heidelberg, 254, 271, 274, 292 host adaptations to, 286 houterzcre. 248 identification of, 249 indica, 248 Infantis, 254, 266-267 Livingstone, 252 marijuana contaminated by, 254 Mbandaka, 274, 530 ntinnesota, 545 molecular genetics of, 254 molecular typing of, 253-255 Montevideo, 252 mouse model for, 298-299, 301-302 Muenster, 268 multidrug-resistant, 287, 290-29 1, 294296, 542, 561, 563 Munchen, 267 Newbrunswick, 268 Newport, 265, 274, 420, 556 Ohio, 266 oranienburg, 542 Panama, 248, 254 Paratyphi, 248, 250, 252, 255, 290, 522, 542 Paratyphi A, 254 Paratyphi B, 252, 266, 528 Paratyphi C, 254 phage typing of, 253-254, 270-271, 294 plasmids of. 254 Pullorum, 248-249, 252, 286, 300 Reading, 266-268, 27 1

Index [Salmonella (salmonellae)] Saintpaul, 248, 254, 271 salami, 248 Sendai, 254 Senftenburg, 252, 254, 286-287, 527 serotypes (serovars, serogroups) of, 248, 251-255, 265-266, 268. 270. 274275, 286-287, 291-292, 294, 301302, 304, 526 Stanley, 542 in swine, 274-275, 296, 300 transovarian (ovarian) transmission of, 253. 270-271 transplacental transmission of, 293 in turkeys, 272, 287, 300 Typhi, 80, 248, 250, 254-255, 286-291, 294-295, 298, 301-304, 522, 539, 602, 631 Typhimurium, 47, 236, 253-254, 265266, 268-269, 273-274, 292-296, 298. 301, 303, 483, 491. 493, 524, 526, 531, 535, 540, 534, 556, 559, 561, 589 Typhimurium (Copenhagen), 266, 268, 274, 452 Vphosa, 248 vaccine against, 271, 275, 289, 291, 298304 virchow, 527, 535 Weltevreden, 294, 524, 540 Worthington, 274 Salmonellosis, 29, 95-96, 255, 268, 292, 300, 349, 521-522, 524, 526-532, 535-536, 540, 543, 545, 554, 556. 559-562, 566, 587, 597 abortion caused by, 266, 268 in animals, 265-275 and antacids, 293-294 and arthritis, 296-298 avian, 269-274, 526 bismuth in treatment of, 291-292 bovine mastitis associated with, 268-269 carriers of, 265, 274-275, 288, 290 in cats, 265 in cattle and dairy cows. 267-269, 300 in companion animals, 265-267, 295 conditions predisposing to, 292-293 economic losses caused by, 268, 275, 300, 565 and endovascular infections, 292 in horses, 266-267 in humans. 275, 286-304, 542

Index [Salmonellosis] immunization against, 269, 300 in immunocompromised hosts, 254, 286, 29 1-292 nontyphoid. 288, 290-293, 304 nosocomial, 293-294, 530 number of cases of, 563-564 and osteomyelitis, 293-294 in poultry, 269-274, 296, 300, 536, 538, 548 and pregnancy, 293 and Reiter's syndrome, 297 restaurant-related, 27 1 and rheumatoid arthritis, 292 and sickle cell disease, 293 and stress, 266-268, 275, 296 symptoms of, 287-288, 291-292 systemic, 287, 291 -292 and systemic lupus erythematosus, 292 treatment of, 291-293, 295-296 treatments predisposing to, 293 Sanger Centre, 495 Sanitation conditions and procedures, 88, 93, 96-99, 173, 175. 177, 274, 289, 331, 388, 399-400,477,498-499, 543, 549, 566, 651 Sanitizers, 48, 79, 86, 397. 424, 480, 498, 538 Sarcina, 346 Scrombotoxin (see Poisoning, scromboid) Seafood products, 592 Aeromonas in, 36 bacteria in, 32, 543-555, 559 Campylobucter in, 92 Clostridium in, 108, 111- 112, 115- 116. 124-125 Listeria monocytogenes in, 225, 229, 559 toxins associated with, 523, 532, 558 Vibrio parahaentolyticus in, 407-425, 533-534, 540 Vibrio spp. in, 388-389, 391-393, 535, 55 1 Vibrio vulnijicus in, 440-463, 540, 543, 56 1 Seattle-King County Department of Health. 85, 90 Serogroups, 85, 172-173, 176, 181, 184, 190, 192, 251, 287, 384-386, 474 Serotypes and serotyping, 63, 71, 86-87, 89, 94, 98, 141, 172-173, 176-177, 182-187. 190, 195, 215, 221, 223-

671 [Serotypes and serotyping] 226, 228, 234, 247, 248, 250, 252, 333, 526, 559 Kauffman-White system, 17 1, 250-25 1 of salmonellae, 25 1-255, 265-266, 268, 270, 274 of Vibrio parahaernolyticus, 412-413 of yersiniae, 472-473, 478 Serpulina hyodysenteriae, 477 Serratia, 28 Shellfish (see Fish and shellfish) Shiga, K., 324 Shigella, 24, 174, 181, 185, 193, 323-336. 489, 522-523, 535-536, 540-541, 543-544, 548-550, 555, 557-558, 561-562, 565, 614, 621 boydii, 324-325, 33 1 detection methods for, 332-335 dysenteriae, 184, 188. 324-325, 328, 331, 333, 548, 549 Jlexneri, 47, 80, 182, 324-329, 331-332, 334-335,492. 538 multidrug-resistant, 336, 563 pathogenic mechanisms of, 325-326 plasmids of, 324 serotypes of, 324 sortnei, 324-325, 328, 331 virulence of, 325-330 Shigellosis, 95, 181, 188, 323-336, 525, 529, 531-532, 538, 548, 550, 556, 562, 587 epidemiology of, 330-332 hemolytic uremic syndrome, 325, 328 in immunocompromised hosts, 325 prevention of, 335-336 and Reiter's syndrome, 325 risk factors for, 331 treatment of, 335-336 Sickle cell disease, 293 SID (see Syndrome, sudden infant death) Silage and listeriosis, 215 Sioux Honey Association of the United States, 118 Smith, Theobald, 84 Snails, 532, 549 Snow, J., 389-390 Soils, bacteria in, 23-24, 72, 90, 109-111, 118, 477 Solar radiation, 548 Sonification, 459, 461 Soy milk, 542 Spltaeroides, 552

672 Spices, bacteria in, 30-31, 69, 149 Spoilage, 98, 120, 124 Spores, bacterial, 109-111, 121, 140, 144148 in feces, 148, 151, 537 in fish, 112 in food, 11 1-1 13, 117-1 18, 126-127, 539 heat-resistant, 126, 144-145, 148, 151, 157 in honey, 537 in soil, 61, 109-111, 118, 537 Sporulation of Bncillrrs cereus, 71 of Clostridiunr pe$ringens, 140, 142, 144, 145-148, 151, 153, 157 carbohydrate effects on, 154 Sprouts, bacteria in, 193, 333-334, 524, 537, 556 Stnphylococcus, 346, 55 1 aureus, 91, 148, 331, 346-372, 517, 522524, 528, 530, 534-535, 537, 539541, 543-545, 550, 552. 554-555, 557-558, 564, 621, 647 carriers of, 348 coagulase produced by, 349-350 enterotoxins produced by, 352-363, 602 epidermidis, 348 extracellular enzymes of, 349-35 1 extracellular toxins of, 350-358 extraintestinal diseases caused by, 348349 hyaluronidase produced by, 35 1 hyicus, 347, 35 1-352 intesmedius, 35 1-352 nosocomial infections caused by, 348 nucleases of, 350-35 1 species of, 346 sublethal injury to, 363-367 toxins produced by, 351-352, 540, 542 water activity of, 349 Stomatococcus, 346 Strawberries, 589 Streptococcus, 126, 346, 525, 555, 558 Group A, 543, 621 pyogenes, 189 Streptolysin, 220 Stx (see Toxin, Shiga) Sudden infant death (SID) syndrome (see Syndrome, sudden infant death) Sugarcane, 533 Surlcus nturinus, 65 Superoxide dismutase (SOD), 221

"

Index Surveillance and other studies of foodborne disease in, 515-567, 597-598 Africa, 545-550 Algeria, 547 Argentina, 552 Asia, 532 Australia, 529-53 1 Bahrain, 545 Bolivia, 553 Brazil, 552-553 Cambodia, 538-539 Canada, 553-556 the Caribbean, 550-55 1 Central African Republic, 546 Central America, 550-553 Chile, 553 China, 533-534 Costa Rica, 553 C&e d'Ivoire, 548-549 countries of the former U.S.S.R., 528-529 and definitions of terms used in, 5 19-521 Egypt, 547 Ethiopia, 547-548 Europe, 560-561 France, 528 Hong Kong, 535 India and neighboring regions and islands, 54 1-543 Indonesia, 539 Iran, 545 Israel, 543-544 Japan, 535-537 Jordan, 544 Kenya, 547-548 Korea, 535-537 KwaZulu-Natal, 549 Lebanon, 544 Liberia, 548-549 Madagascar, 550 Malawi, 546 Malaysia, 539-540 Mexico, 552 Mozambique, 546-547 Nepal, 543 New Guinea, 539 New Zealand, 531-532 Nigeria, 549 and objectives of, 517-519 Oceania, 532 and pathogens encountered in, 523 Peru, 55 1-552 Poland, 528

Index [Surveillance and other studies of foodborne disease in] Saudi Arabia, 544-545 and sentinel studies in, 518, 560-563, 566, 600 Singapore, 540-54 1 South Africa, 549 South America, 550-553 Sudan, 547 Taiwan, 533-544 Tanzania, 547-548 Thailand, 538 the United States, 556-560 Viet Nam, 537-538 Yemen, 545 Zambia, 548 Zimbabwe, 548 Syndrome endotoxic shock, 472, 483 Guillain-Barr&,94, 520, 541, 589 hemolytic uremic (HUS), 183, 185-186, 188-189, 325, 519, 526, 531, 546, 553, 561, 564, 650 predisposing, 454-455, 463 Reiter's, 297, 325, 475 sudden infant death (SID), 140, 148 toxic shock, 35 1-352 Taeniasis, 525 Taenia solium, 539, 542 Temperature effects of on pathogens, 50, 62-63, 70, 85-87, 90-91, 96, 98, 126, 140, 143,146, 149,151, 157, 175,178, 226, 228, 249, 347-348, 388, 393, 409,419,440-441,456-457,461, 478-479,483,494,495, 525, 532, 535, 539, 541, 543, 545-547, 549, 556, 565, 615 low, 31, 44, 87, 91, 98, 153, 388, 395, 409,424,457,461,472,480 Tetrodotoxin, 537-538, 552 Tests and test systems, 51, 70, 288, 297298, 326. 618-619 CAMP, 214-215 latex particle agglutination, 335 McNemar, 6 10-6 11 mouse model for Salmonella, 298-299, 30 1-302 mouse model for yersiniae, 486, 497, 541 mouse protection, 302 mouse virulence, 254, 301, 485

673 [Tests and test systems] multilocus enzyme electrophoresis (MEE), 226, 248 pulsed field gel electrophoresis (PFGE), 616-617 rapid, 52, 71-72. 152, 250, 333-335 Streny, 174, 181, 326, 328, 330, 488, 497 for virulence of yersiniae, 496-498 Therapy with antibiotics, 477 with botulinum neurotoxin, 123 oral rehydration, 178, 392 Toxic Exposure Surveillance System, 5 Toxicant chemicals in food, 522, 622 Toxins, 71, 533-534, 537 accessory cholera (ACE), 393, 395 algal, 542 associated with seafoods, 523, 532, 535, 555, 558 cholera, 41, 175, 178, 291, 384, 386, 392 of Clostridium botulinum, 560 of Clostridium perfj-ingens, 141-142, 148, 152, 154 diarrheic, 63-64, 66, 70, 72 emetic, 62-65, 67, 70, 72-73 of Escherichia coli, 176, 178, 181, 189, 191, 291, 304, 393, 526 fungal, 532, 542, 546 geographic variations in, 179 heat-labile, 178, 291, 393, 552 heat-stable, 178, 191, 349-351, 394-395, 473-474,484, 540, 550 labile (LT), 520, 552 Nag-ST, 394-395 Shiga (Stx), 171, 173, 184, 187-189, 195, 291, 325, 328, 650 Shiga-like (SLT), 181, 184, 325, 395, 520, 531, 552, 646,650 therapeutic uses of, 122-123 of Vibrio, 393-395, 449-454, 463 of yersiniae, 484, 542 zonal occludens (ZOT), 393, 395 Toxoplasma, 530 gondii, 547, 564 Toxoplasmosis, 536, 564 and chorioretinitis, 536 Transmission airborne, 79, 173, 192, 348, 498 by apple cider, 193, 598 of Canlpylobacter, 88, 92, 99, 541 by contaminated food/feed or water, 4849, 192-193, 270, 272, 286, 291-

674 [Transmission] 292, 300, 331-332, 389,400,439463, 471-499, 526,531-533, 538543, 548, 550-551, 553, 598, 650 of diarrheagenic Escherichia coli, 191194, 546-547, 598 by direct contact, 79, 92, 348, 590 fecal-oral, 89, 173, 177, 192-193, 266, 288, 332, 532, 541 foodborne, 590 by fresh produce, 193, 228, 331, 390, 478, 553, 559, 589, 593, 598 by houseflies, 332 by ingestion of contaminated hamburger, 182, 193, 598 nosocomial, 171, 173, 192, 221, 223, 267, 270, 287, 292-294, 331, 348, 525, 530 person-to-person, 89, 148, 186, 18 1, 192193, 286, 292. 294, 324, 331-332, 348, 526, 532. 537-538, 545, 590. 598-600 transovarian, 253 transplacental, 293 waterborne, 590, 598 from wild to domestic animals, 80 Traum, J. E., 79 Treponema lzyodysenteriae, 477 Trichinella, 522 spiralis, 624 Trichinosis, 275, 286, 525, 528-529, 550, 565 Trichothecenes, 542 Tuberculosis, 549 Tung nuts, 535 Turkeys, Salmonella-free, 272 Typhoid fever, 247, 254, 265, 286-291, 294, 298, 300-301, 303-304, 529, 539, 545, 550, 598, 600, 604 and carcinoma, 290 and cholelithiasis, 290 murine, 298, 301 United States Department of Agriculture (USDA), 227-228, 23 1-232, 274, 562, 565-566, 588 University of California, Davis, 267 USDA (see United States Department of Agriculture) Vaccines, 271, 275, 289, 298-304, 392, 400, 553

Index Van Reclilinhausen, 348 V-CJD (see Creutzfeldt-Jakob disease, variant) Vector(s), 288, 332 Vegetables bacteria in, 30, 36, 49, 66, 96, 112-1 17, 193, 222, 228, 478, 525-527, 529, 542-544, 546-548, 552-553, 556, 559, 562, 565, 594, 647, 650 brined, 228 ready-to-use, 29, 30 Vehicles of transmission, 92, 173, 181-182, 191-194, 221, 223, 228, 348. 408, 440,472,498-499, 541, 544, 548, 552-553, 561, 595, 597-598, 611, 624 Verocytotoxin. 650 Verotoxin (VT), 184, 520, 526, 537, 554, 650 Veterinary Medical Teaching Hospital, 267 Viable but nonculturable (VBNC) state, 389, 424,455,459-463 Vibrio, 28, 38, 50, 420, 533. 535-537, 562 biochemical characteristics of species, 444-445 biogroups of, 446, 455 classification of, 385-386, 411, 442 Vibrio alginolyticus, 398, 411, 418, 442, 444-446 Vibrio cnnpbellii, 41 1 Vibrio carchariae, 444-446 Vibrio cholerne, 24, 174-175. 177-178, 303, 324, 383-401,456,462, 540541, 545, 551, 558, 647 antimicrobial effects on, 397 biochemical characterization of, 386-387, 444-446 biotypes of, 384 classical, 384 control of, 399-401 detection of, 397-399 distribution of, 387 ecology of, 388 effects of disinfectants on, 397 El Tor, 384, 397 enterotoxin of, 395 genetics of, 394 non-01, 391, 417, 621 non-01/0139, 391-395, 399 0 1 , 391, 395-399, 551, 621 0139, 385, 388, 397-400 0139 Bengal, 386. 399

Index [Vibrio cholerael pH effects on, 397 relation to plankton, 388, 551 serogroups of, 384-386, 391 susceptibility to antimicrobials, 397 temperature effects on, 395-396 toxins of, 393-394 transmission of, 389-390 viable but nonculturable state (VBNC) of, 389, 424 water requirements of, 396-397 Vibrio cincinnatiensis, 444-446 Vibrio comma, 384 Vibrio damela, 444-446 Vibrio fetus, 84 Vibrio fluvialis, 37, 52, 391. 411, 444-446 Vibrio firnissii, 444-446 Vibrio haneyi, 4 11 Vibrio hollisae, 391, 412, 417, 444-446 Vibrio jejuni, 84 Vibrio ntetschnikovii, 444-446 Vibrio mimicus, 386, 388, 391, 398 biochemical characteristics of, 444-446 enterotoxin of, 386 Vibrionaceae, 37-38, 44, 385-386, 41 1, 442 Vibrio natriegens, 41 1 Vibrio parahaentolyticus, 24, 391, 398, 407425, 442, 456, 462, 525, 530, 533, 535-536, 539-540, 553, 622, 647 adherence to algae, 409 and antibiotics, 420 association with chitin, 410, 42 1 association with mollusks, 409-410, 540 association with seafoods, 407-425 association with zooplankton, 409 biochemical characteristics of, 444-446 and biological inhibitors, 420 and cardiogenic shock, 414 causes disease in shellfish, 418 cell envelope of, 422, 425 cellular activities of, 421 detection of, 411 enterotoxin of, 413 and food additives, 420 gastroenteritis caused by, 410, 413, 556 geographic distribution of, 409 growth requirements of, 418 hemolysin and other toxins produced by, 413-417 identification of, 4 11 Kanagawa phenomenon in, 409-410, 412, 414,416,418,420-421,423

675 [Vibrio parahaemolyticus] needs NaCl, 418, 424 nonpathogenic strains of, 410 pH effects on, 419, 424 and physical inhibitors, 420-421 radiation effects on, 419-420 serological classification of, 4 12-41 3 survival under stress, 423-424 taxonomy of, 41 1 temperature effects on, 409, 419, 424, 556 virulence factors of, 415 water requirements of, 418 Vibrio vulnijicus, 391, 393, 398, 411, 424, 439-463, 622 antibiotic sensitivity of. 458 associated with raw oysters and other seafoods, 440, 448,454, 457-458,462, 535, 540, 543 biochemical characteristics of, 444-446 biogroups of, 442, 446, 448-449, 455 eaten by protozoa, 441 ecology of, 440, 441 effect of food additives on, 458 effect of host gender on pathogenicity, 454 effect of serum iron on, 450-451 effect of starvation on, 458-462 fatal infections caused by, 440, 448, 463 gastroenteritis caused by, 440-463 genetics of, 455 geographic distribution of, 40, 441 growth inhibitors for, 456-458 growth requirements of, 441 hemolysin and other toxins produced by, 449-455, 463 host susceptibility factors, 455-456 isolation of, 442-443, 447 molecular identification of, 447-448 pathogenic for eels, 448-449, 455 predisposing syndromes, 454-455, 463 radiation effects on, 458 requires NaC1, 456 resistance to sonification, 459, 461 septicemia caused by, 440-441, 448, 452, 462 serovars of, 442, 447 survival characteristics of, 441, 463 taxonomy of, 442 temperature effects on, 440-441, 456457,459-462

Index

676 [Vibrio vulr~ijicus] viable but nonculturable (VBNC) state of, 455,459-463 virulence factors for, 449-455, 463 wound infections caused by, 440, 448449, 452,458, 462, 535 Viral hemorrhagic fever, 546 Virulence, 80, 179,182,184,195, 220-221, 252-254, 268, 270, 275, 286, 288, 291,298-300, 304, 326-327, 394395, 408,449-455,463,472, 474, 483-498, 546 Virus(es) enteric, 517, 522, 554, 647 hemorrhagic fever, 546 hepatitis A, 517, 525, 532-533 hepatitis E, 533 human immunodeficiency (HIV), 190, 224, 293, 332, 547, 557 Nonvalk agent, 624 Norwalk-like, 536, 558, 598 small round structured (SRSV), 520, 525, 527, 530 Volunteers, human, tests on, 37, 80, 153, 171,174,177,179-180,185, 190193, 302-303, 324, 391-392, 394395, 417

Water activity, 46,120,123,143,145,226,249, 347, 349, 396,418, 531, 565 Aerornonas in, 36 bacteria in, 24, 61, 90, 177, 192-193, 287, 291, 332,410, 439-463,477-479, 498, 526, 531, 533, 538-543, 548, 550-55 1, 553, 646, 650 effects of chilled, 87-88, 98 purification by solar radiation, 548 Websites DNA sequences, 472 Pathogen Modeling Program, 70 poison center, 10 toxicology, 10 Yersinia pestis gene sequences, 495 WHO (see World Health Organization) WHO/FAO Surveillance Programme for the Control of Foodborne Infections and Intoxications, 521, 567 Working Group on Vacuum Packing and Associated Processes, 126 World Health Organization (WHO), 248, 253, 397, 517, 566, 587-588

Xanthomonas, 28, 489 cantpestris, 489 Xenophon, 588

Yeasts, 125, 554-555 Yellowstone National Park, 80 Yersinia (yersiniae), 24, 174, 254, 326, 47 1499, 529, 561-562 autoagglutination by, 497 binding of Congo Red by, 496 binding of crystal violet, 497 biotypes of, 472 calcium requirements of, 487, 496 cell invasion capabilities of, 485-486, 496 characteristics of, 473 classification of, 474-475 enteropathogenic, 472 flagellar involvement in virulence, 496 growth and survival of, 479-480 identification of species, 472, 482 iron acquisition by, 486-487, 497 iron protein genes in, 487 isolation methods, 480-482 nonpathogenic, 472 outer membrane proteins (Yops), 488 outer proteins (Yops), 488-495 pathogenicity of, 483-498 phage typing of, 474 pH effects on, 482 pyrazinamidase activity in, 497 requirement for iron, 476, 483-484 reservoirs of, 477-479 resistance to effects of serum by, 497 serotypes of, 472, 478 susceptibility to chlorine, 498 temperature effects on, 480, 483, 494-495 tests for pathogenicity of, 496-498, 542 toxins produced by, 484, 542 type I11 protein secretions of, 489-491 virulence attenuation in, 487 virulence gene regulation in, 493-496 virulence plasmids of, 480, 483, 485, 487-489, 497 Yersiniabactin, 486-487 Yersinia bercovierii, 473 Yersinia enterocolitica, 47, 96, 472, 555, 622, 647 associated with pork, 474, 478 biotypes of, 473 gastroenteritis caused by, 474, 542 rodent carriage of, 474 septicemia caused by, 477

Index [Yersinia enterocolitica]

serotypes of, 473-474 temperature effects on, 472, 478-479 virulence plasmids of, 474 Yersinia kristensenii, 473, 485-486, 497 Yersinia molleretti, 473 Yersinia pseudotuberculosis, 472 associated with pork, 479 reservoirs of, 479 serogroups of, 474 temperature effects on, 472 virulence plasmids of, 474 Yersinia pestis, 472, 474, 486-488, 495 Yersinia ruckeri, 474

677 Yersiniosis, 471-499, 525-526, 529, 531, 589 clinical manifestations of, 475-476, 497 control of, 498 foodborne outbreaks of, 476-477 mechanism of infection, 476 mimics acute appendicitis, 475, 477 prevention of, 498 role of arsenicals in, 477-478 Y. pestis Sequencing Group, 495 Zoonosis, 77, 84, 86, 93, 96, 99, 214, 286, 472 Zooplankton, 388, 409, 410