Advances in Food and Nutrition Research, Vol.60

ADVISORY BOARDS KEN BUCKLE University of New South Wales, Australia

MARY ELLEN CAMIRE University of Maine, USA

ROGER CLEMENS University of Southern California, USA

HILDEGARDE HEYMANN University of California, Davis, USA

ROBERT HUTKINS University of Nebraska, USA

RONALD JACKSON Quebec, Canada

HUUB LELIEVELD Global Harmonization Initiative, The Netherlands

DARYL B. LUND University of Wisconsin, USA

CONNIE WEAVER Purdue University, USA

RONALD WROLSTAD Oregon State University, USA

SERIES EDITORS GEORGE F. STEWART

(1948–1982)

EMIL M. MRAK

(1948–1987)

C. O. CHICHESTER

(1959–1988)

BERNARD S. SCHWEIGERT (1984–1988) JOHN E. KINSELLA

(1989–1993)

STEVE L. TAYLOR

(1995–

)

Academic Press is an imprint of Elsevier 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA 32 Jamestown Road, London NW1 7BY, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands Linacre House, Jordan Hill, Oxford OX2 8DP, UK First edition 2010 Copyright # 2010 Elsevier Inc. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material. Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. ISBN: 978-0-12-380944-5 ISSN: 1043-4526 For information on all Academic Press publications visit our website at elsevierdirect.com Printed and bound in USA 10 11 12 10 9 8 7 6 5 4 3 2 1

CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

 Elaine D. Berry

U.S. Department of Agriculture, Agricultural Research Service, U.S. Meat Animal Research Center, Clay Center, Nebraska, USA (67)  Gokhan Boran

Department of Food Engineering, Yu¨zu¨ncu¨ Yıl University, Van, Turkey (119)  Regina M. B. Franco

Departamento de Parasitologia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil (1)  E. C. Henley

EC Henley Consulting; University of Georgia, Athens, Georgia, USA (21)  Diego A. G. Leal

Departamento de Parasitologia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil (1)  S. D. Obukosia

Africa Harvest Biotechnology Foundation International, Nairobi, Kenya (21)  Karen Signori Pereira

Departamento de Engenharia Bioquı´mica, Escola de Quı´mica, Centro de Tecnologia Bloco E – Sala 203, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil (1)  Joe M. Regenstein

Department of Food Science, Cornell University, Ithaca, New York, USA (119)  Maja Rupnik

Institute of Public Health Maribor, Centre for Microbiology; Faculty of Medicine, University of Maribor, Maribor, Slovenia (53)  J. Glenn Songer

Department of Veterinary Microbiology and Preventive Medicine, Department of Veterinary Diagnostic and Production Animal

vii

viii

Contributors

Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa, USA (53)  J. R. N. Taylor

Department of Food Science, University of Pretoria, Pretoria, South Africa (21)  James E. Wells

U.S. Department of Agriculture, Agricultural Research Service, U.S. Meat Animal Research Center, Clay Center, Nebraska, USA (67)

CHAPTER

1 Transmission of Toxoplasmosis (Toxoplasma gondii) by Foods Karen Signori Pereira,*,1 Regina M. B. Franco,† and Diego A. G. Leal†

Contents

Abstract

I. II. III. IV. V. VI. VII. VIII.

Toxoplasmosis Discovery T. gondii Life Cycle Transmission of Toxoplasmosis Pathogenesis and Human Infection Spectra Laboratory Diagnosis and Treatment Toxoplasmosis Transmission by Foods Toxoplasmosis Outbreaks Associated with Water and Foods IX. T. gondii Control (in Foods) References

2 3 3 5 6 8 10 12 13 15

Protozoan foodborne diseases are generally underrecognized. Toxoplasma gondii is the causative agent of toxoplasmosis, one of the most prevalent parasitic infections to humans and domestic animals. The most likely source of T. gondii occurring through food is the consumption of raw or undercooked meat contaminated with tissue cysts. Sporulated T. gondii oocysts, from the feces of infected cats, present in the environment are a potential source of infection. The ingestion of water contaminated with oocysts and

* Departamento de Engenharia Bioquı´mica, Escola de Quı´mica, Centro de Tecnologia Bloco E – Sala 203, {

1

Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil Departamento de Parasitologia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil Corresponding author: Karen Signori Pereira, E-mail address: [email protected]

Advances in Food and Nutrition Research, Volume 60 ISSN 1043-4526, DOI: 10.1016/S1043-4526(10)60001-0

#

2010 Elsevier Inc. All rights reserved.

1

2

Karen Signori Pereira et al.

the eating of unwashed raw vegetables or fruits were identified as an important risk factor in most epidemiological studies. This review presents information and data to show the importance of T. gondii transmission by foods.

I. TOXOPLASMOSIS Toxoplasma gondii is the causative agent of toxoplasmosis, one of the most prevalent parasitic infections that afflict humans and other warm-blooded animals; T. gondii is the only known species associated with toxoplasmosis (Tenter et al., 2000). It is estimated that approximately one-third of the human population worldwide have the parasite. Congenital toxoplasmosis is a special concern related to T. gondii infection which can be especially serious for the fetus if the mother is seronegative, that is, if the mother acquires the primary infection during the pregnancy (Wong and Remington, 1994). The first case of congenital toxoplasmosis in humans was reported by Wolf and Cowen (1937), who identified this protozoan parasite in the brain of a 3-day-old infant with encephalomyelitis. Until the 1940s, there was a scarcity of data of human toxoplasmosis with only a few isolated occurrence reports in children or adults. Improved knowledge about the real prevalence of toxoplasmosis in different regions of the world was made possible with the advent of Sabin–Feldman serologic test, also known as the dye-test, in 1948 (Sabin and Feldman, 1948). Two years later, the protozoan Toxoplasma was associated with an inflammatory disease of the eye (Frenkel and Jacobs, 1958). Over the past several decades, toxoplasmosis has been increasingly recognized as a significant disease with strong implications for public health due to the seriousness of the sequelae derived from the parasitism and associated especially with the congenital or ocular forms. However, the disease has acquired even more importance since 1980, relating to its emergence as an opportunistic pathogen (Wong and Remington, 1993), especially among patients with AIDS, where concomitant infection with the parasite does not often present a favorable prognosis. Moreover, the incidence of toxoplasmosis, either the acquired or congenital forms even in healthy individuals with no immunological impairments, is high in many countries, although most affected individuals do not experience clinical symptoms (Neves et al., 2009). Toxoplasmosis is frequently misdiagnosed or underdiagnosed. Toxoplasmosis is the third most common cause of hospitalization due to foodborne infection overall (Mead et al., 1999).

Transmission of Toxoplasmosis (Toxoplasma gondii) by Foods

3

II. DISCOVERY T. gondii is a coccidian protozoan parasite that belongs to the Phylum Apicomplexa and Family Sarcocystidae, with a worldwide distribution (Smith, 2007). This parasite was described in 1908 by Alfonso Splendore, who identified this organism in a rabbit that died from parasitic disease (Splendore, 1908). In that same year, Nicolle and Manceaux (1909) found the same parasite in an African rodent and denominated this new organism as Leishmania gondii. Subsequently, these same researchers verified that the parasite earlier described as Leishmania did not possess the marked characteristics of protozoan parasites of the Phylum Kinetoplastida and thus they proposed the name T. gondii, based on the shape of the tachyzoite form of this parasite (‘‘toxon: arc; plasma: life’’) (Nicolle and Manceaux, 1909).

III. T. GONDII LIFE CYCLE The life cycle of T. gondii includes felines, the definitive hosts that shed oocysts into the environment (Frenkel et al., 1970). It is well established that oocysts can be stable in the environment for up to a year (Dubey, 1998a). This parasite is also capable of infecting a wide range of other warm-blooded animals (mammals and birds) that are the intermediate hosts. Upon infection, the sporozoites located in two sporocysts inside the oocysts are released and establish a new infection in the enterocytes of both intermediate and definitive hosts. Thus, T. gondii has a life cycle that is comprised of three infective stages: tachyzoites, bradyzoites (in tissue cysts), and sporozoites (in oocysts). The asexual developmental cycle of this protozoan occurs within any nucleated cell of warm-blooded animals. This cycle consists of two infective stages: tachyzoites, which undergo fast multiplication in various host cell types and are typically present in the acute infection, and bradyzoites, which undergo slow multiplication in latent tissue cysts, have a high affinity for neural and muscular tissues and are located predominantly in the central nervous system, the eye, and skeletal and cardiac muscles. The bradyzoites characterize the chronic phase of toxoplasmosis. According to Dubey et al. (1998a,b), cysts can also develop, but to a lesser extent, in any visceral organs, such as lungs, liver, and kidneys. While infected host cells are filled by tachyzoites and are destroyed by their release, the bradyzoites contained in tissue cysts continue the process of multiplication through endodyogeny (Tenter, 2009). The interaction between T. gondii and host cells involves an early stage of adhesion and posterior invasion of the host cell; these steps are both

4

Karen Signori Pereira et al.

crucial for the establishment of parasite infection. The parasitophorous vacuole is an interface between the parasite and host cell functions and facilitates the replication and differentiation of the parasite. In addition, this vacuole provides protection against free radicals, pH, and osmolarity changes, and also assists the parasite in the mechanism of evasion and activation of the host immune system (Laliberte´ and Carruthers, 2008). Intermediate hosts become infected with T. gondii after ingesting infective oocysts that were shed in the environment or tissue cysts from infected animals. Parasite replication in the small intestine eventually leads to the lysis of enterocytes and consequently, tachyzoites disseminate throughout the host (Petersen and Dubey, 2001). By using stagespecific markers such as SAG1 for tachyzoites and BAG1 for bradyzoites, it was possible to follow the life cycle transitions through the acute and chronic phases in live mice (Ferguson, 2009). Briefly, during the early stages of infection, tachyzoites are observed initially in the lymph nodes, spleen, and lungs, and 10 days postinfection, dissemination may occur to all organs of the body, including the brain and heart. The conversion of tachyzoite to bradyzoite occurs between 12 and 15 days postinfection and cyst formation takes place in the brain of the mouse. More tissue cysts are produced in mice that become mildly ill from infection than in those that become highly symptomatic (Weiss and Kim, 2007). In mice, the protozoan showed marked tissue tropism, since no evidence of stage conversion is observed in any other organ (however, there is a variation between different host species; in cats, the majority of tissue cysts are found in the muscles), and individual parasites may express both tachyzoite and bradyzoite antigens simultaneously as revealed by dual labeling techniques (Ferguson, 2009). While some organisms continue to proliferate as tachyzoites before being recognized and destroyed by the host immune response, other parasites invade new cells and develop into bradyzoites contained in tissue cysts. The mechanism that triggers the conversion of tachyzoites into bradyzoites is still not completely understood, but it appears that tissue cyst development is initiated at the time of parasite entry in the cell with the formation of a distinctive vacuole. After approximately 3 months, there is a reduction in the number of dividing organisms. The size of tissue cysts is variable (5–70 mm) and may harbor a few to several hundred bradyzoites (Dubey, 2004). Generally, in most immunocompetent hosts, the immune response controls the replication, disease is limited, and the physiological stress suffered by the parasites causes the differentiation of the tachyzoites into bradyzoite cysts that may persist throughout the host’s life (Tenter et al., 2000). Tissue cysts undergo periodic reactivation, but these events are modulated by an intact immune system. In chronically infected hosts that lose T-cell function, reactivation may lead to disease (Petersen and Dubey,

Transmission of Toxoplasmosis (Toxoplasma gondii) by Foods

5

2001). It is noteworthy that tissue cysts are the terminal life cycle stage in the intermediate hosts and are immediately infectious. If ingested by a definitive host (members of Family Felidae), the bradyzoites initiate another asexual phase of proliferation in the epithelial cells of the small intestine. Then, the sexual phase of the life cycle is initiated with gamogony culminating in the formation of oocysts. Unsporulated oocysts are released into the intestinal lumen and are passed into the environment with the feces of felids. In the case of this apicomplexan protozoan, sporogony occurs outside the host and leads to the development of infectious oocysts (10 mm
12 mm in diameter) which contain two sporocysts, each containing four sporozoites (Dubey et al., 1998a). The sporulation of the oocysts is directly dependent on environmental factors such as temperature and aeration, and after 1–5 days, the oocysts may become infectious (Dubey, 2004). Cats can shed upward of 360 million oocysts in their feces in a single day and oocysts were shed for 4–6 days (Dubey, 2002). These felines are essential in the life cycle of T. gondii (Dubey and Su, 2009).

IV. TRANSMISSION OF TOXOPLASMOSIS Toxoplasma infection can be transmitted by the ingestion of oocysts—shed into the environment from cat feces—which may contaminate water, soil, and vegetables, or also by viable tissue cysts found in raw or undercooked meat of intermediate hosts. Oocysts are highly infectious to herbivores and bradyzoites to cats. Infections caused through the ingestion of oocysts are considered more severe clinically in intermediate hosts than those related through the ingestion of tissue cysts (Hill and Dubey, 2002). Thus, one factor that contributes to the widespread distribution of T. gondii on all continents consists of the successful adaptability and development of different transmission modes exhibited by this protozoa (Dubey and Su, 2009) and that carnivorism and cannibalism contribute to and aggravate the persistence of the protozoan in nature, even in the absence of the felid sexual cycle (Su et al., 2003). As tachyzoites are sensitive to environmental conditions (and they usually die very rapidly outside the host), foodborne transmission of T. gondii via tachyzoite is probably not significant epidemiologically and occurs only infrequently (Tenter, 2009). Tachyzoites of T. gondii have already been detected in body fluids such as saliva, sputum, urine, tears, semen, and milk of several intermediate hosts, including sheep, goats, cows, and camels (Tenter et al., 2000). From literature data, there is great evidence that tachyzoites present in colostrum or maternal milk can infect infants via breast-feeding when mothers have a primary T. gondii infection (Bonametti et al., 1997b;

6

Karen Signori Pereira et al.

Tavares et al., 2006). The lower concentration of proteolytic enzymes in the gastrointestinal tract of a child may explain these cases. In addition, Dubey et al. (1998a,b), reported that tachyzoites may occasionally survive for a short period of time (about 2 h) in acid pepsin solutions. Moreover, oral infections by tachyzoite penetration in the host oral mucosal tissue were hypothesized by Johnson (1997), Riemann et al. (1975), and Sacks et al. (1982). On the other hand, bradyzoites of T. gondii are more resistant to digestive enzymes such as pepsin and trypsin (Dubey, 1998b; Jacobs et al., 1960). It was also demonstrated that T. gondii cysts maintain their infectivity at temperatures of 4  C over a period of 30 days (Tenter, 2009), while heating is the most efficient way to kill T. gondii tissue cysts (Kijlstra and Jongert, 2008a).

V. PATHOGENESIS AND HUMAN INFECTION SPECTRA Infection with T. gondii is an important cause of diseases of the central nervous system and the eye in immunocompromised as well as immunocompetent individuals. When first acquired by the mother, this infection can be transmitted to the fetus. Infants with the most severe clinical signs in the brain and eye are those infected early in pregnancy when fetal immunity is low ( Jamieson et al., 2009). At birth, infants infected in utero may have intracranial calcification, hydrocephalus, convulsions, and ocular diseases such as retinochoroiditis or inflammation of the retina and choroid, with associated vitritis. The severity of disease is influenced by the trimester in which the infection is acquired by the mother (Dunn et al., 1999; Remington et al., 2006). A positive correlation exists between the rate of transmission and infection during the second or third trimesters of pregnancy (Desmonts and Couvreur, 1984; Dunn et al., 1999). In mothers previously exposed to T. gondii, the fetus is very rarely infected (Remington et al., 2006), which suggests that natural maternal immunity against T. gondii is sufficient to protect the fetus from vertical transmission. Untreated acute toxoplasmosis among pregnant women can lead to infection of the fetus via transplacental transmission (Varella et al., 2009.). At first examination, newborns affected by congenital infection may seem normal; however, serious sequelae, such as neurological impairment and blindness, can develop within a few years later (Dunn et al., 1999; Remington et al., 2006; Safadi et al., 2003.). Some reports have suggested that pregnant women diagnosed with acute toxoplasmosis should be treated as soon as possible to reduce the risk and severity of congenital infection (Gilbert et al., 2001; Gras et al., 2005.). Moreover, the gestational age at primary infection is a critical factor that determines the clinical management of pregnant women, since the severity

Transmission of Toxoplasmosis (Toxoplasma gondii) by Foods

7

of toxoplasmosis for the fetus decreases and the transmission rate increases with enhanced gestational age (Beguetto et al., 2003). When the mother is infected in the first trimester of pregnancy, abortion or stillbirth can occur. When mothers acquired their first infection in the second or third trimester, only 15% and 5% of children presented with a subclinical infection form at birth (Gras et al., 2005). Lopes et al. (2009) conducted a study with 492 pregnant women in Londrina, Parana´ State, Brazil, in order to determine the risk factors associated with anti-T. gondii seropositivity, using a multivariate regression analysis approach. Age group, a low level of education, a low per capita income (< US$ 88.23), presence of a cat in the house, and the habit of eating green vegetables were considered important risk factors that might be related to the acquisition of the disease, while Spalding et al. (2005) showed that pregnant women who had contact with soil had the greatest risk of acquiring toxoplasmosis. It is well established that the strain of Toxoplasma can have an effect on the pathology of the infections (Dubey, 1998a). The manifestations of the disease can vary significantly from one host to another and different components contribute to the severity of the disease, including (i) host species, (ii) immune status of host, and (iii) biological and genetic variation within the parasite (Innes, 1997). While asymptomatic infection with T. gondii resulting in a latent infection with tissue cysts is common in humans, symptomatic infection is much less frequent. In immunocompetent individuals, symptoms only occur in 10–20% of the cases; infected people develop chorioretinitis, lymphadenitis, myocarditis, or polymyositis. Although any lymph node may be infected, the most common manifestation is asymptomatic cervical lymphadenopathy (Weiss and Dubey, 2009). In acute phase, the clinical manifestations include a mononucleosislike syndrome, fever, lymph node enlargement, asthenia, and headache (Remington et al., 2006). Neves et al. (2009) related the most frequent signs and symptoms in a cohort of 37 patients that attended Evandro Chagas Clinic Research Institute, FIOCRUZ, Brazil, as follows: lymph node enlargement was the most commonly observed alteration (35/37; 94.6%), abdominal ultrasound reveals liver enlargement in six patients (6/37; 16.2%), and splenomegaly in two patients (2/37; 5.4%). Asthenia (32/37; 86.5%), headache (26/37; 70.3%), fever (25/37; 67.6%), and weight loss (23/27; 62.2%) were the most common symptoms. Immunocompromised patients that develop clinical disease have impairments in T-cell function, thus highlighting the importance of lymphocytes in controlling this persistent infection. In addition, the presence of bradyzoites and their subsequent rupture can cause life-threatening recrudescence of acute infection in immunocompromised individuals (Sullivan et al., 2009). The immune response effectively prevents

8

Karen Signori Pereira et al.

dissemination of this protozoan, but sometimes a spontaneous reactivation of the latent infectious occurs. Production of Interferon-gamma (IFN-g) by T cells, natural killer cells, and various other cell types in the brain protects the host against toxoplasmic encephalitis. The production of IFN-g by microglia during the early stages of tachyzoite proliferation in the brain may be a critical factor in limiting parasitic growth (Wang and Suzuki, 2007). The most vulnerable risk groups to acquire the infection and develop severe toxoplasmosis include persons with primary or acquired immunodeficiency and several deficits in T cell, monocyte, cytokine, and B cell functions; cancer patients with immunosuppressive cancers such as leukemia and lymphoma; transplant patients receiving immunosuppressive drugs and patients with hyper-IgM syndrome as well as those receiving corticosteroids (Weiss and Dubey, 2009). HIV-infected patients have an increased incidence of encephalomyopathy and encephalitis is the most important clinical manifestation of toxoplasmosis in these patients (Dubey, 2004; Horowitz et al., 1983.; Jones et al., 1996.). Nowadays, although widespread use of highly active anti-retroviral therapy (HAART) has led to better control of viral replication promoting an increase in immune function, new immunosuppressed cohorts are continually emerging like patients with autoimmune disorders treated with immunomodulatory factors (Hemmer et al., 2006.). Ocular toxoplasmosis is the most frequent cause of infectious blindness and visual morbidity among young adults in developed countries (Hovakimyan and Cunningham, 2002). The severity of the disease varies greatly between patients. The activation of dormant organisms within the retina causes necrotizing retinopathy and the severity of the disease is probably correlated with the most common causes of retinochoroiditis worldwide, hypersensitivity and inflammation (Garweg and Candolfi, 2009). Recent evidence has identified interleukin (IL)-17 as a marker for disease severity. Other mediators include IL-23 which induces the proliferation of IL-17 producing cells and IL-27 which may regulate TH1-cellmediated responses. CD25(þ) regulatory T cells may control the local inflammatory response and protect the host against collateral inflammatory tissue damage (Garweg and Candolfi, 2009).

VI. LABORATORY DIAGNOSIS AND TREATMENT According to Mozzato and Procianoy (2003), laboratory diagnosis poses a challenge for health care professionals due to the complexity in the interpretation of the results. Moreover, we need to consider that the modern laboratory techniques which have arisen in last decades are not

Transmission of Toxoplasmosis (Toxoplasma gondii) by Foods

9

always available in the national public health care system of the developing countries by comparison to the more developed countries. Generally, the diagnosis of toxoplasmosis in man may be done by serologic tests, PCR (which involves the amplification of specific nucleic acid sequences), histologic demonstration of the parasite, or by isolation of the protozoan that might be done by an animal infectivity assay or inoculation in human tissue cell cultures (Montoya, 2002). Serologic test is the primary method of diagnosis and utilize a specific antibody to T. gondii. Other immunological methods include complement fixation tests, direct agglutination tests, ELISA, indirect agglutination tests, an immunosorbent agglutination test, and a latex agglutination test. Toxoplasmosis is usually diagnosed based on the detection of specific IgG and IgM antibodies; however, the inclusion of other tests is mandatory for a conclusive diagnosis of toxoplasmosis during pregnancy, in HIV/AIDS patients and in neonates ( Jones et al., 2003.). These tests must include measurement of IgG avidity, IgA, IgE, and direct detection by PCR in all cases, including the amniotic fluid. Since IgG can persist for decades, IgM which typically persists for 6–9 months is used as a marker of recent infection. While prenatal diagnosis is based on the detection of T. gondii in the amniotic fluid, neonatal screening is based on the detection of parasites in the placenta and on the detection of IgM and IgA antibodies in newborns. PCR for the detection of parasite DNA in amniotic fluid has improved the sensitivity of prenatal diagnosis (Bessie´res et al., 2009). The accurate diagnosis of congenital toxoplasmosis is essential, since if the mother is treated it would reduce the probability of fetal infection by 50% (Desmonts and Couvreur, 1974). Treatment of toxoplasmosis may vary with the form or the case. Generally, in immunocompetent patients treatment is usually unnecessary since the infection is subclinical and the immune response is able to control it. However, in immunocompromised patients (including HIV and other risk groups), the patients need to be treated and monitored since toxoplasmosis is a major cause of death among AIDS patients (Dubey, 2004). In these patients, the recommended treatment is a combination of two drugs, pyrimethamine (25–100 mg daily) and trisulfapyrimidines (2–6 g daily), administered for 1 month where this combination acts by inhibiting the enzyme, dihydrofolate reductase, of T. gondii preventing the synthesis of DNA and proteins. In some cases, as in cerebral toxoplasmosis, which is frequently seen in HIV patients but may occur with atypical manifestations, it is extremely important to do the differential diagnosis from several other neurological infections like lymphoma and other cancers (Montoya, 2002). The diagnosis of cerebral toxoplasmosis might be done in association with complementary methods such as imaging and immunological

10

Karen Signori Pereira et al.

methods principally on cerebrospinal fluid and blood (Montoya, 2002; Schroeder et al., 2006.). The cerebral biopsy is considered the best and most definitive method of diagnosis for cerebral toxoplasmosis because this method may demonstrate the presence of tachyzoites. However, this approach is used sparingly (Hornef et al., 1999) because it is an invasive procedure. The finding of lesions in the brain demonstrated with imaging methods and a positive serological test for toxoplasmosis must guide the physician to a specific therapeutic approach against the protozoa. In the case of ocular toxoplasmosis where the signs and symptoms may be misdiagnosed as some other disease, the patients may present with a photophobia and see floaters; then a differential diagnosis is also necessary. The ophthalmologic examination with a slit-lamp allows the physician to observe the presence of a granulomatous inflammation and the fundoscopy demonstrates the presence of a yellow focus of retinochoroiditis (Guex-Crosier, 2009). The treatment for ocular toxoplasmosis consists of the administration of sulfadiazine which interferes with the formation of folic acid from para-aminobenzoic acid, associated with pyrimethamine, which interferes with the conversion of folic acid to folinic acid through dihydropteroate synthase (Guex-Crosier, 2009). For many years, ocular toxoplasmosis was traditionally considered a manifestation of congenital and postnatal infections. However, in the last decade, several studies have shown that most of the ocular disease caused by T. gondii is attributed to acquired disease and this fact was also evidenced in the epidemiological study of some waterborne outbreaks (Gilbert and Stanford, 2000; Holland, 1999). Ocular disease may also occur by the reactivation of the parasite when the cysts are present within the retina (Guex-Crosier, 2009). Summarizing, it is reasonable to assume that the investigation of ocular toxoplasmosis does not have to be focused only in pregnant women or in immunocompromised individuals but also must include the immunocompetent people.

VII. TOXOPLASMOSIS TRANSMISSION BY FOODS One-third of the human world population is infected with the protozoan parasite T. gondii. Recent calculations of the disease burden of toxoplasmosis rank this foodborne disease at the same level as salmonellosis or campylobacteriosis (Kijlstra and Jongert, 2008a). T. gondii does not grow in foods or in other environments outside of a suitable host; however, oocysts in environmental can survive for long periods at temperatures ranging from 4 to 37  C. According to Waldeland (1977a,b,c), an oocyst can survive in soil for up to 2 years; so, any fecal material from infected cats will represent a hazard (Nesbakken, 2009).

Transmission of Toxoplasmosis (Toxoplasma gondii) by Foods

11

As noted earlier, toxoplasmosis is acquired by ingesting food and water contaminated with oocysts from feces of infected cats or by the ingestion of raw or undercooked meat, containing tissue cysts (bradyzoites), of an infected intermediary host. Transmission of human toxoplasmosis occurs mainly through the ingestion of food containing cysts of T. gondii, found in sheep, pigs, cows, chickens, and goats. Uncooked pork and its derivatives have been the main foods implicated in outbreaks of toxoplasmosis. However, while consumption of raw or undercooked meat was consistently identified as a risk factor, the relative importance of the risk factor and the type of meat associated with it varied among different countries (Tenter et al., 2000). Belford-Neto et al. (2007)., in Erechim (Rio Grande do Sul state, Brazil), collected samples from porcine tongue and diaphragm obtained in both large and small abattoirs and used molecular biological techniques to determine the prevalence of T. gondii. Seventeen out of 50 (34%) samples from the diaphragm and 33 out of 50 (66%) samples from the tongue demonstrated a positive PCR reaction for T. gondii. In Londrina, Parana´ state of Brazil, 149 samples of sausage were collected from eight factories. Using a mouse bioassay, 13 (8.7%) sausage samples were positive; in one of them T. gondii was isolated and in the other 12, the mice seroconverted (Dias et al., 2005). In the United States, the survey of 698 retail outlets determined the prevalence of viable T. gondii tissue cysts in commercially available fresh pork products to be 0.38% (Dubey et al., 2005). In a study by Warnekulasuriya et al. (1998), in London, T. gondii was detected in ready-to-eat cured meat samples by amplification of the parasite’s P30 gene using PCR. Viable T. gondii was detected in 1 (cured ham) out of 67 ready-to-eat cured meat samples. In 2002, Aspinall et al. (2002) analyzed, using primers specific for the T. gondii SAG2 locus, 71 meat samples obtained from UK retail outlets and found that 27 of the meat samples showed the presence of this parasite. While toxoplasmosis outbreaks have been mainly related to the ingestion of undercook meat, a few outbreaks could be attributed to milk ingestion; T. gondii was isolated in the milk of naturally infected cows (Hiramoto et al., 2001). Unpasteurized goat milk was implicated as a source of infection of T. gondii in several reports (Chiari and Neves, 1984; Riemann et al., 1975; Skinner et al., 1990). So, unpasteurized milk and dairy products could be an important source of human infection with T. gondii. A study from Hiramoto et al. (2001) assessed the infectivity of cysts of the ME-49 strain of T. gondii in artificially infected bovine milk and derived fresh homemade cheese. The infectivity of cysts of the ME-49 strain of T. gondii was maintained in the milk even after storage for 20 days at refrigerator temperatures. Cysts were also able to survive the production process of homemade fresh cheese and storage for a period of 10 days in the same conditions.

12

Karen Signori Pereira et al.

According to Kapperud et al. (1996) oocysts from cat feces can contaminate fields and therefore vegetable products. The eating of unwashed raw vegetables or fruits was identified as an important risk factor in most epidemiological studies (Dorny et al., 2009). Waterborne outbreaks of toxoplasmosis are possible if water is untreated, and consuming seafood from contaminated water basins may pose a theoretical risk. Experimentally, viable T. gondii oocysts were recovered from oysters (Crassostrea virginica; Lindsay et al., 2004) and mussels (Mytilus galloprovincialis; Arkush et al., 2003).

VIII. TOXOPLASMOSIS OUTBREAKS ASSOCIATED WITH WATER AND FOODS Between 1999 and 2008, in Brazil, four toxoplasmosis outbreaks associated with the ingestion of contaminated water and/or foods were registered according to the Brazilian Ministry of Health (Secretaria de Vigilaˆncia em Sau´de (SVS) of Brasil, 2009). According to Mead et al. (1999), T. gondii was associated with the thirdhighest number of deaths caused by foodborne pathogens in the United States. Research from Hoffmann et al. (2007) found that toxoplasmosis is the third most common cause of fatal foodborne disease associated with pork ingestion. In the United States, T. gondii and Listeria monocytogenes are the most important foodborne pathogens in pregnancy, and these organisms can induce death or grave disease in the fetus and newborn (Smith, 1999). Outbreaks of toxoplasmosis are rarely seen since most infected immunocompetent individuals show few or no symptoms. However, 20 toxoplasmosis outbreaks were well described and discussed by Smith in 1993. Of them, 5 were associated with the ingestion of raw goat milk, 11 were associated with the ingestion of raw meat, and 1 was associated with the ingestion of creek water (Smith, 1993). Some outbreaks of toxoplasmosis associated with the ingestion of oocyst-contaminated water have been documented. Benenson et al. (1982) described an outbreak that occurred among British troops in Panama in the jungle associated with the consumption of creek water probably contaminated with oocysts excreted by jungle cats. Another outbreak occurred in British Columbia, Canada in 1995, and 110 acute toxoplasmosis cases were identified. The epidemiological studies demonstrated that the outbreak was consistent with a waterborne source and implicated contaminated municipal drinking water (Bowie et al., 1997). The largest toxoplasmosis outbreak was registered in Santa Isabel do Ivai, Parana´ state, Brazil, and unfiltered, municipally treated water was

Transmission of Toxoplasmosis (Toxoplasma gondii) by Foods

13

the epidemiologically implicated source of T. gondii dissemination. The outbreak peaked between November 2001 and January 2002, and involved at least 426 people (Moura et al., 2006). An outbreak of acquired ocular toxoplasmosis involving 248 people in India was described by Balasundaram et al. (2010). The suspected source of the infection was municipal drinking water. Chiari and Neves (1984) described an acute toxoplasmosis outbreak involving three members of the same family in Minas Gerais State, Brazil. The source of the infection was unpasteurized goat’s milk. Another outbreak, involving 10 people, associated with ingestion of unpasteurized goat’s milk was registered by Sacks et al. (1982) and occurred in California, USA. Despite these episodes, the most likely source of T. gondii infection is the consumption of raw or undercooked meat contaminated with tissue cysts. In 1969, an outbreak of acute toxoplasmosis involved five medical students from Cornell Medical College. The students all ate rare hamburger at the same place, on the same night, and evidence indicates that this was the vehicle for transmission of the infection (Kean et al., 1969). Fertig et al. (1977) related a toxoplasmosis outbreak involving three people in London; there was no direct contact with cats, and the relevant communal meal taken included inadequately grilled lamb. In Brazil, Bandeirantes city (Parana´ State), one registered outbreak of acute toxoplasmosis involved 17 people. The illness was acquired by the ingestion of raw mutton offered during a party in September 1993 (Bonametti et al., 1997a). In 2005, 10 people from the same family acquired toxoplasmosis in Santa Vito´ria do Palmar (Rio Grande do Sul State, Brazil). Epidemiological studies pointed to the source of the outbreak as an industrialized cured meat (copa) (Tavares et al., 2006). Two outbreaks of acute toxoplasmosis in Korea were linked to eating undercooked pork. In the first, three people were infected after eating a meal consisting of raw spleen and liver from a wild pig. In the second outbreak, five soldiers were infected after eating a meal of raw liver from a domestic pig (Choi et al., 1997.; Dawson, 2005). In Australia, a family outbreak of toxoplasmosis involving five members of a Lebanese family was described by De Silva et al. (1984). Kibbi, a traditional Lebanese dish, prepared with raw meat may have been the source of the infection. Another outbreak occurred in Australia and was associated with ingestion of undercooked kangaroo and lamb meat (Robson et al., 1995).

IX. T. GONDII CONTROL (IN FOODS) T. gondii control in foods is primarily associated with adequate cooking and/or prevention of cross-contamination. According to Hillers et al. (2003), the main consumer food-handling behaviors associated with

14

Karen Signori Pereira et al.

prevention are, respectively: (i) use a thermometer to make sure that meat and poultry (including ground) are cooked to safe temperatures, (ii) keep pets out of food preparation areas, (iii) wash hands with warm soapy water before and after handling raw foods, and (vi) knives, cutting boards, and food preparation surfaces should be washed with hot water and soap after contact with raw poultry, meat, and seafood. Washing is effective because the stages of T. gondii in meat are killed by contact with soap and water (Dubey and Beattie, 1988). Commercial procedures of curing with salt or low temperature smoke and the treatment of meat with enhancing solutions such as potassium or sodium lactate, can also kill T. gondii tissue cysts, although the inactivation of these cysts depends of the synergistic interaction between salt concentration, maturation time, and temperature of storage (Kijlstra and Jongert, 2008b). With contaminated pork and pork products, Hill et al. (2004) found that the injection, within 8 h, of 2.0% NaCl or 1.4% or higher lactate-based salt solutions into pork loins containing infective tissue cysts prevented transmission of T. gondii. Storage at meat case temperatures at or below 0  C (32 F) for 7 days also killed T. gondii tissue cysts in pork loins (Hill et al., 2004.). Lunde´n and Uggla (1992) investigated the effects of curing with sodium chloride and sucrose, low-temperature smoking, freezing at  20  C, and cooking in a microwave oven, respectively, on the infectivity of T. gondii encysted in mutton. T. gondii was not isolated from cured, smoked, or frozen meat. However, in two of four steaks processed in a microwave oven, according to a standard household recipe, the parasite remained infective, possibly due to uneven heating of the meat. The tissue cysts of T. gondii are relatively resistant to changes in temperature and remain infectious in refrigerated (1–4  C) carcasses or minced meat for up to 3 weeks (Tenter, 2009). According to Dubey (2004), tissue cysts in meat are killed by heating to an internal temperature of 67  C or by cooling to  13  C. However, occasionally some tissue cysts may survive deepfreezing and it has even been suggested that some strains of T. gondii may be resistant to freezing (Tenter, 2009). More recently, modern food processing technologies using irradiation or high pressure treatment were analyzed for the inactivation of the parasite with promising results (Kijlstra and Jongert, 2008a). High pressure processing (HPP) at 340, 400, 480, and 550 MPa has been shown to inactivate T. gondii oocysts under experimental conditions (Lindsay et al., 2005). T. gondii in tissue cysts and oocysts are also killed by exposure to 0.5 krad of g-irradiation (Dubey, 1998a,b.; Dubey and Thayer, 1994).

Transmission of Toxoplasmosis (Toxoplasma gondii) by Foods

15

REFERENCES Arkush, K. D., Miller, M. A., Leutenegger, C. M., Gardner, I. A., Packham, A. E., Heckeroth, A. R., Tenter, A. M., Barr, B. C., and Conrad, P. A. (2003). Molecular and bioassay-based detection of Toxoplasma gondii oocyst uptake by mussels (Mytilus galloprovincialis). Int. J. Parasitol. 33(10), 1087–1097. Aspinall, T. V., Marlee, D., Hyde, J. E., and Sims, P. F. G. (2002). Prevalence of Toxoplasma gondii in commercial meat products as monitored by polymerase chain reaction – food for thought? Int. J. Parasitol. 32(9), 1193–1199. Balasundaram, M. B., Andavar, R., Palaniswamy, M., and Venkatapathy, N. (2010). Outbreak of acquired ocular toxoplasmosis involving 248 patients. Arch. Ophthal. 128(1), 28–32. Beguetto, E., Buffolano, W., Spadoni, A., Del Pezzo, M., Di Cristina, M., Minenkova, O., Petersen, E., Felici, F., and Gargano, N. (2003). Use of an Immunoglobulin G avidity assay based on recombinant antigens for diagnosis of primary T. gondii infection during pregnancy. J. Clin. Microbiol. 41, 5414–5418. Belford-Neto, R., Nussenblatt, V., Rizzo, L., Muccioli, C., Silveira, C., Nussenblatt, R., Khan, A., Sibley, D., and Belford, R., Jr. (2007). High prevalence of unusual genotypes of Toxoplasma gondii infection in pork meat samples from Erechim, Southern Brazil. An. Acad. Bras. Cieˆnc. 79(1), 111–114. Benenson, M. W., Takafuji, E. T., Lemon, S. M., Greenup, R. L., and Suler, A. J. (1982). Oocysttransmitted toxoplasmosis associated with ingestion of contaminated water. N. Engl. J. Med. 307(11), 666–669. Bessie´res, M. H., Berrebi, A., Cassaing, S., Fillaux, J., Cambus, J. P., Berry, A., Assouline, C., Ayoubi, J. M., and Magnaval, J. F. (2009). Diagnosis of congenital toxoplasmosis: prenatal and neonatal evaluation of methods used in Toulouse University Hospital and incidence of congenital toxoplasmosis. Mem. Inst. Oswaldo Cruz 389–392. Bonametti, A. M., Passos, J. N., da Silva, E. M. K., and Bortoliero, A. L. (1997a). Surto de toxoplasmose aguda transmitida atrave´s da ingesta˜o de carne crua de gado ovino. Rev. Soc. Bras. Med. Trop. 30(1), 21–25 [online]. Bonametti, A. M., Passos, J. N., da Silva, E. M. K., and Macedo, Z. S. (1997b). Probable transmission of acute toxoplasmosis through breast feeding. J. Trop. Pediatr. 43, 116. Bowie, W. R., King, A. E., Werker, D. H., Isaac-Renton, J. L., Bell, A., Eng, S. B., and Marion, S. A. (1997). Outbreak of toxoplasmosis associated with municipal drinking water. The BC Toxoplasma investigation team. Lancet 350(9072), 173–177. Chiari, C. A. and Neves, D. P. (1984). Toxoplasmose humana adquirida atrave´s da ingesta˜o de leite de cabra. Mem. Inst. Oswaldo Cruz 79, 337–340. Choi, W. Y., Nam, H. W., Kwak, N. H., Huh, W., Kim, Y. R., Kang, M. W., Cho, S. Y., and Dubey, J. P. (1997). Foodborne outbreaks of human toxoplasmosis. J. Infect. Dis. 175(5), 1280–1282. Dawson, D. (2005). Foodborne protozoan parasites. Int. J. Food Microbiol. 103(2), 207–227. De Silva, L. M., Mulcahy, D. L., and Kamath, K. R. A. (1984). Family outbreak of toxoplasmosis: a serendipitous finding. J. Infect. 08, 163–167. Desmonts, G. and Couvreur, J. (1974). Toxoplasmosis in pregnancy and its transmission to the fetus. Bull. N. Y. Acad. Med. 50, 146–159. Desmonts, G. and Couvreur, J. (1984). Toxoplasmose conge´nitale. Ann. de Pediatr. (Paris) 31, 805–809. Dias, R. A. F., Navarro, I. T., Ruffolo, B. B., Bugni, F. M., Castro, M. V., and Freire, R. I. (2005). Toxoplasma gondii in fresh pork sausage and seroprevalence in butchers from factories in Londrina, Parana´ state, Brazil. Rev. Inst. Med. Trop. Sao Paulo 47(4), 185–189. Dorny, P., Praet, N., Deckers, N., and Gabriel, S. (2009). Emerging food-borne parasites. Vet. Parasitol. 163(3), 196–206.

16

Karen Signori Pereira et al.

Dubey, J. P. (1998a). Toxoplasma gondii oocysts survival under defined temperatures. J. Parasitol. 84, 862–865. Dubey, J. P. (1998b). Re-examination of resistance of Toxoplasma gondii tachyzoites and bradyzoites to pepsin and trypsin digestion. Parasitology 116, 43–50. Dubey, J. P. (2002). Tachyzoite-induced life cycle of Toxoplasma gondii in cats. J. Parasitol. 88, 713–717. Dubey, J. P. (2004). Toxoplasmosis – a waterborne review. Vet. Parasitol. 126, 57–72. Dubey, J. P. and Beattie, C. P. (1988). Toxoplasmosis of Animals and Man. CRC Press, Boca Raton, FL, 220 pp. Dubey, J. P. and Su, C. (2009). Population biology of Toxoplasma gondii: What’s out and where did they come from? Mem. Inst. Oswaldo Cruz 104, 190–195. Dubey, J. P. and Thayer, D. W. (1994). Killing of different strains of Toxoplasma gondii tissue cysts by irradiation under defined conditions. J. Parasitol. 80, 764–767. Dubey, J. P., Lindsay, D. S., and Speer, C. A. (1998a). Structures of Toxoplasma gondii tachyzoites, bradyzoites and sporozoites and biology and development of tissue cysts. Clin. Microbiol. Rev. 11, 267–299. Dubey, J. P., Thayer, D. W., Speer, C. A., and Shen, S. K. (1998b). Effect of gamma irradiation on unsporulated and sporulated Toxoplasma gondii oocysts. Int. J. Parasitol. 28(3), 369–375. Dubey, J. P., Hill, D. E., Jones, J. L., Hightower, A. W., Kirkland, E., Roberts, J. M., Marcet, P. L., Lehmann, T., Vianna, M. C. B., Miska, K., Sreekumar, C., Kwok, O. C. H., Shen, S. K., and Gamble, H. R. (2005). Prevalence of viable Toxoplasma gondii in beef, chicken, and pork from retail meat stores in the United States; risk assessment to consumers. J. Parasitol. 91, 1082–1093. Dunn, D., Wallon, M., Peyron, F., Petersen, E., Peckham, C., and Gilbert, R. (1999). Mother-tochild transmission of toxoplasmosis: Risk estimates for clinical counseling. Lancet 353, 1829–1833. Ferguson, D. J. P. (2009). Toxoplasma gondii: 1908–2008, homage to Nicolle, Manceaux and Splendore. Mem. Inst. Oswaldo Cruz 104, 133–148. Fertig, A., Selwyn, S., and Tibble, M. J. (1977). Tetracycline treatment in a food-borne outbreak of toxoplasmosis. Br. Med. J. 1, 1064. Frenkel, J. K. and Jacobs, L. (1958). Ocular toxoplasmosis. Arch. Ophthal. 48, 260–279. Frenkel, J. K., Dubey, J. P., and Miller, N. L. (1970). Toxoplasma gondii in cats: Fecal stages identified as coccidian oocysts. Science 167, 893–896. Garweg, J. G. and Candolfi, E. (2009). Immunopathology in ocular toxoplasmosis: Facts and clues. Mem. Inst. Oswaldo Cruz 104, 211–220. Gilbert, R. E. and Stanford, M. R. (2000). Is ocular toxoplasmosis caused by prenatal or postnatal infection? Br. J. Ophtalmol. 84, 224–226. Gilbert, R., Dunn, D., Wallon, M., Hayde, M., Prusa, A., Lebech, M., Kortbeer, T., Peyron, F., Pollak, A., and Petersen, E. (2001). Ecological comparison of the risks of mother-to-child transmission and clinical manifestations of congenital toxoplasmosis according to prenatal treatment protocol. Epidemiol. Infect. 127, 113–120. Gras, L., Wallon, M., Pollak, A., Cortina-Borja, M., Evengard, B., Hayde, M., Petersen, E., and Gilbert, R. (2005). Association between prenatal treatment and clinical manifestations of congenital toxoplasmosis in infancy: A cohort study in 13 European centres. Acta Paediatr. 94, 1721–1731. Guex-Crosier, Y. (2009). Update on the treatment of ocular toxoplasmosis. Int. J. Med. Sci. 6, 140–142. Hemmer, B., Frohman, E., Hartung, H. P., and Stiive, O. (2006). Central nervous system infections – a potential complication of systemic immunotherapy. Curr. Opin. Neurol. 19, 271–276. Hill, D. and Dubey, J. P. (2002). Toxoplasma gondii: Transmission, diagnosis and prevention. Clin. Microbiol. Infect. 8, 634–640.

Transmission of Toxoplasmosis (Toxoplasma gondii) by Foods

17

Hill, D. E., Sreekumar, C., Gamble, H. R., and Dubey, J. P. (2004). Effect of commonly used enhancement solutions on the viability of Toxoplasma gondii tissue cysts in pork loin. J. Food Prot. 67(10), 2230–2233. Hillers, V. N., Medeiros, L., Kendall, P., Chen, G., and DiMascola, S. (2003). Consumer foodhandling behaviors associated with prevention of 13 foodborne illnesses. J. Food Prot. 66 (10), 1893–1899. Hiramoto, R. M., Mayrbaurl-Borges, M., Galisteo, A. J., Jr., Meireles, L. R., Macre, M. S., and Andrade, H. F., Jr. (2001). Infectivity of cysts of the ME-49 Toxoplasma gondii strain in bovine milk and homemade cheese. Rev. Sau´de Pu´blica 35(2), 113–118. Hoffmann, S., Fischbeck, P., Krupnick, A., and McWilliams, M. (2007). Using expert elicitation to link foodborne illnesses in the United States to foods. J. Food Prot. 70(5), 1220–1229. Holland, G. N. (1999). Reconsidering the pathogenesis of ocular toxoplasmosis. Am. J. Ophthalmol. 128, 502–505. Hornef, M. W., Iten, A., Maeder, P., Villemure, J. G., and Regli, L. (1999). Brain biopsy in patients with acquired immunodeficiency syndrome. Arch. Intern. Med. 159, 2590–2596. Horowitz, S. L., Bentson, J. R., Benson, F., Davos, I., Pressman, B., and Gotlieb, M. S. (1983). CNS toxoplasmosis in acquired immunodeficiency syndrome. Arch. Neurol. 40, 649–652. Hovakimyan, A. and Cunningham, E. T., Jr. (2002). Ocular toxoplasmosis. Ophthalmol. Clin. North Am. 15, 327–332. Innes, E. A. (1997). Toxoplasmosis: comparative species susceptibility and host immune response. Comp. Immunol. Microbiol. Infect. Dis. 20, 131–138. Jacobs, L., Remington, J. S., and Melton, M. L. (1960). The resistance of the encysted form of Toxoplasma gondii. J. Parasitol. 46, 11–21. Jamieson, S. E., Cordell, H., Petersen, E., Mcleod, R., Gilbert, R. E., and Blackwell, J. M. (2009). Host genetic and epigenetic factors in toxoplasmosis. Mem. Inst. Oswaldo Cruz 104, 162–169. Johnson, A. M. (1997). Speculation on possible life cycles for the clonal lineages in the genus Toxoplasma. Parasitol. Today 13, 393–397. Jones, J. L., Hanson, D. L., Chu, S. Y., Ciesielski, C. A., Kaplan, J. E., Ward, J. W., and Navin, T. R. (1996). Toxoplasmic encephalitis in HIV-infected persons: Risk factors and trends. The adult/adolescent spectrum of disease group. AIDS 10, 1393–1399. Jones, J. L., Lopez, A., Wilson, M., Schulkin, J., and Gibbs, R. (2003). Congenital toxoplasmosis: A review. Obstet. Gynecol. Surv. 56, 296–305. Kapperud, G., Jenum, P. A., Stray-Pedersen, B., Melby, K. K., Eskild, A., and Eng, J. (1996). Risk factors for Toxoplasma gondii infection in pregnancy. Results of a prospective casecontrol study in Norway. Am. J. Epidemiol. 144, 405–412. Kean, B. H., Kimball, A. C., and Christenson, W. N. (1969). An epidemic of acute toxoplasmosis. JAMA 208(6), 1002–1004. Kijlstra, A. and Jongert, E. (2008a). Control of the risk of human toxoplasmosis transmitted by meat. Int. J. Parasitol. 38, 1359–1370. Kijlstra, A. and Jongert, E. (2008b). Toxoplasma-safe meat: Close to reality? Trends Parasitol. 25, 18–21. Laliberte´, J. and Carruthers, V. B. (2008). Host cell manipulation by the human pathogen Toxoplasma gondii. Cell. Mol. Life Sci. 65, 1900–1915. Lindsay, D. S., Collins, M. V., Mitchell, S. M., Wetch, C. N., Rosypal, A. C., Flick, G. J., Zajac, A. M., Lindquist, A., and Dubey, J. P. (2004). Survival of Toxoplasma gondii oocysts in Eastern oysters (Crassostrea virginica). J. Parasitol. 90(5), 1054–1057. Lindsay, D. S., Collins, M. V., Jordan, C. N., Flick, G. J., and Dubey, J. P. (2005). Effects of high pressure processing on infectivity of Toxoplasma gondii oocysts for mice. J. Parasitol. 91(3), 699–701. Lopes, F. M. R., Mitsuka-Bregano´, R., Gonc¸alves, D. D., Freire, R. L., Karigyo, C. G. T., Wedy, G. F., Matsuo, T., Reiche, E. M. V., Morimoto, H. K., Capobiango, J. D., Inoue, I. T., Garcia, J. L., and Navarro, I. T. (2009). Factors associated with seropositivity

18

Karen Signori Pereira et al.

for anti-Toxoplasma gondii antibodies in pregnant women of Londrina, Parana´, Brazil. Mem. Inst. Oswaldo Cruz 104, 378–382. Lunde´n, A. and Uggla, A. (1992). Infectivity of Toxoplasma gondii in mutton following curing, smoking, freezing or microwave cooking. Int. J. Food Microbiol. 15, 357–363. Mead, P. S., Slutsker, L., Dietz, V., McCaig, L. F., Bresee, J. S., Shapiro, C., Griffin, P. M., and Tauxe, R. V. (1999). Food-related illness and death in the United States. Emerging Infect. Dis. 5(5), 606–625. Montoya, J. G. (2002). Laboratory diagnosis of Toxoplasma gondii infection and toxoplasmosis. J. Infect. Dis. 185, S73–S82. Moura, L., de Moura, Lenildo, Bahia-Oliveira, L. M. G., Wada, M. W., Jones, J. L., Tuboi, S. H., Carmo, E. H., Ramalho, W. M., Camargo, N. J., Trevisan, R., Grac¸a, R. M. T., Silva, A. J., et al. (2006). Waterborne toxoplasmosis, Brazil, from field to Gene. Emerging Infect. Dis. 12 (2), 326–329. Mozzato, L. and Procianoy, R. S. (2003). Incidence of congenital toxoplasmosis in Southern Brazil: A prospective study. Rev. Inst. Med. Trop. 45, 147–151. Nesbakken, T. (2009). Food safety in a global market – Do we need to worry? Small Rumin. Res. 86(1), 63–66. Neves, E. S., Bicudo, L. N., Al Curi, E., Carregal, E., Bueno, W. F., Ferreira, R. G., Amendoeira, M. R., Benchimol, E., and Fernandes, O. (2009). Acute toxoplasmosis: clinical-laboratorial aspects and ophthalmologic evaluation in a cohort of immunocompetent patients. Mem. Inst. Oswaldo Cruz 104, 393–396. Nicolle, C. and Manceaux, L. (1909). Sur un protozoaire nouveau du gondi. C. R. Acad. Sci. 148, 369. Petersen, E. and Dubey, J. P. (2001). Biology of Toxoplasmosis. In ‘‘Toxoplasmosis’’, (D. H. M. Johnson and T. G. Wreghitt, eds), pp. 1–42. Cambridge University Press, Cambridge. Remington, J. S., McLeod, R., Thulliez, P., and Desmonts, G. (2006). Toxoplasmosis. In ‘‘Infectious Diseases of the Fetus and Newborn Infant’’, ( J. S. Remington, J. O. Klein, C. B. Wilson, and C. J. Baker, eds), 6th edn, pp. 947–1091. Philadelphia: Elsevier Saunders. Riemann, H. P., Meyer, M. E., Theis, J. H., Kelso, G., and Behymer, D. E. (1975). Toxoplasmosis in an infant fed unpasteurized goat milk. J. Pediatr. 87, 573–576. Robson, J. M. B., Wood, R. N., Sullivan, J. J., Nicolaides, N., and Lewis, B. R. (1995). A probable foodborne outbreak of toxoplasmosis. Commun. Dis. Intell. 19, 517–522. Sabin, A. A. B. and Feldman, H. A. (1948). Dyes as microchemical indicators of a new immunity phenomenon affecting a protozoan parasite (Toxoplasma). Science 108, 660–663. Sacks, J. J., Roberto, R. R., and Brookes, N. F. (1982). Toxoplasmosis infection associated with raw goat’s milk. J. Am. Med. Assoc. 248, 1728–1732. Safadi, M. A., Berezin, E. N., Farhat, C. K., and Carvalho, E. S. (2003). Clinical presentation and follow up of children with congenital toxoplasmosis in Brazil. Braz. J. Infect. Dis. 7, 325–331. Schroeder, P. C., Post, M. J. D., Oschatz, E., Stadler, A., Bruce-Gregorios, J., and Thurnher, M. M. (2006). Analysis of the utility of diffusion-weighted MRI and apparent diffusion coefficient values in distinguishing central nervous system toxoplasmosis from lymphoma. Neuroradiology 48, 715–720. Secretaria de Vigilaˆncia em Sau´de (SVS) of Brasil (2009). Doenc¸as Transmitidas por Alimentos. Ana´lise Epidemiolo´gica dos Surtos de Doenc¸as Transmitidas por Alimentos no Brasil. http://portal.saude.gov.br/portal/arquivos/pdf/surtos_dta_15.pdf, (Accessed 12 September 2009). Skinner, L. J., Timperley, A. C., Wightman, D., Chatterton, J. M., and Ho-Yen, D. O. (1990). Simultaneous diagnosis of toxoplasmosis in goats and goatowner’s family. Scand. J. Infect. Dis. 22, 359–361.

Transmission of Toxoplasmosis (Toxoplasma gondii) by Foods

19

Smith, J. L. (1993). Documented outbreaks of toxoplasmosis transmission of Toxoplasma gondii to humans. J. Food Prot. 56(7), 630–639. Smith, J. L. (1999). Foodborne infections during pregnancy. J. Food Prot. 62(7), 818–829. Smith, D. D. (2007). The Sarcocystidae: Sarcocystis, Frenkelia, Toxoplasma, Besnoitia, Hammondia, and Cystoisospora. J. Eukaryot. Microbiol. 28(2), 262–266. Splendore, A. (1908). Un nuovo protozoa parassita deconigli d’une malattia che ricorda in molti punti il kala-azar dell’uoma. Nota preliminare. Rev. Soc. Sci. Sa˜o Paulo 3, 109–112. Spalding, S. M., Amendoeira, M. R. R., Klein, C. H., and Ribeiro, L. C. (2005). Serological screening and toxoplasmosis exposure factors among pregnant women in South of Brazil. Rev. Soc. Bras. Med. Trop. 38, 173–177. Su, C., Evans, D., Cole, R. H., Kissinger, J. C., Ajioka, J. W., and Sibley, L. D. (2003). Recent expansion of Toxoplasma through enhanced oral transmission. Science 299, 414–416. Sullivan, W. J., Jr., Smith, A. T., and Joyce, B. R. (2009). Understanding mechanisms and the role of differentiation in pathogenesis of Toxoplasma gondii – A review. Mem. Inst. Oswaldo Cruz 104, 155–161. Tavares, A. L. C., Souza, A., Fabres, A. D., Almeida, M. A. B., Junior, L. R. A., Carmo, G. M. I., Arau´jo, W. N., Garcia, M. H. O., Reis, A. K. V., Figueiredo, D. M. S., Branco, N., Franco, R. M. B., and Hatch, D. L. (2006). Surto intra-familiar de toxoplasmose, Santa Vito´ria do Palmar, RS. Bol. Eletroˆnico Epidemiol. http://portal.saude.gov.br/portal/ arquivos/pdf/boletim_03_06.pdf, (Accessed 12 December 2009). Tenter, A. M. (2009). Toxoplasma gondii in animals used for human consumption. Mem. Inst. Oswaldo Cruz 104, 364–369. Tenter, A. M., Heckeroth, A. R., and Weiss, L. M. (2000). Toxoplasma gondii: From animals to humans. Int. J. Parasitol. 30(12–13), 1217–1258. Varella, I. S., Canti, I. C. T., Santos, B. R., Coppini, A. Z., Argondizzo, L. C., Tonin, C., and Wagner, M. B. (2009). Prevalence of acute toxoplasmosis infection among 41, 112 pregnant women and the mother-to-child transmission rate in a public hospital in South Brazil. Mem. Inst. Oswaldo Cruz 104, 383–388. Waldeland, H. (1977a). Toxoplasmosis in sheep I. Long-term epidemiological studies in four breeding flocks. Acta Vet. Scand. 18(2), 227–236. Waldeland, H. (1977b). Toxoplasmosis in sheep. II. Influence of various factors on the antibody contents. Acta Vet. Scand. 18(2), 237–247. Waldeland, H. (1977c). Toxoplasmosis in sheep. III. Hematological, serological and parasitological studies. Acta Vet. Scand. 18(2), 248–256. Wang, X. and Suzuki, Y. (2007). Microglia produces IFN-gamma independently from T-cells during acute toxoplasmosis in the brain. J. Interferon Cytokine Res. 27, 599–695. Warnekulasuriya, M. R., Johnson, J. D., and Holliman, R. E. (1998). Detection of Toxoplasma gondii in cured meats. Int. J. Food Microbiol. 45(3), 211–215. Weiss, L. M. and Dubey, J. P. (2009). Toxoplasmosis: A history of clinical observations. Int. J. Parasitol. 39, 895–901. Weiss, L. M. and Kim, K. (2007). Bradyzoite Development. In ‘‘Toxoplasma gondii. The Model Apicomplexan – Perspectives and Methods’’, (L. M. Weiss and K. Kim, eds), pp. 341–366. Elsevier. Wolf, A. and Cowen, D. (1937). Granulomatous encephalomyelitis due to an encephalitozoon (encephalitozic ancephalomyelitis): A new protozoan disease of man. Bull. Neurol. Inst. NY. 6, 306–335. Wong, S. Y. and Remington, J. S. (1993). Biology of Toxoplasma gondii. AIDS 7, 299–316. Wong, S. Y. and Remington, J. S. (1994). Toxoplasmosis in pregnancy. Clin. Infect. Dis. 18, 853–861.

CHAPTER

2 The Importance of Dietary Protein in Human Health: Combating Protein Deficiency in Sub-Saharan Africa through Transgenic Biofortified Sorghum E. C. Henley,*,† J. R. N. Taylor,‡,1 and S. D. Obukosia§

Contents

I. Introduction II. Role and Importance of Protein in Human Health A. Importance of muscle mass B. Human protein requirements III. Protein Quality and Its Measurement IV. Sorghum Protein Quality A. Protein content and composition B. Protein digestibility V. Research to Improve Sorghum Protein Quality VI. Will Protein Biofortification of Sorghum Make a Difference? A. Potential impact of biofortified sorghum VII. Conclusions Acknowledgments References

22 24 26 28 31 32 32 36 39 42 44 46 47 47

* EC Henley Consulting, Athens, Georgia, USA { { } 1

University of Georgia, Athens, Georgia, USA Department of Food Science, University of Pretoria, Pretoria, South Africa Africa Harvest Biotechnology Foundation International, Nairobi, Kenya Corresponding author: J. R. N. Taylor, E-mail address: [email protected]

Advances in Food and Nutrition Research, Volume 60 ISSN 1043-4526, DOI: 10.1016/S1043-4526(10)60002-2

#

2010 Elsevier Inc. All rights reserved.

21

22 Abstract

E. C. Henley et al.

Child malnutrition is increasing in Africa. Protein deficiency is an important cause since protein is essential for both growth and maintenance of muscle mass. Sorghum is a major staple food in Africa on account of its hardiness as a crop. However, sorghum protein is very deficient in the indispensable amino acid lysine and on cooking has poor protein digestibility. This results in sorghum having a very low Protein Digestibility Corrected Amino Acid Score (PDCAAS). The Africa Biofortified Sorghum project, a Grand Challenges in Global Heath project, is undertaking research to biofortify sorghum in terms of protein and micronutrient quality using genetic engineering. Lysine and protein digestibility have been improved by suppression of synthesis of the kafirin storage proteins. Transgenic biofortified sorghum has double the PDCAAS of conventional sorghum. This improvement should enable a young child to meet most of its protein and energy requirements from biofortified sorghum porridge. This together with the improvement in micronutrients could provide the basis of a sustainable and broadly comprehensive solution to child malnutrition in many African countries.

I. INTRODUCTION Globally, the incidence of childhood malnutrition is declining. De Onis et al. (2004) reported on trends and prevalence for the years 1990–2005. His group looked at underweight and stunting data using World Health Organization (WHO) developed methodology to plot and predict trends at country levels. They found that stunting and underweight prevalences declined from 34% to 27% and 27% to 22%, respectively. However, in Africa the situation is not improving. The numbers of stunted and underweight children increased from 40 to 45 million and 25 to 31 million, respectively. Africa and subregions have extensive protein–energy malnutrition (PEM) in children under 5 years of age (FAO, 2008a, 2009). According to FAO 2009 food security statistics (FAO, 2009), the percentage of these children who are moderately and severely malnourished in the underweight category in Burkina Faso, Kenya, and South Africa, representing countries from west, east, and southern Africa, are 32%, 25%, and 15%, respectively. In the stunting category, the combined percents of children who are moderately and severely malnourished are 36%, 50%, and 39% and in the category of wasting, the combined percents of moderately and severely malnourished children are 19%, 7%, and 7%, respectively. Beyond growth retardation, under-nourished children are at risk for infectious diseases, diarrhea, and diminished mental development. De Onis et al. (2004) speculate that the lack of progress in Africa may be due in part to the impact of the human immunodeficiency virus (HIV),

Sorghum Protein Biofortication for Africa

23

which causes the condition AIDS. In sub-Saharan Africa, an estimated 333,000 children below 5 years of age died in 1999 with HIV infection and an estimated 11 million were orphaned because of AIDS. The United Nations International Children’s Fund (UNICEF, 2009) reported that in sub-Saharan Africa in 2007, the annual number of under-5 deaths was 4,480,000 and the life expectancy at birth was only 50 years. There are many reasons for deaths in children under 5 years of age, shortened life spans, stunting, and underweight, but major among them is an inadequate diet including protein intake. The major staple foods in Africa are cassava (105 million tons), maize (48 million tons), yam (45 million tons), sorghum (26 million tons), plantain (24 million tons), rice (21 million tons), wheat (19 million tons), millets (18 million tons), sweet potato (12 million tons), and banana (12 million tons) (FAOSTAT, 2007). Among these, sorghum occupies a unique position on account of its hardiness as a crop, including low water usage, drought-tolerance, and resistance to water-logging (Doggett, 1988). Hence, it is a staple food of many of Africa’s most food-insecure people, who live in the desert-margin, semiarid tropics, some 300 million people (ICRISAT, 2009). The Grand Challenges in Global Health are an initiative of the Bill and Melinda Gates Foundation (BMGF). They were announced in 2003, having been developed by a team of 20 scientists and public health experts from 13 countries (Varmus et al., 2003). Fourteen Grand Challenges were identified in seven areas. Grand Challenge 9 (GC 9) is to ‘‘Create a full range of optimal bioavailable nutrients in a single staple plant species’’ with the aim of improving nutrition to promote health. Today in 2010, there are four GC 9 projects, all of which are supported by the BMGF. They are concerned with biofortification of major staple foods in developing countries. Biofortification can be defined as a process to increase the bioavailability and the concentration of nutrients in crops through both conventional plant breeding (White and Broadley, 2005) and recombinant DNA technology (genetic engineering) (Zimmermann and Hurrell, 2002). Polleti et al. (2004) in a review of the progress made in the nutritional fortification of cereals stated that effective biofortification of cereal staples can reach the poor in rural areas, has low recurrent costs, is sustainable in the long term, and in the case of genetic improvement, it only requires an up-front investment. The GC 9 projects are Micronutrient Improved Bananas (Grand Challenges in Global Health, 2010), Biocassava Plus (2010), Golden Rice (2010), and Biosorghum (2010). The Biosorghum project differs somewhat from the other CG 9 projects in that it has aimed to improve the quality and availability of the macronutrient protein as well as the quantity and availability of micronutrients. As will be seen, protein is a uniquely important issue in sorghum, compared to other cereals. The Biosorghum

24

E. C. Henley et al.

project is a consortium, the Africa Biofortified Sorghum (ABS) consortium, of some 10 organizations in Africa and the United States (Biosorghum, 2010; Zhao, 2007). It is led by the Africa Harvest Biotechnology Foundation International based in Nairobi, Kenya. The specific aims of the Biosorghum project were to increase iron and zinc availability by 50%, to increase provitamin A levels to up to 20 mg/kg, to increase lysine content by 80–100%, to increase tryptophan and threonine by 20%, to concomitantly decrease leucine by 15%, and to improve protein digestibility from its current to approx. 60–80% (Grand Challenges in Global Health, 2010). These improvements in sorghum nutrient content and availability are in comparison with current average levels in sorghum. This chapter sets out the role and importance of protein in human health, human protein requirements, and the measurement of food protein quality. Specifically, focus is given to the need to improve sorghum protein quality and the developments that have already taken place in this area are reviewed. Lastly, the improvements in sorghum protein quality achieved to date in the Biosorghum project are presented and on the basis of these data, the potential of biofortified sorghum to improve children’s nutritional status is evaluated.

II. ROLE AND IMPORTANCE OF PROTEIN IN HUMAN HEALTH Protein is essential for life from the beginning of gestation through old age (WHO/FAO/UNU Expert Consultation, 2007). Dietary protein recommendations are based on the requirements for indispensable amino acids, conditionally indispensable amino acids and nitrogen that is needed for syntheses of dispensable amino acids, and other critical nonprotein nitrogen-containing molecules necessary to support growth, tissue repair, and maintenance (Institute of Medicine of the National Academies, 2005). The indispensable amino acids, up to nine, cannot be synthesized by humans and therefore must be consumed in diets. With adequate precursors, the dispensable amino acids and conditionally indispensable amino acids can be synthesized by the human body. These latter amino acids are needed from dietary sources during rapid growth or during physiological stress when synthesis cannot keep up with demand. Table 2.1 shows the classification for amino acids by dispensability (Laidlaw and Kopple, 1987). Of the indispensable amino acids, lysine is present in human muscle at the highest level, at 9.78% (Wolfe and Chinkes, 2005), indicating its critical nature both in amino acid metabolism and in diets. In cereal-based diets, lysine is normally the most limiting indispensable amino acid, as cereals

Sorghum Protein Biofortication for Africa

25

TABLE 2.1 Indispensable, dispensable, and conditionally indispensable amino acids in the human diet Indispensable

Dispensable

Conditionally Precursors of conditionally indispensable indispensable

Histidine

Alanine

Arginine

Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine

Aspartic acid Asparagine Glutamic acid Serine

Cysteine Glutamine Glycine Proline Tyrosine

Glutamine/glutamic acid/aspartic acid Methionine, serine Glutamic acid/ammonia Serine, choline Glutamic acid Phenylalanine

Laidlaw and Kopple (1987).

have an average lysine content of 31 mg/g protein compared to 65 mg/g in legumes and 85 mg/g in animal foods (Young et al., 1998). A typical well-nourished 70-kg male contains about 11 kg of protein, with a little less than half as skeletal muscle (43%) (Lentner, 1981). Structural tissues such as skin and blood each constitute about 15% of total body protein. The metabolically active tissues such as liver and kidney (only about 10% together) contain relatively little protein. Other organs such as heart, brain, lungs, and bone account for the rest. These relative amounts change with age. For example, infants have significantly less muscle, proportionately, and more brain and visceral protein than adults. Interestingly, almost 50% of the protein within humans is composed of just four types: myosin, actin, collagen, and hemoglobin. Myosin and actin are muscle proteins and collagen is a structural protein. In the human body, protein and other nitrogen-containing compounds are continuously broken down and contribute to an amino acid/nitrogen pool from which precursors and amino acids are reused to synthesize enzymes, hormones, lean tissue, immune function proteins, muscle mass, bone matrix, and other essential compounds (Institute of Medicine of the National Academies, 2005). Maintenance of the protein content of certain tissues and organs, such as skin, brain, heart, liver, and kidneys, is essential for survival. In chronic protein deficiency, lean tissue and muscle mass are sacrificed to provide amino acid precursors for synthesis of critical compounds, such as insulin and hemoglobin (Hoffer, 1994).

26

E. C. Henley et al.

To compound the situation, when caloric intake is inadequate, amino acids are oxidized and used to synthesize glucose as an energy source for those tissues such as red blood cells that only use glucose (Guyton and Hall, 1996). If fat stores are available, fat can be used for much of the body’s energy demands. However, in stunted and wasted children and adults suffering from inadequate dietary protein, calories, and other nutrients, fat stores are limited and muscle mass is tapped for amino acids to synthesize both essential protein molecules and glucose for energy. Picou et al. (1966) pointed out that in induced malnutrition, the proportion of body collagen can rise to 50% because of the substantial loss of noncollagen proteins, presumably muscle.

A. Importance of muscle mass Clinical signs of protein–calorie malnutrition such as marasmus, kwashiorkor, or marasmus–kwashiorkor and various degrees of wasting, stunting, or weight loss are indicators that malnutrition is affecting muscle mass (Torun and Chew, 1994). While it is well recognized that muscle is important in the performance of the activities of daily life, Wolfe (2006) emphasizes the role of muscle in health and disease, pointing out that altered muscle metabolism plays a key role in the genesis of many common pathologic conditions. Under normal circumstances, the demands for amino acids in most organs and tissues do not vary significantly from the fasted state to the fed state because little surplus protein is accumulated in the body. According to animal studies (Swick and Benevenga, 1977), a labile protein reserve that can be gained or lost from the body for emergencies or to account for the day-to-day variations in dietary protein intake is contained in the liver and visceral tissues. Rapid starvation or dietary protein depletion results in a loss of the reserve by as much as 40%, while skeletal muscle drops much more slowly. Thus, protein breakdown becomes the source of indispensable amino acids needed for synthesis of critical proteins necessary for body functions (Reeds et al., 1994) and provides amino acids as precursors for energy production. Of the energy nutrients (protein, fat, and carbohydrate), only fat can be stored by the body in unlimited amounts. Carbohydrate is stored in limited amounts primarily in the form of glycogen in the liver and to a lesser extent in muscle where it provides a ready supply of energy for muscle movement (Guyton and Hall, 1996). Clearly, glycogen storage in muscle is limited by the amount of total muscle mass. Amino acids will be deaminated (nitrogen removed) and used as precursors to provide energy in a caloric deficit, thus the need for adequate calories to spare protein. In the course of normal events, amino acids are stored in muscle up to a limited amount to replace the amino acids lost in the previous fasting

Sorghum Protein Biofortication for Africa

27

state. Excess amino acids not needed for the body’s amino acid repletion or energy demands are finally deaminated with the nitrogen from the amino acid used to synthesize urea for urinary excretion. The remaining carbon skeleton can be stored via synthesis of glycogen or fat. Under normal situations and normal dietary intake, gains and losses of amino acids from muscles are in equilibrium. However, during physiological stress such as chronic hunger, pregnancy, burns, HIV/AIDS, cancer, starvation, and trauma, precursor amino acid demands are increased to provide for synthesis of acute-phase proteins, immune function proteins, and proteins needed for wound healing, etc. This results in rapid depletion of the reserve, which accounts for only about 1% of total body protein (Young et al., 1968). The protein lost during fasting is functional body protein and therefore unlike fat or carbohydrate (glycogen) stores. There is no evidence for a protein reserve that serves only as a store to meet future amino acid/nitrogen needs. Kotler et al. (1998) reported a strong association between the depletion of body cell mass (apparently reflecting muscle loss) and the length of survival of seriously ill patients with AIDS. Studies conducted in the Warsaw ghetto between February and July 1942 suggest that death from starvation with no complicating illness occurs when muscle protein breakdown becomes inadequate to maintain the necessary supply of precursor amino acids to make glucose (Winick, 1979). Extensive work by Keys et al. (1950) also concludes that the depletion of muscle mass is the cause of death in human starvation. The relationships of muscle mass to obesity, insulin resistance, and bone health are further indications of how important it is to protect muscle mass (Wolfe, 2006). For individuals in underresourced countries, suffering from wasting-malnutrition with less than optimum muscle mass and strength, bone modeling and remodeling are at risk, since mechanical force (exerted by muscle mass and strength) on bone is essential for the processes that increase bone strength and bone mass. Changes in bone mass and muscle strength track together over the life span (Frost, 1997). Szulc et al. (2005) reported that skeletal muscle mass was correlated positively with bone mineral content and bone mineral density in the Mediterranean Intensive Oxidant Study (MINOS). MINOS was a prospective study of osteoporosis and its determinants that showed that men with the least skeletal mass also had increased risks of falls due to impaired static and dynamic balance. The relative importance of muscle compared with hormonal and other nutritional effects on bone health may be argued in the MINOS study because factors such as dietary protein, insulin growth factor, and testosterone also affect bone directly, but chronically low muscle mass may provide a visible indicator of bone mass and bone density risk. Osteopenia and osteoporosis are seen as problems in elderly people. In countries with shortened life spans, bone health issues may go unnoticed.

28

E. C. Henley et al.

Short-term changes in diets suggest that the main loss of protein is from the viscera (De Blaauw et al., 1996). However, in chronic illness, skeletal muscle, which comprises over 40% of the protein mass of a healthy individual, becomes the largest single contributor to protein loss (Hansen et al., 2000). While several factors play a role in maintaining muscle mass (exercise, hormones, and diet), clearly the importance of adequate dietary protein intake cannot be debated. Not only health, but activities of daily living, quality of life, and productivity are affected (FAO, 2009; Winick, 1979).

B. Human protein requirements Protein requirements, or more specifically amino acid and nitrogen needs, vary depending on age, body size, gender, physiological states (including pregnancy, illnesses, and fitness), and possibly environment. Research, including nitrogen balance studies, and tracer isotope methodologies are used to determine amino acid and protein requirements for various ages, gender, gestation and lactation, and physiologic conditions (exercise, burns, and illnesses) (Institute of Medicine of the National Academies, 2005). Official amino acid/protein requirements are made for healthy persons of various ages, each trimester of pregnancy, and the initial and latter periods of lactation. Nutrition (protein) security planning for populations requires that in addition to the needs for maintaining health, consideration must be given to the prevalence and increased amino acid/protein requirements of individuals with special needs such as protein malnutrition (stunting and wasting) and illnesses. In 2002, an expert consultation of international amino acid/protein scientists was convened by the United Nations World Health Organization, Food and Agriculture Organization, and University (WHO/FAO/UNU) to evaluate studies to-date and make recommendations for human protein intake (WHO/FAO/UNU Expert Consultation, 2007). That body had previously asked Rand et al. (2003) to prepare a meta-analysis of nitrogen balance studies. The meta-analysis, consisting of 235 individual subjects across 19 studies was used in the 2007 report, along with other research to make these current WHO/FAO/UNU Expert Consultation (2007) recommendations. The amino acid requirements for adults in the previous FAO/WHO/UNU Expert Consultation Report in 1985 (FAO/WHO/UNU Expert Consultation, 1985) had been taken directly from the 1973 FAO/ WHO report (FAO/WHO, 1973). However, since 1985 concern had arisen regarding the previously derived values and that the values for adults were too low (WHO/FAO/UNU Expert Consultation, 2007). Table 2.2 shows the 2007 WHO/FAO/UNU adult indispensable amino acid requirements compared to the 1985 FAO/WHO/UNU requirements. It should be noted that the levels of intake for indispensable

Sorghum Protein Biofortication for Africa

29

TABLE 2.2 Comparison of the 1985 FAO/WHO/UNU and 2002 WHO/FAO/UNU Expert Consultationsa mean adult indispensable amino acid requirements without the coefficient of variation added

Amino Acid

2002 2002 (mg/kg body (mg/g weight/day) proteinb)

Histidine 10 Isoleucine 20 Leucine 39 Lysine 30 Methionine þ cysteine 15 Methionine 10 Cysteine 4 Phenylalanine þ tyrosine 25 Threonine 15 Tryptophan 4 Valine 26 Total indispensable amino 184 acids a b c

15 30 59 45 22 16 6 38 23 6 39 277

1985 1985 (mg/kg body (mg/g weight/day) protein)b

8–12 10 14 12 13 —c — 14 7 3.5 10 93.5

15 15 21 18 20 — — 21 11 5 15 141

1985, FAO/WHO/UNU Expert Consultation (1985), 2002, WHO/FAO/UNU Expert Consultation (2007). Mean nitrogen requirement of 105 mg nitrogen/kg per day (0.66 g protein/kg per day). Not given separately.

amino acids shown in the first column of the table do not take into account the variability of individual requirements. If these are taken into account, the safe levels of intake across populations for indispensable amino acids are 24% higher than shown. The 24% calculation is based on the coefficient of variation of the requirements for protein of 12%. For example, in Table 2.2 the mg indispensable amino acids/g protein is based on an intake of 0.66 g protein/kg/day, but the recommended safe level of protein for adults across genders and ages is currently 0.83 g/kg/day (WHO/FAO/UNU Expert Consultation, 2007). This safe level protein recommendation takes into account the 12% coefficient of variation so as to cover 97.5% of the population and is based on a Protein Digestibility Corrected Amino Acid Score (PDCAAS) of 1.0, the highest possible protein quality score since values over 1.0 are rounded down. The issue of protein quality is discussed in detail later. Concerning the major differences in recommendations, the increases of 150% in mg/kg body weight/day adult requirement for lysine and 114% increase in requirement for threonine between the two report recommendations are of particular significance. Cereal proteins are particularly deficient in these amino acids (Young et al., 1998) and as shown earlier, cereals are the major dietary staple across Africa.

30

E. C. Henley et al.

TABLE 2.3 Amino acid scoring patterns mg/g protein requirements of infants, children, adolescents and adults over 18 (genders combined)a

a

Amino Acid

0.5 year

1–2 years

3–10 years

11–14 years

15–18 years

> 18 years

Histidine Isoleucine Leucine Lysine Methionine þ cysteine Phenylalanine þ tyrosine Threonine Tryptophan Valine

20 32 66 57 28 52 31 8.5 43

18 31 63 52 26 46 27 7.4 42

16 31 61 48 24 41 25 6.6 40

16 30 60 48 23 41 25 6.5 40

16 30 60 47 23 40 24 6.3 40

15 30 59 45 22 38 23 6.0 39

WHO/FAO/UNU Expert Consultation (2007).

Table 2.3 shows the amino acid scoring patterns based on the requirements for dietary indispensable amino acids for humans of different ages as determined by the 2002 WHO/FAO/UNU Expert Consultation (2007). Cysteine is paired with methionine since methionine can be used by the body to synthesize cysteine. Phenylalanine is paired with tyrosine since phenylalanine can be used by the body to make tyrosine. The progressively higher requirement for better quality protein for youths, adolescents, preadolescents, preschool children, and infants compared to adults, on account of formers’ requirement for growth as well as maintenance put them at particular nutritional risk. Hence, the appropriate amino acid scoring pattern needs be used in evaluating protein quality and in the determination of PDCAAS of food proteins for people of different ages. When determining the protein adequacy of particular diets, the most at-risk consuming that diet should be taken into account. Special populations such as pregnant and lactating women, infants, preschool children, and the elderly are at nutritional risk under the best of circumstances, but their vulnerability increases when diseases such as HIV/AIDS, or poverty, civil conflicts, and drought are superimposed. During pregnancy and lactation, both protein and energy requirements are increased. The additional safe intake for protein during pregnancy in g/day increases from 1 g/day in the first trimester to 10 g/day in the second trimester to 31 g/day in the third trimester. For example, a 50-kg adult woman, in the third trimester, will require 72.5 g of good quality protein per day (Institute of Medicine of the National Academies, 2005; WHO/FAO/UNU Expert Consultation, 2007). Pregnant teens require even more protein as they will have continued growth requirements during gestation.

Sorghum Protein Biofortication for Africa

31

III. PROTEIN QUALITY AND ITS MEASUREMENT Dietary protein adequacy (aside from the energy/protein relationship) is a function of several protein-dependent factors: the protein content of the food, the digestibility/bioavailability of the protein/amino acids within the food, the quantity of indispensable amino acids within the protein, and the relative ratio of the amino acid content to the requirements of the person(s) or amino acid standard in question. The final protein quality score is dependent on the lowest level of bioavailable indispensable amino acid, the limiting amino acid. Over the years, different methods have been used for the determination of protein quality for humans and production animals, with the general standard method being the Protein Efficiency Ratio (PER) (FAO/WHO Expert Consultation, 1991). PER can be defined as weight gain of test group/protein consumed by test group. However, according to the 1991 FAO/WHO Expert Consultation, a major criticism of the PER assay is its inability to credit protein used for maintenance purposes. For example, a protein source may not support growth and therefore have a PER of zero, but yet it may be adequate for maintenance purposes. The Expert Consultation proposed that PER therefore be replaced by an assay based on measures of the digestibility (bioavailability) of the protein in a food and the amino acid composition of its protein, the PDCAAS. PDCAAS predicts the biological value (BV) of a food, the effectiveness with which absorbed dietary nitrogen can be utilized (WHO/FAO/UNU Expert Consultation, 2007). In turn, PDCAAS takes into account human indispensable amino acid requirements. The FAO/WHO Expert Consultation (1991) proposed PDCAAS as a means of assessing protein quality of both dietary mixtures and individual food proteins. Shortly afterwards, PDCAAS was adopted by the United States Food and Drug Administration for its 1993 nutrition labeling regulations (Henley and Kuster, 1994). PDCAAS was further endorsed by the WHO/FAO/UNU Expert Consultation (2007) and in South Africa, for example, has been adopted for food labeling legislation (South African Department of Health, 2002). PDCAAS is calculated as true protein digestibility (TD)  Amino Acid Score (AAS) (WHO/FAO/UNU Expert Consultation, 2007). True protein ðNÞ digestibility ð%Þ ¼

I  ðF  Fk Þ  100 I

where I is the nitrogen intake, F is the fecal nitrogen loss on the test diet, and Fk is the fecal nitrogen loss on a protein-free diet. TD is determined by rat bioassay (AOAC International, 2000), although as will be seen the rat may not be an appropriate model to estimate the protein digestibility of sorghum in humans.

32

E. C. Henley et al.

AAS ¼ mg of limiting amino acid in 1 g test protein/mg amino acid in required pattern (WHO/FAO/UNU Expert Consultation, 2007). As an example, Table 2.4 shows PDCAAS calculations for sorghum for different age groups based on their indispensable amino acid requirements according to the WHO/FAO/UNU Expert Consultation (2007). Since the AAS shows that lysine is the most limiting amino acid in sorghum, in this case PDCAAS is based on lysine. A TD value of 74% (Daniel et al., 1966; Hopkins, 1981) obtained from a child feeding intervention study was applied. However, as will be seen below, this figure is probably an overestimate for sorghum. Irrespective of this, the PDCAAS measurement reveals that sorghum protein is highly inadequate for all age groups, being less than one-third of the maximum value of 1.0 of proteins such as casein and egg white (WHO/FAO/UNU Expert Consultation, 2007). The value of the PDCAAS is that if the score is 1.0, policy planners are assured that the mg amino acids per g protein in that food meets 100% of the indispensable amino acid requirements for an age group. Protein requirements are based on body mass among other factors, so the absolute amount of protein required for adults will exceed that required for children, even though the amino acid requirements per kilogram body weight are greater for infants and children than for adults (Table 2.3) and that is the rationale for different amino acid patterns for PDCAAS values (Table 2.4). A thorough discussion of the relative requirements for growth versus maintenance for different ages is given in the 2002 WHO/FAO/UNU Expert Consultation (2007) report. Another application of the PDCAAS calculation is that one can easily determine how much of a food will be required in a day to meet the amino acid requirements for an individual. For that calculation, the quantity of protein in a food must be considered.

IV. SORGHUM PROTEIN QUALITY A. Protein content and composition The average protein content of sorghum grain is about 11% and ranges between 7% and 16% (Serna-Saldivar and Rooney, 1995). This percentage is in the same range as other major cereals, including wheat, maize, rice, barley, and pearl millet (Table 2.5). However, the protein quality of sorghum is generally substantially inferior to them, with the lysine content of its protein being substantially lower, 35–90% of the other cereals (Table 2.5). The lower lysine content occurs because the major proteins of sorghum, the kafirin prolamin storage proteins, are essentially lysine-free (reviewed by Belton et al., 2006). Additionally, in comparison with maize whose zein prolamins are very similar, sorghum has a relatively smaller

TABLE 2.4 AAS and PDCAAS values for sorghum calculated using the 2002 WHO/FAO/UNU Expert Consultation recommended scoring patterns for indispensable amino acidsa

a b c

Age group reference

Lysine scoring pattern

AASb

Threonine scoring pattern

AAS

Tryptophan scoring pattern

AAS

PDCAAS (based on lysine)c

0.5 year 1–2 years 4–18 years >18 years

57 52 48 45

0.35 0.38 0.42 0.44

31 27 25 23

1.00 1.15 1.24 1.35

8.5 7.4 6.5 6.0

1.29 1.49 1.69 1.83

0.26 0.28 0.31 0.33

WHO/FAO/UNU Expert Consultation (2007) mg/g protein requirement. Calculated from USDA (2009) National Nutrient Database for Standard Reference NDB No. 20067, where lysine is 20, threonine 31, and tryptophan 11 mg/g protein, respectively. Based on a TD of 74% (Daniel et al., 1966; Hopkins, 1981).

TABLE 2.5 Protein content, protein digestibility, lysine and leucine contents and PDCAASs of normal and improved protein quality lines of sorghum, compared to wheat, maize, barley, and pearl millet (data from several sources as indicated)

Protein (g/100 g flour) Protein digestibility (wet-cooked values, unless indicated otherwise) (%) Lysine (g/100 g flour) Leucine (g/100 g flour) Lysine (mg/g protein)

Sorghum cv. Maciaa

Sorghum cv. P890812 (parent of ABS032)a

Normal

Normal

10.6 (0.0)

Sorghum cv. ABS032a

Sorghum cv. BTX 436 (parent of Sorghum cv. 04CS11249- 04CS112491xTX436)a 1xTX436a

Sorghum cv. P521 opaqueb

Sorghum (USDA 20067)c

Wheat (hard red winter) (USDA 20072)c

Maize (corn white) USDA (20314)c

Rice (brown long grain) USDA (20037)c

Barley (pearled) (USDA 20006)c

Pearl milletd

High protein digestibility mutant

HighNormal lysine Mutant

11.9 (0.1)

Normal High lysine, high protein digestibility transgenic biofortified sorghum 12.8 (0.2) 12.1 (0.1)

11.9 (0.0)

10.6

12.4

14.5

10.5

9.6

7.24

14.5

59.8e (0.7)

47.4e (4.8)

73.7e (2.5)

36.4e (1.7)

51.9e (5.6)

63.2e,f 56.7e,g

74i 59.0e,f 59.8e,f 50.6e,g

86i (not stated) 85.5e,f

85h (not stated) 85.3e,f (yellow maize)

90j (not stated)

74.8e,f

0.19 (0.02)

0.25 (0.01)

0.41 (0.01)

0.18 (0.02) 0.26 (0.02)

0.31

0.25

0.39

0.30

89i (polished, not stated 83.8e,f (rice type not stated) 72j 0.37

0.27

0.48

1.43 (0.07)

1.63 (0.02)

1.33 (0.04)

1.77 (0.12) 1.50 (0.06)

1.29

1.64

0.98

1.29

0.80

0.50

1.77

17.9 (2.1)

21.0 (0.5)

32.0 (0.6)

14.9 (1.2)

29.5

20.2

26.9

28.6

38.5

37.3

33.1

21.8 (1.8)

134.9 (6.3) Leucine (mg/g protein) AAS (based on 0.34 lysine) PDCAASk 0.21

a b c d e f g h i j k

137.0 (1.9)

104.0 (3.2)

131.1 (9.0) 126.0 (4.9)

122

132.3

67.6

122.9

83.3

69.1

122.1

0.40

0.62

0.29

0.42

0.57

0.39

0.52

0.55

0.74

0.72

0.64

0.19

0.45

0.10

0.22

0.36 0.32

0.29 0.23 0.23 0.20

0.44 0.44

0.47 0.47

0.66 0.62 0.53

0.65

0.48

Own data, unpublished. Means and standard deviations (in parentheses) of four independent replicate analyses. Guiragossian et al. (1978), except where indicated otherwise. USDA (2009) National Nutrient Database for Standard Reference, except where indicated otherwise. Serna-Saldivar and Rooney (1995), except where indicated otherwise. In vitro pepsin method. Mertz et al. (1984). Axtell et al. (1981). True digestibility (FAO/WHO Expert Consultation, 1991). Hopkins (1981). South African Department of Health (2002). PDCAAS calculated as protein digestibility  AAS for 3–10-year old children (based on lysine) using the WHO/FAO/UNU Expert Consultation (2007), using lysine and protein digestibility values in this table in order given.

36

E. C. Henley et al.

germ and hence a lower level of the lysine-rich germ proteins (Taylor and Schu¨ssler, 1986). Hence, the AAS for lysine in sorghum is only between approximately 35% and 44% of the 2002 WHO/FAO/UNU Expert Consultation (2007) recommendations for the various age groups. Sorghum protein also appears to have low levels of the indispensable amino acids threonine and tryptophan and high levels of leucine (reviewed by Klopfenstein and Hoseney, 1995). However, as shown in Table 2.4, contrary to earlier suggestions (Klopfenstein and Hoseney, 1995), threonine does not seem to be deficient in sorghum protein. In sorghum, there is a low ratio of tryptophan relative to leucine. The leucine content of sorghum protein, approximately 13 g/100 g, is approximately twice the 2002 WHO/FAO/UNU Expert Consultation (2007) scoring pattern. Tryptophan is believed to have a sparing effect on niacin and the levels of leucine in some sorghum diets may impair this sparing effect and result in the occurrence of pellagra (reviewed by Klopfenstein and Hoseney, 1995). Evidence from rat feeding trials supports the pellagra risk theory concerning sorghum diets (Salter et al., 1985). However, Young and Fukagawa (1988) disputed this risk after analysis of data from several studies.

B. Protein digestibility A second and important issue with regard to sorghum protein quality is that the digestibility of sorghum proteins is lower than that of maize (Elmalik et al., 1986; reviewed by Duodu et al., 2003), despite the fact that the proteins are very similar. Additionally, and of particular significance, sorghum protein digestibility is substantially reduced after wet cooking (Axtell et al., 1981; Taylor and Taylor, 2002), as occurs when sorghum flour is for example made into porridge, the most common form of sorghum foods in Africa. Duodu et al. (2003) showed that the reduction of in vitro protein digestibility that occurs when sorghum is wet cooked has been found by many workers to be substantially greater than that which occurs when maize is wet cooked. As a consequence, sorghum foods have much lower protein digestibility than maize, wheat, rice, and pearl millet foods (Mertz et al., 1984). Research over more than 20 years has provided compelling evidence that, although the causes of the reduction in sorghum protein digestibility are multifactorial (Duodu et al., 2003), the major cause is cross-linking of the kafirin prolamin proteins due to disulfide bonding (Ezeogu et al., 2005; Hamaker et al., 1987; Rom et al., 1992). Disulfide bonding seems to specifically involve the cysteine-rich g- and b-kafirin species (Oria et al., 1995) which are concentrated at the surface of the endosperm protein bodies, the organelles of kafirin storage (Shull et al., 1992). The cross-linking

Sorghum Protein Biofortication for Africa

37

appears to render the major kafirin species, a-kafirin, which is located in the center of the protein bodies (i.e., beneath the g-kafirin), less accessible to protein hydrolysis. Additionally, the kafirin proteins seem to undergo a more severe change in secondary structure on cooking than zeins, from a-helical to b-sheet conformation, which also seems to adversely affect their digestibility (Emmambux and Taylor, 2009). The poor protein digestibility of sorghum seems to relate directly to its inferior human nutritional value, although not surprisingly in view of ethical issues, there has been very limited controlled experimental research in this area. Probably the clearest demonstration was the work of MacLean et al. (1981). The authors fed children, average age of 17 months who were recovering from PEM, a wet-cooked whole grain sorghum-based diet. Four varieties of sorghum were used, two normal (21–22 mg lysine/g protein) and two high-lysine (29–30 mg lysine/g protein). Because of poor weight gain (or weight loss) and inadequate nitrogen retention, most of the children did not complete the 7–9 day experimental diet sequence. The mean absorption and retention of nitrogen from 26 6-day sorghum dietary periods were 46% and 14% of intake, compared with preceding casein mean control values of 81% and 49%. Of particular relevance is the fact that there were no differences between the high-lysine and normal-lysine sorghum diets. The authors also compared these findings with other comparable data for wheat, rice, potato, and maize diets where the absorption and retention of nitrogen ranged from 66% to 81% and 20% to 34%, respectively. Further, the children’s stool weight and energy losses for the sorghum diets were 2.5–3 times those of the casein diets and substantially higher than values from wheat, rice, potato, and maize diets. Additionally, the work showed that with all the sorghum diets, 3-h postprandial levels of plasma lysine did not increase compared to fasting levels and declined compared to fasting levels at 4 h, indicative of lysine deficiency. A later study (MacLean et al., 1983) appeared to give contradictory results in that children fed a diet containing sorghum that had been decorticated (debranned) and processed by extrusion cooking showed very much higher nitrogen absorption and higher nitrogen retention than in the previous study. These findings have to be seen in context. Subsequent work showed that extrusion cooking (essentially a dry type of cooking), unlike wet cooking, does not reduce protein digestibility (Hamaker et al., 1994) and confirms that removal of the outer layers of the sorghum grain improves protein digestibility (reviewed by Duodu et al., 2003). However, at-risk people consume conventionally wet-cooked sorghum and often it is prepared from milled whole grain (personal observations). Further, the findings of MacLean et al. (1981) confirm earlier work. Kurien et al. (1960) replaced rice in the diet of seven normal boys, aged

38

E. C. Henley et al.

10–11, with various proportions of sorghum and found that the apparent protein digestibility of the diet fell from 75% to 69% to 64% to 55% as the proportion of sorghum increased from 0–25% to 50–100%. Nicol and Phillips (1978)fed adult Nigerian men diets based on sorghum, cassava, and rice and compared them to egg protein. The true digestibility of the sorghum diet was found to be lower than that of any of the others and its BV was superior only to that of cassava. The evidence of these studies was summed up by Millward (1999) in a review of the nutritional value of plant-based diets in relation to human requirements, who stated ‘‘digestibility does seem to present a problem for many mixed plant-based diets and some cereals (e.g., millet [apparently referring to finger millet (also known as ragi) (Eleucine coracana)] and sorghum), and this may be a major problem for children in the developing countries.’’ Results from rat feeding experiments, however, have not always agreed with the human studies. Axtell et al. (1981) using three of the four same sorghum varieties as MacLean et al. (1981) found similarly high protein digestibility (N consumed minus N in feces) with all gruels made from all the sorghums, both normal and high lysine. Further, the protein digestibility of the gruels was similar to that which had been found earlier in their laboratory when rats were fed uncooked sorghum flours. They proposed that the young rat is much more efficient at digesting sorghum proteins than children and therefore is not a good model. The findings of Axtell et al. (1981) are explained by a comprehensive sorghum food rat feeding study by Bach Knudsen and coworkers (Bach Knudsen and Munck, 1985; Bach Knudsen et al., 1988a,b; Eggum et al., 1983). These authors fed rats diets comprising neutral, acidic, and alkaline porridges made from non-tannin and polyphenol-rich (tannin) sorghums, and the uncooked sorghum flours. With all the types of porridges, there was a substantial decrease in TD and generally an increase in BV compared to the uncooked flours, with a great increase with high-tannin sorghum porridges. Further, with the high-tannin sorghum porridge diets, there was a substantial reduction in net protein utilization (NPU) but no effect with the non-tannin and low-tannin sorghum porridge diets. The authors attributed the reduction in TD to the kafirin proteins being rendered unavailable by cooking. They proposed that the higher BV was due to the undigested kafirins serving as a nitrogen source for bacteria in the rat hind gut. This, plus fermentation by the rat of resistant starch formed during sorghum processing, was suggested as the reasons for the difference in sorghum food feeding values between rats and people. The authors further proposed that the reason that the effects of the nutritional parameters were exacerbated in rats fed the high-tannin sorghum diets was due to complexation between the tannins and kafirins, the net effect being a change in nitrogen excretory routes from the urine to the feces.

Sorghum Protein Biofortication for Africa

39

V. RESEARCH TO IMPROVE SORGHUM PROTEIN QUALITY Research to improve sorghum protein quality has been ongoing since the 1970s, especially at Purdue University. Singh and Axtell (1973) identified high lysine lines from Ethiopia. However, these have very poor functional quality as the grains are shrunken. By chemical mutagenesis of normal sorghum using diethyl sulfate, a high-lysine sorghum mutant called P721opaque was developed. This has up to 60% higher lysine content due to a reduction in the relative amount of kafirins (Guiragossian et al., 1978). However, as described, when both of these types of high-lysine sorghums were used in human feeding trials, there was no difference in performance between them and normal sorghums (MacLean et al., 1981). As a result of the evidence that low protein digestibility is the major cause of sorghum’s poor nutritional performance, improved protein digestibility types were sought. By crossing P721-opaque with normal sorghums, lines were obtained that had substantially improved protein digestibility, 10–15% higher in uncooked flour and 25% higher in cooked flour (Weaver et al., 1998). These lines also have somewhat elevated levels of lysine. The improved protein digestibility appears to be due to change in the shape of the kafirin protein bodies from spherical to invaginated (Fig. 2.1) (Oria et al., 2000). This change in shape results in the g-kafirin species being concentrated at the bottom of invaginations where they should not interfere with the digestion of the a-kafirin. Tesso et al. (2006) identified a novel sorghum mutant with both high protein digestibility and high-lysine traits, and a relatively hard endosperm. This mutant was an F6 generation of crosses between P721-opaque and hard endosperm sorghum lines. It has some 44% higher lysine than normal A

B CW PB CW

PB

2 µm

2 µm

FIGURE 2.1 Transmission electron micrographs of protein bodies from (A) normal sorghum and (B) high protein digestibility mutant sorghum. PB ¼ kafirin protein body, CW ¼ cell wall.

40

E. C. Henley et al.

sorghum and 20% higher protein digestibility. Very unusually, the grains had areas of densely packed starch granules without the usual endosperm protein matrix between them. The denser structure seemed to be responsible for the relative hardness of the grain. Data on the in vivo effects of the high protein digestibility sorghum are very limited, but what there is does not show any nutritional improvement. Nyannor et al. (2007) investigated the effects of two different high protein digestibility sorghum lines, and normal sorghum and maizebased diets on pigs (sorghum or maize only in the diets) and broiler chickens (sorghum or maize, plus soya in the diets). It was found with the pigs that there was no difference in the apparent ileal or total tract digestibility of dry matter, energy, or nitrogen among the diets. Further, the estimated digestible energy did not differ among the diets. With the chickens, there was no difference in ileal digestibility for any of the nutrients among the different diets. Total tract nitrogen retention was better with maize than with any of the sorghums, although one of the high protein digestibility lines was better in this respect than the other and normal sorghums. There was no difference in the apparent metabolizable energy content among any of the sorghum diets but metabolizable energy was lower than in the maize diet, except for the one high protein digestibility line. The findings of Nyannor et al. (2007) and those of MacLean et al. (1981) are consistent, showing the need to develop sorghum lines that have both substantially improved protein quality and digestibility. The ABS project is employing recombinant DNA technology to improve both sorghum lysine content and wet-cooked protein digestibility. These improvements are being achieved by suppressing the synthesis of specific kafirin species that are very low in lysine and that are responsible for poor protein digestibility (i.e., suppression of the synthesis of various combinations of a-, g-, and d-kafirins) using RNA interference (RNAi) technology (Jung, 2008), as has been demonstrated with the maize zein prolamins (reviewed by Shewry, 2007). Additionally, where appropriate, the synthesis of lysine-rich proteins, such as HT12, an analog of barley hordothionin, is being expressed (Zhao et al., 2003) and the catabolism of lysine by the enzyme lysine ketoreductase (LKR) is suppressed. Sorghum transformation is brought about using a ‘‘super-binary’’ Agrobacterium vector (Zhao et al., 2003). Preliminary data indicate that the kafirin protein bodies in the endosperm of early transgenic biofortified sorghum lines are irregular in shape, that is similar to those of the high protein digestibility mutant (Fig. 2.1). The irregular shape is presumably a reflection of suppression of kafirin synthesis. Table 2.5 compares the protein quality in terms of lysine, leucine, protein digestibility, and PDCAAS of different types of sorghum, including an early transgenic biofortified sorghum line and other major cereals:

Sorghum Protein Biofortication for Africa

41

wheat, maize, rice, barley, and pearl millet. It should be noted that most of the protein digestibility values were obtained using the in vitro pepsin method developed by Chibber et al. (1980). As can be seen, the in vitro protein digestibility values from the Purdue University group (Axtell et al., 1981; Mertz et al., 1984) seem to be in good agreement with the FAO/WHO Expert Consultation (1991) TD values, which came from Hopkins (1981). The only exception is the sorghum value of 74%, which in turn came from Daniel et al. (1966), which is rather higher. As stated earlier, many factors affect sorghum protein digestibility, these include exogenous factors such as grain organizational structure, polyphenols, phytic acid, cell wall components, and starch (reviewed by Duodu et al., 2003), which can vary among individual samples. In fact, in the discussion following the conference reading of the Hopkins (1981) paper, reference was made to the effect of various factors on protein digestibility and there was some criticism of these factors not being taking into account (Hopkins, 1981). In response, Hopkins stated with specific reference to sorghum and millet ‘‘with some of the largely vegetarian diets in some of the developing countries, it is a different story [i.e., different from the situation with regard to USA diets] and protein digestibility is of concern’’. Of relevance to this, Hopkins (1981) reported data that the true digestibility of sorghum diets of Indian children was 66%, similar to values for finger millet (ragi), 65–68%. MacLean et al. (1981) found nitrogen absorption in sorghum diets to be 46  17%, which is of the same order as the Purdue pepsin in vitro digestibility values (Axtell et al., 1981; Mertz et al., 1984), and on this basis, Axtell et al. (1981) stated that the pepsin in vitro method gave similar values to those obtained in human studies. With regard to the accuracy of our own sorghum protein digestibility data in Table 2.5, it is important to note that our in vitro pepsin protein digestibility results seem to be in close agreement with those of the Purdue group (Axtell et al., 1981; Mertz et al., 1984). For example, Macia, a white tan-plant sorghum, had a protein digestibility of 59.8%, the same as Dabar another white tan-plant sorghum (Mertz et al., 1984). Table 2.5 shows that the lysine content of transgenic biofortified sorghum protein was 52–115% higher than that of normal sorghums, 47% higher than the high protein digestibility mutant, and 8% higher than the high-lysine mutant. In comparison with the other major cereals, the protein lysine content of transgenic biofortified sorghum was somewhat higher than that of maize and wheat, similar to pearl millet (Pennisetum glaucum), but lower than that of pearled barley and rice. Thus, the AAS, based on protein lysine content, of transgenic biofortified sorghum was some 50–100% higher than that of normal sorghum and high protein digestibility sorghum, and slightly higher than that of the high-lysine mutant, maize, and wheat, similar to that of pearl millet but lower than

42

E. C. Henley et al.

that of pearled barley and rice. The protein leucine content of transgenic biofortified sorghum was some 20% lower than in normal sorghum, and 15–17% lower than in the high protein digestibility and high-lysine mutants. The wet-cooked in vitro protein digestibility of transgenic biofortified sorghum was 23–102% higher than that of the normal sorghums and 8% and 42% higher, respectively, than the corresponding levels in the high-lysine sorghum and high protein digestibility sorghum mutants. Importantly, the wet-cooked protein digestibility of transgenic biofortified sorghum was raised to levels approaching those of the other major cereals. In this regard, it is important to point out that this early version of transgenic biofortified sorghum was in a low-tannin sorghum (Type 2 sorghum) background. The tannins in tannin sorghum reduce protein digestibility, both in vitro (Chibber et al., 1980) and in vivo (Bach Knudsen et al., 1988a), primarily through complexing with the proline-rich kafirins (Butler et al., 1984; Taylor et al., 2007). This kafirin–tannin complexation is indicated by the relatively low protein digestibility of the transgenic biofortified sorghum parent cultivar P890812. Thus, it is probable that the wet-cooked protein digestibility will be improved somewhat further when the protein quality traits are in non-tannin sorghum lines. The effect of improving both protein lysine content and protein digestibility on PDCAAS is substantial. The PDCAAS of transgenic biofortified sorghum for 1–2-year-old children is double that of normal sorghum and the high protein digestibility sorghum, and 25–40% higher than the high-lysine sorghum. Significantly, the PDCAAS of transgenic biofortified sorghum is the same level as maize, wheat, and pearl millet.

VI. WILL PROTEIN BIOFORTIFICATION OF SORGHUM MAKE A DIFFERENCE? The limited research that has been carried out on the effects of protein and lysine biofortication/fortication of cereal staples on the nutritional status of children has yielded mixed results. Daniel et al. (1966) carried out a short-term study where a sorghum-rich diet was fortified with lysine or lysine plus threonine in the form of free amino acids (i.e., not in proteins) and given to 11–12-year-old girls from a low income group in India. Fortification with lysine resulted in a small increase in nitrogen retention, BV, and NPU. Fortification with lysine plus threonine resulted in a much larger improvement in these parameters. It is noteworthy that the nitrogen retention of the unfortified sorghum-rich diet was only 8.6% compared to the referenced skim milk-rich diet of 32.9%. Supplementation with lysine plus threonine increased nitrogen retention to 21.8%.

Sorghum Protein Biofortication for Africa

43

King et al. (1963) fortified wheat bread with the indispensable amino acid lysine and undertook a yearlong study with chronically undernourished children in Haiti aged 6–18 years. The experimental design was such that there were eight groups of children divided according to age and sex. A significant improvement in corpuscular hemoglobin concentration was observed for three of the eight groups receiving the lysinefortified bread compared to the unfortified bread group and a significant increase in height was observed in two of the groups. However, compared to a control group, there was only an increase in hemoglobin concentration in two of the groups. Significantly, the authors suggested that other limiting factors such as threonine and vitamin A and riboflavin would need to be addressed if optimal growth and hemoglobin responses were to be obtained. Subsequently, in the 1970s, three major, long-duration studies were undertaken where cereal-rich diets (rice, wheat, and maize) of people in developing countries were fortified with lysine and other vitamins and minerals (reviewed by Pellett and Ghosh, 2004). None of the studies reported any significant health benefits, and a task force investigating the reasons found that there were serious flaws in the design, methods, or analysis in the studies (Latham, 1988). More recently, two large, long-term, double-blind studies were undertaken by Scrimshaw and coworkers, in which families in Pakistan and China were given wheat flour fortified with lysine (0.6 g/100 g and 0.3 g/ 100 g, respectively) or control flour to make into bread-type products as their major dietary staple (Hussain et al., 2004; Zhao et al., 2004). A range of anthropometric measurements and blood tests were performed and the results analyzed according to men, women, and children. In both studies, the height and weight increases of the children in the lysine-fortified flour group were significantly higher than in the control groups. In the Pakistan study (Hussain et al., 2004), transferrin levels increased significantly in men, women, and children receiving lysine-fortified flour and hemoglobin increased in the women. In the China study (Zhao et al., 2004), hemoglobin levels were not affected. However, perhaps of importance with respect to HIV/AIDS, CD3 T cell levels increased significantly in women and children receiving the lysine-fortified flour and the increases in the three immunoglobulins IgG, IgA, and IgM were highly significant (p < 0.01) in the children. With respect to the mixed findings on fortification of cereals with free lysine, attention was drawn by the 2002 WHO/FAO/UNU Expert Consultation (2007) to the issue of Maillard reactions between amino acids, especially lysine, and sugars that can occur during food processing. Maillard reactions adversely affect the bioavailability of amino acids. Lysine, as a free amino acid, is particularly susceptible to loss through the Maillard reaction (Ajandouz and Puigserver, 1999).

44

E. C. Henley et al.

The work of MacLean et al. (1981) where there was no difference in the effects of high-lysine sorghum compared to normal sorghum has already been described. The findings of this study contrast sharply with those of Graham et al. (1990) on high-lysine maize. These authors worked with young children in Peru, aged 13–29 months, who were recovering from malnutrition. The children were fed a diet comprising solely a porridge of Quality Protein Maize (QPM) for a period of up to 90 days. QPM is a highlysine mutant and also contains elevated levels of tryptophan and lower levels of leucine. The lysine level in the QPM cultivars used was 38– 40 mg/g protein, that is, 20% higher than in current transgenic biofortified sorghum lines (Table 2.5). In the diets, QPM provided all the protein and lipid and 90% of the energy, the remainder coming from sucrose. The diet was also supplemented with a full complement of vitamins. The responses of the children on the QPM diet were compared to those of similar children receiving a modified cow’s milk formula (CMF). Significantly, the weight gain and height growth were the same between the QPM and CMF groups. The QPM showed a small reduction in serum albumin levels and the plasma-free total essential amino acids and ratio of these to total essential amino acids was lower in the QPM group. With reference to this, the authors stated that the lower plasma albumin levels were a characteristic of vegetable (plant) protein diets. Concerning the dramatic difference in response of the children fed high-lysine maize in this study to those receiving high-lysine sorghum in the study of MacLean et al. (1981), this seems to be related to the fact that, when sorghum is wet cooked, its protein digestibility is considerably reduced, whereas this is not the case with maize, as described above. In this respect, it is relevant that, in another study by Graham and coworkers involving feeding recovering malnourished children QPM, it was found that the children fed QPM had significantly higher nitrogen retention than children fed normal maize (p < 0.01) (Graham et al., 1989). Hence, the fact that transgenic biofortified sorghum has improved protein digestibility as well as increased lysine is probably critical to it making a difference to children’s nutritional status.

A. Potential impact of biofortified sorghum The potential impact of biofortified sorghum can be evaluated by taking Burkina Faso as an example. Sorghum is the most important staple in Burkina Faso, accounting for 40% of cereal production (FAOSTAT, 2007). There are serious child nutrition and health problems in Burkina Faso. According to statistics from the Ministry of Health, in 2007, the average incidence of malnutrition for children under the age of 5 years was 10.9%, with 1.4% of children being severely malnourished (Burkina Faso Ministry of Health, 2008). With respect to malnutrition-related diseases

Sorghum Protein Biofortication for Africa

45

in the children, 5200 cases of anemia and 591 cases of xerophthalmia were reported and PEM accounted for 4% of the principal causes of hospital admissions. With respect to protein intake in Burkina Faso, in 2003–2005, sorghum contributed 28%, pearl millet 19%, maize 13%, groundnuts 11%, pulses 9%, rice 4%, meat 6%, milk 2%, and wheat 1% (FAO, 2008b). The total cereal contribution to protein intake is 65% with PDCAAS values ranging from approximately 0.28 (sorghum) to 0.66 (rice) (Table 2.5), using the 2002 WHO/FAO/UNU Expert Consultation (2007) reference pattern for 1–2-year-old children. Only 8% (milk þ meat) of the dietary protein intake sources has a PDCAAS of 1.0 (FAO/WHO Expert Consultation, 1991), yet the protein recommendations are based on a PDCAAS of 1.0. Thus, with respect to sorghum meeting 28% of a 2-year-old child’s (median weight 12.3 kg; FAO/WHO/UNU Expert Consultation, 1985) safe requirement of 0.97 g protein/kg body weight per day (WHO/FAO/ UNU Expert Consultation, 2007), that is 3.3 g protein, the child would have to consume 3:3 

100 1:00 ¼ 90 g sorghum grain a 12:4 0:28b

a

USDA (2009) National Nutrient Database for Standard Reference protein content of sorghum grain b PDCAAS of normal sorghum (Table 2.5) The solids content of the sorghum Toˆ, the gel-like porridge, consumed in Burkina Faso and other Sahel countries, is approximately 20% (Scheuring et al., 1982). Thus, the child would have to consume some 475 g of Toˆ made from normal sorghum per day, a very large amount. The situation would be even worse in the case where sorghum was the sole cereal consumed, which is often the case in the period before harvest where other foodstuffs have all been consumed, as sorghum’s PDCAAS is only approximately half that of the other cereals (Table 2.5). Then the child would have to consume approximately 1.1 kg of sorghum Toˆ per day, equivalent to 9% of its body weight. The requirement in high cereal diets for extremely high cereal intakes to satisfy children’s protein requirement is in agreement with analysis by Millward (1999) of dietary data from West Bengal, India, published by Pellett (1996). In the West Bengal diet, cereals supplied 90% of protein. Millward showed that the AAS for all preschool children was less than 1.0. He concluded that the children, when weaned, would need either extra milk or pulses if the energy density of the diet did not allow them to increase food intake sufficiently. In this context, it should be noted that the PDCAAS of milk and pulses are

46

E. C. Henley et al.

much higher, 1.00 and 0.77 (chickpea) for 2-year-old children (WHO/ FAO/UNU Expert Consultation, 2007) than those of cereals (Table 2.5). The key issue is thus that with transgenic biofortified sorghum, the fact that PDCAAS is doubled means that half the amount of Toˆ or other sorghum cereal food needs to be consumed by the children in order to meet their safe protein intake. This is based on the assumption that the diet sufficiently meets their energy requirements. On the basis of the findings of Graham et al. (1990) where QPM porridge met all the protein requirements and 90% of the energy requirements of children of this age, it would seem to be the case. As indicated above, dietary protein cannot be considered in isolation from dietary micronutrients. Widespread micronutrient malnutrition is acknowledged in Africa. The major global micronutrient deficiencies identified in a United States Institute of Medicine publication (Committee on Micronutrient Deficiencies, 1998) were vitamin A, iron, iodine, and zinc. It was pointed out that the high intake of phytate in grain-based diets affects mineral status. Among the various options for addressing micronutrient deficiencies, supplementation, fortification, and food-based approaches, the food-based approach attempts to correct the underlying causes of micronutrient deficiencies and is most effective where there is widespread availability, variability, adequacy, and acceptability. Periodic micronutrient supplementation programs have been successful but are not sustainable (Committee on Micronutrient Deficiencies, 1998). Tang et al. (2009) demonstrated that the biofortified Golden Rice, which contains 1.6–35 mg b-carotene (provitamin A) per gram dry rice, is effectively converted to vitamin A in humans. With respect to transgenic biofortified sorghum, preliminary data from early generation lines show reduced levels of phytate, indicating higher iron and zinc bioavailability and similar levels of provitamin A to Golden Rice (Dr Zuo-Yu Zhao, research scientists, Pioneer Hi-Bred International). As an indication of the potential of provitamin A biofortification, Van Jaarsveld et al. (2005) found in a controlled experiment that children consuming orange-fleshed sweet potato containing approximately 100 mg b-carotene per gram showed improved vitamin A status compared to those consuming normal white-fleshed sweet potato. Through consumption of the orange-fleshed sweet potato, there was an increase from 78% to 87% in the percentage of children with normal vitamin A status.

VII. CONCLUSIONS Biofortified sorghum, developed using recombinant DNA technology, has substantially improved protein quality in terms of lysine content and wet-cooked protein digestibility compared to normal sorghum.

Sorghum Protein Biofortication for Africa

47

In terms of PDCAAS, it is similar to other major cereals. In addition, the biofortified sorghum will have higher bioavailability of iron and zinc and significant levels of provitamin A. Thus, cultivation and consumption of biofortified sorghum, Biosorghum, could provide a sustainable and broadly comprehensive solution to the protein and certain micronutrient deficiencies suffered by young children in sub-Saharan African countries that are dependent on cereals as their staple food.

ACKNOWLEDGMENTS Dr Janet Taylor for our protein and protein digestibility data, Ms Annette Exley for our amino acid data, and Mrs Laura da Silva for the electron micrographs; Dr Dirk Hays of Texas A&M University and Dr Zuo-Yu Zhao of Pioneer Hi-Bred International for supplying the high protein digestibility sorghum and transgenic biofortified sorghum lines, respectively; and the Bill and Melinda Gates Foundation for funding through the Grand Challenges in Global Health initiative are acknowledged.

REFERENCES Ajandouz, E. H. and Puigserver, A. (1999). Nonenzymatic browning reaction of essential amino acids: Effect of pH on caramelization and Maillard reaction kinetics. J Agric. Food Chem. 47, 1786–1793. AOAC International (2000). AOAC Official method 991.29 True protein digestibility of foods and food ingredients: Rat bioassay first action 1991. In ‘‘Official Methods of AOAC International’’, (W. Horwitz, Ed.), 17th edn, Vol. II, pp. 66–67. AOAC International, Gaithersburg, MD (Chapter 45). Axtell, J. D., Kirleis, A. W., Hassen, M. M., D’Croz Mason, N., Mertz, E. T., and Munck, L. (1981). Digestibility of sorghum proteins. Proc. Natl. Acad. Sci. USA 78, 1333–1335. Bach Knudsen, K. E. and Munck, L. (1985). Dietary fibre contents and compositions of sorghum and sorghum-based foods. J. Cereal Sci. 3, 153–164. Bach Knudsen, K. E., Munck, L., and Eggum, B. O. (1988a). Effect of cooking, pH and polyphenol level on carbohydrate composition and nutritional quality of a sorghum (Sorghum bicolor (L.) Moench) food, ugali. Br. J. Nutr. 59, 31–47. Bach Knudsen, K. E., Kirleis, A. W., Eggum, B. O., and Munck, L. (1988b). Carbohydrate composition and nutritional quality for rats of sorghum toˆ prepared from decorticated white and whole grain red flour. J. Nutr. 118, 588–597. Belton, P. S., Delgadillo, I., Halford, N. G., and Shewry, P. R. (2006). Kafirin structure and functionality. J. Cereal Sci. 44, 272–286. Biocassava Plus (2010). www.biocassavaplus.org (Accessed January 2010). Biosorghum (2010). www.biosorghum.org (Accessed January 2010). Burkina Faso Ministry of Health (2008). Annuiare Statistique Sante´ – 2007. Ministere de la Sante´, Ouagadougou. Butler, L. G., Riedl, D. J., Lebryk, D. G., and Blytt, H. J. (1984). Interaction of proteins with sorghum tannin: Mechanism, specificity and significance. J. Am. Oil Chem. Soc. 61, 916–920. Chibber, B. A. K., Mertz, E. T., and Axtell, J. D. (1980). In vitro digestibility of high-tannin sorghum at different stages of dehulling. J. Agric. Food Chem. 26, 160–161.

48

E. C. Henley et al.

Committee on Micronutrient Deficiencies (1998). In ‘‘Prevention of Micronutrient Deficiencies: Tools for Policymakers and Public Health Workers’’, (C. P. Howson, E. T. Kennedy, and A. Horwitz, Eds), National Academy Press, Washington, DC. Daniel, V. A., Leela, R., Doraiswamy, T. R., Rajalakshmi, D., Venkat Rao, S., Swaminathan, M., and Parpia, H. A. B. (1966). The effect of supplementing a poor kaffir corn (Sorghum vulgare) diet with L-lysine and DL-threonine on the digestibility coefficient, biological value and net utilization of proteins and retention of nitrogen in children. J. Nutr. Diet. 3, 10–14. De Blaauw, I., Deutz, N. E. P., and Von Meyenfeldt, M. F. (1996). In vivo amino acid metabolism of gut and liver during short and prolonged starvation. Am. J. Physiol. 270, G298–G306. De Onis, M., Blossner, B., Borghi, E., Morris, R., and Frongillo, E. A. (2004). Methodology for estimating regional and global trends of child malnutrition. Int. J. Epidemiol. 33, 1260–1270. Doggett, H. (1988). Sorghum, 2nd edn. Longman Scientific and Technical, London, pp. 1–3. Duodu, K. G., Taylor, J. R. N., Belton, P. S., and Hamaker, B. R. (2003). Mini review: Factors affecting sorghum protein digestibility. J. Cereal Sci. 38, 117–131. Eggum, B. O., Monowar, L., Bach Knudsen, K. E., Munck, L., and Axtell, J. (1983). Nutritional quality of sorghum and sorghum foods from Sudan. J. Cereal Sci. 1, 127–137. Elmalik, M., Klopfenstein, C. F., Hoseney, R. C., and Bates, L. S. (1986). Digestibility and nutritional value of sorghum grain with contrasting kernel characteristics. Nutr. Rep. Int. 34, 811–821. Emmambux, M. N. and Taylor, J. R. N. (2009). Properties of heat-treated sorghum and maize meals and their prolamin proteins. J. Agric. Food Chem. 57, 1045–1050. Ezeogu, L. I., Duodu, K. G., and Taylor, J. R. N. (2005). Effects of endosperm texture and cooking conditions on the in vitro starch digestibility of sorghum and maize flours. J. Cereal Sci. 42, 33–44. FAO (2008a). Food Security Statistics. Food Consumption, Dietary Energy, Proteins, Fat. www.fao.org/economic/ess/foodsecurity-statistics/en/ (Accessed January 2010). FAO (2008b). Food Security Statistics. Consumption Pattern of Main Food Crops-Dietary Protein. www.fao.org/economic/ess/foodsecurity-statistics/en/ (Accessed January 2010). FAO (2009). Food Security Statistics. Child Nutrition Status. www.fao.org/economic/ess/ foodsecurity-statistics/en (Assessed January 2010). FAO/WHO (1973). Energy and protein requirements. Food and Agriculture Organization and World Health Organization, Rome and Geneva, Report of a joint FAO/WHO Ad Hoc Expert Committee. FAO Nutrition meetings Report Series, No. 52, and WHO Technical Report Series, No 522. FAO/WHO Expert Consultation (1991). Protein Quality Evaluation. Food and Agriculture Organization, Rome, Report of Joint FAO/WHO Expert Consultation, FAO Food and Nutrition Paper 51. FAO/WHO/UNU Expert Consultation (1985). Energy and Protein Requirements. World Health Organization, Geneva Report of a Joint FAO/WHO/UNU Expert Consultation. World Health Organization Technical Report Series 724. FAOSTAT (2007). FAOSTAT-Agriculture, Production, Crops. http://faostat.fao.org/ (Accessed November 2009). Frost, H. M. (1997). On our age-related bone loss: Insights from a new paradigm. J. Bone Miner. Res. 12, 1–9. Golden Rice (2010). www.goldenrice.org (Accessed January 2010). Graham, G. G., Lembcke, J., Lancho, E., and Morales, E. (1989). Quality protein maize: Digestibility and utilization by recovering malnourished infants. Pediatrics 83, 416–421. Graham, G. G., Lembcke, J., and Morales, E. (1990). Quality-protein maize as the sole source of dietary protein and fat for rapidly growing young children. Pediatrics 85, 85–91.

Sorghum Protein Biofortication for Africa

49

Grand Challenges in Global Health (2010). Optimisation of Bioavailable Nutrients in Transgenic Bananas. www.grandchallenges.org/ImproveNutrition/Challenges/NutrientRichPlants (Accessed January 2010). Guiragossian, V., Chibber, B. A. K., Van Scoyoc, S., Jambunathan, R., Mertz, E. T., and Axtell, J. D. (1978). Characteristics of proteins from normal, high lysine, and high tannin sorghums. J. Agric. Food Chem. 26, 219–223. Guyton, A. C. and Hall, J. E. (1996). Textbook of Medical Physiology, 9th edn W.B. Saunders Company, Philadelphia, PA, pp. 863, 980, 880, 881. Hamaker, B. R., Kirleis, A. W., Butler, L. G., Axtell, J. D., and Mertz, E. T. (1987). Improving the in vitro protein digestibility of sorghum with reducing agents. Proc. Natl. Acad. Sci. USA 84, 626–628. Hamaker, B. R., Mertz, E. T., and Axtell, J. D. (1994). Effect of extrusion on sorghum kafirin solubility. Cereal Chem. 71, 515–517. Hansen, R. D., Raja, C., and Allen, B. J. (2000). Total body protein in chronic diseases and in aging. Ann. N Y Acad. Sci. 904, 345–352. Henley, E. C. and Kuster, J. M. (1994). Protein quality evaluation by protein digestibilitycorrected amino acid scoring. Food Technol. 48(4), 74–77. Hoffer, L. J. (1994). Starvation. In ‘‘Modern Nutrition in Health and Disease’’, (M. E. Shils, J. A. Olson, and M. Shike, Eds), 8th edn, pp. 927–949. Lea & Febiger, Philadelphia, PA. Hopkins, D. T. (1981). Effects of variation in protein digestibility. In ‘‘Protein Quality in Humans: Assessment and in vitro Estimation’’, (C. E. Bodwell, J. S. Adkins, and D. T. Hopkins, Eds), pp. 169–193. AVI, Westport, CT. Hussain, T., Abbas, S., Khan, M. A., and Scrimshaw, N. S. (2004). Lysine fortification of wheat flour improves selected indices of the nutritional status of predominantly cereal-eating families in Pakistan. Food Nutr. Bull. 25, 114–122. ICRISAT (2009). Sorghum. www.icrisat.org/newsite/crop-sorghum.htm (Accessed November 2009). Institute of Medicine of the National Academies (2005). Dietary Reference Intakes for Energy, Carbohydrate, Fiber Fat, Fatty Acids, Cholesterol, Protein and Amino Acids. National Academies Press, Washington, DC, www.nap.edu (Accessed May, 2009). Jung, R. (2008). Grain quality improvement through altered expression of seed proteins. United States Patent Application 20080134361. Keys, A., Brozek, J., Henshel, A., Mickelsen, O., and Longstreet, T. H. (1950). The Biology of Human Starvation. University of Minnesota Press, Minneapolis, MN. King, K. W., Sebrell, W. H., Jr., Severinghaus, E. L., and Storvick, W. O. (1963). Lysine fortification of wheat bread fed to Haitian School children. Am. J. Clin. Nutr. 12, 36–47. Klopfenstein, C. F. and Hoseney, R. C. (1995). Nutritional properties of sorghum and the millets. In ‘‘Sorghum and Millets: Chemistry and Technology’’, (D. A. V. Dendy, Ed.), pp. 125–168. American Association of Cereal Chemists, St. Paul, MN. Kotler, D. P., Tierney, A. R., and Wang, J. (1998). The magnitude of body cell mass depletion determines the timing of death from wasting in AIDS. Am. J. Clin. Nutr. 50, 444–447. Kurien, P. P., Narayanarao, M., Swaminathan, M., and Subrahmanan, V. (1960). The metabolism of nitrogen, calcium and phosphorus in undernourished children. 6. The effect of complete replacement of rice in poor vegetarian diets by kaffir corn (Sorghum vulgare). Br. J. Nutr. 14, 339–345. Laidlaw, S. A. and Kopple, J. D. (1987). Newer concepts of the indispensable amino acids. Am. J. Clin. Nutr. 46, 593–605. Latham, M. C. (1988). Amino Acid Fortification of Cereals: Results and Interpretation of Trials in Three Countries. In ‘‘Cornell International Nutrition Monograph 20.’’ Cornell University, Ithaca, NY. Lentner, C. (1981). Units of Measurement, Body Fluids, Composition of the Body, Nutrition. In ‘‘Geigy Scientific Tables,’’ 8th edn. Ciba-Geigy Corporation, West Caldwell, NJ.

50

E. C. Henley et al.

MacLean, W. C., Jr., Lo´pez, de Romanˇa, G., Placko, R. P., and Graham, G. C. (1981). Protein quality and digestibility of sorghum in preschool children: balance studies and plasma free amino acids. J. Nutr. 111, 128–136. MacLean, W. C., Jr., Lo´pez de Romanˇa, G., Castanˇaduy, A., and Graham, G. G. (1983). The effect of decortication and extrusion on the digestibility of sorghum by preschool children. J. Nutr. 113, 2171–2177. Mertz, E. T., Hassen, M. M., Cairns-Whittern, C., Kirleis, A. W., Tu, L., and Axtell, J. D. (1984). Pepsin digestibility of proteins in sorghum and other major cereals. Proc. Natl. Acad. Sci. USA 81, 1–2. Millward, D. J. (1999). Meat or wheat for the next millennium? The nutritional value of plantbased diets in relation to human amino acid and protein requirements. Proc. Nutr. Soc. 58, 249–260. Nicol, B. M. and Phillips, P. G. (1978). The utilization of proteins and amino acids in diets based on cassava (Manihot utilissima), rice sorghum (Sorghum sativa) by young Nigerian men of low income. Br. J. Nutr. 39, 271–286. Nyannor, E. K. D., Adedokun, S. A., Hamaker, B. R., Ejeta, G., and Adeola, O. (2007). Nutritional evaluation of high-digestible sorghum for pigs and broiler chicks. J. Anim. Sci. 85, 196–203. Oria, M. P., Hamaker, B. R., and Shull, J. M. (1995). Resistance of sorghum a-, b- and g-kafirins to pepsin digestion. J. Agric. Food Chem. 43, 2148–2153. Oria, M. P., Hamaker, B. R., Axtell, J. D., and Huang, C.-P. (2000). A highly digestible sorghum mutant cultivar exhibits unique fold structure of endosperm protein bodies. Proc. Natl. Acad. Sci. USA 97, 5065–5070. Pellett, P. L. (1996). World essential amino acid supply with attention to South-East Asia. Food Nutr. Bull. 17, 204–234. Pellett, P. L. and Ghosh, S. (2004). Lysine fortification: Past, present and future. Food Nutr. Bull. 25, 107–113. Picou, D., Halliday, D., and Garrow, J. S. (1966). Total body protein, collagen and noncollagen protein in infantile protein malnutrition. Clin. Sci. 30, 345–351. Polleti, S., Gruissem, W., and Sauter, C. (2004). Nutritional fortification of cereals. Curr. Opin. Biotechnol. 15, 162–165. Rand, W. M., Pellett, P. L., and Young, V. R. (2003). Meta-analysis of nitrogen balance studies for estimating protein requirements in healthy adults. Am. J. Clin. Nutr. 77, 109–127. Reeds, P. J., Field, C. R., and Jahoor, F. (1994). Do the differences between the amino acid compositions of acute-phase and muscle proteins have a bearing on nitrogen loss in traumatic states? J. Nutr. 124, 906–910. Rom, D. L., Shull, J. M., Chandrashekar, A., and Kirleis, A. W. (1992). Effects of cooking and treatment with sodium bisulfite on in vitro digestibility and microstructure of sorghum flour. Cereal Chem. 69, 178–181. Salter, M., Bender, D. A., and Pogson, C. I. (1985). Leucine and tryptophan metabolism in rats. Biochem. J. 225, 277–281. Scheuring, J. F., Sidibe, S., and Kante, A. (1982). Sorghum alkali toˆ: Quality considerations. In ‘‘International Symposium on Sorghum Grain Quality’’, (L. W. Rooney and D. S. Murty, Eds), pp. 24–31. International Crops Research Institute for the Semi-Arid Tropics, Patancheru, India. Serna-Saldivar, S. and Rooney, L. W. (1995). Structure and chemistry of sorghum and millets. In ‘‘Sorghum and Millets: Chemistry and Technology’’, (D. A. V. Dendy, Ed.), pp. 69–124. American Association of Cereal Chemists, St. Paul, MN. Shewry, P. R. (2007). Improving the protein content and composition of cereal grain. J. Cereal Sci. 46, 239–250. Shull, J. M., Watterson, J. J., and Kirleis, A. W. (1992). Purification and immunocytochemical localization of kafirins in Sorghum bicolor (L. Moench) endosperm. Protoplasma 171, 64–74.

Sorghum Protein Biofortication for Africa

51

Singh, R. and Axtell, J. D. (1973). High lysine mutant gene (hl) that improves the quality and nutritional value of grain sorghum. Crop Sci. 13, 535–539. South African Department of Health (2002). Regulations Relating to Labelling and Advertising of Foodstuffs, Regulation 1055. www.doh.gov.za/docs/regulations/2002/reg1055. html (Accessed January 2010). Swick, R. W. and Benevenga, N. J. (1977). Labile protein reserves and protein turnover. J. Dairy Sci. 60, 505–515. Szulc, P., Beck, T. J., Marchand, F., and Delmas, P. D. (2005). Low skeletal muscle mass is associated with poor structural parameters of bone and impaired balance in elderly men – the MINOS study. J. Bone Miner. Res. 20, 721–729. Tang, G., Qin, J., Dolnikowski, G. G., Russell, R. M., and Grusak, M. A. (2009). Golden rice is an effective source of vitamin A. Am. J. Clin. Nutr. 89, 1776 1183. Taylor, J. R. N. and Schu¨ssler, L. (1986). The protein compositions of the different anatomical parts of sorghum grain. J. Cereal Sci. 4, 361–369. Taylor, J. and Taylor, J. R. N. (2002). Alleviation of the adverse effects of cooking on protein digestibility in sorghum through fermentation in traditional African porridges. Int. J. Food Sci. Technol. 37, 129–138. Taylor, J., Bean, S. R., Ioerger, B. P., and Taylor, J. R. N. (2007). Preferential binding of sorghum tannins with gamma-kafirin and the influence of tannin binding on kafirin digestibility and biodegradation. J. Cereal Sci. 46, 22–31. Tesso, T., Ejeta, G., Chandrashekar, A., Huang, C.-P., Tandjung, A., Lewamy, M., Axtell, J. D., and Hamaker, B. R. (2006). A novel modified endosperm texture in a mutant high-protein digestibility/high-lysine sorghum (Sorghum bicolor (L.) Moench). Cereal Chem 83, 194–201. Torun, B. and Chew, F. (1994). Protein-energy malnutrition. In ‘‘Modern Nutrition in Health and Disease’’, (M. E. Shils, J. A. Olson, and M. Shike, Eds), 8th edn, pp. 950–976. Lea & Febiger, Philadelphia, PA. UNICEF (2009). The State of the World’s Children. www.unicef.org (Accessed May 2009). USDA (2009). National Nutrient Database for Standard Reference. www.nal.usda.gov/fnic/ foodcomp/search (Accessed November 2009). Van Jaarsveld, P. J., Faber, M., Tanumihardjo, S. A., Nestel, P., Lombard, C. J., and Spinnler Benade´, A. J. (2005). b-carotene-rich orange-fleshed sweet potato improves the vitamin A status of primary school children assessed with the modified-relative-dose response test. Am. J. Clin. Nutr. 81, 1080–1087. Varmus, H., Klausner, R., Zerhouni, E., Acharya, T., Daar, A. S., and Singer, P. A. (2003). Public health: Grand challenges in global health. Science 302, 398–399. Weaver, C. A., Hamaker, B. R., and Axtell, J. D. (1998). Discovery of grain sorghum germ plasm with high uncooked and cooked in vitro protein digestibility. Cereal Chem. 75, 665–670. White, P. J. and Broadley, M. R. (2005). Biofortifying crops with essential mineral elements. Trends Plant Sci. 10, 587–595. WHO/FAO/UNU Expert Consultation (2007). Protein and Amino Acid Requirements in Human Nutrition. World Health Organization, Geneva Report of a Joint WHO/FAO/ UNU Expert Consultation. World Health Organization Technical Report No. 935. Winick, M. (1979). Hunger disease. Studies by the Jewish Physicians in the Warsaw Ghetto. John Wiley & Sons, New York. Wolfe, R. R. (2006). The underappreciated role of muscle in health and disease. Am. J. Clin. Nutr. 84, 475–482. Wolfe, R. R. and Chinkes, D. L. (2005). Isotope Tracers in Metabolic Research: Principles and Practice of Kinetic Analysis. 2nd edn John Wiley & Sons, New York. Young, V. R. and Fukagawa, N. K. (1988). Amino acid interactions: A selective review. In ‘‘Nutrient Interactions’’, (C. E. Bodwell and J. W. Erdman, Eds), pp. 27–71. Marcel Dekker, New York.

52

E. C. Henley et al.

Young, V. R., Hussein, M. A., and Scrimshaw, J. S. (1968). Estimate of loss of labile body nitrogen during acute protein deprivation in young adults. Nature 218, 568–569. Young, V. R., Scrimshaw, N. S., and Pellet, P. L. (1998). Significance of dietary protein source in world nutrition: animal and/or plant protein? In ‘‘Feeding a World Population of More Than 8 Billion People: A Challenge to Science’’, ( J. C. Waterlow, D. G. Armstrong, L. Fowden, and R. Riley, Eds), pp. 205–212. Oxford University Press, New York. Zhao, Z. (2007). The Africa Biofortified Sorghum Project – Applying Biotechnology to Develop Nutritionally Improved Sorghum in Africa. In ‘‘Biotechnology and Sustainable Agriculture 2006 and Beyond’’, (Z. Xu, J. Li, Y. Xue, and W. Yang, Eds), pp. 273–277. Springer, Dordrecht. Zhao, Z., Glassman, K., Sewalt, V., Wang, N., Miller, M., Chang, S., Thompson, T., Catron, S., Wu, E., Bidney, D., Kebede, Y., and Jung, R. (2003). Nutritionally improved transgenic sorghum. In ‘‘Plant Technology 2002 and Beyond’’, (I. K. Vasil, Ed.), pp. 413–416. Kluwer, Dordrecht. Zhao, W., Zhai, F., Zhang, D., An, Y., Liu, Y., He, Y., and Ge, K. (2004). Lysine-fortified wheat flour improves the nutritional and immunological status of wheat-eating families in northern China. Food Nutr. Bull. 25, 123–129. Zimmermann, M. B. and Hurrell, R. F. (2002). Improving iron, zinc and vitamin A nutrition through plant biotechnology. Curr. Opin. Biotechnol. 13, 142–145.

CHAPTER

3 Clostridium difficile: Its Potential as a Source of Foodborne Disease Maja Rupnik*,†,1 and J. Glenn Songer‡,2

Contents

I. Introduction II. Methods for Detection of C. difficile in Food: We Lack a Standard Approach III. C. difficile in Meat and Meat Products: Isolation in Multiple Locations in the United States and Europe Establish this as a Widespread Phenomenon IV. C. difficile in Other Foods: Possible Association with Environmental Strains or Organisms from Animal Feces V. C. difficile in Companion Animal Feed: Animal Exposure may have Far-Reaching Effects on Human Disease VI. Possible Sources of Food Contamination: C. difficile is Widespread in Animals, Humans, and the Environment VII. Occurrence of Common Genotypes in Animals, Humans, and Foods: Crossover is Very Common VIII. C. difficile in Food as a Source of Human Colonization: Contaminated Food may be a Source of the Organism in the Hospital and the Community IX. Conclusions: We have Much to Learn References

54 55

57

57

60

60 61

62 63 63

* Institute of Public Health Maribor, Centre for Microbiology, Maribor, Slovenia { {

1 2

Faculty of Medicine, University of Maribor, Maribor, Slovenia Department of Veterinary Microbiology and Preventive Medicine, Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa, USA Corresponding author: Maja Rupnik, E-mail address: [email protected] Corresponding author: J. Glenn Songer, E-mail address: [email protected]

Advances in Food and Nutrition Research, Volume 60 ISSN 1043-4526, DOI: 10.1016/S1043-4526(10)60003-4

#

2010 Elsevier Inc. All rights reserved.

53

54

Abstract

Maja Rupnik and J. Glenn Songer

Clostridium difficile has been recognized as an important human pathogen for several decades, but its importance as an agent of animal disease was established only recently. The number of reports on C. difficile in food is rising, but the findings vary among studies. In North America, the prevalence of contamination in retail meat and meat products ranges from 4.6% to 50%. In European countries, the percentage of C. difficile positive samples is much lower (0–3%). This chapter summarizes current data on association of C. difficile with different foods and the difficulties associated with isolation of the organism, and discusses the potential of C. difficile as a food-transmitted pathogen.

I. INTRODUCTION Clostridium difficile has been traditionally regarded as a nosocomial human pathogen (Rupnik et al., 2009). Hospitals were the main reservoir for infection that occurred either in outbreaks or as isolated cases. Since 2003, the severity and mortality of C. difficile infection (CDI) have risen dramatically in North America and several European countries (Kuijper et al., 2006; Loo et al., 2005). The epidemiology has changed in parallel. Community-associated (CA)-CDI has, in the past, been considered a minor issue (in comparison to healthcare facility-associated (HA)-CDI), due to the mild nature of these infections. However, both incidence and severity of CA-CDI are changing, and new populations at risk are emerging (Chernak et al. 2005; Limbago et al., 2009). C. difficile has been isolated from many domestic and wild animals, including camels, horses, donkeys (Hafiz and Oakley, 1976), dogs and cats (O’Neill et al., 1993; Riley et al., 1991; Weber et al., 1989), domestic fowl, seals, and snakes (Levett, 1986). It has been found rarely in septicemias and pyogenic infections in domestic animals (Hirsh et al., 1979). There have been sporadic reports of disease in wild species, including cases in a Kodiak bear (Orchard et al., 1983), a rabbit (Rehg and Shoung, 1981), a penguin (Hines and Dickerson, 1993), and captive ostriches (Frazier et al., 1993). The organism is an important cause of diarrhea and fatal necrotizing enterocolitis in foals (Jones et al., 1988a,b; Traub-Dargatz and Jones, 1993) and nosocomial diarrhea in adult horses (Baverud, 2002). Isolation of C. difficile from substantial numbers of normal or diseased food animals was reported relatively recently. Species infected most commonly include piglets (Songer and Anderson, 2006), calves (Hammitt et al., 2008; Rodriguez-Palacios et al., 2006), and poultry (Shimango and Mwakurudza, 2008; Zidaric et al., 2008).

Clostridium difficile as a Cause of Foodborne Disease

55

This observation has logically led to studies on C. difficile in meat and meat products. First reports on possible foodborne transmission of the organism date to almost thirty years ago (Borriello et al., 1983; Gurian et al., 1982), but the first of the very few published studies of C. difficile in meats came from Canada (Rodriguez-Palacios et al., 2006). However, interest in C. difficile in animal-derived and other foods is growing. Our aim here is to give an overview of current knowledge of the association of C. difficile with foods and to comment on isolation methods and interpretation of the public health relevance of the results.

II. METHODS FOR DETECTION OF C. DIFFICILE IN FOOD: WE LACK A STANDARD APPROACH Sample preparation may be by maceration (e.g., in a Stomacher (Seward, Bohemia, NY, USA) or similar apparatus), grinding in a blender, or equivalent. Direct bacteriologic culture for C. difficile has been accomplished by plating on commercial C. difficile plates with cefoxitin and cycloserine (CCFA; Indra et al., 2009) or moxalactam and norfloxacin (CDMN; Weese et al., 2009) and usually supplemented with 5% blood (horse or sheep). Enrichment broths have consisted of brain heart infusion (BHI) with cysteine and yeast extracts (Songer et al., 2009) and Oxoid C. difficile medium without agar (Rodriguez-Palacios et al., 2009; Weese et al., 2010; Table 3.1). These enrichment media may be supplemented with cefoxitin (
16 mg/ml) and cycloserine (
500 mg/ml) or moxalactam (32 mg/ml) and norfloxacin (12 mg/ml). Alcohol shock and subculture on commercial solid media described above follow incubation under anaerobic conditions for 2–12 days. Duplicate cultures (e.g., heat-shocked (80  C, 10 min) and non-heatshocked) can improve recovery rates (Rodriguez-Palacios et al., 2009; Songer et al., 2009). In work to date, specimens were culture positive in enrichments subjected to one treatment or the other, but no specimen was positive with both. The same method used on two duplicate samples had culture sensitivities of 39% and 23%, respectively (Rodriguez-Palacios et al., 2009). Comparison of recovery rates from 214 samples examined by three methods (differing in meat sample preparation, selective supplement in enrichment broth, and agar media for subculturing of enrichments), one of them in duplicate to document reproducibility, revealed C. difficile recovery rates from 1.4% to 2.3%. However, there was no correlation of culture results among methods, and results varied from one repetition to the next (Rodriguez-Palacios et al., 2009). This is clearly a potential growth

TABLE 3.1 Comparison of isolation methods Selective supplement in enrichment

Meat sample/medium

Incubation

Reference

Ingredients of Oxoid selective agar (except agar), 0.1% sodium taurocholate

CDMN supplement (Oxoid)-moxalactam, norfoxacin

4–5 g meat 20 ml prereduced CDMN broth

Rodriguez-Palacios et al. (2007), Weese et al. (2010)

Ingredients of Oxoid selective agar (except agar), 0.1% sodium taurocholate

Cefoxitine, cycloserine

2 g meat

BHI with 0.5% yeast extract, 0.05% cysteine, 0.1% taurocholate BHI

None reported (selection as heat shock at 80  C for 10 min) CDMN supplement (Oxoid)-moxalactam, norfoxacin None reported

1 g meat in 10 ml medium

2–15 days EtOH shock on 2 ml enrichment broth Sediment on selective agar 7 days EtOH shock on 2 ml enrichment broth Sediment on selective agar 1–3 days

25 g in 50 ml medium

10–12 days

von Abercron et al. (2009)

5 g in 20 ml medium

12 days EtOH shock Inoculation on C. difficile selective agar (bioMerieux)

Indra et al. (2009)

Medium

Thioglycolate broth

Rodriguez-Palacios et al. (2009)

Songer et al. (2009)

Clostridium difficile as a Cause of Foodborne Disease

57

area in study of C. difficile biology. Improved selective isolation methods, as well as media for enrichment, direct culture, and subculture, would facilitate study of the organism in CDI, the carrier state, the environment, and in foods.

III. C. DIFFICILE IN MEAT AND MEAT PRODUCTS: ISOLATION IN MULTIPLE LOCATIONS IN THE UNITED STATES AND EUROPE ESTABLISH THIS AS A WIDESPREAD PHENOMENON C. difficile has been reported in meat and meat products in Canada, the United States, France, Austria, and Sweden (Table 3.2). The first publication from Canada reported a high percentage of culture positive samples (Rodriguez-Palacios et al., 2007), although the same group reported only 6% of samples to be culture positive in a subsequent study in 2008 (Rodriguez-Palacios et al., 2009). Yet, other Canadian studies reported that 12% of samples of beef and pork ground meat were culture positive (Weese et al., 2010) and the prevalence in chicken meat was 12.8% (Weese et al., 2010). The prevalence of C. difficile in meat and meat products was much higher (> 40%) in a U.S. study geographically limited to Arizona (Songer et al., 2009). There are few data for C. difficile presence in retail meats in EU countries, but the percentage of positive samples has generally been < 5% (Table 3.2) (Bouttier et al., 2007; Indra et al., 2009; Jo¨bstl et al., 2010; von Abercron et al., 2009). Ground beef is the meat specimen most likely to contain C. difficile, followed by other types of ground meat (pork, turkey, chicken). Seasonality in occurrence of C. difficile in meat has been observed, with 11.5% of samples culture positive in January and February, but only 4% positive from March to August (Rodriguez-Palacios et al., 2009).

IV. C. DIFFICILE IN OTHER FOODS: POSSIBLE ASSOCIATION WITH ENVIRONMENTAL STRAINS OR ORGANISMS FROM ANIMAL FECES Al Saif and Brazier (1996) reported C. difficile in different nonhospital, nonhuman sources, such as soil and water. Three hundred raw vegetable samples (on sale in retail premises) were examined by impression of unwashed surfaces onto plates of selective medium. Seven culture positive raw vegetable samples included cucumber (n ¼ 1), onion (n ¼ 1), potato (n ¼ 2), mushroom (n ¼ 1), carrot (n ¼ 1), and radish (n ¼ 1). Tomato, cabbage, and lettuce did not contain C. difficile on their surfaces. A very recent study of ready-to-eat salads, in Scotland (none of which

TABLE 3.2

Detection of C. difficile in meat and meat products Number of tested samples/number of positive samples

Sampling time interval

Type of meat/ meat product

Canada

Jan to Oct 2005

Canada

Jan to Aug 2008

Retail ground 12 of 60 (20%) meat samples Ground beef 10/149 (6.7%) Veal chop 3/65 (4.6%)

Canada

Aug 2008 to Nov 2008

Canada

Nov 2008 to Jun 2009 Jan to Apr 2007

Country

United States (Tucson, Arizona)

Austria

Feb to Apr 2008

All 28/230 (12%) Ground beef 14/115 (12%) Ground pork 14/115 (12%) Chicken meat 26/203 (12.8%) Ground beef 13/26 (50%) Ground pork 3/7 (42.9%) 4/9 (44.4%) Ground 17/46 (36.9%) turkey Meat productsa Beef, pork, 0/84 chicken

Genotype (PCR ribotypes or PFGE type or toxinotype)

Ribotype/toxinotype (M26/tox-, 077/0, M31/III, 014/0) Ribotypes/toxinotype (M26/tox-, 077/0, J/III, 014/0, C/nd, F/VIII, H/0, K/III) Ribotype 078, 027

Ribotype 078 PFGE types/toxinotypes (NAP1/III, NAP1-related/III, NAP7/V, NAP8/V)

Reference

RodriguezPalacios et al. (2006) RodriguezPalacios et al. (2009) Weese et al. (2010) Weese et al. (2010) Songer et al. (2009)

Indra et al. (2009)

Austria

France

Sweden a

Jul 2007 to Feb 2008

Ground meat Beef (n ¼ 30) Beef and pork (n ¼ 70) (2006 no Vacuum precise packed data given) ground beef pork sausage Apr to Sep Ground beef 2008

Ribotypes AI-57, 053

Jo¨bstl et al. (2010)

2 of 60 (3%) 0/50

Toxinotype 0

Bouttier et al. (2007)

2.4%

Not done

von Abercron et al. (2009)

Beef only 0% Beef and pork 3%

Meat products included summer sausage (ready to eat (RTE)), braunschweiger (RTE), chorizo (uncooked), and pork sausage (uncooked).

60

Maja Rupnik and J. Glenn Songer

originated in the United Kingdom), revealed that 3 of 40 (7.5%) were culture positive (Bakri et al., 2009). Salad mixes (
10%) and bagged spinach (
7%) in the United States have also been culture positive (M. Lloyd and J.G. Songer, unpublished). Analysis of raw milk samples did not reveal contamination with C. difficile (Jo¨bstl et al., 2010).

V. C. DIFFICILE IN COMPANION ANIMAL FEED: ANIMAL EXPOSURE MAY HAVE FAR-REACHING EFFECTS ON HUMAN DISEASE C. difficile was isolated from two samples of vacuum-packed meat intended for dogs (Broda et al., 1996). An additional report on C. difficile in raw turkey dog food was published more recently (Weese et al., 2005). No C. difficile was found in 10 samples of feline raw diet (Bouttier et al., 2007).

VI. POSSIBLE SOURCES OF FOOD CONTAMINATION: C. DIFFICILE IS WIDESPREAD IN ANIMALS, HUMANS, AND THE ENVIRONMENT Sources of C. difficile food contamination might include transfer of spores from the gut to the musculature of healthy or diseased animals, contamination at slaughter, contamination at processing and packaging, or contamination in the local retail market. In fact, each of these sources may contribute to a greater or lesser extent to the final contamination level in meat and meat products. Muscle tissue from healthy horses (Vengust et al., 2003) and cows (unpublished data reported in Rodriguez-Palacios et al., 2009) can contain low numbers of clostridial spores. The high rate of contamination of pork braunschweiger (Songer et al., 2009) suggests that C. difficile spores may localize in Kupffer cells in liver antemortem. Clostridia are members of the gut microbiome in animals and comprise one of the main groups participating in natural carcass degradation. Colonization of muscle tissue in the form of dormant spores could provide a selective advantage, both by augmenting the success of transmission to new hosts (via consumption of contaminated meat) and by precolonization of tissues to facilitate their eventual degradation. Carcass contamination by gut contents at slaughter probably contributes most to the presence of C. difficile in meat and meat products. Slaughter techniques differ from country-to-country, with those in the United States being more of the ‘quick and dirty’ variety than in the EU. This coincides with the high percentage of positive meat samples (Songer et al., 2009).

Clostridium difficile as a Cause of Foodborne Disease

61

However, data on C. difficile in animals prior to slaughter are scarce. An Austrian study that examined cows, pigs, and broilers in the time interval from March to July, 2008, found C. difficile in feces or gut contents of cows (4.5%), pigs (3.3%), and broilers (5%) (Indra et al., 2009). Feedlot calves are commonly infected with C. difficile as neonates, and even normal cattle can be culture positive through the end of the feeding period. Hammitt et al. (2008) investigated the possible role of C. difficile in calf enteritis. C. difficile and toxins were found in 25.3% and 22.9%, respectively, of stool samples from diarrheic calves. These findings agree with those of others (Porter et al., 2002; Rodriguez-Palacios et al., 2006) and strengthen the hypothesis that CDI can manifest as calfhood colitis and diarrhea and that calves are, at the very least, multiplying hosts for this organism. Shimango and Mwakurudza (2008) collected feces from live broilers sold in food markets in Zimbabwe and examined them by bacteriologic culture without enrichment. C. difficile was isolated from samples (and was, hence, present in birds) in five of six markets. Interestingly, 40% of chickens reared at homesteads in Zimbabwe were culture positive and 20% of samples from chickens reared on commercial farms were positive. There is, to date, no published study on presence of C. difficile in slaughter house or packing plant environments, or on equipment or the hands of the workers.

VII. OCCURRENCE OF COMMON GENOTYPES IN ANIMALS, HUMANS, AND FOODS: CROSSOVER IS VERY COMMON C. difficile genotypes are currently determined mostly by PCR ribotyping or pulsed-field gel electrophoresis (PFGE) (Rupnik et al., 2009). Most strains isolated from food sources have a genotype identical to those of human and animal isolates from the same geographic area or worldwide. Two Canadian studies (Rodriguez-Palacios et al., 2006, 2009) reported substantial heterogeneity in strains recovered from meats. In the more recent study, 28 isolated strains belonged to 8 genotypes. Of these, seven were toxinogenic and identical to well-known human and previous animal isolates by PFGE and ribotyping (North American pulsed-field type (NAP) 1/ribotype 027, NAP2/ribotype 077, and NAP4/ribotype 014). These were reported in CA-CDI in the United States (Limbago et al., 2009) and were among the predominant types in the 2005 EU prevalence study (Barbut et al., 2007). Type 014 is the most prevalent type in the current EU surveillance study (Bauer et al., 2010). Ribotypes 077 and 014 were isolated from calves in Canada (Avbersek et al., 2009; RodriguezPalacios et al., 2006) and type 077 from dogs in the United States (Keel et al., 2007).

62

Maja Rupnik and J. Glenn Songer

The American study reported high percentages of culture positives among retail meats, but genotypes were more homogenous (Songer et al., 2009). Thirty-seven strains belonged to two ribotypes only (027 and 078), although both were further divided by PFGE typing (027/NAP1; 027/ NAP1-like; 078/NAP7; 078/NAP8). All of these, with the exception of the NAP1-like strains, are frequently found in animals, especially pigs and cows (Keel et al., 2007; Rodriguez-Palacios et al., 2006). Comparison of human and animal strains via PFGE and MLVA indicates that strains from both sources are indistinguishable (Debast et al., 2009; Jhung et al., 2008). Genotypes found in salad mixes (ribotypes 017 and 001) (Bakri et al., 2009) are very common among human isolates in the United Kingdom (Brazier et al., 2008) and elsewhere (Barbut et al., 2007). They are rare, but nonetheless present, in animals and were detected in calves (017; Rodriguez-Palacios et al., 2006) and horses (001; Keel et al., 2007).

VIII. C. DIFFICILE IN FOOD AS A SOURCE OF HUMAN COLONIZATION: CONTAMINATED FOOD MAY BE A SOURCE OF THE ORGANISM IN THE HOSPITAL AND THE COMMUNITY The human infectious dose for C. difficile is not known. However, given the biology of this organism, the dose of C. difficile ingested may have little to do with the likelihood of developing CDI. Ingested spores emerge as vegetative cells upon exposure to germinants in the intestine and multiply to a greater or lesser extent, depending upon conditions. Furthermore, evidence strongly suggests that these altered conditions are necessary for development of disease. Thus, a large dose of spores, ingested under normal intestinal conditions, might have no effect. A small dose, ingested under altered conditions, might be the primary inciting factor in a case of CDI. Thus, the greatest risk imposed by C. difficile contamination of foods is that people may be exposed continuously to larger or smaller numbers of the organism. This would have the effect of maintaining colonization for times when intestinal conditions are amenable to establishment of the organism and production of clinical signs and lesions. The numbers of C. difficile spores in food are low, and have been detected mainly by enrichment and subculture. A recent study (Weese et al., 2009) enumerated C. difficile in retail ground beef and ground pork in four Canadian provinces. The organism was isolated by direct culture from 28% of ground beef samples (20 spores/g in two samples and 120 and 240 spores/g in one sample each). Twenty-eight percent of ground pork samples were also positive by direct culture (20 spores/g in three samples and 60 spores/g in one). Ribotype 078 was predominant, but

Clostridium difficile as a Cause of Foodborne Disease

63

ribotype 027/NAP1 strains were also identified in both beef and pork (Weese et al., 2009). Additional work with traditional or molecular methods may allow increased sensitivity and specificity, and better estimation of contamination levels. Most meats are cooked before eating, but the recommended internal temperature is 70  C. Results of work by Rodriguez-Palacios et al. (2007), albeit in buffer rather than in meat, suggest that C. difficile strains from meat would survive cooking. This is to say nothing of ready-to-eat meat products, which are also contaminated with C. difficile (Songer et al., 2009), and salad products (Bakri et al., 2009), most of which are consumed raw.

IX. CONCLUSIONS: WE HAVE MUCH TO LEARN Observation of food contamination with C. difficile and consequent discussion of the contribution of food transmission to the increase of C. difficile infection rates is only recent. The percentages of contaminated food samples differ greatly among studies between and within countries and with different sampling intervals. However, detection methods still need optimization and standardization. The number of spores is low but the infectious dose and the role of low dose exposure in establishing infection are unknown. The large overlap of genotypes from food, food animals, and humans suggests a contribution of food to the infection process.

REFERENCES Al Saif, N. and Brazier, J. S. (1996). The distribution of Clostridium difficile in the environment of South Wales. J. Med. Microbiol. 45(2), 133–137. Avbersek, J., Janezic, S., Pate, M., Rupnik, M., Zidaric, V., Logar, K., Vengust, M., Zemljic, M., Pirs, T., and Ocepek, M. (2009). Diversity of Clostridium difficile in pigs and other animals in Slovenia. Anaerobe 15, 252–255. Bakri, M. M., Brown, D. J., Butcher, J. P., and Sutherland, A. D. (2009). Clostridium difficile in ready-to-eat salads, Scotland. Emerging Infect. Dis. 15, 817–818. Barbut, F., Mastrantonio, P., Delme´e, M., Brazier, J., Kuijper, E., and Poxton, I. (2007). European study group on Clostridium difficile (ESGCD). Prospective study of Clostridium difficile infections in Europe with phenotypic and genotypic characterisation of the isolates. Clin. Microbiol. Infect. 13, 1048–1057. Bauer, M. P., Notermans, D. W., van Benthem, B. H., Brazier, J. S., Wilcox, M., Rupnik, M., Monnet, D. L., van Dissel, J. T., and Kuijper, E. J. (2010). Final results of the first pan-European Clostridium difficile infection survey. Clin. Microbiol. Infect. 16, S38. Baverud, V. (2002). Clostridium infections in animals with special reference to the horse. Vet. Q. 24(4), 203–219. Borriello, S. P., Honour, P., and Barclay, F. (1983). Foodborne transmission of Clostridium difficile. Gastroenterology 84(1), 201.

64

Maja Rupnik and J. Glenn Songer

Bouttier, S., Barc, M. C., Felix, B., lambert, S., Torkat, A., Collignon, A., and Barbut, F. (2007). Screening for Clostridium difficile in Meat from French Retailers. European Congress of Clinical Microbiology and Infectious Diseases, Munchen. Brazier, J. S., Raybould, R., Patel, B., Duckworth, G., Pearson, A., Charlett, A., and Duerden, B. I. (2008). HPA regional microbiology network. Distribution and antimicrobial susceptibility patterns of Clostridium difficile PCR ribotypes in English hospitals, Euro Surveill. 13(41), pii: 19000. Broda, D. M., DeLacy, K. M., Bell, R. G., Braggins, T. J., and Cook, R. L. (1996). Psychrotrophic Clostridium spp. associated with ‘blown pack’ spoilage of chilled vacuum-packed red meats and dog rolls in gas-impermeable plastic casings. Int. J. Food Microbiol. 29(2–3), 335–352. Chernak, E., et al. (2005). Severe Clostridium difficile-associated disease in populations previously at low risk—Four states. Morb. Mortal. Wkly Rep. 54, 1201–1205. Debast, S. B., van Leengoed, L. A., Goorhuis, A., Harmanus, C., Kuijper, E. J., and Bergwerff, A. A. (2009). Clostridium difficile PCR ribotype 078 toxinotype V found in diarrhoeal pigs identical to isolates from affected humans. Environ. Microbiol. 11(2), 505–511. Frazier, K. S., Herron, A. J., Hines, M. E., II, Gaskin, J. M., and Altman, N. H. (1993). Diagnosis and enterotoxemia due to Clostridium difficile in captive ostriches (Struthio camelus). J. Vet. Diagn. Invest. 5, 623–625. Gurian, L., Ward, T. T., and Katon, R. M. (1982). Possible foodborne transmission in a case of pseudomembranous colitis due to Clostridium difficile: Influence of gastrointestinal secretions on Clostridium difficile infection. Gastroenterology 83, 465–469. Hafiz, S. and Oakley, C. L. (1976). Clostridium difficile: Isolation and characteristics. J. Med. Microbiol. 9, 129–136. Hammitt, M. C., Bueschel, D. M., Keel, M. K., Glock, R. D., Cuneo, P., DeYoung, D. W., Reggiardo, C., Trinh, H. T., and Glenn Songer, J. (2008). A possible role for Clostridium difficile in the etiology of calf enteritis. Vet. Microbiol. 127, 343–352. Hines, R. S. and Dickerson, S. (1993). Pseudomembranous enteritis associated with ciprofloxacin and Clostridium difficile in a penguin (Eudyptes chrysolophus). J. Zoo Wildl. Med. 24, 553–556. Hirsh, D. C., Biberstein, E. L., and Jang, S. S. (1979). Obligate anaerobes in clinical veterinary practice. J. Clin. Microbiol. 10, 188–191. Indra, A., Lassnig, H., Baliko, N., Much, P., Fiedler, A., Huhulescu, S., and Allerberger, F. (2009). Clostridium difficile: A new zoonotic agent? Wien Kln Wochenschr 121, 91–95. Jhung, M. A., Thompson, A. D., Killgore, G. E., Zukowski, W. E., Songer, G., Warny, M., Johnson, S., Gerding, D. N., McDonald, L. C., and Limbago, B. M. (2008). Toxinotype V Clostridium difficile in humans and food animals. Emerging Infect. Dis. 14, 1039–1045. Jo¨bstl, M., Heuberger, S., Indra, A., Nepf, R., Ko¨fer, J., and Wagner, M. (2010). Clostridium difficile in raw products of animal origin. Int. J. Food Microbiol. 138(1–2), 172–175. Jones, R. L., Shideler, R. K., and Cockerell, G. L. (1988a). Association of Clostridium difficile with foal diarrhea. In ‘‘Proc. 5th Int. Conf. Equine Infect. Dis’’. The University Press of Kentucky, Lexington. Jones, R. L., Adney, W. S., Alexander, A. F., Shideler, R. K., and Traub-Dargatz, J. L. (1988b). Hemorrhagic necrotizing enterocolitis associated with Clostridium difficile infection in four foals. J. Am. Vet. Med. Assoc. 193, 76–79. Keel, K., Brazier, J. S., Post, K. W., Weese, S., and Songer, J. G. (2007). Prevalence of PCR ribotypes among Clostridium difficile isolates from pigs, calves, and other species. J. Clin. Microbiol. 45(6), 1963–1964. Kuijper, E. J., Coignard, B., and Tull, P. (2006). Emergence of Clostridium difficile-associated disease in North America and Europe. Clin. Microbiol. Infect. 12(Suppl 6), 2–18.

Clostridium difficile as a Cause of Foodborne Disease

65

Levett, P. N. (1986). Clostridium difficile in habitats other than the human gastro-intestinal tract. J. Infect. 12, 253–263. Limbago, B. M., Long, C. M., Thompson, A. D., Killgore, G. E., Hannett, G. E., Havill, N. L., Mickelson, S., Lathrop, S., Jones, T. F., Park, M. M., Harriman, K. H., Gould, L. H., McDonald, L. C., and Angulo, F. J. (2009). Clostridium difficile strains from communityassociated infections. J. Clin. Microbiol. 47, 3004–3007. Loo, V. G., Poirier, L., Miller, M. A., Oughton, M., Libman, M. D., Michaud, S., Bourgault, A. M., Nguyen, T., Frenette, C., Kelly, M., Vibien, A., Brassard, P., et al. (2005). A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. N. Engl. J. Med. 353, 2442–2449. O’Neill, G., Adams, J. E., Bowman, R. A., and Riley, T. V. (1993). A molecular characterization of Clostridium difficile isolates from humans, animals and their environments. Epidemiol. Infect. 111, 257–264. Orchard, J. C., Fekety, R., and Smith, J. R. (1983). Antibiotic-associated colitis due to Clostridium difficile in a Kodiak bear. Am. J. Vet. Res. 44, 1547–1548. Porter, M. C., Reggiardo, C., Bueschel, D. M., Keel, M. K., and Songer, J. G. (2002). Association of Clostridium difficile with bovine neonatal diarrhea. In ‘‘Proc. 45th Ann. Mtg. Amer. Assoc. Vet. Lab. Diagn., St. Louis, MO’’. Rehg, J. E. and Shoung, L. Y. (1981). Clostridium difficile colitis in a rabbit following antibiotic therapy for pasteurellosis. J. Am. Vet. Med. Assoc. 179, 1296. Riley, T. V., Adams, J. E., O’Neill, G. L., and Bowman, R. A. (1991). Gastrointestinal carriage of Clostridium difficile in cats and dogs attending veterinary clinics. Epidemiol. Infect. 107, 659–665. Rodriguez-Palacios, A., Stampfli, H. R., Duffield, T., Peregrine, A. S., Trotz-Williams, L. A., Arroyo, L. G., Brazier, J. S., and Weese, J. S. (2006). Clostridium difficile PCR ribotypes in calves, Canada. Emerging Infect. Dis. 12, 1730–1736. Rodriguez-Palacios, A., Staempfli, H. R., Duffield, T., and Weese, J. S. (2007). Clostridium difficile in retail ground meat, Canada. Emerging Infect. Dis. 13, 485–487. Rodriguez-Palacios, A., Reid-Smith, R. J., Staempfli, H. R., Daignault, D., Janecko, N., Avery, B. P., Martin, H., Thomspon, A. D., McDonald, L. C., Limbago, B., and Weese, J. S. (2009). Possible seasonality of Clostridium difficile in retail meat, Canada. Emerging Infect. Dis. 15(5), 802–805. Rupnik, M., Wilcox, M. H., and Gerding, D. N. (2009). Clostridium difficile infection: New developments in epidemiology and pathogenesis. Nat. Rev. Microbiol. 7(7), 526–536. Shimango, C. and Mwakurudza, S. (2008). Clostridium difficile in broiler chickens sold at market places in Zimbabwe and their antimicrobial susceptibility. Int. J. Food Microbiol. 124, 268–270. Songer, J. G. and Anderson, M. A. (2006). Clostridium difficile: an important pathogen of food animals. Anaerobe 12, 1–4. Songer, J. G., Trinh, H. T., Killgore, G. E., Thompson, A. D., McDonald, L. C., and Limbago, B. M. (2009). Clostridium difficile in retail meat products, USA, 2007. Emerging Infect Dis. 15(5), 819–821. Traub-Dargatz, J. L. and Jones, R. L. (1993). Clostridia-associated enterocolitis in adult horses and foals. Vet. Clin. North Am. Equine Pract. 9, 411–421. Vengust, M., Arroyo, L. G., Weese, J. S., and Baird, J. D. (2003). Preliminary evidence for dormant clostridial spores in equine skeletal muscle. Equine Vet. J. 35(5), 514–516. von Abercron, S. M., Karlsson, F., Wigh, G. T., Wierup, M., and Krovacek, K. (2009). Low occurrence of Clostridium difficile in retail ground meat in Sweden. J. Food Prot. 72(8), 1732–1734. Weber, A., Kroth, P., and Heil, G. (1989). Occurrence of Clostridium difficile in faeces of dogs and cats. J. Vet. Med. 36, 568–576.

66

Maja Rupnik and J. Glenn Songer

Weese, J. S., Rousseau, J., and Arroyo, L. (2005). Bacteriological evaluation of commercial canine and feline raw diets. Can. Vet. J. 46, 513–516. Weese, J. S., Avery, B. P., Rousseau, J., and Reid-Smith, R. J. (2009). Detection and enumeration of Clostridium difficile spores in retail beef and pork. Appl. Environ. Microbiol. 75(15), 5009–5011. Weese, J. S., Reid-Smith, R. J., Avery, B. P., and Rousseau, J. (2010). Detection and characterization of Clostridium difficile in retail chicken. Lett. Appl. Microbiol. 75(15), 5009–5011. Zidaric, V., Zemljic, M., Janezic, S., Kocuvan, A., and Rupnik, M. (2008). High diversity of Clostridium difficile genotypes isolated from a single poultry farm producing replacement laying hens. Anaerobe 14(6), 325–327.

CHAPTER

4 Escherichia coli O157:H7: Recent Advances in Research on Occurrence, Transmission, and Control in Cattle and the Production Environment Elaine D. Berry*,1 and James E. Wells*

Contents

I. Introduction II. Sources and Transmission of E. coli O157:H7 in Cattle A. Flies B. Feed C. Water D. Feces, manures, and soils III. Factors Affecting the Prevalence and Levels of E. coli O157:H7 in Cattle and the Production Environment A. Seasonality of shedding B. High-level shedders of E. coli O157:H7 C. Diet effects on shedding and persistence D. Animal stress E. Other effectors IV. Preharvest Control of E. coli O157:H7 A. Vaccines B. Probiotics or direct-fed microbials C. Bacteriophage D. Chlorate E. Neomycin sulfate

68 70 71 72 73 75 76 77 80 82 86 88 89 90 91 92 93 94

* U.S. Department of Agriculture, Agricultural Research Service, U.S. Meat Animal Research Center, Clay 1

Center, Nebraska, USA Corresponding author: Elaine D. Berry, E-mail address: [email protected]

Advances in Food and Nutrition Research, Volume 60 ISSN 1043-4526, DOI: 10.1016/S1043-4526(10)60004-6

67

68

Elaine D. Berry and James E. Wells

F. Other dietary supplements G. Manure and cattle pen surface treatments V. Linking Preharvest and Postharvest Reduction of E. coli O157:H7 VI. Conclusions and Future Prospects Acknowledgments References

Abstract

94 95 97 99 100 100

Escherichia coli O157:H7 is a zoonotic pathogen that is an important cause of human foodborne and waterborne disease, with a spectrum of illnesses ranging from asymptomatic carriage and diarrhea to the sometimes fatal hemolytic uremic syndrome. Outbreaks of E. coli O157:H7 disease are often associated with undercooked beef, but there are other sources of transmission, including water, produce, and animal contact, which can often be linked directly or indirectly to cattle. Thus, preharvest control of this pathogen in cattle production should have a large impact on reducing the risk of human foodborne illness. In this review, we will summarize preharvest research on E. coli O157:H7 in cattle and the production environment, focusing on factors that may influence the transmission, prevalence, and levels of this pathogen, such as season, diet, high-level shedders, and animal stress. In addition, we will discuss recent research on the reduction of this pathogen in cattle production, including vaccination, probiotics, bacteriophage, and manure treatments.

I. INTRODUCTION Escherichia coli O157:H7 became recognized as a human pathogen and a cause of foodborne disease in 1982, following two outbreaks of hemorrhagic colitis linked to the consumption of hamburgers (Riley et al., 1983; Wells et al., 1983). Increased surveillance following these discoveries has identified E. coli O157:H7 as an important cause of bacterial diarrhea, and that the spectrum of illnesses caused by this pathogen include asymptomatic carriage, nonbloody diarrhea, hemorrhagic colitis, thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome (Cohen and Giannella, 1992; Griffin et al., 1988; Su and Brandt, 1995). Children and the elderly are particularly at risk for infection and for the associated complications of thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, and death (Cohen and Giannella, 1992; Griffin et al., 1988; Reiss et al., 2006; Su and Brandt, 1995). The Centers for Disease Control and Prevention has estimated that E. coli O157:H7 causes 73,480 cases of human illness annually, including 2168 hospitalizations and 61 deaths (Mead et al., 1999).

Escherichia coli O157 in Cattle Production

69

Human disease caused by E. coli O157:H7 is often associated with contaminated and undercooked beef (Bell et al., 1994; CDC, 1997; Riley et al., 1983; Rodrigue et al., 1995; Slutsker et al., 1998). However, improved outbreak surveillance for this organism has revealed many other modes of transmission of E. coli O157:H7 to humans, many of which can be linked directly or indirectly to cattle. Other bovine-derived food vehicles include unpasteurized milk (Bhat et al., 2007; Keene et al., 1997), raw milk cheese and butter (Rangel et al., 2005), and dry-cured salami (CDC, 1995). Produce has become a common source of foodborne E. coli O157:H7 since the first U.S. outbreaks were reported in 1991 (Rangel et al., 2005); produce items associated with outbreaks include spinach, lettuces, apple cider and juice, coleslaw, and sprouts (Besser et al., 1993; CDC, 2006; Hilborn et al., 1999; Rangel et al., 2005). Both drinking water and recreational waters have been the sources of E. coli O157:H7 that caused human illness outbreaks (Bruce et al., 2003; O’Connor, 2002; Swerdlow et al., 1992). In 2000, an estimated 2300 people became ill and seven died in a large waterborne outbreak in Walkerton, Ontario, Canada, that was caused by both E. coli O157:H7 and Campylobacter jejuni contamination of the municipal water supply by runoff from land-applied bovine manure (O’Connor, 2002). Numerous E. coli O157:H7 outbreaks resulting from contact with cattle or their manure on farms, at fairs, and at petting zoos have also been reported (CDC, 2005; Durso et al., 2005; Rangel et al., 2005). Research on E. coli O157:H7 in cattle intensified following a large multistate outbreak in 1993 that was due to the consumption of undercooked hamburgers (Bell et al., 1994). Early work established that cattle are an important reservoir of this pathogen and that there is a seasonal pattern of shedding, but indicated that prevalence of E. coli O157:H7 was low, being typically less than 5.0% (Hancock et al., 1994, 1997; Wells et al., 1991). However, with the development and use of more sensitive isolation and detection procedures (in particular immunomagnetic separation; Chapman et al., 1994), it was found that prevalence of the pathogen is often much higher than previously thought. Van Donkersgoed et al. (1999) reported E. coli O157:H7 in 7.5% of feces samples collected from cattle at slaughter, with a peak prevalence rate of 19.7% during the months of June to August. Similarly, Elder et al. (2000) isolated E. coli O157 from 28% of feces samples collected from fed beef cattle at slaughter during July and August. In a subsequent study examining fed beef cattle presented for slaughter at three Midwestern U.S. processing plants, Barkocy-Gallagher et al. (2003) observed a peak E. coli O157:H7 fecal prevalence rate of 12.9% in the summer months, in comparison with 6.8%, 0.3%, and 3.9% in the fall, winter, and spring months. Rhoades et al. (2009) recently compiled published data on the prevalence of Shiga toxin-producing E. coli in feces and at other points in beef production.

70

Elaine D. Berry and James E. Wells

E. coli O157:H7 fecal shedding and hide prevalence are correlated with beef carcass contamination (Arthur et al., 2004; Brichta-Harhay et al., 2008; Elder et al., 2000), indicating that control strategies targeted at the live animal should reduce the risk of illness associated with undercooked beef consumption. This is further suggested by model simulations assessing the benefits of preharvest control measures to reduce E. coli O157:H7 contamination of beef carcasses (Jordan et al., 1999). In addition, reducing the load of this pathogen in and on cattle should enhance the effectiveness of postharvest carcass intervention efforts (Brichta-Harhay et al., 2008). The various vehicles and routes by which E. coli O157:H7 is transmitted to humans clearly demonstrate that the problem of E. coli O157:H7 is not confined to beef products, and further suggest that preharvest control of this microorganism in cattle production should have a large impact on the reduction of risk of illness from water, produce, and other environmental sources. Finally, it is important to note that there are a number of other non-O157 Shiga-toxigenic E. coli (non-O157 STEC) that are important human pathogens that have been linked to cattle and bovine food products (Bettelheim, 2007). As reviewed by Bettelheim (2007), the inability of most E. coli O157:H7 to ferment sorbitol has provided a convenient marker for selecting this pathogen; a result of this, at least in part, is that a preponderance of the research on the ecology and distribution of STEC has been focused on E. coli O157:H7. This chapter will provide a summary of research on E. coli O157:H7 in cattle (or E. coli O157, as reported by individual studies), including sources, transmission, and those factors that affect the prevalence and numbers of the pathogen in cattle and the production environment. In addition, we will highlight recent discoveries and review potential strategies for E. coli O157:H7 reduction and control.

II. SOURCES AND TRANSMISSION OF E. COLI O157:H7 IN CATTLE Research efforts to understand the on-farm ecology of E. coli O157:H7 have found this pathogen in dairy and beef cattle, in calves and adult animals, and in cattle in both feedlot-, confinement- and pasture-based production systems (Gannon et al., 2002; Hancock et al., 1994, 1997; Laegreid et al., 1999; Ogden et al., 2004; Synge et al., 2003; Wells et al., 1991). In addition, E. coli O157:H7 has been isolated from feces of both organically raised and naturally raised cattle, and while data yet are limited, there are no apparent differences in the prevalence of this organism in cattle from these niche marketing production systems in comparison with conventionally raised cattle (Fox et al., 2008a; Kuhnert et al., 2005; Reinstein et al., 2009). Numerous nonbovine animals and other sources have been identified as potential reservoirs or vehicles of E. coli O157:H7,

Escherichia coli O157 in Cattle Production

71

including other livestock and domestic animals, many kinds of wild animals and birds, flies, water, feed, and manure. E. coli O157:H7 has been isolated from sheep (Chapman et al., 1997; Kudva et al., 1996), goats (Fox et al., 2007a; Keen et al., 2006a), pigs (Chapman et al., 1997; Doane et al., 2007; Feder et al., 2003), horses (Hancock et al., 1998), dogs (Hancock et al., 1998), chickens (Dipineto et al., 2006; Doane et al., 2007), and turkeys (Doane et al., 2007). The prevalence of this pathogen in sheep and goats and links to human illness suggest that these small ruminants may be a significant reservoir of E. coli O157:H7 (La Ragione et al., 2009). Kudva et al. (1996) reported a peak prevalence of 31% in free-ranging sheep in June. Flock level prevalence of 40% and fecal prevalence of 6.5% was reported for pastured sheep on 15 farms in Scotland, and fecal concentrations as high as 104 CFU/g of E. coli O157 were observed (Ogden et al., 2005). Keen et al. (2006a) isolated E. coli O157 from 11% of sheep and 2% of goats at county and state fairs in the United States. Overall E. coli O157 prevalence in 181 Boer-sired finishing goats during a 105-day feeding period was 4.6% (Fox et al., 2007a). Transmission of the pathogen to humans via consumption of unpasteurized goat’s milk (Bielaszewska et al., 1997; McIntyre et al., 2002), environmental contact with sheep manure (Ogden et al., 2002), and sheep or goat contact at petting zoos (Heuvelink et al., 2002; Payne et al., 2003) has been reported. Other animals demonstrated to carry E. coli O157:H7 include, but are not limited to, both tame and wild deer (Fischer et al., 2001; Heuvelink et al., 2002; Sargeant et al., 1999), rabbits (Scaife et al., 2006), raccoons (Shere et al., 1998), opossums (Renter et al., 2003), and rats (Cizek et al., 1999). Pigeons (Shere et al., 1998), gulls (Wallace et al., 1997), rooks (Ejidokun et al., 2006), and starlings (Nielsen et al., 2004) are among the numerous bird species that have been found positive for E. coli O157 and other STEC, leading to speculation about an important role for birds that frequent feedlots and farms in the transfer and dissemination of this pathogen from cattle (Bach et al., 2002c; Wetzel and LeJeune, 2006).

A. Flies Livestock manure is a favorite developmental site, food source, and landing spot for flies, and flies can subsequently contaminate other surfaces with pathogens from manure by regurgitation, fecal deposition, or mechanical transfer (Graczyk et al., 2001). Several studies have detected E. coli O157:H7 in flies collected from both dairy and beef cattle production environments (Alam and Zurek, 2004; Hancock et al., 1998; Iwasa et al., 1999; Shere et al., 1998). Fly species shown to harbor this pathogen include members of the Muscidae and Calliphoridae families; however, houseflies (Musca domestica) are most typically implicated (Iwasa et al., 1999; Keen et al., 2006a; Moriya et al., 1999; Talley et al., 2009). Alam and

72

Elaine D. Berry and James E. Wells

Zurek (2004) collected houseflies at a Kansas cattle feedlot weekly from June to October. They found an overall E. coli O157:H7 prevalence of 2.2% of individual flies, and found levels of E. coli O157:H7 as high as 1.5
105 CFU per fly. Houseflies were implicated in the transmission of E. coli O157:H7 from cattle to humans via food contamination (Moriya et al., 1999), and in laboratory studies, were demonstrated to transfer the pathogen onto spinach leaves (Talley et al., 2009). Ahmad et al. (2007) demonstrated that houseflies can transmit E. coli O157:H7 to cattle. Similarly, pulsed-field gel electrophoresis patterns of E. coli O157:H7 were sometimes indistinguishable in fly and livestock isolates collected at the same U.S. agricultural fairs, indicating transfer of the pathogen (Keen et al., 2006a). The persistence and proliferation of E. coli O157:H7 in and on houseflies suggested to Kobayashi et al. (1999) that houseflies are more than just mechanical vectors for this pathogen, and that they may play a more significant role in the transmission of this organism in cattle production. The increases in E. coli O157:H7 prevalence in cattle during the warmer summer months coincide with increases in fly populations, though a direct impact of flies on E. coli O157:H7 prevalence in cattle has not been demonstrated (Rasmussen and Casey, 2001). Flies are ubiquitous in cattle production environments and can generally move about the feedlot or farmyard unencumbered. As a result, if flies had a significant effect on the overall incidence of E. coli O157:H7, one might anticipate that the prevalence of the pathogen in cattle would be more homogenous across pens within a feedlot. Instead, a high degree of variation in prevalence across pens typically has been observed (Arthur et al., 2009; LeJeune et al., 2004; Smith et al., 2001; Wells et al., 2009). Thus, while flies can transmit E. coli O157:H7, they do not appear to have a large effect on the overall prevalence of this microorganism in cattle.

B. Feed Animal feed has been suggested to be a vehicle for transmission of E. coli O157:H7 to cattle (Hancock et al., 2001; Lynn et al., 1998; Rice et al., 1999). Shere et al. (1998) found E. coli O157:H7 in 3 of 32 feed samples collected from feed bunks on one of four dairy farms examined; molecular subtyping by pulsed-field gel electrophoresis indicated that the feed isolates were of the same E. coli O157:H7 strain as that found in cattle, flies, and water samples on that farm. While cattle on another of the remaining three farms were also shedding E. coli O157:H7 during the study, the pathogen was not recovered from feed samples collected on that farm (Shere et al., 1998). Dodd et al. (2003) detected E. coli O157:H7 in 14.9% of feed samples from feed bunks from 54 Midwestern feedlots. Van Donkersgoed et al. (2001) found E. coli O157 in 1.7% of feed samples from feed bunks, but not in fresh mixed rations that had been collected

Escherichia coli O157 in Cattle Production

73

prior to feeding. Similar to the observations of Shere et al. (1998), molecular subtyping of E. coli O157:H7 indicated transmission of the pathogen among cattle, water, and feed within the same feedlot (Van Donkersgoed et al., 2001). Davis et al. (2003) found low prevalences of E. coli O157:H7 in feed components (0.2%) and feed mill samples (0.4%). For feed before and after cattle access, Sanderson et al. (2006) reported 1.25% and 3.25% of feed samples positive for E. coli O157, respectively. Similarly, Doane et al. (2007) intermittently isolated E. coli O157:H7 from feed samples, both before distribution to animals and after placement in feeders or troughs. Conversely, other studies did not find this pathogen in cattle feed (Hancock et al., 1998; Lynn et al., 1998). The above studies indicate that recovery of E. coli O157:H7 from feeds prior to exposure to cattle is infrequent. Once placed in the feedbunk, feeds may be contaminated with the pathogen by cattle feces or saliva, birds, or other vermin (Dodd et al., 2003). Thus, while a role for feeds in transmission of E. coli O157:H7 among cattle is plausible, a clear role for feed hygiene controls to reduce this pathogen in cattle production is not apparent. This was indicated by Smith et al. (2001), who examined E. coli O157:H7 prevalence in more than 3100 cattle in 29 pens of five U.S. feedlots, and found no relationship between the prevalence of fecal shedding and the presence of the pathogen in feed.

C. Water Numerous studies have demonstrated that cattle drinking water can be a reservoir of E. coli O157:H7, and a means of disseminating this pathogen to cattle (Faith et al., 1996; Hancock et al., 1998; LeJeune et al., 2001a,b; Shere et al., 1998; Van Donkersgoed et al., 2001). E. coli O157 was isolated from 3.1% of 320 water trough samples collected at feedlots and dairy farms in the northwestern United States (Hancock et al., 1998). LeJeune et al. (2001b) found E. coli O157 in 1.3% of 473 water troughs at 98 different dairy cattle operations and one slaughter plant. Van Donkersgoed et al. (2001) found this organism in 12% of water troughs of feedlot cattle in southern Alberta, and noted a seasonal effect on its prevalence in the troughs. E. coli O157 was recovered from 25% of water samples collected at a beef cattle feedlot from May to August (Sanderson et al., 2006). Isolation of the same or similar molecular subtypes from both cattle and drinking water at the same farms or feedlots indicated a role for water in the transmission of E. coli O157:H7 among cattle (Faith et al., 1996; Shere et al., 1998; Van Donkersgoed et al., 2001). Shere et al. (2002) confirmed that drinking water contaminated with E. coli O157:H7 can disseminate this organism to cattle.

74

Elaine D. Berry and James E. Wells

Water troughs may be contaminated with E. coli O157:H7 by cattle or their immediate environment, via feces, saliva, dust, feed, or bedding material (LeJeune et al., 2001b; Shere et al., 1998). Furthermore, several works have shown that this bacterium can persist for long periods of time in water. E. coli O157:H7 survived for at least 70 days in municipal drinking water at 5  C, but was reduced to below enumerable levels after 42–49 days in the same water at 20  C (Rice et al., 1992). Similarly, Wang and Doyle (1998) showed that E. coli O157:H7 survived at least 91 days in municipal, reservoir, and lake waters at 8  C, and that survival was greater at this lower temperature, in comparison to 15 and 25  C. Rice and Johnson (2000) also reported persistence of this pathogen in cattle drinking water. LeJeune et al. (2001a) found that E. coli O157 survived at least 245 days in the sediments of microcosms simulating cattle water troughs, and was still infectious to calves after 183 days. Transmission of E. coli O157:H7 by drinking water suggested that water may be a potential intervention target to control the pathogen in cattle production. LeJeune et al. (2004) saw no differences in E. coli O157: H7 prevalence in cattle feces or water trough sediments between pens of cattle supplied with chlorinated (1 ppm residual free chlorine) or nonchlorinated drinking water, likely as a result of large loads of organic matter in the troughs. Stevenson et al. (2004) concluded that electrolyzed oxidizing water may be useful for reducing this pathogen from livestock water, but the accumulation of organic material in the troughs (e.g., feces) would likely eliminate the bactericidal activity. Zhao et al. (2006) tested a large variety of chemical treatments, both alone and in combination, for the ability to inactivate E. coli O157:H7, O26:H11, and O111:NM in drinking water contaminated with cattle rumen contents or feces. Combinations of 0.1% lactic acid and 0.9% acidic calcium sulfate, with one of either 0.05% caprylic acid, 0.1% sodium benzoate, 0.5% butyric acid, or 100 ppm chlorine dioxide, were effective in inactivating enterohemorrhagic E. coli in water, but were not palatable to cattle. The addition of sodium caprylate (Amalaradjou et al., 2006) and trans-cinnamaldehyde (Charles et al., 2008) to cattle drinking water reduced E. coli O157:H7, but impacts on cattle water intake were not determined. Ozonation had little effect on inactivation of E. coli O157:H7 in water contaminated with rumen contents (Zhao et al., 2006). Though it has been shown that drinking water is commonly contaminated with E. coli O157:H7 and can transmit this pathogen to cattle, the impacts of measures to reduce E. coli O157:H7 in cattle drinking water on its prevalence in cattle have not been demonstrated. Smith et al. (2001) found that the prevalence of cattle in feedlot pens shedding E. coli O157: H7 was not correlated with the presence of the organism in the drinking water, or with the temperature, pH, or cleanliness of the water. Similarly, improved water trough hygiene did not reduce the risk of E. coli O157 in

Escherichia coli O157 in Cattle Production

75

young cattle (Ellis-Iversen et al., 2008, 2009). Significant reduction of E. coli O157:H7 from cattle and the production environment as a result of drinking water decontamination must be demonstrated before investing in the development and implementation of water management programs for livestock producers (LeJeune and Wetzel, 2007).

D. Feces, manures, and soils It is clear from the above discussion that transmission by either direct or indirect fecal–oral exposure is an important means of dissemination of E. coli O157:H7 in cattle. In this regard, cattle shedding E. coli O157:H7 provide the ‘‘fuel’’ for the maintenance of this pathogen in the environment and for the infection or reinfection of additional animals. Thus, feces, manure, and soils in the production environment are a significant source of transmission of this organism. Horizontal transmission of E. coli O157:H7 among cattle was indicated by the work of Faith et al. (1996), who found that contact with areas previously occupied by cattle shedding the pathogen was an important factor in the spread of the organism in a dairy herd. Cobbold and Desmarchelier (2002) concluded that heavy fecal contamination of pen floors and hides was the primary source of STEC transmission to calves. The results of Bach et al. (2005a) suggested that feces on pen floors were a more likely source of E. coli O157:H7 to cattle than were contaminated feed or drinking water. Smith et al. (2001) observed that higher percentages of cattle in muddy feedlot pens shed E. coli O157:H7 than did cattle in pens in a normal condition, and reasoned that muddy pen soils may facilitate fecal–oral transmission. The importance of horizontal fecal–oral transmission of this pathogen is further suggested by research that has associated the presence of animals shedding high levels of E. coli O157:H7 with higher prevalence of fecal shedding and/or hide contamination in the pen or herd (Arthur et al., 2009; Bach et al., 2005a; Chase-Topping et al., 2007; Cobbold et al., 2007; Matthews et al., 2006b; Stephens et al., 2009). Many of these studies suggest that even very few cattle excreting high levels of the pathogen (‘‘super-shedders’’) are accountable for a large proportion of the total E. coli O157:H7 contamination of the pen and other cattle. Super-shedders of E. coli O157:H7 as effectors of the occurrence and levels of the pathogen in cattle production will be discussed further below. Although the mammalian lower gastrointestinal tract is the primary habitat of E. coli, including E. coli O157:H7, this bacterium can survive for long periods of time in manure, feedlot surface material, and soils. When inoculated at initial levels of 105 CFU/g, E. coli O157:H7 could be recovered for up to 70 days from bovine feces stored at 5  C (Wang et al., 1996). Persistence was reduced at higher temperatures, but the pathogen

76

Elaine D. Berry and James E. Wells

survived in bovine feces for up to 56 and 49 days at 22 and 37  C, respectively, despite reductions in moisture content and water activity of the feces (Wang et al., 1996). E. coli O157:H7 was recoverable by enrichment for 47 days from an aerated manure pile that was constructed of manure from cattle that were inoculated with the pathogen (Kudva et al., 1998). Fukushima et al. (1999) reported that serotypes of STEC other than E. coli O157:H7 also can persist in bovine feces for several weeks. E. coli O157:H7 was shown to persist in feedlot surface material, and also to multiply in feedlot soils of permissible moisture and manure content (Berry and Miller, 2005). The importance of environmental persistence in the maintenance of E. coli O157:H7 in cattle production is further suggested by several studies that have reported that most isolates on a farm or feedlot are of one to a few genetic subtypes of this organism, which may predominate for months or years (Gannon et al., 2002; Lahti et al., 2003; LeJeune et al., 2004; Shere et al., 1998). In their longitudinal study of beef cattle at a commercial feedlot, LeJeune et al. (2004) found that most E. coli O157:H7 cattle isolates were one of four closely related genetic subtypes that persisted throughout the study, in spite of the large turnovers in cattle population. They concluded that the production environment as a reservoir may have a larger role as a source for transmission of the pathogen than do incoming cattle. Similarly, Lahti et al. (2003) observed persistent genetic subtypes of E. coli O157 in a Finnish cattle finishing unit, indicating that the finishing unit, rather than the introduction of new cattle, was the source of E. coli O157 at the farm. The environmental persistence of this pathogen suggests a potential role for manure management or other similar interventions to reduce E. coli O157:H7 in cattle production, and is discussed further below. Managing manure to eliminate pathogens will reduce not only a source of E. coli O157:H7 for the reinfection of cattle, but also the risk of transmission of this organism to the environment, including water and human food and animal feed crops.

III. FACTORS AFFECTING THE PREVALENCE AND LEVELS OF E. COLI O157:H7 IN CATTLE AND THE PRODUCTION ENVIRONMENT The development of control strategies to reduce E. coli O157:H7 will require the identification of biological, environmental, and/or management factors that affect its incidence in cattle and their production environments. Research investigations and epidemiological studies have identified a number of risk factors or management practices that can or may contribute to the occurrence of this pathogen, and that may be exploitable to reduce its numbers, persistence, and transmission in cattle.

Escherichia coli O157 in Cattle Production

77

A. Seasonality of shedding Among those factors thought to affect the prevalence of E. coli O157:H7, only season has been repeatedly and most consistently demonstrated to influence the shedding of this pathogen by cattle (Barkocy-Gallagher et al., 2003; Chapman et al., 1997; Conedera et al., 2001; Mechie et al., 1997; Milnes et al., 2009; Van Donkersgoed et al., 1999). The prevalence of shedding of this pathogen typically increases during the warmer months, and is lowest in the winter. However, there are studies that have not observed this same seasonal pattern of shedding (Alam and Zurek, 2006; Ogden et al., 2004; Sargeant et al., 2000; Synge et al., 2003). The housing of cattle during the winter in Scotland may account for the higher E. coli O157 prevalence observed during the winter months compared to summer months, perhaps by reducing exposure to the outside environment or by bringing the animals into closer proximity to one another (Ogden et al., 2004; Synge et al., 2003). Interestingly, although the prevalence of shedding the pathogen was higher during the cooler months when cattle were housed than during the warmer months when they were not, Ogden et al. (2004) observed that high-shedding cattle appeared to shed greater concentrations of E. coli O157 during the warmer months. As a result of more favorable growth temperatures, the higher prevalence of E. coli O157:H7 in cattle during warmer seasons may be influenced by the ability to replicate in environmental reservoirs such as feed or water (Hancock et al., 2001). Multiplication of the pathogen in cattle feeds has been demonstrated (Fenlon and Wilson, 2000; Lynn et al., 1998). However, other studies have not observed E. coli O157:H7 growth in feeds, and indicate that there are a number of other factors that can influence the ability of this organism to survive or replicate in feeds, including feed medications, moisture content, organic acids, and pH (Bach et al., 2002a; Van Donkersgoed et al., 2001). Although E. coli O157: H7 has been shown to grow in experimental microcosms simulating cattle water troughs (LeJeune et al., 2001b), cooler temperatures enhanced, rather than inhibited, the survival of the pathogen in water (Rice and Johnson, 2000; Rice et al., 1992; Wang and Doyle, 1998). Similarly, numerous studies have shown that cooler temperatures can enhance the persistence of E. coli, including E. coli O157:H7, in manures and soils (Berry et al., 2007; Ishii et al., 2006; Kudva et al., 1998; Topp et al., 2003; Wang et al., 1996). These observations make it interesting to speculate that greater persistence at cooler temperatures may be involved in the maintenance of this organism in the production environment during cooler winter weather when the rate of shedding by cattle typically is low. Warmer temperatures, in comparison to very cold temperatures, may improve the transfer of E. coli O157:H7 in feedlot surface soils to and/or among cattle. Smith et al. (2001) found an association between the

78

Elaine D. Berry and James E. Wells

environmental condition of a pen and the percentage of cattle shedding E. coli O157:H7, with higher prevalence of the organism occurring in cattle in muddy pens. Likewise, a USDA study in Nebraska examining the effects of diets with wet distillers grains with solubles (WDGS) observed that the percentages of E. coli O157:H7 either on hides or in feces of cattle in either treatment was lowest on a sampling day that followed a period of prolonged cold temperatures that kept pen surfaces frozen (Fig. 4.1; Wells et al., 2009). Cattle heat stress has been considered as a potential cause of the increased prevalence of the shedding of E. coli O157:H7 during the warmer seasons. However, clear effects of heat stress on the shedding of the pathogen by cattle have not been demonstrated (Brown-Brandl et al., 2009; Edrington et al., 2004; Fitzgerald et al., 2003). Factors other than temperature may influence the seasonal prevalence of E. coli O157:H7 in cattle. Edrington et al. (2006a) hypothesized that the seasonal variation of shedding is due to physiological responses of the animal as a result of changing day length. An experiment initiated in the early fall used artificial light in treatment pens to add 4–5 h/day to the daily number of hours of natural light, for a period of 60 days (Edrington et al., 2006a). No differences in bovine fecal prevalence of E. coli O157:H7 were seen after 25 days of treatment, but at 53 days, prevalence was higher in the lighted treatment pens in comparison to the control pens, which received natural light only. Prevalence of the pathogen between the treatment and control cattle did not differ at 28 or 43 days following the removal of the light treatment. These results suggested a potential role for day length in the seasonal variation of E. coli O157:H7 in cattle. Follow-up work by these researchers has examined the effects of hormones that are known to respond to changes in day length, but results have been variable. Schultz et al. (2006) reported that melatonin had no effect on growth rates of E. coli O157: H7 in in vitro broth culture, and that there was no effect of exogenous melatonin on the E. coli O157:H7 fecal shedding patterns of sheep that were experimentally infected with the pathogen. In a separate study, there was no effect of a melatonin low-dose treatment of 0.5 mg/kg BW administered daily for 1 week, but when cattle were subsequently treated with a higher daily dose of 5.0 mg/kg BW, fecal prevalence of E. coli O157:H7 was lower in melatonin-treated cattle than in control cattle (Edrington et al., 2008). There was no effect of tryptophan, a melatonin precursor, on fecal shedding of the pathogen (Edrington et al., 2008). Melatonin treatment did not protect sheep from E. coli O157:H7 infection by horizontal transmission or alter fecal shedding of the organism after the sheep were colonized (Edrington et al., 2009a). An involvement of the thyroid and thyroid hormones in seasonal fluctuations of E. coli O157:H7 shedding has also been suggested (Edrington

Escherichia coli O157 in Cattle Production

A

79

Escherichia coli O157:H7 on hides of animals (average percent of pen)

100 80 60 40 20 0

B Escherichia coli O157:H7 in feces of animals (average percent of pen)

50 40 30 20 10 0

C Average daily air temperature (⬚C)

30 20 10 0 −10 −20

0

50

100

150

200

250

Day Nov

Dec

Jan

Feb Mar Apr Month of study

May June

FIGURE 4.1 Percentage of samples positive (open symbols) or enumerable (closed symbols) for Escherichia coli O157:H7 on hides (A), in feces (B), and on daily average temperature (C) over time of the study. Control diet (CON) pen groups are represented by circles (○,d) and wet distillers grains with solubles diet (WDGS) pen groups are represented by squares (□,j). The growing phase comprises days 1–77, and the finishing phase comprises day 78–245 of production. Each month of the study is denoted in the bar across the bottom of the figure. Reprinted from Wells et al. (2009).

et al., 2007; Schultz et al., 2005). Further work is needed to confirm the impacts of day length and day length-responsive hormones on E. coli O157:H7 in cattle.

80

Elaine D. Berry and James E. Wells

As noted earlier, fly populations increase during the warmer seasons, and flies have been demonstrated to transmit E. coli O157:H7 to cattle (Ahmad et al., 2007). However, any influence of flies on the prevalence of the pathogen in cattle or as a cause of seasonal shedding has not been shown. Human foodborne disease caused by E. coli O157:H7 exhibits a seasonal pattern of occurrence that parallels the seasonal increased shedding of the pathogen by cattle (Rangel et al., 2005; Slutsker et al., 1997; Waters et al., 1994). While all or none of these above factors may contribute to the seasonality of shedding of this pathogen, the regularity of this phenomenon suggests that the identification and confirmation of its cause(s) may point to strategies to reduce shedding by cattle, thereby reducing the risk of human infection.

B. High-level shedders of E. coli O157:H7 Recent work has highlighted the impact of high-level shedders of E. coli O157:H7 on the prevalence and transmission of this pathogen. Most cattle shed E. coli O157:H7 in feces at concentrations that are below the detectable levels of enumeration procedures, but levels as high as 105–106 CFU/g of feces have been reported (Brichta-Harhay et al., 2007; Omisakin et al., 2003; Robinson et al., 2004). Cattle that shed high numbers of E. coli O157:H7 ( 103–104 CFU/g of feces) are called ‘‘super-shedders,’’ and this small proportion of super-shedding cattle is responsible for a large proportion of E. coli O157:H7 contamination in a production environment, which may in turn drive the E. coli O157:H7 prevalence of cattle in that environment (Bach et al., 2005a; Chase-Topping et al., 2007, 2008; Cobbold et al., 2007; Low et al., 2005; Matthews et al., 2006a; Stephens et al., 2009). The significance of super-shedders of E. coli O157:H7 in the dissemination and maintenance of this pathogen in cattle production has been substantiated by a number of studies. Bach et al. (2005a) noted that persistent and high-level shedding by individual animals were likely the source of infection for other animals in the pen. Similarly, the presence of high-level shedders of E. coli O157:H7 in feedlot pens was associated with higher prevalence of the pathogen among cattle in the same pen, while cattle that were negative for the pathogen were more likely to have been in a pen that did not have a super-shedding animal (Cobbold et al., 2007). High-level rectal carriage of E. coli O157 in cattle at slaughter was associated with a higher risk for E. coli O157 fecal-positive animals in the same lot (Low et al., 2005). The presence of a high-level shedder of E. coli O157:H7 on a farm was associated with higher prevalence of the pathogen among cattle on that farm (Chase-Topping et al., 2007). Mathematical modeling of prevalence and transmission dynamics of E. coli O157 on cattle farms in Scotland indicates that the distribution of prevalence is best

Escherichia coli O157 in Cattle Production

81

explained when a small proportion of cattle (the super-shedders) are responsible for most of the infections in the rest of the population, and that 80% of the E. coli O157 infections arise from the 20% of cattle that are shedding high levels of the pathogen (Matthews et al., 2006a,b). High-level fecal shedding of E. coli O157:H7 has also been linked to increased hide contamination, which is an important source of this pathogen on beef carcasses at harvest (Arthur et al., 2007, 2009; Stephens et al., 2009). Arthur et al. (2009) suggested that cattle hide contamination could be minimized if fecal concentrations of E. coli O157:H7 were reduced below 200 CFU/g. In a study examining feedlot cattle, Stephens et al. (2009) observed a larger impact of high-level shedders of E. coli O157:H7 on hide prevalence than fecal prevalence of penmates. Additionally, Fox et al. (2008b) recently reported that the probability of carcass contamination with E. coli O157 was significantly associated with the presence of a high-shedding animal within the same truckload of cattle. The association of high-shedding cattle with carcass contamination extends the chain of events linking super-shedding cattle and human disease risk outlined in Fig. 4.2 by Chase-Topping et al. (2008) to include foodborne exposure in addition to environmental exposure. Factors or mechanisms that result in high-level shedding of E. coli O157:H7 by cattle are not fully understood. However, high levels of fecal excretion and longer duration of shedding are associated with colonization at the terminal rectum (Cobbold et al., 2007; Davis et al., 2006; Lim et al., 2007; Low et al., 2005; Naylor et al., 2003). E. coli O157 from high-level shedding cattle in Scotland were more likely to be phage type 21/28 than isolates from low-shedding cattle, identifying a potential pathogenassociated risk factor (Chase-Topping et al., 2007). Further investigation is needed to determine host, microbial, or other environmental factors that influence high-level shedding of E. coli O157:H7; identification of these factors may indicate strategies to reduce this occurrence in cattle. That even very few animals shedding high levels of the pathogen can be responsible for a large proportion of the total E. coli O157:H7 contamination of the pen and other cattle suggests targeting these animals may be an approach to substantially reduce the risk for human illness caused by this pathogen. Potential strategies include detection, removal, and/or application of intervening treatments to high-level shedders before introduction to the herd or before slaughter (Chase-Topping et al., 2008; Cobbold et al., 2007; Davis et al., 2006; Matthews et al., 2006a,b; Naylor et al., 2003, 2007). As colonization of the terminal rectal mucosa is associated with high-level fecal shedding of this pathogen, recent studies have targeted this site to reduce E. coli O157:H7 in cattle. Direct application of polymyxin B or chlorhexidine to the rectal mucosa of experimentally colonized calves reduced E. coli O157:H7 concentrations in feces (Naylor et al., 2007). Similarly, a combination of bacteriophage application to the

82

Elaine D. Berry and James E. Wells

Cattle Some infections colonize terminal rectum (supershedders)

• Cattle factors • Pathogen factors • Environmental factors

More E. coli O157 shed Farm • Management factors More E. coli O157 infection

Human • Spatio-temporal distribution and risk factors

More E. coli O157 in environment

• More human exposure • Increased disease risk

FIGURE 4.2 The chain of events that link super-shedding cattle and the risk of human infection of Escherichia coli O157. Vertical arrows represent the epidemiological processes involved, with larger arrows indicating an increased risk. Dotted arrows indicate factors that influence risk. Adapted by permission from Macmillan Publishers Ltd: ChaseTopping et al. (2008), copyright 2008.

rectoanal junction and oral administration of bacteriophage in drinking water reduced E. coli O157:H7 shedding by cattle, although it did not eliminate the pathogen (Sheng et al., 2006). In a separate study, oral administration of bacteriophage was determined to be more effective than rectal administration for reducing E. coli O157:H7 shedding (Rozema et al., 2009). Chase-Topping et al. (2008) have provided a recent comprehensive review that discusses the implications of super-shedders of E. coli O157: H7 on the transmission dynamics of this pathogen in cattle, the risk of human illness, and the prospects for disease control.

C. Diet effects on shedding and persistence The ruminant animal differs significantly from monogastric animals, such as humans, rodents, and swine, in gastrointestinal tract physiology and digestion. In particular, ruminant animals have a rumen, or pregastric

Escherichia coli O157 in Cattle Production

83

compartment of the stomach, in which symbiotic microbial fermentation occurs and volatile fatty acids are absorbed (Wells and Varel, 2005). As a consequence, ruminant animals are adept at digesting and utilizing complex forages; however, many modern cattle feeding operations primarily feed high-energy, high-starch corn-based diets. Arguments relative to how cattle are fed and the impact on E. coli O157:H7 have been extensively debated, and recent reviews have detailed the results of animal diet studies on E. coli O157:H7 shedding (Callaway et al., 2009; Jacob et al., 2009a). Early work suggested that cattle fed hay would have lower E. coli O157:H7 in feces than cattle fed corn (Diez-Gonzalez et al., 1998). This hypothesis was derived from observations that hay-fed animals had much fewer fecal E. coli and acid-resistant E. coli than animals fed concentrate (high energy) diets, and acid resistance was emphasized. Nutrient utilization and growth and colonization capabilities may be better indicators of fitness for a niche, and in the case of E. coli O157:H7 in feces, the numbers of generic E. coli more so than acid-resistant E. coli may be a better indicator (Berry et al., 2004, 2006). Commensal strains of E. coli and virulent strains of E. coli O157:H7 differed in their abilities to utilize different carbon sources, and in general, commensal E. coli oxidized more substrates than did E. coli O157:H7 (Durso et al., 2004). However, commensal E. coli and E. coli O157:H7 had similar fitness for growth and were similar with regard to physiological parameters associated with colonization of the gastrointestinal tract (Durso et al., 2004; Jacobsen et al., 2009). Steers fed hay or corn silage had similar levels of generic E. coli in ruminal fluid and feces, but generic E. coli levels in ruminal fluid and feces increased for both diets when the same animals were converted to a corn diet (Berry et al., 2006). However, no effects of diet on the prevalence of E. coli O157:H7 in feces were noted. Interestingly, E. coli O157:H7 were rarely found in samples from the rumen over the 9-month duration of the study and the levels of generic E. coli were 100- to 1000-fold lower in the ruminal fluid than the feces, indicating that the rumen is not a likely reservoir for this pathogen. Likewise, in animals inoculated with E. coli O157:H7, Buchko et al. (2000a) noted a rapid elimination of the pathogen from the rumen, and Van Baale et al. (2004) did not recover E. coli O157:H7 from rumen tissues collected 11 weeks after experimental inoculation. The potential effects of bovine diet on E. coli O157:H7 are most likely to be due to changes in the lower gastrointestinal tract where E. coli is better adapted and the niche is expanded. In numerous studies, the potential effects of diet have often been evaluated using cattle experimentally inoculated with E. coli O157:H7 (Jacob et al., 2009a). These studies by design are super-physiological and the pathogen dose typically is in far excess of what the animal would

84

Elaine D. Berry and James E. Wells

ingest in normal transmission; however, they do provide some insight into colonization and persistent shedding of the pathogen. In comparison, studies examining shedding in naturally colonized animals are biologically more relevant to the feedlot system, but often dietary effects are not evident unless a large number of animals have been sampled repeatedly. Needless to say, the potential effects of diet on E. coli O157:H7 shedding should be surmised from a large body of work more so than any single study. Cattle in feedlots are fed energy-dense grain rations to improve growth and produce a high quality product, and in the United States and Canada most feedlot diets are based on grains such as corn or barley. In order to be implemented in a feedlot, any pathogen reduction benefit from a diet should not come at an increased cost in production. Barley is a grain that is grown and fed in Canada and the U.S. Northern Plains region, and in animals experimentally inoculated (Buchko et al., 2000a) and naturally infected (Berg et al., 2004), fecal prevalence was higher for cattle fed barley compared to cattle fed cracked (dry-rolled) corn. Barley has a starch content that is rapidly digested in the rumen, which results in less starch entering the lower digestive tract. Buchko et al. (2000a) observed lower pH in feces of animals fed cracked corn than those fed barley, and hypothesized that lower prevalence seen with corn could be due to a less hospitable fecal environment. Processing of grain to increase surface area can result in a grain that is more digestible in the rumen and passes less starch to the lower intestine, and processing such as steamflaking are common practices in the U.S. Southern Plains region where many feedlots are concentrated. In a study by Fox et al. (2007b), steamflaked grains (sorghum or wheat) were associated with higher fecal prevalence for E. coli O157:H7 compared to dry-rolled equivalents. When steam-flaked corn was fed to naturally infected cattle sampled over time, fecal E. coli O157 prevalence was only slightly higher compared to the dry-rolled corn, and steam-flaking did result in less fecal starch and higher fecal pH (Depenbusch et al., 2008). However, in this latter study, neither fecal starch amount or fecal pH was associated with presence or absence of the pathogen. As a whole, fecal shedding in feedlot cattle may be manipulated by processing of the energy-rich grains fed to cattle, but the mechanism(s) to date to fully explain this phenomenon has remained elusive. In recent years, grains typically fed to cattle have been shifted to the production of ethanol and cattle have been fed the coproducts, collectively known as distillers grains. Corn, in particular, has been a grain most often used for ethanol production and its coproduct is fed either as wet distillers grain, typically with solubles (WDGS), or to a lesser extent, as dried distillers grain with or without solubles (DDGS or DDG). Brewers’ grains are coproduct residues of beer production fed to cattle, and an

Escherichia coli O157 in Cattle Production

85

epidemiological study identified this animal feed as a factor associated with sixfold increased odds of detecting E. coli O157 in fecal samples (Dewell et al., 2005). A preliminary study feeding WDGS at six different levels from 0% to 50% of the diet (dry matter basis) reported no differences in probability for E. coli O157:H7 in feces, but did report differences in the probability for detecting E. coli O157:H7 in terminal rectum tissues of cattle fed different levels of WDGS (Peterson et al., 2007b). Experiments with inoculated animals demonstrated that cattle fed 25% DDG diets consistently shed nearly 2-log higher levels of E. coli O157:H7 over the last 7 days of the 42-day study, and at necropsy, significant numbers of the pathogen were found associated with tissue samples collected throughout the intestinal tract (Jacob et al., 2008c). Additional work by this laboratory observed that feeding 25% DDG (on dry matter basis) to naturally infected cattle more than doubled the prevalence of E. coli O157 in fecal pat samples collected from pen floors over a 12-week study period (Jacob et al., 2008a), and also found that feeding 25% WDGS (on dry matter basis) was associated with increased prevalence at one sample date but there was no difference on a second sample date (Jacob et al., 2008b). A separate study did not observe an association of feeding DDGS and E. coli O157:H7 fecal prevalence in steam-flaked corn-based diets (Jacob et al., 2009b). A recent large feedlot study following the same animals for more than 9 months through growing and finishing phases of production observed a significant increase in fecal prevalence for growing animals fed 14% WDGS (dry matter basis) diet compared to a 0% WDGS (Wells et al., 2009). During the finishing phase, the steers fed 40% WDGS (dry matter basis) diet had higher fecal prevalence for E. coli O157:H7 and a higher percentage of the animals were found to be shedding the pathogen at enumerable levels in the feces. As observed for processed grains fed to cattle, diets high in distillers grains would result in less starch passing to the hindgut and, as a consequence, higher fecal pH compared to a corn diet (dry-rolled or high moisture; Wells et al., 2009). In addition, the diet high in WDGS had lower levels of lactate and total volatile fatty acids in the feces, supporting the hypothesis that WDGS diets could alter the gastrointestinal tract environment and promote a more hospitable environment to support pathogen growth and persistence in the intestine. Cattle fed WDGS had at least twofold higher levels of generic E. coli in the feces compared to cattle fed corn, indicating that the microbial niche may have been expanded due to diet, in addition to diet providing a more favorable environment. How diets with distillers grains may result in an expanded niche for E. coli and higher prevalence for E. coli O157:H7 has yet to be determined, but could be as simple as a component within the distillers grain product or as complex as an alteration in gut chemistry. For example, Fox et al. (2009b) conducted in vitro studies with fecal

86

Elaine D. Berry and James E. Wells

suspensions and noted that mucus constituents stimulated E. coli O157: H7 growth, and as such, diets that alter mucin production in the intestine may alter levels and persistence of the pathogen. Studies examining the effects of cattle diet on E. coli O157:H7 have primarily focused on the gastrointestinal tract and fecal incidence. As a result, the interaction of diet
environment on the levels and persistence of E. coli O157:H7 in cattle production has not been fully appreciated. In addition to influencing the numbers of this pathogen that are shed in the feces, cattle diets can impact the extent to which E. coli O157:H7 can persist in manure or feedlot surface soils. Cattle diets that result in enhanced survival in manure and the production environment can further contribute to the scenarios depicted in Fig. 4.2, by (1) increasing the opportunities for fecal–oral exposures resulting in additional or repeated E. coli O157:H7 cattle infections, and/or (2) increasing the risks for further environmental contamination (e.g., water, soils, food and feed crops), that in turn increase the risk for human exposure and infection. Diets with lower levels of corn increased the persistence of E. coli O157: H7 in bovine feces and manure (Varel et al., 2008, 2010; Wells et al., 2005). At  10  C, E. coli O157:H7 survived longer in feces from barley-fed cattle compared to corn-fed cattle, and at 22  C, it grew to higher numbers in feces from barley-fed cattle compared to corn-fed cattle (Bach et al., 2005b). In diets with dry-rolled corn, both naturally occurring generic E. coli and/or inoculated E. coli O157:H7 persisted in greater numbers longer in manure from cattle fed 20% and 40% WDGS, than in manure from cattle fed 0% WDGS and a higher percentage of dry-rolled corn (Varel et al., 2008, 2010). The extended persistence of E. coli O157:H7 in manure from cattle fed distillers grains may be responsible in part for the higher prevalence of this pathogen on hides and feces of cattle that has been reported for this feedstuff (Jacob et al., 2008a,b; Wells et al., 2009). Indeed, diet effects on the persistence of this pathogen and the subsequent effects on E. coli O157:H7 prevalence may account for some of the inconsistencies regarding the effects of particular dietary components on the prevalence of this pathogen in cattle (e.g., grain vs. forage, grain type and processing, distillers grains; for a recent review, see Jacob et al., 2009a). Thus, studies examining the impact of cattle diets should consider not only the effect of the diet on the prevalence and numbers of E. coli O157: H7 shed in feces, but also the effect of that diet on the numbers and persistence of E. coli O157:H7 in the manure.

D. Animal stress It is commonly assumed that animal stress has a detrimental effect on microbial food safety risk; however, direct cause-and-effect relationships between animal stress responses and increased carriage and shedding of

Escherichia coli O157 in Cattle Production

87

human pathogens have not been clearly demonstrated (Rostagno, 2009). In addition, it is generally accepted that animal stress can suppress or alter the immune response, leading to increased susceptibility to infection (Rostagno, 2009; Salak-Johnson and McGlone, 2007). However, E. coli O157:H7 commonly occurs in cattle without apparent negative effects on health or performance and is not generally considered to be a pathogen of cattle (Berry et al., 2006; Cray and Moon, 1995). Nonetheless, in vitro work has indicated that neuroendocrine hormones and other immune-modulating molecules associated with stress responses may influence the growth of E. coli, including E. coli O157:H7, and its ability to colonize a host, which is likely a reflection of its status as a commensal. The catecholamine norepinephrine is released into the gastrointestinal tract upon animal stress (Rostagno, 2009). E. coli has been demonstrated to take up norepinephrine and to respond to norepinephrine with stimulated growth and expression of molecules associated with virulence and colonization, including adhesin, Shiga-like toxins, and LEE-encoded proteins (Bansal et al., 2007; Freestone et al., 2007, 2008; Kinney et al., 2000; Lyte et al., 1997; Sperandio et al., 2003). Norepinephrine enhanced adherence of E. coli O157:H7 to the intestinal mucosa in a bovine ligated ileal loop model of infection (Vlisidou et al., 2004). The glucocorticoid dexamethasone induces immunosuppression in cattle, and has been used to enhance the susceptibility of cattle to a variety of bacterial, viral, and protozoal diseases for research purposes, including E. coli O157:H7 infections (Dean-Nystrom et al., 2008; Sreerama et al., 2008; Stoffregen et al., 2004). Dexamethasone treatment enhanced the susceptibility of weaned calves to E. coli O157:H7 colonization, and both fecal and intestinal levels of the pathogen were higher in dexamethasonetreated calves than in nontreated calves (Dean-Nystrom et al., 2008). In comparison to these laboratory studies, results of in vivo work investigating the practical implications of animal stress on the prevalence and shedding of E. coli O157:H7 have been generally less definitive, although a number of potential stressors have been identified. Abrupt weaning was associated with a higher prevalence of E. coli O157 in calves (Herriott et al., 1998). Similarly, cattle weaning and movement stress were risk factors for the presence of a high-level shedder of E. coli O157 on a farm (Chase-Topping et al., 2007). Bach et al. (2004) reported that longhaul transport and a lack of preconditioning increased fecal shedding of E. coli O157:H7 in range calves, and concluded that the stresses of weaning, transport, and relocation likely increased their susceptibility to infection. Conversely, Schuehle Pfeiffer et al. (2009) did not observe an increase in fecal shedding of E. coli O157:H7 by feedlot cattle after transportation to a processing facility. The fasting that is associated with the transportation may both make animals more susceptible to E. coli O157:H7 colonization and increase their shedding of the pathogen (Buchko et al., 2000b; Cray

88

Elaine D. Berry and James E. Wells

et al., 1998; Kudva et al., 1995), although not all studies have seen these effects (Harmon et al., 1999). Thus, fasting may be responsible in part for the effects of transport on the shedding of E. coli O157:H7 (Callaway et al., 2009). Transportation to and lairage at beef processing facilities has been associated with increased prevalence of E. coli O157:H7, but this increase may be due to exposure and contact to contaminated fecal material present in the transport truck and lairage environments (Arthur et al., 2007, 2008; Dewell et al., 2008; Fox et al., 2008b), rather than, or in addition to, increased fecal shedding (Bach et al., 2004). Brown-Brandl et al. (2009) did not observe relationships between handling stress of feedlot cattle, as measured by temperament score, and either E. coli O157:H7 prevalence or concentrations or generic E. coli concentrations in their feces. Schuehle Pfeiffer et al. (2009) reported that cattle with calmer temperaments, as compared to cattle with excitable temperaments, had higher fecal prevalence of this pathogen, which is contrary to the typical hypotheses regarding the effects of animal stress on pathogen carriage and shedding. Edrington et al. (2004) did not observe a clear effect of heat stress on the shedding of E. coli O157:H7 or Salmonella in lactating dairy cattle. Similarly, Brown-Brandl et al. (2009) did not find clear trends between the heat stress levels experienced by individual beef heifers with either fecal generic E. coli concentrations or E. coli O157:H7 concentrations or prevalence. As recently reviewed by Rostagno (2009) and Salak-Johnson and McGlone (2007), the relationships between stressors, immunity, and pathogen infection in livestock are complex. Many factors may influence livestock response to stress or the results of studies examining these responses, including stressor type, duration of stress (acute vs. chronic), sample type, or the time of sample collection relative to the onset of stress (Salak-Johnson and McGlone, 2007). Moreover, confounding factors include the methodology of stress assessment or the lack of specificity of the selected parameter to measure, as well as the variability in responses of individual animals to challenges or stressors (Rostagno, 2009). Further research with solid experimental designs and appropriate controls is needed to clarify the relationships between animal stress responses and food safety risk.

E. Other effectors As noted above, norepinephrine and also epinephrine can increase virulence gene expression and colonization by E. coli O157:H7 in vitro (Bansal et al., 2007; Freestone et al., 2007, 2008; Kinney et al., 2000; Lyte et al., 1997; Sperandio et al., 2003). Beta-agonists are synthetic homologues to

Escherichia coli O157 in Cattle Production

89

norepinephrine and epinephrine, and have been approved for dietary use in feedlot cattle to improve lean muscle growth. Increased fecal shedding of E. coli O157:H7 was observed with inoculated lambs treated with betaagonists (Edrington et al., 2006c), but in feedlot studies with cattle, no significant increase in fecal prevalence for E. coli O157:H7 was observed (Edrington et al., 2006b, 2009b). Feedlot cattle have been fed ionophores for decades to alter rumen microbial flora and to improve feed conversions (Russell and Houlihan, 2003), and an early study observed a tendency for increased E. coli O157 herd prevalence when ionophores were fed (Herriott et al., 1998). Grampositive bacteria are more sensitive to ionophores (Russell and Houlihan, 2003), and commonly fed ionophores, such as monensin or lasalocid, have little effect on the Gram-negative E. coli O157:H7 in pure culture studies (Bach et al., 2002b; Edrington et al., 2003). In feedlot studies with cattle fed grain diets, neither monensin or tylosin altered E. coli O157:H7 fecal prevalence (Jacob et al., 2008b; McAllister et al., 2006). In experimentally inoculated steers, E. coli O157:H7 levels in feces from animals fed grain were not different when also fed monensin, but in feces from animals fed forage, the duration of shedding enumerable levels was shorter when monensin was added to the diet (Van Baale et al., 2004). Potential diet and ionophore interactions have not been fully studied, but in vitro incubations with inoculated rumen fluid had lower levels of E. coli O157:H7 when monensin and tylosin were added to rumen fluid from cattle fed forage than in rumen fluid from cattle fed grain (McAllister et al., 2006).

IV. PREHARVEST CONTROL OF E. COLI O157:H7 Because of the linkage of E. coli O157:H7 fecal shedding and hide prevalence to beef carcass contamination, intervention strategies targeted at the live animal are anticipated to reduce the risk of human illness associated with bovine food products. In addition, the application of effective preharvest control measures that reduce E. coli O157:H7 in the live animal should not only reduce the prevalence of this organism in beef and milk, but also reduce the incidence of environmental contamination by this organism via cattle waste, thereby further reducing the risk of human foodborne and waterborne illness. Some preharvest control procedures currently are in use or are available for application, while some procedures will require regulatory approval and/or additional research to determine their effectiveness for reducing E. coli O157:H7 in cattle.

90

Elaine D. Berry and James E. Wells

A. Vaccines Cattle producers may readily adopt an effective vaccine against E. coli O157:H7 because they are already familiar with vaccine use and can easily incorporate a vaccine into existing cattle management systems (Loneragan and Brashears, 2005). Considerable work has examined the ability of a vaccine against E. coli O157:H7 type III secreted proteins (Bioniche Life Sciences, Inc., Belleville, Ontario, Canada) to reduce E. coli O157:H7 in cattle. Type III secreted proteins are critical for E. coli O157:H7 intestinal colonization of cattle, indicating their utility as a vaccine target (Naylor et al., 2005). In early work, vaccination with type III secreted proteins reduced the prevalence, duration, and magnitude of E. coli O157:H7 fecal shedding in experimentally inoculated cattle, and also reduced the fecal prevalence of the pathogen in naturally colonized cattle (Potter et al., 2004). However, the vaccine did not significantly reduce pen prevalence of fecal E. coli O157:H7 in feedlot cattle in a large field trial (Van Donkersgoed et al., 2005). Following reformulation of the vaccine, several studies have shown efficacy of this vaccine to reduce, though not eliminate, E. coli O157:H7 in cattle. Cattle receiving the vaccine were less likely to be colonized at the terminal rectum (Peterson et al., 2007b; Smith et al., 2009b) and less likely to shed E. coli O157:H7 in feces (Moxley et al., 2009; Peterson et al., 2007c; Smith et al., 2009a). A two-dose regimen of the vaccine reduced E. coli O157:H7 colonization of the terminal rectum, from 17.0% of nonvaccinated cattle to 2.9% of vaccinated cattle (Smith et al., 2009b). Smith et al. (2009a) tested the effect of vaccinating all of the cattle within a region of a feedlot (regional vaccination) on E. coli O157:H7 rectal colonization, fecal shedding, and hide contamination, and observed that regional vaccination reduced the probability for cattle to have E. coli O157:H7-positive hides, as a consequence of reduced environmental E. coli O157:H7. This commercial vaccine is fully licensed for use in Canada and Bioniche Life Sciences, Inc. is working to meet the requirement for a U.S. conditional license (Bioniche Life Sciences, Inc., 2008). A vaccine targeting siderophore receptor and porin proteins (Epitopix, LLC, Wilmar, MN) recently received a conditional license for use in cattle in the United States. (Epitopix, LLC, 2009). The vaccine tended to reduce E. coli O157:H7 fecal prevalence and fecal concentrations of the pathogen in E. coli O157:H7-inoculated calves (Thornton et al., 2009). In two large feedlot trials examining two- and three-dose vaccine regimens, Thomson et al. (2009) observed measures of efficacy for both dose regimens, but saw evidence for a greater efficacy for three doses. Vaccination with three doses of the vaccine was associated with a two log reduction of E. coli O157 in feces (Thomson et al., 2009). Subsequent work in naturally colonized feedlot cattle compared the use of 2- and 3-ml doses of the vaccine

Escherichia coli O157 in Cattle Production

91

administered in two-dose regimens, and found that the 3-ml dose effectively reduced the prevalence of E. coli O157, the number of days that animals tested positive, and the number of days that animals were identified as high-shedders of the pathogen (> 103 CFU/g of feces), compared to the placebo (Fox et al., 2009a). Other experimental vaccines have potential to reduce E. coli O157:H7 in cattle. Immunization of the sows using a vaccine containing E. coli O157:H7 intimin protected the suckling piglets from E. coli O157:H7 infection and intestinal damage (Dean-Nystrom et al., 2002). Vaccination of experimentally infected calves with a combination of type III secreted proteins (EspA, intimin, and Tir) reduced E. coli O157:H7 concentrations in feces and the total load of the pathogen that was shed (McNeilly et al., 2010). In contrast, vaccination of calves with EspA alone induced an immune response, but did not protect against E. coli O157:H7 intestinal colonization (Dziva et al., 2007). Intramuscular immunization with H7 flagellin reduced colonization rates and delayed peak shedding of E. coli O157:H7 in orally inoculated calves, but did not reduce total pathogen shedding (McNeilly et al., 2008). A preliminary study indicated that an experimental bacterin vaccine could reduce E. coli O157 prevalence in feces and on hides, but statistical validation is needed (Woerner et al., 2006b).

B. Probiotics or direct-fed microbials The microbial flora is an important component of the gastrointestinal tract, and certain bacteria have long been recognized for beneficial properties and good health (Wells and Varel, 2005). Mechanistically, beneficial bacteria can prevent harmful bacterial colonization by competitively excluding, producing antibacterial compounds, and/or promoting healthy immune function. Probiotics or direct-fed microbials are live bacteria fed to a host to elicit a beneficial response, and are typically, but not limited to, Lactobacillus spp. strains. In cattle, numerous probiotics have been identified and tested for efficacy against E. coli O157:H7 in cattle (as reviewed by Callaway et al., 2009; Loneragan and Brashears, 2005; Oliver et al., 2009; Sargeant et al., 2007). Some effective probiotics reviewed previously include, either individually or in combinations, Enterococcus (Streptococcus) faecium, L. acidophilus, L. casei, L. fermentum, L. gallinarum, L. plantarum, Propionibacterium freudenreichii, and Streptococcus bovis, and these bacterial types interestingly are localized in the rumen or small intestine. As a direct-fed microbial, selected L. acidophilus strains, alone or in combination with P. freundenreichii, have been the most thoroughly studied and often are very effective at reducing the prevalence of fecal shedding of E. coli O157:H7 when dosed at 109 cells per animal daily (Peterson et al., 2007a; Tabe et al., 2008; Younts-Dahl et al., 2005).

92

Elaine D. Berry and James E. Wells

A reduction in E. coli O157 fecal concentrations has been reported in one study where fecal positive samples were subsequently analyzed using a most probable number procedure in combination with immunomagnetic separation (Stephens et al., 2007a). Feeding Lactobacillus-based direct-fed microbials also have been shown to reduce the prevalence of E. coli O157: H7 on cattle hides (Brashears et al., 2003; Stephens et al., 2007b). Commensal E. coli, including colicinogenic strains, have also been tested for probiotic potential against E. coli O157:H7 in inoculated calves, but results have been limited to date (Schamberger et al., 2004; Tkalcic et al., 2003). Laboratory studies indicated that E. coli O157:H7 can become resistant to individual colicins, so effective treatments may require a cocktail of strains producing different colicin types (Schamberger and Diez-Gonzalez, 2005). Recent work using Bacillus subtilis as a direct-fed microbial in cattle did not effect prevalence or levels of E. coli O157:H7 in feces or on hides (Arthur et al., 2010).

C. Bacteriophage Bacteriophages are viruses of bacteria, and their host specificity and ability to destroy their host bacteria (in the case of virulent, or lytic, bacteriophages) in the process of amplifying their own numbers has made bacteriophages attractive candidates for reducing bacterial pathogens in foods and food animals (Greer, 2005; Johnson et al., 2008). Bacteriophages are obligate parasites, and as a consequence, share a common ecology with their bacterial hosts. Bacteriophages that infect E. coli O157: H7 have been found throughout the feedlot environment, including cattle feces, manure slurries, and water troughs (Callaway et al., 2006; Niu et al., 2009b; Oot et al., 2007). Observations of fluctuations in the prevalences of E. coli O157:H7-infecting bacteriophages and E. coli O157:H7, negative correlations between bacteriophage and E. coli O157:H7, and low frequencies of fecal samples containing both E. coli O157:H7-infecting bacteriophages and their host suggest a classical predator–prey relationship between E. coli O157:H7 and its bacteriophages, and indicate the role that bacteriophages may play in the ecology of this pathogen (Callaway et al., 2006; Niu et al., 2009b; Oot et al., 2007). Several in vitro studies have demonstrated the successful elimination of E. coli O157:H7 with bacteriophages (Bach et al., 2003a; Kudva et al., 1999; Raya et al., 2006). The effectiveness of bacteriophage treatments to reduce this pathogen in live animals has been more variable, and elimination of E. coli O157:H7 has not been demonstrated. Although bacteriophage DC22 eliminated E. coli O157:H7 in an artificial rumen system, it did not reduce the levels of the pathogen shed by inoculated lambs when given in a single dose (Bach et al., 2003a). A single oral dose of bacteriophage CEV1 reduced intestinal levels of E. coli O157:H7 by two log units

Escherichia coli O157 in Cattle Production

93

within 2 days, when the animals were euthanized for examination of gut contents (Raya et al., 2006). Multiple oral doses of mixtures or ‘‘cocktails’’ of different bacteriophages may be more effective for reducing E. coli O157:H7 shedding in animals (Bach et al., 2009; Callaway et al., 2008b). Most studies have administered bacteriophages to animals orally by feed or water. However, some recent experiments have administered bacteriophages by direct application to the rectoanal junction as a means of targeting E. coli O157:H7 at its colonization site. As mentioned above, Sheng et al. (2006) both added bacteriophages to the drinking water and applied bacteriophages to the rectoanal junction mucosa; this treatment reduced but did not eliminate E. coli O157:H7 from the majority of the experimentally inoculated steers. Rozema et al. (2009) recently demonstrated that oral administration of multiple doses of a four-strain E. coli O157:H7-specific bacteriophage cocktail to cattle resulted in fewer E. coli O157:H7 positive samples and a lower mean shedding level of the pathogen, compared to rectal administration of the same bacteriophage treatment. Additional work will be needed to develop and verify the use of E. coli O157:H7-infecting bacteriophages as a method to control E. coli O157:H7 in cattle. However, progress in the area is promising, and has identified a number of items for consideration. Continous bacteriophage therapy has been suggested for the successful reduction of E. coli O157:H7 in cattle, provided that the targeted E. coli O157:H7 do not develop resistance (Rozema et al., 2009). Niu et al. (2009a) demonstrated that lytic capability and host range are considerations when selecting bacteriophage, and further established that the use of bacteriophage cocktails is likely the most effective approach to address the resistance that some E. coli O157: H7 strains may have to some phages and/or the development of resistance by E. coli O157:H7 upon phage exposure.

D. Chlorate Enterobacteriaceae is a large family of facultative anaerobic bacteria and includes many pathogens, such as E. coli O157:H7. These bacteria typically utilize oxygen for aerobic respiration, but when oxygen is absent such as in the gastrointestinal tract, they can perform fermentation. However, some of these bacteria, including E. coli, can continue to respire when alternative electron acceptors are available. Exploitation of nitrate reductase to convert chlorate to the lethal ion chlorite has been proposed to control E. coli O157:H7 in the gastrointestinal tract, with little harm to commensal anaerobic bacteria (Anderson et al., 2000). Rapid reductions of 2–3 log10 per gram for coliforms, generic E. coli, and E. coli O157:H7 were documented for rumen and fecal samples collected from E. coli O157:H7inoculated cattle given water supplemented with chlorate (Callaway et al.,

94

Elaine D. Berry and James E. Wells

2002). Chlorate treatments would likely be administered to cattle just prior to shipping (Anderson et al., 2005), and the application of chlorate for this use is pending U.S. Food and Drug Administration review and approval.

E. Neomycin sulfate Neomycin sulfate is an aminoglyoside antibiotic that is approved for use in cattle to treat enteric infections. While not approved for use to reduce E. coli O157:H7 in cattle, research indicates that neomycin sulfate could be an effective intervention. Administration at therapeutic doses reduced fecal shedding of E. coli O157:H7 compared to controls (Elder et al., 2002; Keen et al., 2006b; Woerner et al., 2006b) and also reduced hide prevalence (Woerner et al., 2006b). In contrast, supplementation of milk replacer with oxytetracycline and neomycin may increase the probability of E. coli O157:H7 shedding in very young calves (Alali et al., 2004). Because reduction of the pathogen occurs rapidly, a proposed usage of neomycin sulfate is short-term administration to cattle just prior to harvest (Elder et al., 2002; Keen et al., 2006b). However, concerns about the development of antibiotic resistance may hinder the approval of neomycin sulfate for this use (Callaway et al., 2009).

F. Other dietary supplements Modifications of the cattle diet would be a preferred method to alter pathogen shedding, but to be successful, such modifications must provide long-term benefits without compromising animal productivity. Feeding hay to reduce pathogen shedding was proposed early (DiezGonzalez et al., 1998) and has been much debated (Callaway et al., 2003; LeJeune and Wetzel, 2007), but any benefits to reducing pathogen shedding are likely short-term. Feeding whole cottonseed has been associated with reduced E. coli O157:H7 shedding (Buchko et al., 2000a; Garber et al., 1995), but in other epidemiological studies cottonseed meal or whole cottonseed was not associated with changes in shedding of the pathogen (Dargatz et al., 1997; Sargeant et al., 2004). A number of potential dietary supplements have been tested in vitro with variable success in reducing E. coli O157:H7. The rumen would be a target for most dietary interventions and in vitro tests with E. coli O157: H7-inoculated rumen fluid have shown antimicrobial capabilities for prebiotic sugars (de Vaux et al., 2002), caprylic acid (Annamalai et al., 2004), esculetin and esculin (Duncan et al., 2004), and citrus peel and pulp (Callaway et al., 2008a), whereas known rumen modifiers Saccharomyces cerevisiae (Bach et al., 2003b) and dicarboxylic acids (Nisbet et al., 2009) had no effect on pathogen reduction. Fecal incubations with esculitin and

Escherichia coli O157 in Cattle Production

95

esculin (Duncan et al., 2004), para-coumaric, ferulic, and trans-cinnamic acids (Wells et al., 2005), and sainfoin forage (Berard et al., 2009) reduced levels of inoculated E. coli O157:H7. In animal studies, esculitin and esculin (Duncan et al., 2004) reduced E. coli O157:H7 in inoculated animals, and sainfoin forage (Berard et al., 2009) and tannin extracts (Min et al., 2007) resulted in lower levels of generic E. coli in feces of treated animals. Orally administered egg yolk antibodies against E. coli O157:H7 were effective in reducing the pathogen shedding from inoculated lambs (Cook et al., 2005). Efficacy studies with the above products in feedlot trials targeting E. coli O157:H7 have not been reported to date. A product from brown seaweed, Ascophyllum nodosum, has been shown to reduce E. coli O157 shedding when fed at 2% dry matter intake in challenge studies (Bach et al., 2008) and in feedlot trials (Braden et al., 2004). The brown seaweed product also increased carcass marbling scores (Braden et al., 2007) and although the antimicrobial extract of brown seaweed phlorotannin did reduce starch fermentation at high levels in vivo (Wang et al., 2008), no effect on lamb growth was observed (Bach et al., 2008).

G. Manure and cattle pen surface treatments Most preharvest control research has focused on methods to reduce the prevalence and levels of shedding of E. coli O157:H7 by cattle. However, it is likely that environmental replication and persistence, along with amplification and shedding by cattle, combine to maintain E. coli O157:H7 in cattle production. Thus, strategies to reduce this organism may need to target both shedding by cattle and persistence in manure in order to break the transmission cycle of E. coli O157:H7. Information reporting direct fecal–oral transmission of this pathogen to cattle and work describing its long-term survivability in manures and feedlot surface soils further indicate a role for managing manure to reduce cattle exposures to E. coli O157: H7. Reducing E. coli O157:H7 from manures will have the additional benefit of reducing the potential for the environmental contamination that is associated with transmission of this pathogen from bovine manure to surface and groundwaters and food/feed crops. However, procedures to reduce pathogens from manures should also consider any potential impacts on the other health and nuisance problems that are linked to manure, namely volatile odor emissions and excess nutrients. The periodic cleaning and disinfection of cattle pens has not been demonstrated to reduce E. coli O157:H7 in cattle, likely due to the constant replenishment of fresh feces on the pen floors (Elder and Keen, 1999; Folmer et al., 2003). Providing dry bedding was associated with a reduced burden of E. coli O157 in cattle (Ellis-Iversen et al., 2008); likewise, wet bedding was identified a risk factor for the pathogen (Ellis-Iversen et al.,

96

Elaine D. Berry and James E. Wells

2007), which is consistent with other reports associating wet or muddy manures with higher E. coli O157:H7 incidence (Garber et al., 1999; Smith et al., 2001). Treatment with carbonate and alkali has been demonstrated to inactivate E. coli in cattle manure (Arthurs et al., 2001; Diez-Gonzalez et al., 2000; Jarvis et al., 2001; Park and Diez-Gonzalez, 2003). High pH is a critical feature, as the carbonate anion is responsible for the bactericidal activity (Diez-Gonzalez et al., 2000). Park and Diez-Gonzalez (2003) further examined the abilities of alkali treatment of cattle manure with carbonate and ammonia to reduce E. coli O157:H7. Because of naturally present carbonate and ammonia in mixtures of feces and urine, pH adjustment with sodium hydroxide alone effectively reduced the pathogen. Similarly, the pathogen was inactivated when manure was supplemented with urea, because of an increase in pH as a result of the enhanced production of ammonia and carbonate from urea hydrolysis. Manure treatment with urea, sodium hydroxide, and/or sodium carbonate to reduce pathogens may be relatively simple and cost-effective, but additional studies are needed to determine the effectiveness of these treatments in the animal production environment. Laboratory studies with plant essential oils including carvacrol, eugenol, and thymol, indicated that these antimicrobial compounds are not only effective for inhibiting odor emissions from livestock manure, but they can also reduce or eliminate E. coli and total coliforms (Varel and Miller, 2001, 2004). Thymol (from thyme oil) was incorporated into corncob granules to improve its stability and applied to feedlot pen surfaces (Varel et al., 2006). Manure from thymol granule-treated pens had lower concentrations of both generic E. coli and coliforms in comparison to untreated control pens. Throughout an 8-week study, E. coli O157 was not recovered from manure in any of the thymol granule-treated pens, and was recovered repeatedly from only one untreated pen. In a separate study, direct application of thymol to feedlot pen surfaces reduced E. coli O157:H7 prevalence by 50% in all treated pens (Wells et al., 2006). Addition of urease inhibitors can prolong the retention of urea nitrogen in the manure, thereby improving its fertilizer value and reducing ammonia emissions (Varel et al., 1999). However, by inhibiting urea hydrolysis to ammonia and carbon dioxide, urease inhibitors may enhance the survival of pathogens in manure (Park and Diez-Gonzalez, 2003). In vitro studies using slurries of cattle manure and urine indicated that addition of the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) can extend the survival of coliform bacteria in the manure (Varel et al., 2007a). However, coliform bacteria were rapidly eliminated when the plant oil thymol was used in combination with NBPT in the manure slurries (Varel et al., 2007a). Similarly, the application of NBPT with the plant oil extracts of linalool and pine oil on surfaces of feedlot pens

Escherichia coli O157 in Cattle Production

97

reduced generic E. coli and coliform bacteria in surface manure, in addition to conserving urea nitrogen and controlling odor production (Varel et al., 2007b). Further work is needed to determine whether feedlot pen surface treatments with thymol or other essential oils that reduce E. coli O157:H7 in manure can also reduce the levels or prevalence of the pathogen in cattle. In recent work, we examined the effects of pond ash-surfaced feedlot pens on the prevalence, levels, and persistence of E. coli O157:H7 in 128 beef cattle during an 84-day finishing period (Berry et al., 2010). Pond ash is a low-cost byproduct of coal-fired electricity generation that provides a hard surface when packed into layers (ACAA, 2008). Benefits of pond ash as a feedlot pen surface include the provision of a solid base during wet weather, thus improving footing for cattle, an easier-to-clean surface, and it may also provide a cleaner area for cattle to rest in, thus alleviating some of the problems associated with muddy pens (dirty animals, loss of traction, stress, and effort expended for walking through mud). Given these potential advantages of pond ash in comparison to traditional soil surfaces, we further hypothesized that use of pond ash may affect the transmission of E. coli O157:H7 among cattle, or the ability of E. coli O157: H7 to persist in accumulated manure. The prevalence of E. coli O157:H7 in feces and on hides of the cattle on both pond ash- and soil-surfaced pens decreased during the study period, but there was no detectable effect of pen surface type. Similarly, no differences were seen in either the prevalence of E. coli O157:H7 or the levels of generic E. coli in feedlot surface manure in pens of cattle housed on soil- and pond ash-surfaced feedlot pens, nor in survival of E. coli O157:H7 and generic E. coli in surface manure from the two types of pen surfaces.

V. LINKING PREHARVEST AND POSTHARVEST REDUCTION OF E. COLI O157:H7 Numerous studies have demonstrated that processing practices and antimicrobial intervention procedures applied at slaughter, including hide washes, steam pasteurization, organic acid washes, hot water washes, or combinations of these treatments, substantially reduce E. coli O157:H7 from cattle carcasses (Barkocy-Gallagher et al., 2003; Bosilevac et al., 2005; Elder et al., 2000; Woerner et al., 2006a). Many of these same studies also have shown that the effectiveness of antimicrobial carcass interventions is improved by reducing the pathogen load at previous steps in the process (Arthur et al., 2004; Brichta-Harhay et al., 2008; Woerner et al., 2006a). As mentioned above, high-level fecal shedding of E. coli O157:H7 is associated with increased hide contamination, and hides are an important source of beef carcass contamination at harvest (Arthur et al., 2009;

98

Elaine D. Berry and James E. Wells

Stephens et al., 2009). Thus, it follows that pathogen reduction efforts applied throughout the animal production and processing chain should reduce the risk of E. coli O157:H7 occurrence in the final beef products. However, recent studies suggest that any benefits of preharvest control efforts may be nullified by increases in E. coli O157:H7 infection and hide carriage of cattle that may occur during transportation and lairage. Arthur et al. (2007) found that both prevalence and levels of E. coli O157: H7 on cattle hides increased during transportation and lairage. Pulsedfield gel electrophoresis subtyping of isolates from cattle before and after transportation and from carcasses after processing revealed a large number of unique E. coli O157:H7 subtypes that were not detected at the feedlot, some of which were found in the transport trailers and many of which were likely a result of contamination from the lairage environment (Arthur et al., 2007). Subsequent work observed similar increases in E. coli O157:H7 hide prevalence from the feedlot through transport and lairage, and the pathogen was recovered from 64% of transport trailers and 60% of samples collected from the lairage environment (Arthur et al., 2008). Molecular subtyping of E. coli O157:H7 isolates indicated that cattle hide contamination that occurred in lairage accounted for a larger proportion of the hide and carcass contamination than did contamination from the feedlot (Arthur et al., 2008). Similarly, Mather et al. (2008) found that 84% of cattle at slaughter had E. coli O157 subtypes on their hides that did not match subtypes found previously on the farm of origin. In contrast, Fegan et al. (2009) did not observe increases in either prevalence or levels of E. coli O157 in feces or on hides as a result of transportation and lairage. E. coli O157 prevalence in feces were similar at the feedlot (18%) and after slaughter (12%), and hide prevalence decreased from 31% at the feedlot to 4% after transportation and lairage. Subtyping isolates by pulsed-field gel electrophoresis showed that all E. coli O157 from hides and feces at slaughter were of the same subtype as those collected at the feedlot. Minihan et al. (2003) did not examine hides, but also did not see an increase in E. coli O157 fecal shedding by cattle as a result of transportation and lairage. Reicks et al. (2007) found E. coli O157: H7 on less than 2% of feedlot cattle hides both before and after shipping. Risk factors for E. coli O157:H7 hide contamination during transportation and lairage included holding cattle in E. coli O157:H7 positive lairage pens, holding cattle in feces-contaminated pens, and transportation for distances greater than 160.9 km (Dewell et al., 2008). Mather et al. (2008) identified transport to the processing plant by a commercial hauler, as opposed to the farmer, as a risk factor for cross-contamination of cattle hides. Odds of preevisceration carcasses being positive for E. coli O157:H7 were higher within truckloads of cattle containing at least one animal with fecal E. coli O157:H7, and were particularly high when at least one high-level shedding animal was within the truckload (Fox et al., 2008b).

Escherichia coli O157 in Cattle Production

99

While not in full agreement, results of the cited studies indicate that E. coli O157:H7 prevalence or numbers in and on cattle during transportation or in lairage can increase as a result of contact with one another, or with contaminated feces, transport trailers, or holding pens at lairage. These observations suggest that preservation of E. coli O157:H7 reduction benefits achieved on the feedlot or farm by preharvest control strategies would require the wide adoption and practice of these procedures, and that interventions are needed to limit cattle contamination with this pathogen during transportation and lairage.

VI. CONCLUSIONS AND FUTURE PROSPECTS In the nearly 30 years since E. coli O157:H7 became recognized as a foodborne pathogen, research has revealed much about the occurrence of this pathogen in cattle, the production environment, and the factors that affect its prevalence, levels, and persistence in cattle. Despite this, questions remain unanswered and foodborne disease caused by this pathogen continues to occur. Successful preharvest interventions that have been examined to date, both those in current use and those in experimental development, can reduce but do not eliminate E. coli O157: H7 in cattle. Indeed, it seems clear that elimination of E. coli O157:H7 from cattle is unrealistic; however, evidence indicates that minimizing the levels or concentrations of the pathogen in cattle production will have substantial impact on its prevalence in cattle, the success of postharvest intervention efforts to reduce it, and on its occurrence in final beef products. Furthermore, in addition to lowering the risk of disease associated with beef consumption, reducing the numbers and persistence of E. coli O157:H7 in cattle should also reduce environmental contamination, thereby lowering the risk of water and produce contamination. Thus, research on the preharvest control of this pathogen must continue. Determination of factors that cause super-shedding of E. coli O157:H7 by some cattle are expected to reveal strategies to mitigate this occurrence, and in combination with procedures that reduce the persistence of E. coli O157:H7 in the production environment, should break the infection cycle. Hypotheses regarding the effect of super-shedders on E. coli O157:H7 prevalence in cattle may have economic implications for the cattle industry, and need to be confirmed. The concept that removing or eliminating high-level shedders will reduce E. coli O157:H7 in cattle suggests that limited resources or expensive control measures (e.g., vaccines) can be targeted at these animals, with the benefit extending to the entire herd. Information regarding the significance of E. coli O157:H7 supershedders and pathogen persistence to cattle infection and environmental contamination suggest that the food safety goals should be reductions in

100

Elaine D. Berry and James E. Wells

the levels or loads of the pathogen. Thus, studies measuring the effects of E. coli O157:H7 control procedures should collect not only prevalence data but also quantitative data. The use of enumerative methods has provided more precise evaluation of relative impact of various factors affecting shedding, prevalence, and persistence of E. coli O157:H7, and the combination of prevalence and concentration data provides information regarding both the distribution and the magnitude of the pathogen. Furthermore, microbial risk assessments require data not only on the occurrence, but also the concentration of E. coli O157:H7 in cattle and the environment, for determination of transmission risks from these sources.

ACKNOWLEDGMENTS The authors wish to thank Drs. T. M. Arthur, J. L. Bono, and T. L. Wheeler for their review of the manuscript and Ms. Janel Nierman for secretarial assistance.

REFERENCES Ahmad, A., Nagaraja, T. G., and Zurek, L. (2007). Transmission of Escherichia coli O157:H7 to cattle by house flies. Prev. Vet. Med. 80, 74–81. Alali, W. Q., Sargeant, J. M., Nagaraja, T. G., and DeBey, B. M. (2004). Effect of antibiotics in milk replacer on fecal shedding of Escherichia coli O157:H7 in calves. J. Anim. Sci. 82, 2148–2152. Alam, M. J. and Zurek, L. (2004). Association of Escherichia coli O157:H7 with houseflies on a cattle farm. Appl. Environ. Microbiol. 70, 7578–7580. Alam, M. J. and Zurek, L. (2006). Seasonal prevalence of Escherichia coli O157:H7 in beef cattle feces. J. Food Prot. 69, 3018–3020. Amalaradjou, M. A. R., Annamalai, T., Marek, P., Rezamand, P., Schreiber, D., Hoagland, T., and Venkitanarayanan, K. (2006). Inactivation of Escherichia coli O157:H7 in cattle drinking water by sodium caprylate. J. Food Prot. 69, 2248–2252. American Coal Ash Association. (ACAA), (2008). Frequently Asked Questions. Available at: http://www.acaa-usa.org. Accessed 7 January 2010. Anderson, R. C., Buckley, S. A., Kubena, L. F., Stanker, L. H., Harvey, R. B., and Nisbet, D. J. (2000). Bactericidal effect of sodium chlorate on Escherichia coli O157:H7 and Salmonella typhimurium DT104 in rumen contents in vitro. J. Food Prot. 63, 1038–1042. Anderson, R. C., Harvey, R. B., Byrd, J. A., Callaway, T. R., Genovese, K. J., Edrington, T. S., Jung, Y. S., McReynolds, J. L., and Nisbet, D. J. (2005). Novel preharvest strategies involving the use of experimental chlorate preparations and nitro-based compounds to prevent colonization of food-producing animals by foodborne pathogens. Poult. Sci. 84, 649–654. Annamalai, T., Mohan Nair, M. K., Marek, P., Vasudevan, P., Schreiber, D., Knight, R., Hoagland, T., and Venkitanarayanan, K. (2004). In vitro inactivation of Escherichia coli O157:H7 in bovine rumen fluid by caprylic acid. J. Food Prot. 67, 884–888. Arthur, T. M., Bosilevac, J. M., Nou, X., Shackelford, S. D., Wheeler, T. L., Kent, M. P., Jaroni, D., Pauling, B., Allen, D. M., and Koohmaraie, M. (2004). Escherichia coli O157 prevalence and enumeration of aerobic bacteria, Enterobacteriaceae, and Escherichia coli O157 at various steps in commercial beef processing plants. J. Food Prot. 67, 658–665.

Escherichia coli O157 in Cattle Production

101

Arthur, T. M., Bosilevac, J. M., Brichta-Harhay, D. M., Guerini, M. N., Kalchayanand, N., Shackelford, S. D., Wheeler, T. L., and Koohmaraie, M. (2007). Transportation and lairage environment effects on prevalence, numbers, and diversity of Escherichia coli O157:H7 on hides and carcasses of beef cattle at processing. J. Food Prot. 70, 280–286. Arthur, T. M., Bosilevac, J. M., Brichta-Harhay, D. M., Kalchayanand, N., King, D. A., Shackelford, S. D., Wheeler, T. L., and Koohmaraie, M. (2008). Source tracking of Escherichia coli O157:H7 and Salmonella contamination in the lairage environment at commercial U.S. beef processing plants and identification of an effective intervention. J. Food Prot. 71, 1752–1760. Arthur, T. M., Keen, J. E., Bosilevac, J. M., Brichta-Harhay, D. M., Kalchayanand, N., Shackelford, S. D., Wheeler, T. L., Nou, X., and Koohmaraie, M. (2009). Longitudinal study of Escherichia coli O157:H7 in a beef cattle feedlot and role of high-level shedders in hide contamination. Appl. Environ. Microbiol. 75, 6515–6523. Arthur, T. M., Bosilevac, J. M., Kalchayanand, N., Wells, J. E., Shackelford, S. D., Wheeler, T. L., and Koohmaraie, M. (2010). Evaluation of a direct-fed microbial product effect on the prevalence and load of Escherichia coli O157:H7 in feedlot cattle. J. Food Prot. 73, 366–371. Arthurs, C. E., Jarvis, G. N., and Russell, J. B. (2001). The effect of various carbonate sources on the survival of Escherichia coli in dairy cattle manure. Curr. Microbiol. 43, 220–224. Bach, S. J., McAllister, T. A., Baah, J., Yanke, L. J., Veira, D. M., Gannon, V. P. J., and Holley, R. A. (2002a). Persistence of Escherichia coli O157:H7 in barley silage: Effect of a bacterial inoculant. J. Appl. Microbiol. 93, 288–294. Bach, S. J., McAllister, T. A., Veira, D. M., Gannon, V. P. J., and Holley, R. A. (2002b). Effect of monensin on survival and growth of Escherichia coli O157:H7 in vitro. Can. Vet. J. 43, 718–719. Bach, S. J., McAllister, T. A., Veira, D. M., Gannon, V. P. J., and Holley, R. A. (2002c). Transmission and control of Escherichia coli O157:H7—A review. Can. J. Anim. Sci. 82, 475–490. Bach, S. J., McAllister, T. A., Veira, D. M., Gannon, V. P. J., and Holley, R. A. (2003a). Effect of bacteriophage DC22 on Escherichia coli O157:H7 in an artificial rumen system (Rusitec) and inoculated sheep. Anim. Res. 52, 89–101. Bach, S. J., McAllister, T. A., Veira, D. M., Gannon, V. P. J., and Holley, R. A. (2003b). Effects of a Saccharomyces cerevisiae feed supplement on Escherichia coli O157:H7 in ruminal fluid in vitro. Anim. Feed Sci. Technol. 104, 179–189. Bach, S. J., McAllister, T. A., Mears, G. J., and Schwartzkopf-Genswein, K. S. (2004). Longhaul transport and lack of preconditioning increases fecal shedding of Escherichia coli and Escherichia coli O157:H7 by calves. J. Food Prot. 67, 672–678. Bach, S. J., Selinger, L. J., Stanford, K., and McAllister, T. A. (2005a). Effect of supplementing corn- or barley-based feedlot diets with canola oil on faecal shedding of Escherichia coli O157:H7 by steers. J. Appl. Microbiol. 98, 464–475. Bach, S. J., Stanford, K., and McAllister, T. A. (2005b). Survival of Escherichia coli O157:H7 in feces from corn- and barley-fed steers. FEMS Microbiol. Lett. 252, 25–33. Bach, S. J., Wang, Y., and McAllister, T. A. (2008). Effect of feeding sun-dried seaweed (Ascophyllum nodosum) on fecal shedding of Escherichia coli O157:H7 by feedlot cattle and on growth performance of lambs. Anim. Feed Sci. Technol. 142, 17–32. Bach, S. J., Johnson, R. P., Stanford, K., and McAllister, T. A. (2009). Bacteriophages reduce Escherichia coli O157:H7 levels in experimentally inoculated sheep. Can. J. Anim. Sci. 89, 285–293. Bansal, T., Englert, D., Lee, J., Hegde, M., Wood, T. K., and Jayaraman, A. (2007). Differential effects of epinephrine, norepinephrine, and indole on Escherichia coli O157:H7 chemotaxis, colonization, and gene expression. Infect. Immun. 75, 4597–4607.

102

Elaine D. Berry and James E. Wells

Barkocy-Gallagher, G. A., Arthur, T. M., Rivera-Betancourt, M., Nou, X., Shackelford, S. D., Wheeler, T. L., and Koohmaraie, M. (2003). Seasonal prevalence of Shiga toxin-producing Escherichia coli, including O157:H7 and non-O157 serotypes, and Salmonella in commercial beef processing plants. J. Food Prot. 66, 1978–1986. Bell, B. P., Goldoft, M., Griffin, P. M., Davis, M. A., Gordon, D. C., Tarr, P. I., Bartleson, C. A., Lewis, J. H., Barrett, T. J., and Wells, J. G. (1994). A multistate outbreak of Escherichia coli O157:H7-associated bloody diarrhea and hemolytic uremic syndrome from hamburgers. The Washington experience. J. Am. Med. Assoc. 269, 1349–1353. Berard, N. C., Holley, R. A., McAllister, T. A., Ominski, K. H., Wittenberg, K. M., Bouchard, K. S., Bouchard, J. J., and Krause, D. O. (2009). Potential to reduce Escherichia coli shedding in cattle feces by using sainfoin (Onobrychis viviifolia) forage, tested in vitro and in vivo. Appl. Environ. Microbiol. 75, 1074–1079. Berg, J., McAllister, T., Bach, S., Stilborn, R., Hancock, D., and LeJeune, J. (2004). Escherichia coli O157:H7 excretion by commercial feedlot cattle fed either barley- or corn-based finishing diets. J. Food Prot. 67, 666–671. Berry, E. D. and Miller, D. N. (2005). Cattle feedlot soil moisture and manure content: II. Impact on Escherichia coli O157. J. Environ. Qual. 34, 656–663. Berry, E. D., Barkocy-Gallagher, G. A., and Siragusa, G. R. (2004). Stationary-phase acid resistance and injury of recent bovine Escherichia coli O157 and non-O157 biotype Escherichia coli isolates. J. Food Prot. 67, 583–590. Berry, E. D., Wells, J. E., Archibeque, S. L., Ferrell, C. L., Freetly, H. C., and Miller, D. N. (2006). Influence of genotype and diet on steer performance, manure odor, and carriage of pathogenic and other fecal bacteria. II. Pathogenic and other fecal bacteria. J. Anim. Sci. 84, 2523–2532. Berry, E. D., Woodbury, B. L., Nienaber, J. A., Eigenberg, R. A., Thurston, J. A., and Wells, J. E. (2007). Incidence and persistence of zoonotic bacterial and protozoan pathogens in a beef cattle feedlot runoff control-vegetative treatment system. J. Environ. Qual. 36, 1873–1882. Berry, E. D., Wells, J. E., Arthur, T. M., Woodbury, B. L., Nienaber, J. A., Brown-Brandl, T. M., and Eigenberg, R. A. (2010). Soil vs. pond ash surfacing of feedlot pens: Occurrence of Escherichia coli O157:H7 in cattle and persistence in manure. J. Food Prot. (In press). Besser, R. E., Lett, S. M., Weber, J. T., Doyle, M. P., Barrett, T. J., Wells, J. G., and Griffin, P. M. (1993). An outbreak of diarrhea and hemolytic uremic syndrome from Escherichia coli O157:H7 in fresh-pressed apple cider. J. Am. Med. Assoc. 269, 2217–2220. Bettelheim, K. A. (2007). The non-O157 Shiga-toxigenic (Verocytotoxigenic) Escherichia coli; under-rated pathogen. Crit. Rev. Microbiol. 33, 67–87. Bhat, M., Denny, J., MacDonald, K., Hofmann, J., Jain, S., and Lynch, M. (2007). Escherichia coli O157:H7 infection associated with drinking raw milk—Washington and Oregon, November—December 2005. MMWR Morb. Mortal. Wkly. Rep. 56, 165–167. Bielaszewska, M., Janda, J., Blahova, K., Minarikova, H., Jikova, E., Karmali, M. A., Laubova, J., Sikulova, J., Preston, M. A., Khakhria, R., Karch, H., Klazarova, H., et al. (1997). Human Escherichia coli O157:H7 infection associated with the consumption of unpasteurized goat’s milk. Epidemiol. Infect. 119, 299–305. Bioniche Life Sciences, Inc. (2008). World’s First Cattle Vaccine to Reduce E. coli O157 Threat Receives Full Licensing Approval in Canada. Available at: http://www.bioniche.com/ news_item.cfm?id¼2193. Accessed 15 January 2010. Bosilevac, J. M., Nou, X., Osborn, M. S., Allen, D. M., and Koohmaraie, M. (2005). Development and evaluation of an on-line hide decontamination procedure for use in a commercial beef processing plant. J. Food Prot. 68, 265–272. Braden, K. W., Blanton, J. R., Jr., Allen, V. G., Pond, K. R., and Miller, M. F. (2004). Ascophyllum nodosum supplementation: A preharvest intervention for reducing Escherichia coli O157:H7 and Salmonella spp. in feedlot steers. J. Food Prot. 67, 1824–1828.

Escherichia coli O157 in Cattle Production

103

Braden, K. W., Blanton, J. R., Jr., Montgomery, J. L., van Santen, E., Allen, V. G., and Miller, M. F. (2007). Tasco supplementation: Effects on carcass characteristics, sensory attributes, and retail display shelf-life. J. Anim. Sci. 85, 754–768. Brashears, M. M., Galyean, M. L., Loneragan, G. H., Mann, J. E., and Killinger-Mann, K. (2003). Prevalence of Escherichia coli O157:H7 and performance by beef feedlot cattle given Lactobacillus direct-fed microbials. J. Food Prot. 66, 748–754. Brichta-Harhay, D. M., Arthur, T. M., Bosilevac, J. M., Guerini, M. N., Kalchayanand, N., and Koohmaraie, M. (2007). Enumeration of Salmonella and Escherichia coli O157:H7 in ground beef, cattle carcass, hide and faecal samples using direct plating methods. J. Appl. Microbiol. 103, 1657–1668. Brichta-Harhay, D. M., Guerini, M. N., Arthur, T. M., Bosilevac, J. M., Kalchayanand, N., Shackelford, S. D., Wheeler, T. M., and Koohmaraie, M. (2008). Salmonella and Escherichia coli O157:H7 contamination on hides and carcasses of cull cattle presented for slaughter in the United States: An evaluation of prevalence and bacterial loads by immunomagnetic separation and direct plating methods. Appl. Environ. Microbiol. 74, 6289–6297. Brown-Brandl, T. M., Berry, E. D., Wells, J. E., Arthur, T. M., and Nienaber, J. A. (2009). Impacts of individual animal response to heat and handling stresses on Escherichia coli and E. coli O157:H7 fecal shedding by feedlot cattle. Foodborne Pathog. Dis. 6, 855–864. Bruce, M. G., Curtis, M. B., Payne, M. M., Gautom, R. K., Thompson, E. C., Bennett, A. L., and Kobayashi, J. M. (2003). Lake-associated outbreak of Escherichia coli O157:H7 in Clark County, Washington, August 1999. Arch. Pediatr. Adolesc. Med. 157, 1016–1021. Buchko, S. J., Holley, R. A., Olson, W. O., Gannon, V. P. J., and Veira, D. M. (2000a). The effect of different grain diets on fecal shedding of Escherichia coli O157:H7 by steers. J. Food Prot. 63, 1467–1474. Buchko, S. J., Holley, R. A., Olson, W. O., Gannon, V. P. J., and Veira, D. M. (2000b). The effect of fasting and diet on fecal shedding of Escherichia coli O157:H7 by cattle. Can. J. Anim. Sci. 80, 741–744. Callaway, T. R., Anderson, R. C., Genovese, K. J., Poole, T. L., Anderson, T. J., Byrd, J. A., Kubena, L. F., and Nisbet, D. J. (2002). Sodium chlorate supplementation reduces E. coli O157:H7 populations in cattle. J. Anim. Sci. 80, 1683–1689. Callaway, T. R., Elder, R. O., Keen, J. E., Anderson, R. C., and Nisbet, D. J. (2003). Forage feeding to reduce preharvest Escherichia coli populations in cattle, a review. J. Dairy Sci. 86, 852–860. Callaway, T. R., Edrington, T. S., Brabban, A. D., Keen, J. E., Anderson, R. C., Rossman, M. L., Engler, M. J., Genovese, K. J., Gwartney, B. L., Reagan, J. O., Poole, T. L., Harvey, R. B., et al. (2006). Fecal prevalence of Escherichia coli O157, Salmonella, Listeria, and bacteriophage infecting E. coli O157:H7 in feedlot cattle in the Southern Plains region of the United States. Foodborne Pathog. Dis. 3, 234–244. Callaway, T. R., Carroll, J. A., Arthington, J. D., Pratt, C., Edrington, T. S., Anderson, R. C., Galyean, M. L., Ricke, S. C., Crandall, P., and Nisbet, D. J. (2008a). Citrus products decrease growth of E. coli O157:H7 and Salmonella typhimurium in pure culture and in fermentation with ruminal microorganisms in vitro. Foodborne Pathog. Dis. 5, 621–627. Callaway, T. R., Edrington, T. S., Brabban, A. D., Anderson, R. C., Rossman, M. L., Engler, M. J., Carr, M. A., Genovese, K. J., Keen, J. E., Looper, M. L., Kutter, E. M., and Nisbet, D. J. (2008b). Bacteriophage isolated from feedlot cattle can reduce Escherichia coli O157:H7 populations in ruminant gastrointestinal tracts. Foodborne Pathog. Dis. 5, 183–191. Callaway, T. R., Carr, M. A., Edrington, T. S., Anderson, R. C., and Nisbet, D. J. (2009). Diet, Escherichia coli O157:H7, and cattle: A review after 10 years. Curr. Issues Mol. Biol. 11, 67–80. Centers for Disease Control and Prevention. (CDC) (1995). Escherichia coli O157:H7 outbreak linked to commercially distributed dry-cured salami—Washington and California, 1994. MMWR Morb. Mortal. Wkly. Rep. 44, 157–160.

104

Elaine D. Berry and James E. Wells

Centers for Disease Control and Prevention. (CDC) (1997). Escherichia coli O157:H7 infections associated with eating a nationally distributed commercial brand of frozen ground beef patties and burgers—Colorado, 1997. MMWR Morb. Mortal. Wkly. Rep. 46, 777–778. Centers for Disease Control and Prevention (CDC) (2005). Outbreaks of Escherichia coli O157: H7 associated with petting zoos—North Carolina, Florida, and Arizona, 2004 and 2005. MMWR Morb. Mortal. Wkly. Rep. 54, 1277–1280. Centers for Disease Control and Prevention (CDC) (2006). Ongoing multistate outbreak of Escherichia coli serotype O157:H7 infections associated with consumption of fresh spinach—United States. MMWR Morb. Mortal. Wkly. Rep. 55, 1045–1046. Chapman, P. A., Wright, D. J., and Siddons, C. A. (1994). A comparison of immunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli O157 from bovine faeces. J. Med. Microbiol. 40, 424–427. Chapman, P. A., Siddons, C. A., Cerdan Malo, A. T., and Harkin, M. A. (1997). A 1-year study of Escherichia coli O157 in cattle, sheep, pigs and poultry. Epidemiol. Infect. 119, 245–250. Charles, A. S., Baskaran, A., Murcott, C., Schreiber, D., Hoagland, T., and Venkitanarayanan, K. (2008). Reduction of Escherichia coli O157:H7 in cattle drinking-water by trans-cinnamaldehyde. Foodborne Pathog. Dis. 5, 763–771. Chase-Topping, M. E., McKendrick, I. J., Pearce, M. C., MacDonald, P., Matthews, L., Halliday, J., Allison, L., Fenlon, D., Low, J. C., Gunn, G., and Woolhouse, M. E. J. (2007). Risk factors for the presence of high-level shedders of Escherichia coli O157 on Scottish farms. J. Clin. Microbiol. 45, 1594–1603. Chase-Topping, M., Gally, D., Low, C., Matthews, L., and Woolhouse, M. (2008). Supershedding and the link between human infection and livestock carriage of Escherichia coli O157. Nat. Rev. Microbiol. 6, 904–912. Cizek, A., Alexa, P., Literak, I., Hamrik, J., Novak, P., and Smola, J. (1999). Shiga toxinproducing Escherichia coli O157 in feedlot cattle and Norwegian rats from a large-scale farm. Lett. Appl. Microbiol. 28, 435–439. Cobbold, R. and Desmarchelier, P. (2002). Horizontal transmission of Shiga toxin-producing Escherichia coli within groups of dairy calves. Appl. Environ. Microbiol. 68, 4148–4152. Cobbold, R. N., Hancock, D. D., Rice, D. H., Berg, J., Stilborn, R., Hovde, C. J., and Besser, T. E. (2007). Rectoanal junction colonization of feedlot cattle by Escherichia coli O157:H7 and its association with supershedders and excretion dynamics. Appl. Environ. Microbiol. 73, 1563–1568. Cohen, M. B. and Giannella, R. A. (1992). Hemorrhagic colitis associated with Escherichia coli O157:H7. Adv. Intern. Med. 37, 173–195. Conedera, G., Chapman, P. A., Marangon, S., Tisato, E., Dalvit, P., and Zuin, A. (2001). A field study of Escherichia coli O157 ecology on a cattle farm in Italy. Int. J. Food Microbiol. 66, 85–93. Cook, S. R., Bach, S. J., Stevenson, S. M. L., DeVinney, R., Frohlich, A. A., Fang, L., and McAllister, T. A. (2005). Orally administered anti-Escherichia coli O157:H7 chicken egg yolk antibodies reduce fecal shedding of the pathogen by ruminants. Can. J. Anim. Sci. 85, 291–299. Cray, W. C., Jr. and Moon, H. W. (1995). Experimental infection of calves and adult cattle with Escherichia coli O157:H7. Appl. Environ. Microbiol. 61, 1586–1590. Cray, W. C., Casey, T. A., Bosworth, B. T., and Rasmussen, M. A. (1998). Effect of dietary stress on fecal shedding of Escherichia coli O157:H7 in calves. Appl. Environ. Microbiol. 64, 1975–1979. Dargatz, D. A., Wells, S. J., Thomas, L. A., Hancock, D. D., and Garber, L. P. (1997). Factors associated with the presence of Escherichia coli O157 in feces of feedlot cattle. J. Food Prot. 60, 466–470. Davis, M. A., Hancock, D. D., Rice, D. H., Call, D. R., DiGiacomo, R., Samadpour, M., and Besser, T. E. (2003). Feedstuffs as a vehicle of cattle exposure to Escherichia coli O157:H7 and Salmonella enterica. Vet. Microbiol. 95, 199–210.

Escherichia coli O157 in Cattle Production

105

Davis, M. A., Rice, D. H., Sheng, H., Hancock, D. D., Besser, T. E., Cobbold, R., and Hovde, C. J. (2006). Comparison of cultures from rectoanal-junction mucosal swabs and feces for detection of Escherichia coli O157 in dairy heifers. Appl. Environ. Microbiol. 72, 3766–3770. de Vaux, A., Morrison, M., and Hutkins, R. W. (2002). Displacement of Escherichia coli O157: H7 from rumen medium containing prebiotic sugars. Appl. Environ. Microbiol. 68, 519–524. Dean-Nystrom, E. A., Gansheroff, L. J., Mills, M., Moon, H. W., and O’Brien, A. D. (2002). Vaccination of pregnant dams with intiminO157 protects suckling piglets from Escherichia coli O157:H7 infection. Infect. Immun. 70, 2414–2418. Dean-Nystrom, E. A., Stoffregen, W. C., Bosworth, B. T., Moon, H. W., and Pohlenz, J. F. (2008). Early attachment sites for shiga-toxigenic Escherichia coli O157:H7 in experimentally inoculated weaned calves. Appl. Environ. Microbiol. 74, 6378–6384. Depenbusch, B. E., Nagaraja, T. G., Sargeant, J. M., Drouillard, J. S., Loe, E. R., and Corrigan, M. E. (2008). Influence of processed grains on fecal pH, starch concentration, and shedding of Escherichia coli O157 in feedlot cattle. J. Anim. Sci. 86, 632–639. Dewell, G. A., Ransom, J. R., Dewell, R. D., McCurdy, K., Gardner, I. A., Hill, A. E., Sofos, J. N., Belk, K. E., Smith, G. C., and Salman, M. D. (2005). Prevalence of and risk factors for Escherichia coli O157 in market-ready beef cattle from 12 U.S. feedlots. Foodborne Pathog. Dis. 2, 70–76. Dewell, G. A., Simpson, C. A., Dewell, R. D., Hyatt, D. R., Belk, K. E., Scanga, J. A., Morley, P. S., Grandin, T., Smith, G. C., Dargatz, D. A., Wagner, B. A., and Salman, M. D. (2008). Impact of transportation and lairage on hide contamination with Escherichia coli O157 in finished beef cattle. J. Food Prot. 71, 1114–1118. Diez-Gonzalez, F., Callaway, T. R., Kizoulis, M. G., and Russell, J. B. (1998). Grain feeding and the dissemination of acid-resistant Escherichia coli from cattle. Science 281, 1666–1668. Diez-Gonzalez, F., Jarvis, G. N., Adamovich, D. A., and Russell, J. B. (2000). Use of carbonate and alkali to eliminate Escherichia coli from dairy cattle manure. Environ. Sci. Technol. 34, 1275–1279. Dipineto, L., Santaniello, A., Fontanella, M., Lagos, K., Fioretti, A., and Menna, L. F. (2006). Presence of Shiga toxin-producing Escherichia coli O157:H7 in living layer hens. Lett. Appl. Microbiol. 43, 293–295. Doane, C. A., Pangloli, P., Richards, H. A., Mount, J. R., Golden, D. A., and Draughon, F. A. (2007). Occurrence of Escherichia coli O157:H7 in diverse farm environments. J. Food Prot. 70, 6–10. Dodd, C. C., Sanderson, M. W., Sargeant, J. M., Nagaraja, T. G., Oberst, R. D., Smith, R. A., and Griffin, D. D. (2003). Prevalence of Escherichia coli O157 in cattle feeds in Midwestern feedlots. Appl. Environ. Microbiol. 69, 5243–5247. Duncan, S. H., Leitch, E. C., Stanley, K. N., Richardson, A. J., Laven, R. A., Flint, H. J., and Stewart, C. S. (2004). Effects of esculin and esculetin on the survival of Escherichia coli O157 in human faecal slurries, continuous-flow simulations of the rumen and colon and in calves. Br. J. Nutr. 91, 749–755. Durso, L. M., Smith, D., and Hutkins, R. W. (2004). Measurements of fitness and competition in commensal Escherichia coli and E. coli O157:H7 strains. Appl. Environ. Microbiol. 70, 6466–6472. Durso, L. M., Reynolds, K., Bauer, N., Jr., and Keen, J. E. (2005). Shiga-toxigenic Escherichia coli O157:H7 infections among livestock exhibitors and visitors at a Texas county fair. Vector Borne Zoonotic Dis. 5, 193–201. Dziva, F., Vlisidou, I., Crepin, V. F., Wallis, T. S., Frankel, G., and Stevens, M. P. (2007). Vaccination of calves with EspA, a key colonisation factor of Escherichia coli O157:H7, induces antigen-specific humoral responses but does not confer protection against intestinal colonisation. Vet. Microbiol. 123, 254–261.

106

Elaine D. Berry and James E. Wells

Edrington, T. S., Callaway, T. R., Varey, P. D., Jung, Y. S., Bischoff, K. M., Elder, R. O., Anderson, R. C., Kutter, E., Brabban, A. D., and Nisbet, D. J. (2003). Effects of the antibiotic ionophores monensin, lasalocid, laidlomycin propionate and bambermycin on Salmonella and E. coli O157:H7 in vitro. J. Appl. Microbiol. 94, 207–213. Edrington, T. S., Schultz, C. L., Genovese, K. J., Callaway, T. R., Looper, M. L., Bischoff, K. M., McReynolds, J. L., Anderson, R. C., and Nisbet, D. J. (2004). Examination of heat stress and stage of lactation (early versus late) on fecal shedding of E. coli O157:H7 and Salmonella in dairy cattle. Foodborne Pathog. Dis. 1, 114–119. Edrington, T. S., Callaway, T. R., Ives, S. E., Engler, M. J., Looper, M. L., Anderson, R. C., and Nisbet, D. J. (2006a). Seasonal shedding of Escherichia coli O157:H7 in ruminants: A new hypothesis. Foodborne Pathog. Dis. 3, 413–421. Edrington, T. S., Callaway, T. R., Ives, S. E., Engler, M. J., Welsh, T. H., Hallford, D. M., Genovese, K. J., Anderson, R. C., and Nisbet, D. J. (2006b). Effect of ractopamine HCl supplementation on fecal shedding of Escherichia coli O157:H7 and Salmonella in feedlot cattle. Curr. Microbiol. 53, 340–345. Edrington, T. S., Callaway, T. R., Smith, D. J., Genovese, K. J., Anderson, R. C., and Nisbet, D. J. (2006c). Effects of ractopamine HCl on Escherichia coli O157:H7 and Salmonella in vitro and on intestinal populations and fecal shedding in experimentally infected sheep and pigs. Curr. Microbiol. 53, 82–88. Edrington, T. S., Callaway, T. R., Hallford, D. M., Anderson, R. C., and Nisbet, D. J. (2007). Influence of exogenous triiodothyronine (T3) on fecal shedding of Escherichia coli O157 in cattle. Microb. Ecol. 53, 664–669. Edrington, T. S., Callaway, T. R., Hallford, D. M., Chen, L., Anderson, R. C., and Nisbet, D. J. (2008). Effects of exogenous melatonin and tryptophan on fecal shedding of E. coli O157: H7 in cattle. Microb. Ecol. 55, 553–560. Edrington, T. S., Farrow, R. L., Genovese, K. J., Callaway, T. R., Anderson, R. C., and Nisbet, D. J. (2009a). Influence of exogenous melatonin on horizontal transfer of Escherichia coli O157:H7 in experimentally infected sheep. Foodborne Pathog. Dis. 6, 729–731. Edrington, T. S., Farrow, R. L., Loneragan, G. H., Ives, S. D., Engler, M. J., Wagner, J. J., Corbin, M. J., Platter, W. J., Yates, D., Hutcheson, J. P., Zinn, R. A., Callaway, T. R., et al. (2009b). Influence of b-agonists (ractopamine HCl and zilpaterol HCl) on fecal shedding of Escherichia coli O157:H7 in feedlot cattle. J. Food Prot. 72, 2587–2591. Ejidokun, O. O., Walsh, A., Barnett, J., Hope, Y., Ellis, S., Sharp, M. W., Paiba, G. A., Logan, M., Willshaw, G. A., and Cheasty, T. (2006). Human Vero cytotoxigenic Escherichia coli (VTEC) O157 infection linked to birds. Epidemiol. Infect. 134, 421–423. Elder, R. O. and Keen, J. E. (1999). Effects of pen cleaning and group vs. individual penning on fecal shedding of naturally-acquired enterohemorrhagic E. coli (EHEC) O157 in beef feedlot cattle. In ‘‘Proceedings of the 80th Annual Meeting of the Conference of Research Workers in Animal Disease, Chicago, IL’’. (Abstract Number 73). Elder, R. O., Keen, J. E., Siragusa, G. R., Barkocy-Gallagher, G. A., Koohmaraie, M., and Laegreid, W. W. (2000). Correlation of enterohemorrhagic Escherichia coli O157 prevalence in feces, hides, and carcasses of beef cattle during processing. Proc. Natl. Acad. Sci. USA 97, 2999–3003. Elder, R. O., Keen, J. E., Wittum, T. E., Callaway, T. R., Edrington, T. S., Anderson, R. C., and Nisbet, D. J. (2002). Intervention to reduce fecal shedding of enterohemorrhagic Escherichia coli O157:H7 in naturally infected cattle using neomycin sulfate. J. Anim. Sci. 80 (Suppl. 1), 151. Ellis-Iversen, J., Smith, R. P., Snow, L. C., Watson, E., Millar, M. F., Pritchard, G. C., Sayers, A. R., Cook, A. J. C., Evans, S. J., and Paiba, G. A. (2007). Identification of management risk factors for VTEC O157 in young-stock in England and Wales. Prev. Vet. Med. 82, 29–41.

Escherichia coli O157 in Cattle Production

107

Ellis-Iversen, J., Smith, R. P., Van Winden, S., Paiba, G. A., Watson, E., Snow, L. C., and Cook, A. D. J. (2008). Farm practices to control E. coli O157 in young cattle—A randomised controlled trial. Vet. Res. 39, 3. Ellis-Iversen, J., Cook, A. J. C., Smith, R. P., Pritchard, G. C., and Nielen, M. (2009). Temporal patterns and risk factors for Escherichia coli O157 and Campylobacter spp. in young cattle. J. Food Prot. 72, 490–496. Epitopix, LLC (2009). Epitopix Receives USDA Conditional License for America’s First E. coli O157 Vaccine for Cattle. Available at: http://www.epitopix.com/news_display.aspx? id¼45 Accessed 15 January 2010. Faith, N. G., Shere, J. A., Brosch, R., Arnold, K. W., Ansay, S. E., Lee, M.-S., Luchansky, J. B., and Kaspar, C. W. (1996). Prevalence and clonal nature of Escherichia coli O157:H7 on dairy farms in Wisconsin. Appl. Environ. Microbiol. 62, 1519–1525. Feder, I., Wallace, F. M., Gray, J. T., Fratamico, P., Fedorka-Cray, P. J., Pearce, R. A., Call, J. E., Perrine, R., and Luchansky, J. B. (2003). Isolation of Escherichia coli O157:H7 from intact colon fecal samples of swine. Emerging Infect. Dis. 9, 380–383. Fegan, N., Higgs, G., Duffy, L. L., and Barlow, R. S. (2009). The effects of transport and lairage on counts of Escherichia coli O157:H7 in the feces and on the hides of individual cattle. Foodborne Pathog. Dis. 6, 1113–1120. Fenlon, D. R. and Wilson, J. (2000). Growth of Escherichia coli O157:H7 in poorly fermented laboratory silage: A possible environmental dimension in the epidemiology of E. coli O157. Lett. Appl. Microbiol. 30, 118–121. Fischer, J. R., Zhao, T., Doyle, M. P., Goldberg, M. R., Brown, C. A., Sewell, C. T., Kavanaugh, D. M., and Bauman, C. D. (2001). Experimental and field studies of Escherichia coli O157:H7 in white-tailed deer. Appl. Environ. Microbiol. 67, 1218–1224. Fitzgerald, A. C., Edrington, T. S., Looper, M. L., Callaway, T. R., Genovese, K. J., Bischoff, K. M., McReynolds, J. L., Thomas, J. D., Anderson, R. C., and Nisbet, D. J. (2003). Antimicrobial susceptibility and factors affecting the shedding of E. coli O157:H7 and Salmonella in dairy cattle. Lett. Appl. Microbiol. 37, 392–398. Folmer, J., Macken, C., Moxley, R., Smith, D., Brashears, M., Hinkley, S., Erickson, G., and Klopfenstein, T. (2003). Intervention strategies for reduction of E. coli O157:H7 in feedlot steers. In ‘‘2003 Nebraska Beef Cattle Report’’, pp. 22–23. University of Nebraska Cooperative Extension MP 80-A, Lincoln, Nebraska. Fox, J. T., Corrigan, M., Drouillard, J. S., Shi, X., Oberst, R. D., and Nagaraja, T. G. (2007a). Effects of concentrate level of diet and pen configuration on prevalence of Escherichia coli O157 in finishing goats. Small Rumin. Res. 72, 45–50. Fox, J. T., Depenbusch, B. E., Drouillard, J. S., and Nagaraja, T. G. (2007b). Dry-rolled or steam-flaked grain-based diets and fecal shedding of Escherichia coli O157 in feedlot cattle. J. Anim. Sci. 85, 1207–1212. Fox, J. T., Reinstein, S., Jacob, M. E., and Nagaraja, T. G. (2008a). Niche marketing production practices for beef cattle in the United States and prevalence of foodborne pathogens. Foodborne Pathog. Dis. 5, 559–569. Fox, J. T., Renter, D. G., Sanderson, M. W., Nutsch, A. L., Shi, X., and Nagaraja, T. G. (2008b). Associations between the presence and magnitude of Escherichia coli O157 in feces at harvest and contamination of preintervention beef carcasses. J. Food Prot. 71, 1761–1767. Fox, J. T., Thomson, D. U., Drouillard, J. S., Thornton, A. B., Burkhardt, D. T., Emery, D. A., and Nagaraja, T. G. (2009a). Efficacy of Escherichia coli O157:H7 siderophore receptor/ porin proteins-based vaccine in feedlot cattle naturally shedding E. coli O157. Foodborne Pathog. Dis. 6, 893–899. Fox, J. T., Drouillard, J. S., Shi, X., and Nagaraja, T. G. (2009b). Effects of mucin and its carbohydrate constituents on Escherichia coli O157 growth in batch culture fermentation with ruminal or fecal microbial inoculum. J. Anim. Sci. 87, 1304–1313.

108

Elaine D. Berry and James E. Wells

Freestone, P. P. E., Walton, N. J., Haigh, R. D., and Lyte, M. (2007). Influence of dietary catechols on the growth of enteropathogenic bacteria. Int. J. Food Microbiol. 119, 159–169. Freestone, P. P. E., Sandrini, S. M., Haigh, R. D., and Lyte, M. (2008). Microbial endocrinology: How stress influences susceptibility to infection. Trends Microbiol. 16, 55–64. Fukushima, H., Hoshina, K., and Gomyoda, M. (1999). Long-term survival of Shiga toxinproducing Escherichia coli O26, O111, and O157 in bovine feces. Appl. Environ. Microbiol. 65, 5177–5181. Gannon, V. P. J., Graham, T. A., King, R., Michel, P., Read, S., Zeibell, K., and Johnson, R. P. (2002). Escherichia coli O157:H7 infection in cows and calves in a beef cattle herd in Alberta, Canada. Epidemiol. Infect. 129, 163–172. Garber, L. P., Wells, S. J., Hancock, D. D., Doyle, M. P., Tuttle, J., Shere, J. A., and Zhao, T. (1995). Risk factors for fecal shedding of Escherichia coli O157:H7 in dairy calves. J. Am. Vet. Med. Assoc. 207, 46–49. Garber, L., Wells, S., Schroeder-Tucker, L., and Ferris, K. (1999). Factors associated with fecal shedding of verotoxin-producing Escherichia coli O157 on dairy farms. J. Food Prot. 62, 307–312. Graczyk, T. K., Knight, R., Gilman, R. H., and Cranfield, M. R. (2001). The role of non-biting flies in the epidemiology of human infectious diseases. Microbes Infect. 3, 231–235. Greer, G. G. (2005). Bacteriophage control of foodborne bacteria. J. Food Prot. 68, 1102–1111. Griffin, P. M., Ostroff, S. M., Tauxe, R. V., Greene, K. D., Wells, J. G., Lewis, J. H., and Blake, P. A. (1988). Illnesses associated with Escherichia coli O157:H7 infections. A broad clinical spectrum. Ann. Intern. Med. 109, 705–712. Hancock, D. D., Besser, T. E., Kinsel, M. L., Tarr, P. I., Rice, D. H., and Paros, M. G. (1994). The prevalence of Escherichia coli O157:H7 in dairy and beef cattle in Washington state. Epidemiol. Infect. 113, 199–207. Hancock, D. D., Rice, D. H., Thomas, L. A., Dargatz, D. A., and Besser, T. E. (1997). Epidemiology of Escherichia coli O157 in feedlot cattle. J. Food Prot. 60, 462–465. Hancock, D. D., Besser, T. E., Rice, D. H., Ebel, E. D., Herriott, D. E., and Carpenter, L. V. (1998). Multiple sources of Escherichia coli O157 in feedlots and dairy farms in the Northwestern USA. Prev. Vet. Med. 35, 11–19. Hancock, D., Besser, T., LeJeune, J., Davis, M., and Rice, D. (2001). The control of VTEC in the animal reservoir. Int. J. Food Microbiol. 66, 71–78. Harmon, B. G., Brown, C. A., Tkalcic, S., Mueller, P. O. E., Park, A., Jain, A. V., Zhao, T., and Doyle, M. P. (1999). Fecal shedding and rumen growth of Escherichia coli O157:H7 in fasted calves. J. Food Prot. 62, 574–579. Herriott, D. E., Hancock, D. D., Ebel, E. D., Carpenter, L. V., Rice, D. H., and Besser, T. E. (1998). Association of herd management factors with colonization of dairy cattle by Shiga toxin-positive Escherichia coli O157. J. Food Prot. 61, 802–807. Heuvelink, A. E., van Heerwaarden, C., Zwartkruis-Nahuis, J. T. M., van Oosterom, R., Edink, K., van Duynhoven, Y. T. H. P., and De Boer, E. (2002). Escherichia coli O157 infection associated with a petting zoo. Epidemiol. Infect. 129, 295–302. Hilborn, E. D., Mermin, J. H., Mshar, P. A., Hadler, J. L., Voetsch, A., Wojtkunski, C., Swartz, M., Mshar, R., Lambert-Fair, M. A., Farrar, J. A., Glynn, M. K., and Slutsker, L. (1999). A multistate outbreak of Escherichia coli O157:H7 infections associated with consumption of mesclun lettuce. Arch. Intern. Med. 159, 1758–1764. Ishii, S., Ksoll, W. B., Hicks, R. E., and Sadowsky, M. J. (2006). Presence and growth of naturalized Escherichia coli in temperate soils from Lake Superior watersheds. Appl. Environ. Microbiol. 72, 612–621. Iwasa, M., Makino, S., Asakura, H., Kobori, H., and Morimoto, Y. (1999). Detection of Escherichia coli O157:H7 from Musca domestica (Diptera: Muscidae) at a cattle farm in Japan. J. Med. Entomol. 38, 108–112.

Escherichia coli O157 in Cattle Production

109

Jacob, M. E., Fox, J. T., Drouillard, J. S., Renter, D. G., and Nagaraja, T. G. (2008a). Effects of dried distillers’ grain on fecal prevalence and growth of Escherichia coli O157 in batch culture fermentations from cattle. Appl. Environ. Microbiol. 74, 38–43. Jacob, M. E., Fox, J. T., Narayanan, S. K., Drouillard, J. S., Renter, D. G., and Nagaraja, T. G. (2008b). Effects of feeding wet corn distillers grains with solubles with or without monensin and tylosin on the prevalence and antimicrobial susceptibilities of fecal foodborne pathogenic and commensal bacteria in feedlot cattle. J. Anim. Sci. 86, 1182–1190. Jacob, M. E., Parsons, G. L., Shelor, M. K., Fox, J. T., Drouillard, J. S., Thomson, D. U., Renter, D. G., and Nagaraja, T. G. (2008c). Feeding supplemental dried distiller’s grains increases faecal shedding of Escherichia coli O157 in experimentally inoculated calves. Zoonoses Public Health 55, 125–132. Jacob, M. E., Callaway, T. R., and Nagaraja, T. G. (2009a). Dietary interactions and interventions affecting Escherichia coli O157 colonization and shedding in cattle. Foodborne Pathog. Dis. 6, 785–792. Jacob, M. E., Fox, J. T., Drouillard, J. S., Renter, D. G., and Nagaraja, T. G. (2009b). Evaluation of feeding dried distiller’s grains with solubles and dry-rolled corn on the fecal prevalence of Escherichia coli O157:H7 and Salmonella spp. in cattle. Foodborne Pathog. Dis. 6, 145–153. Jacobsen, L., Durso, L., Conway, T., and Nickerson, K. W. (2009). Escherichia coli O157:H7 and other E. coli strains share physiological properties associated with intestinal colonization. Appl. Environ. Microbiol. 75, 4633–4635. Jarvis, G. N., Fields, M. W., Adamovich, D. A., Arthurs, C. E., and Russell, J. B. (2001). The mechanism of carbonate killing of Escherichia coli. Lett. Appl. Microbiol. 33, 196–200. Johnson, R. P., Gyles, C. L., Huff, W. E., Ojha, S., Huff, G. R., Rath, N. C., and Donoghue, A. M. (2008). Bacteriophages for prophylaxis and therapy in cattle, poultry, and pigs. Anim. Health Res. Rev. 9, 201–215. Jordan, D., McEwen, S. A., Lammerding, A. M., McNab, W. B., and Wilson, J. B. (1999). Preslaughter control of Escherichia coli O157 beef cattle: A simulation study. Prev. Vet. Med. 41, 55–74. Keene, W. E., Hedberg, K., Herriott, D. E., Hancock, D. D., McKay, R. W., Barrett, T. J., and Fleming, D. W. (1997). A prolonged outbreak of Escherichia coli O157:H7 infections caused by commercially distributed raw milk. J. Infect. Dis. 176, 815–818. Keen, J. E., Wittum, T. E., Dunn, J. R., Bono, J. L., and Durso, L. M. (2006a). Shiga-toxigenic Escherichia coli O157 in agricultural fair livestock, United States. Emerging Infect. Dis. 12, 780–786. Keen, J. E., Durso, L. M., Bono, J. L., and Laegreid, W. W. (2006b). Oral neomycin therapy reduces Shiga-toxigenic E. coli (STEC) O157 fecal shedding in naturally-infected beef cattle. In ‘‘Proceedings of the 87th Annual Meeting of the Conference of Research Workers in Animal Disease’’. Chicago, IL, p.148. Kinney, K. S., Austin, C. E., Norton, D. S., and Sonnenfeld, G. (2000). Norepinephrine as a growth stimulating factor in bacteria-mechanistic studies. Life Sci. 67, 3075–3085. Kobayashi, M., Sasaki, T., Saito, N., Tamura, K., Suzuki, K., Watanabe, H., and Agui, N. (1999). Houseflies: Not simple mechanical vectors of enterohemorrhagic Escherichia coli O157:H7. Am. J. Trop. Med. Hyg. 61, 625–629. Kudva, I. T., Hatfield, P. G., and Hovde, C. J. (1995). Effect of diet on the shedding of Escherichia coli O157:H7 in a sheep model. Appl. Environ. Microbiol. 61, 1363–1370. Kudva, I. T., Hatfield, P. G., and Hovde, C. J. (1996). Escherichia coli O157:H7 in microbial flora of sheep. J. Clin. Microbiol. 34, 431–433. Kudva, I. T., Blanch, K., and Hovde, C. J. (1998). Analysis of Escherichia coli O157:H7 survival in ovine or bovine manure and manure slurry. Appl. Environ. Microbiol. 64, 3166–3174. Kudva, I. T., Jelacic, S., Tarr, P. I., Youderian, P., and Hovde, C. J. (1999). Biocontrol of Escherichia coli O157 with O157-specific bacteriophages. Appl. Environ. Microbiol. 65, 3767–3773.

110

Elaine D. Berry and James E. Wells

Kuhnert, P., Dubosson, C. R., Roesch, M., Homfeld, E., Doherr, M. G., and Blum, J. W. (2005). Prevalence and risk-factor analysis of Shiga toxigenic Escherichia coli in faecal samples of organically and conventionally farmed dairy cattle. Vet. Microbiol. 109, 37–45. La Ragione, R. M., Best, A., Woodward, M. J., and Wales, A. D. (2009). Escherichia coli O157: H7 colonization in small domestic ruminants. FEMS Microbiol. Rev. 33, 394–410. Laegreid, W. W., Elder, R. O., and Keen, J. E. (1999). Prevalence of Escherichia coli O157:H7 in range beef calves at weaning. Epidemiol. Infect. 123, 291–298. Lahti, E., Ruoho, O., Rantala, L., Ha¨nninen, M.-L., and Honkanen-Buzalski, T. (2003). Longitudinal study of Escherichia coli O157 in a cattle finishing unit. Appl. Environ. Microbiol. 69, 554–561. LeJeune, J. T. and Wetzel, A. N. (2007). Preharvest control of Escherichia coli O157 in cattle. J. Anim. Sci. 85(E. Suppl.), E73–E80. LeJeune, J. T., Besser, T. E., and Hancock, D. D. (2001a). Cattle water troughs as reservoirs of Escherichia coli O157. Appl. Environ. Microbiol. 67, 3053–3057. LeJeune, J. T., Besser, T. E., Merrill, N. L., Rice, D. H., and Hancock, D. D. (2001b). Livestock drinking water microbiology and the factors influencing the quality of drinking water offered to cattle. J. Dairy Sci. 84, 1856–1862. LeJeune, J. T., Besser, T. E., Rice, D. H., Berg, J. L., Stilborn, R. P., and Hancock, D. D. (2004). Longitudinal study of fecal shedding of Escherichia coli O157:H7 in feedlot cattle: Predominance and persistence of specific clonal types despite massive cattle population turnover. Appl. Environ. Microbiol. 70, 377–384. Lim, J. Y., Li, J., Sheng, H., Besser, T. E., Potter, K., and Hovde, C. J. (2007). Escherichia coli O157:H7 colonization at the rectoanal junction of long-duration culture-positive cattle. Appl. Environ. Microbiol. 73, 1380–1382. Loneragan, G. H. and Brashears, M. M. (2005). Pre-harvest interventions to reduce carriage of E. coli O157 by harvest-ready feedlot cattle. Meat Sci. 71, 72–78. Low, J. C., McKendrick, I. J., McKechnie, C., Fenlon, D., Naylor, S. W., Currie, C., Smith, D. G. E., Allison, L., and Galley, D. L. (2005). Rectal carriage of enterohemorrhagic Escherichia coli O157 in slaughtered cattle. Appl. Environ. Microbiol. 71, 93–97. Lynn, T. V., Hancock, D. D., Besser, T. E., Harrison, J. H., Rice, D. H., Stewart, N. T., and Rowan, L. L. (1998). The occurrence and replication of Escherichia coli in cattle feeds. J. Dairy Sci. 81, 1102–1108. Lyte, M., Arulanandam, B., Nguyen, K., Frank, C., Erickson, A., and Francis, D. (1997). Norepinephrine induced growth and expression of virulence associated factors in enterotoxigenic and enterohemorrhagic strains of Escherichia coli. Adv. Exp. Med. Biol. 412, 331–339. Mather, A. E., Reid, S. W. J., McEwen, S. A., Ternent, H. E., Reid-Smith, R. J., Boerlin, P., Taylor, D. J., Steele, W. B., Gunn, G. J., and Mellor, D. J. (2008). Factors associated with cross-contamination of hides of Scottish cattle by Escherichia coli O157. Appl. Environ. Microbiol. 74, 6313–6319. Matthews, L., Low, J. C., Gally, D. L., Pearce, M. C., Mellor, D. J., Heesterbeek, J. A. P., ChaseTopping, M., Naylor, S. W., Shaw, D. J., Reid, S. W. J., Gunn, G. J., and Woolhouse, M. E. J. (2006a). Heterogeneous shedding of Escherichia coli O157 in cattle and its implications for control. Proc. Natl. Acad. Sci. USA 103, 547–552. Matthews, L., McKendrick, I. J., Ternent, H., Gunn, G. J., Synge, B., and Woolhouse, M. E. J. (2006b). Super-shedding cattle and the transmission dynamics of Escherichia coli O157. Epidemiol. Infect. 134, 131–142. McAllister, T. A., Bach, S. J., Stanford, K., and Callaway, T. R. (2006). Shedding of Escherichia coli O157:H7 by cattle fed diets containing monensin or tylosin. J. Food Prot. 69, 2075–2083. McIntyre, L., Fung, J., Paccagnella, A., Isaac-Renton, J., Rockwell, F., Emerson, B., and Preston, T. (2002). Escherichia coli O157 outbreak associated with the ingestion of unpasteurized goat’s milk in British Columbia, 2001. Can. Commun. Dis. Rep. 28, 6–8.

Escherichia coli O157 in Cattle Production

111

McNeilly, T. N., Naylor, S. W., Mahajan, A., Mitchell, M. C., McAteer, S., Deane, D., Smith, D. G. E., Low, J. C., Gally, D. L., and Huntley, J. F. (2008). Escherichia coli O157: H7 colonization in cattle following systemic and mucosal immunization with purified H7 flagellin. Infect. Immun. 76, 2594–2602. McNeilly, T. N., Mitchell, M. C., Rosser, T., McAteer, S., Low, J. C., Smith, D. G. E., Huntley, J. F., Mahajan, A., and Gally, D. L. (2010). Immunization of cattle with a combination of purified intimin-531, EspA and Tir significantly reduces shedding of Escherichia coli O157:H7 following oral challenge. Vaccine 28, 1422–1428. Mead, P. S., Slutsker, L., Dietz, V., McCaig, L. F., Bresee, J. S., Shapiro, C., Griffin, P. M., and Tauxe, R. V. (1999). Food-related illness and death in the United States. Emerging Infect. Dis. 5, 607–625. Mechie, S. C., Chapman, P. A., and Siddons, C. A. (1997). A fifteen month study of Escherichia coli O157:H7 in a dairy herd. Epidemiol. Infect. 118, 17–25. Milnes, A. S., Sayers, A. R., Stewart, I., Clifton-Hadley, F. A., Davies, R. H., Newell, D. G., Cook, A. J. C., Evans, S. J., Smith, R. P., and Paiba, G. A. (2009). Factors related to the carriage of Verocytotoxigenic E. coli, Salmonella, thermophilic Campylobacter and Yersinia enterocolitica in cattle, sheep, and pigs at slaughter. Epidemiol. Infect. 137, 1135–1148. Min, E. R., Pinchak, W. E., Anderson, R. C., and Callaway, T. R. (2007). Effect of tannins on the in vitro growth of Escherichia coli O157:H7 and in vivo growth of generic Escherichia coli excreted from steers. J. Food Prot. 70, 543–550. Minihan, D., O’Mahony, M., Whyte, P., and Collins, J. D. (2003). An investigation on the effect of transport and lairage on the faecal shedding prevalence of Escherichia coli O157 in cattle. J. Vet. Med. Ser. B 50, 378–382. Moriya, K., Fujibayashi, T., Yoshihara, T., Matsuda, A., Sumi, N., Umezaki, N., Kurahashi, H., Agui, N., and Wada, A. (1999). Verotoxin-producing Escherichia coli O157:H7 carried by the housefly in Japan. Med. Vet. Entomol. 13, 214–216. Moxley, R. A., Smith, D. R., Luebbe, M., Erickson, G. E., Klopfenstein, T. J., and Rogan, D. (2009). Escherichia coli O157:H7 vaccine dose—Effect in feedlot cattle. Foodborne Pathog. Dis. 6, 879–884. Naylor, S. W., Low, J. C., Besser, T. E., Mahajan, A., Gunn, G. J., Pearce, M. C., McKendrick, I. J., Smith, D. G. E., and Gally, D. L. (2003). Lymphoid follicle-dense mucosa at the terminal rectum is the principal site of colonization of enterohemorrhagic Escherichia coli O157:H7 in the bovine host. Infect. Immun. 71, 1505–1512. Naylor, S. W., Roe, A. J., Nart, P., Spears, K., Smith, D. G., Low, J. C., and Gally, D. L. (2005). Escherichia coli O157:H7 forms attaching and effacing lesions at the terminal rectum of cattle and colonization requires the LEE4 operon. Microbiology 151, 2773–2781. Naylor, S. W., Nart, P., Sales, J., Flockhart, A., Gally, D. L., and Low, J. C. (2007). Impact of the direct application of therapeutic agents to the terminal recta of experimentally colonized calves on Escherichia coli O157:H7 shedding. Appl. Environ. Microbiol. 73, 1493–1500. Nielsen, E. M., Skov, M. N., Madsen, J. J., Lodal, J., Jespersen, J. B., and Baggesen, D. L. (2004). Verocytotoxin-producing Escherichia coli in wild birds and rodents in close proximity to farms. Appl. Environ. Microbiol. 70, 6944–6947. Nisbet, D. J., Callaway, T. R., Edrington, T. S., Anderson, R. C., and Krueger, N. (2009). Effects of the dicarboxylic acids malate and fumarate on E. coli O157:H7 and Salmonella enterica Typhimurium populations in pure culture and in mixed ruminal microorganism fermentations. Curr. Microbiol. 58, 488–492. Niu, Y. D., Johnson, R. P., Xu, Y., McAllister, T. A., Sharma, R., Louie, M., and Stanford, K. (2009a). Host range and lytic capability of four bacteriophages against bovine and clinical human isolates of Shiga toxin-producing Escherichia coli O157:H7. J. Appl. Microbiol. 107, 646–656. Niu, Y. D., McAllister, T. A., Xu, Y., Johnson, R. P., Stephens, T. P., and Stanford, K. (2009b). Prevalence and impact of bacteriophages on the presence of Escherichia coli O157:H7 in feedlot cattle and their environment. Appl. Environ. Microbiol. 75, 1271–1278.

112

Elaine D. Berry and James E. Wells

O’Connor, D. R. (2002). Part One: A Summary. Report of the Walkerton Inquiry: The Events of May 2000 and Related Issues. Ontario Ministry of the Attorney General, Toronto, Ontario pp. 1–35. Ogden, I. D., Hepburn, N. F., MacRae, M., Strachan, N. J. C., Fenlon, D. R., Rusbridge, S. M., and Pennington, T. H. (2002). Long-term survival of Escherichia coli O157 on pasture following an outbreak associated with sheep at a scout camp. Lett. Appl. Microbiol. 34, 100–104. Ogden, I. D., MacRae, M., and Strachan, N. J. C. (2004). Is the prevalence and shedding concentrations of E. coli O157 in beef cattle in Scotland seasonal? FEMS Microbiol. Lett. 233, 297–300. Ogden, I. D., MacRae, M., and Strachan, N. J. C. (2005). Concentration and prevalence of Escherichia coli O157:H7 in sheep faeces at pasture in Scotland. J. Appl. Microbiol. 98, 646–651. Oliver, S. P., Patel, D. A., Callaway, T. R., and Torrence, M. E. (2009). ASAS Centennial Paper: Developments and future outlook for preharvest food safety. J. Anim. Sci. 87, 419–437. Omisakin, F., MacRae, M., Ogden, I. D., and Strachan, N. J. C. (2003). Concentration and prevalence of Escherichia coli O157 in cattle feces at slaughter. Appl. Environ. Microbiol. 69, 2444–2447. Oot, R. A., Raya, R. R., Callaway, T. R., Edrington, T. S., Kutter, E. M., and Brabban, A. D. (2007). Prevalence of Escherichia coli O157 and O157:H7-infecting bacteriophages in feedlot cattle feces. Lett. Appl. Microbiol. 45, 445–453. Park, G. W. and Diez-Gonzalez, F. (2003). Utilization of carbonate and ammonia-based treatments to eliminate Escherichia coli O157:H7 and Salmonella Typhimurium DT104 from cattle manure. J. Appl. Microbiol. 94, 675–685. Payne, C. J. I., Petrovic, M., Roberts, R. J., Paul, A., Linnane, E., Walker, M., Kirby, D., Burgess, A., Smith, R. M. M., Cheasty, T., Wilshaw, G., and Salmon, R. L. (2003). Vero cytotoxin-producing Escherichia coli O157 gastroenteritis in farm visitors, North Wales. Emerging Infect. Dis. 9, 526–530. Peterson, R. E., Klopfenstein, T. J., Erickson, G. E., Folmer, J., Hinkley, S., Moxley, R. A., and Smith, D. R. (2007a). Effect of Lactobacillus acidophilus strain NP51 on Escherichia coli O157: H7 fecal shedding and finishing performance in beef feedlot cattle. J. Food Prot. 70, 287–291. Peterson, R. E., Klopfenstein, T. J., Moxley, R. A., Erickson, G. E., Hinkley, D., Bretschneider, G., Berberov, E. M., Rogan, D., and Smith, D. R. (2007b). Effect of a vaccine product containing type III secreted proteins on the probability of Escherichia coli O157:H7 fecal shedding and mucosal colonization in feedlot cattle. J. Food Prot. 70, 2568–2577. Peterson, R. E., Klopfenstein, T. J., Moxley, R. A., Erickson, G. E., Hinkley, D., Rogan, D., and Smith, D. R. (2007c). Efficacy of dose regimen and observation of herd immunity from a vaccine against Escherichia coli O157:H7 for feedlot cattle. J. Food Prot. 70, 2561–2567. Potter, A. A., Klashinsky, S., Li, Y., Frey, E., Townsend, H., Rogan, D., Erickson, G., Hinkley, S., Klopfenstein, T., Moxley, R. A., Smith, D. R., and Finlay, B. B. (2004). Decreased shedding of Escherichia coli O157:H7 by cattle following vaccination with type III secreted proteins. Vaccine 22, 362–369. Rangel, J. M., Sparling, P. H., Crowe, C., Griffin, P. M., and Swerdlow, D. L. (2005). Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982–2002. Emerging Infect. Dis. 11, 603–609. Rasmussen, M. A. and Casey, T. A. (2001). Environmental and food safety aspects of Escherichia coli O157:H7 infections in cattle. Crit. Rev. Microbiol. 27, 57–73. Raya, R. R., Varey, P., Oot, R. A., Dyen, M. R., Callaway, T. R., Edrington, T. S., Kutter, E. M., and Brabban, A. D. (2006). Isolation and characterization of a new T-Even bacteriophage CEV1, and determination of its potential to reduce Escherichia coli O157:H7 levels in sheep. Appl. Environ. Microbiol. 72, 6405–6410.

Escherichia coli O157 in Cattle Production

113

Reicks, A. L., Brashears, M. M., Adams, K. D., Brooks, J. C., Blanton, J. R., and Miller, M. F. (2007). Impact of transportation of feedlot cattle to the harvest facility on the prevalence of Escherichia coli O157:H7, Salmonella, and total aerobic microorganisms on hides. J. Food Prot. 70, 17–21. Reinstein, S., Fox, J. T., Shi, X., Alam, M. J., Renter, D. G., and Nagaraja, T. G. (2009). Prevalence of Escherichia coli O157:H7 in organically and naturally raised beef cattle. Appl. Environ. Microbiol. 75, 5421–5423. Reiss, G., Kunz, P., Koin, D., and Keeffe, E. B. (2006). Escherichia coli O157:H7 infection in nursing homes: Review of literature and report of recent outbreak. J. Am. Geriatr. Soc. 54, 680–684. Renter, D. G., Sargeant, J. M., Oberst, R. D., and Samadpour, M. (2003). Diversity, frequency, and persistence of Escherichia coli O157 strains from range cattle environments. Appl. Environ. Microbiol. 69, 542–547. Rhoades, J. R., Duffy, G., and Koutsoumanis, K. (2009). Prevalence and concentration of verocytotoxigenic Escherichia coli, Salmonella enterica, and Listeria monocytogenes in the beef production chain: A review. Food Microbiol. 26, 357–376. Rice, E. W. and Johnson, C. H. (2000). Short communication: Survival of Escherichia coli O157: H7 in dairy cattle drinking water. J. Dairy Sci. 83, 2021–2023. Rice, E. W., Johnson, C. H., Wild, D. K., and Reasoner, D. J. (1992). Survival of Escherichia coli O157:H7 in drinking water associated with a waterborne outbreak of hemorrhagic colitis. Lett. Appl. Microbiol. 15, 38–40. Rice, D. H., McMenamin, K. M., Pritchett, L. C., and Hancock, D. D. (1999). Genetic subtyping of Escherichia coli O157 isolates from 41 Pacific Northwest USA cattle farms. Epidemiol. Infect. 122, 479–484. Riley, L. W., Remis, R. S., Helgerson, S. D., McGee, H. B., Wells, J. G., Davis, B. R., Hebert, R. J., Olcott, E. S., Johnson, L. M., Hargrett, N. T., Blake, P. A., and Cohen, M. L. (1983). Hemorrhagic colitis associated with a rare Escherichia coli serotype. N. Engl. J. Med. 308, 681–685. Robinson, S. E., Wright, E. J., Hart, C. A., Bennett, M., and French, N. P. (2004). Intermittent and persistence shedding of Escherichia coli O157 in cohorts of naturally infected calves. J. Appl. Microbiol. 97, 1045–1053. Rodrigue, D. C., Mast, E. E., Greene, K. D., Davis, J. P., Hutchinson, M. A., Wells, J. G., Barrett, T. J., and Griffin, P. M. (1995). A university outbreak of Escherichia coli O157:H7 infections associated with roast beef and an unusually benign clinical course. J. Infect. Dis. 172, 1122–1125. Rostagno, M. H. (2009). Can stress in farm animals increase food safety risk? Foodborne Pathog. Dis. 6, 767–776. Rozema, E. A., Stephens, T. P., Bach, S. J., Okine, E. K., Johnson, R. P., Stanford, K., and McAllister, T. A. (2009). Oral and rectal administration of bacteriophages for control of Escherichia coli O157:H7 in feedlot cattle. J. Food Prot. 72, 241–250. Russell, J. B. and Houlihan, A. J. (2003). Ionophore resistance of ruminal bacteria and its potential impact on human health. FEMS Microbiol. Rev. 27, 65–74. Salak-Johnson, J. L. and McGlone, J. J. (2007). Making sense of apparently conflicting data: Stress and immunity in swine and cattle. J. Anim. Sci. 85(E. Suppl.), E81–E88. Sanderson, M. W., Sargeant, J. M., Shi, X., Nagaraja, T. G., Zurek, L., and Alam, M. J. (2006). Longitudinal emergence and distribution of Escherichia coli O157 genotypes in a beef feedlot. Appl. Environ. Microbiol. 72, 7614–7619. Sargeant, J. M., Hafer, D. J., Gillespie, J. R., Oberst, R. D., and Flood, S. J. (1999). Prevalence of Escherichia coli O157:H7 in white-tailed deer sharing rangeland with cattle. J. Am. Vet. Med. Assoc. 215, 792–794. Sargeant, J. M., Gillespie, J. R., Oberst, R. D., Phebus, R. K., Hyatt, D. R., Bohra, L. K., and Galland, J. C. (2000). Results of a longitudinal study of the prevalence of Escherichia coli O157:H7 on cow-calf farms. Am. J. Vet. Res. 61, 1375–1379.

114

Elaine D. Berry and James E. Wells

Sargeant, J. M., Sanderson, M. W., Smith, R. A., and Griffin, D. D. (2004). Associations between management, climate, and Escherichia coli O157 in the faeces of feedlot cattle in the Midwestern USA. Prev. Vet. Med. 66, 175–206. Sargeant, J. M., Amezcua, M. R., Rajic, A., and Waddell, L. (2007). Pre-harvest interventions to reduce the shedding of E. coli O157 in the faeces of weaned domestic ruminants: A systematic review. Zoonoses Public Health 54, 260–277. Scaife, H. R., Cowan, D., Finney, J., Kinghorn-Perry, S. F., and Crook, B. (2006). Wild rabbits (Oryctolagus cuniculus) as potential carriers of verocytotoxin-producing Escherichia coli. Vet. Rec. 159, 175–178. Schamberger, G. P. and Diez-Gonzalez, F. (2005). Assessment of resistance to colicinogenic Escherichia coli by E. coli O157:H7 strains. J. Appl. Microbiol. 98, 245–252. Schamberger, G. P., Phillips, R. L., Jacobs, J. L., and Diez-Gonzalez, F. (2004). Reduction of Escherichia coli O157:H7 populations in cattle by addition of colicin E7-producing E. coli to feed. Appl. Environ. Microbiol. 70, 6053–6060. Schuehle Pfeiffer, C. E., King, D. A., Lucia, L. M., Cabrera-Diaz, E., Acuff, G. R., Randel, R. D., Welsh, T. H., Jr., Oliphint, R. A., Curley, K. O., Jr., Vann, R. C., and Savell, J. W. (2009). Influence of transportation stress and animal temperament on fecal shedding of Escherichia coli O157:H7 in feedlot cattle. Meat Sci. 81, 300–306. Schultz, C. L., Edrington, T. S., Schroeder, S. B., Hallford, D. M., Genovese, K. J., Callaway, T. R., Anderson, R. C., and Nisbet, D. J. (2005). Effect of the thyroid on faecal shedding of E. coli O157:H7 and Escherichia coli in naturally infected yearling beef cattle. J. Appl. Microbiol. 99, 1176–1180. Schultz, C. L., Edrington, T. S., Callaway, T. R., Schroeder, S. B., Hallford, D. M., Genovese, K. J., Anderson, R. C., and Nisbet, D. J. (2006). The influence of melatonin on growth of E. coli O157:H7 in pure culture and exogenous melatonin on faecal shedding of E. coli O157:H7 in experimentally infected wethers. Lett. Appl. Microbiol. 43, 105–110. Sheng, H., Knecht, H. J., Kudva, I. T., and Hovde, C. J. (2006). Application of bacteriophages to control intestinal Escherichia coli O157:H7 levels in ruminants. Appl. Environ. Microbiol. 72, 5359–5366. Shere, J. A., Bartlett, K. J., and Kaspar, C. W. (1998). Longitudinal study of Escherichia coli O157:H7 dissemination on four dairy farms in Wisconsin. Appl. Environ. Microbiol. 64, 1390–1399. Shere, J. A., Kaspar, C. W., Bartlett, K. J., Linden, S. E., Norell, B., Francey, S., and Schaefer, D. M. (2002). Shedding of Escherichia coli O157:H7 in dairy cattle housed in a confined environment following waterborne inoculation. Appl. Environ. Microbiol. 68, 1947–1954. Slutsker, L., Ries, A. A., Greene, K. D., Wells, J. G., Hutwagner, L., and Griffin, P. M. (1997). Escherichia coli O157:H7 diarrhea in the United States: Clinical and epidemiologic features. Ann. Intern. Med. 126, 505–513. Slutsker, L., Ries, A. A., Maloney, K., Wells, J. G., Greene, K. D., and Griffin, P. M., Escherichia coli O157:H7 Study Group (1998). A nationwide case-control study of Escherichia coli O157:H7 infection in the United States. J. Infect. Dis. 177, 962–966. Smith, D., Blackford, M., Younts, S., Moxley, R., Gray, J., Hungerford, L., Milton, T., and Klopfenstein, T. (2001). Ecological relationships between the prevalence of cattle shedding Escherichia coli O157:H7 and characteristics of the cattle or conditions of the feedlot pen. J. Food Prot. 64, 1899–1903. Smith, D. R., Moxley, R. A., Klopfenstein, T. J., and Erickson, G. E. (2009a). A randomized longitudinal trial to test the effect of regional vaccination within a cattle feedyard on Escherichia coli O157:H7 rectal colonization, fecal shedding, and hide contamination. Foodborne Pathog. Dis. 6, 885–892. Smith, D. R., Moxley, R. A., Peterson, R. E., Klopfenstein, T. J., Erickson, G. E., Bretschneider, G., Berberov, E. M., and Clowser, S. (2009b). A two-dose regimen of a

Escherichia coli O157 in Cattle Production

115

vaccine against Type III secreted proteins reduced Escherichia coli O157:H7 colonization of the terminal rectum in beef cattle in commercial feedlots. Foodborne Pathog. Dis. 6, 155–161. Sperandio, V., Torres, A. G., Jarvis, B., Nataro, J. P., and Kaper, J. B. (2003). Bacteria–host communication: The language of hormones. Proc. Natl. Acad. Sci. USA 100, 8951–8956. Sreerama, S., Sanderson, M. W., Wilkerson, M., and Nagaraja, T. G. (2008). Impact of dexamethasone-induced immunosuppression on the duration and level of shedding of Escherichia coli O157:H7 in calves. Curr. Microbiol. 56, 651–655. Stephens, T. P., Loneragan, G. H., Chichester, L. M., and Brashears, M. M. (2007a). Prevalence and enumeration of Escherichia coli O157 in steers receiving various strains of Lactobacillus-based direct-fed microbials. J. Food Prot. 70, 1252–1255. Stephens, T. P., Loneragan, G. H., Karunasena, E., and Brashears, M. M. (2007b). Reduction of Escherichia coli O157 and Salmonella in feces and on hides of feedlot cattle using various doses of a direct-fed microbial. J. Food Prot. 70, 2386–2391. Stephens, T. P., McAllister, T. A., and Stanford, K. (2009). Perineal swabs reveal effect of super shedders on the transmission of Escherichia coli O157:H7 in commercial feedlots. J. Anim. Sci. 87, 4151–4160. Stevenson, S. M. L., Cook, S. R., Bach, S. J., and McAllister, T. A. (2004). Effects of water source, dilution, storage, and bacterial and fecal loads on the efficacy of electrolyzed oxidizing water for the control of Escherichia coli O157:H7. J. Food Prot. 67, 1377–1383. Stoffregen, W. C., Pohlenz, J. F. L., and Dean-Nystrom, E. A. (2004). Escherichia coli O157:H7 in the gallbladders of experimentally infected calves. J. Vet. Diagn. Invest. 16, 79–83. Su, C. and Brandt, L. J. (1995). Escherichia coli O157:H7 infections in humans. Ann. Intern. Med. 123, 698–714. Swerdlow, D. L., Woodruff, B. A., Brady, R. C., Griffin, P. M., Tippen, S., Donnell, H. D., Jr., Geldreich, E., Payne, B. J., Meyer, A., Jr., Wells, J. G., Greene, K. D., Bright, M., et al. (1992). A waterborne outbreak in Missouri of Escherichia coli 0157:H7 associated with bloody diarrhea and death. Ann. Intern. Med. 117, 812–819. Synge, B. A., Chase-Topping, M. E., Hopkins, G. F., McKendrick, I. J., Thomson-Carter, F., Gray, D., Rusbridge, S. M., Munro, F. I., Foster, G., and Gunn, G. J. (2003). Factors influencing the shedding of verocytotoxin-producing Escherichia coli O157 by beef suckler cows. Epidemiol. Infect. 130, 301–312. Tabe, E. S., Oloya, J., Doetkott, D. K., Bauer, M. L., Gibbs, P. S., and Khaitsa, M. L. (2008). Comparative effect of direct-fed microbials on fecal shedding of Escherichia coli O157:H7 and Salmonella in naturally infected feedlot cattle. J. Food Prot. 71, 539–544. Talley, J. L., Wayadande, A. C., Wasala, L. P., Gerry, A. C., Fletcher, J., DeSilva, U., and Gilliland, S. E. (2009). Association of Escherichia coli O157:H7 with filth flies (Muscidae and Calliphoridae) captured in leafy greens fields and experimental transmission of E. coli O157:H7 to spinach leaves by house flies. (Diptera: Muscidae). J. Food Prot. 72, 1547–1552. Thomson, D. U., Loneragan, G. H., Thornton, A. B., Lechtenberg, K. F., Emery, D. A., Burkhardt, D. T., and Nagaraja, T. G. (2009). Use of a siderophore receptor and porin proteins-based vaccine to control the burden of Escherichia coli O157:H7 in feedlot cattle. Foodborne Pathog. Dis. 6, 871–877. Thornton, A. B., Thomson, D. U., Loneragan, G. H., Fox, J. T., Burkhardt, D. T., Emery, D. A., and Nagaraja, T. G. (2009). Effects of a siderophore receptor and porin proteins-based vaccination of fecal shedding of Escherichia coli O157:H7 in experimentally inoculated cattle. J. Food Prot. 72, 866–869. Tkalcic, S., Zhao, T., Harmon, B. G., Doyle, M. P., Brown, C. A., and Zhao, P. (2003). Fecal shedding of enterohemorrhagic Escherichia coli in weaned calves following treatment with probiotic Escherichia coli. J. Food Prot. 66, 1184–1189.

116

Elaine D. Berry and James E. Wells

Topp, E., Welsh, M., Tien, Y.-C., Dang, A., Lazarovits, G., Conn, K., and Zhu, H. (2003). Strain-dependent variability in growth and survival of Escherichia coli in agricultural soils. FEMS Microbiol. Ecol. 44, 303–308. Van Baale, M. J., Sargeant, J. M., Gnad, D. P., DeBey, B. M., Lechtenberg, K. F., and Nagaraja, T. G. (2004). Effect of forage or grain diets with or without monensin on ruminal persistence and fecal Escherichia coli O157:H7 in cattle. Appl. Environ. Microbiol. 70, 5336–5342. Van Donkersgoed, J., Graham, T., and Gannon, V. (1999). The prevalence of verotoxin, Escherichia coli O157:H7, and Salmonella in the feces and rumen of cattle at processing. Can. Vet. J. 40, 332–338. Van Donkersgoed, J., Berg, J., Potter, A., Hancock, D., Besser, T., Rice, D., LeJeune, J., and Klashinsky, S. (2001). Environmental sources and transmission of Escherichia coli O157 in feedlot cattle. Can. Vet. J. 42, 714–720. Van Donkersgoed, J., Hancock, D., Rogan, D., and Potter, A. A. (2005). Escherichia coli O157: H7 vaccine field trial in 9 feedlots in Alberta and Saskatchewan. Can. Vet. J. 46, 724–728. Varel, V. H. and Miller, D. N. (2001). Plant-derived oils reduce pathogens and gaseous emissions from stored cattle waste. Appl. Environ. Microbiol. 67, 1366–1370. Varel, V. H. and Miller, D. N. (2004). Eugenol stimulates lactate accumulation yet inhibits volatile fatty acid production and eliminates coliform bacteria in cattle and swine waste. J. Appl. Microbiol. 97, 1001–1005. Varel, V. H., Nienaber, J. A., and Freetly, H. C. (1999). Conservation of nitrogen in cattle feedlot waste with urease inhibitors. J. Anim. Sci. 77, 1162–1168. Varel, V. H., Miller, D. N., and Berry, E. D. (2006). Incorporation of thymol into corncob granules for reduction of odor and pathogens in feedlot cattle waste. J. Anim. Sci. 84, 481–487. Varel, V. H., Wells, J. E., and Miller, D. N. (2007a). Combination of a urease inhibitor and a plant essential oil to control coliform bacteria, odour production and ammonia loss from cattle waste. J. Appl. Microbiol. 102, 472–477. Varel, V. H., Wells, J. E., and Berry, E. D. (2007b). Effect of N-(n-butyl) phosphoric triamide (NPBT) and a linalool extract or pine oil extract on urea concentration, odorants, and coliform bacteria when these additives are applied to a cattle feedlot surface. In ‘‘CDROM Proceedings of the International Symposium on Air Quality and Waste Management for Agriculture, Broomfield, CO’’. American Society of Agricultural and Biological Engineers, St. Joseph, MI (ASABE Publication Number 701P0907cd). Varel, V. H., Wells, J. E., Berry, E. D., Spiehs, M. J., Miller, D. N., Ferrell, C. L., Shackelford, S. D., and Koohmaraie, M. (2008). Odorant production and persistence of Escherichia coli in manure slurries from cattle fed zero, twenty, forty, or sixty percent wet distillers grains with solubles. J. Anim. Sci. 86, 3617–3627. Varel, V. H., Wells, J. E., Berry, E. D., and Miller, D. N. (2010). Manure odor potential and Escherichia coli concentrations in manure slurries of feedlot steers fed 40% corn wet distillers grains. J. Environ. Qual. (In press). Vlisidou, I., Lyte, M., van Diemen, P. M., Hawes, P., Monaghan, P., Wallis, T. S., and Stevens, M. P. (2004). The neuroendocrine stress hormone norepinephrine augments Escherichia coli O157:H7-induced enteritis and adherence in a bovine ligated ileal loop model of infection. Infect. Immun. 72, 5446–5451. Wallace, J. S., Cheasty, T., and Jones, K. (1997). Isolation of Vero cytotoxin-producing Escherichia coli O157 from wild birds. J. Appl. Microbiol. 82, 399–404. Wang, G. and Doyle, M. P. (1998). Survival of enterohemorrhagic Escherichia coli O157:H7 in water. J. Food Prot. 61, 662–667. Wang, G., Zhao, T., and Doyle, M. P. (1996). Fate of enterohemorrhagic Escherichia coli O157: H7 in bovine feces. Appl. Environ. Microbiol. 62, 2567–2570.

Escherichia coli O157 in Cattle Production

117

Wang, Y., Xu, Z., Bach, S. J., and McAllister, T. A. (2008). Effects of phlorotannins from Ascophyllum nodosum (brown seaweed) on in vitro ruminal digestion of mixed forage or barley grain. Anim. Feed Sci. Technol. 145, 375–395. Waters, J. R., Sharp, J. C., and Dev, V. J. (1994). Infection caused by Escherichia coli O157:H7 in Alberta, Canada, and in Scotland: A five-year review, 1987–1991. Clin. Infect. Dis. 19, 834–843. Wells, J. E. and Varel, V. H. (2005). Animal/microbial symbiosis. In ‘‘Encyclopedia of Animal Science’’, (W. G. Pond and A. W. Bell, Eds), pp. 449–452. Marcel Dekker, Inc., New York. Wells, J. G., David, B. R., Wachsmuth, I. K., Riley, L. W., Remis, R. S., Sokolow, R., and Morris, G. K. (1983). Laboratory investigation of hemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype. J. Clin. Microbiol. 18, 512–520. Wells, J. G., Shipman, L. D., Greene, K. D., Sowers, E. G., Green, J. H., Cameron, D. H., Downes, F. P., Martin, M. L., Griffin, P. M., Ostroff, S. M., Potter, M. E., Tauxe, R. V., et al. (1991). Isolation of Escherichia coli serotype O157:H7 and other Shiga-like-toxin-producing E. coli from dairy cattle. J. Clin. Microbiol. 29, 985–989. Wells, J. E., Berry, E. D., and Varel, V. H. (2005). Effects of common forage phenolic acids on Escherichia coli O157:H7 viability in bovine feces. Appl. Environ. Microbiol. 71, 7974–7979. Wells, J. E., Berry, E. D., and Varel, V. H. (2006). Effects of essential oils on viability of Escherichia coli O157:H7 in treated beef cattle manure slurries and on prevalence from treated feedlot surfaces. J. Anim. Sci. 84(Suppl. 1), 356–357. Wells, J. E., Shackelford, S. D., Berry, E. D., Kalchayanand, N., Guerini, M. N., Varel, V. H., Arthur, T. M., Bosilevac, J. M., Freetly, H. C., Wheeler, T. L., Ferrell, C. L., and Koohmaraie, M. (2009). Prevalence and level of Escherichia coli O157:H7 in feces and on hides of feedlot steers fed diets with or without wet distillers grains with solubles. J. Food Prot. 72, 1624–1633. Wetzel, A. N. and LeJeune, J. T. (2006). Clonal dissemination of Escherichia coli O157:H7 subtypes among dairy farms in Northeast Ohio. Appl. Environ. Microbiol. 72, 2621–2626. Woerner, D. R., Ransom, J. R., Sofos, J. N., Dewell, G. A., Smith, G. C., Salman, M. D., and Belk, K. E. (2006a). Determining the prevalence of Escherichia coli O157 in cattle and beef from the feedlot to the cooler. J. Food Prot. 69, 2824–2827. Woerner, D. R., Ransom, J. R., Sofos, J. N., Scanga, J. A., Smith, G. C., and Belk, K. E. (2006b). Preharvest processes for microbial control in cattle. Food Prot. Trends 26, 393–400. Younts-Dahl, S. M., Osborn, G. D., Galyean, M. L., Rivera, J. D., Loneragan, G. H., and Brashears, M. M. (2005). Reduction of Escherichia coli O157 in finishing beef cattle by various doses of Lactobacillus acidophilus in direct-fed microbials. J. Food Prot. 68, 6–10. Zhao, T., Zhao, P., West, J. W., Bernard, J. K., Cross, H. G., and Doyle, M. P. (2006). Inactivation of enterohemorrhagic Escherichia coli in rumen content- or feces-contaminated drinking water for cattle. Appl. Environ. Microbiol. 72, 3268–3273.

CHAPTER

5 Fish Gelatin Gokhan Boran*,1 and Joe M. Regenstein†

Contents

Abstract

120 121 124 126 127

I. General Information on Gelatin A. The parent molecule: Collagen B. Collagen–gelatin conversion C. The mechanism of gelation D. The characteristics of gelatin E. Sources of raw material used in gelatin manufacturing F. The gelatin market G. Industrial applications of gelatin II. Fish Gelatin A. Common and potential sources B. Properties of fish gelatin C. Other factors affecting quality D. Quality of fish gelatin compared to mammalian gelatins III. Methodological Challenges in Quality Determination of Gelatin IV. Conclusions and Suggested Readings References

127 128 128 129 129 130 135 138 139 140 140

Gelatin is a multifunctional ingredient used in foods, pharmaceuticals, cosmetics, and photographic films as a gelling agent, stabilizer, thickener, emulsifier, and film former. As a thermoreversible hydrocolloid with a narrower gap between its melting and gelling temperatures, both of which are below human body temperature,

* Department of Food Engineering, Yu¨zu¨ncu¨ Yıl University, Van, Turkey { 1

Department of Food Science, Cornell University, Ithaca, New York, USA Corresponding author: Gokhan Boran, E-mail address: [email protected]

Advances in Food and Nutrition Research, Volume 60 ISSN 1043-4526, DOI: 10.1016/S1043-4526(10)60005-8

#

2010 Elsevier Inc. All rights reserved.

119

120

Gokhan Boran and Joe M. Regenstein

gelatin provides unique advantages over carbohydrate-based gelling agents. Gelatin is mostly produced from pig skin, and cattle hides and bones. Some alternative raw materials have recently gained attention from both researchers and the industry not just because they overcome religious concerns shared by Jews and Muslims but also because they provide, in some cases, technological advantages over mammalian gelatins. Fish skins from a number of fish species are among the other sources that have been comprehensively studied as sources for gelatin production. Fish skins have a significant potential for the production of high-quality gelatin with different melting and gelling temperatures over a much wider range than mammalian gelatins, yet still have a sufficiently high gel strength and viscosity. Gelatin quality is industrially determined by gel strength, viscosity, melting or gelling temperatures, the water content, and microbiological safety. For gelatin manufacturers, yield from a particular raw material is also important. Recent experimental studies have shown that these quality parameters vary greatly depending on the biochemical characteristics of the raw materials, the manufacturing processes applied, and the experimental settings used for quality control tests. In this review, the gelatin quality achieved from different fish species is reviewed along with the experimental procedures used to determine gelatin quality. In addition, the chemical structure of collagen and gelatin, the collagen–gelatin conversion, the gelation process, and the gelatin market are discussed.

I. GENERAL INFORMATION ON GELATIN Gelatin is a term used for a class of protein fractions that have no existence in nature. Gelatin is derived from collagen, which is a natural structural protein, predominantly found in the connective tissues of animals (Balian and Bowes, 1977; Belitz and Schieberle, 2004; DeMan, 1999) although also found in many other tissues. Collagen is the most ubiquitous of animal proteins. Gelatin is one of the most widely used biopolymers and is added to foods, drugs, cosmetics, photographic films, and other products, including paints, matches, and fertilizers, as a gelling agent, foam stabilizer, and structure enhancer (Gudmundsson, 2002; Karim and Bahat, 2009; Yang et al., 2007; Zhou and Regenstein, 2004). Gelatin is able to form a high viscosity solution in warm water, which sets to a gel on cooling. The chemical composition of gelatin is, in many respects, similar to that of collagen, its parent molecule. Gelatin is, however, not composed of one size of collagen fraction or peptide chain but is a combination of many fractions varying in size, including the whole a-chain of the tropocollagen molecule (a trimer of around 330 kDa that aggregates to form the larger collagen structures) and hydrolytic fragments of parts of the

Fish Gelatin

121

a-chains of different lengths (Eastoe and Leach, 1977). Gelatin gels have relatively lower melting temperatures compared to the gels of other gelling agents (Williams, 2007). Gelatin gels generally have a melting temperature below 35
C, that is, below human body temperature, which makes gelatin unique in terms of its sensory aspects, especially flavor release, which is particularly desired for some food applications (Baziwane and He, 2003; Boran and Regenstein, 2009; Choi and Regenstein, 2000). Other gelling agents such as starch, alginate, pectin, and agar are carbohydrates and their gels cannot melt below body temperature as most have much higher melting temperatures (Williams, 2007). Gelatin is obtained from the skins and bones of pigs and cattle, but mostly from pig skin. However, there are alternative raw materials that can be used in gelatin manufacturing, including by-products from the chicken and fish processing industries. Fish skins have received attention from researchers as an alternative raw material having the potential for the production of large amounts of high-quality gelatin. Therefore, recent studies with fish skin gelatin have focused on the evaluation of different fish species as an alternative raw material for gelatin production and the quality of the extracted gelatins in comparison with commercial gelatins from conventional sources (Boran and Regenstein, 2009). In this review, gelatin production and processing, the raw materials used in gelatin production, the technological attributes of gelatin, the gelatin market, and the market specifically for fish gelatin are discussed. In addition, the most critical factors affecting the quality of gelatin are discussed. For this purpose, the chemical structure of collagen is reviewed in detail to take a closer look at the possible factors affecting the properties of the resultant gelatin. The conversion process of collagen into gelatin and the gelation mechanism are discussed to show which driving forces are involved in gelation, which factors might affect the solution–gel and gel–solution transitions, and how extraction conditions might affect the final product, gelatin. The methods being currently used to determine the quality of gelatin are also reviewed.

A. The parent molecule: Collagen Collagen is the most abundant protein in the animal body (DeMan, 1999). Collagen is part of the connective tissue in muscles and many other organs, including the skin, bones, teeth, and tendons. Collagen fibrils normally have a regular periodicity of 64 nm when stained for microscopy, which can be increased to 400 nm with r tension (DeMan, 1999). Collagen molecules are arranged head-to-tail, with a 35-nm gap between molecules, and are found in larger structures having staggered bundles, that is, adjacent collagen molecules are not aligned with each other. Charged and uncharged residues are found to be periodically clustered

122

Gokhan Boran and Joe M. Regenstein

along the sequence of collagen at about every 230 residues, which is around 64 nm, although this distance may vary somewhat among different tissue sources of collagen. This suggests that the collagen molecules are aligned such that the maximum electrostatic and hydrophobic interactions occur between different molecules (Fig. 5.1). Collagen constitutes 20–25% of the total protein in mammals and has a unique amino acid composition, which includes two modified amino (imino) acids, hydroxyproline and hydroxylysine (Belitz et al., 2004). Its molecular structure is mainly the multiple repetition of a ‘‘Glycine-X-Y’’ sequence, where ‘‘X’’ is often proline and ‘‘Y’’ is often hydroxyproline. Collagen has a unique triple helix structure that is based on a special helix of three polypeptide chains with high levels of imino acids. Each polypeptide chain is left handed and has three amino acids per turn. These three polypeptide chains, called a-chains, are supertwisted around one another and form a superhelix that is right handed (Nelson and Cox, 2005). The basic structural unit of the collagen superhelix is called tropocollagen. It has a molecular weight of approximately 330 kDa, with a length of approximately 300 nm and a diameter of 1.5 nm (Belitz et al., 2004).

Collagen molecule 1.5 nm 300 nm

64 nm Collagen fibril 10−300 nm

FIGURE 5.1

Schematic representations of collagen molecules and a collagen fibril.

Fish Gelatin

123

When hydrolyzed, the collagen can give three different fractions: independent a-chains, which may have slightly different molecular weights, a b-chain, that is, two a-chains linked to each other by covalent bonds, and a g-chain, that is, the three a-chains linked to one another by covalent bonds. These fractions differ in molecular size: a-chains correspond to a molecular weight of 80–125 kDa, b-chains correspond to a molecular weight of 160–250 kDa, and g-chains correspond to a molecular weight of 240–375 kDa, which is very similar to the molecular size of collagen (Imeson, 1997). Collagen typically contains about 35% glycine, 11% alanine, and 21% proline and hydroxyproline, the amount of which varies somewhat among different species although the high content of proline and hydroxyproline seems to be characteristics of collagen structure regardless of the source (Balian and Bowes, 1977). Hydroxyproline is a rare amino acid that is absent in nearly all other proteins and so its presence can be used to determine the presence of collagen in the presence of other proteins, and with proper calibration (i.e., the conversion factor is at a minimum species specific), the amount of collagen or gelatin (Engel and Bachinger, 2005) in the mixture. Another protein containing hydroxyproline is elastin, but the amount of hydroxyproline in elastin is very low and the amount of elastin in most tissues is also very low when compared to that of collagen (Nelson and Cox, 2005). Collagen is generally considered to be an incomplete protein since the concentration of some essential amino acids is low in collagen and consequently, in gelatin (Belitz et al., 2004; Nelson and Cox, 2005). However, when eaten as part of a meal, the contribution of gelatin to the amino acid ingestion needs to be considered. The amounts of the aromatic and sulfur-containing amino acids are low (0–0.6%) in collagen, that is, tryptophan and cysteine are mostly absent in collagen (Balian and Bowes, 1977). Cysteine is usually absent in collagen, therefore, there are usually no disulfide bonds involved in collagen structure although there are some collagens that have cysteine (Engel and Bachinger, 2005). For those, disulfide bonds are also involved in the formation of intermolecular cross-links (disulfide bonds) and provide additional stabilizing effects for the structure. The structure of collagen provides an explanation of why glycine is the most abundant amino acid and why proline and hydroxyproline are found so often in collagen. Only glycine residues, as the smallest of the hydrophobic amino acids, can fit into the very tight central core between the individual a-chains, while proline and hydroxyproline residues permit sharp twists of the collagen helix allowing for the relatively low three amino acids per turn (Nelson and Cox, 2005). The collagen molecule is primarily stabilized by hydrogen bonds between the backbone amino group of glycine and the backbone carboxyl

124

Gokhan Boran and Joe M. Regenstein

group of a residue in the X position of a neighboring a-chain, which is usually a proline. Proline in the Y position is generally hydroxylated posttranslation into hydroxyproline, which also plays an important role in the formation of intra- and intermolecular hydrogen bonds. Hydroxyproline is, therefore, important for both the structure of the collagen molecule and of the collagen fibrils (Brinckmann, 2005). During maturation or aging of the living animal, collagen fibers strengthen and are further stabilized primarily by complex covalent bonds. Lysine, hydroxylysine, and histidine residues are heavily involved in the formation of these covalent bonds, that is, aldimine bonds between lysine and lysine or hydroxylysine (Balian and Bowes, 1977; Belitz et al., 2004; Engel and Bachinger, 2005; Eyre and Wu, 2005; Nelson and Cox, 2005) that lead to the formation of desmosine and isodesmosine, which are unusual in that they involve the participation of four amino acids in the reaction. This is possible because of the rigid geometry of the collagen structures and the long times available for these reactions to occur. All the fibril-forming collagen types (type I, II, III, V, XI, XXIV, and XXVII collagens) are cross-linked through a mechanism based on the reactions of aldehydes derived from some lysine (or hydroxylysine) side chains. Histidine might also participate in the formation of a trivalent cross-link by reacting with an aldimine bond formed between a lysine aldehyde and hydroxylysine residue. With respect to tissue source, type I collagen is the most widely occurring collagen found in skin, tendon, bone, cornea, lung, and the vasculature, while type II collagen has a more specific tissue distribution being limited essentially to cartilage, while type III is found in relatively elastic tissues such as embryonic skin, lung, and blood vessels (Hulmes, 2008). For most nonfibrillar collagens (e.g., type IV, VI, and VII collagens), disulfide bonds may be the only source of intra- and intermolecular covalent bonds. There are usually no lysine-mediated cross-links in these collagens (Eyre and Wu, 2005). The best known nonfibrillar collagen is type IV collagen, which is a basement membrane collagen that forms specialized structures found at tissue boundaries, and in fat, muscle, and nerve cells. Collagen VI, on the other hand, is important in maintaining tissue integrity (Hulmes, 2008).

B. Collagen–gelatin conversion There are several methods used by the industry to manufacture gelatin from collagen. The main purpose of the gelatin production process is to first remove unwanted materials that will interfere with the gelatin extraction and then to convert collagen that is insoluble in water into gelatin that is soluble in water, while obtaining maximum yield and superior functional properties (Hinterwaldner, 1977). In general, gelatin

Fish Gelatin

125

is obtained using a sequence of three types of processing steps: pretreatments to remove noncollagen impurities and prepare the collagen for extraction, one or more water extraction steps to convert collagen into gelatin, and finally, a series of refinement and recovery processes to get a highly purified dried gelatin. A limited amount of gelatin has been marketed in liquid form to avoid the drying step (J.M. Regenstein, personal communication). In the first step, raw materials are water washed to remove obvious impurities and then treated with alkali and/or acid to weaken the collagen structure by breaking intramolecular cross-linkages including covalent and hydrogen bonds and to release other impurities. Some size reduction may also be applied to increase the efficiency of the process. In the second step, the actual water extraction is performed at warm temperatures for an appropriate period of time. In the last step, extracted gelatin is subjected to several separation methods, including filtration, evaporation, and deionization, followed by drying and grinding (Hinterwaldner, 1977). Gelatins are classified according to whether an acid or an alkali is used in the final preextraction step. If an acid solution is used as the final solvent, type-A gelatin (acid process) is obtained. In case of alkali as the final solvent, type-B gelatin (alkali process) is obtained (Hinterwaldner, 1977). Type-A gelatin’s isoelectric point is higher compared to that of type-B gelatin, as a milder acid process does not remove the amide nitrogen of glutamine and asparagine, therefore, the resulting gelatin’s isoelectric point might be as high as 9.4. If a more severe acid treatment is required, then some of the amide groups are hydrolyzed and the isoelectric point would be similar to that of the original collagen molecule, which generally lies between 6 and 8. Type-B gelatin’s isoelectric point might be as low as 4.8, as the alkali process results in the loss of the amide groups (Eastoe and Leach, 1977). In the acid process, the bones and skins are treated in a vessel containing a dilute solution of acid for a predetermined period of time. Then, the acid is washed out with cold water. In the alkali process, the demineralized bones (demineralization is mostly done with acid solutions to remove calcium and other salts from the bone to prepare the collagenrich bone material known as ossein) are placed in liming pits and soaked in a lime suspension for longer than 60 days. For the hides or skins, a caustic soda solution is used for a shorter period of time. After this treatment, the raw material is washed thoroughly to remove any residual lime. The acid pretreatment is mostly used for skin, while the alkali pretreatment is mostly used for bones (Petersen and Yates, 1977). The actual extraction method used for both acid and alkali pretreated raw materials is similar. The main extraction step is done using hot water at controlled temperatures, mostly higher than 40
C, and it is the most

126

Gokhan Boran and Joe M. Regenstein

important step in gelatin production. In the industry, the extraction step is usually multiple extractions performed with gradually increasing temperatures beginning from 50 to 60
C and going up to the boiling temperatures, usually in 5–10
C temperature increment. Gelatins are collected so that the lower temperature fractions have minimal degradation and the higher temperature fractions have more variable molecular weights (Hinterwaldner, 1977). The dilute gelatin solution from the extraction process is clarified using lamellar separators (this equipment is built as a set of plates or discs that are arranged at such an angle that the solids can slide off into the sludge chamber, thus achieving clarification) and filtered using self-cleaning centrifugal filters or cellulosic filters. After that, gelatin solutions are deionized by passing through ion exchangers and concentrated, usually in a multiple effect vacuum evaporator. The concentrated solution is then sterilized by hot air in batch driers, and then cooled or chilled to rapidly form a gel. Then, the gel obtained is extruded to get gelatin noodles (i.e., continuous strands), followed by a final drying and grinding process. After all these treatments, gelatin granules or powder are obtained. Acid or alkali pretreatments designed to destroy or weaken cross-linkages between the a-chains or between tropocollagen molecules need to be adjusted not only in terms of concentration but also treatment time to avoid extensive degradation of collagen, which might result in lower quality gelatin. But at the same time, enough degradation is needed to be able to get a higher yield and acceptable gel strength (Holzer, 1996).

C. The mechanism of gelation Hydrogen bonds certainly play an important role in gelation (Johns and Courts, 1977). Gelation can be considered as gelatin regaining its collagen structure, but this would not be exactly correct, because the conversion of collagen into gelatin is an irreversible process although gelatin can partially regain some collagen structure by recovering some crosslinkages. The greater the amount of cross-linkages recovered, the higher the gel strength and viscosity along with the melting and gelling temperatures (Belitz et al., 2004). The concentration of a-chains and the cooling rate are the most important factors affecting the final gelation. At high gelatin concentrations intermolecular bond formation would occur with multiple strands, while the same process is more likely to occur as intramolecular bonds within a single strand at low concentrations. Similarly, slow rates of cooling allow more intra- and intermolecular cross-link formation, while rapid cooling does not allow that to happen (Belitz et al., 2004).

Fish Gelatin

127

D. The characteristics of gelatin Gelatin is a gelling agent that is able to form thermoreversible gels, which means that when the gelatin gel is heated, it softens and again turns into a liquid. Then, it is able to return to the gel form when the solution is cooled again (Stainsby, 1977). Being able to melt below human body temperature makes its use very favorable in the food industry since gelatin is able to melt and release flavor when it is taken into the mouth, which is desired in terms of the sensorial properties of food products (Choi and Regenstein, 2000). Another important characteristic of gelatin is that its gel strength is relatively higher than most of the common gelling agents, which are usually carbohydrates and obtained from vegetable sources (Badii and Howell, 2006). The gap between melting and gelling temperature of gelatin gels is smaller than that of other gelling agents, which is desired for some particular applications, that is, food applications including jellies and custards (Jones, 1977).

E. Sources of raw material used in gelatin manufacturing Pork skin is the most abundantly used raw material in gelatin production. About 45% of the world’s total gelatin production is obtained from pork skin, followed by bovine hides with almost 30% (Karim and Bahat, 2009), and 23% of gelatin is obtained from bovine and porcine bones. Other sources include chicken and fish, but these account for only 1.5% of the world’s annual gelatin production. In Europe, pork skin is the most abundantly used raw material for gelatin production, accounting for around 80% of the total, followed by cattle skin with 15% of the total gelatin production. The remaining 5% is from pork and cattle bones, fish, and chicken. Recent studies have shown that fish skin, especially, might be an alternative source for gelatin production. Fish skin gelatins may provide better alternatives for some applications because of their, at times, relatively lower gel strengths and melting temperatures compared to that of mammalian gelatins. These characteristics may be desired in some food systems for ease of flavor release, leading to better sensory characteristics (Boran and Regenstein, 2009; Choi and Regenstein, 2000). In addition, obtaining valuable by-products from the fishery industry and reducing waste have made it an attractive research topic. As the issues of sustainability and the better use of harvested resources become more critical, the need to use fish waste more productively will only increase. Many fish species have been investigated as a raw material for gelatin extraction and the properties of the gelatins obtained from these sources have also been reported.

128

Gokhan Boran and Joe M. Regenstein

The waste from fish processing after filleting can account for as much as 75% of the total catch weight (Shahidi, 1995). It includes the heads, skin, and scales, guts/internal organs, frames (bone rack with adhering meat), and trim (pieces cut from the fillets during processing) (Regenstein, 2004). About 30% of such waste consists of skin, bone, and scale with high collagen content that could be used to produce collagen and gelatin (Go´mez-Guille´n et al., 2002; Sadowska et al., 2003; Young and Lorimer, 1960). Very little work has been done on fish bone gelatin (Muyonga et al., 2004), while a little more work has been done on scale gelatin. Fish scales gelatin has been obtained from sardine (Harada et al., 2007), Asian carp (Wang and Regenstein, 2009), and lizardfish (Wangtueai and Noomhorm, 2009).

F. The gelatin market The world’s total gelatin production is close to 350,000 tons annually, accounting for the market’s economic value being over US$ 2 billion. Again, gelatin is used in many products, including foods, pharmaceuticals, cosmetics, photographic films, paints, fertilizers, and many more, as a gelling agent, stabilizer, and structure enhancer (Jones, 1977).

G. Industrial applications of gelatin Gelatin’s largest single use is in food products, more specifically, water gel desserts. Gelatin desserts might consist of several other gelling agents (i.e., agar, carrageenan) along with gelatin; however, gelatin gels usually melt at temperatures that are lower than the body temperature, which makes gelatin favorable compared to other gelling agents in food applications. In some parts of the world, water dessert gels are made from carrageenan and these have to be chewed as they do not melt in the mouth. Thus, though both are called water dessert gels, they have very different sensory properties. Gelatin is used in dairy products as a stabilizer and as an ingredient to modify the texture. It is used in yogurt, ice cream, and other dairy products. Gelatin is added to yogurt to reduce syneresis and to increase firmness. Gelatin is an ingredient compatible with milk proteins and improves the sensory perception by not masking the product flavor as much as some other gums (Jones, 1977). The use of different concentrations of gelatin would provide the manufacturer with the possibility of obtaining a wide range of textures in food products. Gelatin is widely used in confectionery products, including soft gummy-type candies and marshmallows. Gelatin is the main gelling agent in gummy-type candies. Marshmallows usually contain about 3% gelatin, in which gelatin serves as a stabilizer and whipping agent ( Jones, 1977).

Fish Gelatin

129

Gelatin is also used in nonfood products, including drugs, cosmetics, photographic films, and paper and paint products. Pharmaceutical gelatin accounts for a significant proportion of the total production and it is used in the production of capsules, tablets, and pastilles (Wood, 1977). Gelatin is used for both soft and hard capsules. The gelatin protects the drugs during distribution and they are not released until after they are in the stomach. Gelatin acts as a binding agent in tablets. It is also used for tablet coating to reduce dusting, to mask unpleasant tastes, and to allow printing and color coatings for product identification. Gelatin has been used for photographic emulsions for more than 100 years. It is still the principal constituent of the binder in the most commercial photographic films and papers (Kragh, 1977).

II. FISH GELATIN In the last decade, gelatin extraction from fish skin has been intensively investigated. The physicochemical, textural, rheological, and sensory properties of extracted fish gelatin have also been studied in comparison with mammalian gelatin. The overall results suggest that fish skin might be an alternative raw material for high-quality gelatin production, eliminating religious concerns shared by the Jewish and Muslim communities and also providing an alternative and more lucrative way to use some fishery by-products (Boran and Regenstein, 2009), thus also improving the overall sustainability of the fishing industry. Some of the fish species investigated include Atlantic salmon (Arnesen and Gildberg, 2007), cod (Gudmundsson and Hafsteinsson, 1997), sin croaker and short fin scad (Cheow et al., 2007), Alaska pollock (Zhou and Regenstein, 2004), big eye snapper and brown stripe red snapper (Jongjareonrak et al., 2006), yellow fin tuna (Cho et al., 2005), Nile perch (Muyonga et al., 2004), black and red tilapia (Jamilah and Harvinder, 2002), grass carp (Kasankala et al., 2007), and silver carp (Boran and Regenstein, 2009).

A. Common and potential sources Gelatin can be obtained from various marine and freshwater sources. The species available for gelatin production are divided into three categories: marine invertebrates, sea mammals, and fishes. Based on their living environments, fishes are usually subdivided into four groups: hot-water fish, warm-water fish, cold-water fish, and ice-water fish. Cold-water fishes, such as pollock, cod, and salmon, account for a large part of the commercial fish capture. They are often processed into skinned and boneless fillets, leaving large amounts of fish skin, scales, and bones as waste or as a raw material that is sometimes wasted, sometimes used for

130

Gokhan Boran and Joe M. Regenstein

fish meal, occasionally for leather, and which would be appropriate for gelatin production. These by-products, especially the skin, usually contain a large amount of protein, most of which is collagen. Warm-water fishes account for most freshwater fish aquaculture production, and currently, many commercial fish gelatins come from these fish species.

B. Properties of fish gelatin 1. Physical attributes a. Gel strength Gel strength is one of the most important quality characteristics used in the gelatin industry to differentiate gelatins. As measuring gel strength is very popular, there is a standard method. According to the standard method (Gelatin Manufacturers Institute of America, GMIA), gel strength must be measured at 10
C on a gelatin sample prepared at 6.67% concentration of protein (w/v). Thus, if the gelatin is contaminated with other proteins, the amount of actual gelatin evaluated would be less that 6.67%. For practical applications, this does not matter as it allows the user to evaluate the ‘‘gelatin’’ they are using. For scientific purposes, however, this may underestimate the quality of the actual gelatin extracted. Another concern is with the protein determination itself. Because gelatin has a unique amino acid composition, a special conversion factor is needed for the Kjeldahl test, and work has shown that calibration of the Biuret or Lowry procedures may also require adjustments for an accurate measurement (Zhou and Regenstein, 2006). The methods for dissolving gelatin in water is not standardized and there are variations in the procedures used, that is, different water temperatures, different durations of sitting at the higher temperature before cooling, with or without stirring, etc. Maturation time and temperature are standardized and are generally followed by most investigators, that is, 16–18 h at 10
C. A particular jar is used for this measurement, called a ‘‘bloom jar’’ (Fig. 5.2), it requires about 155 ml of gelatin solution, which corresponds to about 10 g of gelatin. However, this particular jar cannot be regularly used in many scientific studies because it requires such a substantial amount of sample, which is often limited in scientific studies. Therefore, many scientists use other containers that differ in size and shape leading to significant differences in the results, making the data incomparable among the different studies. Because of sample limitations, in some cases, a lower percentage of gelatin is used, often 3.33%. The test settings are also standardized: It is the force required for a 12.7-mm diameter flat probe to penetrate 4 mm into the gel that is being lowered into the sample at a speed of 1 mm/s. This force, expressed in grams, is then reported as the gel strength. If done totally according to the official methodology, the resulting gel strength may be called the ‘‘Bloom strength.’’

Fish Gelatin

131

FIGURE 5.2 Standard ‘‘bloom jar’’ provided by Texture Technologies Corporation (Scarsdale, NY).

There are different instruments that can be used to handle the probe and the different instruments may give different results (Table 5.1).

b. Viscosity Viscosity is generally measured using tubular glass viscometers as they are relatively inexpensive and easy to use compared to expensive and complicated computer-controlled instruments. Although advanced viscosity instruments might provide higher reproducibility and accuracy, the tubular glass viscometers also give high precision. Compared to gel strength, viscosity is not as well correlated with textural properties. The viscosity is mostly affected by molecular weight distribution. Gelatin samples with high molecular weight fractions give high viscosity but that does not necessarily mean that their gel strengths would also be high. Gelatin samples from fish skin, for example, give unexpectedly high viscosity while giving low gel strength compared to that of pork skin gelatin due to the carefully controlled extraction conditions and consequently, the presence of higher molecular weight protein fractions (Boran and Regenstein, 2009). Arnesen and Gildberg (2007) reported that Atlantic salmon and Atlantic cod skin gelatin had higher viscosities than pork skin gelatin while giving lower gel strengths than pork skin gelatin. Generally, fish skin gelatins are expected to have a lower viscosity compared to that of gelatins obtained from porcine and bovine sources with

132

Gokhan Boran and Joe M. Regenstein

TABLE 5.1 Gel strength of a commercial gelatin measured using different instruments and probes in either a standard bloom jar or a 15-ml capacity small plastic jar Measurement details

Average SD

Standard bloom jar, TAXT2 texture analyzer, spherical probe Standard bloom jar, TAXT2 texture analyzer, cylindrical probe Standard bloom jar, Stevens texture analyzer, spherical probe Standard bloom jar, Stevens texture analyzer, cylindrical probe Small plastic jar, TAXT2 texture analyzer, spherical probe Small plastic jar, TAXT2 texture analyzer, cylindrical probe Small plastic jar, Stevens texture analyzer, spherical probe Small plastic jar, Stevens texture analyzer, cylindrical probe

242

5.7

523

2.1

213

1.2

466

3.8

320 814 294 746

9.2 13.6 2.5 9.6

SD: standard deviation.Same sample (Knox Gelatin, Kraft Foods Global, Inc., Glenview, IL, USA) used for the measurements: 6.67% gelatin, dissolved at 50
C for 30 min in distilled water, matured at 4
C for 16–18 h. The measurements are done at 4
C using the following settings: 4 mm penetration with 12.7 mm diameter probe (either spherical or cylindrical) with 1 mm/s penetration speed. Gel strength is given as g force required penetrating the probe onto the sample (N ¼ 3).

similar molecular weight distributions. However, most investigators do not look at the molecular weight distribution beyond looking at the SDSPAGE electrophoretigram. Because the distribution of peptides can be so great, much of it is ‘‘smeared’’ over the gel and cannot be easily quantitated. To do more is a lot of work and would likely be different for each preparation, so it has not been done critically.

c. Melting and gelling temperature Rheological methods have recently gained importance and have found applications in the determination of gelatin quality. Rheological measurements of both melting and gelling temperatures give highly reproducible results. A temperature sweep test is performed for this purpose. Heating or cooling is required to determine the melting and gelling temperature, respectively. The gelatin gel sample is prepared at a certain concentration and matured at a certain temperature for a certain period of time to standardize the procedure to be able to discriminate the samples based on their chemical differences (Chiou et al., 2006; Cho et al., 2006; Fernandez-Diaz et al., 2003; Kasankala et al., 2007). Prior to rheological determinations of melting and gelling temperatures, the droplet method was used as a standard method for determining the melting temperature. However, the rheological methods have replaced

Fish Gelatin

133

this older method (Wainewright, 1977), which was less precise and more laborious. A drop of an organic solvent with a dye in it, originally carbon tetrachloride, but more recently chloroform, with the recognition of the toxicity of carbon tetrachloride, has been used. The temperature when the droplet fell is taken as the melting temperature—but is it the beginning of the fall, the middle or when it touches the bottom. This is not well spelled out. Obviously, the rate of heating will also affect the results both because of differences in heat penetration and in the accuracy of the reading; slower being better but taking longer! The rheological test measured the stress/strain as a function of time and temperature. Again, an arbitrary heating rate is selected. The midpoint of the transition is usually considered to be the melting or gelling temperature. The amount of cooperativity of the system can be determined by the width of the curve (in degrees) usually measured from the 25% point of transition to the 75% point of transition. The greater the cooperativity (or uniformity of the material), the smaller the width observed. Other rheological tests, including time sweep, frequency sweep, stress sweep, and strain sweep, have also found applications in determination of gelatin quality, as they allow researchers to discriminate the gelatin gels according to their strength and elasticity. Stress and strain sweep tests are used to determine the linear viscoelastic region of the gels. Frequency sweep tests are useful to determine if the gelatin gels change with the changing frequency of the stress applied. Time sweep tests are used to determine if the gelatin gels’ viscoelastic properties change with time at a controlled temperature and at a set level of stress applied. The recent literature on fish gelatin includes some examples of these tests used to make comparisons among gelatin samples from different sources. Chiou et al. (2006) used temperature sweep tests to determine the melting and gelling temperature of gelatin gels. They also used time sweep tests to show the increasing elastic modulus at different temperatures with an increasing concentration of glutaraldehyde added to the gelatin gels. Gudmundsson (2002) used frequency sweep tests successfully to differentiate the gelatin gels based on their elastic moduli and the temperature sweep tests to determine the melting temperature of blended fish gelatin gels. Zhou and Regenstein (2007) used temperature sweep tests to compare the melting temperatures of gelatin gels from different sources. In another study, Zhou et al. (2006) used strain sweep and frequency sweep tests to compare the gelatin gels from different sources based on their viscoelastic properties. Recent studies gave good examples of how rheological measurements had strong correlations with conventional parameters, including gel strength and viscosity (Gilsenan and Ross-Murphy, 2000; Gudmundsson, 2002; Zhou et al., 2006).

134

Gokhan Boran and Joe M. Regenstein

2. Chemical characteristics a. Amino acid composition Chiou et al. (2006) showed that differences in the amino acid composition have significant effects on the melting and setting temperatures of gelatin obtained from different sources. According to their results, the higher proline and hydroxyproline content of pork gelatin correlated with stronger gels having higher gelling temperatures. Proline and hydroxyproline are, however, not the only amino acids having significant effects on gelatin structure. The content of glutamic acid, aspartic acid, lysine, hydroxylysine, arginine, and histidine are also important in cross-link formation and electrostatic interactions. As collagen usually lacks cystine, there are no disulfide bonds in the collagen structure. Collagen is mostly stabilized by hydrogen bonds formed between side chains of the amino acids and water in addition to the twisted structure enforced by the high content of proline and hydroxyproline along with intra- and intermolecular cross-links (Engel and Bachinger, 2005).

b. Peptide size in relation to quality Collagen-containing tissues are treated with acid and/or alkali followed by a heat treatment in the presence of water to break the structure of the collagen fibrils irreversibly to obtain gelatin (Eastoe and Leach, 1977). While the molecular weight of the collagen molecule is about 330 kDa, gelatin is generally considered to be all collagen fractions with a molecular weight higher than an arbitrary minimum of 30 kDa. When most fractions are below 30 kDa, the product is usually called a gelatin hydrolysate, as such products are not able to form a gel, although these peptides are believed to participate in gel formation (Eastoe and Leach, 1977). A heat treatment of about 40
C breaks hydrogen and electrostatic bonds in newly formed collagen molecules releasing single a-chains, but this is insufficient to break the cross-links and covalent bonds in the collagen structure of mature collagen (Eastoe and Leach, 1977). With treatments at higher temperatures, on the other hand, those covalent bonds, including intermolecular cross-links and peptide bonds, break down and therefore, smaller a-chain fractions are obtained (Eastoe and Leach, 1977). The position of the bond breaks determines the molecular weight and the number of polypeptide chains. As amino acid sequence and composition of collagens from different sources vary greatly, bond breaks appear to be relatively random and these random bond breakdowns are the main cause of molecular heterogeneity in gelatin (Eastoe and Leach, 1977). The raw materials used in gelatin production contain a variety of substances that are the source of organic and/or inorganic impurities in gelatin. Noncollagen protein fractions, lipids, nucleic acids, and other cell

Fish Gelatin

135

components are among the organic impurities. Inorganic impurities include naturally present minerals such as calcium, sodium, potassium, and iron along with those derived from substances added for gelatin preparation, that is, acid and/or alkali with their impurities (Eastoe and Leach, 1977). Finally, commercial gelatin products contain a substantial amount of water usually as the second largest component in the whole and its amount varies greatly based on the drying process applied, the nature of the raw material, and the temperature and relative humidity of storage. Generally speaking, the water in gelatin is between 9% and 14% with occasional samples outside of this range (Eastoe and Leach, 1977).

C. Other factors affecting quality There are several factors that significantly affect the properties of gelatin (Cho et al., 2006). The raw materials used in gelatin manufacture have obvious effects on gelatin, mostly originating from differences in the amino acid composition of the collagen of the raw material. Also, variations in processing conditions such as extraction time, extraction temperature, and concentration of acid or alkali dramatically affect the product (Boran and Regenstein, 2009; Cho et al., 2006; Hinterwaldner, 1977; Zhou and Regenstein, 2005). For example, longer extraction temperatures and/ or higher extraction temperatures cause excessive damage to the collagen molecule and the resulting gelatins form weak gels and have low viscosity. Similarly, excessive concentrations of acid and/or alkali can cause degradation of collagen structure giving a gelatin with lower functional values.

1. The effects of processing conditions The extraction conditions greatly affect the quality of gelatin as shown by many researchers. Therefore, optimization of extraction is of great interest to obtain the best possible product by eliminating and/or minimizing the excessive effects of extraction treatments causing extensive damage to collagen molecules. The most important extraction parameters are acid and/or alkali treatments, more specifically, the concentration of acid and/or alkali and duration of the treatment; extraction temperature and duration, and the conditions of refining and drying processes. A general flowchart of gelatin extraction from fish skin is given later and it shows that those treatments greatly affect the quality of the gelatin (Fig. 5.3).

a. Alkali and acid treatments The gel strength of gelatin is greatly influenced by the concentration of acid and/or alkali, the duration of the acid and/or alkali treatment, and possibly the treatment temperature. A study

136

Gokhan Boran and Joe M. Regenstein

Fish skin Washing (cold water) Removal of impurities Washing (cold water) Storage until processing (−20 ⬚C) Cutting skin Alkali treatment Washing (cold water)

Filtration Acid treatment

Washing (cold water)

Filtration Water extraction (over 40 ⬚C) Filtration Drying Gelatin sheets

FIGURE 5.3

A general flowchart of gelatin extraction from fish skin.

by Gudmundsson and Hafsteinsson (1997) showed that high concentrations of alkali or acid increased the gelatin yield while decreasing gel strength. These results have also been confirmed by Zhou and Regenstein (2005). Another study done by Cho et al. (2006) showed that alkali concentration up to 1.5% increased gelatin yield significantly. Zhou and Regenstein (2004) confirmed that the concentrations of acid or alkali have a significant effect on gelatin yield, gel strength, and viscosity. Acid treatment is also important for the sensory aspects of gelatin, appearance and smell, as the acid treatment effectively removes odors and color that originate from the raw material (Boran and Regenstein, 2009; Zhang et al., 2007). Alkali treatment is, similarly, important and responsible for removal of possible impurities from the raw material and also for weakening the collagen structure, leading to higher yield and superior quality. In addition, alkali treatment causes glutamine and asparagine to lose their amine groups, converting them to glutamic and aspartic acid residues, respectively, lowering the isoelectric point of collagen (Johns and Courts, 1977). Therefore, both acid and alkali treatments need to be optimized for pH, duration, and temperature of extraction. Zhou and Regenstein (2005) also did their pretreatment at low temperatures, which led to a much higher amount of the full a-chains. They suggest that the low temperature may have prevented any native proteases/collagenases from degrading the collagen prior to the extraction.

Fish Gelatin

137

The main extraction step although normally done in water can be done at using mild acid or alkali conditions. Acid or alkali treatments are useful for a more effective extraction, increasing the yield and shortening the extraction time. Zhou and Regenstein (2005) showed that acidic conditions are more favorable for higher gelatin yield. However, acidic conditions also cause low gel strength, which is not desired in most gelatin applications. The isoelectric point of collagen is around 6–6.5, depending on the amino acid composition, specifically the content of the acidic and basic amino acids of collagen, which vary both due to source and to processing conditions, for example, due to the impact of processing on glutamine and asparagine. The isoelectric point of purified collagen is difficult to measure because collagen is difficult to isolate in its natural form as it is not readily soluble in water at room temperature and when it is dissolved with the help of heat treatment, collagen loses its natural state. Therefore, the isoelectric point measured does not reflect the physiological isoelectric point of the collagen, but many researchers agree on the approximate value of 7.0 for the isoelectric point of collagen under physiological conditions (Johns and Courts, 1977). Neutral extracts of untreated tissues of pork and rabbit skin, for example, had isoelectric points in the range of pH 5.6 and 6.8, respectively (Johns and Courts, 1977). A pH that is higher or lower than the isoelectric point results in higher extraction yield as collagen is less tightly bound at pH values different from its isoelectric point. The net charge of the collagen molecule is zero at the isoelectric point where there are an equal number of positive and negative charges on the molecule allowing it to form the maximum number of intermolecular salt bonds and electrostatic interactions, which strengthen and stabilize the structure of the collagen. However, the isoelectric point also reflects in part the binding of other ions that change the isoelectric point. The most accurate figure would be the isoionic point, which would be the extrapolation of the isoelectric point to pure water (where the isoelectric point measurement is difficult because there is no charge). According to the application in which gelatin is used, the effect of pH on gelatin needs to be carefully considered and the pH of the extraction solution needs to be adjusted to get a high quality gelatin. For example, as type-A gelatin has a higher isoelectric point, its use is favorable in those applications that require low pH at which the gelatin would be conducive to forming gel networks. Similarly, as type-B gelatin has a low isoelectric point, it is used in those applications that require a higher pH at which the gelatin is readily available for formation of the gel network.

b. Extraction temperature and duration Different temperatures and times are used in gelatin manufacturing but most extractions are between 45 and 60
C. Temperatures from 50 until 80
C can promote intramolecular bond formation between strands and consequently gelatin with stronger gelling ability can be obtained (Djagny et al., 2001). Higher

138

Gokhan Boran and Joe M. Regenstein

temperatures over 80
C, however, result in fracturing of intramolecular chains giving gelatin having a weaker gelling ability. Lower extraction temperatures, on the other hand, lead to low yields but a superior quality. Similarly, longer extraction times give better yield, while the extracted material suffers from low gel strength and viscosity due to excessive damage to collagen fractions with longer heating and possibly extraction of other proteins. Therefore, it is necessary to balance both extraction temperature and duration of the extraction, to get the best possible outcome. For this purpose, a few modern optimization studies have been done on gelatin extraction from skins of different fish species (Boran and Regenstein, 2009; Kasankala et al., 2007; Zhou and Regenstein, 2004).

c. Storage and transportation There are many other factors affecting gelatin properties. Going into detail for each one of them is beyond the scope of this chapter. To be brief, every processing step, especially if heat is involved, has an effect on gelatin properties including yield, gel strength, melting and gelling temperatures, and viscosity. Raw materials are important with respect to purity and ease of processing. The actual type of the acid and/or alkali used in the pretreatment and/or the extraction is also important. Freshness and storage of raw materials, any possible microbial contamination or the presence of microbial or metabolic enzymes, the water content of gelatin, and transportation conditions are other factors that can affect the quality of gelatin.

D. Quality of fish gelatin compared to mammalian gelatins Arnesen and Gildberg (2007) studied the skins of Atlantic salmon and Atlantic cod for gelatin production and reported that Atlantic salmon skin gelatins had higher gel strength and gelling temperatures than Atlantic cod skin gelatins. The gel strength of the salmon and cod were found to be 108 and 71 g, respectively, while their gelling temperatures were 12 and 10
C, respectively. Arnesen and Gildberg (2007) also reported that the gel strength of the gelatins obtained increased with storage time and higher extraction temperature resulted in lower gel strength. Gudmundsson and Hafsteinsson (1997) also studied cod skin as a raw material for gelatin production, reporting that the proline and hydroxyproline content of cod (a cold-water species) skin gelatin ( 18%) was lower compared to that of tilapia (a warm-water species) skin gelatin ( 25%), resulting in relatively lower gel strength and viscosity. According to their results, tilapia skin gelatin gave 260 g bloom strength, while cod skin gelatin had 180 g bloom under the best extraction conditions reported. Choi and Regenstein (2000) compared various gelatin samples from different sources in terms of their physicochemical and sensory properties and reported that Alaska pollock gelatin had lower gel strength along with lower melting temperature

Fish Gelatin

139

compared to that of pork skin gelatin. Alaska pollock gelatin melted at 24
C while the pork skin gelatin melted at 29
C (Choi and Regenstein, 2000). They also compared the sensory properties of gelatin gels prepared from Alaska pollock and pork skin gelatins and reported that a low melting temperature and gel strength might be useful in creating products with a faster and stronger flavor release. Chiou et al. (2006) studied Alaska pollock and Alaska pink salmon for gelatin production and the quality of the gelatin obtained in comparison with pork skin gelatin. They reported that Alaska pollock and Alaska pink salmon skin gelatins had lower melting and gelling temperatures along with lower gel strength compared to that of pork skin gelatin due to the lower proline and hydroxyproline content of skin gelatins obtained from these fish species. They reported that the pollock and salmon skin gelatins had gelling temperatures of 7 and 5
C, respectively, while pork skin was reported to have a gelling temperature of 24
C, which was attributed to the high content of proline and hydroxyproline of the pork skin gelatin (Chiou et al., 2006). Kasankala et al. (2007) studied grass carp skin as an alternative raw material for gelatin production and reported that the hydroxyproline content of grass carp skin gelatin (11.3%) was slightly higher than that of bovine skin gelatin (11.2%) and a little lower than that of pork skin gelatin (13.2%). They also reported high gel strength, melting and gelling temperatures for grass carp skin gelatin compared to that of gelatins obtained from other fish species. According to their results, the carp skin gelatin had a gelling temperature around 19
C and a melting temperature around 26
C, which was a little lower than that of pork skin (25 and 31
C, respectively) and bovine gelatins (21 and 30
C, respectively) (Kasankala et al., 2007). Boran and Regenstein (2009) also reported similar results for skin gelatin obtained from silver carp, another Asian carp species, that is, it had high gel strength (600 g for optimized gelatin) possibly due to the high hydroxyproline content ( 11%). Therefore, it does appear that the assumption that there is a strong connection between the content of hydroxyproline and proline and the physicochemical properties of gelatins continues to hold with the more recent research with fish gelatins.

III. METHODOLOGICAL CHALLENGES IN QUALITY DETERMINATION OF GELATIN Both commercial gelatin powders and those produced on a small scale for research purposes have an amount of water that varies due to the differences in processing and drying methods (Eastoe and Leach, 1977). Water content of gelatin is important for both ease and duration of storage, as high water content favors microbial spoilage. In addition, higher water containing gelatin formulations can be sold for less. The drying method is

140

Gokhan Boran and Joe M. Regenstein

the major factor affecting the water content of gelatin products. Heat drying and freeze drying are two of the most common methods used to remove water from gelatin preparations. Heat drying is generally done at low temperatures between 40 and 60
C for several hours to several days (Hinterwaldner, 1977). Freeze drying is a much faster method compared to heat drying and able to remove water while causing less damage to the gelatin, but it is generally more expensive. The gelatin powder obtained is generally not tested for its water content, and even when determined, this information is not generally included in the calculations when preparing samples for testing, that is, the gelatin is simply weighed out. This might lead to a lack of agreement between data from different sources. To prevent confusion and to get comparable data, water content of gelatin samples should be determined and included in the calculations to make sure that the actual gelatin amount is the same in each sample being compared for their characteristics. After maturation, making a direct comparison of two gelatin samples with different amounts of gelatin for gel strength would be erroneous as the actual gelatin concentration of the samples is different.

IV. CONCLUSIONS AND SUGGESTED READINGS Previous studies done on gelatin have shown that there are clear connections between gelatin’s functional properties and the extraction conditions. While higher extraction temperatures and durations result in higher yield, the gelatin obtained is of poorer quality due to damage to the collagen fractions. Similarly, higher acid and/or alkali concentrations result in higher yield along with purer material, but the gelatin obtained lacks necessary functional properties. Therefore, an optimization of manufacturing process of gelatin is needed to get a final product with desired properties. This chapter only covers general information on collagen and gelatin and a limited introduction to fish gelatin. For a thorough understanding of collagen and gelatin, additional sources should be consulted. For this purpose, an excellent book of contributed chapters edited by Ward and Courts (1977), ‘‘The Science and Technology of Gelatin,’’ covers almost every area related to gelatin and is still a very useful source of information for current collagen and gelatin producers and users, and for researchers.

REFERENCES Arnesen, J. A. and Gildberg, A. (2007). Extraction and characterization of gelatine from Atlantic salmon (Salmo salar) skin. Bioresour. Technol. 98, 53–57. Badii, F. and Howell, N. K. (2006). Fish gelatin: Structure, gelling properties and interaction with egg albumen proteins. Food Hydrocolloid. 20, 630–640.

Fish Gelatin

141

Balian, G. and Bowes, J. H. (1977). The structure and properties of collagen. In ‘‘The Science and Technology of Gelatin’’, (A. G. Ward and A. Courts, Eds), pp. 1–27. Academic Press, New York. Baziwane, D. and He, Q. (2003). Gelatin: The paramount food additive. Food Rev. Int. 19(4), 423–435. Belitz, H. D., Grosch, W., and Schieberle, P. (2004). Food Chemistry. 3rd revised edn. Springer, New York, pp. 579–586. Boran, G. and Regenstein, J. M. (2009). Optimization of gelatin extraction from silver carp skin. J. Food Sci. 74(8), E432–E441. Brinckmann, J. (2005). Collagens at a glance. In ‘‘Collagen: Primer in Structure, Processing and Assembly’’, ( J. Brinckmann, H. Notbohm, and P. K. Mu¨ller, Eds), pp. 1–6. Springer, New York. Cheow, C. S., Norizah, M. S., Kyaw, Z. Y., and Howell, N. K. (2007). Preparation and characterization of gelatins from the skins of sin croaker ( Johnius dussumieri) and short fin scad (Decapterus macrosoma). Food Chem. 101, 386–391. Chiou, B. S., Bustillos, R. J. A., Shey, J., Yee, E., Bechtel, P. J., Imam, S. H., Glenn, G. M., and Orts, W. J. (2006). Rheological and mechanical properties of cross linked fish gelatins. Polymer 47, 6379–6386. Cho, S. M., Gu, Y. S., and Kim, S. B. (2005). Extracting optimization and physical properties of yellowfin tuna (Thunnus albacares) skin gelatin compared to mammalian gelatins. Food Hydrocolloid. 19, 221–229. Cho, S. H., Jahncke, M. L., Chin, K. B., and Eun, J. B. (2006). The effect of processing conditions on the properties of gelatin from skate (Raja kenojei) skins. Food Hydrocolloid. 20, 810–816. Choi, S. S. and Regenstein, J. M. (2000). Physicochemical and sensory characteristics of fish gelatin. J. Food Sci. 65(2), 194–199. DeMan, J. M. (1999). Proteins: Animal proteins. In ‘‘Principles of Food Chemistry’’, pp. 147–149. Aspen Publishers, Inc., Gaithersburg, MD. Djagny, K. B., Wang, Z., and Xu, S. (2001). Gelatin: A valuable protein for food and pharmaceutical industries: Review. Crit. Rev. Food Sci. 41(6), 481–492. Eastoe, J. E. and Leach, A. A. (1977). Chemical constitution of gelatin. In ‘‘The Science and Technology of Gelatin’’, (A. G. Ward and A. Courts, Eds), pp. 73–105. Academic Press, New York. Engel, J. and Bachinger, H. P. (2005). Structure, stability and folding of the collagen triple helix. In ‘‘Collagen: Primer in Structure, Processing and Assembly’’, ( J. Brinckmann, H. Notbohm, and P. K. Muller, Eds), pp. 8–24. Springer, New York. Eyre, D. R. and Wu, J. J. (2005). Collagens crosslinks. In ‘‘Collagen: Primer in Structure, Processing and Assembly’’, ( J. Brinckmann, H. Notbohm, and P. K. Mu¨ller, Eds), pp. 208–225. Springer, New York. Fernandez-Diaz, M. D., Montero, P., and Go´mez-Guille´n, M. C. (2003). Effect of freezing fish skins on molecular and rheological properties of extracted gelatin. Food Hydrocolloid. 17, 281–286. Gilsenan, P. M. and Ross-Murphy, S. B. (2000). Rheological characterization of gelatins from mammalian and marine sources. Food Hydrocolloid. 14, 191–195. Go´mez-Guille´n, M. C., Turnay, J., Ferna´ndez-Dı´az, M. D., Ulmo, N., Lizarbe, M. A., and Montero, P. (2002). Structural and physical properties of gelatin extracted from different marine species: A comparative study. Food Hydrocolloid. 16, 25–34. Gudmundsson, M. (2002). Rheological properties of fish gelatins. J. Food Sci. 67(6), 2172–2176. Gudmundsson, M. and Hafsteinsson, H. (1997). Gelatin from cod skins as affected by chemical treatments. J. Food Sci. 62(1), 37–39. See also p. 47. Harada, O., Kuwata, M., and Yamamoto, T. (2007). Extraction of gelatin from sardine scales by pressurized hot water. Nippon Shokuhin Kagaku Kogaku Kaishi 54, 261–265.

142

Gokhan Boran and Joe M. Regenstein

Hinterwaldner, R. (1977). Technology of gelatin manufacture. In ‘‘The Science and Technology of Gelatin’’, (A. G. Ward and A. Courts, Eds), pp. 315–361. Academic Press, New York. Holzer, D. (1996). Gelatin Production. United States Patent and Trademark Office (USPTO), Alexandria, VA, USA. United States Patent No: 5,484,888. Hulmes, D. J. S. (2008). Collagen diversity, synthesis, and assembly. In ‘‘Collagen, Structure and Mechanics’’, (P. Fratzl, Ed.), pp. 16–22. Springer, New York. Imeson, A. (1997). Thickening and Gelling Agents for Food. Springer, New York, p. 146. Jamilah, B. and Harvinder, K. G. (2002). Properties of gelatins from skins of fish: Black tilapia (Oreochromis mossambicus) and red tilapia (Oreochromis nilotica). Food Chem. 77, 81–84. Johns, P. and Courts, A. (1977). Relationship between collagen and gelatin. In ‘‘The Science and Technology of Gelatin’’, (A. G. Ward and A. Courts, Eds), pp. 138–168. Academic Press, New York. Jones, N. R. (1977). Uses of gelatin in edible products. In ‘‘The Science and Technology of Gelatin’’, (A. G. Ward and A. Courts, Eds), pp. 366–392. Academic Press, New York. Jongjareonrak, A., Benjakul, S., Visessanguan, W., and Tanaka, M. (2006). Skin gelatin from big eye snapper and brown stripe red snapper: Chemical compositions and effect of microbial transglutaminase on gel properties. Food Hydrocolloid. 20, 1216–1222. Karim, A. A. and Bahat, R. (2009). Fish gelatin: Properties, challenges, and prospects as an alternative to mammalian gelatins. Food Hydrocolloid. 23(3), 563–576. Kasankala, L. M., Xue, Y., Weilong, Y., Hong, S. D., and He, Q. (2007). Optimization of gelatin extraction from grass carp (Catenopharyngodon idella) fish skin by response surface methodology. Bioresour. Technol. 98(17), 3338–3343. Kragh, A. M. (1977). Swelling, adsorption and the photographic uses of gelatin. In ‘‘The Science and Technology of Gelatin’’, (A. G. Ward and A. Courts, Eds), pp. 439–474. Academic Press, New York. Muyonga, J. H., Cole, C. G. B., and Duodu, K. G. (2004). Extraction and physico-chemical characterization of Nile perch (Lates niloticus) skin and bone gelatin. Food Hydrocolloid. 18, 581–592. Nelson, D. L. and Cox, M. M. (2005). Lehninger’s Principles of Biochemistry. 4th edn. WH Freeman and Company, New York, pp. 127–129. Petersen, B. R. and Yates, J. R. (1977). Gelatin Extraction. United States Patent No: 4,064,008. Regenstein, J. M. (2004). Total utilization of fish. Food Tech. 58, 28–30. Sadowska, M., Kolodziejska, I., and Niecikowska, C. (2003). Isolation of collagen from the skins of Baltic cod (Gadus morhua). Food Chem. 81, 257–262. Shahidi, F. (1995). Seafood processing by-product. In ‘‘Seafood: Chemistry, Processing Technology and Quality’’, (F. Shahidi and J. R. Botta, Eds), pp. 320–334. Kluwer Academic Publishers, New York. Stainsby, G. (1977). The gelatin gel and the sol–gel transformation. In ‘‘The Science and Technology of Gelatin’’, (A. G. Ward and A. Courts, Eds), pp. 179–206. Academic Press, New York. Wainewright, F. W. (1977). Physical tests for gelatin and gelatin products. In ‘‘The Science and Technology of Gelatin’’, (A. G. Ward and A. Courts, Eds), pp. 529. Academic Press, New York. Wang, Y. and Regenstein, J. M. (2009). Effect of EDTA, HCl, and citric acid on Ca salt removal from Asian (silver) carp scales prior to gelatin extraction. J. Food Sci. 74(6), C426–C431. Wangtueai, S. and Noomhorm, A. (2009). Processing optimization and characterization of gelatin from lizardfish (Saurida spp.) scales. LWT-Food Sci. Technol. 42, 825–834. Ward, A. G. and Courts, A. (1977). The Science and Technology of Gelatin. Academic Press, New York, pp. 564. P. A. Williams, A. (Ed.) (2007). Handbook of Industrial Water Soluble Polymers WileyBlackwell, New York, pp. 75–76.

Fish Gelatin

143

Wood, P. D. (1977). Technical and pharmaceutical uses of gelatin. In ‘‘The Science and Technology of Gelatin’’, (A. G. Ward and A. Courts, Eds), pp. 413–437. Academic Press, New York. Yang, H., Wang, Y., Jiang, M., Oh, J. H., Herring, J., and Zhou, P. (2007). 2-step optimization of the extraction and subsequent physical properties of channel catfish (Ictalurus punctatus) skin gelatin. J. Food Sci. 72(4), C188–C195. Young, G. E. and Lorimer, J. W. (1960). The acid-soluble collagen of cod skin. Arc. Biochem. Biophys. 88, 373–381. Zhang, S., Wang, Y., Herring, J. L., and Oh, J. H. (2007). Characterization of edible film fabricated with channel catfish (Ictalurus punctatus) gelatin extract using selected pretreatment methods. J. Food Sci. 72(9), C498–C503. Zhou, P. and Regenstein, J. M. (2004). Optimization of extraction conditions for pollock skin gelatin. J. Food Sci. 69(5), 393–398. Zhou, P. and Regenstein, J. M. (2005). Effects of alkaline and acid pretreatments on Alaska pollock skin gelatin extraction. J. Food Sci. 70(6), 392–396. Zhou, P. and Regenstein, J. M. (2006). Determination of total protein content in gelatin solutions with the Lowry or Biuret assay. J. Food Sci. 71(8), 474–479. Zhou, P., Mulvaney, S. J., and Regenstein, J. M. (2006). Properties of Alaska pollock skin gelatin: A comparison with tilapia and pork skin gelatins. J. Food Sci. 71(6), 313–321. Zhou, P. and Regenstein, J. M. (2007). Comparison of water gel desserts from fish skin and pork gelatins using instrumental measurements. J. Food Sci. 72(4), C196–C201.

INDEX A Acute toxoplasmosis, 6 Alaska pink salmon, 139 Alaska pollock gelatin, 138–139 Animal feed C. difficile, 60 E.coli O157:H7, pathogen transmission, 72–73 rat feeding experiments, 38 Asthenia, 7 Atlantic cod, 138 Atlantic salmon, 138 B Bacteriophage high-level shedders, 81–82 preharvest control, 92–93 Bill and Melinda Gates Foundation (BMGF), 23 Biofortification anthropometric measurements and blood tests, 43 Burkina Faso, 44–45 cereal, 42–43 CMF, 44 definition, 23 lysine plus threonine, 42 micronutrient deficiency, 46 PDCAAS, 45–46 QPM, 44 transferrin and hemoglobin levels, 43 wheat bread, 43 Biosorghum project, 23–24 Bradyzoites, 3–5 C Carcass contamination C. difficile, 60–61 E. coli O157:H7 fecal shedding and hide prevalence, 70, 89 probability, 81 Carp skin gelatin, 139 C. difficile infection (CDI), 54

Cerebral biopsy, 10 Cerebral toxoplasmosis, 9–10 Clostridial spores, 60 Clostridium difficile animal feed, 60 detection, food bacteriologic culture, 55 enrichment broths, 55–56 recovery rates, 55, 57 environmental strains/organisms, 57, 60 food contamination, 60–61 genotypes, 61–62 human colonization, 62–63 isolation, 54 meat and meat products, 57–59 Cold-water fishes, 129–130 Collagen a-chains, 122–123 amino acids, 123 b-and g-chain, 123 fibrils, 121–122 gelatin conversion alkali and acid process, 125 drying and grinding process, 126 extraction method, 125–126 pretreatment process, 125 removal of unwanted materials, 124 hydroxyproline, 123 isoelectric point, 137 molecular structure, 122 molecules arrangement, 121–122 stabilization, hydrogen bonds, 123–124 structural protein, 25 triple helix structure, 122 tropocollagen, 122 types, 124 Cow’s milk formula (CMF), 44 D DDG. See Dried distillers grain with/without solubles Direct/indirect fecal–oral exposure, 75 Dried distillers grain with/without solubles (DDGS), 84–85

145

146

Index

E Electrolyzed oxidizing water, 74 Enterobacteriaceae, 93 Escherichia coli O157:H7 animal stress catecholamine norepinephrine, 87 heat stress, 88 immune response, 87 livestock response, 88 microbial food safety risk, 86 practical implications, 87 weaning, 87 antimicrobial carcass interventions, 97 bacterial diarrhea, 68 beta-agonists, 88–89 diet effects, shedding and persistence barley grain, 84 cattle fed barley vs. cattle fed cracked, 84, 86 distillers grains, 84 energy-dense grain rations, 84 gastrointestinal tract and fecal incidence, 86 potential effects, 83–84 ruminal fluid, 83 ruminant animals, 82–83 WDGS and DDGS, 84–85 fecal shedding, 70 feedlot cattle, 89 high-level shedders bacteriophage, 81–82 cattle hide contamination, 81 chain of events, 81–82 colonization, 81 feedlot pens, 80 mathematical modeling, 80–81 super-shedders, 80, 82 ionophores, 89 modes of transmission, 69 outbreak, water, 69 preharvest control bacteriophage, 92–93 brown seaweed product, 95 chlorate, 93–94 cottonseed, 94 esculitin and esculin, 95 manure and cattle pen surface treatments, 95–97 neomycin sulfate, 94 probiotics/direct-fed microbials, 91–92 rumen modifiers, 94 vaccines, 90–91

risk factors, 98 seasonality of shedding cattle feeds, 77 cooler temperatures, 77–78 heat stress, cattle, 78 human foodborne disease, 80 melatonin, 78 percentage of samples, 78–79 physiological responses, animal, 78 prevalence, 77 sorbitol fermentation, 70 sources and transmission, cattle animal feed, 72–73 drinking water, 73–75 feces, manures, and soils, 75–76 flies, 71–72 on-farm ecology, 70 potential reservoirs/vehicles, 70–71 prevalence, 71 transportation and lairage, 98–99 F Fecal–oral transmission, 75 Feedlot calves, 61 Fish gelatin chemical characteristics amino acid composition, 134 peptide size, quality, 134–135 common and potential sources, 129–130 effects of processing conditions alkali and acid treatments, 135–137 extraction temperature and duration, 137–138 gelatin extraction flowchart, 135–136 storage and transportation, 138 physical attributes gel strength, 130–132 melting and gelling temperature, 132–133 viscosity, 131–132 quality vs. mammalian gelatins, 138–139 Freeze drying method, 140 Frequency sweep tests, 133 G Gelatin biopolymers, 120 characteristics, 127 chemical composition, 120 collagen–gelatin conversion alkali and acid process, 125

Index

drying and grinding process, 126 extraction method, 125–126 pretreatment process, 125 removal of unwanted materials, 124 gelatin market, 128 gelation mechanism, 126 industrial applications, 128–129 manufacturing, raw material, 127–128 parent molecule, collagen a-chains, 122–123 amino acids, 123 b-and g-chain, 123 fibrils, 121–122 hydroxyproline, 123 molecular structure, 122 molecules arrangement, 121–122 stabilization, hydrogen bonds, 123–124 triple helix structure, 122 tropocollagen, 122 types, 124 quality determination, 139–140 Gelatin hydrolysate, 134 Gelation mechanism, 126 Gel strength bloom jar, 130–131 bloom strength, 130 different instruments and probes, 131–132 measurement, standard method, 130 Glucocorticoid dexamethasone, 87 Grand Challenges in Global Health, 23 H Headache, 7 Heat drying method, 140 High-level shedders bacteriophage, 81–82 cattle hide contamination, 81 chain of events, 81–82 colonization, 81 feedlot pens, 80 mathematical modeling, 80–81 super-shedders, 80, 82 Houseflies, 71–72 Hydroxyproline, 123–124, 134 I Isoionic point, 137 K Kafirin–tannin complexation, 42

147

L Leishmania gondii, 3 Lymph node enlargement, 7 M Mediterranean Intensive Oxidant Study (MINOS), 27 Multivariate regression analysis approach, 7 Muscle mass amino acids, 26–27 death, human starvation, 27 maintenance, 28 malnutrition, 26 metabolism, genesis, 26 MINOS (see Mediterranean Intensive Oxidant Study) obesity relationship, 27 protein loss, 28 rapid starvation/dietary protein depletion, 26 N Neomycin sulfate, 94 N-(n-butyl) thiophosphoric triamide (NBPT), 96 O Ocular toxoplasmosis, 8, 10 Oocysts sporulation, 5 Osteopenia, 27 P Parasitophorous vacuole, 4 Pharmaceutical gelatin, 129 Preharvest control bacteriophage, 92–93 brown seaweed product, 95 chlorate, 93–94 cottonseed, 94 esculitin and esculin, 95 manure and cattle pen surface treatments, 95–97 neomycin sulfate, 94 probiotics/direct-fed microbials, 91–92 rumen modifiers, 94 vaccines, 90–91 Proline, 123–124, 134, 139 Protein content and composition PDCAAS calculation, sorghum, 33, 36 sorghum vs. other cereals, 32, 34–35 tryptophan, 36

148

Index

Protein digestibility changes, protein body, 39 cross-linking, kafirin prolamin proteins, 36–37 diets, 38, 41 disulfide bonding, 36 grain improvement, 37 gruels, 38 high-vs. normal-lysine sorghum, 37 nutritional parameters, 38 rat feeding experiments, 38 sorghum lines, 39–40 transgenic biofortified sorghum, 41–42 wet cooked sorghum, 36 Protein Digestibility Corrected Amino Acid Score (PDCAAS) biological value prediction, 31 calculation, sorghum, 31–33 protein quality, 32, 34–35 transgenic biofortified sorghum, 42 Protein Efficiency Ratio (PER), 31 Pulsed-field gel electrophoresis (PFGE) C. difficile genotypes determination, 61–62 patterns, 72 Q Quality Protein Maize (QPM), 44 R Rheological method, 132–133 Ribotyping, 61–62 S Sorghum protein biofortification anthropometric measurements and blood tests, 43 Burkina Faso, 44–45 cereal, 42–43 CMF, 44 definition, 23 lysine plus threonine, 42 micronutrient deficiency, 46 PDCAAS, 45–46 QPM, 44 transferrin and hemoglobin levels, 43 wheat bread, 43 human health amino acids classification, 24–25 cereal, 29

1985 FAO/WHO/UNU vs. 2002 WHO/FAO/UNU requirements, 28–29 fat, 26 indispensable amino acids, 24 meta-analysis, 28 muscle mass, 26–28 nitrogen and amino acid needs, 28 nitrogen-containing compounds, 25 nutrition security planning, 28 pregnancy and lactation period, 30 protein types, 25 safe level protein recommendation, 29 scoring patterns, amino acid, 30 quality chemical mutagenesis, 39 in vitro pepsin method, 41 lysine-rich protein synthesis, 40 and measurement, 31–32 protein body change, 39 protein content and composition, 32, 34–36 protein digestibility, 36–39 sorghum lines, 39–40 sorghum vs. other cereals, 34–35, 40–41 stunted and underweight children, Africa, 22–23 Stress and strain sweep tests, 133 T Tachyzoites, 3–4 Tannins, 42 Temperature sweep tests, 133 Thermoreversible gels, 127 Thymol, 96 Tilapia skin gelatin, 138 Time sweep tests, 133 Toxoplasma gondii. See also Toxoplasmosis control in foods, 13–14 life cycle asexual developmental cycle, 3–4 felines, 3 interaction, host cells, 3–4 sexual phase, 5 stagespecific markers, 4 Toxoplasmosis congenital toxoplasmosis, 2 discovery, 3 laboratory diagnosis and treatment cerebral toxoplasmosis, 9–10 differential diagnosis, 10 DNA detection, PCR, 9

Index

IgG and IgM antibodies detection, 9 immunocompetent vs. immunocompromised patients, 9 ophthalmologic examination, 10 serologic tests, 9 misdiagnosis/underdiagnosis, 2 outbreaks, 12–13 pathogenesis and human infection spectra asymptomatic infection, 7 immunodeficiency, 8 infection, pregnancy, 6–7 interferon-gamma (IFN-g) production, 8 ocular toxoplasmosis, 8 risk factors, 7–8 transmission cyst infection, ME-49 strain, 11

149

foodborne transmission, 5 food ingestion, 11 oocysts ingestion, 5 sausage samples, 11 tachyzoite, 5–6 undercook meat, 11 unpasteurized milk, 11 Trimester, 6–7 U Unpasteurized milk, 11, 69 W Water dessert gels, 128 Water troughs, 73–74 Wet distillers grain with solubles (WDGS), 84–85