The Pathogenic Enteric Protozoa:: Giardia, Entamoeba, Cryptosporidium and Cyclospora (World Class Parasites)

THE PATHOGENIC ENTERIC PROTOZOA: Giardia, Entamoeba, Cryptosporidium and Cyclospora

World Class Parasites VOLUME 8

Volumes in the World Class Parasites book series are written for researchers, students and scholars who enjoy reading about excellent research on problems of global significance. Each volume focuses on a parasite, or group of parasites, that has a major impact on human health, or agricultural productivity, and against which we have no satisfactory defense. The volumes are intended to supplement more formal texts that cover taxonomy, life cycles, morphology, vector distribution, symptoms and treatment. They integrate vector, pathogen and host biology and celebrate the diversity of approach that comprises modern parasitological research.

Series Editors Samuel J. Black, University of Massachusetts, Amherst, MA, U.S.A. J. Richard Seed, University of North Carolina, Chapel Hill, NC, U.S.A.

THE PATHOGENIC ENTERIC PROTOZOA: Giardia, Entamoeba, Cryptosporidium and Cyclospora

edited by

Charles R. Sterling and

Rodney D. Adam University of Arizona Tucson, Arizona

KLUWER ACADEMIC PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

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1-4020-7878-1 1-4020-7794-7

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TABLE OF CONTENTS List of contributors

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Preface

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Section 1 – Epidemiology 1. Epidemiology and zoonotic potential of Giardia infections R.C. Andrew Thompson

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2. Entamoeba histolytica and Entamoeba dispar, the non-identical twins C. Graham Clark

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3. Epidemiology and strain variation of Cryptosporidium Rachel M. Chalmers and David P. Casemore

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4. Cyclospora cayetanensis: An emergent and still perplexing coccidian parasite Charles R. Sterling and Ynes R. Ortega

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Section 2 – Host parasite interactions 5. Antigenic variation of the VSP genes of Giardia lamblia Rodney D. Adam and Theodore E. Nash

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6. Pathogenesis and immunity to Entamoeba histolytica Jessica L. Tarleton and William A Petri Jr

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7. Innate and T cell-mediated immune responses in cryptosporidiosis Carol R. Wyatt and Vincent McDonald

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Section 3 – Treatment and Control 8. Rationale approaches to treating Cryptosporidium, Cyclospora, Giardia and Entamoeba Jan R. Mead and Pablo Okhuysen

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9. Inactivation and removal of enteric protozoa in water Frank W. Schaefer, III, Marilyn M. Marshall and Jennifer L. Clancy

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10. Monitoring of Giardia and Cryptosporidium in water in the UK and US Jennifer L. Clancy and Paul R. Hunter

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Section 4 - Genomics 11. Entamoeba histolytica genome James J. McCoy and Barbara J. Mann

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12. Cryptosporidium parvum genomics: Impact on research and control Guan Zhu and Mitchell S. Abrahamsen Index

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CONTRIBUTORS Mitchell S. Abrahamsen Associate Professor Department of Veterinary Pathobiology College of Veterinary Medicine University of Minnesota St. Paul, MN 55108 Rodney D. Adam Professor Dept of Medicine and Microbiology/Immunology University of Arizona College of Medicine Tucson, AZ 85719 David P. Casemore Senior Research Fellow Centre for Research into Environment & Health University of Wales Aberystwyth, SY23 2DB, UK Rachel M Chalmers Head, Cryptosporidium Reference Unit National Public Health Service Microbiology Swansea Singlton Hospital Swansea SA2 8QA, UK Jennifer L. Clancy President Clancy Environmental Consultants, Inc. PO Box 314 St. Albans, VT 05478 C. Graham Clark Senior Lecturer, Department of Infectious and Tropical Diseases London School of Hygiene and Tropical Medicine Keppel Street, London, WC1E 7HT, UK

Paul Hunter Professor of Health Protection School of Medicine, Health Policy and Practice University of East Anglia Norwich NR4 7TJ, UK Barbara J. Mann Associate Professor Departments of Internal Medicine and Microbiology University of Virginia School of Medicine Charlottesville, VA 22908 Marilyn M. Marshall Quality Assurance Officer University of Arizona 1203 N. Mountain Tucson, AZ 85721-0471 James J. McCoy Research Scientist Department of Internal Medicine University of Virginia School of Medicine Charlottesville, VA 22908 Vincent McDonald Centre for Adult and Paediatric Gastroenterology, Barts and the London School of Medicine Queen Mary College University of London Turner St London E1 2AD, UK Jan R. Mead Associate Professor Atlanta Veterans Affairs Medical Center and Department of Pediatrics Emory School of Medicine Atlanta, GA 30033

Theodore E. Nash Head, Gastrointestinal Parasites Section Laboratory of Parasitic Diseases National Institutes of Allergy and Infectious Diseases National Institutes of Health Bethesda, MD 20892 Pablo C. Okhuysen Associate Professor of Medicine Division of Infectious Diseases Program Director, University Clinical Research Center The University of Texas Health Sciences Center Houston Medical School and School of Public Health Houston, TX 77030 Ynes R. Ortega Assistant Professor University of Georgia CFS, Dept. Food Science and Technology 1109 Experiment St. Griffin, GA 30223 William A. Petri, Jr. Professor and Chief Division of Infectious Diseases and International Health University of Virginia School of Medicine Charlottesville, VA 22908-1340 Frank W. Schaefer, III Microbiologist National Exposure Research Laboratory U.S. Environmental Protection Agency 26 West Martin Luther King Drive Cincinnati, Ohio 45268-1320 Charles R. Sterling Professor Department of Veterinary Science and Microbiology University of Arizona 1117 E. Lowell Tucson, AZ 85721

Jessica L. Tarleton Undergraduate Student University of Virginia Charlottesville, VA 22908 RC Andrew Thompson Professor WHO Collaborating Centre for the Molecular Epidemiology of Parasitic Infections School of Veterinary and Biomedical Sciences Murdoch University Murdoch, Western Australia 6150 Carol R. Wyatt Associate Professor Department of Diagnostic Medicine/Pathobiology College of Veterinary Medicine Kansas State University Manhattan, KS 66506-5705 Guan Zhu Assistant Professor Department of Veterinary Pathobiology College of Veterinary Medicine Texas A&M University College Station, TX 77843-4467

PREFACE Giardia duodenalis (=G. lamblia), Entamoeba histolytica, Cryptosporidium parvum and Cyclospora cayetanensis are more than just a mouthful for most who might encounter them. These protozoan parasitic agents contribute significantly to the staggering caseload of diarrheal disease morbidity encountered in developing world nations. Compounding the issue of their mere presence is the fact that standard ova and parasite exams frequently do not detect these infections. Detectable stages may be shed intermittently or require specialized staining procedures. Added to this is the often large number of asymptomatic carriers who serve as reservoirs for infecting others. These parasites are also not strangers to more developed nations, having responsibility for both small and large-scale disease outbreaks. In such settings they may be even more difficult to detect simply because they are frequently overlooked in the grand scheme of disease causing possibilities. They share common features; all are Protozoa, all possess trophic stages that inhabit the gastrointestinal tract, all have the ability to produce disease and in some instances death, and all produce environmentally stable cysts or oocysts, which ensure their transmissibility. In other ways, these organisms are profoundly different. Giardia is a flagellate that inhabits the gut lumen in close association with enterocytes. Entamoeba is an amoeba that preferentially inhabits the mucosal region of the gut lumen, but which may, under certain circumstances, become invasive. Cryptosporidium and Cyclospora are obligate intracellular coccidians, each taking up a unique niche within their respective host enterocytes. Many other differences have been observed in these organisms and have come to light because of recent biological, molecular, and immunological studies. These differences likely contribute to unique mechanisms of disease production and host responsiveness, many of which remain to be fully defined. Giardia owns the distinction of having been described by the amateur Dutch scientist Leeuwenhoek (1632-1723) who described many unicellular microorganisms from a variety of sources including Giardia from his own stool samples. Giardia was long thought a strict commensal, but its frequent association with waterborne and day care center disease outbreaks, high prevalence in developing countries, especially among children, and relation to travel-associated diarrhea have all helped to change that picture. Despite advances in our knowledge of Giardia and giardiasis, this organism remains one of the most poorly understood protozoan parasites. Why does it possess two nuclei and why does it display antigenic variability? What are the immune mechanisms behind clearance and why do some individuals develop chronic, long-lasting infections? Does this organism have true zoonotic

potential, and if so, what are the responsible genotypes and hosts. Are there strain differences that influence pathogenicity? Finally, what is the phylogenetic relationship of this organism to other putatively basal eukaryotes? Infection caused by Entamoeba histolytica severely compromises the lives of some 50 million individuals, largely from developing nations. More than 100,000 individuals will die annually from invasive amoebiasis. It is the third leading cause of death among parasitic infections, being overshadowed only by malaria and schistosomiasis. The ability to distinguish E. histolytica from the morphologically similar, but non-pathogenic E. dispar has assisted greatly in defining the epidemiology of amebic disease since the latter accounts for approximately 90% of all Entamoeba infections. The advent of new models of invasive amebic infection has provided important insights into the pathophysiology of amoebiasis, but has also raised numerous important questions. What is the molecular basis for amebic invasion and the host inflammatory response? Does the host response contribute to the disease process? What specific cytokines, chemokines or other inflammatory mediators participate in the invasion and extraintestinal phases of disease and how are they modulated? Does ameba induced apoptosis play a role in amebic liver abscess progression? Finally, is there such a thing as protective immunity to amoebiasis, and, if so, how is it mediated and can it be induced artificially via vaccination? Cryptosporidium parvum became recognized as a medically important parasite in humans following its discovery in AIDS patients and subsequently in young children of developing nations. Further studies have demonstrated its zoonotic potential as well as its ubiquitous presence in numerous animal species and the environment. It accounts for up to 20% of diarrheal episodes in children of developing countries and is a major contributor of diarrheal episodes in young farm animals worldwide. The largest documented waterborne parasitic disease outbreak in history is attributed to this organism and to date it remains refractory to all conventional therapies. It is also extremely resistant to disinfection. The unique intracellular but extracytoplasmic developmental location of this parasite prompts numerous questions. Why has this parasite chosen this location for its development? Does this location somehow offer shelter from antimicrobial therapy? How does Cryptosporidium obtain nutrients from its host cell or the immediate environment? What immune effector mechanisms are operative against this organism at its intracellular and extracytoplasmic location? In addition, does the existence of human and zoonotic genotypes have implications for organism virulence? Cyclospora cayetanensis is the newcomer on the block. Its identity eluded the scientific community for almost a decade before its coccidian

nature was recognized. It is now seen as a disease-causing agent in AIDS patients, children of developing nations and in immunocompetent individuals who are exposed to it. Several recent food borne disease outbreaks in the United States, arising from imported fruit, have heightened awareness of this organism’s existence. Despite what we have learned, this organism remains an enigma. What are its principal transmission routes? Why does it appear to be markedly seasonal? How does its apparently prolonged sporulation time relate to the previous two questions? How is intestinal inflammation induced in the apparent presence of very few organisms? Finally, are humans the only susceptible host? Past studies have enhanced our understanding of the biology, epidemiology and host-parasite relationship of these complex organisms. This, in turn, has led to the development of new strategies aimed at preventing, controlling and treating infections caused by these protozoan parasites. Despite these efforts, however, the organisms that constitute the framework for this book remain problematic. The numerous questions raised in this preface are addressed in chapters dealing with the respective organisms along with issues of a broader nature that encompass epidemiology, chemotherapy, biochemistry and genomics. These chapters, written by acknowledged experts, are intended to provide an overview of the current state of knowledge with respect to select topics, to stimulate thinking about the complex issues that face both parasite and host in such a relationship, to present fresh and new approaches at detection, treatment and control, and to make everyone aware that we have yet to gain the upper hand against these ubiquitous denizens of our gastrointestinal tract.

Charles R. Sterling Rodney D. Adam

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EPIDEMIOLOGY AND ZOONOTIC POTENTIAL OF GIARDIA INFECTIONS

RC Andrew Thompson WHO Collaborating Centre For The Molecular Epidemiology Of Parasitic Infections and Western Australian Biomedical Research Institute, Division Of Veterinary And Biomedical Sciences, Murdoch University, Murdoch, Western Australia, 6150

ABSTRACT Determining the source of infection is central to an understanding of the epidemiology of giardiasis. In this respect, the role of zoonotic transmission has been a matter of controversy for many years. This has been complicated by the fact that the causative agent of giardiasis, Giardia duodenalis, is a common parasite of people, domestic animals and wildlife. The development and application of molecular epidemiological tools has now made it possible to directly genotype Giardia isolated from animals and environmental samples. These studies have shown that many species of mammals are susceptible to infection with zoonotic and host-adapted genotypes of G. duodenalis and that they are often present in the same endemic foci. Recent studies have also demonstrated that zoonotic transmission does occur in nature. However, available data suggests that zoonotic transmission does not appear to play a major role in waterborne outbreaks of giardiasis. More studies are required on the molecular epidemiology of Giardia infections in order to more accurately determine the frequency of zoonotic transmission in localised endemic foci and in outbreak situations. Key Words: Giardia; taxonomy; epidemiology; zoonoses; molecular epidemiology.

INTRODUCTION Members of the genus Giardia are ubiquitous, affecting the intestinal tracts of numerous vertebrate species (Thompson et al.,1993). They are flagellated protozoans belonging to the Class Zoomastigophorea and Order Diplomonadida. However, the phylogenetic affinities of Giardia have been a matter of controversy for many years. Giardia has a very simple intracellular organization, with no mitochondria or peroxisomes and is thought to represent an early branching eukaryote lineage that diverged before the acquisition of mitochondria (Simpson et al., 2002). Giardia has therefore become a key

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organism in attempts to understand the evolution of eukaryotic cells. In this respect, recent research has revealed that Giardia has a primitive vesicular secretory system that has been proposed as the archetype of the Golgi secretory apparatus in higher organisms (Marti et al., 2003a, b). The protozoa that collectively comprise the genus Giardia have intrigued biologists and clinicians for over 300 years, ever since Antony van Leeuwenhoek first discovered the organism (Meyer, 1994). Despite its long history, our understanding of Giardia’s taxonomy, pathogenicity and relationship with its hosts are still poorly understood. Giardia is not invasive and lives and multiplies by asexual multiplication on the lumenal surface of the small intestine of its vertebrate host. Giardia has a very simple two-stage life cycle. The organism produces environmentally resistant cysts which are voided in the faeces and transmitted directly, or via water or food, to another host with infection resulting from ingestion. Exposure first to an acidic environment in the stomach and then bile salts in the proximal small intestine stimulates release of trophozoites from the cyst which attach to and colonise the mucosal surface. As trophozoites pass through the small intestine they encyst and are passed in the faeces. The pathogenesis of Giardia is not clearly understood and symptoms which include persistent diarrhoea, abdominal pain and rapid weight loss, are highly variable (Thompson et al., 1993) and may not be evident in a significant proportion of infected individuals (Rodriguez-Hernandez et al., 1996). The risk factors for clinical giardiasis, particularly in humans, have yet to be resolved but clearly involve host and environmental factors, as well as the ‘strain’ of the parasite. Although species of Giardia inhabit the intestinal tracts of virtually all classes of vertebrates, G. duodenalis (syn G. intestinalis; G. lamblia) is the only species found in humans and most other mammals including dogs, cats and livestock (Thompson, 1998; Olson et al., 1995; Pavlaseck et al., 1995; Xiao and Herd, 1994; Xiao et al., 1994). G. duodenalis has a global distribution and is the most common intestinal parasite of humans in developed countries. In Asia, Africa and Latin America, about 200 million people have symptomatic giardiasis with some 500,000 new cases reported each year (WHO, 1996). It is also a frequently encountered parasite of domestic animals and livestock. Giardiasis is the most frequently diagnosed waterborne disease and along with cryptosporidiosis, is the major public health concern of water utilities in developing nations (Levine et al., 1990; Thurman et al., 1998). The role of animals in water borne transmission has been difficult to determine. This is because, until recently, it has not been possible to ‘type’ isolates of the parasite obtained during outbreak situations as a means of determining the source of contamination; i.e. whether the ‘strain’ of Giardia is of animal or

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human origin (see Thompson, 1998, 2000; Thompson et al., 1990; Erlandsen, 1994; Thompson and Boreham, 1994). However, whether animals serve as the original source of contamination or amplify the numbers of the originally contaminating isolate, or both, remains to be determined (Bemrick and Erlandsen, 1988; Thompson et al., 1990; Thompson, 1998). Similarly, although diagnosis of Giardia by traditional microscopic methods remains a reliable indicator of infection, the detection of G. duodenalis by microscopy or more sensitive techniques such as faecal ELISA are of limited epidemiological value, especially in terms of the source of infection, since they do not provide information on strain/genotype.

TAXONOMY AND HOST-SPECIFICITY Five species of Giardia are currently recognised (Table 1). This represents a comprehensive taxonomic rationalisation proposed by Filice in 1952 and since accepted by most authorities. The schemes proposed by Filice reflected a lack of morphological distinctness between most of the species described earlier in Giardia and doubts over their assumed host specificity. When Filice proposed the G. duodenalis morphological grouping, he was well aware that it was a temporary ‘holding’ place for a diverse group of phenotypically variable yet morphologically uniform organisms. However, at the time, the methodology was not available to reliably discriminate between these variants or ‘strains’.

The recent application of PCR-based procedures which circumvent the need for laboratory amplification using in vitro culture has enabled the characterization of previously inaccessible genotypes and thus the genetic characteristics of morphologically similar variants/strains (Van Keulen et al.,

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1998; Monis et al., 1998; Hopkins et al., 1997, 1999; Thompson et al. 1999). Using PCR-based procedures, in conjunction with analysis of a variety of genetic loci such as rDNA, elongation factor 1- alpha triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh), and with much larger data sets, it has been possible to elucidate the fundamental genetic divisions within the G. duodenalis morphological group (Table 2; Thompson et al.,1999; Monis and Thompson, 2003; Thompson 2003a).

Giardia isolates recovered from humans and many other mammalian species fall into one of the two major genotypic assemblages, A or B (Table 2). Molecular analyses have shown that the genetic distance separating these two assemblages exceeds that used to delineate other species of protozoa (Andrews et al., 1989; Mayerhofer et al., 1995; Monis et al., 1996). Molecular studies have also demonstrated the existence of genetic subgroups within each of these assemblages. Assemblage A consists of isolates that can be grouped into two distinct clusters; AI consists of a mixture of closely-related animal and human isolates which are geographically widespread and most attention regarding the zoonotic potential of Giardia has focused on this AI subgroup. In contrast, the second subgroup, A II consists entirely of human isolates.

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Assemblage B comprises a genetically more diverse group of predominantly human isolates although some animal genotypes have been included (Monis et al., 1996,1998; Ey et al., 1997). Some of these genetic divisions, or genotypic groupings, appear to be confined to specific animal hosts. Giardia genotypes exhibiting a limited host range include those recovered from cats, dogs, rats, voles/muskrats and livestock (Table 2). Unlike the uncertainty regarding the taxonomic status of genotypic assemblages A and B, there is probably sufficient data supporting the restricted host range of these genotypes to warrant species designation (Monis and Thompson, 2003).

CYCLES OF TRANSMISSION Although the World Health Organization has considered Giardia to have zoonotic potential for over twenty years, either through direct faecal-oral or waterborne routes of transmission, direct evidence has been lacking (Thompson, 1998, 2000). Clearly, the greatest zoonotic risk is from those genotypes of Giardia in genotypic assemblage A, particularly those in the AI subgroup, and to a lesser extent genotypes in Assemblage B. In contrast, the animal-specific genotypes appear to be host adapted, restricted to livestock, dogs, cats and rodents (Table 2). There is no epidemiological evidence to suggest that they occur frequently in the human population and thus their zoonotic risk appears minimal. However, from the point of view of zoonotic potential the finding that similar genotypes are dispersed in different hosts is not by itself conclusive evidence that zoonotic transmission is taking place. We therefore need to understand how the four major cycles of transmission that maintain the parasite in mammalian hosts involving transmission between humans, livestock, dogs/cats or wildlife, may interact (Figure 1), and determine the frequency of transmission of zoonotic genotypes. A better assessment for this will come from studies that examine the dynamics of Giardia transmission between hosts living in the same locality or endemic focus. Human to human transmission of Giardia can occur indirectly through the accidental ingestion of cysts in contaminated water or food, or directly in environments where hygiene levels may be compromised, such as in day care centres or among the inhabitants of disadvantaged communities. A number of studies have been undertaken comparing the frequency of occurrence of Assemblage A and B genotypes in different populations of patients (Thompson, 2003b). Assemblage B appears to be more common than Assemblage A, and interestingly the latter is more commonly associated with symptomatic infections.

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In terms of livestock, cattle are most commonly infected and most studies have concentrated on this species. Giardia is very common in both beef and dairy cattle throughout the world, and longitudinal studies have consistently demonstrated prevalence rates of 100% (O’Handley, 2002; O’Handley et al., 1999; Ralston et al., 2003; Xiao and Herd, 1994). Transmission occurs among infected calves as well as chronically infected adults, but the frequency of transmission is particularly high amongst dairy calves (Xiao and Herd 1994; O'Handley et al., 1999; 2000). Recent studies have demonstrated that calves in dairy and beef herds may harbour one of two genotypes of G. duodenalis. Although the livestock genotype (Assemblage E) of Giardia appears to occur most frequently in cattle, studies in Canada and Australia have shown that a small proportion of cattle in a herd may harbour genotypes in Assemblage A, the most common genotypes affecting humans (O’Handley et al., 2000; Appelbee et al., 2003). However, the livestock genotype may also occur to the exclusion of the zoonotic genotype (Thompson, 2003a). Recent studies in Australia have found that G. duodenalis is the most common enteric parasite of domestic dogs and cats (Bugg et al., 1999; McGlade et al., 2003), although it is rarely associated with clinical disease. It

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is also widely prevalent in dogs and cats in the USA and has been shown to be common in pets in other countries (Thompson and Robertson, 2003). However, it has been suggested that prevalence rates of Giardia in companion animals are often underestimated because of the low sensitivity of conventional detection methods, the fact that the parasite may be present at subclinical levels and the intermittent nature of cyst excretion (McGlade et al., 2003). Molecular epidemiological studies have shown that dogs may be infected with their own, host adapted genotype of Giardia (C/D Table 2), as well as with zoonotic genotypes (A/B Table 2). Under natural, pristine conditions, what evidence there is available suggests that wildlife harbour their own genotypes/species of Giardia and not G. duodenalis. However, recent studies have confirmed that beavers in the wild can harbour infections with zoonotic genotypes of G. duodenalis (Appelbee et al., 2002).

ZOONOTIC TRANSMISSION The water connection The consumption of unfiltered/untreated drinking water represents a significant risk for giardiasis (Hoque et al., 2002; Jakubowski and Craun, 2002). The majority of waterborne giardiasis outbreaks in humans have occurred in unfiltered surface or groundwater systems impacted by surface run off or sewage discharges (Jakubowski and Craun, 2002). Irrigation waters used for food crops that are traditionally consumed raw may also represent a high risk as a source of Giardia (Thurston et al., 2002). Environmental contamination of such water systems and supplies may result from human, agricultural and wildlife sources (Heitman et al., 2002).

Wildlife The occurrence of Giardia in wildlife, particularly of isolates that are morphologically identical to G. duodenalis, has been the single most important factor incriminating Giardia as a zoonotic agent. However, there is little evidence to support the role of wildlife as a source of disease in humans, even though the role of wildlife has dominated debate on the zoonotic transmission of Giardia especially when water is the vehicle for such transmission. It was the association between infected animals such as beavers and waterborne outbreaks in people that led the WHO (1979) to categorise Giardia as a zoonotic parasite. It is therefore surprising that so little information is available on the genotypes of Giardia affecting wildlife, as well as in people infected with Giardia as a result of a waterborne outbreak. Although wildlife, particularly aquatic mammals, are commonly infected with Giardia there is little evidence to implicate such infections as the original contaminating source in water borne outbreaks. It would appear that such animals are more likely to have become infected from water

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contaminated with faecal material of human, or less likely, domestic animal origin. Wildlife thus serve to amplify the numbers of the originally contaminating isolate (Bemrick and Erlandsen, 1988; Monzingo & Hibbler, 1987; Thompson, 1998; Thompson et al., 1990). Some studies (eg. Isaac Renton et al., 1993) have genetically characterised isolates associated with waterborne outbreaks, but the typing schemes used did not allow correlation with the currently recognised assemblages. The one study that did genotype Giardia of beaver origin, confirmed previous suggestions that the source of Giardia infection in beavers was likely to be of human origin (Dixon et al., 2002; Monzingo and Hibbler, 1987; Rickard et al., 1999). In this study, 12 of 113 (10.6%) beaver faecal samples from 6 of 14 different riverbank sites in southern Alberta, Canada, were positive for Giardia, and all those genotyped using the16SrRNA gene belonged to the zoonotic genotype, Assemblage A (Appelbee et al., 2002).

Cattle Although the transmission process is complex and the risk is low, there is clearly a definite potential for microbial contamination of ground and surface waters from livestock operations (Donham, 2000). Cattle are susceptible to infection with zoonotic genotypes of Giardia and it has been shown that calves infected with Giardia commonly shed from to cysts per gram of faeces (Xiao, 1994; O’Handley et al., 1999). Thus, even a few calves infected with genotypes in Assemblage A could pose a significant public health risk directly to handlers or indirectly as an important reservoir for human waterborne outbreaks of giardiasis. This is of potential public health significance and may put producers, and other members of the community, at risk. However, longitudinal studies in Australia suggest that zoonotic genotypes may only be present transiently in cattle under conditions where the frequency of transmission with the livestock genotype is high and competition is thus likely to occur. The public health risk from cattle appears to be minimal, at least based on studies in North America and Australia where genotyping has been undertaken and has shown that the livestock genotype appears to predominate in cattle (O’Handley et al., 2000; Hoar et al., 2001). However, under certain circumstances, where Giardia infections may not previously have occurred or been common, an introduced genotype may establish and be perpetuated in the absence of competing genotypes. For example, a recent molecular epidemiological study showed that humans appear to have introduced Giardia into a remote national park in Uganda and are also thought to have been the source of zoonotic genotypes of Giardia in a small number of cohabiting dairy cattle (Graczyk, et al., 2002).

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Pets Although the clinical significance of Giardia in dogs and cats appears to be minimal, the public health significance of such infections in pets has been the subject of much debate and is still a question of uncertainty for veterinarians. In domestic, urban environments of Australia, for example, zoonotic genotypes from Assemblage A and the ‘dog’ genotype, Assemblage D, are both equally common in dogs (Thompson et al., 1999). It is therefore considered that two cycles of transmission probably operate in domestic urban environments with the possibility of zoonotic transmission of Assemblage A genotypes between pets and their owners. This was highlighted in the study by Bugg et al. (1999) which found that dogs from multi-dog households were more commonly infected with Giardia than dogs in single-dog households, emphasising the potential ease with which Giardia can be spread to in-contact animals and therefore presumably humans (Bugg et al., 1999). In contrast, a recent survey of domestic dogs in Japan found all isolates to belong to the dog-specific genotype, Assemblage D (Abe et al., 2003). Molecular epidemiological studies in localised endemic foci, where the frequency of transmission of zoonotic and non-zoonotic genotypes is high, will provide more useful information on the frequency of zoonotic transmission. For example, studies in Aboriginal communities in Australia have shown that the dog genotype predominates in infected dogs (Hopkins et al., 1997). In contrast, in remote tea growing communities in Assam northeast India, where Giardia occurs in both humans and their dogs, 20% of dogs were found to be infected with Giardia, but they were all infected with zoonotic genotypes, mostly from Assemblage A (Traub et al., 2003). This difference may reflect a closer association between individual dogs and their owners in the tea growing communities, and the frequency with which dogs are able to eat human faeces in these communities (Traub et al., 2002). In Aboriginal communities in Australia, such behaviour by dogs is less common and the dogs tend to stay together in packs for much of the time. In environments where the infection pressure is less, such as domestic households in urban settings, dogs are just as likely to harbour zoonotic genotypes of Giardia from Assemblage A as they are their own dog genotype (Assemblage D). The study in Assam, India by Traub et al., (2003), has provided the first direct evidence of zoonotic transmission between dogs and humans, by finding the same genotype of Giardia in people and dogs, not only in the same village, but also in the same household. Giardia isolates were characterised at three different loci; the SSU-rDNA, elongation factor 1- alpha and triose phosphate isomerase (tpi) gene. Evidence for zoonotic transmission was supported by strong epidemiological data showing a highly significant association between the prevalence of Giardia in humans and the presence of

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a Giardia positive dog in the same household. A major finding of this study was the importance of using multiple loci when inferring genotypes to Giardia in epidemiological investigations (Traub et al., 2003).

IN CONCLUSION Domestic animals and wildlife appear to harbour their own host adapted genotypes/species of Giardia although they are susceptible to infection with zoonotic genotypes, principally from Assemblage A. However, what data that is available suggests that the occurrence of such zoonotic genotypes is not common, and they are likely to be quickly diluted and excluded by competitive interactions with host adapted genotypes. The public health risk of zoonotic genotypes in animals would appear to be through direct transmission. There is no convincing evidence that zoonotic transmission impacts significantly on the aetiology of waterborne outbreaks of giardiasis. Giardia of human origin appears to be the main source of water contamination and as such may impact negatively on ecosystem health leading to infections in aquatic wildlife. Recent studies have demonstrated that filter-feeding molluscs are useful indicators of the presence of waterborne pathogens. Genotypic characterisation was recently utilised in a study that isolated Giardia cysts from clams in an estuary in North America (Graczyk, et al., 1999). All isolates were identified as belonging to genotype Assemblage A, highlighting contamination with faeces of mammalian origin, most probably human, that contained G. duodenalis cysts of public health importance. Such filter-feeding molluscan shellfish can concentrate waterborne pathogens and thus in combination with appropriate genotyping procedures can serve as biological indicators of contamination with Giardia cysts and can thus be used for sanitary assessment of water quality. Further studies are needed on the molecular epidemiology of Giardia infections in order to determine the frequency of zoonotic transmission in localised endemic foci and in outbreak situations, and to better understand the interaction between the major cycles of Giardia transmission.

REFERENCES Abe, N., Kimata I., and Iseki M. 2003. Identification of genotypes of Giardia intestinalis isolates from dogs in Japan by direct sequencing of the PCR amplified glutamate dehydrogenase gene. Journal of Veterinary Medicine and Science 61: 29-33. Appelbee, A., Thorlakson, C., and Olson, M.E. 2002. Genotypic characterization of Giardia cysts isolated from wild beaver in southern Alberta, Canada. In: Olson, B.E., Olson, M.E., Wallis, P.M. (Eds.), Giardia: The cosmopolitan parasite. CAB International, Wallingford, UK, pp 299-300. Appelbee, A.J., Frederick, L.M., Heitman, T.L. and Olson M.E. 2003. Prevalence and genotyping of Giardia duodenalis from beef calves in Alberta, Canada. Veterinary Parasitology 112: 289-294.

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Andrews, R.H., Adams, and M. Boreham. P.F.L. et al. 1989. Giardia intestinalis: electrophoretic evidence for a species complex. International Journal for Parasitology 19:183-190. Bemrick, W.J. and Erlandsen, S.L. 1988. Giardiasis - is it really a zoonosis? Parasitology Today 4:69-71. Bugg, R.J., Robertson, I.D., Elliot, A.D. and Thompson, R.C.A. 1999. Gastrointestinal parasites of urban dogs in Perth, Western Australia. Veterinary Journal 57:295-301. Dixon, B.R., Bussey, J., Parrington, L., Parenteau., Moore, R., Jacob, J., Parenteau, M.-P. and Fournier, J. 2002. A preliminary estimate of the prevalence of Giardia sp. in Beavers in Gatineau Park, Quebec, using flow cytometry. In: Olson, B.E., Olson, M.E., Wallis, P.M. (Eds.), Giardia: The cosmopolitan parasite. CAB International, Wallingford, UK, pp 71-79. Donham KJ. 2000. The concentration of swine production. Effects on swine health, productivity, human health, and the environment. Veterinary Clinics of North. America. Food Animal. Practice. 16: 559-597. Erlandsen, S.L. 1994. Biotic transmission - is giardiasis a zoonosis? in Giardia: from Molecules to Disease, (eds R.C.A. Thompson, J.A. Reynoldson and A.J. Lymbery), CAB International, Wallingford, pp. 83-97. Ey, P.L., Mansouri, M., Kulda, J. et al. 1997. Analysis of Giardia from hoofed animals reveals Articodactyl-specific and potentially zoonotic genotypes. Journal of Eukaryotic Microbiology 44:626-635. Filice, F.P. 1952. Studies on the cytology and life history of a Giardia from the laboratory rat. University of California Publications in Zoology 57:53-146. Graczyk, T.K., Thompson, R.C.A., Fayer, R., Adams, P., Morgan., U.M and Lewis, E.J. 1999. Giardia duodenalis cysts of genotype A recovered from clams in the Chesapeake Bay subestuary, Rhode river. American Journal of Tropical Medicine and Hygiene 61:526-529. Graczyk, T.K., Bosco-Nizeyi, J., Ssebide, B., Thompson, R.C.A., Read, C. and Cranfield, M R. 2002. Anthropozoonotic Giardia duodenalis genotype (assemblage) A infections in habitats of free-ranging human-habituated gorillas, Uganda. Journal of Parasitology 88: 905-909. Heitman, T.L., Frederick, L.M., Viste, J.R., Guselle, N.J., Cooke, S.E., Roy, L., Morgan, U.M., Thompson, R.C.A. and Olson, M.E. 2002. Prevalence of Giardia and Cryptosporidium and characterisation of Cryptosporidium spp. isolated from wildlife, human and agricultural sources of the North Saskatchewan River basin in Alberta, Canada. Canadian Journal of Microbiology 48: 530-541. Hoar, B.R., Atwill, E.R., Elmi, C. and Farver, T.B. 2001. An examination of risk factors associated with beef cattle shedding pathogens of potential zoonotic concern. Epidemiology and Infection 127: 147-155. Hopkins, R.M., Meloni, B.P., Groth, D.M., Wetherall, J.D., Reynoldson, J.A. and Thompson, R.C.A. 1997. Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality. Journal of Parasitology 83:44-51. Hopkins, R.M., Constantine, C.C., Groth, D.M., Reynoldson, J.A. and Thompson, R.C.A. 1999. DNA fingerprinting of Giardia duodenalis isolates using the intergenic rDNA spacer. Parasitology 118:531-539. Hoque, M.E., Hope, V.T., Kjellstrom, T., Scragg, R., Lay-Yee, R., 2002. Risk of giardiasis in Aucklanders: a case-control study. International Journal of Infectious Diseases. 6: 191 Isaac-Renton, J.L., Cordeiro, C., Sarafis, K. et al. 1993. Characterization of Giardia duodenalis isolates from a waterborne outbreak. Journal of Infectious Diseases 167:431-40. Jakubowski, W. and Graun, G.F. 2002. Update on the control of Giardia in water supplies. In: Olson, B.E., Olson, M.E., Wallis, P.M. (Eds.), Giardia: The cosmopolitan parasite. CAB International, Wallingford, UK, pp 217-238.

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Levine, W.C., Stephenson, W.T. and Craun, G.F. 1990. Waterborne disease outbreaks, 19861988. Morbidity and Mortality Weekly Report 39:1-13. Marti, M., Li, Y., Schraner, E.M., Wild, P., Kohler, P. and Hehl, A.B. 2003a. The secretory apparatus of an ancient eukaryote: protein sorting to separate export pathways occurs before formation of transient golgi-like compartments. Molecular Biology of the Cell 14: 14331447. Marti, M., Regos, A., Li, Y., Schraner, E.M., Wild, P., Muller, N., Knopf, L.G. and Hehl, A.B. 2003b. An ancestral secretory apparatus in the protozoan parasite Giardia intestinalis. Journal of Biological Chemistry 278: 24837-24848. Mayrhofer, G., Andrews, R.H., Ey, P.L. et al. 1995. Division of Giardia isolates from humans into two genetically distinct assemblages by electrophoretic analysis of enzymes encoded at 27 loci and comparison with Giardia muris. Parasitology 111:11-17. McGlade, T.R., Robertson, I.D., Elliott, A.D. and Thompson, R.C.A. 2003. High prevalence of Giardia detected in cats by PCR. Veterinary Parasitology. 110: 197-205. Meyer, E.A. 1994. Giardia as an organism. In Giardia: from Molecules to Disease, (eds R.C.A. Thompson, J.A. Reynoldson and A.J. Lymbery), CAB International, Wallingford, pp. 3-15. Monis, P.T. and Thompson, R.C.A. 2003. Cryptosporidium and Giardia - zoonoses: fact or fiction? Infection, Genetics and evolution (in press). Monis, P.T., Mayrhofer, G., Andrews, R.H. et al. 1996. Molecular genetic analysis of Giardia intestinalis isolates at the glutamate dehydrogenase gene. Parasitology 112:1-12. Monis, P.T., Andrews, R.H., Mayhofer, G. et al. 1998. Novel lineages of Giardia intestinalis identified by genetic analysis of organisms isolated from dogs in Australia. Parasitology 116:7-19. Monzingo, D.L. Jr. and Hibler, C.P., 1987. Prevalence of Giardia sp. in a beaver colony and the resulting environmental contamination. Journal of Wildlife Diseases. 23: 576-585. O’Handley, R.M., 2002. Giardia in farm animals. In: Olson, B.E., Olson, M.E., Wallis, P.M. (Eds.), Giardia: The cosmopolitan parasite. CAB International, Wallingford, UK, pp 97-105. O'Handley R, Cockwill C, McAllister TA, et al. 1999. Duration of naturally acquired giardiasis and cryptosporidiosis in dairy calves and their association with diarrhoea. Journal of the Americam Veerinary Medical Association 214:391-396. O'Handley RM, Olson ME, Fraser D, et al. 2000. Prevalence and genotypic characterisation of Giardia in dairy calves from Western Australia and Western Canada. Veterinary Parasitology 90:193-200. Olson, M.E., McAllister, T.A., Deselliers, L. et al. 1995. Effects of giardiasis on production in a domestic ruminant (lamb) model. American Journal of Veterinary Research 56:1470-1474. Pavlasak, I., Hess, L., Stehlik, I. et al. 1995. The first detection of Giardia spp. in horses in the Czech Republic. Veterinariya Meditsina (Praha), 40:81-86. Ralston, B.J. McAllister T.A. and Olson M.E. 2003. Prevalence and infection pattern of naturally acquired giardiasis and cryptosporidiosis in range beef calves and their dams. Veterinary Parasitology 114: 113-122. Rickard, L.G., Siefker, C., Boyle, C.R., Gentz, E.J., 1999. The prevalence of Cryptosporidium and Giardia spp. in fecal samples from free-ranging white-tailed deer (Odocoileus virginianus) in the southeastern United States. Journal of Veterinary Diagnostic Investigation 11:65-72. Rodriguez-Hernandez J., Canut-Blasco A. and Martin-Sanchez A.M. 1996. Seasonal prevalence’s of Cryptosporidium and Giardia infections in children attending day care centres in Salamanca (Spain) studied for a period of 15 months. European Journal of Epidemiology 12:291-295. Simpson, A.G., Roger, A.J., Silberman, J.D., Leipe, D.D., Edgcomb, V.P., Jermiin, L.S., Patterson, D.J. and Sogin, M.L. 2002. Evolutionary history of "early-diverging" eukaryotes:

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the excavate taxon Carpediemonas is a close relative of Giardia. Molecular Biology and Evolution 19: 1782-91. Thurman, R., Faulkner, B., Veal, D. et al. 1998. Water quality in rural Australia. Journal of Applied Microbiology 84:627-632. Thompson, R.C.A. 1998. Giardia infections. In: Zoonoses: Biology, Clinical Practice and Public Health Control. (eds S.R. Palmer, E.J.L. Soulsby, and D.I.H. Simpson), Oxford University Press, Oxford, pp. 545-561. Thompson, R.C.A. 2000. Giardiasis as a re-emerging infectious disease and its zoonotic potential. International Journal for Parasitology 30:1259-1267. Thompson, R.C.A., 2002. Towards a better understanding of host specificity and the transmission of Giardia: The impact of molecular epidemiology. In: Giardia: Olson, B.E., Olson, M.E., Wallis, P.M. (Eds.), The cosmopolitan parasite. CAB International, Wallingford, UK, pp 55-69. Thompson, R.C.A. 2003a. Molecular epidemiology of Giardia and Cryptosporidium infections. Journal of Parasitology, 89: S134-S140. Thompson, R.C.A. 2003b The Zoonotic Significance and Molecular Epidemiology of Giardia and Giardiasis Veterinary Parasitology (in press). Thompson, R.C.A, and Boreham, P.F.L. 1994. Biotic and abiotic transmission, In: Giardia: from molecules to disease, (eds R.C.A. Thompson, J.A. Reynoldson and A.J. Lymbery), CAB International, Wallingford, pp. 131-136. Thompson, R.C.A., Lymbery, A.J. and Meloni, B.P. 1990. Genetic variation in Giardia Kunstler, 1882: taxonomic and epidemiological significance. Protozoological Abstracts 14:1-28. Thompson, R.C.A., Reynoldson, J.A. and Mendis, A.H.W. 1993. Giardia and giardiasis. Advances in Parasitology 32:71-160. Thompson, R.C.A., Hopkins, R.M. and Homan, W.L. 1999. Nomenclature and genetic groupings of Giardia infecting mammals. Parasitology Today 16: 210-213. Thurston-Enriquez, J.A., Watt, P., Dowd, S.E., Enriquez, R., Pepper, I.L. and Gerba, C.P. 2002. Detection of protozoan parasites and microsporidia in irrigation waters used for crop production. Journal of Food Protection 65: 378-382. Traub, R.J., Robertson, I.D., Irwin, P., Mencke, N. and Thompson, R.C.A., 2002. The role of dogs in transmission of gastrointestinal parasites in a remote tea-growing community in northeast India. American Journal of Tropical Medicine and Hygiene 67: 539-45. Traub, R.J., Monis, P., Robertson, I., Irwin, P., Mencke, N. and Thompson, R.C.A. 2003. Epidemiological and molecular evidence supports the zoonotic transmission of Giardia among humans and dogs living in the same community. Parasitology (in press). Van Keulen, H., Feely, D.E., and Macechko, P.T. et al. 1998. The sequence of Giardia small subunit rRNA shows that voles and muskrats are parasitized by a unique species Giardia microti. Journal or Parasitology 84:294-300. WHO, 1979. Parasitic Zoonoses. Report of a WHO Expert Committee with the participation of FAO. Technical Report Series No. 637. World Health Organization, Geneva. WHO, 1996. The World Health Report 1996. Fighting Disease Fostering Development. World Health Organization, Geneva. Xiao L. 1994. Giardia infection in farm animals. Parasitology Today 10:436-438. Xiao, L. and Herd, R.P. 1994. Infection pattern of Cryptosporidium and Giardia in calves. Veterinary Parasitology 55:257-262. Xiao, L., Herd, R.P. and McClure, K.E. 1994. Periparturient rise in the excretion of Giardia sp. cysts and Cryptosporidium parvum oocysts as a source of infection for lambs. Journal of Parasitology 80:55-59.

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ENTAMOEBA HISTOLYTICA AND ENTAMOEBA DISPAR, THE NON-IDENTICAL TWINS

C. Graham Clark Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine

ABSTRACT Over the past 25 years a fundamental change has taken place in our understanding of amebiasis, through the recognition of Entamoeba dispar as a species that is distinct from Entamoeba histolytica but is morphologically indistinguishable. This change in taxonomy has significant implications for the diagnosis and treatment of infections, as well as for our ability to interpret the earlier literature. The defining characteristic of the two species remains the ability of E. histolytica to cause invasive disease while E. dispar cannot, but the underlying genetic differences between the two that are responsible for this remain to be defined. The ongoing comparative genome sequencing will hopefully shed light on the dichotomy. Key words: Entamoeba histolytica, Entamoeba dispar, amoebiasis, isoenzymes, monoclonal antibodies, DNA sequencing.

INTRODUCTION In 1875, Fedor Lösch described the first known case of disease caused by an ameba (Lösch,1875, 1978). The remarkable diagrams he produced leave no doubt that he was looking at what is now known as Entamoeba histolytica. The typical nucleus and ingested red blood cells are easily recognisable. Lösch went on to reproduce the disease by infecting dogs and, although he was cautious in his interpretation of the results, it seems clear that he believed that the amebae were responsible for causing the disease. Lösch referred to the organisms as Amoeba coli, a descriptive rather than a taxonomic term. 2003 was the 100th anniversary of the naming of Entamoeba histolytica by Fritz Schaudinn (Schaudinn, 1903, 1978) and, while some aspects of Schaudinn's description are very strange, the name of the organism he described has been retained. The naming of E. histolytica by Schaudinn was far from the end of the taxonomic story, however. Over the next fifteen years or so a large number of additional species of enteric ameba were described, but their relationships to

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E. histolytica were often unclear. In 1919 Clifford Dobell (Dobell, 1919) reviewed the existing literature and concluded that there were only two species of Entamoeba infecting the human colon - E. histolytica producing cysts with 4 nuclei and E. coli producing cysts with 8 nuclei. In Dobell's view at that time, E. histolytica was an obligate tissue parasite. He was later forced to change this view when in 1925 E. histolytica was first grown in culture (Boeck and Drbohlav, 1925). The gradual acceptance over the past 25 years that the organism known as Entamoeba histolytica was in fact made up of two distinct species represents one of the most dramatic changes in human parasitology to take place during that time period. The story of this development starts in 1925 when the eminent French parasitologist Emile Brumpt published a preliminary report (Brumpt, 1925) describing a new species that was morphologically indistinguishable from E. histolytica but was incapable of causing disease. He gave it the name Entamoeba dispar. His evidence consisted of observations on infected patients and on experimental infections of kittens. The latter were used as a very sensitive model for intestinal amebiasis at the time but showed no tissue invasion when infected with the new organism. Why was Brumpt's work not accepted by his contemporaries (Brumpt, 1928)? There appear to have been two primary reasons. The first is that morphology was the accepted basis of all species descriptions at the time and in this case there were no differences. The second is that it had already been established by the seminal work of Walker and Sellards (Walker and Sellards, 1913) that not everyone infected with E. histolytica derived from a 'convalescent carrier' would go on to develop disease, so Brumpt's patients were not distinguishable from these asymptomatic experimental infections. Despite further experimentation by Brumpt (1926) and his student Tschedomir Simic (1931a, 1931b, 1935), their work was essentially ignored for the next 50 years. The next evidence of two groups within E. histolytica did not emerge until 1972 when it was shown that amebae isolated from individuals with disease had different lectin agglutination properties to those isolated from asymptomatically infected individuals (Martínez-Palomo et al. 1973). The basis of this difference has recently been elucidated experimentally. The surface of E. histolytica is covered with a dense layer of lipophosphoproteoglycan, which is absent from E. dispar (EspinosaCantellano et al. 1998; Bhattacharya et al. 2000). In the initial publication the surface properties were linked to differences in virulence among the strains in experimental models but not, at this stage, to species differences. In 1978, Sargeaunt and Williams published the first of a long series of articles on their isoenzyme studies in Entamoeba (Sargeaunt et al. 1978).

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Their examination of a large number of isolates in culture again differentiated two groups of organisms based on the migration of enzymes in gels. One of the groups was primarily isolated from symptomatic patients while the other was never isolated from individuals with invasive disease (Sargeaunt, 1987). The two types were named 'pathogenic' and 'nonpathogenic' E. histolytica. Within 5 years Sargeaunt and Williams were already raising the possibility in print that Brumpt had been right all along and that the 'nonpathogenic' group they were detecting should be identified as E. dispar (Sargeaunt et al. 1982). The basis of this differentiation by isoenzymes has now been shown to be due to sequence differences in the genes, at least for the most widely used enzyme, hexokinase (Ortner et al. 1997). The main reason why Sargeaunt and Williams' proposal to resurrect the name E. dispar was not accepted more quickly is that, starting in 1986, a number of reports were published that seemed to indicate that interconversion between the two forms could take place (Mirelman et al. 1986a, 1986b; Andrews et al. 1990; Mukherjee et al. 1993; Vargas and Orozco, 1993). This phenomenon was observed during attempts to grow the 'nonpathogenic' form under axenic culture conditions. The results implied that the 'nonpathogenic' form was somehow activated, leading to a change in gene expression or protein modification to give the 'pathogenic' isoenzyme phenotype and a virulent organism capable of causing disease. Not surprisingly this caused a lot of controversy in the field of amebiasis research and generated a lot of investigation into the observations. During this same time period the first monoclonal antibodies and the first gene sequences were obtained from these organisms. Monoclonal antibodies often identified two groups of isolates that correlated with their isoenzyme patterns (Strachan et al. 1988; Petri et al. 1990). Likewise, DNA from the two forms was also shown to be distinct (Garfinkel et al. 1989; Tannich et al. 1989). An extensive series of experiments attempting to replicate the conversion phenomenon were unsuccessful (Clark et al. 1992). As the experimental data accumulated, it became more and more difficult to accommodate the interconversion observations within known biological processes - PCR could not detect the presence of both gene sequence types within the same organism for example. Finally, the first DNA-based typing system for Entamoeba isolates showed that the genotypes of 'converted' organisms matched those of laboratory reference strains implying that some form of cross-contamination was the most likely explanation for the observed changes (Clark and Diamond, 1993a, 1993b). It has subsequently become clear that E. dispar cannot be made to grow axenically under the standard conditions used for E. histolytica (Clark, 1995; Kobayashi et al. 1998). Even the smallest number of E. histolytica cells will outgrow E. dispar under these

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conditions, leading to an apparent conversion of isoenzyme patterns (Clark and Diamond, 1993b). The evidence supporting the existence of two groups coupled with a likely explanation for the conversion phenomenon led to the redescription of E. histolytica in 1993 to separate it from E. dispar (Diamond and Clark, 1993). Although initially controversial, this change of nomenclature was quickly accepted and in 1997 was given WHO approval (Anonymous, 1997).

IMPLICATIONS OF THE RECOGNITION OF E. DISPAR AS A DISTINCT SPECIES The splitting of E. histolytica and the acceptance of E. dispar as a different species is far from being simply an intellectual exercise in taxonomy and classification. It has real and significant implications for diagnosis and treatment of infections as well as interpretation of published data. Indeed the change has made the work of the diagnostic lab and the clinician much more difficult. Since Lösch's day, the primary method for identification of Entamoeba infections has been light microscopy. Under the microscope, the cysts and trophozoites of E. histolytica and E. dispar appear identical irrespective of the methods of preparation and staining used. The only exception to this is in cases of amebic colitis where trophozoites filled with red blood cells may be seen and these are indicative of an E. histolytica infection (González-Ruiz et al. 1993). In most samples, however, only cysts will be observed and the species involved will remain unidentifiable. When reading the literature from before 1980, and in many subsequent publications, we cannot in most cases identify the species present when microscopy was the only method used for diagnosis. Therefore interpretation of the data retrospectively is not possible. This will certainly be the case in population surveys where prevalence figures are given. We are now starting to obtain new prevalence data in which the species are separately identified. However, the data remain patchy and relatively few countries have been re-surveyed using species-specific technologies. To date most studies have concentrated on defined and geographically restricted populations. Broader studies have relied on less random sampling, studying hospital patients, for example, who may not be representative of the whole population. In general, E. dispar is found to be the more common species, in a ratio of up to 10:1 in some areas. However this is not always the case. Recently E. histolytica was shown to be the more prevalent in a specific region of Vietnam (Blessmann et al. 2002). Retrospective interpretation of the literature may be possible in the case of serological surveys. It appears from most studies that E. dispar infection does not lead to seropositivity, at least when assayed using E. histolytica

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antigen. Those seropositive individuals infected with E. dispar are likely to have been infected previously with E. histolytica (Gathiram and Jackson, 1987). Interpretation of the infection rate relies on a clear picture of the persistence of seropositivity and of infections in the absence of treatment. At present the data appear to indicate that seropositivity persists for a year or more after an infection is eradicated (Haque et al. 1999; Valenzuela et al. 2001). The persistence of infection in the absence of treatment is less clear as data from different studies do not agree. Children in Bangladesh appear to clear E. histolytica infections quickly but often become reinfected (Haque et al. 2002), while adult infections in Vietnam have a half-life of more than one year (Blessmann et al. 2003). If the latter proves to be the case in Mexico, the serological survey conducted there (which found 5.9% seropositivity) would indicate a much lower rate of new infection than previously suspected. Microscopy continues to be the diagnostic method used in most laboratories around the world. Despite the commercial availability of specific diagnostic tests that allow the differentiation of E. histolytica and E. dispar using ELISA or PCR, the cost of reagents and equipment remains beyond the reach of laboratories in most countries where the infection is prevalent. Recognising this, the WHO recommended the reporting of microscopy-based diagnosis of such infections as "E. histolytica /E. dispar" and suggested that in the absence of proof or a strong suspicion that the organism being seen is E. histolytica the infected individuals should not be treated (Anonymous, 1997). This latter recommendation is based on the relative prevalence data (E. dispar making up an estimated 90% of the cysts reported as E. histolytica /E. dispar) and on the observation that the vast majority of those infected with E. histolytica (in its redefined sense) never go on to develop invasive disease (Gathiram and Jackson, 1987; Haque et al. 1997, 2002; Blessmann et al. 2003). Treatment of asymptomatic individuals is therefore likely to be unnecessary, and when the potential side effects of drug treatment and the expense involved are considered it is difficult to justify this course of action. Ultimately, however, the decision on whether to treat a patient must be left up to the individual physician. The prospects of a short-term solution to the diagnostic problem are slim for developing countries, as it would require a low technology, inexpensive method. At present the two commercially available diagnostic products do not match this description. The first to be marketed was based on the existence of antigenic differences in the Galactose/N-Acetyl galactosamine-specific lectin found on the surface of both species of amebae (Haque et al. 1995, 2000) (Techlab, Inc., Blacksburg, VA, USA). This protein is involved in both ameba attachment to the mucus layer of the colon and in binding of bacteria for ingestion. During invasion, its properties lead the cell to bind host epithelial and other cells, which it then lyses. The diagnostic

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method is based on capturing the lectin from stool samples and detecting its presence using species-specific monoclonal antibodies in an ELISA. Presumably the lectin is shed from the surface of the ameba or is derived from lysed cells. It has not been reported on the surface of cysts to my knowledge. The method is relatively simple to perform and does not need complex and expensive equipment, but the test itself is probably too expensive for widespread use in many countries. The second diagnostic method has not been used widely to date (Blessmann et al. 2002) and is even less accessible to developing countries, relying as it does on Real-Time PCR (Artus Biotech, Hamburg, Germany). Here both the reagents and the equipment are expensive. However, the sensitivity and specificity of the method appear to be even greater than for the ELISA and the test may therefore find a niche in some N. American and European diagnostic laboratories. Despite the 'inconvenience' caused to diagnostic laboratories by the redescription of E. histolytica as a result of the acceptance of E. dispar's existence, the recognition that there are two species involved should lead ultimately to a reduction in the unnecessary use of medication. The increased awareness of E. dispar as will also, hopefully, lead to a reduction in the incorrect attribution of many intestinal problems to 'amebiasis' just because an ameba resembling E. histolytica is present in the stool of an individual with gastrointestinal complaints.

HOW DO E. DISPAR AND E. HISTOLYTICA DIFFER One of the problems with diagnosis of amebae is there are few morphological characteristics to use. The lack of distinguishing morphology is probably the primary reason it took so long for the existence of E. dispar to be accepted. Nevertheless, when molecular characteristics are studied the two species become easily distinguishable. How different they are depends on what measure you use. The differences initially identified and that led to the redescription of E. histolytica fell into three categories (Diamond and Clark, 1993). The first was isoenzyme differences. The initial work of Sargeaunt and Williams, using three and then four enzymes, was later supplemented with additional enzymes by Blanc (Sargeaunt et al. 1982; Sargeaunt, 1987; Blanc, 1992). However the overall picture remained the same - two clearly identifiable groups of organisms persisted. Next came antigenic differences, where monoclonal antibodies identified two groups of organisms that correlated with the isoenzyme patterns obtained from the same isolates (Strachan et al. 1988). Shortly thereafter came DNA differences - from Southern blot analysis then DNA sequencing (Tannich et al. 1989). These again identified two groups that

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correlated with the isoenzyme patterns. It is in the field of DNA analysis that most of the subsequent differences have been detected. No protein coding gene sequence has proven to be identical between the two species. In fact the percentage sequence identity in coding regions of orthologous genes averages only 95% while in non-coding regions it drops to 80% (Tannich et al. 1991; Willhoeft et al. 1999a). Variation within each species has not been widely examined but appears to be significantly less than 1% (Ghosh et al. 2000). This has allowed the design and testing of many different PCR-based diagnostic methods in laboratories around the world. No organisms with characteristics intermediate between the two species have been identified. Thus, although E. histolytica and E. dispar are each other's closest relative within the genus Entamoeba (Silberman et al. 1999), they are clearly distinct and discrete species. Qualitative analyses of their genomes are incomplete although comparative genome sequencing is underway. Several significant differences have been found so far although the significance of most is as yet unclear. The Short Interspersed Nuclear Element (SINE) known as IE or Ehapt2 is abundant in E. histolytica but rare or absent in E. dispar (Willhoeft et al. 2002). The E. histolytica gene family encoding a surface protein known as Ariel also appears to be absent in E. dispar (Willhoeft et al. 1999b). The difference that has generated the most excitement is the absence in E. dispar of a functional gene homologous to the cysteine proteinase known as EhCP5 in E. histolytica (Willhoeft et al. 1999a). The E. dispar chromosomal locus homologous to that in which EhCP5 is found has been sequenced, and a degenerate version of the gene was found that contained numerous mutations and had no possibility of encoding a protein. The corresponding protein in E. histolytica is found on the surface of trophozoites (Jacobs et al. 1998) and is therefore suspected of playing a role in tissue invasion. It is anticipated that additional differences will be uncovered as genome sequencing progresses. While it is still true to say that the two organisms appear identical under the light microscope, using electron microscopy morphological differences can be detected. The early cell surface difference detected by lectin agglutination was later identified as being due to the absence of lipophosphoproteoglycan and this can be visualised directly in transmission electron microscopy of the E. dispar cell surface. A thick surface coat seen on E. histolytica cells is missing from E. dispar leading to a difference in cell surface charge (Espinosa-Cantellano et al. 1998). There have also been differences in the organisation of intramembrane particles reported (Pimenta et al. 2002). Another inter-specific difference involves the ingestion of bacteria. While bacteria ingested by E. dispar are found individually in vacuoles with membranes tightly delimiting the bacterium, in E. histolytica several bacteria are found in the same phagocytic vacuole and with no close

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apposition of the membrane (Pimenta et al. 2002). One note of caution however; the structural differences reported are based on the study of only one isolate of E. histolytica in each paper and the same isolate of E. dispar in both, so the possibility of these being strain differences rather than species differences cannot be excluded.

CONCLUSIONS It is simplistic to say that the major difference between E. histolytica and E. dispar is that one causes disease and the other does not. However, ultimately this will always be the characteristic that defines the two organisms for most people. The genetic basis of this phenotypic difference remains to be established but it is clearly a major goal in current amebiasis research. Our understanding of the genetic differences between E. histolytica and E. dispar is likely to change fundamentally within the next year as the comparative genome analysis reaches fruition. The genome information by itself will not provide the complete picture, however, and it will need to be followed by transcription and protein analyses as important differences may prove to be quantitative rather than qualitative. Indeed, cell biological studies hint at this already as E. dispar is able to kill cells in culture (EspinosaCantellano et al. 1998), including neutrophils, almost as efficiently as E. histolytica. From what we know at present, E. histolytica and E. dispar are rather similar organisms. They inhabit the same niche, eat the same food, are transmitted in the same way, and are genetically closely related. Yet one is capable of causing a serious disease that is often fatal if not treated, while the other is apparently benign. Understanding the reasons for this difference is likely to remain a challenge for several years to come.

REFERENCES Andrews, B.J., L. Mentzoni and B. Bjorvatn. 1990. Zymodeme conversion of isolates of Entamoeba histolytica. Transactions of the Royal Society of Tropical Medicine and Hygiene 84: 63-65. Anonymous. 1997. WHO/PAHO/UNESCO report. A consultation with experts on amoebiasis. Epidemiological Bulletin PAHO 18: 13-14. Bhattacharya, A., R. Arya, C.G. Clark and J.P. Ackers. 2000. Absence of lipophosphoglycanlike glycoconjugates in Entamoeba dispar. Parasitology 120: 31-35. Blanc, D.S. 1992. Determination of taxonomic status of pathogenic and nonpathogenic Entamoeba histolytica zymodemes using isoenzyme analysis. Journal of Protozoology 39: 471-479. Blessmann, J., H. Buß, P.A. Ton Nu, B.T. Dinh, Q.T. Viet Ngo, A. Le Van, M.D. Abd Alla, T.F.H.G. Jackson, J.I. Ravdin and E. Tannich. 2002a. Real-time PCR for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in fecal samples. Journal of Clinical Microbiology 40: 4413-4417.

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Blessmann, J., P. Van Linh, P.A. Nu, H.D. Thi, B. Muller-Myhsok, H. Buß and E. Tannich. 2002b. Epidemiology of amebiasis in a region of high incidence of amebic liver abscess in central Vietnam. American Journal of Tropical Medicine and Hygiene 66: 578-583. Blessmann, J., I.K.M. Ali, P.A. Ton Nu, B.T. Dinh, T.Q. Viet Ngo, A. Le Van, C.G. Clark and E. Tannich. 2003. Longitudinal study of intestinal Entamoeba histolytica infections in asymptomatic adult carriers. (submitted). Boeck, W.C. and J. Drbohlav. 1925. The cultivation of Entamoeba histolytica. American Journal of Hygiene. 5: 371-407. Brumpt, E. 1925. Étude sommaire de l' "Entamoeba dispar" n. sp. Amibe à kystes quadrinucléés, parasite de l'homme. Bulletin de l'Academie de Médecine (Paris) 94: 943-952. Brumpt, E. 1926. Individualité de l'Entamoeba dispar. Présentation de piéces. Bulletin de la Société de Pathologié Exotique 19: 399-404. Brumpt, E. 1928. Differentiation of human intestinal amoebae with four-nucleated cysts. Transactions of the Royal Society of Tropical Medicine and Hygiene 22: 101-114, Discussion pp. 115-124. Clark, C.G. 1995. Axenic cultivation of Entamoeba dispar Brumpt 1925, Entamoeba insolita Geiman and Wichterman 1937 and Entamoeba ranarum Grassi 1879. Journal of Eukaryotic Microbiology 42: 590-593. Clark, C.G., C.C. Cunnick and L.S. Diamond. 1992. Entamoeba histolytica : is conversion of nonpathogenic amebae to the pathogenic form a real phenomenon? Experimental Parasitology 74: 307-314. Clark, C.G. and L.S. Diamond. 1993a. Entamoeba histolytica: a method for isolate identification. Experimental Parasitology 77: 450-455. Clark, C.G. and L.S. Diamond. 1993b. Entamoeba histolytica: an explanation for the reported conversion of "nonpathogenic" amebae to the "pathogenic" form. Experimental Parasitology 77: 456-460. Diamond, L.S. and C.G. Clark. 1993. A redescription of Entamoeba histolytica Schaudinn, 1903 (Emended Walker, 1911) separating it from Entamoeba dispar Brumpt, 1925. Journal of Eukaryotic Microbiology 40: 340-344. Dobell, C. 1919. The amoebae living in man. A zoological monograph. J. Bale, Sons, and Danielson, London., 155p. Espinosa-Cantellano, M., A. González-Robles, B. Chávez, G. Castañon, C. Argüello, A. Lázaro-Haller and A. Martínez-Palomo. 1998. Entamoeba dispar : ultrastructure, surface properties, and cytopathic effect. Journal of Eukaryotic Microbiology 45: 265-272. Garfinkel, L.I., M. Giladi, M. Huber, C. Gitler, D. Mirelman, M. Revel and S. Rozenblatt. 1989. DNA probes specific for Entamoeba histolytica possessing pathogenic and nonpathogenic zymodemes. Infection and Immunity 57: 926-931. Gathiram, V. and T.F.H.G. Jackson. 1987. A longitudinal study of asymptomatic carriers of pathogenic zymodemes of Entamoeba histolytica. South African Medical Journal 72: 669672. Ghosh, S., M. Frisardi, L. Ramirez-Avila, S. Descoteaux, K. Sturm-Ramirez, O.A. NewtonSanchez, J.I. Santos-Preciado, C. Ganguly, A. Lohia, S. Reed and J. Samuelson. 2000. Molecular epidemiology of Entamoeba spp.: evidence of a bottleneck (Demographic sweep) and transcontinental spread of diploid parasites. Journal of Clinical Microbiology 38: 38153821. González-Ruiz, A., M.A. Miles and D.C. Warhurst. 1993. Predictive value of diagnostic tests and prevalence of invasive Entamoeba histolytica infection. Journal of Infectious Diseases 168: 513-514. Guttiérez, G., A. Ludlow, G. Espinoza, S. Herrera, O. Muñoz, N. Rattoni and B. Sepúlveda. 1992. Encuesta serológica nacional: II. Investigación de anticuerpos contra Entamoeba histolytica en la República Mexicana. Salud Publica de Mexico 34: 242-254.

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Haque, R., K. Kress, S. Wood, T.F.H.G. Jackson, D. Lyerly, T. Wilkins and W.A. Petri Jr. 1993. Diagnosis of pathogenic Entamoeba histolytica infection using a stool ELISA based on monoclonal antibodies to the galactose-specific adhesin. Journal of Infectious Diseases 167: 247-249. Haque, R., L.M. Neville, P. Hahn and W.A. Petri Jr. 1995. Rapid diagnosis of Entamoeba infection by using Entamoeba and Entamoeba histolytica stool antigen detection kits. Journal of Clinical Microbiology 33: 2558-2561. Haque, R., A.S.G. Faruque, P. Hahn, D.M. Lyerly and W.A. Petri Jr. 1997. Entamoeba histolytica and Entamoeba dispar infection in children in Bangladesh. Journal of Infectious Diseases 175: 734-736. Haque, R., I.K.M. Ali and W.A. Petri Jr. 1999. Prevalence and immune response to Entamoeba histolytica infection in preschool children in Bangladesh. American Journal of Tropical Medicine and Hygiene 60: 1031-1034. Haque, H., N.U. Mollah, I.K.M. Ali, K. Alam, A. Eubanks, D. Lyerly and W.A. Petri Jr. 2000. Diagnosis of amebic liver abscess and intestinal infection with the TechLab Entamoeba histolytica II antigen detection and antibody tests. Journal of Clinical Microbiology 38: 32353239. Haque, R., P. Duggal, I.K.M. Ali, M.B. Hossain, D. Mondal, R.B. Sack, B.M. Farr, T.H. Beaty and W.A. Petri Jr. 2002. Innate and acquired resistance to amebiasis in Bangladeshi children. Journal of Infectious Diseases 186: 547-552. Jacobs, T., I. Bruchhaus, T. Dandekar, E. Tannich and M. Leippe. 1998. Isolation and molecular characterization of a surface-bound proteinase of Entamoeba histolytica. Molecular Microbiology 27: 269-276. Kobayashi, S., E. Imai, H. Tachibana, T. Fujiwara and T. Takeuchi. 1998. Entamoeba dispar. cultivation with sterilized Crithidia fasciculata. Journal of Eukaryotic Microbiology 45: 3S8S. Lösch, F. 1875. Massenhafte Entwicklung von Amöben im Dickdarm. Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin, von Rudolf Virchow 65: 196-211. Lösch, F.A. 1978. Massive development of amoebae in the large intestine (Translation). In Tropical medicine and parasitology. Classical investigations, B.H. Kean, K.E. Mott and A.J. Russell (eds.). Cornell University Press, Ithaca, NY, p. 71-79. Martínez-Palomo, A., A. González-Robles and M. De la Torre. 1973. Selective agglutination of pathogenic strains of Entamoeba histolytica induced con A. Nature New Biology 245: 186187. Mirelman, D., R. Bracha, A. Chayen, A. Aust-Kettis and L.S. Diamond. 1986a. Entamoeba histolytica: Effect of growth conditions and bacterial associates on isoenzyme patterns and virulence. Experimental Parasitology 62: 142-148. Mirelman, D., R. Bracha, A. Wexler and A. Chayen. 1986b. Changes in isoenzyme patterns of a cloned culture of nonpathogenic Entamoeba histolytica during axenization. Infection and Immunity 54: 827-832. Mukherjee, R.M., K.C. Bhol, S. Mehra, T.K. Maitra and K.N. Jalan. 1993. Zymodeme alteration of Entamoeba histolytica isolates under varying conditions. Transactions of the Royal Society of Tropical Medicine and Hygiene 87: 490-491. Ortner, S., C.G. Clark, M. Binder, O. Scheiner, G. Wiedermann and M. Duchêne. 1997. Molecular biology of the hexokinase isoenzyme pattern that distinguishes pathogenic Entamoeba histolytica from nonpathogenic Entamoeba dispar. Molecular and Biochemical Parasitology 86: 85-94. Petri, W.A., Jr., T.F.H.G. Jackson, V. Gathiram, K. Kress, L.D. Saffer, T.L. Snodgrass, M.D. Chapman, Z. Keren and D. Mirelman. 1990. Pathogenic and nonpathogenic strains of

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Entamoeba histolytica can be differentiated by monoclonal antibodies to the galactosespecific adherence lectin. Infection and Immunity 58: 1802-1806. Pimenta, P.F., L.S. Diamond and D. Mirelman. 2002. Entamoeba histolytica Schaudinn, 1903 and Entamoeba dispar Brumpt, 1925: differences in their cell surfaces and in the bacteriacontaining vacuoles. Journal of Eukaryotic Microbiology 49: 209-219. Sargeaunt, P.G. 1987. The reliability of Entamoeba histolytica zymodemes in clinical diagnosis. Parasitology Today 3: 40-43. Sargeaunt, P.G., J.E. Williams and J.D. Grene. 1978. The differentiation of invasive and noninvasive Entamoeba histolytica by isoenzyme electrophoresis. Transactions of the Royal Society of Tropical Medicine and Hygiene 72: 519-521. Sargeaunt, P.G., J.E. Williams, R. Bhojnani, J. Kumate and E. Jimenez. 1982. A review of isoenzyme characterization of Entamoeba histolytica with particular reference to pathogenic and non-pathogenic stocks isolated in Mexico. Archivos de Investigación Médica (México) 13 (suppl 3): 89-94. Schaudinn, F. 1903. Untersuchungen über die Fortpflanzung einiger Rhizopoden. (Vorläufige Mittheilung). Arbeiten der Kaiserlichen Gesundheitsamte 19: 547-576. Schaudinn, F. 1978. On the development of some rhizopods. (Preliminary report) (Translation). In Tropical medicine and parasitology. Classical investigations, B.H. Kean, K.E. Mott and A.J. Russell (eds.). Cornell University Press, Ithaca, NY, p. 110-118. Silberman, J.D., C.G. Clark, L.S. Diamond and M.L. Sogin. 1999. Phylogeny of the genera Entamoeba and Endolimax as deduced from small subunit ribosomal RNA gene sequence analysis. Molecular Biology and Evolution 16: 1740-1751. Simic, T. 1931a. Étude expérimentale complémentaire de l’Entamoeba dispar Brumpt, de Skoplje, sur le chat. Annales de Parasitologie Humaine et Comparée 9: 497-502. Simic, T. 1931b. Infection expérimentale de l'homme par Entamoeba dispar Brumpt. Annales de Parasitologie Humaine et Comparée 9: 385-391. Simic, T. 1935. Infection expérimentale du chat et du chien par Entamoeba dispar et Entamoeba dysenteriae. Réinfection et immunité croisée du chien. Annales de Parasitologie Humaine et Comparée 13: 345-350. Strachan, W.D., W.M. Spice, P.L. Chiodini, A.H. Moody and J.P. Ackers. 1988. Immunological differentiation of pathogenic and non-pathogenic isolates of Entamoeba histolytica. Lancet i: 561-563. Tannich, E., R.D. Horstmann, J. Knobloch and H.H. Arnold. 1989. Genomic DNA differences between pathogenic and nonpathogenic Entamoeba histolytica. Proceedings of the National Acadademy of Sciences USA 86: 5118-5122. Tannich, E., H. Scholze, R. Nickel and R.D. Horstmann. 1991. Homologous cysteine proteinases of pathogenic and nonpathogenic Entamoeba histolytica. Journal of Biological Chemistry 266: 4798-4803. Valenzuela, O., F. Ramos, P. Morán, E. González, A. Valadez, A. Gómez, E.I. Melendro, M. Ramiro, O. Muñoz and C. Ximénez. 2001. Persistence of secretory antiamoebic antibodies in patients with past invasive intestinal or hepatic amoebiasis. Parasitology Research 87: 849852. Vargas, M.A. and E. Orozco. 1993. Entamoeba histolytica: changes in the zymodeme of cloned nonpathogenic trophozoites cultured under different conditions. Parasitology Research 79: 353-356. Walker, E.L. and A.W. Sellards. 1913. Experimental entamoebic dysentery. Philippine Journal of Science B Tropical Medicine 8: 253-331. Willhoeft, U., L. Hamann and E. Tannich. 1999a. A DNA sequence corresponding to the gene encoding cysteine proteinase 5 in Entamoeba histolytica is present and positionally conserved but highly degenerated in Entamoeba dispar. Infection and Immunity 67: 5925-5929.

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Willhoeft, U., H. Buß and E. Tannich. 1999b. DNA sequences corresponding to the ariel gene family of Entamoeba histolytica are not present in E. dispar. Parasitology Research 85: 787789. Willhoeft, U., H. Buß and E. Tannich. 2002. The abundant polyadenylated transcript 2 DNA sequence of the pathogenic protozoan parasite Entamoeba histolytica represents a nonautonomous non-long-terminal-repeat retrotransposon- like element which is absent in the closely related nonpathogenic species Entamoeba dispar. Infection and Immunity 70: 67986804.

EPIDEMIOLOGY AND STRAIN VARIATION OF CRYPTOSPORIDIUM

R.M. Chalmers1 and D.P. Casemore2 1

Head, PHLS Cryptosporidium Reference Unit, Swansea PHL, Singleton Hospital, Swansea SA2 8QA, UK; 2Senior Research Fellow, Centre for Research into Environment & Health, University of Wales, Aberystwyth, SY23 2DB, UK.

ABSTRACT Cryptosporidium parvum emerged in the 1970s as a common enteric pathogen of young livestock and other animals and as an opportunistic and sometimes fatal infection in humans, primarily affecting the immunocompromised. Since then it has become recognised as a worldwide cause of acute, self-limiting diarrhoeal disease in otherwise healthy humans. It is a common cause of waterborne disease. The highest incidence is among children under 5 years in developed countries, with a younger peak in developing countries. There are multiple sources and routes of infection, indicated initially by field epidemiology studies but subsequently confirmed by phenotypic and genotypic (molecular) methods. Such typing analyses have shed new light on biology and epidemiology, providing a better understanding of the aetiology and public health control of cryptosporidiosis and also on the investigation of potential drug therapy. The last twenty years have thus been a period of exciting advance across many fields. Key words: Cryptosporidium, epidemiology, typing, public health, waterborne disease.

INTRODUCTION Human cryptosporidiosis – or its recognition - typifies the paradigm of a disease whose time had come. Cryptosporidium parvum was discovered in the early 1900s but was not described in humans until 1976 (Current, 1998; Fayer, 1997). It then emerged primarily as a cause of potentially fatal gastrointestinal disease in immunocompromised patients, especially in the then newly emerging condition, AIDS. It was seen initially, therefore, as a rare opportunistic infection. At the same time it was also identified increasingly widely as an enteric pathogen in livestock animals. In the early 1980’s independent studies in several parts of the world showed that it was in fact a common cause of acute self-limiting gastro-enteritis in otherwise healthy

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people, especially children (Casemore, 1990; Cordell and Addiss, 1994; Current, 1998; Griffiths, 1998; Palmer & Biffin, 1990). Increasingly widespread diagnosis and epidemiological investigation soon led to the recognition of waterborne disease as a significant public health problem (Meinhardt et al., 1996; Rose et al., 1997). It was assumed initially that all human infections were zoonotic and indeed infection from direct contact with livestock is common (Casemore et al., 1997). This interpretation led, however, to some curious questions of biological plausibility, but the hypothesis that many infections were non-zoonotic (Casemore and Jackson, 1984) remained unprovable until the emergence of suitable typing and tracing methods.

SOURCES AND TRANSMISSION The epidemiology of cryptosporidiosis is complex, involving both direct and indirect routes of transmission from animals to man and from person to person (Casemore et al., 1997). Cryptosporidium has been reported worldwide and is common in man, in livestock animals and in wildlife; domestic pets were thought to be an uncommon source of infection. Zoonotic infection by direct contact with mammalian livestock, especially lambs and calves, is common, particularly in urban children visiting educational farms (Casemore, 1989). Indirect transmission, especially through water is also common. Indeed, the widespread epidemic in the U.K. of foot and mouth disease during 2001, and consequent control measures, led to a measurable decline in incidence (estimated 35% overall) of cryptosporidiosis in the human population (Smerdon et al., 2003). Direct faecal-oral transmission is common in children attending playgroups and daycare centers (Casemore, 1990; Cordell & Addiss, 1994), although the infection may be introduced, in the first instance, through zoonotic contact (Casemore, 1989; Palmer & Biffin, 1990). Cryptosporidium is a common cause of traveller’s diarrhoea, including that acquired during vacation in the same country, probably due to increased and varied exposures. Hospital (nosocomial) transmission has been reported between patients and sometimes also to health care workers (Casemore et al., 1994). In HIVpositive patients increased risk is thought to be greatest from sexual high-risk behaviours (Hunter and Nichols, 2002; Kim et al., 1998; Matos et al., 1998; Pedersen et al., 1996). There is no evidence of transmission across the placenta. In livestock animals, oocyst excretion in dams is known to increase during the period around birth of offspring (Xiao et al., 1994). Foodborne infection appears to be uncommon but has been associated with, for example, consumption of apple juice (Millard et al., 1995), unpasteurized milk, uncooked (non-fermented) sausages and salad (Casemore 1990; Palmer and Biffin, 1990).

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Water presents a major route of transmission, both drinking water and through recreational use (Meinhardt et al., 1996; Rose et al., 1997). Several outbreaks in the U.K. have involved around 500 laboratory confirmed cases, while an outbreak in Milwaukee in the U.S.A involved and estimated 403,000 cases and cost the community millions of dollars. Outbreaks occur every year associated with potable public drinking supplies, including some associated with ground water sources previously believed to be safe. The incidence of waterborne infection may be amplified by secondary spread although the extent of this, and thus to some extent the size and duration of an outbreak, will reflect both the infecting strain and population immunity. (Frost et al., 2001; Meinhardt et al., 1996; Osewe et al., 1996). These factors may also influence the outcome of epidemiological studies (Harrison et al., 2003; Hunter and Quigley, 1998). It is difficult to assess the contribution of water to sporadic or endemic infection, although such transmission undoubtedly occurs. Concern over waterborne infection has led to the setting up of official advisory groups and issuing of advice in several countries (Harrison et al., 2003; Hunter 2000; Rose et al., 1997). Swimming pools are a significant risk for transmission (Furtado et al., 1998; Meinhardt et al., 1996; Rose et al., 1997). Recent prospective population studies from Australia failed to confirm association between water consumption and endemic infection but did show the importance of swimming pool use (Hellard et al. 2000; Robertson et al., 2002). Transmission associated with swimming pools result from faecal contamination of the pool by users rather than of the mains water supply. Water quality parameter failures (e.g. raised turbidity) associated with unusual levels of challenge to treatment and/or defects in treatment have been noted in many outbreaks (Meinhardt et al., 1996; Rose et al., 1997). In view of the frequency of heavy rainfall prior to many outbreaks it is interesting to speculate on the potential effect of global climate change. Reports suggest that this may lead to increased incidence in foodborne and waterborne infections, and increased monitoring and control may need to be considered (Anon, 2002a; Rose et al., 2001). Private supplies may represent a particular risk, especially for sporadic infection in visitors. These supplies generally serve smaller numbers of consumers (Furtado et al. 1998; Meinhardt et al., 1996).

EPIDEMIOLOGY – PERSON, TIME AND PLACE In developing countries, infection is common in infants aged less than 1 year, while in developed countries infection is most common in children aged from 1 to 5 years, with a secondary peak in young, mainly urban, adults (Casemore 1990; Palmer & Biffin, 1990). A relative increase in incidence in adults is often seen in waterborne outbreaks (Meinhardt et al., 1996). Males and females are generally affected with equal frequency although there is

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evidence from some studies in developing countries of a preponderance of male children, an observation common to a number of infectious diseases. Infection in children in developing countries may be associated with enteropathy and exacerbation of the effects of malnutrition, including immune dysfunction (Agnew et al., 1998; Clark, 1999; Checkley et al., 1998; Griffiths, 1998). There is evidence of seasonal peaks in several studies worldwide, particularly in spring and autumn, which do not necessarily both occur in any one locality, nor recur year by year (Casemore, 1990). They coincide generally with lambing and calving, with other farming events such as muck spreading, with maximal rainfall, and with peak foreign travel. Published reports show that the infection ranks about fourth in the list of pathogens detected in stools submitted to the laboratory. Among young children in the U. K. cryptosporidiosis is more common than salmonella infection, and during peak periods detection rates may exceed 20 per cent (Casemore 1990; Palmer & Biffin 1990). Cryptosporidiosis is generally one of the most common causes of diarrhoea in AIDS patients and in some studies prevalence exceeded 50 per cent (Pedersen et al., 1996; Clark, 1999; Hunter and Nichols, 2002;). Rates and/or severity of disease have declined recently in the developed world, reflecting more effective anti-AIDS therapy. Infection rates and severity are not generally increased for other immunocompromised groups unless profoundly compromised.

MOLECULAR STUDIES – STRAIN VARIATION Molecular methods have answered many of the questions raised by earlier field epidemiology since investigation of strain variation began in the late 1980s and early 1990s. This included the observations that different isolates have varying infective dose size and clinical responses (Fayer and Ungar, 1986). Phenotyping tools tell us something about the characteristics an organism expresses as a distinguishable trait. Those applied to C. parvum include protein analysis and antigenic diversity (Mead et al., 1988; McDonald et al., 1991; McLauchlin et al., 1998; Nichols et al., 1991; Nina et al., 1992), and isoenzyme typing using various housekeeping enzymes (Ogunkolade et al., 1993). These showed that there were consistencies in the nature of variation detected, which had biological and/or epidemiological significance. Differences between species were reported and some differences within the species C. parvum, reflecting “animal types” and “human types” were indicated (McDonald and Awad-el-Kariem, 1995). However, large numbers of oocysts were required from each source and since it is difficult to amplify Cryptosporidium oocyst numbers in the laboratory, only isolates being shed in large numbers by acutely infected animals or people could be studied:

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samples from sub-clinical infections and environmental samples simply did not contain enough oocysts for the methods to be applied. The application of the polymerase chain reaction (PCR), in which the amount of targeted genetic material is amplified from a theoretically very low starting point to produce huge amounts of replicated DNA, has provided researchers with sufficient material to further investigate variation within Cryptosporidium (Morgan and Thompson, 1998). For this reason, almost all of the genetic methods applied to investigate strain variation within the genus rely on initial amplification of targeted gene loci by PCR. In addition, PCR detection of Cryptosporidium in human and animal samples has been shown to be more sensitive and specific than traditional diagnostic microscopy (Webster, 1993; Morgan et al., 1998). The main challenges to the application of PCR to Crypto-sporidium are extracting DNA from the sporozoites within the robust oocysts and avoiding the effect of any inhibitors that might be present in the initial sample. Furthermore, the selection of the appropriate polymerase for a particular biological sample type may be critical since polymerases can be differentially denatured, or inhibited by, for example, proteinases, phenols and detergents present in the sample matrix. Over-coming inhibitors has been achieved by the use of preliminary purification steps to remove sample matrix material. Purification methods include floating oocysts from faecal matter using two-phase systems (e.g. saturated salt solutions) or recovering oocysts from the sample matrix by immunomagnetic separation. Boiling the sample destroys inhibitors, while DNA extraction methods and kits remove inhibitory substances (Boom et al., 1990; Elwin et al., 2001; Xiao et al., 2001a). While direct comparison of nucleotide sequences is the ultimate method or gold standard for detecting DNA sequence variation, the identification of consistent markers provides less complex tools for application to large numbers of samples required for epidemiological investigations. Such tools have been applied to identify species / genotypes within Cryptosporidium and include the investigation of randomly amplified polymorphic DNA, restriction fragment length polymorphisms (RFLP) following locus-specific amplification by PCR, single-strand conformation polymorphism analysis, and the application of real time PCR. PCR-RFLP has been widely used and while it has limitations, it provides a reliable, specific and rapid species / genotype identification particularly if applied with quality control standards. The most widely targeted gene loci have been the small subunit ribosomal DNA (ssu rDNA or 18s) and the Cryptosporidium oocyst wall protein (COWP) gene. Other target gene loci also include thrombospondin-related adhesive proteins (TRAP-C1 and TRAP-C2), dihydrofolate reductase-thymidylate synthase (dhfr-ts), ribonuclease reductase, internal transcribed rDNA spacers (ITS1 and ITS2), acetyl-CoA

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synthetase, beta tubulin and the 70kDa heat shock protein (hsp70) gene (Clark, 1999; Morgan et al., 1999a; Fayer et al., 2000). Using molecular tools to investigate strain variation, it has been shown that many of the differences within C. parvum actually represent two different species: genotype 1, the “human” type, for which the name C. hominis has been proposed, and genotype 2, the “animal” or “cattle” type, which has retained the name C. parvum (Morgan-Ryan et al., 2002). While it is evident that some primer pairs amplify all species within the genus (e.g. those for the ssu rRNA and COWP genes), others are more specific (such as those for TRAP-C2 which are specific for C. parvum and C. hominis) (Elwin et al. 2001). However, others also amplify DNA from related protozoan parasites, and some PCR-RFLP protocols differentiate species / genotypes more readily than others (Sulaiman et al., 1999). Primer pairs must therefore be chosen according to the question being asked, as must the restriction enzymes applied since additional enzymes may be required to differentiate all species. The importance of sequence confirmation of RFLP patterns was illustrated by Chalmers and colleagues (2002a) who identified a novel RFLP pattern, very similar to C. hominis, in the COWP gene of isolates from sheep, but sequence data clearly differentiated the isolate. Therefore, careful primer selection and PCR product analysis is required for detection and characterization, particularly from environmental specimens where a wide range of cryptosporidia and other organisms may be present. These broad methods have helped clarify the taxonomy within the genus (although questions still exist above that level) and establish, with principles of classical parasitology, that there are 13 currently recognised species (Fayer et al., 2000; Fayer et al., 2001; Alvarez-Pellitero and SitjàBobadilla, 2002; Morgan-Ryan et al., 2002) (Table 1). An increasing number of C. parvum genotypes have also been identified, some of which appear to be host-adapted since they have only been detected in a limited range of host species. An example is the marsupial genotype (Xiao et al., 1999) which has, so far, only been detected in marsupials. Some of these genotypes may warrant species status. Declaration of species within the protozoa has traditionally relied upon classical criteria encompassing morphological and ultrastructural data and life cycle characteristics including host range. Additional data are now available from genetic analyses, and these must be considered as complementary to the classical criteria during assignation of species status.

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Whichever methods have been used to characterise isolates of Cryptosporidium at the species level have consistently told us that the vast majority of human infection are caused by C. hominis and C. parvum. It has been speculated that an intact immune system maintains host specificity since four other species have been detected in immunocompromised patients (Morgan et al., 1999b; Gatei et al., 2002). However, three of these species have now also been detected in clinical specimens from a small number of immunocompetent patients (Table 1), indicating that they are circulating in the community and may pose an emerging public health risk, particularly since little is known of their epidemiology, sources and transmission (Chalmers et al., 2002b). In Peru, these three other species were also found in both diarrhoeic and non-diarrhoeic children who had no evidence of HIV infection (Xiao et al., 2001b). Interestingly, in environmental studies of surface water and waste water samples in the USA, a cocktail of Cryptosporidium species was detected using IMS-PCR-RFLP, including C.

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parvum, C. hominis, C. felis, C. andersoni, C. muris, C. baileyi and a range of C. parvum genotypes (Xiao et al., 2001b). Mixed infections have also been noted in waterborne outbreaks (Patel et al., 1998). Molecular tools confirm that people are exposed to a variety of potentially infective organisms from environmental sources, as might be predicted (Meinhardt et al., 1996). C. parvum has not only been detected in humans but also in a wide range of livestock and wild animals, while C. hominis appears to be largely restricted to humans, although there are published reports of natural infection in a non-human primate and a dugong (Morgan et al., 2000) and experimental infections in pigs (Widmer et al., 2000), lambs (Giles et al., 2001) and calves (Akiyoshi et al., 2002). Despite this, C. hominis has a far more restricted host range than C. parvum and the detection of C. hominis in a sample indicates a high probability of a human source (Patel et al., 1998; Harrison et al., 2003). Enhanced surveillance and molecular epidemiology have further elucidated the epidemiology of human cryptosporidiosis and shown that regional and seasonal differences exist that may reflect differing exposures and behaviours (McLauchlin et al., 2000; Anon, 2002b). For example, regional differences may reflect urban/rural or human/zoonotic cycles of transmission. Seasonal differences may be linked to animal husbandry and reproduction, resulting in a spring increase in human C. parvum infections and to recent foreign travel which mainly occurs following the summer holidays resulting in an increase in C. hominis infections. This, however, is worthy of further investigation to identify more precisely the risks during foreign travel. Furthermore, species variations are observed when the data are analysed by countries visited (Anon, 2002b). Thus far, little is known of the epidemiology of non-C. parvum, non-C. hominis infections in humans, of which C. meleagridis predominates. Analysis of outbreak samples has confirmed that urban trans-mission is not restricted to C. hominis but can occur with C. parvum. For example, in an outbreak associated with an indoor swimming pool in England, where the likely source of contamination was human faecal material, C. parvum was confirmed in 34/41 cases (Anon, 2000). Outbreak investigations have also benefited from the identification of the species causing human illness in the form of the provision of advice regarding appropriate control measures. For example, an outbreak in Belfast, Northern Ireland, was epidemiologically linked to the drinking water supply and the source of contamination was initially assumed to be livestock since the source water arose in a rural area and flowed through an aqueduct under agricultural land prior to distribution. However, PCR-RFLP of the COWP gene identified the human cases as C. hominis indicating human sanitation failure as the source of contamination and infection (Glaberman et al., 2002). Inspection of the aqueduct showed a point of ingress of domestic sewage and remedial action was taken.

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Recent studies have further identified separate risk factors for C. hominis and C. parvum. In a case control study of sporadic cases in Wales and the North West of England, risk factors for cryptosporidiosis where C. hominis was detected were travel abroad, changing the nappies (diapers) of children under five, and having contact with another infected person, while for C. parvum, the main risk factor was contact with a farmed animals (Hunter, 2003). This work has shown that there is epidemiological importance to considering the two infections separately. Differing clinical pictures may also emerge, since studies in Peru have shown that oocysts were shed for longer and in greater numbers in C. hominis infections than C. parvum (Xiao et al., 2001b). Differences in pathogenesis between the two species have been observed in the pig model, in which C. parvum had a shorter pre-patent period and resulted in more severe diarrhoea than C. hominis (Periera et al., 2002). These results are consistent with the hypothesis that C. hominis is more adapted to the human host and C. parvum to animal hosts, and consistent with results of infection of human cell lines (Hijjawi et al., 2001). This perhaps gives an explanation for the findings in an outbreak that involved water contaminated from both human and agricultural sources, in which the majority of cases showed infection with C. hominis (Patel et al., 1998). Different isolates of C. parvum also vary in their infectivity for humans (Okhuysen et al., 1997), and such differences in strain infectivity, combined with differing levels of population immunity make it difficult to develop meaningful health-risk based standards for water. These studies, combining clinical evidence, field epidemiology and genotyping, show that, despite some of the limitations of PCR-RFLP as a diagnostic tool, a biologically plausible and consistent epidemiological picture has emerged. This, importantly, provides a key to determining the sources of infection, routes of transmission and relevant interventions. However, it is clear from identifying separate species that further differences are present within the strains circulating in host populations. It can be hypothesised that this is most likely within C. parvum for which both a human cycle and a zoonotic cycle exists. Although a clonal population structure has been previously suggested for Cryptosporidium (Gibbons et al., 1998), this is unlikely since the life cycle incorporates a sexual stage: the apparent absence of recombinants of C. parvum and C. hominis supports their separate species status rather than clonal population expansion. The presence of recombinants within these species and a better understanding of population structures are worthy of further investigation and have implications for understanding the epidemiology and control of Cryptosporidium (Morgan et al., 1999a). To further investigate intra-species variation, sub-typing or “fingerprinting” methods need to be applied. Such tools will also help better identify co-infections of mixed Cryptosporidium species or subtypes. A

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variety of approaches have been investigated to fingerprint Cryptosporidium isolates, mostly based upon non-coding sequences that display a higher level of polymorphism than ssu rDNA or genomic sequences. While introns and intergenic regions may be appropriate targets for some organisms, within Cryptosporidium these are short or rare. However, sequence repeats occurring as mini and microsatellite DNA are common in the Cryptosporidium genome, and have been used as genetic markers in other related protozoan parasites such as Plasmodium falciparum since microsatellite sequences show a higher level of polymorphism than coding sequences. Analysis of microsatellite markers in the U.K., Italy and Denmark, has shown variation within C. parvum and C. hominis, and the technique can be used to demonstrate links between cases and sources of infection (Enemark et al., 2002; Mallon et al, 2002; Caccio, 2003). Studies in Australia have used microsatellite-telomere markers to demonstrate the consistency of predominant strains of C. parvum in cattle on individual farms (Blasdall et al., 2002), and the technique has potential for application to human epidemiological studies. The discovery of dsRNAs in C. parvum and C. hominis isolates from natural, experimental and cell culture infections (Khramtsov et al., 1997) may represent species-specific markers (Khramtsov et al., 2000). While Khramtsov and colleagues found that cDNA sequences of 306nt and 257nt of the ds-RNA differentiated between C. parvum and C. hominis (suggesting coevolution with the Cryptosporidium host cells), other workers sequencing a 173bp fragment identified wide variation but lack of specificity at the species level and concluded that while the ds-RNA typing tool may offer utility as a tracking tool for investigating the source of infection, it must be used in conjunction with other species / genotyping tools (Xiao et al., 2001c). Singlestrand conformation polymorphism (SSCP) analysis and denaturing polyacrylamide gel electrophoresis have been used to identify C. parvum and C. hominis and intra-species variation in human isolates, with SSCP appearing to offer superior intra-species variation (Gasser et al., in press). DNA sequence analysis of the GP60 gene (Strong et al., 2000) has also provided useful epidemiological information, with respect to analysis of strains circulating in the community and to outbreaks, albeit currently in retrospective analysis (Sulaiman et al., 2002; Peng et al., 2002; Glaberman et al., 2002). To provide practical tools for public health investigations and intervention strategies for control, methods identifying strain variation need to be relatively rapid and simple in application. These will help, particularly at the local level, elucidate routes of transmission, compare the relative importance of zoonotic and human transmission, track the spread of virulent strains and establish the importance of parasite heterogeneity to the local community. However, discriminatory methods with a multi-locus approach

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will provide both species confirmation and a better evaluation of parasite population structure. In many other parasite groups, genomic variation indicates differences in virulence, host specificity and drug susceptibility (Thompson and Lymberry, 1996), which has potential importance for both the clinical management of infection and control and for predictive epidemiology, so why not in Cryptosporidium? Application of typing methods to infecting strains may also help elucidate some of the problems of identifying specific therapy. Other questions, including those of cross-immunity between infecting isolates, as yet remain unanswered. However, the advances in identification of strain variation have highlighted differing epidemiological pictures that can be interpreted and explored further for application to the control of the spread of this fascinating parasite.

REFERENCES Agnew, D.G., A.A.M. Lima, R.D. Newman, T. Wuhib, R.D. Moore, R.L. Guerrant, and C. Sears. 1998. Cryptosporidiosis in Northwestern Brazilian children: association with increased diarrhea morbidity. Journal of Infectious Diseases 177:754-760. Akiyoshi, D.E., X. Feng, M.A. Buckholt, G. Widmer, and S. Tzipori. 2002. Genetic analysis of a Cryptosporidium parvum human genotype 1 isolate passaged through different host species. Infection and Immunity 70:5670-5675. Alvarez-Pellitero, P., and A. Sitjà-Bobadilla. 2002. Cryptosporidium molnari n. sp. (Apicomplexa: Cryptosporidiidae) infecting two marine fish species, Sparus aurata L. and Dicentrarchus labrax L. International Journal for Parasitology 32:1007-1021. Anon. 2000. Surveillance of waterborne disease and water quality: January to June 2000, and summary of 1999. Communicable Disease Report Weekly 10:319-322. Anon, 2002a. Health effects of climate change in the UK. Department of Health. London. Anon. 2002b. The development of a national collection for oocysts of Cryptosporidium. Foundation for Water Research, Marlow, Bucks, UK (http://www.fwr.org/). Blasdall, S.A., J.E. Ongerth, and N.J. Ashbolt, 2001. Differentiation of Cryptosporidium parvum subtypes in calves of four dairy herds by a novel microsatellite-telomere PCR with PAGE. Proceedings of Cryptosporidium from Molecules to Disease, 7-12 October, Fremantle, Australia. Boom, R., C.J.A. Sol, M.M.M. Saliman, C.L. Jansen, P.M.E. Wertheim van Dillen, and J. van der Noordaa. 1990. Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology 28:495-503. Caccio, S.M. 2003. Molecular identification of species / genotypes of Cryptosporidium in clinical and environmental samples. Proceedings of Cryptosporidium parvum in food and water, January, Malahide, Dublin. Casemore, D.P. 1989. Sheep as a source of human Cryptosporidiosis. Journal of Infection 19:101-104. Casemore, D.P. 1990. Epidemiological aspects of human Cryptosporidiosis. Epidemiology and Infection 104:1-28. Casemore, D.P., C.A. Gardner, and C. O’Mahoney. 1994. Cryptosporidial infection, with special reference to nosocomial transmission of Cryptosporidium parvum: a review. Folia Parasitologica 41:17-21. Casemore, D.P., and F.B. Jackson. 1984. Hypothesis: cryptosporidiosis in humans is not primarily a zoonosis. Journal of Infection 9:153-156.

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Casemore, D.P., S.E. Wright, and R.L. Coop. 1997. Cryptosporidiosis – human and animal epidemiology. In Cryptosporidium and cryptosporidiosis. Fayer, R. (ed.). CRC Press, Boca Raton, p. 65-92. Chalmers, R.M., K. Elwin, W.J. Reilly, H. Irvine, A.L. Thomas, and P.R. Hunter. 2002a. Cryptosporidium in farmed animals: the detection of a novel isolate in sheep. International Journal for Parasitology 32:21-26. Chalmers, R.M., K. Elwin, A. Thomas, and D.H.M. Joynson. 2002b. Unusual types of cryptosporidia are not restricted to immunocompromised patients. Journal of Infectious Diseases 185:270-271. Clark, D.P. 1999. New insights into human cryptosporidiosis. Clinical Microbiology Reviews 12:554-563. Checkley, W., L.D. Epstein, R.H. Gilman, R.E. Black, L. Cabrera, and C.R. Sterling. 1998. Effects of Cryptosporidium parvum infection in Peruvian children: growth faltering and subsequent catch-up growth. American Journal of Epidemiology 148:497-506. Cordell, R.L., and D.G. Addiss. 1994. Cryptosporidiosis in childcare settings: a review of the literature and recommendations for prevention and control. Pediatric Infectious Diseases Journal 13:310-317. Current, W.L. 1998. Cryptosporidiosis. In Topley & Wilson’s Microbiology and microbial infections, 9th Edition. Volume 5, Parasitology. F.E.G. Cox, J.P. Kreier, and D. Waklin. (eds.). Edward Arnold, London, p. 329-347. Elwin, K., R.M. Chalmers, R. Roberts, E.C. Guy, and D.P. Casemore. 2001. The modification of a rapid method for the identification of gene-specific polymorphisms in Cryptosporidium parvum, and application to clinical and epidemiological investigations. Applied and Environmental Microbiology 67:5581-5584. Enemark, H.L., P. Ahrens, C.J. Lowery, S.M. Thamsborg, J.M.D. Enemark, V. Bille-Hansen, and P. Lind. 2002. Cryptosporidium andersoni from a Danish cattle herd: identification and preliminary characterisation. Veterinary Parasitology. 107:37-49. Fayer, R. (ed.). 1997. Cryptosporidium and cryptosporidiosis. CRC Press, Boca Raton. Fayer, R., and B.L.P. Ungar. 1986. Cryptosporidium spp. and cryptosporidiosis. Microbiological Reviews 50:458-483. Fayer, R., U. Morgan, and S.J. Upton. 2000. Epidemiology of Cryptosporidium: trans-mission, detection and identification. International Journal for Parasitology 30:1305-1322. Fayer, R., J.M. Trout, L. Xiao, U.M. Morgan, A.A. Lal, and J.P. Dubey. 2001. Cryptosporidium canis n. sp from domestic dogs. Journal of Parasitology 87:1415-1422. Frost, F.J., T. Muller, G.F. Craun, R.L. Calderon, and P.A. Roeffer. 2001. Paired city Cryptosporidium serosurvey in the southwest USA Epidemiology and Infection 126:301-307. Furtado, C., G.K. Adak, J.M. Stuart, P.G. Wall, H.S. Evans, and D.P. Casemore. 1998. Outbreaks of waterborne infectious intestinal disease in England and Wales, 1992-5. Epidemiology and Infection 121:109-119. Gasser, R.B., Y.G. Abs El-Osta, and R.M. Chalmers. An electrophoretic analysis of genetic variability within Cryptosporidium parvum from imported and autochthonous cases of human cryptosporidiosis in the United Kingdom. Applied and Environmental Microbiology, in press. Gatei, W., R.W. Ashford, N.J. Beeching, S. Kang’ethe Kamwati, J. Greensill, and C.A. Hart. 2002. Cryptosporidium muris infection in an HIV-infected adult, Kenya. Emerging Infectious Diseases 8:204-206. Gibbons, C.L., B.G. Gazzard, M.A.A. Ibrahimn, S. Morris-Jones, C.S.L. Ong, and F.M. AwadEl-Kariem. 1998. Correlation between markers of strain variation in Crypto-sporidium parvum: evidence for clonality. Parasitology International. 47:139-147. Giles, M., K.A. Webster, J.A. Marshall, J. Catchpole, and T.M. Goddard. 2001. Experimental infection of a lamb with Cryptosporidium parvum genotype 1. Veterinary Record 149:523525.

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Glaberman, S., J.E. Moore, C.J. Lowery, R.M. Chalmers, I. Sulaiman, K. Elwin, P.J. Rooney, B.C. Millar, J.S. Dooley, A.A. Lal, and L. Xiao. 2002. Three drinking-water-associated cryptosporidiosis outbreaks, Northern Ireland. Emerging Infectious Diseases 8:631-633. Griffiths, J,K. 1998. Human cryptosporidiosis: epidemiology, transmission, clinical disease, treatment and diagnosis. Advances in Parasitology 40:37-85. Harrison, S.L., R. Nelder, L. Hayek, I.F. Mackenzie, D.P. Casemore, and D.Dance. 2003. Managing a large outbreak of cryptosporidiosis: how to investigate and when to decide to lift a ‘boil water’ notice. Communicable Disease and Public Health 5:230-239. Hellard, M.E., M.I. Sinclair, C.K. Fairley, R.M. Andrews, M. Bailey, J. Black, S.C. Dharmage, and M.D. Kirk. 2000. An outbreak of cryptosporidiosis in an urban swimming pool: why are such outbreaks difficult to detect? Australian and New Zealand Journal of Public Health 24:272-275. Hijjawi, N.S., B.P. Meloni, U.M. Morgan, and R.C.A. Thompson. 2001. Complete development and long term maintenance of Cryptosporidium parvum human and cattle genotypes in cell culture. International Journal for Parasitology 31:1048-1055. Hunter, P.R. 2000. Advice on the response from public and environmental health to the detection of cryptosporidial oocysts in treated drinking water. Communicable Disease and Public Health 3:24-27. Hunter, P.R. 2003. A case control study of sporadic cryptosporidiosis conducted in Wales and the North West region of England. PHLS Standing Conference on Water and the Environment, Colindale, London, March. Hunter, P.R., and G.L. Nichols. 2002. Epidemiology and clinical features of Cryptosporidium infection in immunocompromised patients. Clinical Microbiology Reviews 15:145-154. Hunter, P.R., and C. Quigley. 1998. Investigation of an outbreak of cryptosporidiosis associated with treated surface water finds limits to the value of case control studies. Communicable Disease and Public Health 1:234-238. Khramtsov, N.V., K.M. Woods, M.V. Nesterenko, C.C. Dykstra, and S.J. Upton. 1997. Viruslike, double stranded RNAs in the parasitic protozoan, Cryptosporidium parvum. Molecular Microbiology 26:289-300. Khramtsov, N.V., P.A. Chung, C.C. Dykstra, J.K. Griffiths, J.K., U.M. Morgan, M.J. Arrowood, and S.J. Upton. 2000. Presence of double-stranded RNAs in human and calf isolates of Cryptosporidium parvum. Journal of Parasitology 86:275-282. Kim, L.S., J. Stansell, J.P. Cello, and J. Koch. 1998. Discrepancy between sex- and waterassociated risk behaviours for cryptosporidiosis among HIV-infected patients in San Francisco. Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology 19:44-49. Mallon, M., A. Mcleod, H.V. Smith, J.M. Wastling, W.J. Reilly, and A.Tait 2001. Multilocus genotyping of Cryptosporidium using micro- and minisatellite markers. Proceedings of Cryptosporidium from Molecules to Disease, 7-12 October, Fremantle, Australia. Matos, O., A. Tomas, P. Aguiar, D.P. Casemore, and F. Antunes. 1998. Prevalence of cryptosporidiosis in AIDS patients with diarrhoea in Santa Maria Hospital, Lisbon. Folia Parasitologica 45:163-166. McDonald, V., and F.M. Awad-el-Kariem. 1995. Strain variation in Cryptosporidium parvum and evidence for distinctive human and animal strains. In Protozoan Parasites and Water The Royal Society of Chemistry Cambridge. pp 104-107. McDonald, V., R.M.A. Deer, J.M.S. Nina, S. Wright, P.L. Chiodini, and K.P.W.J. McAdam. 1991. Characteristics and specificity of hybridoma antibodies against oocyst antigens of Cryptosporidium parvum from man. Parasite Immunology 13:251-259. McLauchlin, J., C. Amar, S. Pedraz-Diaz, and G. Nichols. 2000. Molecular epidemiological analysis of Cryptosporidium spp. in the United Kingdom: results of genotyping

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Cryptosporidium spp. in 1,705 fecal samples from humans and 105 fecal samples from livestock animals. Journal of Clinical Microbiology 38:3984-3990. McLauchlin, J., D.P. Casemore, S. Moran, and S. Patel. 1998. The epidemiology of cryptosporidiosis: application of experimental sub-typing and antibody detection systems to the investigation of water-borne outbreaks. Folia Parasitologica 45:83-92. Mead, J.R., M.J. Arrowood, W.L. Current, and C.R. Sterling. 1988. Field inversion gel electrophoretic separation of Cryptosporidium spp. chromosome sized DNA. Journal of Parasitology 74:366-369. Meinhardt, P.L., D.P. Casemore, and K.B. Miller. 1996 Epidemiologic aspects of human cryptosporidiosis and the role of waterborne transmission. Epidemiologic Reviews 18:118136. Millard, P.S., K.F. Gensheimer, D.G. Addiss, D.M. Sosin, G.A. Beckett, A. Houck-Jankoski, and A. Husdon. 1994. An outbreak of cryptosporidiosis from fresh-pressed apple cider. Journal of American Medical Association 272:1592-1596. Morgan, U.M., and R.C.A. Thompson. 1998. PCR detection of Cryptosporidium: the way forward? Parasitology Today 14:241-245. Morgan, U.M., L. Pallant, B. Dwyer, D.A. Forbes, G. Rich, and R.C.A. Thompson. 1998. Comparison of PCR versus microscopy for the detection of Cryptosporidium – a clinical trial. Journal of Clinical Microbiology 36:995-998. Morgan, U.M., L. Xiao, R. Fayer, A.A. Lal, and R.C.A.Thompson. 1999a. Variation in Cryptosporidium: towards a taxonomic revision of the genus. International Journal for Parasitology 29:1733-1751. Morgan, U.M., L. Xiao, I. Sulaiman, R. Weber, A.A. Lal, R.C. Thompson, and P. Deplazes. 1999b. Which genotypes / species of Cryptosporidium are humans susceptible to? Journal of Eukaryotic Microbiology 46:44-45. Morgan, U.M., L. Xiao, B.D. Hill, P. O’Donoghue, J. Limor, A.A. Lal, and R.C.A.Thompson. 2000. Detection of the Cryptosporidium parvum “human” genotype in a dugong (Dugong dugon). Journal of Parasitology 86:1352-1354. Morgan-Ryan, U.M., A. Fall, L.A. Ward, N. Hijjawi, I. Sulaiman, R. Fayer, R.C.A. Thompson, M. Olson , A. Lal, and L. Xiao. 2002. Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae) from Homo sapiens. The Journal of Eukaryotic Microbiology 49:433440. Nichols, G.L., J. McLauchlin, and D.Samuel. 1991. A technique for typing Cryptosporidium isolates. Journal of Protozoology 38:237s-240s. Nina, J.M.S., V. McDonald, R.M.A. Deer, S.E. Wright, D.A. Dyson, P.I. Chiodini, and K.P.W.J. McAdam. 1992. Comparative study of the antigenic composition of oocyst isolates of Cryptosporidium parvum from different hosts. Parasite Immunology 14:227-232. Ogunkolade, B.W., H.A. Robinson, V. McDonald, K. Webster, and D.A. Evans. 1993. Isoenzyme variation within the genus Cryptosporidium. Parasitology Research 79:385-388. Okhuysen, P.C., C.L. Chappell, J.H. Crabb, C.R. Sterling, and H.L. DuPont. 1999. Virulence of three distinct Cryptosporidium parvum isolates for healthy adults. Journal of Infectious Diseases 180:1275-1281. Osewe, P., D.G. Addiss, K.A. Blair, A. Hightower, M.L. Kamb, and J.P. Davis. 1996. Cryptosporidiosis in Wisconsin: a case-control study of post-outbreak transmission. Epidemiology and Infection 117:297-304. Palmer, S.R., and A.H. Biffin. 1990. Cryptosporidiosis in England and Wales: prevalence and clinical and epidemiological features. British Medical Journal 300:774-777. Patel, S., S. Pedraza-Diaz, J. McLauchlin, and D.P. Casemore. 1998. The molecular characterisation of Cryptosporidium parvum from two large suspected waterborne outbreaks. Communicable Disease and Public Health 1:231-233.

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Pedersen C., S. Danner, A. Lazzarin, M.P. Glauser, R. Weber, C. Katlama, S.E. Barton, and J.D. Lundgren. 1996. Epidemiology of cryptosporidiosis among European AIDS patients. Genitourinary Medicine 72:128-131. Peng, M.M., O. Matos, W. Gatei, D. Pradeep, M. Stantic-Pavlinic, C. Bern, I.M. Sulaiman, S. Glaberman, A.A. Lal, and L. Xiao. 2002. A comparison of Cryptosporidium subgenotpyes from several geographic regions. Journal of Eukaryotic Microbiology; supplement 28s-31s. Periera, S.J., N.E. Ramirez, L. Xiao, and L.A. Ward. 2002. Pathogenesis of human and bovine Cryptosporidium parvum in gnotobiotic pigs. Journal of Infectious Diseases 186: 715-718. Robertson, B., M.I. Sinclair, A.B. Forbes, M. Veitch, M. Kirk, D. Cunliffe, J. Willis, and C.K. Fairley. 2002. Case-control studies of sporadic cryptosporidiosis in Melbourne and Adelaide, Australia. Epidemiology and Infection 128:419-431. Rose, J.B., J.T. Lisle, and M. LeChevallier. 1997. Waterborne cryptosporidiosis: incidence, outbreaks, and treatment strategies. In Cryptosporidium and cryptosporidiosis. Fayer, R. (ed.). CRC Press, Boca Raton, p. 93-109. Rose, J.B., P.R. Epstein, K. Lipp, B.H. Sherman, S.M. Bernard, and J.A. Patz. 2001. Climate variability and change in the United States: potential impacts on water- and foodborne diseases caused by microbiologic agents. Environmental Health Perspectives Supplement 2. 109:211-221. Smerdon, W.J., T. Nichols, R.M. Chalmers, H. Heine, and M. Reacher. 2003. Foot and mouth disease in livestock and reduced cryptosporidiosis in humans, England and Wales. Emerging Infectious Diseases 19:2-5. Strong, W.B., J. Gut, and R.G. Nelson (2000). Cloning and sequence analysis of a highly polymorphic Cryptosporidium parvum gene encoding a 60-kilodalton glycoprotein and characterization of its 15- and 45-kilodalton zoite surface antigen products. Infection & Immunity 68:4117-4134. Sulaiman, I.M., L. Xiao, and A.A. Lal. 1999. Evaluation of Cryptosporidium parvum genotyping techniques. Applied and Environmental Microbiology 65:4431-4435. Sulaiman, I.M., A. A. Lal, and L. Xiao. A population genetic study of the Cryptosporidium parvum human genotype parasites. 2002. Journal of Eukaryotic Microbiology; supplement 24s-27s. Thompson, R.C.A., and A.J. Lymberry. 1996. Genetic variability in parasites and host-parasite reactions. Parasitology 112; s7-s22. Webster, K.A. 1993. Molecular methods for the detection and classification of Cryptosporidium. Parasitology Today 9:263-266. Widmer, G., D. Akiyoshi, M.A. Buckholt, X. Feng, S.M. Rich, K.M. Deary, C.A. Bowman, P. Xu, Y Wang, X. Wang, G.A. Buck, and S. Tzipori. 2000. Animal propagation and genomic survey of a genotype 1 isolate of Cryptosporidium parvum. Molecular Biochemistry and Parasitology 108:187-197. Xiao, L., R.P. Herd, and K.E. McClure. 1994. Periparturient rise in the excretion of Giardia sp. Cysts and Cryptosporidium parvum oocysts as a source of infection for lambs. Journal of Parasitology 80:55-59. Xiao, L., U. Morgan, J. Limor, A. Escalante, M. Arrowood, W. Shulaw, R.C.A. Thompson, R. Fayer, and A.A. Lal. 1999. Genetic diversity within Cryptosporidium parvum and related Cryptosporidium species. Applied and Environmental Microbiology 65:3386-3391. Xiao, L., C. Bern, J. Limor, I. Sulaiman, J. Roberts, W. Checkley, L. Cabrera, R.H. Gilman, and A.A. Lal. 2001a. Identification of 5 types of Cryptosporidium parasites in children in Lima, Peru. Journal of Infectious Diseases 183:492-497. Xiao, L., A. Singh, J. Limor, T.K. Graczyk, S. Gradus, and A.A.Lal. 2001b. Molecular characterisation of Cryptosporidium oocysts in samples of raw surface water and wastewater. Applied and Environmental Microbiology 67:1097-1101.

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Xiao, L., J. Limor, C. Bern, and A.A. Lal. 2001c. Tracking Cryptosporidium parvum by sequence analysis of small double-stranded RNA. Emerging Infectious Diseases 7:141-145.

CYCLOSPORA CAYETANENSIS: AN EMERGENT AND STILL PERPLEXING COCCIDIAN PARASITE

Charles R. Sterling1 and Ynes R. Ortega2 1

Department of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ 85721 2 Center for Food Safety, Dept. Food Science and Technology, 1109 Experiment St, University of Georgia, Griffin, GA 30223

ABSTRACT: Cyclospora infecting humans emerged as a pathogen in the late 1970s and has since been largely associated with disease of children in the tropics, travelers and expatriates to developing countries, and the immunocompromised. It has gained recent notoriety because of foodborne disease outbreaks that have sparked much interest in trying to better define the epidemiology of this most intriguing parasite. It is clear from studies performed on Cyclospora that much further research is required to better understand certain facets of this parsite’s life cycle, interactions of the parasite with its host and interactions of the parasite with the environment. Key words: Cyclosora, epidemiology, foodborne disease, detection.

BACKGROUND Ashford reported on the coccidian identity of what is now known as Cyclospora cayetanensis in the late 1970s when he observed spherical oocysts measuring in diameter from 3 patients of Papua, New Guinea (Ashford, 1979). Ashford also reported on the delayed sporulation of isolated oocysts and the eventual formation of 2 sporocysts, but was unable to clearly delineate the eventual number of sporozoites per sporocyst. Thinking that 4 might be present per sporocyst, he felt the oocysts could belong to an unnamed species of the genus Isospora, Toxoplasma,or Hammondia. As more reports of similar organisms from around the world appeared in the literature, confusion as to its true taxonomic identity arose. Common names that appeared included Cyanobacterium-like body (a blue-green alga), Coccidian-like body and large Cryptosporidium, the latter deriving from its acid-fast staining characteristics (Naranjo et al., 1989; Hart et al., 1990; Long

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et al., 1990; Long et al., 1991; Shlim et al., 1991). Further sporulation studies finally demonstrated that isolated oocysts contained 2 sporocysts, each with 2 sporozoites (Ortega et al., 1993). This clearly placed the organism within the genus Cyclospora, with the species name cayetanensis finally being added in 1994 (Ortega et al., 1994). C. cayetanensis has gained a measure of notoriety as an important pathogen of the immunocompromised, children of developing countries, expatriates living in developing countries, travelers to developing countries and more recently as the causative agent associated with several large foodborne outbreaks of disease in the United States, Canada and elsewhere (Herwaldt, 2000). While much has been learned about this parasite recently, there remain substantial gaps in our knowledge. This chapter describes our current understanding of C. cayetanensis and attempts to identify gaps in knowledge that should be filled so we may better understand this emergent and still perplexing coccidian parasite.

TAXONOMY AND PHYLOGENY Morphological characteristics of fully sporulated oocysts have led to C. cayetanensis being taxonomically placed in the subphylum Apicomplexa, subclass Coccidiasina, order Eucoccidiorida and family Eimeriidae. This taxonomic placement within the Eimeriidae was substantiated by molecular studies of the 18SssrDNA that aligned C. cayetanensis closely with the genus Eimeria (Relman et al., 1996). So much so, that some have posed the question of whether Cyclospora should be considered a mammalian Eimeria species (Relman et al., 1996; Pieniazek and Herwaldt, 1997). Sequence data obtained from Cyclospora isolated from Ethiopian monkeys and Tanzanian baboons have demonstrated differences with C. cayetanensis from humans and differences in the organisms from the respective primate hosts from which oocysts were isolated (Eberhard et al., 1999a; Lopez et al., 1999). This has led to the naming of three new Cyclospora species: C. colibi, C. papionis and C. cercopitheci. The C. papionis from Ethiopian monkeys is considered likely to be the same species of Cyclospora observed in Tanzanian baboons based on morphology and gene sequences. Phylogenetic trees for sequenced Cyclospora and Eimeria species demonstrate a great deal of relatedness between the genera, with the Cyclospora species representing a distinct monophyletic grouping (Olivier et al., 2001). Cyclospora species, therefore, appear to be more closely related to each other than to Eimeria. What is missing from this equation is sequence data from the other named Cyclospora species that constitute an interesting group of organisms reported from reptiles, myriapods, insectivores and one murine host. It has been argued that phenotypic-based traditional taxonomic schemes are complex and unsatisfactory and that molecular methods are arguably the best techniques available for studying the relatedness among organisms. We would argue that

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both methods have served us well so far in helping to recognize Cyclospora and delineate the relatedness of species to each other and to the genus Eimeria. As such, traditional taxonomy and molecular phylogeny both have their place in resolving Cyclospora’s true taxonomic placement.

BIOLOGY AND LIFE CYCLE C. cayetanensis is like the majority of eimerid coccidians in that the end result of infection in its host is the production of an oocyst that undergoes sporogony outside the host’s body. What sets this parasite apart from the majority of Eimeriidae is the apparent length of time it takes to complete sporogony. Parasites such as Toxoplasma gondii and Eimeria tenella usually complete sporogony within 1-5 days, the process being dependent on oxygen and temperature (Frenkel et al., 1970; Norton and Chard, 1983). Under favorable laboratory conditions, C. cayetanensis completes sporogony in from 8 – 14 days (Ortega et al., 1993; Smith et al., 1997) while a baboon isolate sporulated more rapidly (5 days) (Smith et al., 1997). Sporulation times and conditions that may affect them are not known for oocysts passed into the environment. Other cyclosporans, C. caryolytica and C. talpae, both of moles, have been reported to complete Sporulation in 4-5 and 12-14 days respectively (Ortega et al., 1998). Unfortunately, animal models for C. cayetanensis do not exist, so it is unknown whether oocysts that have completed Sporulation were infectious or not. There is an obvious need to study the parameters that affect Sporulation since this impacts on the epidemiology of disease transmission. Directly passed unsporulated oocysts are non-infectious and, therefore, direct person-to-person transmission is unlikely. One has to wonder then how contamination of raspberries occurred in association with the well-documented cases of foodborne disease outbreaks associated with consumption of this fruit. Raspberries must be shipped under conditions that would not favor rapid Sporulation and must usually be consumed within a few days after they are placed on the market. The assumption being made is that fully sporulated oocysts must have somehow found their way onto this delicate fruit (Herwaldt et al., 1997). Seasonality patterns of infection observed in many places suggest that oocysts may survive for extended periods in the environment. It is not known how long or under what conditions this survival might transpire. An issue for consideration is whether or not there are specific environmental cues that trigger the final events of Sporulation that translate into oocyst infectivity. Humans appear to be the only host for C. cayetanensis (Eberhard et al., 2000; Eberhard and Arrowood, 2002). Reports have appeared in the literature of Cyclospora oocysts that might be the same as C. cayetanensis being described from a variety of host animals, but other than the recent reports of oocysts from primates, representing distinct species, they have not

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been confirmed. In this regard, C. cayetanensis, like many Eimeriidae, probably displays host specificity. Ingestion of sporulated and infectious oocysts leads to parasite colonization of the jejunum by sporozoites (Ortega et al., 1997a). The infectious oocyst dose is not known, but as for Cryptosporidium and Giardia, is presumed to be low. Parasites take up residence within an intracellular location within a parasitophorous vacuole. Several studies have confirmed the presence of distinctive intracellular asexual merozoite and sexual gametocyte stages, requisite forms for completion of the life cycle within a single host (Bendall et al., 1993; Sun et al., 1996; Nhieu et al., 1996; Ortega et al., 1997a). The lack of in vitro models of cultivation and other experimental hosts has clearly limited further studies on the biology and life cycle of this parasite.

DISEASE POTENTIAL, IMMUNITY AND TREATMENT The range of symptoms caused by infection with C. cayetanensis, as with many intestinal protozoan pathogens, can be highly variable and depend on a variety of population and environmental factors. Individuals likely to exhibit symptoms of disease include young children of developing world communities, naïve individuals visiting or living as expatriates in developing countries, naïve individuals of developed countries exposed to imported foods and the immunocompromised, particularly individuals with AIDS. Symptoms may develop abruptly or gradually and may be of relatively short duration or last an average of 7 weeks in immunocompetent individuals. This latter contrasting situation was observed in noting symptoms of children living within an endemic country, Peru, versus adult travelers and expatriates of a foreign country visiting or living within another endemic country, Nepal (Hoge et al., 1993; Madico et al., 1997). Interestingly, adult patients from Peru who live in upper class communities display symptoms similar to adult travelers and expatriates. It is quite probable that these individuals had no prior exposure to this organism because of their socioeconomic status. In many endemic settings where poor sanitary conditions prevail, the number of asymptomatically infected individuals usually is higher than those displaying symptoms. In addition, there are indications that in some settings, such as the pueblo jóvenes of Peru, infection early in life predisposes to some type of immunity since infections have not been detected in adults from the same setting (Madico et al., 1997). Symptoms, when they do occur, are quite similar to those brought on by infection with Cryptosporidium. Watery diarrhea, mild to severe nausea, anorexia and abdominal cramping are the chief complaints (Shlim et al., 1991; Wurtz; 1994; Herwaldt, 2000). Adult patients may experience weight loss of 5-10% and diarrhea alternating with constipation has been commonly reported (Ortega et al., 1998).

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C. cayetanensis has been recognized with increasing frequency from patients with AIDS (Long et al., 1990; Pape et al., 1994); Sifuentes-Osornio et al., 1995). This is particularly true in individuals living within or who have traveled to developing countries. The symptoms are identical to those seen in immunocompetent individuals, but may be prolonged. The prolonged course of infection experienced in these patients is likely the result of continued replication of first generation meront stages in the absence of effective intestinal immunity. A lower than expected prevalence of C. cayetanensis infection in AIDS patients is observed in some developing countries, such as Peru. This may be due to the prophylactic use of TMP-SMX against possible Pneumocystis carinii infection (Ortega et al., 1998). The rather high prevalence rate reported in adult AIDS patients of Haiti may be due to an infrequent use of TMP-SMX prophylaxis in that country (Pape et al., 1994). Immunity to C. cayetanensis has not been extensively studied. IgM and IgG antibodies are detected in response to infection and these antibodies recognized a wide range of parasite antigens, many of which are shared by Cryptosporidium (Ortega, et al., 1998). An interesting feature of this infection that has been noted is the profound inflammation seen in histologic sections taken from intestinal biopsies and the paucity of parasite developmental stages encountered (Ortega et al., 1997a). This raises the questions as to whether the parasite might stimulate inflammation by modulating certain pro-inflammatory cytokine responses. Otherwise, the similarities noted by host response, or lack thereof, to C. cayetanensis and Cryptosporidium infections in immunocompetent and AIDS patients might indicate that immune responses operate similarly towards these parasites. Early studies aimed at elucidating the identity of Cyclospora also noted a high degree of infection resistance to conventional antimicrobial therapy. In an initial report from Peru, subjective treatment with trimethoprim-sulfamethoxazole (TMP-SMX) resulted in symptom cessation in one adult and four children after 4 days (mean) of treatment (Madico et al., 1993). The effectiveness of this treatment was confirmed in double-blind randomized placebo-controlled trials involving expatriates in Nepal and children in Peru (Hoge et al., 1995; Madico et al., 1997). TMP, 160mg, and SMX, 800mg, bid for 7-10 days remains the recommended drug treatment for this infection. In one randomized-controlled trial in patients infected with C. cayetanensis, ciprofloxacin, although not as effective, was deemed to be an acceptable drug for patients who could not tolerate TMP-SMX (Verdier et al., 2000).

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SEASONALITY

AND

OUTBREAK

Infections with C. cayetanensis have been reported from around the world, with most occurring in developing countries of the tropics and subtropics (Sterling and Ortega, 1999). Where surveys have been conducted in temperate zoned developed countries, infection rates have been exceedingly low (<0.5%) and have largely been attributed to international travel or consumption of imported food products. Indigenous transmission within the U.S., with no apparent foodborne association, has been suggested in a number of reports. In these instances, the suggested vehicle of transmission was water from 4 different sources: tap, lake, run-off and well (Hale et al., 1994; Wurtz, 1994; Huang et al., 1995; Oii et al., 1995). In another report from the U.S., infection was linked to gardening and working with soil (Koumans et al., 1996). In no instance, however, was there any direct evidence that infection occurred via these contact routes. Oocysts of Cyclospora have been isolated from water sources of Nepal, Peru and Guatemala, where infections in the general population are more common, suggesting that water plays an import role in transmission (Rabold et al., 1994; Sturbaum et al., 1998; Bern et al., 1999; Sherchand et al., 1999; Sherchand and Cross, 2001). An interesting feature associated with C. cayetanensis infections from endemic areas is the rather marked seasonality. In Nepal infections are most common prior to and during the warm monsoon months, but decrease before the rains end (Shlim et al., 1991). In Guatemala they follow a somewhat similar pattern, however the temperature is more moderate (Bern et al., 1999; Bern et al., 2000). In Haiti, the infections were recorded as most common during the drier and cooler months (Eberhard et al., 1999b), while in Lima, Peru, which is very dry throughout the year, infections usually occur during the warmer months (Ortega et al., 1993; Madico et al., 1997). Finally, in Indonesia, cases commonly appear during the cooler wet months (Fryauff et al., 1999). Presumably, factors such as moisture and temperature affect oocyst sporulation and survival. If so, in what way and how does one account for the variations in rather marked seasonality observed in these diverse areas of the world? Could factors such as the amount of sunlight or UV radiation over extended periods of time affect sporulation or oocyst survival? These are important questions since they would likely affect transmission patterns of this parasite. Water and food have been identified as two sources of infection in association with disease outbreaks caused by C. cayetanensis. Zoonotic and person-to-person transmission is unlikely since suspected animal reservoirs of this parasite have not been confirmed as carrying this parasite and since oocyst sporulation requires times of a week or longer under favorable conditions.

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The first outbreak of cyclosporiasis in the US occurred in 1990 among residents of a physicians’ dormitory in Chicago and it was hypothesized that water from rooftop reservoirs was the contaminating source (Huang et al., 1995). The reservoirs received water from a municipal source, were covered with canvas and located in an unprotected building roof area. In addition, there had been a recent pump failure, resulting in stagnating water. People who became ill during this outbreak had attended a catered party, but had also consumed tap water in the dormitory. Because information regarding the food eaten was not obtained, it was not known if there might have been an association between a given food source and the attack rates observed. There was, on the other hand, a high correlation between attack rates and the consumption of tap water coming from the reservoirs. A definite association of disease with water consumption was made in Nepal in 1994 among British expatriates associated with a military facility (Rabold et al., 1994). Twelve of 14 persons became ill and oocysts were identified in the stools from 6 of 8. Oocysts were identified in grab samples from a sealed tank receiving a mixture of river and municipal water that was chlorinated. Although data are lacking, oocysts of Cyclospora, like those of Cryptosporidium, are thought to be highly chlorine resistant. Consumption of untreated water had earlier been identified as a risk factor in association with illness seen at outpatient clinics serving expatriates in Nepal (Hoge et al., 1993). Organisms resembling Cyclospora were identified from the untreated tap water of a home of one of the study patients. C. cayetanensis oocysts have also been isolated from wastewater of sewage lagoons near an area of endemic disease in Lima, Peru (Sturbaum et al., 1998). While not implicated in direct waterborne transmission in this instance, water from these lagoons was used to irrigate certain food crops. The association of C. cayetanensis with foodborne disease transmission was first suggested in 1995 by illness in an airline pilot who had consumed food prepared in a Haitian kitchen and brought on board the airplane (Connor and Shlim, 1995). Foodborne association was again implicated in 1995 in small outbreaks in the United States (Koumans et al., 1998; Herwaldt, 2000). Larger outbreaks, occurring in 1996 and 1997 in both the United States and Canada contributed to this organism’s notoriety and called attention to the fact that imported foodstuffs facilitated in the distribution of this parasite (Herwaldt et al., 1997, 1999). Investigations associated with initial outbreaks in 1995 and 1996 implicated a variety of sources, including raspberries and strawberries, as possibly being the responsible agents of disease transference. Interestingly, the implication that strawberries might have been responsible caused considerable consternation and economic loss among US growers through early 1996 because strawberries were being pulled from the shelves of supermarkets in several

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states (anon, 1996). They were subsequently dissociated from the outbreaks when ultimately it was shown that raspberries were more significantly associated with illness by multivariate analysis of the data from the Florida outbreak of 1995 and outbreaks of early 1996 (Koumans et al., 1998). In addition, it was shown that strawberries were just as likely to be distributed to parts of the country where disease was not occurring as they were to parts of country where disease outbreaks occurred. The outbreaks of 1996 involving C. cayetanensis were instrumental to our understanding of foodborne transmission and the source of contamination. A total of 1,465 cases of cyclosporiasis were reported to the CDC and almost half were event associated (Herwaldt et al., 1997). Cases were reported from 20 states, the District of Columbia and two Canadian provinces. In 55 event related clusters, raspberries were definitely served at 50 and possibly at 4 more. Traceback data, which is often complicated by complex distribution and handling of food sources, pointed to Guatemala and a number of Guatemalan farms as the source of infected fruit. Because of the manner by which raspberries are grown and shipped, it was hypothesized that the berries had been sprayed with insecticides or fungicides prepared using oocyst contaminated water (Herwaldt et al., 1997). While not proven, this raises the question as to the potential effect of such agents on oocyst viability? The outbreaks of 1996 and 1997 led the US Food and Drug Administration to restrict the importation of raspberries during 1998. Canada did not follow this course of action and experienced yet more outbreak clusters (Herwaldt, 2000). Other imported fruits and vegetables have also been linked to foodborne outbreaks of cyclosporiasis in the United States. Blackberries from Guatemala, mesclun lettuce from Peru and basil from Mexico were suspected of being associated with outbreaks from Georgia, Ontario, Florida, the D.C. area and Missouri from 1997-2000 (Herwaldt, 2000). It was noted that the vegetables involved could have come from the United States, and in the case of basil, the outbreak in the D.C. area could have been due to contamination from food-handlers since several were ill at the time of the outbreak. In the Missouri outbreak involving basil, oocysts were actually detected by both microscopy and PCR in frozen leftovers (chicken pasta salad) from one of the parties. The first foodborne outbreak of cyclosporiasis from central Europe was reported in 2002 (Doller et al., 2002). In this instance, an attack rate of 85% was noted in attendees of 4 independent luncheon parties. The suspected source of contamination was one of several types of lettuce grown in either the south of France or in Italy. One of the big questions relating to foodborne transmission is how and in what manner did the suspect food become contaminated? Was it use of contaminated water or human feces used as fertilizer that contained fully sporulated oocysts that served as the primary source of contamination? Given

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the large quantities of consumable vegetables and fruits that are imported into the United States, are there measures that can be taken to prevent further foodborne disease outbreaks of cyclosporiasis? Despite attempted control measures aimed at improving hygiene, sanitation and water quality, outbreaks linked to imported Guatemalan raspberries continued after the 1997 season. Studies using gamma irradiated Toxoplasma and Eimeria have demonstrated that dose levels of 0.5 and 1.0 kGy, respectively, are effective in killing oocysts of these species seeded onto fruit as judged by animal infectivity studies (Dubey et al., 1998; Lee and Lee, 2001). These studies also point to the utility of these organisms as models for studies with Cyclospora since animal models for the latter do not exist.

DETECTION Oocysts of C. cayetanensis have been detected from fecal, environmental and food samples by microscopy (Eberhard et al., 1997; Visvesvara et al., 1997; Sturbaum et al., 1998; Ortega et al., 1997b; Lopez et al., 2001). This has not been without challenges, however, since special stains or filters must be used to enhance their detection. Detection in feces is usually accomplished by means of employing a modified acid-fast stain that must be requested, as for detection of Cryptosporidium. A microscope equipped with an ocular micrometer should be used since acid-fast Cyclospora oocysts measure whereas those of Cryptosporidium measure Ultraviolet fluorescence microscopy is useful in confirming the presence of Cyclospora oocysts since they will autofluoresce green using a 450-490 nm dichroic filter and blue using a 365nm dichroic filter (Berlin et al., 1998; Ortega et al., 1998). This latter method has been used to identify Cyclospora oocysts from food and water, but is deemed extremely labor intensive and insensitive to likely have much utility. Unfortunately, monoclonal antibody based fluorescent detection or immunomagnetic separation techniques have not been developed to enhance detection of this parasite from fecal, environmental or food sources. Environmental and food samples present challenges of oocyst isolation and identification not likely to be encountered in fecal samples. The presence of plant and animal debris and a multitude of chemicals and protozoan organisms that may be genetically quite similar in such samples are likely to lead to disappointing diagnostic outcomes when applied to the detection of Cyclospora. These problems dictate that various sample collection, concentration, purification, differential diagnostic and often viability detection steps have to be applied to samples to come up with a desired diagnosis. The steps to be employed in achieving a diagnostic outcome are also often confounded due to the presence of small oocyst numbers in such matrices. Initially, recovery methods for Cyclospora oocyst

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detection derived from those used to detect Cryptosporidium. Acknowledging that these techniques are cumbersome and that antibodybased reagents are unavailable for Cyclospora, investigators have turned to molecular-based approaches to address this deficiency. The use of such techniques, however, still requires use of specific recovery and concentration steps before they can be applied. It is beyond the scope of this chapter to evaluate all of these and the reader is referred to the excellent review of Shields and Olson (2003). Use of the polymerase chain reaction (PCR) following the use of specific recovery and concentration steps has the potential to be more sensitive and specific when applied to C. cayetanensis detection from virtually any matrix. Initial primers developed for the PCR reaction showed cross reactivity with Eimeria (Relman et al., 1996). Further primer modifications and the addition of restriction fragment length polymorphism (RFLP) analysis permitted a more definitive diagnosis of C. cayetanensis (Jinneman et al., 1996; Jinneman et al., 1998; Sturbaum et al., 1998). It is acknowledged, however, that the primers used would not permit C. cayetanensis to be distinguished from other Cyclospora species. Analysis of the internal transcribed spacer ITS-1 region has been used to distinguish human and primate species of Cyclospora and has demonstrated that this region is highly variable within and between samples (Adam et al., 2000; Olivier et al, 2001). In the majority of samples tested this variability did not correlate with geographic origin of the samples and, therefore, may not be a suitable marker for molecular epidemiology studies. Despite the observed variability, however, conserved species-specific ITS-1 sequences showed consistent and remarkable diversity among Cyclospora spp. ITS-1 sequences argue for polyparasitism and simultaneous transmission of multiple strains rather than multiple and different copies in one organism. This may explain the presence of a single ITS-1 sequence in epidemiologically associated isolates (Adam et al., 2000). Both conditions seem to occur in Cryptosporidium. Two calf-propagated Cryptosporidium contained two different types of ssrDNA sequences whereas a human isolate contained only one (Le Blancq et al., 1997, Widmer et al., 1999). Examining more outbreak associated isolates and propagating the organism in animal models could resolve the issue of polyparasitism, or multiple copies in one organism. An oligoligation assay, used to distinguish PCR products, has been developed to differentiate Cyclospora from Eimeria (Jinneman et al., 1999). This technique may have utility in differentiating Cyclospora species once their hyper-variable regions have been determined. A quantitative real-time PCR assay using both a species-specific primer set and a dual fluorescentlabeled C. cayetanensis hybridization probe has recently been developed to detect this parasite (Varma et al., 2003). DNA from as few as 1 oocyst per

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reaction volume could be detected. It was acknowledged that a more robust testing of the DNA extraction method would be required to ensure suitability for a wide variety of environmental or clinical samples and that further improvements in DNA extraction would enhance the overall efficiency of the assay. The viability of recovered C. cayetanensis oocysts has not been extensively studied. Oocysts can be induced to sporulate and following treatment with bile salts, taurocholate and mechanical pressure can be excysted (Ortega et al., 1993). At present it is unknown if excysted sporozoites can infect cell cultures or cause actual infections in humans. Excystation, and therefore viability, may not be related to infectivity. A novel technique, electrorotation, that is able to detect changes in the physiochemical and morphologic properties of an oocyst, has also been used to indicate an oocyst’s viability (Dalton el al., 2001). Oocysts rotate dependent upon their conductivity and permittivity in an electric field and the resulting electrorotation spectra measurements determine whether the oocyst is viable or not.

CONCLUSIONS Outbreaks of cyclosporiasis, particularly those resulting from consumption of imported foods, have brought C. cayetanensis our attention. Numerous questions relative to the life cycle, biology, immune responsiveness, epidemiology and detection of this organism have been posed in the foregoing chapter and require addressing if we are to better understand and control this emergent and still perplexing coccidian parasite.

REFERENCES: Adam, R.D., Y.R. Ortega, R.H. Gilman, and C.R. Sterling. 2000. Intervening transcribed spacing region 1 variability in Cyclospora cayetanensis. Journal of Clinical Microbiology. 38: 2339-2343. Anon. 1996. Cyclospora gives scientists the raspberry. Food Protection Report. 12(7-8): 5-6. Ashford, R.W. 1979. Occurrence of an undescribed coddidian in man in Papua New Guinea. Annals of Tropical Medicine and Parasitology. 73: 497-500. Bendall, R.P., S. Lucas, A. Moody, G. Tovey, and P.L. Chiodini. 1993. Diarrhoea associated with Cyanobacterium-like bodies: a new coccidian enteritis of man. Lancet. 341:590-592. Berlin, O.G.W., J.B. Peter, C. Cagne, C.N. Conteas, and L.R. Ash. 1998. Autofluorescence and the detection of Cyclospora oocysts. Emerging Infectious Diseases. 4:127-128. Bern, C., B. Hernandez, M.B. Lopez, M.J. Arrowood, M.A. de Mejia, A.M. de Merida, A.W. Hightower, L. Venczel, B.L. Herwaldt, R.E. Klein. 1999. Epidemiologic studies of Cyclospora cayetanensis in Guatemala. Emerging Infectious Diseases. 5:766-774. Bern, C., B. Hernandez, M.B. Lopez, M.J. Arrowood, A.M. de Merida, and R.E. Klein. 2000. The contrasting epidemiology of Cyclospora and Cryptosporidium among outpatients in Guatemala. American Journal of Tropical Medicine and Hygiene. 63:231-235. Connor, B.A., and D.R. Shlim. 1995. Foodborne transmission of Cyclospora. Lancet. 346:1634.

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Dalton, C., A.D. Goater, R. Pethig, and H.V. Smith. 2001. Viability of Giardia intestinalis cysts and viability and sporulation state of Cyclospora cayetanensis oocysts determined by electrorotation. Applied and Environmental Microbiology. 67:586-590. Doller, P.C., K. Dietrich, N. Filipp, S. Brockmann, C. Dreweck, R. Vonthein, C. WagnerWiening, and A. Wiedenmann. 2002. Cyclosporiasis outbreak in Germany associated with the consumption of salad. Emerging Infectious Diseases. 8: 992-994. Dubey, J.P., D.W. Thayer, C.A. Speer, and S.K. Shen. 1998. Effect of gamma irradiation on unsporulated and sporulated Toxoplasma gondii oocysts. International Journal for Parasitology. 28:369-375. Eberhard, M.L., and M.J. Arrowood. 2002. Cyclospora spp. Current Opinion in Infectious Diseases. 15:519-522. Eberhard, ML, A.J. DaSilva, B.G. Lilley, and N.J. Pieneazek. 1999a. Morphologic and molecular characterization of new Cyclospora species from Ethiopian monkeys: C. cercopitheci sp.n., C. colobi sp.n., and C. papionic. Emerging Infectious Diseases. 5:651658. Eberhard, M.L., E.K. Nace, A.R. Freeman, T.G. Streit, A.J. Da Silva, and P.J. Lammie. 199b. Cyclospora cayetanensis infections in Haiti: a common occurrence in the absence of watery diarrhea. American Journal of Tropical Medicine and Hygiene. 60:584-586. Eberhard, M.L., Y.R. Ortega, D.E. Hanes, E.K. Nace, R.Q. Do, M.G. Robl, K.Y. Won, C. Gavidia, N.L. Sass, K. Mansfield, A. Gozalo, J. Griffiths, R. Gilman, C.R. Sterling, and M.J. Arrowood. 2000. Attempts to establish experimental Cyclospora cayetanensis infection in laboratory animals. Journal of Parasitology. 86:577-582. Eberhard, M.L., N.J. Pieniazek, and M.J. Arrowood. 1997. Laboratory diagnosis of Cyclospora infections. Archives of Pathology and Laboratory Medicine. 121:797-797. Frenkel, J.K., J.P. Dubey, and N.L. Miller. 1970. Toxoplasma gondii in cats: fecal stages identified as coccidian oocysts. Science. 167:893-896. Fryauff, D.J., R. Krippner, P. Prodjodipuro, C. Exald, S. Kawengian, K. Pegelow, T. Yun, C. von Heydwolff-Wehnert, B. Oyofo, and R. Gross. 1999. Cyclospora cayetanensis among expatriate and indigenous populations of West Java, Indonesia. Emerging Infectious Diseases. 5:585-588. Hale, D., W. Aldeen, and K. Carroll. 1994. Diarrhea associated with cyanobacteria-like bodies in an imunocoompetent host. An unusual epidemiological source. Journal of the American Medical Association. 271:144-145. Hart, A.S., M.T. Ridinger, R. Soundarajan, C.S. Peters, A.L. Swiatlo, and E. Kocka. 1990. Novel organisms associates with chronic diarrhea in AIDS. Lancet. 335:169-170. Herwaldt, B.L. 2000. Cyclospora cayetanensis: A review, focusing on the outbreaks of cyclosporiasis in the 1990s. Clinical Infectious Diseases. 31:1040-1057. Herwaldt, B.L., M-L. Ackers, and the Cyclospora Working Group. 1997. An outbreak in 1996 of cyclosporiasis associated with imported raspberries. New England Journal of Medicine. 336:1548-1556. Herwaldt, B.L., M.J. Beach, and the Cyclospora Working Group. 1999. The return of Cyclospora in 1997: another outbreak of cyclosporiasis in North America associated with imported raspberries. Annals of Internal Medicine. 130:210-220. Hoge, C.W., D.R. Shlim, M. Ghimire, J.G. Rabold, P. Pandey, A. Walch, R. Rajah, P. Gaudio, and P. Echeverria. 1995. Placebo-controlled trial of co-trimoxazole for Cyclospora infections among travelers and foreign residents in Nepal. Lancet. 345:691-693. Hoge, C.W., D.R. Shlim, R. Rajah, J. Triplett, M. Shear, J.G. Rabold, and P. Echeverria. 1993. Epidemiology of diarrhoeal illness associated with coccidian-like organism among travelers and foreign residents in Nepal. Lancet. 341:1175-1179.

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Huang, P., J.T. Weber, D.M. Sosin, P.M. Griffin, E.G. Long, J.J. Murphy, F. Kocka, C. Peters, and C. Kallick. 1995. The first reported outbreak of diarrheal illness associated with Cyclospora in the United States. Annals of Internal Medicine. 123:409-414. Jinneman, K.C., J.H. Wetherington, A.M. Adams, J.M. Johnson, B.J. Tenge, N-L. Dang, and W.E. Hill. 1996. Differentiation of Cyclospora sp. and Eimeria sp. By using the polymerase chain reaction amplification products and restriction fragment length polymorphisms. Food and Drug Administration Laboratory Information Bulletin LIB No. 4044. Jinneman, K.C., J.H. Wetherington, W.E. Hill, A.M. Adams, J.M. Hohnson, F.J. Tenge, N-L. Dang, R.L. Manger, and M.M. Wekell. 1998. Template preparation for PCR and RFLP of amplification products for the detection and identification of Cyclospora sp. and Eimeria spp. oocysts directly from raspberries. Journal of Food Protection. 61:1497-1503. Jinneman, K.C. J.H. Wetherington, W.E. Hill, C.J. Omiescinski, A.M. Adams, J.M. Johnson, B.J. Tenge, N-L. Dang, and M.M. Wekell. 1999. An oligonucleotide-ligation assay for the differentiation between Cyclospora and Eimeria spp. polymerase chain reaction amplification products. Journal of Food Protection. 62:682-685. Koumans, E.H.A., D.J. Katz, J.M. Malecki, S. Kumar, S.P. Wahlquist, M.J. Arrowood, A.W. Hightower, and B.L. Herwaldt. 1998. An outbreak of cyclosporiasis in Florida in 1995: a harbinger of multistate outbreaks in 1996 and 1997. American Journal of Tropical Medicine and Hygiene. 59:235-242. Koumans, E.H., D. Katz, J. Malecki, S. Wahlquist, S. Kumar, A. Hightower, et al. 1996. Novel parasite and mode of transmission: Cyclospora infection-Florida. Annual Epidemic Intelligence service Conference 45:60. Le Blancq, S.M., N.V. Khramtsov, F. Aamani, S.J. Upton, and T.W. Wu. 1997. ribosomal RNA gene organization in Cryptosporidium parvum. Molecular and Biochemical Parasitology. 90:463-478. Lee, M.B., and E.H. Lee. 2001. Coccidial contamination of raspberries: mock contamination with Eimeria acervulina as a model for decontamination treatment studies. Journal of Food Protection. 64:1854-1857. Long, E.G., A. Ebrahimzadeh, E.H. White, B. Swisher, and C.S. Callaway. 1990. Alga associated with diarrhea in patients with acquired immunodeficiency syndrome and in travelers. Journal of Clinical Microbiology. 28:1101-1104. Long, E.G., E.H. White, W.W. Carmichael, R.R. Quinlisk, B.L. Swisheer, H. Daugharty, and M.T. Cohen. 1991. Morphologic and staining characteristics of a Cyanobacterium-like organism associated with diarrhea. Journal of Infectious Diseases. 164:199-202. Lopez, A.S., D.R. Dodson, M.J. Arrowood, P.A. Orlandi Jr., A.J. Da Silva, J.W. Bier, S.D. Hanauer, R.L. Kuster, S. Oltman, M.S. Baldwin, K.Y. Won, E.M. Nace, M.L. Eberhard, and B.L. Herwaldt. 2001. Outbreak of cyclosporiasis associated with basil in Missouri in 1999. Clinical Infectious Diseases. 32:1010-1017. Lopez, F.A., J. Manglicmot, T.M. Schmidt, C. Yeh, H.V. Smith, and D.A. Relman. 1999. Molecular characterization of Cyclospora-like organisms from baboons. Journal of Infectious Diseases. 179:670-676. Madico, G., R.H. Gilman, E. Miranda, L. Cabrera, and C.R. Sterling, 1993. Treatment of Cyclospora infections with co-trimoxazole. Lancet. 342:122-123. Madico, G., J. McDonald, R.H. Gilman, L. Cabrera, and C.R. Sterling. 1997. Epidemiology and treatment of Cyclospora cayetanensis infection in Peruvian children. Clinical Infectious Diseases. 24:977-981. Narango, J., C. Sterling, R. Gilman, E. Miranda, F. Diaz, M. Cho, and A. Benel. 1989. Cryptosporidium muris-like objects from fecal samples of Peruvians (abstract 324, In: Program and abstracts of the Annual Meeting of the American Society of Tropical Medicine and Hygiene (Honolulu), 10-14 December, 1989.

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Nhieu J.T., R. Nin, J. Fleury-Feith, M.T. Chaumette, A. Schaeffer, and S. Bretagne. 1996. Identification of intracellular stages of Cyclospora species by light microscopy of thick sections using hematoxylin. Human Pathology. 27:1107-1109. Norton, C.C., and M.J. Chard. 1983. The oocyst sporulation time of Eimeria species from the fowl. Parasitology 86:193-198. Oii, W.W., S.K. Zimmerman, and C.A. Needham. 1995. Cyclopora species as a gastrointestinal pathogen in immunocompetent hosts. Journal of Clinical Microbiology. 33:1267-1269. Olivier, C., S. van de Pas, P.W. Lepp, K. Yoder, and D.A. Relman. 2001. Sequence variability in the first internal transcribed spacer region within and among Cyclospora species is consistent with polyparasitism. International Journal for Parasitology. 31:1475-1487. Ortega, Y.R., R.H. Gilman, and C.R. Sterling. 1994. A new coccidian parasite (Apicomlexa: Eimeriidae) from humans. Journal of Parasitology. 80:625-629. Ortega, Y.R., R. Nagle, R.H. Gilman, J. Watanabe, J. Miyagui, H. Quispe, P. Kanagusuku, C. Rojas, and C.R. Sterling. 1997a. Pathologic and clinical findings in patients with cyclosporiasis and a description of intracellular parasite life-cycle stages. The Journal of Infectious Diseases. 176:1584-1589. Ortega, Y.R., C.R. Roxas, R.H. Gilman, N.J. Miller, L. Cabrera, C. Taquiri, and C.R. Sterling. 1977b. Isolation of Cryptosporidium parvum and Cyclospora cayetanensis from vegetables collected in markets of an endemic region in Peru. American Journal of Tropical Medicine and Hygiene. 57:683-686. Ortega, Y.R. C.R. Sterling, and R.H. Gilman. 1998. Cyclospora cayetanensis. Advances in Parasitology. 40:399-418. Ortega, Y.R., C.R. Sterling, R.H. Gilman, V.A. Cama, and F. Diaz. 1993. Cyclospora species: a new protozoan pathogen of humans. New England Journal of Medicine. 328:1308-1312. Pape, J.W., R.-L. Verdier, M. Boncy, J. Boncy, and W.D. Johnson. 1994. Cyclospora infection in adults infected with HIV. Clinical manifestations, treatment and prophylaxis. Annals of Internal Medicine. 121:654-657. Pieniazek, N.J., and B.L. Herwaldt. 1997. Reevaluating the molecular taxonomy: is the human associated Cyclospora a mammalian Eimeria species? Emerging Infectious Diseases. 3:381383. Rabold, J.G., C.W. Hoge, D.R. Shlim, C. Kefford, R. Rajah, and P. Echevenia. 1994. Cyclospora outbreak associated with chlorinated drinking water. Lancet. 344:1360-1361. Relman, D.A., T.S. Schmidt, A. Gajadhar, M. Sogin, J. Cross, K. Yoder, O. Sethabutr, and P. Echeverria. 1996. Molecular phylogenetic analysis of Cyclospora, the human intestinal pathogen, suggests that it is closely related to Eimeria species. Journal of Infectious Diseases. 173:440-445. Sherchand, J.B., and J.H. Cross. 2001. Emerging pathogen Cyclospora cayetanensis infection in Nepal. Southeast Asian Journal of Tropical Medicine and Public Health. 32:143-150. Sherchand. J.B., J.H. Cross, M. Jimba, S. Sherchand, and M.P. Shrestha. 1999. Study of Cyclospora cayetanensis in health care facilities, sewage water and green leafy vegetables in Nepal. Southeast Asian Journal of Tropical Medicine and Public Health. 30:58-63. Shields, J.M., and B.H. Olson. 2003. Cyclospora cayetanensis: a review of an emerging parasitic coccidian. International Journal for Parasitology. 33:371-391. Shlim, D.R., M.T. Cohen, M. Eaton, R. Tajah, E.G. Long, and B.L.P. Ungar. 1991. An algalike organism associated with an outbreak of prolonged diarrhea among foreigners in Nepal. American Journal of Tropical Medicine and Hygiene. 45:383-389. Sifuentes-Osornio, J., G. Porras-Cortes, R.P. Bendall, F. Morales-Villarreal, G. Reyes-Teran, and G.M. Ruiz-Palacios. 1995. Cyclospora cayetanensis infection in patients with and without AIDS: biliary disease as another clinical manifestation. Clinical Infectious Diseases. 21:1092-1097.

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Smith, H.V., C.A. Paton, M.M.A. Mtambo, and R.W.A. Girdwood. 1997. Sporulation of Cyclospora sp. Oocysts. Applied and Environmental Microbiology. 63:1631-1632. Sterling, C.R., and Y.R. Ortega. 1999. Cyclospora: an enigma worth unraveling. Emerging Infectious Diseases. 5:48-53. Sturbaum, G.D., Y.R. Ortega, R.H. Gilman, C.R. Sterling, L. Cabrera, and D.A. Klein. 1998. Detection of Cyclospora cayetanensis in wastewater. Applied and Environmental Microbiology. 64:2284-2286. Sun, T., C.F. Illardi, D. Asnis, A.R. Bresciani, S. Goldenberg, B. Roberts, and S. Teichberg. 1996. Light and electron microscopic identification of Cyclospora species in the small intestine. Evidence of the presence of asexual life cycle in the human host. American Journal of Clinical Pathology. 105:216-220. Varma, M., J.D. Hester, F.W. Schaefer III, M.W. Ware, and H.D. Lindquist. 2003. Detection of Cyclospora cayetanensis using a quantitative real-time PCR assay. Journal of Microbiological Methods. 53:27-36. Verdier, R.I., D.W. Fitzgerald, W.D. Johnson Jr., and J.W. Pape. 2000. Trimethoprimsulfamethoxazole compared with ciprofloxacin for treament and prophylaxis of Isospora belli and Cyclospora cayetanensis infection in HIV-infected patients. A randomized controlled trial. Annals of Internal Medicine. 132:885-888. Visvesvara, G.S., H. Moura, E. Kovacs-Nace, S. Wallace, and M.L. Eberhard. 1997. Uniform staining of Cyclospora oocysts in fecal smears by a modified safranin technique with microwave heating. Journal of Clinical Microbiology. 35:730-733. Widmer, G., E.A. Orbacz, and S. Tzipori. 1999. Constitutive expression of small subunit ribosomal RNA transcripts in Cryptosporidium parvum oocysts and intracellular stages. Journal of Parasitology. 85:229-233. Wurtz, R. 1994. Cyclospora: A newly identified intestinal pathogen of humans. Clinical Infectious Diseases. 18:620-623.

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ANTIGENIC VARIATION OF THE VSP GENES OF GIARDIA LAMBLIA

Rodney D. Adam1 and Theodore E. Nash2

1

Dept of Medicine and Microbiology/Immunology, University of Arizona College of Medicine, Tucson, AZ, USA 2 Laboratory of Parasitic Diseases, National Institutes of Health, Bethesda, MD, USA

ABSTRACT Giardia lamblia, a protozoan parasite inhabiting the small intestine, is a common infection worldwide that frequently results in chronic diarrhea, malabsorption and upper gastrointestinal symptoms. Giardia undergoes surface antigenic variation in humans and animal model infections, a phenomenon that may account for both chronicity of infections and the relatively broad mammalian host specificity with genotypically identical organisms found in humans, cats, beavers, and other mammals. The variantspecific surface proteins (VSPs) are an unusual family of related cysteine-rich proteins, from 50 kD to over 200 kD in size, that coat the surface of the trophozoite. Only one VSP of the estimated 150 or more vsp genes is expressed on an individual at any specific time. However, the repertoires of vsp genes may differ depending on the genetic group. VSPs switch spontaneously every 6-12 generations although some Giardia also switch during encystation/excystation. All VSPs have a high cysteine content of about 12 % cysteines that are mostly present as CXXC motifs as well as a highly conserved C-terminus, a surface Zn finger motif, a GGCY motif and other common features. The biological function(s) of VSPs are uncertain but they undergo both immune and biological selection. The molecular mechanism(s) involved in antigenic variation are unknown but because there is the absence of gene movement or gene mutations and only one of 4 practically identical alleles (Giardia are tetraploid) is expressed; epigenetic processes are likely involved. Further studies of the mechanism of antigenic variation and the biological role of the VSPs promise to contribute to our understanding of G. lamblia and its pathogenesis.

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DISCOVERY AND DOCUMENTATION OF ANTIGENIC VARIATION Antigenic variation of major surface proteins or glycoproteins has emerged as a major virulence factor in the protozoan pathogens, as well as in bacteria and viruses. In Giardia, the possibility of antigenic variation was first suggested by the marked difference in surface antigens (Nash and Keister, 1985) among isolates that were quite similar genetically (Nash et al., 1985). These surface antigens are secreted into culture medium in large quantities and are the dominant molecules found when trophozoites are surface labeled. They were initially called excretory-secretory products (Nash et al., 1983; Nash and Keister, 1985), and are now called variant-specific surface proteins (VSP) (Mowatt et al., 1991). Further evaluation of the surface antigens was possible when a monoclonal antibody (MAb 6E7) was produced for a major 170 kD surface antigen of the WB isolate (Nash and Aggarwal, 1986). “Mutants” were subsequently identified that had lost reactivity to MAb 6E7. Subsequently, the WB isolate, which had already been doubly cloned in soft agar, was again cloned twice by limiting dilution. The resulting cloned organisms (WBA6) expressed a 170 kD surface antigen (initially called CRP170 and subsequently VSPA6) that was reactive with MAb6E7. MAb6E7, which is cytotoxic for organisms expressing VSPA6 (Nash and Aggarwal, 1986) was used to select for organisms that were resistant to the antibody and no longer expressed VSPA6. These organisms were cloned and characterized. One of these cloned lines expressed a 64 kD surface antigen (CRP64, VSP1267), while another expressed a 68 kD surface antigen (CRP68, VSP1269). The process of antigenic variation was then carried out one step farther when a MAb for VSP1267 (MAb5C1) was produced and used to select variants that expressed neither VSPA6 nor VSP1267). This variation occurs spontaneously at a rate of approximately once every six to 13 generations (Nash et al., 1990a). Presumably, any given population of trophozoites is dominated by whatever VSP type is favored by the current growth conditions. Giardia lamblia isolates that commonly infect humans fall into two major genotypes (Adam, 2001). The first (Genotype A; Nash Groups 1 and 2, Mayrhofer Assemblage A) includes the WB isolate in which antigenic variation was first documented. Antigenic variation has also been documented in the other major genotype (Genotype B; Nash Group 3, Mayrhofer Assemblage B), of which the GS isolate is the most studied member. The vsp gene repertoires of these two genotypes are very different in terms of antigenicity and sequence similarity. Nonetheless, the general features of the VSPs and vsp genes are the same for both genotypes.

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CHARACTERISTICS OF THE VSPs The VSPs are encoded by a repertoire of genes estimated at 150 in number (Nash et al., 1990a) and perhaps even larger. All of the VSPs are rich in cysteines, which make up approximately 12% of the amino acid content and are frequently found in a CXXC motif (Adam et al., 1988). Earlier work had suggested the presence of free thiol groups on the surfaces of Giardia trophozoites (Gillin et al., 1984), so it was of interest to determine whether the cysteines of the VSPs were present with disulfide bonds or free thiol groups. In the case of TSA417, it was found that all or nearly all the cysteines were present as intrachain disulfide bonds, both in intact trophozoites and in the solubilized protein (Aley and Gillin, 1993). There was no evidence for interchain disulfide bonds. In nonreducing conditions, TSA417 was partially resistant to trypsin, but reduced protein was fully susceptible, suggesting that the disulfide-bonded cysteines played a role in protection from proteases. These findings have also been verified for another VSP (VSP4A1) (Papanastasiou et al., 1997a). The N-termini of the VSPs are signaling peptides approximately 14 (Lujan et al., 1995b) to 17 (Aley and Gillin, 1993) amino acids in length. The remainder of the N-terminal regions of the VSPs are quite variable and are likely responsible for most of the antigenic variability of VSPs. Of those Mabs for which the recognized epitopes have been determined, they react with the N-terminal region or a repeat (in repeat-containing VSPs such as VSPA6). The approximately 38 amino acids comprising the C terminus are highly conserved, with greater than 90% identity among VSPs, and end in an absolutely conserved CRGKA (Mowatt et al., 1991). Approximately 36 AA of the C terminus is cleaved from the secreted protein (Papanastasiou et al., 1996). Therefore, much of this conserved region appears to be involved in membrane anchoring, while the CRGKA is a cytoplasmic tail. VSPs have a highly conserved GGCY motif of unknown function which is generally found toward the C-terminal end, but not in the conserved portion (Nash et al., 1995). VSPs have a number of common motifs including a Zn finger binding motif that consists of a combination of LIM and RING finger Zn finger motifs (Nash and Mowatt, 1993). It is the only known Zn finger motif on the surface of any organism. In other organisms, LIM and Ring finger proteins have diverse functions. Although initially described as DNA binding proteins, most are associated with protein-protein interaction. Many RING finger proteins in higher eukaryotes play a role in ubiquination (Joazeiro and Weissman, 2000). RING proteins self assemble and are able to cataylze or inhibit particular reactions depending on the protein (Kentsis et al., 2002). The potential role of the RING motif in Giardia is unclear but it is possible that VSP bind to one another on the surface or alternatively there are

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RING-like interactions between VSP and gut. Although the biological role of this motif in VSPs is not known, similar motifs in higher mammalian cells play a role in DNA binding and protein-protein interactions. RING finger proteins are capable of interacting between themselves and self-assembly suggesting that VSPs interact between each other on the surface or interact with particular proteins on the surface of the small intestine. In vitro, Zn finger motifs (Nash and Mowatt, 1993; Zhang et al., 1993) are capable of binding Zn and other cations (Lujan et al., 1995b). Surprisingly, Zn binding is not limited to the Zn finger, but is dependent on the presence of free thiol groups (Nash and Mowatt, 1993; Papanastasiou et al., 1997a). The dependence on free thiol groups for binding of zinc to purified VSP suggests that binding is mediated by the cysteines. There are numerous CXXC groups whose spacing suggests they can bind metals. The lack of binding specificity raises questions about biological role of zinc binding for VSP function, but it should be emphasized that the metals naturally bound by zinc fingers have uncommonly been determined in other systems. In the case of the VSPs, it may be that Zn is not the only cation that is present naturally. In fact, differences in cation binding by different VSPs could contribute to their biological diversity. It is also possible that as VSP are shed in rather enormous amounts, smaller peptides are formed that bind metals and contribute to Zn deficiency.

PROTEIN TRAFFICKING AND POST-TRANSLATION MODIFICATION OF THE VSPs The VSPs are found in the ER and lysosome-like vacuoles, as well as diffusely coating the surface of the trophozoite (Pimenta et al., 1991; McCaffery et al., 1994)(Fig. 1). Even though the vegetative trophozoites do

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not have Golgi that can be identified by EM, they do exhibit complex trafficking of proteins (McCaffery et al., 1994), as well as Brefeldin A (BFA)inhibited protein transport, suggesting that functional Golgi may exist (Lujan et al., 1995a). In fact, VSP trafficking was also inhibited by BFA, suggesting the possibility that the VSPs are transported and processed by Golgi or a Golgi-like organelle (Lujan et al., 1995a). This possibility is supported by the finding that VSPs using their own signal peptide are transported through the Golgi in COS cells and become surface-localized (Nash, Conrad, Kulakova, unpublished). The full transmembrane portion of the peptide is required for proper transport of VSPs to the surface (Nash and Kulakova, unpublished). Addition of a peripheral vacuole (PV) localization signal diverts the VSP to the PV suggesting that the default pathway of membrane bound molecules is to the surface unless other signals are present (Touz et al., 2003). Mutation of the Zn finger results in the VSP being stuck in the ER. When the highly conserved GGCYmotif is deleted, the VSP cannot be detected, suggesting either loss of antigenicity or early destruction (Nash and Kulkova, unpublished results). Glycosylation has been proposed for one VSP (Papanastasiou et al., 1997b), but has not been found in two other VSPs (Nash et al., 1983; Lujan et al., 1995b; Marti et al., 2002). There is also evidence for a palmitoylation site which is probably in the conserved C-terminus that is cleaved upon secretion from the membrane (Papanastasiou et al., 1997b; Hiltpold et al., 2000; Papanastasiou et al., 1996)

FEATURES OF THE VSP GENES The vsp genes demonstrate a number of important similarities, but have an equally important list of differences. The first vsp gene to be described (vspA6 or CRP170) consists of a 99 bp 5’ region followed by about 21 to 23 copies of a 195 bp tandem repeat, then 1338 bp at the 3’ end (Adam et al., 1992; Yang and Adam, 1994). The 3’ 120-130 bp are highly conserved for all vsp genes, in keeping with the amino acid conservation in that region. The putative polyadenylation signal (AGTPuAAPy) found in all Giardia genes is immediately preceded by “PuCTPyAGPuT”, which may begin in the stop codon. Giardia is polyploid (most likely tetraploid), so there are approximately four alleles of each gene. One of the interesting characteristics of the Giardia genome is the remarkably small degree of allelic heterozygosity; this holds true for the vsp genes as well. The only example of allelic sequence heterozygosity documented to date for a vsp gene is the observation that the expressed allele of the vspA6 gene (only one of the four alleles is expressed; see below) has eight nucleotide substitutions in comparison to the other alleles. The other difference between the expressed and other alleles is that the

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expressed allele has approximately 22 copies of the repeat while the nonexpressed alleles have 8 or 9 copies. It is likely that the repeats encode an immunologically important region, since the cytotoxic monoclonal antibody (MAb6E7) reacts with the repeat region (Mowatt et al., 1994). Some other vsp genes also contain tandem repeats. The vspC5 gene contains a 66 bp 5’region, approximately 26 copies of a 105 bp repeat in the expressed allele, and a 135 bp 3’ region comprised primarily of the conserved region. Like the vspA6 gene, the expressed allele has more copies of the repeat than do the nonexpressed alleles (20 or 21), but in contrast, there are no sequence differences among the alleles. CRP136 has 23 copies of a 120 bp repeat (Chen et al., 1995) and CRP65 has four copies of a 228 bp repeat (Upcroft et al., 1997). However, the presence of tandem repeats is not universal and in fact may represent the exception rather than the rule. When repeats are found, they begin near the N terminus and are highly immunogenic, but their biologic functions are not known. There are a number of examples of vsp genes that are highly similar throughout the entire reading frame, suggesting that the vsp gene repertoire has been expanded by duplication and divergence. Perhaps the most remarkable example is the vsp1267 gene, which consists of two identical copies in a tail-to-tail arrangement approximately three kb apart. Another example of gene duplication is the vspG3M-B gene (Mowatt et al., 1994), also called vspA6-S1 (Yang and Adam, 1995b), which is highly similar to the vspA6 gene, except that it contains between one and two copies of the 195 bp repeat and is located on a different chromosome. Interestingly, much of the gene has been sequenced from the WB isolate (probably from Afghanistan) (Yang and Adam, 1995b) and the G3M isolate (Mowatt et al., 1994) (from Peru) and the sequences were identical. This observation suggests that the divergence is not occurring rapidly. In other cases, vsp genes are highly similar in the 5’ region followed by substantial divergence, suggesting that the repertoire has been expanded by recombination (Yang and Adam, 1995a). Two WB-derived genes, vsp9B10A and B encode proteins that react with the same MAb. Yet, the genes have enough sequence differences to allow ready distinction (Nash et al., 2001; Carranza et al., 2002). These observations indicate that similar vsp genes may encode VSPs with cross-reactive epitopes. From the GS isolate, the H7 and H7-1 genes (Nash et al., 1995) as well as another member of the H7 gene family (Nash, unpublished) provide an example from the GS isolate, indicating that this expansion of the repertoire also occurs in the genotype B (Group 3) organisms. Unlike the African trypanosomes, telomeric sites are not required for vsp gene expression. The genomic locations of two vsp genes (vspA6 and vspC5) have been determined in isolates expressing these genes and in isolates which have lost expression (Yang and Adam, 1994; Yang and Adam, 1995a). In

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both cases, they were in a single chromosome-internal site which did not vary in the gain or loss of expression. A telomeric location has been documented for some vsp genes (Upcroft et al., 1997; Arkhipova and Morrison, 2001), but the expression competency for these genes has not yet been determined. Thus, the vsp genes are dispersed throughout the genome, being found in chromosome-internal sites as well as telomeric sites and are found on most or all of the chromosomes.

CONTRO OF EXPRESSION OF THE VSPs Lack of DNA sequence alterations or rearrangements associated with antigenic variation - No examples have yet been documented in which a DNA sequence alteration or DNA rearrangement has been associated with gain or loss of expression of a vsp gene. A number of vsp genes have been sequenced from variants in which the gene is expressed and one in which it is not expressed; to date, no sequence alterations or rearrangements have been correlated with gain or loss of expression. In the initial description of antigenic variation (Adam et al., 1988), there was significant variation in the Southern blots of DNA from several cloned lines which either expressed or did not express vspA6 (CRP170). However, there was no clear correlation between the pattern and whether the vspA6 gene was expressed in that particular cloned line. In retrospect, the variation was due do variation of repeat copy number of a 195 bp repeat found in the vspA6 gene. Allele-specific expression - As tetraploid organisms, G. lamblia trophozoites are expected to have four copies of each allele of a vsp gene. In the cases of the vsp genes that do not contain tandem repeats, there have been no examples of allelic sequence heterozygosity, so the alleles cannot be distinguished from each other. However, in the case of the vspA6 gene, which contains a 195 bp repeat, the alleles can be distinguished from each other because of differing repeat copy numbers (8, 9, or approximately 23). Therefore, the alleles can be distinguished by Southern blotting and transcripts can be distinguished by Northern blotting, leading to the observation that only the allele containing 23 repeats is expressed (Yang and Adam, 1994). Interestingly, there are eight nucleotide (five amino acid) substitutions in the open reading frame of the expressed allele compared to the nonexpressed alleles, but no detected changes in the upstream or downstream noncoding regions. vspC5 contains a 105 bp repeat (Yang et al., 1994). The alleles have 26, 21, or 20 copies of the repeat, and again, the allele with the largest number of repeats is expressed. The allele-specific expression of these vsp genes suggests an epigenetic form of control for their expression. More direct evidence for lack of gene movement has been provided by the integration of a tagged vsp into the genome. Southern blots indicate the lack

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of gene movement during gain or loss of expression (Nash and Kulakova, unpublished). Loss of expressed allele - In one case of antigenic variation, the expressed vspA6 allele was lost from the variant that had lost vspA6 expression (Adam et al., 1992). Whether this resulted from subchromosomal deletion or loss of an entire chromosome has not yet been determined. It should also be noted that the exact relationship between loss of the expressed vsp allele and loss of expression has not been determined. It may be a common or uncommon etiology of loss of expression, or may simply be an epiphenomenon. Antigenic variation with encystation and excystation - When exposed to conditions favoring the formation of cysts, trophozoites rapidly differentiate into cysts. During the process of encystation using trophozoites expressing the VSP, TSA417, the VSP disappears from the cell surface (McCaffery et al., 1994). During excystation of these encysted cells, TSA417 is then found in the peripheral vacuoles, suggesting that they might be endocytosed (Svärd et al., 1998). Within 90 minutes after cells are induced to excyst, TSA417 transcripts disappear and are replaced by other vsp transcripts, of which there is one predominant vsp and others found in lesser amounts. This suggests a population shift induced by the process of encystation and excystation, in contrast to the selection of an alternative vsp followed by preferential replication as occurs during vegetative growth. The mechanism by which this process occurs is not known, but does not appear to involve gross DNA rearrangements. In another study using a WB line expressing VSP1267 (WB1267), the proportion of trophozoites expressing VSP1267 decreased from 99% to 94.2% during encystation, while the proportion expressing a different VSP (VSP9B10B) increased from 0 to 38.1%. The numbers add up to greater than 100% because some of the trophozoites expressed more than one VSP while switching. Interestingly, VSP switching during encystation and excystation does not occur with vspH7 of the GS isolate in vitro (Svärd et al., 1998), in mice (Gottstein and Nash, 1991), or in humans (Nash et al., 1990b).

ANTIGENIC VARIATION IN VITRO AND IN VIVO: CLUES TO THE BIOLOGICAL ROLE OF ANTIGENIC VARIATION Since surface antigen variation was initially identified as an in vitro phenomenon of Giardia, a subsequent determination of the biologic role of antigenic variation has been of special interest. Individual trophozoites studied during in vitro growth express only one VSP on their surface at any time except during switching when two VSPs can be transiently detected.

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Rates of switching are both VSP and isolate dependent. Selection of trophozoites following random expression of VSPs appears to be the major way trophozoites expressing certain VSPs predominate. The course of in vivo infection and VSP expression over time is imperfectly documented and depends upon a number of factors including the host species and maturity of their immune system, type of Giardia isolate and particular VSP expressed. Most experimental studies have employed a culture of the GS isolate expressing VSPH7 as the predominant VSP. In humans VSPH7 is expressed until day 17 post-infection when switching to other VSPs begins (Nash et al., 1990b). The course of infection after 21 days has not been studied. In the adult mouse the infection peaks around day 6-10, then decreases so that by day 21 there are barely detectable numbers of organisms. VSP switching occurs at the time humoral responses are detected during the second week of infection (Byrd et al., 1994). The pattern of infection in neonatal mice is similar to that of adult mice, where switching and decreased numbers are evident by day 14; thereafter small numbers of organisms are likely maintained (Gottstein et al., 1990). The study of antigenic variation in gerbils employed different isolates and clones and therefore cannot be directly compared (Aggarwal and Nash, 1988). None of these experimental models show the expected waves of intestinal parasites expressing one and then another VSP that has been characteristic of some blood borne parasites undergoing antigenic variation. Both immunological and non immunological processes have been identified that act as selection factors in vivo. Therefore, the remainder of the discussion will center on the selection of spontaneously occurring variants. The role of adaptive immunity - Adaptive immunity plays an important role in VSP selection. Specific anti-VSP antibodies are easily detected as a result of infection and clearly have growth inhibitory and/or parasiticidal effects on organisms expressing VSPs specific to these antibodies (Nash et al., 1990b; Gottstein et al., 1990; Stager et al., 1997b; Stager et al., 1997a; Stager and Muller, 1997; Stager et al., 1998; Nash and Aggarwal, 1986; Hemphill et al., 1996). Some VSP specific monoclonal antibodies (Mabs) cause complement independent killing of trophozoites in vitro (Nash and Aggarwal, 1986), and depending on the concentration, almost all VSP specific polyclonal or Mab antibodies cause agglutination and growth inhibition. A number of model systems have suggested a role of surface-reactive intestinal IgA antibodies, including anti-VSP antibodies, in controlling infections (Heyworth and Vergara, 1994; Langford et al., 2002; Gottstein et al., 1993; Stager and Muller, 1997; Stager et al., 1998). Following experimental infections of immunocompetent mice with GS/H7, humoral responses to the VSPH7 develop at about the same time that the number of trophozoites in the intestine declines in mice (Byrd et al.,

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1994). Similarly, experimental infections in humans demonstrated VSP switching at the same time as an antibody response to the original VSP was mounted (Nash et al., 1990b). Experimental infections using B cell deficient mice confirm the importance of humoral antibodies in VSP selection in vivo (Singer et al., 2001; Stager and Muller, 1997; Langford et al., 2002). B cell deficient mice infected with GS/VSPH7 are able to control infection, but the organisms do not undergo antigenic variation (Singer et al., 2001), implying that in this model control of infection is T cell mediated but VSP expression is controlled by antibodies or biological selection (see below). In contrast, another study of B cell deficient adult mice concluded that humoral responses were essential to control infections (Langford et al., 2002). Despite the different conclusions of these studies, they confirm that humoral responses to the VSPs occur and that these humoral responses can negatively select particular VSPs. Although the humoral immune response has generally been considered the most important means of controlling Giardia infections, there is increasing evidence of the importance of the T cell response in Giardia muris infections (Stevens et al., 1978; Roberts-Thomson and Mitchell, 1978) and in mice infected by G. lamblia (Singer and Nash, 2000; Gottstein and Nash, 1991). The relative importance of humoral and cell mediated immunity in the resolution of human infections is not known. The role of biological selection - There is an increasing body of evidence suggesting that non-immunological mechanisms also play an important role VSP selection (biological selection). The effects of proteases on the VSP expression and survival of Giardia in vitro was the first indication of the importance of biological selection (Nash et al., 1991). When trophozoites were grown in high concentrations of either trypsin or chymotrypsin, clones were either protease-resistant and survived, or protease-sensitive and died, being replaced by protease-resistant trophozoites that subsequently regrew and repopulated the culture. These organisms expressed a new VSP resistant to the same concentration of protease that killed the earlier population. The existence of protease sensitive and protease resistant VSPs underscores sequence diversity of the VSPs which results in varied biological characteristics. Biological selection has been demonstrated in vivo during the course of experimental infections. Human volunteers were inoculated with trophozoites of the GS isolate expressing low numbers of many different VSPs, including VSPH7 and another VSP recognized by MAb3F6 (Nash et al., 1987; Nash et al., 1990b). Within the first two weeks; before the adaptive immune system could play a role, VSP expression changed so that most now expressed VSPH7 and essentially none expressed the VSP recognized by MAb3F6. Similarly, gerbils inoculated with clones of the WB isolate expressing

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primarily one VSP, began expressing many VSPs within a few days, before the adaptive immune responses could possibly play a role. The change of VSP expression before an adaptive immune response indicates that biological selection had occurred. The clearest example of biological selection in vivo was seen in experimentally infected SCID mice and irradiated immunodeficient gerbils (Singer et al., 2001). In the absence of an adaptive immune system, differences in VSP expression after inoculation of clones expressing one VSP cannot be due to acquired immune responses. Clones of trophozoites, each expressing a different VSP, were inoculated into SCID mice. In contrast to normal mice who are able to suppress the level of infection to barely detectable numbers by 2-3 weeks post inoculation, these animals maintain high numbers of trophozoites in the small intestine. Preferences for certain VSPs were noted. While certain VSP expressing clones were maintained and highly expressed in the intestines during the course of the infection, other VSPs were not maintained, but were replaced by other VSP expressing trophozoites. Parallel experiments using the same clones were performed in gerbils that were immunosuppressed by irradiation, and VSP preferences were also found, but were not identical to those of mice. These results suggest that one of the roles for diversity of VSP expression may be to broaden the host range of the parasite. Therefore the presence of specific VSPs in a host is determined by multiple factors. The expressed VSP must be suitable to the particular intestinal environment and must not be eliminated by the host immune response. It is likely that the number of VSPs that fulfill these criteria for each host is much smaller than the entire VSP repertoire. The precise biological roles of VSPs are not known. The dramatic protease resistance of certain VSPs suggests that they may protect the parasite from the harsh intestinal environment. However, the mechanism by which the particular structure and unusual motifs are beneficial to the parasite is unclear. Although antigenic variation per se is commonly said to be a mechanism designed to escape the host’s immune response, this is not their only role since biological selection also occurs. When we understand the reason for the large repertoire of vsp genes and the important differences and similarities of these genes, we will likely have a much better understanding of the relationship between Giardia and its hosts.

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SUMMARY Table 1: Features of antigenic variation in Giardia lamblia High frequency of change (every 6 to 13 generations) Large repertoire of vsp genes (estimated at 150) Most or all vsp genes appear to have full open reading frames Sequence alterations have not been associated with antigenic variation DNA rearrangements do not appear to be associated with antigenic variation 6. Expression is allele-specific 7. Vsp gene repertoires differ among different genotypes, but are highly similar within genotypes 8. Telomeric location is not required 9. Switching during encystations/excystation has been documented but is not universal

1. 2. 3. 4. 5.

REFERENCES Adam,R.D. 2001. Biology of Giardia lamblia . Clinical Microbiology Reviews 14: 447-475. Adam,R.D., A. Aggarwal, A.A. Lal, V.F. de la Cruz, T. McCutchan, and T.E. Nash. 1988. Antigenic variation of a cysteine-rich protein in Giardia lamblia. Journal of Expimental Medicine 167: 109-118. Adam,R.D., Y.M. Yang, and T.E. Nash. 1992. The cysteine-rich protein gene family of Giardia lamblia: Loss of the CRP170 gene in an antigenic variant. Moleculal and Cellular Biology 12: 1194-1201. Aggarwal,A., and T.E. Nash. 1988. Antigenic variation of Giardia lamblia in vivo. Infection and Immunity 56: 1420-1423. Aley,S.B., and F.D. Gillin. 1993. Giardia lamblia: Post-translational processing and status of exposed cysteine residues in TSA 417, a variable surface antigen. Experimental Parasitology 77: 295-305. Arkhipova,I.R., and H.G. Morrison. 2001. Three retrotransposon families in the genome of Giardia lamblia: two telomeric, one dead. Proceedings of the Natlional Academy of Sciences U. S. A 98: 14497-14502. Byrd,L.G., J.T. Conrad., and T.E. Nash. 1994. Giardia lamblia infections in adult mice. Infection and Immunity 62: 3583-3585. Carranza,P.G., G. Feltes, A. Ropolo, S.M. Quintana, M.C. Touz, and H.D. Lujan. 2002. Simultaneous expression of different variant-specific surface proteins in single Giardia lamblia trophozoites during encystation. Infection and Immunity 70: 5265-5268. Chen,N., J.A. Upcroft, and P. Upcroft. 1995. A Giardia duodenalis gene encoding a protein with multiple repeats of a toxin homologue. Parasitology 111: 423-431. Gillin,F.D., D.S. Reiner, R.B. Levy, and P.A. Henkart. 1984. Thiol groups on the surface of anaerobic parasitic protozoa. Molecular and Biochemical Parasitology 13: 1-12.

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Gottstein,B., P. Deplazes, and I. Tanner. 1993. In vitro synthesized immunoglobulin A from nu/+ and reconstituted nu/nu mice against a dominant surface antigen of Giardia lamblia. Parasitology Research 79: 644-648. Gottstein,B., G.R. Harriman, J.T. Conrad, and T.E. Nash. 1990. Antigenic variation in Giardia lamblia: Cellular and humoral immune response in a mouse model. Parasite Immunology 12: 659-673. Gottstein,B., and T.E. Nash. 1991. Antigenic variation in Giardia lamblia: Infection of congenitally athymic nude and scid mice. Parasite Immunology 13: 649-659. Hemphill,A., S. Stager, B. Gottstein, and N. Muller. 1996. Electron microscopical investigation of surface alterations on Giardia lamblia trophozoites after exposure to a cytotoxic monoclonal antibody. Parasitology Research 82: 206-210. Heyworth,M.F., and J.A. Vergara. 1994. Giardia muris trophozoite antigenic targets for mouse intestinal IgA antibody. Journal of Infectious Diseases 169: 395-398. Hiltpold,A., M. Frey, and P. Köhler. 2000. Glycosylation and Palmitoylation are Common Modifications of Giardia Variant Surface Proteins. Molecular and Biochemical Parasitology 109: 61-65. Joazeiro,C.A., and A.M. Weissman. 2000. RING finger proteins: mediators of ubiquitin ligase activity. Cell 102: 549-552. Kentsis,A., R.E. Gordon, and K.L. Borden. 2002. Self-assembly properties of a model RING domain. Proceedings of the National Academy of Sciences U. S. A 99: 667-672. Langford,T.D., M.P. Housley, M. Boes, J. Chen, M.F. Kagnoff, F.D. Gillin, and L. Eckmann. 2002. Central importance of immunoglobulin A in host defense against Giardia spp. Infectection and Immunity 70: 11-18. Lujan,H.D., A. Marotta, M.R. Mowatt, N. Sciaky, J. Lippincott-Schwartz, and T.E. Nash. 1995a. Developmental induction of Golgi structure and function in the primitive eukaryote Giardia lamblia. Journal of Biological Chemistry 270: 4612-4618. Lujan,H.D., M.R. Mowatt, J.J. Wu, Y. Lu, A. Lees, M.R. Chance, and T.E. Nash. 1995b. Purification of a variant-specific surface protein of Giardia lamblia and characterization of its metal-binding properties. Journal of Biological Chemistry 270: 13807-13813. Marti,M., Y. Li, P. Kohler, and A.B. Hehl. 2002. Conformationally correct expression of membrane-anchored Toxoplasma gondii SAG1 in the primitive protozoan Giardia duodenalis. Infection and Immunity 70: 1014-1016. McCaffery,J.M., G.M. Faubert, and F.D. Gillin. 1994. Giardia lamblia: Traffic of a trophozoite variant surface protein and a major cyst wall epitope during growth, encystation, and antigenic switching. Experimental Parasitology 79: 236-249. Mowatt,M.R., A. Aggarwal, and T.E. Nash. 1991. Carboxy-terminal sequence conservation among variant-specific surface proteins of Giardia lamblia. Molecular and Biochemical Parasitology 49: 215-227. Mowatt,M.R., B.Y. Nguyen, J.T. Conrad, R.D. Adam, and T.E. Nash. 1994. Size heterogeneity among antigenically related Giardia lamblia variant-specific surface proteins is due to differences in tandem repeat copy number. Infection and Immunity 62: 1213-1218. Nash,T.E., and A. Aggarwal. 1986. Cytotoxicity of monoclonal antibodies to a subset of Giardia isolates. Journal of Immunology 136: 2628-2632. Nash,T.E., S.M. Banks, D.W. Alling, J.W. Merritt, Jr., and J.T. Conrad,. 1990a. Frequency of variant antigens in Giardia lamblia. Experimental Parasitology 71: 415-421. Nash,T.E., J.T. Conrad, and M.R. Mowatt. 1995. Giardia lamblia: Identification and characterization of a variant-specific surface protein gene family. Journal of Eukaryotic Microbiology 42: 604-609. Nash,T.E., F.D. Gillin, and P.D. Smith. 1983. Excretory-secretory products of Giardia lamblia. Journal of Immunology 131: 2004-2010.

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Nash,T.E., D.A. Herrington, M.M. Levine, J.T. Conrad, and J.W. Merritt, Jr. 1990b. Antigenic variation of Giardia lamblia in experimental human infections. Journal of Immunology 144: 4362-4369. Nash,T.E., D.A. Harrington, G.A. Losonsky, and M.M. Levine. 1987. Experimental human infections with Giardia lamblia. Journal of Infectious Diseases 156: 974-984. Nash,T.E., and D.B. Keister. 1985. Differences in excretory-secretory products and surface antigens among 19 isolates of Giardia. Journal of Infectious Diseases 152: 1166-1171. Nash,T.E., H.T. Lujan, M.R. Mowatt, and J.T. Conrad. 2001. Variant-specific surface protein switching in Giardia lamblia. Infection and Immunity 69: 1922-1923. Nash,T.E., T. McCutchan, D. Keister, J.B. Dame, J.D. Conrad, and F.D. Gillin. 1985. Restriction-endonuclease analysis of DNA from 15 Giardia isolates obtained from humans and animals. Journal of Infectious Diseases 152: 64-73. Nash,T.E., J.W. Merritt, Jr., and J.T. Conrad. 1991. Isolate and epitope variability in susceptibility of Giardia lamblia to intestinal proteases. Infection and Immunity 59: 13341340. Nash,T.E., and M.R. Mowatt. 1993. Variant-specific surface proteins of Giardia lamblia are zinc- binding proteins. Proceedings of the National Academy of Science. USA 90: 54895493. Papanastasiou,P., T. Bruderer, Y. Li, C. Bommeli, and P. Köhler. 1997a. Primary structure and biochemical properties of a variant- specific surface protein of Giardia. Molecular and Biochemical Parasitology 86: 13-27. Papanastasiou,P., A. Hiltpold, C. Bommeli, and P. Köhler. 1996. The release of the variant surface protein of Giardia to its soluble isoform is mediated by the selective cleavage of the conserved carboxy-terminal domain. Biochemistry 35: 10143-10148. Papanastasiou,P., M.J. McConville, J. Ralton, and P. Köhler. 1997b. The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein. Biochemical Journal 322: 49-56. Pimenta,P.F., P.P. da Silva, and T.E. Nash. 1991. Variant surface antigens of Giardia lamblia are associated with the presence of a thick cell coat: Thin section and label fracture immunocytochemistry survey. Infection and Immunity 59: 3989-3996. Roberts-Thomson,I.C., and G.F. Mitchell. 1978. Giardiasis in mice. I. Prolonged infections in certain mouse strains and hypothymic (nude) mice. Gastroenterology 75: 42-46. Singer,S.M., H.G. Elmendorf, J.T. Conrad, and T.E. Nash. 2001. Biological selection of variant-specific surface proteins in Giardia lamblia. Journal of Infectious Diseases 183: 119124. Singer,S.M., and T.E. Nash. 2000. T-cell-dependent control of acute Giardia lamblia infections in mice. Infection and Immunity 68: 170-175. Stager,S., R. Felleisen, B. Gottstein, and N. Muller. 1997a. Giardia lamblia variant surface protein H7 stimulates a heterogeneous repertoire of antibodies displaying differential cytological effects on the parasite. Molecula and Biochemical Parasitology 85: 113-124. Stager,S., B. Gottstein, and N. Muller. 1997b. Systemic and local antibody response in mice induced by a recombinant peptide fragment from Giardia lamblia variant surface protein (VSP) H7 produced by a Salmonella typhimurium vaccine strain. International Journal for Parasitology 27: 965-971. Stager,S., B. Gottstein, H. Sager, T.W. Jungi, and N. Muller. 1998. Influence of antibodies in mother's milk on antigenic variation of Giardia lamblia in the murine mother-offspring model of infection. Infection and Immunity 66: 1287-1292. Stager,S., and N. Muller. 1997. Giardia lamblia infections in B-cell-deficient transgenic mice. Infection and Immunity 65: 3944-3946. Stevens,D.P., D.M. Frank, and A.A. Mahmoud. 1978. Thymus dependency of host resistance to Giardia muris infection: studies in nude mice. Journal of Immunology 120: 680-682.

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Svärd,S.G., T.C. Meng, M.L. Hetsko, J.M. McCaffery, and F.D. Gillin. 1998. Differentiationassociated surface antigen variation in the ancient eukaryote Giardia lamblia. Molecular Microbiology 30: 979-989. Touz,M.C., H.D. Lujan, S.F. Hayes, and T.E. Nash. 2003. Sorting of encystation-specific cysteine protease to lysosome-like peripheral vacuoles in Giardia lamblia requires a conserved tyrosine-based motif. Journal of Biological Chemistry 278: 6420-6426. Upcroft,P., N. Chen, and J.A. Upcroft. 1997. Telomeric organization of a variable and inducible toxin gene family in the ancient eukaryote Giardia duodenalis. Genome Research 7: 37-46. Yang,Y.M., and R.D. Adam. 1994. Allele-specific expression of a variant-specific surface protein (VSP) of Giardia lamblia. Nucleic Acids Research 22: 2102-2108. Yang,Y., and R.D. Adam. 1995a. A group of Giardia lamblia variant-specific surface protein (VSP) genes with nearly identical 5' regions. Molecular and Biochemical Parasitology 75: 69-74. Yang,Y.M., and R.D. Adam. 1995b. Analysis of a repeat-containing family of Giardia lamblia variant-specific surface protein genes: Diversity through gene duplication and divergence. Journal of Eukaryotic Microbiology 42: 439-444. Yang,Y.M., Y. Ortega, C. Sterling, and R.D. Adam. 1994. Giardia lamblia trophozoites contain multiple alleles of a variant-specific surface protein gene with 105-base pair tandem repeats. Molecular and Biochemical Parasitology 68: 267-276. Zhang,Y.Y., S.B. Aley, S.L. Stanley, Jr., and F.D. Gillin. 1993. Cysteine-dependent zinc binding by membrane proteins of Giardia lamblia. Infection and Immunity 61: 520-524.

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PATHOGENISIS AND IMMUNITY TO ENTAMOEBA HISTOLYTICA

Jessica L. Tarleton and William A. Petri, Jr. University of Virginia Division of Infectious Diseases

ABSTRACT Diseases caused by the parasite Entamoeba histolytica disproportionately affect residents of underdeveloped areas, afflicting places lacking sufficient sanitation, hygiene, and water processing especially. While the recent differentiation between Entamoeba histolytica and the morphologically identical but completely asymptomatic Entamoeba dispar based on genetic and biochemical analyses has enhanced the study of the disease-causing parasite, a mystery still exists as to the factors which still cause about 90% of Entamoeba histolytica infections to remain asymptomatic after colonization. Researchers delving into these areas have identified several features of the parasite—including the galactose and Nacetylgalactosamine inhibitable adherence lectin, proteinases, and amoebapores, all virulence factors in Entamoeba—and of the host, including the association of anti-lectin IgA with resistance to disease, which may determine whether infection is invasive. As specific and easy diagnostics are developed to aid in identifying more precisely the health burden caused by E. histolytica, vaccine candidates are under evaluation. Key words: Entamoeba histolytica, amebiasis, antigen detection test, amebic colitis, amebic liver abscess, proteinase, amoebapore

INTRODUCTION The World Health Organization’s latest estimate implicates Entamoeba histolytica in 50 million infections and 100,000 deaths each year (WHO 1997). Unfortunately, the majority of this burden falls on peoples of underdeveloped regions. Poverty, crowding, poor sanitation and hygiene—a reality in many of the world’s developing areas—foster the growth and spread of this parasite. In these endemic areas, such as Dhaka, Bangladesh, where a major epidemiological survey is underway, as many as 1 in 10 children will die before their birthday, 30% of those deaths attributed to diarrheal diseases (Petri et al. 2000).

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While research has recently made large advances towards a greater understanding of this and other diseases that mainly plague the developing world, it has yet to reveal a large-scale solution to for those afflicted by E. histolytica. Infection with the parasite may manifest itself in several symptom patterns—or usually, not at all: 90% of individuals colonized with the parasite exhibit no symptoms of disease whatsoever (Ayeh-Kumi et al. 2001). Of those who do, amebic colitis and amebic liver abscess represent the most common health problems caused by E. histolytica. Amebic colitis is characterized by a gradual, several-week onset of weight loss and diarrhea, with 70% of patients finding blood in stools (Petri and Singh 1999). Colonic ulcers of these patients usually take on a flask shape characteristic of necrosis caused by E. histolytica. Amebic liver abscess (ALA), which usually occurs independently of colitis, may present acutely or with a several week onset, with symptoms including weight loss, fever, and abdominal pain. Uncommon clinical symptoms of E. histolytica infection include acute necrotizing colitis, ameboma—masses of tissue in the colon, and rectovaginal fistulas, and extraintestinally, cutaneous amebiasis, splenic abscess, and brain abscess (Petri and Singh 1999; Devilliers and Durra 1998). Only the newest epidemiological studies have accurately estimated the impact of E. histolytica on health, for only recently was E. histolytica differentiated from Entamoeba dispar, a morphologically identical but nonpathogenic species (Diamond and Clark 1993; Haque et al. 1998). Previously, E. dispar was simply identified as noninvasive infection by E. histolytica. While it remains virtually impossible to distinguish between E. histolytica and E. dispar microscopically, the two species have been found to deviate both genomically and biochemically (reviewed in Tanyuksel et al. 2001). Four glycolytic enzymes, glucose-phosphate isomerase, phosphoglucomutase, hexokinase, and malic enzyme, vary between the species, which can be demonstrated by gel electrophoresis (Diamond and Clark 1993). Furthermore, E. histolytica and E. dispar differ in DNA restriction patterns and can be differentiated by PCR analysis (Tachibana et al. 1991; Novati et al. 1996) and by E. histolytica-specific monoclonal antibodies (Haque et al. 1998). Even with this recent differentiation it is clear that most E. histolytica infections never induce symptoms. Now, with this recent reclassification, researchers can begin to unravel confusing issue of invasive and noninvasive infections by E. histolytica. Do variations between parasites, between hosts, or a combination of theses factors account for invasiveness and severity of infection? Regions of endemicity for E. histolytica usually include those with inadequate sanitation and water supplies, poor hygiene, crowding, and warmer, wetter climates (Walsh 1988); clearly, temperate developing areas, in

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combination with lack of health resources, are the hardest hit by this infection. In such areas, several host factors, such as sex, immunocompromised state, sexual activity, and even genetic susceptibility may make infection and disease more likely (Jalan and Mailtry 1988). First, the prevalence of amebic liver abscess (ALA) is at least 10 times higher in males than females (Petri and Singh 1999; Hughes and Petri 2000). Heavy alcohol consumption also may be a risk factor for ALA (Petri and Singh 1999); some researchers suggest that a higher tendency towards heavy alcohol use in males could partially explain this observance (Seeto and Rockey 1999; Hughes and Petri 2000). In addition, a large review of 86 papers about E. histolytica written between 1925-1997 reports that, cumulatively, reviewed articles indicate a higher incidence in males than females of all invasive E. histolyltica infections except fulminating amebic colitis, but not a higher incidence of asymptomatic E. histolytica infection (Acuna-Soto et al. 2000). Men who have sex with men are at a higher risk for E. histolytica infection due to a higher frequency of fecal-oral contact (Seeto and Rockey 1999). Recently more attention has focused on E. histolytica infection in HIV infected persons, both in conjunction with higher observed risk in men who have sex with men and also with regards to immunosuppression. Most patients with ALA in two San Francisco hospitals from 1979-1994 who had not lived in or traveled to an endemic area were immunosuppressed, including HIV infection. While cases of amebiasis concurrent with HIV infection continue to be reported (Ohnishi, Murata, and Okuzawa 1994; Fatkenheuer et al. 1997), and immunosuppression has been associated with higher incidence of infection (Seeto and Rockey 1999), no study has conclusively explained a link between HIV and invasive E. histolytica infection. Finally, recent research has revealed a genetic component to disease susceptibility. In children from Dhaka, Bangladesh, where a large epidemiological survey in children is underway, a higher incidence of infection and reinfection correlated with presence of anti-trophozoite IgG in serum. Furthermore, the study showed that anti-trophozoite IgG can be inherited, with siblings of children with the antibody showing 4.8 times greater odds of possessing the same antibody. Finally, most children do not convert from anti-trophozoite IgG negative to positive upon new infection. The authors suggest that serum anti-trophozoite IgG may serve as a marker of genetic deficiencies in innate or acquired immune responses (Haque et al. 2002). In the United States, where, in general, water sanitation and public health are optimal, immigration from an endemic area or recent travel to an endemic area prove to be high risk factors for E. histolytica infection (Seeto and Rockey 1999; Hughes and Petri 2000). Infection with E. histolytica

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usually presents clinically within a year of immigration to the U.S (Petri and Singh 1999). In light of the differentiation of the species E. histolytica and E. dispar, diagnosis by microscopy is no longer sufficient. The TechLab E. histolytica II kit relies on antigen detection sensitive for a surface lectin unique to E. histolytica. Antigen detection tests may occur on either stool or serum samples, but serum testing has proven more sensitive and specific (Haque et al. 2000; 2001). In addition, PCR can be used to detect E. histolytica; though proven less sensitive than antigen detection, it carries the potential advantage of distinguishing between strains (Mirelman et al. 1997; Tanyuksel et al. 2001). However, this technology is not feasible for widespread use in developing countries where diagnosis is needed most. The World Health Organization emphasizes the importance of using diagnostics specific for E. histolytica to improve treatment and management of the disease (WHO 1997). PATHOGENESIS The life cycle of the parasite consists of two stages: the invasive trophozoite and the infectious cyst. In terms of human disease, the trophozoite is responsible for tissue invasion and damage, while the cyst is the means of human transmission (Lushbaugh et al. 1988). Cysts may reside in contaminated food in water, which human hosts ingest (WHO 1997). When the ingested cyst, which can withstand harsh environmental conditions, reaches the small intestine, it excysts into a quadranucleate ameba, which then divides into eight uninuclear trophozoites (Lushbaugh et al. 1988). Evidence from studies with the reptilian parasite Entamoeba invadens imply that the trophozoite may monitor the galactose and N-acetylgalactosamine in its intestinal environment to determine the most favorable time to encyst (Eichinger 2001). A cell surface protein of the trophozoite plays a primary role in this sensory activity as well as critical adhesion activities of the trophozoite. The Gal/GalNAc lectin recognizes galactose (Gal) and N-acetylgalactosamine (GalNAc) (Ravdin and Guerrant 1981; Petri et al. 1987) found on human colonic mucin glycoproteins. Interaction between the lectin of the trophozoite and the host glycoproteins is required for adherence and cytolysis, so that if either is lacking or inhibited, the trophozoite is rendered benign (Ravdin and Guerrant 1981; Le et al. 1988). The Gal/GalNAc lectin is unique to E. histolytica, and it is, in fact, this antigen that the TechLab stool antigen detection test recognizes. The Gal/GalNAc lectin has thus far been characterized in great detail because of its critical role in adherence and cytolysis and its vaccine candidacy. It consists of three subunits (Petri et al. 1989); the heavy (Hgl)

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and light (Lg1) subunits are connected by disulfide bonds (Mai et al. 1999). A third, intermediate subunit (Igl) is noncovalently associated with the Hgl/Lgl dimer (Cheng et al. 1998) (Figure 1).

The carbohydrate recognition domain (CRD), the region of the lectin that binds Gal and GalNAc has been localized to the Hgl (Dodson et al. 1999). The importance of this region lies in its potential as a site for directing inhibitory vaccines or drugs (Petri et al. 2002). Much less is known about the functions of Lgl and Igl in virulence. One study used antisense RNA to inhibit production of Lgl in order to discern its role in virulence. Ameba deficient in Lgl demonstrated reduced cytotoxicty and cytopathogenicity. However, the loss of Lgl may have disrupted the Hgl/Lgl dimer enough to effect virulence for reasons not isolated to the function of the Lgl (Ankri et al. 1999). Because of our current inability to produce knockout mutant ameba, the antisense RNA approach may prove very useful in isolating the functions of many ameba proteins (Ramakrishnan and Petri 2001). The lectin is not only involved in adherence but has been directly identified as a player in cell cytolysis. While it has been shown that hostparasite contact via the Gal/GalNAc lectin is required for cytolysis (Ravdin and Guerrant 1981), the lectin itself contributes directly to the cytolytic activity. Anti-lectin monoclonal antibody (mAb) against epitope 1 of Hgl

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inhibited cytotoxicity but not adherence, indicating disparate mechanisms of the two functions (Saffer and Petri 1991). Among the lectin’s plurality of functions, its cytoplasmic tail is also suspected to have intracellular signaling functions. Amebae expressing a fusion protein containing the cytoplasmic domain of the Hgl subunit have a decreased adherence and lysis ability in vitro as well as decreased severity of liver abscess in an animal model (Vines et al. 1998). The interplay between the extracellular sensory function of the lectin and the internal arrangement of the cytoskeleton may play a large role in pathogenicity. The E. histolytica trophozoite moves by forming a pseudopod in front, into which its streaming cytoplasm moves, and a posterior foot called a uroid; the membrane moves towards the posterior uroid (Calderon et al. 1980). This locomotion is most likely powered by movement and synthesis of the actin cytoskeleton (Bailey 1988). The mechanism called capping occurs when the ameba, as its membrane streams backwards, collects surface antigens on the uroid and then releases the “cap” along with the collected antigens, including the Gal/GalNAc lectin and a 96-kDa surface protein (Arhets et al. 1998). It has been suggested that this shedding of antigens and their attached host antibodies may aid the ameba in evading host immune defenses (Arhets et al. 1995). However, more recent research shows that the cytoskeleton plays a more direct role in ameba pathogenesis: experiments show that ameba with defective cytoskeletons not only fail to form uroids or undergo capping but also fail to kill target cells in vitro, and Arhets et al. suggest that the cytoskeleton plays a critical role in contact-dependent cytotoxicity (Arhets et al. 1998). The cysteine proteinases, secreted into the extracellular environment of an amoeba, have also been identified as major virulence factors of E. histolytica. The proteinases have the ability to degrade elements of the extracellular matrix, including purified fibronectin, laminin, and type I collagen, which would allow the parasite direct access to its target cell (Keene et al. 1986). Furthermore, the cysteine proteinases interfere with both the complement pathway and humoral response of the human immune system. The enzymes can cleave the complement C3 in such a way as to activate the complement pathway, causing lysis of E. dispar but not E. histolytica (Reed and Gigli 1990). The Gal/GalNAc lectin inhibits complement-mediated lysis of E. histolytica by this specific mechanism by cross-reacting with CD59, a membrane inhibitor of C5b-9 in human blood cells (Braga et al. 1992). The contrasting responses of E. dispar and E. histolytica to the proteinases may partially explain their contrasting invasiveness. The parasite does not only activate but may conversely evade complement activity: the proteinases are capable of degrading and inactivating C3 and C5 so as to circumvent this host immune response to the parasite (Reed et al. 1995). In addition, the

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proteinases are also primarily culpable for E. histolytica’s degradation of secretory IgA and IgG, which may limit host humoral immune responses (Kelsall and Ravdin 1993; Tran et al. 1998). One particular proteinase, CP5, has been identified in E. histolytica but is absent from E. dispar. Antisense RNA inhibition of CP5 did not cause a significant decrease in the destruction of cell monolayers by intact trophozoites, but interestingly decreased the trophozoites’ erythrophagocytic ability for unknown reasons (Ankri et al. 1998) E. histolytica also secretes a pore-forming protein, called amoebapore, important in the pathogenesis of disease. Three isoforms, A, B, and C, exist. The peptide can insert ion channels into artificial membranes and depolarize and may also be cytolytic to eukaryotic cells (Lynch et al. 1982; Rosenberg et al. 1989; Leippe et al. 1994). The amoebapores have antibacterial properties, disrupting membrane integrity of Gram-positive bacteria (Leippe et al. 1994). It has been suggested that the primary function of amoebapores is the killing of ingested bacteria (Leippe et al. 1994). IMMUNITY Human immune defenses against Entamoeba histolytica begin with the mucus lining the intestinal wall. Conflictingly, while mucus serves to restrict the ameba’s access to colonic epithelial cells (Chadee et al. 1987), it also provides a rich environment for colonization. Mucins, making up a portion of human colonic mucus, produce the O-linked proteins, galactose and N-acetylgalactosamine, to which the Gal/GalNAc lectin specifically binds (Ravdin et al. 1985; Chadee et al. 1988). The relative benefit to burden ratio of colonic mucin glycoproteins, as to whether they help in dispelling the parasite or encourage colonization and therefore invasive infection, is still not clear (Tse and Chadee 1991). Inside the intestine, secretory antibodies are produced against parasites. Specifically, IgA is produced in response to invasive infection by E. histolytica. Anti-lectin IgA has been found in human milk (Grundy et al. 1983), serum (Abu-El-Magd et al. 1996), and saliva, and a saliva antibody test has even been proposed, though not widely adopted, for diagnosis of infection (Del Muro et al. 1990). Recently, secretory IgA against the Gal/GalNAc lectin has been associated with defense against infection (see below). The inflammatory response provides a third mechanism of human defense against the parasite, and recent research has taken us a step closer to understanding this means of protection as well. Until recently, the dynamic between leukocytes recruited by the immune system and amebic trophozoites remained unclear: different research reported that polymorphonuclear neutrophils killed trophozoites but also that the reverse was true (Gillin et al. 1988). In intestinally derived cell lines, Entamoeba histolytica induces the

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production of certain pro-inflammatory cytokines and chemokines such as and IL-6 and IL8 (Kim et al. 1995; Yu et al. 1997; reviewed in Stanley 2001). Eckmann et al. (1995) demonstrated the variety of cytokines, including and IL-6, secreted from cultured human cells upon contact with trophozoites; the release of these cytokines appear to be mediated by production of cytolytically released Yu and Chadee (1997) found that upon stimulation with amebic proteins, amebic secretory proteins, or live trophozoites, colonic cells produced IL-8, even without cellcell contact or colonic cell damage. In order to determine whether such cytokine production occurs in vivo, the severe combined immunodeficiency disease mouse with a human intestinal xenograft (SCID-HU-INT mouse) provided an opportunity to use an animal model to produce a human immune response in the region of human tissue, the intestine. Seydel et al.(1997) determined that when the human intestinal tissue of these mice was infected with trophozoites, the xenografts produced the human inflammatory cytokines and IL-8 in direct response to E. histolytica, and that cytokine production occurred in regions other than those in direct contact with the parasites. However, while this research showed definitively that cytokines contributed to the inflammatory response seen in infected tissues, the exact cause of the inflammation and tissue damage for which E. histolytica earned its name was still up in the air. Several more studies began to illuminate this issue. By inhibiting transcription factor, which controls expression of IL-1, IL-6, and IL-8, cytokine production and gut inflammation in E. histolytica infected tissue was greatly reduced. Interestingly, tissue damage was also greatly reduced, possibly implicating the host inflammatory response in its own tissue damage (Seydel et al. 1998). Employing the SCID-HU-INT mouse model again, it was shown that mice whose neutrophils had been depleted before infection with E. histolytica suffered significantly less tissue damage (Seydel et al. 1998). It appears as though trophozoites, when lysing host neutrophils, spill their toxic contents into host tissues and create damage not directly caused by the parasites (Jarumilinta and Kradolfer 1964; Guerrant et al. 1981). Further elucidation of human immune response came with Campbell et al.’s (2000) findings that the 170 kDa subunit of the Gal/GalNAc lectin induced IL-12 production in human THP-1 macrophages; the researchers even localized the immunogenic region of the subunit to between amino acids 596998. As IL-12 promotes Th1 cytokine differentiation and in turn macrophage protection, the induction of IL-12 by the lectin figures significantly in the vaccine search. In the hopes of designing a vaccine for amebiasis, the role of acquired immunity to E. histolytica infection bears much importance, and huge strides

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have been made in this area recently. Clinical observations have noticed more severe cases of amebiasis in patients in immunocompromised states, including patients taking corticosteroids (Ratcliffe 1988). Animal models in various states of immunosuppression have also displayed more severe infections (Ghadirian and Meerovitch 1981a; Ghadirian and Meerovittch 1981b; Ghadirian and Kongshavn 1985; reviewed in Huston and Petri 1998). A recent study by Haque et al. (2001) on a large group of children in Dhaka, Bangladesh, an endemic area, has greatly advanced our understanding about protective immunity against amebiasis: they found that the presence of anti-lectin IgA provides a marker of acquired immunity. Evidence of this lies in several specific findings: first, none of 64 children in the initial survey who were found to have stool anti-lectin IgA were colonized with E. histolytica; second, after a one-year prospective study, children with stool anti-lectin IgA showed 64% fewer new E. histolytica infections (while 39% of children showed new infections during the one-year period). Finally, IgA appeared in the system concurrently with resolution of infection. A subsequent study has pinpointed the area of the anti-lectin response to the carbohydrate recognition domain (CRD), refining the marker of acquired immunity to IgA produced against the CRD of the Gal/GalNAc lectin (see Fig. 2). In addition, the possibility of anti-trophozoite IgG marking genetic insufficiency of immune defenses will surely be filled out in much more detail in the future (Haque et al. 2002).

The human immune responses to Entamoeba histolytica infection remain only generally defined, but the hope of a vaccine pushes research further into knowledge of this subject. Vaccine development appears a distant but feasible goal. (Table 1).

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New information on the existence of acquired immunity has supported that hope, and growing knowledge of the highly- conserved and pathogenically critical Gal/GalNAc lectin has made it the object of several vaccine tests (Petri and Ravdin 1991). Several other amebic proteins present possible vaccine candidates as well. The serine-rich Entamoeba histolytica protein (SREHP) is also wellconserved, immunogenic, and has not been found in other Entamoeba species, including E. dispar (Stanley et al. 1990). Immunization with recombinant SREHP protected 100% of gerbils from ALA when administered through a single intradermal injection (Zhang 1994). A 29-kDa cysteine rich antigen has also been shown to elicit a protective response in animal models (Soong 1995). In addition, 150- and 170-kilodalton surface antigens of E. histolytica have conferred protection against ALA in hamsters (Cheng and Tachibana 2001). The amoebapore and a few cysteine proteinases have also been considered as possible vaccine candidates (Huston and Petri 1998). Clearly, many issues should be considered when seeking a solution to the health problems caused by E. histolytica—it is imperative that we treat the overarching health risks posed by poverty and poor sanitation, as well as research further the nature of human immunity, the variations in virulence among parasite strains, and vaccine candidates. Diseases of poverty pose multifaceted challenges encompassing both science and society. As research into each moves forward, the pool of information from which we can draw upon and combine will grow, leading us toward better health solutions to amebic diseases.

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REFERENCES Abu-El-Magd, I., C.J.G. Soong, A.M. El-Hawey, and J.I. Ravdin. 1996. Humoral and mucosal IgA antibody response to a recombinant 52-kDa cysteine-rich portion of the Entamoeba histolytica galactose-inhibitable lectin correlates with detection of native 170-kDa lectin antigen in serum of patients with amebic colitis. Journal of Infectious Diseases 174: 157162. Acuna-Soto, R., J.H. Maguire, and D.F. Wirth. 2000. Gender distribution in asymptomatic and invasive amebiasis. American Journal of Tropical Medicine and Hygiene 62:733-739. Ankri, S., F. Padilla-Vaca, T. Stolarsky, L. Koole, U. Katz, and D. Mirelman. 1999. Antisense inhibition of expression of the light subunit (35 kDa) of the Gal/GalNAc lectin complex inhibits Entamoeba histolytica. Molecular Microbiology 33:327-337. Ankri, S., T. Stolarsky, and D. Mirelman. 1998. Antisense inhibition of expression of cysteine proteinases does not affect Entamoeba histolytica cytopathic or haemolytica activity but inhibits phagocytosis. Molecular Microbiology 28:777-785. Arhets, P., J.C. Olivo, P. Gounon, P. Sansonetti, and N. Guillen. 1998. Virulence and functions of myosin II are inhibited by overexpression of light meromysin in Entamoeba histolytica. Infection and Immunity 9:1537-1547. Arhets, P., P. Gounon, P. Sansonetti, and N. Guillen. 1995. Myosin II is involved in capping and uroid formation in the human pathogen Entamoeba histolytica. Infection and Immunity 63:4358-4367. Ayeh-Kumi, P.F., A.M. Ibnekarim, L.A. Lockhart, C.A. Gilchrist, W.A. Petri, Jr., and R. Haque. 2001. Entamoeba histolytica: Genetic diversity of clinical isolates from Bangladesh as demonstrated by polymorphisms in the serine-rich gene. Experimental Parasitology 99:80-88. Bailey, G. 1988. Chemotaxis by Entamoeba histolytica. In Amebiasis, J.I. Ravdin (ed.). John Wiley & Sons, New York, p. 347-50. Braga, L., H. Ninomiya, J. J. McCoy, S. Eacker, T. Wiedmer, C. Pham, S. Wood, P. J. Sims, and W. A. Petri. 1992. Inhibition of the complement membrane attack complex by the galactose-specific adhesin of Entamoeba histolytica. Journal of Clinical Investigation 90:1131-1137. Calderon, J., M.L. Munoz, and H.M. Acosta. 1980. Surface redistribution and release of antibody-induced caps in Entamoeba. Journal of Experimental Medicine 151:184. Campbell, D., B.J. Mann, and K. Chadee. 2000. A subunit vaccine candidate region of the Entamoeba histolytica galactose-adherence lectin promotes interleukin-12 gene transcription and protein production in human macrophages. European Journal of Immunology 30:423430. Chadee, K., M.L. Johnson, E. Orozco, W.A. Petri, Jr., and J.I. Ravdin. 1988. Binding and internalization of rat colonic mucins by the Gal/GalNAc adherence lectin of Entamoeba histolytica. Journal of Infectious Diseases 158: 398-406. Chadee, K., W.A. Petri, Jr., D.J. Innes, and J.I. Ravdin. 1987. Rat and human colonic mucins bind to and inhibit adherence lectin of Entamoeba histolytica. Journal of Clinical Investigation 80: 1245-1254. Cheng, X.J. and H. Tachibana. 2001. Protection of hamsters from amebic liver abscess formation by immunization with the 150- and 170-kDa surface antigens of Entamoeba histolytica. Parasitology Research 87:126-130. Cheng, X.J., H. Tsukamoto, Y. Kaneda, and H. Tachibana. 1998. Identification of the 150 kDa surface antigen of Entamoeba histolytica as a galactose- and N-acetyl-D-galactosamineinhibitable lectin. Parasitology Research 84: 632-639. De Villiers, J.P. and G. Durra. 1998. Case report: amoebic abscess of the brain. Clinical Radiology 53:307-309.

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Del Muro, R., E. Acosta, E. Merino, W. Glender, and L. Ortiz-Ortiz. 1990. Diagnosis of intestinal amebiasis using salivary IgA antibody detection. Journal of Infectious Diseases 162: 1360-1364. Diamond, L.S. and C.G. Clark. 1993. A rediscription of Entamoeba histolytica Shaudinn 1903 (amended Walker 1903) separating it from Entamoeba dispar (Brumpt 1925). Journal of Eukaryote Microbiology 40:340-344. Dodson, J.M., P.W. Lenkowski, Jr., A.C. Eubanks, T.F. Jackson, J. Napodeno, D.M. Lyerly, L.A. Lockhart, B.J. Mann, and W.A. Petri, Jr. 1999. Infection and immunity mediated by the carbohydrate recognition domain of the Entamoeba histolytica Gal/GalNAc lectin. Journal of Infectious Diseases 179: 460-466. Eckmann, L., S.L. Reed, J.R. Smith, and M.F. Kagnoff. 1995. Entamoeba histolytica trophozoites induce an inflammatory cytokine response by cultured human cells through the paracrine action of cytolytically released Journal of Clinical Investigation 96: 1269-1279. Eichinger, D. 2001. A role for a galactose lectin and its ligand during encystment of Entamoeba histolytica. Journal of Eukaryotic Microbiology 48: 17-21. Fatkenheuer, G., G. Arnold, H.M. Steffen, C. Franzen, M.Schrappe, V. Diehl, and B. Salzberger. 1997. Invasive amoebiasis in two patients with AIDS and cytomegalovirus colitis. Journal of Clinical Microbiology 35:2168-2169. Ghadirian, E. and E. Meerovitch. 1981a. Effect of immunosuppression on the size and metastatis of amoebic liver abscesses in hamsters. Parasite Immunology 3:329-338. Ghadirian, E. and E. Meerovitch. 1981b. Effect of splenectomy on the size of amoebic liver abscesses and metastic foci in hamsters. Infection and Immunity 31:571-573. Ghadirian, E. and P.A. Kongshaven. 1985. The effect of spelectomy on resistance of mice to Entamoeba histolytica infection. Parasite Immunology 7:479-487. Gillin, F.D., D.S. Reiner, and A. Zenian. 1988. Interaction of Entamoeba histolytica with nonimmune and immune intestinal defenses. In Amebiasis, J.I. Ravdin (ed.). John Wiley & Sons, New York, p. 444-45. Grundy, M.S., L. Cartwright-Taylor, L. Lundin, C. Thors, and G. Huldt. 1983. Antibodies against Entamoeba histolytica in human milk and serum in Kenya. Journal of Clinical Microbiology 7: 753-758. Guerrant, R.L., J. Brush, J.I. Ravdin, J.A. Sullivan, and G.L. Mandell. 1981. Interaction between Entamoeba histolytica and human polymorphonuclear neutrophils. Journal of Infectious Diseases 143: 83-93. Haque R., I.K.M. Ali, Z. Akther, and W.A. Petri, Jr. 1998. Comparison of PCR, isoenzyme analysis, and antigen detection for diagnosis of Entamoeba histolytica infection. Journal of Clinical Microbiology 36: 449-452. Haque, R., P. Duggal, I.M. Ali, M.B. Hossain, D. Mondal, R.B. Sack, B.M. Farr, T.H. Beaty, and W.A. Petri, Jr. 2002. Innate and acquired resistance to amebiasis in Bangladeshi children J Infect Dis 2002; 186:547-552. Haque, R., M.A. Ibnekarim, R.B. Sack, B.M. Farr, G. Ramakrishnan, and W.A. Petri, Jr. 2001. Amebiasis and mucosal IgA antibody against Entamoeba histolytica adherence lectin in Bangladeshi children. Journal of Infectious Diseases 183:1787-1793. Haque, R., N.U. Mollah, I.K.M. Ali, K. Alam, A. Eubanks, D. Lyerly, and W.A. Petri Jr. 2000. Diagnosis of amebic liver abscess and intestinal infection with the TechLab Entamoeba histolytica II antigen detection and antibody tests. Journal of Clinical Microbiology. 38: 3235-3239. Hughes, M.A. and W.A. Petri, Jr. 2000. Amebic liver abscess. Infections of the Liver 14:565582.

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Huston, C.D. and W.A. Petri, Jr. 1998. Host-pathogen interaction in amebiasis and progress in vaccine development European Journal of Clinical Microbiology and Infectious Diseases 17:601-614. Jalan, K.N. and T.K. Maitra. 1988. Amebiasis in the Developing World The Morphology of Entamoeba histolytica. In Amebiasis, J.I. Ravdin (ed.). John Wiley & Sons, New York, p. 535-555. Jarumilinta, R. and F. Kradololer. 1964. The toxic effect of Entamoeba histolytica on leukocytes. Annual Trop. Med. Parasitol. 58:375-381. Keene, W. E., M. G. Pettit, S. Allen, and J. H. McKerrow. 1986. The major neutral proteinase of Entamoeba histolytica. J. Exp. Med. 163:536-549. Kelsall, B. L., and J. I. Ravdin. 1993. Degradation of human immunoglobulin A by Entamoeba histolytica. Journal of Infectious Diseases 168:1319-1322. Kim, J.M., H.C. Jung, K. Im, Y.J. Cho, and C.Y. Kim. 1995. Interleukin-8 gene expression in the human colon epithelial cell line, HT-29, exposed to Entamoeba histolytica. Korean Journal of Parasitology 33:357-364. Le, E., A. Becker, and S.L. Stanley. 1988. Use of Chinese hamster ovary cells with altered glycosylation patterns to define the carbohydrate specificity of Entamoeba histolytica adhesions. Journal of Experimental Medicine 167:1725-1730. Leippe, M., J. Andrä, R. Nickel, E. Tannich and H.J. Müller-Eberhard. 1994. Amoebapores, a family of membranolytic peptides from cytoplasmic granules of Entamoeba histolytica: isolation, primary structure, and pore formation in bacterial cytoplasmic membranes. Molecular Microbiology 14: 895-904. Lushbaugh, W.B, and J.H. Miller. 1988. The Morphology of Entamoeba histolytica. In Amebiasis, J.I. Ravdin (ed.). John Wiley & Sons, New York, p. 41-42. Lynch, E.C., I.M. Rosenberg, C. Gitler. 1982. An ion-channel forming protein produced by Entamoeba histolytica. The EMBO Journal 1:801-804. Mai, Z., S. Ghosh, M. Frisardi, B. Rosenthal, R. Rogers, and J. Samuelson. 1999. Hsp60 is targeted to a cryptic mitochondrion-derived organelle (“Crypton”) in the microaerophilic protozoan parasite Entamoeba histolytica. Molecular and Cellular Biology 19: 2198-2205. Mirelman D., Y. Nuchamowitz, and T. Stolarsky. 1997. Comparison of use of enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes for simultaneous detection of Entamoeba histolytica and E. dispar. Journal of Clinical Microbiology 35: 2405-2407. Novati S, Sironi M, Granata S, Bruno A, Gatti S, Scaglia M, Bandi C. 1996. Direct sequencing of the PCR amplified SSU rRNA gene of Entamoeba dispar and the design of primers for rapid differentiation from Entamoeba histolytica. Parasitology 112: 363-369. Ohnishi K., M. Murata, and E. Okuzawa. 1994. Symptomatic amebic colitis in a Japanese homosexual AIDS patient. Internal Medicine 33: 120-122. Petri, W.A., Jr., M.D. Chapman, T. Snodgrass, B.J. Mann, J. Broman, and J.I. Ravdin. 1989. Subunit structure of the galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica. Journal of Biological Chemistry 264: 3007-3012. Petri, W.A., Jr., R. Haque, D. Lyerly, and R.R. Vines. 2000. Estimating the impact of amebiasis on health. Parasitology Today 16:320-321. Petri, W.A., R. Haque, and B.J, Mann. 2002. The bittersweet interface of parasite and host: Lectin-carbohydrate interactions during human invasion by the parasite Entamoeba histolytica. Annual Review of Microbiology 56:39-64. Petri, W.A., Jr. and J.I. Ravdin. 1991. Protection of gerbils from amebic liver abscess by immunization with the galactose-specific adherence lectin of Entamoeba histoytica. Infection and Immunity 59: 97-101. Petri, W.A., Jr. and U. Singh. 1999. Diagnosis and management of amebiasis. Clinical Infectious Diseases 29:1117-1125.

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Petri, W.A., Jr., R.D. Smith, P.H. Schlesinger, C.F. Murphy, and J.I. Ravdin. 1987. Isolation of the galactose binding adherence lectin of Entamoeba histolytica. Journal of Clinical Investigation. 80: 1238-1244. Ramakrishnan, G. and W.A. Petri, Jr. 2001. Getting sense and finding function in protozoa. Nature Biotechnology 19: 213-215. Ratcliffe, G.E. 1988. Amoebic disease precipitated by corticosteroids prescribed for tuberculous pleural effusions. Tubercule 69: 219-221. Ravdin, J.I. and R.L. Guerrant. 1981. Role of adherence in cytopathogenic mechanisms of Entamoeba histolytica. Journal of Clinical Investigation 68: 1305-1313. Ravdin, J.I., J.E. John, L.I. Johnston, D.J. Innes, and R.L. Guerrant. 1985. Adherence of Entamoeba histolytica to rat and human colonic mucosa. Infection and Immunity 48:292297. Reed, S. L., J. A. Ember, D. S. Herdman, R. G. DiScipio, T. E. Hugli, and I. Gigli. 1995. The extracellular neutral cysteine proteinase of Entamoeba histolytica degrades anaphylatoxins C3a and C5a. Journal of Immunology 155:266-274. Reed, S. L., and I. Gigli. 1990. Lysis of complement-sensitive Entamoeba histolytica by activated terminal complement components. Initiation of complement activation by an extracellular neutral cysteine proteinase. Journal of Clinical Investigation 86:1815-1822. Rosenberg I., D. Bach, L.M. Loew, and C. Gitler. 1989. Isolation, characterization and partial purification of a transferable membrane channel (amoebapore) produced by Entamoeba histolytica. Molecular and Biochemical Parasitology 33:237-247. Saffer, L.D. and W.A. Petri, Jr. 1991. Role of the galactose lectin of Entamoeba histolytica in adherence-dependent killing of mammalian cells. Infection and Immunity 59: 4681-4683. Seeto, R.K., and D.C. Rockey. 1999. Amebic liver abscess: epidemiology, clinical features, and outcome. Western Journal of Medicine 170:104-109. Seydel, K.B., E. Li, P.E. Swanson, and S.L. Stanley, Jr. 1997. Human intestinal epithelial cells produce pro-inflammatory cytokines in response to infection in a SCID mouse-human intestinal xenograft model of amebiasis. Infection and Immunity 65: 1631-1639. Seydel, K.B., E. Li, Z. Zhang, and S.L. Stanley, Jr. 1998. Epithelial cell-initiated inflammation plays a crucial role in early tissue damage in an amebic infection of human intestine. Gastroenterology 115: 1446-1453. Soong, C.G., B.E. Torian, M.D. Abd-Alla, T.F.H.G. Jackson, V. Gathiram, and J.I. Ravdin. 1995. Protection of gerbils from amebic liver abscess by immunization with recombinant Entamoeba histolytica 29-kilodalton antigen. Infection and Immunity 63:472-477. Stanley, S.L. 2001. Pathophysiology of amebiasis. Trends in Parasitology 17:280-285. Stanley, S.L., A. Becker, C. Kunz-Jenkins, L. Foster, and E. Li. 1990. Cloning and expression of a membrane antigen of Entamoeba histolytica possessing multiple tandem repeats. Proceedings of the National Academy of Sciences USA 87:4976-4980. Tachibana H, Ihara S, Kobayashi S, Kaneda Y, Takeuchi T, and Watanabe Y. 1991. Differences in genomic DNA sequences between pathogenic and nonpathogenic isolates of Entamoeba histolytica identified by polymerase chain reaction. Journal of Clinical Microbiology 29:2234-2239. Tannich, E., R.D. Horstmann, J. Knobloch, and H.H. Arnold. 1989. Genomic DNA differences between pathogenic and nonpathogenic Entamoeba histolytica. Proceedings of the National Academy of Science USA 86:5118-5122. Tanyuksel, M., H Tachibana, and W.A. Petri, Jr. 2001. Amebiasis, an emerging disease. In Emerging Diseases 5 ed., W.M. Scheld, W.A Craig, and J.M. Hughes (eds.), ASM Press, Washington, D.C., p.197-212. Tran, V. Q., D. S. Herdman, B. E. Torian, and S. L. Reed. 1998. The neutral cysteine proteinase of Entamoeba histolytica degrades IgG and prevents its binding. Journal of Infectious Diseases 177:508-511.

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Tse, S.K. and K. Chadee. 1991. The interaction between intestinal mucus glycoproteins and enteric infections. Parasitology Today 7: 163-172. Vines, R.R., G. Ramakrishnan, J.B. Rogers, L.A. Lockhart, B.J. Mann, and W.A. Petri, Jr. 1998. Regulation of adherence and virulence by the Entamoeba histolytica lectin cytoplasmic domain, which contains a beta2 integrin motif. Molecular Biology of the Cell 9: 2069-2079. Walsh, J.A. 1988. Prevalence of Entamoeba histolytica infection. In Amebiasis, J.I. Ravdin (ed.). John Wiley & Sons, New York, p.93-105. World Health Organization. 1997. Amoebiasis. W. H. O. Weekly Epidemiological Record 72:97-100. Yu, Y. and K. Chadee. 1997. Entamoeba histolytica stimulates interleukin 8 from human colonic epithelial cells without parasite-enterocyte contact. Gastroenterology 112: 15361547. Zhang, T., P.R. Cieslak, and S.L. Stanley. 1994. Protection of gerbils from amebic liver abscess by immunization with a recombinant Entamoeba histolytica antigen. Infection and Immunity 62:1166-1170.

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INNATE AND T CELL-MEDIATED IMMUNE RESPONSES IN CRYPTOSPORIDIOSIS

Carol R. Wyatt1 and Vincent McDonald2 1

Department of Diagnostic Medicine/Pathogiology, Mosier Hall, Kansas State University, Manhattan, KS 66506-5705 2 Barts and the London School of Medicine and Dentistry, Department of Adult and Paediatric Gastroenterology, DDRC, Turner St, London E1 2AD, UK

ABSTRACT A variety of innate immune responses may help to control early parasite replication, initiate inflammation and generate signals for T cell activation. Ultimately, elimination of infection involves cells that are of the Th1 (cell-mediated immunity) phenotype, but there may be a protective role for lymphocytes of the Th2 type (antibody-dependent responses). Intraepithelial lymphocytes have increased activity as a result of infection and may be important in the anti-cryptosporidial immune response. Key words: Cryptosporidium, innate immunity, T cells, intraepithelial lymphocytes, cytokines, antibodies

INTRODUCTION Since its recognition as an important zoonotic pathogen, much research on Cryptosporidium parvum has focused on host responses to the parasite. Responses that lead to disease and that end the infection are important to understand because they offer clues that can lead to effective control of cryptosporidiosis. This chapter describes our current understanding of the contributions of the host to disease pathogenesis and to clearance of C. parvum infection.

INNATE IMMUNITY Innate immunity comprises T cell-independent immune responses that provide at least partial protection against infection. Non-lymphoid cells and natural killer (NK) cells may be involved. Products from these cells such as cytokines, complement or antibiotic peptides may be important in microbicidal mechanisms.

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Infection of epithelial cells by C. parvum activates a key inflammatory transcription factor (Chen et al. 2001). Expression of the proinflammatory chemokines IL-8, and RANTES, is dependent, and increased expression of these molecules has been observed in infected cell lines (Gargalla et al. 1997; Laurent et al. 1997). Treatment of cells with a specific inhibitor of reduced secretion of IL-8 (Chen et al. 2001). also promotes production of cationic antimicrobial peptides such as defensins and, in bovine C. parvum infection, increased expression of a was demonstrated (Tarver et al. 1998). Synthesized anti-microbial peptides were previously shown to inhibit the viability of C. parvum sporozoites (Arrowood et al. 1991). The initial stimulus for activation is unclear but parasite products might interact with Toll-like receptor (TLR) molecules or intracellular proteins such as NOD-1 (Philpott et al. 2001) which recognize evolutionarily conserved bacterial products. Cryptosporidial infection of epithelial cells also induces prostaglandin production (Laurent et al. 1998). Prostaglandins may have different protective effects because they can modulate T cell activation and decrease inflammation. Prostaglandins might also act by promoting increased mucin production and contributing to mechanisms of diarrhea. An increased incidence of cryptosporidiosis has been reported in HIV patients with mutations in the mannose-binding lectin (MBL) gene (Kelly et al. 2001). MBL activates complement via lectin-associating serine proteases that cleave C4 and C2. MBL and C4 adhered to the surface of sporozoites in vitro and complement components and MBL were detected in the gut lumen of AIDS patients with diarrhea (Kelly et al. 2001). MBL may block parasite attachment to host cells or induce the complement membrane attack complex. A crucial innate response to intracellular pathogens is production by NK cells. NK cell activation is induced by proinflammatory cytokines from accessory cells including IL-12 and and is inhibited by IL-10 (Tripp et al. 1993). Cryptosporidial antigen can generate a similar response in splenic NK cells from SCID mice which lack T + B cells (McDonald et al. 2000). can partially protect against C. parvum infection of SCID and T celldeficient nude mice. Affected mice develop progressive, chronic, and sometimes fatal infections (Ungar et al. 1990; Mead et al. 1991; reviewed by Theodos, 1998). Treatment with neutralizing antibodies exacerbates infection and hastens the onset of morbidity (Ungar et al. 1991; Chen et al. 1993; McDonald and Bancroft, 1994; Urban et al. 1996). mice have heavier infections and greater morbidity than do SCID mice with an intact gene (Hayward et al. 2000). Induction of in neonatal SCID mice

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involves IL-12 since injection with the cytokine can suppress C. parvum development. IL-12 treatment increases expression in the ileum, and administration of neutralizing anti- IL-12 to SCID mice increases parasite reproduction. directly inhibits C. parvum reproduction in human enterocyte cell lines by preventing sporozoite invasion and by depleting intracellular Fe availablility (Pollok et al. 2001). It is not clear, however, if NK cells are the source of in these infection models, scid-nu/nu mice carrying the Beige mutation, making them deficient in NK cell function, had more intense C. parvum infections than did SCID mice (Mead et al. 1991). Other attempts to demonstrate NK involvement were unsuccessful, however. SCID mice given anti-ASGMl antibodies to deplete NK cells, or IL-2 to stimulate NK cell maturation, were not made more susceptible to infection (Rohlman et al. 1993; McDonald and Bancroft, 1994). And, no role for in activation of production was seen, as injection with neutralizing antibodies had no effect on parasite reproduction (Chen et al. 1993; McDonald and Bancroft, 1994).

IMMUNOPATHOGENESIS Dysregulation of the mucosal immune response is critical to the pathogenesis of numerous intestinal diseases. For example, Crohn’s is a Th1 (see later) inflammatory condition associated with T cell and macrophage infiltration of the lamina propria and expression of the proinflammatory cytokines IL-12,IL-18, and (Garside, 2000) that can induce villous atrophy and crypt hyperplasia. Indeed, treatment of Crohn’s with antiTNF monoclonal antibodies can be a highly effective therapy. Interestingly, C. parvum infection increases susceptibility of mice to develop spontaneous colitis similar to ulcerative colitis (Sacco et al. 1998). Villous atrophy and crypt hyperplasia are common features during cryptosporidial infection and lymphocytes, macrophages and neutrophils infiltrate the infection site (Tzipori, 1988). C. parvum-infected calves have increased numbers of villous and cell subpopulations (Abrahamsen et al. 1997). In mice, cattle and humans, infection leads to increased intestinal expression of (Kapel et al. 1996; White et al. 2000; Wyatt et al. 2001). Induction of other proinflammatory cytokines such as IL12 in cattle (Payer et al. 1998) and (Lacroix et al. 2001) in mice has also been described. Cytokines activate intestinal metalloproteases, which damage the tissue (MacDonald et al. 1999), and may also impair ion movement or induce epithelial cell apoptosis. Inflammation increases production of secretagogues, including prostaglandins, neural peptides and reactive nitrogen

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or oxygen intermediates (Gaginella et al. 1995). Secretagogue activity and damage to the epithelium that prevents absorption are likely to be involved in the development of cryptosporidial diarrhea. The regulation of the inflammatory response is poorly understood. In calves, the numbers of and cells subsides in parallel with recovery from a primary C. parvum infection (Abrahamsen et al. 1997), suggesting the infection, at least in part, drives the inflammatory response. In murine C. parvum infection, plasticity in the Th response was reported as a waning of a Th1 response late in infection as a Th2 response emerged (Aguirre et al. 1998). This change in polarity may allow Th1 and Th2 cytokines to downregulate each other’s inflammatory functions. Intestinal regulatory cells (Th3 or Tr1) that produce the antiinflammatory cytokines or IL-10 prevent inflammatory bowel disease in mice (Groux and Powrie, 1999). inhibits the severe intestinal inflammation normally observed in C57BL/6 mice infected orally with Toxoplasma gondii (Buzoni-Gatel et al. 2001). Intraepithelial lymphocytes (IEL) produce and the cytokine may inhibit chemokine production by enterocytes. Significantly, expression is increased in intestinal biopsies from humans infected with C. parvum (Robinson et al. 2000) and a similar finding has been made in necropsies from infected mice (McDonald et al., in preparation). In addition to its direct anti-inflammatory properties, can ameliorate damage to epithelial barrier function caused by in vitro following C. parvum infection (Roche et al. 2000).

T CELLS INVOLVED IN ADAPTIVE IMMUNITY Much of the data on the role of T cells in immunity to cryptosporidial infection has been obtained from murine infection models. T cells are essential for parasite clearance as chronic C. parvum or C. muris infections are observed in T cell-deficient mice whereas infections in normal mice are selflimiting (reviewed by McDonald and Bancroft, 1998). Infections in immunocompromised mice can be cleared by injection of T cell-containing lymphoid cells from normal mice (reviewed by Theodos, 1998). In addition, a study with T cell receptor (TCR) knockout mice suggested that cells are necessary for recovery whereas the cells are not essential but may provide some protection in neonates (Waters and Harp, 1996). Within the cell population, the two major cell subsets are the Thelper cells and the cytotoxic cells. Cryptosporidial infection is exacerbated in mice deficient in cells through a mutation in MHC class II expression, or by treatment with anti-CD4 (Aguirre et al. 1994; Ungar

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et al. 1991; McDonald et al. 1994). Also, SCID mice injected with lymphoid cells from normal mice do not recover from infection if cells are depleted from the donor cells (Chen et al. 1993; McDonald and Bancroft 1994; McDonald et al. 1994; Perryman et al. 1994). In human disease, lifethreatening infection is common in HIV patients with low cell counts (Blanshard et al. 1992). Restoration of cells following antiretroviral therapy confers resistance to C. parvum infection (Farthing, 2000). cells are, therefore, essential for elimination of Cryptosporidium infection. The cell subpopulation appears to be less important in immunity. Mice deficient in MHC-class I expression, and so lacking cells, were no more susceptible to infection than control mice (Aguirre et al. 1994). Similarly, in mice depletion of cells by antibody, the ability to control infection was either unaffected or only mildly diminished (Ungar et al. 1990, 1991; Chen et al. 1993; McDonald and Bancroft, 1994; McDonald et al. 1994, 1996; Perryman et al. 1994). Splenic lymphocytes incubated with C. parvum antigens contained proliferating but not cells (Harp et al. 1994). cells are activated by MHC class I-restricted presentation of peptides normally originating from proteins present in the cell cytoplasm (Williams et al. 1996). The extracytoplasmic location of Cryptosporidium may mean that the class I presentation pathway is deficient.

TH1 AND TH2 RESPONSES In response to infection, cells differentiate into Th1 or Th2 cells that mediate cell-mediated (Th1) or humoral (Th2) responses are defined by patterns of cytokine production; IL-2, IL-12, and are associated with Th1 and IL-4, IL-5, and IL-10 with Th2. Intracellular pathogens induce Thl responses, while extracellular pathogens promote Th2 responses. Th1 and Th2 responses can cross-regulate each other, and the outcome of an infection can depend upon the timing and magnitude of each response. C57BL/6 mice are susceptible to C. parvum, and either die or remain persistently infected, suggesting a role for in clearance. In infected neonatal C57BL/6 mice, mucosal IL-4 and IL-10 are expressed, while in neonatal parental strain mice, which clear the infection, both Th1 and Th2 cytokines are expressed (Lacroix et al. 2001). Splenocytes from C57BL/6 mice stimulated in vitro with a sporozoite antigen preparation express IL-5, but not IL-2, IL-4, or suggesting a bias toward production of IgA antibodies (Mead and You, 1998). In contrast, C57BL/6 mice have prolonged infections compared with the parent strain (Aguirre et al. 1998). Administration of neutralizing results in

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increased numbers of shed oocysts but does not prolong infection in adult C57BL/6 mice while anti-IL-4 treatment prolongs the infection. BALB/c mice infected with C. parvum clear the infection (Mead and You, 1998), and repeated IL-12 injection increases oocyst output and prevents recovery (Smith et al. 2001). Splenocytes from BALB/c mice stimulated in vitro with sporozoite antigens express IL-2, IL-4, IL-5, and and those stimulated with a recombinant 23 kDa sporozoite surface protein express IL-2, IL-5, and but not IL-4 and IL-12, mRNA (Bonafonte et al. 2000), suggesting that C. parvum infection induces Th1- and Th2-like responses in BALB/c mice. Neonatal BALB/c mice also clear the infection; however, they shed more oocysts compared with parental strain mice (McDonald, unpublished observations).

GUT MUCOSAL LYMPHOCYTES Mucosal lymphocytes are the first lymphoid cells to contact mucosal pathogens. Intraepithelial lymphocytes (IEL) are located at the base of the villi, beneath epithelial cell tight junctions, while lamina propria lymphocytes (LPL) lie within the villi beneath the lamina propria membrane (reviewed by Kraehenbuhl and Neutra, 1992). Both IEL and LPL have been implicated in immune responses to cryptosporidial infection. induction of chemokine expression in intestinal epithelial cells from C. parvum-infected mice results in recruitment of T cells and macrophages into the mucosa (Lacroix-Lamande et al. 2002). Consistent with this observation, neonatal calf ileal explants inoculated in vitro with C. parvum oocysts accumulate mucosal and lymphocytes around epithelial cells when compared with control explants (Wyatt et al. 1999). IEL from 3 day infected calves express IL-10 mRNA in conjunction with the expression of several sporozoite epitopes, and also express combinations of and (Wyatt et al. 2002). LPL have increased numbers of and lymphocytes compared with controls (Abrahamsen et al. 1997). During cryptosporidial diarrhea, neonatal calf IEL have increased percentages of lymphocytes and lymphocytes and express (Wyatt et al. 1997). Calves that have recovered from diarrhea have elevated and and LPL that express but not IL10, IL-4, or IL-2 (Wyatt et al. 2001). Adoptive transfer of IEL from primed BALB/c donor mice to SCID mice was shown to clear C. muris infection in a response requiring cells and (McDonald et al. 1996; Culshaw et al. 1997. Subsequent similar work

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using C. parvum confirmed that IEL from primed donor mice can clear a persistent infection in SCID mice (Adjei et al. 2000).

HUMORAL IMMUNITY Antibodies can be important in the immune response to a pathogen. Antibodies that fix complement can promote lysis while those that bind molecules critical to host cell invasion can neutralize the infectivity of the pathogen. However, the role of antibodies in immunity to C. parvum is unclear. C. parvum infected mice excrete fecal antibodies to sporozoite antigens, and IgA isotype antibodies can reduce the severity of infection in neonatal mice (reviewed by Wyatt, 2000). However, HIV-1 infected patients with chronic cryptosporidiosis and low cell counts make serum IgM, IgG, and IgA, and salivary IgA, antibodies to C. parvum, but these antibodies do not clear the infection (Cozon et al. 1994). P23 is an immunodominant sporozoite surface glycoprotein that contains at least two epitopes sensitive to antibody neutralization. In cattle, antibody-rich colostrum generated by immunizing cattle with recombinant p23 protects calves against cryptosporidial diarrhea, but does not clear the infection (reviewed by Wyatt, 2000). C. parvum infected calves excrete antibodies to p23 (Wyatt et al. 2000). IgA antibodies are prominent 5-6 days after inoculation, and IgM and peak 7 days after inoculation while the calves have diarrhea. antibodies peak as calves recover from diarrhea. Bovine antibodies are regulated by IL-4, while antibodies are regulated by (reviewed by Wyatt, 2000), and calves recovering from cryptosporidial diarrhea have LPL that contain and switched B cells, and express but not IL-4 (Wyatt et al. 2001). Thus, calves recovering from cryptosporidiosis have LPL capable of generating an immune response that includes antibodies and

CONCLUSIONS These observations raise several issues. First is the dual role of the Th1 response. Th1 cytokines mediate the inflammation that leads to disruption of intestinal architecture and cell functions, resulting in diarrhea. Yet Th1 cytokines are also necessary to clear C. parvum infection. Thus, any intervention designed to stimulate a protective Th1 response must also control the inflammatory effects on the gut. Second is the apparent protective role of Th2 cytokines such as IL-4. As an intracellular pathogen, control of Cryptosporidium should not require a Th2

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response. However, antibodies that can prevent entry of the pathogen into epithelial cells can decrease the level of infection in the gut, thereby lessening disease manifestations. Perhaps IL-4 induces production and elaboration into the gut lumen of antibodies that prevent sporozoites and merozoites from infecting additional epithelial cells. Third is the role of cells that infiltrate the gut. If MHC I presentation is ineffective due to the extracytoplasmic location of the parasite, then what is the role of cells? An answer might lie in the type of T cell expressing the CD8 molecule. In several models, gut mucosal cells express CD8. These cells are skewed toward developing a Th1-like response (Yin et al. 2000), and they can downregulate inflammatory responses (Egan and Carding, 2000). Indeed, if gut mucosal cells can modulate inflammation within the mucosa during C. parvum infection, then manipulation of these cells might minimize inflammation while the infection is being controlled.

REFERENCES Abrahamsen M.S., C.A. Lancto, B. Walcheck, W. Layton, M.A. Jutila. 1997. Localization of and lymphocytes in Cryptosporidium parvum-infected tissues in naïve and immune calves. Infection and Immunity 65:2428-2433. Adjei A.A., A.K. Shrestha, M. Castro, F.J. Enriquez. 2000. Adoptive transfer of immunity with intraepithelial lymphocytes in Cryptosporidium parvum-infected severe combined immunodeficient mice. American Journal of Medical Science. 320:304-309. Aguirre S.A., P.H. Mason, and L.E. Perryman. 1994. Susceptibility of major histocompatibility (MHC) class I- and class II-deficient mice to Cryptosporidium parvum infection. Infection and Immunity 62: 697-699. Aguirre S.A., L.E. Perryman, W.C. Davis, and T.C. McGuire 1998. IL-4 protects adult C57BL/6 mice from prolonged Cryptosporidium parvum infection: analysis of and lymphocytes in gut-associated lymphoid tissue during resolution of infection. Journal of Immunology 161: 1891-1900. Arrowood M.J., J.M. Jaynes, and M.C. Healey. 1991. In vitro activities of lytic peptides against sporozoites of Cryptosporidium parvum. Antimicrobial Agents and Chemotherapy 35:224-227. Blanshard C., A.M. Jackson, D.C. Shanson, N. Francis, and B.G. Gazzard.1992. Cryptosporidiosis in HIV seropositive patients. Quarterly Journal of Medicine 85:813-823. Bonafonte M.-T., L.M. Smith, and J.R. Mead. 2000. A 23-kDa recombinant antigen of Cryptosporidium parvum induces a cellular immune response on in vitro stimulated spleen and mesenteric lymph node cells from infected mice. Experimental Parasitology. 96:32-41. Buzoni-Gatel D., H. Debbabi, J. Menneciat, J. Martin, A.C. Lepage, J.D. Schwartzman, and L.H. Kasper. 2001. Murine ileitis after intracellular parasite infection is controlled by TGFintraepithelial lymphocytes. Gastroenterology 120:914-924. Chen W., J.A. Harp, and A.G. Harmsen. 1993. Requirements for CD4+ cells and gamma interferon in resolution of established Cryptosporidium parvum infection in mice. Infection and Immunity 61: 3928-3932. Chen X.-M., S. Levine, P.L. Splinter, P.S. Tietz, A.L.Ganong, C.Jobin, G.J.Gores, C.V. Paya,

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and N.F. LaRusso. 2001. Cryptosporidium parvum activates nuclear factor in biliary epithelia preventing epithelial cell apoptosis. Gastroenterology 120:1774-1783. Culshaw R.J., G.J. Bancroft, and V. McDonald. 1997. Gut intraepithelial lymphocytes induce immunity against Cryptosporidium infection through a mechanism involving gamma interferon production. Infection and Immunity. 65:3074-3079. Egan P.J., and S.R. Carding. 2000. Downmodulation of the inflammatory response to bacterial infection by gammadelta T cells cytotoxic for activated macrophages. Journal of Experimental Medicine. 191:2145-2158. Farthing M.J.G. 2000. Clinical aspects of human cryptosporidiosis. Contributions to Microbiology 6:50-74. Fayer R., L. Gasbarre, P. Pasquale, A. Canals, S. Almeria, and D. Zarlenga. 1998. Cryptosporidium parvum infection in bovine neonates: dynamic clinical, parasitic and immunologic patterns. International Journal for Parasitology 28:49-56. Gaginella T.S., J.F. Kachur, H. Tamai, and A. Keshavarzian. 1995. Reactive oxygen and nitrogen metabolites as mediators of secretory diarrhea. Gastroenterology 109:2019-2028. Gargala G., A. Delaunay, L. Favennec, P. Brasseur, and J.J. Ballet. 1997. In vitro interactions of human peripheral blood and intestinal intraepithelial lymphocytes with Cryptosporidium parvum and C. parvum permissive cell lines. Journal of Eukaryotic Microbiology 44: 71S72S. Garside P. 2001. A role for IL-18 in intestinal inflammation. Gut 47:6-9. Groux H., and Powrie F. 1999. Regulatory T cells and inflammatory bowel disease. Immunology Today 20: 442-445. Harp J.A., Whitmire W.H., and Sacco R. 1994. In vitro proliferation by murine CD4+ cells in response to Cryptosporidium parvum antigen. Journal of Parasitology 80:67-72. Hayward A.R., K. Chmura, and M. Cosyns. 2000. is required for innate immunity to Cryptosporidium parvum in mice. Journal of Infectious Diseases 182:1001-1004. Kapel N., Y. Benhamou, M. Buraud, D. Magne, P. Opolon, and J.-G.Gobert. 1996. Kinetics of mucosal ileal gamma-interferon response during cryptosporidiosis in immunocompetent neonatal mice. Parasitology Research 82: 664-667. Kelly P., D.L. Jack, A. Naeem, B. Mandana, R.C.G. Pollok, N.J. Klein, M.W. Turner, and M.J.G. Farthing. 2000. Mannose-binding lectin is a component of innate mucosal defense against Cryptosporidium parvum. Gastroenterology 119:1236-1242. Kraehenbuhl J.-P., and M.R. Neutra. 1992. Molecular and cellular basis of immune protection of mucosal surfaces. Physiological Reviews. 72:853-879. Lacroix S, R. Mancassola, M. Naciri, and F. Laurent. 2001. Cryptosporidium parvum-specific mucosal response in C57BL/6 mice and gamma interferon-deficient mice: role of tumor necrosis factor alpha in protection. Infection and Immunity 69:1635-1642. Lacroix-Lamande S., R. Mancassola, M. Naciri, and F. Laurent. 2002. Role of gamma interferon in chemokine expression in the ileum of mice and in a murine intestinal epithelial cell line after Cryptosporidium parvum infection. Infection and Immunity. 70:2090-2099. Laurent, F., L. Eckmann, T.C. Savidge, C. Morgan, C. Theodos, M. Naciri, and M.F. Kagnoff. 1997. Cryptosporidium parvum infection of human intestinal epithelial cells induces polarized secretion of C-X-C chemokines. Infection and Immunity 65:5067-5073. Laurent F,M.F. Kagnoff, T.C. Savidge, M. Naciri, and L. Eckmann. 1998. Human intestinal epithelial cells respond to Cryptosporidium parvum infection with increased prostaglandin H synthase 2 expression and prostaglandin and production. Infection and Immunity 66:1787-1790.

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MacDonald T.T., M. Bajaj-Elliott, and S.L.F. Pender. 1999. T cells orchestrate intestinal mucosal shape and integrity. Immunology Today 20:505-510 McDonald V., and G.J. Bancroft. 1994. Mechanisms of innate and acquired immunity in SCID mice infected with Cryptosporidium parvum. Parasite Immunology 16:315-320. McDonald V., and G.J. Bancroft. 1998. Immunological control of Cryptosporidium infection. Chemical Immunology 70: 103-123. McDonald V., H.A. Robinson, J.P. Kelly, and G.J. Bancroft. 1994. Cryptosporidium muris in adult mice: adoptive transfer of immunity and roles of CD4 versus CD8 cells. Infection and Immunity 62: 2289-2294. McDonald V., H.A. Robinson, J.P. Kelly, and G.J. Bancroft. 1996. Immunity to Cryptosporidium muris in mice is expressed through gut CD4+ intraepithelial lymphocytes. Infection and Immunity 64: 2556-2562. McDonald V., R. Smith, H. Robinson, and G. Bancroft. 2000. Host immune responses against Cryptosporidium. Contributions to Microbiology 6: 75-91. Mead J.R., M.J. Arrowood, R.W. Sidwell, and M.C. Healey. 1991. Chronic Cryptosporidium parvum infections in congenitally immunodeficient SCID and nude mice. Journal of Infectious Diseases 163:1297-1304. Mead, J.R., and X. You. 1998. susceptibility differences to Cryptosporidium parvum infection in two strains of gamma interferon knockout mice. Journal of Parasitology. 84:1045-1048. Perryman L.A., P.H. Mason, and C.E. Chrisp. 1994. Effect of spleen cell populations in resolution of Cryptosporidium parvum infection in SCID mice. Infection and Immunity 62: 1474-1477. Pollok R.C.G., M.J.G. Farthing, M. Bajaj-Elliott, I.R. Sanderson, and V. McDonald.2001. Interferon gamma induces enterocyte resistance against infection by the intracellular pathogen Cryptosporidium parvum. Gastroenterology 120:99-107. Philpott D.J., S.E. Girardin, and P.J. Sansonetti. 2001. Innate immune responses of epithelial cells following infection with bacterial pathogens. Current Opinion in Immunology 13:410416. Roche J.K., C.A.P. Martins, R. Cosme, R. Fayer, and R.L. Guerrant. 2000. Transforming growth factor ameliorates intestinal epithelial barrier disruption by Cryptosporidium parvum in vitro in the absence of mucosal T lymphocytes. Infection and Immunity 68:56355644. Robinson P., P.C. Okhuysen, C.L. Chappell, D.E. Lewis, I. Shahab, S. Lahoti, and A.C. White Jr. 2000. Transforming growth factor is expressed in the jejunum after experimental Cryptosporidium parvum infection in humans. Infection and Immunity 68:5405-5407. Rohlman V., T. Kuhls, D. Mosier, D. Crawford, and R. Greenfield. 1993. Cryptosporidium parvum infection after abrogation of natural killer cell activity in normal and severe combined immunodeficiency mice. Journal of Parasitology 79: 295-297. Sacco R.E, J.S. Haynes, J.A. Harp, W.R. Waters, and M.J. Wannemuehler. 1998. Cryptosporidium parvum initiates inflammatory bowel disease in germfree T cell receptormice. American Journal of Pathology 153:1717-1722. Smith, L.M., M.-T. Bonafonte, L.D. Campbell, and J.R. Mead. 2001. Exogenous interleukin12 (IL-12) exacerbates Cryptosporidium parvum infection in gamma interferon knockout mice. Experimental parasitology. 98:123-133. Tarver AP, D.P. Clark, G. Diamond, J.P. Russell, H. Erdjument-Bromage, P. Tempst, K.S. Cohen, D.E. Jones, R.W. Sweeney, M. Wines, S. Hwang, and C.L. Bevins. 1998. Enteric

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defensin: molecular cloning and characterization of a gene with inducible intestinal epithelial cell expression associated with Cryptosporidium parvum infection. Infection and Immunity 66: 1045-1056. Theodos C.M. 1998. Innate and cell-mediated immune responses to Cryptosporidium parvum. Advances in Parasitology 40:87-119. Tripp C.P., S.F. Wolf, and E.R.Unanue. 1993. Interleukin-12 and tumor necrosis factor alpha are costimulators of interferon gamma production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin-10 is a physiologic antagonist. Proceedings of the National Academy of Sciences 90:3725-3729. Tzipori S. 1988. Cryptosporidiosis in perspective. Advances in Parasitology 27:63-129.Ungar B.L.P., J.A. Burris, C.A. Quinn, and F.D. Finkelman.1990. New mouse models for chronic Cryptosporidium infection in immunodeficient hosts. Infection and Immunity 58: 961-969. Ungar B.L.P., T.-C. Kao, J.A. Burris, and F.D. Finkelman. 1991 Cryptosporidium infection in an adult mouse model. Independent roles for IFN-g and CD4+ T lymphocytes in protective immunity. Journal of Immunology 147:1014-1022. Urban J.F. Jr., R. Fayer, S.-J. Chen, W.C. Gause, M.K. Gately, and F.D. Finkelman. 1996. IL-12 protects immunocompetent and immunodeficient neonatal mice against infection with Cryptosporidium parvum. Journal of Immunology 156:263-268. Waters W.R., and J.A. Harp. 1996. Cryptosporidium parvum infection in T-cell receptor and mice. Infection and Immunity 64:1854-1857. White A.C., P. Robinson, P.C. Okhuysen, D.E. Lewis, I. Shahab, S. Lahoti, H.L. Dupont, and C.L. Chappell. 2000. expression in jejunal biopsies in experimental human cryptosporidiosis correlates with prior sensitization and control of oocyst excretion. The Journal of Infectious Diseases 181:701-709. Wyatt, C.R. 2000. Cryptosporidium parvum and mucosal immunity in neonatal cattle. Animal Health Research Reviews. 1:25-34. Wyatt, C.R., W.J. Barrett, E.J. Brackett, D.A. Schaefer, and M.W. Riggs. 2002. Association of IL-10 expression by mucosal lymphocytes with increased expression of Cryptosporidium parvum epitopes in infected epithelium. Journal of Parasitology. 88:281-286. Wyatt, C.R., E.J. Brackett and W.J. Barrett. 1999. Accumulation of mucosal T lymphocytes around epithelial cells after in vitro infection with Cryptosporidium parvum. Journal of Parasitology. 85:765-768. Wyatt, C.R., E.J. Brackett, P.H.Mason, J. Savidge, and L.E. Perryman. 2000. Excretion patterns of mucosally delivered antibodies to p23 in Cryptosporidium parvum infected calves. Veterinary Immunology and Immunopathology. 76:309-317. Wyatt, C.R., E.J. Brackett, L.E. Perryman, A. C. Rice-Ficht, W.C. Brown, and K.I. O’Rourke. 1997. Activation of intestinal intraepithelial T lymphocytes in calves infected with Cryptosporidium parvum. Infection and Immunity. 65:185-190. Wyatt C.R., J. Bracket, and J. Savidge. 2001. Evidence for the emergence of a type -1-like immune response in intestinal mucosa of calves recovering from cryptosporidiosis. Journal of Parasitology 87:90-95. Williams D.B., Vassilakos A., and W.K Suh. 1996. Peptide presentation by MHC class I molecules. Trends in Cellular Biology 6:267-273. Yin, Z., D.-H. Zhang, T. Welte, G. Bahtiyar, S. Jung, L. Liu, X.-Y. Fu, A. Ray, and J. Craft. 2000. Dominance of IL-12 over IL-4 in cell differentiation leads to default production of failure to down-regulate IL-12 receptor expression. Journal of Immunology. 164:3056-3064.

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RATIONALE APPROACHES TO TREATING CRYPTOSPORIDIUM, CYCLOSPORA, GIARDIA AND ENTAMOEBA

Jan R. Mead1 and Pablo Okhuysen2 1

Atlanta Veterans Affairs Medical Center and Department of Pediatrics, Emory School of Medicine, Atlanta, GA 30033 2 Department of Medicine, Division of Infectious Diseases and School of Public Health, University of Texas Health Science Center in Houston, Houston, TX 77030

ABSTRACT Treatment options for diarreal illness caused by intestinal protozoa differ depending on the particular protozoa. Cryptosporidium parvum, an important parasite among the immunocompromised and young children of developing nations remains refractory to all conventional therapies. Cyclospora cayetanensis, an emerging pathogen can be treated with trimethoprim-sulfamethoxazole, however the discovery of alternative treatments remains hampered by the inability to cultivate the parasite or establish an animal model. Despite being a frequent cause of diarrheal illness throughout the world, relatively few agents outside of metronidazole are used in therapy against Giardia lamblia. While metronidazole is the agent of choice for the treatment of most amoebic infections, proper diagnosis is important to distinguish between pathogenic and non-pathogenic Entamoeba species. Key words: Cryptosporidium parvum, Cyclospora, Giardia, Entamoeba histolytica

CRYPTOSPORIDIUM Several groups could benefit from an effective therapy against cryptosporidiosis: children, the elderly, and immunocompromised groups such as, organ transplant recipients, and cancer patients. Chemotherapy would also be useful among the immunocompetent in more severe cases and in outbreak situations to curb the spread of disease. Among the more important groups in need of therapy are individuals infected with human immunodeficiency virus (HIV). While cryptosporidiosis is serious in immunocompetent people, it can be devastating to those with AIDS.

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The use of potent highly active anti-retroviral therapy (HAART) in patients with advanced HIV infection can improve or lead to the clearance of C. parvum from the stools. Patients treated with double anti-retroviral therapy or protease inhibitors have demonstrated excellent responses and sustained therapeutic effects after follow-up (Ives and Easterbrook, 2001; Miao, et al., 2000). Although HAART is not thought to have a direct effect on C. parvum, its indirect activity is apparently a result of improved immunological status, related to increased cell counts rather than to modulation of the viral load. Few options exist for patients with AIDS in whom highly active antiretroviral therapy fails or is not an option. Controlled treatment trials have provided some useful information but have not resulted in identifying an effective therapeutic agent. While many compounds have demonstrated activity using in vitro assays, fewer therapeutic agents have demonstrated significant potency using animal models. Paromomycin has been one of the most widely used agents to treat cryptosporidial infections in AIDS patients. It is a poorly absorbed aminoglycoside that is related to neomycin and kanamycin. It achieves high concentrations in the gut, in part due to poor bio-availability. It has shown efficacy in animal models (Healey et al., 1995; Tzipori et al., 1995) and as a prophylactic treatment in neonatal calves (Fayer and Ellis, 1993), lambs (Viu et al., 2000) and goats (Johnson et al., 2000; Mancassola et al., 1995). In humans, the drug was considered partially effective in that decreases in symptoms (frequency of stools) were noted but the parasite was not eradicated (Flannigan and Soave, 1993). In a placebo-controlled, double blind study, treatment was found to be partially effective (White et al., 1994). However, a more recent study showed no significant difference between the treated and placebo groups (Hewitt, et al., 2000). Consequently, many patients initially respond to paromomycin treatment with a decrease in diarrhea but then relapse. Combination therapy with paromomycin and azithromycin demonstrated some efficacy in relieving symptoms and parasitic burden. In a open-label, combination study (Smith et al., 1998) patients with AIDS, chronic cryptosporidiosis, and low cell counts were given 1.0 gram of paromomycin b.i.d. plus 600 mg of azithromycin once per day for 4 weeks, followed by paromomycin alone for 8 weeks. Both partial clinical and parasitological responses were observed, as treatment of cryptosporidiosis with azithromycin and paromomycin was associated with significant reduction in oocyst excretion and some clinical improvement. Nitazoxanide, a broad-spectrum antiparasitic agent, was found to be effective in a clinical trials performed in Mali, Mexico and Egypt (Doumbo et al., 1997; Rossignol et al., 1998; Rossignol et al., 2001); however, clinical trials in the U.S. have not been encouraging, and nitazoxanide has not been

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approved by the US Food and Drug Administration. In a double-blind, placebo-controlled study, patients were randomly treated with either 500 or 1000 mg nitazoxanide, or placebo orally b.i.d. for 14 days (Rossignol et al., 1998). Patients on nitazoxanide then crossed over to placebo while the placebo patients crossed over to nitazoxanide therapy at either the high or low dose depending on their randomization. Both doses of nitazoxanide produced parasitological cure rates superior to the placebo responses (12/19 [63%, P = 0.016] for patients receiving 1 g/d and 10/15 [67%, P = 0.013] for those receiving 2 g/d). In a recent study, a 3-day course of nitazoxanide significantly improved the resolution of diarrhea, parasitological eradication and mortality in HIV-seronegative, but not HIV-seropositive children (Amadi et al., 2002). Other therapies have been evaluated with various degrees of reported efficacy. None have been proven totally efficacious. These include spiramycin, (Portnoy et al., 1984; Saez-Llorens et al., 1989; Vargas et al., 1993), clarithromycin, (Holmberg et al., 1998; Jordan et al., 1996), rifabutin, (Fichtenbaum et al., 2000), roxithromycin (Uip et al., 1998) and the use of hyperimmune bovine anti-Cryptosporidium colostrum (Fries et al., 1994; Okhuysen et al, 1998). In the absence of an effective therapy for cryptosporidiosis, fluid support and maintaining electrolyte balance is of paramount importance for severe cases among immunocompetent and immunocompromised individuals.

A CHALLENGING RESISTANCE

PARASITE

WITH

INNATE

Why are so many therapies not effective against this parasite? It seems that C. parvum has a natural resistance to drug therapy. Several factors may contribute to this lack of efficacy. These include: 1) the parasite’s unique location in the host cell which may affect drug concentration (transported from the host cell across to the parasite); 2) lack of specific targets or differences in targets either at the molecular or structural levels; 3) differences in biochemical pathways; 4) existence of transport proteins or efflux pumps that transport drugs out of the parasite or into the host cell. In the last few years the establishment of large scale sporozoite-expressed sequence tag (EST) and genomic sequence tag (GST) projects have aided in the identification of C. parvum genes and in the understanding of phylogenetic similarities and diversities within the genome. C. parvum, it appears, is more divergent and less related to the other coccidia. It has been proposed that C. parvum is more closely related to the gregarine parasites within the Apicomplexa. Comparisons of small-subunit ribosomal RNA gene sequences have demonstrated that the gregarine/Cryptosporidium clade is

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separate from the other major apicomplexan parasites (Carreno et al., 1999). Another study compared six different proteins along with the SSU rRNA and concluded that the parasite should be placed at an early emerging branch of the Apicomplexa (Zhu et al, 2000a). Other structural differences have been noted. Unlike most of the Apicomplexa, C. parvum appears to lack a plastid genome (Riordan et al, 1999; Zhu et al., 2000b). In general, macrolide antibiotics have not demonstrated consistent efficacy even after long-term administration. While this lack of efficacy may or may not be related to the lack of a plastid, drug development targeting a plastid genome or metabolic pathway associated with it may not be useful. In addition, evidence suggests that C. parvum has a putative mitochondrion and mitochondrion-associated enzymes markedly different in structure from those of its nearest relatives (Riordan et al, 1999) but lacks much of the electron transport chain. Some parasite targets (e.g. enzymes, structural proteins) are different than those of other related parasites. For example, the dihydrofolate reductase (DHFR) gene of C. parvum differs from the DHFR of Plasmodium. Sequencing of the dihydrofolate reductase gene indicated that the enzyme may be intrinsically resistant to 2,4-diaminopyrimidine inhibitors (Vasquez et al., 1996). The C. parvum DHFR active site contained novel residues at several positions analogous to those at which point mutations have been shown to produce antifolate resistance in other DHFRs (Vasquez et al., 1996). This may in part explain why C. parvum is resistant to clinically used antibacterial and anti-protozoal anti-folates. It has also been determined that C. parvum differs fundamentally in polyamine metabolism from other eukaryotes (Bacchi and Yarlett, 1995). Polyamine biosynthesis in C. parvum occurs via a pathway used by plants and certain bacteria, in which arginine is converted to agmatine by the action of arginine decarboxylase (Keithly et al., 1997). Neither arginine decarboxylase nor agmatine is found in other parasitic protozoa, which make this a unique target. A factor that may contribute to the ineffectiveness of many drugs is the unusual location of the parasite in the host cell. The parasite has a unique niche inside the cell in that it is intracellular but extracytoplasmic. It has been postulated that the basal membranes modulate the transport of certain drugs, so drugs entering the cytoplasm of the host cell may not be transported across to the parasite. There is evidence to suggest that this is the case for geneticin and paromomycin, a clinically relevant drug (Griffiths et al., 1998). In cell culture studies, apical but not basolateral exposure of these drugs led to significant parasite inhibition. The existence of multi-drug resistant (MDR) transporters might facilitate resistance and may also be involved in the rapid efflux of drugs as well as nutrient uptake. P-glycoproteins and MDRs are members of the ATP-binding

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cassette (ABC) superfamily that are responsible for drug resistance by extruding drugs against a concentration gradient. ABC transporters with considerable homology to the mammalian MDR-associated protein (MRP) and at least one grouped with the multidrug resistance protein (MDR) have been identified in C. parvum (Perkins et al., 1999; Zapata et al., 2002; Strong and Nelson, 2000).

NEW AREAS OF DRUG RESEARCH A number of drug targets have been proposed over the years but have not been pursued either because the presumed drug target could not be found, toxicity of the drug was too great, or because the drug lacked efficacy in animal models or in clinical trials. Some of the following therapies remain promising because the target is selective or unique and efficacy has been demonstrated either in vitro or in vivo against the target. The shikimate pathway is an important pathway in plants, bacteria, and fungi for the synthesis of aromatic compounds, amino acids, ubiquinone compounds, and folate. Several apicomplexans, including C. parvum, demonstrated evidence for the presence of enzymes of the shikimate pathway (Roberts et al., 1998). A fatty acid synthase gene (CpFAS1) identified in C. parvum encodes a multifunctional polypeptide which differs from the organellar type II fatty acid enzymes identified in T. gondii and P. falciparum (Zhu et al., 2000c). As mentioned above, arginine, an essential amino acid and precursor of the polyamine pathway, is converted by C. parvum to agmatine by the action of arginine decarboxylase (Keithly et al., 1997). Inhibitors of arginine decarboxylase (ADC) significantly reduce intracellular growth of C. parvum, whereas inhibitors of ornithine had no effect upon ADC activity or upon growth of the parasite (Water et al., 1997; Waters et al., 2000). Anti-tubulin agents such as the benzimidazoles and the dinitroaniline herbicides have demonstrated efficacy against a number of parasitic agents (Roos et al., 1997; Stokkermans et al., 1996; Traub-Cseko, et al. 2001). Although benzimidazoles are widely used against helminth infections and some protozoa, C. parvum did not have the predicted amino acids for benzimidazole drug sensitivity in the tubulin-coding gene (Katiyar et al., 1994; Edlind, et al., 1994). In a subsequent study, benzimidazole treatment was not effective against C. parvum when evaluated in mice (Fayer et al, 1995). Conversely, dinitroaniline herbicides were effective against C. parvum in vitro (Arrowood et al, 1995). The efficacy of this class of compounds has been determined in neonatal mice (Armson et al., 1999). In addition, several newly synthesized drugs have recently demonstrated anticryptosporidial activity. These include several lipophilic DHFR inhibitors (Brophy et al., 2000) and acridinic thioethers and aurone analogs (Kayser et

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al., 2001). These latter compounds have structural similarities to the plantderived phenolic chalcones that are active against other parasites such as Plasmodium, Leishmania and Trypanosomes. Despite the lack of response with immune colostrum in clinical trials, immunotherapy may still be useful in conjunction with conventional drug therapy or as a mechanism to decrease the severity of infection in neonatal animals or moderately immunocompromised individuals. The ability of monoclonal antibodies to neutralize C. parvum infectivity and control infection in vivo has been demonstrated (Langer et al., 2001; Riggs et al., 1997). Colostrum or monoclonal antibodies directed at neutralizing epitopes or antigens involved in attachment or invasion of host cells may be more effective than broadly generated immunotherapies. Not only is it important to develop better, more efficacious drugs, but also to improve drug transport and delivery to the parasite in vivo. Nanosuspensions are drug nanoparticles dispersed in a liquid phase, leading to increased solubility and dissolution velocity. Alternatively, the use of mucoadhesive polymers may help to increase drug retention time in the gut. Mucoadhesive drug delivery systems are also attractive since they attach to the intestinal wall and are in close proximity to the parasite. It was shown that a mucoadhesive nanosuspension bupravaquone formulation cleared the infection more effectively from the intestinal tract than the unmodified nanosuspension (Kayser et al., 2001a). How can we develop efficacious drugs? The continuing increase in genome sequence data should aid in the identification of new enzymes and biochemical pathways and increase our understanding of host/parasite interactions. However, the inability to cryopreserve the parasite and propagate the parasite continuously in vitro hinders many avenues of chemotherapeutic research. Cryopreservation of the parasite would establish standard isolates that could be used for multiple studies with consistency and reduced variability. The ability to propagate the parasite continuously in vitro could lead to establishment of a genetic model that would be useful for mutant/transfection studies. These studies would then facilitate identifying molecules as viable drug targets. In addition, the lack of effective drugs against these targets severely hampers the ability to validate these targets. Identification of unique targets in this challenging parasite is an important first step in developing effective therapeutic agents.

CYCLOSPORA Cyclospora cayetanensis is a protozoan parasite of humans causing diarrheal illness after colonizing the mucosal epithelium of the small intestine. C. cayetanensis is a parasite that has been described world-wide and identified in water and food. Cyclosporiasis causes “flu-like” symptoms

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including profound diarrhea lasting 1 to 3 weeks. Clinical signs include weight loss, nausea, anorexia, vomiting, and abdominal cramping (Herwalt, 2000). Among immunocompromised individuals (e.g. HIV-infected), cyclosporiasis symptoms may be exacerbated. However, drug treatment is generally effective in controlling infection and relapse (or reinfection) can be averted by maintenance or prophylactic drug dosages (Pape et al., 1994; Verdier, et al., 2000). Several studies have demonstrated that trimethoprim-sulfamethoxazole (TMP-SMX) is the drug of choice. In a placebo-controlled study in Nepal, TMP-SMX (160/800 mg) taken twice daily for 7 days was effective at treating Cyclospora infections (Hoge et al., 1995). Treatment was also found to be effective in a study of HIV-infected patients, when TMP-SMX was given 4 times a day for 10 days, followed by secondary prophylaxis (Pape et al., 1994). Among individuals that cannot be treated with TMP-SMZ (e.g. sulfa drug allergies), treatment options are limited and infections may be prolonged. Most alternative treatments such as albendazole, TMP alone, azithromycin, nalidixic acid, norfloxacin, tinidazole, metronidazole, quinacrine, tetracycline, and diloxanide furoate, do not seem to be effective against the parasite (Pape et al., 1994). One alternative, ciprofloxacin, has demonstrated moderate activity in HIV-infected patients when a 500 mg dose was administered twice daily. In a randomized, controlled study comparing trimethoprim-sulfamethoxazole or ciprofloxacin, patients who received trimethoprim-sulfamethoxazole, diarrhea ceased in 3.0 days, whereas those patients treated with ciprofloxacin had cessation of diarrhea at 4.0 days (Verdier et al., 2000). While active, ciprofloxacin was not as effective as TMP-SMZ in this population and has not shown comparable activity in immunocompetent patients (Herwalt, 2000). Little is known about potential virulence, pathogenicity or drug susceptibility differences among isolates involved in outbreaks and in endemic environments. One of the major challenges for research into the biology of Cyclospora is the lack of in vitro or in vivo models. Until an infectivity model is available that supports the entire life cycle of the parasite, many of these characteristics will remain obscure. Attempts to establish an animal model of cyclosporiasis using human-derived oocysts has not yet been achieved (Eberhard et al., 2000).

GIARDIA The antiparasitic agent of choice is metronidazole administered 250 mg t.i.d. for 5 days with an efficacy approximating 80 to 85% (Garner 2002). Patients should be advised of the disulfiram-like reaction that may occur with the concomitant use of alcohol. Isolates with decreased susceptibility to metronidazole have been described in vitro (Upcroft 2001) and clinical

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experience suggests that this may be relevant in practice. Albendazole at a dose of 400 mg daily for 5 days is an alternative to therapy and appears to be effective. Other effective agents not available for use in the US are tinidazole administered as a single dose (also effective in 90% of cases) and quinacrine 100 mg orally three times a day after meals for 5 days. Treatment of pregnant women poses special problems due to the potential mutagenicity of metronidazole although this has not been well documented in humans. In these cases, treatment should be delayed until at least after the first trimester, provided the patient’s hydration and nutritional status can be maintained. If therapy is indicated in this setting then paromomycin, a non-absorbable aminoglycoside can be considered for use at a dose of 500 mg four times daily if the patient’s renal function is normal. Uncommonly, one faces refractory cases in which a combination of metronidazole and quinacrine can be used for a longer period of time (14 day course). Nitazoxanide, a broad spectrum antiparasitic agent and its metabolite tizoxanide are more potent than metronidazole in vitro even in metronidazole resistant isolates and can be an alternative treatment when used at a dose of 1.5 gm po bid for 30 days in areas of the world where this drug is approved for use (Abboud, 2001; Adagu, 2002; Rossignol 2001; Ortiz 2001). Newer targets will undoubtedly be identified as the Giardia genome is analyzed (Adam, 2000). Given the ubiquity of giardiasis in certain environments such as day care centers, treatment of asymptomatic Giardia infections is not routinely recommended since healthy children do not appear to suffer deleterious effects with chronic asymptomatic cyst passage and therapy may expose them to unnecessary medication related side effects.

ENTAMOEBA HISTOLYTICA Therapy of Entamoeba infection should take in consideration not only the location and severity of the infection but the species causing infection. It is now well recognized that infections with E. dispar are asymptomatic and that therapy is not warranted. For asymptomatic cyst passers of E. histolytica, paromomycin at a dose of 500 mg tid for 7 days has shown to be more effective than diloxanide furoate when administered for 10 days (Blessman, 2002). Symptomatic infections should always be treated. E. histolytica can cause symptomatic intestinal syndromes including the following: 1) a dysenteric syndrome with production of small volumes of bloody, mucoid stools without fecal leukocytes, 2) colitis characterized by ulcerations of the colonic mucosa with typical flask-shaped abscesses, or 3) the formation of a fibrotic mass in the intestinal wall (ameboma). Metronidazole is the agent of choice for the treatment of amoebic colitis. Tinidazole is a reasonable alternative with the advantage that single dose therapy can be equally efficacious.

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Intestinal infections should also be treated with an intraluminal agent to prevent future invasion of remaining cysts. Chronic amoebic colitis is clinically indistinguishable from inflammatory bowel disease and those receiving corticosteroids are at risk for toxic megacolon and perforation and may sometimes necessitate parenteral therapy when patients are unable to tolerate the oral route. Infective trophozoites can migrate hematogenously to the right lobe of the liver, causing abscess formation, abdominal pain, jaundice and fever. Adjacent anatomical structures, such as the pulmonary parenchyma, peritoneum and pericardium can become involved. Amoebae can also disseminate to the brain. Immunosuppressed or malnourished individuals, those at the extremes of age, patients with malignancy, and women during pregnancy and post-partum stages are especially at risk for invasive amebiasis. Metronidazole followed by a luminal agent is the therapy of choice in extraintestinal disease. Since amebomas can mimic adenocarcinoma, a biopsy may be needed to differentiate disease. Indications for surgical drainage of an amoebic abscess include large dimensions, impending rupture, left lobe location, or lack of therapeutic response to metronidazole.

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chemoprophylaxis in HIV disease. HIV Outpatient Study (HOPS) Investigators. Journal of the American Medical Association 279: 384-386. Ives, N., B. Gazzard, and P. Easterbrook. 2001. The changing pattern of AIDS-defining illnesses with the introduction of highly active antiretroviral therapy (HAART) in a London clinic. Infection 42:134-139. Johnson, E., J. Windsor, D. Muirhead, G. King, and R. Al-Busaidy. 2000. Confirmation of the prophylactic value of paromomycin in a natural outbreak of caprine cryptosporidiosis. Veterinary Research Communications 24: 63-67. Jordan, E. C. 1996. Clarithromycin prophylaxis against Cryptosporidium enteritis in patients with AIDS. Journal of the National Medical Association 188: 425-427. Katiyar, S. K., V. R. Gordon, G. L. McLaughlin, and T. D. Edlind. 1994. Antiprotozoal activities of benzimidazoles and correlations with beta-tubulin sequence. Antimicrobial Agents and Chemotherapy 38:2086-2090. Kayser, O. 2001. A new approach for targeting to Cryptosporidium parvum using mucoadhesive nanosuspensions: research and applications. International Journal of Pharmaceutics 214: 83-85. Kayser, O., R. Waters, K. Woods, S. Upton, J. Keithly, and A. Kiderlen. 2001. Evaluation of in vitro activity of aurones and related compounds against Cryptosporidium parvum. Planta Medica 67: 1-4. Keithly, J., G. Zhu, S. Upton, K. Woods, M. Martinez, and N. Yarlett. 1997. Polyamine biosynthesis in C. parvum and its implications for chemotherapy. Molecular and Biochemical Parasitology 88: 35-42. Langer, R., D. Schaefer, and M. Riggs. 2001. Characterization of an intestinal epithelial cell receptor recognized by the Cryptosporidium parvum sporozoite ligand CSL. Infection and Immunity 69: 1661-1670. Lopez, A. S., D. R. Dodson, M. J. Arrowood, P. A. Orlandi Jr, A. J. da Silva, J. W. Bier, S. D. Hanauer, R. L. Kuster, S. Oltman, M. S. Baldwin, K. Y. Won, E. M. Nace, M. L. Eberhard, and B. L. Herwaldt. 2001. Outbreak of cyclosporiasis associated with basil in Missouri in 1999. Clinical Infectious Diseases 32: 1010-1017. Mancassola, R., J. Reperant, M. Naciri, and C. Chartier. 1995. Chemoprophylaxis of Cryptosporidium parvum infection with paromomycin in kids and immunologicalstudy. Antimicrobial Agents and Chemotherapy 39: 75-78. Miao, Y., F. Awad-El-Kariem, C. Franzen, D. Ellis, A. Muller, H. Counihan, P. Hayes, and B. Gazzard. 2000. Eradication of cryptosporidia and microsporidia following successful antiretroviral therapy. AIDS 25: 124-129. Okhuysen, P., C. Chappell, J. Crabb, L. Valdez, E. Douglass, and H. DuPont. 1998. Prophylactic effect of bovine anti-Cryptosporidium hyperimmune colostrum immunoglobulin in healthy volunteers challenged with Cryptosporidium parvum. Clinical Infectious Diseases 26: 1324-1329. Ortiz, J.J., A. Ayoub, G. Gargala, N.L. Chegne, and L. Favennec. 2001. Randomized clinical study of nitazoxanide compared to metronidazole in the treatment of symptomatic giardiasis in children from Northern Peru. Alimentary Pharmacology and Therapeutics 15:1409-1415. Pape, J. W., R. I. Verdier, M. Boncy, J. Boncy, and W. D. Johnson. 1994. Cyclospora infection in adults infected with HIV. Annals of Internal Medicine 121: 654-657. Perkins, M., T. Wu, and S. Le Blancq. 1999. Cyclosporin analogs inhibit in vitro growth of Cryptosporidium parvum. Antimicrobial Agents and Chemotherapy 42: 843-848. Portnoy, D., M. Whiteside, E. R. Buckley, and C. MacLeod. 1984. Treatment of intestinal cryptosporidiosis with spiramycin. Annals of Internal Medicine 101: 202-204. Riggs, M., A. Stone, P. Yount, R. Langer, M. Arrowood, and D. Bentley. 1997. Protective monoclonal antibody defines a circumsporozoite-like glycoprotein exoantigen of

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INACTIVATION AND REMOVAL OF ENTERIC PROTOZOA IN WATER

F.W. Schaefer, III1, M.M. Marshall2 and J.L. Clancy3 1

U.S. Environmental Protection Agency, Cincinnati, OH 45268 University of Arizona, Tucson, AZ 85721 3 Clancy Environmental Consultants, Inc., St. Albans, VT 05478 2

ABSTRACT Protozoan parasites including Giardia, Cryptosporidium, and Entamoeba can be transmitted through water and cause disease in humans and animals. Control of waterborne infection can be accomplished through a variety of physical and chemical means, resulting in the production of safe drinking water and protection of public health. Coagulation and filtration are the most commonly employed methods for physical removal of parasites, while chlorine-based compounds, ozone, and ultraviolet light are used for inactivation. Combinations of treatment technologies can result in parasite removal/inactivation greater than 6-log, resulting in reliable public health protection. Key words: Cryptosporidium, Giardia, Entamoeba, coagulation, slow sand filtration, sedimentation, diatomaceous earth filtration, multi-medium filtration, dissolved air flotation, chlorine, chloramine, chlorine dioxide, ozone, ultraviolet light

INTRODUCTION Giardia, Entamoeba, and Cryptosporidium are enteric pathogenic protozoa known to infect the intestinal tract of numerous mammals including man. Unlike many parasites, these organisms have direct life cycles involving vegetative, developmental stages that ultimately encyst in the host intestinal tract before passage in the fecal material. The Giardia and Entamoeba cyst as well as the Cryptosporidium oocyst are environmentally resistant transmission stages known to be transmitted by ingestion of water. These transmission stages can be detected in most surface and finished water at some time or other necessitating control to assure public health (LeChevallier et al., 1991a, b). Cysts and oocysts are known to be more resistant to disinfection than pathogenic bacteria. Theory suggests that the wall of the cyst and oocyst

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protects the parasite from the disinfectant. In developed nations, waterborne outbreaks of giardiasis and cryptosporidiosis have occurred numerous times over the latter part of the century. Waterborne amebiasis, on the other hand, rarely has been reported, with the largest outbreak occurring in Chicago in 1933 (Craun, 1986). However, this outbreak was the result of a sewage cross-connection to a hotel water system rather than inadequate water treatment. Besides studies on physical removal, studies on the efficacy of free chlorine, hypochlorite, chloramine, chlorine dioxide, ozone, and ultraviolet light (UV) have been done on Giardia cysts and Cryptosporidium oocysts.

PHYSICAL REMOVAL OF PARASITES Two important factors in physical removal of cysts and oocysts in the water treatment process are size and composition of the wall. The spherical E. histolytica cysts range in size from 5 to The oval G. lamblia cysts range in size from 8 to in length and 7 to in breadth. Spherical C. parvum oocysts range in size from 4 to The general rule is that the smaller the cyst or oocyst the more difficult it is to remove using conventional water treatment technology. This concept has been documented over the latter part of the century (Logsdon and Hoff, 1986). Consequently, using this rationale, C. parvum oocysts are the most difficult of the aforementioned parasites to physically filter from water. Detailed information on the exact composition of cysts and oocysts is lacking. Little is known about the Entamoeba cyst wall. Giardia lamblia cysts contain significant amounts of carbohydrate and protein in a 3:2 ratio (w/w). The carbohydrate component has been identified as (Gerwig et al., 2002). Although studies on the oocyst wall of C. parvum have been done, about all that can be said is the oocyst wall is composed of glycoprotein (Bonnin et al., 1991). While protein differences and their uneven distribution throughout the oocyst wall have been explored as a way to speciate Cryptosporidium, definitive studies defining the exact composition of the oocyst wall remain to be completed (Harris and Petry, 1999; Jenkins et al., 1999). Cysts and oocysts are naturally electronegative (Ongerth and Hutton, 1997). However, if they are not purified from fecal material properly avoiding extremes of pH, inactivation with formaldehyde, and exposure to potassium dichromate, their surface charge will not mimic the natural condition (Schaefer, 2001). Experiments designed to study the interaction of oocyst surface charge (zeta potential), glass bead packed columns, natural organic matter, biofilm, and alum as coagulant demonstrated that both biofilm on the glass bead packed columns and natural organic matter significantly decreased the column removal

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efficiency from 51% to 14% (Dai and Hozalski, 2002). The electronegative zeta potential of natural organic matter treated oocysts was significantly increased in addition to increasing their hydrophobicity. When alum was used as a coagulant, it counteracted the high electronegative oocyst surface charge resulting in a removal efficiency of 73%. The physical removal results reported below must be viewed with caution, for many of the investigators did not use properly prepared or treated transmission stages. The most popular techniques to physically remove particulates and microorganisms from water include rapid granular media (sand, dual media, or mixed media) filters, membranes, slow sand filters, or diatomaceous earth (DE) filters. Coagulation, flocculation, sedimentation or some combination thereof usually precedes the rapid granular media filters (Logsdon and Hoff, 1986). The physical removal of cysts and oocysts and the way in which they interact with filter media and coagulants is related to a number of factors including the surface characteristics of the cyst or oocyst, the amount of other particulates in the water, the physical characteristics of the water, the size of the filter media, design of the filter, operation of the filter, and the size of the cyst or oocyst. Slow sand filters, as the name implies, are beds of sand through which water slowly passes usually at a velocity around 0.1 m/hr in a 1 m deep filter. As the filter matures, the schmutzdecke, a microbiologically active scum layer, develops on the top and increases the efficiency of the filter. Better than 2-log (99.0%) removal of Giardia cysts is possible with slow sand filters (Bellamy et al., 1985). Cryptosporidium oocyst are efficiently removed using slow sand filtration with better than 99.997 % reduction (Timms et al., 1995). Jar tests of gravel pit water with turbidities ranging between 1 and 2 nephelometric turbidity units (ntu) showed about 90% or higher removal of Giardia cysts in the pH range of 6 to 9. In an experiment with the alum coagulant at 22 mg/L, the Giardia cysts passing the filter were at a density of 80 cysts/L. However, when the alum coagulant feed was interrupted, the Giardia cyst density passing through the filter rose to 1,760 cysts/L (Aronzarena, 1979). Turbidity breakthrough at the end of a rapid filter run has been shown to be indicative of passage of large numbers of Giardia cysts (Logsdon et al., 1981). Clearly the optimal operation of coagulant feed and the rapid filter are critical to removal of Giardia cysts. The Mobile Water Purification Unit, Model 1940, was tested by the U.S. Army for removal of E. histolytica cysts. When alum and soda ash were used for coagulation 98.5 % and 99.8% of the applied E. histolytica cysts were removed during sedimentation (U.S. Army, 1944). Several studies have studied optimization of coagulation and rapid filtration processes for Cryptosporidium oocyst removal. In the first study, the best oocyst removals were ~3-log and were

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achieved with ferric chloride coagulant in combination with pre-oxidation, cationic polymer and filter aid addition and tri-media filtration (Yates et al., 1997). The second study investigated the effect of pH on the coagulants ferric chloride, alum, and polyaluminum chloride. The three coagulants were effective in achieving 4.3-log removal of oocysts (States et al., 2001). Ongerth and Pecorano (1995) conducted trials with multiple runs over several months in the northwestern US using river sources to examine oocyst removal. The treatment system characteristics were direct filtration with in-line flocculation; multi-media filters; low-turbidity, low-alkalinity raw water with optimal alum coagulation; and constant rate operation at 5 gallons per minute per square foot They achieved 2.7- to 3.1-log removal of Cryptosporidium oocysts in this pilot-scale direct filtration plant, which was only slightly lower than the concurrent removal of Giardia cysts. Dissolved air flotation (DAF) was shown to achieve a >2-log removal of oocysts in bench-scale trials under a variety of operating conditions (Plummer et al., 1995). The objective of the DAF study was to determine the effect of specific design and operating variables on oocyst removal. The source water was from a reservoir in upstate New York. The study showed that coagulant dosage had a significant effect on oocyst removal by DAF. At a dose of 5 mg/L ferric chloride, oocyst removal was 3.7-log, but was reduced to 2.0-log at 3 mg/L, and to 0.38-log at 2 mg/L. Acceptable turbidity, and dissolved organic carbon (DOC) were noted at a dosage of 3.5 mg/L, indicating that higher coagulant doses may be needed to provide optimal oocyst removal. In another DAF study, Cryptosporidium removals ranged from 2.9log to 4.0-log, with granular activated carbon (GAC) and dual media filters providing the highest removals (Hall et al., 1995). Nieminski and Ongerth conducted a two-year study at both a pilot- and full-scale plant treating river water in Utah. The pilot plant processes included a flash mixer, four-stage flocculation basins, and two sedimentation basins with dual media filtration. The pilot plant had two treatment trains, one of which was converted to direct filtration. The full scale plant was a 900 gpm plant which operated both as a conventional plant for some runs, and as a direct plant for others by bypassing the settling basin and routing flocculated water to the filters. For each operational mode, 10 seeding runs were conducted for the pilot plant and four seeding runs were conducted for the fullscale plant. Results showed that Cryptosporidium oocyst removal was 2.9log in both the conventional and direct modes in the pilot plant; at full scale, oocyst removal was 2.3-log by conventional treatment to 2.8-log by direct filtration. Cyst-sized particles, as determined by optical particle counters and turbidity were shown to be indicators of oocyst removal.

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DE filters have been shown to remove certain viruses, bacteria, and protozoa. The removal efficiency of this filter type is dependent upon the pore grade of the diatomaceous earth, precoat rate, body feed rate, whether the diatomaceous earth has been coated with hydrous oxides of iron or cationic polymer and the filtration rate. Studies have shown more than 99% of G. muris cysts are removed by DE filters provided the filter septum is precoated properly (Logsdon et al., 1981). Similar results were obtained for G. lamblia cyst removal (DeWalle et al., 1984). Entamoeba histolytica cysts are completely removed by a properly operated DE filter (U.S. Army, 1944). When properly operated, DE filters can produce up to 6-log Cryptosporidium oocyst removal (Ongerth and Button, 1997; Ongerth and Hutton, 2001). Membrane processes - ultrafiltration and microfiltration - have been shown to provide high levels of cyst and oocyst removal (>6-log). In these studies, cysts and oocysts are rarely seen in the filter permeate, and removals are generally determined based upon the initial seed concentration (Jacangelo et al., 1995). Physical straining of cysts and oocysts from the feed water appears to be the removal mechanism. These processes are widely used in the pharmaceutical, electronics, and food industries for removal of sub-micron particles so highly effective cyst and oocyst removal is expected.

DISINFECTION The effectiveness of chemical disinfection is dependent upon the disinfectant, pH, temperature, disinfectant demand in the water, and the organism being inactivated. Free chlorine, the most frequently used disinfectant in water treatment, has several species in water depending on the pH. The more active species, HOCl, is found around pH 6 to 7, while the less active species, is present at higher pH of 8 to 10. In contrast to HOCl, is most effective as an undissociated gas in the pH range 6 to 9. Chloramine, the weakest of the water treatment disinfectants, requires a pH around 8 or higher to ensure monochloramine is the predominant species, when chlorine and ammonia are mixed together in equimolar proportions. Ozone is independent of pH in the ranges encountered in water treatment. Because of the high reactivity and volatility of ozone, controlling water disinfection presents challenges. As water temperature increases, ozone disinfection efficacy increases. The effect of temperature and pH on the effectiveness of disinfectants on Giardia and other microorganisms is shown in Table 1. Compared to the other microorganisms listed in the Table 1, Giardia and Entamoeba cysts are more resistant to water treatment disinfection than bacteria or viruses. G. muris appears to be more resistant to ozone and chlorine than G. lamblia.

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UV light is the most recent addition to the arsenal of disinfectants for protozoan parasites. Earlier studies of UV effectiveness on Giardia and Cryptosporidium indicated that very high UV doses were required for inactivation, but these studies were misleading. In vitro excystation was used as the indicator of inactivation, and data showed that little inactivation occurred at reasonable UV doses (Rice and Hoff, 1981; Karanis et al., 1992; Ransome et al., 1993; Finch et al., 1997; Clancy et al., 1998). However, when animal infectivity was used to assess UV efficacy, a dramatic reduction in infectivity was noted (Bukhari et al., 1999). Animal infectivity measures the ability of the cyst or oocyst to complete the infection cycle, whereas in vitro excystation measures metabolic activity. It is now understood that although cysts and oocysts treated with UV may still exhibit metabolic activity, they are unable to cause infection in a susceptible host, and therefore, have been successfully inactivated. Once this was known, a rapid succession of studies was undertaken to determine the susceptibility of Cryptosporidium in particular. Studies on UV inactivation of Giardia and Cryptosporidium are presented in Table 2.

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Chlorine is effective against Giardia, and water treatment processes are designed to provide sufficient contact time such that cysts remaining in the water after filtration will be inactivated by chlorine or other disinfectant exposure. However, Cryptosporidium is resistant to chlorine-based disinfectants and oocysts escaping filtration could remain viable after treatment. Ozone can also be used to control Cryptosporidium in water, but the levels of inactivation are nowhere near those seen with UV. Temperature plays an important role in ozone inactivation, and water systems in colder climates cannot achieve the required levels of Cryptosporidium inactivation with ozone. The CT for ozone inactivation of Cryptosporidium is approximately 25 to 35 timers higher than that required for ozone inactivation of Giardia. Ozone has a high potential for bromate ion formation, a potential human carcinogen, when employed at the significantly higher CT levels to inactive C. parvum oocysts (Federal Register, 1996). Goals of 0.5- to 1-log inactivation of Cryptosporidium may be achievable depending on many factors including water quality, plant design and operational flexibility, limiting ozone as a choice for Cryptosporidium control to specific sites. UV remains the most effective choice for Cryptosporidium control, both in terms of efficacy and cost effectiveness.

CONCLUSIONS Current water treatment processes can be highly effective for control of protozoan parasites in drinking water. Combinations of physical removal through coagulation and filtration, coupled with disinfection can result in log reductions ranging from 2- to over 6-log. Protozoan parasites are resistant to standard chemical disinfection using chlorine-based compounds, so physical removal plays an important role in the treatment process for parasite control. UV light is the latest and most effective method for control of both Giardia and Cryptosporidium, and is being implemented worldwide for control of these pathogens. Production of safe drinking water relies on the multiple barrier approach to drinking water treatment. This begins with source water protection to prevent pollution, followed by appropriate treatment, and maintenance of water quality through proper storage and distribution to the consumer. This holistic approach permits water suppliers to provide safe drinking water and a high level of public health protection.

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Rice, E.W., J.C. Hoff, and F.W. Schaefer, III. 1982. Inactivation of Giardia cysts by chlorine. l Applied and Environmental Microbiology 43:250-251. Roy, D., R.S. Engelbrecht, and E.S.K. Chian. 1982. Comparative inactivation of six enteroviruses by ozone. Journal American Water Works Association 74(12):660-664. Scarpino, P.V., M. Lucas, D.R. Dahling, G. Berg, and S.L. Chang. 1974. Effectiveness of hypochlorous acid and hypochlorite ion in destruction of virues and bacteria, pp. 359-368. In A. J. Rubin (ed.), Chemistry of Water Supply, Treatment, and Distribution Ann Arbor Science Publishers, Woburn, Massachusetts. Schaefer, F.W. 2001. Can we believe our results?, pp. 155-161. In M. Smith and K.C. Thompson (ed.), Cryptosporidium: The analytical challenge. Cambridge, UK. Snow, W.B. 1956. Recommended chlorine residuals for military water supplies. Journal American Water Works Association 48(12): 1510-1514. Shin, G-A, K.G. Linden, G. Faubert, M.D. Sobsey. 2000. Low pressure UV inactivation of Cryptosporidium parvum and Giardia lamblia based on infectivity assays and DNA repair of UV-irradiated Cryptosporidium parvum oocysts. Proceedings AWWA Water Quality Technology Conference 2000, Salt Lake City, UT. States, S., R. Tomko, M. Scheming, and L. Casson. Enhanced coagulation and removal of Cryptosporidium in drinking water treatment. 2001. Proceedings AWWA Water Quality Technology Conference. November 2001, Nashville, TN. Timms, S., J.S. Slade, and C.R. Fricker. 1995. Removal of Cryptosporidium by slow sand filtration. Water Science and Technology 31:81-84. U.S. Army. 1944. Efficiency of standard Army water purification equipment and of diatomite in removing cysts of Entamoeba histolytica from water. Report 834, 63-82. Wickramanayake, G.B., A.J. Rubin, and O.J. Sproul. 1984. Inactivation of Giardia lamblia cysts with ozone. Applied and Environmental Microbiology 48:671-672. Wickramanayake, G.B., A.J. Rubin, and O.J. Sproul. 1985. Effects of ozone and storage temperature on Giardia cysts. Journal American Waterworks Association 77(8):74-77. Yates, R.M., J.F. Green, S. Liang, R.P. Merlo, and R. DeLeon. 1997. Optimizing coagulation/filtration processes for Cryptosporidium removal, pp. 281-290. In C.R. Fricker, J.L. Clancy, and P.A. Rochelle (ed.), Proceedings 1997 International Symposium on Waterborne Cryptosporidium. American Water Works Association, Denver, CO.

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MONITORING OF GIARDIA AND CRYPTOSPORIDIUM IN WATER IN THE UK AND US J.L. Clancy1 and P.R. Hunter2

1

Clancy Environmental Consultants, Inc., PO Box 314, St. Albans, VT 05478 School of Medicine, Health Policy and Practice, University of East Anglia, Norwich NR4 7TJ, United Kingdom 2

ABSTRACT Regulatory agencies in the UK and US approach monitoring water for Giardia and Cryptosporidium quite differently. Water suppliers in the US are not required to monitor for Giardia and Cryptosporidium in water, but largeand most medium-sized water utilities monitor regularly. Monitoring is done because there are testing methods available, the public expects testing to be done, and utilities may be vulnerable to litigation if outbreaks occur in the absence of testing. Consumers, regulators, and elected officials often view parasite monitoring as a proactive way to protect public health. Routine parasite monitoring provides no public health protection, as it is not sensitive enough to predict or detect contamination events leading to negative public health outcomes. In England and Wales there is a legal requirement for all water utilities to conduct a risk assessment of each supply. If a particular supply is deemed to be at risk of contamination by Cryptosporidium oocysts, then continuous monitoring of the supply for oocysts needs to be carried out where water is filtered at a rate of not less than 40 L/hr. It is a criminal offense for a supply to exceed 1 oocyst/10L over 24 hours. The regulations apply only to treated water systems where an adequate treatment system is in place. There is no particular requirement for testing for Giardia, though some water utilities have tested their supplies. Key words: Giardia; Cryptosporidium; water supplies; monitoring; public health; regulations

INTRODUCTION In this chapter, we review the different approaches used in the United Kingdom (UK) and United States (US) for monitoring water supplies for Cryptosporidium and Giardia. Although there are similarities between the approaches of the two countries, the UK has developed a more legalistic approach involving continuous monitoring of supplies considered to be at

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significant risk. We first consider the situation in the US and then turn our attention to the UK setting.

EARLY HISTORY OF WATER TESTING FOR GIARDIA AND CRYPTOSPORIDIUM IN THE US Microbiologists have been concerned with monitoring for pathogens in water for over a century. Suckling (1910) noted that the search for pathogenic organisms is “...beset with difficulties and is seldom successful.” He noted low pathogen numbers, large sample volumes, cumbersome procedures, and the time required to complete analyses as relevant issues. Nearly 100 years later, in spite of the advances made in microbiological testing, these same concerns remain. This has not, however, dampened the development of methods for the recovery and detection of Giardia cysts and Cryptosporidium oocysts in water, nor impeded widespread testing in the absence of regulatory requirements. From 1971 to 1985, 92 outbreaks of waterborne giardiasis occurred in the US (Craun, 1986). The first detection of Giardia cysts in water was made during an outbreak in Rome, NY in 1975 by filtering ~1 million L of Rome drinking water through a swimming pool filter, extracting the sediment, and feeding it to two beagle pups. The pups developed giardiasis, and a single cyst was noted microscopically in the sediment. This method of sample collection was impossible to use routinely, and in 1989, the United States Environmental Protection Agency (USEPA) developed a portable system for sample collection that consisted of a nominal porosity string wound filter for sample collection (Jakubowski and Hoff, 1979). The processing involved washing the filter with distilled water; centrifugation or overnight settling of the extracted material; centrifugation of the settled material; further concentration of the sediment by centrifuging in Lugol’s iodine; separation of the cysts from the sediment using zinc sulfate flotation; and microscopic examination. This method was not used routinely, but was used to examine water supplies suspected of causing a giardiasis outbreak. Over the next decade the method was greatly improved by introducing a Percoll-sucrose separation step to replace the zinc sulfate, and incorporating an immunofluorescent monoclonal antibody (mAb) stain for cyst detection. Since there was also a mAb for Cryptosporidium, this was incorporated into the method and it became a single analysis that could recover and detect both parasites (LeChevallier et al., 1990). LeChevallier and colleagues used this new method, nicknamed the IFA method as it used immunofluorescence for parasite detection, in national surveys of source and finished drinking water samples from 66 surface water plants. Giardia and

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Cryptosporidium were found in the raw waters at frequencies of 87% and 97%, respectively (LeChevallier et al., 1991a). Giardia was detected in 17% and Cryptosporidium in 27% of the finished water samples (LeChevallier et al., 1991b). From 1988 to 1993, a survey of 347 samples from 72 surface water treatment plants showed Giardia present in 54% of the samples with a 60% prevalence of Cryptosporidium (LeChevallier et al., 1995). This period coincided with a number of outbreaks of waterborne cryptosporidiosis in both the US and UK, and included the 1993 Milwaukee outbreak affecting over 400,000 people. Although few water utilities had done testing of their supplies for Giardia and Cryptosporidium, the Milwaukee outbreak brought attention to the possibility of widespread oocyst contamination of source waters. Due to the high level of water industry interest in determining the concentrations of Giardia and Cryptosporidium in source and finished water, the IFA method came into widespread use. This was the beginning of voluntary national testing for Giardia and Cryptosporidium. The IFA method was used in several studies and became the standard method for protozoa analysis, although it had never been collaboratively tested to determine its precision and bias. Clancy et al (1994) were the first to conduct a collaborative study of the IFA method, demonstrating in an evaluation of 16 laboratories that overall performance was poor. Recoveries were low for both parasites, and false positives and negatives were reported commonly. Additional studies followed that confirmed these findings; samples seeded with >9,000 cysts and oocysts were reported as non-detects by expert laboratories (Clancy et al., 1999). The IFA method suffered from poor reproducibility, poor sensitivity, high detection limit (>100 organisms/L), inability to differentiate cysts or oocysts using IFA-based technology, high false positive rate and high false negative rate. Although testing for protozoan parasites is not mandated, Giardia is regulated under the 1986 amendments to the Safe Drinking Water Act, the Surface Water Treatment Rule (SWTR). The USEPA set a maximum contaminant level of zero for Giardia, but regulates this contaminant through treatment technology and not by direct measurement. The SWTR requires that surface water treatment plants achieve a 99.9% or 3-log removal and inactivation of Giardia cysts. Conventional filtration receives 2.5-log credit and the additional 0.5-log is achieved through disinfection. Plants meeting these operational standards are said to be in compliance with the regulation.

THE USEPA INFORMATION COLLECTION RULE In 1997, the USEPA instituted a monitoring regulation, the Information Collection Rule (ICR). The ICR required utilities serving greater than 100,000 people and using surface water to monitor source waters for

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Giardia cysts and Cryptosporidium oocysts for 18 months. Those with source waters found to be positive for either protozoan at a concentration L were required to initiate finished water monitoring. In spite of the lack of robustness of the IFA method, it was altered slightly and was the specific method required by USEPA for this monitoring. The IFA method is now referred to as the ICR method (USEPA, 1996). Analysis of the 18 months of ICR data showed that of 5,829 samples analyzed for the protozoa, 93 percent of the samples were non-detects for Cryptosporidium and 81 percent were negative for Giardia. When cysts or oocysts were detected, it was generally one or two organisms observed in a small subsample. These observed numbers were then extrapolated to 100 L for source water or 1000 L for treated water. The final result was often a very high reported number based upon this extrapolation. The ICR data fall generally into two broad categories – either non-detects or very high reported levels based on low analyzed sample volume. For example, from a 100 L sample, a portion equivalent to ~2.5 L is actually examined microscopically due to method limitations. If 1 oocyst is observed, then the reported value is 40 oocysts per 100 L, assuming incorrectly that oocysts are evenly distributed in a sample. The non-detect data are equally absurd. Using the same example, if no oocysts were detected in the 2.5 L equivalent volume, the count is reported as <40 oocysts per 100 L. This means the count could be 39 or 0 or any number in between. The method is so poor that actual count data as well as non-detect data are unreliable (Allen et al. 2000). The ICR data are in contrast to the high levels noted in the previous studies by LeChevallier et al (1991a,b; 1995). One possible explanation is that the early versions of the IFA method did not require confirmation of cysts and oocyst, but relied on IFA identification alone, and so overestimates (false positives were likely to be reported.

USEPA METHODS 1622 and 1623 Over the years, several laboratories tried to improve the ICR method, but these attempts were futile. Shortly after the ICR monitoring began, the USEPA began working on an improved method for Cryptosporidium analysis. This effort did not focus on improving the ICR method; rather it abandoned the ICR method altogether and used a new approach. The approach taken in developing this new method was to evaluate each step in the method sampling, processing, and assay - for its ability to permit recovery of spiked oocysts in reagent water. Each individual step was optimized in reagent water, and after optimization, the steps were combined into a full method that was single laboratory validated in two laboratories (Clancy et al., 1999). Important advances in the new method include: 1) use of sampling filters that permit 100% capture of oocysts and permit high and consistent oocyst

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recovery (~80%), 2) replacement of buoyant density gradient centrifugation with immunomagnetic separation (IMS) for significantly improved recovery of target organisms from sediment, 3) use of well slides in place of membranes for sample preparation for microscopy, 4) inclusion of 4’-6’diamidino-2-phenylindole (DAPI) staining in conjunction with mAb staining for enhanced confirmation, and 5) the requirement of observing oocysts at 1000 X using epifluorescence, UV, and Nomarski differential interference contrast optics for better identification. When a Giardia IMS kit became available, the assay was expanded to include both parasites. These methods have been improved slightly since their initial development and are now finalized as USEPA Method 1622: Cryptosporidium in Water by Filtration/IMS/FA and Method 1623: Giardia and Cryptosporidium in Water by Filtration/IMS/FA (USEPA, 2001a, b). These methods are available at www.epa.gov/microbes. Methods 1622 and 1623 are still characterized by high variability, but are much more sensitive than previous methods. In laboratories with welltrained analysts, recoveries can be 50% or greater routinely with a seed dose of 100 organisms. With the improvements in sampling, separation, and staining, false positive and false negative rates are lower. These methods are significant improvements over the ICR and other methods, and recognizing this, many countries have adopted them for use as the standard for protozoan analysis of water. Originally developed for 10 L source water samples, the methods have been adapted for higher volume source (50 L) and finished water (1000 L) monitoring (McCuin and Clancy, 2003). LeChevallier et al (2003) repeated the earlier studies of source water occurrence and added a cell culture- polymerase chain reaction (CC-PCR) assay to determine infectivity of recovered oocysts. The addition of the infectivity assay allows risk assessment for the first time since water supply monitoring began. Without infectivity, risk assessment was based on IFA staining and microscopy alone, and included measurements of true positives, false positives, and non-viable oocysts. The USEPA plans to use Method 1622 in another temporary monitoring program slated for 2004 under the Long Term 2 Enhanced Surface Water Treatment Rule (LT2). Like Giardia in the SWTR, Cryptosporidium will be regulated through treatment technology and not through testing. In the LT2 monitoring scheme, utilities serving greater than 10,000 people will be required to measure the Cryptosporidium concentration in their source waters in order to next determine the level of treatment needed to protect public health. Utilities with higher Cryptosporidium levels will need to provide additional treatment through removal or inactivation of the parasite. The monitoring is for the presence of oocysts only, detected using IFA and confirmed with DIC and DAPI staining. For regulatory purposes, the total number of oocysts will be used to determine occurrence and hence risk; no

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differentiation as to viability or infectious potential of oocysts will be measured.

CURRENT US MONITORING PRACTICES Although method development has progressed significantly, the usefulness of routine monitoring data - a sample collected monthly or less frequently - is limited. However, this has not stopped most US water utilities from monitoring their raw and finished drinking water for Giardia and Cryptosporidium in the absence of regulatory requirements. While significant improvements, Methods 1622 and 1623 still have limitations: 1) the sample volumes are relatively small (10-50 L for source water, up to 1000 L for finished water), 2) the method does not predict viability of cysts or oocysts, so risk assessment is not possible, 3) variability is high due to sample matrix effects, 4) a negative result does not mean cysts or oocysts are not present in the water, 5) a positive result does not mean the water presents a public health concern, and 6) the data are not real-time, often unavailable to the utility for days or weeks. For these reasons the data cannot be used to make public health decisions, but are used to satisfy public expectations and management directives.

THE BACKGROUND TO WATER TESTING FOR GIARDIA AND CRYPTOSPORIDIUM IN THE UK The approach to testing treated waters in the UK differs significantly from that in the US. Before discussing the UK approach, it is important to understand the different legal and political environment found in the UK. The UK legislation on the quality of water intended for consumption is itself governed by European legislation. Until recently this was the Directive 80/778/EEC (Anon 1980). A more recent directive is now being enacted into UK legislation (Anon 1998). Neither of these European directives has set standards for either Cryptosporidium or Giardia. However, both require that water intended for human consumption “is free from any micro-organisms and parasites and from any substance which, in numbers or concentrations, constitute a danger to human health”. The problem was and remains that it is difficult to agree on a health standard for Cryptosporidium. What effectively changed water quality legislation in the UK was an outbreak of cryptosporidiosis that occurred in the South West Region of England in August 1995 (Harrison et al., 2002; Waite and Jiggins, 2002). Some 575 cases were identified as being part of this outbreak. This number is probably an underestimate as the area affected was a popular tourist destination and increased reporting was noted from many regions in the UK coincident with this outbreak (Nichols, 2002). Descriptive epidemiology

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linked the outbreak to drinking water from a particular water treatment works. This same treatment works had been associated with another outbreak some three years earlier and the UK Drinking Water Inspectorate (DWI) considered that there were grounds for prosecution for providing water unfit for human consumption. The evidence on which the case was brought was the report of the Outbreak Control Team (OCT). This prosecution failed over the admissibility of the epidemiological evidence. In brief the presiding judge ruled that the report of the OCT was not admissible as evidence as epidemiological studies do not allow a complete forensic chain of evidence (Waite and Jiggins, 2002). This left the DWI in the position that it may never be able to prosecute water utilities for providing unfit water where the only evidence came for epidemiological studies. This was the main driver for the new legislation, discussed below, for monitoring drinking water supplies for Cryptosporidium oocysts. The background for this legislation is discussed in more detail by Waite and Jiggins (2002). Unlike the situation for Cryptosporidium there is currently no legislative pressure for monitoring treated water supplies for Giardia in the UK. In large part this reflects the very different experience with waterborne giardiasis in the UK compared to the US. The UK has only ever seen one outbreak of giardiasis linked to mains drinking water compared to over 20 outbreaks of cryptosporidiosis (Hunter, 1997). The one outbreak of giardiasis was in 1985 and, although a case control study found an association with a water supply, the exact problem was not identified (Jephcott et al., 1986).

UK MONITORING OF CRYPTOSPORIDIUM IN TREATED WATER UNDER THE REGULATIONS The regulations for testing water supplies for Cryptosporidium were originally set out in the Water Supply (Water Quality) (Amendment) Regulations (1999). These regulations were then incorporated into the Water Supply (Water Quality) Regulations (2000). These regulations require water utilities to undertake a number of steps. The first of these is to conduct a risk assessment. Under the regulations a water undertaker is required to conduct a risk assessment to determine whether each supply is at significant risk from Cryptosporidium. The report of that risk assessment then has to be submitted to the Secretary of State who will then make a judgment on whether the risk assessment has been adequately carried out. The regulations do not proscribe how the risk assessment is carried out though guidance is available (Drinking Water Inspectorate, 1999). However, there are certain circumstances that would always mean that the water treatment works is at significant risk: Direct abstraction or with an average storage of seven days or i) less from a river or stream.

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ii)

Evidence of rapid river or surface water connection to the aquifer demonstrated by the confirmed presence of faecal coliform bacteria in the raw water. Past history of an outbreak of cryptosporidiosis associated iii) with the water supply where the reason is unexplained and no specific steps have been taken to prevent a recurrence. If a water supply is deemed to be at increased risk of Cryptosporidium oocysts, then the water undertaker is required to put in place a water treatment process that will guarantee an oocyst count of <1/10 L and have monitoring in place to demonstrate that this standard is being met. It should be noted that the standard was chosen not because of health criteria, but that it was considered that this standard should be achievable in any adequately managed water treatment works. Under these regulations the sampling process is continuous and at least 40 L/hr needs to be filtered. A gap in monitoring of no more than one hour is allowed while filters are changed. Usually the collection device should be changed at least once a day. The regulations allow three days to analyze each filter, though if there is a significant rise in turbidity, or if there are other reasons to suspect that oocyst counts may have risen, the filter should be changed and examined as soon as possible. The analysis has to be carried out in accredited laboratories using approved standard operating procedures (SOPs). Laboratories have to participate in an external quality assurance scheme. These SOPs cover aspects of sample collection, transport, storage, analysis and reporting. The SOPs are available on the DWI website (http://www.dwi.gov.uk/regs/crypto/mainindex.htm). As the results may be used to support a criminal prosecution, the guidance documents specify a forensic degree of chain of evidence, using tamper proof containers and high levels of documentation. All results should be reported to the DWI on a monthly basis, though abnormally high results must be reported without delay. These results are also in the public domain and are available to consumers should they wish to see them. Abnormally high results should also be reported to the relevant Health Authority who will then make an appropriate assessment of whether this poses a threat to Public Health and take the necessary steps to mitigate that threat. The UK Public Health Laboratory Service issued advice on the interpretation of these results when the regulations came into force (Hunter, 2000). Under these regulations, risk assessments were conducted on 1,481 treatment works and some 332 (22.4%) (158 treated surface waters and 174 treated groundwaters) were deemed to be at significant risk. Many of these atrisk treatment works have subsequently been taken out of service. In the year

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from April 2000, 36,916 samples were taken from 188 treatment works of which 2,755 (7.5%) were positive with one or more oocysts/1000 L and seven exceeded the standard of <1/10 L. In the following year, 51,168 samples were taken from 166 sites and only 1,676 (3.3%) were positive with at least one oocyst. None have exceeded the standard (Information available from http://www.fwr.org/crypnote.htm). As yet there have been no outbreaks linked to drinking water in England and Wales since the regulations were implemented.

UK MONITORING OF CRYPTOSPORIDIUM AND GIARDIA IN TREATED WATER NOT UNDER THE REGULATIONS The discussion so far has been concerned with analyses under the “Cryptosporidium regulations”. These regulations apply only to England and Wales, though both Scotland and Northern Ireland have similar regulations or directions for their water undertakers. Even in England and Wales, not all water supplies are covered by this legislation. For example, some large surface water supplies still remain unfiltered and, as there is no process for removing oocysts, the regulations do not require continuous monitoring. All of these supplies should have appropriate water treatment plants in place in the near future. In many cases, however, the water undertaker still monitors the supply on a continuous basis as if it were covered by the regulations. This type of monitoring is known as operational as opposed to regulatory monitoring. In addition, many water utilities monitor Cryptosporidium and Giardia in raw waters from time to time using 10 L “grab samples”. Grab samples are sometimes used to assist in the investigation of outbreaks of waterborne disease (Howe et al., 2002), in the examination of raw water and occasional other purposes. Alternate analytical methods for both Cryptosporidium and Giardia are recommended by the UK Standing Committee of Analysts (1999).

CONCLUSIONS As has been seen, there are both similarities and differences between the US and UK approaches. The main difference is the use to which most results are put. In the UK, monitoring for Cryptosporidium is largely covered by legislation and follows risk assessment. In the US, monitoring of oocyst levels is done to inform risk assessment. Much of the technology and methods are the same and differ largely because of the requirement by the UK authorities for continuous monitoring. The focus in the US is on source water testing while the UK prescribes finished water monitoring. A major problem

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remains with oocyst monitoring that reflects the situation with pathogen monitoring of water in general. Although significantly improved over the last ten years, the methods are still not robust and lack sensitivity and specificity. Information on viability, and hence potential to affect public health, is not known. Also methods in common use are unable to distinguish between C. parvum and many other species that have little public health significance. It is important to have sensitive and specific methods for Cryptosporidium and Giardia detection in water, but their application is most appropriate for research studies, watershed evaluations to target point sources of fecal contamination, outbreak investigations, etc. Even under these circumstances, the limits of the method must be fully understood and the data reported to reflect specificity, sensitivity, and reproducibility of the method.

REFERENCES Allen, M.J., J.L. Clancy, and E.W. Rice. 2000. The plain, hard truth about pathogen monitoring. Journal American Water Works Association 92 (9):94-76. Anon. 1980. Council Directive 80/778/EEC of 15 July 1980 relating to the quality of water intended for human consumption. Official Journal of the European Community L229:11-29. Anon. 1998. Council Directive 98/83/EC of 13 November 1998 relating to the quality of water intended for human consumption. Official Journal of the European Community L330:32-54. Clancy, J.L., W.D. Gollnitz, and Z. Tabib. 1994. Commercial labs: how accurate are they? Journal American Water Works Association 86 (9):89-97. Clancy, J.L., Z. Bukhari, R.M. McCuin, Z. Matheson, and C.R. Fricker. 1999. USEPA Method 1622. Journal American Water Works Association 91 (9): 60-68. Craun, G.F. 1986. Waterborne disease in the United States. CRC Press, Boca Raton, FL. Drinking Water Inspectorate. 1999. Guidance on assessing risk from Cryptosporidium oocysts intreated water supplies to satisfy the Water Supply (Water Quality) (Amendment) Regulations. 1999. SI 1524. Department of the Environment, Transport and the Regions, London. Harrison, S.L., R. Nelder, L.Hayek, I.F. Mackenzie, D.P. Casemore and D. Dance. 2002. Managing a large outbreak of cryptosporidiosis: how to investigate and when to decide to lift a ‘boil water’ notice. Communicable Disease and Public Health 5: 230-239. Howe, A.D., S. Forster, S. Morton, R. Marshall, K. Osborn, Wright, P. and Hunter, P.R. 2002. Cryptosporidium oocysts in a water supply associated with an outbreak of cryptosporidiosis. Emerging Infectious Diseases 8: 619-624. Hunter, P.R. 1997. Waterborne Disease: Epidemiology and Ecology. John Wiley, Chichester, UK. Hunter, P.R. 2000. Advice on the response to reports from public and environmental health to the detection of cryptosporidial oocysts in treated drinking water Communicable Disease and Public Health 3:24-27. Jakubowski, W. and T.H. Erickson. 1979. Methods for detection of Giardia cysts in water supplies. In Waterborne transmission of giardiasis, W. Jakubowski and J.C. Hoff (eds.). US Environmental Protection Agency, 600/9-79-001, Cincinnati, OH. Jephcott, A.E., N.T. Begg and I.A. Baker. 1986. Outbreak of giardiasis associated with mains water in the united kingdom. Lancet i: 730-732. LeChevallier, M.W., T.M. Trok, M.O. Burns, and R.G. Lee. 1990. Comparison of the zinc sulfate and immunofluorescence techniques for detecting Giardia and Cryptosporidium. Journal American Water Works Association 82 (9):75.

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LeChevallier, M.W., W.D. Norton, and R.G. Lee. 1991a. Occurrence of Giardia and Cryptosporidium spp. in surface water supplies. Applied and Environmental Microbiology 57:2610-2616. LeChevallier, M.W., W.D. Norton, and R.G. Lee. 1991b. Giardia and Cryptosporidium spp. in filtered drinking water supplies. Applied and Environmental Microbiology 57:2617-2621. LeChevallier, M. W. and W.D. Norton. 1995. Giardia and Cryptosporidium in raw and finished water. Journal American Water Works Association 87 (9): 54-68. LeChevallier, M.W., G.D. DiGiovanni, J.L. Clancy, Z. Bukhari, S. Bukhari, J.S. Rosen, J. Sobrinho, and M.M. Frey. 2003. Comparison of Method 1623 and cell culture-PCR for detection of Cryptosporidium in source waters. Applied and Environmental Microbiology 69: 971-979. McCuin, R.M. and J.L. Clancy. 2003. Modifications to USEPA methods 1622 and 1623 for detection of Cryptosporidium oocysts and Giardia cysts in water. Applied and Environmental Microbiology 69:267-274. Nichols, G. 2002. Using existing surveillance-based data. In: Drinking Water and Infectious Disease: Establishing the Links. CRC Press, Boca Raton, FL., 131 -141. Standing Committee of Analysts. 1999. Isolation and Identification of Cryptosporidium oocysts and Giardia cysts in waters: SCA Blue Book No 172. Environment Agency London. (http://www.environment-agency.gov.uk). Suckling, E.V. 1910. The Examination of Waters and Water Supplies. ed. The Blakiston Co, Philadelphia. USEPA. 1996. Information Collection Rule. United States Environmental Protection Agency. Office of Research and Development, Washington, DC. ICR Microbial Laboratory Manual. EPA/600/R-95/178. USEPA. 2001a. USEPA Method 1622: Cryptosporidium in Water by Filtration/IMS/FA. U. S. Environmental Protection Agency. Office of Water, Washington, DC. EPA 821-R-01-026. USEPA. 2001b. USEPA Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. U.S. Environmental Protection Agency. Office of Water, Washington, DC. EPA 821-R-01-025. Waite, M. and P. Jiggins. 2002. Cryptosporidium in England and Wales. In: Drinking Water and Infectious Disease: Establishing the Links. CRC Press, Boca Raton, FL., 119-126. Water Supply (Water Quality) (Amendment) Regulations. 1999. SI 1524. Stationery Office, London Water Supply (Water Quality) Regulations. 2000. SI 3184. Stationery Office, London

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ENTAMOEBA HISTOLYTICA GENOME

J.J. McCoy1 and B.J. Mann1,2 1

Departments of Internal Medicine and 2Microbiology, University of Virginia School of Medicine

ABSTRACT E. histolytica is a human parasite that is a significant cause of worldwide morbidity and mortality. This parasite is also of interest because it is an early branching amitochondriate eukaryote. The genome project should be completed by the end of 2003, however, the current release and preliminary annotation has already confirmed some previous observations and revealed new interesting aspects of the genome. The genome size is estimated to be 20 Mb with an overall AT content of 78%. The exact number of chromosomes is not resolved. Estimates range from 6 to 14. The genome includes numerous extrachromosomal circular elements including an episome with the rDNA genes. The chromosomal genome also contains a variety of other repeated elements including degenerate transposon-like elements. The promoter elements of E. histolytica have some unique features including a novel "GAAC" element. Introns are estimated to occur in 15% of protein encoding genes. A limited number of genomic sequences of other Entamoeba species have also been released. A comparison of these genomes, along with the nonpathogenic "cousin" of E. histolytica should help to discern the basis of virulence and the phylogenetic relationship of these and other species. Key words: Entamoeba histolytica, E. dispar, E. terrapinae, E. invadens, E. moshovskii, genome, rDNA, episomes

INTRODUCTION The human intestinal protozoan parasite E. histolytica is estimated to infect 50 million people and result in approximately 100,000 deaths per year from ulcerative colitis and amebic liver abscesses (WHO, 1995). Infection with E. histolytica, or amebiasis, is found worldwide, but is most common in developing countries. In most cases, E. histolytica infections are symptomless, but approximately 10% of infected individuals develop colitis (Reed, 2000). Most cases are seen in the very young and the very old, pregnant women, corticosteroid-treated individuals, and the malnourished (Seeto et al., 1999). Human to human transmission of E. histolytica occurs by a fecal-oral route beginning with the ingestion of the tetra-nucleated cyst in contaminated food or

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water. The trophozoite form colonizes the bowel lumen and can invade through intestinal epithelium to cause colitis, liver abscesses, or form cysts that are excreted to begin a new round of infection (Clark et al., 2000).

GENOME PROJECT There are two E. histolytica Genome Projects. One, funded by the National Institutes of Health, is at the Institute for Genomic Research (TIGR) (www.tigr.org/tdb/e2k1/eha1/) and the other is at the Wellcome Trust Sanger Institute Pathogen Sequencing unit (www.sanger.ac.uk/Projects/E_histolytica/). In October 2002 sequence information from the two projects was merged to create a 7-fold coverage of the genome. Automated annotation for the E. histolytica assemblies of >10 kb, representing approximately 12 Mb of unique sequence coverage and a majority of the E. histolytica coding regions, became available in January 2003. A database of the upstream and downstream sequences between predicted coding regions from the automated annotation of the 7X assembly is also available on the TIGR website for searching and download. A comparative Entamoeba genome project is also underway at the Wellcome Trust Sanger Institute in collaboration with Graham Clark, London School of Tropical Medicine and Hygiene. Sequences of E. terrapinae, a parasite of turtles, E. moshovskii, a free-living organism, and E. invadens, a reptilian parasite, are available on the Sanger site. The eventual plan is to sequence 21,000 shotgun clones of each parasite. Entamoeba invadens has been used a model of encyst- and excystment because its lifecycle can be completed in culture, and E. histolytica does not readily form cysts in culture (Eichinger 2001). A dataset of 16,000 Entamoeba invadens sequence reads is also available at the TIGR site. The E. histolytica genome is ~78 % AT rich overall and highly repetitive. These features of the genome have made assembly difficult (Brendan Loftus, TIGR, Neil Hall, personal communication). It is likely that the genome will not be completely closed, although investigators in the field have discussed closing at least one chromosome (Mann, 2002).

CHROMOSOMES The chromatin organization, karyotype and ploidy of E. histolytica have been difficult to determine and somewhat controversial. Analysis of chromatin spreads by electron microscopy revealed nucleosome-like structures (TorresGuerrero et al., 1991). The chromatin contains basic DNA-binding proteins that are different from other known eukaryotic histones (Torres-Guerrero et al., 1991), however genes encoding homologs to histones H1 (Scharfetter et al.,

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1997), H2B (Sanchez et al., 1994), H3 (Fodinger et al., 1993), and H4 (Binder et al., 1995) have been identified. The failure of the E. histolytica genome to condense at metaphase has hindered karyotypic characterization, however light microscopy studies estimate 6 chromosomes in E. histolytica nuclei (Arguello et al., 1992; Gomez-Conde et al., 1998) while electron microscopy suggests 9-12 chromosome-like structures (Torres-Guerrero et al., 1991). The pulsed-field gel electrophoresis technique has not unambiguously resolved E. histolytica chromosomes. This may possibly be due to abundant endogenous amebic nucleases, and the molecular mixture of linear and circular molecules, which generates broad bands (Orozco et al., 1993; Petter et al., 1993; Riveron et al., 2000). Willhoeft and Tannich used rotating-field electrophoresis (ROFE) to establish electrophoretic karyotypes for three different E. histolytica isolates, HM1-IMSS, 200:NIH, and HK-9 (Willhoeft et al., 1999b). Using this technique, 3135 chromosomes were identified that ranged in size from 0.3 to 2.2 Mb. These authors also identified 14 linkage groups using 68 independent cDNA probes. Although size polymorphisms were identified, linkage groups were conserved between the different isolates. Several probes bound to as many as four different bands, suggesting a ploidy of 4n (Figure 1). By adding the sizes of the largest chromosomes from each of the 14 linkage groups, the haploid genome size was estimated to be approximately 20 Mb.

GENE ORGANIZATION Based on published sequence, intergenic regions appear to be relatively short, ranging in size from 400 bp-2.3 kb (Bruchhaus et al., 1993; Petter et al.,

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1992; Willhoeft et al., 1999a). One pair of genes, pak and mcm3, have overlapping transcripts, with the 3’ untranslated region (UTR) of pac sharing 40 nucleotides with the 5’ UTR of mcm3 (Gangopadhyay et al., 1997). Initially introns were found in only a few of the early reported gene sequences (Lohia et al., 1993; Plaimauer et al., 1994; Sanchez-Lopez et al., 1998; Urban et al., 1996; Willhoeft et al., 1999a) suggesting a paucity of introns in E. histolytica. However, a more thorough and recent study identified an additional nine introncontaining genes in E. histolytica and the closely-related species Entamoeba dispar (Wilihoeft et al., 2001). The identified introns contain between 46 and 115 nucleotides, and have an average AT content of 83%, higher than the 73% found in the corresponding coding sequences and 78% in the E. histolytica genome overall. Conserved motifs were identified at the 5’ (GTTTGT) and 3’ (TAG) ends of the introns, corresponding to the GT and AG intron splice signals of most eukaryotic genes. Evidence for functional splicing machinery in E. histolytica was the identification of a U6 small nuclear RNA gene (Miranda et al., 1996). The U6 gene was expressed and present as a single copy in the E. histolytica genome. An analysis of an estimated 2-fold coverage of the E. histolytica genome found introns in 15% of genes with homologies to known proteins, an unexpectedly high percentage based on previously reported introncontaining genes (Loftus, unpublished, Mann, 2002). The final analysis of the entire genome should eventually give the precise percentage. The sequences of 5’ and 3’ UTRs of protein-encoding genes in E. histolytica have been analyzed for their influence on gene expression (Bruchhaus et al., 1994; Gilchrist et al., 1997; Ortiz et al., 2000; Purdy et al., 1996; Hidalgo et al, 1997; Schaenman et al., 1998). Conserved 5’ UTR sequences that have been identified include an unusual TATA-like site 30 nucleotides upstream of the transcription start site, an initiator element at the transcription start site, and a novel “GAAC” element, located at variable distances between the TATA-like site and the transcription start site (Singh et al. 1997). The 3’ UTRs contain a conserved pentanucleotide TAA/TTT, which acts as the transcription termination signal and a polypyrimidine region at the end of transcribed sequences (Bruchhaus et al., 1993; Singh et al., 1997). It has been proposed that these transcribed sequences determine mRNA secondary structure, which in turn, contributes to the binding of proteins that regulate gene expression (Ortiz-Garcia et al., 1997). The gene encoding a homolog of the 54 KDa subunit of the signal recognition particle (SRP54) gene provided evidence that E. histolytica caps mRNAs in a manner similar to other eukaryotes (Ramos et al., 1997). Capping of the SRP54 transcript was supported by the identification of an extra G residue at the cDNA transcription start site that was not present in the genomic sequence. Another unusual characteristic of E. histolytica is that transcription of protein-coding genes is (Lioutas et al., 1995b), suggesting

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either that E. histolytica RNA polymerase II is different from other eukaryotic RNA polymerases II, or that in E. histolytica, protein-encoding genes may be transcribed by RNA polymerase I, such as is the case for the PARP and VSG genes in trypanosomes.

EPISOMES Early attempts to resolve individual E. histolytica chromosomes by PFGE revealed a smear of fuzzy bands and several distinct high molecular weight bands. It was proposed that at least some of these high molecular weight bands represented circular DNA. Electron microscopy verified the presence of circular episomes of 4-50 kb in E. histolytica strain HM-1:IMSS (Dhar et al., 1995; Lioutas et al., 1995a). The most abundant and best-characterized episome is the 24.5 kb rDNA (EhR1) molecule that is present in about 200 copies per haploid genome equivalent. The EhR1 molecules account for over 80% of E. histolytica circular DNAs and 5-10% of total cellular DNA. Each EhR1 molecule in strain HM1:IMSS contains two 5.2 kb inverted repeats encoding ribosomal RNA (Bhattacharya et al., 1989). A 3.7 kb downstream spacer and a 9.2 kb upstream spacer separate the two units (Sehgal et al., 1994). The rRNA genes are believed to be present exclusively on the EhR1 plasmid, because no chromosomal copies of ribosomal genes have been identified. No proteins appear to be encoded on EhR1, although a single polyadenylated 0.7 kb RNA was identified by Northern blots. In contrast to naturally occurring plasmids in most prokaryotes and eukaryotes, two-dimensional gel electrophoresis and electron microscopic studies identified multiple replication sites on the EhR1 episome (Dhar et al., 1996). The smaller episomes do not appear be derivatives of the rDNA plasmid because EcoR1 fragments of the rDNA plasmid do not hybridize with these episomes on Southern blots (Dhar et al., 1995). The DNA sequences of these smaller episomes have not been determined so their coding potential and functions are unknown.

REPEATED SEQUENCES The genome of E. histolytica is highly repetitive and contains several types of repetitive DNA. The most abundant repeated DNA is found on the ribosomal plasmid EhR1. Two families, the 170 bp DraI repeats and the 144 bp ScaI repeats, comprise the 3.7 kb downstream intergenic spacer, while the 9.2 kb upstream spacer contains ScaI, 145 bp PvuI, and HinfI repeats (Bhattacharya et al., 1998). Long interspersed elements (LINEs) and short interspersed elements (SINEs) are autonomous and non-autonomous transposable elements, respectively. By analyzing sequences generated by the E. histolytica genome

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project, Van Dellen et al described three families of LINEs, named EhLINEs, which include the previously reported EhRLE (Sharma et al., 2001) and HMc (Mittal et al., 1994) repeated sequences, and two families of SINE-like sequences named EhSINEs (Van Dellen et al., 2002). One family, EhLINE1, shared a common 3’ end with IE, a previously reported highly transcribed 0.55 kb repetitive element (Cruz-Reyes et al., 1995). Individual EhLINE/EhRLE elements do not contain any open reading frames but a comparison of a number of elements suggests that they contain degenerate nucleic acid-binding motifs, restriction enzyme-like endonuclease domains, and reverse transcriptase domains (Sharma et al., 2001; Van Dellen). They also share 3’ end sequences with EhSINEs. It is estimated that LINEs and SINEs comprise 6% of E. histolytica DNA, second only to rDNA episomes in their abundance in the genome. Zaki and Clark isolated two other novel multicopy loci, designated locus 1-2 and locus 5-6, that contain internal repeats from E. histolytica, in an effort to isolate microsatellites (Zaki et al., 2001). While microsatellites were not found, these loci may have the potential to be used as polymorphic molecular markers to investigate E. histolytica epidemiology and intraspecies variations. Examination of sequences from the E. histolytica Genome project has identified many other groups of previously unidentified repetitive DNA (Van Dellen et al., 2002). The functions of these repetitive sequences are not yet known.

EVOLUTIONARY POSITION In the colonic lumen E. histolytica is an obligate fermentor, lacking proteins of the mitochondrial electron transport chain and tricarboxylic acid cycle. Organelles of higher eukaryotes, such as mitochondria, rough endoplasmic reticulum, Golgi apparatus, centrioles, and microtubules, have not been identified by electron microscopy (Clark et al., 2000). Because of this apparent lack of higher eukaryotic machinery, combined with the presence of bacterial-like fermentation enzymes, early evolutionary theories considered E. histolytica to be a transitional eukaryote that diverged from other eukaryotes prior to the acquisition of mitochondria and other organelles. (For a more complete review, see Samuelson, 2002). However, evidence supporting secondary loss of mitochondria has been presented and may explain the apparent relative simplicity of E. histolytica compared to higher eukaryotes. The E. histolytica genome contains at least three genes, pyridine nucleotide transhydrogenase (PNT), the mitochondrial chaperonin cpn60, and a mitochondrial-type hsp70, normally found on the mitochondrial genome (Bakatselou et al., 2000; Clark et al., 1995). The identification of these genes in E. histolytica supports the secondary loss hypothesis. The loss may have resulted from adaptation of the parasite to the gut (Boore et al., 1999; Clark et al., 2000). In addition, the identification of a mitochondrion-derived organelle, (the crypton or mitosome) (Mai et al., 1999; Tovar et al., 1999) and Golgi (Ghosh et al.,

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1999), along with genes of higher eukaryotes such as ras-family signal transduction proteins (Lohia et al., 1994; Lohia et al., 1996), ABC-family drug transporters (Descoteaux et al., 1995), and introns (Wilihoeft et al., 2001), suggest a common eukaryotic ancestor.

BACTERIAL-LIKE GENES IN E. histolytica Three hypotheses have been proposed that might explain the presence of bacterial-like anaerobic fermentation enzymes in E. histolytica and other protozoans. The amitochondriate fossil hypothesis suggests that genes encoding fermentation enzymes were present prior to the acquisition of mitochondrion, and then lost in most eukaryotes (Reeves, 1984). The hydrogen hypothesis suggests that fermentation genes were acquired simultaneously with the mitochondrial endosymbiont (Martin et al., 1998; Rotte et al., 2000). The lateral transfer hypothesis proposes that the fermentation genes were directly acquired from anaerobic prokaryotes (de Koning et al., 2000; Doolittle, 1999; Field et al., 2000; Rosenthal et al., 1997). Phylogenetic evidence supports the lateral transfer hypothesis in that 1) the sequences of several E. histolytica fermentation genes are more similar to those of bacteria than to those of phylogenetically similar protozoan parasites, 2) E. histolytica fermentation enzyme gene homologs are absent from most higher eukaryotes, and 3) E. histolytica possesses several other bacterial genes besides those involved in anaerobic fermentation (Rosenthal et al., 1997). Molecular techniques have made it possible to test evolutionary hypotheses based on phenotypic traits. Completion of the E. histolytica genome project is certain to lead to a better understanding of the parasite’s evolution and phylogenetic relationship with other protests.

Entamoeba histolytica vs. Entamoeba dispar E. dispar is closely related to E. histolytica, both morphologically and genetically, yet it has never been associated with disease (Diamond, 1993). The genomes of these two organisms are highly similar and apparently syntenic (Willhoeft, 1999a). Homologs for several of the better-studied E. histolytica virulence factors are also found in E. dispar, but some differences exist. E. dispar contains at least two genes encoding the galactose/N-acetyl Dgalactosamine (Gal/GalNAc)-inhibitable lectin heavy subunit and four genes encoding the lectin light subunit (Dodson, 1997). The Gal/GalNAc lectin is the major surface adhesin of the parasite. Lectin heavy and light subunit homologs showed 86% and 79% amino acid identity between the two species. Homologs for all three isoforms (A,B,C) of the amebic pore-forming protein, amoebapore are also present in E. dispar (Leippe et al., 1993). Analyses of amebic cysteine proteinases found that two of the most highly expressed and active cysteine proteinase genes (ehcp1 and ehcp5), which account for over 70% of cysteine proteinase activity in E. histolytica, are not expressed in E. dispar (Bruchhaus et

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al., 1996; Jacobs et al., 1998). A pseudogene corresponding to ehcp5 is present and positionally conserved in E. dispar (Willhoeft et al., 1999a). A direct comparison of these two genomes, along with microarray analysis should help to uncover differences in homologous genes and gene expression.

CONCLUSION The E. histolytica genome project along with genomic sequence from other Entamoeba species should provide the tools to identify key virulence factors and the mechanisms pathogenicity. This should lead to better understanding of host-parasite relationships, and potentially new drugs and vaccines. The genome sequence should also help to establish phylogenetic relationships between early branching eukaryotes and a greater understanding of the evolution of eukaryotic organisms.

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Willhoeft, U., L. Hamann, and E. Tannich. 1999a. A DNA sequence corresponding to the gene encoding cysteine proteinase 5 in Entamoeba histolytica is present and positionally conserved but highly degenerated in Entamoeba dispar. Infection and Immunity 67:5925-5929. Willhoeft, U., and E. Tannich. 1999b. The electrophoretic karyotype of Entamoeba histolytica. Molecular and Biochemical Parasitology 99:41-53. . Zaki, M., and C. G. Clark. 2001. Isolation and characterization of polymorphic DNA from Entamoeba histolytica. Journal of Clinical Microbiology 39:897-905.

CRYPTOSPRORIDIUM PARVUM GENOMICS: IMPACT ON RESEARCH AND CONTROL

G. Zhu1 and M. S. Abrahamsen2 1

Department of Veterinary Pathobiology, Texas A & M University, College Station, TX Department of Veterinary Pathobiology, University of Minnesota, St. Paul, MN

2

ABSTRACT Cryptosporidium parvum is a well-recognized cause of diarrhea in humans and animals throughout the world, and is associated with a substantial degree of morbidity and mortality in patients with acquired immunodeficiency syndrome (AIDS). Despite intensive efforts over the past 20 years, there is currently no effective therapy for treating or preventing infection by C. parvum. Until recently, the development of effective anticryptosporidial therapies has been hindered by the paucity of biological targets for structurebased drug design. With the pending completion of sequencing of the entire C. parvum genome, there should be no lack of potential biological targets for development of specific inhibitors. What are needed are focused efforts to identify essential parasite biochemical pathways for which specific inhibitors can be developed that are safe to the host. Analysis of the currently available sequences has identified specific aspects of C. parvum biochemistry that are unique relative to mammals. In addition, the ongoing genome efforts are providing a clearer picture of the biology of the apicomplexan parasites. It is becoming evident that C. parvum uses distinct approaches to solve basic metabolic needs as compared to Plasmodium falciparum and Toxoplasma gondii. These differences suggest that therapies developed for these model apicomplexans may not necessarily be effective against cryptosporidiosis, and emphasizes the importance of understanding the unique biology of C. parvum. Key Words: Genomics, DNA replication, fatty acid biosynthesis, glycolysis, purine salvage, metabolism

INTRODUCTION The life cycle of C. parvum is similar to that of other apicomplexans, and is composed of multiple asexual and sexual developmental stages (Fayer et al., 1997). Currently, it is not known precisely how the genome content changes in the different developmental stages, nor are the basic mechanisms

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of DNA replication or translocation during development understood. It is evident, however, that for C. parvum to complete its life cycle, it must faithfully replicate its genome a significant number of times and thus must have a high metabolic demand for nucleotides throughout its development. Further, as an obligate intracellular organism C. parvum must have unique means of generating energy and acquiring or synthesizing the basic cellular components required to accomplish this rapid cell multiplication and extensive remodeling of the parasite’s cell structure during completion of its life cycle. A major limitation to understanding the basic biology of C. parvum is the inability to obtain purified samples of the various asexual, sexual, and intracellular developmental stages of the parasite. As a consequence, few molecular or biochemical studies have been directed toward understanding the mechanisms associated with the pathogenesis of C. parvum or the complicated developmental biology during its intracellular development. To address this limitation, several large-scale sequencing projects have been initiated (Liu et al., 1999; Strong and Nelson, 2000), including a project to sequence the complete C. parvum genome (CpGP) that is nearing completion (Abrahamsen, 1999; Widmer et al., 2002). These efforts have dramatically increased our understanding of this important pathogen and have demonstrated that C. parvum differs from the related apicomplexan pathogens Toxoplasma gondii and Plasmodium falciparum in several basic biological processes. The molecular divergence of C. parvum from other apicomplexans is also congruent with the phylogenetic position of the Cryptosporidium genus as an early emerging branch within the Apicomplexa (Zhu et al., 2000a), or furthermore, as a sister to the Class Gregarina (Carreno et al., 1999). In this chapter, we will discuss key insights into DNA replication, purine metabolism, fatty acid biosynthesis and energy metabolism that are unique to C. parvum relative to its mammalian host that may ultimately provide new biological targets for development of specific inhibitors.

DNA REPLICATION PROTEINS The life cycles of all apicomplexans consist of at least 3 distinct developmental processes: sporogony, merogony, and gamogony. Both sporogony and merogony are cell multiplication processes that produce more than 2 daughter cells in each cell cycle and differ from the host cell somatic duplication, which suggests a unique mechanism may be involved in the DNA replication in apicomplexan parasites. However, little was known about the molecules that are involved in the process and regulation of the apicomplexan DNA replication. Recently, the rapid development in molecular biology and genomics has provided us opportunities in the discovery and characterization of DNA replication proteins in C. parvum and other apicomplexans.

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One important class of DNA replication molecules is the replication protein A (RPA) that belongs to the eukaryotic single stranded DNA (ssDNA) binding proteins involved in the DNA replication, repair and recombination (Wold, 1997; Iftode et al., 1999). In animals, plants and fungi, RPA is a heterotrimeric protein of approximately 70, 32 and 14 kDa subunits. The large subunit (RPA1) consists of an N-terminal domain for interacting with various factors involved in the regulation of DNA replication, repair or recombination, a middle section with two ssDNA-binding sites, and a Cterminal region for binding the other two subunits. We first discovered the CpRPA1 gene from the parasite genome by sequencing the downstream region near the CpFAS1 locus. Surprisingly CpRPA1 encodes a short-type 54-kDa RPA1 subunit that differs from its host and lacks the N-terminal protein-interacting domain that is present in animal, plant and fungal RPA1s, indicating that C. parvum may utilize a distinct pathway for regulatory factors to interact with RPA during DNA metabolism (Zhu et al., 1999). Interestingly, from the ongoing CpGP we have identified a second RPA1 gene (CpRPA1B) that differs significantly not only from its host, but also from CpRPA1 (Millership and Zhu, 2002). Although CpRPA1B appears to encode a “full-size” RPA1 (i.e., its ORF predicts a 75.5 kDa protein), western blotting analysis detects only a ~45 kDa band, indicating that either CpRPA1B is translated at an alternative start codon, or the protein is cleaved following translation. Therefore, both RPA1 proteins in C. parvum belong to the short-type subunits. Short-type RPA1 subunits have also been identified from the genomes of P. falciparum (PfRPA1) and T. gondii (TgRPA1). Like CpRPAlB, the native PfRPAl protein is short (54 kDa), although its ORF predicts a 134-kDa polypeptide (Voss et al., 2002). In contrast, TgRPA1 is apparently encoded by a short ORF (data not shown). In addition, the CpRPA1 and CpRPA1B proteins are differentially distributed in C. parvum sporozoites and during its life cycle, suggesting different roles for these two proteins in the parasite DNA metabolism. In addition, a homologue to the middle subunit (CpRPA2) was recently identified from the CpGP. Despite the low homology among all RPA2 subunits, we were able to confirm that CpRPA2 protein was indeed an ssDNA-binding protein and could be regulated by phosphorylation. The phosphorylation of CpRPA2 completely abolished the ssDNA-binding property. Similar to other DNA replication proteins, the expression of CpRPA2 gene is regulated by the cell cycle and its protein is distributed more intensively in the C. parvum oocysts, meronts and gamonts than other life cycle stages (Millership and Zhu, unpublished data). RPA proteins are essential to all eukaryotes and several anticancer compounds have been shown to target these proteins (Basilion et al., 1999; Peters et al., 2001). As apicomplexan RPA proteins differ from their hosts in

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both structure and function, data-mining of the CpGP and functional analysis to identify other elements in the RPA-associated DNA metabolic pathway could provide us new opportunities to study the process and regulation of the unique parasite life/cell cycles, and to explore this distinct pathway as a novel drug target against apicomplexans

PURINE METABLOISM/SALVAGE The enzymes of the nucleotide biosynthesis pathway are essential for supporting cell proliferation. In mammals, nucleotides may be synthesized through one of two pathways. In the de novo synthesis pathway, the purine ring system is assembled in a step-wise manner. In the salvage pathway, preformed nucleotides, nucleosides and nucleobases are recycled. The extent of utilization of each pathway in mammals is dependent on the cell type and growth state of the cell. In contrast to their mammalian hosts, all parasitic protozoa examined to date are incapable of synthesizing the purine ring de novo, but depend on preformed nucleotides that they purportedly obtain from the host using specific salvage pathways (Berens et al., 1995). The apicomplexans P. falciparum and T. gondii share similar purine salvage pathways with other human protozoan pathogens including Entamoeba histolytica and Giardia lamblia. In the case of P. falciparum and T. gondii it has been demonstrated that both hypoxanthine–guanine–xanthine phosphoribosyltransferase (HXGPRT) and adenosine kinase are key enzymes for purine salvage and that disruption of this pathway is inhibitory to parasite growth (Shi et al., 1999; Pfefferkorn et al., 2001; Kicska et al., 2002). To date, few studies have investigated purine metabolism in Cryptosporidium. A recent study presented biochemical evidence that C. parvum lacks the ability for de novo purine nucleotide synthesis (Doyle et al., 1998). The authors hypothesized that similar to Toxoplasma, C. parvum may have a single HXGPRT enzyme involved in purine salvage. Recently, we attempted to clone the putative C. parvum HXGPRT gene by genetic complementation in T. gondii (Striepen et al., 2002). This system takes in which the HXGPRT gene has advantage of a T. gondii line been inactivated (Donald et al., 1996). Without HXGPRT activity, parasite survival is dependent on inosine monophosphate dehydrogenase (IMPDH) to convert IMP to XMP to supply GMP for DNA synthesis. In the presence of mycophenolic acid (MPA), a potent inhibitor of IMPDH, only T. gondii tachyzoites that have been transformed with a vector containing a functional HXGPRT gene are viable. To isolate the putative C. parvum HXGPRT gene, randomly sheared C. parvum genomic DNA was used to construct a complementation library in an empty T. gondii expression vector. tachyzoites were transfected with this heterologous library and cultured in the presence of MPA. Following selection, drug resistant parasites

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emerged, however, the conceptual translation of the ORF contained in the recovered C. parvum genomic sequence revealed clear similarity to IMPDH, not HXGPRT. Surprisingly, the similarity of the isolated CpIMPDH gene was much greater to prokaryotic than to eukaryotic IMPDH. All phylogenetic methods support the specific grouping of C. parvum IMPDH with the homologs (Striepen et al., 2002). CpIMPDH was never grouped with other eukaryotic IMPDHs including the IMPDH from the apicomplexan P. falciparum. Our findings indicate that CpIMPDH is not of eukaryotic nuclear descent or even mitochondrial origin (since it is not specifically related to the but instead was likely obtained by lateral gene transfer (LGT) from a Since bacterial IMPDH enzymes are much less sensitive to MPA, the relationship of CpIMPDH also clarifies its insensitivity to MPA used in selective medium. Moreover, subsequent experimental approaches and extensive searching of the available C. parvum genomic and EST databases using T. gondii and P. falciparum HXGPRT failed to identify any C. parvum homologs, strongly suggesting that C. parvum lacks this enzyme. In contrast, in silico analysis identified putative C. parvum homologs for all of the enzymes necessary for the salvage and conversion of adenosine to GMP: a 5’ nucleotidase, an adenosine transporter, adenosine kinase, AMP deaminase, IMPDH and GMP synthase. These findings indicate that C. parvum purine salvage is distinct from other apicomplexans and the only source of purines for C. parvum is dependent on the adenosine salvage pathway. Therefore, it would be predicted that all of the enzymes within the adenosine salvage pathway, including IMPDH, are essential for C. parvum. Thus, inhibition of these enzymes would lead to a depletion in the supply of nucleotides for DNA synthesis, resulting in the inhibition of parasite growth. CpIMPDH would be particularly attractive target due to its apparent bacterial origin and clear difference from eukaryotic IMPDH homologs.

ENERGY METABOLISM Early biochemical analyses indicated that C. parvum relies mainly on glycolysis as an energy source (Denton et al., 1996; Entrala and Mascaro, 1997). A great number of enzymatic activities within the pathway have been detected in oocysts and free sporozoites, almost all of which were present in the cytosol. There are many enzymes necessary for glycolysis. Malate and lactate dehydrogenases (MDH and LDH), both of which are cytosolic, have been reported as partial sequences in the C. parvum EST and GSS projects (Liu et al., 1999; Strong and Nelson, 2000). The complete CpLDH1 gene was determined by an independent cloning approach (Zhu and Keithly, 2002), while CpMDH1 was identified from a contig in the ongoing CpGP. Unlike

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LDH from T. gondii and Plasmodium spp. that contain a short amino acid insertion at the active site loop, CpLDH1 does not possess such an insertion. Phylogenetic analysis indicates that these two enzymes in C. parvum and other apicomplexans are sisters to MDH, differing from the majority of other eukaryotic MDH and LDH enzymes. This observation suggests that apicomplexan MDH and LDH enzymes might have been acquired from (Zhu and Keithly, 2002). In addition to MDH and LDH, a number of other enzymes within the glycolytic pathway have been identified by the complete EST and GSS projects. In fact, by data-mining the C. parvum contigs deposited in the GenBank by the ongoing CpGP, we were able to identify almost all genes (except for one) encoding C. parvum glycolytic enzymes previously detected with biochemical assays (Entrala and Mascaro, 1997). These enzymes include: phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), pyrophosphate-dependent phospho-fructokinase (PPi-PFK, two homologues), triosephosphate isomerase (TIM), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGM), enolase, pyruvate kinase (PK), LDH, phosphoenolpyruvate carboxylase (PEPCL), MDH, malic enzyme (ME), and adenylate kinase (AK). Among them, the PPi-PFK is typical to anaerobic microorganisms and has been proven to be a potential drug target for the phosphonic acid analogs in T. gondii (Peng et al., 1995). The only enzyme absent in the GenBank C. parvum contigs at the moment is the aldolase. In contrast to P. falciparum, but in agreement with early observations that C. parvum lacks an active tricarboxylic acid (Krebs’) cycle (Denton et al., 1996; Entrala and Mascaro, 1997), no enzymes within the Krebs’ cycle could be identified from all available C. parvum genomic data. Another important enzyme associated with anaerobic metabolism is the oxygen-sensitive pyruvate-ferredoxin oxidoreductase (PFO) that converts pyruvate to acetyl CoA and with the reduction of ferredoxin. Cryptosporidium PFO homologues were first found in the GSS entries from GenBank. Subsequent cloning and sequencing efforts revealed that the C. parvum PFO was part of a pyruvate-NADP oxidoreductase PNO (CpPNO) fused with a cytochrome P450 reductase (CPR) (Rotte et al., 2001). This type of gene fusion was coincidently found in another unrelated protist, Euglena gracilis. It is interesting that the E. gracilis PNO contains a mitochondrial transit sequence. However, CpPNO lacks such a mitochondria transit sequence, suggesting it could be cytosolic. Phylogenetic analysis has suggested a common ancestry of PFO in amitochondriate protists with Euglena mitochondrial PNO and apicomplexan CpPNO, which provides a new insight at the evolution of these unique proteins (Rotte et al., 2001). Furthermore, it has been suggested that CpPNO might be responsible for the

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parasite’s sensitivity to 5’-nitrothiazole (NTZ) that has been in human clinical trials for treating chronic cryptosporidiosis (Coombs and Muller, 2002). The complete sequencing of CpPNO gene makes it possible to truly test this hypothesis.

FATTY ACID SYNTHETIC PATHWAY Fatty acids are one of the major components of biomembranes. At least 7 enzymes are involved in the synthesis of fatty acids (Smith, 1994): ketoacyl synthase (KS), acyl transferase (AT), ketoacyl reductase (KR), dehydrase (DH), enoyl reductase (ER), acyl carrier protein (ACP) and thioesterase (TE). In animals and fungi, these enzymes are fused into one or two multifunctional polypeptides, referred to as Type I fatty acid synthase (FAS). In bacteria and plants, these proteins are discrete, monofunctional enzymes, termed Type II FAS. In eukaryotes, Type I FAS proteins are cytosolic, while Type II FAS enzymes are localized in the plant plastid. Our understanding of fatty acid metabolism in the Apicomplexa began with the discovery of several Type II FAS enzymes in T. gondii and P. falciparum and a giant 25-kb Type I FAS gene in C. parvum (CpFAS1) (Waller et al., 1998; Zhu et al., 2000c). Type II FAS genes were originally identified from T. gondii and P. falciparum EST by the presence of plastid transit sequences, and subsequent analyses confirmed that these enzymes were indeed targeted to the apicoplast. In contrast, CpFAS1 polypeptide was localized in the cytosol. It contains 21 domains distributed as a loading unit (ligase-ACP), 3 chain elongation modules (KS-AT-DH-ER-KR-ACP), and a C-terminal reductase. This architecture differs from animal FAS, which is fused with 7 enzymes (KS-AT-DH-ER-KR-ACP-TE) (Smith, 1994). Does C. parvum differ from other apicomplexans by lacking any Type II FAS genes? Although this question cannot be ultimately answered until the entire C. parvum genome is sequenced and annotated, current molecular, biochemical and genomic data apparently support this hypothesis. Firstly, in vitro drug tests indicated that C. parvum was insensitive to thiolactomycin (a Type II FAS-specific inhibitor that could inhibit the growth of T. gondii and P. falciparum) (Zhu et al., 2000c). Secondly, C. parvum apparently lacks the plastid genome, suggesting that a plastid is likely absent to harbor Type II FAS (Zhu et al., 2000b). Thirdly, from all currently available C. parvum genome sequences in the GenBank, we are unable to identify any Type II FAS homologues, nor the plastid genome. In addition to CpFAS1, a 40-kb ORF encoding the first known protist polyketide synthase (CpPKS1) gene was identified and characterized from C. parvum (Zhu et al., 2002). It was originally presented as 3 separate GSS entries homologous to CpFAS1 in the GenBank. About 38-kb nucleotides of CpPKS1 were sequenced from overlapping gDNA clones, and the N-terminal

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~2-kb gap was closed by taking advantage of the CpGP. Like CpFAS1, CpPKS1 contains a loading unit, 7 elongation modules and a reductase. However, most of the elongation modules in CpPKS1 contain only 2-6 enzymes (incomplete sets), suggesting that their intermediate and/or final products may retain keto, hydroxyl groups and/or double bands. This is characteristic to the Type I PKS identified from bacteria and fungi (Rawlings, 1997; Staunton and Weissman, 2001). The discovery of CpFAS1 and CpPKS1 genes provides us with a new opportunity to study the fatty acid/polyketide biosynthesis in C. parvum. In addition, these distinct mega-synthases may also serve as novel targets in drug discovery against cryptosporidiosis. Indeed, FAS enzymes have been recognized as a new class of drug target against a variety of tumors (Pizer et al., 1996; Pizer et al., 1998) and pathogens that include bacteria (Mdluli et al., 1998; Heath et al., 1999; McMurry et al., 1999) and fungi (Broedel et al., 1996; Zhao et al., 1996). This notion is further supported by the inhibitory studies of FAS inhibitors in C. parvum, T. gondii and P. falciparum (Waller et al., 1998; Zhu et al., 2000c). In addition to CpFAS1 and CpPKS1, several other genes encoding fatty acid metabolic enzymes have been identified from the ongoing CpGP. These include: 1) the Ppant transferase that activates ACP by transferring a 4’phosphopantethein prosthetic group from CoA to the Ser residue in ACP; 2) three independent fatty acyl-CoA ligases (ACL) for the activation of fatty acids to form fatty acyl-CoA; 3) acyl-CoA binding protein (ACBP) responsible for the transport of fatty acyl-CoA; 4) acetyl-CoA carboxylase (ACC) for the synthesis of malonyl-CoA; and 5) fatty acyl elongase that is typically associated with microsomes in other eukaryotes. It is clear that a more complete picture will soon arise with the completion of the CpGP.

SUMMARY The impact on basic research and target discovery of a complete C. parvum genome project (CpGP) will be dramatic. It is predictable that all classical or specific energy metabolic enzymes and isoenzymes will be identified from the complete parasite genome. However, some enzymes exclusive to C. parvum and/or the Apicomplexa may require additional functional studies to determine their biochemical features. The availability of the complete genome sequence will clearly accelerate the discovery and reconstruction of various pathways, both in silico and in vitro. From the complete genome, one can identify genes of interest to study their functions and pharmaceutical values, determine if there are alternative pathways for the drug targets, and study the dynamics of metabolic enzymes during the intracellular life cycle stages (which is almost impossible by traditional biochemical analysis of crude parasite extract). Moreover, the complete C.

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parvum genome will ultimately clarify whether a speculative gene or drug target is absent or present, and will allow for a better understanding of the evolution and relationship of apicomplexan pathogens.

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Wold, M.S., 1997. Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annual Review of Biochemistry 66: 61-92. Zhao, X.J., G.E. McElhaney-Feser, W.H. Bowen, M.F. Cole, S.E. Broedel, Jr. and R.L. Cihlar, 1996. Requirement for the Candida albicans FAS2 gene for infection in a rat model of oropharyngeal candidiasis. Microbiology 142: 2509-2514. Zhu, G. and J.S. Keithly, 2002. Alpha-proteobacterial relationship of apicomplexan lactate and malate dehydrogenases. Journal of Eukaryotic Microbiology 49: 255-261. Zhu, G., J.S. Keithly and H. Philippe, 2000a. What is the phylogenetic position of Cryptosporidium? International Journal of Systematic and Evolutionary Microbiology 50: 1673-1681. Zhu, G., M.J. LaGier, F. Stejskal, J.J. Millership, X. Cai and J.S. Keithly, 2002. Cryptosporidium parvum: the first protist known to encode a putative polyketide synthase. Gene 298: 79-89. Zhu, G., M.J. Marchewka and J.S. Keithly, 1999. Cryptosporidium parvum possesses a shorttype replication protein A large subunit that differs from its host FEMS Microbiology Letters 176: 367-372. Zhu, G., M.J. Marchewka and J.S. Keithly, 2000b. Cryptosporidium parvum appears to lack a plastid genome. Microbiology 146: 315-321. Zhu, G., M. J. Marchewka, K.M. Woods, S. J. Upton and J.S. Keithly, 2000c. Molecular analysis of a Type I fatty acid synthase in Cryptosporidium parvum. Molecular & Biochemical Parasitology 105: 253-260.

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INDEX

A

Acquired immunodeficiency syndrome (AIDS) amebiasis in, 77 cryptosporidiosis in, 30, 92, 104–105 cyclosporiasis in, 47, 109 Acridinic thioethers, in cryptosporidiosis, 107–108 AIDS. See Acquired immunodeficiency syndrome (AIDS) Albendazole, in giardiasis, 110 Alcohol use, amebiasis and, 77 Amebiasis, 75–84. See also Entamoeba dispar; Entamoeba histolytica in AIDS, 77 alcohol use and, 77 antibody production in, 81, 83 cytokines in, 81–82 death from, 75, 141 diagnosis of, 78 extraintestinal, 111 IgA in, 81, 83 immune evasion in, 80 immune response to, 81–84 inflammatory response in, 81–82 interleukin-12 in, 82 lectin-based test in, 19–20 lectin-glycoprotein interaction in, 78–79 microscopy in, 18, 19 mucus in, 81 pathogenesis of, 78–81 polymerase chain reaction test in, 20 proteinases in, 80–81 risk factors for, 76–78, 141–142 in SCID mouse, 82 serology in, 18–19 serum anti-trophozoite IgG and, 77 symptoms of, 76 treatment of, 19, 110–111 vaccine against, 83–84 virulence factors in, 79–81 Amoebapore, 81 Antibody in amebiasis, 81, 83 in cryptosporidiosis, 97

in cyclosporiasis, 47 in giardiasis, 67–68 Arginine decarboxylase inhibitors, in cryptosporidiosis, 107 ATP-binding cassette transporters, in Cryptosporidium parvum, 106–107 Aurone analogs, in cryptosporidiosis, 107–108 Azithromycin, in cryptosporidiosis, 104 B

Basil, cyclosporiasis with, 50 Benzimidazoles, in cryptosporidiosis, 107 Blackberries, cyclosporiasis with, 50 Bupravaquone, in cryptosporidiosis, 108 C

Cat, giardiasis in, 6–7, 9–10 Chromosomes, of Entamoeba histolytica, 142–143 Ciprofloxacin, in cyclosporiasis, 109 Clarithromycin, in cryptosporidiosis, 105 CpFAS1 gene, 156–160 CpPKS1 gene, 156–160 Cryptosporidiosis. See also Cryptosporidium parvum adaptive immunity in, 94–95 age-related incidence of, 29–30 in AIDS, 30, 92, 104–105 antibody in, 97 cytokines in, 95–96 drug delivery systems in, 108 drug resistance in, 105–107 foodborne, 28 immunopathogenesis of, 93–94 inflammatory response in, 93–94 innate immunity to, 91–93 in, 92–93, 95–96 mucosal lymphocytes in, 96–97 natural killer cell response in, 92–93 in, 92 p23 in, 97

166 pathogenesis of, 35 prostaglandin production in, 92 risk factors for, 35 in SCID mice, 92–93, 96–97 seasonal incidence of, 30 in T cell–deficient mice, 94–95 T cells in, 94–96 transforming growth in, 94 transmission of, 28–29 treatment of, 103–108 villous atrophy in, 93 waterborne, 29, 34, 129–138. See also Water monitoring Cryptosporidium spp., 33–34 DNA fingerprinting of, 35–36 water monitoring for, 129–138. See also Water monitoring Cryptosporidium hominis, 32, 33, 34, 35

DNA fingerprinting of, 35–36 Cryptosporidium parvum, 27–37 animal type, 32 ATP-binding cassette transporters in, 106–107 CpFAS1 gene of, 156–160 CpPKS1 gene of, 156–160 dehydrogenases of, 157–158 dihydrofolate reductase of, 106 DNA fingerprinting of, 35–36 DNA replication proteins of, 154–156 drug resistance of, 105–107 energy metabolism of, 157–159 enzymes of, 107, 156–160 fatty acid metabolism in, 159–160 fatty acid synthase gene of, 107 genome of, 153–161 glycolytic enzymes of, 157–159 host-adapted genotypes of, 32 human type, 32 life cycle of, 153–154 molecular studies of, 30–37 polymerase chain reaction test of, 31–32 purine metabolism in, 156–157 pyruvate-ferredoxin oxidoreductase of, 158–159 transmission of, 28–29 CXXC motif, of Giardia, 61, 62 Cyclospora cayetanensis, 43–53 detection of, 51–53 identification of, 43–44 life cycle of, 45–46 taxonomy of, 44–45 vs. Eimeria, 52–53 Cyclosporiasis, 43–53

Index in AIDS, 47, 109 ciprofloxacin in, 109 diagnosis of, 51–53 foodborne, 45, 48, 49–51 immunity to, 46, 47 microscopy in, 51 polymerase chain reaction test in, 52 seasonality of, 48 symptoms of, 46 timethoprim-sulfamethoxazole in, 109 treatment of, 47, 108–109 waterborne, 48, 49 Cytokines in amebiasis, 81–82 in cryptosporidiosis, 95–96 D

Dehydrogenases, of Cryptosporidium parvum, 157–158 Dihydrofolate reductase, of Cryptosporidium parvum, 106 Dinitroaniline herbicides, in cryptosporidiosis, 107 DNA fingerprinting, of Cryptosporidium. 35–36 DNA replication proteins, of Cryptosporidium parvum, 154–156 Dogs, giardiasis in, 6–7, 9–10 Drug resistance, in cryptosporidiosis, 105–107 E

Eimeria, vs. Cyclospora, 52–53 Energy metabolism, of Cryptosporidium parvum, 157–159 Entamoeba dispar, 15–22. See also Amebiasis bacterial ingestion by, 21–22 electron microscopy of, 21–22 enzymes of, 17–18, 20–21, 76 genome sequencing of, 21 proteinases of, 80–81 vs. E. histolytica, 20–22, 147–148 Entamoeba histolytica, 15–22. See also Amebiasis amoebapore of, 81 antibody to, 81 bacterial ingestion by, 21–22 bacterial-like genes in, 147–148 chromosomes of, 142–143 electron microscopy of, 21–22

Index enzymes of, 17–18, 20–21, 75 episomes of, 145 evolutionary position of, 146–147 Gal/GalNAc lectin of, 78–80, 82 gene expression in, 144–145 genome of, 21, 141–148 locomotion of, 80 long interspersed elements of, 145–146 proteinases of, 80–81 repeated sequences of, 145–146 serine-rich protein of, 84 short interspersed elements of, 145–146 vs. E. dispar, 20–22, 147–148 Enzymes of Cryptosporidium parvum, 107, 156–160 of Entamoeba dispar, 17–18, 20–21, 76 of Entamoeba histolytica, 17–18, 20–21, 75 Episomes, of Entamoeba histolytica, 145

167 water monitoring for, 129–138. See also Water monitoring Zn finger motif of, 62 Giardiasis. See also Giardiasis animal role in, 2–3 antibody response to, 67–68 biological selection in, 68–69 in domestic dogs and cats, 6–7, 9–10 experimental, 67–69 in immunodeficient gerbils, 69 in livestock, 6, 8 non-immunological mechanisms in, 68–69 pathogenesis of, 2 in SCID mice, 69 T cell response in, 68 transmission of, 5–7 treatment of, 109–110 VSP expression in, 67 waterborne, 7, 129–138 in wildlife, 7–8 GP60 gene, 36

F

Fatty acid metabolism, in Cryptosporidium parvum, 159–160 Fatty acid synthase gene, of Cryptosporidium parvum, 107

H Highly active anti-retroviral therapy, in cryptosporidiosis, 104

G

I

Gal/GalNAc lectin, of Entamoeba histolytica, 78–80, 82 Genome of Cryptosporidium parvum, 153–161 of Entamoeba histolytica, 21, 141–148 GGCY motif, of Giardia, 61 Giardia, 1–10 antigenic variation in, 60, 66–70 assemblage A, 4–5, 60 assemblage B, 4–5, 60 CXXC motif of, 61, 62 encystation of, 66 excystation of, 66 GGCY motif of, 61 host specificity of, 2, 3–5 life cycle of, 2 post-translation modification in, 62–63 RING motif of, 61–62 secretory system of, 2 species of, 2, 3–5 transmission of, 5–7 variant-specific surface proteins of, 59–70. See also Variant-specific surface proteins; vsp genes

Immune system in amebiasis, 81–84 in cryptosporidiosis, 91–95 in cyclosporiasis, 46, 47 Immunoglobulin A in amebiasis, 81, 83 in cryptosporidiosis, 97 Immunoglobulin G in amebiasis, 77 in cryptosporidiosis, 97 in cyclosporiasis, 47 Immunoglobulin M, in cyclosporiasis, 47 Immunotherapy, in cryptosporidiosis, 108 Inflammatory response in amebiasis, 81–82 in cryptosporidiosis, 93–94 in cryptosporidiosis, 92–93, 95–96 Interleukin-12, in amebiasis, 82 L

Lectin-based test, in amebiasis, 19–20 Livestock, giardiasis in, 6, 8

168 Long interspersed elements, of Entamoeba histolytica, 145–146

M Mesclun lettuce, cyclosporiasis with, 50 Metronidazole in amebiasis, 1 1 0 – 1 1 1 in giardiasis, 109–110 Mucus, in amebiasis, 81

N Natural killer cells, in cryptosporidiosis, 92–93 in cryptosporidiosis, 92 Nitazoxanide in cryptosporidiosis, 104–105 in giardiasis, 110

P P23, in cryptosporidiosis, 97 Paromomycin in cryptosporidiosis, 104 in giardiasis, 110 Polymerase chain reaction test in amebiasis, 20 of Cryptosporidium parvum, 31–32 in cyclosporiasis, 52 Prostaglandins, in cryptosporidiosis, 92 Proteinases in amebiasis, 80–81 of Entamoeba dispar, 80–81 of Entamoeba histolytica, 80–81 Purine metabolism, in Cryptosporidium parvum, 156–157 Pyruvate-ferredoxin oxidoreductase, of Cryptosporidium parvum, 158–159

R Raspberries, cyclosporiasis with, 45, 49–50 Replication protein A, of Cryptosporidium parvum, 155–156 Rifabutin, in cryptosporidiosis, 105 RING motif, of Giardia, 61–62 Roxithromycin, in cryptosporidiosis, 105

S SCID mouse amebiasis in, 82

Index cryptosporidiosis in, 92–93, 96–97 giardiasis in, 69 Serine-rich Entamoeba histolytica protein, 84 Short interspersed elements, of Entamoeba histolytica, 145–146 Spiramycin, in cryptosporidiosis, 105

T Tinidazole, in amebiasis, 110–111 Transforming growth in cryptosporidiosis, 94 Trimethoprim-sulfamethoxazole in cyclosporiasis, 47, 109

V Vaccine, against amebiasis, 83–84 Variant-specific surface proteins, 59–70 antibodies to, 67–68 biological function of, 66–69 CXXC motif of, 61 during encystation, 66 genes for, 63–65. See also vsp genes in gerbils, 68–69 GGCY motif of, 61 identification of, 60 in mice, 67–68, 69 post-translation modification of, 62–63 protease effect on, 68 regulation of, 65–66 RING motif of, 61–62 selection of, 66–69 trafficking of, 62–63 Zn finger motif of, 62 Villous atrophy, in cryptosporidiosis, 93 vsp genes, 63–65 allele loss from, 66 allele-specific expression of, 65–66 alleles of, 63–64 encystation and, 66 expression of, 64–66 similarities among, 64 tandem repeats of, 64

W Water monitoring, 129–138 grab samples in, 137 Information Collection Rule in, 131–132 Surface Water Treatment Rule in, 131 in United Kingdom, 134–137 in United States, 130–134

Index USEPA Method 1622 in, 133–134 USEPA Method 1623 in, 133–134 Water Supply Regulations in, 135–137 Water treatment, 117–124 bromate ion formation with, 124 chemical disinfection for, 121–124 chloramine disinfection for, 121, 122 chlorine disinfection for, 124 coagulants for, 119–120 DE filters for, 121 dissolved air flotation for, 120

169 membranes for, 121 ozone disinfection for, 121, 122, 124 physical processes for, 118–121 rapid granular filter for, 119–120 slow sand filters for, 119 ultraviolet light disinfection for, 122, 123 Wildlife, giardiasis in, 7–8

Z

Zn finger motif, of Giardia, 62