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

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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 (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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REFERENCES Aronzarena, M.M. Removal of Giardia muris cysts by granular media filtration. 34 pp. 1979. U. of Cincinnati. Thesis/Dissertation. Bellamy, W.D., G.P. Silverman, D.W. Hendricks, and G.S. Logsdon. 1985. Removing Giardia cysts with slow sand filtration. Journal American Water Works Association 77(2):52-60. Bonnin, A., J.F. Dubremetz, and P. Camerlynck. 1991. Characterization and immunolocalization of an oocyst wall antigen of Cryptosporidium parvum (Protozoa: Apicomplexa). Parasitology 103:171-177. Bukhari, Z., J.L. Clancy, M. Marshall, D. Korich, H.V. Smith, J. O’Grady, N. Sykes, and C. Fricker. 1998. UV inactivation of Cryptosporidium with pulsed UV light. Proceedings American Water Works Association Water Quality Technology Conference, San Diego, CA. Bukhari, Z., T.M Hargy, J.R. Bolton, B. Dussert, and J.L. Clancy. 1999. Medium-pressure UV light for oocyst inactivation. Journal American Water Works Association 91(3):86-94. Clancy, J.L., Z. Bukhari, T.M. Hargy, J.R. Bolton, B. Dussert, and M.M. Marshall. 2000. Using UV to inactivate Cryptosporidium. Journal American Water Works Association 92(9): 97-104. Clancy, J.L., T.M. Hargy, D.G. Korich, M.M. Marshall and W.L. Nicholson 2002. Susceptibility of multiple strains of Cryptosporidium parvum oocysts to UV light. AwwaRF, Denver, CO. ISBN 1-58321-261-2 Craik, S.A., D. Weldon, G.R. Finch, J.R. Bolton, and M. Belosevic. 2001. Inactivation of Cryptosporidium parvum oocysts using medium- and low-pressure ultraviolet radiation. Water Research 35:1387-1398. Craun, G.F. 1986. Statistics of Waterbome Outbreaks in the U.S. (1920-1980), pp. 73-159. In G. F. Craun (ed.), CRC Waterborne Diseases in the United States CRC Press, Boca Raton, Florida. Dai, X., and R.M. Hozalski. 2002. Effect of NOM and biofilm on the removal of Cryptosporidium parvum oocysts in rapid filters. Water Research. 36:3523-3532. DeWalle, F.B., J. Engeset, and W. Lawrence. Removal of Giardia lamblia cysts by drinking water treatment plants. EPA--600/2-84-069. 1984. Cincinnati, Ohio, U.S. Environmental Protection Agency. Federal Register 1996. 63(99): 24169390-24169476. Finch, G.R., L.L. Gyurek, L.R.J. Liyanage, and M. Belosevic, 1997. Effect of various disinfection methods on the inactivation of Cryptosporidium. AwwaRF, Denver, CO. ISBN 0-89867-926-5. Finch, G.R. and M. Belosevic, 1999. Inactivation of Cryptosporidium parvum and Giardia muris with medium pressure ultraviolet radiation. Proceedings USEPA Workshop on UV Disinfection of Drinking Water, Arlington, VA. Gerwig, G.J., J.A. Kuik, B.R. Leeflang, J.P. Kamerling, J.F.G. Vliegenthart, C.D. Karr, and E.L. Jarroll. 2002. The Giardia intestinalis filamentous cyst wall contains a novel beta (13)-N-acetyl-D-galactosamine polymer: a structural and conformational study. Glycobiology 12:499-505. Hall, T., J. Pressdee, R. Gregory, and K. Murray. 1995. Cryptosporidium removal during water treatment using dissolved air flotation. Water Science and Technology 31:125-135. Hargy, T.M., J.L. Clancy, Z. Bukhari, M.M. Marshall. Shedding UV light on the Cryptosporidium threat. Journal of Environmental Health, July/August 2000, 19-22. Harris, J.R., and F. Petry. 1999. Cryptosporidium parvum: structural components of the oocyst wall. Journal Parasitology 85:839-849.

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Hayes, S.L., E.W. Rice, M.W. Ware, and F.W. Schaefer. 2003. Low pressure ultraviolet studies for inactivation of Giardia muris cysts. Journal of Applied Microbiology 94:54-59. Hoff, J.C. Inactivation of microbial agents by chemical disinfectants. EPA/600/2-86/067,44 p. 1986. Washington, D.C., U.S. Environmental Protection Agency. Jacangelo, J.G., S.S. Adham, and J.-M. Laine. 1995. Mechanism of Cryptosporidium, Giardia and MS2 virus removal by MF and UF. Journal American Water Works Association 87(9):107-121. Jenkins, M.C., J. Trout, C. Murphy, J.A. Harp, J. Higgins, W. Wergin, and R. Fayer. 1999. Cloning and expression of a DNA sequence encoding a 41-kilodalton Cryptosporidium parvum oocyst wall protein. Clinical Diagnostic Laboratory Immunology 6:912-920. Karanis, P., W.A. Maier, H.M. Seitz, D. Schoenen. 1992. UV sensitivity of protozoan parasites. Journal Water Supply Research and Technology-Aqua. 41(2):95-100. Ketzenelson, E., B. Kletter, and H.I. Shuval. 1974. Inactivation kinetics of viruses and bacteria in water by use of ozone. Journal American Water Works Association 66(12):725729. Klevens, C.M. 2001. Parameters for UV disinfection of surface water. Poster Session, First International Congress On Ultraviolet Technologies. Washington, D.C. Leahy, J. G. Inactivation of Giardia muris cysts by chlorine and chlorine dioxide. 115 p. 1985. Ohio State University; Dept. of Civil Engineering. Thesis/Dissertation LeChevallier, M.W., W.D. Norton, and R.G. Lee. 1991a. Giardia and Cryptosporidium spp. in filtered drinking water supplies. Applied and Environmental Microbiology 57:26172621. LeChevallier, M.W., W.D. Norton, and R.G. Lee. 1991b. Occurrence of Giardia and Cryptosporidium spp. in surface water supplies. Applied and Environmental Microbiology 57:2610-2616. Logsdon, G.S. and J.C. Hoff. 1986. Barriers to the transmission of waterborne disease, pp. 255-276. In G.F. Craun (ed.), CRC Waterborne Diseases in the United States CRC Press, Boca Raton, Florida. Logsdon, G.S., J.M. Symons, R.L. Hoye Jr, and M.M. Arozarena. 1981. Alternative filtration methods for removal of Giardia cysts and cyst models. Journal American Water Works Association 73(2):111-118. Mackey, E.D., H.B. Wright, T.M. Hargy, J. Malley, Jr., and R.S. Cushing. 2002. Comparing Cryptosporidium and MS2 bioassays – implications for comparing UV reactor validation. Journal American Water Works Association 94(2): 62-69. Nieminski, E. and J. Ongerth. 1995. Removing Giardia and Cryptosporidium by conventional treatment and direct filtration. Journal American Water Works Association 87(9): 96-106. Ongerth, J. and J.P. Pecoraro. 1995. Removing Cryptosporidium using multimedia filters. Journal American Water Works Association 87(12): 83-89. Ongerth, J.E. and P.E. Hutton. 1997. Diatomaceous earth filtration for removal of Cryptosporidium oocysts. Journal American Water Works Association 89 (12):39-46. Ongerth, J.E. and P.E. Hutton. 2001. Testing of diatomaceous earth filtration for removal of Cryptosporidium oocysts. Journal American Water Works Association 93 (12): 53-56. Plummer, J.D., J.K. Edzwald, and M.B. Kelley. 1995. Removing Cryptosporidium by dissolved-air flotation. Journal American Water Works Association 87(9):85-95. Ransome, M.E., T.N. Whitmore, E.G. Carrington, 1993. Effect of disinfectants on the viability of Cryptosporidium parvum oocysts. Water Supply (Amsterdam) 11: 75-89. Rice, E.W., and J.C. Hoff. 1981. Inactivation of Giardia lamblia cysts by ultraviolet irradiation. Applied and Environmental Microbiology 42:546-547.

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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