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17 Parasites: Cryptosporidium, Giardia and Cyclospora as foodborne pathogens Dr Rosely Nichols and Professor Huw Smith, Scottish Parasite Diagnostic Laboratory, UK 17.1 Introduction Giardia, Cryptosporidium and Cyclospora are intestinal protozoan parasites that parasitise both human and non-human hosts. Increasing evidence since 1970 has implicated these organisms as significant contaminants of food. Their life cycles consist of reproductive stages, which infect the intestine, and transmissive stages (cysts of Giardia and oocysts of Cryptosporidium and Cyclospora [(oo)cysts]) which are excreted in the faeces of infected hosts. Of great importance is that (oo)cysts are environmentally robust, being capable of prolonged survival in moist dark environments. Whereas cysts of Giardia and oocysts of Cryptosporidium are infectious to susceptible hosts immediately following excretion, oocysts of Cyclospora are not infectious when excreted and require a period of maturation in the environment before they become infective to other hosts. Of the various species of Giardia, Cryptosporidium and Cyclospora, Giardia duodenalis, Cryptosporid- ium parvum and Cyclospora cayetanensis are significant pathogens of humans. 17.2 Description of the organisms 17.2.1 Life cycles Giardia The genus Giardia consists of five species: G. agilis, infecting amphibians, G. muris, infecting rodents, G. duodenalis, infecting mammals, G. psittaci, infecting budgerigars and parakeets, and G. ardeae, infecting great blue herons. The parasites that infect humans are also known as G. intestinalis (= lamblia) and are ascribed to the duodenalis species. G. intestinalis is regarded by some authori- ties as a race of G. duodenalis. Giardia parasites infecting humans can also infect non-human hosts. In this chapter we use the species name duodenalis to describe those duodenalis ‘type’ parasites which infect both human and non-human hosts. Exposure to the acidity of the stomach and the alkalinity of the jejunum induces the cyst to excyst, producing two pyriform (pear-shaped) G. duodenalis tropho- zoites which attach onto the apical surfaces of enterocytes and divide by binary fission. Detachment from enterocytes, together with exposure to increased con- centration of bile salts and elevated pH during passage through the lumen of the small intestine cause trophozoites to encyst into ovoid cysts which are excreted in faeces. The life cycle of Giardia is presented in Fig. 17.1. 454 Foodborne pathogens ORGANISMS IN EXTERNAL ENVIRONMENT Cyst Disintegrates Ingested Excreted Trophozoite Cyst Cyst Trophozoites on mucosa of small intestine Excystation in upper small intestine Multiplication by binary fission in small intestine ORGANISMS IN HUMANS Fig 17.1 Life cycle of Giardia. The life cycle is direct, requiring no intermediate host, and the parasite exists in two distinct morphological forms, namely the cyst and trophozoite. Redrawn from Meyer and Jarroll (1980). Cryptosporidium Originally described by Tyzzer (1910, 1912), Cryptosporidium has emerged as an important pathogen of human beings in the last 25 years. Although more than 20 ‘species’ of this coccidian parasite have been described on the basis of the animal hosts from which they were isolated, host specificity as a criterion for spe- ciation appears to be ill founded as some ‘species’ lack such specificity. Currently, there are ten ‘valid’ species: C. parvum, C. andersoni and C. muris which infect mammals; C. baileyi and C. meleagridis which infect birds; C. serpentis and C. nasorum which infect reptiles and fish respectively; C. wrairi has been described in guinea pigs; C. felis in cats and C. saurophilum in lizards. Cryptosporidium felis has also been identified as a cause of infection in humans, in a small number of cases. The discovery of DNA sequence-based differences within the riboso- mal RNA (rRNA) gene repeat unit between individual isolates within a ‘valid’ species means that the taxonomy of the genus remains under revision. Recently, C. meleagridis has been described from 6 immunocompetent (out of 1735 speci- mens) human patients. Purified oocysts from the patient’s faecal material were indistinguishable from C. parvum by conventional methods, but showed geneti- cal identity to C. meleagridis determined by polymerase chain reaction restric- tion fragment length polymorphism (PCR-RFLP) of the COWP gene and sequencing of the COWP, TRAP-C1 and 18S rRNA PCR gene fragments (Pedraza-Diaz et al., 2000). The life cycle of C. parvum is complex (Fig. 17.2), comprising asexual, sexual and transmissive stages in a single host (monoxenous). The spherical oocyst measures 4.5–5.5mm in diameter and contains four naked (not within a sporo- cyst) crescentic sporozoites (Table 17.1; Fig. 17.2). Fayer et al. (1990) provide a good account of the biology of Cryptosporidium. Two genotypes of C. parvum have been identified: genotype 1, found primar- ily in humans, and genotype 2 with a much broader host range, including humans, and other mammals. As yet, no recombinant of these two genotypes has been identified, suggesting that they maintain separate reproductive strategies. Cyclospora Recently identified as a coccidian parasite, Cyclospora organisms have been implicated in human enteritis since 1977. Prior to 1992, their classification remained in doubt, being referred to, among others, as ‘cyanobacterium-like bodies’ and ‘coccidia-like bodies’. The species that infects humans, Cyclospora cayetanensis (Ortega et al., 1993), is closely related to the genus Eimeria (Relman et al., 1996). Eleven species of Cyclospora have been described from moles, rodents, insectivores, snakes and humans. Recently, three new species of Cyclospora isolated from monkeys and baboons from western Ethiopia have been proposed: C. cercopitheci from green monkeys, C. colobi from colobus monkeys and C. papionis from baboons (Eberhard et al., 1999). Cyclospora oocysts are spherical, measuring 8–10mm in diameter, and are excreted unsporulated. The life cycle of Cyclospora is not fully understood, but involves both sexual and asexual stages of development in a single host. As for Giardia and Cryptosporidium, Giardia and Cyclospora 455 Cryptosporidium, exposure to the acidity of the stomach and the alkalinity of the jejunum causes the sporozoites contained within sporocysts to excyst. Two types of meronts and sexual stages were observed in the jejunal enterocytes of biopsy material from oocyst excreting humans (Ortega et al., 1997a). Under laboratory conditions, 40% of oocysts exposed to temperatures of 25–30°C sporulated after 1–2 weeks, each oocyst containing two sporocysts, with two sporozoites within each sporocyst (Ortega et al., 1993; Smith et al., 1997). 456 Foodborne pathogens Sporozoites Trophozoite Reinfection Schizogony Schizont with 8 merozoites Microgametocyte Macrogametocyte Microgametes Zygote Oocyst Autoinfection Faeces Resistant oocyst Ingestion Intestinal epithelial cells Fig. 17.2 Life cycle of Cryptosporidium. Reproduced with permission from Smith and Rose (1980). 17.3 Symptoms caused in humans 17.3.1 Giardiasis Giardiasis is self-limiting in most people. The short-lived acute phase is charac- terised by flatulence with sometimes sulphurous belching and abdominal disten- sion with cramps. Diarrhoea is initially frequent and watery but later becomes bulky, sometimes frothy, greasy and offensive. Stools may float on water. Blood Cryptosporidium, Giardia and Cyclospora 457 Table 17.1 Characteristic features of G. duodenalis cysts and C. parvum and C. cayeta- nensis oocysts by epifluorescence microscopy and Nomarski differential interference con- trast (DIC) microscopy Appearance of G. duodenalis cysts and C. parvum oocysts: under the FITC (fluorescein isothiocyanate) filters of an epifluorescence microscope The putative organism must conform to the following fluorescent criteria: uniform apple green fluorescence, often with an increased intensity of fluorescence on the outer perimeter of an object of the appropriate size and shape (see below). Appearance of C. cayetanensis oocysts: under the UV filters of an epifluorescence microscope The putative organism must conform to the following fluorescent criteria: uniform sky blue autofluorescence on the outer perimeter of an object of the appropriate size and shape (see below). Appearance under Nomarski differential interference contrast (DIC) microscopy Giardia duodenalis Cryptosporidium parvum Cyclospora cayetanensis cysts oocysts oocysts • Ellipsoid to oval, • Spherical or slightly • Spherical, smooth, smooth walled, ovoid, smooth, thick thin walled, colourless colourless and walled, colourless and refractile refractile and refractile • 8–12 ¥ 7–10mm • 4.5–5.5mm • 8–10mm (length ¥ width) • Mature cysts • Sporulated oocysts • Unsporulated oocysts contain four nuclei contain four nuclei contain developing displaced to one sporocysts pole of the organism • Axostyle (flagellar • Four elongated, naked • Sporulated oocysts axonemes) lying (i.e. not within a contain two ovoid diagonally across sporocyst(s)) sporocysts, each the long axis of sporozoites and a containing two the cyst cytoplasmic residual sporozoites body within the oocyst • Two ‘claw-hammer’- shaped bodies lying transversely in the mid-portion of the organism and mucus are usually absent and pus cells are not a feature on microscopy. In chronic giardiasis, malaise, weight loss and other features of malabsorption may become prominent. Stools are usually pale or yellow and are frequent and of small volume and, occasionally, episodes of constipation intervene with nausea and diarrhoea precipitated by the ingestion of food. Malabsorption of vitamins A and B 12 and d-xylose can occur. Disaccharidase deficiencies (most commonly lactase) are frequently detected in chronic cases. In young children, ‘failure to thrive’ is frequently due to giardiasis, and all infants being investigated for causes of mal- absorption should have a diagnosis of giardiasis excluded (Smith et al., 1995a; Girdwood and Smith, 1999a). Cyst excretion can approach 10 7 /g faeces (Danciger and Lopez, 1975). The prepatent period (time from infection to the initial detection of parasites in stools) is on average 9.1 days (Rendtorff, 1979). The incubation period is usually 1–2 weeks. As the prepatent period can exceed the incubation period, initially a patient can have symptoms in the absence of cysts in the faeces. 17.3.2 Cryptosporidiosis In immunocompetent patients Cryptosporidium is a common cause of acute self-limiting gastroenteritis, symp- toms commencing on average 3–14 days post-infection. Symptoms include a ’flu- like illness, diarrhoea, malaise, abdominal pain, anorexia, nausea, flatulence, malabsorption, vomiting, mild fever and weight loss (Fayer and Ungar, 1986). From 2 to more than 20 bowel motions a day have been noted, with stools being described as watery, light-coloured, malodorous and containing mucus (Case- more, 1987). Severe, cramping (colicky) abdominal pain is experienced by about two-thirds of patients and vomiting, anorexia, abdominal distension, flatulence and significant weight loss occur in fewer than 50% of patients. Gastrointestinal symptoms usually last about 7–14 days, unusually 5–6 weeks, while persistent weakness, lethargy, mild abdominal pain and bowel looseness may persist for a month (Casemore, 1987). In young malnourished children, symptoms may be severe enough to cause dehydration, malabsorption and even death. Histopathol- ogy of infected intestinal tissue reveals loss of villus height, villus oedema and an inflammatory reaction. Mechanisms of severe diarrhoea are primarily conse- quences of malabsorption, possibly due to a reduction of lactase activity. The ratio of symptomatic to asymptomatic cases is not known. Illness and oocyst excretion patterns may vary owing to factors such as immune status, infective dose, host age and possible variations in the virulence of the organism; however, oocyst shedding can be intermittent and can continue for up to 50 days after the cessation of symptoms (mean: 7 days). In humans, the prepatent period is between 7 and 28 days. The mean incubation period (time from infection to the manifestation of symptoms) is 7.2 days (range 1–12) with a mean duration of illness of 12.2 days (range 2–26) (Jokipii and Jokipii, 1986). As the prepatent period can exceed the incubation period, initially a patient can have symptoms in the absence of oocysts in the faeces. 458 Foodborne pathogens Oocyst excretion by either human or non-human hosts can be up to 10 7 /g during the acute phase of infection. Infected calves and lambs excrete up to 10 9 oocysts daily for up to 14 days (Blewett, 1989). In immunocompromised patients In patients with Acquired Immune Deficiency Syndrome (AIDS), other acquired abnormalities of T lymphocytes, congenital hypogammaglobulinaemia, severe combined immunodeficiency syndrome, those receiving immunosuppressive drugs and those with severe malnutrition, symptoms include very frequent episodes of watery diarrhoea (between 6 and 25 bowel motions daily, passing between 1 and 20 litres of stool daily). Associated symptoms include cramping, upper abdominal pain, often associated with meals, profound weight loss, weak- ness, malaise, anorexia and low-grade fever (Whiteside et al., 1984). Infection can involve the pharynx, oesophagus, stomach, duodenum, jejunum, ileum, appendix, colon, rectum, gall bladder, bile duct, pancreatic duct and the bronchial tree (Soave and Armstrong, 1986; Cook, 1987). Except in those individuals in whom suppression of the immune system can be relieved by discontinuing immunosuppressive therapies, symptoms can persist unabated until the patient dies (Soave and Armstrong, 1986). Cryptosporidiosis in the immunocompro- mised can be a common and life-threatening condition in developing countries, causing profuse intractable diarrhoea with severe dehydration, malabsorption and wasting. AIDS triple therapies can reduce the severity of the clinical conse- quences of cryptosporidiosis. Oocyst excretion can continue for 2–3 weeks after the disappearance of symptoms (Soave and Armstrong, 1986). 17.3.3 Cyclosporiasis Cyclosporiasis is a ’flu-like illness, and diarrhoea with weight loss, low-grade fever, fatigue, anorexia, nausea, vomiting, dyspepsia, abdominal pain and bloat- ing have been described as symptoms (Ortega et al., 1993; Huang et al., 1995; Fleming et al., 1998). The incubation period is between 2 and 11 days (Soave, 1996) with moderate numbers of unsporulated oocysts being excreted for up to 60 days or more. In immunocompetent individuals the symptoms are self- limiting and oocyst excretion is associated with clinical illness, whereas in immunocompromised individuals diarrhoea may be prolonged. The self-limiting watery diarrhoea can be explosive, but leukocytes and erythrocytes are usually absent. Often, diarrhoea can last longer than 6 weeks in immunocompetent indi- viduals. The diarrhoeal syndrome may be characterised by remittent periods of constipation or normal bowel movements (Ortega et al., 1993). Malabsorption with abnormal d-xylose levels has also been reported (Connor et al., 1993). 17.4 Infectious dose and treatment The infectious dose to human beings is between 25 and 100 cysts for G. intesti- nalis (Rendtorff, 1954, 1979), although a volunteer study demonstrated that a Cryptosporidium, Giardia and Cyclospora 459 human-source isolate can vary in its ability to colonise other humans (Nash et al., 1987), suggesting that certain isolates may be less infectious to some humans than others. For Cryptosporidium, human volunteer studies indicate that the infec- tious dose varies from isolate to isolate, being between 30 and 132 oocysts for the Iowa (bovine, genotype 2, originally isolated by Dr H Moon, University of Iowa, from a calf and passaged in calves at the Sterling Parasitology Laboratory, University of Arizona) isolate of C. parvum (DuPont et al., 1995), 1042 oocysts for the UCP (UCP = Ungar C. parvum; bovine, genotype 2 received from Dr Beth Ungar in 1989, originally from Dr R. Fayer at the United States Department of Agriculture and passaged in calves by ImmuCell Corp., Maine) isolate, and nine oocysts for the TAMU (Texas A & M University; equine, genotype 2, isolated from a human exposed to an infected foal and passaged in calves) C. parvum isolate (Okhuysen et al., 1999). An infective dose between ten and 100 has been suggested for C. cayetanensis (Adams et al., 1999). While effective chemotherapy is available for giardiasis (nitroimidazole com- pounds, quinacrine, furazolidone, albendazole and mebendazole), cyclosporiasis (trimethoprim-sulfamethoxazole, excluding those who are intolerant to sulpha drugs), no effective chemotherapy is available for cryptosporidiosis. 17.5 Current levels of incidence Contamination of fresh produce, especially fruit, vegetables, salads and other foods consumed raw or lightly cooked, with viable (oo)cysts has been respon- sible for several outbreaks of giardiasis, cryptosporidiosis and cyclosporiasis (Tables 17.2–17.4). Other food types known to have been contaminated or epi- demiologically associated with outbreaks include Christmas pudding, home- canned salmon, chicken salad, sandwiches, fruit salad, ice, raw sliced vegetables, cold pressed (non-alcoholic) apple cider, raspberries, noodle salad, basil pesto pasta salad and mesclun lettuce (Tables 17.2–17.4). Our knowledge of incidence is scarce owing to the lack of a reproducible, sensitive detection method (see Table 17.5). Infectious (oo)cysts can be transmitted to a susceptible host via any faecally contaminated matrix, including water, aerosol, food and transport hosts. Food products can became contaminated with (oo)cysts in a variety of ways, and it is likely that more than one route may be involved in transmission, particularly in endemic areas. • Person to person (anthroponotic) transmission. Anthroponotic transmission has been documented particularly for foods that are intended to be consumed raw, or for those that are handled after being cooked. Direct contamination, by symptomatic or asymptomatic (oo)cyst excretors, during food preparation, or following food handler contact with (oo)cyst excretors are frequently reported routes for foodborne giardiasis and cryptosporidiosis (Tables 17.2 and 17.3), and are due to poor personal hygiene standards of that food handler. The hygienic practice of washing hands before preparing food can minimise (oo)cyst contamination and transmission. Guidelines exist for food handlers 460 Foodborne pathogens suffering diarrhoea, or those with recent symptoms. The most recently docu- mented foodborne outbreak of cryptosporidiosis, involving 88 cases, origi- nated from a food handler who continued to work in spite of having gastroenteritis (Quiroz et al., 2000). Washing uncooked fruit and vegetables before consumption is also recommended; however, one study indicates that washing is not sufficient to remove all C. parvum oocysts seeded onto lettuce surfaces (Ortega et al., 1997b). • Animal to person (zoonotic) transmission. There are no recorded outbreaks of zoonotic foodborne transmission of Giardia or Cyclospora. Direct contact of food with bovine faeces was the suggested cause of the largest foodborne out- break of cryptosporidiosis, which occurred in Maine, USA. In this outbreak, apples collected from an orchard in which a Cryptosporidium-infected calf grazed were made into non-alcoholic cider (Millard et al., 1994) (Table 17.3). Cryptosporidium, Giardia and Cyclospora 461 Table 17.2 Some documented foodborne outbreaks of giardiasis No. of persons Suspected Probable/possible Reference affected food-stuff source of infection 3 Christmas pudding Rodent faeces Conroy (1960) 29 Home-canned salmon Food handler Osterholm et al. (1981) 13 Noodle salad Food handler Petersen et al. (1988) 88 Sandwiches – White et al. (1989) 10 Fruit salad Food handler Porter et al. (1990) – Tripe soup Infected sheep Karabiber and Aktas (1991) 27 Ice Food handler Quick et al. (1992) 26 Raw sliced vegetables Food handler Mintz et al. (1993) Table 17.3 Some documented foodborne outbreaks of cryptosporidiosis No. of persons Suspected food-stuff Probable/possible Reference affected source of infection 160 Cold pressed (non- Contamination of Millard et al. (1994) alcoholic) apple fallen apples from cider infected calf 25 Cold pressed (non- ? Contaminated Anon. (1997a) alcoholic) apple water used to cider wash apples 15 Chicken salad Food handler Anon. (1996) 54 Not identified Common food Anon. (1998a) ingredient 152 Eating in one of two Food handler Quiroz et al. (2000) university campus cafeterias 17.5.1 Foodborne giardiasis Foodborne transmission was suggested in the 1920s (Musgrave, 1922; Lyon and Swalm, 1925) when water, vegetables and other foods were found to be conta- minated with cysts. Since then, cysts have been detected on vegetables including lettuce (Mastrandrea and Micarelli, 1968; Barnard and Jackson, 1980) and soft fruit (e.g. strawberries, Kasprzak et al., 1981; Barnard and Jackson, 1980). One report identifies the possibility of offal (tripe) being intrinsically infected (Kara- biber and Aktas, 1991). The remaining seven documented outbreaks presented in Table 17.2 occurred from 1977 onwards. 17.5.2 Foodborne cryptosporidiosis Five outbreaks of foodborne transmission have been documented, all of which occurred in the USA (Table 17.3). Two occurred following the consumption of non-alcoholic, pressed apple cider, in 1993 and 1996 affecting a total of 185 individuals. In the first outbreak, apples were collected from an orchard in which an infected calf grazed. Some apples had fallen onto the ground (windfalls) and had probably been contaminated with infectious oocysts then (Millard et al., 1994). The source of oocysts in the second outbreak is less clear as windfalls were not used and waterborne as well as other routes of contamina- tion were suggested (Anon., 1997a). A foodborne outbreak, which affected 15 individuals, occurred in 1995 with chicken salad, contaminated by a food handler, being the probable vehicle of transmission (Anon., 1996). In 1997, an outbreak was documented in Spokane, Washington. Among 62 attendees of a banquet dinner, 54 (87%) became ill. Eight of 10 stool specimens obtained from ill banquet attendees were positive for Cryptosporidium. Epidemiological investi- gation suggested that foodborne transmission occurred through a contaminated ingredient in multiple menu items (Anon., 1998a). All Cryptosporidium faecal samples from this outbreak were of genotype 1 (Quiroz et al., 2000). During September and October 1998, a cryptosporidiosis outbreak, affecting 462 Foodborne pathogens Table 17.4 Some documented foodborne outbreaks of cyclosporiasis No. of persons Suspected Probable/possible source Reference affected food-stuff of infection 1465 Guatemalan ? Aerosolisation of oocysts Herwaldt et al. raspberries during application of (1997) insecticides or fungicides 1450 Guatemalan ? Aerosolisation of oocysts Anon. (1998b) raspberries during application of insecticides or fungicides 48 Basil pesto Unknown Anon. (1997b) pasta salad Unknown Mesclun lettuce Unknown Anon. (1997c)