Parasites: Cryptosporidium, Giardiaand Cyclosporaas foodborne pathogens

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 4.5–5.5 mm 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 8–10 mm 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 ¥ 7–10 mm
•
4.5–5.5 mm
•
8–10 mm
(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)