Download Waterborne transmission of Cryptosporidium and Giardia

Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
A. Méndez-Vilas (Ed.)
_______________________________________________________________________________________
Waterborne transmission of Cryptosporidium and Giardia: detection,
surveillance and implications for public health
D. Carmena
MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College, Hammersmith Hospital Campus, Du Cane Road,
London W12 0NN, UK
Protozoan enteroparasites of the genera Cryptosporidium and Giardia have emerged over the past decades as major
waterborne pathogens. Both parasites are among the major causative agents of gastroenteritis and nutritional disorders in
humans, with tens of millions of new cases occurring every year. Although cryptosporidiosis and giardiasis are normally
self-limiting in immune-competent individuals, both are potential severe, life-threatening diseases in immunecompromised patients. Both pathogens are transmitted via the faecal-oral route, with the consumption of contaminated
drinking water and use of recreational waterways being significant avenues for acquisition of infection. Because infected
humans and animals can seed enormous amounts of the transmission stages of these parasites (Cryptosporidium oocysts
and Giardia cysts), both micro-organisms are ubiquitous in the aquatic environment. In addition, (oo)cysts may remain
viable for several months under a range of environmental conditions, their small size allow them to pass through
conventional water plant filters, both are resistant to disinfectants at the concentrations and exposure times commonly
used, and have low infectious doses in humans. Taken together, these data indicate that Cryptosporidium and Giardia
represent a significant threat to public health. Indeed an increasing number of waterborne outbreaks of cryptosporidiosis
and giardiasis have been reported worldwide, with most cases occurring in the USA and UK. This situation has become a
major concern for water utilities and sanitary authorities that are responsible for providing safe drinking water supplies for
human consumption. In this chapter relevant aspects of the biology and epidemiology of Cryptosporidium and Giardia
from the aquatic environmental perspective are discussed, including pathology, treatment, transmission dynamics,
detection, prevention and control measures.
Keywords Cryptosporidium, Giardia, drinking water, recreational water, surveillance, public health, diarrhoea, zoonoses
1. Introduction
Organisms of the genera Cryptosporidium and Giardia are protozoan parasites that infect the gastrointestinal tract of
vertebrate animals, including mammals, birds, reptiles, amphibians and in the case of Cryptosporidium, fish. Species
within these genera cause human cryptosporidiosis and giardiasis, which constitute the most common causes of
protozoan diarrhoea and lead to considerable morbidity and mortality. About 200 million people have symptomatic
giardiasis worldwide [1], with prevalences of 2–5% in industrialized countries and 20–30% in developing regions of
Asia, Africa and Latin America. Transmission of cryptosporidiosis and giardiasis is typically associated with poor
faecal-oral hygiene, being completed by any mechanism by which material contaminated with faeces containing
infectious (oo)cysts can be swallowed by a susceptible host. Water and food are the most common transmission
vehicles, although person-to-person or animal-to-person direct contacts are also important routes of infection. Immunecompromised individuals (i.e. AIDS and cancer patients, the very young or the elderly) are most at risk from the clinical
consequences of these diseases. Because Cryptosporidium and Giardia infections cause considerable socio-economic
burden in developing countries, both pathogens were included in the World Health Organization’s Neglected Disease
Initiative in 2004 [2].
Cryptosporidium is currently classified within the phylum Apicomplexa, class Sporozoae, subclass Coccidia, order
Eucoccidiida, suborder Eimeriina and family Cryptosporidiidae. To date at least 20 Cryptosporidium species have been
recognized (see Table 1), among which eight (C. hominis, C. parvum, C. meleagridis, C. andersoni, C. suis, C. muris,
C. canis and C. felis) have a zoonotic potential. Furthermore, nearly 61 Cryptosporidium genotypes with uncertain
species status have also been reported [3,4]. Cryptosporidium was initially described as an intracellular parasite
infecting the gastric glands of experimentally infected mice at the beginning of the last century. The first cases of
human cryptosporidiosis were not reported until 1976, and soon afterwards the AIDS pandemic brought
Cryptosporidium to the forefront as an opportunistic parasite infecting immune-compromised patients. However, its
relevance in public health acquired a new dimension twenty years later, when Cryptosporidium began to be recognized
as an important waterborne pathogen.
The genus Giardia falls within the phylum Sarcomastigophora, class Zoomastigophora, order Diplomonadida and
family Hexamitidae. It includes six valid species that inhabit the intestinal tracts of virtually all classes of vertebrates,
with G. duodenalis (syn. G. intestinales, G. lamblia) being the only species found in humans (Table 2). Members of the
G. duodenalis group should actually be regarded as a species complex with little morphological variation among them,
yet can be assigned to at least seven genotypes or ‘assemblages’ (A-G) [5,6]. Giardia has been known since the
seventeenth century work by Antony van Leeuwenhoek, although for most of this time this protozoa has been
considered a harmless intestinal commensal, primarily because the parasite was identified in asymptomatic individuals.
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Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
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Méndez-Vilas (Ed.)
_______________________________________________________________________________________
It was not until 1987 that the pathogenic nature of Giardia was definitively confirmed in a study of infection in human
volunteers [7]. As in the case of Cryptosporidium, Giardia is also an important cause of waterborne illness.
Table 1 Cryptosporidium species (adapted from references [4], [8] and [9]).
Species
Main host
C. andersoni
C. baileyi
C. bovis
C. canis
C. fayeri
C. felis
C. fragile
C. galli
C. hominis
C. macropodum
C. meleagridis
C. molnari
C. muris
C. parvum
C. ryanae
C. scophthalmi
C. serpentis
C. suis
C. varanii
C. wrairi
Cattle
Chicken
Cattle
Dog
Kangaroo
Cat
Toad
Chicken
Humans
Kangaroo
Turkey
Marine fish
Mouse
Mouse
Cattle
Cultured fish
Snake
Pig
Lizard
Guinea pig
Reported
infections
Yes
No
No
Yes
No
Yes
No
No
Yes
No
Yes
No
Yes
Yes
No
No
No
Yes
No
No
in
human
Reported in waterborne
outbreaks
No
No
No
No
No
No
No
No
Yes
No
Yes
No
No
Yes
No
No
No
No
No
No
Reported in water (nonoutbreak conditions)
Yes
Yes
No
Yes
No
Yes
No
No
Yes
No
Yes
No
Yes
Yes
No
No
No
Yes
No
No
2. Life cycle of Cryptosporidium and Giardia
Members of the genus Cryptosporidium are able to infect and multiply in a wide range of vertebrate hosts, including
humans, mice, rats, cats, dogs, sheep, cattle, horses, pigs, poultry, birds, reptiles, and fish. Cryptosporidium is an
obligate intracellular parasite that requires a single host (monoxenous) to complete its life cycle. Infected hosts shed
oocysts, the infective stage of the parasite, contaminating pastures, soil and water. Once the oocyst is accidentally
ingested by a new host, exposure to stomach acid, bile salts and host metabolic temperature promotes thinning of the
oocyst wall (excystation), resulting in release of four motile sporozoites into the small intestine (Fig 1A). This requires
different factors derived from both the parasite and the host cell, including expression of receptors on the surface of
oocysts, trypsin and other enzymes for sporozoite excystation [10]. Released sporozoites contain secretary apical
organelles required for adhesion, penetration and inclusion in host epithelial cells lining the luminal surfaces of the
digestive and respiratory tracts. After this process the parasite nestles itself within a parasitophorous vacuole
(intracellular but extracytoplasmatic) where it is protected from the hostile gut environment and is supplied by the host
cell with energy and nutrients. The sporozoites undergo then asexual reproduction cycle (merogony), involving
differentiation and, sequentially, trophozoite, Type I meront and merozoite production. Some of these merozoites can
cause autoinfection by attaching and invading neighbouring epithelial cells (perpetuating the asexual cycle) whereas
others become Type II meronts, which contain 4 Type II merozoites. Released Type II merozoites infect adjacent host
epithelial cells and differentiate into either macrogamonts or microgamonts, initiating the parasite’s sexual phase
(gametogony). Microgamonts differentiate into microgametes, which after being released can penetrate and fertilise the
macrogamonts, producing a zygote. Following meiosis, the zygote differentiates into four naked sporozoites that then
develop into cysts of two types: 20% of oocysts have thin walls, enabling autoinfection of the host by rupturing and
releasing new sporozoites. The remaining thick-walled oocysts (environmentally-resistant and immediately infective)
are shed in the faeces, usually in large numbers (Fig 1B-D). A new generation of parasites can develop and mature in as
little as 12–14 hours.
Giardia has a simple, non-invasive life cycle that involves two well-differentiated forms: the trophozoite
(proliferative stage) and the cyst (resilient to environmental stressors and infective stage). Cysts are passed in the faeces
of infected hosts, contaminating food, soil and water. Fortuitous ingestion of cysts by a new susceptible host leads to the
release of the trophozoites after exposure to stomach acid and bile salts. Trophozoites are teardrop shaped, binucleate,
with four pairs of flagella and have a ventral sucking disc that allow them to adhere and colonise the brush border of the
intestinal epithelium (Fig 2A). The trophozoites absorb their nutrients by pinocytosis from the lumen of the small
intestine and undergo asexual replication through longitudinal binary fission. As faeces enter the colon and begin to
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A. Méndez-Vilas (Ed.)
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dehydrate, the trophozoite-to-cyst transformation (encystation) occurs, being the newly formed and readily infective
cysts (Fig 2B-D) excreted with the faeces to re-initiate the cycle. Although Giardia is considered to be strictly asexual,
recent evidence suggests that exchange of genetic material may take place during the excystation and encystation
processes in G. duodenalis [11].
Table 2 Giardia species and assemblages (adapted from references [5] and [9]).
Species
Main host
G. duodenalis
G. agilis
G. muris
G. ardeae
G. psittaci
G. microti
Mammals
Amphibians
Rodents
Birds
Birds
Rodents
Reported
infections
Yes
No
No
No
No
No
Assemblages
A (= G. duodenalis)
B (= G. enterica)
C/D (= G. canis)
E (= G. bovis)
F (= G. cati)
G (= G. simondi)
Mammals
Mammals
Dog
Cattle
Cat
Rat
Yes
Yes
No
No
No
No
in
human
Reported in waterborne
outbreaks
Yes
No
No
No
No
No
Reported in water (nonoutbreak conditions)
Yes
No
No
No
No
No
Yes
No
No
No
No
No
Yes
Yes
No
No
No
No
3. Cryptosporidium and Giardia infections in humans
3.1 Pathology and clinical manifestations
At least eight Cryptosporidium species (see Table 1) and two Cryptosporidium genotypes (cervine and monkey) have
been found to infect humans, with C. hominis and C. parvum accounting for more than 90% of the cases [12]. The main
site of the infection is the small intestine (jejunum and ileum), although the parasite may also spread throughout the
gastrointestinal and respiratory tracts. Invasion of host cells is restricted to the luminal border and leads to loss of the
surface epithelium, causing villous disruption and atrophy, blunting and crypt cell hyperplasia, mononuclear cell
infiltration in the lamina propia and eventually enterocyte death [13]. Tissue damage triggers the immunological and
inflammatory responses of the host, inducing the recruitment of prostaglandin-secreting inflammatory cells. Crypt cells
and prostaglandin in turn stimulate chloride ion secretion and inhibits NaCl absorption. As a consequence of the
impaired intestinal absorption of fluids and nutrients and the disruption in the normal ion flux, secretory diarrhoea
occurs. Extra-gastrointestinal cryptosporidiosis has been reported in both immune-competent individuals and immunecompromised patients, affecting pancreas, liver and bile ducts. Tracheo-bronchial involvement and even sinusitis have
been described in severe cases. The precise pathogenic mechanisms of cryptosporidiosis have not yet been fully
elucidated, although in vitro experiments using Caco-2 cells suggest that enterotoxins may be involved in the process
[14].
The duration and severity of cryptosporidiosis in humans is directly related to the immunological status of the host,
with CD4+ T lymphocyte counts and gamma interferon levels playing an important role in fighting the infection [15]. In
immune-competent individuals the infection and colonization of the pathogen within the intestinal tract can be
asymptomatic or develop illness causing an acute self-limiting gastroenteritis with a median duration of 9–15 days.
Children (particularly those under two years old) are most frequently affected, the disease being associated with
impaired growth, malnutrition and significant morbidity and mortality in developing countries. The major symptom is
watery diarrhoea associated with abdominal pain, anorexia, weight loss, nausea, fatigue and low-grade fever [16]. In
immune-compromised people including those with haematological malignancies, AIDS patients with CD4+ lymphocyte
counts of <50/mm3 and patients undergoing cancer chemotherapy or chemo-suppressive treatment associated with
organ transplantation, the intestinal infection is chronic or intractable and can affect atypical and extra-intestinal sites.
G. duodenalis is the only specie within the genus Giardia able to infect humans. Giardia infection is not invasive and
often asymptomatic: as many as 50% to 75% of infected persons may not develop any symptoms of the disease [17].
The main site of the infection is the small intestine (jejunum and duodenum), being extra-intestinal invasion infrequent.
The pathogenesis of giardiasis is not completely understood but recent evidence suggests that the parasite’s trophozoites
trigger a multifactorial pathophysiological process involving damage to the intestinal brush border and mucosa,
induction of the host immune and inflammatory responses, impairment in pancreatic function and alteration of duodenal
flora [18]. Cases of urticaria and reactive arthritis have also been associated to Giardia infections. In addition, altered
epithelial permeability caused by cytopathic molecules of parasitic origin, disruption of tight junction-associated protein
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zonula occludin-1 (ZO-1) and apoptosis leads to both digestive and absorptive changes that induce acute or chronic
diarrhoea [19]. Other common symptoms include abdominal pain, steatorrhea, bloating, flatulence and weight loss, with
nausea, vomiting and fever being less frequent. Similarly to Cryptosporidium infection, the duration and severity of
giardiasis may vary depending upon the immunological, developmental and nutritional status of the host and the
parasite strain infectivity and virulence. In immune-competent individuals giardiasis is short-lasting and resolves
spontaneously in 1–4 weeks with symptoms lasting for only 3–4 days. Immune-compromised individuals are more
susceptible to symptomatic infection and have more severe and prolonged disease, lasting even for months if treatment
is not provided.
A
B
C
D
Fig. 1 Cryptosporidium parvum oocysts are spheroidal objects, 4 to 6 microns in diameter, and may contain as many as 4 banana
shaped sporozoites. A) Scanning electron micrograph of a human intestinal biopsy infected with C. parvum. B) Nomarski differential
interference contrast microscopy image. C) Immunofluorescence microscopy image. D) Immunofluorescence microscopy image with
DAPI staining, specific for nucleic acids. DAPI stained sporozoites appear as intense bright blue spots. Figures B, C and D
correspond to the same field of view. Image credits: M.V. McCrossan EM Unit, London School of Hygiene & Tropical Medicine,
UK (image A) and H.D.A Lindquist, U.S. EPA (images B, C and D)
3.2 Treatment
Despite recognition of Cryptosporidium and Giardia as important human pathogens, there are a relatively limited
number of agents available for chemotherapeutical purposes, and more research is required in order to identify existing
or new compounds that may be effective against cryptosporidiosis and giardiasis. To date nitazoxanide is the only drug
licensed in the United States for treating cryptosporidiosis in immune-competent individuals, being able to reduce the
duration of diarrhoea and oocyst shedding in several clinical trials (revised in reference [20]). Nitazoxanide has also
been demonstrated effective in well controlled clinical trials with AIDS patients, achieving eradication of the parasite in
up to 62% of the treated people. A dose of 2-g per day during 12 weeks is recommended for treating this type of patient.
A wider spectrum of compounds is currently available for the treatment of giardiasis. The drugs of choice are the
derivatives of nitroimidazol, including metronidazole, tinidazole and ornidazole, all well known inhibitors of nucleic
acid synthesis. Metronidazole at doses of 250 mg two to three times a day for 7–10 consecutive days has a median
efficacy over 90%, but some severe side effects such as pancreatitis, central nervous system toxicity and potential
carcinogenesis have been reported. Tinidazole at a 2-g single dose in adults achieves levels of efficacy ranging from
80% to 100% and reduce greatly the adverse side effects of metronidazole. Benzimidazole carbamates including
mebendazole and albendazole have also been successfully used for the treatment of giardiasis with rates of efficacy
from 62% to 95%, and also other drugs such as quinacrine, furazolidone and paromomycin [21].
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4. Cryptosporidium and Giardia transmission
Cryptosporidium and Giardia can be transmitted to humans through any mechanism by which material contaminated
with infectious (oo)cysts is swallowed by a susceptible host. Both parasites are maintained in a variety of transmission
cycles, including human-to-human, animal-to-human, and environmental transmission involving appropriate vehicles,
mainly water and food. Whatever the case, it is of capital importance to understand the host range of different species
and strains/genotypes, how they are maintained in nature and their zoonotic potential in domestic and wildlife animals.
Experimental cross-transmission studies have been carried out in order to investigate the host specificity of a given
strain/genotype, but very often the relevance of the obtained results is limited by incomplete molecular characterization
of the source (oo)cysts, uncertainty about their viability or the parasite-free status of the challenged hosts and the use of
high doses of (oo)cysts that are unlikely to represent a natural infection [19].
A
B
C
D
Fig. 2 Giardia duodenalis cysts are ovoid or ellipsoidal objects, 8 to 14 microns in length, and may contain as many as 4 nuclei and
structures from their trophozoites. A) Transmission electron micrograph of trophozoites infecting the epithelial surface of the
intestine. B) Nomarski differential interference contrast microscopy image. C) Immunofluorescence microscopy image. D)
Immunofluorescence microscopy image with DAPI staining, specific for nucleic acids. DAPI stained nuclei appear as intense bright
blue spots. Figures B, C and D correspond to the same field of view. Image credits: Reference [22], with kind permission of Springer
Science and Business Media (image A) and H.D.A. Lindquist, U.S. EPA (images B, C and D).
4.1 Direct transmission between humans
Person-to-person transmission is an important route of spread of Cryptosporidium and Giardia, being usually associated
with poor hygiene and sanitation. Thus, cryptosporidiosis and giardiasis are common in day-care centres, with diaperchanging and mouthing behaviour being important causes of infection. Transmission of these diseases are also frequent
in nosocomial settings including hospitals, nursing homes and other institutions, where both transmission from patients
to health care staff and patient-to-patient transmission have been reported. Cryptosporidium has also been discovered in
both sputum and vomits, and these body fluids may prove to be additional vehicles of transmission [23]. Transmission
can also occur through certain types of sexual contact.
4.2 Transmission between animals and humans
Cryptosporidium and Giardia have been identified in numerous mammalian (including livestock and house pets), avian,
reptilian, and piscine hosts worldwide [24,25]. Because the extent of cross-transmission to humans appears to be
genotype-dependent, molecular characterization of animal isolates is crucial to evaluate their epidemiological relevance.
Both the bovine genotype of C. parvum, and to a much lesser extent C. andersoni have been demonstrated to be
zoonotic (see Table 1). Because newborn calves are predominantly infected with C. parvum whereas older animals are
infected primarily with C. andersoni, young animals are the principal source of zoonotic C. parvum. Transmission may
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happen either by direct contact with infected young cattle or by consumption of livestock-contaminated drinking water
[26]. Indeed cattle have been clearly implicated as the source of at least one waterborne outbreak [27]. C. parvum
infection is less common in sheep and goats, although direct transmission of C. parvum from lambs to children and
handlers have been reported [28]. Although evidence of contamination of water supplies by Cryptosporidium from
infections in sheep and goats is limited, this possibility should not be ruled out completely. Regarding companion
animals, dogs and cats do not seem to be important zoonotic reservoirs of Cryptosporidium, as they are most commonly
infected with their host-adapted species C. canis and C. felis, respectively [29]. These species, together with C. suis and
C muris may cause sporadic infections in humans, most likely through direct contact.
Genotypes of Giardia of assemblage A and to a lesser extent of assemblage B pose the greatest zoonotic risk (see
Table 2). In cattle the livestock genotype (assemblage E) occurs most frequently, although a small proportion of animals
may harbour genotypes of assemblage A [30]. In any case the potential risk of zoonotic transmission of Giardia
infections between cattle and humans is probably minimal. In sheep and goats Giardia isolates of assemblage E are also
predominant, with zoonotic assemblages A and B being detected in 30% of infected animals [31]. Transmission to
humans can occur either directly or by environmental contamination, with few reports proposing sheep and goats as
sources of Giardia contamination of water supplies. Similarly, dogs and cats are both susceptible to infection with
zoonotic genotypes of Giardia [32], but the chances of a contamination event from a dog or cat leading to a waterborne
outbreak seem unlikely [30].
Wildlife in water catchments have often been identified as potential sources of Cryptosporidium and Giardia
infections in humans. Beavers have being involved in a number of Giardia waterborne outbreaks, although recent
research has evidenced that beavers can actually become infected with Giardia of human origin from contaminated
municipal water supplies. Besides, C. parvum can be mechanically passed to water by birds in faeces, suggesting that
birds, particularly waterfowl, can serve both as reservoir host and vectors [33]. Evidence from the limited number of
molecular studies conducted to date support the idea that wildlife genotypes are host-specific, non-human adapted and
do not seem to represent a major public health concern.
4.3 Transmission through water
Because infected animals (and humans) can shed massive amount of infective (oo)cysts to the environment (see subsection 5.1) Cryptosporidium and Giardia are ubiquitous. In aquatic environments both pathogens have been isolated
worldwide from rivers, lakes or reservoirs used as source waters for human consumption or recreation, and also from
treated drinking water supplies and recreational waters facilities, including swimming pools and water parks. In
addition, (oo)cysts may reach groundwater supplies through infiltration of contaminated surfaces waters. Commonly
reported (oo)cyst concentrations are in the range of 0.01–150 per litre, but higher concentrations have been found in
agricultural run-off and urban wastewater effluents. Not surprisingly, Cryptosporidium and Giardia have been
frequently associated with waterborne illness in the last 30 years. The most remarkable episode happened in 1993, when
a massive Cryptosporidium waterborne outbreak was registered in Milwaukee, Wisconsin (USA), affecting an
estimated 403,000 people including 4,400 people hospitalized and more than 100 deaths [34]. The total cost of
outbreak-associated illness was estimated at $96.2 million, including $31.7 million in medical costs and $64.6 million
in productivity losses [35]. Since then at least 165 Cryptosporidium waterborne outbreaks have been recorded in
developed countries, with North American and European outbreaks accounting for the vast majority [36,37,38]. To date
only three Cryptosporidium species (C. hominis, C. parvum and C. meleagridis) have been isolated from water-related
outbreaks, although several others have been reported in surface waters under non-outbreak conditions (see Table 1).
Giardia is more frequently found in surface waters than Cryptosporidium, and it has been associated with at least 132
waterborne outbreaks worldwide [36]; the most important ocurred in Norway in 2004 and affected more than 1,500
persons [39]. Generally most human infections are due to the consumption of water from unfiltered surface water
sources, shallow wells and during water recreational activities. Giardia has been the most commonly identified
pathogen in waterborne outbreaks reported in the United States since 1971, with almost 28,000 cases during the period
1965 to 1996 [21]. Despite this little is known about the Giardia assemblages present in the aquatic environment, due to
the scarcity of genotyping surveys conducted.
Although most of the waterborne outbreaks of cryptosporidiosis and giardiasis have been associated with
contamination of municipal drinking water systems, exposure to contaminated recreational waters (i.e. swimming and
wading pools) were also significant sources of infection in humans. Contamination of the source water may be caused
by a wide range of events including the dispersion and transportation of (oo)cysts after heavy rainfall or melting snow,
sewage contamination of surface water and wells, inadequate treatment practices and deficiencies in water treatment
systems or combination of these factors [40]. The relative significance of these contamination sources may differ
depending on the particular characteristics of each water catchment and supply network.
4.4 Transmission through food
Foodborne outbreaks of cryptosporidiosis and giardiasis are less frequently reported in the literature than waterborne
outbreaks, probably due the lower evidence of parasitic infection and the lack of standardized detection methods [41].
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Raw milk and meat, farm-made apple cider, fermented milk, salads and raw vegetables are among the food products
where the presence of (oo)cysts has been documented. Food can be contaminated by infected food handlers, irrigation
with contaminated water or manure. Although there is evidence of zoonotic transmission in certain cases, more research
(particularly genotyping studies) is needed to correctly assess the public health relevance of these findings. In USA
approximately 10% of all cryptosporidiosis and giardiasis cases are thought to be foodborne.
5. Cryptosporidium and Giardia are particularly suited for waterborne transmission
(Oo)cysts of Cryptosporidium and Giardia share a number of biochemical and physical features that make them
especially robust to a wide range of ecological stressors and favour their successful dispersal in the aquatic
environment.
5.1 (Oo)cysts are shedding in high numbers
Both Cryptosporidium and Giardia have adopted high fecundity rates as adaptative strategy to enhance their
probabilities of survival and transmission. Cattle and humans per se are regarded as the most important reservoirs of
Cryptosporidium and Giardia infection in humans, respectively. Mean intensity of infection by Cryptosporidium in
cattle has been estimated to be in the range between 2–1,000 oocysts/g of faeces [42], with maximum shedding of 106-8
oocysts/g of faeces at the peak of the infection. Considering that cattle may shed up to 40 kg of faeces a day and that
oocyst excretion last 3–12 days in infected animals, these data clearly illustrate the huge impact of cattle on the load of
infective Cryptosporidium to the environment, including watersheds. Humans are also major contributors to the
environmental (oo)cyst pool that causes contamination of surface waters. Therefore, infected persons can shed up to
105-7 Cryptosporidium oocysts/g of faeces during infection and up to 109 Giardia cysts in a single day.
5.2 Persistence in the aquatic environment
Cryptosporidium oocysts and Giardia cysts can survive for six months and 2–3 months, respectively, suspended in
natural surface waters [9]. The survival of both pathogens in the aquatic environment is significantly affected by
temperature, with (oo)cysts infectivity decreasing as temperature increases. In reservoir waters at 22 °C and 30 °C, 45
and 11 days, respectively, were estimated for a 2 log reduction of C. parvum infectious oocysts assessed by cell culture
[43]. Similarly, survival of Giardia cysts declines to less than a month at 21°C. (Oo)cysts are extremely susceptible to
temperatures above 37 °C and heat-shocks, with complete loss of infectivity after incubation at 64 °C for 5 min in the
case of Cryptosporidium and 54 °C for 10 min in the case of Giardia. In addition, Cryptosporidium oocysts are able to
retain infectivity after freezing at –10 °C for up to 168 h, but not after 24 h at –20 °C. Giardia cysts are more sensitive
to extreme low temperatures and can not survive freezing conditions. Desiccation, freeze-thaw cycles and solar
radiation are other environmental stresses that can threat the viability and infectivity of (oo)cysts [44].
5.3 Small size
Because of their small size (see Figs 1B and 2B), Cryptosporidium oocysts and Giardia cysts have very low specific
gravities, with settling velocities in water ranging from 0.03 to 5-10 m/day [45]. Some authors have proposed that
sedimentation rates are significantly increased when (oo)cysts attach to other suspended particles, whereas others found
that this is not the case and (oo)cysts remain free for considerable periods of time, a fact that facilitates their transport
and diffusion in a water body [44]. While sedimentation of (oo)cysts may remove them temporarily from host ingestion,
turbulence generated by underflow events can result in sediment re-suspension, with deposited (oo)cysts re-joining the
pool of suspended particles. These characteristics have important implications for the water industry, as (oo)cysts
removal by sedimentation would be insignificant even after long periods of reservoir storage, and (oo)cysts can pass
through most conventional filters used in water treatment facilities. Even well-designed and operated water treatment
systems allow the passage of infectious (oo)cysts, which might expose the community to outbreaks of giardiasis and
cryptosporidiosis [2].
5.4 Resistance to chemical disinfection
Both Cryptosporidium and Giardia (particularly the former) are resistant to chlorine-based disinfectants at the
concentrations and exposure times commonly used in the water industry [46], and using increased chlorine
concentrations may lead to generation of toxic disinfection by-products such as trihalomethanes or nitrite. In practice
this means that water treatments based only on chlorination are not sufficient to inactivate potential infective (oo)cysts.
Effective physical removal or inactivation of Cryptosporidium and Giardia requires the right combination of water
filtration and disinfection (including ozonation and ultraviolet irradiation) procedures operated under optimum
conditions [21,40].
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5.5 High infectivity
Exposure to Cryptosporidium and Giardia will result in infection or illness depending on a number of pathogen-, hostand environment-derived factors, including the strain genotype of the parasite involved and the susceptibility and
immune status of the host. Whatever the case the infectivity of (oo)cysts is considered high: as little as 10–30
Cryptosporidium oocysts have been proven to induce infection in 20–40% of human volunteers, with ID50 in the range
of 9–1,042 oocysts [47–49]. Similarly, only 10 Giardia cysts were sufficient to produce infection in another two human
volunteers. The median infectious dose for Giardia has been estimated to be in the range of 25–100 cysts [9]. Although
it is unclear if the number of (oo)cysts generally present in drinking water constitute a sufficient dose to cause illness in
humans, research using mathematical models (see also section 7) has demonstrated that some fraction of the population
could become infected with a single Cryptosporidium oocyst [40].
6. Detection of Cryptosporidium and Giardia in water
Due to their small size and frequently low number in water samples, detection of Cryptosporidium oocysts and Giardia
cysts is difficult and requires time consuming and labour intensive work and also well trained and experienced
personnel. Under these circumstances, optimized and standardized techniques for (oo)cysts monitoring in surface and
drinking waters are essential. Current recommended and most common used methods are based on four sequential
steps: i) mechanical filtration of small-large volumes (1–1,000 L) of water to maximize (oo)cysts recovery and ensure a
representative sample; ii) purification and concentration of (oo)cysts by immunomagnetic separation (IMS); iii) staining
with specific fluorescent antibodies (FA); and iv) enumeration using fluorescence (see Figs 1C and 2C) and differential
interference contrast microscopy [50]. Other techniques that can be also used for (oo)cysts purification purposes include
density gradient, saturated-salt solution centrifugation, continuous flow centrifugation and flow cytometry with cell
sorting [51].
6.1 Spatial and temporal distribution of (oo)cysts
The environmental load of Cryptosporidium oocysts and Giardia cysts in surface water bodies is discontinuous in
nature and depends on factors such as livestock contamination (particularly during the periods of calving and lambing),
agricultural practices, sewage contamination, heavy rainfall and flooding or snow and ice melting. However,
operational deficiencies and sub-optimal processes in water treatment facilities rather than meteorological-related
events account for the majority of the waterborne outbreaks reported. Taken together, these data indicate clearly that
spatial and temporal variations in the (oo)cysts distribution are expected in both untreated and finished waters, with
(oo)cysts contents fluctuating in a spiking pattern. This fact needs to be considered in the sampling design of studies
intended to assess the presence of Cryptosporidium and Giardia in water samples. Indeed, extensive monitoring
programs have shown that prolonged periods with zero or very low (oo)cysts counts are followed by peaks of high
counts but short duration [40]. Thus, spot sampling tends to underestimate the real prevalence of these pathogens in
water, and more importantly, tends also to miss those ‘rare but all important’ high count samples. In fact, as many as 10
samples may be required to describe the range of (oo)cysts concentrations that a community is experiencing in its water
supply [52]. In a simulated model for a waterborne outbreak of cryptosporidiosis, nine out of every ten 100-L spot
samples underestimated the risk to the population to some degree, with 33% containing no oocysts [53]. According to
this model, there was a one in three chance of a 100-l spot sample suggesting zero risk when in fact there was an
outbreak across the population.
6.2 Recovery efficiency
(Oo)cysts recovery levels can be influenced by a large number of factors, including the physicochemical and organic
properties or the water matrix (turbidity, pH, inorganic compounds, clays and suspended algae), the age and condition
of the (oo)cysts, the type of cartridge/capsule filter and the quality of the reagents employed. As a consequence, every
method used for the detection of (oo)cysts in water samples must be appropriately validated and its initial precision and
recovery efficiency determined in spiking trials in the laboratory with known amounts of (oo)cysts [54]. To estimate the
actual concentration of (oo)cysts in an environmental sample, the (oo)cyst counts need to be corrected for the recovery
efficiency of the used method. This is complicated by the fact that the recovery efficiency can vary between samples.
Typical (oo)cysts recoveries based on the filtration/IMS/FA system range between 10–70% [54–57], although recent
improvements in this methodology have allowed values up to 80% [58]. Reported recovery levels for Giardia cysts
usually exceed those for Cryptosporidium oocysts.
6.3 Viability and infectivity
From a public health perspective, the relevance of the detection of (oo)cysts in a water sample will depend on their
viability and infectivity, as an (oo)cyst that is apparently intact and metabolically sound but incapable of excystation is
of no more importance that a dead (oo)cyst . The term ‘viable’ has been defined as ‘capable of reproducing under
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appropriate conditions’ [59], although other authors have suggested that viability should be better used to reflect the
(oo)cyst metabolic activity and integrity [60]. Thus, a viable (oo)cyst can be infective or non-infective, and if noninfective is of no public health concern. Viability has been widely assessed by microscopy-based methods reliant on
vital dye uptake such as propidium iodine, 4’6-diamidino-2-phenyl indole (DAPI, see Figs 1D and 2D), and SYTO-59,
but they have proven to be relatively unreliable, tend to overestimate the infective potential of (oo)cysts and
differentiation of (oo)cysts from debris and/or other micro-organisms may be difficult [51]. Similar problems have been
found using in vivo excystation assays (as (oo)cysts that are capable of excysting might not be capable of initiating
infection) and also with fluorescent in situ hybridization techniques. Therefore, results obtained with these methods
must be interpreted with caution, particularly if risk assessment or treatment evaluation is under consideration [60]. To
date infectivity studies in animal models or humans volunteers and cell culture assays (including human epithelial and
intestinal cell lines) are regarded as the most consistent approaches to assess (oo)cysts viability and/or infectivity. Thus
infectivity assays remain the gold standard as they are the only method that provides definitive evidence of the (oo)cyst
capability to initiate and cause infection in an specific host. Cell culture assays (often coupled to PCR, reverse
transcription PCR or immunofluorescence) offer equivalent results to those from infectivity studies when (oo)cysts not
exposed to disinfectants or environmental stressors are used. However both methods are costly, labour-intensive, timeconsuming and require large numbers of (oo)cysts for analyses (particularly the infectivity assays), features that make
them unsuitable for routine testing of environmental samples [40,51,60].
6.4 Molecular characterization
The most commonly found Cryptosporidium species and genotypes in surface and waste waters include the two major
Cryptosporidium pathogens of humans (C. parvum and C. hominis) in addition to the cattle species C. andersoni,
whereas only zoonotic Giardia assemblages A and B have been reported to date [9]. However, infected livestock,
wildlife and migratory animals are also important contributors to the (oo)cysts pool found in surface waters, although
many of them are not pathogenic to humans (see sub-sections 4.2 and 4.3) and consequently do not represent a public
health threat. Thus, molecular characterization of Cryptosporidium and Giardia isolates from water samples at the
species/assemblage level is essential to understand not only their potential infectivity to humans, but also their
environmental transmission. Additional typing to the subspecies level to describe variation between Cryptosporidium
species and Giardia assemblages can provide further information to address contamination source and disease tracking
during outbreaks [26,41]. A wide range of Cryptosporidium target genes are available for amplification-based
techniques, including nuclear small subunit ribosomal DNA (18S rDNA), heat shock protein 70 (Hsp70),
Cryptosporidium oocyst wall protein (COWP), internal transcribed spacer 1 and 2 (ITS1-2), actin, β-tubulin,
glycoprotein 60 (Gp60), and micro- and mini-satellite multilocus repeats, with a sensitivity as low as a single oocyst
[26,61]. For instance, PCR-restriction fragment length polymorphism (PCR-RFLP) of the COWP gene allows
differentiation among C. parvum, C. hominis and C. wrairi, whereas PCR-RFLP of the 18S rDNA discriminates among
C. parvum, C. muris and C baileyi [61]. On the other hand, Gp60 sequence and micro- and mini-satellite repeats are
used to cluster Cryptosporidium strains into subtypes [41,51]. For Giardia, PCR amplification of gene markers such as
18S rDNA, glutamate dehydrogenase (GDH), triose phosphate isomerase (TPI), β-giardin and elongation factor 1α (EF1α) has been used for speciation and assemblage/sub-assembalge determination [26,61].
7. Surveillance and control measures
Waterborne cryptosporidiosis and giardiasis have become a major concern for the sanitary authorities and the water
industry responsible for providing safe drinking water supplies for human consumption. Different strategies are
available for the surveillance of Cryptosporidium oocysts and Giardia cysts in raw and finished waters. Thus, current
regulations of countries such as USA [62], Canada [63], Australia and New Zealand [64] establish that water treatment
facilities must comply with specific goals for treatment performance, including (oo)cysts removal for filter systems,
turbidity monitoring and estimation of pathogen inactivation. None of the regulatory agencies of these countries
recommend (oo)cysts monitoring on the basis that current methods for the detection of (oo)cysts suffer from low
recovery rates, do not provide any information on their viability or human infectivity and there is a lag time between
sample collection and test result. On the contrary, continuous Cryptosporidium oocysts monitoring is compulsory in
England and Wales [65] and Northern Ireland [66]. Regulations in these countries set a treatment standard of an average
of less than one oocyst in 10 L of water supplied from a water treatment facility. The standard does not take into
account different species of Cryptosporidium, nor whether any oocysts detected are viable or infective to humans. In
addition, detection of Cryptosporidium oocysts at any level above one per 10 L would constitute a criminal offence.
Because this approach was not set on human health criteria, its usefulness and cost-effectiveness have been questioned
by some authors [67].
Based on (oo)cysts measurements made before and after detected waterborne outbreaks of cryptosporidiosis and
giardiasis, some researchers have estimated (oo)cysts levels that, if exceeded, may lead the possibility of an outbreak.
Therefore, concentrations of 10–30 oocysts and 3–5 cysts per 100 L of treated water have been proposed as ‘action
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levels’ for Cryptosporidium and Giardia, respectively [68,69]. Authors recommended that a single sample containing
(oo)cysts at concentrations above the action threshold should be evaluated immediately in order to initiate appropriate
action without delay. However, as discussed before, action levels are hampered by the highly variable sensitivities of the
methods available for (oo)cysts detection, so obtaining reliable counts may be a difficult task.
Integrated risk models based on mathematical and statistical methods have been proven to be useful tools to assess
the probability of waterborne cryptosporidiosis and giardiasis outbreaks. These models take into account environmental
(source, concentration, distribution and viability/infectivity of pathogens in water), technical (water treatment
efficiency, performance of detection methods), medical (virulence, dose-response relationship, immune status of the
population), and exposure factors, considering also the natural variation and uncertainties associated to some of those
variables [70–72].
Prevention measures are by far the most practical approach to reduce the risk of cryptosporidiosis and giardiasis, as
demonstrated during an epidemic of foot and mouth disease in livestock in England and Wales in 2001: limiting visitors
to the countryside to reduce direct and indirect exposure to livestock resulted in a marked attenuation (81.8%) of
Cryptosporidium reports as compared to the previous year [73]. Because contamination of water supplies is a major
source of human infection, implementation of measures to decrease the spread of (oo)cysts in the environment and to
improve the performance of the water treatment processes are critical. To achieve this goal the following aspects need
consideration:
a) Good farming and agricultural practices. Management strategies should be designed to minimize direct livestock
contamination of surface waters. This include adequate housing, thorough cleaning, disinfection and drying of
animal pens, appropriate removal or inactivation (i.e. composting) of fresh manure, prevention of runoff from
animal housing, isolation of ill animals (particularly calves), and implementation of measures that reduce
transmission between animals (i.e reducing stocking density).
b) Water catchment protection. Practices such as restricting access of livestock to water bodies by fencing, access
ramps, rotational grazing, or off-site watering, and active control of herds to prevent extensive surface water
contact may significantly reduce (oo)cysts contamination. Grass or riparian filter strips acting as buffer zones
have also been proven effective in reducing (oo)cysts from overland flow generated during low or moderate
precipitation [12]. Intake points from surface water supplies should be kept well away from other contamination
sources, such as sewer overflows or discharges of untreated and treated sewage.
c) Improved detection methods. New, practical and cost-effective technologies with higher and more consistent
sensitivities and with the ability to differentiate species and (oo)cysts viability/infectivity are needed to obtain
reliable (oo)cysts concentration measures in water samples and to assess the risk of human infection.
d) Optimized water treatment. Water treatment plants should be operated in accordance with good practice at all
times, monitoring the performance of the system and introducing corrective measures in those circumstances
when the treatment is compromised. Additional second-line treatments including membrane filtration, UV
irradiation and ozonization can further improve the efficiency of the system in removing/inactivating (oo)cysts.
Acknowledgements This work was partially supported by a grant from the Department of Health, Basque Government, Spain (Exp.
No. 2007111002). The author is very grateful to Richard G.E. Chamberlain for his advice with the English language editing.
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