Page 3: Guidelines for Canadian Drinking Water Quality: Guideline Technical Document - Enteric Protozoa: Giardia and Cryptosporidium

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Part II. Science and Technical Considerations

4.0 Description

Protozoa are a diverse group of eukaryotic, typically unicellular, microorganisms. The majority of protozoa are free-living organisms that can reside in fresh water and pose no risk to human health. However, some protozoa are pathogenic to humans. These protozoa fall into two functional groups: enteric parasites and free-living protozoa. Human infections caused by free-living protozoa are generally the result of contact during recreational bathing (or domestic uses of water other than drinking); as such, this group of protozoa is addressed in the Guidelines for Canadian Recreational Water Quality (Health Canada, 2012a). Enteric protozoa, on the other hand, have been associated with several drinking water-related outbreaks, and drinking water serves as an important route of transmission for these organisms; as such, a discussion of enteric protozoa is presented here.

Enteric protozoa are common parasites in the gut of humans and other mammals. They, like enteric bacteria and viruses, can be found in water following direct or indirect contamination by the faeces of humans or other animals. These microorganisms can be transmitted via drinking water and have been associated with several waterborne outbreaks in North America and elsewhere (Schuster et al., 2005; Karanis et al., 2007). The ability of this group of microorganisms to produce (oo)cysts that are extremely resistant to environmental stresses and conventional drinking water disinfection has facilitated their ability to spread and cause illness.

The enteric protozoa that are most often associated with waterborne disease in Canada are Cryptosporidium and Giardia. These protozoa are commonly found in source waters: some strains are highly pathogenic, can survive for long periods of time in the environment and are highly resistant to chemical disinfection. Thus, they are the focus of the following discussion. A brief description of other enteric protozoa of human health concern (i.e., Toxoplasma gondii, Cyclospora cayetanensis and Entamoeba histolytica) is provided in Appendix C.

4.1 Giardia

Giardia is a flagellated protozoan parasite (Phylum Protozoa, Subphylum Sarcomastigophora, Superclass Mastigophora, Class Zoomastigophora, Order Diplomonadida, Family Hexamitidae). It was first identified in human stool by Antonie van Leeuwenhoek in 1681 (Boreham et al., 1990). However, it was not recognized as a human pathogen until the 1960s, after community outbreaks and its identification in travellers (Craun, 1986; Farthing, 1992).

4.1.1 Life cycle

Giardia inhabits the small intestines of humans and other animals. The trophozoite, or feeding stage, lives mainly in the duodenum but is often found in the jejunum and ileum of the small intestine. Trophozoites (9-21 µm long, 5-15 µm wide and 2-4 µm thick) have a pear-shaped body with a broadly rounded anterior end, two nuclei, two slender median rods, eight flagella in four pairs, a pair of darkly staining median bodies and a large ventral sucking disc (cytostome). Trophozoites are normally attached to the surface of the intestinal villi, where they are believed to feed primarily upon mucosal secretions. After detachment, the binucleate trophozoites form cysts (encyst) and divide within the original cyst, so that four nuclei become visible. Cysts are ovoid, 8-14 µm long by 7-10 µm wide, with two or four nuclei and visible remnants of organelles. Environmentally stable cysts are passed out in the faeces, often in large numbers. A complete life cycle description can be found in a review paper by Adam (2001).

4.1.2 Species

The taxonomy of the genus Giardia is rapidly changing as emerging data on the isolation and identification of new species and genotypes, strain phylogeny and host specificity become available. The current taxonomy of the genus Giardia is based on the species definition proposed by Filice (1952), who defined three species: G. duodenalis (syn. G. intestinalis, G. lamblia), G. muris and G. agilis, based on the shape of the median body, an organelle composed of microtubules that is most easily observed in the trophozoite. Other species have subsequently been described on the basis of cyst morphology and molecular analysis. Currently, six Giardia species are recognized (Table 1) (Thompson, 2004; Thompson and Monis, 2004). These six species have been reported from mammals, birds, rodents and amphibians and are not easily distinguished. Their host preferences have been widely debated--except for G. agilis, which is morphologically different, has been reported only from amphibians and is not regarded as infective to humans (Adam, 1991).

The name G. lamblia is commonly applied to isolates from humans, although this species is capable of infecting a wide range of mammals. Molecular characterization of this species has demonstrated the existence of genetically distinct assemblages: assemblages A and B infect humans and other mammals, whereas the remaining assemblages C, D, E, F and G have not yet been isolated from humans and appear to have restricted host ranges (and likely represent different species or groupings) (Table 1) (Adam, 2001; Thompson, 2004; Thompson and Monis, 2004; Xiao et al., 2004; Smith et al., 2007). In addition to genetic dissimilarities, these variants also exhibit phenotypic differences, including differential growth rates and drug sensitivities (Homan and Mank, 2001; Read et al., 2002). These genetic differences have been exploited as a means of distinguishing human-infective Giardia from other strains or species (Amar et al., 2002; Cacciò et al., 2002; Read et al., 2004); however, the applicability of these methods to analysis of Giardia within water has been limited (see Section 6.6). Thus, at present, it is necessary to consider that any Giardia cysts found in water are potentially infectious to humans.

4.2 Cryptosporidium

Cryptosporidium is a protozoan parasite (Phylum Apicomplexa, Class Sporozoasida, Subclass Coccodiasina, Order Eucoccidiorida, Suborder Eimeriorina, Family Cryptosporidiidae) that was first recognized as a potential human pathogen in 1976 in a previously healthy 3-year-old child (Nime et al., 1976). A second case of cryptosporidiosis occurred 2 months later in an individual who was immunosuppressed as a result of drug therapy (Meisel et al., 1976). The disease became best known in immunosuppressed individuals exhibiting the symptoms now referred to as acquired immunodeficiency syndrome, or AIDS (Hunter and Nichols, 2002).

4.2.1 Life cycle

The recognition of Cryptosporidium as a human pathogen led to increased research into the life cycle of the parasite and an investigation of the possible routes of transmission. Cryptosporidium has a multi-stage life cycle, typical of an enteric coccidian. The entire life cycle takes place in a single host and evolves in six major stages: 1) excystation, where sporozoites are released from an excysting oocyst; 2) schizogony (syn. merogony), where asexual reproduction takes place; 3) gametogony, the stage at which gametes are formed; 4) fertilization of the macrogametocyte by a microgamete to form a zygote; 5) oocyst wall formation; and 6) sporogony, where sporozoites form within the oocyst (Current, 1986). A complete life cycle description and diagram can be found in a review paper by Smith and Rose (1990). Syzygy, a sexual reproduction process that involves association of the pre-gametes end to end or laterally prior to the formation of gametes, was recently described in two species of Cryptosporidium, C. parvum and C. andersoni,providing new information regarding Cryptosporidium's biology (life cycle) and transmission (Hijjawi et al., 2002; Rosales et al., 2005).

As a waterborne pathogen, the most important stage in Cryptosporidium's life cycle is the round, thick-walled, environmentally stable oocyst, 4-6 µm in diameter. There is sometimes a visible external suture line, and the nuclei of sporozoites can be stained with fluorogenic dyes such as 4′,6-diamidino-2-phenylindole (DAPI). Upon ingestion by humans, the parasite completes its life cycle in the digestive tract. Ingestion initiates excystation of the oocyst and releases four sporozoites, which adhere to and invade the enterocytes of the gastrointestinal tract (Spano et al., 1998a; Pollok et al., 2003). The resulting parasitic vacuole contains a feeding organelle along with the parasite, which is protected by an outer membrane. The outer membrane is derived from the host cell (intracellular). The sporozoite undergoes asexual reproduction (schizogony), releasing merozoites that spread the infection to neighbouring cells. Sexual multiplication (gametogony) then takes place, producing either microgametes ("male") or macrogametes ("female"). Microgametes are then released to fertilize macrogametes and form zygotes. A small proportion (20%) of zygotes fail to develop a cell wall and are termed "thin-walled" oocysts. These forms rupture after the development of the sporozoites, but prior to faecal passage, thus maintaining the infection within the host. The majority of the zygotes develop a thick, environmentally resistant cell wall and four sporozoites to become mature oocysts, which are then passed in the faeces.

4.2.2 Species

Our understanding of the taxonomy of the genus Cryptosporidium is continually being updated. Cryptosporidium was first described by Tyzzer (1907), when he isolated the organism, which he named Cryptosporidium muris, from the gastric glands of mice. Tyzzer (1912) found a second isolate, which he named C. parvum, in the intestine of the same species of mice. This isolate was considered to be structurally and developmentally distinct by Upton and Current (1985). Although numerous species names have been proposed based on the identity of the host, most isolates of Cryptosporidium from mammals, including humans, are similar to C. parvum as described by Tyzzer (1907, 1912). At present, 20 valid species have been recognized (Table 2) (Egyed et al., 2003; Thompson and Monis, 2004; Xiao et al., 2004; Fayer et al., 2008; Jirků et al., 2008; Power and Ryan, 2008; Ryan et al., 2008).

With the advent of molecular techniques, several genotypes of Cryptosporidium have been proposed among various animal groups, including rodents, marsupials, reptiles, wild birds and primates, and research suggests that these genotypes vary with respect to their development, drug sensitivity and disease presentation (Chalmers et al., 2002; Xiao and Lal, 2002; Thompson and Monis, 2004; Xiao et al., 2004). To date, over 40 genotypes have been identified (Fayer, 2004; Xiao et al., 2004; Feng et al., 2007; Fayer and Xiao, 2008; Fayer et al., 2008). The molecular analysis of C. parvum human and bovine isolates, linked to human cryptosporidiosis outbreaks, indicates the existence of two predominantly distinct genotypes in humans (Morgan et al., 1997; Peng et al., 1997; Spano et al., 1998b; Sulaiman et al., 1998; Widmer et al., 1998; Awad-El-Kariem, 1999; Ong et al., 1999; Cacciò et al., 2000; McLauchlin et al., 2000; Xiao et al., 2001). Genotype 1 (syn. genotype I, genotype H and C. hominis) isolates are limited, for the most part, to humans, whereas genotype 2 (syn. genotype II and genotype C) isolates are zoonotic and have been reported in calves and other ruminants/ungulates, mice and humans. Genotype 1 was subsequently recognized as a new species, C. hominis (Morgan-Ryan et al., 2002). Further studies have identified additional genotypes in humans. Pieniazek et al. (1999) identified two novel Cryptosporidium genotypes, similar to a dog and a cat genotype, in persons infected with human immunodeficiency virus (HIV). Two new Cryptosporidium  genotypes have been identified in humans, one similar to a cervine (deer) isolate (Ong et al., 2002) and a not-yet-identified genotype (i.e., not been previously identified in humans or other animals) (Wong and Ong, 2006). These findings have important implications for communities whose source water may be contaminated by faeces from wildlife. The epidemiological significance of these genotypes is still unclear, but findings suggest that certain genotypes are adapted to humans and transmitted (directly or indirectly) from person to person. Numerous other Cryptosporidium genotypes, for which a strain designation has not been made, have also been identified (Feng et al., 2007; Smith et al., 2007; Fayer et al., 2008; Xiao and Fayer, 2008).

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