Giardia is a flagellated, teardrop-shaped parasite which has only two life forms, the trophozoite and the cyst (Figures 10.1A,B). It belongs to the class Zoomastigophorea, the order Diplomona-dida and the family Hexamitidae (Meyer, 1990). It is one of the oldest eukaryotic organisms, based on the sequence analysis of its small 16S ribosomal RNA (Sogin et al., 1989). It also lacks many of the organelles typical of higher eukar-yotes, such as mitochondria, peroxisomes and a typical Golgi apparatus (Adam, 1991; Gillin et al., 1996; Roger et al., 1998). Because of its early evolutionary status, the study of Giardia can yield valuable insight into eukaryotic development.
The trophozoite, which measures 9-21 ^ in length x 5-15 ^ in width (Figure 10.1A), contains four sets of posteriorly directed flagella, which aid in the parasite's movement. The most prominent feature of the trophozoite is the ventral disk, which may help Giardia to attach
Principles and Practice of Clinical Parasitology
Edited by Stephen Gillespie and Richard D. Pearson © 2001 John Wiley & Sons Ltd
to intestinal epithelial cells (Figure 10.2). This disk is composed of a tight, clockwise spiral of microtubules, bound together by microribbons (Feely et al., 1990; Adam, 1991; Thompson et al., 1993; Gillin et al., 1996; Upcroft and Upcroft,
1998). In the disk are the prominent antigens of tubulin within microtubules and giardins within the microribbons (Peattie, 1990; Marshall and Holberton, 1993). The microtubules are critical to the functioning of the disk, as well as to the
Fig. 10.1 (opposite) (A) A trophozoite and (B) a cyst are pictured in a trichrome stain of a stool sample. The teardrop shape and two nuclei of the trophozoite with central karyosomes are readily apparent. The median body lies centrally. Trophozoites measure 5-15 |im wide and 9-21 |im long. In the cyst, the cytoplasm has separated from the smooth cyst wall. Centrally located axonemes, a transversely placed, claw-like median body, and two eccentrically located nuclei can be detected. Cysts measure 6-10 |im wide and 8-12 |im long movement of the flagella. There are two apparently equal nuclei which, on stained preparations, create the characteristic face-like image (Kabnick and Peattie, 1990).
There have traditionally been only a few species designated for Giardia, based on host restriction and microscopic morphology; G. lamblia in humans and G. muris in rodents have been studied most thoroughly. G. agilis, an amphibian species, is also recognized. Morphologically, G. lamblia differs from G. muris by its shape and the pattern of the median bodies— centrally located tight collections of micro-tubules. The median bodies of G. lamblia lie transversely in a claw-like shape compared with the round median bodies in G. muris. The trophozoites of G. muris are also more rounded.
Only G. lamblia has been cultured in vitro (Keister, 1983). The ability to culture G. lamblia has allowed a detailed characterization of parasites. Although the Giardia that infect humans appear morphologically identical, they are quite heterogeneous when carefully analyzed. Studies on parasites have included analysis of surface antigens and isoenzymes (Meloni et al., 1988; Homan et al., 1992; Thompson et al., 1993), restriction fragment length polymorphism (Nash et al., 1985), sequence differences in the 16S subunit rRNA (Weiss et al., 1992; van Keulen et al., 1995), ability to express surface proteins (Nash and Mowatt, 1992) and the nature of these proteins (Ey et al., 1996), gene products (Monis et al., 1996) and sequence differences in the triose phosphate isomerase genes (Baruch et al., 1996). Based on these analyses, investigators are beginning to classify Giardia spp. into groups or assemblages (Nash and Mowatt, 1992; Weiss et al., 1992; Ey et al., 1996; Monis et al., 1996; Lu et al., 1998). It is possible that differences between groups may translate into a phenotypic difference in the ability of a given strain to cause diarrhea (Nash et al., 1987; Paintlia et al., 1998). These analyses have also led to the determination that Giardia which infect other non-human mammalian species may be similar, and at times identical, to those that infect humans
(Strandén et al., 1990; Thompson et al., 1993; Baruch et al., 1996; Ey et al., 1996). Giardia has at léast fivé séts of chromosomés pér nucléus, with a total génomic sizé of approximatély 1.2 x 107 basé pairs (Adam et al., 1988; Upcroft and Upcroft, 1998).
Requirements for in vitro growth of G. lamblia are an anaerobic or microaerophilic environment and exogenous cysteine (Gillin et al., 1996). Growth is enhanced by biliary lipids and intestinal mucous. Given the location of Giardia in the host and the absence of mitochondria, it is not surprising that it relies upon anaerobic metabolic pathways for energy production (Brown et al., 1998). Alanine is the predominant end-product of carbohydrate metabolism under these anaerobic growth conditions (Adam, 1991; Thompson et al., 1993). Giardia uses glucose as a major energy source to produce ethanol and acetate and C02 (Adam, 1991). It may also generate ATP via the arginine dihydrolase pathway (Edwards et al., 1992). It can reduce 02 to water by the action of an NADH oxidase (Upcroft and Upcroft, 1998). Because of an inability to synthesize cellular lipids and nucleic acid precursors, it must scavenge phospholipids, fatty acids, purines and pyrimidines from intestinal contents (Adam, 1991; Thompson et al., 1993; Stevens et al., 1997). It divides by binary fission and has a doubling time in culture of 9-12 hours.
One of the most interesting biologic properties of Giardia is the ability to vary its surface proteins, both in culture and during infection (Nash, 1997). Giardia is covered by one of a family of variant specific surface proteins (VSPs), which are rich in cysteine and may also contain zinc and iron. These change spontaneously in vitro and in vivo in both human and animal infection (Aggarwal and Nash, 1988; Gottstein et al., 1990; Nash et al., 1990; Byrd et al., 1994). A potential role of the VSPs could be to help
Giardia to survive in vivo by protecting it against the action of intestinal proteases (Gillin et al., 1990; Nash et al., 1991). Changes in VSPs could also help Giardia to evade immune recognition, although there is only limited experimental data to support this (Nash, 1997; Stager and Muller, 1997).
As Giardia pass through the small bowel to the colon they encyst, forming a rigid, filamentous shell that allows them to survive outside the host (Campbell and Faubert, 1994). In vitro, encystation was first accomplished in 1987 (Gillin et al., 1987) and since then the process has been well defined. It can be induced by culturing trophozoites in a milieu of reduced bile acid and decreased cholesterol concentration, followed by the presence of excess bile salts in an alkaline environment (Gillin et al., 1996; Lujan et al., 1997). After induction of encystation, cyst wall proteins (CWPs) are transcribed and secreted into newly formed encystment-specific vesicles (ESVs), which develop just under the dorsal surface of the trophozoite (Mowatt et al., 1995). During this differentiation into cysts, there is the induction of Golgi-like enzyme activities (Lujan et al., 1995; Gillin et al., 1996). The proteins then become incorporated into the cyst wall in a fibrous, filamentous layer. One of these CWPs may be detected in a stool ELISA assay (Rosoff and Stibbs, 1986; Rosoff et al., 1989; Boone et al., 1999). A prominent sugar associated with CWPs is N-acetylgalactosamine (Das and Gillin, 1996). In vitro, the process from intracellular production of the CWPs to their assembly into the cyst wall takes 14-16 hours (Erlandsen et al., 1996). As the cyst matures, there is a single trophozoite division.
After ingestion by a host, excystation occurs when the cysts are exposed to gastric acid, pancreatic enzymes and the induction of a parasite-derived cysteine protease (Gillin et al., 1996; Hetsko et al., 1998). The process of excystation is a highly coordinated sequence of structural, physiological and molecular events, initiated when the parasite detects the appropriate environmental stimuli (Hetsko et al., 1998).
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