Info

or mice

Genotype 1 (human)

Humans

No*

Genotype 2 (bovine)

Humans/animals

Yes*

*Neonatal pigs and one macacaque have been reported infected with a genotype 1 strain. Genotype 1 and 2 strains will both infect INFy knockout mice.

*Neonatal pigs and one macacaque have been reported infected with a genotype 1 strain. Genotype 1 and 2 strains will both infect INFy knockout mice.

Excitation

Auto infection Ingestion

Excitation

Type II mer ont

Mature oocyst

Type II mer ont

Macrogamont

Fig. 6.2 Life-cycle of C. parvum. Mature, fully infective oocysts are released from the intestinal epithelial cells to exit in stool or initiate host autoinfection. Ingestion of mature excreted oocysts results in excystation (under the influence of gastric acid and proteases in the small bowel) and release of four sporozoites, which invade the apical microvillous membrane of small intestinal epithelial cells to form trophozoites. Trophozoites undergo two rounds of merogony, with production of either Type I or Type II meronts. Type II meronts are thought to initiate sexual reproduction, with formation of microgametes (males) and macrogametes (females). Fertilization yields immature oocysts, which sporulate in situ rapidly yielding mature oocysts. Reproduced by permission from Fayer et al. (2000)

thromobospondin-related anonymous protein 2 (Peng et al., 1997). In general, animal/bovine isolates are infectious to both humans and animals, whereas human isolates are only infectious for humans. This latter fact has hampered in-depth studies of human isolates because of the inability to propagate large numbers of these oocysts experimentally. A recent review analyzing 12 studies, in which human C. parvum stool isolates (n = 173; ~60% from AIDS patients) were genotyped revealed that 78% of all endemic and epidemic human isolates examined were the human genotype (Clark, 1999). Improved understanding of the genetic variability of Cryptosporidium is essential for evaluation of potentially distinct species, to pinpoint outbreak sources, to delineate virulence factors and ultimately to identify drug and vaccine targets.

Life-cycle

The life-cycle of Cryptosporidium is monoxenous, completed within the gastrointestinal tract of a single host (Figure 6.2) (Current and Haynes, 1984; Current et al., 1986; Fayer et al., 2000). The oocyst is the only exogenous stage and is approximately 4-6 ^ in diameter, with distinct inner and outer layers and four fully developed and infectious sporozoites. Ingestion of oocysts initiates infection. After exposure to gastric acid, bile salts and/or proteolytic enzymes in the upper gastrointestinal tract, excystation of the sporo-zoites occurs through a small suture in the end of the oocyst wall. Released motile sporozoites probe and attach with their apical membrane selectively to the apical membrane (luminal surface) of enterocytes. Infection (and hence presumably excystation) has also been reported in other sites (often contiguous with the intestinal tract), such as the biliary tract, pancreatic ducts, sinuses and respiratory tract, which are also lined with epithelial cells. Release of presumably membrane-lysing molecules by the apical complex of sporozoites cause the host cell membrane and microvilli to indent and fold around the sporozoite, which ultimately places it in an intracellular but extracytoplasmic compartment below the cell's outer membrane, termed the

'parasitophorous vacuole'. A 'feeder organelle' (located at the base of the parasitophorous vacuole) forms between the developing intra-cellular parasite and the host cell. This distinctive electron-dense structure is presumed to permit exchange of molecules with the host cell (see also Pathogenesis: Intestinal Disease).

After invasion, sporozoites differentiate into rounded trophozoites, which, after asexual reproduction (merogony or schizogony), become type I meronts (or schizonts) with six to eight merozoites. Similar to sporozoites, merozoites are curved parasites with a double inner membrane and an apical complex of rings and micronemes. The rupture of type I meronts releases mature merozoites, which can further invade adjacent epithelial cells and become either type I or II meronts. The cycling of type I meronts is thought to partially explain the ability of C. parvum to persist in the human host. Type II meronts have four merozoites that invade host cells to undergo sexual reproduction (gameto-gony) and become male or female gamonts, which can be seen as early as 36 hours postinfection. Mature micro- (male) and macro-(female) gamonts attach and fuse and form the zygote, which develops into either a thick-walled or thin-walled oocyst, each with four fully infectious sporozoites. Thin-walled oocysts are associated with autoinfection of the intestine, providing a second mechanism by which C. parvum can auto-infect the host, resulting in persistent infection. In contrast, thick-walled oocysts are capable of surviving for long periods of time in the environment. The prepatent period, or time from oocyst ingestion to the excretion of infectious oocysts, is approximately 4-22 days for humans.

PATHOGENESIS: INTESTINAL DISEASE

The mechanisms by which C. parvum cause diarrhea are not well understood, although available data suggest that C. parvum alters intestinal epithelial cell function as well as the enteric immune and nervous systems. The outcome of C. parvum infection, asymptomatic colonization vs. diarrheal disease, can be expected to be dependent on both parasite virulence

Fig. 6.3 Intestinal disease pathogenesis. Adhesion/invasion of C. parvum sporozoites/merozoites to the apical membrane of intestinal epithelial cells with trophozoite formation appears to stimulate the activity of several cellular kinases that participate in the cytoskeletal rearrangement associated with C. parvum invasion of the cell. Cellular invasion also stimulates the intestinal epithelial cells to produce prostaglandin synthase, IL-8 and TNFa. Recruitment of polymorphonuclear leukocytes (by IL-8), activation of macrophages (by TNFa), production of prostaglandins (by prostaglandin synthase) and alteration in the function of ion transporters (by cellular kinases) would be predicted to stimulate intestinal secretion in response to cellular infection with C. parvum. Cellular invasion also results in flattened and fused small intestinal villi, possibly secondary to cell infection and/or in response to the submucosal immunologic response. This morphologic picture is associated with malabsorption, which contributes to C. parvum diarrheal disease. Additionally, a subset of cells infected by C. parvum, which undergo apoptotic cell death, and the enteric nervous system are also probably contributing to the pathophysiology of disease. Reproduced by permission from Sears (2000)

Fig. 6.3 Intestinal disease pathogenesis. Adhesion/invasion of C. parvum sporozoites/merozoites to the apical membrane of intestinal epithelial cells with trophozoite formation appears to stimulate the activity of several cellular kinases that participate in the cytoskeletal rearrangement associated with C. parvum invasion of the cell. Cellular invasion also stimulates the intestinal epithelial cells to produce prostaglandin synthase, IL-8 and TNFa. Recruitment of polymorphonuclear leukocytes (by IL-8), activation of macrophages (by TNFa), production of prostaglandins (by prostaglandin synthase) and alteration in the function of ion transporters (by cellular kinases) would be predicted to stimulate intestinal secretion in response to cellular infection with C. parvum. Cellular invasion also results in flattened and fused small intestinal villi, possibly secondary to cell infection and/or in response to the submucosal immunologic response. This morphologic picture is associated with malabsorption, which contributes to C. parvum diarrheal disease. Additionally, a subset of cells infected by C. parvum, which undergo apoptotic cell death, and the enteric nervous system are also probably contributing to the pathophysiology of disease. Reproduced by permission from Sears (2000)

factors and the intestinal response to the infection. However, despite the appreciation that C. parvum isolates are genetically diverse, there is as yet no delineation of specific virulence factors of C. parvum, nor genetic means to create defined mutants for pathogenetic analysis. Thus, insight into the pathogenesis of this infection currently arises from evaluation of the intestinal pathology of human and animal infections and from studies of in vitro and in vivo disease models (reviewed in Clark and Sears, 1996; Sears, 2000; Sears and Guerrant, 1994). Based on these data, Figure 6.3 proposes a model by which C. parvum infection may result in diarrheal disease.

Attachment of C. parvum sporozoites to intestinal epithelial and/or biliary cells appears to be a specific host-parasite interaction requiring both Gal/GalNAc epitopes on intestinal epithelial cell glycoproteins and on the sporozoite surface (Chen and LaRusso, 2000; Joe et a/., 1994, 1998). Subsequent intestinal epithelial cell invasion by C. parvum sporozoites and merozoites has been shown to be dependent on remodeling of host cell actin (Chen and LaRusso, 2000; Elliot and Clark, 2000; Forney et a/., 1999) but not tubulin, resulting in a plaquelike actin structure at the host-parasite interface. It is of interest that, in addition to actin and the actin binding protein a-actinin (Elliott and Clark, 2000), a putative C. parvum transport protein termed CpABC localizes to the host cell-parasite boundary, where it is postulated to play a role in exporting molecules from the parasite to the cell or vice versa (Perkins et al., 1999). The exact mechanisms by which C. parvum cellular invasion results in actin rearrangement are unknown but current data suggest involvement of host cell kinase signaling pathways (Forney et al., 1999). C. parvum cellular invasion also appears to trigger new protein synthesis, including prostaglandin H synthase 2, proinflammatory cytokines/chemokines [tumor necrosis factor a (TNFa), interleukin-8 (IL-8), GRO-a and possibly interleukin-1p (IL-1 p)] and the mucosal antibiotic peptide, p-defensin, all potentially contributors to C. parvum disease pathogenesis, as outlined below (Laurent et al., 1997, 1998; Seydel et al., 1998; Tarver et al., 1998).

The histopathology resulting from invasion of the intestinal epithelium by C. parvum varies. Information on human intestinal pathology is primarily available from biopsies in AIDS patients with cryptosporidiosis and chronic diarrhea (Genta et al., 1993; Goodgame et al., 1993, 1995; Lumadue et al., 1998). In general, higher-intensity infections, as assessed by histo-pathology and number of stool oocysts, are accompanied by more severe gut injury, including villous atrophy and fusion, crypt hyperplasia and cellular submucosal infiltration (including both mononuclear cells and polymorphonuclear leukocytes), and are associated with evidence of carbohydrate, protein and vitamin (e.g. B12) malabsorption. Reduced activity of brush border enzymes (e.g. lactase, sucrase) occurs and is likely of clinical importance. However, no association between stool volume in AIDS patients and the intensity of infection by biopsy has been identified to date (Genta et al., 1993; Goodgame et al., 1995; Lumadue et al., 1998; Manabe et al., 1998). Furthermore, severe diarrhea is reported in some patients with low-intensity infections and normal duodenal histology. This latter observation could be due, for example, to severe infection in an unbiopsied site, unrecognized co-pathogens and/ or variations in the virulence of C. parvum strains.

In addition to malabsorption, several other potential mechanisms are postulated to contri bute to the development of intestinal symptoms (particularly diarrhea) in individuals with C. parvum infection. First, physiologic studies of C. parvum-infected intestinal tissue of mice and piglets and of human intestinal epithelial cell monolayers suggest that C. parvum infection may alter intestinal ion transport and/or increase gut permeability (Adams et al., 1994; Argenzio et al., 1990, 1993, 1994; Griffiths et al., 1994; Kapel et al., 1997; Moore et al., 1995). In the animal models, impaired absorption of sodium coupled to glucose occurs whether or not symptomatic disease results (Argenzio et al., 1990; Kapel et al., 1997; Moore et al., 1995). In contrast, glutamine-stimulated sodium absorption appears to remain largely intact, suggesting that glutamine-based oral rehydration solutions may be superior to glucose-based oral rehydra-tion solutions in the treatment of C. parvum-induced diarrhea (Argenzio et al., 1990; Kapel et al., 1997; Levine et al., 1994). In more severe disease with diarrhea in piglets, prostanoid-dependent secretion may occur and it can be postulated that the kinases activated by cellular invasion by C. parvum may also act to stimulate intestinal secretion (Argenzio et al., 1990; Forney et al., 1999). Of note, consistent with the available in vitro results, studies of AIDS patients with C. parvum have also provided evidence of reduced intestinal barrier function (Goodgame et al., 1995; Lima et al., 1997). Second, elevated levels of the neuroactive prostaglandin, PGI2, are present in C. parvum-infected piglet intestinal tissue and inhibitor analyses suggest that the enteric nervous system contributes to secretion in C. parvum disease (Argenzio et al., 1996, 1997). Third, pro-inflammatory cytokines (e.g. TNFa, IL-8) are expected to stimulate mucosal recruitment of leukocytes, with production of inflammatory mediators such as prostaglandins (Kandil et al., 1994; Laurent et al., 1997, 1998; Seydel et al., 1998). These inflammatory mediators generated in response to C. parvum infection are known to stimulate intestinal secretion. Consistent with the potential importance of inflammation in the pathogenesis of diarrhea in C. parvum infection, up to 75% of symptomatic, but not asymptomatic, Brazilian children with C. parvum infection had evidence of fecal leukocytes in their stools (Newman et al., 1999). Fourth, cellular injury and apoptosis have been

Table 6.2 C. parvum: a very infectious parasite

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