Molecular Epidemiological Studies

To determine the patterns of transmission of Toxoplasma requires intensive studies which employ polymorphic markers to track strains in contiguous space and time. Such studies are rare, but perhaps the best examples are the careful studies of marine mammals, in which close typing of isolates shows infection by both type II strains and by isolates clustered into a new strain, type X (Miller et al., 2004). The risk of infection in sea otters was related to the level of freshwater runoff (Miller et al., 2002), and a hotspot of type X strain transmission was mapped to a single site (Morro Bay) on the California coast (Miller et al., 2004). It is proposed that oocysts in runoff from water fall into marine sediments and may be concentrated in mollusks - a common food source for the otters. Although Toxoplasma can survive in bivalves (Lindsay et al., 2004), there is as yet no evidence that this occurs in nature and the terrestrial source of this unusual genotype has still to be discovered. However, extension of this approach to the region inland from Morro Bay should throw light on these questions and perhaps provide the first genetically defined transmission network for Toxoplasma.

A second system which has been subject to molecular epidemiological study is the transmission of Toxoplasma in sheep. A longitudinal study of UK sheep revealed consistently high levels of congenital transmission, with over 40 percent of live-born lambs affected (Duncanson et al., 2001; Williams et al., 2005). Analysis of 10-year pedigree records proved supportive of vertical transmission, finding that abortion and Toxoplasma infection are more common in some maternal lineages (Morley et al., 2005). Mobile genetic element markers (Terry et al., 2001) have been used to characterize the strains circulating in the flock, and these found evidence of a clonal genotype in longitudinal studies (Figure 3.3). Occasionally,

FIGURE 3.3 PCR amplification using the single primer extension with repA and repB MGE primer of T. gondii from ovine chord samples. Samples were taken from Charollais sheep on a Worcester farm in March 1999 (1-4), January 2000 (5-8), and March 2000 (9), and from a Lancaster farm in March 2000 (10-12). The majority of isolates within a flock are clonal, but a few variant strains are found.

FIGURE 3.3 PCR amplification using the single primer extension with repA and repB MGE primer of T. gondii from ovine chord samples. Samples were taken from Charollais sheep on a Worcester farm in March 1999 (1-4), January 2000 (5-8), and March 2000 (9), and from a Lancaster farm in March 2000 (10-12). The majority of isolates within a flock are clonal, but a few variant strains are found.

new genotypes were seen, perhaps reflecting infection from oocysts. Evidence for vertical transmission has also been reported in other species, including mice and rats (Beverley, 1959; Dubey et al., 1997; Owen and Trees, 1999; Marshall et al., 2004). It is characteristic that vertical transmission may be overlooked, as parasites adapted to this route are predicted to have low pathogenicity and therefore to be cryptic (Dunn and Smith, 2001).

The cat is believed to play a central role in the transmission of Toxoplasma, yet, ironically, there are no genetic data that demonstrate oocyst-induced infection. The most frequently cited evidence relates to the serious outbreak of toxoplasmosis in Vancouver Island (Bowie et al., 1997). This epidemic lasted for 6 months between 1994 and 1995, and caused 100 cases of clinical human disease. Mapping the sites of infection led to the conclusion that the outbreak was caused by oocyst contamination of the local water reservoir. Infected feral cats and cougars were both found in the watershed area (Aramini et al., 1998), but attempts to extract oocysts from the water supply were unsuccessful. The only isolate found in the neighborhood was isolated from cougar feces and was found to have an atypical genotype (Lehmann et al., 2000; Grigg et al., 2001a). This isolate was supposed to be at the origin of the outbreak. Similarly, other reported oocyst-associated infections also rely on circumstantial evidence (Benenson et al., 1982; Bahia-Oliveira et al., 2003).

In summary, although population-based studies are revealing large-scale patterns of genetic variation in Toxoplasma, more intensive molecular epidemiological studies are required to elucidate transmission networks, and this is clearly a priority if we are to understand and control the transmission of the parasite.

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