TRICHINELLA SPECIES AND TOXOPLASMA GONDII IN WOLVERINE (GULO GULO) FROM NORTHWESTERN CANADA

Trichinella spp. and Toxoplasma gondii are important foodborne parasites world-wide, and are especially important in wildlife harvested for food and fur in Canada. Due to their top position in the food web as well as their scavenging behavior, large carnivores such as wolverines may be more exposed to these parasites through foodborne routes. Limited information is available on these parasites in wildlife from northwestern Canada, and such information is critical to control and prevent infections in animals and people. To conduct surveillance studies, the first and foremost step is to select samples suitable for detection of the parasites. I first compared tongues and diaphragms of wolverine harvested for fur for the detection of Trichinella species (Chapter 2), and found tongue could act as a better sampling site; therefore, further epidemiological studies on Trichinella used tongue tissues (Chapter 3). Using a double separatory funnel digestion method, we found larvae of Trichinella in the tongue of 78% (95% CI = 73–82) of 338 wolverine from the Yukon Territory. The mean intensity of infection was 22.6 ± 39 (SD, range 0.1–295) larvae per gram of muscle tissue. Species or genotypes of Trichinella circulating in the wildlife of the Yukon are not known. Using multiplex PCR, Trichinella T6 was revealed as the predominant genotype (76%), followed by T. nativa (8%); mixed infections with Trichinella T6 and T. nativa (12%) were observed. While confirming multiplex genotyping by DNA sequencing, I unexpectedly discovered a previously undescribed species of Trichinella in a wolverine from the Canadian North that appeared identical to T. nativa in multiplex PCR. Therefore, I investigated the phylogeny, geography and host range of this undescribed species in Chapter 4. My findings showed that this unknown species of Trichinella, to be designated initially as the T13 genotype, is in fact ancient, and putative sister of the lineages including T. patagoniensis and the remaining species [(sister-clade (T1 + T7) and sister-clade (T2, T6, T3, T8, T5, T9)] of the encapsulated group. A deep divergence for T13 indicates it is the oldest endemic lineage in North America and comparative historical biogeographic studies are necessary to resolve a history of distribution and evolution at high latitudes. Based on my field survey, T13 has only been found in wolverines, and further studies are required to understand the limits of geographic distribution and host range in North America. Overall, wolverines have a high prevalence, intensity, and diversity of Trichinella spp. This work demonstrated that wolverine can host at least 5 species of Trichinella (Trichinella T6, T. nativa, the new species – T13, T. pseudospiralis, and T. spiralis). To find out the suitable sampling sites for detecting T. gondii in wolverines, I used magnetic capture-qPCR to compare tissue prevalence and intensity of DNA of T. gondii in heart and brain (known predilection sites in other species) (Chapter 5). Tissue prevalence (16 positives) and infection intensity (1221 TEG) was higher in heart compared to brain (10 positives, 347 TEG); thus, heart was selected for the next objective (Chapter 6). I also showed that heart fluid (HF) and filter paper eluates (FE) of chest fluid performed equally well in Enzyme Linked Immunosorbent Assay (ELISA) and Indirect Fluorescent Antibody test (IFAT), and HF performed better with modified agglutination test (MAT). IFAT and ELISA had higher sensitivity, specificity, and accuracy compared to MAT (Chapter 5). I used this information to fulfill the next knowledge gap; lack of information on the status of T. gondii in the wildlife of northwestern Canada. I used an ELISA to determine the prevalence of antibodies, and a sequence-specific magnetic capture DNA extraction and real-time PCR (MC-qPCR) to detect DNA of T. gondii in wolverines in the Yukon. Antibodies to T. gondii were detected in 27.5% (95% CI 23.0-32.5%) of 338 heart fluid samples. Eighty-one wolverines were positive on both ELISA and MC-qPCR. Strong correlation between the results of ELISA and MC-qPCR was observed. Overall, prevalence of T. gondii in wolverines in the Yukon was lower than that reported in this species elsewhere in northern Canada, which corresponds with lower seroprevalence in people in the western vs central and eastern North American Arctic. Due to their scavenging habits, apex position in the food chain and wide home range, wolverine hosted high prevalence, high larval intensity, and multiple species of Trichinella, and also showed moderate prevalence and intensity of T. gondii infections. Therefore, wolverine should be considered as a sentinel species for surveys for Trichinella spp. and T. gondii across their distributional range, and as potential indicators of human risk of exposure through foodborne routes.

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