Toxoplasma gondii and related Sarcocystidae parasites in harvested caribou from Nunavik, Canada.

Caribou are keystone species important for human harvest and of conservation concern; even so, much is unknown about the impact of parasites on caribou health and ecology. The aim of this study was to determine the seroprevalence, tissue prevalence, and diversity of tissue-dwelling coccidian parasites (including Toxoplasma gondii, Neospora caninum and Sarcocystis spp.) in 88 migratory caribou (Rangifer tarandus) harvested for human consumption in two communities in Nunavik, Québec, Canada. Both T. gondii and N. caninum have potential to cause abortions and neurological disease in caribou. Seroprevalence for antibodies to T. gondii using ELISA on fluid from thawed hearts was 18% overall, and no DNA of T. gondii was detected in tissues, which has positive implications for food safety since this parasite is zoonotic. Seroprevalence for antibodies to N. caninum using competitive ELISA was 5%, and DNA of N. caninum was detected in only one heart sample. DNA of Sarcocystis, a non-zoonotic, related coccidian, was detected in tissue samples from 85% of caribou, with higher prevalence in heart (82%) than skeletal muscle (47%). This is the first time that Sarcocystis spp. from caribou in Canada have been identified to species level, many of which have been described in reindeer from Fennoscandia. The high prevalence and diversity of Sarcocystis spp. suggests intact trophic relationships between canids and caribou in Nunavik. Besnoitia spp. was serendipitously detected in three muscle samples, a parasite previously associated with skin lesions in caribou in Nunavik. Community-level differences in T. gondii exposure and prevalence of Sarcocystis spp. in skeletal muscle tissues may reflect differences in hunter selection of individual animals and muscles, or possibly regional differences in the ecology of carnivore definitive hosts for these parasites. Further work is needed to explore effects of tissue coccidians in caribou, their taxonomic classifications, and community level differences in parasite prevalence and diversity.

A new multi-host species indirect ELISA using protein A/G conjugate for detection of anti-Toxoplasma gondii IgG antibodies with comparison to ELISA-IgG, agglutination assay and Western blot.

Toxoplasma gondii is a zoonotic protozoan parasite which can cause significant disease and losses in livestock and wild animals. It is increasingly recognized as an important foodborne pathogen in a broad range of food animals and products. Effective control strategies require rapid, reliable and cost-effective detection methods for large scale surveys and diagnostic applications in a broad range of warm-blooded animals. To overcome one or more of these shortcomings in the currently available detection methods for T. gondii infection a non-species-specific protein A/G conjugate was used in the development of an indirect ELISA (ELISA-A/G) for the detection of IgG antibodies in serum samples obtained from experimentally infected pigs. The performance of the assay was evaluated using serum samples from pigs, cats, mice and seals with known positive or negative status for T. gondii infection. Results of the ELISA-A/G obtained with pig serum samples were compared with those generated by traditional ELISA using host specific IgG conjugate (ELISA-IgG), modified agglutination test (MAT) and Western blot analysis (WB). Using protein A/G conjugate, comparative analysis of results from 77 samples obtained from T. gondii infected pigs showed excellent agreement between the ELISA-A/G and in-house ELISA-IgG (0.917 κ). Similar agreements were also observed when these samples were tested by a commercial ELISA kit (0.816 κ), MAT (0.816 κ) and WB (0.79 κ). A total of 86 serum samples obtained from cats, mice and seals experimentally infected with T. gondii and tested by the ELISA-A/G as well as MAT for the presence of anti-Toxoplasma IgG antibodies yielded Kappa value of 1.0 for cats and mice and 0.79 for seals. These results show that the ELISA-A/G is a suitable method for serological detection of T. gondii infection in multiple host species and has the potential for testing samples from a broad range of domestic, wild, and aquatic mammalian host species. Simultaneous testing of samples from multiple host species on the same ELISA plate, and the use of multiple plates in a single run for large scale screening will enhance the cost effectiveness and speed of the test in the control and management of toxoplasmosis. This study also shows the effectiveness of the protein A/G conjugate in a modified WB assay for confirmation of T. gondii infection in mammalian hosts. Appropriate validation studies using field samples from various host species to validate the performance of ELISA-A/G is recommended prior to its application for diagnostic and surveillance programs.

Rapid discrimination of Salmonella isolates by single-strand conformation polymorphism analysis.

A molecular typing technique was developed for the differentiation of Salmonella isolates based on single-strand conformation polymorphism (SSCP) analysis of amplicons generated by PCR. Amplicons from parts of the fimA (both the 5' and 3' ends), mdh, invA, and atpD genes were generated separately from a panel of Salmonella strains representing Salmonella bongori, and four subspecies and 17 serovars of Salmonella enterica. These amplicons were subjected to SSCP analysis for differentiation of the salmonellae on the basis of different conformational forms arising due to nucleotide sequence variations in the target genes. Several distinct SSCP banding patterns (a maximum of 14 each for atpD and fimA 3' end) were observed with this panel of Salmonella strains for amplicons generated from each target gene. The best discrimination of Salmonella subspecies and serovar was achieved from the SSCP analysis of a combination of at least three gene targets: atpD, invA, and either mdh or fimA 3' end. This demonstrates the applicability of SSCP analysis as an important additional method to classical typing approaches for the differentiation of foodborne Salmonella isolates. SSCP is simple to perform and should be readily transferable to food microbiology laboratories with basic PCR capability.