ABSTRACTApplication of Diagnostic Assays for the Detection of Mycobacterium bovis Infection in Suspect Michigan Reservoir SpeciesByKarla Ann FentonMycobacterium bovis, the etiologic agent of bovine tuberculosis, is present on multiple continents and has one of the broadest host ranges of any known zoonotic pathogen. The broad host range of this microbe has created a complex ecosystem, in which diagnostic assay development, disease discovery, epidemiological characterization, surveillance, and control have proven to be a global challenge. In the state of Michigan, an endemic focus of bovine tuberculosis is maintained within the sole wildlife reservoir, the white-tailed deer; however, additional surveillance and assay application for the discovery of other potential wild and domestic animal reservoirs are currently being researched. This dissertation discusses four projects in which traditional diagnostic assays and three new assays utilizing a macrophage cell line and two antibody based assays; the multiantigen print immunoassay (MAPIA) and rapid test (RT), were implemented in the surveillance and disease characterization of Mycobacterium bovis in potential Michigan reservoir species. The first study was an evaluation of the immune response in specific pathogen-free (SPF) cats stimulated with Sensitinogen, a heat killed Mycobacterium bovis product, using the RT, MAPIA, and bovine purified protein derivative (bPPD) single skin test. The bPPD test at 72 hours had a mean skin thickness of 0.3 mm for stimulated cats and 0.1 mm for controls. Rapid test identified 4 of 6 stimulated cats after bPPD injection. The MAPIA detected antibody against MPB83, 16/83, 16kDa, and M. bovis culture filtrate (MBCF) antigens. All assays differentiated between stimulated and control cats; however 7 of 49 non-SPF control cats had a reaction for either antigen MBCF or 16/83. These preliminary studies show potential for antemortem detection of M. bovis among domestic cats. The second study was an evaluation of disease spread by experimentally assessing intra-species lateral transmission of virulent M. bovis among four replicate inoculated/exposed, co-habitating wild caught Didelphis virginiana (Virginia opossum). Gross and histologic examinations were consistent with caseogranulomatous pneumonia in all four inoculated opossums. Additionally, these four inoculated opossums had a positive test band on the RT and were M. bovis culture positive. The exposed and control groups were unremarkable on gross, histology, rapid test, and culture. These findings support that there was no appreciable lateral transmission of M. bovis after aerosol inoculation and 45 days of cohabitation between opossum. The third study was a surveillance of 124 hunter harvested feral in the state of Michigan from November of 2006 until September 2010. Three swine were noted having gross lesions of tuberculosis; however, these were not confirmed to be tuberculous lesions histologically. All other animals in the study had no evidence grossly or histologically of tuberculosis. The fourth study utilized the monocytic cell line TIB-202 to propagate M. bovis in order to investigate a more rapid, sensitive, flexible, and cost effective diagnostic culture method. The phagocytic cells were incubated with virulent M. bovis, lysed, and evaluated for M. bovis propagation by PCR amplification of the IS6110. The preliminary PCR results were able to detect a marked increase in amplifiable DNA within lysated cells after six days of incubation, supporting idea that this cell line could potentially be used as a rapid detection diagnostic assay; however, continued research is needed. In conclusion, domestic cats, Virginia opossum, and feral swine appear to have a minimal impact on bovine tuberculosis spread and persistence in the state of Michigan. However minimal the current impact maybe, continued research and surveillance is empirical for disease characterization and the ultimate global eradication of tuberculosis.
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