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United States Department of Agriculture

Agricultural Research Service

Research Project: IMMUNOLOGY AND INTERVENTION STRATEGIES FOR JOHNE'S DISEASE Title: Use of ethanol extract of Mycobacterium bovis for detection of specific antibodies in sera of farmed red deer (cervus elaphus) with bovine tuberculosis

Authors
item Wadhwa, Ashutosh -
item Johnson, Rachel -
item Mackintosh, Colin -
item Griffin, J -
item Waters, Wade
item Bannantine, John
item Eda, Shigetoshi -

Submitted to: BioMed Central (BMC) Veterinary Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 6, 2013
Publication Date: December 17, 2013
Repository URL: http://handle.nal.usda.gov/10113/58286
Citation: Wadhwa, A., Johnson, R.E., Mackintosh, C.G., Griffin, J.F., Waters, W.R., Bannantine, J.P., Eda, S. 2013. Use of ethanol extract of Mycobacterium bovis for detection of specific antibodies in sera of farmed red deer (Cervus elaphus) with bovine tuberculosis. BioMed Central (BMC) Veterinary Research. 9(1):256.

Interpretive Summary: Bovine tuberculosis outbreaks occur in cattle and deer with transmission occurring back and forth between these species. The goal of this study is to evaluate the diagnostic testing of Mycobacterium bovis, the bacterium that causes bovine tuberculosis, in red deer species. We have developed an antibody-based test called ethanol vortex ELISA that has shown great promise in testing cattle for Johne’s disease. The novelty of the test lies in the way the antigen is prepared. Because mycobacteria have a high lipid content in their cell walls, a gentle extraction in 80% ethanol removes many of these immunogenic molecules from the surface of the bacteria. The same principles that worked for the mycobacteria that causes Johne’s disease have been applied to M. bovis testing in red deer. We discovered that the test works best if serum samples from red deer are first pretreated with a protein extract from a different Mycobacterium species to remove non-specific antibodies from those samples. When this pretreatment step is performed, the test’s sensitivity and specificity for detecting bovine tuberculosis in red deer were measured at 90% and 93.3%, respectively. Therefore, the results of this study suggest a new and improved antigen-based diagnostic test will identify M. bovis infection in deer populations.

Technical Abstract: Bovine tuberculosis (bTB) in wildlife species poses a threat to domestic livestock in many situations. Control programs for bTB in livestock depend on testing and slaughtering the positive animals; however, the currently available diagnostic tests often have poor specificity. In our previous study, we developed a sensitive enzyme linked immunosorbent assay (ELISA), called ethanol vortex ELISA (EVELISA), using surface antigens of MAP extracted by briefly agitating the bacilli in 80% ethanol solution. The objective of this study is to examine whether EVELISA technique could be used to detect anti- Mycobacterium bovis (MB) antibodies in the serum of infected farmed red deer (Cervus elaphus) in New Zealand. In this study, we tested a total of 50 red deer serum samples, divided in 4 groups - animals uninfected with MB and unvaccinated with MAP (n = 15), experimentally infected with MB (n = 15), naturally infected with MB (n = 5) and animals vaccinated with a commercial MAP vaccine (n = 15). The presence of anti-MB antibodies was tested using an ethanol extract of MB. Without absorption of anti-MAP cross reactive antibodies, it was found that 13 out of the 15 animals that received a MAP vaccine showed high antibody binding. Using heat killed MAP as absorbent of cross reactive antibodies, the tentative diagnostic sensitivity and specificity of the EVELISA was estimated to be 90% and 93.3%, respectively. The results from this study suggest that EVELISA may form a basis for a sensitive and specific test for the diagnosis of bTB.

Last Modified: 12/19/2014
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