Submitted to: Clinical and Diagnostic Laboratory Immunology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/24/2005
Publication Date: 1/20/2006
Citation: Waters, W.R., Palmer, M.V., Slaughter, R.E., Jones, S.L., Pitzer, J.E., Minion, F.C. 2006. Diagnostic Implications of Antigen-induced Gamma interferon production by blood leukocytes from mycobacterium bovis-infected Reindeer (Rangifer Tarandus). Clinical and Diagnostic Laboratory Immunology. 13:37-44. Interpretive Summary: Reindeer are routinely tested for tuberculosis by tuberculin skin testing as outlined in the USDA uniform methods and rules for the eradication of bovine tuberculosis in the United States. However, skin testing has an apparent lack of specificity as numerous reindeer are classified as reactors yet the bacterium is not isolated from tissues when the animal is killed. In the present study, a new tuberculosis test for use with samples from deer species was evaluated with samples from experimentally infected and non-infected reindeer. The test proved beneficial and more reliable than current approved techniques. Findings from this study will be useful for defining improved protocols for tuberculosis surveillance of reindeer and will likely have impact on tuberculosis testing procedures used with other deer species.
Technical Abstract: Reindeer (Rangifer tarandus) are routinely tested for tuberculosis by tuberculin skin testing (i.e., a measure of delayed type hypersensitivity) as outlined in the USDA uniform methods and rules for the eradication of bovine tuberculosis in the US. However, skin testing has an apparent lack of specificity as numerous reindeer are classified as reactors yet M. bovis is not isolated from tissues upon necropsy. An in vitro interferon (IFN)-g-based method of tuberculosis diagnosis has been developed and approved for use in cattle in the US. A similar test (i.e., Cervigam') for use in Cervidae is in development and appears promising with samples from white-tailed deer. Our objective was to evaluate the ability of the Cervigam' assay to detect IFN-y produced by blood leukocytes from M. bovis-infected reindeer to mycobacterial antigens. Thirteen male reindeer of ~9 months of age were inoculated with 10**5 cfu Mycobacterium bovis into their tonsillar crypts and 4 age-matched male reindeer served as non-infected controls. Blood was collected periodically for in vitro stimulation and IFN-g analysis. Polyclonal stimulation of whole blood cultures with mitogen resulted in significant (P < 0.05) production of IFN-g as compared to non-stimulated samples at all time points. As early as 30d after inoculation and at most time points thereafter, responses by infected reindeer to M. bovis PPD exceeded (P < 0.05) those by non-infected reindeer. Despite differences in responses to M. bovis PPD by the two infection groups, 4/4 reindeer within the non-infected group had positive responses (i.e., > 0.1 deltaOD) to M. bovis PPD. ESAT-6 and CFP-10 are proteins expressed by tuberculosis complex Mycobacteria spp., but not by M. bovis BCG and most other non-tuberculous Mycobacteria spp.. Mean responses by infected reindeer to rCFP-10 and rESAT-6:CFP-10 fusion protein exceeded (P < 0.01) respective responses by non-infected reindeer at all time points. In contrast to M. bovis PPD, responses to rCFP-10 and rESAT-6:CFP-10 by 3/4 non-infected reindeer were considered negative (i.e., < 0.1 deltaOD) at each time point. Responses were also evaluated with samples processed immediately or after a 24 hr delay at room temperature. The 24 hr delay in processing did not alter (P > 0.05) the response, indicating that delays associated with overnight delivery will not interfere with the test. Together, these findings indicate that the Cervigam' assay should prove useful for TB surveillance of reindeer. However, use of specific antigens such as rESAT-6:CFP-10 may improve specificity.