Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 5/22/2008
Publication Date: 5/22/2008
Citation: Waters, W.R., Palmer, M.V., Thacker, T.C., Nonnecke, B.J., Lyashchenko, K., Schiller, I., Marg, B., Oesch, B., Vordermeier, M., O'Brien, D., Schmitt, S., Estes, M. 2008. Tuberculosis Diagnosis: Assay Optimization, Validation, and Antigens for Specific Diagnosis. [abstract].
Technical Abstract: Interferon (IFN)-gamma release assays (i.e. Bovigam®, Prionics AG) are components of tuberculosis (TB) eradication programs in many countries. Because this test relies on functional leukocytes, environmental conditions before and during the in vitro culture period have the potential to influence the outcome. Effects of stimulation vessel geometry, incubation conditions, and antigen/ IFN-gamma stability at different temperatures were evaluated using blood from experimentally- and naturally-infected cattle. Whole blood was stimulated in 24- (standard), 48- and 96-well culture plates with Mycobacterium bovis purified protein derivative (PPD), M. avium PPD, rESAT-6:CFP-10, Staphylococcus Enterotoxin B, or pokeweed mitogen. Stimulation was similar between formats, demonstrating the potential for use of 96-well plates to minimize reagent use and streamline large scale testing. Likewise, stimulation at 37°C and 33°C were equivalent; however, IFN-gamma production was reduced at 29°C and undetectable at 25°C and 22°C. CO2 was not required for optimal stimulation. Antigens are usually stored at 2-8°C (PPD) or at -80°C (recombinant proteins). Responses to recombinant antigens (i.e., rESAT-6:CFP-10, TB10.4, TB27.4, MPB83) stored at 4°C for 24h and at 20°C for 8h were comparable to that of antigens thawed immediately from -80°C. However, prolonged (i.e. 70 months) storage of PPD’s at 4°C decreased their stimulatory capacity. Repeated freeze-thaw cycles may degrade proteins within samples; however, natural IFN-gamma concentrations within plasma supernatants were not impacted by up to 5 freeze-thaw cycles. Additionally, storage of plasma at 4°C for up to 28 days did not affect IFN-gamma concentrations. These findings offer technical opportunities and identify potential limitations for testing of new antigens under field conditions. Free-ranging white-tailed deer are reservoirs of M. bovis infection for cattle in Michigan, USA. A capture, test, and cull strategy is being evaluated as a method for limiting transmission of TB from deer to cattle. A prompt and accurate animal-side test is required for this strategy to be feasible. To test the feasibility of candidate tests, 760 serum samples were collected from hunter-killed white-tailed deer and evaluated using a panel of antibody-based tests. Sensitivities ranged from 46 to 68%, while specificities and negative predictive values were all > 92%; thus demonstrating the potential for reasonably accurate results, even under extreme field conditions. Ultimately, test and cull strategies in conjunction with vaccine strategies could be used to improve control of TB in this reservoir species. Cross-reactive responses elicited by exposure to non-tuberculous mycobacteria often confound interpretation of traditional ante-mortem TB tests. Use of specific proteins, such as ESAT-6, CFP-10, and MPB83, may improve test specificity in areas where exposure to environmental non-tuberculous mycobacteria is common. Using an intratonsilar challenge model, calves infected/sensitized with M. avium subsp. avium or M. avium subsp. paratuberculosis did not respond to rESAT-6:CFP-10 or MPB83 despite developing robust responses to complex mycobacterial antigen preparations. In contrast, calves infected/sensitized with M. kansasii developed an antibody response to MPB83 and IFN-gamma response to rESAT-6:CFP10, as well as robust (cellular, humoral and delayed type hypersensitive) responses to heterogeneous mycobacterial antigen preparations. Despite these robust responses, M. kansasii was not detected upon necropsy even within lymph nodes draining the site of inoculation. These findings demonstrate the potential for confounding responses to specific antigens elicited by certain "environmental" mycobacteria even when there is no gross, microscopic or bacteriological evidence of exposure to the sensitizing agent. Despite th