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United States Department of Agriculture

Agricultural Research Service

Research Project: COUNTERMEASURES TO PREVENT AND CONTROL TUBERCULOSIS IN CATTLE AND WILDLIFE RESERVOIRS Title: Tuberculosis Diagnosis: Assay Optimization, Validation, and Antigens for Specific Diagnosis

Authors
item WATERS, WADE
item PALMER, MITCHELL
item THACKER, TYLER
item NONNECKE, BRIAN
item Lyashchenko, Konstantin - CHEMBIO DIAGNOSTICS
item Schiller, Irene - PRIONICS AG
item Marg, Beatrice - PRIONICS AG
item Oesch, Bruno - PRIONICS AG
item Vordermeier, Martin - VET. LAB. AGENCIES, UK
item O'Brien, Dan - DNR, LANSING, MI
item Schmitt, Steve - DNR, LANSING, MI
item Estes, Mark - UNIV. OF TX MED. BRANCH

Research conducted cooperatively with:
item Prionics Ag

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: May 22, 2008
Publication Date: May 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 these minor limitations, M. tb complex specific antigens generally provide improved specificity over heterogeneous mycobacterial antigens such as PPD’s. Together, these findings describe advances in ante-mortem TB testing and target areas for improvement. Further, studies with samples from cattle and other hosts of TB may prove useful for the discovery and validation of improved antigens and readouts of infection, such as granulysin, IL-21, nitric oxide and other biomarkers.

Last Modified: 8/27/2014
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