Submitted to: Clinical and Vaccine Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/19/2009
Publication Date: 8/1/2009
Publication URL: //cvi.asm.org/cgi/reprint/16/8/1196
Citation: Schiller, I., Waters, W.R., Vordermeier, H.M., Nonnecke, B.J., Welsh, M., Keck, N., Whelan, A., Sigafoose, T., Stamm, C., Palmer, M.V., Thacker, T.C., Hardegger, R., Marg-Haufe, B., Raeber, A., Oesch, B. 2009. Optimization of a Whole-Blood Gamma Interferon Assay for Detection of Mycobacterium bovis-Infected Cattle. Clinical and Vaccine Immunology. 16(8):1196-1202. Interpretive Summary: Bovine tuberculosis (TB), caused by Mycobacterium bovis, has an important and adverse effect on socioeconomic, public health and trade of animals and animal products. The eradication of bovine TB in cattle is based on the detection and slaughter of infected animals or whole herds. Control of bovine TB is hindered by the presence of numerous wildlife reservoirs such as white-tailed deer, European badgers, and brush-tailed possums. Reasons for the failure to eradicate the disease are multi-factorial. The Bovigam™ assay is approved for use within the United States as a complimentary test for bovine TB. The BOVIGAM® assay constitutes a laboratory-based TB test and is widely used complementary to the tuberculin skin test as it offers national TB control programs and industry an additional tool to curtail the spread of TB in cattle and other Bovidae. The assay critically depends on the sample quality, culture conditions and quality control of stimulation reagents. In the present study, test applications and laboratory variables were evaluated to improve test specificity and sensitivity. Knowledge obtained from this study may lead to an improved test for tuberculosis, thereby, advancing the national tuberculosis eradication program.
Technical Abstract: Antigens of Mycobacterium bovis elicit a cell-mediated immune response upon intradermalinjection in cattle. In vitro, such antigens stimulate the production of interferon (IFN)-gamma by bovine T-cells in whole blood culture (IFN-gamma assay). We have analyzed various parameters of the in vitro IFN-gamma assay ranging from blood sampling to execution of the IFN-gamma test in view of potential simplifications of the assay. Here we show that IFN-gamma responses may be reduced under certain animal handling/holding conditions and a delayed time from blood collection to culture may lead to a reduced in vitro IFN-gamma response. Delayed initiation of culture in a PPD34 based assay (24h compared to 8h after blood collection) however, resulted in a significant improvement of specificity (97% compared to 85%), whereas there was only a modest reduction of sensitivity (from 96% to 90%), which was statistically not significant. Furthermore, we show that the stimulation temperature needs to be 33°C or higher, carbon dioxide is not required for stimulation, and that various plate formats ranging from 24- to 96-well plates can be utilized. The produced IFN-gamma is stable at 4°C for 28 days as well as after repeated freeze-thaw cycles. Thus, stimulation of samples may be initiated in the field without the need for a carbon dioxide source and bovine IFN-gamma is stable under various routine laboratory temperature scenarios. These findings demonstrate opportunities for improvements in the bovine IFN-gamma test platform and flexibilities in test application.