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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Infectious Bacterial Diseases Research » Research » Publications at this Location » Publication #215900

Title: Effects of Culture Conditions and Tuberculin Source on Interferon-gamma production in Whole Blood Cultures from Mycobacterium bovis Infected Cattle

item Waters, Wade
item Palmer, Mitchell
item Nonnecke, Brian

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 11/19/2007
Publication Date: 11/19/2007
Citation: Schiller, I., Waters, W.R., Vordermeier, M., Palmer, M.V., Egnuni, T., Hardegger, R., Kyburz, A., Nonnecke, B.J., Raeber, A., Oesch, B. 2007. Effects of Culture Conditions and Tuberculin Source on Interferon-gamma production in Whole Blood Cultures from Mycobacterium bovis Infected Cattle [abstract]. United States Animal Health Association. p. 140.

Interpretive Summary:

Technical Abstract: The BOVIGAM® interferon (IFN) - gamma assay constitutes an ante-mortem, in vitro laboratory-based tuberculosis test and is used complementary to the tuberculin skin test. The assay is performed in two stages: firstly, whole blood is cultured with antigens stimulating blood leucocytes to produce IFN-gamma which is quantified by ELISA in a second step. Environmental conditions before and during the culturing of the leucocytes influence the efficacy of in vitro IFN-gamma production. Optimal conditions are therefore essential. In this study we analyzed the effect of stimulation vessel geometry, temperatures during stimulation and the stability of antigens stored at different temperatures. Blood from experimentally infected cattle and from tuberculosis-negative cattle was stimulated in 24-well tissue culture trays (standard), 48-well and 96-well culture plates with the following antigens: Purified protein derivate from Mycobacterium bovis (PPD-B) and from Mycobacterium avium (PPD-A), a fusion protein from early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10), and pokeweed mitogen. Stimulation was equally efficient in all three plate formats. The results with specific antigens correlate with mitogen induced stimulation. CO2 is not required during incubation, as cultures from an incubator with 5% CO2 produced similar amounts of IFN-gamma as without CO2. However, the temperature used for stimulation was critical: Stimulation at 37°C and 33°C were equally efficient, but a culture temperature of 29°C reduced IFN-gamma production significantly. At 25°C and 22°C no stimulation was detectable. Antigens are usually stored at 2-8°C (tuberculins) or at -80°C (recombinant proteins) until usage. We tested in parallel antigen storage of recombinant proteins (ESAT-6:CFP-10 fusion protein, TB10.4, TB27.4, MPB83) at 4°C for 24h or at 20°C for 8h prior to use in cell culture. Our results show that antigens may be stored at either of these conditions without affecting the efficacy of stimulation. Finally, we compared the activities of tuberculins from five different sources in naturally infected cattle (n=10). Matched PPD-B and PPD-A tuberculins were used at eight dilutions each. Relative potency 30 (RP30) was defined as the tuberculin concentration required to induce 30% of the peak response values. RP30 differed by a factor of more than 10 between the PPD-B with the highest and lowest potency. Therefore tuberculins of different sources may give different results and the overall assay performance may be improved by optimizing tuberculin concentrations.