Publications | Improved diagnostic tools

THEMATIC AREA 3

Improved diagnostic tools

• Optimization of a whole-blood gamma interferon assay for detection of Mycobacterium bovis-infected cattle
• Assessment of Mycobacterium tuberculosis OmpATb as a novel antigen for the diagnosis of bovine tuberculosis
• Comparison of tuberculin activity in the interferon-gamma assay for the diagnosis of bovine tuberculosis

Optimization of a whole-blood gamma interferon assay for detection of Mycobacterium bovis-infected cattle Antigens of Mycobacterium bovis elicit a cell-mediated immune response upon intradermal injection in cattle. In vitro, such antigens stimulate the production of gamma 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 that a delayed time from blood collection to culture may lead to a reduced in vitro IFN-gamma response. Delayed initiation of culture in a purified-protein-derivative-based assay (24 h compared to 8 h 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 degrees C or higher; that carbon dioxide is not required for stimulation; and that various plate formats, ranging from 24 to 96 wells per plate, can be utilized. The produced IFN-gamma is stable at 4 degrees 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

Schiller I., Waters W.R., Vordermeier H.M., Nonnecke B., Welsh M., Keck N., Whelan A., Sigafoose T., Stamm C., Palmer M., Thacker T., Hardegger R., Marg-Haufe B., Raeber A., Oesch B. Optimization of a whole-blood gamma interferon assay for detection of Mycobacterium bovis-infected cattle. Clin. Vaccine Immunol. 16 (8):1196-202. 2009

Assessment of Mycobacterium tuberculosis OmpATb as a novel antigen for the diagnosis of bovine tuberculosis In the search for better tools to control bovine tuberculosis, the development of diagnostic tests with improved specificity and sensitivity has a high priority. We chose to search for novel immunodiagnostic reagents. In this study, Rv0899 (outer membrane protein A of Mycobacterium tuberculosis [OmpATb]) was evaluated as a stimulation antigen in a gamma interferon (IFN-gamma) release assay to diagnose bovine tuberculosis. OmpATb induced IFN-gamma responses in cattle experimentally infected with M. bovis as early and as persistently as ESAT-6 and CFP-10, the current lead diagnostic antigens. In naturally infected cattle, OmpATb stimulated IFN-gamma production in 22 of 26 animals (85%). Importantly, OmpATb detected a portion of M. bovis-infected cattle which did not respond to ESAT-6 and CFP-10 (five of six cattle). The combined diagnostic sensitivity of OmpATb, ESAT-6, and CFP-10 for a preselected group consisting of naturally infected cattle with an overrepresentation of ESAT-6/CFP-10 nonresponders was 96% (25 of 26 animals). The specificity of OmpATb for uninfected cattle was 100% (27 cattle were tested; 12 of them gave false-positive results with tuberculins). In summary, our results indicate that OmpATb has the potential to enhance the sensitivity of previously described diagnostic tests based on ESAT-6 and CFP-10 and that the combined use of OmpATb, ESAT-6, CFP-10, and other proteins may achieve at least equal sensitivity to that obtained with purified protein derivative, but at a higher specificity. Further studies evaluating the diagnostic performance of OmpATb in combination with other proteins are ongoing

Schiller I., Vordermeier H.M., Waters W.R., Palmer M., Thacker T., Whelan A., Hardegger R., Marg-Haufe B., Raeber A., Oesch B. Assessment of Mycobacterium tuberculosis OmpATb as a novel antigen for the diagnosis of bovine tuberculosis. Clin. Vaccine Immunol. 16 (9):1314-21. 2009

Comparison of tuberculin activity in the interferon-gamma assay for the diagnosis of bovine tuberculosis Schiller I., Vordermeier H.M., Waters W.R., Cagiola M., Whelan A., Palmer M.V., Thacker T.C., MeIjlis J., Carter C., Gordon S., Egnuni T., Hardegger R., Marg-Haufe B., Raeber A., Oesch B. Comparison of tuberculin activity in the interferon-gamma assay for the diagnosis of bovine tuberculosis. Veterinary Record, Publication Acceptance Date: June 9, 2009