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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVED TB TEST FOR CATTLE Title: Comparative Analysis of the Bovigam Interferon-Gamma Elisa for Bovine Tuberculosis Using Alternative Antigens or Tuberculins for Blood Stimulation in Field Trials and Experimental Infections

Authors
item Schroder, Bjorn -
item Hardegger, Roland -
item Purro, Mario -
item Moyen, Jean-Louis -
item Gormley, Eamonn -
item WATERS, WADE
item PALMER, MITCHELL
item THACKER, TYLER
item Whelan, Adam -
item Vordermeier, H -
item Raeber, Alex -

Research conducted cooperatively with:
item Prionics Ag

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: July 1, 2010
Publication Date: September 15, 2010
Citation: Schroder, B., Hardegger, R., Purro, M., Moyen, J., Gormley, E., Waters, W.R., Palmer, M.V., Thacker, T.C., Whelan, A., Vordermeier, H.M., Raeber, A.J. 2010. Comparative Analysis of the Bovigam Interferon-Gamma Elisa for Bovine Tuberculosis Using Alternative Antigens or Tuberculins for Blood Stimulation in Field Trials and Experimental Infections [abstract].

Technical Abstract: Bovine Tuberculosis (bTB) caused by Mycobacterium bovis (M. bovis) is a major infectious disease found worldwide in domestic animals, particularly cattle, as well as in certain wildlife populations and is a re-emerging disease in many countries that have been officially bTB-free for decades. The eradication of bTB in cattle is based on the detection and slaughter of infected animals. The standard test for the detection of bTB is the tuberculin skin test. The BOVIGAM® interferon (IFN)-gamma assay constitutes a laboratory-based bTB test and is widely used complementary to the tuberculin skin test (1). The assay consists of a first step culturing whole blood with antigens and stimulating leucocytes to produce IFN-gamma which is quantified by ELISA in a second step. Traditional use of BOVIGAM® is based on measuring the difference in IFN-gamma production between stimulation with bovine and avian tuberculin (PPD). The main benefits of BOVIGAM® compared to skin testing are increased sensitivity, possibility of more rapid repeat testing, no need for a second visit to the farm and more objective test procedure and interpretation. In areas of low bTB prevalence, concerns exist about BOVIGAM® specificity, as well as for skin test. To improve the specificity of BOVIGAM®, defined mycobacterial antigens such as ESAT-6 and CFP-10 have been evaluated for stimulation of whole blood cultures. We have analysed the performance of BOVIGAM® in field studies and experimental infections using defined antigen cocktails and compared it with BOVIGAM® using bovine and avian PPD for stimulation. Materials & methods Evaluation of diagnostic specificity was performed in 63 cattle from a herd free of bTB in Switzerland. Diagnostic sensitivity was assessed in 49 cattle from a herd in a region in France with low bTB prevalence. Both France and Switzerland are officially free of bTB according to OIE guidelines. Experimental infection with M. bovis was conducted at the National Animal Disease Center, Ames, Iowa (NADC) in a biosafety level 3 (BL-3) facility. Calves received M. bovis strain 95-1315 by aerosol and blood samples were taken repeatedly up to 49 days post-infection as described previously (2). Blood collection, stimulation of blood with avian and bovine PPD or with defined antigen cocktails and determination of the bovine IFN-gamma levels in stimulated plasma with the BOVIGAM® ELISA were conducted according to the manufacturer’s instructions for use. Two antigen cocktails (PC4 and PC7) were compared that contained overlapping peptides covering defined sequences of ESAT-6 and CFP-10 (3) as well as peptides derived from one (PC7) and 4 (PC4) additional mycobacterial antigens (4,5). Results All 49 animals of the French herd from a region with low bTB prevalence were tested using the single intradermal comparative Table 1: Comparison of diagnostic performance of BOVIGAM® with two antigen peptide cocktails PC4 and PC7 and PPD. Diagnostic <p>Diagnostic Sensitivity <p>Specificity (n=49) 95% CI (n=63) 95% CI PPD 81% 68-95% 89% 81-97% PC4 75% 60-90% 100% 89-100% PC7 81% 68-95% 100% 89-100% cervical tuberculin test and 32 of the 49 animals were found to be reactors. This correponds to a sensitivity of the skin test of 65%. In comparison, BOVIGAM® with PPD and peptide cocktails showed sensitivities of 81%, 75% and 81%, respectively. Specificity for the BOVIGAM® was assessed in a bTB free herd in Switzerland and the results showed a sensitivtiy of 89% using PPD and 100% using peptide cocktails (Table 1). Experimental infection with M.Bovis was performed to assess the earliest time-point post-infection at which a T-cell mediated IFN-gamma response was detectable in blood following stimulation with the different antigens. Figure 1 shows the time-course of one of a total of 8 cattle experimentally infected with M.Bovis. (Please view attachment. Figure does not copy to ARIS) Figure 1: Time-course showing optical density (OD450nm) readings in plasma as measured by the BOVIGAM IFN-gamma assay following stimulation of blood with peptide cocktail (PC11)(diamond), bovine PPD (CSL B20) (triangle) and control stimulations with PBS (cross) and RPMI (circles). Responses were first detected between day 10 and 14 post-infection and reached a saturation level at day 17 independent of the antigen used for stimulation. Discussion & conclusions These studies show that in combination with antigen cocktails used for stimulation of the cell mediated immune response, a higher diagnostic specificity and equivalent analytical and diagnostic sensitivity in comparison with PPDs could be achieved with the BOVIGAM® ELISA.

Last Modified: 9/10/2014
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