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Title:   Author  item
                                            SCHRODER, BJORN - Prionics Agitem
                                            HARDEGGER, ROLAND - Prionics Agitem
                                            PURRO, MARIO - Prionics Agitem
                                            MOYEN, JEAN-LOUIS - Department Of Agriculture Analysis Laboratoryitem
                                            GORMLEY, EAMONN - Department Of Agriculture Analysis Laboratoryitem
                                            Waters, Wadeitem
                                            Palmer, Mitchellitem
                                            Thacker, Tyleritem
                                            WHELAN, ADAM - Veterinary Laboratories Agency (VLA)item
                                            VORDERMEIER, H - Veterinary Laboratories Agency (VLA)item
                                            RAEBER, ALEX - Prionics Ag
  Submitted to: Meeting Abstract
                                    Publication Type: Abstract Only
                                    Publication Acceptance Date: 7/1/2010
                                    Publication  Date: 9/15/2010
                                    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].Interpretive Summary:
                                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                  Diagnostic 
          Sensitivity                  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


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