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United States Department of Agriculture

Agricultural Research Service

Research Project: COUNTERMEASURES TO PREVENT AND CONTROL TUBERCULOSIS IN CATTLE AND WILDLIFE RESERVOIRS Title: Field evaluation of alternative antigens in the Bovigam assay

Authors
item Waters, Wade
item Orloski, Kathy -
item Francisco, T -
item Antognoli, C -
item Meyer, R -

Submitted to: Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: November 15, 2010
Publication Date: November 15, 2010
Citation: Waters, W.R., Orloski, K., Francisco, T., Antognoli, C., Meyer, R. 2010. Field evaluation of alternative antigens in the Bovigam assay. In: Proceedings of the US Animal Health Association Tuberculosis Scientific Advisory Subcommittee Meeting, November 15, 2010, Minneapolis, Minnesota. p. 346.

Technical Abstract: Bovine tuberculosis (TB) was detected in a Holstein cow upon routine slaughter inspection in March 2010. Initial ante-mortem testing indicated that the herd of origin (~900 Holsteins, Colorado) had a relatively high within herd prevalence of bovine TB (11 - 25%). The owner agreed to additional TB test evaluation including skin test and collection of blood for blood-based tests. The herd was subsequently depopulated with all animals evaluated for tuberculous lesions upon slaughter. With this herd, three independent studies were undertaken: evaluation of the accuracy of Bovigam and emerging serologic assays, comparison of Bovigam and comparative cervical test as confirmatory tests, and evaluation of alternative antigens for potential use in the Bovigam assay (the topic of this report). For the study to evaluate use of alternative antigens in the Bovigam assay, the objective was to evaluate interferon (IFN)-gamma responses to ESAT-6/CFP10 peptide cocktail (EC) and purified protein derivatives (PPD) from various manufacturers, as well as to evaluate interactions based upon responses to pokeweed mitogen (PWM, indicative of cell viability) or infection status. The study was performed in collaboration with Prionics Ag, Schlieren, Switzerland (manufacturers of the Bovigam) and the Texas Animal Health Commission (TAHC) diagnostic laboratory in Austin, Texas. Blood was collected from 126 animals immediately prior to necropsy and 8 hr after transport by truck to the slaughter facility. Whole blood samples were delivered overnight to the TAHC diagnostic laboratory in Austin, Texas for determination of IFN-gamma responses using the Bovigam assay. Whole blood culture treatments included: no stimulation (NS, culture medium only), CSL M. bovis origin PPD (PPDb, 20 ug/ml), CSL M. avium origin PPD (PPDa, 20 ug/ml), Lelystad PPDb (300 IU/ml), Lelystad PPDa (250 IU/ml), EC, and PWM (5 ug/ml), each provided by Prionics Ag, Schlieren, Switzerland. Stimulation was in 96 well tissue culture plates at 37 degrees C for 20-24 hrs; plasma was separated via centrifugation and harvested; and plasma IFN-gamma concentrations determined by routine ELISA. Animals were considered: infected (n = 54, M. bovis cultured from tissues or detected by PCR on histocompatible lesions, exposed (n = 69, no isolation made or no gross lesions), or PCR pending (n =3). Without regards to infection status, delta IFN-gamma responses (i.e., response to antigen minus NS) to Lelystad PPDb exceeded (p < 0.001, n = 126) corresponding responses to CSL PPDb. Despite differences in magnitude, relative responses to Lelystad and CSL PPDb were highly correlated (r**2 = 0.7, n = 126). Responses to Lelystad and CSL PPDa were similar in magnitude and highly associated (r**2 = 0.8, n = 126). PWM responses were lower (mean = 0.77) than expected suggesting potential effects of animal transport and associated stress. The magnitude of responses to PWM did not correlate (r**2 = 0.2 – 0.31) with corresponding responses to antigen (i.e., EC and PPDb); however, exclusion of PWM low responders (i.e., < 0.5 OD) resulted in a greater percentage of antigen responses considered positive (i.e., > 0.1 Delta OD). With regards to infection status, responses to EC by infected animals exceeded (p = 0.04) that of exposed animals and responses by infected animals to Lelystad PPDb tended to exceed (p = 0.1) that of exposed animals. Responses to EC were generally robust (mean = 0.33, n = 126) and comparable to responses Lelystad PPDb (r**2 = 0.77). These findings demonstrate the necessity to critically evaluate the choice of antigens for use in the Bovigam' assay.

Last Modified: 9/2/2014
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