ABSTRACTA peptide cocktail derived from the mycobacterial antigens ESAT-6, CFP-10, and Rv3615c allowed differentiation betweenMycobacterium bovis-infected and M. bovis bacillus Calmette-Guérin (BCG)-vaccinated cattle when used as a skin test reagent for a “DIVA” test (i.e., a test capable ofdifferentiatinginfected and uninfectedvaccinatedanimals). Addition of the antigen Rv3020c improves the diagnostic sensitivity without compromising specificity in the face of BCG or Johne's disease vaccination.

Mycobacterium bovis BCG vaccination sensitizes cattle to bovine tuberculin, which compromises the use of the current bovine tuberculosis (TB) surveillance tests. Although the performance of a blood test (that utilizes antigens expressed by Mycobacterium bovis but not by BCG) capable of discriminating infected from vaccinated animals (DIVA interferon gamma test [DIT]) has been evaluated in naturally infected TB field reactors, there is a need to perform similar analysis in a BCG-vaccinated M. bovis-infected population. Furthermore, we explored different scenarios under which a DIT may be implemented alongside BCG vaccination: (i) serial testing to resolve potential false-positive skin test results or (ii) a standalone test to replace the single intradermal comparative cervical tuberculin (SICCT) skin test. Our results demonstrated significantly better relative test sensitivity when the DIT was evaluated in a serial test scenario. Direct comparison of pre-and post-skin test blood samples revealed that the SICCT test induced significant boosting of the gamma interferon response in M. bovis-infected animals to both the ESAT-6 -CFP-10 and Rv3615c peptide cocktails that comprise the DIT, which persisted for the ESAT-6 -CFP-10 reagent for at least 14 days. Importantly, no similar boosting effects were observed in noninfected BCG vaccinates, suggesting that DIVA blood testing after a recent skin test would have minimal impact on test specificity.KEYWORDS blood test, bovine tuberculosis, DIVA, gamma interferon, Mycobacterium bovis, tuberculin skin test B ovine tuberculosis (TB) in cattle, caused predominantly by infection with Mycobacterium bovis, poses a significant economic animal health problem, where it has been estimated that globally the disease costs $3 billion U.S. annually (1). In Great Britain, bovine TB control measures are based on a test and cull program using the single intradermal comparative cervical tuberculin (SICCT) skin test as a primary diagnostic test, but also under certain circumstances the whole-blood interferon gamma (IFN-␥) release assay as an ancillary test to maximize detection of M. bovis-infected cattle. Despite this, the annual number of herd breakdowns remains high in the West of England and Wales, and thus new control strategies, including cattle vaccination, are under consideration. Studies with cattle have shown that M. bovis BCG vaccination significantly protected cattle following experimental infection with M. bovis, with some animals displaying no evidence of disease pathology in either the lungs or associated lymph nodes (2-4). However, these studies also highlight that BCG is not fully protective in all vaccinated animals, and as such, should BCG vaccination be implemented as

Bacillus Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis (M. bovis), is the lead candidate vaccine for control of bovine tuberculosis (TB) in cattle. However, BCG vaccination sensitises cattle to bovine tuberculin, thus compromising the use of the current bovine TB surveillance tests. To address this, we have developed a diagnostic skin test that is not compromised by BCG vaccination and is able to detect BCG vaccinated animals that subsequently develop bovine TB following exposure to M. bovis. Building on previous work using ‘in house’ formulated protein cocktail reagents, we herein present test performance data for a single fusion protein (DST-F) containing the mycobacterial antigens ESAT-6, CFP-10 and Rv3615c formulated as a ‘ready to use’ reagent by a commercial manufacturer. Our results demonstrate that, unlike tuberculin reagents, a diagnostic skin test using DST-F maintained high specificity in BCG vaccinated animals. Furthermore, the DST-F skin test demonstrated a high relative sensitivity in identifying M. bovis infected animals, including those where BCG vaccination failed to prevent bovine TB pathology following experimental exposure to M. bovis. The DST-F is currently undergoing field trials in Great Britain to support its licensure and commercialisation.