Vaccination with Bacillus Calmette–Guérin (BCG) could be an additional tool to control bovine tuberculosis in cattle. However, to continue with ‘test-and-cull’ control programmes, BCG-compatible tests to distinguish infected amongst vaccinated animals (DIVA) are required. We summarise herein our recent progress towards the development of a DIVA skin test.
Cattle vaccination could be added to existing control strategies, but the only cattle vaccine candidate available, BCG, does not protect all vaccinated animals and compromises the utility of tuberculin purified protein derivative (PPD) in diagnostic tests. To apply BCG alongside PPD-based test-and-cull approaches, e.g. based on the single intradermal comparative cervical tuberculin test (SICCT), requires replacing or supplementing PPD with BCG-compatible tests to detect infected animals within vaccinated populations.
The discovery that a number of gene regions were deleted from the BCG genome during its attenuation allowed a rational search for DIVA antigens based on antigens encoded by these ‘regions of difference’. Two such antigens, ESAT-6 and CFP-10, were shown to fulfil the DIVA criteria . However, although these two DIVA antigens were highly specific in cattle, their sensitivity was inferior to that of PPD. Subsequently, an ‘omics’-based antigen mining programme identified the antigen Rv3615c, which when used to complement ESAT-6 and CFP-10 provided significant additional sensitivity without reducing specificity . However, matching DIVA specificity in BCG-vaccinated animals to SICCT specificity in unvaccinated cattle using the blood test format resulted in loss of sensitivity. We correctly hypothesised that the required high specificity could be achieved by using a cocktail of the three antigens as skin test antigens . This cocktail displayed comparable sensitivity to the SICCT, while its specificity in BCG-vaccinated animals matched that of the SICCT in unvaccinated cattle .
Further product development led to the generation of a fusion protein composed of all three antigens  which exhibited performance equal to that of the protein cocktail, but with a better production and stability profile. A parallel development led to a peptide cocktail representing the same proteins. The next stage in the development of these potentially groundbreaking DIVA reagents is to validate them to OIE standards .