Development of diagnostic reagents to differentiate between Mycobacterium bovis BCG vaccination and M-bovis infection in cattle

In Great Britain a recent independent scientific review far the government has concluded that the development of a cattle vaccine against Mycobacteriu m bovis holds the best long-term prospect for tuberculosis control in Briti sh herds. A sine qua non for vaccination is the development of a complement ary diagnostic test to differentiate between vaccinated animals and those i nfected with M. bovis so that test-and-slaughter-based control strategies c an continue alongside vaccination. In order to assess the feasibility of de veloping a differential diagnostic test for a live vaccine, we chose M. bov is BCG Pasteur as a model system. Recombinant forms of antigens which are e xpressed in M. bovis but not, or only at low levels, in BCG Pasteur (ESAT-6 , MPB64, MPB70, and MPBS3) were produced. These reagents were tested either alone or in combination by using peripheral blood mononuclear cells from M . bovis-infefted, BCG-vaccinated, and Mycobacterium avium-sensitized calves . All four antigens induced in vitro proliferation and gamma interferon res ponses only in M. bovis-infected animals. A cocktail composed of ESAT-6, MP B64, and MPB83 identified infected animals but not those vaccinated with BC G. In addition, promiscuous T-cell epitopes of ESAT-6, MPB64, and MPB83 wer e formulated into a peptide cocktail. In T-cell assays with this peptide co cktail, infected animals were identified with frequencies similar to those obtained in assays with the protein cocktail, while BCG-vaccinated or M. av ium-sensitized animals did not respond. In summary, our results suggest tha t peptide and protein cocktails can be designed to discriminate between M. bovis infection and BCG vaccination.