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

Abstract

In Great Britain a recent independent scientific review for the government has concluded that the development of a cattle vaccine against Mycobacterium bovis holds the best long-term prospect for tuberculosis control in British herds. A sine qua non for vaccination is the development of a complementary diagnostic test to differentiate between vaccinated animals and those infected with M. bovis so that test-and-slaughter-based control strategies can continue alongside vaccination. In order to assess the feasibility of developing a differential diagnostic test for a live vaccine, we chose M. bovis BCG Pasteur as a model system. Recombinant forms of antigens which are expressed in M. bovis but not, or only at low levels, in BCG Pasteur (ESAT-6, MPB64, MPB70, and MPB83) were produced. These reagents were tested either alone or in combination by using peripheral blood mononuclear cells from M. bovis-infected, BCG-vaccinated, and Mycobacterium avium-sensitized calves. All four antigens induced in vitro proliferation and gamma interferon responses only in M. bovis-infected animals. A cocktail composed of ESAT-6, MPB64, and MPB83 identified infected animals but not those vaccinated with BCG. In addition, promiscuous T-cell epitopes of ESAT-6, MPB64, and MPB83 were formulated into a peptide cocktail. In T-cell assays with this peptide cocktail, infected animals were identified with frequencies similar to those obtained in assays with the protein cocktail, while BCG-vaccinated or M. avium-sensitized animals did not respond. In summary, our results suggest that peptide and protein cocktails can be designed to discriminate between M. bovis infection and BCG vaccination.

FIG. 1

Recognition of mycobacterial proteins. PBMC isolated from one SICTT-positive field reactor (M. bovis), three BCG-vaccinated cattle (BCG), and one animal with PPD-A-biased responses (PPD-A biased) were incubated with recombinant mycobacterial proteins and avian and mammalian tuberculin at 10 μg/ml for 6 days. Results are expressed as means ± standard errors.

FIG. 2

Proliferative responses induced by protein (A) and peptide (B) cocktails. PBMC isolated from a SICTT-positive reactor were incubated with recombinant ESAT-6, MPB64, or MPB83 individually (10 μg/ml) or a pool of all three (5 μg/ml each). PBMC from the same reactor animal were incubated with peptides p1 to p4 individually (10 μg/ml) or a pool of all four peptides (10 μg/ml each). Results are expressed as means ± standard errors.

FIG. 3

Proliferative responses induced by protein and peptide cocktails. PBMC isolated from 3 SICTT-positive field reactor cattle (SICTT+), 2 cattle experimentally infected with M. bovis (M. bovis exp.), 7 BCG-vaccinated cattle (BCG), and 13 cattle with either PPD-A-biased responses or negative SICTT responses (PPD-A/uninfected) were incubated with either the protein cocktail (5 μg/ml each) or the peptide cocktail described in Table 1 (10 μg/ml each). Results are expressed as mean proliferative responses ± standard errors.

FIG. 4

IL-2 production as a surrogate marker for proliferation. Whole-blood cultures in the presence of either the protein or the peptide cocktail were performed with samples obtained from 9 SICTT-positive field reactors and from 10 negative control animals from farms with no history of TB. Supernatants were collected after 24 h, and IL-2 production was determined. Filled circles, samples from cattle recognizing the cocktails in LTAs; filled squares, samples from cattle not responding to cocktails in LTAs.