Use of bacterial whole-genome sequencing to investigate local persistence and spread in bovine tuberculosis.

Mycobacterium bovis is the causal agent of bovine tuberculosis, one of the most important diseases currently facing the UK cattle industry. Here, we use high-density whole genome sequencing (WGS) in a defined sub-population of M. bovis in 145 cattle across 66 herd breakdowns to gain insights into local spread and persistence. We show that despite low divergence among isolates, WGS can in principle expose contributions of under-sampled host populations to M. bovis transmission. However, we demonstrate that in our data such a signal is due to molecular type switching, which had been previously undocumented for M. bovis. Isolates from farms with a known history of direct cattle movement between them did not show a statistical signal of higher genetic similarity. Despite an overall signal of genetic isolation by distance, genetic distances also showed no apparent relationship with spatial distance among affected farms over distances <5 km. Using simulations, we find that even over the brief evolutionary timescale covered by our data, Bayesian phylogeographic approaches are feasible. Applying such approaches showed that M. bovis dispersal in this system is heterogeneous but slow overall, averaging 2 km/year. These results confirm that widespread application of WGS to M. bovis will bring novel and important insights into the dynamics of M. bovis spread and persistence, but that the current questions most pertinent to control will be best addressed using approaches that more directly integrate WGS with additional epidemiological data.

Development of a skin test for bovine tuberculosis for differentiating infected from vaccinated animals.

The tuberculin skin test has been used for the diagnosis of bovine and human tuberculosis (TB) for over a hundred years. However, the specificity of the test is compromised by vaccination with the Mycobacterium bovis-derived vaccine strain bacille Calmette-Guérin (BCG). Since current promising vaccines against bovine TB are based on heterologous prime-boost combinations that include BCG, there is a need for diagnostic tests for differentiating infected from vaccinated animals (DIVA). The application of antigens such as ESAT-6 and CFP-10 for DIVA has so far been realized largely through their application in the blood-based gamma interferon release assay. In the current study, we have reassessed the potential of such antigens as skin test reagents for DIVA in cattle. A cocktail of the Mycobacterium tuberculosis complex recombinant protein antigens ESAT-6, CFP-10, MPB70, and MPB83 elicited delayed-type hypersensitivity (DTH) skin test responses in 78% of naturally infected tuberculin-positive cattle. Importantly, this cocktail induced no skin responses in BCG-vaccinated cattle despite them being sensitized for strong tuberculin responses. Further optimization of skin test antigen combinations identified that the inclusion of Rv3615c (Mb3645c) enhanced skin test sensitivity in naturally infected cattle without compromising specificity. In addition, we demonstrate for the first time the utility of synthetic peptides as promising skin test antigens for bovine TB for DIVA. Our data provide a promising basis for the future development of skin tests for DIVA with practical relevance for TB diagnosis in both veterinary and clinical settings.