Expression and field evaluation of new Mycobacterium bovis antigens

Bovine tuberculosis (bTB) represents a threat to livestock production. Mycobacterium bovis is the main causative agent of bTB and a pathogen capable of infecting wildlife and humans. Eradication programs based on surveillance in slaughterhouses with mandatory testing and culling of reactive cattle have failed to eradicate bTB in many regions worldwide. Therefore, developing effective tools to control this disease is crucial. Using a computational tool, we identified proteins in the M. bovis proteome that carry predictive binding peptides to BoLADRB3.2 and selected Mb0309, Mb1090, Mb1810 and Mb3810 from all the identified proteins. The expression of these proteins in a baculovirus-insect cell expression system was successful only for Mb0309 and Mb3810. In parallel, we expressed the ESAT-6 family proteins EsxG and EsxH in this system. Among the recombinant proteins, Mb0309 and EsxG exhibited moderate performance in distinguishing between cattle that test positive and negative to bTB using the official test, the intradermal tuberculin test (IDT), when used to stimulate interferon-gamma production in blood samples from cattle. However, when combined as a protein cocktail, Mb0309 and EsxG were reactive in 50 % of positive cattle. Further assessments in cattle that evade the IDT (false negative) and cattle infected with Mycobacterium avium paratuberculosis are necessary to determine the potential utility of this cocktail as an additional tool to assist the accurate diagnosis of bTB.