Display of antigens on polyester inclusions lowers the antigen concentration required for a bovine tuberculosis skin test

The tuberculin skin test is the primary screening test for the diagnosis of bovine tuberculosis (TB) and use of this test has been very valuable in control of this disease in many countries. However, the test lacks specificity when cattle have been exposed to environmental mycobacteria or vaccinated with Mycobacterium bovis bacille Calmette-Guerin (BCG). Recent studies have shown that use of three or four recombinant mycobacterial proteins, including ESAT6, CFP10, Rv3615c and Rv3020c or a peptide cocktail derived from these proteins, in the skin test greatly enhanced test specificity with minimal loss in test sensitivity. These proteins are present in members of the pathogenic Mycobacterium tuberculosis complex, but absent or not expressed by the majority of environmental mycobacteria or by the BCG vaccine strain. To produce a low cost skin test reagent these proteins were displayed at high density on polyester beads by translational fusion to a polyhydroxyalkanoate (PHA) synthase which mediated formation of antigen displaying inclusions in recombinant Escherichia coli. Display of the proteins on the polyester beads greatly increased their immunogenicity, allowing for the use of very low concentrations of proteins in the skin test, between 0.1 to 3 mug of mycobacterial protein/inoculum. Polyester beads simultaneously displaying all four proteins were produced in a single fermentation process. The polyester beads displaying three or four mycobacterial proteins were shown to have a high sensitivity for detection of M. bovis-infected cattle and induced minimal response in animals exposed to environmental mycobacteria or vaccinated with BCG.