New skin test for detection of bovine tuberculosis based on antigen-displaying polyester inclusions produced by recombinant Escherichia coli.

Applied and environmental microbiology

PubMedID: 24532066

Chen S, Parlane NA, Lee J, Wedlock DN, Buddle BM, Rehm BH. New skin test for detection of bovine tuberculosis based on antigen-displaying polyester inclusions produced by recombinant Escherichia coli. Appl Environ Microbiol. 2014;.
The tuberculin skin test for diagnosing tuberculosis (TB) in cattle lacks specificity if animals are sensitized to environmental mycobacteria as some antigens in purified protein derivative (PPD) prepared from Mycobacterium bovis are present in non-pathogenic mycobacteria. Three immunodominant TB antigens, ESAT6, CFP10 and Rv3615c, are present in members of the pathogenic Mycobacterium tuberculosis complex, but absent from the majority of environmental mycobacteria. These TB antigens have the potential to enhance skin test specificity. To increase their immunogenicity these antigens were displayed on polyester beads by translationally fusing them to a polyhydroxyalkanoate (PHA) synthase which mediated formation of antigen displaying inclusions in recombinant Escherichia coli. The most common form of these inclusions is poly(3-hydroxybutyric acid) (PHB). The respective fusion proteins displayed on these PHB inclusions (beads) were identified using tryptic peptide fingerprinting analysis in combination with MALDI-TOF/MS. The surface exposure and accessibility of antigens was assessed by ELISA. Polyester beads displaying all three TB antigens showed greater reactivity with TB antigen-specific antibody than beads displaying only one TB antigen. This was neither due to cross-reactivity of antibodies with the other two antigens, nor due to differences in protein expression levels between beads displaying single or three TB antigens. The triple antigen displaying polyester beads were used for skin testing of cattle and detected all cattle experimentally-infected with M. bovis with no false positive reactions observed in those sensitised to environmental mycobacteria. The results suggested applicability of TB antigen-displaying polyester inclusions as diagnostic reagents for distinguishing TB-infected from non-infected animals.