A fluorescence polarization assay using recombinant protein ESAT-6 for the detection of antibodies against pathogenic Mycobacterium bovis in bovine

Bovine tuberculosis (bTB) is a major bacterial disease that causes significant economic disruption across the globe.

Our study was based on using a fluorescence polarization assay (FPA) that used fluorescein-labeled ESAT-6 protein to detect Mycobacterium bovis antibodies in bovine serum.

The ESAT-6 protein was used in a FPA. Positive TB reactors were determined by the comparative intradermal test (CID) and interferon gamma test (IFN-γ). Antibodies against M. bovis were detected using a fluorescein isothiocyanate (FITC) labeled tracer and a whole culture FITC labeled tracer in the positive cattle.

Of the 192 animals tested for bTB, 37 were found to be positive by either the CID or IFN-γ assays. Using the mP values from five culture-positive serum samples, a cutoff value of more than >127 mp provided the best discrimination between positive reactors and negative bTB animals. The ESAT-6 results of FPA in comparison with CID results revealed sensitivity of 92.9% and specificity of 64.6%, and in comparison with results IFN-γ, showed sensitivity of 95.7% and specificity of 49%. FPA using FITC labelled ESAT-6 as a tracer has better sensitivity (95.7%) and specificity (49.1%) than IFN-γ test in humoral immune response in animals.

This work revealed that the ESAT-6 protein as an antigen can be used in diagnosing bTB using a practical and sensitive humoral test.