Evaluation of an ESAT-6 Recombinant Skin Test Reagent for Bovine Tuberculosis Diagnosis in Guinea Pigs and Cattle

Bovine purified protein derivative (PPD-B), a crude protein extract from <i>Mycobacterium bovis</i> cultures, has been the standard reagent for delayed-type hypersensitivity (DTH) testing in cattle, but its undefined composition and variability compromise reproducibility and specificity. To address these limitations, we developed and evaluated RRbTB-E, a recombinant fusion protein comprising ESAT-6, as a defined alternative for skin testing. RRbTB-E was produced in <i>Escherichia coli</i>, purified by affinity chromatography, and characterized by SDS-PAGE and Western blot. In <i>M. bovis</i>-sensitized guinea pigs, RRbTB-E induced robust DTH reactions comparable to PPD-B, with consistent performance across six independent experiments and long-term stability after storage at 4-8 °C for more than 900 days. Furthermore, RRbTB-E did not induce significant reactions in non-sensitized or <i>Mycobacterium avium</i>-sensitized animals, confirming antigenic specificity. RRbTB-E also elicited DTH responses in <i>Mycobacterium tuberculosis</i>-sensitized guinea pigs. In naturally infected cattle, it triggered responses similar in magnitude to PPD-B, while remaining negative in non-infected animals. These findings support RRbTB-E as a stable, reproducible, and specific candidate for standardized intradermal testing in bovine tuberculosis diagnosis.