The Establishment of a Novel γ-Interferon In Vitro Release Assay for the Differentiation of IMycobacterial Bovis/I-Infected and BCG-Vaccinated Cattle

Bovine tuberculosis (bTB) is a complex zoonotic disease that poses challenges in its management throughout numerous countries. The potential of Bacille Calmette-Guérin (BCG) vaccination as a preventative measure against bTB is explored by many researchers. However, traditional tuberculin skin test methods cannot differentiate infected from vaccinated animals (DIVA). In this study, we established a novel interferon-gamma in vitro release assay (IGRA) for the bTB DIVA test by expressing a recombinant fusion protein named RCE that contains three differential antigens (Rv3872, CFP-10, and ESAT-6) that are present in virulent M. bovis but lacking in BCG genomes. Using a commercial IGRA bTB diagnostic kit based on several RCE stimulators of peripheral blood mononuclear cells as the references for testing 97 cattle, the RCE optimal concentration and cut-off value were measured using the receiver operator curve (ROC). After that, we utilized RCE-IGRA on calves that had received a BCG vaccination to demonstrate that it could be an ideal DIVA method. Therefore, this study is significant for the application of BCG vaccination and the more effective prevention and control of bTB. BCG vaccination is increasingly reconsidered in the effective prevention of bovine tuberculosis (bTB). However, the primary challenge in BCG vaccination for cattle is the lack of a technique for differentiating between infected and vaccinated animals (DIVA). This study aimed to establish a novel DIVA diagnostic test based on an interferon-gamma in vitro release assay (IGRA). The plasmid encoding three differential antigens (Rv3872, CFP-10, and ESAT-6) absent in BCG genes but present in virulent M. bovis was previously constructed. Thus, a recombinant protein called RCE (Rv3872, CFP-10, and ESAT-6) was expressed, and an RCE-based DIVA IGRA (RCE-IGRA) was established. The RCE concentration was optimized at 4 μg/mL by evaluating 97 cattle (74 of which were bTB-positive, and 23 were negative) using a commercial IGRA bTB diagnostic kit. Further, 84 cattle were tested in parallel with the RCE-IGRA and commercial PPD-based IGRA (PPD-IGRA), and the results showed a high correlation with a kappa value of 0.83. The study included BCG-vaccinated calves (n = 6), bTB-positive cattle (n = 6), and bTB-negative non-vaccinated calves (n = 6). After 3 months post-vaccination, PPD-IGRA generated positive results in both vaccinated and infected calves. However, RCE-IGRA developed positive results in infected cal