Integrated Bioinformatics Analysis for Target Identification and Evaluation of Recombinant Protein as an Antigen for Intradermal Skin Test in Bovine Tuberculosis Diagnosis

Bovine tuberculosis (bTB) is a respiratory disease caused by <i>Mycobacterium bovis</i>, posing a significant threat to animal health and the livestock industry. Current control strategies for bTB rely on diagnostic tests and slaughter policies. However, the limitations of existing diagnostic methods, which depend on PPD antigens, necessitate the exploration of alternative antigens to enhance the accuracy and reliability of bTB diagnosis. This study aimed to identify, produce, and evaluate novel antigens for use in the intradermal skin test for bTB diagnosis. A pangenome analysis of four <i>Mycobacterium</i> species identified 12 unique genes specific to <i>M. bovis</i> SP38. Further integrated bioinformatic analysis revealed 224 genomic islands associated with virulence and pathogenesis. Among these, a highly antigenic protein, termed HP28, was selected for in vivo testing. The recombinant HP28 protein (rHP28) was expressed in <i>E. coli</i> and assessed for its ability to induce intradermal skin reactions in guinea pigs. The rHP28 protein elicited a skin reaction of 6.6 mm at 72 h post-injection, whereas negative controls showed no reaction. This study presents a pipeline for the selection of antigens using integrated bioinformatic analysis to identify diagnostic targets that can effectively distinguish between sensitized and non-sensitized animals, offering a promising approach for improving bTB diagnostics.