Identification of conserved vaccine targets in Mycoplasma bovis through integrated pan-genome and reverse vaccinology approaches with in vivo immunogenicity and safety evaluation.

Mycoplasma bovis is a major etiological agent of bovine respiratory disease, causing substantial economic losses to the cattle industry. To address this challenge, we performed a pan-genome analysis of 80 globally sourced M. bovis genomes and identified 1058 core genes. Reverse vaccinology and immunoinformatics approaches were subsequently integrated to screen three potential vaccine candidates: namely lipoate protein ligase 2 (LplA), elongation factor 4 (LepA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Codon-optimized gene sequences were cloned into the pET-28a vector and successfully expressed and purified using E. coli expression system. Thirty female BALB/c mice were randomized into five groups (n = 6 per group). For primary immunization, each mouse received 100 μg of recombinant protein emulsified in Freund's complete adjuvant via subcutaneous injection, followed by booster immunizations with 50 μg of protein formulated with Freund's incomplete adjuvant at weeks 2 and 4. Fourteen days after primary immunization, all vaccinated groups exhibited significantly elevated antigen-specific antibody levels, which remained high through week 6. Notably, rLplA and rGAPDH induced higher antibody titers than rLepA. Analysis of the IgG2a/IgG1 subtype ratio indicated that rLplA, rGAPDH, and rLepA predominantly elicited Th2-type immune responses. In addition, all three recombinant proteins significantly increased cytokine production. Moreover, no adverse physiological reactions or histopathological abnormalities were observed throughout the immunization period, confirming the favorable safety profile of the candidates. Collectively, these findings identify promising targets for the development of M. bovis subunit vaccines to address pathogen variability, antibiotic resistance, and the limited efficacy of current vaccines.