Development and Evaluation of Artificially Attenuated Corynebacterium pseudotuberculosis, Trueperella pyogenes, and Staphylococcus aureus Strains as Live Vaccine Candidates Against Ovine Caseous Lymphadenitis

Caseous lymphadenitis is a chronic infectious disease causing substantial economic losses to the sheep and goat industry worldwide. The disease is associated with a polymicrobial etiology involving C. pseudotuberculosis, T. pyogenes, and S. aureus. While inactivated vaccines provide partial protection, live attenuated vaccines offer the advantage of inducing both humoral and cell-mediated immune responses. In this study, we aimed to develop live attenuated vaccine candidates for the three major caseous lymphadenitis-associated pathogens through serial subculturing. Primary strains CpS1-1 (C. pseudotuberculosis), TpS1-1 (T. pyogenes), and SaS9-1 (S. aureus) were subjected to continuous in vitro passage to generate attenuated derivatives CpS1-500 (500 passages), TpS1-260 (260 passages), and SaS9-220 (220 passages). Pathogenicity evaluation in mice and goats demonstrated that all three passaged strains exhibited significantly reduced virulence compared to their parental counterparts, with attenuated local abscess formation, pyrexia, growth retardation, and mortality. Immunization with the attenuated strains elicited robust humoral and cell-mediated immune responses, as evidenced by elevated specific antibody levels and increased IFN-γ, TNF-α, and IL-12 production. Challenge experiments revealed that CpS1-500 conferred 83.3% (5/6) protection against virulent C. pseudotuberculosis challenge, while TpS1-260 and SaS9-220 provided 100% (6/6) protection against their respective virulent strains. These findings demonstrate that serial subculturing is an effective approach for generating live attenuated vaccine candidates against caseous lymphadenitis-associated pathogens, with the passaged strains showing promise for further development as multivalent live vaccines for caseous lymphadenitis prevention.