Background: Caseous lymphadenitis (CLA) is a significant infectious disease in sheep and goats, traditionally associated with Corynebacterium pseudotuberculosis. However, recent evidence highlights the polymicrobial nature of CLA, with Trueperella pyogenes and Staphylococcus aureus frequently co-isolated from lesions. Current vaccines mainly target single pathogens, and combined inactivated vaccines against all three bacteria are lacking. Methods: We developed a trivalent inactivated vaccine using strains CpS1, TpS1, and SaS9, inactivated with 0.2% formalin and formulated with either PBS, Freund’s adjuvant, or aluminum hydroxide adjuvant. Vaccine quality, safety, and immunogenicity were evaluated in experimental goats. The aluminum hydroxide-adjuvanted vaccine was further assessed for protective efficacy via challenge with individual pathogens and in field trials on goat and sheep farms. Results: All formulations induced specific antibodies against the three pathogens, with Freund’s adjuvant eliciting the highest level, while aluminum hydroxide provided a favorable safety profile and induced antibody levels that were numerically lower than but not statistically distinguishable from the Freund’s adjuvant group at most time points; however, the small group size (n = 6) limited the statistical power to detect differences between adjuvants. The aluminum hydroxide-adjuvanted vaccine conferred protection rates of 66.7% against C. pseudotuberculosis, 83.3% against T. pyogenes, and 83.3% against S. aureus in challenge trials. In field studies, vaccination was associated with a lower observed incidence of CLA, from 12.5% in unvaccinated controls to 2.5% in vaccinated goats and from 8% to 2% in vaccinated sheep within 90 days post-immunization. Conclusions: The trivalent inactivated vaccine with aluminum hydroxide adjuvant showed promising immunogenicity and preliminary protective efficacy against experimental challenge and was associated with a lower observed overall abscess incidence under the investigated field conditions.

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.