Trueperella pyogenes is a significant opportunistic pathogen that causes substantial economic losses in animal agriculture due to its ability to infect various animal tissues and organs. Limited research has been conducted on the prevalence and biological characteristics of T. pyogenes isolated from sheep and goats. This study aimed to isolate T. pyogenes from clinical samples of sheep and goats in western China, examining genetic evolutionary relationships, antibiotic resistance, and virulence genes. Between 2021 and 2023, standard bacteriological methods were used to isolate and identify T. pyogenes from 316 samples (209 from goats and 107 from sheep) collected from 39 farms. Susceptibility to 14 antibiotics was tested using broth microdilution per CLSI guidelines, and PCR detected eight virulence genes. Whole-genome sequencing analyzed genetic relationships and gene carriage status in 39 isolates. The results indicated that 86 strains of T. pyogenes were isolated from 316 samples, yielding an isolation rate of 27.2% (goats n = 47, 22.5%; sheep n = 39, 36.4%). The virulence genes plo, cbpA, nanH, nanP, fimA, fimC, and fimE were present in 100%, 66.7%, 64.1%, 71.8%, 69.2%, 59.0%, and 82.1% of isolates, respectively, with none carrying the fimG gene. The dominant virulence genotype was plo/nanH/nanP/fimA/fimC/fimE. The isolates exhibited resistance to erythromycin (44.2%, 38/86), gentamicin (38.4%, 33/86), sulfamethoxazole/trimethoprim (37.2%, 32/86), tetracycline (32.6%, 28/86), and streptomycin (32.6%, 28/86), and low resistance to chloramphenicol (14.0%, 12/86), ciprofloxacin (7.0%, 6/86), penicillin (5.8%, 5/86), and clindamycin (4.7%, 4/86). All isolates were susceptible to cefotaxime, vancomycin, and linezolid. Among the 86 isolates, 37 (43.0%) displayed multidrug resistance (MDR) characteristics. The whole genome sequencing of 39 isolates identified eight types of resistance genes, including ant(2″)-Ia, ant(3″)-Ia, cmlA1, cmx, erm(X), lnu(A), sul1, and tet(W). Except for tet(W), erm(X), and sul1, the other resistance genes were reported for the first time in T. pyogenes isolated in China. The drug susceptibility test results and resistance gene detection for the isolated strains were consistent for tetracycline, erythromycin, gentamicin, and sulfisoxazole. Similar allelic profiles and genetic evolutionary relationships were found among isolates from different farms. This study highlights the antibiotic resistance status and virulence gene-carrying rate of Trueperella pyogenes, providing a basis for clinical medication.

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.

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.