African Swine Fever (ASF) is a highly lethal viral disease in swine. The emergence and rapid spread of African Swine Fever virus (ASFV) in China, since 2018 have caused significant economic losses to the pig farming industry. The complexity of ASFV has impeded the development of effective vaccines, and with no commercial vaccines currently available in China, highlighting the urgent need for safe and efficacious vaccine candidates. In this study, we utilized a highly immunogenic quadruple‐gene‐deleted recombinant pseudorabies virus (PRV) strain (rPRV SX‐10ΔUL24/TK/gI/gE) as a vector to construct two recombinant viral strains expressing ASFV p54, p72, CD2v, and pp62 proteins using the HDR‐CRISPR/Cas9 system. These strains, rPRV‐p54+p72 and rPRV‐CD2v+pp62, demonstrated stable genetic characteristics and efficiently expressed and delivered heterologous proteins while maintaining biological properties similar to their parental strain. Safety evaluation revealed that both recombinant strains exhibited favorable safety profiles in immunized mice and piglets. Furthermore, the strains induced robust humoral and cellular immune responses, as evidenced by specific antibody enzyme‐linked immunosorbent assay (ELISA), lymphocyte proliferation assays, and analysis of CD3+, CD4+, and CD8+ T lymphocytes. These findings suggest that rPRV‐p54+p72 and rPRV‐CD2v+pp62 are promising bivalent vaccine candidates for protecting against both PRV and ASFV infections.

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.

Newcastle disease (ND), a highly contagious poultry disease caused by NDV, primarily triggers gastrointestinal lesions. Microecological preparations, novel biological additives for restoring intestinal microbiota diversity, improve nutrient absorption, reinforce intestinal barrier function, and modulate host immune responses. This study investigated the effects of a compound microecological preparation on intestinal pathogenicity in chickens infected with genotype VII Newcastle disease virus (NDV). SPF chickens were allocated to four dietary groups with or without a compound microecological preparation, followed by NDV challenge in two groups. Survival, intestinal morphology, and transcriptomic responses were assessed. The results showed that chickens fed with the compound microecological preparation exhibited improved intestinal development. Following NDV infection, these chickens displayed milder cecal lesions without obvious hemorrhage and a higher survival rate. Furthermore, differential gene transcription analysis revealed that supplementation with the compound microecological preparation regulated the expression of genes associated with metabolic processes, biological regulation, immune response, and growth and development pathways, consistent with the clinical findings. In conclusion, we demonstrated that the compound microecological preparation promotes intestinal development in chickens, delays disease progression following NDV infection, and alleviates pathological damage. Transcriptomic analysis further indicated that the preparation enhances intestinal mucosal immunity by stimulating IgA production and strengthening the immune response against genotype VII NDV. These findings provide a scientific basis for the application of compound microecological preparations in regulating the intestinal immune system and in the prevention of Newcastle disease.

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.