Poultry serve as natural reservoirs for Salmonella spp., and the consumption of Salmonella-contaminated poultry products remains a leading cause of infection in both developed and developing countries. The irrational use of antimicrobials in the poultry industry has contributed to the global emergence of multidrug-resistant (MDR) Salmonella strains. This study aimed to evaluate whether Salmonella enterica serovar Typhimurium (S. typhimurium) outer membrane vesicles (OMVs) could serve as a broad-spectrum immunogen, providing protection against the most prevalent MDR Salmonella serovars. The immunogenicity and protective efficacy of S. typhimurium OMVs were assessed in both specific-pathogen-free (SPF) mice and chickens challenged with S. typhimurium infection. In addition, the cross-protective efficacy of OMVs against infections with heterologous serovars, specifically S. montevideo and S. albany, was also evaluated in chickens. Our results demonstrated that S. typhimurium OMVs elicited a robust humoral immune response and conferred significant immune protection in both mice and chickens. Following challenges with MDR S. montevideo and S. albany strains, immunized birds exhibited reduced levels of fecal shedding and liver invasion. Furthermore, in vitro studies revealed that S. typhimurium OMVs also possess cross-immunogenicity against MDR S. Enteritidis and S. gallinarum. In conclusion, S. typhimurium OMVs provide cross-protection against heterologous MDR Salmonella infections both in vitro and in vivo. Our study suggests that OMVs derived from S. typhimurium strains hold promise as novel subunit vaccine candidates for preventing MDR heterologous serovar infections in animals, offering a potential solution to the growing challenge of antimicrobial resistance in the poultry industry.

The endemicity of H9N2 avian influenza viruses (AIVs), particularly the Y280 lineage, poses persistent challenges to the poultry industry due to the limitations of inactivated vaccines, such as interference by maternally derived antibodies (MDAs) and incomplete suppression of viral replication. This study evaluated the immunogenicity and protective efficacy of a novel recombinant turkey herpesvirus vaccine expressing the hemagglutinin gene of H9N2/Y280 (rHVT-H9/Y280) in commercial Hy-Line Brown layers with high-MDA backgrounds. In a comparative challenge study, the rHVT-H9/Y280 vaccine induced complete protection against a homologous Y280 strain challenge at 4 weeks of age, whereas commercial inactivated vaccines failed to completely block replication, showing virus isolation rates of 16.7–25%. Long-term serological monitoring demonstrated that the rHVT-H9/Y280 vaccine elicited a robust humoral response characterized by persistent maintenance of high HI titers (>8.0 log₂) up to 39 weeks post-vaccination. These findings confirm that rHVT-H9/Y280 effectively overcomes MDA interference and provides protection by inhibition of viral replication in layer chickens, making it a promising candidate for the effective control of H9N2 AIV in endemic regions.

The widespread panzootic of clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 necessitates the development of vaccine platforms capable of rapid adaptation to emerging antigenic variants. Although commercial recombinant turkey herpesvirus (rHVT) vaccines are available, they often utilize heterologous inserts that may fail to optimally limit viral shedding of novel field strains. Here, we report the rapid construction of a homologous rHVT-H5 vaccine expressing the hemagglutinin (HA) gene of a representative clade 2.3.4.4b isolate via CRISPR/Cas9-mediated non-homologous end joining (NHEJ). In vitro characterization confirmed stable HA surface expression and growth kinetics comparable to the parental virus. In specific-pathogen-free (SPF) chickens, rHVT-H5 elicited robust hemagglutination inhibition (HI) antibody titers. Following lethal challenge with a homologous clade 2.3.4.4b H5N1 virus, the vaccine conferred 100% protection against mortality and clinical signs while significantly reduced oropharyngeal sheddings and completely inhibited viral shedding in cloacal samples. These findings demonstrate that an antigenically matched rHVT-H5 constitutes a promising strategy for mitigating the ongoing global threat posed by clade 2.3.4.4b HPAI H5N1.