Outer membrane vesicles from Aeromonas veronii : biological properties and synergistic immunity enhancement with inactivated bacteria in crucian carp

ABSTRACT Aeromonas veronii ( A. veronii ) causes hemorrhagic septicemia and enteritis in aquatic animals, posing a threat to global aquaculture. Conventional vaccines are inadequate due to short immune duration, narrow antigenic spectrum, and insufficient commercialization. Outer membrane vesicles (OMVs), naturally secreted by Gram-negative bacteria, hold significant potential in vaccine development due to their enrichment with immunologically active components. This study aimed to characterize the biological properties of OMVs derived from the A. veronii TH0426 strain and evaluate their potential as a vaccine. Characterization revealed a diameter distribution of 10 to 300 nm. Proteomic analysis identified 76 proteins, including conserved antigens such as outer membrane channel-forming protein II (OmpII; 80% coverage) and outer membrane protein A (OmpA; 44% coverage). Evaluation in a crucian carp ( Carassius auratus ) model demonstrated that OMVs alone or combined with inactivated whole vaccine (Av) cells significantly enhanced serum antibody titers, serum bactericidal activity, and related immune enzymes ( P < 0.05). Furthermore, they upregulated the expression of immune factors (TGF-β, TNF-α, IL-10, IL-1β) in tissues such as the liver and spleen ( P < 0.05). In the challenge test, the relative percent survival of the OMV + Av group reached 66%. These results indicate that A. veronii OMVs possess application potential as vaccines, can effectively enhance the immune protection efficacy of aquatic animals, and provide novel insights for the development of next-generation vaccines against A. veronii . IMPORTANCE This study demonstrated the biological characteristics and application potential of OMVs derived from the A. veronii TH0426 strain as a vaccine. OMVs exhibited favorable safety profiles in crucian carp and significantly enhanced serum bactericidal activity. Furthermore, OMVs displayed potent immunogenicity, effectively elevating the levels of key serum immune enzymes, immune factors, and IgM, thereby significantly boosting both the innate and adaptive immune responses in crucian carp. Critically, OMVs effectively enhanced the immunoprotective efficacy conferred by inactivated A. veronii whole cells, significantly improving the resistance of crucian carp to A. veronii infection. Future studies will explore the broader application value of OMVs and further investigate the feasibility of utilizing OMVs from the A. veronii TH0426 strain as a potential vaccine.