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

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 OMVs + 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.