The quadruple gene mutant (aerA-ahh1-rtxA-th) of Aeromonas dhakensis shows reduced virulence and promising vaccine development potential

Aeromonas dhakensis is a zoonotic pathogen responsible for severe infections in both humans and aquatic organisms. The increasing incidence of antibiotic resistance in A. dhakensis strains underscores the urgent need for alternative control strategies, such as vaccines. In this study, a quadruple gene deletion mutant of A. dhakensis Δ aart was generated by targeting the virulence genes: aer A, ahh 1, rtx A, and th . The Δ aart exhibited significantly reduced virulence in hemolytic activity and cytotoxicity, whereas no impairment in bacterial growth. Using zebrafish model, the LD 50 of Δ aart was 136-fold higher than that of the wild-type strain. Following immunization, the peak expression of innate immune-related genes ( SOD , CAT , LZM , TNF-α , IL-6 , IL-1β ) in the kidney was observed on day 3, followed by a decline; MHC-I peaked on day 7, and IL-10 on day 14, with similar trends noted in the intestine, while MHC-I did not show significant changes. Notably, IgM levels in the immunized zebrafish increased significantly at day 7 post-immunization, peaked at day 14, and remained at a high level at day 28. Furthermore, the Δ aart provided substantial protection against wild-type A. dhakensis infection, with a relative percent survival (RPS) of 56.67%, and less lesion was observed in intestines and kidney histopathology. These findings suggest that the Δ aart mutant holds promise as a safe and effective live-attenuated vaccine candidate for controlling A. dhakensis infections.