Pathogenic Aeromonas veronii with the cheZ-mshK-aerA triple-gene mutant is attenuated and exhibits a potential candidate as a live attenuated vaccine.

Aeromonas veronii is a zoonotic pathogen that is commonly found in various aquatic environments and causes serious damage to the aquaculture industry. Anti-virulence strategies based on mutating the virulence factors are important antibiotic alternative methods against A. veronii infection. The type Ⅳ pili, polar flagella, and aerolysin are considered to be the major virulence factors. In this study, we constructed a triple-gene deletion mutant strain (cheZ/mshK/aerA, ΔCMA-AV) and evaluated its potential as a live attenuated vaccine (LAV) candidate. Pathogenicity test showed that the LD of ΔCMA-AV in crucian carp (Carassius auratus) was 5.34 times higher than that of WT-AV. Further research found that the decline of pathogenicity was associated with the decrease of hemolysis, biofilm formation ability and expression levels of virulence-related genes (type IV pili and flagella). Our preliminary results on ΔCMA-AV as a LAV showed that crucian carp immunized with ΔCMA-AV at a concentration of 3 × 10 CFU/mL did not show any evident pathological alterations or clinical symptoms. Non-specific immune indicators, including serum immune-related enzyme activities and the expression of immune-related genes in immune organs, showed an up-regulation trend in the ΔCMA-AV group. A. veronii specific antibody levels increased significantly from 2 - 4 weeks. The relative percent survival in ΔCMA-AV groups was 73.97% ± 3.57 %, significantly higher than that of the inactivated A. veronii groups (53.73% ± 10.87 %). These results indicate that the three genes (cheZ/mshK/aerA) play an important role in the pathogenicity of A. veronii, in additionΔCMA-AV could induce an effective immune response and provide strong protection against A. veronii in crucian carp without adverse effects.