ΔFleQ of Aeromonas hydrophila generated as a live attenuated vaccine in common carp (Cyprinus carpio)

Aeromonas hydrophila is a pathogen responsible for motile Aeromonas septicemia (MAS) in fish and an opportunistic pathogen that poses a significant threat to aquaculture and human health. Live attenuated vaccines are considered to be an effective and environmentally friendly method for the prevention and control of bacterial infectious diseases in aquaculture. The pathogenic mechanisms of bacterial flagella include assisting bacterial movement and attachment to the host's intestinal tract, as well as promoting biofilm formation. FleQ is a key gene that regulates flagellar assembly in A. hydrophila. In this study, we constructed the A. hydrophila ΔFleQ mutant and investigated its phenotypic characteristics, degree of attenuation, and immunogenicity in common carp through immersion immunization. The results indicated that the ΔFleQ mutant exhibited significantly diminished motility, hemolytic activity, biofilm formation, antibiotic resistance, capacity for intestinal colonization, and attenuated virulence compared to the wild-type (WT). RNA-seq analysis revealed that 3281 genes were differentially expressed in ΔFleQ mutant, including 356 upregulated and 302 downregulated genes. KEGG enrichment analysis and qRT-PCR validation indicated that genes associated with flagellar assembly, the two-component system, tyrosine metabolism, bacterial chemotaxis pathways were downregulated. The LD₅₀ value of ΔFleQ (8.91 × 10⁸ CFU/mL) was 28.2 times higher than that of the WT (3.16 × 10⁷ CFU/mL). Fish immunized by immersion with ΔFleQ showed increased lgM responses and upregulated cytokine gene such as TNF-α, IL-1β, IL-6. Additionally, the relative percent survival (RPS) of ΔFleQ was 78.8 % in common carp following two immersion immunizations. These findings suggest that ΔFleQ could serve as a potential vaccine candidate for the prevention of diseases caused by A. hydrophila in common carp.