Decreased pathogenicity of triple-mutant of Aeromonas hydrophila flgK, flgL, flgE suggests its potential as a live attenuated vaccine for Carassius auratus.

Aeromonas hydrophila is one of the top pathogenic bacteria that causes serious economic losses in aquaculture. In this study, we constructed a live attenuated vaccine (LAV) candidate strain by mutating the flagella genes flgK, flgL, and flgE in A. hydrophila (AH-ΔKLE). This strain exhibited sufficient attenuation with a significant reduction of 35.91-fold in pathogenicity. Compared to wild type strain of A. hydrophila (AH-WT), AH-ΔKLE exhibited flagellar loss, reduced motility, diminished hemolytic activity, decreased biofilm formation, and down-regulation of pathogenicity-related genes. 144 h after immunization of Carassius auratus with AH-ΔKLE, the activities of ACP, AKP, LZM, SOD, CAT, GSH, MDA and T-AOC were significantly increased. Additionally, the expression levels of TNF-α, MHCI, IL-8, IL-10, TGF-β and IL-4 were significantly upregulated from 6 to 48 h post immunization in gill, spleen and head kidney tissues. Furthermore, the specific antibody IgM levels were elevated in the AH-ΔKLE group at 4 weeks post immunization. Fish vaccinated via intraperitoneal injection with the AH-ΔKLE strain exhibited a significantly higher relative percent survival (RPS) of 78.33 % than 45.33 % of the inactive vaccine. Compared with the control group, AH-ΔKLE showed effective protection without causing obvious pathological changes in the gill, intestine and spleen. To sum up, our results demonstrate that AH-ΔKLE represents a safe and efficacious LAV candidate strain for clinical prevention of A. hydrophila infection in Carassius auratus.