Mutagenesis of virulence genes in an epidemic strain of Aeromonas hydrophila ML09-119 for development of an attenuated vaccine strain

Abstract

The epidemic strain of Aeromonas hydrophila caused a devastating outbreak of motile Aeromonads septicemia of catfish (MAS) on the fish farms of Southeastern United States in 2009. A.hydrophila ML09-119 was reported to cause severe mortality in commercial catfish farms. Research has shown on the virulence of this epidemic strain on channel catfish (Ictalurus punctatus) including molecular identification of the specific strain and unique DNA sequences. However, no research has been done so far on the virulence of this specific strain to blue and hybrid catfish. In this study, intraperitoneal injection (IP) challenge method combining cohabitation culture was used to detect the susceptibility of channel, blue (Ictalurus furcatus) and hybrid catfish (blue catfish × channel catfish) to A. hydrophila ML09-119. Our results showed that the virulence of ML09-119 to channel catfish is significantly higher than it is to blue and hybrid catfish (p=0.0156 < 0.05, a = 0.05). No significant differences were observed between blue and hybrid catfish (p=0.0801>0.05, a=0.05). Significant differences in survival were observed for bacterial strains ML09-119, ZC1, and AL06-06 (P < 0.0001) and between fish species in the ML09-119 group (P = 0.0126). No interaction effect between variables of bacterial strains and fish species was observed (Pfish species × bacterial strains=0.1002). The difference in survival between channel catfish and grass carp injected with ML09-119 was significant (P = 0.0069). No significant differences in survival between channel catfish and grass carp intraperitoneally injected with ZC1 or AL06-06 were observed, but there was a significant difference between strains ZC1 and AL06-06 in both channel catfish (P = 0.0075) and grass carp (P = 0.0089). Previously our lab sequenced 11 A. hydrophila isolates, 6 of which are epidemic stains, while the others were historical A. hydrophila isolates not affiliated with an epidemic outbreak of disease is described as “reference” strains. A comparative genomic analysis indicated that 53 epidemic-associated genetic regions with 313 predicted genes were uniquely present in the epidemic isolates but absent from the reference isolates. Thirty four genes from this region were predicted to be related to the virulence of the epidemic strains. A functional metabolic island that encodes a complete pathway for myo-inositol catabolism was identified and demonstrated to be functional based on the ability of epidemic A. hydrophila isolate ML09-119 to use myo-inositol as a sole carbon source while the reference strain AL06-06 cannot. A novel O-antigen cluster was found in all the epidemic isolates and one reference isolates. In this study, the gene iolA coding for the enzyme aldehyde dehydrogenase for myo-inositol catabolism was inactivated by traditional allelic exchange to generate the A. hydrophila ΔiolAtra mutant. The iolA-iolR genetic region was also mutagenized using a recombineering technique generating ΔiolArec mutants. An in vivo challenge in channel catfish showed that there was no mortality in the channel catfish that were challenged with ΔiolAtra mutant, but there was mortality in the channel catfish challenged with ΔiolArec mutants similar to wild type ML09-119. Eight mutants were created by knocking out the upstream of the iolA gene in the iolA-iolR promoter region. Results of the in vivo challenge in channel catfish showed that ΔiolArec3, ΔiolArec4 exhibited some decrease in mortality, but there were no significant difference in the mortality between the channel catfish challenged with ΔiolArec3, ΔiolArec4 and the channel catfish challenged with the wild type ML09-119. ELISA antibody titers of the survivors of the ΔiolAtra after 21 days showed that ΔiolAtra can induce strong antibody response against the wild type A. hydrophila ML09-119, indicating that this mutant can serve as a promising vaccine candidate against the epidemic A. hydrophila. Lipid A-Core ligase (waaL) and O-antigen polymerase (wzy) mutants were created by both traditional splicing PCR and conjugation technique and recombineering technique respectively, ΔwaaLtra or ΔwaaLRec, Δwzytra or ΔwzyRec. An in vivo channel catfish challenge study was committed on channel catfish to study the role of O-antigen in the virulence of the epidemic strain of A. hydrophila. The results shows that the channel catfish that were challenged with ΔwaaLtra, Δwzytra had 100% survival rate, but 0% survival rate was observed in the channel catfish that were challenged with ΔwaaLRec, ΔwzyRec. A ΔymcA mutant was created by knocking out the ymcA gene by recombineering technique to study the role of ymcA gene of the O-antigen in the virulence of A. hydrophila. 68.13 ± 16.75% survival rate was observed in the channel catfish that were challenged with ΔymcA mutant. Sub-challenge of the survivors of ΔymcA treatment group 21 days post first challenge showed that 90.48 ± 8.25% survival rate was observed. Significant difference was observed between the ΔymcA treatment group and the positive control group which were naive channel catfish challenged with wild type. ELISA antibody titers of the survivors of the ΔymcA treatment group 21 days post first challenge showed that ΔymcA induced strong antibody response against the wild type A. hydrophila ML09-119 indicating that ΔymcA mutant can serve as a promising vaccine candidate.