Development of attenuated Vibrio parahaemolyticus mutant strains as potential live vaccines

Abstract

Vibrio parahaemolyticus is a pathogenic Gram-negative bacterium that commonly infects numerous species of marine fishes. In this study, we obtained eight attenuated mutant strains by the selection of rifampicin-resistant V. parahaemolyticus from three wild-type strains. Among eight selected strains, four strains (L4650, A650, L4200 and A400) were widely attenuated by virulence analysis. Sequences of two genes, tlh and toxR, that related to virulence were analyzed. The results showed that seven attenuated strains were different in sequences of the tlh gene while there was no difference in sequence of toxR gene in comparison with that of wild-type strains. Three attenuated strains (L4650, L4200 and A650) had the nucleotide changes in tlh gene that resulted to the changes in the size of deduced amino acid sequences. In addition, rpoB gene coding for -subunit of RNA polymerase that related to Rifampicin resistance was analyzed for all selected strains. Two strains (A650 and L4650) had the nucleotide changes in ropB sequence. To assess the potential use of the L4650 strain as a live attenuated vaccine, the groupers (Epinephelus coioides) were intraperitoneally injected with the L4650 strain and later challenged with the virulent wild-type strain at three different doses. Results showed the protection for L4650 strain vaccinated-fish produced relatively earlier than usual at 15 days post-vaccination with relative percent survival (RPS) values from 96.91-100%. At 60 days post-vaccination, the RPS values of vaccinated fish were 96.27-100%. Our results suggest the L4650 strain could become a potential vaccine candidate against V. parahaemolyticus for future studies.