Identification of pathogenicity, investigation of virulent gene distribution and development of a virulent strain-specific detection PCR method for Vibrio harveyi isolated from Hainan Province and Guangdong Province, China

A collection of 46 Vibrio harveyi strains were isolated from Epinephelus spp., Lutjanus erythopterus, and other maricultured fish in coastal areas of Hainan Province and Guangdong Province, China, between 2011 and 2013. Eighteen strains were determined to be pathogenic via artificial infection of healthy Epinephelus coioides at 107 colony-forming units (CFU) mL− 1. Mortality occurred within 2 to 6 h after injection. Genotypic assays of the 46 strains by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) revealed a similar genotype profile, referred to as the ERIC-1 profile, for all 18 pathogenic strains. This finding indicates that pathogenic V. harveyi strains in south China have similar genetic backgrounds and might be representative pathogenic strains of this region. All 46 strains were screened for the presence of virulence genes typical of V. harveyi, of zoonotic Vibrio species such as V. cholerae, V. parahaemolyticus, and V. vulnificus and of the aquatic pathogen V. anguillarum. Virulence genes were amplified by PCR using specific primers, and five typical virulence genes of the Harveyi clade, luxR, toxRvh, chiA, serine protease and vhh, were detected in all pathogenic isolates. Non-pathogenic strains carried only 1 to 4 of these genes, indicating that these five genes might be the main virulence genes of ERIC-1 strains. Strain-specific PCR primers were designed based on the sequences of distinct ERIC-PCR bands for the 18 pathogenic strains. Species-specific primers exhibited high specificity and sensitivity. This study demonstrates that bacteria that are highly important to mariculture could be specifically detected using ERIC-PCR fingerprint-based amplification.