Characterization of human-and shrimp-pathogenic vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus is a halophilic bacterium distributed in marine and estuarine environments worldwide. V. parahaemolyticus may causes gastroenteritis after consumption of contaminated seafood. The pathogenicity of this bacterium in humans is associated to the tdh and trh genes, which are considered to be the major virulence genes in the pathogenesis. The trh gene possesses a significantly broader nucleotide sequence variation and can be subdivided in two main subtypes (trhl and trh2) which share 84% identical in sequence. In order to characterize V. parahaemolyticus carrying the trh gene, 73 clinical isolates of trh V. parahaemolyticus obtained previously from various countries were investigated. No significant difference was observed in the urease production between tdh trhl* and tdh trh2 (p = 0.063) and between the tdh trhl and tdh trh2 isolates (p = 0.788). This implied that the tdh and trh genes of V. parahaemolyticus were not involved in the urease production. The isolates carrying only the trh gene showed variation in their haemolytic activity that might correlate to the sequence variation in trh1 and trh2 genes. The ratio of urease production and haemolytic activities between the trhl and trh2+ isolates and biofilm formation of trh V. parahaemolyticus isolates were not significantly different. Out of 16 of 34 isolates (47.0%) of trh V. parahaemolyticus gave positive for CRISPR detection. CRISPR-virulence typing of all CRISPR-positive isolates was constructed and compared to profiles obtained by CRISPR typing alone. The discriminatory power index (DI) of CRISPR-virulence typing was higher than the CRISPR typing alone. V. parahaemolyticus causes acute hepatopancreatic necrosis disease (AHPND) in shrimp. To clarify the pathogenicity of AHPND V. parahaemolyticus in shrimp, all 33 AHPND V. parahaemolyticus isolates obtained in this study were characterized. All isolates were O1 serotype but possessed different K antigens. DNA profiles of AHPND V. parahaemolyticus isolates were similar, but were distinct from those obtained from clinical and environmental isolates. This indicates the causative agent of AHPND might be originated from one clone and then subsequently different K antigens have been developed. In this study, two sets of primers (LAMP-A2 and LAMP-A3) specific to the unique DNA sequences derived from the plasmid and toxin gene of AHPND V. parahaemolyticus were developed and evaluated for LAMP assay to identify AHPND V. parahaemolyticus. In pure culture and in spiked shrimp experiments, the LAMP assay was superior to PCR for the detection of AHPND V. parahaemolyticus. In pure cultures, the detection limit of LAMP-A3 was 53 cfu/ml or 0.1 cfu/reaction, whereas in spiked shrimp experiments, the detection limit was 4.4x105 cfu/ml or 8.8x102 cfu/reaction. This technique could also detect AHPND V. parahaemolyticus in shrimp, sediment and water samples collected from a shrimp farm. The results suggested that the LAMP- A3- based LAMP assay was suitable for the identification of AHPND V. parahaemolyticus in shrimp aquaculture. To control AHPND V. parahaemolyticus, Bdellovibrio and like organisms (BALOS) were isolated and the capability to reduce AHPND V. parahaemolyticus was evaluated. Nine isolates of BALOs specific to AHPND V. parahaemolyticus were obtained and were identified as Bacteriovorax spp. The ability of BALOS designated as BV- A to reduce numbers of AHPND V. parahaemolyticus in vitro was observed in co-culture after incubation for 2 days and continued until 7 days of incubation period. In vivo, BV-A could reduce the mortality of shrimp post-larvae infected with AHPND V. parahaemolyticus. This study indicates that Bacteriovorax can be used as a biocontrol agent of AHPND V. parahaemolyticus in shrimp aquaculture.