Conjugative plasmid diversity of Vibrio parahaemolyticus in the sediment of shrimp farm and its association with bacterial virulence

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus is an emerging bacterial disease in cultured shrimp. The disease has caused the huge economic losses to the global shrimp aquaculture industry, with annual losses of over 1 billion. All AHPND related V. parahaemolyticus contain a 70 kb plasmid with genes encoding homologues of the Photorhabdus insect-related (Pir) binary toxin PirAB^vp, which is key virulence factor for shrimp. In order to clarify the genetic diversity of the conjugative plasmid of the V. parahaemolyticus strain that causes AHPND in the shrimp culture environment and understand the prevalence of APPND-associated plasmid, this study was conducted in five coastal provinces in China. A total of 100 sediment samples were collected from the shrimp farms in the five coastal provinces. PCR was then conducted to amplify the pirAB and conserved genes encoding the conjugative transfer protein on the plasmids. V. parahaemolyticus strains were then isolated from the pirAB-positive sediment samples to obtain the plasmid. Sanger sequencing was then conducted to obtain the sequence of the plasmid. Finally comparative sequence analysis was performed to assess the plasmid diversity. The results showed that 39 out of 100 samples contained plasmid conjugative transfer protein fragments. 15 strains of V. parahaemolyticus were isolated from 100 sediment samples, 13 of which contained 1-2 plasmids. The results of plasmid sequencing showed that these plasmids can be divided into 8 types/profiles, of which 7 do not carry pirAB gene, but all contain gene clusters encoding conjugation transfer. According to the isolation of 8 types of plasmids carried by V. parahaemolyticus, 8 strains of V. parahaemolyticus were selected for prawn challenge experiments. It was found that the toxicity of these strains to prawns was significantly different, and the mortality rate of prawns was between 15%-100%. Only pirAB positive strains will produce AHPND symptoms, and the mortality rate of shrimp is 100%. The analysis of the plasmid composition shows that the genetic material exchange between plasmids is frequent, and the genetic composition of most of the plasmids comes from a 183 kb super-large plasmid pVP2HP. In this study, we found that the conjugative plasmids isolated from the sediments of shrimp farms had rich genetic diversity. Most of the plasmids are genetically composed of plasmid pVP2HP, and only pirAB positive strains will produce AHPND symptoms. This study improved understanding of plasmid genetic diversity of V. parahaemolyticus and its relationship with AHPND, and provided theoretical guidance for the prevention and control of AHPND.