Isolation And Identification Of Pathogen Of An Acute Hepatopancreatic Necrosis Disease Case From Litopenaeus Vannamei And Functional Analysis Of Pirb Protein

In August 2016,an outbreak of shrimp death occurred in a shrimp farming area in Shandong Province,having brought serious economic loss to local farmers.A bacterium named VP-1 was isolated from the hepatopancreas of dying shrimps.Artificial infection test and LD₅₀ experiments confirmed that pathogenicity of VP-1.VP-1 was identified as Vibrio parahaemolyticus by physiological and biochemical tests and 16S rDNA identification.Drug sensitivity test is demonstration of the VP-1 was sensitive to three antibacterials containing florfenicol,ofloxacin and enrofloxacin.In order to study the pathological damage of shrimp caused by VP-1,the observation of histopathological and ultrastructural on naturally infected shrimps was studied.Histopathologically,hepatopancreas and muscle have obviously damages such as degeneration,dissolved and necrosis.In addition,the intestine and gill has also injuried at different levels.Ultrastructural observation demonstration of that mitochondria swelling,the structure of mitochondrial ridge rupture,mitochondria vacuolation and forming cavity,nucleus chromatin dissolved and marginalization,rough endoplasmic reticulum distention,microvilli broken in intestinal epithelial.Acute hepatopancreatic necrosis disease?AHPND?is caused by Vibrio parahaemolyticus,which carries a virulent plasmid?pVH plasmid?of about 69 k bp.The genome of the plasmid can encode Pir toxin protein.In order to obtain the plasmid genome of VP-1 isolate,the whole gene of VP-1 was sequencing by method of second-generation genome sequencing.The results revealed that VP-1 had a high similarity with the plasmid genome from Vibrio spp which has published at NCBI?access number:KM067908?.19 Scaffods of VP-1 matching the published plasmid genome of Vibrio parahaemolyticus?GenBank accession no.KM067908?which as reference sequence by using Geneious.Then,the genome circle map of the plasmid was aquired by BRIG software.It is concluded that VP-1 also carries a virulent plasmid encoding Pir protein,causing AHPND in shrimp.In order to know the effect of Pir-like protein on shrimp,the purified PirB was successfully expressed by prokaryotic expression and then was injected to crayfish intramuscularly.The hispathology,mortality,blood cells morphology,immune parameters?SOD,POD,PO?tests were carried out.The results showed that PirB protein could cause AHPND,casusing low cumulative mortality rate?30%?and could change the blood cells'morphology.SOD activity increased firstly but decreased lately while POD and PO activities increased.