A Preliminary Study On The Effect Of Different Strains Carrying PirAB Gene On The Pathogenicity And Intestinal Flora Of Healthy Litopenaeus Vannamei

Litopenaeus vannamei is the largest farmed shrimp species in China.However,with the rapid expansion of Litopenaeus vannamei farming scale,breeding deterioration of ecological environment and coupled shrimp quality problems,the incidence of shrimp diseases is gradually increasing and has become one of the main constraints restricting the healthy sustained and rapid development of the Litopenaeus vannamei aquaculture industry.Acute hepatopancreatic necrosis disease(AHPND)is one of the most serious bacterial diseased that harm the breeding of shrimp in recent years,which can cause large scale deaths and reduce reduction of shrimps,causing great economic losses to the Litopenaeus vannamei culture industry.The current study believes that Vibrio cayying pirA and pir B gene is the direct cause of the outbreak of AHPND in shrimps.In order to explore the main pathogenic species of AHPND of Litopenaeus vannamei in Zhoushan area,30 suspected strains of AHPND pathogenic bacteria isolated from cultured Litunaeus vannamei were detected by pirAB virulence gene and species identification.Four representative strains were produced for pirA and pir B gene full length analysis and physiological and biochemical characteristics.The pathogenicity of different strains carrying pirAB gene to healthy Litopenaeus vannamei and the histopathological changes caused by them were investigated by artificial infection experiment.We investigate the bacterial species composition and diversity in the intestine of Litopenaeus vannamei after infection with Vibrio parahaemolyticus and Vibrio owensi carrying pirAB gene by using Illumina sequencing,and explored the effects of different AHPND pathogenic bacteria on the intestinal flora of healthy shrimp.It is expected to provide a scientific basis for the prevention and control of the disease from the perspective of intestinal microecological regulation.The results were as follows:1、According to the Nested-PCR method of OIE"Diagnosis Manual of Aquatic Animal Diseases",30 strains of bacteria were tested for virulence genes.Methods and Results 16S r RNA,gyr B,rpo A genes of these isolates were obtained by PCR,sequenced and analyzed.The results showed that 10 strains of 30 strains carried the pirAB virulence gene,4 strains of Vibrio parahaemolyticus,4 strains of Vibrio campbellii and 2 strains of Vibrio owensii.2、Combined with the results of molecular biology identification of pathogenic bacteria,four representative strains of pirAB virulence gene positive were selected(1strain of Vibrio parahaemolyticus,2 strains of Vibrio campbellii,1 strain of Vibrio owensi)after the analysis of virulence genes and physiological and biochemical characteristics,an artificial infection test was carried out.Through experiments,it was found that shrimp became ill and died after being infected by 4 strains of pirAB~+bacteria,which were mainly manifested as the specific symptoms of AHPND,but infection with pirAB~-strains did not cause shrimps to become ill,and there was no AHPND symptom.The cumulative mortality of experimental shrimp exposed to AHPND-positive strains were higher than the short exposure group.Subsequent histopathological analyses revealed that the challenged shrimp,48h after infected with the bacteria,showed histological lesions of AHPND,such as massive sloughing of hepatopancreatic tubule epithelial cells,intestinal villi shedding.The most serious pathological changes in the hepatopancreas of shrimp infected by Vibrio parahaemolyticus.The results of ultramicroscopic pathology are mainly epithelial cell lysis and necrosis,indicating that different strains have different pathogenicity to shrimp.3、Illumina Mi Seq high-throughput sequencing platform was used to the diversity and composition of intestinal microflora in Litopenaeus vannamei infected with Vibrio parahaemolyticus and Vibrio owensi were compared and analyzed.The Alpha diversity index in the shrimp intestines of the infected group was significantly lower than that of the control group,and the Alpha diversity index of the shrimp in the Vibrio owensi infection group was higher than that of the Vibrio parahaemolyticus infection group.Analysis of the structure of intestinal flora at the phylum level showed that Proteobacteria,Bacteroidetes,Firmicutes were the three most dominant categories in the intestinal microflora of shrimp infected by highly pathogenic Vibrio parahaemolyticus,and Proteobacteria,Bacteroidetes,Firmicutes and Cyanobacteria were the most dominant groups in the intestinal flora of shrimp infected by Vibrio owensi.Compared with the control group,the relative abundance of conditional pathogenic bacteria such as Proteobacteria and Bacteroides in the infected group was significantly increased,but the normal flora such as Firmicutes and Cyanobacteria were significantly reduced.At the genus level,the relative abundance of conditional pathogens such as Vibrio in the intestinal tract of Vibrio parahaemolyticus infection was significantly higher than that of Vibrio owensi at each time period of infection,on the other hand,the relative abundance of Lactobacillus in the intestine of shrimp in the other infection group began to recover 12 h after infection.