Etiological Study And Effective Drug Screening Of Hepatopancreatic Necrosis Syndrome In Penaeus Vannamei

Penaeus vannamei is one of the most important economic shrimp in the world,and it is also the main species of shrimp cultured in China.In recent years,hepatopancreatic necrosis commonly breaks out in Penaeus vannamei farms in southern China after 30 days of culturing.Hepatopancreatic Necrosis Syndrome(HPNS)has caused great economic losses to the shrimp culture industry.Up to now,the etiology of HPNS in Penaeus vannamei is not clear.This study was carried out to clarify the pathogen of HPNS,diseased shrimp were collected from a shrimp pond in Guangxi province.The pathogen of HPNS was determined by pathological diagnosis,pathogenic analysis,bacterial isolation and identification test,and regression infection experiment.Then the effective bacteriostatic drugs of the main pathogenic bacteria of HPNS were screened through in vitro bacteriostatic test,so as to provide a basis for the rational prevention and control of HPNS in the production and breeding of Penaeus vannamei.Methods:(1)In this study,the diseased prawns were collected from the shrimp farm of Qinzhou,Guangxi,and their clinical symptoms were observed.The pathomorphology of the hepatopancreas was observed,and the biochemical indexes of hepatopancreas were detected to support diagnose the disease of Penaeus vannamei.(2)The infectivity of HPNS was analyzed by feeding experiment.Eleven common viruses of diseased shrimp were detected by PCR method.The bacteria were isolated,cultured and identified,and the biochemical characteristics of the dominant strains were determined by micro-biochemical reaction tube.The virulence genes of the dominant strains were detected and sequenced by PCR method,and the sequencing results were compared by NCBI.(3)To identify the main pathogens of HPNS,the dominant strains isolated from HPNS diseased shrimp were used for regression infection experiment.Shrimps were infected by Vibrio parahaemolyticus,Vibrio cholerae and Proteus vulgaris via injection and immersion,then the clinical symptoms,the histopathology of the hepatopancreas and the biochemical indexes of hepatopancreas of infected prawn were observed.Otherwise,the bacterias in the intestine and hepatopancreas of infected prawn were isolated and identified.In order to analysis the pathogenicity of HPNS to experimental animals,healthy mice were injected intraperitoneally with Vibrio parahaemolyticus,Vibrio cholerae and Proteus vulgaris via,and the mortality,pathological changes and target bacteria of the dead mice were analyzed.(4)The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of antibiotics commonly used in aquaculture,Chinese herbal medicine “sanhuanglian” mixture,“chaiyinhugan” mixture and Polygonum Hydropiper flavonoids extract against Vibrio parahaemolyticus,Vibrio cholerae and Proteus vulgaris were determined by tube double dilution method and drug disc agar diffusion method respectively,and the time-kiling curve was drawn.Results:(1)The diseased Penaeus vannamei showed decreased food intake and slower eating speed,diffused hepatopancreas,empty stomach,jejunum or broken intestine;the histopathological observation of hepatopancreas in diseased shrimps showed that the hepatopancreatic tubules were atrophic,and the absorption cells appeared pathological changes or necrosis;the hepatopancreas function of diseased shrimps was decreased,as indicated by biochemical index,including the activities of total protein,alkaline phosphatase(AKP),acid phosphatase(ACP),alanine aminotransferase(ALT),aspartate aminotransferase(AST),total superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in hepatopancreas were significantly lower than those in the blank control group.(2)The results of infectivity analysis showed that the pathogen of HPNS was a biological pathogen with infectivity.The results of viral pathogens test were negative,41 kinds of bacteria were isolated from 15 diseased shrimp samples of HPNS,which belonged to nine genera.Vibrio accounted for 35.90% and Proteus20.51%.None of the bacteria was isolated from all 15 shrimps.The identification of physiological characteristics of bacteria showed that the main pathogens were Vibrio parahaemolyticus,Vibrio cholerae and Proteus vulgaris.The virulence genes of Vibrio parahaemolyticus were Tox R and TLH,omp W is the virulence gene of Vibrio cholerae,rpo A is the virulence gene of Proteus vulgaris.(3)The results of anaphylaxis experiment showed that all the three dominant strains could infect Penaeus vannamei,which resulted in the infection of hepatopancreatic necrosis syndrome,indicating that hepatopancreatic necrosis syndrome is a bacterial disease.The isolated dominant strain could infect mice and cause mouse death,indicating that the pathogen is pathogenic to experimental animals.(4)Three kinds of Chinese herbal medicine“Chaiyinhugan” mixture,“Sanhuanglian” mixture and Polygonum hydropiper flavonoids extract all had certain inhibitory effects on the growth of the three dominant strains.The minimum inhibitory concentrations of “Chaiyin hugan”mixture on the three dominant strains were 15.625 mg/m L,7.8125 mg/m L and31.25 mg/m L.The minimum inhibitory concentrations of “Sanhuanglian mixture” on the three dominant strains were 15.625 mg/m L and 31.25 mg/m L.The minimum inhibitory concentration of Polygonum Hydropiper flavonoids extract against three dominant strains was 15.625 mg/m L.Conclusion: The disease of Penaeus vannamei cultured in a pond of Shabu Town,Qinnan District,Qinzhou City,Guangxi Province was diagnosed as HPNS.The pathogen of HPNS was bacterial,and Vibrio parahaemolyticus,Vibrio cholerae and Proteus vulgaris were the main pathogens of HPNS.The“chaiyinhugan” mixture,Sanhuanglian mixture and Polygonum hydropiper flavonoids extract and have bacteriostatic effect on the main pathogenic bacteria of HPNS,which can be applied to the reasonable control of HPNS in the production and culture of Penaeus vannamei.