Molecular Typing And Screening Of Antagonistic Bacteria Of Vibrio Hepatopancreas Necrosis Strain Of Litopenaeus Vannamei In Jiangsu Province

Acute hepatopancreas necrosis syndrome(AHPNS)has been an important disease affecting the development of shrimp farming in recent years.Since 2009,the production of Asian shrimp has been greatly reduced by AHPNS,which has brought enormous economic losses to the shrimp industry in Asia.The shrimp farming industry in China,dominated by Litopenaeus vannamei,has also been greatly impacted.In 2013,the Global Aquaculture Alliance(GAA)issued a statement that the pathogen of AHPNS was Vibrio parahaemolyticus.The pathogen produces virulence through effector proteins PirA and PirB.The digestive organs of diseased shrimp are pale,the liver and pancreas are atrophy,with the jejunum and stomach,and the mortality rate reaches 100% within 1 to 3 days from onset.Previously,the pathogenesis of AHPNS has not been clearly studied,and there is no effective prevention and treatment methods reported.In this paper,the pathogenic bacteria of AHPNS of Litopenaeus vannamei from different sources were isolated and molecular typed to obtain the molecular epidemiological data of acute hepatopancreatic necrosis syndrome in Jiangsu province.The screening of antagonistic bacteria of pathogenic Vibrio was studied to provide technical data for the ecological control of the disease.From 2017 to 2018,shrimp farms in Lianyungang Guanyun,Dongtai,Yancheng and Rudong counties(cities and districts)in Jiangsu coastal areas suffered from acute death in varying degrees.The shrimp was characterized by jejunum,jejunum,atrophy or swelling of liver,pancreas and pancreas,which showed typical symptoms of AHPNS.A total of 642 strains of bacteria were isolated from the liver and pancreas of Litopenaeus vannamei with different seedling sources,different growth stages,different morbidity characteristics,different culture modes and suspected AHPNS.The pathogenicity-related primers AP3 and 16 S rRNA universal primers were used for amplification,and 28 strains of vannamer were identified and obtained.Thepathogenic Vibrio parahaemolyticus of shrimp AHPNS was classified according to different source patterns.Molecular typing of 28 strains of pathogenic Vibrio parahaemolyticus isolated from shrimp AHPNS patients in Jiangsu Province was studied by pulsed field electrophoresis(PFGE),Finally,four main PFGE clusters(G 1-G 4)of Vibrio parahaemolyticus were obtained,and 24 bands were identified.The similarity between bands was from 86.1 % to 100 %,indicating that the strains were closely related to each other.The results of PFGE typing were correlated with the culture patterns,symptoms,seedling sources and growth stages of shrimp.It indicated that the occurrence of acute hepatopancreatic necrosis syndrome of Litopenaeus vannamei caused by Vibrio parahaemolyticus had an epidemic trend in Jiangsu Province.There were some differences among the four PFGE clusters,which were related to the origin of 28 strains of bacteria,breeding patterns,symptoms and different growth stages of infected shrimp.In this study,a PFGE database of Vibrio parahaemolyticus from Litopenaeus vannamei acute hepatopancreatic necrosis in Jiangsu Province was preliminarily established to compare the PFGE bands of Vibrio parahaemolyticus from different pathogenic or suspected shrimp samples with the results of this study.According to the principle that shrimp individuals from the same source have common genetic material and show the same fingerprint in PFGE band,the genetic relationship among strains is analyzed,the source of infection and epidemic scope of disease are studied,On this basis,early warning and prevention of disease are carried out,and effective measures are taken to prevent and control acute hepatopancreatic necrosis syndrome of Litopenaeus vannamei.252 strains of bacteria were isolated from the sediments of the southern coastal beach of Jiangsu Province.Fourteen strains of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis of Litopenaeus vannamei were used as indicator bacteria.After screening and re-screening,a strain H 0011 with good antagonistic effect was obtained.The strain was identified as Providencia by morphological examination,physiological and biochemical characteristics and 16 S rDNA gene sequence.Litopenaeus vannamei were exposed to high density stress with Providencia H 0011 to test their safety.During the experiment,Litopenaeus vannamei in bathing group and feeding group were in good condition without morbidity or death.After optimizing the growth conditions of H 0011,the optimum growth temperature was30 ℃,salinity was 10,and pH was 8 ‰.It entered logarithmic growth stage at 4 hand reached its peak at 20 h.In this paper,the pathogenic Vibrio of AHPNS was collected and typed for the first time in different seedlings,different regions,different growing periods,different breeding patterns and different death characteristics of Litopenaeus vannamei in Jiangsu area,which provided some technical support for further obtaining molecular epidemiological data of acute hepatopancreatic necrosis syndrome in Jiangsu area;and for the first time,the acute hepatopancreatic necrosis syndrome of Litopenaeus vannamei was obtained.Hepatopancreatic necrosis has a highly effective antagonistic bacteria,which provides a new idea for the prevention and control of shrimp acute hepatopancreatic necrosis syndrome.