Multilocus Sequence Typing And Antibiotic Resistance Anlysis Of AHPND-causing Vibrio Parahaemolyticus

Acute hepatopancreatic necrosis disease?AHPND?first outbreak in China in 2009,and then it spread successively to Vietnam?2010?,Malaysia?2011?,Thailand?2012?,Mexico?2013?and the Philippines?2015?.It's a bacterial disease,with a high shrimp mortality?100%?.By 2013,AHPND had caused unprecedented harm to the shrimp farming industry in Asia.Causing economic losses of more than$1 billion a year,and there is still a trend of further expansion.In 2013,Lightner et al have studied and discovered that the culprit of AHPND disease is a mutant vibrio parahaemolyticus which has mutated after infection with bacteriophage virus.In 2015,a research group in Taiwan has selected mutant strain of virulence gene of pirA and pirB with natural deletion and experimental deletion,and found that the pathogenicity of both mutants was eliminated,so as to further confirm that toxin of PirA and PirB is the ultimate cause of acute hepatopancreatic necrosis disease.On the basis of existing research,to develop an effective technique for the diagnosis,prevention and control of acute hepatopancreatic necrosis disease of shrimp,is the key to reducing the risk of the AHPND epidemic.This subject starts with the diagnosis of AHPND.In this project,A fluorescence in situ hybridization assay was established,by a visual monitoring environment and pathogenic bacteria in prawn tissue,a further confirmation that the pathogenicity of AHPND strains preserved was made in our laboratory.The next is to use the AHPND-causing strain as the experimental material.And download the housekeeper gene information from other regions of the world to analyze the epidemic and genetic relationship of AHPND pathogenic strains in the world.Finally,we studied the antibiotic resistance of AHPND pathogenic strains from genotype and phenotype.And provide a theoretical basis for promoting the healthy and sustainable development of shrimp farming industry,this is of great practical significance to the aquatic ecological safety and economic development of our country.1.Establishment of fluorescence in situ hybridization assay of VP_AHPNDThis study is based on gene design fluorescence in situ hybridization probe of pirA,hence,established a fluorescence in situ hybridization assay for the detection of acute hepatopancreatic necrosis.The existing pirA gene was found to be conserved without mutation by sequence comparison software ClustalX.The probe was designed with Primer Premier 5 software.The probe nucleic acid sequence was not matched with other non-AHPND strains by NCBI database BLAST option.In order to further verify its specificity,21 Non AHPND pathogenic strain and 12 AHPND pathogenic strain were selected to conduct a validation.Twelve positive samples of AHPND could be identified by fluorescence in situ hybridization,which was consistent with the results of PCR detection.When pirA probe is hybridized with VP_AHPND,The fluorescence signal was clear and the shape of the body was obvious.At the same time,it was observed that there were obvious pathogenic bacteria in the hepatopancreas sections of the diseased prawn with the pirA probe.Fluorescence in situ hybridization can be used to detect vibrio parahaemolyticus pathogenicity of AHPND in environment and shrimp tissues,and provide more tools for better understanding of the harm of these environmental microorganisms to aquatic products.2.New sequence type isolates of AHPND-causing Vibrio parahaemolyticus from Litopenaeus vannamei by multilocus sequence typingIn this study,the genetic characteristics of Vibrio parahaemolyticus with acute hepatopancreatic necrosis disease were analyzed by the multilocus sequence typing method.Seven housekeeping genes of Vibrio parahaemolyticus were selected as:dnaE,gyrB,recA,dtdS,pntA,pyrC and tnaA.The AHPND pathogenic bacteria were amplified and sequenced.The nucleic acid sequence was uploaded to PubMLST database for alignment and the sequence type of each strain was obtained.MLST data of AHPND-causing V.parahaemolyticus from other areas were collected and the genetic evolution was analyzed by MEGA 5.0 and eBURST V3 software.The results showed that the pathogenic strain of AHPND isolated from Guangdong Province of China belonged to a new sequence type ST1710?42,134,99,79,141,41,51?.Compared with other sequence types in PubMLST database,it has the high homology with ST415 and ST975.The 9 STs of AHPND-causing V.parahaemolyticus were separated by eBURST into two doublets and five singletons.Phylogenetic tree analysis showed that the new sequence type ST1710 was similar to the singletons ST975.The sequence of AHPND type and its related non AHPND sequence type strains were from the environment,and it is closely related to shrimp aquaculture.Our study is the first report of molecular epidemiology of AHPND causing V.parahaemolyticus strains in China using multilocus sequence typing,and enriches the PubMLST database and provides an insight into their evolutionary mechanisms.3.Antibiotic resistance research on isolate strain of acute hepatopancreatic necrosis disease in Guangdong,ChinaIn the fourth chapter,the antibiotic resistance of AHPND pathogens was studied from two aspects:genotype and phenotype,so as to provide help for monitoring the emergence of antibiotic resistance bacteria,it has important practical significance for aquatic ecological security and economic development in China.AHPND pathogenic strains isolated from Guangdong were resistant to ampicillin?AMP?.As for aminoglycoside?AK,CN,K,S?,?-lactam?AMC?,cephalosporins?KZ?,there is always partial strain with a medium sensitivity.The others are more sensitive.Its antibiotic resistance gene type is only a little,only aminoglycoside resistant genes as aadA2,and sulfanilamide resistance gene as sul?,sul?,therefore,the AHPND strains isolated in our laboratory showed a relatively low antibiotic resistance,with a high sensitivity to many antibiotics,and a few resistant genes.To some extent,this also indicated that the drug use in the breeding base was good,but due to the large number of moderately sensitive strains,it was necessary to continue to pay attention to the drug resistance of shrimps.