Study On The Isolation,Identification And Pathogenesis Of Vibiro Parahaemolyticus Causing Shrimp Early Mortality Syndrome

An unusual disease outbreak in shrimp cultivation began in China in 2009 and spread Vietnam,Malaysia,Thailand and Mexico,which leading to a rapid and severe mortality in the early cultivation stage of approximately 35 days.This disease was called early mortality syndrome(EMS),but a more descriptive name of acute hepatopancreatic necrosis syndrome(AHPNS)was proposed.In early 2013 the causal agent was identified as a specific type of Vibrio parahaemolyticus.It is commonly admitted the agent is a novel strain of Vibiro parahaemolyticus harboring an extrachromosomal plasmid with a gene that encodes a homologue of the insecticidal Photorhabdus insect-related binary toxin Pir AB.However the pathogenic mechanism of this insect-related toxin is still unclear.This research isolates its pathogen from the cultivation pond of shrimp in the south west coastal aera of Guangdong Province,PRC.These isolates were deeply identified by multiple molecular biological technique.White shrimp animal model was established for further challenge assay.The toxic gene expression level and virulence of these isolates were detected.The details are as follows:1.Isolation of pathogen of EMSVibrio parahaemolyticus food microbiological testing standard GB/T 4789.7-2013 and US Food and Drug Administration(FDA)Bacteriological Analytical Manual Online were used as guideline in the isolation from shrimp cultivation ponds in outbreak area of EMS.Healthy,dying,dead shrimp,water,and mud sample were collected for isolation of pathogens.A total of 81 strains of bacteria were isolated from 8 ponds,and 39 of them were identified as Vibrio parahaemoyticus.Further identification were performed by three authoritative primer pairs,strains showed all positive result by all three of them were picked out as suspected pathogen.A total of 12 strains of bacteria were screened out.2.Identification of pathogen of EMSThese 12 strains of suspected pathogens were identified by multiple molecular biological technique.Conventional virulence gene test revealed that all of them contains tlh gene,none of them harbored tdh or trh gene.API20 E microbial identification test was used for species identification.16 S r RNA gene were sequenced for phylogenetic analysis.Result showed that all of these suspected bacteria are Vibrio parahaemolyticus.ERIC-PCR and plasmids fingerprinting were used for diversity analysis,and it showed that the diversity of these strains are quiet simple.Toxic gene pir A were amplified and sequenced,the sequences of them showed highly homology.Total RNA of these strains were extracted and their pir A gene expression level were measured.The result indicated major difference among strains,the highest value was approximately 13 fold of the lowest value.SDS-PAGE and Western Blotting method were employed for toxin detection.These suspected strains showed similar test result with 3HP strain which is a corroborative pathogen of EMS.Above all,these suspected strains were deeply identified from the species information,diversity analysis,plasmid fingerprinting,toxic gene,toxic gene transcription and protein expression.3.Establishment of white shrimp animal model and challenge assayWhite shrimp animal model was established for challenge assay.The equipment of this animal model could control the temperature from roomtemperature to 34 oC.the monitoring factor of aquiculture water were p H(6.7~8.5)?nitrite(less than 0.1 m M)and ammonia(less than 0.35 m M).The maximum volume of experiment tank is 50 L.The challenge mode and dose were verified by 3HP strain.Immersion challenge test was chosen for 15 min,the dosage of bacteria cultivation was 106 CFU/m L.Nonpathogenic strain S02 and sterile TSB+ were used as negative control for challenge test.Every 12 h observation after infection,and collect the prawn body death,organize pathology slice observation diagnosis.The results showed that 12 strains have the virulence of prawn with early mortality syndrome,except the F5 and F18 strains,the rest of the 10 strains can achieve 100% mortality rate,and most of the strains can lead to the infection prawn's death within 48 h.By tissue section identification can also be found these prawn HP necrosis,prawns with typical symptoms of early mortality syndrome characteristics.With non-toxic S02 strain as a negative control group only 1 died prawns,(no shrimp death TSB+ medium control),no symptoms of HP necrosis showed.These results are in conformity with molecular biology identification results.Through the above experiment can be confirmed,these 12 strains of Vibrio parahaemolyticus are pathogenic strains of early mortality syndrome.4.The toxins and virulence gene expression and the influence of temperatureIt is found that the isolation of 12 strains of pathogens in shrimp early mortality syndrome,the corresponding situation between toxin genes expression and virulence is not good.Is found after preliminary analysis that may be caused by the difference of temperatures in toxin infection process and gene expression,different stress response to toxin in prawns led to this phenomenon.So we designed experiment for discussing the impact of different temperature on strain infection capacity.The experiment selected F12 strain for its hightest virulence gene expression level under 37?,and G10 for its lowest gene expression level,as the research object.Chose temperature range from 20 to 32? which is the suitable growth temperature for prawn,set a gradient every 3?,of two strains of bacteria in 5 different temperatures the virulence of toxicity test.Results showed that the experimental setup of five temperature gradient within the scope of the virulence of pathogenic strain with the increase of temperature increased,both of two strains first appeared death prawns and achieved 100% fatality rate at 32?.Comparison between strains found that the death rate of F12 strain is higher than the G10 strain in every temperature gradients,the results are in conformity with the toxin gene expression results.In a certain temperature range,virulence is different between strains,differences in gene expression is the main reason lead to this phenomenon.