Characterization And Pathogenicity Analysis Of Vibrio Parahaemolyticus Isolated From Penaeid Shrimps In China(2013-2016)

Part 1.Vibrio parahaemolyticus is found to be the earliest Vibrio pathogen of shrimp,causing huge losses to the shrimp aquaculture industry.In this study,we identified the bacteria strains that isolated from diseased shrimp in coastal areas of China from 2013 to 2016,and 151 strains were identified as V.parahaemolyticus,totally.Among them,34 strains from Hebei,13 strains from Tianjin,21 strains from Shandong,7 strains from Jiangsu,29 strains from Zhejiang,18 strains from Guangdong,24 strains from Guangxi,and 5 strains from Hainan.Three isolated from Fenneropenaeus chinensis,7 isolated from Marsupenaeus japonicus,and other 141 isolated from Litopenaeus vannamei.Nearly 33.11% of them carried virulence protein genes pir AVP and pir BVP,which were related to hepatopancreatic necrosis disease(AHPND).The result of O-antigen serotype showed that there were 2 strains of O untypeable in 151 V.parahaemolyticus,and the identification rate was 98.68%,which concentrated in O1,O8,O3,O2.However,the identification rate of K-antigen serotype was only41.06%(62strains).Among 151 V.parahaemolyticus,only 60 strains were fully typed by O/K-antigen serotype with the total type rate of 39.74%.The O:K serotype of the 60 strains were divided into 24 types,which were relatively dispersed.The preponderance type was O1:K25(14 strains,23.33%),and there was no O3:K6.Drug sensitivity analysis results: 151 V.parahaemolyticus showed varying degrees of resistance to 14 kinds of antibiotics.Tacrolimus and cefalotin were resisted seriously with a resistance rate of 53.64% and 39.74%,respectively.Multiple drug resistance is very serious.55.63%(84/151)of V.parahaemolyticus showed resistance to more than two drugs,22.52%(34/151)of V.parahaemolyticus showed resistance to more than three drugs,10strains(6.62%)were resistant to four or more drugs,and there were two strains were resistant to eight drugs.All strains were fully sensitive to piperacillin,gentamicin,ciprofloxacin,amoxicillin/clavulanic acid,piperacillin/tazobactam and neramine.There were significant regional differences in antibiotic resistance of V.parahaemolyticus.Part 2.AHPND of shrimps broke out in 2010.It caused tremendous losses onshrimp culture worldwide.It has been reported that L.vannamei and P.monodon is susceptible species of AHPND,and the susceptibility of F.chinensis to the disease is unclear.In this study,a strain 20140723005 was isolated from F.chinensis,which was suspected to be suffering from AHPND.The isolate was identified as V.parahaemolyticus with AHPND related virulence protein gene pir AVP and pir BVP based on the 16 S r DNA and gro EL gene sequencing analysis,biochemical characteristics and PCR.The antibiotic sensitivity test showed that the isolate was resistant to cefalotin and cefuroxime,but was sensitive to amoxicillin,gentamicin,ciprofloxacin and other 16 kinds of tested antibiotics.The pathogenicity of the isolate to F.chinensis was analyzed by immersion infection.The results showed that infected shrimps presented empty gut,pale hepatopancreas,and up to 100% mortality in 18 h.Subsequent histological analyses showed that the hepatopancreas tubules collapsed with massive sloughing of hepatopancreas epithelial cells,which was the typical pathological characteristics of AHPND.This study demonstrated that F.chinensis was also the susceptible host species of AHPND.Part 3.In this study,a bacterial strain 20160303005-1 isolated from the hepatopancreas tissue of L.vannamei suffered from EMS,was identified as V.parahaemolyticus according to its physiological and biochemical characteristics and analysis of both 16 S r RNA and gro EL gene sequence.The serotype of the bacterium is O1:KUT(Kuntypeable).It revealed positive amplification of the pir AVP and pir BVP genes which related to cause AHPND in a virulence plasmid harbored by this strain.However,the isolate examined gave negative amplification results for the virulent clinical V.parahaemolyticus strains markers thermostable direct hemolysin gene tdh and TDH-related hemolysin gene trh.Immersion challenge tests with L.vannamei were also employed for pathogenicity and histopathology studies.The results showed the isolatewas high virulent,with a value of the median lethal dose(LD50)of 7.96×103CFU/ml.The empty gut in shrimp was observed at 6 h post-challenged.The hepatopancreas seemed to be pale andatrophy at 9 h.More than half of shrimps died at12 h,and up to 100% mortality achieved at 24 h.Histopathological research revealed that guts,gills,and hepatopancreas of infected shrimps were damaged.The hepatopancreas tubules collapsed with massive sloughing of hepatopancreas epithelial cells,which was the typical pathological characteristics of AHPND.Among 21 antibiotics tested,the isolate was resistant to amoxicillin,cefalotin,ticarcillin,cefuroxime,and cotrimoxazol,but was sensitive to gentamicin,ciprofloxacin,and other14 kinds of tested antibiotics.These above results provided basic data for epidemiology and drug control research for V.parahaemolyticus in aquaculture.Part 4.In this study,a strain 20151116002-3 isolated from the hepatopancreas tissue of M.japonicus,withpir AVP and pir BVP genes related to the pathogenesis of AHPND were positive in the pathogen detection of DNA samples from the hepatopancreas of diseased M.japonicus from Guangxi.Strain 20140722001-1,isolated from the hepatopancreas of diseased M.japonicus from Hebei,with the positive of acute hepatopancreatic necrosispathogen Vibrio(Vahpn)test.The pathogenicity of these two isolates to M.japonicus was analyzed by acute immersion infection.The results showed that 20151116002-3 infected shrimps presented empty gut,pale hepatopancreas in 9 h,and up to 100% mortality in 72 h,but 20140722001-1 infected shrimps presented pale hepatopancreas in 30 h,and up to 100% mortality in 96 h.Histopathological research revealed that 20151116002-3 infected shrimps presented hepatopancreas tubules collapsed with massive sloughing of hepatopancreas epithelial cells,which was the typical pathological characteristics of AHPND,but 20140722001-1 infected shrimps presented no typical pathological characteristics of AHPND.Isolate 20151116002-3 and20140722001-1were identified as V.parahaemolyticus according to its physiological and biochemical characteristics and analysis of both 16 S r RNA and gro EL gene sequence.The serotype of the bacterium is O1:KUT(Kuntypeable)and O3: K31,respectively.Both revealed positive amplification of the pir AVPand pir BVPgenes which related to cause AHPND in a virulence plasmid harbored by this strain.The antibiotic sensitivity test showed that the isolate20151116002-3 was resistant to ticarcillin,cefotaxime,cefoxitin,and 20140722001-1 was only resistant tocefotaxime.