凡纳滨对虾急性肝胰腺坏死病(AHPND)病原分离鉴定及其致病性分析
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摘要
本研究从患急性肝胰腺坏死病(Acute hepatopancreatic necrosis disease,AHPND)的凡纳滨对虾(Litopenaeus vannamei)肝胰腺中分离到一株优势菌,编号为20160303005-1,通过16S rRNA和分子伴侣蛋白groEL基因序列分析,并结合生理生化特征,将该细菌鉴定为副溶血弧菌(Vibrio parahaemolyticus),其血清型为O1:KUT(K untypeable)。基因分析结果显示,该菌株携带可引起对虾AHPND的相关毒力蛋白基因pirA~(VP)和pirB~(VP),但不携带副溶血弧菌临床菌株毒力基因:耐热直接溶血毒素(Thenmostable direct hemolysin,tdh)和相对耐热直接溶血毒素(TDH-related hemolysin,trh)基因。菌株对凡纳滨对虾具有较强的致病性,浸泡感染的半数致死剂量(LD_(50))为7.96×10~3 CFU/ml。对虾急性感染后,6 h肝胰腺颜色变浅,肠胃变空;9 h肝胰腺呈浅白色,萎缩变小。9 h死亡数过半,24 h全部死亡。组织病理学分析显示,感染后对虾肝胰腺小管崩塌,上皮细胞严重脱落,呈现出典型的AHPND病理症状。药敏实验结果显示,该菌对庆大霉素、环丙沙星和头孢他啶等16种药物敏感,对阿莫西林、替卡西林和头孢噻吩等5种药物表现为耐药。上述研究可为该病原的流行病学及药物防控研究提供基本数据。
Acute hepatopancreatic necrosis disease(AHPND) is an emerging shrimp disease causing great losses for the shrimp culture industry worldwide since 2010. In the present study, a bacterial strain 20160303005-1 was isolated from the hepatopancreas tissue of Litopenaeus vannamei with early mortality syndrome(EMS), and was identified as Vibrio parahaemolyticus based on its physiological and biochemical characteristics; and the analysis of both 16 S rRNA and gro EL gene sequence. The serotype of the bacterium is O1:KUT(K untypeable). It revealed positive amplification of the genes pirA~(VP) and pirB~(VP) which is related to cause AHPND in a virulence plasmid harboring this strain. However, the isolate examined showed negative amplification results for the virulent clinical V. parahaemolyticus strain markers—thermostable direct hemolysin gene tdh and TDH-related hemolysin gene trh. The immersion challenge test with L. vannamei was also employed to study pathogenicity and histopathology. The results showed that the isolate was highly virulent, with a median lethal dose(LD_(50)) value of 7.96×10~3 CFU/ml. The empty gut in shrimp was observed at 6 h post-challenged. The hepatopancreas appeared pale and atrophic at 9 h. More than half of the shrimps died at 12 h, and up to 100% died at 24 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. Among 21 antibiotics tested, the isolate was resistant to amoxicillin, cefalotin, ticarcillin, cefuroxime, and cotrimoxazol; however, it was sensitive to gentamicin, ciprofloxacin, and other 14 antibiotics tested. These results provide basic data for epidemiology and drug control research on V. parahaemolyticus in aquaculture.
Acute hepatopancreatic necrosis disease(AHPND) is an emerging shrimp disease causing great losses for the shrimp culture industry worldwide since 2010. In the present study, a bacterial strain 20160303005-1 was isolated from the hepatopancreas tissue of Litopenaeus vannamei with early mortality syndrome(EMS), and was identified as Vibrio parahaemolyticus based on its physiological and biochemical characteristics; and the analysis of both 16 S rRNA and gro EL gene sequence. The serotype of the bacterium is O1:KUT(K untypeable). It revealed positive amplification of the genes pirA~(VP) and pirB~(VP) which is related to cause AHPND in a virulence plasmid harboring this strain. However, the isolate examined showed negative amplification results for the virulent clinical V. parahaemolyticus strain markers—thermostable direct hemolysin gene tdh and TDH-related hemolysin gene trh. The immersion challenge test with L. vannamei was also employed to study pathogenicity and histopathology. The results showed that the isolate was highly virulent, with a median lethal dose(LD_(50)) value of 7.96×10~3 CFU/ml. The empty gut in shrimp was observed at 6 h post-challenged. The hepatopancreas appeared pale and atrophic at 9 h. More than half of the shrimps died at 12 h, and up to 100% died at 24 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. Among 21 antibiotics tested, the isolate was resistant to amoxicillin, cefalotin, ticarcillin, cefuroxime, and cotrimoxazol; however, it was sensitive to gentamicin, ciprofloxacin, and other 14 antibiotics tested. These results provide basic data for epidemiology and drug control research on V. parahaemolyticus in aquaculture.
引文
Ahmed R,Rafiquzaman SM,Hossain MT,et al.Speciesspecific detection of Vibrio alginolyticus in shellfish and shrimp by real-time PCR using the gro EL gene.Aquaculture International,2016,24(1):157-170
Bennett PM.Plasmid encoded antibiotic resistance:Acquisition and transfer of antibiotic resistance genes in bacteria,British Journal of Pharmacology,2008,153(S1):S347-S357
Bureau of Fisheries and Fisheries Law Enforcement,Ministry of Agriculture.China fishery statistical yearbook.Beijing:China Agriculture Press,2016,28,30,71[农业部渔业渔政管理局.中国渔业统计年鉴.北京:中国农业出版社,2016,28,30,71]
Chen Y,Chen X,Yu F,et al.Serology,virulence,antimicrobial susceptibility and molecular characteristics of clinical Vibrio parahaemolyticus strains circulating in southeastern China from 2009 to 2013.Clinical Microbiology and Infection,2016,22(3):258(e9-e16)
COFI Sub-committee on Aquaculture.Aquatic biosecurity:A key for sustainable aquaculture development.Phuket:Co FISub-Committee on Aquaculture,2010[FAO渔业委员会水产养殖分委员会.水生生物安保:可持续水产养殖发展的一个关键.普吉:FAO渔业委员会水产养殖分委员会,2010]
Dabu IM,Lim JJ,Arabit PMT,et al.The first record of acute hepatopancreatic necrosis disease in the Philippines.Aquaculture Research,2017,48(3):792-7998
de la Pe?a LD,Cabillon NA,Catedral DD,et al.Acute hepatopancreatic necrosis disease(AHPND)outbreaks in Penaeus vannamei and P.monodon cultured in the Philippines.Diseases of Aquatic Organisms,2015,116(3):251-254
Dong X,Wang HL,Xie GS,et al.An isolate Vibrio campbellii carrying the pirVP gene causes acute hepatopancreatic necrosis disease.Emerging Microbes and Infections,2017,6(1):e2
Elmahdi S,Da Silva LV,Parveen S.Antibiotic resistance of Vibrio parahaemolyticus and Vibrio vulnificus in various countries:A review.Food Microbiology,2016,57:128-134
Flegel TW.Historic emergence,impact and current status of shrimp pathogens in Asia.Journal of Invertebrate Pathology,2012,110(2):166-173
Freel KC,Millán-Agui?aga N,Jensen PR.Multilocus sequence typing reveals evidence of homologous recombination linked to antibiotic resistance in the genus Salinispora.Applied and Environmental Microbiology,2013,79(19):5997-6005
Han JE,Tang KF,Tran LH,et al.Photorhabdus insect-related(Pir)toxin-like genes in a plasmid of Vibrio parahaemolyticus,the causative agent of acute hepatopancreatic necrosis disease(AHPND)of shrimp.Diseases of Aquatic Organisms,2015,113(1):33-40
Honda T,Yuxin NI,Miwatani T.Purification and characterization of a hemolysin produced by a clinical isolate of Kanagawa phenomenon-negative Vibrio parahaemolyticus and related to the thermostable direct hemolysin.Infection and Immunity,1988,56(4):961-965.
Hossain MT,Kim EY,Kim YR,et al.Application of gro EL gene for the species-specific detection of Vibrio parahaemolyticus by PCR.Letters in Applied Microbiology,2012,54(1):67-72
Hossain MT,Kim EY,Kim YR,et al.Development of a gro ELgene-based species-specific multiplex polymerase chain reaction assay for simultaneous detection of Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus.Journal of Applied Microbiology,2013,114(2):448-456
Huang J,Zeng LB,Dong X,et al.Trend analysis and policy recommendation on aquatic biosecurity in China.Engineering Sciences,2016,18(3):15-21[黄倢,曾令兵,董宣,等.水产生物安保发展趋势与政策建议.中国工程科学,2016,18(3):15-21]
Ishibashi M,Ohta K,Shimada T,et al.Current status of OKserotype combinations of Vibrio parahaemolyticus.Nippon Saikingaku Zasshi,2000,55:539-541
Joshi J,Srisala J,Truong VH,et al.Variation in Vibrio parahaemolyticus isolates from a single Thai shrimp farm experiencing an outbreak of acute hepatopancreatic necrosis disease(AHPND).Aquaculture,2014,428-429:297-302
Kondo H,Van PT,Dang LT et al.Draft genome sequence of non-Vibrio parahaemolyticus acute hepatopancreatic necrosis disease strain KC13.17.5,isolated from diseased shrimp in Vietnam.Genome Announcements,2015,3(5):e00978-15
Kongrueng J,Yingkajorn M,Bunpa S,et al.Characterization of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease in southern Thailand.Journal of Fish Diseases,2015,38(11):957-966
Lai HC,Ng TH,Ando M,et al.Pathogenesis of acute hepatopancreatic necrosis disease(AHPND)in shrimp.Fish and Shellfish Immunology,2015,47(2):1006-1014
Lee CT,Chen IT,Yang YT,et al.The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin.Proceedings of the National Academy of Sciences of the United States of America,2015,112(34):10798-10803
Liu LY,Xiao JZ,Xia XM,et al.Draft genome sequence of Vibrio owensii strain SH-14,which causes shrimp acute hepatopancreatic necrosis disease.Genome Announcements,2015,3(6):e01395-15
NACA.Report of the Asia Pacific emergency regional consultation on the emerging shrimp disease:Early mortality syndrome(EMS)/acute hepatopancreatic necrosis syndrome(AHPNS).2012
Nunan L,Lightner D,Pantoja C,et al.Detection of acute hepatopancreatic necrosis disease(AHPND)in Mexico.Diseases of Aquatic Organisms,2014,111(1):81-86
Rahimi E,Ameri M,Doosti A,et al.Occurrence of toxigenic Vibrio parahaemolyticus strains in shrimp in Iran.Foodborne Pathogens and Disease,2010,7(9):1107-1111
Reed LJ,Muench H.A simple method of estimating fifty percent endpoints.American Journal of Hygiene,1938,27(3):493-497
Soto-Rodriguez SA,Gomez-Gil B,Lozano-Olvera R,et al.Field and experimental evidence of Vibrio parahaemolyticus as the causative agent of acute hepatopancreatic necrosis disease of cultured shrimp(Litopenaeus vannamei)in Northwestern Mexico.Applied and Environmental Microbiology,2015,81(5):1689-1699
Sujeewa AKW,Norrakiah AS,Laina M.Prevalence of toxic genes of Vibrio parahaemolyticus in shrimps(Penaeus monodon)and culture environment.International Food Research Journal,2009,16:89-95
Tey YH,Jong KJ,Fen SY,et al.Genetic variation in Vibrio parahaemolyticus isolated from the aquacultural environments.Letters in Applied Microbiology,2015,60(4):321-327
Thitamadee S,Prachumwat A,Srisala J,et al.Review of current disease threats for cultivated penaeid shrimp in Asia.Aquaculture,2016,452:69-87
Tran L,Nunan L,Redman RM,et al.Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp.Diseases of Aquatic Organisms,2013,105(1):45-55
World Organization for Animal Health(OIE).Aquatic animal health code.Paris:OIE,2015
Xu X,Cheng J,Wu Q,et al.Prevalence,characterization,and antibiotic susceptibility of Vibrio parahaemolyticus isolated from retail aquatic products in North China.BMCMicrobiology,2016,16:32-41
Bennett PM.Plasmid encoded antibiotic resistance:Acquisition and transfer of antibiotic resistance genes in bacteria,British Journal of Pharmacology,2008,153(S1):S347-S357
Bureau of Fisheries and Fisheries Law Enforcement,Ministry of Agriculture.China fishery statistical yearbook.Beijing:China Agriculture Press,2016,28,30,71[农业部渔业渔政管理局.中国渔业统计年鉴.北京:中国农业出版社,2016,28,30,71]
Chen Y,Chen X,Yu F,et al.Serology,virulence,antimicrobial susceptibility and molecular characteristics of clinical Vibrio parahaemolyticus strains circulating in southeastern China from 2009 to 2013.Clinical Microbiology and Infection,2016,22(3):258(e9-e16)
COFI Sub-committee on Aquaculture.Aquatic biosecurity:A key for sustainable aquaculture development.Phuket:Co FISub-Committee on Aquaculture,2010[FAO渔业委员会水产养殖分委员会.水生生物安保:可持续水产养殖发展的一个关键.普吉:FAO渔业委员会水产养殖分委员会,2010]
Dabu IM,Lim JJ,Arabit PMT,et al.The first record of acute hepatopancreatic necrosis disease in the Philippines.Aquaculture Research,2017,48(3):792-7998
de la Pe?a LD,Cabillon NA,Catedral DD,et al.Acute hepatopancreatic necrosis disease(AHPND)outbreaks in Penaeus vannamei and P.monodon cultured in the Philippines.Diseases of Aquatic Organisms,2015,116(3):251-254
Dong X,Wang HL,Xie GS,et al.An isolate Vibrio campbellii carrying the pirVP gene causes acute hepatopancreatic necrosis disease.Emerging Microbes and Infections,2017,6(1):e2
Elmahdi S,Da Silva LV,Parveen S.Antibiotic resistance of Vibrio parahaemolyticus and Vibrio vulnificus in various countries:A review.Food Microbiology,2016,57:128-134
Flegel TW.Historic emergence,impact and current status of shrimp pathogens in Asia.Journal of Invertebrate Pathology,2012,110(2):166-173
Freel KC,Millán-Agui?aga N,Jensen PR.Multilocus sequence typing reveals evidence of homologous recombination linked to antibiotic resistance in the genus Salinispora.Applied and Environmental Microbiology,2013,79(19):5997-6005
Han JE,Tang KF,Tran LH,et al.Photorhabdus insect-related(Pir)toxin-like genes in a plasmid of Vibrio parahaemolyticus,the causative agent of acute hepatopancreatic necrosis disease(AHPND)of shrimp.Diseases of Aquatic Organisms,2015,113(1):33-40
Honda T,Yuxin NI,Miwatani T.Purification and characterization of a hemolysin produced by a clinical isolate of Kanagawa phenomenon-negative Vibrio parahaemolyticus and related to the thermostable direct hemolysin.Infection and Immunity,1988,56(4):961-965.
Hossain MT,Kim EY,Kim YR,et al.Application of gro EL gene for the species-specific detection of Vibrio parahaemolyticus by PCR.Letters in Applied Microbiology,2012,54(1):67-72
Hossain MT,Kim EY,Kim YR,et al.Development of a gro ELgene-based species-specific multiplex polymerase chain reaction assay for simultaneous detection of Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus.Journal of Applied Microbiology,2013,114(2):448-456
Huang J,Zeng LB,Dong X,et al.Trend analysis and policy recommendation on aquatic biosecurity in China.Engineering Sciences,2016,18(3):15-21[黄倢,曾令兵,董宣,等.水产生物安保发展趋势与政策建议.中国工程科学,2016,18(3):15-21]
Ishibashi M,Ohta K,Shimada T,et al.Current status of OKserotype combinations of Vibrio parahaemolyticus.Nippon Saikingaku Zasshi,2000,55:539-541
Joshi J,Srisala J,Truong VH,et al.Variation in Vibrio parahaemolyticus isolates from a single Thai shrimp farm experiencing an outbreak of acute hepatopancreatic necrosis disease(AHPND).Aquaculture,2014,428-429:297-302
Kondo H,Van PT,Dang LT et al.Draft genome sequence of non-Vibrio parahaemolyticus acute hepatopancreatic necrosis disease strain KC13.17.5,isolated from diseased shrimp in Vietnam.Genome Announcements,2015,3(5):e00978-15
Kongrueng J,Yingkajorn M,Bunpa S,et al.Characterization of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease in southern Thailand.Journal of Fish Diseases,2015,38(11):957-966
Lai HC,Ng TH,Ando M,et al.Pathogenesis of acute hepatopancreatic necrosis disease(AHPND)in shrimp.Fish and Shellfish Immunology,2015,47(2):1006-1014
Lee CT,Chen IT,Yang YT,et al.The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin.Proceedings of the National Academy of Sciences of the United States of America,2015,112(34):10798-10803
Liu LY,Xiao JZ,Xia XM,et al.Draft genome sequence of Vibrio owensii strain SH-14,which causes shrimp acute hepatopancreatic necrosis disease.Genome Announcements,2015,3(6):e01395-15
NACA.Report of the Asia Pacific emergency regional consultation on the emerging shrimp disease:Early mortality syndrome(EMS)/acute hepatopancreatic necrosis syndrome(AHPNS).2012
Nunan L,Lightner D,Pantoja C,et al.Detection of acute hepatopancreatic necrosis disease(AHPND)in Mexico.Diseases of Aquatic Organisms,2014,111(1):81-86
Rahimi E,Ameri M,Doosti A,et al.Occurrence of toxigenic Vibrio parahaemolyticus strains in shrimp in Iran.Foodborne Pathogens and Disease,2010,7(9):1107-1111
Reed LJ,Muench H.A simple method of estimating fifty percent endpoints.American Journal of Hygiene,1938,27(3):493-497
Soto-Rodriguez SA,Gomez-Gil B,Lozano-Olvera R,et al.Field and experimental evidence of Vibrio parahaemolyticus as the causative agent of acute hepatopancreatic necrosis disease of cultured shrimp(Litopenaeus vannamei)in Northwestern Mexico.Applied and Environmental Microbiology,2015,81(5):1689-1699
Sujeewa AKW,Norrakiah AS,Laina M.Prevalence of toxic genes of Vibrio parahaemolyticus in shrimps(Penaeus monodon)and culture environment.International Food Research Journal,2009,16:89-95
Tey YH,Jong KJ,Fen SY,et al.Genetic variation in Vibrio parahaemolyticus isolated from the aquacultural environments.Letters in Applied Microbiology,2015,60(4):321-327
Thitamadee S,Prachumwat A,Srisala J,et al.Review of current disease threats for cultivated penaeid shrimp in Asia.Aquaculture,2016,452:69-87
Tran L,Nunan L,Redman RM,et al.Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp.Diseases of Aquatic Organisms,2013,105(1):45-55
World Organization for Animal Health(OIE).Aquatic animal health code.Paris:OIE,2015
Xu X,Cheng J,Wu Q,et al.Prevalence,characterization,and antibiotic susceptibility of Vibrio parahaemolyticus isolated from retail aquatic products in North China.BMCMicrobiology,2016,16:32-41