多种细菌与凡纳滨对虾肝胰腺坏死症(HPNS)爆发有关
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摘要
近年来,在中国南方超过50%以上的凡纳滨对虾养殖场在养殖30 d后爆发1种疾病,导致80%以上的养殖对虾死亡。患病对虾临床症状为肝胰腺萎缩坏死、活动力减弱、对虾在池塘底部死亡,发病初期水面和池塘边观察不到病虾,因此养殖者通常将该病称为"偷死病"。患病对虾肝胰腺萎缩坏死是主要症状,因此,我们称这种对虾疾病为对虾肝胰腺坏死症(hepatopancreas necrosis syndrome,HPNS)。2012年3月-2013年10月,我们在中国南方广东、海南和广西3个省的12个养殖地区采集具有HPNS症状的凡纳滨对虾进行了组织病理观察、病毒检测与人工感染、细菌分离鉴定与人工感染研究。组织病理学研究表明具有HPNS症状的病虾肝胰腺坏死,在部分区域肝胰腺管细胞消失,肝胰腺小管间结缔组织减少。对305尾HPNS对虾进行11种对虾病毒PCR检测,并采用人工病毒感染方式感染健康对虾,感染对虾不表现HPNS症状。从63尾HPNS病虾的肝胰腺、血淋巴和肠道分离鉴定383株细菌,这些细菌分别属于10个属,49种细菌,每尾对虾均混合感染多种细菌,其中38尾对虾中分离到副溶血弧菌,34尾对虾中分离到蜡样芽孢杆菌,20尾对虾中分离到苏云金芽孢杆菌,19尾对虾中分离到霍乱弧菌。选取副溶血弧菌和苏云金芽孢杆菌分别采用注射和浸泡方式感染健康对虾,感染对虾均出现HPNS症状。结果表明多种细菌与凡纳滨对虾HPNS爆发有关。
In recent years,a disease outbreak occurred in > 50 percent of Litopenaeus vannamei farms at about 30 days after frys are released into the ponds in southern China. About 80% culture shrimps were dead in culture period. The clinical symptoms of the diseased shrimps include atrophy and necrosis of thehepatopancreas,weakened activity,and shrimp found dead in the bottom of the pond. The farmers in China usually called this symptom as"hidden death disease". Since hepatopancreas atrophy and necrosis are the main symptoms of the disease,so we called this disease as hepatopancreas necrosis syndrome( HPNS). In March 2012 to October 2013,we have collected the diseased L. vannamei with the symptoms of HPNS in 12 farms from Guangdong,Hainan and Guangxi provinces,China. Histopathology showed necrosis of hepatopancreas of the diseased shrimps,disappearance of some hepatopancreatic epidermal cells,and connective tissue. We used PCR to detect the presence of 11 viruses in 305 HPNS shrimps and performed artificial viral infection to healthy shrimps. The artificially infected shrimps did not show symptoms of HPNS. A total of 383 bacteria strains were identified from hepatopancreas,hemolymph and intestine of 63 diseased shrimps. These bacteria belong to 10 genera,49 species. We isolated Vibrio parahaemolyticus from 38 shrimps,Bacillus cereus from 34 shrimps,B. thuringiensis from 20 shrimps and V. cholera from 19 shrimps. Injection and immersion of V. parahaemolyticus and B. thuringiensis can cause symptoms of HPNS. The results suggest that multiple bacteria were involved in HPNS of L. vannamei.
In recent years,a disease outbreak occurred in > 50 percent of Litopenaeus vannamei farms at about 30 days after frys are released into the ponds in southern China. About 80% culture shrimps were dead in culture period. The clinical symptoms of the diseased shrimps include atrophy and necrosis of thehepatopancreas,weakened activity,and shrimp found dead in the bottom of the pond. The farmers in China usually called this symptom as"hidden death disease". Since hepatopancreas atrophy and necrosis are the main symptoms of the disease,so we called this disease as hepatopancreas necrosis syndrome( HPNS). In March 2012 to October 2013,we have collected the diseased L. vannamei with the symptoms of HPNS in 12 farms from Guangdong,Hainan and Guangxi provinces,China. Histopathology showed necrosis of hepatopancreas of the diseased shrimps,disappearance of some hepatopancreatic epidermal cells,and connective tissue. We used PCR to detect the presence of 11 viruses in 305 HPNS shrimps and performed artificial viral infection to healthy shrimps. The artificially infected shrimps did not show symptoms of HPNS. A total of 383 bacteria strains were identified from hepatopancreas,hemolymph and intestine of 63 diseased shrimps. These bacteria belong to 10 genera,49 species. We isolated Vibrio parahaemolyticus from 38 shrimps,Bacillus cereus from 34 shrimps,B. thuringiensis from 20 shrimps and V. cholera from 19 shrimps. Injection and immersion of V. parahaemolyticus and B. thuringiensis can cause symptoms of HPNS. The results suggest that multiple bacteria were involved in HPNS of L. vannamei.
引文
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[10]ANDERSON J,VALDERRAMA D.Production:Global shrimp review-disease drops production,but recovery anticipated[J].Global Aquaculture Advocate,2013,November/December:12-13.
[11]BONDAD-REANTASO M G,SUBASINGHE R P,JOSUPEIT H,et al.The role of crustacean fisheries and aquaculture in global food security:past,present and future[J].Journal of Invertebrate Pathology,2012,110:158-165.
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[13]STENTIFORD G D,NEIL D M,PEELER E J,et al.Disease will limit future food supply from the global crustacean fishery and aquaculture sectors[J].Journal of Invertebrate Pathology,2012,110:141-157.
[14]EDUARDO M L,MOHAN C V.Early mortality syndrome threatens asia's shrimp farms[J].Global Aquaculture Advocate,2012,July/August:38-39.
[15]TRAN L,NUNAN L,REDMAN R M,et al.Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp[J].Diseases of Aquatic Organisms,2013a,105:45-55.
[16]TRAN L,NUNAN L,REDMAN R M,et al.EMS/AHPNS:infectious disease caused by bacteria[J].Global Aquaculture Advocate,2013b,July/August:18-20.
[17]HAN J E,TANG K F,TRAN L H,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[J].Diseases of Aquatic Organisms,2015,113(1):33-40.doi:10.3354/dao02830.
[18]BELL T A,LIGHTNER D V.A handbook of normal shrimp histology[R].Special Publication No.1.World Aquaculture Society.Baton Rouge,LA,1988.
[19]LIGHTNER D V.A handbook of shrimp pathology and diagnostic procedures for diseases of cultured penaeid shrimp[R].World Aquaculture Society.Baton Rouge,LA,1996.
[20]KCHL S,NIEDERSTTTER H,PARSON W.DNA extraction and quantitation of forensic samples using the phenol-chloroform method and real-time PCR[J].Methods in Molecular Biology,2005,297:13-30.
[21]HOLT J G.Bergey's Manual of Determinative Bacteriology[M].9th ed.Baltimore:Williams&Wilkins,1984.
[22]HORZ H P,VIANNA M E,GOMES B P F A,et al.Evaluation of universal probes and primer sets for assessing total bacterial load in clinical samples:general implications and practical use in endodontic antimicrobial therapy[J].Journal of Clinical Microbiology,2005,43:5332-5337.
[2]GAA.Cause of EMS shrimp disease identified[J].Global Aquaculture Advocate,2013,July/August:8.
[3]FAO.Report of the FAO/MARD technical workshop on early mortality syndrome(EMS)or acute hepatopancreatic necrosis syndrome(AHPND)of cultured shrimp(under TCP/VIE/3304)[R].FAO Fisheries and Aquaculture Report No.1053.Rome.Hanoi,Viet Nam,2013:54.
[4]LIGHTNER D V,REDMAN R M,PANTOJA C R,et al.Historic emergence,impact and current status of shrimp pathogens in the Americas[J].Journal of Invertebrate Pathology,2012,110:174-183.
[5]MOONEY A.An emerging shrimp disease in Vietnam,microsporidiosis or liver disease?[EB/OL].[2012-02-24].http:∥aquatichealth.net/issues/38607.
[6]NACA-FAO.Quarterly aquatic animal disease report(Asia and Pacific Region)[R].NACA,Bangkok,Thailand,2011.
[7]ANUPARP P A,THITAMADEE S,SRIURAIRATANA S,et al.Shotgun sequencing of bacteria from AHPNS,a new shrimp disease threat for Thailand[Z].Poster,National Institute for Aquaculture Biotechnology,Mahidol University.Bangkok,Thailand,2012.
[8]FLEGEL T W.Historic emergence,impact and current status of shrimp pathogens in Asia[J].Journal of Invertebrate Pathology,2012,110:166-173.
[9]LEAO E M,MOHAN C V.Early mortality syndrome threatens Asia's shrimp farms[J].Global Aquaculture Advocate,2012,July/Aug:38-39.
[10]ANDERSON J,VALDERRAMA D.Production:Global shrimp review-disease drops production,but recovery anticipated[J].Global Aquaculture Advocate,2013,November/December:12-13.
[11]BONDAD-REANTASO M G,SUBASINGHE R P,JOSUPEIT H,et al.The role of crustacean fisheries and aquaculture in global food security:past,present and future[J].Journal of Invertebrate Pathology,2012,110:158-165.
[12]NEWMAN S G.Impacts of acute hepatopancreatic necrosis syndrome-short-term market dynamics affect long-term practices[J].Global Aquaculture Advocate,2013,May/June:16-17.
[13]STENTIFORD G D,NEIL D M,PEELER E J,et al.Disease will limit future food supply from the global crustacean fishery and aquaculture sectors[J].Journal of Invertebrate Pathology,2012,110:141-157.
[14]EDUARDO M L,MOHAN C V.Early mortality syndrome threatens asia's shrimp farms[J].Global Aquaculture Advocate,2012,July/August:38-39.
[15]TRAN L,NUNAN L,REDMAN R M,et al.Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp[J].Diseases of Aquatic Organisms,2013a,105:45-55.
[16]TRAN L,NUNAN L,REDMAN R M,et al.EMS/AHPNS:infectious disease caused by bacteria[J].Global Aquaculture Advocate,2013b,July/August:18-20.
[17]HAN J E,TANG K F,TRAN L H,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[J].Diseases of Aquatic Organisms,2015,113(1):33-40.doi:10.3354/dao02830.
[18]BELL T A,LIGHTNER D V.A handbook of normal shrimp histology[R].Special Publication No.1.World Aquaculture Society.Baton Rouge,LA,1988.
[19]LIGHTNER D V.A handbook of shrimp pathology and diagnostic procedures for diseases of cultured penaeid shrimp[R].World Aquaculture Society.Baton Rouge,LA,1996.
[20]KCHL S,NIEDERSTTTER H,PARSON W.DNA extraction and quantitation of forensic samples using the phenol-chloroform method and real-time PCR[J].Methods in Molecular Biology,2005,297:13-30.
[21]HOLT J G.Bergey's Manual of Determinative Bacteriology[M].9th ed.Baltimore:Williams&Wilkins,1984.
[22]HORZ H P,VIANNA M E,GOMES B P F A,et al.Evaluation of universal probes and primer sets for assessing total bacterial load in clinical samples:general implications and practical use in endodontic antimicrobial therapy[J].Journal of Clinical Microbiology,2005,43:5332-5337.