海南省南美白对虾锡那罗州弧菌的分离鉴定
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摘要
为了有效防治海南儋州南美白对虾病害,试验首先采用常规方法鉴定从病虾肝胰腺分离的优势菌株的形态、培养特性、生理生化反应等,并用聚合酶链式反应(PCR)扩增16S r DNA序列;其次,用人工感染试验检测分离菌株的致病性;最后,通过体外裂解试验测定噬菌体(噬菌28TM)对分离株的裂解能力。结果表明:该分离株HYRJ2013为锡那罗州弧菌(Vibrio sinaloensis)。肌肉注射HYRJ2013菌株感染南美白对虾,出现与自然发病虾相似症状,测得其半数致死量(LD50)为6.68×107cfu/m L。噬菌体(噬菌28TM)可裂解该菌株,用于生物防治。说明锡那罗州弧菌是南美白对虾的条件致病菌。
To effective prevent and control of the disease of Penaeus vannamei Boone( P. vannamei Boone) in Hainan province,the morphology,cultural characteristics,physiological and biochemical reactions of the dominant strain isolated from the hepatopancreas tissue of diseased P.vannamei Boone were identified by routine methods,and 16 S r DNA sequence was amplified by polymerase chain reaction( PCR) for sequencing analysis. Furthermore,the artificial experimental infection was conducted to detect the pathogenicity of the isolate. Finally,the lytic ability of phage Shijun28 TMagainst the isolate was detected by in vitro lysis test. The results showed that the isolate( HYRJ2013) was identified as Vibrio sinaloensis. There were similar symptoms with the natural incidence of P. vannamei Boone when the isolate HYRJ2013 was used to infect P. vannamei Boone by muscular injection. The median lethal dose( LD50) was measured as 6. 68 × 107 cfu / m L. The phage Shijun28 TMcould lyse the isolate and be used for biological control. The results indicate that Vibrio sinaloensis is an opportunistic pathogen to P. vannamei Boone.
To effective prevent and control of the disease of Penaeus vannamei Boone( P. vannamei Boone) in Hainan province,the morphology,cultural characteristics,physiological and biochemical reactions of the dominant strain isolated from the hepatopancreas tissue of diseased P.vannamei Boone were identified by routine methods,and 16 S r DNA sequence was amplified by polymerase chain reaction( PCR) for sequencing analysis. Furthermore,the artificial experimental infection was conducted to detect the pathogenicity of the isolate. Finally,the lytic ability of phage Shijun28 TMagainst the isolate was detected by in vitro lysis test. The results showed that the isolate( HYRJ2013) was identified as Vibrio sinaloensis. There were similar symptoms with the natural incidence of P. vannamei Boone when the isolate HYRJ2013 was used to infect P. vannamei Boone by muscular injection. The median lethal dose( LD50) was measured as 6. 68 × 107 cfu / m L. The phage Shijun28 TMcould lyse the isolate and be used for biological control. The results indicate that Vibrio sinaloensis is an opportunistic pathogen to P. vannamei Boone.
引文
[1]沈文英,阳会军,尹军霞.南美白对虾的病害及防治研究现状[J].水利渔业,2004,24(1):58-60.
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[5]金春英.南美白对虾弧菌病病原的分离和鉴定[J].海洋环境科学,2013,32(5):647-651.
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[14]CHIMETTO L A,CLEENWERCK I,MOREIRA A P,et al.Vibrio variabilis sp.nov.and Vibrio maritimus sp.nov.,isolated from Palythoa caribaeorum[J].Int J Syst Evol Microbiol,2011,61(12):3009-3015.
[15]GOMEZ-GIL B,FAJER-AVILA E,PASCUAL J,et al.Vibrio sinaloensis sp.nov.,isolated from the spotted rose snapper,Lutjanus guttatus Steindachner,1869[J].Int J Syst Evol Micr,2008,58(7):1621-1624.
[16]苏杭.养殖凡纳滨对虾病原菌(哈维氏弧菌)的分离鉴定与防治[D].成都:四川农业大学,2010.
[17]FLORES-MIRANDA M D C,LUNA-GONZALEZ A,CAMPACORDOVA A I,et al.Microbial immunostimulants reduce mortality in whiteleg shrimp(Litopenaeus vannamei)challenged with Vibrio sinaloensis strains[J].Aquaculture,2011,320(1/2):51-55.
[18]STALEY C,CHASE E,HARWOOD V J.Detection and differentiation of Vibrio vulnificus and V.sinaloensis in water and oysters of a Gulf of Mexico estuary[J].Environ Microbiol,2013,15(2):623-633.
[19]刘晓贺,顾敬敏,韩文瑜,等.应用噬菌体GH15和K治疗金黄色葡萄球菌感染[J].微生物学报,2013,53(5):498-506.
[2]SOTO-RODRIGUEZ S A,GOMEZ-GIL B,LOZANO R,et al.Virulence of Vibrio harveyi responsible for the“Bright-red”Syndrome in the Pacific white shrimp Litopenaeus vannamei[J].J Invertebr Pathol,2012,109(3):307-317.
[3]PHUOC L H,CORTEEL M,NAUWYNCK H J,et al.Increased susceptibility of white spot syndrome virus-infected Litopenaeus vannamei to Vibrio campbellii[J].Environ Microbiol,2008,10(10):2718-2727.
[4]SELVIN J,LPITON A P.Vibrio alginolyticus associated with white spot disease of Penaeus monodon[J].Dis Aquat Organ,2003,57(1/2):147-150.
[5]金春英.南美白对虾弧菌病病原的分离和鉴定[J].海洋环境科学,2013,32(5):647-651.
[6]东秀珠.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[7]MA F M,ANTONIO L G,CRDOVA A I C,et al.Isolation and charaterization of infectious Vibrio sinaloensis strains from the Pacific shrimp Litppenaeus vannamei(Decapoda:Penaeidae)[J].Rev Biol Trop,2012,60(2):567-576.
[8]张宝存,刘飞,边慧慧,等.一株凡纳滨对虾病原菌的分离、鉴定及其致病力分析[J].渔业科学进展,2012,33(2):56-62.
[9]SAMOCHA T M,LAWRENCE A L,COLLINS C A,et al.Production of the Pacific white shrimp,Litopenaeus vannamei,in highdensity greenhouse-enclosed raceways using low salinity groundwater[J].J Appl Aquacult,2004,15(3):1-19.
[10]ESIOBU N,AMENTA L,IKE J.Antibiotic resistance in soil and water enviroments[J].Int J Environ Health Res,2002,12(2):133-144.
[11]AKINBOWALE O L,PENG H,BARTON M D.Antimicrobial resistance in bacteria isolated from aquaculture sources in Australia[J].J App Microbiol,2006,100(5):1103-1113.
[12]吴后波,潘金培.海水养殖真鲷弧菌病病原菌外毒素的理化特性[J].水生生物学报,2004,28(4):409-412.
[13]KUSHMARO A,BANIN E,LOYA Y,et al.Vibrio shiloi sp nov.,the causative agent of bleaching of the coraloculina patagonica[J].Intl J Syst Evol Microbiol,2001,51(4):1383-1388.
[14]CHIMETTO L A,CLEENWERCK I,MOREIRA A P,et al.Vibrio variabilis sp.nov.and Vibrio maritimus sp.nov.,isolated from Palythoa caribaeorum[J].Int J Syst Evol Microbiol,2011,61(12):3009-3015.
[15]GOMEZ-GIL B,FAJER-AVILA E,PASCUAL J,et al.Vibrio sinaloensis sp.nov.,isolated from the spotted rose snapper,Lutjanus guttatus Steindachner,1869[J].Int J Syst Evol Micr,2008,58(7):1621-1624.
[16]苏杭.养殖凡纳滨对虾病原菌(哈维氏弧菌)的分离鉴定与防治[D].成都:四川农业大学,2010.
[17]FLORES-MIRANDA M D C,LUNA-GONZALEZ A,CAMPACORDOVA A I,et al.Microbial immunostimulants reduce mortality in whiteleg shrimp(Litopenaeus vannamei)challenged with Vibrio sinaloensis strains[J].Aquaculture,2011,320(1/2):51-55.
[18]STALEY C,CHASE E,HARWOOD V J.Detection and differentiation of Vibrio vulnificus and V.sinaloensis in water and oysters of a Gulf of Mexico estuary[J].Environ Microbiol,2013,15(2):623-633.
[19]刘晓贺,顾敬敏,韩文瑜,等.应用噬菌体GH15和K治疗金黄色葡萄球菌感染[J].微生物学报,2013,53(5):498-506.