红鳍东方鲀病原鱼肠道弧菌的生物学特性研究
STUDIES ON BIOLOGICAL CHARACTERISTICS OF PATHOGENIC VIBRIO ICHTHYOENTERI ISOLATED FROM PUFFERFISH (TAKIFUGU RUBRIPS L. )
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摘要: 对引起红鳍东方鲀发病死亡的病原细菌进行了分离和主要生物学特性研究,包括病原性、形态特征、理化特性、16S rRNA基因序列及其系统发育学分析、胞外酶及溶血素活性、K抗原及耐药性等。结果表明,引起红鳍东方鲀发病死亡的病原细菌为弧菌属(VibrioPacini 1854)的鱼肠道弧菌(V.ichthyoenteri Ishimaru,et al.1996),2株代表菌株16S rRNA基因序列(GenBank登录号分别为:EF611424和EF635304)与GenBank数据库中鱼肠道弧菌的同源性在98%—100%,且在构建的MP系统发生树中与鱼肠道弧菌聚为一个分支。分离菌不具有淀粉酶、蛋白酶、脂肪酶、DNA酶、脲酶、明胶酶和卵磷脂酶活性,且在含7%家兔脱纤血液营养琼脂培养基上不溶血。不具有K抗原。人工感染试验中分离菌对红鳍东方鲀表现出明显的致病性。药敏试验结果显示,4株分离菌对供试37种抗菌药物中的苯唑青霉素和杆菌肽2种耐药。Abstract: Pufferfish(Takifugu rubrips L.) of the Order Tetraodontiformes is a mariculture fish with high economic value in the aquaculture industry in northern China.Presently,commercial production of pufferfish in China is hampered by variable mortalities and this may be due,at least in part,to pathogenic bacteria.In November 2005,our laboratory examined diseases occurring in pufferfish,and Vibrio ichthyoenteri was isolated and identified based on its extensive phenotypic informations.In the present paper,the isolation and characterization of the pathogenic bacteria obtained from pufferfish are described.Biological study was carried out on pathogenic bacteria isolated from infected individuals using traditional and modern molecular techniques in morphology,physiology and biochemistry.Molecular identification of the isolates was conducted,the 16S rRNA genes were partially sequenced and compared with those of databases,and a molecular phylogenetic tree was constructed.The representative strain(HC051105-1) was used in experimental infection of selected pufferfish to confirm pathogenicity.In addition,extracellulase activity,hemolytic activity and drug resistance of isolates were all examined,and the isolates were checked for the presence of K-antigen by slide agglutination and tube agglutination using rabbit antiserum.Bacteriological cultures on nutrient agar and blood agar from the liver,kidney and spleen of moribund(or dead) pufferfish yielded an apparently pure culture.Four strains(HC051105-1,HC051105-3,HC051105-5 and HC051105-7) isolated from liver and kidney were randomly selected and examined and found quite similar in morphology,being Gram-negative,rod-shaped(some slightly curved),single with round ends,no-endospores,and(0.4―0.6)μm×(1.0―2.5)μm in size.The sequenced 16S rRNA gene of represent strains HC051105-1 and HC051105-3(GenBank accession No.EF611424 and EF635304) were 1452 bp and 1461 bp in length respectively,and the two strains shared high similarity(98%―100%) with the 16S rRNA gene of V.ichthyoenteri from GenBank database.The results showed that the identified strain belonged to V.ichthyoenteri(Ishimaru,et al 1996) in terms of morphology,physiology and biochemistry up with the regarding criteria,and the results of molecular identification(16S rRNA partial sequence).The phenotypic determination of extracellulase activity and hemolytic activity showed the isolates have not diastase,proteinase,Lipase,DNase,lecithinase,urease and gelatinase,and could not produce haemolysin.Detection of K-antigen revealed the isolates have not K-antigen.In addition,an artificial infection experiment of strain(HC051105-1) revealed correspondent pathogenicity of V.ichthyoenteri to pufferfish.The susceptibility of isolates to 37 antimicrobial agents showed that 4 strains were all resistant to oxacillin and bacitracin.The study further demonstrated the significance of V.ichthyoenteri as pathogenic bacteria and extensive pathogenicity in the fish species.
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