摘要
为研究虹鳟Oncorhynchus mykiss致病性杀鲑气单胞菌Aeromonas salmonicida的致病机理,利用微生物学及分子生物学方法,从患皮肤溃疡病的虹鳟病灶处分离纯化获得1株优势菌株RBWF-1,通过人工感染试验证实其具有较强的致病性。结果表明:菌株RBWF-1对虹鳟的半致死浓度(LD50)为5.5×10~6CFU/m L;通过对菌株RBWF-1 16S r DNA基因序列扩增、测序及Blast对比,发现其与杀鲑气单胞菌杀日本鲑亚种Asalmonicida subsp.masoucida菌株NBRC 13748及无色亚种A.salmonicida subsp.achromogens菌株NCIMB 1110的16S rDNA基因序列同源性最高,一致性均为99.6%;构建系统发育树显示,菌株RBWF-1与杀鲑气单胞菌杀鲑亚种A.salmonicida subsp.salmonicida、杀日本鲑亚种、无色亚种和史氏亚种A.salmonicida subsp.smithia聚为一支;为进一步明确菌株的分类地位,利用Biolog微生物鉴定系统结合传统生理生化试验对菌株RBWF-1各项理化指标进行检测,经对比,与杀鲑气单胞菌杀日本鲑亚种吻合度最高,因而判断该分离株为杀鲑气单胞菌杀日本鲑亚种;同时,对分离菌株的气溶素、溶血素、封闭带毒素、DNA酶、丝氨酸蛋白酶、磷脂酶、S层蛋白7个毒力相关基因进行了PCR检测,检测结果均呈阳性;酶活性检测结果显示,菌株RBWF-1胞外产物具有蛋白酶、淀粉酶、脂酶、DNA酶和溶血活性。本研究结果可为中国虹鳟养殖过程中杀鲑气单胞菌的感染特征、分类鉴定和疫苗研制提供必要的数据和参考。
One dominant bacterial strain RBWF-1 was isolated from the skin ulcer lesion of diseased rainbow trout Oncorhynchus mykiss,and its virulence was proved to be high to healthy rainbow trout by artificial LD50 value of 5.5×10~6 CFU/m L infection test to elucidate the mechanism of Aeromonas salmonicida in rainbow thout. 16S rDNA gene of strain RBWF-1 was amplified and sequenced,and was highly homologous with Aeromonas salmonicida subsp. masoucida strain NBRC 13748 and A. salmonicida subsp. achromogenes strain NCIMB 1110,with similarity of99.6%. Phylogenetic tree based on 16S rDNA gene revealed that strain RBWF-1 clustered together with A.salmonicida subsp. salmonicida,A. salmonicida subsp. masoucida,A. salmonicida subsp. achromogenes and A. salmonicida subsp. smithia. Biolog's identification system combined with physiological and biochemical test indicated that strain RBWF-1 had the highest similarity with those of A.salmonicida subsp. masoucida,and was identified to be A. salmonicida subsp. salmonicida. Meanwhile,7 virulence-related genes including haemolysin,serine protease,lipase,DNases,aerolysin,zonula occludens toxin and S-layer protein,were all detected in the isolated strain. The enzymatic activity test showed that the extracellular products of strain RBWF-1 had the activities of protease,lipase,amylase,DNase and hemolysin. The findings provide the necessary data and information for the infectious characteristics,taxonomic identification and vaccine development of A.salmonicida in rainbow trout cultured in China.
引文
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