诸氏鲻虾虎鱼致病性创伤弧菌的分离与鉴定
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摘要
从患病的诸氏鲻虾虎鱼分离病原,研究其致病性。通过形态学观察、生理生化特性、HSP60序列扩增及系统发育树分析等方法系统鉴定,并进行了人工感染和毒力基因检测。结果表明,从患病的诸氏鲻虾虎鱼肝脏组织分离到1株革兰阴性杆菌,编号PYMc14-2,回归感染试验证实该菌株为诸氏鲻虾虎鱼的病原菌,毒力基因hemo检测结果阳性。生化特性结果与创伤弧菌类似,PCR扩增获得的486bp的HSP60基因序列与创伤弧菌(登录号AF230955.1)同源性达94%;系统进化树分析表明,PYMc14-2与创伤弧菌(登录号AF230955.1)聚为一簇,初步鉴定该菌为创伤弧菌。
In order to isolate and identify the pathogenic bacteria in diseased Mugilogobius chulae,and analyse the pathogenicity of the bacteria,the bacterial pathogens were isolated from the liver tissue of diseased M.chulae,and then traditional physiological,biochemical experiments and HSP60 molecular method were done,while the virulence-related gene(hemolysin)of Vibrio vulnificuss was investigated by PCR.Strain PYMc14-2was confirmed to be the pathogen of the disease by challenge test.The results showed that strain PYMc14-2was similar to V.vulnificusin most of the phenotypes.In order to investigate the phylogenetic position of the pathogen,HSP60 gene partial sequences of strain PYMc14-2was determined.The results showed that the HSP60 gene sequence of strain PYMc14-2was 486 bp in length and its similarity with V.vulnificus(AF230955.1)in GenBank was 94%.Phylogenetic tree of Vibrio based on HSP60 genes showed that strain PYMc14-2clustered together with V.vulnificus(AF230955.1).Based on the results obtained,the strain PYMc14-2was identified as V.vulnificus.
In order to isolate and identify the pathogenic bacteria in diseased Mugilogobius chulae,and analyse the pathogenicity of the bacteria,the bacterial pathogens were isolated from the liver tissue of diseased M.chulae,and then traditional physiological,biochemical experiments and HSP60 molecular method were done,while the virulence-related gene(hemolysin)of Vibrio vulnificuss was investigated by PCR.Strain PYMc14-2was confirmed to be the pathogen of the disease by challenge test.The results showed that strain PYMc14-2was similar to V.vulnificusin most of the phenotypes.In order to investigate the phylogenetic position of the pathogen,HSP60 gene partial sequences of strain PYMc14-2was determined.The results showed that the HSP60 gene sequence of strain PYMc14-2was 486 bp in length and its similarity with V.vulnificus(AF230955.1)in GenBank was 94%.Phylogenetic tree of Vibrio based on HSP60 genes showed that strain PYMc14-2clustered together with V.vulnificus(AF230955.1).Based on the results obtained,the strain PYMc14-2was identified as V.vulnificus.
引文
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[14]刘秀珍,邹晓理,莫小燕,等.海水网箱养殖石斑鱼病原菌研究[J].热带海洋,1994,13(1):81-86.
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[16]黎炯,叶星,卢迈新,等.罗非鱼创伤弧菌的分离鉴定和药敏试验[J].江西农业大学学报,2011,33(5):965-970.
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[18]吴后波,潘金培.弧菌属细菌及其所致海水养殖动物疾病[J].中国水产科学,2001,8(1):89-93.
[2]李建军,杨笑波,魏社林,等.裸项栉虾虎鱼的全人工繁殖及其胚胎发育[J].中国实验动物学报,2008,16(2):111-116.
[3]李建军,吴美慧,叶惠欣,等.不同发育期诸氏鲻虾虎鱼对钻井液的敏感性比较[J].中国比较医学杂志,2013,23(4):48-51.
[4]李建军,林忠婷,陈小曲,等.四种重金属离子对诸氏鲻虾虎鱼的单一和联合毒性[J].海洋环境科学,2014,33(2):236-241.
[5]余露军,李宁求,刘礼辉,等.日本鳗鲡混合感染迟缓爱德华氏菌与创伤弧菌的分离与鉴定[J].中国人兽共患病学报,2009,25(8):799-803.
[6]兰云,胡秀彩,沈晓静,等.黄喉拟水龟腐皮病病原菌的分离鉴定及药敏试验[J].动物医学进展,2014,35(9):127-131.
[7]胡秀彩,边延峰,赵腊梅,等.鱼源维氏气单胞菌的分离鉴定[J].动物医学进展,2013,34(12):232-235.
[8]Goh S H,Facklam R R,Chang M.Identification of Enterococcus species and phenotypically similar Lactococcus and Vagococcus species by reverse checkerboard hybridization to chaperonin 60gene sequences[J].J Clin Microbiol,2000,38(11):3953-3959.
[9]Kowk A Y,Chow A W.Phylogenetic study of Staphylococcus and Macrococcus species based on partial Hsp60gene sequences[J].Int J Syst Evol Microbiol,2003,53(1):87-92.
[10]李宁求,白俊杰,吴淑勤,等.斜带石斑鱼3种致病性弧菌的分子生物学鉴定[J].水产学报,2005,29(3):356-361.
[11]钟英斌,张晓华,陈吉祥,等.哈维氏弧菌溶血素基因vhhA在大肠杆菌中的表达及活性研究[J].中国海洋大学学报,2007,37(1):97-102.
[12]王远微,汤承,于学辉,等.三重PCR检测鱼类致病性嗜水气单胞菌[J].微生物学报,2008,48(7):947-951.
[13]许斌福,林天龙,董传甫.鳗鲡创伤弧菌的分子鉴定[J].中国人兽共患病学报,2005,21(11):995-980.
[14]刘秀珍,邹晓理,莫小燕,等.海水网箱养殖石斑鱼病原菌研究[J].热带海洋,1994,13(1):81-86.
[15]马爱敏,闫茂仓,常维山,等.黄姑鱼创伤弧菌的分离和鉴定[J].中国人兽共患病学报,2008,24(10):960-964.
[16]黎炯,叶星,卢迈新,等.罗非鱼创伤弧菌的分离鉴定和药敏试验[J].江西农业大学学报,2011,33(5):965-970.
[17]张晓君,毕可然,阎斌伦,等.矛尾复虾虎鱼病原哈氏弧菌的鉴定及特异性检测方法的建立[J].水产科学,2011,30(12):759-763.
[18]吴后波,潘金培.弧菌属细菌及其所致海水养殖动物疾病[J].中国水产科学,2001,8(1):89-93.