鲮鱼致病性迟钝爱德华氏菌的鉴定及药敏分析
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摘要
自广东省清远市某养殖场患病鲮鱼的肝、肾、脾组织分离到两株优势菌QY15814、QY15815,通过细菌生化鉴定管与16SrDNA序列分析对这两株菌进行鉴定,采用纸片扩散法进行药敏试验,并测定了这两株菌对鲮鱼的半致死剂量。试验结果显示,这两株菌为革兰氏阴性短杆菌,兼性厌氧,有动力,能利用蔗糖、甘露糖、麦芽糖,硝酸盐还原、吲哚、MR试验均为阳性;根据其生理生化特性,鉴定为迟钝爱德华氏菌,通过PCR扩增其16SrDNA基因,经测序及NCBI比对,结果与迟钝爱德华氏菌的同源性高达99%。这两株致病菌对头孢噻肟、头孢吡肟、头孢曲松、头孢西丁、头孢哌酮、头孢呋辛等抗生素高度敏感,对链霉素、克拉霉素、红霉素中度敏感,对阿米卡星、复方新诺明、大观霉素、克林霉素不敏感。这两株菌对鲮鱼具有较强的致病力,半致死剂量分别为1.93×105cfu/g和9.65×104cfu/g。
Two dominant bacterial strains(QY15814and QY15815)were isolated from hepatopancreas,kidney and spleen of diseased mud carp(Cirrhinus molitorella)in an aquafarm in Qingyuan city,Guangdong province.They were identified through biochemical identification tubes and 16 SrDNA sequence analysis,and antibiotic susceptibility test was performed through Kirby-Bauer disk diffusion method.Moreover,the median lethal dose values(LD50)for the isolates to mud carp were determined.The results showed that the isolates were facultative anaerobes which were gram negative,rod in shape,and motile.Their indole,MR and nitrate reduction test were positive,and they utilized sucrose,mannose and maltose.The isolates were identified as Edwardsiella tarda according to their physiological and biochemical characteristics.The 16 SrDNA sequences of the isolates showed a high similarity(99%)to that of E.tardavia BLAST network services in NCBI.The isolates were highly susceptible to cefotaxime,cefepime,ceftriaxone,cefoxitin,cefoperazone,cefuroxime and the others,intermediately susceptible to phytomycin,clarithromycin,erythromycin,and not susceptible to amikacin,complex sinomin,spectinomycin,and clindamycin.The LD50 values for the isolates to healthy mud carps were 1.93×105cfu/g and 9.65×104cfu/g,respectively,indicating that they had strong pathogenicity to mud carp.
Two dominant bacterial strains(QY15814and QY15815)were isolated from hepatopancreas,kidney and spleen of diseased mud carp(Cirrhinus molitorella)in an aquafarm in Qingyuan city,Guangdong province.They were identified through biochemical identification tubes and 16 SrDNA sequence analysis,and antibiotic susceptibility test was performed through Kirby-Bauer disk diffusion method.Moreover,the median lethal dose values(LD50)for the isolates to mud carp were determined.The results showed that the isolates were facultative anaerobes which were gram negative,rod in shape,and motile.Their indole,MR and nitrate reduction test were positive,and they utilized sucrose,mannose and maltose.The isolates were identified as Edwardsiella tarda according to their physiological and biochemical characteristics.The 16 SrDNA sequences of the isolates showed a high similarity(99%)to that of E.tardavia BLAST network services in NCBI.The isolates were highly susceptible to cefotaxime,cefepime,ceftriaxone,cefoxitin,cefoperazone,cefuroxime and the others,intermediately susceptible to phytomycin,clarithromycin,erythromycin,and not susceptible to amikacin,complex sinomin,spectinomycin,and clindamycin.The LD50 values for the isolates to healthy mud carps were 1.93×105cfu/g and 9.65×104cfu/g,respectively,indicating that they had strong pathogenicity to mud carp.
引文
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[15]王方华,邹为民,李安兴.广东贺江水域野生鲮鱼体表寄生虫典型海弯水虱的种群动态[J].动物学报,2008,54(3):407-415.
[16]刘礼辉,张德锋,李宁求,等.鲮鱼源无乳链球菌的鉴定、血清型分析及药敏试验[J].南方农业学报,2015,46(11):2053-2058.
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[21]Grimont P A D,Grimont F,Richard C,et al.Edwardsiella hoshinae,a new species of Enterobacteriaceae[J].Current Microbiology,1980,4(6):347-351.
[22]王波,莫照兰.迟缓爱德华氏菌及其致病机理[J].海洋科学集刊,2007(48):133-139.
[23]刁菁,杨秀生,叶海斌,等.一种水产迟钝爱德华氏菌快速药敏检测方法的研究[J].水产科学,2014,33(1):22-28.
[24]王燕,张晓华,吕俊超,等.养殖大菱鲆病原菌迟缓爱德华氏菌的分离鉴定及其疫苗研制[J].中国水产科学,2009,16(3):394-403.
[25]张晓君,战文斌,陈翠珍,等.牙鲆迟钝爱德华氏菌感染症及其病原的研究[J].水生生物学报,2005,29(1):31-37.
[26]杨春志,王秀华,黄倢.养殖大菱鲆迟缓爱德华氏菌的分离鉴定与系统发育分析[J].上海水产大学学报,2008,17(3):280-284.
[27]葛慕湘,靳晓敏,张艳英,等.迟钝爱德华氏菌耐药表型及4种耐药基因检测[J].水产科学,2015,34(5):300-304.
[28]Xiao J F,Wang Q Y,Liu Q,et al.Isolation and identification of fish pathogen Edwardsiella tarda from mariculture in China[J].Aquaculture Research,2009,40(1):13-17.
[29]肖颖,李晓玥,张亚宁,等.牙鲆迟钝爱德华氏菌的分离及其鉴定[J].河北师范大学学报:自然科学版,2010,34(4):481-486.
[30]刘春,李凯彬,王庆,等.斑马鱼迟缓爱德华氏菌的鉴定、致病性及药物敏感性[J].华中农业大学学报:自然科学版,2013,32(3):105-111.
[2]朱新平,刘家照,夏仕玲,等.鲤鱼DNA对鳗鱼耐寒性能的影响[J].淡水渔业,1996,26(3):14-16.
[3]陈永乐,陈奋昌,钟海浪.西江鲮鱼年龄与生长的研究[J].水产学报,1990,14(3):198-205.
[4]卫二建,朱新平,郑光明,等.珠江鲮的形态变异及与人工繁殖群体的比较[J].大连水产学院学报,2007,22(5):357-360.
[5]毛永庆,蔡发盛,林鼎.鲮鱼最适生长的营养素需要量研究[J].水生生物学报,1985,9(3):213-223.
[6]Zhang D C,Huang Y Q,Shao Y Q,et al.Molecular cloning,recombinant expression,and growth-promoting effect of mud carp(Cirrhinus molitorella)insulin-like growth factor-I[J].General and Comparative Endocrinology,2006,148(2):203-212.
[7]Zhang D,Guo H,Zhu C,et al.The complete mitochondrial genome of mud carp Cirrhinus molitorella(Cypriniformes,Cyprinidae)[J].Mitochondrial DNA,2013,26(1):149-150.
[8]Nguyen T T T,Sunnucks P.Strong population genetic structure and its management implications in the mud carp Cirrhinus molitorella,an indigenous freshwater species subject to an aquaculture and culturebased fishery[J].Journal of Fish Biology,2012,80(3):651-668.
[9]Cheng F,Ye W,Ye F L.Isolation of microsatellite DNA and preliminary genomic analysis of mud carp(Cirrhina molitorella)[J].Zoological Research,2007,28(2):119-125.
[10]Yang C,Zhu X P,Sun X W.Development of microsatellite markers and their utilization in genetic diversity analysis of cultivated and wild populations of the mud carp(Cirrhina molitorella)[J].Journal of Genetics and Genomics,2008,35(4):201-206.
[11]方苹,陈静,李婧慧,等.鲮鱼“红鳃病”病原的分离鉴定与药物敏感性试验研究[J].科学养鱼,2010(1):54-55.
[12]郎所.中华鲮鱼的寄生单殖吸虫[J].上海师范大学学报:自然科学版,1978(1):87-93.
[13]陈如作.寄生于鲮鱼(Cirrhinus nolitorella)粘孢子虫的一新种[J].暨南理医学报,1988(1):115-117.
[14]华鼎可.广东鲮鱼鱼种的粘孢子虫病[J].动物学杂志,1979,14(2):43-47.
[15]王方华,邹为民,李安兴.广东贺江水域野生鲮鱼体表寄生虫典型海弯水虱的种群动态[J].动物学报,2008,54(3):407-415.
[16]刘礼辉,张德锋,李宁求,等.鲮鱼源无乳链球菌的鉴定、血清型分析及药敏试验[J].南方农业学报,2015,46(11):2053-2058.
[17]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:76.
[18]李杰.致病性迟缓爱德华氏菌的鉴定、PCR检测及esaC基因的功能鉴定[D].青岛:中国海洋大学,2008.
[19]Wikler M A.M100-S18.Performance standards for antimicrobial susceptibility testing:Eighteenth Informational Supplement[S].USA:Clinical and Laboratory Standards Institute(CLSI),2008.
[20]Santos Y,Toranzo A E,Barja J L,et al.Virulence properties and enterotoxin production of Aeromonas strains isolated from fish[J].Infection and Immunity,1989,56(12):3285-3293.
[21]Grimont P A D,Grimont F,Richard C,et al.Edwardsiella hoshinae,a new species of Enterobacteriaceae[J].Current Microbiology,1980,4(6):347-351.
[22]王波,莫照兰.迟缓爱德华氏菌及其致病机理[J].海洋科学集刊,2007(48):133-139.
[23]刁菁,杨秀生,叶海斌,等.一种水产迟钝爱德华氏菌快速药敏检测方法的研究[J].水产科学,2014,33(1):22-28.
[24]王燕,张晓华,吕俊超,等.养殖大菱鲆病原菌迟缓爱德华氏菌的分离鉴定及其疫苗研制[J].中国水产科学,2009,16(3):394-403.
[25]张晓君,战文斌,陈翠珍,等.牙鲆迟钝爱德华氏菌感染症及其病原的研究[J].水生生物学报,2005,29(1):31-37.
[26]杨春志,王秀华,黄倢.养殖大菱鲆迟缓爱德华氏菌的分离鉴定与系统发育分析[J].上海水产大学学报,2008,17(3):280-284.
[27]葛慕湘,靳晓敏,张艳英,等.迟钝爱德华氏菌耐药表型及4种耐药基因检测[J].水产科学,2015,34(5):300-304.
[28]Xiao J F,Wang Q Y,Liu Q,et al.Isolation and identification of fish pathogen Edwardsiella tarda from mariculture in China[J].Aquaculture Research,2009,40(1):13-17.
[29]肖颖,李晓玥,张亚宁,等.牙鲆迟钝爱德华氏菌的分离及其鉴定[J].河北师范大学学报:自然科学版,2010,34(4):481-486.
[30]刘春,李凯彬,王庆,等.斑马鱼迟缓爱德华氏菌的鉴定、致病性及药物敏感性[J].华中农业大学学报:自然科学版,2013,32(3):105-111.