鲟源致病性鲁氏耶尔森菌的分离、鉴定及药敏研究
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摘要
2017年,新疆阜康市某鲟鱼养殖场的西伯利亚鲟出现大批死亡,主要症状为生殖孔红肿外突,腹部有点状出血。自患病西伯利亚鲟肝脏、脾脏分离到一株优势菌株XB2,通过革兰氏染色、生理生化试验、药敏试验,并对其16S rRNA基因序列进行测定,用Mega 6.0对序列进行分析。生理生化试验结果显示,菌株XB2为革兰氏阴性短杆菌,单独或成对出现;产酸不产气,V-P反应呈阳性,能利用蔗糖、果糖、葡萄糖、明胶、枸橼酸;在含1%(m/V)氯化钠中能生长,高于3%(m/V)氯化钠中不生长;在4℃不生长,20～30℃均能生长,但在37℃不生长;不利用乳糖、水杨苷、纤维二糖、硫化氢、酒石酸、阿拉伯糖。进化树显示,菌株XB2与鲁氏耶尔森菌KJ812974同源性达99%。通过回感试验确定其半致死密度为2.37×104 cfu/mL。结合生理生化鉴定结果和16S rRNA基因序列分析结果,确定菌株XB2为鲁氏耶尔森菌。药敏结果显示,鲁氏耶尔森菌XB2对氟苯尼考、左氧氟沙星、哌拉西林、阿洛西林等9种药物敏感,对麦迪霉素、大观霉素、链霉素、氨苄西林等15种药物耐药。
In 2017,Siberian sturgeon(Acipenser baerii)were found a massive death in a sturgeon culture farm in Fukang City,Xinjiang,and had clinical symptom of haemorrhage in red and swollen anus and punctate hemorrhage in abdomen.A dominant bacterium named XB2 was isolated from the liver and spleen of the diseased Siberian sturgeon,and identified through gram stain,physiology,biochemistry,and 16S rRNA gene sequences analysis by Mega 6.0.The results showed that XB2 was a gram-negative brevibacterium,either alone or in pairs,V-P reaction positive,and used sucrose,fructose,glucose,gelatin,and citric acid,with production of acid in stead of gas.XB2 grew in 1%(m/V)sodium chloride and did not grow in more than 3%(m/V)sodium chloride,with growth at 20—30℃and without growth at 4°C and 37°C.XB2 did not utilize lactose,salicin,cellobiose,hydrogen sulfide,tartaric acid and arabinose.Phylogenetic tree indicated that XB 2had homology of 99% with Yersinia ruckeri(KJ812974),with LD50 of 2.37×104cfu/mL.The drug sensitivity test showed that XB 2is sensitive to Florfenicol,Levofloxacin,Piperacillin and Azlocillin,and resistant to Midecamycin,Spectinomycin,Streptomycin and Ampicillin.
In 2017,Siberian sturgeon(Acipenser baerii)were found a massive death in a sturgeon culture farm in Fukang City,Xinjiang,and had clinical symptom of haemorrhage in red and swollen anus and punctate hemorrhage in abdomen.A dominant bacterium named XB2 was isolated from the liver and spleen of the diseased Siberian sturgeon,and identified through gram stain,physiology,biochemistry,and 16S rRNA gene sequences analysis by Mega 6.0.The results showed that XB2 was a gram-negative brevibacterium,either alone or in pairs,V-P reaction positive,and used sucrose,fructose,glucose,gelatin,and citric acid,with production of acid in stead of gas.XB2 grew in 1%(m/V)sodium chloride and did not grow in more than 3%(m/V)sodium chloride,with growth at 20—30℃and without growth at 4°C and 37°C.XB2 did not utilize lactose,salicin,cellobiose,hydrogen sulfide,tartaric acid and arabinose.Phylogenetic tree indicated that XB 2had homology of 99% with Yersinia ruckeri(KJ812974),with LD50 of 2.37×104cfu/mL.The drug sensitivity test showed that XB 2is sensitive to Florfenicol,Levofloxacin,Piperacillin and Azlocillin,and resistant to Midecamycin,Spectinomycin,Streptomycin and Ampicillin.
引文
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[15]邓梦玲,耿毅,汪开毓,等.西伯利亚鲟海豚链球菌的分离鉴定及毒力基因检测[J].水产学报,2015,39(1):127-135.
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[17]储卫华,于勇.鲟鱼嗜水气单胞菌的分离与鉴定[J].淡水渔业,2003,33(2):16-17.
[18]杨移斌,夏永涛,杨先乐,等.鲟源病原菌HD0824的分离鉴定及药敏特性研究[J].西南农业学报,2017,30(5):1239-1244.
[19]杨移斌,胡鲲,杨先乐,等.鲟源鲁氏耶尔森氏菌的分离鉴定及药敏特性研究[J].水生生物学报,2013,37(2):393-398.
[20]李琳,李瑾年,余为一.细菌分类鉴定方法的研究概况[J].安徽农业科学,2004,32(3):549-551.
[21]李爱华,蔡桃珍,吴玉深,等.我国鱼类病原—嗜水气单胞菌的耐药性研究[J].微生物学通报,2001,28(1):58-63.
[22]Ellis A E.Immunity to bacteria in fish[J].Fish and Shellfish Immunology,1999,9(4):291-308.
[23]Martin K R,Kurt B.Innate immune response in rainbow trout(Oncorhynchus mykiss)against primary and secondary infections with Yersinia ruckeri O1[J].Developmental and Comparative Immunology,2008,33(1):35-45.
[24]陶健,李绍戊,刘红柏.中草药与复方对鲁氏耶尔森菌的体外抑菌作用[J].水产学杂志,2013,26(6):40-43.
[25]杨移斌,杨先乐,曹海鹏.41种中草药对3种鲟源病原菌的体外抑菌效果[J].淡水渔业,2013,43(4):80-84.
[2]孙大江,曲秋芝,张颖,等.中国的鲟鱼养殖[J].水产学杂志,2011,24(4):67-70.
[3]王吉桥,姜志强,胡红霞.主要养殖鲟鱼的生物学特性[J].水产科学,1998,17(6):34-38.
[4]田甜,杨元金,王京树,等.鲟鱼病害研究进展[J].湖北农业科学,2012,51(3):559-563.
[5] Furones M D,Rodgers C J,Munn C B.Yersinia ruckeri,the causal agent of enteric red mouth disease(ERM)in fish[J].Annual Review of Fish Diseases,1993(3):105-125.
[6] Rucker R R.Red mouth disease of rainbow trout(Salmo gairdneri)[J].Bulletin Office International Desiese Epizooties,1965,65(5):825-830.
[7]范方玲,汪开毓,耿毅.斑点叉尾(Ictalurus punctatus)源鲁氏耶尔森氏菌的分离鉴定及系统发育分析[J].海洋与湖沼,2010,41(6):862-868.
[8]连浩淼,卢彤岩,刘红柏,等.虹鳟肠炎红嘴病病原菌的确定及其生长特性研究[J].大连海洋大学学报,2015,30(5):498-504.
[9]方苹,丁彩霞,吴亚锋,等.鲢鱼、鳙鱼源鲁氏耶尔森氏菌的分离鉴定及系统发育分析[J].淮海工学院学报:自然科学版,2017,26(2):84-87.
[10]马英,关瑞章,郭松林,等.鳗鲡病原菌16SrRNA基因序列测定及系统进化分析[J].华中农业大学学报,2010,29(6):758-763.
[11]房海,陈翠珍.水产养殖动物病原细菌学[M].北京:中国农业出版社,2010:198-203.
[12]尚红,汪毓三,申子瑜.全国临床检验操作规程[M].北京:人民卫生出版社,2015:578-594.
[13] Matthew A W,Franklin R C,Karen B,et al.CLSI VET01-A4,performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals[M].Wayne:Clinical and Laboratory Standards Institute,2013:50-53.
[14]潘厚军,刘晓勇,常藕琴,等.西伯利亚鲟停乳链球菌的分离、鉴定与致病性[J].中国水产科学,2009,16(6):891-904.
[15]邓梦玲,耿毅,汪开毓,等.西伯利亚鲟海豚链球菌的分离鉴定及毒力基因检测[J].水产学报,2015,39(1):127-135.
[16]赵凤岐,曹谨玲,刘青.西伯利亚鲟败血症病理学观察与病原学研究[J].水生生物学报,2009,33(2):316-323.
[17]储卫华,于勇.鲟鱼嗜水气单胞菌的分离与鉴定[J].淡水渔业,2003,33(2):16-17.
[18]杨移斌,夏永涛,杨先乐,等.鲟源病原菌HD0824的分离鉴定及药敏特性研究[J].西南农业学报,2017,30(5):1239-1244.
[19]杨移斌,胡鲲,杨先乐,等.鲟源鲁氏耶尔森氏菌的分离鉴定及药敏特性研究[J].水生生物学报,2013,37(2):393-398.
[20]李琳,李瑾年,余为一.细菌分类鉴定方法的研究概况[J].安徽农业科学,2004,32(3):549-551.
[21]李爱华,蔡桃珍,吴玉深,等.我国鱼类病原—嗜水气单胞菌的耐药性研究[J].微生物学通报,2001,28(1):58-63.
[22]Ellis A E.Immunity to bacteria in fish[J].Fish and Shellfish Immunology,1999,9(4):291-308.
[23]Martin K R,Kurt B.Innate immune response in rainbow trout(Oncorhynchus mykiss)against primary and secondary infections with Yersinia ruckeri O1[J].Developmental and Comparative Immunology,2008,33(1):35-45.
[24]陶健,李绍戊,刘红柏.中草药与复方对鲁氏耶尔森菌的体外抑菌作用[J].水产学杂志,2013,26(6):40-43.
[25]杨移斌,杨先乐,曹海鹏.41种中草药对3种鲟源病原菌的体外抑菌效果[J].淡水渔业,2013,43(4):80-84.