牛源A型多杀性巴氏杆菌分离鉴定及毒力基因检测
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摘要
为了确定黑龙江省两个规模化奶牛场犊牛急性呼吸道感染的病因,进行了病原的分离纯化,细菌16S rDNA基因和多杀性巴氏杆菌特异性基因OmpH鉴定、生化试验、药敏试验、半数致死量试验、荚膜血清型分型及22种毒力因子的检测,确定主要病原的种类、型别及生物学特性。结果表明,从死亡牛肺脏和患病牛鼻拭子分离的两株病原菌均为荚膜A型多杀性巴氏杆菌,分别被命名为WC-16551和LY-01,序列分析结果表明其与牛源多杀性巴氏杆菌同源性达到99%以上,药敏试验显示两株菌均对恩诺沙星、环丙沙星高度敏感,半数致死量结果表明LY-01菌株的毒力明显强于WC-16551菌株,两株分离菌均携带19种毒力因子,未检出hgbB、toxA和nanB三种基因。研究表明导致两个牛场发病的主要病原菌为荚膜血清型A型的多杀性巴氏杆菌。
In order to determine the cause of acute respiratory infection of calves in two large-scale dairy farms in Heilongjiang province,the bacterial pathogen was isolated and purified,and the bacterial 16 S rDNA gene and Pasteurella multocida specific gene OmpH identification,biochemical test,drug sensitivity test,The median lethal dose test,detection of capsular serotyping and 22 virulence factors to identified the species,type and biological characteristics of major pathogens.The results indicated that the two bacterial pathogen isolated from the dead bovine lung and the diseased bovine nasal swab were all Pasteurella multocida capsular Serotype A,named WC-16551 and LY-01.The sequence analysis showed that it was associated with Pasteurella multocida.The homology of bovine Pasteurella multocida was more than 99%.The susceptibility test showed that both strains were sensitive to enrofloxacin and ciprofloxacin.The half lethal dose showed that the LY-01 toxicity was significantly stronger than WC-16551.They all carry 19 virulence factors,three genes hgbB,toxA and nanB were not detected.Studies have shown that the main pathogen causing the onset of two cattle farms is the capsular serotype A of Pasteurella multocida.
In order to determine the cause of acute respiratory infection of calves in two large-scale dairy farms in Heilongjiang province,the bacterial pathogen was isolated and purified,and the bacterial 16 S rDNA gene and Pasteurella multocida specific gene OmpH identification,biochemical test,drug sensitivity test,The median lethal dose test,detection of capsular serotyping and 22 virulence factors to identified the species,type and biological characteristics of major pathogens.The results indicated that the two bacterial pathogen isolated from the dead bovine lung and the diseased bovine nasal swab were all Pasteurella multocida capsular Serotype A,named WC-16551 and LY-01.The sequence analysis showed that it was associated with Pasteurella multocida.The homology of bovine Pasteurella multocida was more than 99%.The susceptibility test showed that both strains were sensitive to enrofloxacin and ciprofloxacin.The half lethal dose showed that the LY-01 toxicity was significantly stronger than WC-16551.They all carry 19 virulence factors,three genes hgbB,toxA and nanB were not detected.Studies have shown that the main pathogen causing the onset of two cattle farms is the capsular serotype A of Pasteurella multocida.
引文
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[3]刘朋,陈萌,程子龙,等.奶牛荚膜血清A型巴氏杆菌的分离鉴定及致病性[J].中国兽医学报,2018,38(8):1548-1552.
[4] MARCHESI J R,SATO T,WEIGHTMAN A J,et al.Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA[J].Applied and Environmental Microbiology,1998,64(2):795-799.
[5]胡旭,梁宏儒,姜东君,等.牛多杀性巴氏杆菌外膜蛋白H的表达与纯化[J].中国畜牧兽医,2013,40(11):38-41.
[6]彭忠,梁婉.多杀性巴氏杆菌分子分型方法简述[J].微生物学报,2016,56(10):1521-1529.
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[10]贺奋义,魏玉明,郭慧琳,等.河西走廊牛源A型多杀性巴氏杆菌灭活疫苗的制备及临床效果检测观察[J].畜牧兽医杂志,2014,33(3):17-20.
[11] KATSUDA K,HOSHINOO K,UENO Y,et al.Virulence genes and antimicrobial susceptibility in Pasteurella multocida isolates from calves[J].Veterinary Microbiology,2013,167(3-4):737-741.
[12]林立山,黄秦,才灵杰,等.牛源多杀性巴氏杆菌的分离与初步鉴定[J].微生物学报,2018,58(12):2240-2248.
[13] VERMA S,SHARMA M,KATOCH S,et al. Profiling of virulence associated genes of Pasteurella multocida isolated from cattle[J].Veterinary Research Communications,2013,37(1):83-89.
[14] BOSCH M,GARRIDO M E,LLAGOSTERA M,et al.Characterization of the Pasteurella multocida hgbA gene encoding a Hemoglobin-Binding Protein[J].Infection and Immunity,2002,70(11):5955-5964.
[15] C OX A J,HUNT M L,BOYCE J D,et al. Functional characterization of HgbB,a new hemoglobin binding protein of Pasteurella multocida[J].Microbial Pathogenesis,2003,34(6):287-296.
[16]王羽,董文龙,王巍,等.牛源多杀性巴氏杆菌荚膜血清型及毒力基因检测[J].中国兽医杂志,2017,53(9):16-19.
[17]王蕾,邬琴,柴迎锦,等.羊源D型多杀性巴氏杆菌的分离鉴定及毒力基因检测[J].动物医学进展,2019,40(1):25-30.
[18]梁禄,徐晨,成子强,等.牛源A型多杀性巴氏杆菌的生物学特性鉴定与致病基因检测[J].中国兽医科学,2018,48(8):1011-1018.
[19] SHIVACHANDRA S B,VISWAS K N,KUMAR A A.A review of hemorrhagic septicemia in cattle and buffalo[J].Animal Health Research Reviews,2011,12(1):67-82.
[2] CARTER G R.The type specific capsular antigen of Pasteurella multocida[J].Canadian Journal of Medical Sciences,1952,30(1):48-53.
[3]刘朋,陈萌,程子龙,等.奶牛荚膜血清A型巴氏杆菌的分离鉴定及致病性[J].中国兽医学报,2018,38(8):1548-1552.
[4] MARCHESI J R,SATO T,WEIGHTMAN A J,et al.Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA[J].Applied and Environmental Microbiology,1998,64(2):795-799.
[5]胡旭,梁宏儒,姜东君,等.牛多杀性巴氏杆菌外膜蛋白H的表达与纯化[J].中国畜牧兽医,2013,40(11):38-41.
[6]彭忠,梁婉.多杀性巴氏杆菌分子分型方法简述[J].微生物学报,2016,56(10):1521-1529.
[7] FAHAM K,HASSAN M,MORTEZA A M. Occurrence of virulence factors and antimicrobial resistance in Pasteurella multocida strains isolated from slaughter cattle in Iran[J].Frontiers in Microbiology,2014(5):536.
[8]马文戈,于力.牛源荚膜血清A型多杀性巴氏杆菌的分离鉴定[J].中国预防兽医学报,2008,30(10):747-750.
[9]马晓菁,王静梅,剡根强.犊牛肺炎多杀性巴氏杆菌的分离与鉴定[J].中国兽医学报,2010,30(9):1193-1196.
[10]贺奋义,魏玉明,郭慧琳,等.河西走廊牛源A型多杀性巴氏杆菌灭活疫苗的制备及临床效果检测观察[J].畜牧兽医杂志,2014,33(3):17-20.
[11] KATSUDA K,HOSHINOO K,UENO Y,et al.Virulence genes and antimicrobial susceptibility in Pasteurella multocida isolates from calves[J].Veterinary Microbiology,2013,167(3-4):737-741.
[12]林立山,黄秦,才灵杰,等.牛源多杀性巴氏杆菌的分离与初步鉴定[J].微生物学报,2018,58(12):2240-2248.
[13] VERMA S,SHARMA M,KATOCH S,et al. Profiling of virulence associated genes of Pasteurella multocida isolated from cattle[J].Veterinary Research Communications,2013,37(1):83-89.
[14] BOSCH M,GARRIDO M E,LLAGOSTERA M,et al.Characterization of the Pasteurella multocida hgbA gene encoding a Hemoglobin-Binding Protein[J].Infection and Immunity,2002,70(11):5955-5964.
[15] C OX A J,HUNT M L,BOYCE J D,et al. Functional characterization of HgbB,a new hemoglobin binding protein of Pasteurella multocida[J].Microbial Pathogenesis,2003,34(6):287-296.
[16]王羽,董文龙,王巍,等.牛源多杀性巴氏杆菌荚膜血清型及毒力基因检测[J].中国兽医杂志,2017,53(9):16-19.
[17]王蕾,邬琴,柴迎锦,等.羊源D型多杀性巴氏杆菌的分离鉴定及毒力基因检测[J].动物医学进展,2019,40(1):25-30.
[18]梁禄,徐晨,成子强,等.牛源A型多杀性巴氏杆菌的生物学特性鉴定与致病基因检测[J].中国兽医科学,2018,48(8):1011-1018.
[19] SHIVACHANDRA S B,VISWAS K N,KUMAR A A.A review of hemorrhagic septicemia in cattle and buffalo[J].Animal Health Research Reviews,2011,12(1):67-82.