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中文题名：

 致肉牛运输热溶血曼氏杆菌的分离鉴定及某些生物学特性研究    

姓名：

 韩小丽    

学号：

 20162013041    

学科代码：

 090602    

学科名称：

 预防兽医学    

学生类型：

 硕士    

院系：

 动物科技学院    

研究方向：

 动物疾病诊断与防治    

第一导师姓名：

 剡根强    

第一导师单位：

 新疆石河子大学    

完成日期：

 2019-06-10    

外文题名：

 Isolation Identification and Partial Biological Characteristics of Mannheimia haemolytica in Shipping Fever of Beef Cattle    

中文关键词：

  关键词：运输热 ; 牛源溶血曼氏杆菌 ; 生物学特性    

外文关键词：

  Key words:shipping fever ; bovine Mannheimia haemolytica ; Biological characteristics    

中文摘要：

目的：溶血曼氏杆菌（Mannheimia haemolytica）是引起运输热（shipping fever）的主要致病菌。该菌是一种机会性致病菌，当宿主遭受到运输、环境应激、呼吸道病原感染等情况时会导致抵抗力下降，进而细菌迅速增殖引起菌血症，在受感染的牛只中引起出血性、纤维素性肺炎。近年来，随着新疆肉牛业的发展，频繁性地调运牛只，由运输压力引发致病病原感染导致调运牛只死亡的数量日趋增多，给肉牛的调运带来较大的经济损失。2017年4月，新疆某牛场在调运的一批肉牛中疑似爆发巴氏杆菌感染，造成近30余头发病牛死亡，为查明致肉牛病死原因，本研究采集两头病死牛心、肝、脾、肺组织进行病原体的分离鉴定及部分生物学特性研究，为该疾病的诊断及有效预防控制提供病原学基础。

方法：无菌采集病死牛心血、肺脏、肝脏、脾脏病变组织，进行涂片染色镜检、细菌分离培养、生化检测、血清型和PCR鉴定；通过活菌计数法和菌液吸光光度法测定各个分离株的生长曲线并将其比较；参照白细胞毒素LktC、外膜脂蛋白相关基因PlpB、gs60、转铁蛋白结合蛋白相关基因tbpB、铁结合蛋白相关基因fbpA、参与糖酵解相关基因gapA、参与复制相关基因dnaN、参与四型菌毛相关基因ptfA及粘附相关基因fimA基因序列设计特异性引物，并以分离株的DNA为模板进行PCR扩增；通过腹腔注射小鼠，测定7株分离菌株的小鼠半数致死量（LD50）；分别采用纸片扩散法和试管法对菌株进行了19种抗菌药物及5种常用化学消毒剂的敏感性检测。

结果：分离获得7株分离株，命名为Mh1至Mh7。分离株的形态及生长特性、生化鉴定及PCR鉴定结果与溶血曼氏杆菌特性一致；其血清型鉴定为荚膜血清A2型溶血曼氏杆菌。分离菌均含有四型菌毛相关基因ptfA、参与复制相关基因dnaN、白细胞介素相关基因LktC三种毒力基因。分离株Mh1、Mh2、Mh3、Mh4、Mh5、Mh6、Mh7对小鼠的LD50分别为2.25×108.50、1.30×108.33、3.45×107.83、1.60×108.00、3.60×107.83、2.65×108.17、1.90×108.33。所以菌株均对氨苄西林、环丙沙星、氟苯尼考、头孢拉定、头孢西丁、头孢噻肟、头孢噻吩、新霉素、万古霉素、替米考星等大部分抗菌药物具有较强敏感性；Mh3、Mh4、Mh5三株菌株均在0.02%次氯酸钠、0.1%氢氧化钠、0.01%碘液和0.01%过氧乙酸消毒液中1 min可全部被杀死。

结论：引起某牛场肉牛运输热的病原为荚膜血清A2型溶血曼氏杆菌；7株分离株主要携带ptfA、dnaN、LktC三种毒力基因，对小鼠均有较强的致病性；分离株对大部分市场常用抗生素及化学消毒剂敏感。试验结果进一步确定了该牛场肉牛死亡的病因，为疾病的有效防治提供了科学依据，同时也丰富了新疆致肉牛运输热溶血曼氏杆菌的生物学特性研究。

关键词：运输热；牛源溶血曼氏杆菌；生物学特性

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外文摘要：

Object: Mannheimia haemolytica is a key pathogen causing shipping fever. It is an opportunistic pathogen that changes from the role of host commensal bacteria to pathogenic bacteria when the host decreases resistance to respiratory tract viruses or mycoplasma due to transportation, environmental stress or infection with respiratory viruses or mycoplasma, and the bacteria proliferate rapidly and massively proliferate and causing bacteremia, and it can cause haemorrhagic bronchopneumonia, fibroid bronchopneumonia in cattle infection. In recent years, with the development of beef cattle industry in Xinjiang, there are more and more cases of death caused by pathogen infection by transportation stress, which brings great economic loss to the transfer of beef cattle. In April 2017,suspected Pasteurella infection broke out in a batch of beef cattle dispatched in a cattle farm in Xinjiang, and resulting in the death of more than 30 sick cattle. In order to find out the causes of death of beef cattle, in this study ,heart, liver, spleen and lung tissues were collected from two diseased cattle for isolation and identification of pathogens,and some biological characteristics of the isolated strains were analyzed, which provided the etiological basis for the diagnosis and effective prevention and control of the disease.

Methods：The tissues of heart blood, liver, spleen and lung of the diseased cattle were collected aseptically for smear staining, isolation and culture of bacteria, biochemical test, serotype identification and PCR identification.The growth curve of each isolated was tested by the method of viable count (CFU) and absorbance (OD) and that of different strains were compared;The gene sequence of the leukotoxin related gene LktC,the outer membrane lipoprotein-related gene PlpB,gs60,the transferrin-binding protein-related gene tbpB,the iron-binding protein-related gene fbpA,and the glycolytic-related gene gapA,Participating in the replication-related gene dnaN,participating in the four-type pili-related gene ptfA and the adhesion-related gene fimA were loaded and designed specific primers, the DNA of the isolated strain used as template for PCR amplification;The mouse half-lethal dose (LD50) of seven Mh strains were determined by intraperitoneal injection in mice.Sensitivity detection of 19 antibacterial drugs and 5 commonly used chemical disinfectants were carried out by using the paper diffusion method and the test tube method.

Results: Seven isolates were isolated and named as Mh1 to Mh7.The results of The characteristics of morphological and growth,biochemical identification and PCR identification of the strain were consistent with the characteristics of Mannheimia haemolytica,and the results of the serotype identification showed that the isolates were capsular serum A2 type Mannheimia haemolytica.All isolates contained four types of fimbriae associated gene ptfA,involved in replication related gene dnaN,leukotoxin related gene LktC three virulence genes.The LD50 assay showed that the LD50 of isolated strain Mh1, Mh2, Mh3, Mh4, Mh5, Mh6, Mh7 were 2.25×108.50, 1.30×108.33, 3.45×107.83, 1.60×108.00, 3.60×107.83, 2.65×108.17, 1.90×108.33, respectively.All 7 strains were sensitive to most drugs such as ampicillin, ciprofloxacin,florfenicol, cefradine, cefoxitin, cefotaxime, cefotaxime, neomycin, vancomycin, and tilmicosin. Three strains of Mh3, Mh4, Mh5 were killed in 0.02%sodium hypochlorite,0.1% sodium hydroxide, 0.01% iodine solution and 0.01% peracetic acid disinfectant solution for one minute.

Conclusion:The pathogen causing shipping fever of beef cattle in a cattle farm is capsular serum A2 type hemolyti

Object: Mannheimia haemolytica is a key pathogen causing shipping fever. It is an opportunistic pathogen that changes from the role of host commensal bacteria to pathogenic bacteria when the host decreases resistance to respiratory tract viruses or mycoplasma due to transportation, environmental stress or infection with respiratory viruses or mycoplasma, and the bacteria proliferate rapidly and massively proliferate and causing bacteremia, and it can cause haemorrhagic bronchopneumonia, fibroid bronchopneumonia in cattle infection. In recent years, with the development o

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