【作者】 刘辉；

【导师】 杨增岐；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2011， 硕士

【摘要】 副猪嗜血杆菌病,又称猪革拉瑟氏病(Gl?sser’s Disease),是由副猪嗜血杆菌(Haemophilus parasuis, Hps)引起的主要导致保育期仔猪发生多发性浆膜炎、关节炎和脑膜炎的一种细菌性疾病。近年来,该病在世界范围内广泛流行,且发病率和死亡率呈现显著的上升趋势,给我国的养猪业也造成了巨大的经济损失。随着该病危害的不断加重,人们对其研究也在逐渐增多,国内部分省市已成功分离获得了当地的副猪嗜血杆菌菌株,并进行了流行病学研究,但对陕西省副猪嗜血杆菌的流行情况及流行菌株的分子特点则报道较少。本研究从陕西省关中地区分离鉴定出了5株副猪嗜血杆菌菌株,建立了较稳定的副猪嗜血杆菌保存方法,对分离株进行了常见药物的敏感性试验,并通过针对副猪嗜血杆菌tbpA基因的PCR-RFLP分型方法对5株临床分离株进行了基因分型,为陕西省副猪嗜血杆菌的分子流行病学研究以及该病的治疗提供了一定实验依据,主要研究结果如下:1.为了检测副猪嗜血杆菌在陕西省部分猪场的感染情况,本试验从陕西省关中地区14个患多发性浆膜炎和关节炎猪场的30个样品中分离到5株细菌,并对其进行了形态观察、菌落培养和生化特性的鉴定;再根据副猪嗜血杆菌的16S rRNA序列设计引物对分离菌株进行PCR扩增,将扩增产物连接T载体后测序并进行序列分析,发现获得的基因片段与GenBank中国内外副猪嗜血杆菌16S rRNA序列的同源性均在99%～100%之间,确定分离菌株为副猪嗜血杆菌。该结果表明,关中地区存在副猪嗜血杆菌病的流行。2.对副猪嗜血杆菌的保存方法进行了探索。通过将菌株接种于不同的培养基待其生长良好后置于不同的条件下保存,并间隔一定时间将保存的菌种复苏,观察其是否依然存活来寻求较适合的副猪嗜血杆菌保存条件。结果发现,将副猪嗜血杆菌菌株通过TSB培养基扩大培养两次后加入终浓度为20%的灭菌甘油置于-70℃保存,是副猪嗜血杆菌较适合的保存方法。3.用药敏纸片法检测了5株副猪嗜血杆菌临床分离株对9种药物的敏感性。结果显示,所有的菌株对氟嗪酸、妥布霉素和卡那霉素均100%敏感,对利福平的敏感率也较高(40%),而对于头孢呋新、羧苄青霉素的耐药率则分别高达100%和80%。这个结果表明,5株副猪嗜血杆菌临床分离株的耐药性均比较强,且不同菌株对同一抗生素的敏感性也不同,从而为关中地区副猪嗜血杆菌病的治疗提供了用药依据。4.利用Redondo等建立的针对副猪嗜血杆菌的PCR-RFLP基因分型方法,通过三种限制性内切酶TaqⅠ、AvaⅠ、AfaⅠ对5株副猪嗜血杆菌临床分离株的转铁结合蛋白A编码基因(tbpA)进行了PCR-RFLP基因分型,得到了4种不同的RFLP基因图谱,分别为EBC、BBJ、EBJ、EBJ、EAD,再将这四种基因型与国内外其它相关研究所得到的基因型进行比较分析,结果显示,副猪嗜血杆菌在陕西省关中地区存在多种基因型菌株,且各基因型具有明显的地域特点,为陕西省副猪嗜血杆菌病的分子流行病学研究提供了理论依据。本试验从陕西省关中地区分离鉴定出了5株副猪嗜血杆菌菌株,并对其进行了药物敏感性检测,同时也对分离菌株进行了基于tbpA基因的PCR-RFLP基因分型,为进一步开展本地区副猪嗜血杆菌病的病原分离鉴定、分子流行病学和预防治疗方面的研究提供了一定依据。更多还原

【Abstract】 Gl?sser’s Disease is a typical bacterial disease, which is caused by Haemophilus parasuis, always causes serofibrinous pleurisy, pericarditis, peritonitis, arthritis and meningitis in piglets. In recent years, the incidence and mortality of the disease increased significantly in the world, creating huge economic losses to the pig industry. Nowadays, many institutions have isolateded lots of Hps isolates and obtained some achievements in its epidemiology. However, these studies only focused in some certain area. The study related to Shaanxi province is still rare. In this research, 5 strains of Haemophilus parasuis were isolated and identified from GuangZhong District of Shaanxi province, and the appropriate preservation conditions for Haemophilus parasuis strains were explored and obtained. Further we performed drug sensitivity test against common antibiotic drugs to the isolates. The PCR-RFLP analysis based on the tbpA gene was used for genetyping of the 5 Haemophilus parasuis isolates. Not only can the isolation, identification and molecular epidemiology of Haemophilus parasuis provide guidance for controlling the disease, but also help revealing its prevalent pattern in the district. The main results are as follows:1. To certify whether the Gl?sser’s disease existed in Guanzhong District, from Shaanxi province, we isolated 5 bacteria from the swine which were characterized by polyserositis and fibrinopurrlent polyarthritis. Then we identified them based on the cultural characteristics, morphologic characteristics, biochemical characteristics; primers were designed according to the sequences of Haemophilus parasuis and PCR was conducted to amplify the isolated strains. Amplification product was connected to the T vector and sequenced. Homology of 16S rRNA sequences between the sequences of the isolates and those from GenBank was between 99%～100%. All the above results conformed the isolates to be Haemophilus parasuis. Our findings also suggested that Haemophilus parasuis widely existed in this area, providing certain experimental basis for studies of Haemophilus parasuis disease in this area.2. The conservation conditions of the 5 isolates were also explored. This was performed through inoculating well-growing strains in different mediums and solutions at different temperatures. The preserved strains were recovered and detected whether they still survived every certain time. The results showed that inoculating Haemophilus parasuis strains in TSB medium twice and preserving them in sterilization glycerin with final concentration of 20% at -70℃was the best way of preservation, laying a certain foundation for the further study of Haemophilus parasuis.3. Drug susceptibility pieces method was used to detect sensitivity of the 5 isolates of Haemophilus parasuis against nine common drugs. All of the strains were 100% sensitive to Ofloxacin, Tobramycin and Kanamycin, as well as Rifabutin (40%), while Cefuroxime and Carbenicillin was not sensitive at all. Through which, we found that all the 5 strains were not highly sensitive against the common drugs and different isolates also showed different sensitivity to the same drug, which provided basis for the treatment of Haemophilus parasuis in this region.4. PCR-RFLP genotyping method, which was created by Redondo et al., was used in this research. Three restriction enzymes, TaqⅠ, AvaⅠand AfaⅠ, were used for PCR-RFLP genotyping the turn iron binding protein protein A (tbpA) genes of the 5 strains of Haemophilus parasuis. Four different RFLP gene patterns were obtained, which were named EBC, BBJ, EBJ, EBJ and EAD, respectively. We further compared and analyzed the four genotypes and other genotypes obtained by relevant research institutes, the results showed that the prevalent genotypes of Haemophilus parasuis isolates in Shaanxi province presented high diversity. Our results provided theory basis for molecular epidemiology investigation of Haemophilus parasuis disease in Shaanxi province.We isolated and identified 5 Haemophilus parasuis isolates and took the drug sensitive tests of them. Then the isolates were subjected to be ananlyzed using PCR-RFLP based on the tbpA gene. Through this work, we provided an effective method to study the isolation and identification, molecular character and the control of Haemophilus parasuis disease.更多还原