【作者】 陈凡；

【导师】 何启盖； 陈焕春；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 胸膜肺炎放线杆菌是猪传染性胸膜肺炎的病原，感染猪出现最急性型、急性型和慢性型等3种临床症状，造成急性死亡或生长缓慢，耐过猪成为未免疫猪群发生感染的传染源，给世界养猪业带来严重经济损失。本病原共有15种血清型，分别属于生物Ⅰ型和生物Ⅱ型，其中生物Ⅰ型菌株致病力较强。不同血清型的菌株毒力高低不同，引起疾病的严重程度也有差异。通过血清学和病原学流行病学调查，了解当地流行的优势血清型菌株，有助于了解疾病来源动态，研制特异性强的疫苗并推广应用，最终控制该病。然而，我国目前尚无用于胸膜肺炎放线杆菌分离菌株血清型和血清学调查的型特异性血清和型特异性抗原，而这是本病前期研究的基础。为此，开展分型血清及定型抗原的研制和分型方法的研究与应用，显得十分必要。 本试验中，将引进的胸膜肺炎放线杆菌13种血清型标准菌株，在TM／SN培养基上增殖，收获菌体，灭活后用于免疫家兔，制备了分型血清。分别应用玻片凝集试验、琼脂扩散试验和免疫电泳试验，对抗血清的特异性、效价和保存期进行了监测，结果表明，制备的抗血清有较好的特异性，在4℃下保存1个月、-20℃下8个月和-80℃下1年其效价无明显变化。用制备的分型血清采用琼脂扩散试验对PCR阳性的胸膜肺炎放线杆菌地方分离菌株进行了鉴定，分别为血清2、3和8型，说明抗血清可用于分离菌株的鉴定分型。 为了能通过检测送检血清了解动物感染病原的血清型，将增殖并收获的胸膜肺炎放线杆菌13个血清型标准菌株，采用洗涤直接离心、超声波处理和酚抽提等方法，制备不同血清型抗原，通过玻片凝集试验、琼脂扩散试验和对流免疫电泳，以标准抗血清作为参考，评价各种诊断抗原的特异性和灵敏性。分别应用研制的3种抗原对来自湖北、湖南、江西、河南和安徽等五省的155份送检血清进行检测，初步结果表明定型抗原及其检测方法能准确地进行血清学鉴别诊断和血清分型。 基因分型是近几年来用于细菌分型的新技术，能克服经典的血清学分型方法对部分菌株不能进行分型的缺点，已经应用于其它细菌的分型。本研究中，根据胸膜肺炎放线杆菌各血清型之间某些毒力相关因子的基因差异，设计不同的引物，分别对各血清型外毒素(Apx)、外膜蛋白(OmlA)、转铁蛋白B(TbpB)进行PCR扩增，得到不同的特异性片段，可区分胸膜肺炎放线杆菌生物Ⅰ型13个标准菌株血清型中的8个血清型，建立了基因分型方法，常见病原菌如大肠杆菌、沙门氏菌、巴氏杆菌、波氏杆菌和链球菌在apx-PCR中不能扩增出特异性片段。对分离的12株野生菌用此方法进行分型，其结果与常规的血清学分型结果一致，为血清2、3和10型。将此分型系统用于临床送检的126份肺脏和42份扁桃体检测，结果表明，可直接检测出胸膜肺炎放线杆菌感染，并能判定感染病原的血清型。本方法还可以将一些尚华中农业大学硕士学位论文未定型的菌株进行归类,弥补了血清学方法的不足,而且,缩短了诊断的时间。 总之,本研究制备了13个血清型的胸膜肺炎放线杆菌分型血清和诊断抗原,提出了适用的血清学检测方法:在分子水平上,建立了胸膜肺炎放线杆菌基因分型方法,结果与经典的血清学分型方法相符,同时还弥补了经典的血清学方法对部分分离株不能分型的不足。这些方法为进一步开展病原分离鉴定、血清流行病学和分子流行病学提供了有效的手段。关健词:胸膜肺炎放线杆菌;血清学分型;基因分型;病原学检侧更多还原

【Abstract】 Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the etiological agent of porcine contagious pleuropneumoniae, which is characterized by sudden death or growth retardation during the peracute, acute and chronical course of disease. Pigs survived infection may serve as source for nonimmune pig herd. To date, 15 serovars of bivar I and biovar II were documented and stronger virulence was observed in biovar I . The difference of virulence among the isolates contributes to the different severity of the disease. To control and prevent the disease, it will be helpful to know the disease origin and dynamics, and production and application of serotype specific vaccine, which is based on serological and etiological investigations which provide information about predominant serovars. Unfortunately, the commercial availability of serotyping sera and antigens, the fundamental materials for A.pleuropneumoniae research, is very limited currently in China. Thus, the preparation of the above reagents and development of typing system are very important in disease control and prevention.In this report, to prepare the sera for serotyping, 13 serotypes of A. pleuropneumoniae reference strains were cultured, harvested and inactivated prior to being emulsed with adjuvants for raising antibodies in rabbits. The specificity, titer and shelf life of the 13 sera were measured by slide agglutination test, agar diffusion test and counter immunodiffusion assay. The results indicated that the sera possessed good specificity, and can be stored at 4℃ for 1 month, at -20℃ and -80℃ for one year without significant variation in quality. The materials and method were applied to verify five PCR positive field strains and were shown to be serotype 2, 3 and 8. These results revealed that the prepared serum and relevant assay could be used for serotyping field isolates.For the purpose of serologically investigating and typing the possible prevalent A.pleuropneumoniae strains via serum detection, the serotype specific antigens of 14 serotype of A. pleuropneumoniae reference strains were produced from antigen containing supernatant after washing and centrifugtion, sonication or phenol extraction. The titration and specificity of antigens were assessed by slide agglutination test, agar diffusion test and counter immunodiffusion assay. 155 serum samples collected from Hubei, Hunan, Jiangxi, Henan and Anhui provinces were assayed with newly prepared antigens. The results revealed the antigens can be used to differentiate serotypes of A. pleuropneumoniae.Genotyping is a newly developed technique for bacterial typing and could be used totype bacteria, in particular those untypable strains in conventional serological typing formats. In this test, a PCR system was developed on basis of genes encoding the three RTX (apx), outer membrane proteins (omlA), and transferring binding protein (tbpB) of A. pleuropneumoniae. We found that the resultant patterns could distinguish the reference strains for serovars 1, 2, 3, 4, 5, 6, 10, 12 and 13 of biovar I of A. pleuropneumoniae. However, neither the reference strains for serovars 7? 8 and 15 nor the reference strains for serovars 9 and 11 could be distinguished. No amplified products were found when a field isolate of each of the following were examined - Escherichia coli, Bordetella bronchiseptica, Salmonella spp, Pasteurella spp and Streptococcus suis. Complete agreement was obtained between serotyping and genotyping when 12 field isolates of serovars 2, 3 and 10 were examined. The method can also be used to directly detect A. pleuropneumoniae infection in lung and tonsil tissue. The detection results of total 42 tonsils and 126 lungs showed the potential clinical application of the genotyping system because it could not only detect infection but also verify the serotype of A. pleuropneumoniae in tissues. Besides, the ability to sort the untypable strains may be complementary to the serotyping system. Inaddition, the detection is performed in shorter time than serological test.In conclusion, the serotyping sera更多还原