【作者】 刁有祥；

【导师】 崔治中；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2005， 博士

【摘要】 猪传染性胸膜肺炎(Porcine pleuropneumonia)是由猪胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)引起的一种高度传染性、致死性呼吸道病,以急性出血和慢性的纤维素性坏死性胸膜炎病变为主要特征。本病对各种猪均易感,在新引进猪群多呈急性爆发,其发病率和死亡率常在20%以上,最急性型的死亡率可高达80%～100%。该病在我国的发生呈逐年上升趋势,已成为危害养猪业最严重的疾病之一,给我国养猪业造成严重的经济损失。本研究根据胸膜肺炎放线杆菌ApxⅣ基因设计一对引物,扩增特异的650bp 核酸片段,建立了PCR 检测猪胸膜肺炎放线杆菌的方法。特异性试验结果表明,12 个血清型的放线杆菌参考菌株均能扩增出650bp 特异性的核酸片段,而大肠杆菌、多杀性巴氏杆菌、猪肺炎支原体、伤寒沙门氏菌和支气管败血性波氏杆菌的扩增结果均为阴性。敏感性试验结果表明,PCR 的最低检出限量为500 个放线杆菌,表明我们建立的PCR 检测方法敏感性高、特异性强。利用建立的PCR 检测方法对22 株从山东省不同地区分离的疑似放线杆菌菌株进行检测,结果14 株为阳性。对感染猪病变组织的检测结果表明,病变部位不同,胸膜肺炎放线杆菌的检出率不同,其中以扁桃体的检出率最高。该方法的建立为胸膜肺炎放线杆菌的诊断和鉴定提供了良好的方法。利用PCR 反应扩增出胸膜肺炎放线杆菌ApxⅣ基因650bp 的DNA,回收并纯化PCR产物,用地高辛标记,制备出地高辛标记的核酸探针。该探针与不同血清型的胸膜肺炎放线杆菌均能发生特异性杂交,而与大肠杆菌、多杀性巴氏杆菌、沙门氏菌、葡萄球菌、猪肺炎支原体、支气管败血波氏杆菌等细菌的核酸杂交均为阴性。对胸膜肺炎放线杆菌的最低检出限量为10×102个放线杆菌。对疑似胸膜肺炎放线杆菌感染病变组织DNA 检测结果表明,在扁桃体、鼻腔、气管、肺脏均可检测出胸膜肺炎放线杆菌,以扁桃体的检出率最高。该研究为猪胸膜肺炎放线杆菌的诊断和流行病学调查提供了良好的方法。猪胸膜肺炎放线杆菌有12个血清型,不同国家和地区流行的APP 血清型不同,而不同血清型的APP 所产生的Apx 毒素不同。本研究从山东省不同地区采集的85 份具有严重呼吸道症状病死猪的肺脏、气管和扁桃体进行胸膜肺炎放线杆菌(APP)的分离鉴定,采用凝集试验和PCR 方法对分离菌株进行血清型和Apx 毒素型的检测。结果从山东省不同地区分离到20 株分属5 个血清型的胸膜肺炎放线杆菌。其中,血清7 型6更多还原

【Abstract】 The ApxIVA gene , a recently discovered RTX determinant of Actinobacillus pleuropneumoniae, was shown to be species-specific. A pair primer were designed to specifically amplified a 650 bp fragment.The PCR result of specificity assay showed that the reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae were positive, No positive results appeared in other bacterial species closely related to A. pleuropneumoniae.The sensitivity result showed that as little as 500 of Actinobacillus pleuropneumoniae could be detected by PCR.The detection results for clinical samples tissue showed that Actinobacillus pleuropneumoniae DNA could be found in trachea, nasal cavitiesor and tonsils.The high diagnostic sensitivity and specificity of PCR will make it useful in field diagnostic work and epidemic investigation. The ApxIV gene , a recently discovered RTX determinant of Actinobacillus pleuropneumoniae, was shown to be species-specific. A pair of primers were designed for amplifying this 650bp fragment in PCR experiments.The PCR product was labeled with digoxigenin as DNA probe for detection of Actinobacillus pleuropneumoniae.The hybridization assay result showed that the reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae were positive, No cross-hybridization was detected with DNAs from other bacterial species closely related to A. pleuropneumoniae.The sensitivity result showed that as few as 10×102 of Actinobacillus pleuropneumoniae could be detected by DIG-labeled probe. The detection results for clinical samples tissue showed that Actinobacillus pleuropneumoniae could be found in tonsils ,trachea and lung.The high sensitivity and specificity of the DIG-labeled probe will make it useful in field diagnostic work and epidemic investigation. 20 strains of Actinobacillus pleuropneumoniae were isolated from different districts of Shandong province. Medicine sensitivity results showed that most of the isolated strains were sensitive to cephalothin , amikacinum,gentamicin ,and kanamycini. resistance to penicillin , nitrofurantoinum and vancomycin. This study provide theoretical foundation for prevention and treatment for Porcine pleuropneumonia. 20 field strains of Actinobacillus pleuropneumoniae were isolated in lung, tonsils and trachea from 85 pigs with severe pleuropneumonia in different districts of Shandong province. Slide agglutination test and PCR was used to determine their serotype and Apx toxins type respectively. The results of slide agglutination test were indicated that there were 5 serotypes among the 20 field strains, serotype 7(6/20) and serotype 5 (5/20) were the dominant , the other serotype were serotype 3(4/20),serotype 4(3/20) and serotype 8(2/20).The PCR results indicated that there were four kinds of Apx toxins in the 20 field strains , among them, serotype 3,4,8 contain ApxⅡ,ApxⅢand ApxⅣ, serotype 5 contain ApxⅠ, ApxⅡand ApxⅣ, serotype 7 contain ApxⅡand ApxⅣ, ApxⅡand ApxⅣexisted in all the 20 field strains .The results of Apx sequence analysis indicated that the homogeneity of Apx gene were higher than 99% in different serotype APP if they had same type of Apx. The pathogenicity was different when mice inoculated with APP which contain different Apx , serotype 5 APP was very virulent ,for it contain ApxI and ApxⅡ.This study establish the foundation for control Porcine infectious pleuropneumonia in immune prevention , subunit vaccine and PCR diagnosis based on ApxⅣgene. PCV-2 (Porcine circovirus type-2, PCV-2) and PRRSV (Porcine reproductive and respiratory syndrome virus , PRRSV) were detected by PCR from 253 porcine pleuropneumonia samples and 125 clinically healthy lung samples were collected from different districts of Shandong province .The results showed that 171 samples for PCV-2 and 101 samples for PRRSV were positive. The positive ratio were 67.5% and 40% , respectively. the co-infection number of PCV-2 and PRRSV was 68 in 253 samples, the positive ratio was 26.8%. While in the clinically healthy samples, only 21 positive samples for PCV-2 and 12 samples for PRRSV were detected, the positive ratio were 16.8% and 9.6% , respectively , no co-infection samples were found. Statistical result showed significant difference between positive ratio for PCV-2 and PRRSV in Porcine pleuropneumonia samples and in clinically healthy samples. The above results demonstrated that there maybe some relationship between the infection of porcine pleuropneumonia and PCV-2 and PRRSV in pigs.更多还原