【摘要】：豬肺炎支原體(Mycoplasma hyopneumoniae, Mhp)是引發(fā)陸川豬支原體肺炎(Mycoplasmal pneumonia of swine, MPS)的主要原因。本研究旨在對(duì)廣西陸川豬Mhp進(jìn)行病原分離鑒定及其P97 R1區(qū)基因序列分析,為進(jìn)一步了解和研究廣西陸川豬Mhp的病原特性、流行病學(xué)以及該病的綜合防制提供理論依據(jù)。方法：(1)采集廣西陸川豬疑似患有MPS的肺組織,常規(guī)Mhp病原分離培養(yǎng)后進(jìn)行涂片染色和PCR方法鑒定,篩選陽(yáng)性病料；(2)常規(guī)病理組織切片,HE染色觀察組織病理學(xué)變化特征。采用免疫組織化學(xué)方法對(duì)組織中病原分布進(jìn)行定位；用原位雜交技術(shù)對(duì)組織中Mhp的核酸進(jìn)行檢測(cè)、定位；(3)參考Mhp基因組序列設(shè)計(jì)擴(kuò)增P97基因R1區(qū)片段的一對(duì)引物,提取基因組DNA,PCR擴(kuò)增黏附因子P97基因R1區(qū),對(duì)PCR產(chǎn)物進(jìn)行測(cè)序,并將廣西陸川豬Mhp不同地方流行毒株的P97基因R1區(qū)序列的堿基組成和推導(dǎo)的氨基酸序列進(jìn)行比較。結(jié)果：(1)采用液體培養(yǎng)方法成功從疑似患有MPS的廣西陸川豬肺組織中分離到M11p野毒株,液體培養(yǎng)物通過瑞氏一吉姆薩染色鏡檢,觀察到環(huán)狀、兩極狀等多形態(tài)的菌體；PCR檢測(cè)液體培養(yǎng)物能擴(kuò)增出目的片段；(2)Mhp陽(yáng)性肺的病理組織HE染色結(jié)果顯示,病變肺組織呈現(xiàn)典型的間質(zhì)性肺炎以及融合性支氣管肺炎變化,肺泡隔增厚,間質(zhì)有大量炎性細(xì)胞增生和浸潤(rùn),肺泡腔狹窄、縮小。細(xì)支氣管管壁增厚,上皮細(xì)胞脫落,大量炎性細(xì)胞滲出,細(xì)支氣管周圍淋巴小結(jié)樣增生。免疫組化結(jié)果顯示,Mhp陽(yáng)性信號(hào)主要分布在細(xì)支氣管管壁柱狀上皮細(xì)胞胞質(zhì)和黏膜,管腔中的分泌物以及周圍的炎性細(xì)胞胞漿中：原位雜交結(jié)果顯示,Mhp DNA主要分布在氣管和支氣管黏膜表面。(3)廣西陸川豬Mhp P97 R1區(qū)基因的堿基組成和推導(dǎo)的氨基酸序列比較結(jié)果顯示,所擴(kuò)增的15株廣西陸川豬Mhp毒株與3株廣西二元雜商品豬Mhp毒株的P97基因R1區(qū)多處堿基發(fā)生變異,通過推導(dǎo)氨基酸序列統(tǒng)計(jì)P97基因R1區(qū)氨基酸重復(fù)基序(AAKPV/E)數(shù)為9-18,平均值為12,TN重復(fù)數(shù)在1-4,發(fā)現(xiàn)廣西陸川豬Mhp流行毒株變異使其的毒力和黏附能力增強(qiáng)。結(jié)論：本研究成功從疑似患有MPS的廣西陸川豬肺組織中分離到Mhp野毒株,并定位了肺組織中Mhp病原菌和核酸的分布情況：此外,15株廣西陸川豬Mhp毒株與3株廣西二元雜商品豬的Mhp毒株的P97基因R1區(qū)序列分析、比較結(jié)果表明,廣西陸川豬MhpP97基因R1區(qū)發(fā)生變異。
[Abstract]:Mycoplasma hyopneumoniae (MHP) is the main cause of Mycoplasma pneumonia of swine, pneumonia. The purpose of this study was to identify the pathogen of Mhp in Luchuan pig in Guangxi and to analyze the gene sequence of P97 R1 region in order to provide a theoretical basis for further understanding and studying the pathogenic characteristics, epidemiology and comprehensive prevention and control of MHP in Luchuan pig in Guangxi. Methods: (1) lung tissues of Guangxi Luchuan pig suspected to have MPs were collected, smear staining and PCR method were performed after isolation and culture of Mhp, and positive venereal disease materials were screened. (2) histopathological changes were observed by HE staining in routine histopathological sections. Immunohistochemical method was used to locate the pathogen in tissues; in situ hybridization was used to detect and locate Mhp nucleic acid; (3) a pair of primers were designed to amplify the R1 region of P97 gene with reference to the MHP genome sequence. The R1 region of P97 gene was amplified by PCR from genomic DNA. The PCR products were sequenced. The base composition and deduced amino acid sequence of R1 region of P97 gene of different endemic strains of Mhp in Luchuan pig in Guangxi were compared. Results: (1) the M11p strain was isolated successfully from the lung tissue of Guangxi Luchuan pig with MPs by liquid culture method. (2) HE staining showed that the pathological tissue of MHP positive lung showed typical interstitial pneumonia and fusion bronchopneumonia, and the alveolar septum thickened. A large number of inflammatory cells proliferate and infiltrate in the interstitium, and the alveolar lumen is narrow and narrowed. The wall of the bronchiole thickened, the epithelial cells fell off, a large number of inflammatory cells exudated, and lymphoid nodule hyperplasia around the bronchioles. Immunohistochemical results showed that MHP positive signals were mainly distributed in the cytoplasm and mucous membrane of columnar epithelial cells in the wall of bronchioles. Secretion in the lumen and the cytoplasm of the surrounding inflammatory cells: in situ hybridization results showed that the Mhp DNA was mainly distributed on the surface of trachea and bronchi mucosa. (3) the base composition and deduced amino acid sequence of Mhp P97 R1 region gene of Guangxi Luchuan pig Column comparison shows that, The P97 gene R1 region of 15 strains of Guangxi Luchuan pig Mhp virus and 3 Guangxi dualistic commercial pig MHP strains were mutated. According to the deduced amino acid sequence, the number of amino acid repeat motifs (AAKPVP / E) in R1 region of P97 gene was 9-18, with an average of 12 TN repeats in 1-4. It was found that the variation of Mhp epidemic strain of Guangxi Luchuan pig enhanced its virulence and adhesion ability. Conclusion: Mhp wild strain was isolated from lung tissue of Guangxi Luchuan pig with suspected MPs in this study. The distribution of Mhp pathogenic bacteria and nucleic acid in lung tissue was also determined. In addition, the R1 region of P97 gene of 15 strains of Guangxi Luchuan pig Mhp virus and 3 Guangxi dualistic commercial pigs were analyzed. The R1 region of MhpP97 gene in Luchuan pig was mutated.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.62

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