【作者】 陈江；

【导师】 王嘉福； 冉雪琴；

【作者基本信息】 贵州大学 ， 微生物学， 2016， 硕士

【摘要】 微生态学的理论与实践推动着人们对呼吸道中固有微生物群落的研究与认识。上呼吸道的鼻腔黏膜表面和其他与外界相通的黏膜组织一样,定植着结构相对稳定和组成十分复杂的微生物菌群。鼻腔由于受到组织结构、生理功能以及外界诸多因素的影响,构成了有别于呼吸道其他部位的独特生态环境。本研究采用PCR-DGGE和Real-time PCR的方法,结合Quantity one图象分析软件对DGGE图谱进行分析和UPGMA进行聚类分析,并计算多样性指数(香浓指数、均匀性指数、丰富度指数和优势度指数)。数据使用SPSS19.0进行分析来测定群落结构之间的相关性。得到以下结果:1.通过DNA酶结合CTAB法提取DNA更能客观反映鼻腔黏膜上定植的微生物多样性的真实情况;2.菌群指纹图谱中反应了香猪鼻腔内微生物菌群的动态变化情况,不同生长阶段的香猪鼻腔真菌、细菌菌群组成稳定差异不显著(P>0.05),同时感染组与健康组香猪鼻腔内真菌和细菌群落组成差异不显著(P>0.05).3.处于微生物定植高峰期的二月龄香猪鼻腔黏膜内,细菌群落多样性指数明显高于其它月龄;稳定期的五月龄香猪,鼻腔细菌群落多样性指数最低;4.DGGE图谱中检测到大量未培养真菌和细菌,也检测到莫拉克斯氏菌属(Moraxella sp.)、肺炎链球菌(Streptococcus pneumoniae)、副猪嗜血杆菌属(H.parasuis)、金黄色酿脓葡萄球菌属(Staphylococcus aureus)、流感嗜血杆菌属(Haemophilus influenza)、白色念珠菌属(Candida.sp)等常见的条件致病性微生物。5.群落测序结果中的微生物主要分为六大类:酵母亚门(Saccharomycotina)、子囊菌门(Ascomycota)、放线菌门(Actinobacteria)、盘菌亚门(Pezizomycotina)、厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)。6.感染组中香猪鼻腔内,金黄色葡萄球菌(Staphylococcus aureus)的拷贝数相对比例明显增加,可以作为鼻腔内呼吸道感染的致病性指示细菌。通过对香猪鼻腔中真菌和细菌群落结构的多样性、动态变化和稳定性进行分析,筛选生境中导致香猪呼吸道感染的致病性指示微生物,为香猪呼吸道疾病的诊断和预防提供一定的理论参考。更多还原

【Abstract】 Aims: Theory and practice of micro-ecology is driving people to understand the respiratory tract microflora. Nasal mucosal surface of the upper respiratory tract and other mucosal tissues with the outside world, planting the relatively stable structure and composing of very complex microbial flora. Due to the organizational structure, functions and the impact of external factors, many physiological form is different from other parts of the respiratory tract in the unique ecological environment.Methods and results: Through PCR-DGGE and Real-time PCR method combined with image analysis software Quantity one, DGGE profiles and UPGMA cluster were analyzed, and calculate the diversity index(Shannon index, evenness index, richness index and dominance index). DNA was extracted from the nasal mucosa more objectively reflect the real situation of the diversity of microbial colonization by DNA enzyme binding CTAB method; Pigs get the nasal flora of PCR-DGGE fingerprinting atlas reflects the dynamic changes in the nasal cavity microflora, different growth stages of pigs nasal fungal, bacterial flora structural stability was not significantly different(P> 0.05), Meanwhile fungal and bacterial community structure in the infected and healthy pigs nasal group was also not significant(P> 0.05).; Internal colonization in the peak of the two-month-old pig nasal mucosa, bacterial diversity index was significantly higher than other months, when the stage of May, the lowest nasal microbial diversity index;Conclusion: DGGE profiles of bands clone sequencing detected a large number of uncultured bacteria and fungi, but also to detect common condition of pathogenic microorganisms: Streptococcus pneumoniae, Staphylococcus pyogenes, Staphylococcus aureus, Moraxella sp., Haemophilus influenza, H. parasuis, Candida albicans.; Cloning and sequencing results microorganisms mainly attributable to:Ascomycota, Pezizomycotina, saccharomycotina, Bacteroides, Firmicutes, Actinobacteria. Infection in pigs within the nasal cavity, Staphylococcus aureus is the relative copy number was significantly increased, as the nasal cavity respiratory tract infection pathogenic indicator bacteria.Significance and Impact of Study: Through the diversity of fungi and bacteria of community structure in the nasal cavity pigs, dynamics, and stability analysis, screening habitats cause respiratory infections in pigs pathogenic indicator organisms for the diagnosis and prevention of respiratory disease in pigs provide theoretical reference.更多还原