羊源嗜吞噬细胞无浆体遗传特征分析

发布时间：2018-04-27 11:59

本文选题：绵羊 + 山羊　；参考：《河南农业大学》2015年硕士论文

【摘要】：嗜吞噬细胞无浆体(Anaplasma phagocytophilum),是一种专性细胞内寄生的革兰阴性菌,主要寄生于中性粒细胞。A.phagocytophilum可引起新发的经蜱传播的人兽共患自然疫源性疾病,不仅对人类健康构成严重威胁,而且对畜牧业经济效益造成重大影响。另外,A.phagocytophilum分离株在多种宿主动物和蜱均存在遗传多样性。本研究旨在应用分子生物学技术对我国羊源A.phagocytophilum的遗传特征进行分析。1.为了解我国部分地区奶山羊嗜吞噬细胞无浆体病的流行情况,并为该病的防控提供科学的参考和依据,促进奶山羊业的健康发展,本研究于2012年7月至2014年9月,采用巢式PCR检测方法,对采自河南省(洛阳宜阳、洛阳涧西区、新密市、南阳市西峡县、开封、平顶山、商丘宁陵县)、云南省(昆明)、陕西省(富平县、麟游县、泾阳县、龙陵县)奶山羊血液样品300份、羊奶样品204份进行了嗜吞噬细胞无浆体感染情况调查。结果仅在羊血液样本中检测出A.phagocytophilum,阳性率为18%(54/300),而羊奶中未检出A.phagocytophilum。不同地区奶山羊的A.phagocytophilum感染情况不同。洛阳宜阳地区奶山羊感染A.phagocytophilum阳性率达100%(5/5),陕西麟游阳性率较高为76.67%(46/60),平顶山的阳性率为12.50%(2/16),陕西高陵为2.86%(1/35),而洛阳涧西、郑州新密、南阳西峡、开封、宁陵、云南昆明、陕西泾阳、陕西富平均未见感染;不同品种奶山羊A.phagocytophilum的感染情况不同,杂交奶山羊A.phagocytophilum感染率最高,为83.87%(26/31),关中奶山羊阳性率较高,为22.83%(21/92),其次是本地奶山羊,阳性率为11.11%(7/63),而未见萨能奶山羊感染A.phagocytophilum;不同饲养模式下的奶山羊A.phagocytophilum感染情况也不同,放牧奶山羊的阳性率高达38.85%(54/139),而舍饲条件下的奶山羊未见感染A.phagocytophilum。另外,16S rRNA基因序列分析显示,本研究中的54条A.phagocytophilum序列中有一条序列出现了3个碱基的差异。遗传进化分析显示,羊源A.phagocytophilum菌株和人分离株(U02521)关系较近,同源性为99.1%~99.5%,存在潜在的人兽共患风险。2.为了解我国羊源嗜吞噬细胞无浆体的遗传特征,是否存在人兽共患风险,本研究于2012年5月至2014年9月,应用巢式PCR方法,对采自河南省(洛阳、郑州、开封、南阳、新乡、济源、平顶山、三门峡、驻马店)、云南省(昆明)、辽宁省(朝阳)、山东省(临朐)、贵州省(贵阳、晴隆)、山西省(晋城)、黑龙江省(黑龙江)、陕西省(富平县、麟游县、泾阳县、龙陵县)140份羊源A.phagocytophilum DNA阳性样品进行16S rRNA、MSP4(主要膜蛋白)和groESL(热休克蛋白)基因的扩增、测序以及采用Mega5.05软件进行遗传进化分析。结果显示,140条16S rRNA基因序列出现了5种不同的序列类型,分别命名为基因型A-E;且基因型D与人分离株(U02521)关系较近,同源性高达99.8%,仅存在一个碱基差异;不同基因型之间存在2~7个碱基差异;5个基因型在我国不同地区之间分布情况存在差异,基因型A存在于调查的所有省份,而基因型B-E仅存在于个别省份,如基因型B只存在陕西;基因型C只存在于河南洛阳;基因型D只存在于山西、黑龙江和河南洛阳;基因型E只存在于河南郑州。MSP4基因进化树显示47条序列出现了3类群共7个基因型,分别命名为基因型Ⅰa、Ⅰb、Ⅰc、Ⅰd、Ⅱa、Ⅱb和Ⅲ。7个基因型在我国不同地区之间分布情况存在差异,有的地区存在7个MSP4基因型,而有的地区仅存在1~2个基因型。130条groESL基因序列出现9种序列类型,分别命名为基因型A-I型;我国不同地区的基因型存在差异,河南省发现了A-G共7个基因型;而陕西省发现了A、C、D、E、H、I共6个基因型;山西省发现了A、B、D、F、G共5个基因型;贵州和黑龙江省均只发现基因型A;山东和辽宁省分别仅发现了基因型C和E。本研究应用PCR方法对我国部分地区奶山羊血液和羊奶样品进行了检测,结果在血液样品中发现A.phagocytophilum阳性,表明我国奶山羊存在A.phagocytophilum感染。本研究首次采用PCR方法基于16S rRNA、MSP4和groESL基因对我国羊源A.phagocytophilum遗传特征进行分析,结果共发现5个16S rRNA基因型,7个MSP4基因型,9个groESL基因型,说明我国羊源嗜吞噬细胞无浆体存在遗传多样性;另外,16S rRNA基因序列分析显示羊源的嗜吞噬细胞无浆体菌株和人源分离株(U02521)关系较近,同源性高达99.1%~99.8%,其中,基因型D和U02521仅存在一个碱基差异,对人类健康存在潜在威胁。本研究为进一步开展人兽共患病的监测和公共卫生安全研究奠定了基础。
[Abstract]:Phagocytic cells (Anaplasma phagocytophilum), a specific cell parasitic Gram-negative bacteria, mainly parasitic on neutrophils.A.phagocytophilum can cause new tick borne human zoonotic natural foci, which not only poses a serious threat to human health, but also contributes to the economic benefits of animal husbandry. In addition, A.phagocytophilum isolates have genetic diversity in various host animals and ticks. The aim of this study is to analyze the genetic characteristics of A.phagocytophilum in our sheep source using molecular biology technology.1. to understand the epidemic situation of phagocytic phagocytosis in dairy goats in some areas of our country and to provide the prevention and control of the disease. For scientific reference and basis, the healthy development of dairy goats was promoted. From July 2012 to September 2014, the nested PCR detection method was adopted in Henan province (Luoyang Yiyang, Luoyang Jianxi District, Xinmi City, Nanyang City, Xixia County, Kaifeng, Pingdingshan, Shangqiu, Lingxian County), Yunnan province (Kunming County, Lin You County, Jingyang County). The blood samples of 300 milk goats and 204 samples of goat milk samples were investigated in the phagocytic phagocytic cell without plasma infection. The results showed that the positive rate of A.phagocytophilum was 18% (54/300) in sheep blood samples, while the A.phagocytophilum infection of milk goats in different areas of goat milk was different. Luoyang should be different. The positive rate of A.phagocytophilum infection of milk goats in Yang area was 100% (5/5), the positive rate of Shaanxi Lin tour was 76.67% (46/60), the positive rate of Pingdingshan was 12.50% (2/16), Gaoling of Shaanxi was 2.86% (1/35), while Luoyang Jianxi, Zhengzhou Xinmi, Nanyang Xixia, Kaifeng, Ningling, Yunnan Kunming, Jingyang, and rich milk mountains The infection rate of sheep A.phagocytophilum was different, the A.phagocytophilum infection rate of hybrid dairy goats was the highest, 83.87% (26/31), the positive rate of milk goats in Guanzhong was 22.83% (21/92), followed by local milk goats, the positive rate was 11.11% (7/63), but no milk goat infected A.phagocytophilum, and milk goat A.phagocytoph in different feeding mode. The positive rate of Ilum infection was also different, the positive rate of grazing dairy goats was 38.85% (54/139), while the dairy goats under the condition of feeding were not infected with A.phagocytophilum.. The sequence analysis of 16S rRNA gene showed that one of the 54 A.phagocytophilum sequences in this study showed the difference of 3 bases. Genetic evolution analysis showed that the sheep source A.ph was A.ph. The relationship between the agocytophilum strain and the human isolate (U02521) is close, the homology is 99.1%~99.5%, the potential human zoonosis risk is.2. to understand the genetic characteristics of the phagocytic phagocytic phagocyte in our country, and whether there is the risk of human zoonosis. From May 2012 to September 2014, the nested PCR method was applied to Henan province (Luoyang, Zhengzhou). Kaifeng, Nanyang, Xinxiang, Jiyuan, Pingdingshan, Sanmenxia, Zhumadian), Yunnan province (Kunming), Liaoning province (Chaoyang), Shandong province (Linqu), Guizhou province (Zhumadian County, Jingyang County, Jingyang County, Jingyang County, Jingyang County, Jingyang County, Jingyang County, dragon Ling county), 140 samples of 16S rRNA, MSP4 (main MSP4) The genes of membrane protein) and groESL (heat shock protein) were amplified, sequenced and Mega5.05 software was used to carry out genetic evolution analysis. The results showed that 140 16S rRNA sequences had 5 different sequence types, respectively named genotype A-E, and the genotype D was closely related to human isolates (U02521), and the homology was up to 99.8%, only one of them existed. Differences in base bases; there are 2~7 base differences between different genotypes; the distribution of 5 genotypes exists in different regions in China, genotype A exists in all provinces of investigation, and genotype B-E exists only in some provinces, such as genotype B only exists in Shaanxi; genotype C exists only in Luoyang, Henan; genotype D exists only in Shanxi, Heilongjiang and Luoyang, Henan; genotype E only existed in the.MSP4 gene evolution tree of Zhengzhou, Henan, which showed that there were 3 groups of 7 genotypes, which were named genotype I a, I B, I C, I C, I d, II A, II B and III.7 genotypes in different regions in China, and there were 7 MSP4 genotypes in some regions, and some in some regions. There are only 9 genotypes of 1~2 genotype.130 groESL sequence, named A-I type. There are different genotypes in different regions of our country. In Henan Province, there are 7 genotypes in A-G, while A, C, D, E, H, I are found in 6 genotypes in Shaanxi province; Guizhou and black dragon have been found in Guizhou and black dragon. Only genotypic A was found in Jiangan Province, and only genotypic C and E. were found in Shandong and Liaoning province. PCR method was used to detect the blood and goat milk samples in some areas of China. The results showed that A.phagocytophilum was positive in the blood samples, indicating that the milk goats in our country were infected with A.phagocytophilum. This study was the first time to use PC in the study. The R method was based on the 16S rRNA, MSP4 and groESL genes to analyze the genetic characteristics of sheep origin A.phagocytophilum in China. The results showed that 5 16S rRNA genotypes, 7 MSP4 genotypes and 9 groESL genotypes showed that the genetic diversity of the phagocytic phagocytic body in our sheep was found. Furthermore, the 16S rRNA gene sequence analysis showed the phagocytic fines of the sheep source. The relationship between the cytoplasmic and human source isolates (U02521) is close, and the homology is up to 99.1%~99.8%. Among them, there is only one base difference in genotype D and U02521, and there is a potential threat to human health. This study lays a foundation for further research on zoonosis and public health security.

【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.6

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