本文選題：綿羊 + 山羊�。� 參考：《河南農(nóng)業(yè)大學》2015年碩士論文

【摘要】：嗜吞噬細胞無漿體(Anaplasma phagocytophilum),是一種專性細胞內(nèi)寄生的革蘭陰性菌,主要寄生于中性粒細胞。A.phagocytophilum可引起新發(fā)的經(jīng)蜱傳播的人獸共患自然疫源性疾病,不僅對人類健康構成嚴重威脅,而且對畜牧業(yè)經(jīng)濟效益造成重大影響。另外,A.phagocytophilum分離株在多種宿主動物和蜱均存在遺傳多樣性。本研究旨在應用分子生物學技術對我國羊源A.phagocytophilum的遺傳特征進行分析。1.為了解我國部分地區(qū)奶山羊嗜吞噬細胞無漿體病的流行情況,并為該病的防控提供科學的參考和依據(jù),促進奶山羊業(yè)的健康發(fā)展,本研究于2012年7月至2014年9月,采用巢式PCR檢測方法,對采自河南省(洛陽宜陽、洛陽澗西區(qū)、新密市、南陽市西峽縣、開封、平頂山、商丘寧陵縣)、云南省(昆明)、陜西省(富平縣、麟游縣、涇陽縣、龍陵縣)奶山羊血液樣品300份、羊奶樣品204份進行了嗜吞噬細胞無漿體感染情況調(diào)查。結果僅在羊血液樣本中檢測出A.phagocytophilum,陽性率為18%(54/300),而羊奶中未檢出A.phagocytophilum。不同地區(qū)奶山羊的A.phagocytophilum感染情況不同。洛陽宜陽地區(qū)奶山羊感染A.phagocytophilum陽性率達100%(5/5),陜西麟游陽性率較高為76.67%(46/60),平頂山的陽性率為12.50%(2/16),陜西高陵為2.86%(1/35),而洛陽澗西、鄭州新密、南陽西峽、開封、寧陵、云南昆明、陜西涇陽、陜西富平均未見感染;不同品種奶山羊A.phagocytophilum的感染情況不同,雜交奶山羊A.phagocytophilum感染率最高,為83.87%(26/31),關中奶山羊陽性率較高,為22.83%(21/92),其次是本地奶山羊,陽性率為11.11%(7/63),而未見薩能奶山羊感染A.phagocytophilum;不同飼養(yǎng)模式下的奶山羊A.phagocytophilum感染情況也不同,放牧奶山羊的陽性率高達38.85%(54/139),而舍飼條件下的奶山羊未見感染A.phagocytophilum。另外,16S rRNA基因序列分析顯示,本研究中的54條A.phagocytophilum序列中有一條序列出現(xiàn)了3個堿基的差異。遺傳進化分析顯示,羊源A.phagocytophilum菌株和人分離株(U02521)關系較近,同源性為99.1%~99.5%,存在潛在的人獸共患風險。2.為了解我國羊源嗜吞噬細胞無漿體的遺傳特征,是否存在人獸共患風險,本研究于2012年5月至2014年9月,應用巢式PCR方法,對采自河南省(洛陽、鄭州、開封、南陽、新鄉(xiāng)、濟源、平頂山、三門峽、駐馬店)、云南省(昆明)、遼寧省(朝陽)、山東省(臨朐)、貴州省(貴陽、晴隆)、山西省(晉城)、黑龍江省(黑龍江)、陜西省(富平縣、麟游縣、涇陽縣、龍陵縣)140份羊源A.phagocytophilum DNA陽性樣品進行16S rRNA、MSP4(主要膜蛋白)和groESL(熱休克蛋白)基因的擴增、測序以及采用Mega5.05軟件進行遺傳進化分析。結果顯示,140條16S rRNA基因序列出現(xiàn)了5種不同的序列類型,分別命名為基因型A-E;且基因型D與人分離株(U02521)關系較近,同源性高達99.8%,僅存在一個堿基差異;不同基因型之間存在2~7個堿基差異;5個基因型在我國不同地區(qū)之間分布情況存在差異,基因型A存在于調(diào)查的所有省份,而基因型B-E僅存在于個別省份,如基因型B只存在陜西;基因型C只存在于河南洛陽;基因型D只存在于山西、黑龍江和河南洛陽;基因型E只存在于河南鄭州。MSP4基因進化樹顯示47條序列出現(xiàn)了3類群共7個基因型,分別命名為基因型Ⅰa、Ⅰb、Ⅰc、Ⅰd、Ⅱa、Ⅱb和Ⅲ。7個基因型在我國不同地區(qū)之間分布情況存在差異,有的地區(qū)存在7個MSP4基因型,而有的地區(qū)僅存在1~2個基因型。130條groESL基因序列出現(xiàn)9種序列類型,分別命名為基因型A-I型;我國不同地區(qū)的基因型存在差異,河南省發(fā)現(xiàn)了A-G共7個基因型;而陜西省發(fā)現(xiàn)了A、C、D、E、H、I共6個基因型;山西省發(fā)現(xiàn)了A、B、D、F、G共5個基因型;貴州和黑龍江省均只發(fā)現(xiàn)基因型A;山東和遼寧省分別僅發(fā)現(xiàn)了基因型C和E。本研究應用PCR方法對我國部分地區(qū)奶山羊血液和羊奶樣品進行了檢測,結果在血液樣品中發(fā)現(xiàn)A.phagocytophilum陽性,表明我國奶山羊存在A.phagocytophilum感染。本研究首次采用PCR方法基于16S rRNA、MSP4和groESL基因對我國羊源A.phagocytophilum遺傳特征進行分析,結果共發(fā)現(xiàn)5個16S rRNA基因型,7個MSP4基因型,9個groESL基因型,說明我國羊源嗜吞噬細胞無漿體存在遺傳多樣性;另外,16S rRNA基因序列分析顯示羊源的嗜吞噬細胞無漿體菌株和人源分離株(U02521)關系較近,同源性高達99.1%~99.8%,其中,基因型D和U02521僅存在一個堿基差異,對人類健康存在潛在威脅。本研究為進一步開展人獸共患病的監(jiān)測和公共衛(wèi)生安全研究奠定了基礎。
[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.

【學位授予單位】：河南農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.6

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