本文選題：信鴿 + 宏基因組學(xué)　； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文

【摘要】：信鴿與人類接觸密切,信鴿飼養(yǎng)在我國有著悠久的歷史和廣泛的群眾基礎(chǔ),并主要集中在大中城市等人口密集地區(qū)。信鴿攜帶疫病存在公共衛(wèi)生安全風(fēng)險。本課題圍繞幾種信鴿重要新型病原體,從病原宏基因組學(xué)研究、新型圓環(huán)病毒全基因組序列分析、出血性大腸桿菌活體成像模型、新型多重耐藥海豚葡萄球菌分離鑒定等方面開展了系統(tǒng)研究。病原宏基因組學(xué)研究在本研究中利用以二代測序技術(shù)為基礎(chǔ)的宏基因組學(xué)研究方法,對長春地區(qū)2015年3月到2016年1月間采集的信鴿與野化家鴿樣本進行了病原篩查研究。共采集信鴿肛咽拭子1375份,血清樣本157分,健康信鴿78只,病鴿及病死信鴿610余只,野化家鴿35只。樣品采集地點包括長春市的綠園、二道、凈月、寬城、九臺、新立城鎮(zhèn)和范家屯鎮(zhèn)等地區(qū)。上述樣本經(jīng)過宏基因組學(xué)前期建庫、測序及序列拼接后一共得到8,774,818個讀長(reads),總共251,328,000個堿基(nt),經(jīng)過拼接得到8,054,530個重疊序列,能拼接的序列占總序列的91.79%,Overlap Reads平均長度為181 nt。經(jīng)過數(shù)據(jù)庫注釋,有32.956%的序列注釋到了真菌,16.136%的序列注釋到了細菌、2.201%的序列注釋到了病毒、余下48.707%的為未知序列。其中病毒可以進一步分成22個科和兩個亞科,37個屬,9個科11個屬的病毒。與禽類相關(guān)的病毒主要包括有:A型輪狀病毒、鴿圓環(huán)病毒、海鷗圓環(huán)病毒、燕八哥圓環(huán)病毒、雞圓環(huán)病毒、痘病毒、鴨乙型肝炎病毒。通過對比信鴿與野化家鴿的病毒組,發(fā)現(xiàn)兩種不同種類的鴿子所攜帶的病毒種類存在較大的差異。A型輪狀病毒在四組信鴿樣品中均有出現(xiàn),野化家鴿(feral pigeon)沒有reads注釋到輪狀病毒。腺聯(lián)病毒、禽冠狀病毒、腸道病毒B、禽白血病病毒、鴿細小病毒等病毒序列則只出現(xiàn)在野化家鴿樣品中。只有鴿圓環(huán)病毒(columbid circovirus)同時出現(xiàn)在信鴿與野化家鴿樣品中。實驗發(fā)現(xiàn)圓環(huán)病毒和輪狀病毒為長春地區(qū)信鴿感染的主要病原。通過對比不同分組信鴿圓環(huán)病毒reads注釋情況,發(fā)現(xiàn)YPDS鴿樣品的PiCV序列數(shù)量和豐富度均高于健康鴿。新型圓環(huán)病毒全基因組序列分析。根據(jù)宏基因組測序的reads,選取了圓環(huán)病毒、輪狀病毒進行進一步的深度分析。針對宏基因組測序得到的reads設(shè)計引物,獲得了兩株信鴿PiCV的基因組全序列。分析發(fā)現(xiàn)兩株吉林PiCV處在一個進化分支,與來自歐美的6株鴿圓環(huán)病毒與1株雞圓環(huán)病毒共處在一個大的分支。而中國的鴿圓環(huán)病毒浙江株和福建株則處在另一個大的分支上,因此可以懷疑,這兩株信鴿圓環(huán)病毒很可能是隨著信鴿進口貿(mào)易一并引進的外來病原。本實驗成功的分離鑒定到一株信鴿輪狀病毒,通過與9株已報道的輪狀病毒部分VP2基因的對比和遺傳進化分析,最后發(fā)現(xiàn)該毒株與北京地區(qū)環(huán)頸斑鳩的輪狀病毒關(guān)系最近,而與其他鴿源輪狀病毒關(guān)系較遠,這提示,信鴿和環(huán)頸斑鳩這兩種鳩鴿科的鳥類可以發(fā)生了病毒的交叉?zhèn)鞑�。出血性大腸桿菌活體成像模型在關(guān)注宏基因組測序結(jié)果中病毒注釋情況的同時,還對樣品序列中的細菌序列做了注釋和分析。發(fā)現(xiàn)注釋到大腸桿菌、葡萄球菌、沙門氏菌和巴氏桿菌的reads數(shù)量很多。為了探究大腸桿菌感染鴿子的過程,構(gòu)建了發(fā)光大腸桿菌感染鴿子的生物發(fā)光活體成像模型。通過腹腔注射感染幼齡信鴿,通過生物發(fā)光信號來觀察細菌在信鴿體內(nèi)的定殖和擴散形況。在該實驗中成功觀察到了生物發(fā)光大腸桿菌通過鴿消化道上行感染并引起幼鴿嘔吐的整個過程,并且在有嘔吐癥狀幼鴿的嗉囊和嘔吐物中檢測到了生物發(fā)光信號,而嘔吐恰也是目前在賽鴿中廣泛流行的幼鴿疾病綜合癥(YPDS)的典型癥狀之一。新型多重耐藥海豚葡萄球菌分離鑒定在樣本收集的過程中,還對部分有特征性病癥的信鴿進行了致病菌的分離。在眾多的細菌分離株中,選取了一株具有多重耐藥性的葡萄球菌進行了進一步的實驗分析。經(jīng)過形態(tài)學(xué)觀察、生化特性與耐藥性分析、Partial kat(Catalase)基因與16S rRNA基因序列分析,確定該菌為凝固酶陽性、β內(nèi)酰胺酶陽性海豚葡萄球菌,該菌具有多重耐藥特性。動物實驗證實,該菌株可以引起幼鴿的肺炎和眼部炎癥。該菌株的16S rRNA基因與土耳其家鴿分離株(HQ452509.1)16S rRNA基因的核苷酸同源性最高,為100%。而Partial kat(Catalase)基因則與俄羅斯家鴿源海豚葡萄球菌(KU641399.1)的同源性最高,為99%。然而該菌具有其它家鴿分離株所不具備的多種抗生素的耐藥性,該病菌對氨芐西林、青霉素G、復(fù)方磺胺惡唑、甲氧芐氨嘧啶、克林霉素、紅霉素、達福普丁、環(huán)丙沙星、四環(huán)素均耐藥,對阿米卡星、慶大霉素、妥布霉素、頭孢西丁、苯唑西林、替考拉寧、萬古霉素、利奈唑胺、莫匹羅星、利福平敏感。綜上所述,本研究利用宏基因組學(xué)方法和生物發(fā)光模型等新技術(shù),結(jié)合傳統(tǒng)的病原分離鑒定手段,針對長春地區(qū)信鴿新型病原進行了系統(tǒng)研究。成功獲得了兩株圓環(huán)病毒的全基因組序列,鑒定分離一株信鴿源A型輪狀病毒和一株多重耐藥的海豚葡萄球菌;對上述幾株新型病原的分離鑒定研究工作將在一定程度上彌補了國內(nèi)信鴿疫病研究的不足,為今后信鴿疫病的防控提供了參考。成功建立出血性大腸桿菌活體成像模型,感染模型的建立將有效提高對鴿源病原感染機制的研究提供了一種新的手段,同時為信鴿其它疫病感染模型建立提供新的思路。
[Abstract]:Pigeon is closely related to human beings, and the breeding of pigeons has a long history and extensive mass base in our country. It is mainly concentrated in the densely populated areas such as large and medium-sized cities. The carrier pigeon carries the risk of public health safety. This topic is based on several important new pathogens of the pigeon, from the study of the pathogenic macrogenomics and the new circic virus. Genomic sequence analysis, hemorrhagic Escherichia coli living imaging model, new multidrug resistant Staphylococcus aureus isolation and identification were systematically studied. In this study, the etiological macrogenomics study used the two generation sequencing technology based metagenomics research method to collect the Changchun region from March 2015 to January 2016. The pigeon and wild pigeon samples were screened for pathogen screening. 1375 samples of pigeon anoropharyngeal swabs, 157 serum samples, 78 healthy pigeons, 610 sick pigeons and 610 dead pigeons, 35 wild pigeons, including the green garden in Changchun, two, the net month, the wide city, nine stations, Xinli Town and fan Jia Tun Town, were collected. After the construction of the metagenomic pre-school period, 8774818 reading lengths (reads) and 251328000 bases (NT) were obtained after the sequence and sequence splicing, and 8054530 overlapping sequences were obtained by splicing. The sequence of splicing was 91.79% of the total sequence, and the average length of Overlap Reads was 181 nt. through the database annotation, 32.956% of the sequence annotated. Fungi, 16.136% of the sequence annotated to the bacteria, 2.201% of the sequence annotated to the virus, the remaining 48.707% of the unknown sequence. The virus can be further divided into 22 families and two subfamilies, 37 genera, 9 families and 11 genera of the virus. The viruses associated with poultry include: A rotavirus, pigeon circovirus, seagull circovirus, Yan eight Coronavirus, chicken circovirus, pox virus, duck hepatitis B virus. By comparing the virus groups of homing pigeons and wild pigeons, the species of two different types of dove were found to have a large difference in the type of.A rotavirus in four groups of pigeon samples, and the wild pigeon (Feral pigeon) did not have reads annotation to rotaform disease. Virus, avian coronavirus, enterovirus B, avian leukosis virus, pigeon parvovirus and other virus sequences only appeared in the wild pigeon samples. Only the pigeon circovirus (columbid circovirus) appeared in the homing pigeon and the wild pigeon samples. The experiment found that circovirus and rotavirus were the main diseases of pigeon infection in Changchun area. By comparing the reads annotation of different group pigeon circovirus, it was found that the number and richness of the PiCV sequence of the YPDS pigeon samples were higher than that of the healthy pigeons. The whole genome sequence analysis of the new-type circovirus. According to the reads of the macrogenome sequencing, the circovirus was selected and the rotavirus was further analyzed. The sequence of the macrogenome was sequenced. The reads design primers obtained the total genomic sequence of two homing pigeon PiCV. The analysis found that two strains of Jilin PiCV were in one evolutionary branch, with 6 pigeon circoviruses from Europe and the United States and 1 chicken circoviruses in a large branch. China's pigeon circovirus Zhejiang strain and Fujian strain were in another large branch. It is doubted that the two strains of the pigeon circovirus are likely to be a foreign pathogen introduced in the import trade of the pigeon. This experiment was successfully isolated and identified as a pigeon rotavirus. Through comparison with 9 reported rotavirus VP2 genes and genetic evolution analysis, it was found that the strain was found in the Beijing region. The relationship between rotavirus and other pigeon rotavirus is far closer. This suggests that the two species of dove pigeondoves can cross spread the virus. The hemorrhagic Escherichia coli living body imaging model also focuses on the bacterial sequence in the sample sequence while focusing on the release of the virus in the genome sequencing results. Annotated and analyzed. It was found that there were a lot of reads in Escherichia coli, Staphylococcus, Salmonella and Pasteurella. In order to explore the process of infection of dove in Escherichia coli, a bioluminescent living body imaging model of Escherichia coli infected with dove was constructed. By intraperitoneal injection of infecting young pigeons, bioluminescent signals were used. The colonization and diffusion of bacteria in the pigeon were observed. In this experiment, the whole process of the infection of the bioluminescent Escherichia coli through the pigeon digestive tract and the vomiting of the young pigeons was observed successfully, and the biological light signal was detected in the crop and vomit of the young pigeons with vomit symptoms, and the vomiting was just the present in the pigeon. One of the typical symptoms of the widely popular young pigeon disease syndrome (YPDS). The isolation and identification of a new type of multidrug resistant Staphylococcus aureus in the process of collection and isolation of pathogenic pigeons in some characteristic diseases. In a number of bacterial isolates, a strain of staphylococci with multiple resistance was selected. Further experimental analysis. After morphological observation, biochemical characteristics and drug resistance analysis, Partial Kat (Catalase) gene and 16S rRNA gene sequence analysis, the bacteria were determined to be coagulase positive, beta lactamase positive Staphylococcus, and the bacteria had multiple resistance characteristics. Animal experiment confirmed that the strain could cause pneumonia and eye of young pigeons. The 16S rRNA gene of this strain has the highest nucleotide homology with the Turkey pigeon isolate (HQ452509.1) 16S rRNA gene, and the Partial Kat (Catalase) gene has the highest homology with the Russian pigeon source Staphylococcus aureus (KU641399.1), which is 99%. but it has a variety of resistance to other pigeon isolates. The resistance to ampicillin, penicillin G, compound sulfamethoxazole, trimethoprim, clindamycin, erythromycin, Dafoe Putin, ciprofloxacin, tetracycline were all resistant to Amikacin, gentamicin, tobramycin, cefoxinin, zoxoxinine, teicoplanin, vancomycin, linezolidamine, mopiopine, and rifampicin. To sum up, this study, using the new techniques such as macrogenomics and bioluminescence, combined with the traditional pathogen isolation and identification methods, systematically studied the new pathogens of Changchun pigeons, and successfully obtained the whole genome sequence of two circovirus strains, identified as a strain of a pigeon source A rotavirus and one strain of multiple tolerance. The study on the isolation and identification of these new pathogens will make up for the deficiency of the domestic pigeon blight and provide reference for the prevention and control of the pigeon blight in the future. The imaging model of the hemorrhagic Escherichia coli is established successfully, and the establishment of the infection model will effectively improve the pathogen infection of the pigeon source. The study of mechanism provides a new means, and provides a new idea for establishing other infectious disease models of pigeons.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.39

【參考文獻】

相關(guān)期刊論文 前4條

1 傅業(yè)濤;玉峰;;中國信鴿價值評估方法初探[J];中國農(nóng)業(yè)會計;2016年07期

2 王原;曹俊敏;陳益民;許江燕;;BD PHOENIX-100全自動微生物鑒定儀對葡萄球菌鑒定能力的評價[J];浙江中醫(yī)藥大學(xué)學(xué)報;2009年02期

3 張曉楠;肖玉;黃勇;張秀敏;王濤;王麗;關(guān)璐媛;趙瑋欽;;影響大腸桿菌電轉(zhuǎn)化效率因素的探討[J];中國現(xiàn)代醫(yī)學(xué)雜志;2008年04期

4 高文通，高喬婉，，范懷忠;番木瓜環(huán)斑病毒（PRV）提純的研究[J];中國病毒學(xué);1994年02期

相關(guān)博士學(xué)位論文 前1條

1 何彪;蝙蝠病毒組學(xué)及其新病毒的發(fā)現(xiàn)與鑒定[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2014年

相關(guān)碩士學(xué)位論文 前2條

1 張曉院;農(nóng)村慢性鼻—鼻竇炎患者分泌物病原菌培養(yǎng)分析[D];安徽醫(yī)科大學(xué);2015年

2 李金松;應(yīng)用非序列依賴單引物PCR（SISPA-PCR）技術(shù)篩查腹瀉相關(guān)的病毒[D];中國疾病預(yù)防控制中心;2010年

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