几种信鸽新型病原分析与感染模型研究

发布时间：2018-06-25 12:14

本文选题：信鸽 + 宏基因组学　；参考：《中国人民解放军军事医学科学院》2017年硕士论文

【摘要】：信鸽与人类接触密切,信鸽饲养在我国有着悠久的历史和广泛的群众基础,并主要集中在大中城市等人口密集地区。信鸽携带疫病存在公共卫生安全风险。本课题围绕几种信鸽重要新型病原体,从病原宏基因组学研究、新型圆环病毒全基因组序列分析、出血性大肠杆菌活体成像模型、新型多重耐药海豚葡萄球菌分离鉴定等方面开展了系统研究。病原宏基因组学研究在本研究中利用以二代测序技术为基础的宏基因组学研究方法,对长春地区2015年3月到2016年1月间采集的信鸽与野化家鸽样本进行了病原筛查研究。共采集信鸽肛咽拭子1375份,血清样本157分,健康信鸽78只,病鸽及病死信鸽610余只,野化家鸽35只。样品采集地点包括长春市的绿园、二道、净月、宽城、九台、新立城镇和范家屯镇等地区。上述样本经过宏基因组学前期建库、测序及序列拼接后一共得到8,774,818个读长(reads),总共251,328,000个碱基(nt),经过拼接得到8,054,530个重叠序列,能拼接的序列占总序列的91.79%,Overlap Reads平均长度为181 nt。经过数据库注释,有32.956%的序列注释到了真菌,16.136%的序列注释到了细菌、2.201%的序列注释到了病毒、余下48.707%的为未知序列。其中病毒可以进一步分成22个科和两个亚科,37个属,9个科11个属的病毒。与禽类相关的病毒主要包括有:A型轮状病毒、鸽圆环病毒、海鸥圆环病毒、燕八哥圆环病毒、鸡圆环病毒、痘病毒、鸭乙型肝炎病毒。通过对比信鸽与野化家鸽的病毒组,发现两种不同种类的鸽子所携带的病毒种类存在较大的差异。A型轮状病毒在四组信鸽样品中均有出现,野化家鸽(feral pigeon)没有reads注释到轮状病毒。腺联病毒、禽冠状病毒、肠道病毒B、禽白血病病毒、鸽细小病毒等病毒序列则只出现在野化家鸽样品中。只有鸽圆环病毒(columbid circovirus)同时出现在信鸽与野化家鸽样品中。实验发现圆环病毒和轮状病毒为长春地区信鸽感染的主要病原。通过对比不同分组信鸽圆环病毒reads注释情况,发现YPDS鸽样品的PiCV序列数量和丰富度均高于健康鸽。新型圆环病毒全基因组序列分析。根据宏基因组测序的reads,选取了圆环病毒、轮状病毒进行进一步的深度分析。针对宏基因组测序得到的reads设计引物,获得了两株信鸽PiCV的基因组全序列。分析发现两株吉林PiCV处在一个进化分支,与来自欧美的6株鸽圆环病毒与1株鸡圆环病毒共处在一个大的分支。而中国的鸽圆环病毒浙江株和福建株则处在另一个大的分支上,因此可以怀疑,这两株信鸽圆环病毒很可能是随着信鸽进口贸易一并引进的外来病原。本实验成功的分离鉴定到一株信鸽轮状病毒,通过与9株已报道的轮状病毒部分VP2基因的对比和遗传进化分析,最后发现该毒株与北京地区环颈斑鸠的轮状病毒关系最近,而与其他鸽源轮状病毒关系较远,这提示,信鸽和环颈斑鸠这两种鸠鸽科的鸟类可以发生了病毒的交叉传播。出血性大肠杆菌活体成像模型在关注宏基因组测序结果中病毒注释情况的同时,还对样品序列中的细菌序列做了注释和分析。发现注释到大肠杆菌、葡萄球菌、沙门氏菌和巴氏杆菌的reads数量很多。为了探究大肠杆菌感染鸽子的过程,构建了发光大肠杆菌感染鸽子的生物发光活体成像模型。通过腹腔注射感染幼龄信鸽,通过生物发光信号来观察细菌在信鸽体内的定殖和扩散形况。在该实验中成功观察到了生物发光大肠杆菌通过鸽消化道上行感染并引起幼鸽呕吐的整个过程,并且在有呕吐症状幼鸽的嗉囊和呕吐物中检测到了生物发光信号,而呕吐恰也是目前在赛鸽中广泛流行的幼鸽疾病综合症(YPDS)的典型症状之一。新型多重耐药海豚葡萄球菌分离鉴定在样本收集的过程中,还对部分有特征性病症的信鸽进行了致病菌的分离。在众多的细菌分离株中,选取了一株具有多重耐药性的葡萄球菌进行了进一步的实验分析。经过形态学观察、生化特性与耐药性分析、Partial kat(Catalase)基因与16S rRNA基因序列分析,确定该菌为凝固酶阳性、β内酰胺酶阳性海豚葡萄球菌,该菌具有多重耐药特性。动物实验证实,该菌株可以引起幼鸽的肺炎和眼部炎症。该菌株的16S rRNA基因与土耳其家鸽分离株(HQ452509.1)16S rRNA基因的核苷酸同源性最高,为100%。而Partial kat(Catalase)基因则与俄罗斯家鸽源海豚葡萄球菌(KU641399.1)的同源性最高,为99%。然而该菌具有其它家鸽分离株所不具备的多种抗生素的耐药性,该病菌对氨苄西林、青霉素G、复方磺胺恶唑、甲氧苄氨嘧啶、克林霉素、红霉素、达福普丁、环丙沙星、四环素均耐药,对阿米卡星、庆大霉素、妥布霉素、头孢西丁、苯唑西林、替考拉宁、万古霉素、利奈唑胺、莫匹罗星、利福平敏感。综上所述,本研究利用宏基因组学方法和生物发光模型等新技术,结合传统的病原分离鉴定手段,针对长春地区信鸽新型病原进行了系统研究。成功获得了两株圆环病毒的全基因组序列,鉴定分离一株信鸽源A型轮状病毒和一株多重耐药的海豚葡萄球菌;对上述几株新型病原的分离鉴定研究工作将在一定程度上弥补了国内信鸽疫病研究的不足,为今后信鸽疫病的防控提供了参考。成功建立出血性大肠杆菌活体成像模型,感染模型的建立将有效提高对鸽源病原感染机制的研究提供了一种新的手段,同时为信鸽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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.39

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