钦州地区禽白血病的流行调查及其分离株env基因的分析

发布时间：2018-04-13 11:54

本文选题：钦州 + 禽白血病　；参考：《广西大学》2015年硕士论文

【摘要】：禽白血病(Avian leukosis, AL)是由统称为禽白血病病毒(Avian leukosis virus, ALV)的一群病毒引起的多种肿瘤病的总称。ALV给养禽业造成巨大的经济损失,国内外都给予很大关注,目前在全国范围内已经开展流行病学调查和净化工作。广西自开展AL的研究和净化工作以来,对玉林、桂林、百色、南宁等主要养禽地区的流调工作已基本完成。本课题选择广西的临海地区——钦州进行相关的流行病学调查以及病毒的分离和基因序列测定与分析,旨在完善全区范围内的主要地方品种鸡及其周围饲养的水禽及其他禽类的流行病学资料,并通过病毒分离和对分离株重要基因进行测序分析与比较,进一步掌握当地禽类感染ALV的亚型及分离株的遗传变异情况,为当地的净化与防控工作提供依据。首先,课题采集钦州地区饲养的铁脚麻鸡、“海鸭蛋”麻鸭、狮头鹅、鸽子和火鸡5个主要家禽品种的肛拭、蛋清和血清共计953份样品,应用IDEXX生产的商品化ALV-p27抗原和A/B、J抗体ELISA检测试剂盒进行检测。结果显示,除了铁脚麻鸡的肛拭样品之p27抗原阳性率为13.04%(12/92)、蛋清样品之p27抗原阳性率为3.26%(3/92)及血清样品之A/B抗体阳性率为5.08%(3/59)之外,其它样品及项目的检测的阳性率均为0%。其次,课题对抗原或抗体检测为阳性的16份样品、铁脚麻鸡检测阴性的16份样品以及火鸡检测阴性的33份样品共65份样品相对应的禽只的血清进行DF-1细胞培养的病毒分离,再用ELISA和PCR的方法分别检测其F2代的细胞培养液和培养物,经鉴定为阳性的样品再进行病毒e7nv基因的扩增及其序列的测定与分析。结果从65份样品中分离出2株J亚群ALV(分别命名为QZ2和QZ14),QZ2和QZ14的env基因核苷酸同源性很高(95.7%)；它们gp37基因核苷酸及其编码的氨基酸与J亚群参考株的同源性都在90%以上,env与gp85基因核苷酸与J亚群参考株(除ADOL-7501外)的同源性也都在90%以上,而gp85基因编码的氨基酸与J亚群参考株的同源性在70.5%～90.9%之间,与原型株HPRS103的达90%以上,U3区基因核苷酸与J亚群参考株的同源性在87.6%～92.9%之间,QZ2的E元件基本保持完整,而QZ14有5个碱基的缺失。本课题研究的结果表明,钦州地区5个主要禽类中仅铁脚麻鸡存在ALV的感染,经分离鉴定为J亚群,基因序列分析表明QZ2和QZ14的不同基因已发生变异。
[Abstract]:Avian leukemia (ALV) is caused by a group of viruses called avian leukemia virus (ALV). ALV has caused enormous economic losses to poultry industry, and has attracted great attention both at home and abroad.At present, epidemiological investigation and purification have been carried out nationwide.Since the research and purification of AL in Guangxi, the flow regulation of the main poultry breeding areas such as Yulin, Guilin, Baise and Nanning has been basically completed.In this study, we selected Linhai region of Guangxi, Qinzhou to carry on the related epidemiology investigation, the virus isolation and the gene sequence determination and analysis.The aim of this study was to improve the epidemiological data of major local chickens and their surrounding waterfowl and other birds in the whole region, and to analyze and compare the important genes of the isolates by virus isolation and sequencing.To further understand the subtypes of ALV infection and the genetic variation of isolates in local poultry, to provide the basis for local purification, prevention and control.First of all, 953 samples of egg white and serum were collected from five main poultry breeds in Qinzhou, namely, hens, sea duck, lion goose, pigeon and turkey.The commercial ALV-p27 antigen produced by IDEXX and the ELISA kit of A / B J antibody were used for detection.The results showed that the positive rate of p27 antigen in anus swabs was 13.04 / 92%, that of egg white was 3.26 / 92) and the positive rate of A / B antibody in serum was 5.08 / 3 / 59), the positive rates of other samples and items were all 0% (P < 0.05), the results showed that the positive rate of p27 antigen in anus swab samples was 13.04% / 92%, 3.26% / 92% in egg white samples, and 5.08% / 59 in serum samples respectively.Secondly, 16 samples with positive antigens or antibodies, 16 samples with negative detection of iron-footed hens and 33 samples with negative turkey were isolated from sera of poultry corresponding to DF-1 cell culture.ELISA and PCR were used to detect the culture medium and culture of F2 passage respectively. The positive samples were amplified by e7nv gene and sequenced.Results two strains of subgroup J ALV (named QZ2 and QZ14, QZ2 and QZ14) were isolated from 65 samples. The nucleotide homology of gp37 gene and its encoded amino acid was 90%.The nucleotides of gp85 gene and the reference strain of subgroup J (except ADOL-7501) were all more than 90% homology.The homology between amino acids encoded by gp85 gene and reference strain of subgroup J was between 70.5% and 90.9%, and the homology of E element of QZ2 between gp85 gene and the reference strain of subgroup J was 87.6% and 92.9%, respectively.QZ14 has a 5-base deletion.The results of this study showed that ALV was isolated and identified as subgroup J among the five main poultry species in Qinzhou area. The gene sequence analysis showed that the different genes of QZ2 and QZ14 had been mutated.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.3

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