宁夏禽流感流行病学调查及病毒HA基因遗传进化分析

发布时间：2018-02-15 12:18

本文关键词： 宁夏禽流感流行病学调查风险因素分析遗传变异 TaqMan探针荧光RT-PCR　出处：《甘肃农业大学》2017年博士论文　论文类型：学位论文

【摘要】：禽流感(avian Influenza,AI)是由禽流感病毒(Avian influenza viruse,AIV)引起的一种禽类疾病综合征。该病毒属于正黏病毒科(Orthomyxoviridae)甲型流感病毒属(Influenzavirus A),呈世界性分布,能感染许多的禽类。水禽是禽流感病毒的贮存库,一旦禽流感病毒从水禽传播给家禽,可引起致死性的疾病毁灭性的后果。还有一少部分禽流感病毒可由禽类传播给人,甚至导致人的死亡。因此,开展禽流感流行病学调查和分子遗传进化分析研究,对禽流感的防控及人-禽间的流感大流行预测具有重要意义。本研究通过利用简单随机的抽样方法,对宁夏2013~2015年采集的33个规模场、9个活禽市场和3个屠宰场的2700份血清以及18个市场(次)、143个养殖场、5个屠宰场共计297个家禽群体的5840份样品,分别进行血清学和病原学检测,同时利用统计学方法对规模场禽流感的感染风险因素进行分析。血清学检测结果表明,H5和H9亚型禽流感免疫抗体总体个体合格率分别为84.41%和94.3%,场群合格率分别为86.67%和98.89%;规模场免疫抗体水平总体高于活禽市场和屠宰场;H7亚型禽流感感染抗体个体阳性率和场群阳性率均为0。病原学检测结果表明,各监测场点主要以H9亚型禽流感病原为主。此外,在1个县的规模场检测到了H5N6亚型高致病性禽流感,在1个市场中检测到了H7N9亚型禽流感,在1个野鸟栖息地中检测到了H3N2亚型禽流感;10月份至次年5月份是病毒的活跃期;从不同监测品种来看,黄羽肉鸡病原学阳性高于其它品种的禽类;从不同地区检测结果来看,各地均检测到了禽流感病毒,其中,中卫市和固原市为禽流感流行的主要区域。规模场感染风险因素分析表明,执行全进全出制度、安装严密的防鸟网、病死禽及时无害化处理、单批次养殖、人员出入严格消毒、不进出活禽市场和不与外部人员接触可以有效预防规模场禽流感的感染;利用logistic回归建立回归模型,并绘制ROC曲线,结果表明,模型拟合度和预测规模场禽流感传播风险的能力均较好。利用暴发调查方法,对一起规模场肉杂鸡感染H5N6亚型禽流感病毒疫情开展了紧急流行病学调查。结果表明,引起此次疫情的原因是,由携带有H5N6亚型禽流感的野鸟传播给散养肉杂鸡后,经过养殖场人员与病死禽接触将疫情带入该规模场。对该起疫情通过紧急扑杀和无害化处理后,有效控制了本病的传播和蔓延。针对调查结果,建议养殖场应加强生物安全建设,转变饲养模式,依法科学开展养殖;同时政府应加大科技研发的投入产出力度,推动科技工作者及时研制高效匹配的疫苗,以用于禽流感的防控。为了研究宁夏禽流感病毒遗传进化特性,对分离的16株禽流感病毒开展了HA基因的遗传进化分析。结果显示,分离的1株野鸟源H3N2亚型禽流感与蒙古国A/duck/Mongolia/199/2015(H3N8)株的核苷酸同源性为最高,为98.6%,提示该毒株由外源传入;分离的1株H5N6亚型禽流感病毒属于Clad 2.3.4.4分支,较2015年之前在宁夏分离到的3株H5亚型禽流感毒核苷酸同源性发生了很大的变异,提示要及时利用匹配的疫苗来开展预防和控制;分离到1株H7N9流感病毒与2013新发H7N9亚型流感病毒的表面基因高度相似,与人源代表株A/Anhui/1/2013(AH/1)的HA基因的核苷酸同源性同样高度相似,达到98.7%,提示要密切加强对该病毒的监测,关注此类病毒变异情况;分离的13株H9N2亚型禽流感病毒与疫苗毒株的核苷酸同源性在88.7%~91.4%之间,说明分离到的H9亚型毒株的HA基因序列已经发生了变异,推测当前使用的疫苗难以起到有效的保护作用。同时,为满足禽流感病毒的检测需要,本研究建立了H5和H9亚型禽流感病毒TaqMan MGB荧光RT-PCR检测方法。与病毒分离鉴定、商品化试剂盒一致性检验结果相比,本研究建立的H5亚型和H9亚型禽流感病毒TaqMan MGB探针荧光RT-PCR检测方法特异性强,敏感性高,能快速、准确地检测禽咽喉-泄殖腔拭子、病理组织和粪便中H5和H9亚型禽流感病毒核酸,可为禽流感诊断、疫病监测、流行病学调查等方面提供有力的科学依据。
[Abstract]:Avian influenza (avian Influenza, AI) by avian influenza virus (Avian influenza, viruse, AIV) a poultry disease caused by the syndrome. The virus belongs to the Orthomyxoviridae (Orthomyxoviridae) influenza A virus (Influenzavirus A), with a worldwide distribution, can infect many birds. Waterfowl storage bird influenza virus, avian influenza virus from birds once spread to poultry, can cause fatal disease with devastating consequences. There is a small part of the bird flu virus from poultry to human transmission, even cause people's death. Therefore, to carry out bird flu epidemiological investigation and molecular phylogenetic analysis research on avian influenza the prevention and control of avian and human influenza pandemic the prediction has important significance. This research by using simple random sampling method, the Ningxia 2013~2015 acquisition of 33 field scale, 9 live poultry markets and 3 slaughter 2700 blood Clear and 18 markets (Times), 143 farms, 5840 samples of 5 slaughterhouses totaling 297 poultry populations, respectively, serological and pathogenic detection, and use the statistical method to the scale of flu infection risk factors were analyzed. The serological detection results show that H5 and H9 subtype of avian influenza antibody individual overall pass rates were 84.41% and 94.3%, a group of qualified rate were 86.67% and 98.89%; the scale of the field level of antibody is higher than that of the live poultry market and slaughterhouse; the positive rate of antibody positive rate of group and individual field infection of H7 avian influenza are 0. pathogen detection results show that the monitoring points mainly H9 subtype avian influenza pathogen. In addition, the scale of field detection in the 1 counties of the H5N6 subtype of highly pathogenic avian influenza, in 1 the market was detected in the H7N9 subtype of avian influenza, in 1 wild bird habitats were detected in H3N2 subtype Avian influenza virus; from October to May is the period of active monitoring; from different species, was higher than that of the other varieties of pathogen of Huang Yu broiler poultry; from the detection results of different regions, all were detected in the avian influenza virus, the central city and Guyuan city as the main area. The scale of the field of avian influenza infection the risk factor analysis showed that the implementation of the whole system installed in all out, bird proof rigorous, poultry timely harmless treatment, single batch culture, personnel entry and strict disinfection, no import of live bird markets and contact with external personnel can effectively prevent infection of avian influenza field scale; regression using logistic regression models, and draw the results show that the ROC curve model fitting and prediction of field scale spread of avian influenza risk ability are good. The outbreak investigation method, on field scale of chickens infected with H5N6 avian influenza The virus carried out emergency epidemiological investigation. The results showed that the cause of the outbreak is carried by wild birds spread of H5N6 avian influenza to backyard chickens, and poultry farms through personnel contact will be brought into the field of epidemic scale. The epidemic through emergency culling and harmless treatment after the control of the spread of the disease and spread effectively. According to the results of the survey, suggestions of farms should strengthen the biological safety construction, change the feeding mode, in accordance with the law to carry out scientific breeding; at the same time the government should increase the input and output of science and technology research and development efforts to promote the development of high technology workers timely, vaccine for the prevention and control of avian influenza in order to. Study on the genetic characteristics of avian influenza virus in Ningxia, on 16 strains of avian influenza virus to carry out genetic evolution analysis of HA gene. The results showed that 1 strains isolated from wild birds in the H3N2 subtype of avian flu and Mongolia The ancient A/duck/Mongolia/199/2015 (H3N8) nucleotide homology was the highest, 98.6%, suggesting that the strain by exogenous afferents; 1 strains of H5N6 subtype avian influenza virus belongs to the Clad 2.3.4.4 branch in Ningxia before 2015, compared with 3 isolated strains of H5 subtype avian influenza virus nucleotide homology has great variation, suggesting that to timely use, vaccine to carry out prevention and control; surface genes isolated from 1 strains of H7N9 influenza virus and 2013 new H7N9 influenza viruses are highly similar, and human strains A/Anhui/1/2013 (AH/1) HA gene nucleotide homology is also highly similar to 98.7%, suggesting that closely to strengthen the monitoring of the virus, paid attention to this kind of virus mutation; 13 strains of H9N2 subtype avian influenza virus vaccine strain and the nucleotide homology between 88.7%~91.4%, HA gene sequence from H9 strains Has been changed, that vaccines currently in use is difficult to play an effective role in the protection. At the same time, in order to meet the needs of detection of avian influenza virus, this study established a method for the determination of H5 and H9 subtype avian influenza virus TaqMan MGB fluorescence RT-PCR. Identification and virus isolation, commercial kit consistency test results compared this study established a method for detection of H5 subtype and H9 subtype avian influenza virus TaqMan MGB RT-PCR fluorescent probe has strong specificity, high sensitivity, fast and accurate detection of avian throat - cloacal swabs, H5 and H9 subtype avian influenza virus nucleic acid pathological tissues and feces, for the diagnosis of avian influenza, epidemic monitoring and provide the scientific basis for epidemiological investigation.

【学位授予单位】：甘肃农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S855.3

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