禽白血病净化中的种公鸡检测及逆转录酶抑制剂类药物在降低禽白血病垂直传播危害中的应用

发布时间：2018-07-18 15:07

【摘要】：禽白血病病毒(Avian Leukosis Virus,ALV)自首次被报道以来已在世界各地爆发和流行,因其高致病性、高致死率、高肿瘤并发率给各国养禽业造成了巨大的损失。ALV主要通过垂直传播方式呈指数级扩大,种蛋和精液在病毒垂直传播过程中起着决定性的作用。在很长的时期内,我们主要通过检测母鸡的血浆以及种蛋中是否有ALV感染来实施净化,而常常忽略了对公鸡的检测,然而对于净化中公鸡检测究竟以血浆病毒分离为准还是以精液分离为准也无明确结论。另外,ALV经垂直传播感染后,会引起感染鸡体重、产蛋率等各种生产机能下降,也会导致很高的致死率,给鸡群造成很大的危害,对于一些高阳性率的地方品系鸡群还存在过度淘汰影响育种进度的现实问题,我们希望通过多种途径能够降低高阳性率鸡群的ALV带毒率或至少减少由于垂直传播带毒对雏鸡的致病作用,为此本研究通过鸡胚卵黄囊接种模拟雏鸡感染ALV,然后给感染雏鸡添加AZT等逆转录酶抑制剂类药物观察了此类药物对降低ALV垂直传播危害的作用。1.不同种鸡群禽白血病净化中的种公鸡检测本研究对我国两个正在实施禽白血病净化的种鸡场的公鸡分别采取血浆病毒分离和精液病毒分离两种方式观察和比较了两种检测途径对评价种公鸡ALV感染状态时的对应性,并对部分阳性毒株做了序列测定和分析。结果显示,对正在实施净化的某蛋鸡场检测公鸡400份,其中精液样品病毒分离阳性为11份,血浆样品病毒分离阳性为13份,精液和血浆病毒分离同时为阳性的有8份。对正在实施净化的某野山鸡场检测公鸡1800份,其中精液样品病毒分离阳性为45份,血浆样品病毒分离阳性为188份,精液和血浆病毒分离同时为阳性的仅有16份。上述数据说明仅仅通过单纯的血浆病毒分离或者单纯的精液病毒分离均不能有效的检出所有的ALV阳性公鸡,从净化效率角度考虑应该同时对公鸡的血浆和精液做全面的病毒分离来确定阳性个体进而淘汰,这将有助于在最短的时间内淘汰阳性公鸡。对部分毒株gp85序列的测定和同源性分析结果显示,所分离到的20个毒株之间同源性高达99.0%~100%,这些样品与A亚群参考株之间的同源性最高,为88.6%~99.2%,显著高于与B、E、F、J、K亚群的同源性,与B、E、F、J、K等不同亚群同源性分别为83.2%~83.7%、85.4%~89.6%、83.0%~83.9%、50.0%~51.1%和84.8%~85.9%。进化树比对分析也发现,分离到的所有毒株均与A亚群毒株在一个分支上。2.逆转录酶抑制剂类药物对垂直传播感染ALV的SPF鸡的保护作用本研究通过对SPF鸡胚卵黄囊接种NX0101毒株模拟鸡胚经垂直传播感染ALV-J,待鸡胚孵育出壳后,将鸡胚分为两组,只对其中一组在出壳后的连续一周内肌肉注射5mg的Zidovudine(AZT)和2mg的Lamivudine(LAM),另一组在出壳后的连续一周内肌肉注射等剂量的生理盐水,与卵黄囊接种空白DMEM并且出壳后连续一周内肌肉注射等剂量生理盐水的完全空白对照组对比,观察逆转录酶抑制剂类药物对SPF鸡的保护作用以及对降低垂直传播危害的作用。结果显示,未使用药物的病毒感染组其体重、死亡率以及免疫NDV和AIV-H9所产生的抗体水平均显著低于使用药物的病毒感染组,更显著低于未接毒的生理盐水对照组,但是药物的使用未能阻止感染组ALV病毒血症的形成,说明药物无法替代针对禽白血病的净化工作。上述相关数据不仅进一步展示了ALV-J垂直传播对鸡群的危害,也显示了AZT和LAM等逆转录酶抑制剂类药物的使用有助于降低ALV-J垂直传播感染对鸡群的危害。
[Abstract]:Avian Leukosis Virus (ALV) has been outbursts and prevalent all over the world since it was first reported. Because of its high pathogenicity, high mortality, and high incidence of cancer, it has caused huge losses to poultry industry in various countries..ALV is expanded exponentially through vertical transmission, and eggs and semen play a role in the vertical transmission of the virus. In a very long period of time, we have done the purification mainly by detecting the plasma of hens and whether there is ALV infection in the eggs, and we often ignore the detection of the cocks. However, there is no definite conclusion whether the detection of the cock is based on the separation of the plasma virus or the semen separation. In addition, the ALV is vertical. After transmission of infection, it can cause the infection of chicken weight, the production function of laying eggs and other production functions, which will cause a high mortality rate and cause great harm to the chicken group. For some local chicken groups with high positive rates, there are some practical problems affecting the breeding progress. We hope to reduce the high positive rate of chicken through a variety of ways. The virulence rate of ALV in the group or at least reduces the pathogenic effect of the vertical transmission band poison on the chicks. Therefore, the chicken embryo yolk sac was inoculated with the simulated chicks infected with ALV, and then the infected chicks were added AZT and other reverse transcriptase inhibitors to observe the effect of this kind of drug on reducing the vertical transmission of ALV in.1.. In this study, we observed and compared the two methods of plasma virus isolation and separation of semen virus from two roosters who were carrying out avian leukaemia in our country, and compared two detection approaches to the evaluation of the ALV infection status of the cock, and the sequencing and classification of some positive strains. The results showed that 400 cocks were detected in a chicken farm which was being purified, of which 11 were isolated from the semen sample virus, 13 in the plasma sample virus isolation, and 8 in the semen and plasma virus isolation. 1800 roosters were detected in a wild mountain chicken field being purify, of which the semen sample virus was isolated. The positive was 45, the plasma sample virus isolation was 188, the semen and the plasma virus were isolated and positive only 16. The above data showed that all ALV positive cocks could not be detected only by pure plasma virus isolation or pure semen virus isolation, and the cocks should be considered at the same time from the angle of purification efficiency. The plasma and semen were separated from the virus to determine the positive individuals and then eliminated, which would help to eliminate the positive cocks in the shortest time. The determination of the gp85 sequence and the homology analysis showed that the homology of the 20 isolated strains was up to 99.0%~ 100%, and these samples were the same as the A subgroup reference strain. The homology of the subgroups of B, E, F, J, K is the highest, and the homology of the subgroups of B, E, F, J, K, respectively, are 83.2%~83.7%. The protective effect of drugs on SPF chicken with vertical transmission infection ALV was studied by inoculating the NX0101 strain of the yolk sac of the SPF chicken embryo to simulate the vertical transmission of ALV-J in chicken embryo, and the chicken embryo was divided into two groups after the chicken embryo was incubated and incubated, and the Zidovudine (AZT) and 2mg Lamivudine (LAM) of a group of 5mg were injected into the shell for a week. The other group was intramuscularly injected with normal saline within one week after the outer shell, compared with the complete blank control group of the yolk sac inoculated with blank DMEM and the intramuscular injection of normal saline within a week after the outer shell. The protective effect of the reverse transcriptase inhibitor on the SPF chicken and the reduction of the vertical transmission damage were observed. The results showed that the body weight, mortality, and the level of antibody produced by immune NDV and AIV-H9 were significantly lower than those in the virus infected group, which were significantly lower than those in the uninfected saline control group, but the use of the drug failed to prevent the formation of ALV virus in the infection group, indicating that the drug was not possible. Instead of purifying the avian leukaemia, these data not only further demonstrate the harm of vertical transmission of ALV-J to the chickens, but also show that the use of reverse transcriptase inhibitors such as AZT and LAM helps to reduce the harm of vertical transmission of ALV-J to chickens.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.31

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