地方品种芦花鸡禽白血病初步净化研究

发布时间：2018-06-19 05:35

本文选题：芦花鸡 + 禽白血病毒　；参考：《山东农业大学》2015年硕士论文

【摘要】：禽白血病(AL)是由禽C型反转录病毒群引起的禽类多种肿瘤性疾病的统称,可引起鸡的良性和恶性肿瘤以及亚临床感染并导致生长迟缓、产蛋下降和免疫抑制。近年来已在中国鸡群中广泛传播,对地方品种鸡群的安全构成了严重的威胁,并给地方品种鸡保种和选育工作带来很大的困难。目前实施AL净化的国际经典方法常因检测材料或检测方法的单一造成漏检。本研究取地方品种芦花公、母鸡的无菌抗凝血清经细胞培养分离病毒,并用ELISA方法检测细胞上清及母鸡卵白中的ALV-p27抗原,了解芦花鸡群中ALV的感染状态,并随机抽取2份经ELISA检测上清呈阳性的CEF提取前病毒cDNA进行ALV亚型的鉴定;同时用不同检测方法对同一批鸡的不同材料进行检测,探究不同检测材料与不同检测方法之间的相关性,优化ALV检测方法以降低ALV漏检率。并在此基础上对该种鸡群后代进行追踪检测,以较早实现对芦花鸡AL的净化工作。此外,本文还探究了鸡群ALV感染状态与NDV、AIV-H5及AIV-H9疫苗免疫抗体水平相关性以及鸡群ALV感染状态与鸡群鸡白痢阳性率相关性。1芦花鸡群感染状态调查为了解江苏某鸡场芦花鸡ALV的感染状态,对来自该鸡场种鸡群1024枚种蛋(508只鸡两枚,8只鸡一枚)以及对应516只母鸡无菌抗凝血清、232份公鸡无菌抗凝血清及精液接种CEF9d后的细胞上清中的ALV-p27抗原进行ELISA方法检测,结果显示母鸡卵白阳性检出率为20.7%(107/516),母鸡无菌抗凝血阳性检出率为2.52%(13/516)。公鸡精液阳性检出率为15.1%(35/232),无菌抗凝血阳性检出率为1.29%(3/232)。病毒分离鉴定结果显示芦花鸡群存在ALV-J(LH14J)感染,与国内外参考毒株ADOL-7501、NX0101、SD9901、WN100403、HPRS-103的同源性为87.4%-97.8%,其中与国外分离株的同源性最高。检测结果表明该芦花鸡群存在一定程度的外源性ALV感染。母鸡的卵白和无菌抗凝血两种检测材料不能相互替代,均需检测。2 ALV检测材料及方法优化研究为了避免检测材料和方法的单一造成漏检现象,本研究对公母鸡无菌抗凝血清提取了RNA进行斑点杂交试验,另外又采集232份对应公鸡精液接种CEF培养9d后对细胞上清中的ALV-p27抗原进行ELISA检测。结果显示公母鸡斑点杂交的阳性率为5.88%(44/748),其中母鸡血样斑点杂交与卵白的阳性吻合率为30.0%(6/20),与母鸡血样CEF上清的阳性吻合率为30.0%(6/20),公鸡血样斑点杂交与其血样CEF上清阳性吻合率为12.5%(3/24);公鸡精液阳性检出率为15.1%(35/232),与其血样CEF上清阳性吻合率为8.6%(3/35)。本研究结果表明:单一检测卵白、抗凝血或精液并不能检出所有ALV阳性鸡,不同检测材料的相互补充验证才能确保ALV阳性鸡检出率,在ALV的净化进程中除对芦花鸡母鸡卵白、公母鸡抗凝血经细胞培养分离病毒后的ELISA检测这一经典方法之外,可将加测精液并兼顾血清中RNA的提取及斑点杂交等技术手段作为ALV经典净化方法的有力补充。另外,本研究经卵白检出的107只阳性鸡有三种形式,其中两枚蛋均为阳性鸡只93只,占86.9%,两枚蛋一枚阴性一枚阳性的鸡只12只,占11.2%,只有一枚蛋且为阳性鸡只2只,占1.9%。本研究结果显示ALV检测时增加同一宿主的样本数量也可有效降低ALV漏检率。3对种鸡群后代的追踪检测为了了解对该种鸡群AL的净化效果,本研究又采集了1320份该种鸡群后代子鸡群的无菌抗凝血进行病毒分离,结果显示,该子代鸡群的ALV阳性检出率为0.3%(4/1320),与其亲代种鸡群无菌抗凝血ALV阳性检出率2.14%(16/748)相比,阳性率降低非常明显,说明对该芦花鸡鸡群的净化取得了一定的成果。4鸡群ALV感染状态与NDV、AIV-H5及AIV-H9疫苗免疫抗体水平以及鸡群白痢阳性率相关性研究取不同梯度OD值(阴性、OD0.2-0.3、OD0.5-0.6、OD0.8-0.9、OD1-2、OD2以上)ALV-p27抗原阴阳性鸡蛋各15枚,检测各组样品NDV、H5、H9抗体滴度。结果显示芦花鸡群中NDV、H5、H9抗体水平均是只有个别p27抗原阳性组较阴性对照组偏低,差异显著(p≤0.05),其他各组抗体水平无明显相关性。但整体NDV抗体水平ALV阳性鸡蛋较阴性鸡蛋抗体水平偏低(8.9±1.106 VS 9.8±0.145),差异显著,H5抗体水平ALV阳性鸡蛋与阴性鸡蛋抗体水平差异不显著(7.9±0.457 VS 8.2±0.223)。H9抗体水平ALV阳性鸡蛋较阴性鸡蛋抗体水平略微降低(9.8±0.674 VS 10.5±0.192),差异不显著,但是有差异显著趋势(p=0.063)。此外,还对各组鸡蛋进行了鸡白痢感染率检测,结果显示芦花鸡群p27抗原阳性鸡只白痢检出率为39.6%,p27抗原阴性鸡只白痢检出率为22.2%,p27抗原阳性鸡只白痢检出率明显高于阴性鸡只,但不同梯度OD值p27抗原阳性鸡只鸡白痢检出率差异不显著,无明显相关性。
[Abstract]:Avian leukosis (AL) is a common name for a variety of avian tumor diseases caused by avian C retrovirus group. It can cause benign and malignant tumor and subclinical infection of chicken and lead to growth retardation, egg drop and immunosuppression. In recent years, it has been widely spread among Chinese chicken groups and poses a serious threat to the safety of local chicken groups. The international classic methods for AL purification are often caused by the unitary detection of testing materials or detection methods. This study takes the local varieties of aloe, and the aseptic anticoagulant serum of hens to isolate the virus by cell culture, and to detect the cell supernatant and hen egg white by the ELISA method. In the ALV-p27 antigen, we know the infection state of ALV in the aloe chicken group, and randomly select 2 copies of the ALV subtype of the virus cDNA before the ELISA detection of the positive CEF, and detect the different materials of the same batch of chicken by different detection methods, and explore the correlation between the different detection materials and the different detection methods, and optimize the A The LV detection method was used to reduce the ALV leakage rate. On this basis, the descendants of the chicken group were tracked and tested to realize the purification of the AL of the aloe chicken earlier. In addition, the correlation between the ALV infection state of the chicken group and the immune antibody level of NDV, AIV-H5 and AIV-H9 vaccines and the phase of chicken group ALV infection and the positive rate of chicken chicken white dysentery were also investigated. The infection status of the closed.1 aloe chicken group was investigated in order to understand the infection state of the aloe chicken ALV in a chicken farm in Jiangsu, and the ALV-p27 antigen in the cell supernatant after inoculation of the 508 cocks aseptic anticoagulant serum and the semen inoculated CEF9d from the chicken farm of the chicken farm 1024 eggs (508 chickens two, one chicken) and the corresponding 516 hen's aseptic anticoagulant serum, and the semen inoculated with the semen. The positive rate of egg white in hens was 20.7% (107/516), and the positive rate of aseptic anticoagulation was 2.52% (13/516) in hens. The positive rate of rooster semen was 15.1% (35/232), and the positive rate of aseptic anticoagulant was 1.29% (3/232). The results of virus isolation showed that there was ALV-J (LH14J) infection in the aloe chicken group. The homology of ADOL-7501, NX0101, SD9901, WN100403, HPRS-103 is 87.4%-97.8%, and the homology of the isolated strains is the highest. The test results show that the aloe chicken group has some exogenous ALV infection. The egg white and the aseptic anticoagulant two detection materials of the hen can not be replaced by each other, and all the.2 ALV detection materials and sides need to be detected. In order to avoid a single leak detection phenomenon of testing materials and methods, this study was used to extract RNA for speckle hybridization test on male chicken aseptic anticoagulant serum. In addition, the ALV-p27 antigen in cell supernatant was detected by ELISA test after 232 corresponding cock semen inoculated with CEF and 9D. The results showed the blot hybridization of male hens. The positive rate was 5.88% (44/748), which was 30% (6/20) and 30% (6/20), and 12.5% (3/24), the positive rate of cock blood sample dot blot and CEF supernatant was 12.5% (3/24), and the positive rate of cock semen was 15.1% (35/232), and its blood sample CEF supernatant was positive. The rate of sexual anastomosis was 8.6% (3/35). The results of this study showed that a single detection of egg white, anticoagulant or semen could not detect all ALV positive chickens. The detection rate of ALV positive chickens could be ensured by the complementary verification of different testing materials. In the process of purification of ALV, the Chicken Hen Egg White and the ELIS of the male and female chickens were cultured to isolate the virus after the cell culture of the virus. A can be used as a powerful supplement to the classical method of detecting the semen and taking into account the extraction of RNA in serum and dot blot. In addition, there are three forms of 107 positive chickens detected by egg white, of which two eggs are 93 positive chickens, 86.9% and one negative one positive for two eggs. Only 12 chickens, 11.2%, only one egg and 2 positive chickens, accounting for 1.9%., the results of the study show that the increase of the sample number of the same host by ALV detection can also effectively reduce the ALV leakage rate of.3 for the descendants of the chicken group to understand the purification effect of the chicken group AL. This study also collected 1320 chicken fowl descendants. The results showed that the positive rate of ALV was 0.3% (4/1320). Compared with the positive rate of aseptic anticoagulant ALV (16/748) 2.14% (16/748), the positive rate of the group was significantly lower than that of the parent chicken group. It showed that the purification of the chicken flock was a result of ALV infection of.4 chicken group and NDV, AI. The correlation between the immunization antibody level of V-H5 and AIV-H9 vaccine and the positive rate of chicken white dysentery was studied with 15 ALV-p27 antigen Yin and Yang eggs, which were negative, OD0.2-0.3, OD0.5-0.6, OD0.8-0.9, OD1-2, OD2 above, and detected NDV, H5, and H9 antibody titer in each group. The antigen positive group was lower than the negative control group, the difference was significant (P < 0.05). There was no significant correlation between the antibody levels in other groups, but the level of the whole NDV antibody level ALV positive eggs was lower than that of the negative egg antibody (8.9 + 1.106 VS 9.8 + 0.145), the difference was significant, the level of ALV positive egg and negative egg antibody level of H5 antibody level was not significant (7.9 + 0.45). 7 VS 8.2 + 0.223).H9 antibody level ALV positive eggs slightly lower than the negative egg antibody level (9.8 + 0.674 VS 10.5 + 0.192), the difference is not significant, but there is a significant difference (p=0.063). Besides, the infection rate of chicken white dysentery was detected in each group of eggs, and the results showed that the detection rate of white dysentery was 39.6%, p27 The detection rate of only white dysentery in the antigen negative chicken was 22.2%, and the detection rate of white dysentery in chicken with p27 antigen positive was significantly higher than that of the negative chicken, but the difference in the detection rate of chicken white dysentery in chicken with different gradient p27 antigen positive chicken was not significant, and no significant correlation was found.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31

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