禽白血病病毒通过弱毒疫苗污染途径对鸡免疫机能及生产性能的影响

发布时间：2018-06-12 10:38

本文选题：禽白血病病毒 + 弱毒疫苗　；参考：《山东农业大学》2017年硕士论文

【摘要】：禽白血病病毒(Avian leukosis virus,ALV)是引起禽类发生肿瘤性疾病的一种重要病原,该病毒可引起禽的多种具有传染性的良性肿瘤和恶性肿瘤,同时能导致感染机体发生严重的细胞和体液免疫抑制以及生长抑制,进而易与其他病原并发或混合感染,给养禽业带来重大损失。为了防止ALV的流行,很多国家已实施了非常严格的净化措施来控制ALV。但由于ALV传播方式的广泛性,使得ALV感染在我国及其它国家仍然普遍存在。除垂直传播外,使用了被ALV污染的弱毒疫苗被认为是ALV感染的另一重要途径,不管是在中国还是其他国家都曾多次报道过ALV在弱毒疫苗中的污染。使用了被ALV污染的弱毒疫苗会给已经完成ALV净化的养殖企业带来巨大损失。因此我们设计了人工模拟试验,探究使用不同剂量ALV污染疫苗对海兰褐鸡的影响,并对ALV在弱毒疫苗中的污染进行了不同检测方法的比较。1.弱毒疫苗中禽白血病病毒污染对海兰褐鸡免疫机能及生产性能的影响为了探究使用污染ALV的弱毒疫苗后对鸡的生产性能和免疫机能的影响,将不同剂量ALV-A掺入市售合格的马立克氏病疫苗,接种到海兰褐鸡上;七日龄时分别接种ALV-A、ALV-J,并同时免疫NDV和AIV-H9灭活疫苗。结果表明,使用每羽份污染10TCID50 ALV和50 TCID50 ALV两种剂量的MDV疫苗后均造成了海兰褐鸡体重的下降,与使用未污染疫苗组相比差异显著。其中使用污染50 TCID50 ALV的MDV疫苗组明显低于使用污染10 TCID50 ALV的MDV疫苗组。并且使用两种ALV污染剂量的疫苗后MDV核酸含量与使用未污染组相比显著降低。其中使用污染50 TCID50 ALV的MDV疫苗组显著低于使用污染10 TCID50 ALV的MDV疫苗组。同时使用污染两种剂量ALV的疫苗后NDV和AIV-H9的抗体滴度与使用无污染疫苗组相比显著下降。其中使用污染50 TCID50 ALV的MDV疫苗组明显低于使用污染10 TCID50 ALV的MDV疫苗组。并且,七日龄再次感染ALV后,生长及免疫的抑制作用更加明显。其中,再次感染ALV-J的组生长及免疫的抑制作用比再感染ALV-A的组更加明显。本研究通过展示弱毒疫苗中ALV污染对海兰褐鸡生产性能及免疫机能的影响,提示我们在使用弱毒疫苗时要严防外源病毒的污染。2.内源性禽白血病病毒基因对分子生物学方法检测弱毒疫苗中禽白血病病毒污染的干扰及鉴别使用了污染有禽白血病病毒特别是A亚群ALV的弱毒疫苗是ALV感染途径之一,而应用PCR检测弱毒疫苗中ALV污染时通常会受到鸡染色体上内源性ALV基因的干扰。目前国家标准的检验规程是将疫苗经不同预处理后接种至鸡胚成纤维细胞后盲传并测定其细胞上清中p27抗原,但是很多企业不了解这一情况通过使用p27的PCR检测方法检测疫苗污染并引起很多纠纷,为了说明这一方法的不合理性,本研究针对ALV保守的群特异性抗原p27基因设计合成了两对引物,应用两对引物对不同来源SPF鸡胚及其制备的鸡胚成纤维细胞(CEF)进行PCR检测时均为阳性,但鸡胚的蛋清和细胞培养上清经ALV-p27抗原ELISA检测均为阴性;应用上述引物对市售合格的鸡马立克氏病毒(MDV)、鸡传染性法氏囊病毒(IBDV)和禽痘病毒(FPV)等活疫苗进行PCR检测时均为阳性,但应用本实验室研制的鸡外源性禽白血病病毒特异性核酸探针交叉斑点杂交检测试剂盒却均为阴性,并且应用核酸斑点杂交可检测到10 TCID50/1000羽份的人为添加污染,显示了良好的特异性和灵敏度。本研究表明在某些SPF鸡胚及其制品中内源性ALV基因对PCR检测的干扰,也通过比较试验提供了可供参考的解决方案。
[Abstract]:Avian leukosis virus (ALV) is an important cause of the tumorigenesis of birds. The virus can cause a variety of infectious benign tumors and malignant tumors of birds, and can cause severe cellular and humoral immune suppression and growth inhibition in the infected organisms, which are easily associated with other pathogens. Mixed infection brings great loss to the poultry industry. In order to prevent the epidemic of ALV, many countries have carried out very strict purification measures to control ALV., but because of the universality of ALV transmission, ALV infection still exists in our country and other countries. In addition to vertical transmission, the use of ALV contaminated weakly toxic vaccine is considered ALV Another important way of infection, both in China and in other countries, has reported ALV pollution in the weak vaccine. The use of ALV contaminated weakly toxic vaccines will bring huge losses to the enterprises that have finished ALV purification. So we designed artificial simulation tests to explore the use of different doses of ALV contaminated vaccines to the sea. The influence of the blue brown chicken and the different detection methods of ALV in the weak virus vaccine compared the effect of avian leukosis virus contamination on the immune function and production performance of the brown chicken in the.1. weak virus vaccine in order to explore the effects of the weak toxic vaccine on the production performance and immune function of the chicken after the use of the contaminated ALV vaccine, and the different doses of ALV-A were added. The eligible Marek's disease vaccine was inoculated on the brown brown chicken, inoculated with ALV-A, ALV-J, and immunized with NDV and AIV-H9 inactivated vaccine at the age of seven. The results showed that the use of MDV vaccines with two doses of 10TCID50 ALV and 50 TCID50 ALV per plume caused the decline of the body weight of the brown brown chicken and the use of the uncontaminated vaccine group. The MDV vaccine group using 50 TCID50 ALV pollution was significantly lower than the MDV vaccine group using 10 TCID50 ALV, and the MDV nucleic acid content of the two ALV contaminated doses decreased significantly compared with the uncontaminated group. The MDV vaccine group used to pollute 50 TCID50 ALV was significantly lower than the use of pollution 10 TCID50. The antibody titer of NDV and AIV-H9 in the MDV vaccine group of LV was significantly lower than that in the non polluted vaccine group. The MDV vaccine group used to pollute 50 TCID50 ALV was significantly lower than that of the MDV vaccine group using 10 TCID50 ALV, and the growth and immune suppression after re infection of ALV was seven days old. In this study, the effects of ALV pollution on the production performance and immune function of the brownish brown chicken were demonstrated by showing the effect of ALV pollution on the production performance and immune function of ALV-A in the weakly toxic vaccine. This study suggested that we should strictly prevent the exogenous virus contamination of endogenous avian leukosis when using the weak virus vaccine. The virus gene interferes with the detection of avian leukosis virus contamination in the weak virus vaccine by molecular biology methods and differentiating the use of the weakly toxic vaccine with avian leukosis virus, especially the A subgroup ALV, is one of the ALV infection pathways. While the PCR detection of ALV pollution in the weak virus vaccine is usually affected by the endogenous ALV gene on the chicken chromosomes. At present, the national standard test procedure is to inoculate the vaccine after different pretreatments and inoculate the vaccine into the chicken embryo fibroblast and transmit the p27 antigen in the cell supernatant. However, many enterprises do not understand this situation by using the PCR detection method of p27 to detect the vaccine contamination and cause many disputes, in order to explain the irrational nature of this method, Two pairs of primers were designed for the design of the ALV conserved group specific antigen p27 gene. Two pairs of primers were used for PCR detection of SPF chicken embryo and the chicken embryo fibroblast (CEF) from different sources, but the egg white and cell culture supernatant of chicken embryos were all negative by ALV-p27 anti original ELISA, and the above primers were applied to the market. All live vaccines such as chicken Marek's virus (MDV), avian infectious bursal virus (IBDV) and avian pox virus (FPV) were all positive for PCR detection, but the detection kits of specific nucleic acid probe cross spot hybridization for chicken exogenous avian leukosis virus (fowl) developed in our laboratory were negative, and nucleic acid blot hybridization could be used for detection. The anthropogenic addition of 10 TCID50/1000 plumes showed good specificity and sensitivity. This study showed that the interference of endogenous ALV genes to PCR detection in some SPF chicken embryos and their products provided a reference solution by comparison test.
【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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