本文選題：禽白血病病毒 + 弱毒疫苗��； 參考：《山東農(nóng)業(yè)大學》2017年碩士論文

【摘要】：禽白血病病毒(Avian leukosis virus,ALV)是引起禽類發(fā)生腫瘤性疾病的一種重要病原,該病毒可引起禽的多種具有傳染性的良性腫瘤和惡性腫瘤,同時能導致感染機體發(fā)生嚴重的細胞和體液免疫抑制以及生長抑制,進而易與其他病原并發(fā)或混合感染,給養(yǎng)禽業(yè)帶來重大損失。為了防止ALV的流行,很多國家已實施了非常嚴格的凈化措施來控制ALV。但由于ALV傳播方式的廣泛性,使得ALV感染在我國及其它國家仍然普遍存在。除垂直傳播外,使用了被ALV污染的弱毒疫苗被認為是ALV感染的另一重要途徑,不管是在中國還是其他國家都曾多次報道過ALV在弱毒疫苗中的污染。使用了被ALV污染的弱毒疫苗會給已經(jīng)完成ALV凈化的養(yǎng)殖企業(yè)帶來巨大損失。因此我們設計了人工模擬試驗,探究使用不同劑量ALV污染疫苗對海蘭褐雞的影響,并對ALV在弱毒疫苗中的污染進行了不同檢測方法的比較。1.弱毒疫苗中禽白血病病毒污染對海蘭褐雞免疫機能及生產(chǎn)性能的影響為了探究使用污染ALV的弱毒疫苗后對雞的生產(chǎn)性能和免疫機能的影響,將不同劑量ALV-A摻入市售合格的馬立克氏病疫苗,接種到海蘭褐雞上;七日齡時分別接種ALV-A、ALV-J,并同時免疫NDV和AIV-H9滅活疫苗。結(jié)果表明,使用每羽份污染10TCID50 ALV和50 TCID50 ALV兩種劑量的MDV疫苗后均造成了海蘭褐雞體重的下降,與使用未污染疫苗組相比差異顯著。其中使用污染50 TCID50 ALV的MDV疫苗組明顯低于使用污染10 TCID50 ALV的MDV疫苗組。并且使用兩種ALV污染劑量的疫苗后MDV核酸含量與使用未污染組相比顯著降低。其中使用污染50 TCID50 ALV的MDV疫苗組顯著低于使用污染10 TCID50 ALV的MDV疫苗組。同時使用污染兩種劑量ALV的疫苗后NDV和AIV-H9的抗體滴度與使用無污染疫苗組相比顯著下降。其中使用污染50 TCID50 ALV的MDV疫苗組明顯低于使用污染10 TCID50 ALV的MDV疫苗組。并且,七日齡再次感染ALV后,生長及免疫的抑制作用更加明顯。其中,再次感染ALV-J的組生長及免疫的抑制作用比再感染ALV-A的組更加明顯。本研究通過展示弱毒疫苗中ALV污染對海蘭褐雞生產(chǎn)性能及免疫機能的影響,提示我們在使用弱毒疫苗時要嚴防外源病毒的污染。2.內(nèi)源性禽白血病病毒基因?qū)Ψ肿由飳W方法檢測弱毒疫苗中禽白血病病毒污染的干擾及鑒別使用了污染有禽白血病病毒特別是A亞群ALV的弱毒疫苗是ALV感染途徑之一,而應用PCR檢測弱毒疫苗中ALV污染時通常會受到雞染色體上內(nèi)源性ALV基因的干擾。目前國家標準的檢驗規(guī)程是將疫苗經(jīng)不同預處理后接種至雞胚成纖維細胞后盲傳并測定其細胞上清中p27抗原,但是很多企業(yè)不了解這一情況通過使用p27的PCR檢測方法檢測疫苗污染并引起很多糾紛,為了說明這一方法的不合理性,本研究針對ALV保守的群特異性抗原p27基因設計合成了兩對引物,應用兩對引物對不同來源SPF雞胚及其制備的雞胚成纖維細胞(CEF)進行PCR檢測時均為陽性,但雞胚的蛋清和細胞培養(yǎng)上清經(jīng)ALV-p27抗原ELISA檢測均為陰性;應用上述引物對市售合格的雞馬立克氏病毒(MDV)、雞傳染性法氏囊病毒(IBDV)和禽痘病毒(FPV)等活疫苗進行PCR檢測時均為陽性,但應用本實驗室研制的雞外源性禽白血病病毒特異性核酸探針交叉斑點雜交檢測試劑盒卻均為陰性,并且應用核酸斑點雜交可檢測到10 TCID50/1000羽份的人為添加污染,顯示了良好的特異性和靈敏度。本研究表明在某些SPF雞胚及其制品中內(nèi)源性ALV基因?qū)CR檢測的干擾,也通過比較試驗提供了可供參考的解決方案。
[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.
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S858.31

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