本文選題：蘆花雞 + 禽白血病毒　； 參考：《山東農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：禽白血病(AL)是由禽C型反轉(zhuǎn)錄病毒群引起的禽類(lèi)多種腫瘤性疾病的統(tǒng)稱(chēng),可引起雞的良性和惡性腫瘤以及亞臨床感染并導(dǎo)致生長(zhǎng)遲緩、產(chǎn)蛋下降和免疫抑制。近年來(lái)已在中國(guó)雞群中廣泛傳播,對(duì)地方品種雞群的安全構(gòu)成了嚴(yán)重的威脅,并給地方品種雞保種和選育工作帶來(lái)很大的困難。目前實(shí)施AL凈化的國(guó)際經(jīng)典方法常因檢測(cè)材料或檢測(cè)方法的單一造成漏檢。本研究取地方品種蘆花公、母雞的無(wú)菌抗凝血清經(jīng)細(xì)胞培養(yǎng)分離病毒,并用ELISA方法檢測(cè)細(xì)胞上清及母雞卵白中的ALV-p27抗原,了解蘆花雞群中ALV的感染狀態(tài),并隨機(jī)抽取2份經(jīng)ELISA檢測(cè)上清呈陽(yáng)性的CEF提取前病毒cDNA進(jìn)行ALV亞型的鑒定;同時(shí)用不同檢測(cè)方法對(duì)同一批雞的不同材料進(jìn)行檢測(cè),探究不同檢測(cè)材料與不同檢測(cè)方法之間的相關(guān)性,優(yōu)化ALV檢測(cè)方法以降低ALV漏檢率。并在此基礎(chǔ)上對(duì)該種雞群后代進(jìn)行追蹤檢測(cè),以較早實(shí)現(xiàn)對(duì)蘆花雞AL的凈化工作。此外,本文還探究了雞群ALV感染狀態(tài)與NDV、AIV-H5及AIV-H9疫苗免疫抗體水平相關(guān)性以及雞群ALV感染狀態(tài)與雞群雞白痢陽(yáng)性率相關(guān)性。1蘆花雞群感染狀態(tài)調(diào)查為了解江蘇某雞場(chǎng)蘆花雞ALV的感染狀態(tài),對(duì)來(lái)自該雞場(chǎng)種雞群1024枚種蛋(508只雞兩枚,8只雞一枚)以及對(duì)應(yīng)516只母雞無(wú)菌抗凝血清、232份公雞無(wú)菌抗凝血清及精液接種CEF9d后的細(xì)胞上清中的ALV-p27抗原進(jìn)行ELISA方法檢測(cè),結(jié)果顯示母雞卵白陽(yáng)性檢出率為20.7%(107/516),母雞無(wú)菌抗凝血陽(yáng)性檢出率為2.52%(13/516)。公雞精液陽(yáng)性檢出率為15.1%(35/232),無(wú)菌抗凝血陽(yáng)性檢出率為1.29%(3/232)。病毒分離鑒定結(jié)果顯示蘆花雞群存在A(yíng)LV-J(LH14J)感染,與國(guó)內(nèi)外參考毒株ADOL-7501、NX0101、SD9901、WN100403、HPRS-103的同源性為87.4%-97.8%,其中與國(guó)外分離株的同源性最高。檢測(cè)結(jié)果表明該蘆花雞群存在一定程度的外源性ALV感染。母雞的卵白和無(wú)菌抗凝血兩種檢測(cè)材料不能相互替代,均需檢測(cè)。2 ALV檢測(cè)材料及方法優(yōu)化研究為了避免檢測(cè)材料和方法的單一造成漏檢現(xiàn)象,本研究對(duì)公母雞無(wú)菌抗凝血清提取了RNA進(jìn)行斑點(diǎn)雜交試驗(yàn),另外又采集232份對(duì)應(yīng)公雞精液接種CEF培養(yǎng)9d后對(duì)細(xì)胞上清中的ALV-p27抗原進(jìn)行ELISA檢測(cè)。結(jié)果顯示公母雞斑點(diǎn)雜交的陽(yáng)性率為5.88%(44/748),其中母雞血樣斑點(diǎn)雜交與卵白的陽(yáng)性吻合率為30.0%(6/20),與母雞血樣CEF上清的陽(yáng)性吻合率為30.0%(6/20),公雞血樣斑點(diǎn)雜交與其血樣CEF上清陽(yáng)性吻合率為12.5%(3/24);公雞精液陽(yáng)性檢出率為15.1%(35/232),與其血樣CEF上清陽(yáng)性吻合率為8.6%(3/35)。本研究結(jié)果表明:單一檢測(cè)卵白、抗凝血或精液并不能檢出所有ALV陽(yáng)性雞,不同檢測(cè)材料的相互補(bǔ)充驗(yàn)證才能確保ALV陽(yáng)性雞檢出率,在A(yíng)LV的凈化進(jìn)程中除對(duì)蘆花雞母雞卵白、公母雞抗凝血經(jīng)細(xì)胞培養(yǎng)分離病毒后的ELISA檢測(cè)這一經(jīng)典方法之外,可將加測(cè)精液并兼顧血清中RNA的提取及斑點(diǎn)雜交等技術(shù)手段作為ALV經(jīng)典凈化方法的有力補(bǔ)充。另外,本研究經(jīng)卵白檢出的107只陽(yáng)性雞有三種形式,其中兩枚蛋均為陽(yáng)性雞只93只,占86.9%,兩枚蛋一枚陰性一枚陽(yáng)性的雞只12只,占11.2%,只有一枚蛋且為陽(yáng)性雞只2只,占1.9%。本研究結(jié)果顯示ALV檢測(cè)時(shí)增加同一宿主的樣本數(shù)量也可有效降低ALV漏檢率。3對(duì)種雞群后代的追蹤檢測(cè)為了了解對(duì)該種雞群AL的凈化效果,本研究又采集了1320份該種雞群后代子雞群的無(wú)菌抗凝血進(jìn)行病毒分離,結(jié)果顯示,該子代雞群的ALV陽(yáng)性檢出率為0.3%(4/1320),與其親代種雞群無(wú)菌抗凝血ALV陽(yáng)性檢出率2.14%(16/748)相比,陽(yáng)性率降低非常明顯,說(shuō)明對(duì)該蘆花雞雞群的凈化取得了一定的成果。4雞群ALV感染狀態(tài)與NDV、AIV-H5及AIV-H9疫苗免疫抗體水平以及雞群白痢陽(yáng)性率相關(guān)性研究取不同梯度OD值(陰性、OD0.2-0.3、OD0.5-0.6、OD0.8-0.9、OD1-2、OD2以上)ALV-p27抗原陰陽(yáng)性雞蛋各15枚,檢測(cè)各組樣品NDV、H5、H9抗體滴度。結(jié)果顯示蘆花雞群中NDV、H5、H9抗體水平均是只有個(gè)別p27抗原陽(yáng)性組較陰性對(duì)照組偏低,差異顯著(p≤0.05),其他各組抗體水平無(wú)明顯相關(guān)性。但整體NDV抗體水平ALV陽(yáng)性雞蛋較陰性雞蛋抗體水平偏低(8.9±1.106 VS 9.8±0.145),差異顯著,H5抗體水平ALV陽(yáng)性雞蛋與陰性雞蛋抗體水平差異不顯著(7.9±0.457 VS 8.2±0.223)。H9抗體水平ALV陽(yáng)性雞蛋較陰性雞蛋抗體水平略微降低(9.8±0.674 VS 10.5±0.192),差異不顯著,但是有差異顯著趨勢(shì)(p=0.063)。此外,還對(duì)各組雞蛋進(jìn)行了雞白痢感染率檢測(cè),結(jié)果顯示蘆花雞群p27抗原陽(yáng)性雞只白痢檢出率為39.6%,p27抗原陰性雞只白痢檢出率為22.2%,p27抗原陽(yáng)性雞只白痢檢出率明顯高于陰性雞只,但不同梯度OD值p27抗原陽(yáng)性雞只雞白痢檢出率差異不顯著,無(wú)明顯相關(guān)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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S858.31

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本文編號(hào)：2038696