臺灣博碩士論文加值系統

家禽白血病J亞群病毒 (avian leukosis virus J subgroup [ALV-J]) 因在肉雞間的水平傳播快速及會造成生長遲緩而逐受重視。台灣的種雞大部分都是由國外進口的，在國外ALV-J已逐受控制的同時，在台灣的感染情形為如何是我們所想要知道的。此外不同的雞種對ALV-J的抗性亦不同，而台灣的土雞的感染情形並未有報告，因此我們研究目的是了解ALV-J在台灣土雞及白肉雞的分布情形。因現行常用的ELISA檢測方法會有內源性病毒的偽陽性出現，故我們決定採病毒分離及RT-PCR進行檢測。在調查8場的白肉雞場，有5個場呈現ALV-J陽性（62.5%）。在台灣土雞方面，我們總共調查4場的土雞及一場的土種雞，其中只有一黑羽土種雞在經病毒分離及病理學檢查後證實確有感染ALV-J的情形，且呈現病毒陽性抗體陰性 (V+A-) 的高感受性狀態。在序列比對方面由種雞分離到的12株、肉雞分離到的5株及由土雞分離到的4株之序列與國外毒株比較，發現本土毒株間的相似度達94-100﹪。親源性樹狀圖結果顯示台灣的毒株可分成2群的，而且樹狀圖結果顯示各雞場的毒株間及不同來源雞種的毒株間較無關聯性，而與病毒所被分離的時間較有關聯性，且病毒有隨時間而演變的趨勢。在國外經過幾年的撲滅計劃ALV-J已逐受控制，而結果顯示台灣尚受其高度污染，因此尚須更嚴謹的檢測及撲滅計劃來控制ALV-J的感染。

Subgroup J avian leukosis virus (ALV-J) has become more and more important because its rapid horizontal spread, and it causes body weight suppression in commercial broilers. Since Taiwan broiler breeders are imported from other countries, we would like to know the situation of ALV-J infection in Taiwan broilers as the breeding companies claim that they have controlled the ALV-J infection in their breeders. Furthermore, there was no information on ALV-J infection in native chickens in Taiwan. Thus, the objectives of this study were to investigate the situation of ALV-J infection in broilers and native chickens in Taiwan. Because commercial ELISA kit had false positive reaction, virus isolation and RT-PCR were used for identification of ALV-J infection. We had totally investigated 8 commercial broiler flocks from a slaughter house, and 5 of them were infected with ALV-J (62.5%). We had investigated 4 native chicken flocks and 1 native chicken breeder flocks, and only the native chicken breeder showed ALV-J infection. Immunotolerant infection was suspected in the native breeder chickens since they showed viremia and lack of antibody (V+A-). The sequences of the gp85 gene of ALV-J isolates from breeders, broilers and native chickens were 94-100% identity among them. Phylogenetic trees showed that Taiwan isolates fell into two lineages. And it indicated those isolates had no relation to poultry flocks and breeders. Furthermore, Phylogenetic trees showed isolates could be grouped together by time they isolated, and virus variation could be saw during past 3 years (2000-2002). Thought commercial application of the eradication program over several years has resulted in a marked reduction in the prevalence of ALV-J in overseas breeding company, according to our research, Taiwan is still highly contaminated by ALV-J. So we have to control ALV-J infection strictly in Taiwan.

目錄
目錄----------------------------------------------------------I
表次----------------------------------------------------------Ⅳ
圖次----------------------------------------------------------Ⅴ
中文摘要------------------------------------------------------Ⅵ
英文摘要------------------------------------------------------Ⅶ
第一章 緒言---------------------------------------------------Ⅷ
第二章 文獻探討
第一節 歷史背景-----------------------------------------------1
第二節 家禽白血病病毒的分類
2.1 病毒的分類------------------------------------------------2
2.2 病毒亞群的分類--------------------------------------------2
2.3 內源性病毒------------------------------------------------4
第三節 家禽白血病病毒的性狀
3.1 病毒的型態學特徵------------------------------------------6
3.2 病毒的基因體----------------------------------------------6
3.3 病毒的蛋白質----------------------------------------------7
3.4 基因體上未編碼的區域--------------------------------------8
3.5 形成反轉錄病毒的DNA--------------------------------------11
3.6 病毒的轉錄及轉譯-----------------------------------------12
3.7 病毒的env基因片段與病毒的封套----------------------------13
第四節 家禽白血病的流行病學
4.1 病毒的傳播-----------------------------------------------16
4.2 病毒的致病性---------------------------------------------17
4.3 實驗室診斷-----------------------------------------------20
4.4 預防與防治-----------------------------------------------21
第五節 家禽白血病J亞群病毒-----------------------------------23
第三章 材料與方法
第一節 採樣
1.1 血漿樣本-------------------------------------------------28
1.2 腫瘤組織-------------------------------------------------29
第二節 病毒分離與細胞培養
2.1 細胞的繼代-----------------------------------------------29
2.2冷凍與解凍細胞--------------------------------------------30
2.3 病毒的分離-----------------------------------------------31
第三節 血清學的檢測
3.1 家禽白血病GSA抗原之ELISA檢測-----------------------------31
3.2 家禽白血病J亞群抗體之ELISA檢測---------------------------32
第四節 病毒核酸之增殖
4.1 病毒RNA之萃取--------------------------------------------32
4.2 反轉錄反應（RT）-----------------------------------------33
4.3 細胞核酸DNA之抽取----------------------------------------34
4.4 聚合酵素鏈反應（PCR）------------------------------------34
第五節 聚合酵素鏈反應產物的cloning
5.1 聚合酵素鏈反應（PCR）產物之純化--------------------------36
5.2 用TA-cloning kit 進行病毒核酸之cloning-------------------37
5.3 快速質體檢定法-------------------------------------------38
5.4 質體小量抽取法（miniprep）-------------------------------38
5.5 限制酵素切割---------------------------------------------39
第六節 病毒的定序
6.1 核酸序列的定序-------------------------------------------39
6.2 序列之分析-----------------------------------------------40
第四章 結果
第一節 家禽白血病J亞群在種雞場的感染情形---------------------41
第二節 家禽白血病J亞群在台灣雞場之分布情形
2.1 肉雞場之調查情形-----------------------------------------42
2.2 土雞場之調查情形-----------------------------------------45
2.3 蛋雞場之調查情形-----------------------------------------46
第三節 血清學調查
3.1 土雞場之血清學調查---------------------------------------46
3.2 蛋雞場之血清學調查---------------------------------------47
第四節 土雞感染白血病J亞群之病理學特徵-----------------------47
第五節 聚合酵素鏈反應（PCR） 與反轉錄聚合酵素鏈反應（RT-PCR）
5.1 以H5/H7引子增幅ALV-J病毒序列-----------------------------48
5.2 以EV1/EV2引子增幅ALV-J env 病毒序列----------------------48
5.3 以H5/AD1及PA1/PA2 增幅A亞群病毒序列----------------------49
第六節 聚合酵素鏈反應（PCR）產物進行cloning------------------49
第七節 病毒之核酸序列分析
7.1 病毒核酸gp85的序列分析-----------------------------------49
7.2 種雞分離病毒的序列分析-----------------------------------51
7.3 肉雞分離病毒序的列分析-----------------------------------51
7.4 土雞分離病毒序列分析-------------------------------------52
7.5 土雞病毒序列與其它毒株之關係-----------------------------52
第五章 討論--------------------------------------------------64
第六章 參考文獻----------------------------------------------71

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