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研究生:林坤輝
研究生(外文):Lin Kuen-Huei
論文名稱:新城病病毒引發雞冠病變之探討
論文名稱(外文):Studies on the Comb Lesions Associated with Newcastle Disease Virus Infection in Chicken
指導教授:蔡信雄蔡信雄引用關係
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:獸醫學系
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:122
中文關鍵詞:新城病免疫組織化學免疫電子顯微鏡學反轉錄聚合酶鏈鎖反應單管多引子反轉錄聚合酶鏈鎖反應
外文關鍵詞:Newcastle diseaseimmunohistochemical methodimmunoelectron microscopic methodreverse transcription-polymerase chain reactionmultiplex reverse transcription-polymerase chain reaction
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新城病(ND)是一種具高度傳染性及高致死性之惡性傳染病,本病依其病原性分為五型。以往在台灣所發生之ND係由親內臟強毒型病毒(VVNDV)所引起腸炎及腦炎為主徵。NDV引發雞冠的病變,迄今尚未有深入之研究。有鑑於此,本試驗分別以VVNDV強毒株(V158)和弱毒株(B1)點眼感染4隻13週齡無特定病原雞(SPF),並另置2隻SPF雞作為陰性對照組。接種後每隔12小時採取雞冠樣本直至屆滿144小時,試驗期間若有雞隻死亡或屆滿144小時,則立即進行剖檢與採樣。將採集之雞冠分別進行組織病理學、免疫組織化學(IHC)、免疫電子顯微鏡學(IEM)、反轉錄聚合酶鏈鎖反應(RT-PCR)、單管多引子反轉錄聚合酶鏈鎖反應(Multiplex RT-PCR)及病毒分離等。V158株接種雞呈現食慾不振、精神沉鬱及排綠痢等,並於感染後84和96小時各有2隻雞隻相繼死亡,B1株接種雞僅見輕微之沉鬱,而對照組則無任何症狀。組織病理學與免疫組織化學檢查僅於V158株接種雞發現腦炎、腸炎與雞冠內皮細胞增生及肥大的病變,且於增生與肥大之內皮細胞均可見免疫組織化學染色呈現陽性反應,而弱毒株僅可引起內皮細胞輕微之肥大而無增生的現象,但對照組則無任何變化。以RT-PCR檢查V158與B1株,可在接種後24小時檢測出雞冠組織中含有NDV之血球凝集素-神經氨酸酶(HN)基因與融合(F)基因等之核酸片段產物,並利用multiplex RT-PCR分別確認為NDV強毒株及NDV弱毒株。雞冠病毒之回收,僅見於V158接種雞組。以IEM檢查,可發現金離子多出現於血管內皮細胞之細胞膜及鄰近之細胞質間。家禽流行性感冒病毒弱毒株(H6N1)人工接種雞隻的雞冠,經由組織病理學檢查及multiplex RT-PCR檢測,結果均為陰性。本試驗結果顯示VVNDV確實可引起雞冠血管內皮細胞等的增生及肥大。
Newcastle disease (ND) is a highly contagious and lethal disease, divided into 5 pathogenic types. Most of ND outbreak in Taiwan were caused by viscerotropic velogenic NDV (VVNDV) with enteritis and encephalitis. Until now, the comb lesions associated with NDV infection has not been studied in detail. An experimental trial of VVNDV (V158) and lentogenic NDV (B1) in 4 specific pathogenic free (SPF) chicken of 13 week old respectively were carried out via eyedrop inoculation. In addition, 2 SPF chickens with the same age were used as control group. Sampling would be performed at pre-inoculation and every 12 hour interval following post-inoculation. Necropsies were performed immediately as the inoculated chicken died and at expiration of the test. The examination included histopathologic, immunohistochemical (IHC), reverse transcription-polymerase chain reaction (RT-PCR), multiplex reverse transcription-polymerase chain reaction (multiplex RT-PCR), and immunoelectron microscopic (IEM) examinations. The signs of anorexia, depression, and greenish diarrhea were observed in V158 group, mild depression in B1 group, and normal in control group. Two chickens of V158 group were respectively died at 84th and 96th hour following post-inoculation. Encephalitis, enteritis, comb lesion with endotheliosis and positive reaction of IHC were detected in V158 group only. The mild hypertrophy of vascular endothelium was occasionally observed in the combs of B1 group with negative reaction of IHC. No reaction was found in control group. The specific fragmented nucleic acids encorded by HN and F gene were detected by RT-PCR in the both V158 and B1 groups at 24 hour post-inoculation, which could be differentiated into V158 and B1 by multiplex RT-PCR. The NDV could be re-isolated from the combs of V158 group only. The gold particles were scattered in the cytoplastic membrane and neighboring of the comb endothelium. The combs of the SPF chickens inoculated with avian influenza virus (H6N1) showed the negative results to histopathologic and multiplex RT-PCR examinations. Based on the results of our studies, VVNDV has been confirmed to induce hypertrophy and hyperplasia in the endothelial cells of comb in chicken.
中文摘要……………………………………………………... Ⅰ
英文摘要……………………………………………………... Ⅲ
致謝…………………………………………………………... Ⅴ
目錄…………………………………………………………... Ⅶ
圖次…………………………………………………………... ⅩⅡ
表次…………………………………………………………... ⅩⅣ
第一章 前言………………………………………………... 1
第二章 文獻回顧…………………………………………... 4
2.1 新城病的歷史背景…………………………………… 4
2.2 新城病病毒的特性…………………………………… 5
2.2.1 新城病病毒的型態與結構…...………………….. 5
2.2.2 新城病病毒的核酸與蛋白質之性質及功能……. 6
2.2.2.1 核蛋白衣蛋白……………………………….. 7
2.2.2.2 磷酸化蛋白………………………………….. 7
2.2.2.3 大蛋白……………………………………….. 8
2.2.2.4 基質蛋白或膜蛋白………………………….. 8
2.2.2.5 血液凝集神經胺酸酶醣蛋白……………….. 9
2.2.2.6融合醣蛋白…………………………………... 9
2.3新城病病毒之生物合成與生活史……………………. 11
2.3.1 病毒之附著………………………………………. 11
2.3.2 病毒之轉錄與轉譯………………………………. 12
2.3.3 病毒之複製………………………………………. 13
2.3.4 病毒之組合與釋放………………………………. 14
2.4流行病學………………………………………………. 14
2.4.1 感染途徑與傳播方式……………………………. 14
2.4.2臨床症狀………………………………………….. 15
2.4.3 肉眼病變…………………………………………. 16
2.4.4 組織病理學變化…………………………………. 17
2.4.4.1 神經系統病變……………………………….. 17
2.4.4.2 血管系統病變……………………………….. 18
2.4.4.3 淋巴器官病變……………………………….. 18
2.4.4.4 消化道病變………………………………….. 18
2.4.4.5 呼吸道病變………………………………….. 19
2.4.4.6 生殖系統病變……………………………….. 19
2.4.4.7 其他臟器病變……………………………….. 20
2.5 診斷技術之背景資料………………………………… 20
2.5.1 聚合酶鏈鎖反應…………………………………. 20
2.5.1.1反轉錄聚合酶鏈鎖反應……………………... 22
2.5.1.2 多引子反轉錄聚合酶鏈鎖反應…………….. 23
2.5.2 免疫組織化學染色法……………………………. 24
2.5.2.1 歷史與背景………………………………….. 24
2.5.2.2 應用原理…………………………………….. 26
2.5.3 免疫金染色法……………………………………. 28
2.5.3.1 免疫金染色法之歷史背景…………………. 2.5.3.2 免疫金染色技術之原理…………………….. 2831
2.5.4微血管之組織與生理學………………………….. 32
2.5.4.1連續型微血管……………………………….. 33
2.5.4.2 窗型微血管………………………………….. 33
2.5.4.3 間斷型微血管……………………………….. 34
2.5.4.4 雞冠之構造與組織型態…………………….. 35
第三章 材料與方法………………………………………... 36
3.1 病材…………………………………………………… 36
3.1.1 野外病例…………………………………………. 36
3.1.2 實驗設計…………………………………………. 36
3.1.2.1 病毒株來源………………………………… 36
3.1.2.2 新城病病毒組……………………………….. 37
3.1.2.3 家禽流行性感冒病毒組…………………….. 37
3.2 組織病理學…………………………………………… 38
3.2.1組織切片之製備………………………………….. 38
3.2.2 蘇木紫與伊紅染色………………………………. 39
3.3 免疫組織化學染色法………………………………… 39
3.3.1單株抗體與ABC kit之製備……………………. 39
3.3.2 前置處理與注意事項……………………………. 40
3.3.3 染色步驟…………………………………………. 41
3.3.4 呈色劑之配置……………………………………. 42
3.4 免疫金染色法………………………………………… 43
3.4.1 金離子之製備……………………………………. 43
3.4.2 蛋白質A金離子(protein A-gold)之製備……….. 43
3.4.3 超薄切片之製備…………………………………. 44
3.4.4 免疫金染色步驟…………………………………. 45
3.5反轉錄聚合酶鏈鎖反應………………………………. 46
3.5.1病毒核酸之萃取………………………………….. 46
3.5.2 引子的設計………………………………………. 47
3.5.3 反應條件…………………………………………. 48
3.5.4 電泳分析…………………………………………. 48
3.5.5 反轉錄聚合酶鏈鎖反應產物之定序……………. 49
3.5.5.1 反轉錄聚合酶鏈鎖反應產物之選殖……….. 49
3.5.5.2 質體之純化與定序………………………….. 51
3.6 單管多引子反轉錄聚合酶鏈鎖反應………………… 53
3.6.1 病毒核酸之萃取…………………………………. 53
3.6.2 引子之設計………………………………………. 53
3.6.3 反應條件…………………………………………. 54
3.6.4 電泳分析 ………………………………………… 54
3.7 病毒分離……………………………………………… 54
第四章 結果………………………………………………... 56
4.1 病理學檢查…………………………………………… 56
4.1.1 野外病例…………………………………………. 56
4.1.2 實驗動物…………………………………………. 56
4.1.2.1 新城病病毒組……………………………….. 57
4.1.2.2 家禽流行性感冒病毒組…………………….. 57
4.2 免疫組織化學染色法………………………………… 58
4.3 免疫金染色法………………………………………… 58
4.4 反轉錄聚合酶鏈鎖反應……………………………… 59
4.4.1 野外病例…………………………………………. 59
4.4.2 實驗動物…………………………………………. 59
4.4.2.1 新城病病毒組……………………………….. 60
4.5單管多引子反轉錄聚合酶鏈鎖反應…………………. 60
4.5.1 野外病例…………………………………………. 60
4.5.2 實驗動物…………………………………………. 61
4.5.2.1 新城病病毒組……………………………….. 61
4.5.2.2 家禽流行性感冒病毒組…………………….. 61
4.6 病毒分離與定序……………………………………… 62
第五章 討論………………………………………………... 63
參考文獻……………………………………………………... 73
作者簡介……………………………………………………... 108
圖 次
圖一、雞冠。在接種後48小時可於強毒組雞冠的血管內皮細胞發現明顯的增生與肥大(400X;H&E)…….……….. 92
圖二、大腦。腦部血管內皮細胞發生明顯增生及肥大的變化(200X;H&E)…………..………………………………… 92
圖三、小腸。腸管粘膜呈現局部性的壞死與細胞之脫落,且在集淋小結內之淋巴細胞發生廣泛性的壞死(40X;H&E)……………………………………………………...……… 93
圖四、雞冠。弱毒組於接種84小時後僅偶見血管內皮細胞發生肥大,但不見增生現象(400X;H&E)…………….. 93
圖五、雞冠。對照組無任何變化(400X;H&E)…………… 93
圖六、雞冠。ND強毒組之雞冠在接種60小時後,血管內皮細胞呈現陽性反應(1000X;ABC)……………………..... 94
圖七、雞冠。ND弱毒組雞冠血管內皮細胞呈現陰性反應(1000X;ABC)………………………………………….…… 94
圖八、雞冠。ND對照組雞冠血管內皮細胞呈現陰性反應(1000X;ABC)…………………………………………...… 94
圖九、免疫金染色(IGS, Bar=0.5μm)。在ND強毒組雞冠血管內皮細胞的細胞膜與鄰近之細胞質內發現金離子顆粒之浸潤(箭號所指)..…………………………………...... 95
圖十、ND野外病例,可於雞冠偵測出426bp之特異片段(HN gene通用片段;universal primer)….……………..……. 96
圖十一、ND野外病例,可於雞冠偵測出535bp之特異片段(F gene通用片段;universal primer)…………………...… 97
圖十二、於F6、F14、F15之腦、雞冠與脾臟中皆可偵測出NDV強毒株之核酸,且於F15之腦及脾臟還可偵測出AIV之核酸……………………………………….………….. 98
圖十三、ND強毒組皆可增幅出NDV強毒株特異之221bp核酸片段,其餘弱毒組與對照組則均呈陰性反應………… 99
圖十四、由強毒組之雞冠樣本進行病毒再分離,培養於vero cell,在36-72小時,即呈現出特異性的細胞病理變化反應(CPE)(100X)………………………………………… 100
圖十五、F基因(47-422)以DNASTAR軟體John Hein法比對核酸序列後繪製之親緣關係圖…………………………... 101
表 次
表一、罹患ND野外病例之腦、腸管與雞冠組織病理學變化……………………………………………………………... 102
表二、NDV接種組雞隻血管內皮細胞之組織病變……….. 103
表三、利用免疫組織化學染色法(ABC法)偵測實驗雞隻雞冠中NDV蛋白質之結果…………………………….……… 104
表四、利用IGS來偵測實驗雞隻雞冠中NDV之結果……………………………………………………….…….. 105
表五、利用RT-PCR來偵測實驗雞隻雞冠樣本中NDV核酸之結果……………………………….…………………….. 106
表六、利用multiplex RT-PCR偵測實驗雞隻雞冠樣本中NDV核酸之結果………………………..…………………... 107
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