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研究生:陳志專
研究生(外文):TAN CHEE CHUAN
論文名稱:鴿子新城病病毒的分離及鑑定與病原性研究
論文名稱(外文):Isolation, identification and pathogenicity of Newcastle disease virus in pigeons
指導教授:林子恩林子恩引用關係
指導教授(外文):James, A. Lin
學位類別:碩士
校院名稱:國立中興大學
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:2001
畢業學年度:89
語文別:中文
中文關鍵詞:鴿子新城病
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台灣地區新城病目前為止已發生三次大流行,在此流行期間曾由鴿子分離出新城病病毒。台灣飼養鴿子歷史長遠,主要是供為比賽及觀賞之用。目前飼養鴿子的數目龐大且有增加的傾向。1999年十月至2001年四月在台灣的北,中,南各地區陸續發生多項病鴿出現斜頸、共濟失調、扭頸歪頭、單側或雙側翼及腿部麻痺及水樣或綠色下痢便之臨床症狀。本研究的目的是收集上述發病的鴿子由病理、病毒分離和鑑定程序做出最後診斷,由鴿子分離之病毒分離株分別以鴿子與實驗雞隻做病原性接種實驗,並建立完整的鴿子新城病免疫計劃以便對鴿子實施疫苗接種和疾病管制的參考。病鴿在剖檢後並無明顯的肉眼病變,經病理組織學檢查發現病鴿的大腦、小腦血管內皮細胞腫脹、血管周圍有淋巴球浸潤、腎臟有間質性腎炎。採取病鴿的腦、脾製成無菌乳劑接種於9-11日齡雞胚胎尿囊腔。雞胚胎尿囊腔接種後的平均死亡時間為72-96小時,尿囊液以負染色法在穿透式電子顯微鏡下發現類似副黏液病毒之形態。使用多引子反轉錄酶-聚合酶連鎖反應測定結果為新城雞瘟病毒野外株。利用新城病F基因部份位置(47-435bp)進行定序結果確定為新城病病毒。綜合病鴿之臨床症狀、剖檢病變、病理組織學檢查、雞胚胎尿囊腔接種、血球凝集試驗、血球凝集抑制試驗、穿透式電子顯微鏡觀察及多引子反轉錄酶-聚合酶連鎖反應及定序,以此方法分離出四株新城病毒野外分離株,選其中之一病毒分離株(P1/Taiwan/2000)。利用多次的超高速離心及蔗糖梯度比重將病毒純化,得到比重在30-40%蔗糖濃度之間,病毒蛋白濃度為78mg/ml,血球凝集試驗力價為6,400x的純化病毒。感染雞胚胎纖維母細胞之單層細胞後,約三天後開始產生特徵性融合斑。1日齡無特定病原小雞腦內接種指數(ICPI)為1.64。接種於9-11日齡的無特定病原雞胚胎測定其平均致死時間(MDT)小於60小時。顯示此分離株為強毒株。動物接種試驗結果顯示未免疫之幼鴿及成鴿經病毒株攻毒後的死亡率及發病率為100%。並呈現與自然發病相同之臨床症狀及病變。無特定病原小雞經腦內接種後可引起急性死亡、組織病變及剖解病變與新城病例的感染類似。序列比對基因庫中的F基因序列結果發現這四株病毒株P1/Taiwan/2000、P2/ Taiwan/2000、P3/Taiwan/2000及P4/Taiwan/2000皆與新城病毒株Taiwan 95 (登錄號U62620)只有2%差異性。
經免疫四週齡及八週齡分別施打兩劑新城病(A/PMV-1)的油質死毒疫苗或活毒與死毒併用即可達到良好的免疫保獲效果。
Three major outbreaks of Newcastle disease occurred in Taiwan in the last decades (in 1969,1984 and 1995). Pigeons in Taiwan are mostly used for racing activity and breeding. The populations of pigeons tend to become larger in recently years. A contagious disease of racing pigeons with the neurotropic symptoms have been spread through the Taiwan during October 1999 to April 2001. The clinical signs observed were ataxia, torticollis, anorexia, tremor, and inability to peck feed, flying backward in circle, and greenish watery diarrhea. Forty-four submissions were used for the etiology. study In post-mortem examinations, no evident gross lesions were observed. Histopathologically, the predominant lesions were seen in the brains, constituting of endothelial cell proliferation and swelling, lymphocytes perivascular cuffing and interstitial nephritis were also noted. Tissue emulsions from the brains and spleens were used for inoculating into the allantoic cavities the nine-to-ten days old of chicken embryos. The allantoic fluids collected from inoculated embryos showed the hemagglutination phenomenum. The transmissible electron microscopy paramyxovirus-like particles could be noticed and they were also confirmed as Newcastle disease virus (NDV) fields isolates by multiple RT-PCR and nucleotide sequence. Four strains of NDV were isolated and identification from above submissions based on the clinical sign, histopathological, chicken embryo egg inoculation, hemagglutination test, hemagglutination inhibition test, electron microscopy, Multiplex RT-PCR and nucleotide sequence. One of the strains (P1/Taiwan/2000) was used for further study. Different ultracentrifugation and sedimentation through a sucrose gradient (10-50%) were used for purification of the viral fluid. This result showed that the sucrose concentrations, protein contents and the hemagglutination titers of the purified virus were 30-40%, 78mg/ml and 6 logarithm 26 ×100 fold respectively. This isolate have a mean death time less than 60 hours, the intracerebral pathogenicity index was 1.64, all data indicate that this virus isolate are velogenic virus. In the animal pathogenicity experiments of unvaccinated adult and young pigeons, the result showed that mortality and morbidity were 100%, the clinical signs and lesions were same as the field cases. induced Nucleotide sequence analysis of the viral F-gene (47-435bp region), the four strains (P1/Taiwan/2000, P2/Taiwan/2000, P3/Taiwan/2000 and P4/Taiwan/2000 ) were 2% different with the NDV Taiwan/95 strain. Two doses of inactivated oil vaccine (NDV or A/PMV-1) give four weeks apart at four-week old and eight-week old pigeon have a good protection.
目 次
英文摘要…………………………………………………… I
中文摘要…………………………………………………… III
目次……………………………………………………….. IV
表次……………………………………………………….. VII
圖次……………………………………………………….. VIII
第一章 緒言 ………………………………….………..……… 1
第二章 文獻探討……………………………………………….. 2
第一節、新城病病毒的歷史背景…………..………… ...2
第二節、新城病病毒特性…………….…………..……....….3
2-1 分類學 .……………………….. ……..……………..….3
2-2 病毒的形態……………………..……..……………….….3
2-3 病毒的基因體及蛋白質組成成份……………………….….3
2-4 病毒的純化……………………………………………….….5
2-5 病毒的物理特性………………………………………….….6
2-6 病毒株的分類…………………………………………….….6
2-7 病毒的複製、組合及釋放……………………………….….7
2-8 流行病學………………………………………………….….8
2-8-1 自然宿主與傳播方式……….………………………..….8
2-8-2 臨床症狀及肉眼病..….…………………….…….…….10
2-8-3組織病理學..…………..……….……………….……..11
2-9 新城病的毒力分析………….………………………….….11
第三節 新城病的診..…………………………………….…… 12
3-1抗體診斷…………………………………………..………. 14
3-2新城病病毒核酸定序…………………. ……………………14
第四節 預防與控制………………………………………………16
4-1 疫苗種類…………………………………………………… 16
4-2 雞的免疫計劃…………………………………………… ..18
4-3 鴿子的免疫計劃…………………………………………….19
第三章、材料與方法……………………………………………22
第一節、試驗材料
1-1、病材的來源….……………………………………………… 22
1-2、血球凝集抑制試驗的抗原…………………………………. 22
1-3、疫苗………….…………………………………………..… 22
第二節、試驗方法
2-1、細菌學檢查…………………………….………………………22
2-2、組織病理學……………………………..…………………… 22
2-3、抗體偵測……………………………….………………………22
2-4、病毒的增殖(雞胚胎尿囊腔接種)…….………………………23
2-5、紅血球凝集試驗(HA)………………………………….………23
2-6、0.85%雞紅血球懸浮液之製備…………………..….….… 23
2-7、紅血球凝集抑集試驗(HI)…………………………….………24
2-8、平均致死時間(Mean death time MDT)……………….…….24
2-9、腦內接種指數(Intracerebal pathogenicity index,ICPI).24
2-10、雞纖維母細胞的製作………………………………….………24
2-11、病毒的力價測定(TCID50)………………………….…………25
2-12、病毒的純化方法……………………………………………… 25
2-13、電子顯微鏡(負染色法)……………………………………….25
2-13-1、染劑之配製………………………………..……………….25
2-13-2、支持膜之製作………………………………..…………….25
2-13-2、樣品之處理及染色…………..…………………………….26
2-14、病毒RNA核酸的粹取………..…………….……..………….26
2-15 新城病病毒F-基因的定序……..……………………..………28
2-15-1 單管 (RT-PCR)增幅新城病病毒F-基因….….…… ….30
2-15-2 新城病病毒F-基因部份位置定序……………..…..… .31
第三節、鴿子接種試驗………………………………………………31
3-1 接種方式………………..………………………………………31
3-2、臨床症狀觀察………………..……………………………….31
3-3、組織病理學………………………..………………………….32
3-4、病毒的分離……………………………..………………….. 32
3-5、抗體測定及抗原偵測………………………..……………….32
''''
第四節、鴿子免疫計劃之實驗設計…………………. …………32
4-1、抗原的製備……………………………………………………..32
4-2、疫苗的製備…………….…………..………………… ……..32
4-3、實驗分組…………………………………………..33
4-4、抗體湞測………………………………………………………..33
4-5、臨床症狀觀察………………….……………………………….33
4-6、組織病理學………………………….………………………….33
4-7、病毒的分離……………………………………………………..33
4-8、抗體測定及抗原偵測………….………………………………33
第五節、免疫計晝之統計分析……………………… 34
第四章 結果……………………………………………………35
第一節、病鴿病材與病原的分離及鑑定……………………………35
1-1 解剖及病理組織學…………………… .……………………35
1-2細菌學檢查…………………………………………… …………35
1-3 病毒的增殖(雞胚胎尿囊腔接種) 及紅血球凝集試驗(HA)…..35
1-4 雞纖維母細胞病變………………………….…………………..35
1-5電子顯微鏡(負染色法) …………………………………………35
1-6多引子反轉錄-聚合酉每 連鎖反應 ( Multiplex RT-PCR )………36
1-7病毒之純化………………………………………………………36
第二節、病毒之毒力試驗、純化及新城病之定序…………………….36
2-1平均致死時間(MDT) ………………………………… …………36
2-2腦內接種指數(ICPI) …………………………………… ……….36
2-3病毒的力價測定(TCID50) ……………………………… ………37
2-4單管反轉錄-聚合酉每 連鎖反應增幅新城病病毒F-基因及定序..37
第三節、鴿子接種試驗…….…..………………………………………………38
3-1 臨床症狀觀察…………………..……….………………………38
3-2組織病理學…………………………………..………….……….38
3-3病毒的分離……………………………………….……..……….38
3-4 抗體測定及抗原偵測………………………………….……..…38
第四節、鴿子新城病免疫計劃之實驗結果……..…………………39
4-1、免疫反應……………………………………………………….40
4-2、統計分析……………………………………………………….40
第五章 討論……………………………………………………41
參考文獻……………………………………………………………..57
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