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研究生:梁文榮
研究生(外文):Wen-Jung Liang
論文名稱:以鴿高病原性新城病病毒(NDV)株製成不活化疫苗強化幼鴿對NDV之免疫保護力
論文名稱(外文):Improving Immunoprotectivity of the Inactivated Newcastle Disease Virus ( NDV ) Vaccine in Young Pigeons by Using the Antigen Prepared from the Highly Pathogenic Strains of NDV
指導教授:林茂勇
指導教授(外文):Maw-Yeong Lin
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:2005
畢業學年度:93
語文別:中文
論文頁數:56
中文關鍵詞:新城病鴿疫苗
外文關鍵詞:Newcastle diseasepigeonvaccine
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選新城病病毒 ( Newcastle disease virus;NDV ) 之Ishii株、台灣1984年分離之2株 ( TW/84-495和TW/84-486 ) 和台灣1999年分離之5株( TW/99-V154、TW/99-V156B、TW/99-V157、TW/99-V158和TW/99-V173 ) 做成抗原檢測2002年間台灣各地10個鴿群之164隻賽鴿血清之NDV血球抑制凝集反應 ( hemagglutination-inhibition;HI ) 抗體,全部上述第VII基因型急性內臟型NDV ( velogenic viscerotropic NDV;VVNDV ) 所檢測得鴿血清中之NDV-HI抗體力價幾何平均值 ( geometric mean titer;GMT ) 和變異係數(coefficient variation;CV)值均較為高些。
選上述在鴿血清可檢出較高NDV-HI抗體力價GMT和CV值較高的4株 ( TW/99-V156B、TW/99-V157、TW/99-V158和TW/84-486株 ) VVNDV分別口服 ( 1000 MLD ) 感染1組四週齡低NDV-HI抗體幼鴿,發現其死亡率在TW/99-V156B、TW/99-V157、TW/99-V158和TW/84-486分別為100%、60%、20%和0%。各株之毒性與其在臨床檢出的NDV-HI抗體力價GMT和CV值高低頗有正相關聯性。以上述4株VVNDV製成之NDV不活化油質與鋁膠型疫苗,併同市售6種鴿用NDV不活化疫苗分別免疫8組,每組6隻之四週齡幼鴿,於免疫3週後再以該4株NDV混合液口服 ( 1000 MLD ) 攻擊,結果以該4株製作之NDV不活化疫苗
對幼鴿之免疫保護力顯較其他市售鴿用NDV不活化疫苗為高。選用在檢測鴿臨床血清具高抗體之高病原性新分離VVNDV株製成NDV不活化疫苗,可有效強化幼鴿對NDV之免疫保護力。
選本實驗室於2004年分離之4株 ( TW/04-V411、TW/04-V411-2、TW/04-V418-1B和TW/04-V418-2 ) NDV與國內外分離株進行其F基因之核苷酸與胺基酸序列分析,發現新分離之4株NDV之親源性與近5年來台灣分離株頗為相似。
The Ishii strain, 2 1984 local isolates ( TW/84-495 and TW/84-486 ) and 5 1999 local isolates ( TW/99-V154, TW/99-V156B, TW/99-V157, TW/99- V158, TW/99-V173 ) of Newcastle disease virus ( NDV ) were selected as antigen producing strains for detecting the hemagglutination- inhibition ( HI ) antibody titer of the sera collected from 10 flocks of 164 pigeons. All the above genotype VII velogenic viscerotropic NDV ( VVNDV ) isolates provided higher geometric mean titer ( GMT ) and coefficient variation ( CV ) value of NDV-HI antibody titer in the pigeon sera detected.
The 4 isolates ( TW/99-V156B, TW/99-V157, TW/99-V158 and TW/ 84-486 ) of VVNDV with higher GMT and CV value were chosen for orally inoculating in each of the 4 groups, 5 birds per group, of 4-week-old low NDV-HI titer pigeon. The mortality caused by the TW/99-V156B, TW/99- V157, TW/99-V158 and TW/84-486 isolates was 100%, 60%, 20% and 0
% respectively. The virulence of each isolate was found positively correlated with the level of GMT and CV value that each isolate provided in the detection of the above field pigeon sera. The oil-emulsion or gel type inactivated vaccine prepared by the above 4 isolates and 6 commercial inactivated NDV vaccines was taken to immunize 8 groups, 6 birds per group, of 4-week-old pigeons then orally challenged ( 1000 MLD ) with the mixture of the 4 homologous isolates at 3 weeks post vaccination for comparison of its immunoprotectivity. The inactivated vaccine prepared by the above 4 isolates prominently provided higher protectivity level than the commercial vaccines. The inactivated NDV vaccine for pigeons prepared from the high virulent isolates of VVNDV to pigeons with also high in picking up field pigeon serum antibody shall highly improves of its immunoprotectivity to young pigeons.
Phylogenetic analysis the nucleotide of F gene and its deduced amino acid of four isolates ( TW/04-V411、TW/04-V411-2、TW/04-V418-1B and TW/04-V418-2 ) collected in this laboratory during 2004 with many local and international isolates or strains. The 4 new local NDV isolates were found phylogentically close to the last 5 year's local isolates.
中文摘要.............................................................Ⅰ
英文摘要.............................................................Ⅲ
誌謝................................................................Ⅴ
目錄................................................................Ⅵ
圖表目錄..................................................................Ⅸ
第1章 前言.........................................................1
第2章 文獻回顧...................................................................3
2.1 新城病的歷史背景...................................................................3
2.2 新城病病毒的特性...............................................
2.2.1 病毒型態與結構................................................5
2.2.2 病毒核苷酸與蛋白質性能與功能....................................5
2.2.2.1 NDV之核蛋白衣蛋白.............................................6
2.2.2.2 NDV之磷酸化蛋白...............................................6
2.2.2.3 NDV之大蛋白..................................................6
2.2.2.4 NDV之基質蛋白或膜蛋白..........................................7
2.2.2.5 NDV之血球凝集素神經胺酸酶醣蛋白.................................7
2.2.2.6 NDV之融合醣蛋白...............................................7
2.3 新城病病毒之生物合成..........................................10
2.3.1 病毒轉錄與轉譯...............................................10
2.3.2 病毒的複製...................................................10
2.3.3 病毒組合與釋放...............................................10
2.4. 新城病之流行病學.............................................11
2.5 疫苗製造....................................................11
2.5.1 製造用種株...................................................11
2.5.2 NDV之接種...................................................12
2.5.3 NDV之培養...................................................12
2.5.4 NDV之採收...................................................12
2.5.5 處理........................................................13

第3章 材料與方法...................................................15
3.1 強毒NDV ( VVNDV ) 株製成不活化疫苗對幼鴿之免疫保護力研究.........15
3.1.1 新城病病毒之分離、病毒斑點之純化及增殖..........................15
3.1.2 病毒蔗糖梯度離心之純化、濃縮與萃取..............................16
3.1.3 反轉錄聚合酶連鎖反應 ( RT-PCR ) 及其產物之鑑定..................17
3.1.4 NDV-HI抗原..................................................17
3.1.5 賽鴿血清....................................................18
3.1.6 NDV血球凝集試驗 ( NDV-HI ) 試驗..............................18
3.1.7 試驗鴿隻....................................................18
3.1.8 疫苗之選購...................................................18
3.1.9 自製NDV不活化疫苗............................................19
3.1.10 強毒NDV ( VVNDV ) 對幼鴿口服之病原性試驗.......................19
3.1.11 NDV不活化疫苗對幼鴿之免疫保護力評估試...........................19
3.2 鴿新城病病毒F基因選殖、定序及親源關係研究.......................19
3.2.1 病毒之分離、增殖、純化與濃縮...................................19
3.2.2 病毒RNA萃取及純化............................................21
3.2.3 反轉錄聚合酶連鎖反應 ( RT-PCR ) 及其產物之鑑定..................21
3.2.4 PCR產物選殖及定序............................................21
3.2.5 F基因核酸序列及胺基酸序列比較與親源關係分析......................22
第4章 結果........................................................24
4.1 強毒NDV ( VVNDV ) 株製成不活化疫苗對幼鴿之免疫保護力研究.........24
4.1.1 病毒株......................................................24
4.1.2 新城病病毒之RT-PCR結果.......................................24
4.1.3 NDV血球凝集抑制 ( NDV-HI ) 試驗..............................24
4.1.4 強毒NDV ( VVNDV ) 對幼鴿口服之病原性試驗......................24
4.1.5 NDV不活化疫苗對幼鴿之免疫保護力評估試驗.........................26
4.2 鴿新城病病毒F基因選殖、定序及親源關係研究.......................26
4.2.1 病毒株......................................................26
4.2.2 新城病病毒之RT-PCR結果及其產物選殖鑑定.........................28
4.2.3 F基因核酸序列及胺基酸序列比較與親源關係分析......................28
第5章 討論........................................................35
參考文獻............................................................38
中英文對照縮寫.......................................................50
附錄................................................................51
作者簡介............................................................56
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