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研究生:殷際航
研究生(外文):Ji-Hang Yin
論文名稱:雞隻早期給予雞傳染性貧血商業用疫苗之評估
論文名稱(外文):Evaluation on Early Administration of Chicken Infectious Anemia Vaccine
指導教授:歐繕嘉
指導教授(外文):Shan-Chia Ou
口試委員:謝明昆蔡宜倫
口試日期:2016-06-03
學位類別:碩士
校院名稱:國立中興大學
系所名稱:微生物暨公共衛生學研究所
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:2016
畢業學年度:104
語文別:中文
論文頁數:192
中文關鍵詞:雞傳染性貧血疫苗免疫組織化學染色原位雜交反應即時定量聚合酶鏈鎖反應
外文關鍵詞:Chicken infectious anemia (CIA)VaccineImmunohistochemistry Staining (IHC)In Situ Hybridization (ISH)Real-Time PCR (RT-PCR)
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雞傳染性貧血 (Chicken infectious anemia,CIA),為一好發於2至4週齡之雞隻且能造成雞隻產生貧血之臨床表徵、非再生性貧血、全身性淋巴組織流失之一免疫抑制重要性疾病,此疾病對於全世界之家禽產業造成相當大的影響與經濟上之損失,造成此疾病之病毒是由雞傳染性貧血病毒 (Chicken anemia virus,CAV) 感染所導致。雞傳染性貧血病毒廣泛存在於環境中,因病毒不具有封套,故相對之下不易被多數消毒劑清除,此外,目前仍無有效之治療方式給予患病之雞隻。因此大多數家禽業者以給予雞隻雞傳染性貧血商業用疫苗之方式以控制本病所帶來經濟上的損失。本篇論文研究目的為,藉由早期給予雛雞市售商品化疫苗以了解雛雞是否會產生CAV感染後之相對應臨床症狀或病理病變,同時,為模擬並了解自然感染傳播方式之機轉,使用我國已上市之商業用疫苗進行1日齡及3週齡雛雞之免疫接種。為評估與調查雞傳染性貧血病毒疫苗所造成之影響,本次實驗分別將1日齡雞隻與3週齡雞隻各分成口服給予劑量為2x103.5 TCID50 AviPro® Thymovac疫苗組及口服給予滅菌PBS (Phosphate buffer saline) 作為對照組,並於隔周進行雞隻之採血、體重監測及犧牲,並藉由觀察血容比 (Packed cell volume) 之數值以確認雞隻是否有貧血之臨床表徵出現,並於雞隻犧牲後進行肉眼及組織病理學之病變觀察以了解組織是否有因疫苗病毒感染而造成相對應之病變。同時利用即時定量聚合酶鏈鎖反應進行病毒核酸於各臟器之偵測與定量,並使用免疫組織化學染色法及原位雜交試驗分別了解雞傳染性貧血之蛋白與核酸於組織之分布位置。結果顯示,早期給予雛雞市售商品化疫苗,可於1日齡雞隻於給予疫苗7至第35天後觀察到貧血之臨床症狀;於1日齡雞隻及3週齡雞隻之疫苗組,於給予後7至第35天觀察到體重下降;於1日齡雞隻及3週齡雞隻之疫苗組皆於給予後第28天,於胸腺、脾臟、華氏囊及骨髓觀察到有最高之雞傳染性貧血病毒病毒含量;於1日齡雞隻及3週齡雞隻之疫苗組皆於給予後第7天至第35天可觀察到臟器如胸腺、華氏囊及骨髓萎縮,且主要於給予後第7天至第28天以免疫組織化學染色後主要於胸線、華氏囊及骨髓之淋巴球細胞有明顯之陽性反應,另於原位雜交反應下僅有胸腺於給予疫苗後第7天至第28天有明顯之陽性反應。

Chicken anemia virus (CAV), which causes chicken infectious anemia (CIA), is recognized as an important pathogen in poultry disease and poses a considerable threat to worldwide poultry industry. Although to arrive at sufficient maternal antibodies and to prevent the occurrence of CIA for chicks, commercial vaccines are given to the breeders at the adequate time, it is possible for some reasons that the vaccination is executed before the recommended period. The purpose of this study focused on the consequences, in accordance with the CAV infection, that chickens were given commercial vaccine, 2x103.5 TCID50 AviPro® Thymovac, early rather than the recommended period. 1-day-old and 3-week-old chickens were adopted to conduct this study and were separately divided into vaccinated group and control group. Diagnostic methods in this study involved the measurement of packed cell volume and body weight, the observation of gross and histopathological lesions, and the detection of nucleic acid and protein of CAV by real-time PCR (RT-PCR), in situ hybridization (ISH) and immunohistochemistry staining (IHC). The results illustrated that prominent anemia was noticed in 1-day-old vaccinated group from 7 to 35 day post inoculation (DPI). Waned weight was shown from 7 to 35 DPI in both 1-day-old and 3-week-old vaccinated group. Highiest CAV viral load was detected particularly at 28 DPI in both vaccinated groups in the immune system organs such as thymus, spleen, bursa of Fabricius and bone marrow. Atrophy was noticed in thymus, bursa of Fabricius and bone marrow from 7 to 35 DPI in both vaccinated groups. Dominant IHC positive signals were noticed from 7 to 28 DPI in the lymphocytes of thymus, bursa of Fabricius and bone marrow. Only in the thymus did the ISH staining has the positive signals.


中文摘要 i
英文摘要 ii
目次 iii
圖次 viii
表次 xi
附錄 xi
第一章 前言 1
第二章 文獻探討 2
第1節 雞傳染性貧血病毒 (Chicken Anemia Virus, CAV) 2
2.1.1 雞傳染性貧血病毒之歷史背景 2
2.1.2 雞傳染性貧血病毒之病毒特性 3
2.1.2.1 雞傳染性貧血病毒之分類與命名 3
2.1.2.2 雞傳染性貧血病毒之病毒型態 4
2.1.2.3雞傳染性貧血病毒之基因體、複製與表現 4
2.1.2.4雞傳染性貧血病毒之分離 7
2.1.2.5 雞傳染性貧血病毒之對抗物理、化學能力 8
2.1.3 雞傳染性貧血病毒之致病機轉 8
2.1.3.1 傳播方式 8
2.1.3.2 臨床症狀 9
2.1.3.3 病理病變 10
2.1.4 雞傳染性貧血病毒之診斷方式 11
2.1.4.1 核酸偵測 12
2.1.4.1.1 聚合酶鏈鎖反應 ( Polymerase Chain Reaction,PCR) 12
2.1.4.1.2 即時定量聚合酶鏈鎖反應(Quantitative real-time PCR,RT-PCR) 13
2.1.4.1.3 原位雜合反應 (In Situ hybridization,ISH) 13
2.1.4.2 抗原偵測 14
2.1.4.2.1 免疫組織化學染色法 (Immunohistochemical staining,IHC) 14
2.1.4.3 血清學檢查 15
2.1.5 雞傳染性貧血疾病之預防、控制及治療 15
第三章 實驗設計 17
第一部份 18
第四章 材料與方法 19
第1節 雞傳染性貧血病毒之表現載體構築 19
4.1.1 雞傳染性貧血病毒來源 19
4.1.2雞傳染性貧血病毒核酸之萃取 19
4.1.3 雞傳染病貧血病毒之目標基因增幅 20
4.1.3.1 特異性引子 (Primer) 序列設計 20
4.1.3.2 聚合酶鏈鎖反應之目標基因增幅 20
4.1.3.3 聚合酶鏈鎖反應電泳產物分析及確認 21
4.1.3.4 聚合酶鏈鎖反應之產物萃取純化 21
4.1.4 原核表現載體之構築 22
4.1.4.1 原核表現載體之製備 22
4.1.4.2 接合反應 (Ligation) 22
4.1.4.3 轉型作用 (Transformation) 23
4.1.4.4 利用Colony PCR進行重組載體之確認及挑選 23
4.1.4.5 菌落培養及重組質體之抽取 24
4.1.4.6 重組質體之定序 24
4.1.4.7 重組質體之序列分析 25
第2節 雞傳染性貧血病毒之重組蛋白表現及純化 25
4.2.1 勝任細胞 (Competent cell) 之製備 25
4.2.2 重組質體之原核系統表現重組蛋白 25
4.2.3 重組蛋白之蛋白質電泳分析及特異性分析與確認 26
4.2.3.1重組蛋白質之電泳分析 26
4.2.3.1 重組蛋白質之特異性分析及確認 26
4.2.4 重組蛋白之純化 27
4.2.5重組蛋白之定量分析 27
第3節 雞傳染性貧血病毒之單株抗體製備 27
4.3.1 實驗動物 27
4.3.2 免疫計畫 27
4.3.3 骨髓細胞瘤之培養 28
4.3.4 融合瘤之細胞融合 28
4.3.5 融合瘤細胞之培養 29
4.3.6 陽性融合瘤細胞之篩選 29
4.3.6.1酵素連結免疫吸附分析法 (Enzyme-linked immunosorbant assay,ELISA) 29
4.3.6.2西方墨漬法 (Western blot) 30
4.3.7 陽性融合瘤細胞之單株化 31
4.3.7.1 間接螢光抗體檢驗法 (Indirect fluorescent antibody assay,IFA) 31
4.3.8 雞傳染性貧血病毒之單株抗體亞型分析 32
第五章 結果 33
第1節 雞傳染性貧血病毒目標基因片段之選殖及表現載體之構築 33
5.1.1目標基因增幅之電泳產物分析及確認 33
5.1.2 目標基因片段與表現載體之構築 33
5.1.3 序列分析比對 33
5.1.4 重組蛋白之特異性分析及確認 33
5.1.5 純化之重組蛋白之特異性分析及確認 34
第2節 雞傳染性貧血病毒之單株抗體製備結果 34
5.2.1 融合瘤前小鼠脾臟觀察 34
5.2.2融合瘤細胞之篩選 35
5.2.2.1 酵素連結免疫吸附分析法分析結果 35
5.2.2.1.1 Box Titration抗原與抗體之定量結果 35
5.2.2.1.2 陽性融合瘤細胞篩選結果 35
5.2.2.2 西方墨漬法分析結果 35
5.2.2.3 間接螢光抗體檢驗法分析結果 36
5.2.3 單株抗體之亞型分析結果 36
第二部分 47
第六章 材料與方法 48
第1節 雞傳染性貧血之動物試驗 48
6.1.1 實驗動物 48
6.1.2 疫苗免疫計畫及檢驗流程 48
6.1.3 血容比之檢測 49
6.1.4 核酸偵測及核酸定量 49
6.1.4.1臟器乳劑之製作 49
6.1.4.2 核酸之萃取 49
6.1.4.3 聚合酶鏈鎖反應 50
6.1.4.4 即時定量聚合酶鏈鎖反應 51
6.1.4.4.1 即時定量聚合酶鏈鎖反應之特異性引子設計與選擇 51
6.1.4.4.2 引子之特異性偵測 51
6.1.4.4.3 特異性引子之敏感性偵測及標準曲線之建立 51
6.1.4.4.4 即時定量聚合酶鏈鎖反應試劑與步驟 52
6.1.4.4.5 即時定量聚合酶鏈鎖反應之精準性及再現性 53
6.1.5 型態病理學檢查 53
6.1.5.1肉眼病變之觀察及病變指數 53
6.1.5.2 組織病理學檢查 54
6.1.5.2.1石蠟切片之製作 54
6.1.5.2.2免疫組織化學染色法 (Immunohistochemistry staining,IHC) 54
6.1.5.2.3 組織病變之觀察及病變指數 55
6.1.5.2.4 原位雜交反應 (In situ hybridization,ISH) 56
6.1.5.2.4.1 探針 (Probe) 之製備 56
6.1.5.2.4.1.1 特異性引子 (Primer) 設計 56
6.1.5.2.4.1.2 探針之合成 56
6.1.5.3.4.1.3 合成探針之電泳膠體確認與分析 57
6.1.5.3.4.1.4 原位雜交法 57
第2節 統計分析 58
第七章 結果 59
第1節 動物實驗之結果 59
7.1.1 臨床症狀之觀察與血容比檢測 59
7.1.2 雞傳染性貧血病毒核酸檢測 59
7.1.2.1 雞傳染性貧血病毒之核酸分佈 59
7.1.2.2 即時定量聚合酶鏈鎖反應之條件建立 60
7.1.2.2.1 特異性分析 60
7.1.2.2.2標準曲線之建立與敏感性分析 60
7.1.2.2.3 精準性及再現性分析 61
7.1.2.3雞傳染性貧血病毒之核酸定量 61
7.1.3 病理學檢查 62
7.1.3.1 肉眼病變之觀察 62
7.1.3.2 型態病理學檢查 63
7.1.3.2.1 組織病變之觀察結果 63
7.1.3.2.2 免疫組織化學染色法之觀察結果 65
7.1.3.2.3 原位雜交法 66
7.1.3.2.3.1合成探針之電泳膠體確認與分析 66
7.1.3.2.3.2 原位雜交法之觀察結果 66
第八章 討論 137
第1節 口服給予雞隻AviPro® Thymovac之實驗結果 138
8.1.1 臨床表徵 138
8.1.2病理病變 141
8.1.2.1胸腺 142
8.1.2.2.脾臟 144
8.1.2.3華氏囊 144
8.1.2.4骨髓 145
8.1.2.5 肉眼與組織病變指數 146
8.1.3 免疫組織化學染色及原位雜交反應結果之探討 146
8.1.3.1 胸腺 146
8.1.3.2 脾臟 148
8.1.3.3 華氏囊 149
8.1.3.4 骨髓 149
8.1.4即時定量聚合酶鏈鎖反應 149
8.1.5 各診斷工具之比較 151
第2節 提早給予商業用AviPro® Thymovac疫苗之結果 151
第3節 預防及控制 153
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