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研究生:蕭宇辰
研究生(外文):Yu-Chen Shiau
論文名稱:愛德華氏菌及嗜水性產氣單胞菌混合疫苗對日本鰻之保護效果研究
論文名稱(外文):Studies on the protection of divalent vaccine of Edwardsiella tarda and Aeromonas hydrophila in Japanese eel(Anguilla japonica).
指導教授:劉秉忠
指導教授(外文):Ping-Chung Liu
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:89
中文關鍵詞:愛德華氏菌嗜水性產氣單胞菌日本鰻疫苗
外文關鍵詞:Edwardsiella tardaAeromonas hydrophilaAnguilla japonicaJapanese eelvaccine
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本論文使用愛德華氏菌及嗜水性產氣單胞菌之混合疫苗探討其對養殖鰻魚主動免疫的保護效果。本實驗所使用之愛德華氏菌(Edwardsiella tarda strain Et8)菌株系於 2007 年分離自年罹病鰻魚之血液;嗜水性產氣單胞菌(Aeromonas hydrophila strain A1)菌株則是 1999 年分離自潰瘍症鰻魚之表皮。經由福馬林不活化菌體混合魚油,對鰻魚進行不同之疫苗給予方式,測定其血清力價及保護效果。
實驗結果顯示,使用腹腔注射接種疫苗對鰻魚產生對抗愛德華氏菌及嗜水性產氣單胞菌之特異性抗體均有提升的效果;使用愛德華氏菌對經免疫接種鰻魚進行攻擊試驗,其保護效果有逐漸上升的趨勢,到第 16 週保護效果可達 100%。疫苗以口服方式投餵,可提高特異性抗體產生量,抗嗜水性產氣單胞菌及抗愛德華氏菌抗體力價有逐漸上升的趨勢,到第 12 週到可達最高值;試驗鰻魚經嗜水性產氣單胞菌攻擊試驗後,保護效果亦有逐漸上升之趨勢,第 12 週相對活存率達 60%,而使用愛德華氏菌攻擊其保護效果維持在 80%。
綜合以上結果,鰻魚使用腹腔注射接種可以引發較迅速且較高的抗體力價;使用口服投餵鰻魚引發之抗體時間較慢,可以延長投餵時間來提升抗體力價。腹腔注射接種疫苗可以有效保護鰻魚對抗愛德華氏菌,對嗜水性產氣單胞菌則沒有保護效果;口服投餵疫苗可以保護鰻魚對抗愛德華氏菌,對嗜水性產氣單胞菌保護效果有逐漸提升的現象。因此使用口服投餵混合魚油疫苗,是保護鰻魚對抗愛德華氏菌及嗜水性產氣單胞菌感染之可行策略。
This thesis investigated the protection of divalent vaccine of Edwardsiella tarda and Aeromonas hydrophila in Japanese eel (Anguilla japonica). The E. tarda (strain Et8f) and A. hydrophila (strain A1) were previously isolated from blood or skin ulcer of eel in 2007 and 1999, respectively. The vaccines consisted of formalin-inactivated whole bacterial cell with the addition of fish oil were given to eel by different methods. The specific antibody responses and protective efficacies in vaccinated eel were evaluated .
The results revealed that eel immunized with the Et8f- and A1- vaccine via intraperitoneal (i.p.) injection exhibited higher specific antibody titers, and the survival rates after challenge with E. tarda were elevated gradually during the experiment period. The survival rate was 100% at week 16. Fish exhibited greater specific antibody titers by orally vaccination with divalent bacteria (Et8f- and A1-), and could reach to the maximum at week 12. The protective efficacy after challenge with A. hydrophila was gradually elevated, and the survival rate was 60% at week 12. However, the survival rate of the group challenged with E. tarda was above 80% during the experiment period.
The results indicated that using i.p injection could result in higher and faster specific antibody titer in eel, but slower via oral vaccination. Prolonging the feeding period could be used to elevate the specific antibody titer. Fish immunized by i.p. injection exhibited good protective efficacy after challenge with E. tarda, but not for A. hydrophila. In addition, fish immunized orally exhibited good protective efficacy after challenge with E. tarda or A. hydrophila. Therefore, the strategy of oral administration with formalinized bacterial cell supplemented with fish oil could be a good approach in future vaccine development to protect eel from A. hydrophila and E. tarda infection.
謝辭……………………………………………….……………...…………i
中文摘要……………………………………………….……….…………iii
英文摘要……………………………………………….…….……………iv
目錄……………………………………….…………..…...………………vi
表目錄……………………………………………………....……………viii
圖目錄…………………………………………………...…...……………ix
第一章 前言…………………………………………...……..……………1
第二章 文獻整理………………………..…………...……………………3
第三章 材料方法…………………………………...……………………22
3.1 試驗菌株來源及保存……………………...……………………22
3.2 菌株生化特性分析………………………...……………………22
3.2.1 革蘭氏染色………………..………...……………………22
3.2.2 細胞色素氧化酵素………………...………………..……23
3.2.3 磷脂質分解酵素…………………...………………..……23
3.2.4 抗生素敏感試驗……………………….……..……..……23
3.2.5 API 20E 鑑定…………………………………...…………23
3.2.6 Biolog System………………………………..……………24
3.2.7 Polymerase Chain Reaction………………...……………...24
3.3 實驗生物………………………………………...………………25
3.4 菌株毒力試驗…………………………………...………………25
3.4.1 溶血試驗………………………….……...……………….25
3.4.2 菌體攻擊試驗…………………………..………………..26
3.5 疫苗製作及接種……………………….……………...………...26
3.6 抗血清製備…………………………………………...…………27
3.7 抗血清凝集測試……………………………………..…………27
3.7.1 免疫沉澱試驗………………………………...…………..27
3.7.2 免疫火箭電泳………………………………...…………..27
3.8 疫苗保護效果……………………………………...……………28
第四章 結果……...………………………………………………………29
第五章 討論…...…………………………………………………………33
參考文獻…...……………………………………………………………..38
表…….……………………………………………………………………61
圖……...…………………………………………………………………..69
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