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研究生:葉偉生
研究生(外文):Wai-Sang Ip
論文名稱:弧菌二價疫苗及微顆粒包覆口服疫苗應用於養殖石斑魚上之研究
論文名稱(外文):The application of divalent vibrio vaccine and encapsulation of oral vaccine on cultured grouper (Epinephelus sp.)
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:2012
畢業學年度:100
語文別:中文
論文頁數:119
中文關鍵詞:疫苗弧菌石斑魚褐藻酸鈉
外文關鍵詞:vaccinevibriogroupersodium alginate
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本研究旨在以褐藻酸鈉分別包覆溶藻弧菌 (Vibrio alginolyticus) 及鮫弧菌 (Vibrio carchariae) 作為微顆粒口服疫苗,除探討其物理特性及接種石斑魚後之免疫效果外,並探討使用油質佐劑及鋁質佐劑於此二種弧菌之二價注射疫苗對石斑魚的免疫效果。
利用褐藻酸鈉為包覆殼材製備口服疫苗,可有效包覆菌體,且褐藻酸鈉不會與菌體產生化學結構的變化,可保持菌體之完整性;在仿胃液酸鹼值 (pH 3.75) 條件下能有效保護菌體抗原不受破壞,而在仿腸液酸鹼值 (pH 7.4) 條件下可經由微顆粒的膨潤現象,使菌體持續釋放;當以1.5 %褐藻酸鈉、1.5 %氯化鈣及500 rpm之攪拌速度時,所製備之褐藻酸鈉微顆粒具有圓球狀外形、粒徑分佈平均以及釋放效果佳等優點。經口服投餵接種後,對石斑魚的保護效果及血清中抗體力價均能有效提升,在以溶藻弧菌與鮫弧菌攻擊後,最高相對活存率分別可達75 %及88.89 %。
石斑魚經注射接種疫苗後,對抗溶藻弧菌及鮫弧菌的保護效果及抗體力價皆有上升,但油佐劑對石斑魚可誘發較好的抗溶藻弧菌免疫效果,其保護效果可達100 %;而鋁佐劑對石斑魚有較佳的抗鮫弧菌保護效果,相對活存率可達88.89 %,且保護效力皆可持續至24週。

Abstract
The present study was designed to investigate the physical properties and immune effects of oral vaccines using encapsulated Vibrio alginolyticus or Vibrio carchariae as sodium alginate microparticles in vaccinated groupers and also to study the immune response of grouper after vaccination with both oil and aluminum adjuvanted bivalent vaccine of Vibrio alginolyticus and Vibrio carchariae delivered via intraperitoneal injection.

In the preparation of microparticles vaccine, sodium alginate as coating material could effectively coat the bacterial without any changes in the chemical structure between sodium alginate and also bacteria, therefore could maintain the integrity of the bacteria. Under simulation of gastric fluid condition (pH 3.75), sodium alginate could effectively protect the antigen from destruction while swelling phenomenon of microparticles was observed under simulation of intestine fluid condition (pH 7.4), and thus favored the sustained release of bacteria. The prepared sodium alginate microparticles using 1.5% sodium alginate and 1.5% calcium chloride under the stirring speed of 500 rpm have a spherical shape, even distribution of particle size and good release effect.

After vaccination using alginate coated-bacteria microparticle delivered by oral feeding, the protective effect in the grouper and the serum antibody were effectively enhanced with the highest relative percentage survival (RPS) of 75% and 88.89% after challenged with Vibrio alginolyticus (S4Y) and Vibrio carchariae (EMI), respectively.

On the other hand, increased immune effects in grouper against Vibrio alginolyticus and Vibrio carchariae were observed after vaccination via intraperitoneal injection with both oil and aluminium adjuvanted bivalent vaccine. From the results obtained, grouper vaccinated with oil adjuvanted vaccine showed the best protective effect with the relative percentage survival (RPS) up to 100% in grouper against Vibrio alginolyticus while that for aluminum adjuvanted vaccine showed RPS of 88.9% in grouper against Vibrio carchariae, and both protective effects could last continuously up to Weeks 24.

目錄
摘要 I
Abstract II
表目錄 IX
圖目錄 X
第一章、前言 1
第二章、文獻整理 3
2.1 石斑魚簡介 3
2.1.1 石斑魚之養殖 4
2.2 弧菌症 5
2.3 溶藻弧菌 6
2.4 鮫弧菌 7
2.5 魚類免疫 8
2.5.1 非特異性免疫 9
2.5.1.1 表面屏障 9
2.5.1.2 細胞性免疫 10
2.5.1.3 體液性免疫 10
2.5.2 特異性免疫 11
2.6 魚類疫苗 12
2.6.1 注射型疫苗 13
2.6.2 浸泡型疫苗 13
2.6.3 口服型疫苗 13
2.6.3.1 腸溶性包覆疫苗 15
2.7 佐劑 15
2.7.1 油質佐劑 16
2.7.2 鋁質佐劑 17
2.8 微顆粒 19
2.9 褐藻酸鈉 19
2.9.1 褐藻酸鈉結構 19
2.9.2 褐藻酸鈉的凝膠機制 20
第三章、材料與方法 22
實驗架構 22
3.1實驗菌株 23
3.1.1菌株來源 23
3.1.2菌株活化保種 23
3.2 細菌之生化鑑定分析 23
3.2.1 API 20E system 23
3.2.2 Biolog GN system 24
3.3 抗原製備 24
3.4 注射菌苗製備 25
3.5 口服疫苗製備 25
3.5.1 褐藻酸鈉微顆粒製備 25
3.6 口服疫苗物理特性分析 26
3.6.1 褐藻酸鈉微顆粒菌體包覆 26
3.6.2 褐藻酸鈉微顆粒粒徑 26
3.6.3 褐藻酸鈉微顆粒口服疫苗之組成分 26
3.6.4 褐藻酸鈉微顆粒載菌量 26
3.6.5 不同濃度褐藻酸鈉及氯化鈣對微顆粒菌體釋放之影響 27
3.6.6 不同pH值對褐藻酸鈉微顆粒結構變化之影響 27
3.6.7 間接式酵素連結免疫吸附分析 28
3.7 疫苗體內試驗 28
3.7.1 實驗魚隻 28
3.7.2二價疫苗之腹腔注射接種 29
3.7.3 口服疫苗之投餵接種 29
3.7.4 攻擊試驗 30
3.7.5魚隻接種後各階段血清樣本採樣 30
3.7.6血清中蛋白質含量測定 30
3.7.7 血清中抗體力價偵測 31
第四章、結果 32
4.1實驗菌株 32
4.1.2 菌株確認 32
4.2口服疫苗物理特性分析 32
4.2.1褐藻酸鈉微顆粒之外觀形態 32
4.2.2轉速對褐藻酸鈉微顆粒粒徑之影響 32
4.2.3 褐藻酸鈉微顆粒口服疫苗之組成分 33
4.2.4 褐藻酸鈉微顆粒包覆菌體之載菌量 33
4.2.5 不同濃度褐藻酸鈉及氯化鈣對微顆粒菌體釋放之影響 33
4.2.6 不同pH值對褐藻酸鈉微顆粒結構變化之影響 34
4.3菌苗之體內試驗 34
4.3.1 腹腔注射菌苗於石斑魚體內之形態 34
4.3.2 二價疫苗注射接種後之攻擊試驗 35
4.3.3 二價注射接種後之血清抗體力價 36
4.3.4 褐藻酸鈉微顆粒包覆疫苗口服接種後之攻擊試驗 37
4.3.5 褐藻酸鈉微顆粒包覆疫苗口服接種後之血清抗體力價 38
第五章、討論 39
5.1 口服疫苗物理特性分析 39
5.2 注射不同佐劑二價疫苗之體內試驗 43
5.3 口服褐藻酸鈉包覆疫苗之體內試驗 47
第六章、參考文獻 53
第七章、圖表 78

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