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研究生:李柏蒼
研究生(外文):Po-Tsang Lee
論文名稱:應用溶藻弧菌疫苗及其免疫雞蛋黃IgY於石斑魚養殖之研究
論文名稱(外文):The application of Vibrio alginolyticus vaccine and egg yolk IgY against the same vaccine on cultured grouper (Epinephelus coioides)
指導教授:李國誥李國誥引用關係
指導教授(外文):Kuo-Kau Lee
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:2010
畢業學年度:98
語文別:中文
論文頁數:109
中文關鍵詞:溶藻弧菌疫苗IgY
外文關鍵詞:Vibrio alginolyticusvaccineImmunoglobulin Y
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本研究探討 Vibrio alginolyticus 疫苗及其免疫雞蛋粉 IgY 對石斑幼魚之主動與被動免疫的保護效果。Vibrio alginolyticus S4Y 菌株係分離自罹病石斑魚,經強化後作為疫苗抗原製備及攻擊試驗使用。經毒力測試實驗,結果顯示本菌株對石斑魚之半致死劑量 (LD50) 為 2.0×104 CFU/g fish body weight。

在主動免疫試驗方面,腹腔注射含大豆油佐劑之菌苗加脂多醣 (Inject II 組) 與投餵以洋菜膠包覆之疫苗 (Oral) 組保護效果最佳,接種後第 4、8、12 及 16 週時活存率相同,分別為 100%、100%、100% 及 75%;腹腔注射含大豆油佐劑之菌苗 (Inject I) 組次之,分別為 75%、100%、100% 及 50%;浸泡 Bath 組僅在接種後 4 週具有些許的保護效果 (活存率 50%);浸泡 Dip 組在各採樣點皆無發現有保護效果。

被動免疫方面,含 2% 單一 S4Y 蛋粉組保護效果最佳,連續投餵 7 天或 14 天後攻擊,活存率分別為 100% 及 90%;蛋粉添加至 4% 時,活存率分別為 20% 及 20%,蛋粉添加比例的升高無法與保護能力有正相關;含 2% 單一 S4Y 蛋粉混合 1% β-glucan 之活存率分別為 100% 及 80%,無法顯著提升保護力;含 2% 或 4% 混合蛋粉處理組 (V. alginolyticus S4Y 及 V. carchariae EMI) 之活存率分別為 70%、100% 及 80%、100%,顯示飼料中提高蛋粉比例至 4% 並不會提高保護力,反而增加成本,故添加量為 2% 時較為適當。

綜合以上結果,主動免疫以 Inject II 組及 Oral 組能有效的增強石斑魚對抗 V. alginolyticus 之感染,對於未來發展注射及口服疫苗尚具有相當意義,值得繼續深入探討。而被動免疫中,無論是單一抗菌蛋粉組或混合抗菌蛋粉處理組皆以添加量為 2% 時較為適當。此結果顯示應用抗菌蛋黃 IgY 以對抗石斑幼魚弧菌症具相當潛力,值得深入探討。

This study investigated the immunological protection by applying injectable vaccines of Vibrio alginolyticus strain S4Y and yolk IgY against the same vaccines on juvenile grouper. The bacterium originally isolated from diseased groupers was cultured and passed in the fish three times to increase its virulence, and then was used in this study for experimental purposes including vaccine preparation and virulence test. The LD50 value of the bacterium in grouper was 2.0×104 CFU/g fish body weight.

For active immunization, the best protective groups were group Inject II (using soybean oil as adjuvant supplemented with lipopolysaccharide) and Oral (encapsulated with agarose) with a same survival rate after immunization for 4, 8, 12 and 16 weeks prior to a lethal bacterial challenge. Of those, the survival rate were 100, 100, 100 and 75%, respectively. Following the best two protective groups was group Inject I (adjuvanted with soybean oil only) with survival rate of 100, 100, 100 and 75%, respectively. Meanwhile, Only a little protection was achieved in group Bath after immunization for 4 weeks with a survival rate of 50%. In contrast, no protection was achieved in group Dip after immunization.

For passive immunization, the group fed with 2% egg yolk IgY against V. alginolyticus bacterium in feed daily for one week and two weeks exhibited the best protection with survival rates of 100 and 90%, respectively. However, the increase of the IgY against the bacterium to 4% in feed did not enhance the proportion with survival rates of 20 and 20%, respectively. The additional of 1% glucan in the 2% IgY in feed did not exhibit better performance with survival rates of 100 and 80%, respectively. In addition, fish fed with 2% and 4% IgY against divalent bacterium (V. alginolyticus S4Y and V. carchariae EMI) in feed exhibited good protection with survival rates of 70 and 100%, 80 and 100%, respectively. The results indicated that the additional of 2% IgY against V. alginolyticus challenge.

In conclusion, the present results reveal that the use of soybean oil as adjuvant supplemented with lipopolysaccharide and the use of agarose for encapsulation in feed possess potential for further development in injectable vaccine and oral vaccine, respectively. Furthermore, the additional of 2% IgY against V. alginolyticus on divalent bacterin exhibits good potential for passive immune protection against the vibriosis in grouper fry.

謝辭----------------------------------------------------------------------------------I
中文摘要---------------------------------------------------------------------------II
Abstract---------------------------------------------------------------------------IV
目錄-------------------------------------------------------------------------------VI
表目錄----------------------------------------------------------------------------IX
圖目錄-----------------------------------------------------------------------------X
第一章 前言----------------------------------------------------------------------1
第二章 文獻整理----------------------------------------------------------------3
第三章 材料方法---------------------------------------------------------------27
3.1 試驗菌株-------------------------------------------------------------- 27
3.1.1 菌株來源----------------------------------------------------------27
3.1.2 菌株活化保種----------------------------------------------------27
3.1.3 實驗生物----------------------------------------------------------27
3.1.4 養殖系統----------------------------------------------------------27
3.1.5 菌株強化----------------------------------------------------------28
3.1.6 菌株對石斑魚之毒性試驗-------------------------------------28
3.1.7 API 20E system 鑑定--------------------------------------------28
3.1.8 BIOLOG system 鑑定-------------------------------------------29
3.2 抗原製備---------------------------------------------------------------29
3.3 LPS脂多醣萃取-------------------------------------------------------29
3.4 蛋白質含量測定------------------------------------------------------30
3.5 採血及血清製備------------------------------------------------------30
3.6 兔子抗點帶石斑魚全血清之製備---------------------------------30
3.7主動免疫----------------------------------------------------------------30
3.7.1 實驗設計----------------------------------------------------------30
3.7.2 浸泡疫苗----------------------------------------------------------31
3.7.3 腹腔注射疫苗----------------------------------------------------31
3.7.4 口服疫苗----------------------------------------------------------31
3.7.5 攻擊試驗----------------------------------------------------------32
3.7.6 超氧陰離子之測定----------------------------------------------32
3.7.7 化學發光反應----------------------------------------------------32
3.7.8 抗體力價試驗----------------------------------------------------33
3.8 被動免疫---------------------------------------------------------------33
3.8.1 被動免疫實驗設計----------------------------------------------33
3.8.2 蛋粉 IgY 製備--------------------------------------------------34
3.8.3 蛋粉IgY抗體力價測試----------------------------------------34
3.8.4 飼料製備----------------------------------------------------------35
3.8.5 攻擊試驗----------------------------------------------------------35
3.9 統計分析------------------------------------------------------------35
第四章 結果---------------------------------------------------------------------37
4.1 菌株來源與強化-----------------------------------------------------37
4.2 菌株對石斑魚之毒性試驗-----------------------------------------37
4.3 主動免疫--------------------------------------------------------------37
4.3.1 超氧陰離子產生率之測定-------------------------------------37
4.3.2 化學發光反應----------------------------------------------------38
4.3.3 抗體力價試驗----------------------------------------------------39
4.3.4 攻擊試驗----------------------------------------------------------39
4.4 被動免疫--------------------------------------------------------------40
4.4.1 蛋粉 IgY 抗體力價測試---------------------------------------40
4.4.2 攻擊試驗-----------------------------------------------------------40
第五章 討論---------------------------------------------------------------------42
5.1 主動免疫----------------------------------------------------------------42
5.1.1 石斑幼魚菌苗免疫對非特異性免疫因子之影響-----------42
5.1.2 石斑幼魚菌苗免疫對抗體力價之影響-----------------------43
5.1.3 石斑幼魚菌苗免疫效果之試驗--------------------------------45
5.2 被動免疫----------------------------------------------------------------50
5.2.1 石斑幼魚免疫球蛋粉IgY被動免疫效果之試驗-----------50
參考文獻--------------------------------------------------------------------------52
表-----------------------------------------------------------------------------------86
圖-----------------------------------------------------------------------------------94


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