臺灣博碩士論文加值系統

摘要
本實驗室建立Eimeria tenella單一卵囊球蟲株(ET-CH1)，持續感染繼代，維持其活力與毒力，所收集之卵囊皆保存於4℃。已芽胞化卵囊分別不以臭氧處理及臭氧曝氣15分鐘、30分鐘與60分鐘，取1,000顆免疫雞隻進行減毒球蟲對雞群免疫實驗，經50,000顆未處理卵囊感染後，其結果顯示，臭氧處理30分鐘之卵囊相較於15及60分鐘者，在排出卵囊抑制指數或雞群增重上都更具有保護效力。而在其後的免疫劑量之免疫實驗中，得知以500顆卵囊免疫雞隻，相對於100顆及1,000顆卵囊，有較少之卵囊排出及較高的增重。在死亡率方面，各實驗之免疫組皆較未免疫之感染組呈現較低之死亡率，足見以低劑量之球蟲卵囊免疫雞群可避免其後球蟲症之爆發。實驗觀察，以臭氧處理30分鐘之已芽胞化卵囊500顆免疫雞隻所得之免疫效力，在雞隻排出卵囊量、死亡率及平均體重上，均比未經免疫之對照組佳，可見減毒球蟲卵囊可提供雞群保護性並減少雞場卵囊之污染。
在未芽胞化卵囊經臭氧處理之後的芽胞化比率方面，顯示在臭氧處理10分鐘內，對於卵囊芽胞化的影響大約減低其芽胞率20 %左右，但隨著處理時間的增長，卵囊的芽胞率呈現快速的下降，當處理超過60分鐘時，幾乎所有未芽胞化卵囊都無法完成芽胞化，由此可知，臭氧可有效地使未芽胞化卵囊不活化。
將經過臭氧處理的卵囊置於電子顯微鏡下觀察，發現構成卵囊外層之第一卵囊壁形成體（Wall-Forming body I）呈現出比未處理之表面較為光滑；處理20分鐘時，第一卵囊壁形成體似乎變得更薄；處理30分鐘時第一卵囊壁形成體呈現破裂，並使得內層之第二卵囊壁形成體（Wall-Forming body II）裸露；而處理60分鐘、90分鐘及120分鐘，均未顯示第二卵囊壁形成體有減少的趨勢，且觀察不到任何的差異。

Abstract
Chickens were first immunized with ozone-treated Eimeria tenella oocysts and then challenged with E. tenella oocysts originally derived from a single oocyst. The E. tenella single-oocyst strain was designated ET-CH1, that had been successively passaged in chickens. Oocysts collected from feces were first suspended in 2.5 % potassium dichromate solution for 48 hrs, until they sporulated and then transferred to 4℃ for storage until used. The sporulated oocysts in group A were not treated with ozone, but those in group B were treated with ozone for 60 mins, in group C for 30 mins, and in group D for 15 mins. The aforementioned oocysts were then orally inoculated into chickens at an immunizing dose of 1,000 oocysts per chicken. Nineteen days after immunization with the ozone-treated oocysts, all the chickens were challenged with 50,000 non-ozone-treated sporulated oocysts. Chickens in the groups immunized with 30 mins ozone-treated oocysts showed higher protection than the other groups based on O.P.G. (oocysts per gram of feces), body weight, and mortality against the challenge infection. In a dose-effect experiment, groups of chickens were respectively immunized with 100 (group C100), 500 (group C500), and 1,000 (group C1000) oocysts that had been treated with ozone for 30 mins. Chickens in the group immunized with 500 oocysts showed higher protection than those immunized with 100 or 1,000 oocysts. All the immunized groups showed lower mortality than the non-immunized group after challenge with 50,000 oocysts. Therefore, our results showed that chickens immunized with 500 oocysts that had been treated with ozone for 30 mins were best protected than the other groups when evaluated by O.P.G., body weight and mortality.

目 錄
目錄………………………………………………………………………I
圖次……………………………………………………………………IV
表次……………………………………………………………………VI
中文摘要…………………………………………………………………1
英文摘要…………………………………………………………………2
第一章 前言……………………………………………………………4
第二章 文獻探討………………………………………………………7
第一節、 雞之Eimeria屬球蟲之簡介…………………………7
一、Eimeria屬球蟲之分類…………………………………7
二、球蟲卵囊的構造………………………………………7
三、脫囊作用………………………………………………8
四、生活史…………………………………………………9
五、病害……………………………………………………10
第二節、球蟲症之防治………………………………………11
一、抗球蟲藥物……………………………………………11
二、國外市售疫苗之簡介…………………………………11
三、免疫效力評估的方法…………………………………13
第三節、臭氧…………………………………………………16
一、概述……………………………………………………16
二、靛青精（indigo）臭氧含量測試法…………………17
三、臭氧對病毒及細菌方面的研究………………………18
四、原蟲性寄生蟲在臭氧方面的研究……………………19
五、其他寄生蟲在臭氧方面的研究………………………20
六、臭氧其他方面的研究…………………………………20
第三章 材料與方法……………………………………………………22
第一節、環境控制及材料來源………………………………22
一、雞隻來源與飼養管理…………………………………22
二、球蟲來源………………………………………………22
第二節、卵囊收集與處理步驟………………………………23
一、卵囊之繼代與收集……………………………………23
二、球蟲卵囊臭氧曝氣處理………………………………24
第三節、活毒免疫之可行性…………………………………26
一、實驗動物分組…………………………………………26
二、雞隻球蟲免疫及感染實驗……………………………26
三、經臭氧處理卵囊免疫雞隻之免疫劑量實驗…………26
第四節、臭氧處理卵囊之殺滅效果…………………………28
一、未芽胞化卵囊經臭氧處理之芽胞率實驗……………28
二、經臭氧處理卵囊掃瞄式電子顯微鏡觀察……………28
第五節、實驗數據紀錄及統計分析…………………………30
一、每克糞材含有卵囊數與排出卵囊抑制指數…………30
二、死亡率…………………………………………………31
三、體重……………………………………………………31
四、統計分析………………………………………………31
第四章 結果……………………………………………………………32
第一節、活毒免疫之可行性評估………………………………32
一、未經臭氧處理及臭氧處理60與30
分鐘卵囊免疫雞隻後之感染實驗……………………32
二、未經臭氧處理及臭氧處理30與15
分鐘卵囊免疫雞隻後之感染實驗……………………32
三、臭氧處理30分鐘卵囊之免疫劑量實驗……………33
第二節、臭氧處理卵囊之殺滅效果……………………………34
一、未芽胞化卵囊經臭氧處理之芽胞率實驗……………34
二、經臭氧處理卵囊之光學顯微鏡觀察…………………34
三、經臭氧處理卵囊之掃描式電子顯微鏡觀察…………34
第五章 討論……………………………………………………………58
第一節、活毒免疫之可行性………………………………………58
一、實驗數據採納原則……………………………………58
二、數據評估探討…………………………………………58
三、其他……………………………………………………60
第二節、臭氧處理卵囊之殺滅效果………………………………61
一、未芽胞化卵囊經臭氧處理之芽胞率…………………61
二、經臭氧處理卵囊電子顯微鏡觀察……………………61
三、未芽胞化卵囊與已芽胞化卵囊之臭氧
處理殺滅效果…………………………………………62
參考文獻………………………………………………………………63
圖 次
圖一、第一次實驗各組每日O.P.G.折線圖…………………………35
圖二、第一次實驗各組免疫後與感染後總O.P.G.量
及其總量直方圖…………………………………………………35
圖三、第一次實驗各組死亡率直方圖………………………………36
圖四、第一次實驗各組平均體重直方圖……………………………36
圖五、第二次實驗各組每日O.P.G.折線圖…………………………37
圖六、第二次實驗各組免疫後與感染後總O.P.G.量
及其總量直方圖…………………………………………………37
圖七、第二次實驗各組死亡率直方圖………………………………38
圖八、第二次實驗各組平均體重直方圖……………………………38
圖九、第三次實驗各組每日O.P.G.折線圖…………………………39
圖十、第三次實驗各組免疫後與感染後總O.P.G.量
及其總量直方圖…………………………………………………39
圖十一、第三次實驗各組死亡率直方圖……………………………40
圖十二、第三次實驗各組平均體重直方圖…………………………40
圖十三、第三次實驗陰性對照組之糞便……………………………41
圖十四、第三次實驗100顆卵囊免疫組於免疫後
24日齡之下痢便………………………………………………41
圖十五、第三次實驗500顆卵囊免疫組於免疫後
24日齡之下痢便………………………………………………42
圖十六、第三次實驗1,000顆卵囊免疫組於免疫後
24日齡之下痢便………………………………………………42
圖十七、第三次實驗陽性對照組於免疫後24日齡之血痢便………43
圖十八、未芽胞化卵囊經不同時間臭氧處理後之
芽胞率折線圖……………………………………………………43
圖十九、光學顯微鏡觀察經臭氧處理10分鐘卵囊…………………44
圖二十、光學顯微鏡觀察經臭氧處理20分鐘卵囊…………………44
圖二十一、光學顯微鏡觀察經臭氧處理30分鐘卵囊………………45
圖二十二、光學顯微鏡觀察經臭氧處理60分鐘卵囊………………45
圖二十三、光學顯微鏡觀察經臭氧處理90分鐘卵囊………………46
圖二十四、光學顯微鏡觀察經臭氧處理120分鐘卵囊……………46
圖二十五、未經臭氧處理之卵囊於電子顯微鏡下觀察………………47
圖二十六、未經臭氧處理之卵囊於電子顯微鏡下觀察………………47
圖二十七、經臭氧處理10分鐘之卵囊於電子顯微鏡下觀察………48
圖二十八、經臭氧處理10分鐘之卵囊於電子顯微鏡下觀察………48
圖二十九、經臭氧處理20分鐘之卵囊於電子顯微鏡下觀察………49
圖三十、經臭氧處理20分鐘之卵囊於電子顯微鏡下觀察…………49
圖三十一、經臭氧處理30分鐘之卵囊於電子顯微鏡下觀察………50
圖三十二、經臭氧處理30分鐘之卵囊於電子顯微鏡下觀察………50
圖三十三、經臭氧處理60分鐘之卵囊於電子顯微鏡下觀察………51
圖三十四、經臭氧處理60分鐘之卵囊於電子顯微鏡下觀察………51
圖三十五、經臭氧處理90分鐘之卵囊於電子顯微鏡下觀察………52
圖三十六、經臭氧處理90分鐘之卵囊於電子顯微鏡下觀察………52
圖三十七、經臭氧處理120分鐘之卵囊於電子顯微鏡下觀察………53
圖三十八、經臭氧處理120分鐘之卵囊於電子顯微鏡下觀察………53
表 次
表一、第一次實驗中，各組雞隻每日O.P.G之記錄…………………54
表二、第二次實驗中，各組雞隻每日O.P.G之記錄…………………55
表三、第三次實驗中，各組雞隻每日O.P.G之記錄…………………56
表四、三次實驗之排出卵囊抑制指數、死亡率、
平均體重比較表…………………………………………………57

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