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研究生:詹昆衛
研究生(外文):Kun-Wei Chan
論文名稱:臭氧處理對Eimeriatenella卵囊活性與其免疫性之探討
論文名稱(外文):Viability and immunogenicity of Eimeria tenella oocyst treated with ozone
指導教授:王俊秀王俊秀引用關係
指導教授(外文):Jiunn-Shiow Wang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系
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:2002
畢業學年度:90
語文別:中文
論文頁數:90
中文關鍵詞:臭氧Eimeria tenella卵囊免疫性
外文關鍵詞:Eimeria tenellaozoneoocyst
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摘要
近來由於集約式家禽工業式飼養經營的環境模式,導致動物緊迫'及疾病的增加。腸道感染如球蟲症、沙門氏桿菌症、隱胞子蟲症在商用種雞的飼養有逐漸增加趨勢,並導致家禽飼養業中嚴重的經濟損失。目前,化學藥劑仍是廣泛控制球蟲症的主要方式,但由於野外寄生蟲抗藥株的出現,及動物體內藥物殘留的問題,利用免疫及分子控制球蟲症方法應可用於克服這些問題。
本研究以Eimeria tenella單一卵囊增殖法繼代增殖之卵囊分別於飽和臭氧水溶液中曝臭氧,30、60、90、120分鐘後收集卵囊,室溫培養48小時觀察芽胞化比率。結果顯示臭氧處理時間越長,未芽胞化比率越高。此外將試驗分五組,A組(無臭氧處理)、B組(臭氧處理60分鐘)、C組(臭氧處理120分鐘)、陽性對照組(攻毒不免疫)、陰性對照組(不免疫不攻毒)。A、B、C組各以1,000顆卵囊免疫六日齡小雞(Lohwann蛋雞及Arbor Acres肉雞),於二十五日齡以5萬顆球蟲(同株球蟲)攻毒,觀察每克糞中卵囊數(O.P.G.) 及紀錄體重,於三十六日齡犧牲。使用SAS分析系統,正交比較體重與增重之關係。分析蛋雞實驗結果得知,免疫組體重顯著重於未免疫組,而各組攻毒後增重結果,免疫組顯著重於未免疫組,且經活毒免疫雞隻組增重顯著重於減毒免疫雞隻組。此外各組O.P.G.結果顯示,於免疫時期A組O.P.G.最多且時間最長,而在攻毒後,免疫組中以C組最高。肉雞實驗結果顯示,在體重上免疫接種並不會造成組間差異,而各組於攻毒後之增重,免疫組顯著重於未免疫組。攻毒後B、C組O.P.G.較A組高,餘結果和蛋雞相同。綜合實驗結果以體重及增重評估得知,經免疫雞隻較未免疫雞隻表現為佳;活毒卵囊與減毒卵囊對雞隻之保護效力並無差異。此外以O.P.G.配合評估結果得知,以臭氧減毒後之卵囊在免疫接種後對環境的污染較活毒卵囊為低。
Summary(英文摘要)
In recent years, intensive livestock and poultry rearing practices have led to an increase in animal stress and incidence of disease. Intestinal infections such as coccidiosis, salmonellosis, and cryptosporidiosis are becoming increasingly prevalent in commercially bred chickens and inflict severe economic losses in the poultry industry. Currently, chemotherapy is used extensively to control coccidiosis , but drug resistance in field strains of parasites and drug residue problem mandate development of alternative methods to control this disease. Immunological and molecular control strategies, such as vaccine development now appear to be suitable to achieve this goal.
In the first part of this study, the effect of ozone on the sporulation of Eimeria tenella oocysts in vitro was examined. Oocysts were obtained by single oocyst inoculation and maintained by passage in chicken, and then were exposed to ozone for 30, 60, 90, and 120 minutes, respectively. The ozone treated oocysts were then incubated for 48 hours in room temperature for observing their sporulation ratios. It was found that the longer the ozone treatment, the higher the inhibition of the sporulation rate of the oocysts was observed.
In the second part of this thesis, chickens were immunized with ozone-attenuated oocysts and then challenged homologously with 50,000 E. tenella oocysts. Chickens were divided into 5 groups (A to E) of 10 chickens each and treated as follows: Group A: inoculated with 1,000 untreated oocysts at 6 days old and challenged at 25 days old, Group B: inoculated with 1,000 oocysts treated with ozone for 60 min, Group C: inoculated with 1000oocyst treated with ozone for 120 min, Group D: not vaccinated and given only challenge infection (positive control), Group E: not vaccinated and unchallenged (negative control). The challenge infection with 50,000 E. tenella oocysts was carried out for Groups A-E when the chickens were 25 days old. Before being sacrificed at 36 days old, the chickens were weighed and the number of oocysts per gram (OPG) of feces was determined by the McMaster method.
The body weight and weight gains of all the chickens were analyzed by orthogonal contrast and SAS system. The body weights of the vaccinated groups of chickens were significantly greater than the unvaccinated ones. While the negative group (Group E) chickens was greater than the challenged groups (A, B, C, and D). Weight gains of chickens in the vaccinated groups were significantly greater than the unvaccinated one. Weight gain of chicken immunized with non-treated oocysts (A group) was significantly greater the chicken given ozone attenuated oocysts (B, C groups). As for the OPG, chicken of group A showed the highest and longest period of oocysts shedding after the primary infection.
In summary, immunized chickens have higher weight gains and chicken given ozone-attenuated oocysts help to reduce the numbers of oocyst shed into the environment.
目 次
目次…………………………………………………………………I
表次………………………………………………………………Ⅳ
圖次………………………………………………………………Ⅵ
中文摘要…………………………………………………………………1
英文摘要………………………………………………………………3
第一章 前言…………………………………………………………5
第二章 文獻探討………………………………………………………7
第一節、 雞隻球蟲症之生物學特性………………………7
一、雞球蟲症之簡介…………………………………7
二、Eimeria屬球蟲之分類……………………………7
三、雞Eimeria屬球蟲之生活史………………………8
(1)芽胞繁殖期…………………………………………8
(2)裂殖體繁殖期…………………………………………9
(3)配子繁殖期…………………………………………9
四、雞Eimeria屬球蟲之致病性…………………………10
(1)球蟲症的病原性………………………………………10
1、各種別之相關致病性………………………………11
2、各種別在雞隻體內繁殖能力………………………12
3、雞隻食入具有感染性卵囊之數量…………………12
(2)擁擠效應…………………………………………13
五、Eimeria屬球蟲的宿主特異性………………………14
六、Eimeria屬球蟲的免疫原性………………………14
七、球蟲之疫苗控制……………………………………15
(1)現有之球蟲疫苗……………………………………17
(2)活毒疫苗的組成……………………………………18
(3)目前使用中之各種球蟲疫苗介紹 …………………19
1、早熟系之優點…………………………………20
2、E. maxima同種異株之間變異性的問題 ……20
3、未來發展……………………………………21
4、基因重組疫苗………………………………22
第二節、臭氧的基本性質及應用之探討………………30
一、臭氧之物理化學特性………………………………30
二、臭氧對人體之影響…………………………………31
三、臭氧在水中之反應型態與分解反應……………31
四、臭氧之應用………………………………………32
五、臭氧之消毒理論…………………………………34
(1)臭氧對病毒作用之機制……………………………34
(2)臭氧對細菌之作用機制……………………………35
(3)臭氧對原蟲之應用…………………………………36
第三章 材料與方法………………………………………………38
第一節、臭氧對Eimeria tenella芽胞化之影響……38
一、雞隻來源與飼養管理………………………………38
二、球蟲株來源及單一球蟲株之建立…………………38
三、卵囊之增殖、收集、純化及芽胞化之處理………39
四、未芽胞化卵囊之曝氣處理…………………………40
第二節、芽胞化之Eimeria tenella 卵囊經臭氧處理後能否提供雞隻保護性免疫實驗………………………………41
一、材料與方法…………………………………………41
1、雞隻來源與管理…………………………………41
2、球蟲卵囊之製備………………………………41
3、已芽胞化E.tenella卵囊經臭氧處理之過程…41
4、經臭氧處理芽胞化之E. tenella 卵囊後能否提供雞
隻保護性免疫之試驗方法………………………42
5、統計分析方式………………………………………42
第四章 結果…………………………………………………………44
第一節、臭氧對Eimeria tenella芽胞化之影響…44
第二節、芽胞化之Eimeria tenella 卵囊經臭氧處理後能否提
供雞隻保護性免疫實驗……………………………45
一、各次實驗中O.P.G.之結果…………………………45
二、各組體重分析結果………………………………46
三、病理剖檢與病理組織切片之證據………………48
四、以病變程度指數(severity indix)評估各組差異…49
第五章 討論……………………………………………………………72
第一節、臭氧對Eimeria tenella芽胞化之影響………………72
第二節、芽胞化之Eimeria tenella 卵囊經臭氧處理後能否提供雞隻保護性免疫實驗…………………………………………76
參考文獻………………………………………………………………79
表 次
表1 雞隻Eimeria屬球蟲的特徵……………………………25
表2 六種Eimeria屬球蟲實驗接種之建議劑量………………26
表3 Williams RB.以7日齡小雞所做7種Eimeria屬球蟲繁殖趨勢
(reproductive potentials)實驗,所得之結果,與文獻中所推
測之繁殖趨勢理論值比較………………………………27
表4 歐美國家所使用之雞球蟲疫苗……………………………28
表5 各Eimeria種別之野外初代株與其對應之早熟系,於雞隻
體內生活史所持續時間之比較……………………………29
表6 各族群體重與增重之差異,利用SAS系統正交比較
(orthogonal contrast)時,各組所使用之係
數…………………………………………………………………43
表7 蛋雞第一次實驗中,各組雞隻每克糞中卵囊數(O.P.G.)
之記錄………………………………………………………50
表8 蛋雞第二次實驗中,各組雞隻每克糞中卵囊數(O.P.G.)
之記錄…………………………………………………………51
表9 肉雞第一次實驗中,各組雞隻每克糞中卵囊數(O.P.G.)
之記錄…………………………………………………………52
表10 蛋雞第一次實驗中體重與增重記錄………………………53
表11 蛋雞第二次實驗中體重與增重記錄………………………61
表12 肉雞第一次實驗中體重與增重記錄………………………61
表13 蛋雞實驗中,每組10隻雞中任取6隻之切片,作為組織
病理判讀之材料,依病變程度訂出積分,計算出病變程度指
數,以評估各組腸病變程度…………………………………52
表14 肉雞實驗中,將每組雞隻之組織病理切片,作為判讀之材
料,依病變程度訂出積分,計算出病變程度指數,以評估各
組腸管病變程度………………………………………………53
表15 10隻雞接種E. tenella後,平均每隻雞產生之卵囊量,及
因球蟲死亡隻雞數………………………………………75
圖 次
圖1 臭氧處理時間與卵囊芽胞率之關係………………………56
圖2 蛋雞第一次實驗時,各組每克糞中卵囊數之記錄…………56
圖3 蛋雞第二次實驗時,各組每克糞中卵囊數之記錄…………57
圖4 肉雞第一次實驗時,各組每克糞中卵囊數之記錄…………57
圖5 蛋雞第一次實驗,免疫期間與攻毒期間各組O.P.G.總數之記
錄…………………………………………………………………58
圖6 蛋雞第二次實驗,免疫期間與攻毒期間各組O.P.G.總數之記
錄………………………………………………………………58
圖7 肉雞雞第一次實驗,免疫期間與攻毒期間各組O.P.G.總數之
記錄………………………………………………………………58
圖8 蛋雞第一次實驗中中免疫接種卵囊後,各組排出卵囊芽胞
率之記錄……………………………………………………59
圖9 蛋雞第一次實驗中,攻毒接種後,各組排出卵囊芽胞率之
情形………………………………………………………………59
圖10 蛋雞第二次實驗中,免疫接種卵囊後,各組排出卵囊芽胞
率之記錄………………………………………………………59
圖11 蛋雞第二次實驗中,攻毒接種後,各組排出卵囊芽胞率之
情形……………………………………………………………59
圖12 肉雞第一次實驗中,免疫接種卵囊後,各組排出卵囊芽胞
率之記錄………………………………………………………59
圖13 肉雞第一次實驗中,攻毒接種後,各組排出卵囊芽胞率之
記錄…………………………………………………………59
圖14 蛋雞第一次實驗中,各組雞隻於1日齡、7日齡、24日齡、
36日齡之體重記錄…………………………………………60
圖15 蛋雞第一次實驗中,各時期平均日增重之記錄…………60
圖16 蛋雞第二次實驗中,各組雞隻於1日齡、7日齡、24日齡、
36日齡之體重記錄…………………………………………61
圖17 蛋雞第二次實驗中,各時期平均日增重之記
錄………………………………………………………………61
圖18 肉雞第一次實驗中,各組雞隻於1日齡、7日齡、24日齡、
36日齡之體重記錄………………………………………62
圖19 肉雞第一次實驗中,各時期平均日增重之記
錄…………………………………………………………62
圖20 肉雞實驗中,陽性對照組死亡雞隻之盲腸………………63
圖21 肉雞實驗中,於36日齡時犧牲時,陰性對照組雞隻之盲
腸……………………………………………………………63
圖22 肉雞實驗中,於36日齡時犧牲時,陽性對照組雞隻之盲
腸………………………………………………………………64
圖23 肉雞實驗中,於36日齡時犧牲時,A組雞隻之盲
腸………………………………………………………………64
圖24 肉雞實驗中,於36日齡時犧牲時,B組雞隻之盲
腸………………………………………………………………65
圖25 肉雞實驗中,於36日齡時犧牲時,C組雞隻之盲
腸…………………………………………………………………65
圖26 肉雞實驗中,於36日齡時犧牲時,陰性對照組盲腸之組
織切片…………………………………………………………66
圖27 肉雞實驗中,於36日齡時犧牲時,陰性對照組盲腸之組
織切片………………………………………………………66
圖28 肉雞實驗中,陽性對照組於攻毒後死亡雞隻盲腸之組織切
片…………………………………………………………67
圖29肉雞實驗中,陽性對照組於攻毒後死亡雞隻盲腸之組織切
片……………………………………………………………67
圖30 肉雞實驗中,陽性對照組於攻毒後死亡雞隻盲腸之組織切
片………………………………………………………………68
圖31 肉雞實驗中,A組盲腸組織切片低倍視
野……………………………………………………………68
圖32肉雞實驗中,B組盲腸組織切片低倍視
野……………………………………………………………69
圖33肉雞實驗中,C組盲腸組織切片低倍視野
……………………………………………………………69
圖34 肉雞實驗中,A組盲腸組織切片高倍視
野……………………………………………………………70
圖35肉雞實驗中,B組盲腸組織切片高倍視野
……………………………………………………………70
圖36 肉雞實驗中,C組盲腸組織切片高倍視野
……………………………………………………………71
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