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研究生:葉坤松
研究生(外文):Ku-Song Yei
論文名稱:台灣南部地區結核病牛難以自感染場根除之原因探討
論文名稱(外文):Causes of difficult eradication of tuberculosis affected cows from the infected diary herds in southern Taiwan
指導教授:吳永惠吳永惠引用關係
指導教授(外文):Yeong-Huey Wu
學位類別:碩士
校院名稱:國立屏東科技大學
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:日文
論文頁數:85
中文關鍵詞:乳牛結核病流行病學台灣
外文關鍵詞:Dairy cowsTuberculosisEpidemiologyTaiwan
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為究明台灣南部地區結核病牛難以自感染場根除之原因,並比較皮內結核菌素檢驗(intradermal tuberculin test;ITT)、迦瑪干擾素檢驗 (gamma interferon test;IFN-γ test)及雙重引子聚合酶鏈鎖反應(duplex polymerase chain reaction;duplex PCR)等3種檢驗方法,應用於乳牛結核病檢驗之敏感性和特異性,以探討根除本病之有效檢驗方法。一方面對結核病感染場(前次ITT檢驗有陽性牛隻出現之乳牛場)2場215頭ITT陰性乳牛,每3個月1次連續4次,同時進行此3種檢驗法,結果在4次檢查中,全部牛隻ITT均呈現陰性,血液IFN-γ和duplex PCR檢驗則分別有13.0% (28/215)和7.9% (17/215) 呈現陽性;對該17頭ITT陰性而duplex PCR陽性之牛隻,採鼻黏液和乳汁進行duplex PCR,結果鼻黏液有23.5% (4/17) 呈現陽性,而乳汁均呈陰性。另一方面,對另外5場72頭ITT 陽性乳牛,採血進行IFN-γ和duplex PCR檢驗、採鼻黏液和乳汁進行duplex PCR、以及採縱膈和咽背淋巴結進行Mycobacterium bovis分離,結果血液IFN-γ和duplex PCR檢驗呈陽性者分別有95.8% (69/72) 和86.1% (62/72),鼻黏液和乳汁之duplex PCR呈現陽性者分別有66.6% (48/72)和38.6% (17/44);在ITT、IFN-γ和duplex PCR檢驗呈陽性之牛中分別有68.1% (49/72)、68.1% (47/69) 和67.7%(42/62) 可由淋巴結分離到Mycobacterium bovis。由此7場之檢驗結果合併分析各檢驗法之敏感性和特異性,若以ITT結果作為判定標準,則IFN-γ檢驗之敏感性和特異性分別為95.8% (69/72) 和87.0% (187/215),duplex PCR之敏感性和特異性分別為86.1% (62/72) 和92.1% (198/215);若以IFN-γ檢驗結果作為判定標準,則ITT為71.1% (69/97) 和98.4% (187/190),duplex PCR為78.3% (76/97) 和98.4% (187/190);若以duplex PCR結果作為判定標準,則ITT為78.5% (62/97) 和95.2% (198/208),IFN-γ檢驗為96.2% (76/79) 和90.0% (187/208),以上顯示在結核病之敏感性方面,以IFN-γ檢驗為最高,PCR其次,而ITT最低;併用ITT和IFN-γ檢驗,或併用IFN-γ檢驗和duplex PCR,可顯著地 (P<0.05)提高敏感性和特異性。對上述7感染場內之工作人員和其他動物採血進行duplex PCR,結果工作人員有30.0% (3/10) 呈現陽性,而狗10隻、貓4隻和鼠10隻則均為陰性。又逢機取15頭牛和2位工作人員duplex PCR陽性之反應產物片段,連同Mycobacterium bovis標準菌株(ATCC19210)和1999年分離自台灣南部地區之Mycobacterium bovis菌株之PCR產物特異性片段,進行DNA選殖定序,結果全部19個樣本之DNA序列之相似性達94.9-100.0%。綜合以上結果,顯示現行ITT之敏感性偏低,不易將感染牛隻完全檢出,ITT陽性牛或假陰性牛均易藉由鼻黏液或乳汁散佈結核菌,造成其他牛隻之感染,此應是某些感染場難以根除之主因,對這種感染場我們建議併用ITT與IFN-γ,或ITT與PCR檢驗來提高其檢出率。
In order to explore the causes of difficult eradication of tuberculosis affected cows from the infected diary herds in southern Taiwan, intradermal tuberculin test (ITT), gamma interferon test (IFN-γtest) and duplex polymerase chain reaction (duplex PCR) for tuberculosis were conducted in ITT positive farms. The sensitivity and specificity of these methods for tuberculosis were studied. Using these detecting methods, a total of 215 ITT negative cows from two infected diary herds, which have had ITT positive cows in the last three ITT test, were continuously examined four times at interval of three months. The results showed that all 215 cows were negative reactions on ITT test in four times of examinations, but 13.0﹪(28/215) and 7.9﹪(17/215) cows were positive on IFN-γtest or duplex PCR, respectively. Using the duplex PCR, the nasal mucus and milk were examined on the 17 cows, which were negative for ITT but positive for duplex PCR of blood samples. The results revealed that 23.5﹪(4/17) cows were positive on the nasal mucus samples. On the other hand, the blood, nasal mucus and milk samples of 72 ITT positive cows from five other infected farms were examined by above methods, and their mediastinal and dorsal pharynx lymph nodes were sampled for the Mycobacterium bovis isolation. The results showed that 95.8﹪(69/72) and 86.1﹪(62/72) cows were positive on blood samples for tuberculosis by IFN-γtest and duplex PCR, respectively. Sixty six point six percent (48/72) and 38.6﹪(17/44) cows were positive for tuberculosis by duplex PCR on nasal mucus and milk samples, respectively. Mycobacterium bovis were isolation from 68.1﹪(49/72) ITT, 68.1﹪(47/69) IFN-γtest and 67.7﹪(42/62) duplex PCR positive cows, respectively. From above results obtained from these seven infected farms, the sensitivities and specificities of the three methods were statistically studied . The results showed as following. If using ITT results as the diagnostic standard, the sensitivity and specificity were 95.8﹪(69/72) and 87.0﹪(187/215) for IFN-γtest, and 86.1﹪(62/72) and 92.1﹪(198/215) for duplex PCR, respectively. If using IFN-γtest results as the diagnostic standard, the sensitivity and specificity were 71.7﹪(69/97) and 98.4﹪(187/190) for ITT, and 78.3﹪(76/97) and 98.4﹪(187/190) for duplex PCR, respectively. If using duplex PCR results as the diagnostic standard, the sensitivity and specificity were 78.5﹪(62/97) and 95.2﹪(198/208) for ITT, and 96.2﹪(76/79) and 90.0﹪(187/208) for IFN-γtest, respectively. According to the above results, the sensitivity and specificity for detecting bovine tuberculosis is the highest by using IFN-γtest and the lowest by using ITT among IFN-γtest, duplex PCR and ITT . The sensitivity and specificity were significantly (P<0.05) increased if combine ITT and IFN-γtest, or IFN-γtest and duplex PCR . Blood samples collected from 10 herdsmen, 10 dogs, four cats and 10 rats in these seven infected diary herds were examined by duplex PCR, only 30.0﹪(3/10) herdsmen were showed positive for tuberculosis. The duplex PCR products of blood samples from 15 cows and two herdsmen were randomly selected for analyses of DNA sequence and for comparisons of DNA sequences among those Mycobacterium bovis standard strain (ATCC 19210) and a field strain isolated from southern Taiwan in 1999. The similarity of all those DNA sequences is 94.9—100﹪. From the above results, we concluded that the sensitivity of ITT is too low to efficiently eliminate the Mycobacterium bovis infected cows from the farms, and the ITT positive or false—negative cows could easily spread the pathogens through their nasal discharges or milks to the ITT negative cows. This might be the most important causes of difficulty eradicating the tuberculosis from the infected diary herds. For those herds, we suggest that using the ITT and IFN-γtest, or ITT and PCR to improve the detecting rate of tuberculosis in cows.
目 錄
摘要 I
英文摘要 III
誌謝 VI
目 錄 VII
圖 次 X
表 次 XI
第一章 前 言 1
第二章 文獻回顧 4
2.1 結核病發生概況 4
2.2 分枝桿菌之形態及特性 9
2.3 分枝桿菌的分類 10
2.4 分枝桿菌對動物之病原性 12
2.5 結核病之致病機制 13
2.6 皮內結核菌素檢驗 16
2.7 牛迦瑪干擾素檢驗 18
2.8 聚合酶鏈鎖反應偵測結核病感染 19
2.9 台灣乳牛結核病檢驗法之演進 20
第三章 材料與方法 22
3.1 供試動物及其他材料 22
3.2 皮內結核菌素檢驗 23
3.2.1 試劑 23
3.2.2 方法 23
3.3 ITT陽性牛之剖檢及淋巴結之分枝桿菌分離 23
3.4 迦瑪干擾素試驗 24
3.4.1 試劑及製備 24
3.4.2 方法 25
3.5 M. bovis 和M. tuberculosis之標準菌株增菌培養 27
3.5.1 培養基 27
3.5.2 方法 27
3.6 標準菌株、血液、鼻黏液、乳汁檢體中分枝桿菌核酸之萃取 28
3.6.1 試劑及儀器 28
3.6.2 方法 28
3.6.2.1 標準菌株核酸之萃取 28
3.6.2.2 血液白血球分離 29
3.6.2.3 白血球內結核菌DNA之萃取 29
3.6.2.4 鼻黏液、乳汁內結核菌DNA之萃取 30
3.7 PCR引子對設計 30
3.8 聚合酶鏈鎖反應 32
3.8.1 試劑 32
3.8.2 方法 33
3.8.2.1 核酸之增幅 33
3.8.2.2 增幅產物之分析 35
3.8.2.3 聚合酶鏈鎖反應之特異性試驗 35
3.8.2.4 聚合酶鏈鎖反應之敏感性試驗 35
3.9 PCR產物之選殖 36
3.9.1 試劑 36
3.9.2 方法 37
3.9.2.1 電泳膠片PCR產物之純化 37
3.9.2.2 接合作用 40
3.9.2.3 勝任細胞之製備 40
3.9.2.4 轉型作用 40
3.9.2.5 質體之純化 41
3.9.2.6 限制酶切割及確定 41
3.9.2.7 質體DNA定序 42
3.10 污染場內之人員和其他動物PCR檢驗 42
第四章 結 果 44
4.1 雙重引子PCR及巢式雙重引子PCR之特異性分析 44
4.2 雙重引子PCR及巢式雙重引子PCR之敏感性分析 47
4.3 PCR反應產物之選殖定序 50
4.4 感染場之流行病學調查 54
4.5 三種檢驗方法之敏感性及特異性比較 58
4.6 乳汁和鼻黏液之PCR檢驗 65
4.7 感染場內工作人員和牛以外動物之血液PCR檢驗 65
第五章 討 論 66
參考文獻 73
作者簡介 85
圖 次
圖3-1基因選殖流程圖.………………………………………………39
圖4-1雙重引子PCR檢驗之特異性分析電泳圖………………………45
圖4-2巢式雙重引子PCR檢驗之特異性分析電泳圖…………………46
圖4-3雙重引子PCR檢驗之敏感性分析電泳圖………………………48
圖4-4巢式雙重引子PCR檢驗之敏感性分析電泳圖………………..49
圖4-5限制酶切割選殖序列圖…………………………………………51
圖4-6血液PCR呈陽性(ITT陽性牛4隻,ITT陰性牛11隻,工作人員2位)、1999年分離自高雄縣乳牛之M.bovis 、M.bovis標準菌株(ATCC 19210)三者之PCR產物核酸序列比對之親緣圖………………………………52
表 次
表2-1 1947-1999年台灣乳牛皮內結核菌素試驗檢驗結果……………7
表2-2 1997-1999年台灣南部地區某兩縣乳牛結核病感染場,由陽性場轉為陰性場所需之檢驗次數之分布百分率………………………………8
表3-1採血進行結核病Duplex PCR之7場結核病感染場內工作人員及
其他動物採樣分布…………………………………………………………43
表4-1血液PCR陽性樣本(ITT陽性牛4隻,ITT陰性牛11隻,工作人
員2位)、M.bovis標準菌株、分離自高雄縣乳牛場之M.bovis
菌株三者之PCR產物核酸序列之相似性及相異性百分比…….……….53
表4-2兩感染場每隔3個月一次連續4次ITT檢查均呈陰性牛隻215頭
之末梢血液IFN-γ檢驗之變動情形……………………………………...56
表4-3兩感染場每隔3個月一次連續4次ITT檢查均呈陰性牛隻215頭
之末梢血液PCR檢驗之變動情形………………………………………..57
表4-4兩感染場每隔3個月一次連續4次ITT檢查均呈陰性牛隻215頭
之末梢血液IFN-γ檢驗與PCR呈一致性反應之牛隻頭數及其所佔百分比……………………………………………………………………………58
表4-5五場72頭ITT陽性牛之縱膈和咽背淋巴結牛分枝桿菌分離結果,與ITT、IFN-γ及PCR檢驗結果相互間之敏感性和特異性……………………………………………………………………………61
表4-6五場72頭ITT陽性牛之縱膈和咽背淋巴結牛分枝桿菌分離結果,與ITT、IFN-γ及PCR檢驗相互併用結果之敏感性和特異性……………………………………………………………………………62
表4-7以ITT、IFN—γ和PCR檢驗結果相互為判定標準,由7感染場共287頭乳牛之檢驗結果,分析3檢驗法之敏感性及特異性……………………63
表4-8以ITT 、IFN—γ和PCR檢驗結果為判定標準,由7感染場共
287頭乳牛之檢驗結果,分析併用任何二種檢驗法時之敏感性和
特異性……………………………………………………………………64
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