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研究生:陳培源
研究生(外文):Pei­-Yuen Chen
論文名稱:台南市流浪犬鉤端螺旋體之血清學與分子診斷調查
論文名稱(外文):Serology and Molecular Diagnosis Survey of Leptospires of Stray Dogs in Tainan City
指導教授:周世認詹昆衛詹昆衛引用關係
指導教授(外文):Shih-Jen ChouKun-Wei Chan
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:獸醫學系研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
畢業學年度:103
語文別:中文
論文頁數:60
中文關鍵詞:鉤端螺旋體顯微凝集試驗聚合酶鏈鎖反應恆溫式圈環型核酸增幅法台南市流浪犬
外文關鍵詞:Leptospiramicroscopic agglutination testpolymerase chain reactionloop-mediated isothermal amplification testTainan citystray dogs
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鉤端螺旋體是一種細桿狀螺旋型,兩端呈鈎狀的一種細菌。鉤端螺旋體屬(Leptospira)可分為兩群,一為致病性的Leptospira interrogans sensu lato,另一為非致病性的腐生型Leptospira biflexa sensu lato ◦本菌能引起人類及動物的鉤端螺旋體病(leptospirosis),是一種人畜共同傳染病。本菌感染的宿主相當廣泛,包括許多家畜如牛、馬、羊、猪、犬等以及野生動物,甚至一些水生動物。感染鉤端螺旋體的家犬、流浪犬可直接或間接感染於人,在公共衛生上,屬於重要感染病原◦故本研究目的擬進行台南市流浪犬鉤端螺旋體之血清學與分子診斷調查,以瞭解台南市流浪犬鉤端螺旋體之感染情形。本試驗共採集台南市動物收容所流浪犬隻168個血液樣本。應用血清學之顯微凝集試驗 (microscopic agglutination test, MAT)及分子生物學之聚合酶鏈鎖反應 (polymerase chain reaction, PCR)和恆溫式圈環型核酸增幅法 (loop-mediated isothermal amplification, LAMP)等技術,檢測流浪犬血清之鉤端螺旋體抗體陽性率及病原核酸。結果以MAT檢測168隻流浪犬之鉤端螺旋體血清抗體陽性率為41.67 % (70/168)。且在70例抗體陽性檢體中,有25.71 % (18/70)為多重血清型感染。總血清型陽性之反應數為90個,總血清型陽性率為53.25 % (90/168),血清型以Lyme最多 (40 %),其次為Javanica (25.56 %)和Canicola (11.11 %)。陽性率最高之月份為5 - 6月(21.43 %),其次為9 - 10月(20 %)和1 - 2月(20 %)。未結紮犬之陽性率為結紮犬之6倍。以PCR檢測168個血液樣本之血清病原核酸結果皆為陰性。但以11 - 12月和5 - 6月收集之56隻流浪犬的血液樣本進行LAMP檢測,其血清陽性率結果為16.07 % (9/56),可見一般傳統式PCR在僅有少量病原核酸存在之條件下,往往未能偵測到病原,而以敏感度高於傳統式PCR的LAMP進行偵測,得到敏感性較高的實驗結果◦本研究結果可提供動物防疫單位及公共衛生學上之參考資料。
Leptospira is a thin rod-shaped spiral, both ends of the hook-shaped form of a bacterium. Leptospira can be divided into two groups, one is pathogenic Leptospira interrogans sensu lato and another is non-pathogenic saprophytic Leptospira biflexa sensu lato. Leptospirosis is a important zoonosis which causes the clinical signs in humans and animals. The bacteria are infected host quite extensive, including many domestic animals such as cattle, horses, sheep, pigs, dogs, wild animals, and even some aquatic animals. The infected Leptospira dogs and stray dogs can directly or indirectly infect human. Therefore, the purpose of this study to carry out the serological and molecular diagnosis survey of leptospires of stray dogs in Tainan City, to understand the situation of leptospirosis infection. The test were collected 168 stray dogs blood samples in Tainan City animal shelter and leptospiral positive detect rate of examed stray dogs was tested by serological microscopic agglutination test (MAT), polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) test. In MAT to detect, 168 stray dogs of a seroantibody positive rate were 41.67 % (70/168). In 70 cases of antibody positive samples, there are 18 cases 25.71 % (18/70) for serovars of multiple-infected. A total number of samples tested positive to all serovars was 90‚ the positive rate were 53.25 % (90/168). Serovars with Lyme majority (40 %), followed by Javanica (25.56 %) and Canicola (11.11 %). Among the positive cases‚ the highest months were from May to June (21.43 %), followed by September to October (20 %) and January to February (20 %). Non-neutered dogs positive rate was six times the neutered dogs. 168 blood samples by PCR analysis of pathogenic nucleic acid results were negative. However, 56 blood samples were collected in November to December and May to June were LAMP test for detection of stray dogs, presented 16.07 % (9/56) of seropositive rate. Generally traditional PCR in the presence of only a small number of pathogenic nucleic acid, often fail to detect pathogens, and again LAMP test its sensitivity is higher than conventional PCR, get higher sensitivity results. The results of this study reference materials available to the animal epidemic prevention agencies and public health in the future.
中文摘要 …………………………………………………………………… i
英文摘要 …………………………………………………………………… iii
目次 ………………………………………………………………………… v
表次 ………………………………………………………………………… viii
圖次 ………………………………………………………………………… x
第一章 緒言 ………………………………………………………… 12
第二章 文獻探討 …………………………………………………… 14
第一節 歷史背景 …………………………………………………… 14
第二節 鉤端螺旋體病之病原 ……………………………………… 16
第三節 鉤端螺旋體之流行病學 ………………………………… 17
2.3.1 鉤端螺旋體之分佈 ……………………………………… 17
2.3.2 鉤端螺旋體之宿主範圍 ………………………………… 17
2.3.3 鉤端螺旋體之傳播與致病機轉 ………………………… 18
2.3.4 鉤端螺旋體病之臨床症狀與病理變化 …………………… 19
2.3.4.1 臨床症狀 ……………………………………………… 19
2.3.4.2 病理變化 ……………………………………………… 19
第四節 鉤端螺旋體病之診斷、治療與預防 ……………………… 21
2.4.1 鉤端螺旋體病之診斷 ……………………………………… 21
2.4.1.1 血液學檢查 …………………………………………… 22
2.4.1.2 尿液分析 ……………………………………………… 22
2.4.1.3 顯微凝集試驗 ………………………………………… 22
2.4.1.4 酵素連結免疫吸附法 ………………………………… 23
2.4.1.5 聚合酶鏈鎖反應 ……………………………………… 23
2.4.1.6 PCR - 限制性片段長度多態性 ……………………… 24
2.4.1.7 恆溫式圈環型核酸增幅法 …………………………… 24
2.4.1.8 組織病理學 …………………………………………… 25
2.4.2 鉤端螺旋體病之預防與治療 ……………………………… 25
第三章 材料與方法 ………………………………………………… 28
第一節 樣本採集區域及方法 ……………………………………… 28
3.1.1 樣本採集 ………………………………………………… 28
3.1.2 採樣方法 ………………………………………………… 28
第二節 台南市流浪犬鉤端螺旋體之血清學調查 ………………… 29
3.2 顯微凝集試驗 ……………………………………………… 29
3.2.1 操作步驟 ………………………………………………… 29
3.2.2 凝集判讀 ………………………………………………… 29
第三節 台南市流浪犬鉤端螺旋體之分子診斷調查………………… 30
3.3.1 聚合酶鏈鎖反應 ………………………………………… 30
3.3.1.1 血漿樣本之核酸萃取 ………………………………… 30
3.3.1.2 陽性對照菌株來源 …………………………………… 31
3.3.1.3 引子對之選用 ………………………………………… 31
3.3.1.4 反應之總體積 ………………………………………… 32
3.3.1.5 G1/G2引子對之PCR反應條件 ……………………… 32
3.3.1.6 DNA電泳分析 ………………………………………… 32
3.3.1.7 統計分析 ………………………………………………… 33
3.3.2 恆溫式圈環型核酸增幅法 ……………………………… 33
3.3.2.1 血漿樣本之核酸萃取 ………………………………… 33
3.3.2.2 陽性對照菌株來源 …………………………………… 33
3.3.2.3 引子對之選用 ………………………………………… 33
3.3.2.4 反應之總體積 ………………………………………… 34
3.3.2.5 反應條件 ……………………………………………… 34
3.3.2.6 DNA電泳分析 ……………………………………………35
第四章 結果 ………………………………………………………… 36
第一節 台南市流浪犬鉤端螺旋體之血清學調查結果 …………… 36
4.1.1 顯微凝集試驗之結果 ……………………………………… 36
4.1.1.1 血清抗體陽性率與抗體力價 ……………………………36
4.1.1.2 感染之血清型 ……………………………………………36
4.1.1.3 血清抗體陽性率與月份之關係 …………………………37
4.1.1.4 公/母犬血清抗體陽性比例 ……………………………… 38
4.1.1.5 抗體陽性犬隻之平均年齡 …………………………………38
4.1.1.6 抗體陽性犬隻之平均體重 …………………………………38
4.1.1.7 絕育/未絕育犬之血清抗體陽性比例 …………………… 38
第二節 台南市流浪犬鉤端螺旋體之分子診斷調查結果 ………… 39
4.2.1 聚合酶鏈鎖反應之结果 …………………………………… 39
4.2.2 恆溫式圈環型核酸增幅法之結果 ………………………… 39
第五章 討論 ……………………………………………………… 40
第六章 結論 ………………………………………………………… 46
參考文獻 ………………………………………………………………… 48
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