臺灣博碩士論文加值系統

從疑似豬大腸桿菌感染症死亡之豬隻，經剖檢後，從病變部位做大腸桿菌之培養、分離和鑑定，其結果檢測 177頭疑似大腸桿菌感染病豬中有 35 頭為豬溶血性大腸桿菌 ( hemolytic E. coli；HEC )，檢測率為19.7 ％，再將此35株HEC培養後進行其核酸萃取，以大腸桿菌之忌熱性毒素 ( heat-labile enterotoxin；LT ) 及耐熱性毒素 ( heat-stable enterotoxin；ST1、ST2 ) 基因之特定引子對 ( primers ) 進行聚合酶鏈反應 ( polymerase chain reaction；PCR ) 檢測其基因，其增幅的DNA在含有溴化乙錠 ( ethidium bromide；EtBr ) 瓊膠中，進行電泳後，經紫外光的照射，可呈現出LT1 360 bp、 ST1 160 bp、ST2 423 bp、eaeA 241 bp的特異性反應帶，再將此35株HEC之核酸以multiplex PCR檢測其LT1、ST1和ST2之毒素基因，結果僅含有ST1毒素基因為11.43 ％ ( 4／35 )，同時含有LT1、ST2毒素基因為5.71 ％ ( 2／35 )，同時檢出LT1、ST1和ST2毒素基因為82.86 ％ ( 29／35 )。將分離出的HEC進行28種藥物感受性試驗，發現對大部份藥物均有抗藥性 ( Amoxicillin、Ampicillin、Chloramphenicol、Gentamycin、Erythromycin、Ceftiofur、Baytril、Furazolidone、Kanamycin、Cephalothin、Methicillin、Lincomycin、Neomycin、Nalidixic acid、Oxolinic acid、Penicillin G、Streptomycin、Flumequine、Vancomycin、Linco-spectin、Tiamulin + Tetra、Ciprofloxacin和Sarafloxacin HCI )，但Ceftiofur、Nitrofurantoin、Florfenicol、Tetracycline 和Sulfamethoxazole ＋ Trimethoprim、Trimethoprim 等具敏感性。從結果顯示HEC具有LT和ST毒素基因，是造成離乳豬、保育豬及肉豬前期之豬隻死亡原因，且產生抗藥性不易治療。因此，本研究結果可提供仔豬溶血性大腸桿菌症臨床預防治療上參考的依據。

177 Escherichia coli ( E. coli ) -suspected samples from 360 pigs that died from the E. coli suspected infections were cultured to isolate and detect this pathogens. Thirty five samples of 177 E. coli-suspected samples were identified to be hemolytic E. coli ( HEC ) infections. The positive rate was 19.7 ％ ( 35/177 ). HEC nucleic acid was extracted from pure culture colony and amplified by polymerase chain reaction ( PCR ) using the two specific primers that design for amplification of the heat-labile enterotoxin ( LT ) gene 360 bp or heat-stable enterotoxin ( ST ) gene ( ST1 160 bp, ST2 423 bp ), eaeA 241 bp, respectively. Results of the multiplex PCR analysis for detect the LT1, ST1 and ST2 toxic genes of HEC, the only had LT1 gene were 11.43 ％ ( 4／35 ), both had LT1 and ST2 genes were 5.71 ％ ( 2／35 ), and both had the LT1, ST1 and ST2 genes were 82.86 ％ ( 29／35 )。A drug sensitivity test was performed to determine the sensitivity of isolated HEC strains to 28 drugs. HEC had resistant to most commercial drugs ( Amoxicillin, Ampicillin, Chloramphenicol, Gentamycin, Erythromycin, Ceftiofur, Baytril, Furazolidone, Kanamycin, Cephalothin, Methicillin, Lincomycin, Neomycin, Nalidixic acid, Oxolinic acid, Penicillin G, Streptomycin, Flumequine, Vancomycin, Linco-spectin, Tiamulin + Tetra, Ciprofloxacin, Sarafloxacin HCI ), except for Ceftiofur, Nitrofurauntoin, Florfenicol, Tetracycline, Sulfamethoxazole ＋ Trimethoprim, Trimethoprim. The findings of drugs resistance were indicated that neonatal and weaned pigs with colibacillosis caused by HEC was usually difficult to be treated due to drug resistance. The results of the study may provide the information for clinical prevention and treatment of colibacillosis in piglets.

中文摘要....................................................................................................... Ⅰ
英文摘要....................................................................................................... Ⅲ
誌謝............................................................................................................... ˇ
目錄............................................................................................................... Ⅵ
圖表目錄....................................................................................................... Ⅶ
第1章 前言.................................................................................................... 1
第2章 文獻回顧............................................................................................ 3
2. 1 大腸桿菌之分類及疫情........................................................................ 3
2. 2 大腸桿菌之形態學及生化性狀……………………………................ 4
2. 3 大腸桿菌之分離及鑑定……………………………………………… 4
2. 4 大腸桿菌之臨床症狀及組織病理學變化…………………………… 5
2. 5 大腸桿菌之致病機轉………………………………………………… 5
2. 5. 1 Enteropathogenic E. coli ( EPEC ) ................................................ 5
2. 5. 2 Enteroaggregative E. coli ( EAggEC ) .......................................... 5
2. 5. 3 Enterotoxigenic E. coli ( ETEC )…............................................... 6
2. 5. 4 Enterohemolytic E. coli ( EHEC )………………......................... 7
2. 5. 5 Verotoxin........................................................................................ 7
2. 5. 6 Enteroinvasive E. coli ( EIEC )...................................................... 8
2. 5. 7 RTX 家族之介紹.......................................................................... 9
2. 5. 8 clyA 基因之介紹........................................................................... 9
2. 6 聚合酶鏈反應 ( polymerase chain reaction；PCR )........................ 10
第3章 材料與方法..................................................................................... 11
3 1 材料..................................................................................................... 11
3. 1. 1 藥品............................................................................................. 11
3. 1. 2 培養基........................................................................................ 12
3. 1. 3 培養液........................................................................................ 12
3. 1. 4 套組............................................................................................ 12
3. 1. 5 儀器............................................................................................. 12
3. 1. 6 器材............................................................................................ 14
3 2 樣本採取............................................................................................ 14
3 3 溶血性E. coli ( HEC ) 的分離及生化鑑定....................................... 15
3 4 聚合酶鏈反應 ( polymerase chain reaction；PCR ) 檢測毒素基因......................................................................................................... 16
3. 5 溶血性E. coli ( HEC ) 的保存.......................................................... 18
3. 6 多引子對聚合酶鏈反應 ( multiplex PCR ) ...................................... 18
3. 7 培養液中hemolytic E. coli ( HEC ) 最少含量之檢測...................... 20
3. 8 PCR引子對之特異性試驗................................................................. 20
3. 9 ST2毒素基因之定序.......................................................................... 21
3. 9. 1 PCR產物的純化 ( Purification ) ............................................... 21
3. 9. 2 定序............................................................................................. 22
3. 10 藥物敏感試驗................................................................................... 22
第4章 結果................................................................................................. 24
4. 1 每月溶血性E. coli ( HEC ) 病例數統計........................................... 24
4. 2 溶血性E. coli ( HEC ) 的分離........................................................... 26
4. 3 溶血性E. coli ( HEC ) 之生化鑑定................................................... 26
4. 4 聚合酶鏈反應 ( PCR ) 之檢測及定序結果..................................... 27
4. 4. 1 PCR引子對之敏感性試驗......................................................... 29
4. 4. 2 PCR引子對之特異性試驗......................................................... 31
4. 4. 3 多引子對聚合酶鏈反應 ( PCR ) 之檢測結果......................... 34
4. 4. 4 ST2毒素基因之定序.................................................................. 36
4. 5 藥物敏感性試驗................................................................................. 37
第5章 討論................................................................................................. 39
參考文獻........................................................................................................ 42
作者簡介........................................................................................................ 51

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