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研究生:黃心蓀
論文名稱:台灣地區鴿糞中之沙氏桿菌分離調查與類源分析
論文名稱(外文):Prevalece and phylogenetic analysis of Salmonella in domestic pigeons in Taiwan
指導教授:林子恩林子恩引用關係
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:91
語文別:中文
論文頁數:73
中文關鍵詞:沙氏桿菌鴿子定序
外文關鍵詞:Salmonellapigeonsequence
相關次數:
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沙氏桿菌症在幼鴿與成鴿為主要的細菌性疾病,以副傷寒(paratyphoid)為主。本實驗自1999年八月到2001年八月針對台灣北中南各地之私人鴿舍、鴿店調查飼養鴿隻的沙氏桿菌污染情形。共採集泄殖腔拭子與新鮮糞材433個樣本,使用傳統細菌分離流程,Tetrathionate broth(TTI)增菌42℃ 24- 48小時後,分別釣至XLD 與MacConkey agar培養37℃ 24小時,選擇無色的菌落進行GFB-12E電腦密碼系統鑑定,最後再以Biolog系統確認,共確立12株沙氏桿菌,其中11株為Salmonella ser. Typhimurium,分離率為2.8 %。本實驗另由剖檢病鴿之肝臟壞死點、關節病變分離確立7株。使用菌體抗血清鑑定,結果上述由調查分離所得11株為B組,1株為C2組。由剖檢分離得到的7株,菌體抗血清均為B組。所得19株鴿子沙氏桿菌分離株對nitrofurantoin、ampicillin、doxycycline有較高抗藥性,而對ofloxacin、norfloxacin、chloramphenicol有較佳感受性。實驗中應用Cohen等人發表之沙氏桿菌屬別專一性引子(hto gene)與Lin等人發表之鼠傷寒沙氏桿菌專一性引子(mdh gene)進行聚合酵素連鎖反應測試,19株分離株之結果均呈陽性,最後以16S rDNA核酸序列,分析出鴿之沙氏桿菌分離株之類源關係樹狀圖,分離株之間相似性高,比對結果均為Salmonella 菌屬,僅能與Citrobacter標準株區隔成不同群體。
Salmonellosis is an enteric bacterial infection in both young and adult pigeons. Paratyphoid mainly infects pigeons, characterized as subclinical infection in adult pigeons and often an acute fatal disease in young squabs. Prevalence was investigated by fecal sampling from lofts around whole Taiwan during August 1999 to August 2001. The fecal samples were firstly enriched for 24-48 hours in tetrathionate broth, then transfered to XLD and MacConkey agars. The colorless colonies were then identified as Salmonella spp. by GFB-12E and Biolog systems. Among four hundred and thirty-three samples, 12(2.8%)were positive for Salmonella spp.. Seven strains were also identified as Salmonella spp. from necrotic liver and articular cavity of necropsied pigeons. Eleven of twelve strains of fecal samples reacted with antiserum group B, and one isolate was positive with antiserum group C2. All of the seven strains from necropsied pigeons were positive with antiserum group B. The isolates were resistant to nitrofurantoin, ampicillin, and doxycyline, but sensitive to ofloxacin, norfloxacin, and chloramphenicol.
Salmonella genus-specific(hto gene)and Typhimurium-specific (mdh gene)polymerse chain reaction(PCR)primers were used to develope a rapid diagnostic mehod for Salmonella in this study. All isolates showed positive by specific PCR. The pigeon isolates shows highly similarity to Salmonella . 16S rDNA analysis was unable to separate pigeon’s isolates to subspecies level. The phylogenetic relationships allow to discriminate the isolates and Citrobacter only.
第一章 緒言 1
第二章 文獻探討 2
第一節 沙氏桿菌簡介 2
1-1 沙氏桿菌型態與生化特性 2
1-2 血清型分類 4
第二節 沙氏桿菌的分離與鑑定 7
第三節 快速偵測沙氏桿菌 12
3-1 改進傳統分離的技術 12
3-2 非分離性質的快速偵測技術 14
第四節 聚合酵素連鎖反應應用於沙氏桿菌快速鑑
定與核糖體16S核糖核酸基因鑑定原理 17
4-1 聚合酵素連鎖反應應用於沙氏桿菌快速鑑
定 17
4-2 核糖體16S核糖核酸基因鑑定原理 18
第五節 鴿子的沙氏桿菌症 19
5-1 症狀、傳播方式與預防控制 20
5-2 區別診斷 22
第六節 流行病學 23
第三章 材料與方法 26
第一節 鴿糞中之沙氏桿菌調查材料 26
1-1 材料 26
1-2 方法 28
第二節 沙氏桿菌分離株藥物敏感性試驗 30
第三節 特異性聚合酵素連鎖反應應用在鴿糞分離
之沙氏桿菌分離株 31
3-1 細菌去氧核糖核酸DNA之萃取 31
3-2 使用專一性引子確認分離株 31
3-3 聚合酵素連鎖反應之條件設定與產物確認 32
第四節 鴿糞中之沙氏桿菌分離株16S rDNA核酸定
序分析 34
4-1 16S rDNA基因增幅 34
4-2 鴿糞中之沙氏桿菌16S rDNA基因定序 35
第四章 結果 39
第一節 鴿糞中沙氏桿菌細菌分離結果 39
1-1 分離鴿糞中之沙氏桿菌 39
1-2 分離株抗生素敏感性試驗結果 44
第二節 使用專一性引子確認分離菌株 47
第三節 沙氏桿菌分離株之16S rDNA核酸序列分析 52
3-1 16S rDNA序列比對 52
3-2 建立菌種演化親源關係 53
第五章 討論 59
參考文獻 66
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