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研究生:許嘉芳
研究生(外文):Jia-Fang Hsu
論文名稱:台灣不同動物間產氣莢膜梭菌盛行率及流行病學之研究
論文名稱(外文):Study of the Prevalence and Epidemiology of Clostridium perfringens Isolated from Different Animals in Taiwan
指導教授:陳德勛
指導教授(外文):Ter-Hsin Chen
口試委員:郭致榮陳志銘
口試日期:2017-06-30
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫病理生物學研究所
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:2017
畢業學年度:105
語文別:中文
論文頁數:47
中文關鍵詞:產氣莢膜梭菌毒素分型Vero細胞藥物敏感性試驗
外文關鍵詞:Clostridium perfringenstoxinotypesvero cellantimicrobial susceptibility
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產氣莢膜梭菌 (Clostridium perfringens, C. perfringens) 是一種梭菌屬厭氧性革蘭氏陽性菌,此菌廣泛的分布於環境中,在緊迫狀態時會形成芽孢。產氣莢膜梭菌生長快速且分泌許多毒素,依照其產生的毒素可分成A到E五種毒素型,且所產生的毒素在人類和各種動物中會引發腸胃道感染等不同疾病。此外,產氣莢膜梭菌也會造成食物中毒,大多是由A型菌株產生的腸毒素 (Clostridium perfringens enterotoxin, CPE) 所引起。
本研究旨在探討各物種間產氣莢膜梭菌之流行病學、毒力分型、其他毒力因子檢測及Vero細胞毒性試驗,於2015到2017間總共收集1189個糞便檢體,物種來源包含醫院健檢門診民眾、住院下痢病患、伴侶動物 (犬、貓)、經濟動物 (雞、豬) 及大動物 (牛、馬)。結果共分離到419株產氣莢膜梭菌分離株,整體分離率為35%。大部分皆為毒素型type A (n = 414),其中檢測到4株帶有cpe基因,235株帶有β2基因,一株帶有netB基因。此外也有2株菌為毒素型type E分別來自健康人及犬,牛和馬來源的3個分離株檢測不到α、β、β2、ι、ε、cpe、netB及tpeL等毒素基因。另外,根據Vero細胞毒性試驗結果顯示,帶有cpe及部分不帶有cpe的分離株皆可造成Vero細胞很強的細胞毒性,推測部分分離株可能帶有其他致病因子而導致Vero細胞死亡。根據藥物敏感性試驗結果顯示,檢測結果所有分離株對vancomycin具有敏感性 (≤2 μg/ml)。在ampicillin、metronidazole、cefotaxime具有良好的敏感性,而約有25 %分離株對clindamycin具有抗藥性,此結果與獸醫用藥或是飼料中添加抗生素是否有關仍需進一部探討。
Clostridium perfringens is a gram-positive anaerobe which is able to form spore. It is widespread in the environment such as soil, sewage, food and feces. It is not only the normal intestinal flora of humans and animals but also zoonosis. Permitting classification of C. perfringens isolates into five toxinotypes based on the production of four typing toxins; that is alpha (CPA), beta (CPB), epsilon (ETX) and iota (ITX).
The aim of this study was to investigate the prevalence, toxinotype, other toxins, Vero cell cytotoxicity and antimicrobial resistance of C. perfringens isolates from different species such as healthy humans, patients, dogs, cats, pigs, chickens, cows and horses in Taiwan.
We collected 1189 samples from different animals (including healthy humans, patients, dogs, cats, chickens, pigs, cows, and horses from Apr. 2015 to Apr. 2017. The C. perfringens isolation rate were 95/225 (42%) in healthy humans, 58/359 (16%) in patients, 52/65 (80%) in dogs, 26/31 (84%) in cats, 113/192 (59%) in pigs, 18/215 (8%) in chickens, 51/76 (67%) in cows, and 6/26 (23%) in horses. Most of these isolates were toxinotype type A and four of them had cpe gene. Two isolates were toxinotype type E and were isolated from human and dog. Three isolates from two cows and one horse were negative for the eight toxigenic genes.
The result of Vero cell cytotoxicity testing revealed that most of isolates did not cause Vero cell cytotoxicity. However, in some isolates, whether with or without cpe gene, could cause significant cell rounding. All isolates are susceptible to vancomycin. Most of isolates are susceptible to ampicillin、metronidazole、cefotaxime. About 25 % isolates are resistance to clindamycin.
致謝……………………………………………………………………………….……i
中文摘要………………………………………………………………………...….…ii
英文摘要……………………………………………………………………………...iii
目錄…………………………………………………………………………….…..…iv
表目次…………………………………………………………………………….….vii
圖目次……………………………………………………………………………….viii
第一章 前言 1
第二章 文獻探討 2
第一節 產氣莢膜梭菌簡介 2
一、 產氣莢膜梭菌背景資料 2
二、 人類產氣莢膜梭菌症 2
三、 動物產氣莢膜梭菌症 3
第二節 產氣莢膜梭菌的分離與鑑定 5
一、 細菌分離與培養 5
二、 生化特性 6
第三節 產氣莢膜梭菌之毒素分型與毒力因子 6
一、 毒素分型 6
二、 毒力因子機制 7
三、 腸毒素 8
四、 NetB、TpeL及cp-ile毒素 9
第四節 產氣莢膜梭菌的抗藥性 10
第五節 產氣莢膜梭菌流行病學與公共衛生的重要性 11
一、 人類及食源性疾病 11
二、 動物流行病學 12
三、 產氣莢膜梭菌的治療與預防 12
第三章 材料與方法 14
第一節 樣品來源及採樣方法 14
一、 糞便樣品採集 14
二、 人類檢體採集、保存與運送 14
三、 伴侶動物檢體採集、保存與運送 14
四、 經濟動物檢體採集、保存與運送 15
五、 大動物檢體採集、保存與運送 15
第二節 產氣莢膜梭菌之培養分離與鑑定 16
一、 菌株分離培養與保存 16
二、 DNA萃取 16
三、 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 17
四、 電泳分析 17
第三節 毒素分型與其他毒力因子鑑定 17
一、 毒素分型及毒力因子 17
二、 毒素分型多引子聚合酶連鎖反應 18
三、 電泳分析 18
第四節 細胞毒性試驗 19
一、 細胞培養 19
二、 毒素配置 20
三、 細胞毒性試驗 20
第五節 抗生素敏感性試驗 21
一、 藥品 21
二、 抗生素敏感性試驗方法 21
第六節 數據統計分析 22
第四章 實驗結果 23
第一節 產氣莢膜梭菌盛行率 23
第二節 毒素分型與其他毒力因子結果 23
第三節 細胞毒性試驗 24
第四節 抗生素敏感性試驗結果 25
第五章 討論 26
第一節 台灣與國外產氣莢膜梭菌分離率概況 26
第二節 毒素分型與其他毒力因子結果 26
第三節 藥物敏感性試驗結果 27
第六章 總結 29
第七章 參考文獻 30
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