臺灣博碩士論文加值系統

Clostridium perfringens（Cp）在現代化家禽產業中造成壞死性腸炎。本次研究目的針對台灣地區上市前五週齡的健康白肉雞進行Cp的分離率、腸道病變、腸道菌量檢測、毒力分型及抗藥特性調查。從2012年六月至2013年二月，總共調查了98間禽舍共435件迴腸內容物樣品，樣品以厭氧環境下培養於tryptose sulfite cycloserine agar，再挑取黑色菌落劃於血液培養基及egg yolk agar上進行厭氧培養，典型的Cp會分別呈現溶血菌落及黃白色暈圈的菌落。本次採樣的Cp分離件數共43件（9.9%），分離出Cp的場次有29場（29.6%）。採樣的腸道肉眼病變與顯微病變則以分級作為評估，範圍從健康且無明顯病變的腸道為第0級到嚴重的瀰漫出血性壞死性腸炎為第4級，所有腸道樣品的顯微病變平均為2.08；樣品分離出產氣莢膜芽胞梭菌的腸道顯微病變平均為2.58。腸道Cp菌量評估採用SYBR green進行定量聚合酶連鎖反應，檢測範圍可從102 ~ 107 CFU/g (E= 99.6 % R2=0.998)，結果顯示本次腸道分離Cp的菌量範圍從6.85 x 102 ~ 1.61 x 107 CFU/g，平均菌量為3.9 x 106 CFU/g，統計分析顯示由定量聚合酶連鎖反應檢測迴腸的Cp菌量及腸道顯微病變分數具有正相關性（p<0.01, R2=0.58）。統計分析顯示從台灣北部、中部及南部的Cp分離率、場次分離率、腸道菌量、顯微病變均無顯著差異。分型檢測中所有的腸道樣品分離株皆含有alpha毒素，並被歸類於type A，且分離中無檢測出任何腸毒素、NetB毒素及TpeL毒素。將分離株進行最小抑菌濃度檢測，檢測藥品包含amoxicillin、bacitracin chlortetracycline、enrofloxacin、erythromycin、florfenicol及lincomycin，其MIC50分別為 ≤ 0.125、0.5、128、0.25、≥ 256、2、≥ 256 μg/mL。Amoxicillin、bacitracin及enrofloxacin 在本次研究中對Cp具有良好的活性。總結而言，在台灣的分離率較國外研究低，但場次分離率為29.6 %，顯示台灣的Cp廣泛的存在；Cp菌量與腸道顯為病變的正相關性也顯示降低禽舍雞隻以及環境中Cp的菌量應有助於雞隻腸道的健康。

Clostridium perfringens (Cp) causes avian necrotic enteritis in commercial poultry. The aim of this study was to detect the Cp positive rate, intestinal lesions, intestinal bacterial counts, toxintyping, and antimicrobial resistance in prior-marketing 5-week-old healthy broiler chickens in Taiwan. From June 2012 to February 2013, 435 samples of ileum contents from 98 broiler farms were analyzed. The samples were cultivated on tryptose sulfite cycloserine under anaerobic conditions. Black colonies were cultivated on blood agar plate and egg yolk agar, typical Cp colonies displayed hemolysis and zone of yellow to white precipitate forms respectively. The total number of positive samples was 43 (9.9%) and 29 (29.6%) flocks showed positive. For evaluated gross lesions and microscopic lesions, score range from 0 (healthy and no lesions) to 4 (severe, diffuse, hemorrhagic necrotic enteritis). Total intestinal samples mean score were 2.08, and Cp positive samples mean score were 2.58. Intestinal Cp counts were detected by SYBR green for Quantitative PCR, Cp detection range from 102 ~ 107 CFU in Cp positive intestinal samples. Result showed intestinal Cp counts range from 6.85 x 102 ~ 1.61 x 107 CFU/g, and average bacterial counts were 3.9 x 106 CFU/g in this study. Statistical analysis showed positive correlation (p<0.01, R2=0.58) between Cp counts in ileum contents determined by Quantitative PCR and intestinal microscopic lesions score. Statistical analysis showed no significant difference between northern, central and southern Taiwan to Cp positive rate, flock positive rate, bacterial counts and microscopic lesions score. All isolates containing alpha toxin were classified as type A possessing and no enterotoxin, NetB toxin and TpeL toxin were detected. Minimum inhibitory concentration (MIC) of amoxicillin, bacitracin, chlortetracycline, enrofloxacin, erythromycin, florfenical and lincomycin had variable MIC50 of ≤0.125, 0.5, 128, 0.25, ≥256, 2, ≥256 μg/mL, respectively. Cp isolates showed the great activity against amoxicillin, bacitracin and enrofloxacin in this study. In conclusion, the Cp positive rate in Taiwan was lower than other countries. Flock positive rate was 29.6 % suggested that Cp was existed widely in Taiwan. Positive correlation between Cp bacterial counts and microscopic lesions displayed that decreasing Cp counts in chicken and poultry environment may improve chicken intestinal health.

誌謝 I
中文摘要 II
ABSTRACTIV
目次 VI
表次 IX
圖次 X
第一章 緒言 1
第二章 文獻探討 3
第一節 梭菌 3
2.1.1 梭菌屬 3
2.1.2 產氣莢膜芽胞梭菌 3
2.1.3 產氣莢膜芽胞梭菌之分離與鑑定 4
2.1.4 人類產氣莢膜芽胞梭菌症 6
2.1.5 禽類產氣莢膜芽孢梭菌症 7
2.1.6 禽類產氣莢膜芽胞梭菌的治療與預防 8
第二節 產氣莢膜芽胞梭菌之分型與毒力因子 12
2.2.1 分型 12
2.2.2 毒力因子機制 12
2.2.3 NetB及TpeL毒素 14
2.2.4 VirR/VirS系統 15
第三節 產氣莢膜芽胞梭菌之致病機制 17
2.3.1 疾病風險因子 17
2.3.2 梭菌於腸道的黏附性及細菌素的分泌 18
2.3.3 致病機制 19
第四節 產氣莢膜芽胞梭菌之流行病學 20
2.4.1 人類及食品流行病學 20
2.4.2 家禽流行病學 20
第三章 材料與方法 24
第一節 樣本採集及分離鑑定 24
3.1.1 腸道樣品採樣 24
3.1.2 產氣莢膜芽胞梭菌之分離與鑑定 24
3.1.3 實驗材料 25
3.1.4 培養基之配置 25
第二節 腸道內容物即時定量聚合酶連鎖反應檢測 27
3.2.1 糞便的DNA萃取 27
3.2.2 建立標準曲線與樣品檢測 27
3.2.3 特異性檢測 28
第三節 肉眼病變與組織病理學檢查 29
3.3.1 肉眼病變 29
3.3.2 顯微病變 29
第四節 毒力分型與毒力因子鑑定 30
3.4.1 毒力分型及毒力因子 30
3.4.2 電泳分析 30
第五節 最小抑菌濃度檢測 31
3.5.1 藥品 31
3.5.2 最小抑菌濃度（Minimum Inhibitory concentrations；MIC） 31
第六節 統計與分析 32
第四章 結果 33
第一節 產氣莢膜芽胞梭菌分離率 33
第二節 腸內容物定量聚合酶連鎖反應檢測 35
第三節 肉眼病變與顯微病變 37
第四節 毒力分型與毒力因子檢測 39
第五節 最小抑菌濃度檢測 40
第五章 討論 41
第一節 台灣白肉雞產氣莢膜芽胞梭菌分離概況 41
第二節 定量聚合酶連鎖反應檢測 43
第三節 腸道病變 45
第四節 毒力分型與毒力因子檢測 47
第五節 最小抑菌濃度檢測比較及分析 49
第六章 結論 53
參考文獻 55

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