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研究生:何偉瑮
研究生(外文):Wei-Li Ho
論文名稱:Smonellacholeraesuis細胞毒素之產生與限鐵環境對其致病力之影響
論文名稱(外文):Production of cytotoxin by Salmonella choleraesuis and effects of iron-deficiency on the virulence of Salmonella
指導教授:周正俊周正俊引用關係
指導教授(外文):Cheng-Chun Chou
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:167
中文關鍵詞:沙門氏桿菌豬傷寒沙門氏桿菌細胞毒素培養條件致病性
外文關鍵詞:SalmonellaSalmonella choleraesuiscytotoxinculture conditionsironvirulence
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Salmonella choleraesuis細胞毒素之產生與限鐵環境對其致病力之影響
摘 要
本研究乃在探討:(1)沙門氏桿菌在tryptic soy broth (TSB)及乳品中細胞毒素之產生,(2)培養條件對菌體生長及細胞毒素產生之影響,(3)限鐵對菌體細胞毒素產生之影響,(4)缺鐵對S. choleraesuis在老鼠體中致病力之影響。
結果顯示,所測試之12株沙門氏桿菌在TSB及乳品中均生長良好,但僅在TSB中測得細胞毒素之產生;其毒素產量因不同之沙門氏桿菌而異,其中以S. choleraesuis之毒素產量最高,其對Vero 細胞之CD50 值約達 586 CD50/mg lysate protein。在3% 脫脂奶粉中添加tryptone或soytone後,即可測得S. choleraesuis細胞毒素之產生;若再加入葡萄糖則可進一步提高細胞毒素之產生。
利用S. choleraesuis進一步探討,發現細胞毒素產生所需之最適溫度、培養液起始pH值及振盪速率分別為37℃、 pH 6.0~8.0以及100 rpm。在所測試的八種氮源中,以tryptone作為氮源時,S. choleraesuis所產生之細胞毒素量最高。而在九種碳源中,分別以葡萄糖、果糖、半乳糖、山梨醇或甘露醇為碳源時,可獲得較高之毒素產生量。當培養液中食鹽含量達4.0%以上時,即無法測得細胞毒素。 S. choleraesuis培養於含有2% tryptone、0.5% 食鹽、0.25% K2HPO4 以及 0.25% 的碳源之培養基中,於最適條件下經14小時之培養,可得最高量之細胞毒素。在上述條件下培養之S. choleraesuis 的菌體溶出物之Vero 細胞之CD50 達589~759 CD50/mg lysate protein。
Hemoglobin、ferritin 、transferrin、lactoferrin 之添加可激發沙門氏桿菌在限鐵培養基中之生長,惟激發之效果因含鐵化合物之種類、 劑量及菌株之不同而有所差異。在TSB 中添加2,2’-dipyridyl會使S. choleraesuis生長之遲滯期延長,生長速率與定常期最高菌數下降。但EDDA之添加僅造成生長遲滯期之延長。在缺鐵培養基中S. choleraesuis細胞毒素之產量明顯增加。缺鐵的馴化處理可加快S. choleraesuis在老鼠血清中的增殖速率。
在探討缺鐵馴化處理對S. choleraesuis在老鼠體內致病力的影響時,乃將經缺鐵培養基馴化之S. choleraesuis感染老鼠,而以培養於TSB中之S. choleraesuis為對照組。結果發現,在感染24小時後,兩組老鼠之脾臟、腎臟、血液及肝臟中之菌體數量均顯著升高;但對照組菌體的增加幅度高於感染經缺鐵馴化者。此外,老鼠的營養性缺鐵狀態會減弱S. choleraesuis在其體中之致死能力。
Production of cytotoxin by Salmonella choleraesuis and effects of iron-deficiency on the virulence of Salmonella
ABSTRACT
In this study, experiments were conducted to investigate (1) the cytotoxin production by various Salmonella spp. in tryptic soy broth (TSB) and milk products, (2) the growth and cytotoxin production under various culture conditions, (3) the effects of iron-limitation condition on the growth and cytotoxin production, and (4) the effects of iron—deficiency on the virule
nce of S. choleraesuis in mice.
Results revealed that all the twelve Salmonella spp. tested grew well in TSB and the all milk products. However, none of cytotoxin production was noted except in TSB. Amounts of cytotoxin produced varied with strains of Salmonella tested. Among the organisms tested, S. choleraesuis produced the highest amount of cytotoxin. The Vero cell CD50 of S. choleraesuis lysate of cells was a titer of 586 per mg of lysate protein. Addition of tryptone or soytone in the 3% skim milk resulted in the cytotoxin production by S. choleraesuis. Supplementation of glucose in this tryptone-added skim milk further increased the production of cytotoxin by test organism.
Further studies with S. choleraesuis showed that the optimal temperature, initial medium pH and shaking speed for cytotoxin production were at 37℃, pH6.0~8.0, and 100rpm, respectively. Tryptone was the best of the eight nitrogen sources tested for toxin production by S. choleraesuis. While, among nine carbon sources tested, S. choleraesuis produced a higher amount of cytotoxin in media containing glucose, fructose, galactose, sorbitol or mannitol as the carbon source. No toxin was detected when broth contained 4.0% or more sodium chloride. Culture of S. choleraesuis in medium consist of 2 % tryptone, 0.5% NaCl, 0.25% K2HPO4 and 0.25% of the best carbon source under the optimal conditions resulted in the highest cytotoxin production in 14 h. The Vero cell CD50 of S. choleraesuis lysate of cell grown under these optimal conditions was a titer of 589~759 per mg of lysate protein.
Addition of hemoglobin, ferritin, transferrin or lactoferrin was found to stimulate the growth of S. choleraesuis on the iron—limited medium. The stimulating effects varied with the kinds, dosages of these iron-containing compounds and strains of Salmonella. Addition of 2,2’-dipyridyl to TSB resulted in an extended lag period, a reduced growth rate and a less maximum population noted at stationary phase. While, addition of EDDA to TSB only resulted in an extended period of lag phase. A significant increased cytotoxin production was found when S. choleraesuis was cultured in iron-limited TSB. On the other hand, pre-adaptation at iron-limited condition increases the growth rate of S. choleraesuis in mouse serum.
To study the effect of iron-deficiency on the virulence of S. choleraesuis, mice were injected with test organisms which have been pre-cultured in iron-limited TSB, while mice injected with S. choleraesuis grown in TSB as the control group. After 24 h of infection, a marked increase in the bacterial population in spleen, kidney, liver, and blood of the infected mice was noted. However, the magnitude of population increase was more in the mice injected with control S. choleraesuis than in that injected with the iron-limitation adapted S. choleraesuis. Besides, an attenuation of the lethality of S. choleraesuis was noted on mice fed with iron-deficient diets.
封面
貳、材料與方法
一、實驗材料
1. 實驗菌株
2. 實驗細胞株
3. 牛乳製品
4. 藥品
5. 儀器設備
二、實驗方法
1. 菌株之保存
2. 實驗菌株接種源之製備
3. 菌數之測定
4. 菌株於TSB及不同牛乳製品中之生長
5. 培養物濾液之製備
6. 菌體溶出學之製備
7. 細胞株之培養
8. 細胞毒素活性測試
9. 細胞毒素CD50的定義
10. 蛋白質濃度測定
11. 統計分析
參、結果與討論
一、沙門氏桿菌與E.coli O157 : H7細胞毒素對Vero細胞之毒性表現
二、不同沙門氏桿菌在TSB中生長及有細胞毒素之產生
三、沙門氏桿菌於乳製品中之生長及細胞毒素之產生
四、細胞毒素在PBS與牛乳中對熱之敏感性
肆、結論
第三章 : 培養條件對Salmonella Choleraseuis細胞毒素產生之影響
摘要 : 中文
英文
壹、前言
貳、材料與方法
一、實驗材料
1. 實驗材料
2. 實驗細胞株
3. 藥品
4. 儀器設備
二、實驗方法
1. 實驗茵株接種源之製備
2. 培養基成分與環境因子對菌株生長及細胞毒素產生之影響
3. 細胞毒素之製備
4. 細胞毒素之分析
5. 菌數之測定
6. 蛋白質之測定
7. 統計分析
參、結果與討論
一、氮源對S.choleraesuis之細胞毒素產生的影響
二、碳源對S.choleraesuis之細胞毒素產生的影響
三、鹽濃度對S.choleraseuis之細胞毒素產生的影響
四、培養液起始pH值對S.choleraesuis之細胞毒素產生的影響
五、振盪速率對S.choleraesuis之細胞毒素產生的影響
六、培養溫度對S.choleraesuis之細胞毒素產生的影響
肆、結論
第四章 : 限鐵對沙門氏桿菌生長、細胞毒素產生以及抗血清力之影響
摘要 : 中文
英文
壹、前言
貳、材料與方法
一、實驗材料
1. 實驗菌株
2. 實驗細胞株
3. 實驗動物
4. 藥品
5. 儀器設備
二、實驗方法
1. 實驗菌株接種源之製備
2. 含鐵化合物的製備
3. 限鐵培養基中含化合物對沙門氏桿菌生長之激發
4. 鐵鉗合劑對菌體生長影響之測定
5. 鐵對S.choleraesuis之抗血清能力之測定
6. 細胞毒素之製備與毒性分析
7. 菌數之測定
8. 蛋白質之測定
9. 統計分析
參、結果與討論
一、限鐵環境下S.choleraesuis之生長
二、限鐵環境下S.choleraesuis細胞毒素之產生
三、不同含鐵化合物對沙門氏桿菌在限鐵培養基中生長之激發
四、缺鐵馴化對S.choleraesuis在血清中存活能力之影響
肆、結論
第五章 : 缺鐵對S.choleraesuis在老鼠體中致病力之影響
摘要 : 中文
英文
壹、前言
貳、材料與方法
一、實驗材料
1. 實驗菌株
2. 實驗動物
3. 藥品
4. 儀器設備
二、實驗方法
1. 實驗菌株接種源之製備
2. 動物實驗流程
3. 動物飼養
4. 老鼠飼料組成及製備
5. 沙門氏桿菌菌血症之誘發
6. 動物犧牲、組織處理及尿液收集
7. 血液中血紅素濃度分析
8. 肝鐵含量之測定
9. 尿液分析
參、結果與討論
一、缺鐵馴化處理對S.choleraesuis在老鼠體中生長之影響
二、缺鐵馴化處理對S.choleraesuis致病力之影響
三、老鼠體中鐵含量與受S.choleraesuis感染之敏感緎
四、老鼠體中鐵含量與尿液中一氧化氮含量之相關性
肆、結論
總結
第六章 ; 參考文獻
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