張本恒臺灣大學:獸醫學研究所薛人華Hsueh, Jen-HuaJen-HuaHsueh2010-05-042018-07-092010-05-042018-07-092009U0001-0308200923165900http://ntur.lib.ntu.edu.tw//handle/246246/178927本研究首次在台灣從鱘魚 (Acipenser schrencki) 分離出Staphylococcus lugdunensis，自動物園飼養之鱘魚分離Staphylococcus lugdunensis 菌株進行病原性試驗，以人工皮膚割傷後，菌液浸泡幼魚，以8.1×108CFU/mL、1.95×109CFU/mL及1.95×109CFU/mL細菌濃度進行三組重複試驗，第14天累計死亡率分別為83.3％、66.6％及33.3％，顯示該菌對鱘魚之致病性較為慢性，死亡魚隻浮游水表面或沉於底部，魚體呈現彎曲狀，且體表色變淡並附著黏液，於鰓蓋底部、體腹表面及擦傷部位可見明顯潮紅及出血現象。分離株於形態學、生長特性和生理生化特性方面類似標準菌株S. lugdunesis。對大多數抗生素對具有感受性。以葡萄球菌16S rDNA引子，增幅出1320bp片段，與S. lugdunensis有99％相似度。再根據S. lugdunesis之外毒素hemolysin (slush) 基因設計，可增幅出1011bp之PCR片段，與S. lugdunensis hemolysin gene有99％相似度。本研究顯示出鱘魚引起出血敗血病變與S. lugdunensis之病原性具有關聯性，也證實出為台灣首度鱘魚感染S. lugdunensis病例。This study described the first isolation of Staphylococcus lugdunensis from sturgeon, Acipenser schrencki, in Taiwan. The pathogenicity experiments were performed by immersion challenge in juvenile sturgeons. Fish were received 8.1×108CFU/mL, 1.95×109CFU/mL and 1.95×109CFU/mL of bascterial suspension and triple duplicate, and the cumulative mortality was 83%, 66.6％and 33.3％, respectively. It revealed chronic pathogenicity of S. lugdunensis for sturgeons. During the necropsy, the body of dead fish appeared curved shape, discorolation and mucus secretion. The congestion and hemorrhage were present on the base of operculum, the surface of abdmenal skin, and the skin of scraping area. The growth character, morphology and biochemical properties of S. lugdunensis isolated from sturgeons were similar to the reference strain of S. lugdunensis. The 16S rDNA sequence of 1320bp amplicon from isolates has shown 99% identify with S. lugdunensis. The S. lugdunensis hemolysin (SLUSH) sequence of 1011bp amplicon also has shown 99% identify with S. lugdunensis hemolysin gene. The results of this study revealed pathologenicitic and hemorrhagic of S. lugdunensis in sturgeons. This was the first report of S. lugdunensis in sturgeon cases.中文摘要---------------------------------------------- Ⅰ文摘------------------------------------------------ Ⅱ錄-------------------------------------------------- Ⅲ目錄------------------------------------------------ Ⅶ目錄------------------------------------------------ Ⅷ一章 前言------------------------------------------- 1二章 文獻回顧--------------------------------------- 3一節 Staphylococcus lugdunensis之簡介--------------- 3.1命名----------------------------------------------- 3.2細菌特性------------------------------------------- 3.3疾病與致病特徵------------------------------------- 4二節 葡萄球菌對魚類養殖的危害----------------------- 6.1 歷史回顧------------------------------------------ 6.2 臨床症狀及組織病理的變化-------------------------- 7.3 流行病學與防治------------------------------------ 8三節 葡萄球菌分類、分離及鑑定----------------------- 9.1 葡萄球菌之分類特徵-------------------------------- 9.2 葡萄球菌之分離及培養特性-------------------------- 9.3 葡萄球菌之鑑定方法------------------------------- 10四節 葡萄球菌之溶血毒素介紹------------------------ 11.1 外毒素分類--------------------------------------- 11.2 溶血毒素種類------------------------------------- 11五節 鱘魚生物特性之簡介---------------------------- 14三章 實驗材料與方法------------------------------ 16一節 實驗設計及流程圖------------------------------ 16二節 實驗材料-------------------------------------- 17.1 細菌分離株來源----------------------------------- 17.2 葡萄球菌標準株來源------------------------------- 17三節 實驗方法-------------------------------------- 17.1 菌株之分離與鑑定--------------------------------- 17.1.1 細菌分離及培養--------------------------------- 17.1.2 細菌生理生化特性鑑定試驗----------------------- 17.1.2.1 觸酶試驗 (Catalase test)--------------------- 17.1.2.2 氧化酶試驗 (Oxidase test)-------------------- 18.1.2.3 亞硝酸還原試驗(Nitrate reduction test)------- 18.1.2.4 溶血試驗------------------------------------- 18.1.2.5 運動性試驗 (Motility test)------------------- 18.1.2.6 膠質液化反應試驗(Gelatin liquification)------ 18.1.2.7 Lysine Decarboxylase test-------------------- 19.1.2.8 檸檬酸鹽利用試驗 (Simmon citrate test)------- 19.1.2.9 生長溫度試驗--------------------------------- 19.1.2.10 鹽度耐受性試驗------------------------------ 19.1.2.11 pH耐受性試驗-------------------------------- 19.1.2.12 膽汁生長試驗-------------------------------- 19.1.2.13 API staph system生化試驗-------------------- 19.1.2.14 API50醣類試驗------------------------------- 20.1.2.15 藥物敏感性試驗------------------------------ 20.2 16S rDAN鑑定分析--------------------------------- 21.2.1 菌體檢驗--------------------------------------- 21.2.2 DNA萃取與製備---------------------------------- 21.2.3 引子設計--------------------------------------- 21.2.4 16S rDAN聚合酶連鎖反應 (polymerase chain reaction ; PCR)------------------------------------------- 22.2.5 PCR產物之電泳分析------------------------------ 22.2.6 PCR產物純化、定序及比對分析-------------------- 22.3 人工接種試驗------------------------------------- 23.3.1 實驗動物--------------------------------------- 23.3.2 細菌濃度配製----------------------------------- 23.3.3 菌株懸浮液動物接種試驗------------------------- 23.3.4 死亡率觀察------------------------------------- 24.3.5 病理學檢查------------------------------------- 24.3.5.1 外觀及肉眼解剖病變檢查----------------------- 24.3.5.2 組織病理學檢查------------------------------- 24.4 溶血基因序列 (Hemolysin gene)分析---------------- 24.4.1 菌體檢驗--------------------------------------- 24.4.2 DNA萃取與製備---------------------------------- 25.4.3 S. lugdunensis synergistic hemolysin gene 引 子之設計--------------------------------------- 25.4.4 Hemolysin gene聚合酶連鎖反應 (polymerase chain reaction ; PCR)-------------------------- 25.4.4 PCR產物之電泳分析------------------------------ 26.4.5 PCR產物純化、定序及比對分析-------------------- 26四章 結果------------------------------------------ 27一節 細菌分離與鑑定-------------------------------- 27.1 形態學檢查--------------------------------------- 27.2 生理生化特性鑑定試驗----------------------------- 27.3 API staph system生化試驗------------------------- 27.4 API50醣類試驗------------------------------------ 28.5 藥物敏感性試驗----------------------------------- 29.6 16S rDANgene鑑定分析----------------------------- 29二節 人工接種試驗---------------------------------- 30.1 動物接種試驗------------------------------------- 30.2 病理學檢查--------------------------------------- 30三節 溶血基因序列(Hemolysin gene, SLUSH)分析------- 31五章 討論------------------------------------------ 32考文獻 -------------------------------------------- 40application/pdf2320690 bytesapplication/pdfen-US鱘魚Staphylococcus lugdunensis葡萄球菌出血性敗血症SturgeonHemorragic septicema[SDGs]SDG11thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/178927/1/ntu-98-R94629016-1.pdf