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研究生:王宏玲
研究生(外文):HUNG-LING WANG
論文名稱:虹鱒來源鏈球菌於魚體內散播途徑之研究
論文名稱(外文):Studies on the spreading routes of Streptococcus spp. in rainbow trout ( Oncorhynchus mykiss )
指導教授:陳媺玫
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:獸醫學研究所
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:2001
畢業學年度:89
語文別:中文
論文頁數:87
中文關鍵詞:鏈球菌虹鱒免疫組織化學染色
外文關鍵詞:streptococcirainbow trout
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為比較強、弱毒株鏈球菌侵入魚體內的分佈路徑與存活情況,及宿主巨噬細胞清除鏈球菌的能力,本實驗以虹鱒來源鏈球菌為模型,實驗分兩方向進行:(一)菌浴感染虹鱒,定期取樣分離細菌及進行免疫組織化學染色,以觀察鏈球菌侵入魚體路徑及其感染後在臟器及組織中分佈的情形和最終存在的位置(二)以虹鱒的巨噬細胞吞噬鏈球菌,觀察鏈球菌是否能在巨噬細胞中存活並且繁殖。在感染虹鱒方面,結果顯示無論強、弱毒株在人工感染虹鱒後2小時首先在脾臟分離到鏈球菌,到18天仍可在脾臟分離到鏈球菌。臨床症狀則出現在感染後第三天,強毒株感染的虹鱒症狀比弱毒株感染的嚴重。剖檢下顯示感染的虹鱒有脾臟、腎臟的腫大及胃腸道積水,有些魚隻可見肝臟充血或蒼白及清澈樣腹水。組織學病變發現弱毒株RT-2感染的虹鱒除了眼睛的病變、炎症細胞浸潤及腎小管玻璃小滴變性外,無明顯變化,而強毒株R0726感染的虹鱒則還有多處臟器組織都發現有炎症細胞的浸潤,肝細胞腫脹、腎小管玻璃小滴變性、皮膚肌肉出血及壞死、心包膜增厚等,眼睛周圍組織及眼窩處可見大量細胞外的細菌,亦有許多吞噬細胞中含大量的細菌。免疫組織化學染色結果顯示強、弱毒株感染虹鱒由鰓或腸道進入魚體後先分佈到脾臟,然後到肝臟及腎臟,到第三天所有臟器都可看到細菌,即發展成全身性感染,強毒株感染的虹鱒在感染後第24天還可在脾臟及頭腎看到細菌,弱毒株感染的虹鱒在感染後第18天還可在脾臟看到細菌,但在感染後第24天體內以不見有鏈球菌,而在強毒株感染的虹鱒體內菌量遠比弱毒株感染的虹鱒多。以虹鱒的巨噬細胞吞噬鏈球菌實驗方面,結果顯示,強毒株除了在含抗體及補體之虹鱒血清的情況下巨噬細胞中活的菌數會隨時間減少,在其他情況都會有增加的趨勢,而弱毒株在含抗體及補體之虹鱒血清的情況下巨噬細胞中活的菌數亦會隨時間減少,但其他情況則是微減或不變。將含巨噬細胞、白血球及部分紅血球之細胞懸浮液吞噬鏈球菌,進行穿透式電子顯微鏡的觀察,感染後90分鐘可見巨噬細胞除了有許多小型液胞,還有大型的液胞產生,各實驗組都可見吞噬泡有死掉的細菌外型變不規則,內部變空。
In order to realize the spreading routes of streptococci in rainbow trout ( Oncorhynchus mykiss ), we infected high-virulent strain ( R0726 ) and low-virulent strain ( RT-2 ) streptococci into rainbow trout fry experimentally, then isolated and stained immunohiatochemically streptococci at different time. The results showed that 2h postinfection, the streptococci were isolated from spleen of R0726 and RT-2 infected fry firstly. And 72h postinfection, streptococci could be isolated from all organs. According to the result of immunohistochemical staining, the streptococci get into fry by route of gill or gastrointestinal tract and spread to spleen, then liver and kidney, finally it developed systemic infection Besides, streptococci still could be isolated from spleen at 18d postinfection. The clinical signs of R0726 and RT-2 infected fry appeared suddenly at 3d postinfection and most serious from 3d to 6d, but clinical signs of fry infected with R0726 were more serious than those of RT-2 infected fry. The immunohistochemical findings showed that spreading routes of R0726 and RT-2 were similar. However, we found that the amount of R0726 in fry were much more than RT-2. Phagocytosed low-virulent strain ( RT-2 ) were killed whereas intracellular high-virulent strain ( R0726 ) seemed to survive within phagosomes of isolated macrophages of rainbow trout in the absence of anti-streptococci antibody and complement. Opsonisation of the streptococci was found to greatly enhance the killing activity of the rainbow trout macrophages.
摘要...................................................I
英文摘要...............................................II
目錄...................................................III
表目錄.................................................V
圖目錄.................................................VI
第一章前言..........................................1
第二章 文獻探討
第一節鏈球菌
一.歷史背景......................................3
二.鏈球菌的分類..................................4
三.培養特性及分離鑑定方法........................6
(一)生化性狀試驗..................................6
(二)基因鑑定方法..................................7
第二節魚類鏈球菌感染症
一. 臨床症狀與剖檢病變...........................8
二. 組織病理變化.................................9
三. 傳播途徑.....................................12
四. 流行病學.....................................13
五. 治療與防治...................................14
第三節鏈球菌之致病因子
一. 種別性.......................................16
二. 吞噬細胞內之存活能力.........................17
三. 鏈球菌之表面抗原與莢膜.......................18
四. 鏈球菌之外毒素...............................19
第三章材料與方法
實驗流程...............................................22
第一節材料
一. 鏈球菌菌株...................................23
二. 實驗動物.....................................23
三. 細菌培養基...................................24
四. 緩衝溶液及試液之配方.........................24
第二節方法
一. 兔抗鏈球菌血清製備...........................30
二. 酵素連結免疫吸附.............................30
三. 細菌細胞壁抗原之抽取.........................31
四. 蛋白質電泳分析...............................32
五. 西方轉漬法...................................33
六. 實驗感染虹鱒.................................33
七. 鏈球菌之分離.................................34
八. 免疫組織化學染色.............................34
九. 凝集試驗.....................................36
十. 巨噬細胞的分離...............................36
十一 吞噬試驗與細胞內存活試驗..........................37
十二 穿透式電子顯微鏡..................................38
十三 統計..............................................38
第四章 結果.........................................39
第五章 討論.........................................66
參考文獻...............................................78
附錄...................................................87
表目錄
表一 鏈球菌菌株之特徵..................................48
表二 強毒株R0726感染虹鱒之細菌分離結果.................52
表三 弱毒株RT-2感染虹鱒之細菌分離結果..................52
表四 免疫組織化學染色的結果............................55
表五 巨噬細胞吞噬試驗與細胞內存活試驗的結果............62
表六 以R0726進行細胞內存活試驗結果之柱狀圖.............62
表七 以RT-2進行細胞內存活試驗結果之柱狀圖..............63
表八 巨噬細胞吞噬試驗與細胞內存活試驗第一次實驗之結果..63
表九 巨噬細胞吞噬試驗與細胞內存活試驗第二次實驗之結果..64
表十 巨噬細胞吞噬試驗與細胞內存活試驗第三次實驗之結果..64
圖目錄
圖一 以兔抗R0726血清進行西方轉漬法的結果...............48
圖二 以兔抗112601血清進行西方轉漬法的結果..............49
圖三 以兔抗ATCC19433血清進行西方轉漬法的結果...........49
圖四 以兔抗R0726血清進行西方轉漬法的結果...............49
圖五 虹鱒呈現體色變黑、凸眼及眼睛出血..................50
圖六 嚴重凸眼及眼睛出血之虹鱒..........................50
圖七 鰭基部及腹部有出血斑、肛門腫大、腹部擴張之虹鱒....51
圖八 感染虹鱒之剖檢病變................................51
圖九 眼睛組織病變圖....................................53
圖十 肝臟組織病變圖....................................53
圖十一 後腎間質組織病變圖..............................53
圖十二 腦膜組織病變圖..................................53
圖十三 後腸漿膜層增厚..................................54
圖十四 腎小管上皮細胞嚴重玻璃小滴變性及空泡化..........54
圖十五 肌肉纖維變性壞死................................54
圖十六 肝臟組織病變圖..................................54
圖十七 皮膚組織壞死及炎症細胞浸潤......................55
圖十八 心臟組織病變圖..................................55
圖十九 二級鰓絲,免疫組織化學染色......................56
圖二十 腸道內容物有許多鏈球菌,免疫組織化學染色........56
圖二十一 胃黏膜下層中可見鏈球菌,免疫組織化學染色......56
圖二十二 脾臟組織,免疫組織化學染色....................56
圖二十三 肝臟組織,免疫組織化學染色....................57
圖二十四 腎臟間質組織細胞,免疫組織化學染色............57
圖二十五 眼窩,免疫組織化學染色........................57
圖二十六 肝臟,免疫組織化學染色........................57
圖二十七 脾臟,免疫組織化學染色........................58
圖二十八 腎臟,免疫組織化學染色........................58
圖二十九 腎臟間質組織,免疫組織化學染色................58
圖三十 鰓,免疫組織化學染色............................58
圖三十一 腸道,免疫組織化學染色........................59
圖三十二 幽門盲囊,免疫組織化學染色....................59
圖三十三 眼睛組織,免疫組織化學染色....................59
圖三十四 腦部,免疫組織化學染色........................59
圖三十五 肌肉組織,免疫組織化學染色....................60
圖三十六 皮膚病變處,免疫組織化學染色..................60
圖三十七 心包膜,免疫組織化學染色......................60
圖三十八 頭腎的網狀內皮細胞及吞噬細胞,B&B染色.........60
圖三十九 眼睛周圍浸潤之吞噬細胞,B&B染色...............61
圖四十 眼睛周圍,B&B染色...............................61
圖四十一 脾臟,B&B染色.................................61
圖四十二 穿透式電子顯微鏡樣材之Toluidine blue染色......65
圖四十三 巨噬細胞,穿透式電子顯微鏡....................65
圖四十四 巨噬細胞,穿透式電子顯微鏡....................65
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