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研究生:吳佳瑞
研究生(外文):Chia-Ruei Wu
論文名稱:養殖海鱺感染巴斯德桿菌之病理學與細胞凋亡研究
論文名稱(外文):Studies on the Pathology and Apoptosis of Cultured cobia ( Rachycentron canadum) infected by Photobacterium damselae subsp. piscicida
指導教授:劉秉忠
指導教授(外文):Liu Ping-Chung
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:108
中文關鍵詞:海鱺巴斯德桿菌病理學細胞凋亡
外文關鍵詞:cobiaPhotobacterium damselae subsp. piscicidaPathologyApoptosis
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Photobacterium damselae subsp. piscicida被證實是多種魚類的致病菌,也是海鱺養殖中常見的病原菌之一。本論文主要探討Ph. damselae subsp. piscicida感染海鱺後對組織的影響以及是否能誘導海鱺細胞產生細胞凋亡 (Apoptosis),實驗菌株Ph. damselae subsp. piscicida(strain 9205)係分離自屏東地區已出現異狀的瀕死養殖海鱺。
野外自然被Ph. damselae subsp. piscicida感染的海鱺最常發現的症狀為體表出血及體色變深,而在實驗室以活菌攻擊後只觀察到體色變深並無發現出血的情形。在組織病理學 (Histopathology) 研究野外自然感染症與實驗室攻擊感染的病魚的組織發現,最易受影響的組織順序為後腎、脾臟、肝臟、頭腎以及心臟。後腎很容易發現肉芽腫病變(granuloma lesion),罹病的海鱺組織較常發現的病理變化還包括後腎臟的腎小管上皮細胞 (Renal epithelial cells) 壞死 (Necrosis) 及混濁腫脹 (Cloudy swelling ),造血細胞 (Hematopoeitic Cells) 也有局部性的壞死;在肝臟部份,大部分肝臟實質組織出現脂肪變化 (Fatty changes),肝臟細胞質出現空泡化及細胞質混濁的現象,肝細胞間之cell boundary模糊不清,可發現在肝細胞之細胞質或脂肪球中有不正常之嗜酸性物質的沉積;脾臟組織出現壞死 (Necrosis) 及炎症 (Inflammation) 的病理變化,也有液化壞死 (Liquefactive necrosis) 的病理變化出現;頭腎組織可發現有壞死及炎症反應。
本研究也探討Ph. damselae subsp. piscicida(strain 9205)之細胞外產物及兩種分離純化的磷脂質分解酵素對海鱺組織的影響。細胞外產物攻擊後的海鱺,其肝臟有出血、壞死及脂肪變性等病理變化,且在脂肪球內有嗜酸性物質沉積,脾臟的顆粒細胞增加,有炎症發生,紅髓萎縮及白髓壞死的病理變化。腹腔注射phospholipase A2 (58 KDa) 添加 LPS與phospholipase C (27 KDa) 添加 LPS後皆造成海鱺肝臟組織壞死,而壞死區以外的肝臟細胞實質組織出現脂肪變化 (Fatty changes),肝細胞的細胞質有混濁 (Cloudy) 的現象,細胞質不但呈現混濁且出現許多小囊胞或空胞(Vacuoles)。 phospholipase C (27 KDa)則會另外造成肝細胞嚴重出血。
在組織病理的觀察發現細胞核有異常的變化,有出現細胞凋亡的特徵,而以Ph. damselae subsp. piscicida(strain 9205)之細胞外產物進行誘導海鱺的巨噬細胞產生細胞凋亡的實驗中,經以TUNEL染色檢測發現細胞外產生能誘導海鱺產生細胞凋亡。
Photobacterium. damselae subsp. piscicida has been proven to be a pathogenic for various fish, and has also been identified as one of the commonly found pathogen for cultured cobia. This thesis mainly investigated the influence of Ph. damselae subsp. piscicida on cobia tissue and also investigated whether it could induce onset of Apoptosis or not.  The strain 9205 (Ph. damselae subsp. piscicida) isolated from moribund cobia in Pingtung were used in this experiment
The most common symptoms of fish infected by Ph. damselae subsp. piscicida naturally in the field were bleeding on the skin and deep color, but those challenged by bacteria in the lab are only deep color and no bleeding observed. From histopathological studies on the fish tissues infected by Ph. damselae subsp. piscicida naturally or challenged in the lab, the tissues severly affected in order were posterior-kidney, spleen, liver, head-kidney, and heart. Posterior-kidney were infected by the bacterium the occurrence of granuloma lesion. The common pathological changes in cobia tissue included necrosis and cloudy swelling of renal epithelial cells in poserior-kidney, necrosis of a part of hematopoeitic cells, fatty changes of parenchyma in liver, cytoplasm vacuoles and cloudy in liver, cell boundary blur among hepatocytes, and the accumulation of abnormal acidophilus matters in cytoplasm or fat globule in hepatocytes. There were pathological changes of necrosis and inflammation together with liquefactive necrosis in the spleen. In addition, necrosis and inflammation were also bound in head-kidney.
This experiment also investigated the influence of extracellular products (ECP) of Ph. damselae subsp. piscicida(strain 9205)and two types of purified phospholipase on cobia tissues. Pathological changes, such as bleeding, necrosis, and fatty changes, were observed in livers from cobia challenged by ECP. There were pathological changes observed like the accumulation of acidophilus matters in fat globule, increment of granular cells in spleen, inflammation, cataplasia of red pulp, and necrosis of white pulp. Both the injection of phospholipase A2 (58 KDa) (added LPS) and phospholipase C (27 KDa) (added LPS) into cobia intraperitoneally all caused necrosis of liver tissue, and there were fatty changes in parenchyma of hepatocytes. Away from necrosis sites, a cytoplasmic cloudines of hepatocytes were also observed. Except cloudiness, a lot of vesicles or vacuolesobserved in the cytoplasm. Furthermore, phospholipase C (27 KDa) would result in heavily bleeding in hepatocytes.
There were abnormal changes in nucleus in the histopathological observation a symptom of apoptosis. The ECP of Ph. damselae subsp. piscicida (strain 9205)could induce apoptosis of cobia as measured by TUNEL stain.
目 錄

● 謝 辭 ………………………………………………………… Ⅰ
● 中文摘要 ……………………………………………………… Ⅲ
● 英文摘要 ……………………………………………………… Ⅴ
● 目 錄 ………………………………………………………… Ⅶ
● 表目錄 ………………………………………………………… Ⅹ
● 圖目錄 ……………………………………………………… VII

● 第一章、前言 ………………………………………………… 1
● 第二章、文獻整理 …………………………………………… 4
● 第三章、材料與方法 ………………………………………… 17
3.1實驗動物馴養 …………………………………… 16
3.2實驗菌株 ………………………………………… 16
3.2.1菌株來源 ……………………………….. 16
3.2.2菌株的培養與保存 …………………….. 16
3.2.3菌株的確認 …………………………….. 17
3.2.4菌株強化 ……………………………….. 17
3.3細菌生理生化特性之鑑定分析 ………………… 17
3.3.1革蘭氏染色 …………………………….. 17
3.3.2細胞色素氧化酵素 …………………….. 18
3.3.3葡萄糖的利用 ………………………….. 18
3.3.4 MacConkey agar之生長能力 ………….. 18
3.3.5 NaCl之耐受性試驗 ……………………. 19
3.3.6抗生素敏感性試驗 …………………….. 19
3.3.7溶血能力測定 ………………………….. 19
3.3.8快速鑑定系統的鑑定 ………………….. 19
1. API 20E system ……………………….. 19
2. Biolog system …………………………. 20
3. MICROGEN GN-ID …………………... 20
4. BIONOR MONO-Pp rapid diagnostic
test ……………………………………..
20
3.4 Ph. damselae subsp. piscicida(9205)細胞外產物之基本特性分析 ……………………………...
21
3.4.1細胞外產物之製備 …………………….. 21
3.4.2細胞外產物之特性分析 ……………….. 21
1.蛋白質含量的測定 …………………… 22
2.蛋白質分解酵素的測定 ……………… 22
3.溶血活性測定 ………………………… 23
3.5 Ph. damselae subsp. piscicida(9205)對海鱺之毒性與組織病理試驗 …………………………...
22
3.5.1菌體對海鱺組織之毒性與組織病理試驗 22
3.5.2細胞外產物對海鱺之毒性與組織病理
試驗 ……………………………………..
23
3.6 TUNEL assay ……………………………………. 23
3.7 Ph. damselae subsp. piscicida(9205)細胞外產
物中磷脂質分解酵素之分離純化 ……………...
24
3.7.1細胞外產物中磷脂質分解酵素之部份分
離純化 …………………………………..
24
3.8 ECP中的純化物質對海鱺之毒性與組織病理試
驗………………………………………….............
25
3.8.1脂多醣體Lipopolysaccharide(LPS)之
萃 ………………………………….........
25
3.8.2 Ph. damselae subsp. Piscicid(9205)細
胞外產物中蛋白質分解酵素之純化 …..
25
3.8.3磷脂質分解酵素、脂多醣體及蛋白質分
解酵素對海鱺組織之毒性與組織病理試 ..............................................................

26
3.9海鱺脾臟初代細胞的培養……………………… 26
● 第四章、結果 ………………………………………………… 27
4.1細菌生理生化特性之鑑定分析 ………………… 27
4.2野外自然感染巴斯德菌症之組織病理學 ……… 27
4.3 Ph. damselae subsp. piscicida(strain 9205)對海鱺之毒性與組織病理試驗 ………………….......
28
4.4細胞外產物對海鱺之毒性與組織病理試驗 …… 30
4.5磷脂質分解酵素及蛋白質分解酵素對海鱺組織
之毒性與組織病理試驗 ………………………...
30
4.6活體內誘導海鱺細胞凋亡試驗 ………………… 31
4.7細胞外產物對海鱺脾臟初代細胞的影響……….. 32
● 第五章、討論 ………………………………………………… 33
● 參考文獻 ……………………………………………………… 41
● 表 ……………………………………………………………… 54
● 圖 ……………………………………………………………… 65
● 附錄 …………………………………………………………… 89
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