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研究生:陳峻鎰
研究生(外文):Chung-Yi Chen
論文名稱:鯉魚及錦鯉疱疹病毒流行病學、快速診斷方法及病毒基因之親緣關係分析
論文名稱(外文):Epidemiology, Rapid Diagnostic Methods and Phylogenetic
指導教授:陳石柱陳石柱引用關係
指導教授(外文):Shih-Chu Chen
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:獸醫學系所
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:2007
畢業學年度:96
語文別:中文
論文頁數:96
中文關鍵詞:錦鯉疱疹病毒鯉魚錦鯉
外文關鍵詞:Koi herpesvirusCarpKoi carp
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為瞭解錦鯉疱疹病毒 ( koi herpesvirus;KHV ) 對鯉魚之病害性,自2003年至2005年間,共收集臺南地區40件鯉魚 ( Cyprinus carpio carpio ) 及9件錦鯉 ( Cyprinus carpio koi ) 疑似感染KHV之病例,其中經PCR檢測確診為KHV病例共計37件,總陽性率為75.5%,包括鯉魚75% ( 30/40 )、錦鯉77.8% ( 7/9 )。經調查發現其臨床症狀呈現食慾不振、浮頭及游動遲緩。病程約2星期、各場間的累計死亡率介於70~100%,總平均累計死亡率約為82.3% ( 鯉魚場80.3% 及錦鯉場90.7% )。肉眼病變可見全身體表出現明顯的充出血、鰓絲蒼白及壞死。本研究所收集KHV病例之組織病變包括:鰓部鰓薄板上皮細胞增生、融合 ( 86.5% ) 及壞死 ( 35.1% ),並且可見嗜酸性核內包涵體,肝臟局部壞死灶 ( 10.8% )、脾臟局部壞死灶 ( 37.8% ),腎臟出血 ( 13.5% )、腎小管上皮細胞變性 ( 18.9% ) 及壞死 ( 13.5% ),心肌岑克氏壞死 ( 8.1% ) 等。將37件樣品分別以2組PCR引子對 ( KHV TK:410 bp、KHV 9/5:483 bp ) 增幅之特異性DNA片段,經定序比對結果其核酸序列完全相同,顯示臺南地區之錦鯉疱疹病毒症為同一來源。KHV 9/5核酸序列 ( 483 bp ) 與GenBank中的KHV日本株 ( accession number AP008984 ) 相似度100%,與以色列株 ( accession number DQ177346 ) 及美國株 ( accession number
DQ657948 ) 相似度皆為99%,顯示臺南地區之錦鯉疱疹病毒症感染與日本株關係最為密切。本研究調查發現錦鯉疱疹病毒症之好發月份在11月至翌年4月間 ( 溫度17.5~25.3℃,平均溫度20.9℃ ),而氣溫較高的5~10月 ( 平均溫度28.3℃ ) 不易發病。又試驗得知,恆溫式圈環形核酸增幅法 ( loop-mediated isothermal amplification;LAMP ) 檢測較PCR靈敏,為一可用於臨床初步篩檢KHV的快速診斷方法。
Koi herpesvirus ( KHV ) is an important viral disease in koi ( Cyprinus carpio koi ) and common carp ( Cyprinus carpio carpio ). From 2003 to 2005, 40 cases of common carp and 9 cases of koi carp farms were collected in Tainan. Those cases were diagnosed by PCR examination, 37 cases were identified as KHV infection, total positive rate of KHV infection in carp is 75.5% [ carp cases 75% ( 30/40 ) and koi cases 77.8% ( 7/9 ), respectively ]. Through investigating, the clinical signs include lethargic, anorexic, increased respiratory movements and moving slowly in diseased fish. The incubation period of KHV infection is about 2 weeks, and the cumulative mortality of fish infected with koi herpesvirus ( KHV ) were 70~100%, the total cumulative mortality was 82.3% ( carp farms 80.3% and koi farms 90.7% ). The gross finding were hyperemia and haemorrhage on the body, pale and necrosis in gill filaments. The histopathological lesions of total KHV cases of this study include: epithelial hyperplasia, fusion ( 86.5% ) and necrosis ( 35.1% ) in gill filaments, eosinophilic intranuclear inclusion body were also found; focal necrosis lesion in liver ( 10.8% ); focal necrosis lesion in spleen
( 37.8% ); haemorrhage in kidney ( 13.5% ); degeneration ( 18.9% ) and necrosis ( 13.5% ) in renal tubular cells; Zenker’s necrosis in heart muscles ( 8.1% ). Amplify specific DNA fragment of 37 samples by PCR ( KHV TK: 410 bp、KHV 9/5:483 bp ), alignment report present their nucleic acid sequences were identical, koi herpesvirus infection in koi and common carp in Tainan might be the same origins. In KHV 9/5 gene sequences, they exhibited identities of 100% with Japanese strain ( accession number AP008984 ), 99% with Israeli strain ( accession number DQ177346 ) and 99% with USA strain ( accession number DQ657948 ). The koi herpesvirus infection in Tainan were most closely relationship with Japanese strain. This research present koi herpesvirus infection month from November to April next year ( weather temperature 17.5~25.3℃, average temperature 20.9℃ ), and it is only seldom cases in May to October with higher temperature ( average temperature 28.3℃ ). Therefore, these results suggested that the LAMP assay might be more sensitive than PCR test. LAMP is a rapid diagnostic method for primary examined KHV in clinic.
目 錄
摘要......................................................Ⅰ
Abstract..................................................Ⅲ
誌謝......................................................Ⅴ
目錄......................................................Ⅵ
圖表目錄...................................................Ⅸ
第1章 前言................................................1
第2章 文獻回顧.............................................4
2.1 疱疹病毒科 ( Herpesviridae ) 的特性...................4
2.2 鯉科疱疹病毒 ( cyprinid herpesvirus )................5
2.3 錦鯉疱疹病毒症簡介....................................5
2.3.1 病因................................................5
2.3.2 疫學................................................6
2.3.3 宿主範圍.............................................7
2.3.4 臨床症狀.............................................7
2.3.5 病毒在魚組織的分布....................................8
2.3.6 病毒的傳播方式.......................................8
2.3.7 溫度對本病發展過程的影響...............................9
2.4 診斷方法............................................10
2.4.1 組織病理學檢查......................................10
2.4.2 聚合酶連鎖反應 ( polymerase chain reaction;PCR )...10
2.4.3 恆溫式圈環形核酸增幅法 ( loop-mediated isothermal
amplification;LAMP ) 簡介與應用....................11
2.4.4 KHV ( CyHV-3 ) 病毒分離.............................13
2.4.5 電子顯微鏡觀察......................................14
2.5 防治方法............................................14
2.6 區別診斷............................................15
第3章 材料與方法..........................................17
3.1 病例收集............................................17
3.2 臨床病例之診斷檢查...................................17
3.2.1 外觀及解剖病變觀察...................................17
3.2.2 病材採集及保存......................................17
3.3 組織病理學之觀察...................................17
3.4 電子顯微鏡之觀察...................................18
3.4.1 病變組織之電子顯微鏡觀察............................18
3.5 病變組織之核酸萃取.................................18
3.6 核酸濃度之定量.....................................19
3.7 聚合酶鏈鎖反應 ( PCR ).............................19
3.7.1 引子對之選用.......................................19
3.7.2 聚合酶鏈鎖反應方法及條件.............................20
3.7.3 核酸之電泳分析.....................................21
3.7.4 PCR產物純化、定序與分析.............................21
3.8 KHV基因選殖 ( clone )..............................21
3.8.1 PCR反應產物純化....................................21
3.8.2 接合試驗...........................................22
3.8.3 轉形試驗...........................................22
3.8.4 純化質體...........................................22
3.8.5 定序分析...........................................23
3.9 恆溫式圈環形核酸增幅法 ( LAMP ) ....................23
3.9.1 LAMP引子對之選用...................................23
3.9.2 LAMP反應方法及條件.................................23
3.9.3 核酸之電泳分析.....................................24
第4章 結果..............................................25
4.1 病例收集及臨床病例之診斷檢查.........................25
4.2 組織病理學變化.....................................35
4.3 電子顯微鏡之觀察...................................42
4.4 聚合酶鏈鎖反應 ( PCR ) ............................43
4.5 核酸序列的比對分析..................................47
4.5.1 KHV TK gene.......................................47
4.5.2 KHV 9/5 gene......................................52
4.6 KHV基因選殖 ( clone ) .............................64
4.7 恆溫式圈環形核酸增幅法 ( LAMP ) ....................64
4.7.1 KHV LAMP和KHV PCR ( 9/5、TK ) 之靈敏度比較.........65
4.8 臺南地區鯉魚發生KHV與氣溫關係統計結果................67
笫5章 討論.............................................72
參考文獻..................................................78
附錄......................................................86
作者簡介...................................................96
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