臺灣博碩士論文加值系統

第二型犬小病毒 (Canine parvovirus type 2, CPV-2) 為犬隻重要消化道及免疫系統疾病之一，造成犬隻嚴重出血性腸炎及消化道周邊淋巴組織壞死，並有報告指出與幼犬心肌炎有關。CPV-2首次被報告於1978年，隨後於全世界皆有相關的報告發生，在與不同宿主交互作用下，於1980年代出現新的變異株CPV-2a及CPV-2b，並於2000年發現CPV-2c變異株，且新的型別已經取代原始CPV-2。先前研究顯示CPV-2不同變異株在組織分布上並無顯著差異，但在致病性上卻可能有所差別。在診斷上，原位雜合 (In situ hybridization, ISH) 染色曾被報告為一種高度敏感性的診斷方式，且可直接觀測病毒於細胞中感染的情形，但目前仍沒有報告藉由ISH染色探討不同型別之CPV-2造成之顯微病變差異。因此，本研究收集台灣地區自然感染犬小病毒之檢體，藉由核酸定序方式分成2a, 2b及2c三型，合成CPV-2 ISH探針並以帶有CPV-2部分基因質體之勝任細胞加以測試，建立穩定之ISH染色條件後，藉由ISH染色技術，比較不同CPV-2型別之組織病變，並比較各個型別於不同臟器之分布及ISH陽性訊號之差異。本研究共收集24個CPV2感染之剖檢病例 (8例CPV-2a，2例CPV-2b及14例CPV-2c)，肉眼皆呈現不等程度之犬小病毒典型病變，組織病理學亦發現小腸腺窩上皮壞死及腺窩擴張等病變，ISH染色結果發現陽性訊號主要出現於壞死脫落之小腸腺窩上皮及消化道周邊淋巴組織內淋巴球及巨噬細胞。但不同型別犬小病毒感染犬隻並未於組織病理變化及組織分佈上發現顯著差異，推測不同型別犬小病毒自然感染之死亡犬隻造成消化道破壞之程度及病原分佈並未有明顯差別，可能表示CPV-2不同變異株於致病性及致病機轉上並無明顯差異。

Canine parvovirus type 2 (CPV-2) is one of the most important alimentary and immune system diseases in dogs. It induces severe hemorrhagic enteritis and necrosis of peripheral lymphoid tissue of gastrointestinal tract, and was also been reported showing myocarditis in puppies. CPV-2 was first reported in 1978, and had become a world-wide infectious disease throughout these years, the long period of interaction with the different hosts leads to the variation of the virus. In the 1980s, new antigenic type CPV-2a and 2b had been discovered, and CPV-2c was been reported in the year 2000. The prototype CPV-2 is now totally been replaced by these variants. Previous research shows that CPV-2 variant has no significant difference in tissue distribution, but may have some differences in pathogenicity. While during CPV-2 diagnosis, In Situ hybridization has been reported showing high sensitivity, and could directly observe viral infection circumstances within the cells. However, no previous research compared the differences of micro lesions and ISH signal distribution caused by all three types of CPV-2 variants. Thus, the present study collects positive CPV-2 samples from Taiwan, using genetic sequencing technique to classify CPV-2 variants into three groups (2a, 2b and 2c). Synthesis the CPV-2 ISH probe, using the plastid DNA which contain the CPV-2 partial gene with in the competent cell to test the ISH probe, after establish the protocol, ISH staining technique is then used to compare the differences of histopathological changes and the tissue distributions between CPV-2 variants. Twenty four CPV-2 necropsy cases had been collected (8 CPV-2a, 2 CPV-2b, 14CPV-2c) , all of these cases are shows the variant severity of CPV-2 infectious lesion, and the histopathology also shows necrosis of crypts epithelium and crypts dilation. The ISH positive signal are mainly found in the necrotic crypt epithelium, and lymphocytes and macrophages in lymphoid tissues of gastrointestinal tract. But no significant differences were found in histopathology and tissue distribution between the variants, shows that there are no obvious differences of intestinal damage and virus distribution between three variants. The result suggested that there may be no obvious difference of pathogenicity and mechanisms between three variants.

中文摘要 i
Abstract iii
謝誌 v
目錄 vi
圖表目錄 ix
第一章 前言 1
第二章 文獻回顧 3
2.1 犬小病毒 (Canine Parvovirus) 3
2.1.1 歷史 3
2.1.2 分類 (Taxonomy) 3
2.1.3 病毒結構 (Virus structure) 4
2.1.4 基因特性 (Genetic characterization) 5
2.1.5 演化 (Evolution) 5
2.1.6 基因分型 (Genotyping) 6
2.1.7 VP2胺基酸變異 6
2.1.8 抗原型別地理分佈 (Antigenic type distribution) 7
2.2 第二型犬小病毒之感染與疾病 8
2.2.1 致病機轉 8
2.2.2 傳播途徑 8
2.2.3 臨床症狀 9
2.2.4 肉眼病變 9
2.2.5 顯微病變 9
2.2.6 診斷方式 10
2.2.7 不同型別致病性之差異 11
2.2.8 治療與預防 11
2.3 原位雜合染色法 (In situ hybridization; ISH) 12
2.3.1 簡介 12
2.3.2 原位雜合染色使用於CPV-2之診斷 12
2.3.3 原位雜合染色法之限制 13
第三章 材料與方法 14
3.1 第二型犬小病毒原位雜合染色技術建立 14
3.1.1 實驗設計 14
3.1.2 樣本收集與保存 15
3.1.3 檢體資料調查 15
3.1.4 組織病理切片製作 15
3.1.5 原位雜合探針合成 15
3.1.6 陽性及陰性樣本製備 16
3.1.7 ISH探針測試 16
3.1.8 原位雜合染色條件建立 17
3.2 不同型別之第二型犬小病毒原位雜合染色 18
3.2.1 實驗設計 18
3.2.2 核酸萃取 19
3.2.3 第二型犬小病毒陽性篩選 19
3.2.4 第二型犬小病毒型別分析 19
3.2.5 肉眼病變觀察 20
3.2.6 組織病理切片製作及染色 20
3.2.7 組織病理學觀察及評分 21
3.2.8 原位雜合染色及評分 21
3.2.9 資料分析 22
第四章 結果 23
4.1第二型犬小病毒原位雜合染色技術建立 23
4.1.1 樣本收集與紀錄 23
4.1.2 原位雜合探針測試 23
4.1.3 原位雜合染色條件建立 23
4.2不同型別之第二型犬小病毒原位雜合染色 23
4.2.1 第二型犬小病毒型別分析 23
4.2.2肉眼病變觀察 24
4.2.3組織病理學觀察及評分 24
4.2.4原位雜合染色及評分 25
4.2.5 資料分析 25
第五章 討論 44
參考文獻 47
作者簡介 57

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