臺灣博碩士論文加值系統

貓傳染性腹膜炎 (feline infectious peritonitis, FIP) 是由貓冠狀病毒 (feline coronavirus, FCoV) 所引起之免疫性致死性疾病。根據體腔滲出液之與否， FIP 可區分為滲出型與非滲出型；而經由血清學分析， FCoV 可區分為第一型及第二型。為了解滲出型與非滲出型 FIP 貓隻體內病毒感染細胞分佈情況之差異，本研究針對曾就診於台大動物醫院，並經由解剖及組織病理學確診的 FIP 貓隻，挑選其中典型的非滲出型 FIP 五例，以及滲出型 FIP 八例。將其組織切片進行免疫化學染色 (immunohistochemistry, IHC) ，結果在檢測之患貓臟器都可被觀察到 FCoV 感染的細胞。在乾式病例中腎臟 (80%) 與腦 (100%) 的 FCoV 陽性率，分別高於溼式病例之 37.5% 及 40% 。與 FCoV 相關之病灶在數量及嚴重程度上，均有乾式病例甚於溼式病例之趨勢。為了建立原位增幅技術並以此分析不同型別病毒與致病 性之關連性，本研究利用型別特異性引子於相關組織切片上進行直接式原位反轉錄聚合酶鏈鎖反應 (direct in situ RT-PCR) 。由於組織之消化過程對於反應結果影響甚鉅，首先需將其條件最佳化；針對 F96-156 該同時感染第一及第二型 FCoV 之 FIP 病例，其最佳化條件為肝臟切片以 50 μg/mL proteinase K 於 37℃ 作用 1 小時，及肺臟切片以 2 mg/mL pepsin 於室溫下作用 4 小時。以此最佳化消化條件進行後續之 direct in situ RT-PCR ，其肝臟及肺臟均可觀察到第二型 FCoV 所感染的細胞。

Feline infectious peritonitis (FIP) is an immune-mediated disease manifesting clinically as effusive or non-effusive form. The causative agents, feline coronaviruses (FCoVs), are serologically classified into type I and II. In order to understand the differences in the distribution pattern of FCoV-infected cells within the lesions between effusive and non-effusive FIP, cats presented to the Veterinary Hospital at National Taiwan University with the final diagnosis of FIP were selected. Tissue samples from five typical non-effusive cases and eight typical effusive cases were subjected to immunohistochemistry staining. FCoV-infected cells were found in nearly all kinds of the examined organ samples. The FCoV-positive rates of kidney (80%) and brain (100%) in non-effusive FIP were higher than those of effusive FIP (37.5% and 40%, respectively). As comparing to effusive cases, a tendency that the lesions associated with FCoV were more abundant and severe in non-effusive ones was noted. To correlate different type of FCoVs with the development of FIP, in situ amplification of FCoV nucleic acid in affected tissues was attempted using type-specific primers via direct in situ RT-PCR. As the process of digestion might greatly influence the test results, optimization of the assay condition was first established. For F96-156, a FIP case that infected with type I and II FCoVs, incubation with 50 μg/mL of proteinase K at 37℃ for one hour in liver sections, and 2 mg/mL of pepsin at room temperature for four hours in lung sections were found to be optimal. Under these optimized conditions, type II FCoV-infected cells were detected in both liver and lung.

圖目錄 v
表目錄 vi
摘要 vii
Abstract viii
第一章　序言 1
第二章　文獻回顧 3
第一節　貓傳染性腹膜炎研究之歷史背景 3
1.1　貓傳染性腹膜炎的發現 3
1.2　貓冠狀病毒之宿主範圍 3
1.3　治療與疫苗的發展 4
第二節　貓冠狀病毒感染之流行病學 5
第三節　貓冠狀病毒之分型 6
第四節　致病機制與免疫反應 8
第五節　臨床症狀與診斷 10
5.1　臨床症狀 10
5.2　臨床病理學所見 11
5.3　實驗室診斷 11
5.4　病理學診斷 12
第六節　實驗動物感染 14
6.1　貓冠狀病毒分離之概況 14
6.2　實驗動物感染之致病率 14
6.3　實驗動物感染接種途徑之比較 15
6.4　實驗動物感染病變出現之進程 15
第三章　材料與方法 17
第一節　實驗設計 17
第二節　臨床檢體之處理與分析 17
2.1　臨床檢體之處理 17
2.2　貓冠狀病毒 RNA 之萃取 (RNA extraction) 18
2.3　反轉錄反應 (Reverse transcription) 18
2.4　聚合酶鏈鎖反應 (Polymerase chain reaction, PCR) 19
2.5　電泳分析 (Electrophoresis) 20
第三節　臨床檢體之血清型別分型 21
3.1　血清型別分型之反轉錄聚合酶鏈鎖反應 21
3.2　電泳分析 (Electrophoresis) 22
第四節　屍體解剖與採樣 23
第五節　臟器組織乳劑製備 23
第六節　免疫組織化學染色 (Immunohistochemistry) 23
第七節　直接式原位反轉錄聚合酶鏈鎖反應 (Direct in situ RT-PCR) 25
7.1　石蠟包埋組織切片細胞膜之消化 25
7.1.1　消化條件最佳化：陽性對照組 26
7.1.2　消化條件最佳化：陰性對照組 26
7.1.3　消化條件最佳化：實驗組 26
7.2　RNase-free DNase I 之處理 27
7.3　直接式原位反轉錄聚合酶鏈鎖反應 27
7.3.1　原位反轉錄反應 27
7.3.2　原位聚合酶鏈鎖反應 28
7.4　呈色反應 29
第四章　結果 30
第一節　貓傳染性腹膜炎組織病理學特徵 30
第二節　貓冠狀病毒免疫化學染色 30
2.1　FCoV 於各臟器之感染情況 30
2.2　乾式與溼式 FIP 病灶之比較 31
第三節　貓冠狀病毒分型聚合酶鏈鎖反應 32
3.1　臨床檢體分型結果 32
3.2　組織臟器乳劑分型結果 32
第四節　貓冠狀病毒原位反轉錄聚合酶鏈鎖反應 33
4.1　組織細胞消化條件之最佳化 33
4.2　原位反轉錄聚合鏈鎖反應 33
第五章　討論 35
附圖 41
附表 52
參考文獻 57
附錄一　主要溶液之配置 68
附錄二　使用儀器廠商名稱 69

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