鄭謙仁臺灣大學：獸醫學研究所李育宗Lee, Yu-TsungYu-TsungLee2007-11-282018-07-092007-11-282018-07-092005http://ntur.lib.ntu.edu.tw//handle/246246/60006豬增生性壞死性肺炎（Porcine proliferative and necrotizing pneumonia; PNP）最早於1988年加拿大魁北克省發現。顯微鏡下特徵為第Ⅱ型肺泡細胞增生，肺間質變厚且肺泡腔有許多單核細胞及脫落壞死細胞之蓄積，偶爾可見玻璃樣膜之形成及細支氣管壞死；因其顯微病灶呈現特異變化，且不明瞭是由何種病原所引起，故稱之為“豬增生性壞死性肺炎”。 本研究自台灣地區豬場，於臨床呈現呼吸急促、呼吸困難及腹式呼吸等症狀之患豬，從2002至2003年共收集275個病例。以組織病理學進行形態變化之篩選，發現有50個（18%）病例呈現增生性壞死性肺炎之形態；於此50個PNP病例，發現細菌與病毒混合感染病例占49例（98%），僅有1例（2%）為單一病毒感染所致。以聚合酶鏈鎖反應（PCR）偵測第二型豬環狀病毒（PCV2）、豬生殖與呼吸道綜合症病毒（PRRSV）及黴漿菌（Mycoplasma hyopneumoniae）分別有50（100%）、6（12%）及11（22%）個陽性病例；另外細菌分離以沙氏桿菌（Salmonella sp.）所占比例最高，約為22.4%。另運用原位雜交法（ISH）對PCV2、PRRSV及豬流行性感冒病毒（SIV）進行檢測，結果發現PCV2主要分佈於PNP的間質及壞死病灶區，並能於肺泡及終末細支氣管上皮細胞、巨噬細胞與壞死細胞碎片皆可見有病毒核酸；PRRSV則多分佈於間質病灶區及肺泡壁處，能於巨噬細胞與肺泡上皮細胞見到病毒核酸；SIV則存在於間質及壞死病灶區，且能於肺泡及終末細支氣管上皮細胞與巨噬細胞見有病毒核酸。另外，以免疫組織化學染色（IHC）檢測豬小病毒（PPV），其陽性訊號主要分佈於間質及壞死病灶區，此陽性訊號存於巨噬細胞與肺泡及終末細支氣管上皮細胞。由ISH結果顯示，PCV2與SIV兩種病毒多於壞死及間質病灶區見有病毒核酸，但SIV呈現之核酸訊號較少且弱。然而，PRRSV主要分佈位置與間質病灶區之肺泡壁較具有關聯性。PPV常於壞死及間質病灶區內見有陽性訊號，但亦能於終末細支氣管之化膿病灶區見有陽性訊號。以ISH及IHC探討四種不同病毒之分佈位置得知，PNP病灶應由不同病原混合感染所造成；其中又以PCV2及PPV兩種病毒最為重要，此兩種病毒之分佈與PNP病灶位置具有緊密之關聯性。另外，本實驗首次發現台灣地區豬隻具有高盛行率之PPV感染。In the late 1980s, a new type of porcine pneumonia of unknown etiology called proliferative and necrotizing pneumonia (PNP) was described in Quebec, Canada. Histologically, it was characterized by a lymphohistiocytic interstitial pneumonia with a marked proliferation of type 2 pneumocytes and many coagulates of necrotic cells in the alveoli. Occasionally, hyaline membranes and bronchiolar necrosis were also observed. Between 2002 and 2003, 275 cases were collected from pig farms of Taiwan. The main clinical signs of these cases were tachypnoea, dyspnea, and abdominal respiration. Histopathological characteristic lesions of proliferative and necrotizing pneumonia (PNP) were found in 50 cases (18%). Single viral infection was only detected in 1 case (2%), whereas in other 49 cases (98%), a combination of viral and bacterial infection was noted. Polymerase chain reaction (PCR) was used for the detection of porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae, and the frequency of positive results were 50 (100%), 6 (12%) and 11 (22%) cases, respectively. The infection of Salmonella sp. was presented in 12 cases (24%) by bacterial isolation. By in-situ hybridization (ISH), PCV2 signals were chiefly distributed in interstitial and necrotic lesions, and the positive signals could be found in macrophages-like cells and necrotic debris. PRRSV signals were mainly located at interstitial lesions and alveolar wall, and the positive signals could be found in macrophages-like cells and epithelial cells of alveoli. Swine influenza virus (SIV) could be found in interstitial and necrotizing lesions, and the signals could be found in macrophages-like cells, and epithelial cells of terminal bronchiole and alveoli. By immunohistochemistry (IHC), porcine parvovirus (PPV) signals were mainly distributed in interstitial and necrotic lesions, and the positive signals could be found in macrophages-like cells and epithelial cells of terminal bronchiole and alveoli. The ISH results revealed that the signals of PCV2 and SIV were profoundly present in the necrotic and interstitial lesions, but SIV signals were weaker and less often. The signals of PRRSV were scattered and related to the alveolar wall of interstitial lesions. The IHC results showed the signals of PPV were present in the necrotic and interstitial lesions, the signals could occasionally be found in suppurative lesions of the terminal bronchiole. In conclusion, PNP lesions maybe induced by the co-infection of several pathogens, especially PCV2 and PPV. Additionally, this is the first study demonstrates a high prevalence of PPV infection in Taiwan.目錄…………………………………………………………………………Ⅰ 表次…………………………………………………………………………Ⅴ 圖次…………………………………………………………………………Ⅵ 中文摘要……………………………………………………………………Ⅶ 英文摘要……………………………………………………………………Ⅸ 第一章 序言………………………………………………………………...1 第二章 文獻回顧……………………………………………………………3 第一節 肺臟的解剖生理及病理變化之簡介…………………………3 1-1 前言…………………………………………………………...3 1-1-1 肺臟的一般正常結構、形態與生理功能……………..3 1-1-1-1 肺內導氣部………………………………………...4 1-1-1-2 肺的呼吸部………………………………………...4 1-1-2 肺臟組織的再生與衰老變化…………………………..6 1-2 肺炎之分類、組織病理學型態及可能的特殊致病機制……6 1-2-1 支氣管性肺炎…………………………………………..7 1-2-2 間質性肺炎……………………………………………..7 1-2-3 栓子性肺炎……………………………………………..8 1-2-4 肉芽腫性肺炎…………………………………………..9 第二節 豬增生性壞死性肺炎………………………………………...10 2-1 歷史背景與發生概況………………………………………..10 2-2 豬增生性壞死性肺炎之臨床症狀與病理變化……………..10 第三節 豬增生性壞死性肺炎相關病原之特性………………………12 3-1 豬流行性感冒病毒之特性及病理變化……………………..12 3-1-1 豬流行性感冒病毒之特性、型態及構造…………….12 3-1-2 豬流行性感冒病毒之複製…………………………….13 3-1-3 豬流行性感冒病毒之流行病學……………………….13 3-1-4 豬流行性感冒病毒之臨床症狀與病理變化………….14 3-2 豬生殖與呼吸道綜合症病毒之特性及病理變化…………..15 3-2-1 豬生殖與呼吸道綜合症病毒之特性、型態及構造….15 3-2-2 豬生殖與呼吸道綜合症病毒之複製…………………..16 3-2-3 豬生殖與呼吸道綜合症病毒之流行病學……………..16 3-2-4 豬生殖與呼吸道綜合症病毒之臨床症狀與病理變化..17 3-3豬環狀病毒之特性與病理變化……………………………….18 3-3-1 豬環狀病毒之特性、型態及構造………………………18 3-3-2 豬環狀病毒之複製……………………………………...19 3-3-3 豬環狀病毒之流行病學………………………………...20 3-3-4 豬環狀病毒之臨床症狀與病理變化…………………...20 3-4 豬小病毒之特性與病理變化………….……………………...22 3-4-1 豬小病毒之特性、型態及構造…………………………22 3-4-2 豬小病毒之複製………………………………………...23 3-4-3 豬小病毒之流行病學…………………………………...24 3-4-4 豬小病毒之臨床症狀與病理變化……………………...24 3-5相似於豬增生性壞死性肺炎組織病灶之動物或人之疾病….25 3-5-1 Severe acute respiratory syndrome…………...................25 3-5-2 Acute respiratory distress syndrome…………………….26 3-5-3 Acute bovine pulmonary edema and emphysema………26 第四節 原位雜交……………………………………………………….27 第五節 免疫組織化學染色…………………………………………….29 第三章 材料與方法………………………………………………………….30 第一節 實驗設計……………………………………………………….30 第二節 實驗材料……………………………………………………….31 2-1 病材……………………………………………………………31 第三節 實驗方法……………………………………………………….31 3-1 病原診斷………………………………………………………31 3-2 組織病理學診斷………………………………………………32 3-3 豬隻基本資料與診斷之彙整…………………………………32 3-4 探針之製備……………………………………………………32 3-4-1 PCV2探針………………………………………………32 3-4-2 PRRSV探針…………………………………………….33 3-4-2-1 微量質體DNA萃取……………………………….33 3-4-2-2 聚合酶鏈鎖反應…………………………………...33 3-4-2-3 DNA洋菜膠片電泳分析………………………….34 3-4-2-4 PCR產物的直接核苷酸定序……………………..35 3-4-2-5 PRRSV探針之合成……………………………….35 3-4-3 SIV探針………………………………………………...35 3-4-3-1 流行性感冒病毒核酸之萃取……………………...35 3-4-3-2 反轉錄聚合酶鏈鎖反應…………………………...36 3-4-3-3 DNA洋菜膠片電泳分析…………………………..37 3-4-3-4 RT-PCR產物的直接核苷酸定序………………….37 3-4-3-5 SIV探針之合成……………………………………37 第四節 免疫膜點法…………………………………………………….37 第五節 原位雜交……………………………………………………….38 第六節 免疫組織化學染色…………………………………………….39 第四章 結果………………………………………………………………….41 第一節 實驗動物之背景、病原診斷與組織病理型態學之結果…….41 1-1 病原診斷……………………………………………………...41 1-2 組織病理型態學之分類……………………………………...41 第二節 增生性壞死性肺炎病例之組織學變化、病原診斷與年齡分佈情形……………………………………………………………45 2-1組織病理學之變化及特徵……………………………………45 2-2利用特殊染色排除此病灶為細菌團塊或鈣化現象…………45 2-3病原診斷結果及交叉分析……………………………………48 2-4罹病豬隻年齡分佈情形………………………………………48 第三節 探針製備結果………………………………………………….51 3-1 PRRSV之探針製備…………………………………………...51 3-2 AIV之探針製備……………………………………………….51 第四節 原位雜交及免疫組織化學染色結果………………………….56 4-1 PCV2原位雜交結果…………………………………………..56 4-2 PRRSV原位雜交結果………………………………………...63 4-3 SIV原位雜交結果…………………………………………….68 4-4 PPV免疫組織化學染色結果………………………………….73 4-5各病毒於PNP病灶區的相關位置及感染細胞……………....81 第五章 討論………………………………………………………………….83 第六章 參考文獻…………………………………………………………….97 附錄………………………………………………………………………….106 附錄一 50個PNP病例以PCR / RT-PCR、細菌分離與鑑定、原位雜交反應及免疫組織化學染色結果……………………………………….106 附錄二 50個PNP病例以PCR / RT-PCR、細菌分離與鑑定及組織病理學診斷結果…………………………………………………………….1104300650 bytesapplication/pdfen-US豬增生性壞死性肺炎第二型豬環狀病毒豬生殖與呼吸道綜合症病毒豬流行性感冒病毒豬小病毒Porcine Proliferative and Necrotizing PneumoniaPorcine Circovirus type 2Porcine Reproductive and Respiratory Syndrome VirusSwine Influenza VirusPorcine Parvovirusthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/60006/1/ntu-94-R92629016-1.pdf