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研究生:邱慧英
研究生(外文):Hue-Ying Chiou
論文名稱:動物園貓科動物慢性腎炎之微生物性病因探討
論文名稱(外文):Microbial etiological survey of felid chronic nephritis in zoo
指導教授:龐飛龐飛引用關係
指導教授(外文):Victor Fei Pang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:獸醫學研究所
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:2005
畢業學年度:93
語文別:中文
論文頁數:91
中文關鍵詞:貓科動物慢性腎炎鉤端螺旋體貓傳染性腹膜炎
外文關鍵詞:FelidChronic NephritisLeptospiraFeline Infectious Peritonitis
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於歷年執行動物園野生動物疾病監控調查研究計畫中,針對死亡動物進行病理學檢查,發現貓科動物多具有慢性腎炎(Chronic nephritis)之病理變化。慢性腎炎為一常見形態學診斷,肇因於絲球體腎炎(Glomerulonephritis)、間質性腎炎(Interstitial nephritis)及腎盂腎炎(Pyelonephritis)等各種不同腎臟疾病之末期病變。可導致貓腎臟病變之非感染性因子包括先天缺陷、腎動脈血液供應不良、腎毒性化學物質、重金屬、高血壓及食因性因子等。感染性因子則包括貓傳染性腹膜炎病毒(Feline infectious peritonitis virus, FIPV)、貓白血病病毒(Feline leukemia virus, FeLV)、貓免疫缺陷病毒(Feline immunodeficiency virus, FIV)、鉤端螺旋體(Leptospira spp.)、大腸桿菌(Escherichia coli)、Proteus spp.、Pseudomonas spp.、Klebsiella spp.、鏈球菌(Streptococcus spp.)、Bartonella spp.、弓蟲(Toxoplasma gondii)、犬新胞蟲(Neospora caninum)、心絲蟲(Dirofilaria immitis)等。FIP為貓科動物重要傳染性疾病,易感染所有貓科動物,尤其是獵豹。動物園貓科動物感染FeLV及FIV之病例相當罕見,FeLV雖曾被記載發生於美洲山獅、獵豹與雲豹,而感染FIV亦曾發表於非洲獅、老虎及花豹,但是,目前FeLV與FIV感染症在動物園貓科動物的重要性仍不清楚。至於可引起間質性腎炎之鉤端螺旋體,於1978年曾有兩名動物園管理員經由飼養的熊感染發病,而成為近年來再度受到重視的重現性重要人畜共通傳染病。
本研究利用組織化學染色、免疫組織化學染色、聚合酶鏈鎖反應等方法,回溯性探討動物園內死亡貓科動物引發慢性腎炎之可能病因。收集自1989年至2004年台大獸醫學系及台灣動物科技研究所於北、中、南部各公私立動物園及動物展示場剖檢之貓科動物腎臟組織蠟塊,重新製作組織切片,依據H&E染色進行組織病理變化形態學分析;選擇具有腎炎病變之檢體,利用免疫組織化學染色(Immunohistochemistry)、免疫螢光抗體染色法 (Immunofluorescence)及聚合酶鏈鎖反應 (PCR),分別偵測腎臟組織內是否有鉤端螺旋體、貓傳染性腹膜炎病毒、貓白血病病毒及貓免疫缺陷病毒等病原的存在。
由歷年收集之177例動物園貓科動物石蠟包埋腎臟檢體,經重新製作組織切片,進行組織病理學分析,結果發現65.5%(116/177)具有腎炎之病理變化,其中以間質性腎炎(Interstitial nephritis)發生頻次最高,佔97.4%(113/116)。利用Leptospira shermani外膜蛋白多源抗體進行免疫組織化學染色,結果發現其陽性率為24.8%(28/113)。另外,利用TGEV棘蛋白多源抗體進行免疫螢光抗體染色,結果於46例具有膜性及/或增殖性絲球體腎炎病例中,發現6例 (6/46, 13%) 於絲球體中出現陽性反應。由177個石蠟包埋腎臟檢體中成功自70個病例萃取出核酸,分別進行鉤端螺旋體、FeLV及FIV之PCR檢測,結果鉤端螺旋體的陽性率為24.3% (17/70),FeLV的陽性率為2.9% (2/70),而FIV則完全未發現陽性病例 (0/70)。此結果顯示,鉤端螺旋體可能為國內動物園貓科動物慢性腎炎的重要致病因子,至於FIPV、FeLV及FIV等病毒之感染則所佔比率極低。
During routing health monitoring and disease surveillance for zoo animals, it was revealed that chronic interstitial nephritis was a common pathological finding in zoo felids. Chronic interstitial nephritis may represent the end result of several different renal diseases, including glomerulonephritis, interstitial nephritis, and chronic pyelonephritis. The non-infectious causes of chronic renal diseases in the cat include congenital disorders, decreased blood supply to renal arteries, toxic chemicals, heavy metals, hypertension, and dietary; the infectious causes include feline infectious peritonitis virus (FIPV), feline leukemia virus (FeLV), and feline immunodeficiency virus (FIV), Leptospira spp., Escherichia coli, Proteus spp., Pseudomonas spp., Klebsiella spp., Streptococcus spp., Bartonella spp., Toxoplasma gondii, Neospora caninum, and Dirofilaria immitis etc. All felid species are susceptible to FIPV but the disease has a higher prevalence in certain species such as the cheetah. Infections of FeLV and FIV are only rarely reported in exotic feline species, such as African lions, tigers, and panthers. The clinical significance of FIV and FeLV in zoo felids is still unknown. Leptospirosis is a zoonotic emerging infectious diseases causing interstitial nephritis in man and animals and has been reported in zoo workers acquired from bear cubs. This observation suggests that zoonotic leptospiral infection should be considered as a possible pathogen for zoo felids as well.
One hundred and seventy-seven paraffin embedded renal tissues of the zoo felids collected during 1989 and 2004 were subjected to histopathological examination. Immunohistochemistry (IHC) was used to detect Leptospira spp. and immunofluorsecence antibody test (IFA) was used for feline coronavirus. Polymerase chain reaction (PCR) was used to detect Leptospira spp., FeLV, and FIV. In the 177 cases, 65.5%(116/177)showed evidence of nephritis. Within the 116 cases of nephritis, 97.4%(113/116) were classified as interstitial nephritis. By applying the polyclonal antibody against the outer membrane protein of Leptospira shermani by IHC, a 24.8% (28/113) positive rate was revealed. In addition, the analysis of FIPV antigens in the kidneys of 46 felids with membrano- and/or proliferative glomerulonephritis using IFA by a polyclonal antibody against the spike protein of TGEV revealed that 6 out of 46 (6/46, 13%) cases showing positive signals in the glomeruli. Nucleic acid was successfully extracted from the tissue block of 70 out of 177 cases. Seventeen out of the 70 cases (17/70, 24.3%) were positive for Leptospira, 2 out of the 70 cases (2/70, 2.9%) were positive for FeLV, but none of the cases (0/70) was positive for FIV by PCR.
The results suggest that leptospira should be considered as a possible important pathogen in association with the common observed chronic nephritis in zoo felids. The role of FIPV, FeLV, and FIV in chronic nephritis of zoo felids is minor.
目錄 I
表次 IV
圖次 V
中文摘要 VII
英文摘要 IX
第一章 緒言……………………………………………………………………….. 1
第二章 文獻探討………………………………………………………………….. 3
第一節 潛在可能引發動物園貓科動物腎臟病變之病因………………… 3
第二節 腎臟病變之致病機制…………………………………………….. 4
2.1 腎臟的功能…………………………………………………... 4
2.2 腎臟病變之致病機制………………………………….......... 5
2.3 鉤端螺旋體病的致病機制…………………………………... 9
2.4 病毒性疾病引發腎臟病變之致病機制……………………... 10
第三節 鉤端螺旋體在動物間的致病性………………………………….. 10
3.1 鉤端螺旋體的發現…………………………………………... 11
3.2 鉤端螺旋體之分類與特性…………………………………... 11
3.3 鉤端螺旋體在動物間扮演的致病角色……………………... 13
3.4 鉤端螺旋體與動物園圈飼及野生動物之關係……………... 15
第四節 貓傳染性腹膜炎病毒與貓科動物之關係……………………….. 16
4.1 FIP的歷史背景與概況………………………………………. 16
4.2 FIP的病毒特性………………………………………………. 17
4.3 FIPV與動物園貓科動物之關係…………………………….. 20
第五節 貓白血病病毒與動物園貓科動物之關係……………………….. 20
5.1 FeLV的病毒特性……………………………………………. 20
5.2 FeLV與動物園圈飼及野外貓科動物之關係……………….. 21
第六節 貓免疫缺陷病毒與貓科動物之關係…………………………….. 22
6.1 FIV的病毒特性………………………………………………. 22
6.2 FIV與動物園圈飼及野外貓科動物之關係…………………. 23
第三章 材料與方法……………………………………………………………….. 24
第一節 實驗設計………………………………………………………….. 24
第二節 實驗材料………………………………………………………….. 25
2.1 動物園貓科動物腎臟組織蠟塊樣本之收集……………….. 25
2.2 組織化學染色……………………………………………….. 25
2.3 免疫染色……………………………………………………... 26
2.3.1 應用於鉤端螺旋體之免疫組織化學染色檢測試劑……………………………………………………..
26
2.3.2 應用於貓傳染性腹膜炎病毒之免疫螢光染色檢測試劑……………………………………………………..
26
2.3.2.1 直接免疫螢光抗體染色法……………… 26
2.3.2.2 間接免疫螢光抗體染色法……………... 27
2.4聚合酶鏈鎖反應……………………………………………… 27
2.4.1 應用於萃取蠟塊組織中DNA之試劑………………. 27
2.4.2 應用於PCR反應與電泳之試劑……………………. 27
第三節 實驗方法………………………………………………………….. 30
3.1 蠟塊組織之DNA萃取………………………………………. 30
3.2 組織化學染色法與腎臟病變之型別分析…………………… 30
3.2.1 組織化學染色法-蘇木紫與伊紅染色法……………... 30
3.2.2腎臟組織病變之型別分析…………………………….. 31
3.3 免疫染色……………………………………………………… 31
3.3.1 應用免疫組織化學染色法於鉤端螺旋體之檢測……………………………………………………….
31
3.3.2 應用免疫螢光染色法於貓傳染性腹膜炎病原之檢測. 32
3.3.2.1 直接免疫螢光抗體染色法………………. 32
3.3.2.2 間接免疫螢光抗體染色法………………. 33
3.4 聚合酶鏈鎖反應……………………………………………… 33
3.4.1 16s DNA之檢測………………………………………. 33
3.4.2 鉤端螺旋體之檢測……………………………………. 34
3.4.3 貓白血病病毒之檢測…………………………………. 34
3.4.4 貓免疫缺陷病毒之檢測………………………………. 35
3.4.5 洋菜膠電泳……………………………………………. 35
第四章 結果……………………………………………………………………….. 37
第一節 動物園貓科動物背景資料、腎臟病變之型別與年齡分析…………………………………………………………………
37
1.1 背景資料……………………………………………………… 37
1.2 年齡分析……………………………………………………… 39
1.3 貓科動物解剖例之腎炎年發生率…………………………… 41
1.3.1 五月齡以下貓科動物腎臟組織病理學變化與發生頻次……………………………………………………..
44
1.3.2 五月齡至三歲………………………………………… 44
1.3.3 三歲至十歲…………………………………………… 44
1.3.4 十歲以上……………………………………………… 45
1.3.5 年齡不詳……………………………………………… 45
第二節 動物園貓科動物鉤端螺旋體病之陽性率………………………… 48
2.1 應用免疫組織化學染色檢測腎臟組織中Leptospira的抗原及其檢出陽性率……………………………………………..
48
2.2 應用聚合酶鏈鎖反應檢測腎臟組織中Leptospira核酸及其檢出陽性率…………………………………………………..
51
2.3 應用聚合酶鏈鎖反應與免疫組織化學染色檢測Leptospira陽性病例腎臟之病理學特徵………………………………..
53
第三節 應用免疫組織化學染色檢測腎臟組織中貓傳染性腹膜炎 (Feline infectious peritonitis) 病毒抗原及其檢出陽性率………..................................................................................

57
3.1 直接免疫螢光抗體染色法……………………………… 57
3.2 間接免疫螢光抗體染色法……………………………… 57
第四節 應用聚合酶鏈鎖反應檢測腎臟組織中貓白血病病毒 (Feline leukemia virus) 核酸及其檢出陽性率…………………………
61
第五節 應用聚合酶鏈鎖反應檢測腎臟組織中貓免疫缺陷病毒 (Feline immunodeficiency virus) 核酸及其檢出陽性率……………..
63
第五章 討論………………………………………………………………………… 64
參考文獻…………………………………………………………………………….. 74
附錄 81
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