Epidemiological Investigation of Bovine Leptospirosis from Beef Abattoir and Dairy Farms

為了解台灣地區牛鉤端螺旋體症流行之情形。我們自2003年8月至2004年4月期間，由桃園縣某一屠宰場收集480頭牛血液及配對之腎臟組織，及由花蓮縣及台東縣共19處乳牛場採得695頭牛血清。以顯微凝集試驗進行鉤端螺旋體血清抗體檢查，力價≧1:100判定為陽性。腎臟組織則以聚合酶鏈反應及細菌培養進行病原體檢測，而腎臟組織以10%福馬林固定後，製作切片分別以H&E染色、鍍銀染色以及LipL32抗體進行組織學及免疫組織化學檢查。由屠宰場採集之血清樣本，顯微凝集試驗結果有27.5% (132/480)的血清呈現陽性，108頭感染單一血清群，24頭感染兩種以上血清群，抗體力價分佈為1:100至1:3200 (幾何平均力價為1:175)，主要感染之血清群為Pomona。480個配對腎臟組織中只有1例為聚合酶鏈反應陽性，初步鑑定屬於L. interrogans基因種。78個有小白點之腎臟樣本中只有1例以免疫組織化學染色為陽性。培養之結果則皆為陰性。以McNemar試驗檢測相關性，結果腎臟肉眼病變與血清抗體陽性間有顯著相關 (p<0.01)，多發局部間質性腎炎與血清抗體陽性之間亦為顯著相關 (p<0.01)。針對花蓮縣、台東縣19處乳牛場之血清抗體及危險因子進行調查，場陽性率為100%。總抗體陽性率為37.8% (263/695)，抗體力價分佈為1:100至1:400 (幾何平均力價為1:131)，而主要感染之血清群為Shermani。危險因子為牧場週遭有動物出沒 (OR=1.7, p<0.01)。由上述調查結果得知，台灣牛隻並未進行鉤端螺旋體疫苗注射，抗體陽性出現即代表自然感染，可能造成經濟之損失也可能危及人類之健康，因此，我們認為牛鉤端螺旋體症在台灣普遍存在，若要成左滷惆謅To understand the status of bovine leptospirosis in Taiwan, we collected 480 serum samples and corresponding kidney tissues from one abattoir near Taoyuan and 570 serum samples from 15 dairy farms in Hualien and Taitung counties, during the period between August, 2003 and April, 2004. The leptospiral antibody was examined by microscopic agglutination test (MAT). A titer ≧1:100 was considered as positive. The polymerase chain reaction (PCR) and bacteria isolation using EMJH medium were employed for detection of Leptospira bacteria in kidney tissues. The kidneys fixed in 10% formalin were routinely prepared for the sections of H&E and Warthin-Starry staining and immunohistochemically (IHC) stained with LipL32 antibody for detection of Leptospira. Twenty seven point five percent (132/480) of bovine sera from abattoir were positive by MAT. One hundred and eight samples were positive to one, and 24 samples were positive to more than one, which had titres between 1:100 and 1:3200 with geometric mean titer equal to 1:175. The most common serogroup is Pomona. One of four hundred eighty kidney sample was PCR positive, which belongs to genomospecies of L. interrogans after an initial examination. One of the seventy eight kidney samples which have white spot was IHC positive. The results of Warthin-Starry staining and culture are all negative. McNemar test There are significant association between both macroscopic renal lesions, multi-focal interstitial nephritis (MFIN) and anti-leptospiral antibody (p<0.01) by McNemar test. The study in Hualien and Taitung counties, we evaluated the serology and risk factors in 19 dairy farms. The positive rate of all herds were 100%. Total positive rate was 37.8% (263/695), which had titres between 1:100 and 1:400 with geometric mean titer equal to 1:131. The most serogroup was Shermani. Main risk factor associated with seropositive was presence of animals around the farm (OR=1.7; p<0.01). This serological results probably reflected natural exposure because vaccination of cattle against leptospirosis was not practiced in Taiwan. These results represent economic loss and potential threats to human health. From the investigation, bovine leptospirosis is prevalent in Taiwan. A successful control program for bovine leptospirosis should enforce a comprehensive investigation on the infection and the herd management practices.中文摘要 Ⅰ 英文摘要 Ⅱ 目錄 Ⅲ 圖次 Ⅳ 表次 Ⅴ 第一章 緒言 1 第二章 文獻回顧 3 第一節 鉤端螺旋體症之歷史 4 第二節 病性鉤端螺旋體特性與分類 6 第三節 台灣和國際鉤端螺旋體症之流行病學 11 第四節 牛鉤端螺旋體症之臨床症狀、診斷、治療、控制與預防 21 第三章 屠宰場牛鉤端螺旋體症流行病學調查 28 第一節 摘要 29 第二節 前言 31 第三節 材料與方法 33 3-1 樣本收集 33 3-2血清抗體檢測 33 3-3 屠宰場牛隻鉤端螺旋體病原分離 36 3-4 腎臟組織聚合酶鏈反應 36 3-5腎臟組織鍍銀染色 38 3-6 腎臟免疫組織化學染色 39 3-7 統計方法 40 第四節 結果 41 4-1 血清抗體檢測結果 41 4-2 鉤端螺旋體病原分離結果 43 4-3 聚合酶鏈反應結果 43 4-4 鍍銀染色結果 45 4-5 免疫組織化學染色結果 45 4-6 血清學檢測結果與腎臟肉眼病變之相關性 46 4-7 血清學檢測結果與腎臟局部間質性腎炎之相關性 47 第五節 討論 48 第六節 結論 51 第四章 乳牛場牛鉤端螺旋體症流行病學調查 52 第一節 摘要 53 第二節 前言 54 第三節 材料與方法 55 3-1 血清之收集與問卷調查 55 3-2 血清抗體偵測 55 3-3 統計方法 56 第四節 結果 58 4-1 血清學結果 58 4-2 危險因子之探討 60 第五節 討論 62 第六節 結論 64 第五章 總結與展望 65 參考文獻 66 附錄 8