乳牛結核菌素試驗陽性場併用皮內結核菌素試驗、酵素結合免疫吸附法和駢比試驗對檢出感染牛之效果

The Efficacy of Combining Intradermal Tuberculin Test (ITT), Enzyme-linked Immunosorbent Assay and Comparative Test on Detecting Infected Cattles of ITT-positive Dairy Herds

陳振茂(Cheng-Mao Chen)

指導教授 ： 吳永惠

國立屏東科技大學/農學院/獸醫學系所/碩士(2013年)

https://doi.org/10.6346/NPUST.2013.00074

摘要

我國乳牛結核病 ( bovine tuberculosis，TB ) 污染場TB難以清淨原因之一被認為是因為皮內結核菌素試驗 ( intradermal tuberculin test；ITT ) 之敏感性不夠高所致。因此，臨床上常被建議併用ITT與其它檢驗方法以改善檢出率。本研究之目的在評估ITT併用酵素結合免疫吸附法 ( enzyme-linked immunosorbent assay，ELISA ) 或迅速法 ( rapid test，RT ) 是否能有效的檢出結核病感染牛，以及駢比皮內結核菌素試驗 ( 駢比試驗。comparative intradermal tuberculin test；CITT ) 是否能正確的鑑別出‘真正的’牛型結核病感染牛場。總計有248頭來自ITT陽性場牛隻，包括ITT和ELISA均呈陽性反應牛11頭、ITT陽性但ELISA陰性牛218頭、ITT陰性但ELISA陽性牛13頭及ITT和ELISA均陰性牛6頭，被行ITT、ELISA ( 和/或RT )、剖檢檢查咽背和縱膈淋巴結及肺臟有無肉眼病變、以及淋巴結和肺臟病變之牛型分枝桿菌 ( Mycobacterium bovis, M. bovis ) 分離。結果若以肉眼病變為診斷標準 ( gold standard )，相符率在單用一種檢驗法時以RT為最高 ( 77.0%，94/122 )，其次為ELISA ( 70.2%，165/235 )、ITT ( 31.4%，77/245 )；併用兩種檢驗法時，ITT＋ELISA ( 25.9%，56/216 )、ITT＋RT ( 24.4%，40/164 )。敏感性在單用一種檢驗法時，以ITT為最高 ( 86.8%，53/61 ) 為最高，其次為RT ( 45.0%，9/20 )、ELISA ( 13.3%，8/60 )；併用兩種檢驗法時，ITT＋ELISA ( 98.1%，54/55 )、ITT＋RT ( 94.5%，35/37 )。特異性在單用一種檢驗法時，以ELISA ( 89.6%，156/174 ) 為最高，其次為RT ( 83.1%，84/101 )、ITT ( 12.5%，23/183 )；併用兩種檢驗法時，ITT＋ELISA ( 1.9%，3/157 )、ITT＋RT ( 3.1%，4/126 )。另方面，若以M. bovis分離結果作為診斷標準，相符率在單用一種檢驗法時，亦以RT為最高 ( 80.5%，91/113 )，其次為ELISA ( 73.3%，129/176 )、ITT ( 25.3%，45/178 )；在併用兩種檢驗法時，ITT＋ELISA ( 12.8%，14/109 )、ITT＋RT ( 8.5%，8/94 )。敏感性在單用一種檢驗方法時，亦以ITT ( 87.8%，36/41 ) 為最高，其次為RT ( 50.0%，7/14 )、ELISA ( 14.6%，6/41 )；在併用兩種檢驗法時，ITT＋ELISA ( 100%，13/13 )、ITT＋RT ( 100%，7/7 )。特異性在單用一種檢驗法時，亦以ELISA ( 90.2%，121/134 ) 為最高，其次為RT ( 84.6%，83/98 )、ITT ( 8.8%，12/136 )；在併用兩種檢驗法時，ITT＋ELISA ( 1.0%，1/94 )、ITT＋RT ( 1.1%，1/85 )。由以上結果，無論以肉眼病變或M. bovis 分離結果作為診斷標準，敏感性在細胞媒介性免疫 ( cell-mediated immunity ) 檢驗法 ( ITT ) 顯著優於體液性免疫 ( humoral immunity ) 檢驗法 ( ELISA 和RT ) ( P<0.05 )，但相符率則體液性免疫檢驗法顯著優於細胞媒介性免疫檢驗法 ( P<0.05 )，顯示這些ITT陽性檢出牛絕大多數為早期TB感染牛；併用兩種生前檢驗法可顯著提高敏感性到100% ( P<0.05 )。在駢比試驗 ( CITT ) 方面，由18場確認有肉眼病變和/或有分離到M. bovis之ITT陽性場，共151頭包括145頭ITT陽性反應牛和6頭ITT陰性反應牛，在開始行尾根皺襞結核菌素試驗 ( ITT ) 後10天內被作頸部CITT檢查，結果顯示CITT不能完全檢定ITT陽性牛是否有肉眼病變和/或M. bovis感染 ( 分離陽性 )，但可確認CITT陰性牛並不會出現肉眼病變，也不會分離到M. bovis。另方面，自上述18場ITT陽性乳牛場逢機篩選10場10次67頭CITT檢查結果來分析，結果顯示，CITT陽性牛常被發現於各場，CITT疑陽性牛之比率 ( 43.3%，29/67 ) 顯著高於 CITT陰性牛 ( 23.9%，16/67 ) ( P<0.05 )；此外，由8場肉眼病變和/或M. bovis分離均為陰性之首次ITT陽性乳牛場27頭來分析，結果顯示均無CITT陽性牛，CITT陰性牛之比率 ( 66.7%，18/27 ) 顯著高於 CITT疑陽性牛 ( 33.3%，9/27 ) ( P<0.05 )。由以上結果，推測這種新陽性場之ITT陽性牛不是M. bovis感染，而有可能是非結核性分枝桿菌 ( nontuberculous mycobacteria ) 感染。
關鍵字：牛結核病、牛結核菌素試驗、牛結核病酵素免疫結合吸附法、牛結核病快速法、牛皮內結核菌素駢比試驗

關鍵字

牛結核病 ； 牛結核菌素試驗 ； 牛結核病酵素免疫結合吸附法 ； 牛結核病快速法 ； 牛皮內結核菌素駢比試驗

並列摘要

One of the cause to eradicate the tuberculosis ( TB ) with difficulty in the infected dairy herds in Taiwan was considered that the sensitivity of intradermal tuberculin test ( ITT ) is not enough. Therefore, combining the ITT with other diagnostic methods to improve the detecting rate was suggested. The aims of this study are to evaluate the efficacy of combining ITT with enzyme-linked immunosorbent assay ( ELISA ) or rapid test ( RT ) on effectively detecting the TB-infected dairy cattles, and the ability of comparative intradermal tuberculin test ( CITT ) for accurately identifing the ‘true’ bovine tuberculosis-infected herds. A total of 248 cattles from ITT-positive farms, including 11 of ITT- positive and ELISA- positive, 218 of ITT- positive but ELISA- negative, 13 of ITT-negative but ELISA-positive and 6 of ITT-negative and ELISA-negative cattles, were performed with the ITT, ELISA ( and / or RT ), gross lesion examinations of retropharyngeal and mediastinal lymph nodes and lung in the necropsy, and Mycobacterium bovis ( M. bovis ) isolation of the lymph nodes and lung lesions. If using the results of gross lesion as the diagnostic standard, the results showed that the highest coincidence is RT ( 77.0%, 94/122 ), followed by ELISA ( 70.2%, 165/235 ), and ITT ( 31.4%, 77/245 ) in the single diagnostic tests, and ITT+ELISA ( 25.9%, 56/216 ) and ITT+RT ( 24.4%, 40/164 ) in the combination of the two diagnostic tests. The highest sensitivity is ITT ( 86.8%, 53/61 ), follow by RT ( 45.0%, 9/20 ), and ELISA ( 13.3%, 8/60 ) in the single diagnostic tests, and ITT+ELISA ( 98.1%, 54/55 ) and ITT+RT ( 94.5%, 35/37 ) in the combination of the two diagnostic tests. The highest specificity is ELISA ( 89.6%, 156/174 ), followed by RT ( 83.1%, 84/101 ), and ITT ( 12.5%, 23/183 ) in the single diagnostic tests, and ITT+ELISA ( 1.9%, 3/157 ) and ITT+RT ( 3.1%, 4/126 ) in the combination of the two diagnostic tests. On the other hand, if using the results of M. bovis isolation as the diagnostic standard, the highest coincidence is also RT ( 80.5%, 91/113 ), followed by ELISA ( 73.3%, 129/176 ), and ITT ( 25.3%, 45/178 ) in the single diagnostic tests, and ITT+ELISA ( 12.8%, 14/109 ) and ITT+RT ( 8.5%, 8/94 ) in the combination of the two diagnostic tests. The highest sensitivity is also ITT ( 87.8%, 36/41 ), follow by RT ( 50.0%, 7/14 ), and ELISA ( 14.6%, 6/41 ) in the single diagnostic tests, and ITT+ELISA ( 100%, 13/13 ) and ITT+RT ( 100%, 7/7 ) in the combination of the two diagnostic tests. The highest specificity is also ELISA ( 90.2%, 121/134 ), followed by RT ( 84.6%, 83/98 ), and ITT ( 8.8%, 12/136 ) in the single diagnostic tests, and ITT+ELISA ( 1.0%, 1/94 ) and ITT+RT ( 1.1%, 1/185 ) in the combination of the two diagnostic tests. According above results, the coincidence of the humoral immunity tests ( ELISA and RT ) is significantly ( P<0.05 ) better than the cell-mediated immunity test ( ITT ), and the sensitivity of the cell-mediated immunity test ( ITT ) is significantly ( P<0.05 ) better than the humoral immunity tests ( ELISA and RT ), whether using the results of gross lesion or isolation as the diagnostic standard. The results indicated that the majority of these ITT-positive cattles were in the early stages of TB infection. Combining two of antemortem diagnostic tests could significantly increase the sensitivity to 100% ( P<0.05 ). On the results of the CITT test, a total of 151 cattles included 145 ITT- positive and 6 ITT-negative reactors from 18 ITT-positive dairy farms were conducted the cervical CITT test within 10 days of the initial caudal-fold tuberculin test ( ITT ). The results showed that CITT couldn’t completely certified the ITT-positive reactors whether to have gross lesion and/or M. bovis infection ( isolation-positive ), but can confirmed the CITT-negative reactors to haven’t gross lesion and/or M. bovis infection ( isolation-negative ). On the other hand, 10 times of CITT in the 10 farms randomly selected from above 18 ITT-positive dairy farms, a total of 67 cattles were analysed. The results showed that CITT-positive reactors were commonly found in each farm, and the ratio of the CITT-suspected reactors ( 43.3%，29/67 ) is significantly ( P<0.05 ) higher than CITT-negative reactors ( 23.9%，16/67 ). Besides, a total of 27 cattles from 8 initial ITT-positive dairy farms, which no cattle with gross lesion and M. bovis isolation was found, were analysed. The results showed that no CITT-positive reactors was found, and the ratio of the CITT-negative reactors ( 66.7%，18/27 ) was significantly higher than CITT-suspected reactors ( 33.3%，9/27 ) ( P<0.05 ). According to the above results, we presumed the ITT-positive reactor isn’t the M. bovis infection, and maybe the infection of nontuberculous mycobacteria.

Key words：bovine tuberculosis, intradermal tuberculin test, enzyme-linked immunosorbent assay for bovine tuberculosis, rapid test for bovine tuberculosis, comparative intradermal tuberculin test

並列關鍵字

bovine tuberculosis ； intradermal tuberculin test ； enzyme-linked immunosorbent assay for bovine tuberculosis ； rapid test for bovine tuberculosis ； comparative intradermal tuberculin test

參考文獻

行政院農業委員會動植物防疫檢疫局。牛結核病檢驗方法。台北市，行

吳永惠。牛病學。藝軒，台北，141-145，2000。

葉坤松、吳永惠、廖明輝、劉宏仁、張清棟、蕭終融。台灣乳牛場結核病牛難以根除之原因探討。台灣獸醫誌 30：56-63，2004。

蕭終融。結核病。行政院農業委員會家畜衛生試驗所，論文專輯，1991。2010年，引自：

王金和、吳義興、陳素貞。美製純蛋白結核菌素在台灣使用之評估。中華民國獸醫學會雜誌。10：1-4，1984。