臺灣博碩士論文加值系統

目前分布在世界上的分枝桿菌約有一百多種，為數眾多的一部分存在於環境中的水或是土壤等腐生環境當中，稱為環境分枝桿菌，當牛隻暴露在環境分枝桿菌之下，有可能會造成非特異性致敏化，進而干擾結核菌素皮內注射檢測牛隻感染牛型分枝桿菌的診斷。目前台灣在乳牛的結核病根除計畫的執行上仍以結核菌素皮內注射作為標準的檢測工具，只要檢測結果為陽性的牛隻會立即撲殺。本實驗室於2009年4月至2010年8月為止，共針對115頭結核菌素陽性撲殺牛隻進行剖檢及採樣，將咽背淋巴結送往淡水家畜衛生試驗所進行培養之後，依其培養結果將各牛隻所屬的牧場進行環境採樣並分組。
本研究中使用聚合酶鏈鎖反應，針對大部分環境分枝桿菌皆有的熱休克蛋白65 (heat shock protein 65, hsp65)基因片段檢測環境樣本所含的菌叢。共計檢測16個牧場，將其分為三個組別，第一個組別為有檢出結核菌素陽性的牛隻，且牛隻剖檢後於咽背淋巴結可以培養出牛型分枝桿菌的牧場，這類牧場中所採集的環境樣本總數為105個，PCR陽性率為50.58% (53/105)；第二個組別所對應的牧場為該場有結核菌素陽性的牛隻，但是咽背淋巴結分離牛型分枝桿菌的結果為陰性，懷疑這些牛隻有結核菌素偽陽性的情形，這類牧場共採樣194個環境樣本，PCR陽性率為78.35% (152/194)；第三個組別為結核菌素測試為陰性的牧場，總環境樣本數為88個，陽性率為60.23% (53/88)；結果顯示第二個分組的環境分枝桿菌陽性率顯著高於另外兩組 (p<0.05)，牛隻在這類牧場中有較高的機率接觸到環境分枝桿菌，造成非特異性致敏化的風險也較高。另外為了要區分在牧場的欄舍當中到底是哪一個區域有特別高的風險來接觸這些環境中的分枝桿菌，以區域劃分作為分組，可發現各牧場中的運動場區的分枝桿菌檢測的陽性率顯著高於其他畜欄。
將所有的PCR陽性所偵測到的序列定序之後繪製親緣樹狀圖，發現有些菌叢僅存在於特定的牧場分組當中，其中僅B組的牧場能偵測到的菌叢數量特別多，共有14個菌叢，且B組中的6個牧場全部都有這些菌叢的分布，這一些菌叢無法在A、C組別中檢測出來，表示B組環境確實有別於其他分組之處。有些環境樣本序列和結核菌素陽性牛隻組織、血液以及乳汁中所偵測到的序列親緣分析上屬於同一菌叢，也許此菌叢的確即為造成牛隻結核菌素檢測偽陽性的病原，此牧場需特別注意此等菌的分布情形方能進行有效的預防消毒。
結核菌素偽陽性牛隻的存在會造成政府以及酪農的經濟損失，本研究依據每個不同的牧場皆有繪製地圖，評估其高風險菌叢的位置並標示出來，期望對於這些牧場防治牛隻結核菌素的偽陽性有一定的幫助。

Among the more than 100 known species of the genus Mycobacterium, majority of them are described as “environmental mycobacteria” (EM), present as saprophytes in water or soil worldwide. Several species of EM are known as opportunistic pathogens and may lead to false positive diagnosis of bovine tuberculosis by intradermal tuberculin test (ITT). In Taiwan, ITT is a standard diagnostic tool for tuberculosis eradication program in dairy herds and cattle showing positive reaction are culled immediately. However, from April of 2009 to August of 2010, approximately 32％ (37/115) of the cattle culled did not exhibit typical tubercle lesions grossly or histopathologically, nor M. bovis or M. tuberculosis was detected by polymerase chain reaction (PCR).
In the present study, we used PCR to detect the heat shock protein 65 (hsp65) gene fragments of EM present in various environmental samples to evaluate the distribution of EM flora in dairy farms displaying ITT-positive result, as well as in farms with ITT-negative result as the control group. A total of 16 dairy farms were included and divided into three groups. Group A included farms which had ITT-positive history and M. bovis could be isolated from the retropharyngeal lymph node; within the 105 environmental samples of group A, the positive rate of hsp65 PCR was 50.58%. Group B were farms which had ITT-positive history but no M. bovis could be isolated from the retropharyngeal lymph nodes, indicating that these farms might be ITT false-positive; within the 194 environmental samples of group B, the positive rate of hsp65 PCR was 78.35%. Group C were farms in which dairy cattle displayed ITT-negative history; within the 88 environmental samples of group C, the positive rate of HSP65 PCR was 60.23%. The results showed that the positive rate of hsp65 PCR from Group B was clearly higher than those of group A and C (p&lt;0.05), the cattle in these farms might have a higher chance to contact EM resulting in non-specific sensitization. To further investigate where in the farms that the cattle had higher risk of exposing to EM, each farm was divided into several different regions and it was found that the sport fields in these farms had much higher positive rates of hsp65 PCR than in other regions.
The hsp65 PCR products were sequenced and subjected to phylogenetic analysis. It was found that fourteen mycobacterial flora could only be found in all of the six farms of group B, suggesting that the EM in group B was really different from group A and group C. The results of phylogenetic analysis of the sequences of the hsp65 PCR products from environmental samples and from the milk, tissue, and blood of cattle from these farms of group B strongly supported that the EM flora should contribute to the ITT-false positivity.
Such false-positive reactors have caused severe, unnecessary economic losses not only to the producers but also to the government. Therefore, knowing the EM flora present in cattle farms and whether theses EM flora may have influence on the ITT results is rather important.

中文摘要 i
英文摘要 iii

目錄 I
表目錄 IV
圖目錄 V

第一章、前言…………………………………………………………………………1
第二章、文獻回顧……………………………………………………………………3
第一節 分枝桿菌簡介 3
1.1 形態學 3
1.2染色特性 3
1.3 分枝桿菌之區分 3
1.4 分枝桿菌在環境的抵抗性 4
第二節 分枝桿菌的分類 4
2.1 依據生長特性分類 5
2.2 依據致病性以及環境分佈分類 6
2.3 具有致病性之分枝桿菌 7
第三節 傳播途徑 8
第四節 環境分枝桿菌的診治現況 10
第五節 結核菌素試驗 11
5.1 舊式結核菌素 11
5.2 純化蛋白結核菌素 11
5.3 駢比試驗 12
第六節 影響結核菌素反應準確性之原因 12
6.1 偽陰性之發生原因 13
6.2 偽陽性之發生原因 14
第七節 特定分枝桿菌抗原致敏素 14
第八節 分枝桿菌親緣性分析 15
第九節 結核病相關法規及台灣地區乳牛結核病之檢驗 18
第十節 台灣環境分枝桿菌檢測的研究 21
第三章 實驗材料與方法 23
第一節 前言 23
第二節 實驗設計 24
第三節 材料 25
3.1 標準菌株 25
3.2 人工合成引子 25
3.3 反應試劑 25
3.3.1 環境樣本之核酸萃取 25
3.3.2 組織、乳汁以及血液樣本之核酸萃取 26
3.3.3 聚合酶鏈鎖反應 26
3.3.4 DNA洋菜膠片電泳分析 27
3.4 採樣準備用品 27
3.4.1 環境樣本採樣前準備物品 27
3.4.2 組織、血液以及乳汁樣本採樣前準備物品 27
第四節 方法 27
4.1 乳牛牧場分組 27
4.2 結核菌素陽性牛隻咽背淋巴結以及血液和乳汁樣本採樣 28
4.3 環境樣本採樣 28
4.4 結核菌素陽性牛隻咽背淋巴結、乳汁及血液核酸萃取 29
4.5 環境樣本核酸之萃取 30
4.6 聚合酶鏈鎖反應 30
4.7 DNA洋菜膠片電泳分析 31
4.8 統計分析 32
4.9 基因序列之親緣樹狀圖繪製 33
第四章 結果 34
第一節 樣材蒐集 34
1.1 結核菌素陽性牛隻咽背淋巴結、乳汁及血液樣材之蒐集 34
1.2 環境樣本樣材之蒐集 34
第二節 聚合酶鏈鎖反應結果 35
2.1 結核菌素皮內注射陽性牛隻咽背淋巴以及血液樣本 35
2.2 環境樣本 35
第三節 環境樣本hsp65聚合酶鏈鎖反應結果統計分析 36
第四節 基因定序親緣樹狀圖繪製 38
第五節 聚合酶鏈鎖反應及親緣分析結果合併牧場地圖 39
第五章 討論 68
第一節 聚合酶鏈鎖反應 68
1.1 以結核菌素皮內注射以及牛型分枝桿菌培養結果作為分組 68
1.2 以牧場內區域劃分作為分組 69
1.3 各分組之組內比較 69
1.4 以地理位置作為分組 70
第二節 親緣樹狀圖 71
第三節 聚合酶鏈鎖反應及親緣分析結果合併牧場地圖 73
第六章 結論 75
第七章 參考文獻 76
附錄一 84
附錄二 88

余珍芳。1993。圈飼野生動物及乳牛結核病之研究。碩士論文。國立台灣大學獸醫學研究所。
吳雅蕾。1997。台灣地區Mycobacterium fortuitum之生態分佈、藥物感受性試驗、質體相及核糖體DNA限制酶圖譜。碩士論文。國立台灣大學獸醫學研究所。
吳義興。1980。台灣現行牛結核菌素之製造及應用之檢討。碩士論文。國立台灣大學獸醫學研究所。
姜臺生。1993。牛結核菌素皮內反應法與伽瑪干擾素測試法之比較。碩士論文。國立中興大學獸醫學研究所。
陳敬雄、王雅萍。2010。非結核分枝桿菌診治的現況與臨床困境。台灣醫界53：No. 10。
羅永村。1995。乳牛結核菌素非特異性反應之探討。碩士論文。國立台灣大學獸醫學研究所。
Adekambi, T., Drancourt, M., 2004, Dissection of phylogenetic relationships among 19 rapidly growing Mycobacterium species by 16S rRNA, hsp65, sodA, recA and rpoB gene sequencing. Int J Syst Evol Microbiol 54, 2095-2105.
Bechelli, L.M., Barrai, I., Garbajosa, P.G., Uemura, K., Mg Mg, G., Tamondong, C., 1973, Correlation between leprosy rates in villages different distances apart. Bull World Health Organ 48, 257-260.
Berghaus, R.D., Farver, T.B., Anderson, R.J., Jaravata, C.C., Gardner, I.A., 2006, Environmental sampling for detection of Mycobacterium avium ssp. paratuberculosis on large California dairies. J Dairy Sci 89, 963-970.
Biet, F., Boschiroli, M.L., Thorel, M.F., Guilloteau, L.A., 2005, Zoonotic aspects of Mycobacterium bovis and Mycobacterium avium-intracellulare complex (MAC). Vet Res 36, 411-436.
Brown-Elliott, B.A., Wallace, R.J., Jr., 2002, Clinical and taxonomic status of pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria. Clin Microbiol Rev 15, 716-746.
Brown, P.E., Christensen, O.F., Clough, H.E., Diggle, P.J., Hart, C.A., Hazel, S., Kemp, R., Leatherbarrow, A.J., Moore, A., Sutherst, J., Turner, J., Williams, N.J., Wright, E.J., French, N.P., 2004, Frequency and spatial distribution of environmental Campylobacter spp. Appl Environ Microbiol 70, 6501-6511.
Brunello, F., Ligozzi, M., Cristelli, E., Bonora, S., Tortoli, E., Fontana, R., 2001, Identification of 54 mycobacterial species by PCR-restriction fragment length polymorphism analysis of the hsp65 gene. J Clin Microbiol 39, 2799-2806.
Burl, S., Adetifa, U.J., Cox, M., Touray, E., Whittle, H., McShane, H., Rowland-Jones, S.L., Flanagan, K.L., 2010, The tuberculin skin test (TST) is affected by recent BCG vaccination but not by exposure to non-tuberculosis mycobacteria (NTM) during early life. PLoS One 5, e12287.
Chilima, B.Z., Clark, I.M., Floyd, S., Fine, P.E., Hirsch, P.R., 2006, Distribution of environmental mycobacteria in Karonga District, northern Malawi. Appl Environ Microbiol 72, 2343-2350.
Corner, L.A., 2006, The role of wild animal populations in the epidemiology of tuberculosis in domestic animals: how to assess the risk. Vet Microbiol 112, 303-312.
Dai, J., Chen, Y., Dean, S., Morris, J.G., Salfinger, M., Johnson, J.A., 2010, Multiple Genome Comparison Reveals New Loci for Mycobacterium Species Identification. J Clin Microbiol.
Dai, J., Chen, Y., Dean, S., Morris, J.G., Salfinger, M., Johnson, J.A., 2011, Multiple-genome comparison reveals new loci for Mycobacterium species identification. J Clin Microbiol 49, 144-153.
Davis, B. D., R. Dulbecoo and H. N. Eisen, 1990, Microbiology, 4th ed. pp. 647-664. J. B. Cippincott Company, Pennsylvania.
de Lisle, G.W., Bengis, R.G., Schmitt, S.M., O''Brien, D.J., 2002, Tuberculosis in free-ranging wildlife: detection, diagnosis and management. Rev Sci Tech 21, 317-334.
de Lisle, G.W., Mackintosh, C.G., Bengis, R.G., 2001, Mycobacterium bovis in free-living and captive wildlife, including farmed deer. Rev Sci Tech 20, 86-111.
Del Portillo, P., Murillo, L.A., Patarroyo, M.E., 1991, Amplification of a species-specific DNA fragment of Mycobacterium tuberculosis and its possible use in diagnosis. J Clin Microbiol 29, 2163-2168.
Devallois, A., Goh, K.S., Rastogi, N., 1997, Rapid identification of mycobacteria to species level by PCR-restriction fragment length polymorphism analysis of the hsp65 gene and proposition of an algorithm to differentiate 34 mycobacterial species. J Clin Microbiol 35, 2969-2973.
Domenech, P., Jimenez, M.S., Menendez, M.C., Bull, T.J., Samper, S., Manrique, A., Garcia, M.J., 1997, Mycobacterium mageritense sp. nov. Int J Syst Bacteriol 47, 535-540.
Du, Y., Qi, Y., Yu, L., Lin, J., Liu, S., Ni, H., Pang, H., Liu, H., Si, W., Zhao, H., Wang, C., 2011, Molecular characterization of Mycobacterium tuberculosis complex (MTBC) isolated from cattle in northeast and northwest China. Res Vet Sci 90, 385-391.
Edwards, L.B., Acquaviva, F.A., Livesay, V.T., Cross, F.W., Palmer, C.E., 1969, An atlas of sensitivity to tuberculin, PPD-B, and histoplasmin in the United States. Am Rev Respir Dis 99, Suppl:1-132.
Enwall, K., Throback, I.N., Stenberg, M., Soderstrom, M., Hallin, S., 2010, Soil resources influence spatial patterns of denitrifying communities at scales compatible with land management. Appl Environ Microbiol 76, 2243-2250.
Falkinham, J.O., 3rd, 2002, Nontuberculous mycobacteria in the environment. Clin Chest Med 23, 529-551.
Farhat, M., Greenaway, C., Pai, M., Menzies, D., 2006, False-positive tuberculin skin tests: what is the absolute effect of BCG and non-tuberculous mycobacteria? Int J Tuberc Lung Dis 10, 1192-1204.
Fine, P.E., Floyd, S., Stanford, J.L., Nkhosa, P., Kasunga, A., Chaguluka, S., Warndorff, D.K., Jenkins, P.A., Yates, M., Ponnighaus, J.M., 2001, Environmental mycobacteria in northern Malawi: implications for the epidemiology of tuberculosis and leprosy. Epidemiol Infect 126, 379-387.
Francis, J., Choi, C.L., Frost, A.J., 1973, The diagnosis of tuberculosis in cattle with special reference to bovine PPD tuberculin. Aust Vet J 49, 246-251.
Goren, M.B., Cernich, M., Brokl, O., 1978, Some observations of mycobacterial acid-fastness. Am Rev Respir Dis 118, 151-154.
Gutierrez, M., Tellechea, J., Garcia Marin, J.F., 1998, Evaluation of cellular and serological diagnostic tests for the detection of Mycobacterium bovis-infected goats. Vet Microbiol 62, 281-290.
Horsburgh, C.R., Jr., Selik, R.M., 1989, The epidemiology of disseminated nontuberculous mycobacterial infection in the acquired immunodeficiency syndrome (AIDS). Am Rev Respir Dis 139, 4-7.
Jones TC and Hunt RD, 1983, Diseased caused by higher bacteria and fungi. In: Veterinary pathology. 5th ed. pp: 638-718. Philadelphia, Lea and Febiger.
Kelly, G.E., McGrath, G.E., More, S.J., 2010, Estimating the extent of spatial association of Mycobacterium bovis infection in badgers in Ireland. Epidemiol Infect 138, 270-279.
Kerr, W.R., Lamont, H.G., Mc, G.J., 1946, Studies on tuberculin sensitivity in the bovine; introduction. Vet Rec 58, 443-446.
Kim, B.J., Lee, S.H., Lyu, M.A., Kim, S.J., Bai, G.H., Chae, G.T., Kim, E.C., Cha, C.Y., Kook, Y.H., 1999, Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpoB). J Clin Microbiol 37, 1714-1720.
Kim, H., Kim, S.H., Shim, T.S., Kim, M.N., Bai, G.H., Park, Y.G., Lee, S.H., Cha, C.Y., Kook, Y.H., Kim, B.J., 2005a, PCR restriction fragment length polymorphism analysis (PRA)-algorithm targeting 644 bp Heat Shock Protein 65 (hsp65) gene for differentiation of Mycobacterium spp. J Microbiol Methods 62, 199-209.
Kim, H., Kim, S.H., Shim, T.S., Kim, M.N., Bai, G.H., Park, Y.G., Lee, S.H., Chae, G.T., Cha, C.Y., Kook, Y.H., Kim, B.J., 2005b, Differentiation of Mycobacterium species by analysis of the heat-shock protein 65 gene (hsp65). Int J Syst Evol Microbiol 55, 1649-1656.
Kirschner, P., Springer, B., Vogel, U., Meier, A., Wrede, A., Kiekenbeck, M., Bange, F.C., Bottger, E.C., 1993, Genotypic identification of mycobacteria by nucleic acid sequence determination: report of a 2-year experience in a clinical laboratory. J Clin Microbiol 31, 2882-2889.
Kopecky, K.E., Larsen, A.B., Packer, R.A., 1968, Evaluation of an intravenous tuberculin test in cattle. Am J Vet Res 29, 31-38.
Lamont, H.G., 1947, Tuberculin testing. Vet Rec 59, 407-409.
Larsen, A.B., Kopecky, K.E., 1965, An Intravenous Tuberculin Test for Cattle. Am J Vet Res 26, 676-678.
Lee, C.K., Gi, H.M., Cho, Y., Kim, Y.K., Lee, K.N., Song, K.J., Song, J.W., Park, K.S., Park, E.M., Lee, H., Bai, G.H., 2004, The genomic heterogeneity among Mycobacterium terrae complex displayed by sequencing of 16S rRNA and hsp 65 genes. Microbiol Immunol 48, 83-90.
Lepper, A.W., Newton-Tabrett, D.A., Corner, L.A., Carpenter, M.T., Scanlan, W.A., Williams, O.J., Helwig, D.M., 1977, The use of bovine PPD tuberculin the single caudal fold test to detect tuberculosis in beef cattle. Aust Vet J 53, 208-213.
Lin, M.Y., Reddy, T.B., Arend, S.M., Friggen, A.H., Franken, K.L., van Meijgaarden, K.E., Verduyn, M.J., Schoolnik, G.K., Klein, M.R., Ottenhoff, T.H., 2009, Cross-reactive immunity to Mycobacterium tuberculosis DosR regulon-encoded antigens in individuals infected with environmental, nontuberculous mycobacteria. Infect Immun 77, 5071-5079.
Marras, T.K., Chedore, P., Ying, A.M., Jamieson, F., 2007, Isolation prevalence of pulmonary non-tuberculous mycobacteria in Ontario, 1997 2003. Thorax 62, 661-666.
Moore, J.E., Kruijshaar, M.E., Ormerod, L.P., Drobniewski, F., Abubakar, I., 2010, Increasing reports of non-tuberculous mycobacteria in England, Wales and Northern Ireland, 1995-2006. BMC Public Health 10, 612.
Murohashi, T., Kondo, E., Yoshida, K., 1969, The role of lipids in acid-fastness of mycobacteria. Am Rev Respir Dis 99, 794-798.
Ninet, B., Monod, M., Emler, S., Pawlowski, J., Metral, C., Rohner, P., Auckenthaler, R., Hirschel, B., 1996, Two different 16S rRNA genes in a mycobacterial strain. J Clin Microbiol 34, 2531-2536.
Norton, S., Corner, L.A., Morris, R.S., 2005, Ranging behaviour and duration of survival of wild brushtail possums (Trichosurus vulpecula) infected with Mycobacterium bovis. N Z Vet J 53, 293-300.
Palmer, C.E., Long, M.W., 1966, Effects of infection with atypical mycobacteria on BCG vaccination and tuberculosis. Am Rev Respir Dis 94, 553-568.
Paterson, A. B., 1959, Tuberculosis: Bacteriology. In: Stable Forth, A. W., Galloway and I. A. Ed. Infections Disease of animals Disease Due to Bacteria. 2nd ed. pp: 617-687. Butter worths, London.
Pavlik, I., Dvorska, L., Matlova, L., Svastova, P., Parmova, I., Bazant, J., Veleba, J., 2002, Mycobacterial infections in cattle in the Czech Republic during 1990-1999. Vet Med-Czech 47, 241-250.
Pfyffer, G. E., Brown-Elliot, B. A., Wallace, R. J., Jr., 2003, Mycobacterium: general characteristics, isolation, and staining procedures. In: Manual of Clinical Microbiology. 8th ed. pp. 532-559.
Pillai, S.D., Widmer, K.W., Ivey, L.J., Coker, K.C., Newman, E., Lingsweiler, S., Baca, D., Kelley, M., Davis, D.S., Silvy, N.J., Adams, L.G., 2000, Failure to identify non-bovine reservoirs of Mycobacterium bovis in a region with a history of infected dairy-cattle herds. Prev Vet Med 43, 53-62.
Quinn, P.J., Markey BK, Carter ME, Donnelly WJ and Leonard FC, 2002, Mycobacterium species. In: Veterinary microbiology and microbial disease 1st ed. pp. 97-105. Blackwell.
Quinn, P.J., M. E. Carter, B. Markey and G. R. Carter, 1994, Mycobacterium species. In: Quinn, P. J., M. E. Carter, B. Markey and G. R. Carter. Ed. Clinical Veterinary Microbiology. pp. 156-169. Virginia, Wolfe.
Radunz, B.L., Lepper, A.W., 1985, Suppression of skin reactivity to bovine tuberculin in repeat tests. Aust Vet J 62, 191-194.
Rao, S., Kitron, U., Weigel, R.M., 2010, Spatial and genotypic clustering of Salmonella over time in a swine production unit. Prev Vet Med 97, 90-99.
Reischl, U., Feldmann, K., Naumann, L., Gaugler, B.J., Ninet, B., Hirschel, B., Emler, S., 1998, 16S rRNA sequence diversity in Mycobacterium celatum strains caused by presence of two different copies of 16S rRNA gene. J Clin Microbiol 36, 1761-1764.
Robert, G. D., E. W. Koneman and T.K. Kim, 1991, Mycobacterium. In: Manual of clinical microbiology 5th ed. pp. 304-337, AMS, District of Coumbra.
Rodriguez, J.G., Mejia, G.A., Del Portillo, P., Patarroyo, M.E., Murillo, L.A., 1995, Species-specific identification of Mycobacterium bovis by PCR. Microbiology 141 ( Pt 9), 2131-2138.
Runyon, E.H., 1959, Anonymous mycobacteria in pulmonary disease. Med Clin North Am 43, 273-290.
Salman, M., 2010, Preventive veterinary medicine-What has changed in 2009? Prev Vet Med 93.
Schinsky, M.F., Morey, R.E., Steigerwalt, A.G., Douglas, M.P., Wilson, R.W., Floyd, M.M., Butler, W.R., Daneshvar, M.I., Brown-Elliott, B.A., Wallace, R.J., Jr., McNeil, M.M., Brenner, D.J., Brown, J.M., 2004, Taxonomic variation in the Mycobacterium fortuitum third biovariant complex: description of Mycobacterium boenickei sp. nov., Mycobacterium houstonense sp. nov., Mycobacterium neworleansense sp. nov. and Mycobacterium brisbanense sp. nov. and recognition of Mycobacterium porcinum from human clinical isolates. Int J Syst Evol Microbiol 54, 1653-1667.
Stevens, K.B., Del Rio Vilas, V.J., Guitian, J., 2009, Classical sheep scrapie in Great Britain: spatial analysis and identification of environmental and farm-related risk factors. BMC Vet Res 5, 33.
Suther, D.E., Franti, C.E., Page, H.H., 1974, Evaluation of a comparative intradermal tuberculin test in California dairy cattle. Am J Vet Res 35, 379-387.
Sweeney, F.P., Courtenay, O., Hibberd, V., Hewinson, R.G., Reilly, L.A., Gaze, W.H., Wellington, E.M., 2007, Environmental monitoring of Mycobacterium bovis in badger feces and badger sett soil by real-time PCR, as confirmed by immunofluorescence, immunocapture, and cultivation. Appl Environ Microbiol 73, 7471-7473.
Telenti, A., Marchesi, F., Balz, M., Bally, F., Bottger, E.C., Bodmer, T., 1993, Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol 31, 175-178.
Tizard I., 1992, Cell-mediated (type IV) hypersensitivity. In: Veterinary immunology: an introduction . 4th ed. pp. 372-380. Philadelphia, WB Saunders Co.
Wadley, D.E., 1949, The use of bovine PPD in the single caudal fold test to detect tuberculosis in beef cattle. Aust Vet J 53, 208-213.
Watt, B., 1995, Lesser known mycobacteria. J Clin Pathol 48, 701-705.
Webster, G., Newberry, C.J., Fry, J.C., Weightman, A.J., 2003, Assessment of bacterial community structure in the deep sub-seafloor biosphere by 16S rDNA-based techniques: a cautionary tale. J Microbiol Methods 55, 155-164.
Wood, P.R., Corner, L.A., Rothel, J.S., Baldock, C., Jones, S.L., Cousins, D.B., McCormick, B.S., Francis, B.R., Creeper, J., Tweddle, N.E., 1991, Field comparison of the interferon-gamma assay and the intradermal tuberculin test for the diagnosis of bovine tuberculosis. Aust Vet J 68, 286-290.