臺灣博碩士論文加值系統

鳥型分枝桿菌副結核桿菌亞種（Mycobacterium avium subsp. paratuberculosis; MAP）是一個人畜共通並在全世界散佈且造成經濟損失的一種重要的病原，在反芻獸造成Johne''s disease，而每年Johne''s disease在美國會造成15億美元的經濟損失，近幾年來，MAP在台灣及美國的盛行率有逐年升高的趨勢。目前有許多種檢測方法來區分動物是否感染MAP，如PCR及ELISA等，但PCR及ELISA都有敏感度及特異度不夠高的問題，所以臨床上需要發展新的檢測技術。本實驗的目的為以環形恆溫核酸增幅法及nested PCR-ELISA來檢測糞便中的MAP，並以糞便細菌培養為gold standard作比較。檢驗樣本分成兩個部分，第一部分為21個經淡水家衛所以糞便細菌培養檢測證實的樣本，第二部分為11個臨床上疑似感染MAP牛隻的樣本，並經由ELISA檢測結果均為陽性，所有的樣本皆分別以細菌培養、PCR、nested PCR、LAMP及nested PCR-ELISA檢測作比較。結果顯示，PCR檢測的敏感度為100 pg/μl的DNA，約為3,000個MAP的量，而nested PCR與LAMP敏感度相同可以檢測到1 pg/μl的DNA，約為30個MAP的量，而nested PCR-ELISA 的敏感度為0.1 pg/μl的DNA，約為3個MAP的量，在特異度方面，無論是PCR、nested PCR、LAMP及nested PCR-ELISA都不會檢測到E. coli、Staphylococcus sp.、Enterococcus sp.的DNA。在檢測21個實驗室確認的樣本部分，LAMP及nested PCR-ELISA的檢測結果皆與細菌培養相符合（16個陽性及9個陰性），其敏感度及特異度皆為100%。在檢測臨床疑似病例樣本的部分，有9個樣本PCR、nested PCR、LAMP及nested PCR-ELISA的結果為陽性，但細菌培養結果為陰性，顯示檢測結果差異較大，推測可能是因為採集糞便樣本後的保存方式不當影響到細菌分離培養的結果。本實驗之結果顯示，以LAMP及nested PCR-ELISA檢測糞便樣本的MAP的準確度相當高。

Mycobacterium avium subsp. paratuberculosis (MAP) is one of the most widespread and economically important zoonotic pathogens of ruminants and human, which may cause Johne''s disease in ruminants. The cost of Johne''s disease to the cattle industry is staggering with an estimated $1.5 billion loss every year in the USA. In recent years, the prevalence of MAP in the USA and Taiwan had increased remarkably. Accurate diagnostic tests for MAP such as ELISA and PCR are routinely used to identified the disease-free animals, however, the sensitivity and specificity of ELISA and PCR are unsatisfied. The purpose of this study was to detect the presence of MAP in fecal samples of dairy cows by Loop-mediated Isothermal Amplification (LAMP) system and nested PCR-ELISA, which were compared by using bacteriological culture as the gold standard. The samples were collected from two sources, 21 laboratory-confirmed samples and 11 field samples of suspected cases with positive result of ELISA assay. All 32 faecal samples were detected by bacteriological culture, PCR, nested PCR, LAMP and nested PCR-ELISA. The results indicated that PCR can detect 100 pg/μl of MAP, while LAMP and nested PCR can detect 1 pg/μl of MAP. However, nested PCR-ELISA can detect 0.1 pg/μl of MAP. None of the negative controls including E. coli, Staphylococcus sp. and Enterococcus sp. was detected by PCR, nested PCR, LAMP and nested PCR-ELISA. In 16 positive and 9 negative laboratory-confirmed samples, the results from LAMP and nested PCR-ELISA were identical to those from bacteriological culture, which yielded a sensitivity of 100% and specificity of 100%. However, all of the 9 field samples detected by PCR, nested PCR, LAMP and nested PCR-ELISA showed positive results, which were inconsistent with those by bacteriological culture. It is inferred that the storage condition of faecal samples may affect the results of bacteriological culture. In conclusion, the accuracies for detection of MAP in faecal samples by LAMP and nested PCR-ELISA are relatively high as compared to the bacteriological culture.

摘要 i
Abstract iii
目 錄 v
表 次 x
圖 次 xi
第一章 序言 1
第二章 文獻探討 3
第一節 鳥型分枝桿菌副結核桿菌亞種 3
一、 形態學 3
二、 染色特性 5
三、 MAP的抗性 5
四、 MAP的特殊基因片段 6
第二節 Johne’s disease 7
一、 Johne’s disease的歷史 7
二、 Johne’s disease的介紹 7
三、 Johne’s disease的病程分級 8
1. Stage Ⅰ （Silent infection）： 8
2. Stage Ⅱ （Inapparent carrier adult）： 8
3. Stage Ⅲ （Clinical disease） 10
4. Stage Ⅳ （Advanced clinical disease） 10
四、 Johne’s disease的感染途徑及致病機序 11
五、 Johne’s disease的病理學檢查 12
第三節 Johne’s disease的流行病學 13
一、 北美洲的流行病學 13
二、 其他國家的流行病學 17
1. 歐洲地區 17
2. 大洋洲地區 18
3. 南亞地區 19
4. 非洲地區 19
三、 台灣的流行病學 19
第四節 MAP的IS900基因片段 20
第五節 MAP的診斷方法 21
一、 剖檢（Necropsy） 21
二、 腸繫膜淋巴結採樣 24
三、 補體結合試驗（Complement fixation test; CF test） 24
四、 細菌培養（Bacterial culture） 24
1. 糞便及組織細菌培養 24
2. 細菌培養的方法 25
3. 菌落型態 26
4. 細菌培養的樣本來源 26
五、 Ziehl-Neelsen-stained smear（acid-fast stain; 抗酸性染色） 27
六、 酵素免疫分析法（Enzyme-linked immunosorbent assay; ELISA） 27
七、 膠體免疫擴散試驗（Agar gel immunodiffusion test; AGID） 28
八、 Gamma interferon assay （IFN-γ） 28
九、 聚合酶鏈式反應（Polymerase chain reaction; PCR） 28
十、 巢式聚合酶鏈式反應（nested Polymerase chain reaction; nested PCR） 29
第六節 環形恆溫核酸增幅法（LAMP） 29
第七節 巢式聚合酶鏈式反應-酵素免疫分析法（nested PCR-ELISA） 31
第三章 實驗材料與方法 33
第一節 樣本來源 33
一、 標準菌株DNA 33
二、 實驗樣本 33
三、 待測樣本 33
第二節 細菌培養 33
一、 試驗藥品及商品化套組 33
二、 純化糞便（糞便需儲存在- 70 ℃） 34
三、 接種培養基 34
四、 培養及觀察培養基的斜面 34
第三節 DNA之萃取 34
一、 試驗之商品化套組 34
二、 純化糞便（糞便需儲存在- 20℃） 35
三、 使用QIAampR DNA Stool Mini Kit萃取DNA 35
第四節 酵素免疫分析法（ELISA） 36
一、 試驗之商品化套組 36
二、 前置作業 36
三、 ELISA（使用PourquierR ELISA Paratuberculosis Screening） 36
四、 數值分析 37
第五節 聚合酶鏈鎖反應（PCR） 37
一、 試驗藥品及商品化套組 37
二、 PCR反應 38
三、 電泳 38
第六節 巢式聚合酶鏈鎖反應（nested PCR） 40
一、 試驗藥品及商品化套組 40
二、 PCR反應 40
三、 電泳 40
第七節 環形恆溫核酸增幅法（LAMP） 40
一、 試驗藥品及商品化套組 40
二、 LAMP反應 41
三、 電泳 41
四、 反應條件最佳化 41
第八節 巢式聚合酶鏈鎖反應-酵素免疫分析法（nested PCR-ELISA） 42
一、 試驗藥品及商品化套組 42
二、 將solid-phase primer結合到well上 43
三、 nested PCR反應 43
四、 Enzyme-linked immunosorbent assay （ELISA） 44
第九節 TA clone 45
一、 試驗藥品及商品化套組 45
二、 切膠純化產物 46
三、 TA clone 46
四、 Colony PCR 47
五、 保菌及抽取plasmid 47
第十節 敏感度測試 48
一、 定量及連續10倍稀釋 48
二、 PCR、nested PCR、LAMP及nested PCR-ELISA之敏感度測試 49
三、 nested PCR-ELISA數值分析 49
第十一節 特異度測試 49
一、 試驗藥品及商品化套組 49
二、 選取牛隻常見的細菌當作特異度之測試 49
三、 PCR、nested PCR、LAMP及nested PCR-ELISA之特異度測試 50
四、 nested PCR-ELISA數值分析 50
第四章 實驗結果 51
第一節 TA clone 51
一、 Transform 51
二、 Colony PCR 51
三、 酵素切割 52
四、 定序結果 53
第二節 敏感度測試 53
一、 PCR 53
二、 nested PCR 54
三、 LAMP 56
四、 nested PCR-ELISA 56
第三節 特異度測試 57
一、 PCR 57
二、 nested PCR 57
三、 LAMP 58
四、 nested PCR-ELISA 58
五、 Cut-off值 60
第四節 細菌培養 60
一、 實驗樣本（編號1-21） 60
二、 待測樣本（編號22-32） 61
第五節 酵素免疫分析法 61
一、 待測樣本（編號22-32） 61
第六節 聚合酶鏈鎖反應 61
一、 實驗樣本（編號1-21） 61
二、 待測樣本（編號22-32） 61
第七節 巢式聚合酶鏈鎖反應 63
一、 實驗樣本（編號1-21） 63
二、 待測樣本（編號22-32） 63
第八節 環形恆溫核酸增幅法 63
一、 LAMP條件最佳化 63
二、 實驗樣本（編號1-21） 65
三、 待測樣本（編號22-32） 65
第九節 巢式聚合酶鏈鎖反應-酵素免疫分析法 65
一、 實驗樣本（編號1-21） 65
二、 待測樣本（編號22-32） 65
第五章 討論 67
第一節 LAMP條件最佳化 67
第二節 敏感度 68
一、 PCR、nested PCR、LAMP及nested PCR-ELISA敏感度比較 68
二、 nested PCR-ELISA的標準曲線 68
第三節 特異度 69
一、 PCR、nested PCR、LAMP及nested PCR-ELISA的特異度 69
二、 nested PCR-ELISA的cut-off value 69
第四節 實驗樣本及待測樣本之檢測 70
一、 實驗樣本（編號1-21） 70
1. PCR 70
2. nested PCR 70
3. LAMP 71
4. nested PCR-ELISA 71
二、 待測樣本（編號22-32） 72
1. PCR、nested PCR、LAMP及nested PCR-ELISA 72
第五節 檢測方法之比較 73
第六節 結論 75
參考文獻 76

1.郭晉禾。副結核分枝桿菌在台灣地區乳牛群之分佈。碩士論文。國立台灣大學獸醫學研究所。台北市。中華民國。2001。
2.黃春申，蔡洵洵，官南綾，涂堅。台灣地區反芻動物 Mycobacterium avium subsp. paratuberculosis 之盛行率調查。家畜衛試所研報 46：39-44，2011。
3.吳捷華。利用競爭型PCR-ELISA定量nirS型脫硝菌碩士論文，大同大學生物工程研究所碩士論文。台北市。中華民國。2007。
4.Adessi C, Matton G, Ayala G, Turcatti G, Mermod J, Mayer P, and Kawashima E. Solid phase DNA amplification-characterisation of primer attachment and amplification mechanisms. Nucleic Acids Res 28: e87, 2000.
5.Autschbach F, Eisold S, Hinz U, Zinser S, Linnebacher M, Giese T, Loffler T, Buchler MW, and Schmidt J. High prevalence of Mycobacterium avium subspecies paratuberculosis IS900 DNA in gut tissues from individuals with Crohn''s disease. Gut 54: 944-949, 2005.
6.Begg DJ, and Whittington RJ. Experimental animal infection models for Johne''s disease, an infectious enteropathy caused by Mycobacterium avium subsp. paratuberculosis. Vet J 176: 129-145, 2008.
7.Blowey RW, and Weaver AD. Color Atlas of Diseases and Disorders of Cattle. 3rd ed. Mosby, Inc., Edinburgh, Scotland, 53-82, 2011.
8.Bull TJ, Hermon-Taylor J, Pavlik I, El-Zaatari F, and Tizard M. Characterization of IS900 loci in Mycobacterium avium subsp. paratuberculosis and development of multiplex PCR typing. Soc General Microbiol 146: 2185-2197, 2000.
9.Bull TJ, McMinn EJ, Sidi-Boumedine K, Skull A, Durkin D, Neild P, Rhodes G, Pickup R, and Hermon-Taylor J. Detection and Verification of Mycobacterium avium subsp. paratuberculosis in Fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn''s disease. J Clin Microbiol 41: 2915-2923, 2003.
10.Chacon O, Bermudez LE, and Barletta RG. Johne''s disease, inflammatory bowel disease, and Mycobacterium paratuberculosis. Annu Rev Microbiol 58: 329-363, 2004.
11.Chevrier D, Rasmussen S, and Guesdon J. PCR product quantification by non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells. Mol Cell Probes 7: 187-197, 1993.
12.Chiodini RJ. The history of paratuberculosis (Johne''s disease): A review of the literature 1895 to 1992. Intl Assoc Paratuberculosis, Inc, Publ., 658, 1993.
13.Chiodini RJ, Kruiningen HJV, and Merkal RS. Ruminant paratuberculosis (Johne''s disease): the current status and future prospects. Cornell Vet 74: 218-262, 1984.
14.Clark DL Jr, Koziczkowski JJ, Radcliff RP, Carlson RA, and Ellingson JL. Detection of Mycobacterium avium subspecies paratuberculosis: comparing fecal culture versus serum enzyme-linked immunosorbent assay and direct fecal polymerase chain reaction. J Dairy Sci 91: 2620-2627, 2008.
15.Collins MT. Update on paratuberculosis: 1. Epidemiology of Johne''s disease and the biology of Mycobacterium paratuberculosis. Ir Vet J 56: 565-574, 2003.
16.Collins MT, and Sockett DC. Accuracy and economics of the USDA-licensed enzyme-linked immunosorbent assay for bovine paratuberculosis. J Am Vet Med Assoc 203: 1456-1463, 1993.
17.Cousins DV, Condron RJ, Eamens GJ, Whittington RJ, and Lisle GW de. Paratuberculosis (Johne''s Disease). In: ed. Australian and New Zealand Standard Diagnostic Procedures Standing Committee on Agriculture and Resource Management-Commonwealth Scientific and Industrial Research Organisation, Melbourne., 1-21, 2001.
18.Daffe M, and Draper P. The envelope layers of mycobacteria with reference to their pathogenicity. Adv Microb Physiol 39: 131-203, 1998.
19.Dubnau E, Chan J, Raynaud C, Mohan VP, Laneelle MA, Yu K, Quemard A, Smith I, and Daffe M. Oxygenated mycolic acids are necessary for virulence of Mycobacterium tuberculosis in mice. Mol Microbiol 36: 630-637, 2000.
20.Elze J, Liebler-Tenorio E, Ziller M, and Kohler H. Comparison of prevalence estimation of Mycobacterium avium subsp. paratuberculosis infection by sampling slaughtered cattle with macroscopic lesions vs. systematic sampling. Epidemiol Infect 13: 1-9, 2012.
21.En F, Wei X, Jian L, and Qin C. Loop-mediated isothermal amplification establishment for detection of pseudorabies virus. J Virol Methods 151: 35-39, 2008.
22.Enosawa M, Kageyama S, Sawai K, Watanabe K, Notomi T, Onoe S, Mori Y, and Yokomizo Y. Use of Loop-Mediated Isothermal Amplification of the IS900 Sequence for Rapid Detection of Cultured Mycobacterium avium subsp. paratuberculosis. J Clin Microbiol 41: 4359-4365, 2003.
23.Feller M, Huwiler K, Stephan R, Altpeter E, Shang A, Furrer H, Pfyffer GE, Jemmi T, Baumgartner A, and Egger M. Mycobacterium avium subspecies paratuberculosis and Crohn''s disease: a systematic review and meta-analysis. Lancet Infect Dis 7: 607-613, 2007.
24.Giese SB, and Ahrens P. Detection of Mycobacterium avium subsp. paratuberculosis in milk from clinically affected cows by PCR and culture. Vet Microbiol 77: 291-297, 2000.
25.Gilardoni LR, Paolicchi FA, and Mundo SL. Bovine paratuberculosis: a review of the advantages and disadvantages of different diagnostic tests. Rev Argent Microbiol 44: 201-215, 2012.
26.Gwozdz J, Thompson K, Murray A, Reichel M, Manktelow B, and West D. Comparison of three serological tests and an interferon-gamma assay for the diagnosis of paratuberculosis in experimentally infected sheep. Aust Vet J 78: 779-783, 2000.
27.Harris NB, and Barletta RG. Mycobacterium avium subsp. paratuberculosis in Veterinary Medicine. Clin Microbiol Rev 14: 489-512, 2001.
28.Heuer C, Wilson P, and Larking K. Johne’s disease in New Zealand livestock. Vetscript 2: 39-41, 2011.
29.Hope A, Kluver P, Jones S, and Condron R. Sensitivity and specificity of two serological tests for the detection of ovine paratuberculosis. Aust Vet J 78: 850-856, 2000.
30.Iwamoto T, Sonobe T, and Hayashi K. Loop-Mediated Isothermal Amplification for Direct Detection of Mycobacterium tuberculosis Complex, M. avium, and M. intracellulare in Sputum Samples. J Clin Microbiol 41: 2616-2622, 2003.
31.Jubb T, Sergeant E, Callinan A, and Galvin J. Estimate of the sensitivity of an ELISA used to detect Johne’s disease in Victorian dairy cattle herds. Aust Vet J 82: 569-573, 2004.
32.Kaneko H, Kawana T, Fukushima E, and Suzutani T. Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J Biochem Biophys Methods 70: 499-501, 2007.
33.Larsen JWA, Webb Ware JK, and Kluver P. Epidemiology of bovine Johne''s disease (BJD) in beef cattle herds in Australia. Aust Vet J 90: 6-13, 2012.
34.Lee MF, Chen YH, Hsu HJ, and Peng CF. One-tube loop-mediated isothermal amplification combined with restriction endonuclease digestion and ELISA for colorimetric detection of resistance to isoniazid, ethambutol and streptomycin in Mycobacterium tuberculosis isolates. J Microbiol Methods 83: 53-58, 2010.
35.Lee MF, Chen YH, and Peng CF. Evaluation of reverse transcription loop-mediated isothermal amplification in conjunction with ELISA-hybridization assay for molecular detection of Mycobacterium tuberculosis. J Microbiol Methods 76: 174-180, 2009.
36.Lombard JE, Gardner IA, Jafarzadeh SR, Fossler CP, Harris B, Capsel RT, Wagner BA, and Johnson WO. Herd-level prevalence of Mycobacterium avium subsp. paratuberculosis infection in United States dairy herds in 2007. Prev Vet Med 108: 234-238, 2013.
37.Lybeck KR, Storset AK, Djonne B, Valheim M, and Olsen I. Faecal shedding detected earlier than immune responses in goats naturally infected with Mycobacterium avium subsp. paratuberculosis. Res Vet Sci 91: 32-39, 2011.
38.Martinson SA, Hanna PE, Ikede BO, Lewis JP, Miller LM, Keefe GP, and McKenna SLB. Comparison of Bacterial Culture, Histopathology, and Immunohistochemistry for the Diagnosis of Johne''s Disease in Culled Dairy Cows. J Vet Diagn Invest 20: 51-57, 2008.
39.McKenna SLB, Keefe GP, Barkema HW, McClure J, VanLeeuwen JA, Hanna P, and Sockett DC. Cow-Level Prevalence of Paratuberculosis in Culled Dairy Cows in Atlantic Canada and Maine. J Dairy Sci 87: 3770-3777, 2004.
40.Meadus WJ, Gill CO, Duff P, Badoni M, and Saucier L. Prevalence on beef carcasses of Mycobacterium avium subsp. paratuberculosis DNA. Int J Food Microbiol 124: 291-294, 2008.
41.Merkal R. Paratuberculosis: advances in cultural, serologic, and vaccination methods. J Am Vet Med Assoc 184: 939-943, 1984.
42.Metzger-Boddien C, Khaschabi D, Schonbauer M, Boddien S, Schlederer T, and Kehle J. Automated high-throughput immunomagnetic separation-PCR for detection of Mycobacterium avium subsp. paratuberculosis in bovine milk. Int J Food Microbiol 110: 201-208, 2006.
43.Motiwala AS, Strother M, Theus NE, Stich RW, Byrum B, Shulaw WP, Kapur V, and Sreevatsan S. Rapid detection and typing of strains of Mycobacterium avium subsp. paratuberculosis from broth cultures. J Clin Microbiol 43: 2111-2117, 2005.
44.Munch M, Nielsen LP, Handberg KJ, and Jorgensen PH. Detection and subtyping (H5 and H7) of avian type A influenza virus by reverse transcription-PCR and PCR-ELISA. Arch Virol 146: 87-97, 2001.
45.Munster P, Fechner K, Volkel I, von Buchholz A, and Czerny CP. Distribution of Mycobacterium avium ssp. paratuberculosis in a German zoological garden determined by IS900 semi-nested and quantitative real-time PCR. Vet Microbiol 163: 116-123, 2013.
46.Muskens J, Barkema H, Russchen E, van Maanen K, Schukken YH, and Bakker D. Prevalence and regional distribution of paratuberculosis in dairy herds in the Netherlands. Vet Microbiol 77: 253-261, 2000.
47.Nagata Y, Yokota H, Kosuda O, Yokoo K, Takemura K, and Kikuchi T. Quantification of picogram levels of specific DNA immobilized in microtiter wells. FEBS Lett 183: 379-382, 1985.
48.Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, and Hase T. Loop-mediated Isothermal Amplification (LAMP) of DNA. Nucleic Acids Res 28: E63, 2000.
49.OIE. Manual of diagnostic tests and vaccines for terrestrial animals 2008- chapter 2.1.11- Paratuberculosis (Johne''s disease). In OIE Terrestrial Manual (World Organisation for Animal Health) 276-291, 2008.
50.Okuni JB, Loukopoulos P, Reinacher M, and Ojok L. Seroprevalence of Mycobacterium avium subspecies Paratuberculosis Antibodies in Cattle from Wakiso, Mpigi and Luwero Districts in Uganda. Int J Anim Vet Adv 3: 156-160, 2011.
51.Oroskar AA, Rasmussen SE, Rasmussen HN, Rasmussen SR, Sullivan BM, and Johansson A. Detection of immobilized amplicons by ELISA-like techniques. Clin Chem 42: 1547-1555, 1996.
52.Pandey BD, Poudel A, Yoda T, Tamaru A, Oda N, Fukushima Y, Lekhak B, Risal B, Acharya B, Sapkota B, Nakajima C, Taniguchi T, Phetsuksiri B, and Suzuki Y. Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients. J Med Microbiol 57: 439-443, 2008.
53.Pithua P, and Kollias NS. Estimated prevalence of caprine paratuberculosis in boer goat herds in missouri, USA. Vet Med Int 2012: 674085, 2012.
54.Quinn PJ, Markey BK, Leonard FC, Hartigan P, Fanning S, and FitzPatrick ES. Veterinary Microbiology and Microbial Disease. Blackwell Science, Oxford, UK., 97-105, 2012.
55.Raji N, Sadeghizadeh M, Tafreshi K, and Jahanzad E. Detection of human Papillomavirus 18 in cervical cancer samples using PCR-ELISA (DIAPOPS). Iran J Microbiol 3: 177-182, 2011.
56.Rasmussen SR, Larsen MR, and Rasmussen SE. Covalent immobilization of DNA onto polystyrene microwells: The molecules are only bound at the 5'' end. Analytical Anal Biochem 198: 138-142, 1991.
57.Reddacliff LA, Vadali A, and Whittington RJ. The effect of decontamination protocols on the numbers of sheep strain Mycobacterium avium subsp. paratuberculosis isolated from tissues and faeces. Vet Microbiol 95: 271-282, 2003.
58.Risa DR, Hamela KL, and Aylinga JM. The detection of Mycobacterium paratuberculosis in bovine faeces by isolation and the comparison of isolation with the examination of stained smears by light microscopy. N Z Vet J 36: 112-114, 1988.
59.Schonenbrucher H, Abdulmawjood A, Failing K, and Bulte M. New triplex real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in bovine feces. Appl Environ Microbiol 74: 2751-2758, 2008.
60.Sergeant E, and Baldock F. The estimated prevalence of Johne''s disease infected sheep flocks in Australia. Aust Vet J 80: 762-768, 2002.
61.Sergeant ESG, Marshall DJ, Eamens GJ, Kearns C, and Whittington RJ. Evaluation of an absorbed ELISA and an agar-gel immuno-diffusion test for ovine paratuberculosis in sheep in Australia. Prev Vet Med 61: 235-248, 2003.
62.Shanahan F. Crohn''s disease. The Lancet 359: 62-69, 2002.
63.Singh SV, Singh AV, Singh R, Sharma S, Shukla N, Misra S, Singh PK, Sohal JS, Kumar H, Patil PK, Misra P, and Sandhu KS. Sero-prevalence of bovine Johne''s disease in buffaloes and cattle population of North India using indigenous ELISA kit based on native Mycobacterium avium subspecies paratuberculosis ''Bison type'' genotype of goat origin. Comp Immunol Microbiol Infect Dis 31: 419-433, 2008.
64.Sjoroos M, Ilonen J, and Lovgren T. Solid-Phase PCR with Hybridization and Timeresolved Fluorometry for Detection of HLA-B27. Clin Chem 47: 498-504, 2001.
65.Smith B. Large animal internal medicine. 4th ed. Mosby, Inc., St. Louis, U.S.A., 881-887, 2009.
66.Smith SI, West DM, Wilson PR, de Lisle GW, Collett MG, Heuer C, and Chambers JP. Detection of Mycobacterium avium subsp. paratuberculosis in skeletal muscle and blood of ewes from a sheep farm in New Zealand. N Z Vet J 59: 240-243, 2011.
67.Sonawane GG, and Tripathi BN. Comparison of a quantitative real-time polymerase chain reaction (qPCR) with conventional PCR, bacterial culture and ELISA for detection of subsp. infection in sheep showing pathology of Johne''s disease. SpringerPlus 2: 45, 2013.
68.Stabel JR. Host responses to Mycobacterium avium subsp. paratuberculosis: a complex arsenal. Anim Health Res Rev 7: 61-70, 2006.
69.Stringer LA, Wilson PR, Heuer C, Hunnam JC, Verdugo C, and Mackintosh CG. Prevalence of Mycobacterium avium subsp. paratuberculosis in farmed red deer (Cervus elaphus) with grossly normal mesenteric lymph nodes. N Z Vet J 61: 147-152, 2013.
70.Timms VJ, Gehringer MM, Mitchell HM, Daskalopoulos G, and Neilan BA. How accurately can we detect Mycobacterium avium subsp. paratuberculosis infection? J Microbiol Methods 85: 1-8, 2011.
71.Tomita N, Mori Y, Kanda H, and Notomi T. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 3: 877-882, 2008.
72.Tsai SM, Chan KW, Hsu WL, Chang TJ, Wong ML, and Wang CY. Development of a loop-mediated isothermal amplification for rapid detection of orf virus. J Virol Methods 157: 200-204, 2009.
73.USDA. Johne’s Disease on U.S. Dairies, 1991–2007. No N5210408 Fort Collins, Colo: USDA, APHIS, Veterinary Services, Center for Epidemiology and Animal Health 2008.
74.Vergne I, Chua J, Lee HH, Lucas M, Belisle J, and Deretic V. Mechanism of phagolysosome biogenesis block by viable Mycobacterium tuberculosis. Proc Natl Acad Sci USA 102: 4033-4038, 2005.
75.Wang CH, Lien KY, Wu JJ, and Lee GB. A magnetic bead-based assay for the rapid detection of methicillin-resistant Staphylococcus aureus by using a microfluidic system with integrated loop-mediated isothermal amplification. Lab Chip 11: 1521-1531, 2011.
76.Whitlock RH, and Buergelt C. Preclinical and clinical manifestations of paratuberculosis (including pathology). Vet Clin North Am Food Anim Pract 12: 345-356, 1996.
77.Whitlock RH, Wells SJ, Sweeney RW, and Tiem JV. ELISA and fecal culture for paratuberculosis (Johne’s disease)- sensitivity and specificity of each method. Vet Microbiol 77: 387-398, 2000.
78.Whittington RJ, Marsh I, McAllister S, Turner MJ, Marshall DJ, and Fraser CA. Evaluation of modified BACTEC 12B radiometric medium and solid media for culture of Mycobacterium avium subsp. paratuberculosis from sheep. J Clin Microbiol 37: 1077-1083, 1999.
79.Wu CW, Livesey M, Schmoller SK, Manning EJ, Steinberg H, Davis WC, Hamilton MJ, and Talaat AM. Invasion and persistence of Mycobacterium avium subsp. paratuberculosis during early stages of Johne''s disease in calves. Infect Immun 75: 2110-2119, 2007.
80.Zimmer K, Drager KG, Klawonn W, and Hess RG. Contribution to the diagnosis of Johne''s disease in cattle. Comparative studies on the validity of Ziehl–Neelsen staining, faecal culture and a commercially available DNA-Probe test in detecting Mycobacterium paratuberculosis in faeces from cattle. J Vet Med 46: 137-140, 1999.