Prospective comparison of Classical and Modern Molecular mycobacterial detection techniques in cattle infected with M. avium subspecies paratuberculosis
Main Article Content
Abstract
Background: “Classical” mycobacterial identification is difficult to perform, time consuming, inefficient and often insensitive. Modern techniques, incorporating molecular methods, may be more rapid, less expensive, more sensitive and more accurate. We prospectively compared “classical” and modern mycobacterial detection techniques.
Methods: A plaque/qPCR study has been published on the blood of a herd of Johne disease (cattle infected with M. avium subspecies paratuberculosis (MAP.)) Simultaneously aliquots were submitted for “classical” mycobacterial identification. Plaque/qPCR could be completed in <3days. “Classical” cultures were followed for a minimum of 2 years.
Results: Plaque/qPCR MAP positivity were observed in 74% of Johne and 34% of Control animals. “Classical” detection on appropriate culture medium were seen in 19% in Johne disease and 18% Controls.
Conclusions: “Classical” mycobacterial detections methodology has demonstrably been supplanted by modern molecular methods.
Article Details
How to Cite
GREENSTEIN, Robert J.; BROWN, Sheldon T..
Prospective comparison of Classical and Modern Molecular mycobacterial detection techniques in cattle infected with M. avium subspecies paratuberculosis.
Medical Research Archives, [S.l.], v. 13, n. 10, oct. 2025.
ISSN 2375-1924.
Available at: <https://esmed.org/MRA/mra/article/view/6979>. Date accessed: 05 dec. 2025.
doi: https://doi.org/10.18103/mra.v13i10.6979.
Section
Research Articles
The Medical Research Archives grants authors the right to publish and reproduce the unrevised contribution in whole or in part at any time and in any form for any scholarly non-commercial purpose with the condition that all publications of the contribution include a full citation to the journal as published by the Medical Research Archives.
References
1. Hansen GHA. Indberetning til det norske medicinske Selskap i Christiania om en med Understottelse av Selkabet foretage Reise for at anstille Undersogelser angaaende Spedalskhedens Aarsager. Norsk Magazin for Laegevidenskaben 1874;4:1–88. (In Norwegian).
2. Hansen GHA. Bacillus leprae. Quarterly Journal of the Microscopical Science 1880;20:92–102.
3. Koch R. Die Aetiologie der Tuberculose. Berliner Klinische Wochenschrift 1882;19:221–230. (In German).
4. Koch R. Die Aetiologie der Tuberkulose. Mittbeilungen aus dem Kaiserlichen Gesundbeitsamte 1884;2:1–88. (In German).
5. Dulberger CL, Rubin EJ, Boutte CC. The mycobacterial cell envelope — a moving target. Nature Reviews Microbiology 2020;18(1):47–59. Doi: 10.1038/s41579-019-0273-7.
6. Marrakchi H, Lanéelle M-A, Daffé M. Mycolic Acids: Structures, Biosynthesis, and Beyond. Chemistry & Biology 2014;21(1):67–85. Doi: https://doi.org/10.1016/j.chembiol.2013.11.011.
7. Ziehl F. Zur Farbung des Tuberkelbacillus. Dtsch, med Wschr, 1882;8:451.
8. Neelsen FCA. Ein casuistischer Bietrag zur Lehre von der Tuberkulose. Zbl med Wiss 1883;21(497-501).
9. Lowenstein E. Die Zachtung der Tubekelba zillen aus dem stramenden Blute. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg Abt I Orig 1931;1:120–127.
10. Lowenstein E. Der Kulturelle Nachweis von Tuberkelbakterien in Milch auf Malachitgrun Einarboden. Ann Inst Pasteur 1933;50:161.
11. Jensen KA. Rinzuchtung und Typenbestim mung von Tuberkelbazillentammen. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg Abt I Orig 1932;125.
12. Jensen KA. Reinzüchtung und Typenbestimmung von Tuberkelbazillenstämmen: Ein
Vereinfachung der Methoden für die Praxis. Zentralbl Bakt 1932;125:222–239.
13. Knox R. SEMI-SOLID AGAR MEDIA FOR RAPID CULTURE OF TUBERCLE BACILLI. The Lancet 1955;266(6881):110–112. Doi: 10.1016/S0140-6736(55)92114-0.
14. Middlebrook G, Cohn ML. Bacteriology of tuberculosis: laboratory methods. Am J Public Health Nations Health 1958;48(7):844–53. Doi: 10.2105/ajph.48.7.844.
15. Middlebrook G, Reggiardo Z, Tigertt WD. Automatable radiometric detection of growth of Mycobacterium tuberculosis in selective media. The American review of respiratory disease 1977;115(6):1066–9. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=122228).
16. Greenstein RJ, Su L, Haroutunian V, Shahidi A, Brown ST. On the Action of Methotrexate and 6-Mercaptopurine on M. avium subspecies paratuberculosis. PLoS ONE 2007;2(1):e161. (In eng). Doi: 10.1371/journal.pone.0000161.
17. Greenstein RJ, Su L, Shahidi A, Brown WD, Clifford A, Brown ST. Unanticipated Mycobacterium tuberculosis complex culture inhibition by immune modulators, immune suppressants, a growth enhancer, and vitamins A and D: clinical implications. Int J Infect Dis 2014;26C:37–43. Doi: 10.1016/j.ijid.2014.01.026.
18. Shin SJ, Collins MT. Thiopurine drugs azathioprine and 6-mercaptopurine inhibit Mycobacterium paratuberculosis growth in vitro. Antimicrob Agents Chemother 2008;52(2):418–26. (Research Support, Non-U.S. Gov't). Doi: 10.1128/AAC.00678-07.
19. Krishnan MY, Manning EJ, Collins MT. Effects of interactions of antibacterial drugs with each other and with 6-mercaptopurine on in vitro growth of Mycobacterium avium subspecies paratuberculosis. J Antimicrob Chemother 2009;64(5):1018–23. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19759042).
20. Chiodini RJ, Van Kruiningen HJ, Merkal RS, Thayer Jr. WR, Coutu JA. Characteristics of an unclassified Mycobacterium species isolated from patients with Crohn's disease. J Clin Microbiol 1984;20(5):966–971.
21. Wilson SM, al-Suwaidi Z, McNerney R, Porter J, Drobniewski F. Evaluation of a new rapid bacteriophage-based method for the drug susceptibility testing of Mycobacterium tuberculosis. Nat Med 1997;3(4):465–8. Doi: 10.1038/nm0497-465.
22. Jacobs WR, Jr., Barletta RG, Udani R, et al. Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages. Science 1993;260(5109):819–22. Doi: 10.1126/science.8484123.
23. Carrière C, Riska PF, Zimhony O, et al. Conditionally replicating luciferase reporter phages: improved sensitivity for rapid detection and assessment of drug susceptibility of Mycobacterium tuberculosis. Journal of Clinical Microbiology 1997;35(12):3232–3239. Doi: doi:10.1128/jcm.35.12.3232-3239.1997.
24. Swift BM, Rees CE. Detecting mycobacteria in cattle blood. Vet Rec 2013;173(21):522–3. Doi: 10.1136/vr.f7067.
25. Foddai A, Elliott CT, Grant IR. Optimization of a phage amplification assay to permit accurate enumeration of viable Mycobacterium avium subsp. paratuberculosis cells. Appl Environ Microbiol 2009;75(12):3896–902. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19395561).
26. Stewart LD, Foddai A, Elliott CT, Grant IR. Development of a novel phage-mediated immunoassay for the rapid detection of viable Mycobacterium avium subsp. paratuberculosis. J Appl Microbiol 2013;115(3):808–17. (In eng). Doi: 10.1111/jam.12275.
27. Greenstein RJ, Su L, Grant IR, et al. Comparison of a mycobacterial phage assay to detect viable Mycobacterium avium subspecies paratuberculosis with standard diagnostic modalities in cattle with naturally infected Johne disease. Gut pathogens 2021;13(1):30. Doi: 10.1186/s13099-021-00425-5.
28. Pozzato N, Gwozdz J, Gastaldelli M, Capello K, Dal Ben C, Stefani E. Evaluation of a rapid and inexpensive liquid culture system for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faeces. Journal of microbiological methods 2011;84(3):413–7. Doi: 10.1016/j.mimet.2011.01.019.
29. Twort FW, Ingram GLY. Zbl Bakt 1914;73(277).
30. Francis J, Macturk HM, Madinaveitia J, Snow GA. Mycobactin, a growth factor for Mycobacterium johnei. I. Isolation from Mycobacterium phlei. Biochem J 1953;55(4):596–607. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=13115341).
31. Saleeby CW. The Action of Sunlight. Nature 1922;109(2723):11–12. Doi: 10.1038/109011b0.
32. Bruce D, Ooi JH, Yu S, Cantorna MT. Vitamin D and host resistance to infection? Putting the cart in front of the horse. Exp Biol Med (Maywood) 2010;235(8): 921–7. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=20660091).
33. Salahuddin N, Ali F, Hasan Z, Rao N, Aqeel M, Mahmood F. Vitamin D accelerates clinical recovery from tuberculosis: results of the SUCCINCT Study [Supplementary Cholecalciferol in recovery from tuberculosis]. A randomized, placebo-controlled, clinical trial of vitamin D supplementation in patients with pulmonary tuberculosis’. BMC Infectious Diseases 2013;13(1):22. Doi: 10.1186/1471-2334-13-22.
34. Stewart-Tull DES. Mycobacterium leprae - The bacteriologist's enigma. In: Ratledge C, Stanford J, eds. The Biology of the Mycobacteria, Volume 1: Physiology, Identification, and Classification. 1 ed. New York: Academic Press; 1982:273–307.
35. Cole ST, Eiglmeier K, Parkhill J, et al. Massive gene decay in the leprosy bacillus. Nature 2001;409(6823):1007–11. (Research Support, Non-U.S. Gov't) (In eng). Doi: 10.1038/35059006.
36. Ridley DS, Jopling WH. A classification of leprosy for research purposes. Lepr Rev 1962;33:119–28. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14492126).
37. Ridley DS, Jopling WH. Classification of leprosy according to immunity - a five-group system. Int J Lepr Other Mycobact Dis 1966;34:255–273.
38. Lima MIS, Correa MBC, Moraes E, et al. HSP60 mimetic peptides from Mycobacterium leprae as new antigens for immunodiagnosis of Leprosy. AMB Express 2023;13(1):120. Doi: 10.1186/s13568-023-01625-9.
39. Alban SM, de Moura JF, Thomaz-Soccol V, et al. Phage display and synthetic peptides as promising biotechnological tools for the serological diagnosis of leprosy. PLoS One 2014;9(8):e106222. (In eng). Doi: 10.1371/journal.pone.0106222.
40. Sengupta U. Recent Laboratory Advances in Diagnostics and Monitoring Response to Treatment in Leprosy. Indian Dermatol Online J 2019;10(2):106–114. (In eng). Doi: 10.4103/idoj.IDOJ_260_18
2. Hansen GHA. Bacillus leprae. Quarterly Journal of the Microscopical Science 1880;20:92–102.
3. Koch R. Die Aetiologie der Tuberculose. Berliner Klinische Wochenschrift 1882;19:221–230. (In German).
4. Koch R. Die Aetiologie der Tuberkulose. Mittbeilungen aus dem Kaiserlichen Gesundbeitsamte 1884;2:1–88. (In German).
5. Dulberger CL, Rubin EJ, Boutte CC. The mycobacterial cell envelope — a moving target. Nature Reviews Microbiology 2020;18(1):47–59. Doi: 10.1038/s41579-019-0273-7.
6. Marrakchi H, Lanéelle M-A, Daffé M. Mycolic Acids: Structures, Biosynthesis, and Beyond. Chemistry & Biology 2014;21(1):67–85. Doi: https://doi.org/10.1016/j.chembiol.2013.11.011.
7. Ziehl F. Zur Farbung des Tuberkelbacillus. Dtsch, med Wschr, 1882;8:451.
8. Neelsen FCA. Ein casuistischer Bietrag zur Lehre von der Tuberkulose. Zbl med Wiss 1883;21(497-501).
9. Lowenstein E. Die Zachtung der Tubekelba zillen aus dem stramenden Blute. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg Abt I Orig 1931;1:120–127.
10. Lowenstein E. Der Kulturelle Nachweis von Tuberkelbakterien in Milch auf Malachitgrun Einarboden. Ann Inst Pasteur 1933;50:161.
11. Jensen KA. Rinzuchtung und Typenbestim mung von Tuberkelbazillentammen. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg Abt I Orig 1932;125.
12. Jensen KA. Reinzüchtung und Typenbestimmung von Tuberkelbazillenstämmen: Ein
Vereinfachung der Methoden für die Praxis. Zentralbl Bakt 1932;125:222–239.
13. Knox R. SEMI-SOLID AGAR MEDIA FOR RAPID CULTURE OF TUBERCLE BACILLI. The Lancet 1955;266(6881):110–112. Doi: 10.1016/S0140-6736(55)92114-0.
14. Middlebrook G, Cohn ML. Bacteriology of tuberculosis: laboratory methods. Am J Public Health Nations Health 1958;48(7):844–53. Doi: 10.2105/ajph.48.7.844.
15. Middlebrook G, Reggiardo Z, Tigertt WD. Automatable radiometric detection of growth of Mycobacterium tuberculosis in selective media. The American review of respiratory disease 1977;115(6):1066–9. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=122228).
16. Greenstein RJ, Su L, Haroutunian V, Shahidi A, Brown ST. On the Action of Methotrexate and 6-Mercaptopurine on M. avium subspecies paratuberculosis. PLoS ONE 2007;2(1):e161. (In eng). Doi: 10.1371/journal.pone.0000161.
17. Greenstein RJ, Su L, Shahidi A, Brown WD, Clifford A, Brown ST. Unanticipated Mycobacterium tuberculosis complex culture inhibition by immune modulators, immune suppressants, a growth enhancer, and vitamins A and D: clinical implications. Int J Infect Dis 2014;26C:37–43. Doi: 10.1016/j.ijid.2014.01.026.
18. Shin SJ, Collins MT. Thiopurine drugs azathioprine and 6-mercaptopurine inhibit Mycobacterium paratuberculosis growth in vitro. Antimicrob Agents Chemother 2008;52(2):418–26. (Research Support, Non-U.S. Gov't). Doi: 10.1128/AAC.00678-07.
19. Krishnan MY, Manning EJ, Collins MT. Effects of interactions of antibacterial drugs with each other and with 6-mercaptopurine on in vitro growth of Mycobacterium avium subspecies paratuberculosis. J Antimicrob Chemother 2009;64(5):1018–23. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19759042).
20. Chiodini RJ, Van Kruiningen HJ, Merkal RS, Thayer Jr. WR, Coutu JA. Characteristics of an unclassified Mycobacterium species isolated from patients with Crohn's disease. J Clin Microbiol 1984;20(5):966–971.
21. Wilson SM, al-Suwaidi Z, McNerney R, Porter J, Drobniewski F. Evaluation of a new rapid bacteriophage-based method for the drug susceptibility testing of Mycobacterium tuberculosis. Nat Med 1997;3(4):465–8. Doi: 10.1038/nm0497-465.
22. Jacobs WR, Jr., Barletta RG, Udani R, et al. Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages. Science 1993;260(5109):819–22. Doi: 10.1126/science.8484123.
23. Carrière C, Riska PF, Zimhony O, et al. Conditionally replicating luciferase reporter phages: improved sensitivity for rapid detection and assessment of drug susceptibility of Mycobacterium tuberculosis. Journal of Clinical Microbiology 1997;35(12):3232–3239. Doi: doi:10.1128/jcm.35.12.3232-3239.1997.
24. Swift BM, Rees CE. Detecting mycobacteria in cattle blood. Vet Rec 2013;173(21):522–3. Doi: 10.1136/vr.f7067.
25. Foddai A, Elliott CT, Grant IR. Optimization of a phage amplification assay to permit accurate enumeration of viable Mycobacterium avium subsp. paratuberculosis cells. Appl Environ Microbiol 2009;75(12):3896–902. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19395561).
26. Stewart LD, Foddai A, Elliott CT, Grant IR. Development of a novel phage-mediated immunoassay for the rapid detection of viable Mycobacterium avium subsp. paratuberculosis. J Appl Microbiol 2013;115(3):808–17. (In eng). Doi: 10.1111/jam.12275.
27. Greenstein RJ, Su L, Grant IR, et al. Comparison of a mycobacterial phage assay to detect viable Mycobacterium avium subspecies paratuberculosis with standard diagnostic modalities in cattle with naturally infected Johne disease. Gut pathogens 2021;13(1):30. Doi: 10.1186/s13099-021-00425-5.
28. Pozzato N, Gwozdz J, Gastaldelli M, Capello K, Dal Ben C, Stefani E. Evaluation of a rapid and inexpensive liquid culture system for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faeces. Journal of microbiological methods 2011;84(3):413–7. Doi: 10.1016/j.mimet.2011.01.019.
29. Twort FW, Ingram GLY. Zbl Bakt 1914;73(277).
30. Francis J, Macturk HM, Madinaveitia J, Snow GA. Mycobactin, a growth factor for Mycobacterium johnei. I. Isolation from Mycobacterium phlei. Biochem J 1953;55(4):596–607. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=13115341).
31. Saleeby CW. The Action of Sunlight. Nature 1922;109(2723):11–12. Doi: 10.1038/109011b0.
32. Bruce D, Ooi JH, Yu S, Cantorna MT. Vitamin D and host resistance to infection? Putting the cart in front of the horse. Exp Biol Med (Maywood) 2010;235(8): 921–7. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=20660091).
33. Salahuddin N, Ali F, Hasan Z, Rao N, Aqeel M, Mahmood F. Vitamin D accelerates clinical recovery from tuberculosis: results of the SUCCINCT Study [Supplementary Cholecalciferol in recovery from tuberculosis]. A randomized, placebo-controlled, clinical trial of vitamin D supplementation in patients with pulmonary tuberculosis’. BMC Infectious Diseases 2013;13(1):22. Doi: 10.1186/1471-2334-13-22.
34. Stewart-Tull DES. Mycobacterium leprae - The bacteriologist's enigma. In: Ratledge C, Stanford J, eds. The Biology of the Mycobacteria, Volume 1: Physiology, Identification, and Classification. 1 ed. New York: Academic Press; 1982:273–307.
35. Cole ST, Eiglmeier K, Parkhill J, et al. Massive gene decay in the leprosy bacillus. Nature 2001;409(6823):1007–11. (Research Support, Non-U.S. Gov't) (In eng). Doi: 10.1038/35059006.
36. Ridley DS, Jopling WH. A classification of leprosy for research purposes. Lepr Rev 1962;33:119–28. (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14492126).
37. Ridley DS, Jopling WH. Classification of leprosy according to immunity - a five-group system. Int J Lepr Other Mycobact Dis 1966;34:255–273.
38. Lima MIS, Correa MBC, Moraes E, et al. HSP60 mimetic peptides from Mycobacterium leprae as new antigens for immunodiagnosis of Leprosy. AMB Express 2023;13(1):120. Doi: 10.1186/s13568-023-01625-9.
39. Alban SM, de Moura JF, Thomaz-Soccol V, et al. Phage display and synthetic peptides as promising biotechnological tools for the serological diagnosis of leprosy. PLoS One 2014;9(8):e106222. (In eng). Doi: 10.1371/journal.pone.0106222.
40. Sengupta U. Recent Laboratory Advances in Diagnostics and Monitoring Response to Treatment in Leprosy. Indian Dermatol Online J 2019;10(2):106–114. (In eng). Doi: 10.4103/idoj.IDOJ_260_18