Applicability of using 15 MIRU–VNTR loci for genotyping of Mycobacterium avium subsp. paratuberculosis from two cattle farms in Egypt

  • Mohamed Sabry Abd Elraheam ElsayedEmail author
Original Article


Mycobacterium avium subspecies paratuberculosis (MAP) is a notorious infectious agent that causes Johne’s disease which leads to serious economic losses in cattle farms all over the world. The Lack of accurate epidemiological and molecular data is a major barrier to the implementation of disease control strategies. Basically, the tracing of infections requires rapid detection of the widely spreading genotypes with the ability to determine isolates from common and different sources. This study aimed to evaluate the applicability and discriminatory power of 15 mycobacterial interspersed repetitive unit (MIRU)–variable number tandem repeat (VNTR) loci of M. tuberculosis for MAP genotyping. Additionally, detection of the most efficient loci combinations for molecular epidemiological investigations of MAP isolates. The discriminatory capacity and applicability of 15 known loci [2 exact tandem repeat (ETR) loci, 6 MIRU loci, 4 Mtub loci, and 3 Queen’s University of Belfast (QUB) group loci] were assessed using 26 isolates from two cattle herds (Holstein Frisian) in El buhaira and Giza Governorates at north of Egypt. The results proved the presence of 12 different genotypes. All the used loci gave Hunter–Gaston discrimination index of DI = 0.963 while the ten loci (Mtub04, MIRU10, QUB11b, MIRU26, QUB26, QUB4156, MIRU04, ETRC, Mtub30, and Mtub39) were highly discriminating with DI = 0.956. Moreover, the five loci (Mtub21, MIRU31, MIRU16, MIRU40, and ETRA) gave moderate discriminatory power with DI = 0.839. The MIRU31 locus expressed no polymorphism among strains. MIRU–VNTR typing generally proved applicability and high discriminatory power with DI = 0.963. The ten highly discriminating DI = 0.956 proved to be the most suitable for the first-line genotyping of MAP from cattle, with nearly similar resolving ability as all the 15 loci. MIRU–VNTR proved fastness, efficiency, and feasibility in genotyping of MAP from cattle in Egypt.


M. paratuberculosis Cattle MIRU–VNTR genotyping MIRU–VNTR combinations 



The author would like to acknowledge the Culture Affair and Mission Sector in Egypt for supporting the short term scholarship during which this study was fulfilled. The author thanks Prof. Taro Yamamoto and Associate Prof. Takayuki Wada Department of International Health, Institute of Tropical Medicine, Nagasaki University, Japan for their kind help, hosting and guidance during his work.

Compliance with ethical standards

Conflict of interest

The author declares that he has no conflict of interest.

Ethical approval

This research article does not contain any studies conducted on human or animal subjects.


  1. 1.
    Ronai Z, Csivincsik A, Gyuranecz M, Kreizinger Z, Dan A, Janosi S (2014) Molecular analysis and MIRU-VNTR typing of Mycobacterium avium subsp. paratuberculosis strains from various sources. J Appl Microbiol 118:275–283. CrossRefGoogle Scholar
  2. 2.
    Dziedzinska R, Slana I (2017) Mycobacterium avium subsp. paratuberculosis - An Overview of the Publications from 2011 to 2016. Curr Clin Microbiol 4:19–28CrossRefGoogle Scholar
  3. 3.
    Behr MA, Collins DM (eds.) (2010) Paratuberculosis: organism, disease, control. CAB International, Oxfordshire. pp 10–21, 126–137, 294–305Google Scholar
  4. 4.
    Stevenson K, Alvarez J, Bakker D, Biet F, De Juan L, Denham S, Gerlach GF, Heron I, Kopecna M, May L, Pavlik I, Sharp JM, Thibault VC, Willemsen P, Zadoks RN, Greig A (2009) Occurrence of Mycobacterium avium subspecies paratuberculosis across host species and European countries with evidence for transmission between wildlife and domestic ruminants. BMC Microbiol 9:212–224. CrossRefGoogle Scholar
  5. 5.
    Nacy C, Buckley M (2008) Mycobacterium avium paratuberculosis: infrequent human pathogen or publichealth threat? American Academy of Microbiology, Colloquium Report.…/colloquium-reports/…/4507-mycobacterium-avium-paratuber
  6. 6.
    Knupfer E (2010) Within-farm strain dynamics of Mycobacterium avium subsp. paratuberculosis: evidence for limited vertical transmission, Doctoral thesis.
  7. 7.
    Pribylova R, Slana I, Cech S, Kralova A, Pavlik I (2013) Mycobacterium avium subsp. paratuberculosis detected in the reproductive tract of cows from an infected herd. Reprod Domest Anim 48:790–794CrossRefGoogle Scholar
  8. 8.
    Salem M, Zeid AA, Hassan D, El-Sayed A, Zschoeck M (2005) Studies on Johne’s disease in Egyptian cattle. J Vet Med B 52:134–137. CrossRefGoogle Scholar
  9. 9.
    Fawzy A, Fayed A, Youssef H, El-Sayed A, Zschöck M (2016) First report of MIRU-VNTR genotyping of Mycobacterium avium subsp. paratuberculosis isolates from Egypt. IJVR 17:130–133Google Scholar
  10. 10.
    Abdellrazeq GS, El-Naggar MM, Khaliel SA, Gamal-Eldin AE (2014) Detection of Mycobacterium avium subsp. paratuberculosis from cattle and buffaloes in Egypt using traditional culture, serological and molecular based methods. Vet World 7:586–593CrossRefGoogle Scholar
  11. 11.
    El Sayed MSA (2014) LCD array and IS900 efficiency in relation to traditional diagnostic techniques for diagnosis of Mycobacterium avium subspecies paratuberculosis in cattle in Egypt. Int J Mycobacteriol 3:101–107CrossRefGoogle Scholar
  12. 12.
    Amin AS, Hsu CY, Darwish SF, Ghosh P, AbdEl-Fatah EM, Behour TS, Talaat AM (2015) Ecology and genomic features of infection with Mycobacterium avium subspecies paratuberculosis in Egypt. Microbiology 161:807–818. CrossRefGoogle Scholar
  13. 13.
    Fritsch I, Luyven G, Köhler H, Lutz W, Möbius P (2012) Suspicion of Mycobacterium avium subsp. paratuberculosis transmission between cattle and wild-living red deer (Cervus elaphus) by multitarget genotyping. Appl Environ Microbiol 78:1132–1139. CrossRefGoogle Scholar
  14. 14.
    Sohal JS, Arsenault J, Labrecque O, Fairbrother JH, Roy JP, Fecteau G, L’Homme Y (2014) Genetic structure of Mycobacterium avium subsp. paratuberculosis population in cattle herds in Quebec as revealed by using a combination of multilocus genomic analyses. J Clin Microbiol 52:2764–2775. CrossRefGoogle Scholar
  15. 15.
    Thibault VC, Grayon M, Boschiroli ML, Hubbans C, Overduin P, Stevenson K, Gutierrez MC, Supply P, Biet F (2007) New variable number tandem-repeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing. J Clin Microbiol 45:2404–2410. CrossRefGoogle Scholar
  16. 16.
    Reyes JF, Chan CHS, Tanaka MM (2012) Impact of homoplasy on variable numbers of tandem repeats and spoligotypes in Mycobacterium tuberculosis. Infect Genet Evol 12:811–818. CrossRefGoogle Scholar
  17. 17.
    Supply P, Lesjean S, Savine E, Kremer K, van Soolingen D, Locht C (2001) Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units. J Clin Microbiol 39:3563–3571. CrossRefGoogle Scholar
  18. 18.
    Supply P, Allix C, Lesjean S, Cardoso-Oelemann M, Rüsch-Gerdes S, Willery E, Savine E, de Haas P, van Deutekom H, Roring S, Bifani P, Kurepina N, Kreiswirth B, Sola C, Rastogi N, Vatin V, Gutierrez MC, Fauville M, Niemann S, Skuce R, Kremer K, Locht C, van Soolingen D (2006) Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis. J Clin Microbiol 44:4498–4510. CrossRefGoogle Scholar
  19. 19.
    Wada T, Maeda S (2013) Multiplex agarose gel electrophoresis system for variable number of tandem repeats genotyping: analysis example using Mycobacterium tuberculosis. Electrophoresis 34:1171–1174. CrossRefGoogle Scholar
  20. 20.
    Moriyama M, Ogawa K, Nishimori K, Uchiya K, Ito T, Yagi T, Nakashima I, Nakagawa T, Tarumi O, Nikai T (2006) Usefulness of variable numbers of tandem repeats typing in clinical strains of Mycobacterium avium. Kekkaku 81:559–566Google Scholar
  21. 21.
    Kazumi Y, Udagawa T, Maeda S, Murase Y, Sugawara I, Okumura M, Azuma Y, Goto M, Tsunematsu N (2007) Comparison of usefulness between variable numbers of tandem repeats (VNTR) analysis and restriction fragment length polymorphism (RFLP) in the genotyping of Mycobacterium avium. Kekkaku 82:741–748Google Scholar
  22. 22.
    Supply P, Mazars E, Lesjean S, Vincent V, Gicquel B, Locht C (2000) Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Mol Microbiol 36:762–771CrossRefGoogle Scholar
  23. 23.
    Bull TJ, Sidi-Boumedine K, McMinn EJ, Stevenson K, Pickup R, Hermon-Taylor J (2003) Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex. Mol Cell Probes 17:157–164. CrossRefGoogle Scholar
  24. 24.
    Belisle JT, Sonnenberg MG (1998) Isolation of genomic DNA from mycobacteria. Methods Mol Biol 101:31–44. Google Scholar
  25. 25.
    Romano MI, Amadio A, Bigi F, Klepp L, Etchechoury I, Noto Llana M, Morsella C, Paolicchi F, Pavlik I, Bartos M, Leao SC, Cataldi A (2005) Further analysis of VNTR and MIRU in the genome of Mycobacterium avium complex, and application to molecular epidemiology of isolates from South America. Vet Microbiol 110:221–237. CrossRefGoogle Scholar
  26. 26.
    Radomski N, Roguet A, Lucas FS, Veyrier FJ, Cambau E, Accrombessi H, Moilleron R, Behr MA (2013) Moulin L (2013) atpE gene as a new useful specific molecular target to quantify Mycobacterium in environmental samples. BMC Microbiol 13:277. CrossRefGoogle Scholar
  27. 27.
    Inagaki T, Nishimori K, Yagi T, Ichikawa K, Moriyama M, Nakagawa T, Shibayama T, Uchiya K, Nikai T, Ogawa K (2009) Comparison of a Variable-Number Tandem-Repeat (VNTR) method for typing Mycobacterium avium with Mycobacterial Interspersed Repetitive-Unit-VNTR and IS 1245 restriction fragment length polymorphism typing. J Clin Microbiol 47:2156–2164. CrossRefGoogle Scholar
  28. 28.
    Pate M, Kušar D, Zolnir-Dovc M, Ocepek M (2011) MIRU–VNTR typing of Mycobacterium avium in animals and humans: heterogeneity of Mycobacterium avium subsp. hominissuis versus homogeneity of Mycobacterium avium subsp. avium strains. Res Vet Sci 91:376–381. CrossRefGoogle Scholar
  29. 29.
    Harmsen D, Dostal S, Roth A, Niemann S, Rothgänger J, Sammeth M, Albert J, Frosch M, Richter E (2003) RIDOM: comprehensive and public sequence database for identification of Mycobacterium species. BMC Infect Dis 3:26. PMID: 14611664 CrossRefGoogle Scholar
  30. 30.
    Sanderson JD, Moss MT, Tizard MLV, Hermon-Taylor J (1992) Mycobacterium paratuberculosis DNA in Crohn’s disease tissue. Gut 33:890–896CrossRefGoogle Scholar
  31. 31.
    Ryan P, Bennett MW, Aarons S, Lee G, Collins JK, O’Sullivan GC, O’Connell J, Shanahan F (2002) PCR detection of Mycobacterium paratuberculosis in Crohn’s disease granulomas isolated by laser capture microdissection. Inflamatory bowel disease. Gut 51:665–670. CrossRefGoogle Scholar
  32. 32.
    Castellanos E, Romero B, Rodrıguez S, De Juan L, Bezos J, Mateos A, Domınguez L, Aranaz A (2010) Molecular characterization of Mycobacterium avium subspecies paratuberculosis types II and III isolates by acombination of MIRU-VNTR loci. Vet Microbiol 144:118–126. CrossRefGoogle Scholar
  33. 33.
    Kim SH, Chun BW, Jung J, Kemp BM, Kwak KD, Cho NS, Kim JJ, Han MS, Kim W (2010) A preliminary study on the origin of Koreans based on Y-STR variation. Genes Genomics 32:371–377. Google Scholar
  34. 34.
    Hunter PR, Gaston MA (1988) Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity. J Clin Microbiol 26:2465–2466Google Scholar
  35. 35.
    Amonsin A, Li LL, Zhang Q, Bannantine JP, Motiwala AS, Sreevatsan S, Kapur V (2004) Multilocus short sequence sepeat sequencing approach for differentiating among Mycobacterium avium subsp. paratuberculosis strains. J Clin Microbiol 42:1694–1702. CrossRefGoogle Scholar
  36. 36.
    Comas I, Homolka S, Niemann S, Gagneux S (2009) Genotyping of genetically monomorphic bacteria: DNA sequencing in Mycobacterium tuberculosis highlights the limitations of current methodologies. PLoS ONE 4:e7815. CrossRefGoogle Scholar
  37. 37.
    Ahlstrom C, Barkema HW, Stevenson K, Zadoks RN, Biek R, Kao R, Trewby H, Haupstein D, Kelton DF, Fecteau G, Labrecque O, Keefe GP, McKenna SLB, De Buck J (2015) Limitations of variable number of tandem repeat typing identified through whole genome sequencing of Mycobacterium avium subsp. paratuberculosis on a national and herd level. BMC Genomics 16:16. CrossRefGoogle Scholar
  38. 38.
    Overduin P, Schouls L, Roholl P, van der Zanden A, Mahmmod N, Herrewegh A, van Soolingen D (2004) Use of multilocus variable-number tandem-repeat analysis for typing Mycobacterium avium subsp. paratuberculosis. J Clin Microbiol 42:5022–5028. CrossRefGoogle Scholar
  39. 39.
    Yang L, Wang C, Wang H, Meng Q, Wang Q (2015) Evaluation of MIRU-VNTR for typing of Mycobacterium bovis isolated from Sika deer in Northeast China. BMC Vet Res 11:93. CrossRefGoogle Scholar
  40. 40.
    van Hulzen KJE, Heuven HCM, Nielen M, Hoeboer J, Santema WJ, Koets AP (2011) Different Mycobacterium avium subsp. paratuberculosis MIRU-VNTR patterns coexist within cattle herds. Vet Microbiol 148:419–424. CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Bacteriology, Mycology, and Immunology, Faculty of Veterinary MedicineUniversity of Sadat CityMenoufiaEgypt

Personalised recommendations