Bovine Tuberculosis in the Republic of Macedonia: Postmortem, Microbiological and Molecular Study in Slaughtered Reactor Cattle

Iskra Cvetkovikj 1 , Slavcho Mrenoshki 1 , Kiril Krstevski 1 , Igor Djadjovski 1 , Branko Angjelovski 1 , Zagorka Popova 1 , Aleksandar Janevski 1 , Aleksandar Dodovski 1 ,  and Aleksandar Cvetkovikj 1
  • 1 Veterinary Institute, Faculty of Veterinary Medicine, Ss. Cyril and Methodius University in Skopje, Skopje, Macedonia (the former Yugoslav Republic of)


Bovine tuberculosis is a chronic infectious disease in cattle caused mainly by Mycobacterium bovis and to a lesser extent by Mycobacterium caprae. The other members of the Mycobacterium tuberculosis complex (MTBC) can also cause the disease in domestic and wild animals and all of them have a zoonotic potential. The main purpose of the study was to determine the presence and distribution of the tuberculous lesions in reactor cattle, and to isolate and identify the causative agents of bovine tuberculosis in the Republic of Macedonia. Lymph nodes and affected organs from 188 reactor cattle slaughtered due to a positive intradermal comparative cervical tuberculin test were analyzed by detection of tuberculous lesions, followed by isolation and molecular identification of the isolated mycobacteria. The isolation was performed on selective media - Lowenstein Jensen with glycerol, Lowenstein Jensen without glycerol and Stonebrink medium supplemented with pyruvate. The molecular identification of the MTBC members was performed by analysis of the Regions of difference (RD1, RD9 and RD4) and detection of single nucleotide polymorphisms in the lepA gene for Mycobacterium caprae. Typical tuberculous lesions were detected in 62 animals (33.0%) and the lesions were most prevalent in the mediastinal lymph nodes (47.5%). The isolated mycobacteria in the MTBC were identified as Mycobacterium bovis and Mycobacterium caprae and were found in both animals with visible lesions (82.2%) and animals without visible lesions (27.7%). The slaughterhouse postmortem examinations and laboratory investigations should be included on regular bases in order to improve the National eradication program.

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  • 1. O’Reilly, L.M., Daborn, C.J. (1995). The epidemiology of Mycobacterium bovis infections in animals and man. A review. Tuber Lung Dis. 76 (1): 1-46.

  • 2. Pesciaroli, M., Alvarez, J., Boniotti, M.B., Cagiola, M., Di Marco V., Marianelli, C., Pacciarini, M., Pasquali, P. (2014). Tuberculosis in domestic animal species. Res Vet Sci. 97, Suppl. S78-85. PMid:25151859

  • 3. Task force bovine tuberculosis subgroup. Working document on eradication of bovine tuberculosis in the EU accepted by the Bovine tuberculosis subgroup of the Task Force on monitoring animal disease eradication. (2006). SANCO/10200/2006.

  • 4. Brosch, R., Gordon, S., Marmiesse, M., Brodin, P., Buchrieser, C., Eiglmeier, K., Garnier, T., Gutierrez, C., Hewinson, G., Kremer, K., Parsons, L., Pym, A., Samper, S., Soolingen, D., Cole, S. (2002). A new evolutionary scenario for the Mycobacterium tuberculosis complex. P Natl Acad Sci USA. 99 (6): 3684-3689. PMid:11891304 PMCid:PMC122584

  • 5. Schiller, I., Waters, W.R., Vordermeier, H.M., Jemmi, T., Welsh, M., Keck, N., Whelan, A., Gormley, E., Boschiroli, M.L., Moyen, J.L., Vela, C., Cagiola, M., Buddle, B.M., Palmer, M., Thacker, T., Oesch, B. (2011). Bovine tuberculosis in Europe from the perspective of an officially tuberculosis free country: trade, surveillance and diagnostics. Vet Microbiol. 151 (1): 153-159. PMid:21439740

  • 6. Shitaye J.E., Tsegaye W., Pavlik I. (2007). Bovine tuberculosis infection in animal and human populations in Ethiopia: a review. Vet Med (Praha) 52 (8): 317-332.

  • 7. Muller, B., Durr, S., Alonso, S., Hattendorf, J., Laisse, C.J., Parsons, S.D., Van Helden, P.D., Zinsstag, J. (2013). Zoonotic Mycobacterium bovisinduced tuberculosis in humans. Emerg Infect Dis. 19 (6): 899-908. PMid:23735540 PMCid:PMC4816377

  • 8. Gramatikovski, G., Stojanoski, B. (1985). Epidemiological situation of infectious diseases in Socialistic Republic of Macedonia 1927-1977. Veterinary Institute Skopje. (in Macedonian).

  • 9. Nikolovski, G., Petrov, E.A., Cokrevski, S., Arsevska, E., Nikolovska, G. (2012) Bovine tuberculosis in cattle during the implementation of official control measures in Republic of Macedonia for the period 2007-2009. Slov Vet Res 49 (2): 79-87.

  • 10. Food and Veterinary Agency of Republic of Macedonia (2007). Program for eradication of bovine tuberculosis. Official Gazette of Republic of Macedonia No. 22/2007. (in Macedonian).

  • 11. OIE. (2009). Bovine tuberculosis. Manual for diagnostic tests and vaccines for terrestrial animals.

  • 12. Corner, L.A., Gormley, E., Pfeiffer, D.U. (2012). Primary isolation of Mycobacterium bovis from bovine tissues: conditions for maximising the number of positive cultures. Vet Microbiol. 156 (1): 162-171. PMid:22074859

  • 13. Gormley, E., Corner, L.A., Costello, E., Rodriguez- Campos, S. (2014). Bacteriological diagnosis and molecular strain typing of Mycobacterium bovis and Mycobacterium caprae. Res Vet Sci. 97 Suppl, S30-43. PMid:24833269

  • 14. Pinsky, B.A., Banaei, N. (2008). Multiplex real-time PCR assay for rapid identification of Mycobacterium tuberculosis complex members to the species level. J Clin Microbiol. 46 (7): 2241-2246. PMid:18508937 PMCid:PMC2446918

  • 15. Domogalla, J., Prodinger, W.M., Blum, H., Krebs, S., Gellert, S., Muller, M., Neuendorf, E., Sedlmaier, F., Buttner, M. (2013). Region of difference 4 in alpine Mycobacterium caprae isolates indicates three variants. J Clin Microbiol. 51 (5): 1381-1388. PMid:23408688 PMCid:PMC3647898

  • 16. Reddington, K., O’Grady, J., Dorai-Raj, S., Niemann, S., van Soolingen, D., et al. (2011). A novel multiplex Real-Time PCR for the identification of Mycobacteria associated with zoonotic tuberculosis. PLoS One 6 (8): e23481. PMid:21858140 PMCid:PMC3153498

  • 17. Rivière, J., Carabin, K., Le Strat, Y., Hendrikx, P., Dufour, B. (2014). Bovine tuberculosis surveillance in cattle and free-ranging wildlife in EU Member States in 2013: a survey-based review. Vet Microbiol. 173 (3): 323-331. PMid:25205200

  • 18. Schiller, I., Oesch, B., Vordermeier, H.M., Palmer, M.V., Harris, B.N., Orloski, K.A., Buddle,B.M., Thacker, T.C., Lyashchenko, K.P., Waters, W.R. (2010). Bovine tuberculosis. A review of current and emerging diagnostic techniques in view of their relevance for disease control and eradication. Transbound Emerg Dis. 57 (4): 205-220.

  • 19. Fitzgerald, S.D., Hollinger, C., Mullaney, T.P., Bruning-Fann, C.S., Tilden, J., Smith, R., Averill, J., Kaneene, J.B. (2016). Herd outbreak of bovine tuberculosis illustrates that route of infection correlates with anatomic distribution of lesions in cattle and cats. J Vet Diagn Invest. 28 (2): 129-132. PMid:26965232

  • 20. Pritchard, D.G. (1988). A century of bovine tuberculosis 1888-1988: conquest and controversy. Comp Clin Path. 99 (4): 357-399.

  • 21. Whipple, D.L., Bolin, C.A., Miller, J.M. (1996). Distribution of lesions in cattle infected with Mycobacterium bovis. J Vet Diagn Invest. 8 (3): 351-354. PMid:8844579

  • 22. Corner, L.A. (1994). Post mortem diagnosis of Mycobacterium bovis infection in cattle. Vet Microbiol. 40 (1): 53-63.

  • 23. Ministry of Agriculture, Food and Environment (2013). National Eradication Program for Bovine tuberculosis in Spain for 2013. (in Spanish)

  • 24. European Commission Health & Consumer Protection Directorate-General Veterinary and International Affairs Unit G5 - Veterinary Programmes. (2013). Working Document on eradication of Bovine tuberculosis in the EU.SANCO/10067/2013

  • 25. Croatian Ministry of agriculture (2015). Annual order for animal protection from infectious and parasitic diseases for 2016. Official Gazette of Croatia No. 141/2015 (in Croatian)

  • 26. Boritsch, E.C., Supply, P., Honore, N., Seeman, T., Stinear, T. P., Brosch, R. (2014). A glimpse into the past and predictions for the future: the molecular evolution of the tuberculosis agent. Mol. Microbiol. 93 (5): 835-852. PMid:25039682

  • 27. Mostowy, S., Cousins, D., Brinkman, J., Aranaz, A., Behr, M.A. (2002). Genomic deletions suggest a phylogeny for the Mycobacterium tuberculosis complex. J Infect Dis. 186 (1): 74-80. PMid:12089664

  • 28. Huard, R.C., Lazzarini, L.C., Butler, W.R., van Soolingen, D., Ho, J.L. (2003). PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions. J. Clin. Microbiol. 41 (4): 1637-1650. PMid:12682155 PMCid:PMC153936

  • 29. Warren, R.M., Gey van Pittius, N.C., Barnard, M., Hesseling, A., Engelke, E., de Kock, M., Gutierrez, M.C., Chege, G.K., Victor, T.C., Hoal, E.G., van Helden, P.D. (2006). Differentiation of Mycobacterium tuberculosis complex by PCR amplification of geno- mic regions of difference. Int. J. Tuberc. Lung Dis. 10 (7): 818-822. PMid:16850559

  • 30. Pounder, J.I., Anderson, C.M., Voelkerding, K.V., Salfinger, M., Dormandy, J., Somoskovi, A., Heifets, L., Graham, J.J., Storts, D.R., Petti, C.A. (2010). Mycobacterium tuberculosis complex differentiation by genomic deletion patterns with multiplex polymerase chain reaction and melting analysis. Diagn. Microbiol. Infect. Dis. 67 (1): 101-105. PMid:20227227

  • 31. Rettinger, A., Broecki, S., Fink, M., Prodinger, W.M., blum, H., Krebs, S., Domogalla, J., Just, F., Gellert, S., Straubinger, R.K., Buttner M. (2015). The region of difference four is a robust genetic marker for subtyping Mycobacterium caprae Isolates and is linked to spatial distribution of three subtypes. Transbound Emerg Dis. PMid:26518998

  • 32. Rodriguez, S., Bezos, J., Romero, B., de Juan, L., Alvarez, J., Castellanos, E., Moya, N., Lozano, F., Javed, M.T., Saez-Llorente, J.L., Liebana, E., Mateos, A., Dominguez, L., Aranaz, A. (2011). Mycobacterium caprae infection in livestock and wildlife. Spain. Emerg. Infect. Dis. 17 (3): 532-535. PMid:21392452 PMCid:PMC3165998

  • 33. Munyeme, M., Rigouts, L., Shamputa, I.C., Muma, J.B., Tryland, M., Skjerve, E., Djønne, B. (2009). Isolation and characterization of Mycobacterium bovis strains from indigenous Zambian cattle using Spacer oligonucleotide typing technique. BMC microbiology 9 (1):1. PMid:19619309 PMCid:PMC2719650

  • 34. Proano-Pérez, F., Benitez-Ortiz, W., Desmecht, D., Coral, M., Ortiz, J., Ron, L., Portaels, F., Rigouts, L., Linden, A. (2011). Post-mortem examination and laboratory-based analysis for the diagnosis of bovine tuberculosis among dairy cattle in Ecuador. Prev Vet Med. 101 (1): 65-72. PMid:21645934

  • 35. Nassar, A.F.C., Miyashiro, S., Oliveira, C.G., Pacheco, W.A. and Ogata, R.A. (2007). Isolation and identification of bovine tuberculosis in a Brazilian herd (São Paulo). Mem Inst Oswaldo Cruz. 102 (5): 639-642. PMid:17710311

  • 36. Shittu, А., Clifton-Hadley, R.S., Ely, E.R., Upton, P.U., Downs, S.H. (2008). Factors associated with bovine tuberculosis confirmation rates in suspect lesions found in cattle at routine slaughter in Great Britain, 2003-2008. Prev Vet Med. 110 (3): 395- 404.

  • 37. Courcoul, A., Moyen, J.L., Brugere, L., Faye, S., Henault, S., Gares, H., Boschiroli, M.L. (2014). Estimation of sensitivity and specificity of bacteriology, histopathology and PCR for the confirmatory diagnosis of bovine tuberculosis using latent class analysis. PloS one 9(3): p.e90334. PMid:24625670 PMCid:PMC3953111

  • 38. Duignan, A., Good, M., More, S.J. (2012). Quality control in the national bovine tuberculosis eradication programme in Ireland. Rev. Sci. Tech. Off. Int. Epiz. 31, 845-860.

  • 39. Good, M., Duignan, A. (2011). An evaluation of the Irish Single Reactor Breakdown Protocol for 2005 to 2008 inclusive and its potential application as a monitor of tuberculin test performance. Vet. Microbiol. 151 (1): 85 -90. PMid:21441002

  • 40. De la Rua-Domenech, R., Goodchild, A.T., Vordermeier, H.M., Hewinson, R.G., Christiansen, K.H., Clifton-Hadley, R.S. (2006). Ante mortem diagnosis of tuberculosis in cattle: a review of the tuberculin tests, γ-interferon assay and other ancillary diagnostic techniques. Res Vet Sci. 81 (2): 190-210. PMid:16513150

  • 41. Cvetnic, Z., Katalinic-Jankovic, V., Sostaric, B., Spicic, S., Obrovac, M., Marjanovic, S., Benic, M., Kirin, B.K., Vickovic, I. (2007). Mycobacterium caprae in cattle and humans in Croatia. Int J Tuberc Lung Dis. 11 (6): 652-658. PMid:17519097

  • 42. Beširović, H., Alić, A., Špičić, S., Cvetnić, Ž., Prašović, S., Velić, L. (2012). Bovine tuberculosis in Bosnia and Herzegovina caused by Mycobacterium caprae. Vet Arhiv. 82 (4): 341-349.

  • 43. Boniotti, M.B., Goria, M., Loda, D., Garrone, A., Benedetto, A., Mondo, A., Tisato, E., Zanoni, M., Zoppi, S., Dondo, A., Tagliabue, S., Bonora, S., Zanardi, G., Pacciarini, M.L. (2009). Molecular typing of Mycobacterium bovis strains isolated in Italy from 2000 to 2006 and evaluation of variablenumber- tandem-repeats for a geographic optimized genotyping. J Clin Microbiol. 47 (3): 636-644. PMid:19144792 PMCid:PMC2650904

  • 44. Prodinger, W.M., Brandstätter, A., Naumann, L., Pacciarini, M., Kubica, T., Boschiroli, M.L., Aranaz, A., Nagy, G., Cvetnic, Z., Ocepek, M., Skrypnyk, A., Erler, W., Niemann, S., Pavlik, I., Moser, I. (2005). Characterization of Mycobacterium caprae isolates from Europe by mycobacterial interspersed repetitive unit genotyping. J Clin Microbiol. 43 (10): 4984-4992. PMid:16207952 PMCid:PMC1248478

  • 45. Sahraoui, N., Muller, B., Guetarni, D., Boulahbal, F., Yala, D., Ouzrout, R., Berg, S., Smith, N.H., Zinsstag, J. (2009). Molecular characterization of Mycobacterium bovis strains isolated from cattle slaughtered at two abattoirs in Algeria. BMC Vet Res. 5 (1): 4. PMid:19173726 PMCid:PMC2640374

  • 46. Zeng, W., Zhang, Y., Zhao, X., Huang, G., Jiang, Y., Dong, H., Li, X., Wan K., He, C. (2013). Occurrence of non-tuberculous mycobacteria species in livestock from northern China and first isolation of Mycobacterium caprae. Epidemiol Infect. 141 (7): 1545-1551. PMid:23298678

  • 47. Shitaye, J.E., Getahun, B., Alemayehu, T., Skoric, M., Treml, F., Fictum, P., Vrbas, V., Pavlik, I. (2006). A prevalence study of bovine tuberculosis by using abattoir meat inspection and tuberculin skin testing data, histopathological and IS6110 PCR examination of tissues with tuberculous lesions in cattle in Ethiopia. Vet Med (Praha). 51 (11): 512-522.

  • 48. European Food and safety Authority. (2014). The European Union Summary Report on Trends and Sources of Zoonoses. Zoonotic Agents and Foodborne Outbreaks in 2012. EFSA Journal 12 (2): 3547 (pp 312).

  • 49. Rodriguez, E., Sanchez, L.P., Perez, S., Herrera, L., Jimenez, M.S., Samper, S., Iglesias, M.J. (2009). Human tuberculosis due to Mycobacterium bovis and M. caprae in Spain, 2004-2007. Int J Tuberc Lung D. 13 (12): 1536-1541. PMid:19919773


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