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Macedonian Veterinary Review

The Journal of the Faculty of Veterinary Medicine-Skopje at the Ss. Cyril and Methodius University in Skopje

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Application of Fluorescence Based Molecular Assays for Improved Detection and Typing of Brucella Strains in Clinical Samples

Kiril Krstevski
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  • Veterinary Institute, Faculty of Veterinary Medicine, Ss. Cyril and Methodius University in Skopje, Republic of Macedonia
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/ Ivancho Naletoski
  • Animal Production and Health Section, Joint FAO/IAEA Division, International Atomic Energy Agency, Vienna, Austria
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/ Dine Mitrov
  • Veterinary Institute, Faculty of Veterinary Medicine, Ss. Cyril and Methodius University in Skopje, Republic of Macedonia
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/ Slavcho Mrenoshki
  • Veterinary Institute, Faculty of Veterinary Medicine, Ss. Cyril and Methodius University in Skopje, Republic of Macedonia
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/ Iskra Cvetkovikj
  • Veterinary Institute, Faculty of Veterinary Medicine, Ss. Cyril and Methodius University in Skopje, Republic of Macedonia
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/ Aleksandar Janevski
  • Veterinary Institute, Faculty of Veterinary Medicine, Ss. Cyril and Methodius University in Skopje, Republic of Macedonia
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/ Aleksandar Dodovski
  • Veterinary Institute, Faculty of Veterinary Medicine, Ss. Cyril and Methodius University in Skopje, Republic of Macedonia
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/ Igor Djadjovski
  • Veterinary Institute, Faculty of Veterinary Medicine, Ss. Cyril and Methodius University in Skopje, Republic of Macedonia
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Published Online: 2015-11-18 | DOI: https://doi.org/10.14432/j.macvetrev.2015.09.055


Bacteria from the genus Brucella are causative agents of brucellosis - a zoonotic disease which affects many wild and domestic animal species and humans. Taking into account the significant socio-economic and public health impact of brucellosis, its control is of great importance for endemic areas. The chosen control strategy could be successful only if adapted to the current epidemiological situation. This implies that a choice of appropriate diagnostic procedures for detection and typing of Brucella spp. strains are of essential importance. Significant advancement of molecular techniques and their advantages compared to classical methods, give strong arguments in promotion of these techniques as a powerful tool for comprehensive diagnostics of brucellosis. Considering this, the major tasks of the study were to select and implement molecular tests for detection and genotyping Brucella spp. and evaluate their performances using DNA from cultivated brucellae (islolates) and limited number of tissue samples from seropositive animals. The obtained results confirmed that implemented real time PCR for Brucella spp. detection, as well as MLVA-16 used for genotyping, have excellent analytical sensitivity (4.2 fg of Brucella DNA were successfully detected and genotyped). Furthermore, compared to bacteriological cultivation of Brucella spp., real time PCR and MLVA-16 protocols showed superior diagnostic sensitivity and detected Brucella DNA in tissues from which Brucella could not be cultivated. Based on the summarized study results, we propose a diagnostic algorithm for detection and genotyping of Brucella spp. bacteria. Routine use of proposed diagnostic algorithm will improve the effectiveness of infection confirmation and help for accurate evaluation of epidemiological situation.

Keywords: brucellosis; clinical samples; DNA; real time PCR; MLVA-16


  • 1. Scholz, H.C., Vergnaud, G. (2013). Molecular characterisation of Brucella species. Rev. Sci. Tech Off. int. Epiz., 32 (1): 149-162.Google Scholar

  • 2. Whatmore, A. (2011). Current understanding of the genetic diversity of Brucella, an expanding genus of zoonotic pathogens. Infec. Genet. Evol., 9(6): 1168-1184 http://dx.doi.org/10.1016/j.meegid.2009.07.001 PMid:19628055CrossrefWeb of ScienceGoogle Scholar

  • 3. World Health Organization (2005). The control of neglected zoonotic diseases; a route to poverty alleviation. Zoonoses and Veterinary Public Health, WHO, Geneva, Switzerland.Google Scholar

  • 4. European Commission, Directorate General for Health and Consumers (2009). Working Document on Eradication of Bovine, Sheep and Goats Brucellosis in the EU.SANCO/6095/2009. Available at: http:// ec.europa.eu/food/animal/diseases/eradication/ eradication_bovine_sheep_goats_brucellosis_en.pdf. last access on 14/7/2015Google Scholar

  • 5. Scientific Committee on Animal Health and Animal Welfare (2001). Brucellosis in sheep and goat. SANCO.C.2/AH/R23/2001. Available at: http://ec.europa.eu/food/fs/sc/scah/out59_en.pdf. last access on 14/7/2015 Google Scholar

  • 6. Garin-Bastuji, B., Blasco, J.M., Martın, C., Albert, D. (2006). The diagnosis of brucellosis in sheep and goats, old and new tools. Small Ruminant Research 62, 63-70. http://dx.doi.org/10.1016/j.smallrumres.2005.08.004CrossrefGoogle Scholar

  • 7. Poester, F.P, Nielsen, K., Samartino, L.E, Yu, W. L. (2010). Diagnosis of brucellosis. Open Vet. Sci. J., 4, 46-60. http://dx.doi.org/10.2174/1874318801004010046CrossrefGoogle Scholar

  • 8. World Organization for Animal Health - OIE (2014). Chapter 8.4. Infection with Brucellaabortus, B. melitensis and B. suis. In Terrestrial Animal Health Code. OIE, Paris, 2012.Google Scholar

  • 9. Hornitzky, M., Searson, J. (1986). The relationship between the isolation of Brucella abortus and serological status of infected, nonvaccinated cattle. Aust. Vet. J., 63, 172-174. http://dx.doi.org/10.1111/j.1751-0813.1986.tb02966.x PMid:3094489CrossrefGoogle Scholar

  • 10. Fekete, A., Bantlem, J.A., Hallingm, S.M., Sanborn, M.R. (1990). Preliminary development of a diagnostic test for Brucella using polymerase chain reaction. J. Appl. Bacteriol., 69, 216-227. http://dx.doi.org/10.1111/j.1365-2672.1990.tb01512.xCrossrefGoogle Scholar

  • 11. Bounaadja, L., Albert, D., Chenais, B., Henault, S., Zygmunt, M.S., Poliak, S., Garin-Bastuji, B. (2009). Real-time PCR for identfication of Brucella spp.: A comparative study of IS711, bcsp31 and per target genes. Vet. Microbiol., 137, 156-164. http://dx.doi.org/10.1016/j.vetmic.2008.12.023 PMid:19200666CrossrefWeb of ScienceGoogle Scholar

  • 12. Leal-Klevezas, D.S. et al. (1995). Single-step PCR for detection of Brucella spp. from blood and milk of infected animals, J. Clin. Microbiol., 12, 3087.Google Scholar

  • 13. Ouahrani-Bettache, S., Soubrier, M.P., Liautard, J.P. (1996). IS6501-anchored PCR for the detection and identification of Brucella species and strains. J. Appl. Bacteriol., 81, 154-160. http://dx.doi.org/10.1111/j.1365-2672.1996.tb04493.x PMid:8760325CrossrefGoogle Scholar

  • 14. Rijpens, N.P.,Jannes, G., Van Asbroeck, M., Rossau, R., Herman, L.M. (1996). Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes. Appl. Environ. Microbiol., 62(5): 1683-1688. PMid:8633866 PMCid:PMC167942Google Scholar

  • 15. Romero, C.,Gamazo, C., Pardo, M., López-Goñi, I. (1995). Specific detection of Brucella DNA by PCR. J. Clin. Microbiol., 33(3): 615-617. PMid:7538508 PMCid:PMC227999Google Scholar

  • 16. Yu, W.L., Nielsen, K. (2010). Review of detection of Brucella spp. by polymerase chain reaction. Croat. med. J., 51(4): 306-313. http://dx.doi.org/10.3325/cmj.2010.51.306 PMid:20718083 PMCid:PMC2931435CrossrefGoogle Scholar

  • 17. Bricker, B.J., Halling, S.M. (1994). Differentiation of Brucella abortus bv.1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv.1 by PCR. J. Clin. Microbiol., 32, 2660 -2666. PMid:7852552 PMCid:PMC264138Google Scholar

  • 18. Bricker, B.J., Halling, S.M. (1995). Enhancement of the Brucella AMOS-PCR assay for differentiation of Brucella abortus vaccine strains S19 and RB51. J. Clin. Microbiol., 33, 1640-1642. PMid:7650203 PMCid:PMC228233Google Scholar

  • 19. Lopez-Goni, I., Garcia-Yoldi, D., Marin, C.M., de Miguel, M.J., Muñoz, P.M., Blasco, J.M., Jacques, I., Grayon, M., Cloeckaert, A., Ferreira, A.C., Cardoso, R., Corrêa de Sá, M.I., Walravens, K., Albert, D., Garin-Bastuji, B. (2008). Evaluation of a multiplex PCR assay (bruce-ladder) for molecular typing of all Brucella species, including the vaccine strains. J. Clin. Microbiol.,46, 3484-3487. http://dx.doi.org/10.1128/JCM.00837-08 PMid:18716225 PMCid:PMC2566117CrossrefGoogle Scholar

  • 20. García-Yoldi, D., Marín, C.M., de Miguel, M.J., Muñoz, P.M., Vizmanos, J.L., López-Goñi, I. (2006). Multiplex PCR assay for the identification and differentiation of all Brucella species and the vaccine strains Brucella abortus S19 and RB51 and Brucella melitensis. Rev1. Clin Chem., 52(4): 779-781. http://dx.doi.org/10.1373/clinchem.2005.062596 PMid:16595839CrossrefGoogle Scholar

  • 21. Probert, W.S., Schrader, K.N., Khuong, N.Y., Bystrom, S.L., Graves, M.H. (2004). Real-time multiplex PCR assay for detection of Brucella spp., B.abortus, and B. melitensis. J. Clin. Microbiol., 42, 1290-1293. http://dx.doi.org/10.1128/JCM.42.3.1290-1293.2004 PMid:15004098 PMCid:PMC356861CrossrefGoogle Scholar

  • 22. Al Dahouk, S., Le Flèche, P., Nöckler, K. et al. (2007). Evaluation of Brucella MLVA typing for human brucellosis. J. Microbiol. Meth., 69, 137-145. http://dx.doi.org/10.1016/j.mimet.2006.12.015 PMid:17261338CrossrefGoogle Scholar

  • 23. Bricker, B.J., Ewalt, D.R., Halling, S.M. (2003). Brucella “HOOF Prints”: strain typing by multilocus analysis of variable number tandem repeats (VNTRs). BMC Microbiol., 3, 15. http://dx.doi.org/10.1186/1471-2180-3-15 PMid:12857351 PMCid:PMC183870CrossrefGoogle Scholar

  • 24. Le Flèche, P., Jacques, I., Grayon, M., Al Dahouk, S., Bouchon, P., Denoeud, F., Nockler, K., Neubauer, H., Guilloteau, L.A., Vergnaud G. (2006). Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay. BMC Microbiol., 6, 9. http://dx.doi.org/10.1186/1471-2180-6-9 PMid:16469109 PMCid:PMC1513380CrossrefGoogle Scholar

  • 25. Whatmore, A.M., Perrett, L.L., Macmillan, A.P. (2007). Characterisation of the genetic diversity of Brucella by multilocus sequencing. BMC Microbiol., 7, 34. http://dx.doi.org/10.1186/1471-2180-7-34 PMid:17448232 PMCid:PMC1877810 CrossrefGoogle Scholar

  • 26. Ferreira, A.C., Chambel, L., Tenreiro, T., Cardoso, R. et al. (2012). MLVA16 typing of Portuguese human and animal Brucella melitensis and Brucella abortus isolates. PLoS One 7, e42514. http://dx.doi.org/10.1371/journal.pone.0042514 PMid:22905141 PMCid:PMC3419166CrossrefGoogle Scholar

  • 27. Garofolo, G., Di Giannatale, E., De Massis, F., Zilli, K., Ancora, M., et al. (2013): Investigating genetic diversity of Brucella abortus and Brucella melitensis in Italy with MLVA-16. Infect. Genet. Evol.19, 59-70. http://dx.doi.org/10.1016/j.meegid.2013.06.021 PMid:23831636Web of ScienceCrossrefGoogle Scholar

  • 28. Her, M., Kang, S.I., Cho, D.H., Cho, Y.S., Hwang, I.Y., et al. (2009). Application and evaluation of the MLVA typing assay for the Brucella abortus strains isolated in Korea. BMC Microbiol., 9, 230. http://dx.doi.org/10.1186/1471-2180-9-230 PMid:19863821 PMCid:PMC2774859CrossrefGoogle Scholar

  • 29. Jiang, H., Fan, M., Chen, J., Mi, J., Yu, R., et al. (2011). MLVA genotyping of Chinese human Brucella melitensis biovar 1, 2 and 3 isolates. BMC Microbiol., 11, 256. http://dx.doi.org/10.1186/1471-2180-11-256 PMid:22108057 PMCid:PMC3233519CrossrefGoogle Scholar

  • 30. Jiang, H., Wang, H., Xu, L., Hu, G., Ma, J., et al. (2013). MLVA genotyping of Brucella melitensis and Brucella abortus isolates from different animal species and humans and identifi cation of Brucella suis vaccine strain S2 from cattle in China. PLoS One, 8, e76332 http://dx.doi.org/10.1371/journal.pone.0076332Web of ScienceCrossrefGoogle Scholar

  • 31. Kang, S.I., Heo, E.J., Cho, D., Kim, J.W., Kim, J.Y., et al. (2011). Genetic comparison of Brucella canis isolates by the MLVA assay in South Korea. J. Vet. Med. Sci.73, 779-786. http://dx.doi.org/10.1292/jvms.10-0334CrossrefGoogle Scholar

  • 32. Kiliç, S., Ivanov, I.N., Durmaz, R., Bayraktar, M.R., et al. (2011). Multiple-locus variable-number tandemrepeat analysis genotyping of human Brucella isolates from Turkey. J. Clin. Microbiol. 49, 3276-3283. http://dx.doi.org/10.1128/JCM.02538-10 PMid:21795514 PMCid:PMC3165627CrossrefGoogle Scholar

  • 33. Marianelli, C., Petrucca, A., Pasquali, P., Ciuchini, F., Papadopoulou, S., Cipriani, P. (2008). Use of MLVA-16 typing to trace the source of a laboratory-acquired Brucella infection. J. Hosp. Infect. 68, 274-276. http://dx.doi.org/10.1016/j.jhin.2008.01.003 PMid:18289724CrossrefGoogle Scholar

  • 34. Menshawy, A.M., Perez-Sancho, M., Garcia-Seco, T., Hosein, H.I., García, N., Martinez, I., Sayour, A.E., Goyache, J., Azzam, R.A., Dominguez, L. (2014). Assessment of genetic diversity of zoonotic Brucella spp. recovered from livestock in Egypt using multiple locus VNTR analysis. Biomed. Res. Int., 2014:353876.Web of ScienceGoogle Scholar

  • 35. Mick, V., Le Carrou, G., Corde, Y., Game, Y., Jay, M. et al. (2014). Brucella melitensis in France: Persistence in wildlife and probable spillover from alpine ibex to domestic animals. PLoS ONE, 9(4): e94168. http://dx.doi.org/10.1371/journal.pone.0094168 PMid:24732322 PMCid:PMC3986073Google Scholar

  • 36. Bosilkovski, M. (2015). Brucellosis: It is not only Malta! In A. Sing (Ed.), Zoonozes - Infections Affecting Humans and Animals (pp. 287-316). Springer Science+Business Media Dordrecht. ISBN 978-94-017-9456-5. http://dx.doi.org/10.1007/978-94-017-9457-2_11CrossrefGoogle Scholar

  • 37. Newby, D.T., Hadfield, T.L., Roberto, F.F. (2003). Real-time PCR detection of Brucella abortus: a comparative study of SYBR green I, 59-exonuclease, and hybridization probe assays. Appl. Environ. Microbiol., 69, 4753-4759. http://dx.doi.org/10.1128/AEM.69.8.4753-4759.2003 PMid:12902268 PMCid:PMC169142CrossrefGoogle Scholar

  • 38. Redkar, R., Rose, S., Bricker, B., Del Vecchio, V. (2001). Real-time detection of Brucella abortus, Brucella melitensis and Brucella suis. Mol. Cell. Probes, 15, 43-52. http://dx.doi.org/10.1006/mcpr.2000.0338 PMid:11162079CrossrefGoogle Scholar

  • 39. Hinić, V., Brodard, I., Thomann, A., Holub, M., Miserez, R., Abril, C. (2009). IS711-based real-time PCR assay as a tool for detection of Brucella spp. in wild boars and comparison with bacterial isolation and serology. BMC Vet. Res., 5, 22. http://dx.doi.org/10.1186/1746-6148-5-22 PMid:19602266 PMCid:PMC2719624Web of ScienceGoogle Scholar

  • 40. Ilhan, Z., Aksakal, A., Ekin, I.H., Gulhan, T., Solmaz, H., Erdenlig. S. (2008). Comparison of culture and PCR for the detection of Brucella melitensis in blood and lymphoid tissues of serologically positive and negative slaughtered sheep. Lett. Appl. Microbiol., 46(3): 301-306. http://dx.doi.org/10.1111/j.1472-765X.2007.02309.x PMid:18179446 CrossrefWeb of ScienceGoogle Scholar

About the article

Received: 2015-07-16

Revised: 2015-09-04

Accepted: 2015-09-10

Published Online: 2015-11-18

Published in Print: 2015-10-01

Citation Information: Macedonian Veterinary Review, ISSN (Online) 1857-7415, DOI: https://doi.org/10.14432/j.macvetrev.2015.09.055.

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