Original Scientific Article
Application of fluorescence based molecular assays for improved detection and typing of Brucella strains in clinical samples
Kiril Krstevski
*
,
Ivancho Naletoski
,
Dine Mitrov
,
Slavcho Mrenoshki
,
Iskra Cvetkovikj
,
Aleksandar Janevski
,
Aleksandar Dodovski
,
Igor Djadjovski
Received: 16 July 2015
Received in revised form: 04 September 2015
Accepted: 10 September 2015
Available Online First: 16 September 2015
Published on: 15 October 2015
Correspondence: Kiril Krstevski, krstevski@fvm.ukim.edu.mk
Abstract
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
References
1. Scholz H.C, Vergnaud G, Molecular characterisation of Brucella speciesRev. Sci. Tech Off. int. Epiz 2013; 32: 1149-162.
3. World Health Organization. The control of neglected zoonotic diseases;a route to poverty alleviation 2005; WHO, Geneva, Switzerland: Zoonoses and Veterinary Public Health;
8. World Organization for Animal Health –OIE. Chapter 8.4. Infection with Brucellaabortus, B. melitensis and B. suis 2014; Paris, 2012: In Terrestrial Animal Health Code.OIE;
11. Bounaadja L, Albert D, Chenais B, Henault S, Zygmunt M.S, Poliak S, Garin-Bastuji B, Real-time PCR for identfication of Brucella spp.:A comparative study of IS711, bcsp31 and per target genesVet. Microbiol 2009; 137: 156-164. http://dx.doi.org/10.1016/j.vetmic.2008.12.023. PMid:19200666
12. Leal-Klevezas D.S, Single-step PCR for detection of Brucella spp. from blood and milk of infected animalsJ. Clin. Microbiol 1995; 12: 3087-
14. Rijpens N.P, Jannes G, Van Asbroeck M, Rossau R, Herman L.M, Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probesAppl. Environ. Microbiol 1996; 62: 51683-1688.PMid:8633866 PMCid:PMC167942
15. Romero C, Gamazo C, Pardo M, López-Goñi I, Specific detection of Brucella DNA by PCRJ. Clin. Microbiol 1995; 33: 3615-617. PMid:7538508 PMCid:PMC227999
17. Bricker B.J, Halling S.M, Differentiation of Brucella abortus bv.1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv.1 by PCRJ. Clin. Microbiol 1994; 32: 2660-2666.PMid:7852552 PMCid:PMC264138
18. Bricker B.J, Halling S.M, Enhancement of the Brucella AMOS-PCR assay for differentiation of Brucella abortus vaccine strains S19 and RB51J. Clin. Microbiol 1995; 33: 1640-1642.PMid:7650203 PMCid:PMC228233
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, Evaluation of a multiplex PCR assay (bruce-ladder) for molecular typing of all Brucella species, including the vaccine strainsJ. Clin. Microbiol 2008; 46: 3484-3487. http://dx.doi.org/10.1128/JCM.00837-08. PMid:18716225 PMCid:PMC2566117
20. García-Yoldi D, Marín C.M, de Miguel M.J, Muñoz P.M, Vizmanos J.L, López-Goñi I, Multiplex PCR assay for the identification and differentiation of all Brucella species and the vaccine strains Brucella abortus S19 and RB51 and Brucella melitensisRev1. Clin Chem 2006; 52: 4779-781. http://dx.doi.org/10.1373/clinchem.2005.062596. PMid:16595839
21. Probert W.S, Schrader K.N, Khuong N.Y, Bystrom S.L, Graves M.H, Real-time multiplex PCR assay for detection of Brucella spp., B.abortus, and Bmelitensis. J. Clin. Microbiol 2004; 42: 1290-1293. http://dx.doi.org/10.1128/JCM.42.3.1290-1293.2004. PMid:15004098 PMCid:PMC356861
23. Bricker B.J, Ewalt D.R, Halling S.M, Brucella “HOOF Prints”: strain typing by multilocus analysis of variable number tandem repeats (VNTRs)BMC Microbiol 2003; 3: 15-
http://dx.doi.org/10.1186/1471-2180-3-15. PMid:12857351 PMCid:PMC183870
24. Le Flèche P, Jacques I, Grayon M, Al Dahouk S, Bouchon P, Denoeud F, Nockler K, Neubauer H, Guilloteau L.A, Vergnaud G, Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assayBMC Microbiol 2006; 6: 9- http://dx.doi.org/10.1186/1471-2180-6-9. PMid:16469109 PMCid:PMC1513380
25. Whatmore A.M, Perrett L.L, Macmillan A.P, Characterisation of the genetic diversity of Brucella by multilocus sequencingBMC Microbiol 2007; 7: 34-
http://dx.doi.org/10.1186/1471-2180-7-34. PMid:17448232 PMCid:PMC1877810
26. Ferreira A.C, Chambel L, Tenreiro T, Cardoso R, MLVA16 typing of Portuguese human and animal Brucella melitensis and Brucella abortus isolatesPLoS One 2012; 7: e42514- http://dx.doi.org/10.1371/journal.pone.0042514. PMid:22905141 PMCid:PMC3419166
27. Garofolo G, Di Giannatale E, De Massis F, Zilli K, Ancora M, Investigating genetic diversity of Brucella abortus and Brucella melitensis in Italy with MLVA-16Infect. Genet. Evol 2013; 19: 59-70. http://dx.doi.org/10.1016/j.meegid.2013.06.021. PMid:23831636
28. Her M, Kang S.I, Cho D.H, Cho Y.S, Hwang I.Y, Application and evaluation of the MLVA typing assay for the Brucella abortus strains isolated in KoreaBMC Microbiol 2009; 9: 230- http://dx.doi.org/10.1186/1471-2180-9-230. PMid:19863821 PMCid:PMC2774859
29. Jiang H, Fan M, Chen J, Mi J, Yu R, MLVA genotyping of Chinese human Brucella melitensis biovar 1, 2 and 3 isolatesBMC Microbiol 2011; 11: 256-
http://dx.doi.org/10.1186/1471-2180-11-256. PMid:22108057 PMCid:PMC3233519
30. Jiang H, Wang H, Xu L, Hu G, Ma J, MLVA genotyping of Brucella melitensis and Brucella abortus isolates from different animal species and humans and identification of Brucella suis vaccine strain S2 from cattle in ChinaPLoS One 2013; 8: e76332- http://dx.doi.org/10.1371/journal.pone.0076332.
31. Kang S.I, Heo E.J, Cho D, Kim J.W, Kim J.Y, Genetic comparison of Brucella canis isolates by the MLVA assay in South KoreaJ. Vet. Med. Sci 2011; 73: 779-786.
http://dx.doi.org/10.1292/jvms.10-0334.
32. Kiliç S, Ivanov I.N, Durmaz R, Bayraktar M.R, Multiple-locus variable-number tandem-repeat analysis genotyping of human Brucella isolates from TurkeyJ. Clin. Microbiol 2011; 49: 3276-3283. http://dx.doi.org/10.1128/JCM.02538-10. PMid:21795514 PMCid:PMC3165627
33. Marianelli C, Petrucca A, Pasquali P, Ciuchini F, Papadopoulou S, Cipriani P, Use of MLVA-16 typing to trace the source of a laboratory-acquired Brucella infectionJ. Hosp. Infect 2008; 68: 274-276. http://dx.doi.org/10.1016/j.jhin.2008.01.003. PMid:18289724
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, Assessment of genetic diversity of zoonotic Brucella spp. recovered from livestock in Egypt using multiple locus VNTR analysisBiomed. Res. Int 2014; 2014: 353876-
35. Mick V, Le Carrou G, Corde Y, Game Y, Jay M, Brucella melitensis in France:Persistence in wildlife and probable spillover from alpine ibex to domestic animalsPLoS ONE 2014; 9: 4e94168- http://dx.doi.org/10.1371/journal.pone.0094168. PMid:24732322 PMCid:PMC3986073
36. Bosilkovski M, Sing A, Brucellosis:It is not only Malta!Zoonozes – Infections Affecting Humans and Animals 2015; Springer Science+ Business Media Dordrecht; 287-316.ISBN 978-94-017-9456-5 http://dx.doi.org/10.1007/978-94-017-9457-2_11.
37. Newby D.T, Hadfield T.L, Roberto F.F, Real-time PCR detection of Brucella abortus:a comparative study of SYBR green I, 59-exonuclease, and hybridization probe assaysAppl. Environ. Microbiol 2003; 69: 4753-4759. http://dx.doi.org/10.1128/AEM.69.8.4753-4759.2003. PMid:12902268 PMCid:PMC169142
38. Redkar R, Rose S, Bricker B, Del Vecchio V, Real-time detection of Brucella abortus, Brucella melitensis and Brucella suisMol. Cell. Probes 2001; 15: 43-52.
http://dx.doi.org/10.1006/mcpr.2000.0338. PMid:11162079
39. Hinić V, Brodard I, Thomann A, Holub M, Miserez R, Abril C, IS711-based real-time PCR assay as a tool for detection of Brucella spp. in wild boars and comparison with bacterial isolation and serologyBMC Vet. Res 2009; 5: 22- http://dx.doi.org/10.1186/1746-6148-5-22. PMid:19602266 PMCid:PMC2719624
40. Ilhan Z, Aksakal A, Ekin I.H, Gulhan T, Solmaz H, Erdenlig S, Comparison of culture and PCR for the detection of Brucella melitensis in blood and lymphoid tissues of serologically positive and negative slaughtered sheepLett. Appl. Microbiol 2008; 46: 3301-306. http://dx.doi.org/10.1111/j.1472-765X.2007.02309.x. PMid:18179446
Acknowledgment
The authors would like to acknowledge the Joint FAO/IAEA Division and Technical Cooperation Programme of IAEA for the continuous scientific and professional technical support, contributing to the successful upgrading of existing and implementation of new molecular techniques at FVMS.
Copyright
© 2015 Krstevski K. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non Commercial License (http://creativecommons.org), which permits unrestricted non-commercial use, distribution, and reproduction in any medium provided the original work is properly cited.
Conflict of Interest Statement
The authors declared that they have no potential conflict of interest with respect to the authorship and/or publication of this article.
Citation Information
Macedonian Veterinary Review. Volume 38, Issue 2, Pages 223-232, p-ISSN 1409-7621, e-ISSN 1857-7415, DOI:
10.14432/j.macvetrev.2015.09.055, 2015
10.14432/j.macvetrev.2015.09.055
