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

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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.

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

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:19628055

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

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 14/7/2015

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

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.2005.08.004

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/10.2174/1874318801004010046

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.

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:3094489

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.x

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:19200666

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.

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:8760325

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:PMC167942

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:PMC227999

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:PMC2931435

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:PMC264138

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: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. (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:PMC2566117

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:16595839

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:PMC356861

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:17261338

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: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. (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:PMC1513380

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

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:PMC3419166

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:23831636

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:PMC2774859

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:PMC3233519

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.0076332

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.10-0334

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:PMC3165627

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: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. (2014). Assessment of genetic diversity of zoonotic Brucella spp. recovered from livestock in Egypt using multiple locus VNTR analysis. Biomed. Res. Int., 2014:353876.

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:PMC3986073

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_11

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:PMC169142

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:11162079

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:PMC2719624

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

Macedonian Veterinary Review

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