Skip to main content

Advertisement

SpringerLink
Log in

Evaluation of PCR methods for detection of Brucella strains from culture and tissues

  • Regular Articles
  • Published:
Tropical Animal Health and Production Aims and scope Submit manuscript

Abstract

The genus Brucella causes significant economic losses due to infertility, abortion, stillbirth or weak calves, and neonatal mortality in livestock. Brucellosis is still a zoonosis of public health importance worldwide. The study was aimed to optimize and evaluate PCR assays used for the diagnosis of Brucella infections. For this aim, several primers and PCR protocols were performed and compared with Brucella cultures and biological material inoculated with Brucella. In PCR assays, genus- or species-specific oligonucleotide primers derived from 16S rRNA sequences (F4/R2, Ba148/928, IS711, BruP6-P7) and OMPs (JPF/JPR, 31ter/sd) of Brucella were used. All primers except for BruP6-P7 detected the DNA from reference Brucella strains and field isolates. In spiked blood, milk, and semen samples, F4-R2 primer-oriented PCR assays detected minimal numbers of Brucella. In spiked serum and fetal stomach content, Ba148/928 primer-oriented PCR assays detected minimal numbers of Brucella. Field samples collected from sheep and cattle were examined by bacteriological methods and optimized PCR assays. Overall, sensitivity of PCR assays was found superior to conventional bacteriological isolation. Brucella DNA was detected in 35.1, 1.1, 24.8, 5.0, and 8.0% of aborted fetus, blood, milk, semen, and serum samples by PCR assays, respectively. In conclusion, PCR assay in optimized conditions was found to be valuable in sensitive and specific detection of Brucella infections of animals.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  • Al Dahouk, S., Tomaso, H., Nöckler, K., Neubauer, H. and Frangoulidis, D., 2003. Laboratory-based diagnosis of brucellosis—a review of the literature. Part II: serological tests for brucellosis. Clinical Laboratory, 49(11-12), 577–589.

    PubMed  Google Scholar 

  • Al-Ajlan, H.H., Ibrahim, A.S. and Al-Salamah, A.A., 2011. Comparison of different PCR methods for detection of Brucella spp. in human blood samples. Polish Journal of Microbiology, 60, 27–33.

    CAS  PubMed  Google Scholar 

  • Amin, A.S., Hamdy, M.E. and Ibrahim, A.K., 2001. Detection of Brucella melitensis in semen using the polymerase chain reaction assay. Veterinary Microbiology, 83, 37–44.

    Article  CAS  PubMed  Google Scholar 

  • Asaad, A.M. and Alqahtani, J.M., 2012. Serological and molecular diagnosis of human brucellosis in Najran, Southwestern Saudi Arabia. Journal of Infection and Public Health, 5, 189–194.

    Article  PubMed  Google Scholar 

  • Blasco, J.M. and Molina-Flores B., 2011. Control and eradication of Brucella melitensis infection in sheep and goats. Veterinary Clinics of North America, 27 (1), 95–104.

    PubMed  Google Scholar 

  • Bounaadja, L., Albert, D., Chenais, B., Henault, S., Zygmunt, M.S., Poliak, S. and Garin-Bastuji, B., 2009. Real-time PCR for identification of Brucella spp.: a comparative study of IS711, bcsp31 and per target genes. Veterinary Microbiology, 137, 156–164.

    Article  CAS  PubMed  Google Scholar 

  • Bricker, B.J. and Halling, S.M., 1994. Differentiation of Brucella abortus bv. 1, 2 and 4, Brucella melitensis, Brucella ovis bv. 1 by PCR. Journal of Clinical Microbiology, 32, 2660–2666.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bricker, B.J. and Halling, S.M., 1995. Enhancement of Brucella AMOS PCR assay for differentiation of Brucella abortus vaccine strains S19 and RB51. Journal of Clinical Microbiology, 33, 1640–1642.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bricker, B.J., 2002. PCR as a diagnostic tool for brucellosis. Veterinary Microbiology, 90, 435–436.

    Article  CAS  PubMed  Google Scholar 

  • Christopher, S., Umapathy, B.L. and Ravikumar, K.L., 2010. Brucellosis: review on the recent trends in pathogenicity and laboratory diagnosis. Journal of Laboratory Physicians, 2, 55–60.

    Article  PubMed  PubMed Central  Google Scholar 

  • Costa, A., Sant’ana, M., Carvalho, S., Moustacas, S., Silva, S., Paixão, T.A. and Santos, R.L., 2012. Diagnosis of B. ovis infection by serology and PCR in urine samples from naturally infected rams in the State of Piauí. Brazilian Journal of Veterinary and Animal Science, 64, 751–754.

    Google Scholar 

  • Çetinkaya, B., Öngör, H., Muz, A., Ertaş, H.B., Kalender, H. and Erdoğan, H.M. 1999. Detection of Brucella species DNA in the stomach content of aborted sheep fetuses by PCR. Veterinary Record, 144, 239–240.

    Article  PubMed  Google Scholar 

  • Díaz Aparicio, E., 2013. Epidemiology of brucellosis in domestic animals caused by Brucella melitensis, Brucella suis and Brucella abortus. Revue Scientifique et Technique, 32 (1), 53–60.

    Article  Google Scholar 

  • Geresu, M.A. and Kassa, G.M., 2016. A review on diagnostic methods of brucellosis. Journal of Veterinary Science and Technology, 7, 323.

    Google Scholar 

  • Godfroid, J., Scholz, H.C., Barbier, T., Nicolas, C., Wattiau, P., Fretin, D., Whatmore, A.M., Cloeckaert, A., Blasco, J.M., Moriyon, I., Saegerman, C., Muma, J.B., Al Dahouk S., Neubauer, H. and Letesson, J., 2011. Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century. Preventive Veterinary Medicine, 102, 118–131.

    Article  CAS  PubMed  Google Scholar 

  • Herman, L. and de Ridder, H., 1992. Identification of Brucella spp. by using the polymerase chain reaction. Applied and Environmental Microbiology, 58, 2099–2101.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Lucero, N.E., Ayala, S.M., Escobar, G.I. and Jacob, N.R., 2008. Brucella isolated in humans and animals in Latin America from 1968 to 2006. Epidemiology and Infection, 136(4), 496–503.

    Article  CAS  PubMed  Google Scholar 

  • Mahmood, R., Ali, T., Waheed, U., Asif, M. and Khan, Q.M., 2016. Application of serum based PCR and fluorescence polarization assay for diagnosis of brucellosis among people occupationally at risk to disease. International Journal of Agriculture & Biology, DOI: 10.17957/IJAB/15.0099.

    Google Scholar 

  • Mukherjee, F., Jain, J., Patel, V. and Nair, M., 2007. Multiple genus-specific markers in PCR assays improve the specificity and sensitivity of diagnosis of brucellosis in field animals. Journal of Medical Microbiology, 56, 1309–1316.

    Article  CAS  PubMed  Google Scholar 

  • Müştak, H.K., Günaydın, E., Küçükayan, U. and Dakman, A., 2009. Diagnosis of Brucella spp. by conventional cultural method and polymerase chain reaction in stomach contents of aborted fetuses. Journal of Etlik Veterinary Microbiology, 20, 35–38.

    Google Scholar 

  • Navarro, E., Escribano, J., Fernandez, J.A. and Solera, J., 2002. Comparison of three different PCR methods for detection of Brucella spp. in human blood samples. FEMS Immunology and Medical Microbiology, 34, 147–151.

    Article  CAS  PubMed  Google Scholar 

  • OIE terrestrial manual, 2009 bovine brucellosis. Retrieved February 24, 2016 from http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.04.03_BOVINE_ brucell. pdf.

  • Poiester, F.P., Nielsen, K., Samartino, L.E. and Yu, W.L., 2010. Diagnosis of Brucellosis. Open Veterinary Science Journal, 4, 46.

  • Refaat, H.M., Ahmed, O.I., Elabd, S.H. and Sanad, M.M., 2016. Polymerase chain reaction (PCR) technique compared to conventional bacteriological and serological techniques in diagnosis of human brucellosis in Egypt. African Journal of Microbiology, 10(2), 54–61.

    Article  Google Scholar 

  • Ren, H., Yang, M., Zhang, G., Liu, S., Wang, X., Ke, Y., Du, X., Wang, Z., Huang, L., Liu, C. and Chen, Z., 2016. Development of a rapid recombinase polymerase amplification assay for detection of Brucella in blood samples. Molecular and Cellular Probes, 30, 122–124.

    Article  CAS  PubMed  Google Scholar 

  • Rijpens, N.P., Jannes, G., Van Asbroeck, M., Rossau, R. and Herman, L.M., 1996. Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes. Applied and Environmental Microbiology, 62, 1683–1688.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Romero, C., Gamazo, C., Pardo, M. and Lopez-Goni, I., 1995. Specific detection of Brucella DNA by PCR. Journal of Clinical Microbiology, 33, 615–617.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Schrader, C., Schielke, A., Ellerbroek, L. and Johne, R., 2012. PCR inhibitors—occurrence, properties and removal. Journal of Applied Microbiology, 113, 1014–1026.

    Article  CAS  PubMed  Google Scholar 

  • Shakerian, A., Deo, P., Rahimi, E., Shahjavan, A.R. and Khamesipour, F., 2016. Molecular detection of Brucella melitensis in sheep and goat milk in Iran. Tropical Journal of Pharmacological Research, 15(5), 913–918.

    Article  CAS  Google Scholar 

  • Vizcaino, N., Verger, J.M., Grayon, M., Zygmunt, M.S. and Cloeckaert, A., 1997. DNA polymorphism at the OMP-31 locus of Brucella spp.: evidence for a large deletion in Brucella abortus and other species-specific markers. Microbiology, 143, 2913–2921.

    Article  PubMed  Google Scholar 

  • Zamanian, M., Hashemi Tabar, G.R., Rad, M. and Haghparast, A., 2015. Evaluation of different primers for detection of Brucella in human and animal serum samples by using PCR method. Archives of Iranian Medicine, 18(1), 44–50.

    PubMed  Google Scholar 

  • Zerva, L., Bourantas, K., Mitka, S., Kansousidou, A. and Legakis, N.J., 2001. Serum is the preferred clinical specimen for diagnosis of human brucellosis by PCR. Journal of Clinical Microbiology, 39, 1661–1664.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This study was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: VHAG-1902).

Author information

Authors and Affiliations

  1. Department of Microbiology, Faculty of Veterinary Medicine, University of Ondokuz Mayıs, Samsun, Turkey

    Alper Çiftci

  2. Department of Art and Sciences, Faculty of Biology, University of Dumlupınar, Kütahya, Turkey

    Tuba İça

  3. Department of Microbiology, Faculty of Veterinary Medicine, University of Adnan Menderes, Aydın, Turkey

    Serap Savaşan

  4. Department of Microbiology, Faculty of Veterinary Medicine, University of Ankara, Ankara, Turkey

    Barış Sareyyüpoğlu, Mehmet Akan & Kadir Serdar Diker

Authors
  1. Alper Çiftci
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tuba İça
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Serap Savaşan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Barış Sareyyüpoğlu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mehmet Akan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kadir Serdar Diker
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alper Çiftci.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Çiftci, A., İça, T., Savaşan, S. et al. Evaluation of PCR methods for detection of Brucella strains from culture and tissues. Trop Anim Health Prod 49, 755–763 (2017). https://doi.org/10.1007/s11250-017-1256-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11250-017-1256-1

Keywords

Search

Navigation