Skip to content
1887

Journal of Medical Microbiology logo Journal of Medical Microbiology

Volume 56, Issue 10

Multiple genus-specific markers in PCR assays improve the specificity and sensitivity of diagnosis of brucellosis in field animals No Access

Abstract

-specific nucleotide sequences encoding the BCSP 31 kDa protein, Omp2 and the 16S rRNA were employed in three independent diagnostic PCR assays. Results of the three PCR assays on six reference strains of were in complete agreement. The results of PCR assays based on and on 19 Indian field isolates (human, bovine and murine tissues) also agreed completely. However, when the 16S rRNA gene was employed as the diagnostic target in the PCR, only 14 out of these 19 isolates and 2 out of 7 bovine milk isolates were identified as the genus . The bovine blood samples were insensitive to 16S rRNA PCR. The antibody-detecting ELISA results of field samples (=87) from a serologically positive herd in India were compared separately with and PCRs of blood (=62). While the PCR was the most sensitive, the degree of association of ELISA with blood PCR (=0.37 at <0.05) was similar to that with the blood PCR ( =0.34 at <0.05). An improvement in the correlation between ELISA and blood PCR was noticed ( =0.5 at <0.05) when a consensus result of and blood PCR was considered for comparison with ELISA. The use of more than one marker-based PCR gave increased sensitivity and higher specificity and appears to be a more reliable molecular diagnostic approach for screening of field animals.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): OPA, overall proportion of agreement
SGM
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.47160-0
2007-10-01
2025-03-22
Loading full text...

Full text loading...

References

  1. Alton G. G., Jones L. M., Angus R. D., Verger J. M. 1988 Techniques for the Brucellosis Laboratory Paris: Institute National De La Recherche Agronomique;
     [Google Scholar]
  2. Amin A. S., Hamdy M. E., Ibrahim A. K. 2001; Detection of Brucella melitensis in semen using the polymerase chain reaction assay. Vet Microbiol 83:37–44 [CrossRef]
     [Google Scholar]
  3. Ariza J., Pellicer T., Pallares R., Foz A., Gudiol F. 1992; Specific antibody profile in human brucellosis. Clin Infect Dis 14:131–140 [CrossRef]
     [Google Scholar]
  4. Baily G. G., Krahn J. B., Drasar B. S., Stoker N. G. 1992; Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hyg 95:271–275
     [Google Scholar]
  5. Bardenstein S., Mandelboim M., Ficht T. A., Baum M., Banai M. 2002; Identification of the Brucella melitensis vaccine strain Rev.1 in animals and humans in Israel by PCR analysis of the Pst I site polymorphism of its omp2 gene. J Clin Microbiol 40:1475–1480 [CrossRef]
     [Google Scholar]
  6. Baumann P., Furniss A. L., Lee J. V. 1984; Genus I Vibrio Pancini. In Bergey's Manual of Systematic Bacteriology vol. 1 pp 518–538 Edited by Krieg N. R., Holt J. G. Baltimore: Williams & Wilkins;
     [Google Scholar]
  7. Bogdanovich T., Skurnik M., Lubeck P. S., Ahrens P., Hoorfar J. 2004; Validated 5′ nuclease PCR assay for rapid identification of the genus Brucella . J Clin Microbiol 42:2261–2263 [CrossRef]
     [Google Scholar]
  8. Bricker B. J. 2002; PCR as a diagnostic tool for brucellosis. Vet Microbiol 90:435–446 [CrossRef]
     [Google Scholar]
  9. Bricker B. J., Ewalt D. R. 2006; HOOF prints: Brucella strain typing by PCR amplification of multilocus tandem-repeat polymorphisms. Methods Mol Biol 345:141–173
     [Google Scholar]
  10. Bricker B. J., Tabatabai L. B., Deyoe B. L., Mayfield J. A. 1988; Conservation of antigenicity in a 31 kDa Brucella protein. Vet Microbiol 18:313–325 [CrossRef]
     [Google Scholar]
  11. Corbel M. J. 1997; Brucellosis: an overview. Emerg Infect Dis 3:213–221 [CrossRef]
     [Google Scholar]
  12. Corbel M. J., Brinley-Morgan W. J. 1984; Genus Brucella Meyer and Shaw 1920, 173. In Bergey's Manual of Systematic Bacteriology vol 1 pp 377–388 Edited by Krieg N. R., Holt J. G. Baltimore: Williams & Wilkins;
     [Google Scholar]
  13. Cousins D. V., Williams S. N., Ross B. C., Ellis T. M. 1993; Use of a repetitive element isolated from Mycobacterium tuberculosis in hybridization studies with Mycobacterium bovis : a new tool for epidemiological studies for bovine tuberculosis. Vet Microbiol 37:1–17 [CrossRef]
     [Google Scholar]
  14. Da Costa M., Guillou J. P., Garin-Bastuji B., Theibaud M., Dubray G. 1996; Specificity of six gene sequences for the detection of the genus Brucella by DNA amplification. J Appl Bacteriol 81:267–275 [CrossRef]
     [Google Scholar]
  15. Dorsch M., Moreno E., Stackebrandt E. 1989; Nucleotide sequence of the 16S rRNA from Brucella abortus . Nucleic Acids Res 17:1765 [CrossRef]
     [Google Scholar]
  16. Elfaki M. G., Uz-Zaman T., Al-Hokail A. A., Nakeeb S. M. 2005; Detection of Brucella DNA in sera from patients with brucellosis by polymerase chain reaction. Diagn Microbiol Infect Dis 53:1–7 [CrossRef]
     [Google Scholar]
  17. Ewalt D. R., Bricker B. J. 2000; Validation of the abbreviated Brucella AMOS PCR as a rapid screening method for differentiation of Brucella abortus field strain isolates and the vaccine strains, 19 and RB51. J Clin Microbiol 38:3085–3086
     [Google Scholar]
  18. Fekete A., Bantle J. A., Halling S. M. 1992; Detection of Brucella by polymerase chain reaction in bovine fetal and maternal tissues. J Vet Diagn Invest 4:79–83 [CrossRef]
     [Google Scholar]
  19. Ferrao-Beck L., Cardoso R., Munoz P. M., de Miguel M. J., Albert D., Ferreira A. C., Marin C. M., Thiebaud M., Jacques I. other authors 2006; Development of a multiplex PCR assay for polymorphism analysis of Brucella suis biovars causing brucellosis in swine. Vet Microbiol 115:269–277 [CrossRef]
     [Google Scholar]
  20. Fitch T. A., Bearden S. W., Sowa B. A., Adams L. G. 1989; DNA sequence and expression of the 36 kilo-dalton outer membrane protein gene of Brucella abortus . Infect Immun 57:3281–3291
     [Google Scholar]
  21. Gee J. E., De B. K., Levett P. N., Whitney A. M., Novak R. T., Popovic T. 2004; Use of 16S rRNA gene sequencing for rapid confirmatory identification of Brucella isolates. J Clin Microbiol 42:3649–3654 [CrossRef]
     [Google Scholar]
  22. Godfroid J., Saegerman C., Wellemans V., Walravens K., Letesson J. J., Tibor A., McMillan A., Spencer S., Sanna M. other authors 2002; How to substantiate eradication of bovine brucellosis when aspecific serological reactions occur in the course of brucellosis testing. Vet Microbiol 90:461–477 [CrossRef]
     [Google Scholar]
  23. Le Fleche 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 [CrossRef]
     [Google Scholar]
  24. Leal-Klevezas D. S., Martinez-Vaquez I. O., Lopez-Merino A., Martinez-Soriano J. P. 1995; Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. J Clin Microbiol 33:3087–3090
     [Google Scholar]
  25. Leyla G., Kadri G., Umran O. 2003; Comparison of polymerase chain reaction and bacteriological culture for the diagnosis of sheep brucellosis using aborted fetus samples. Vet Microbiol 93:53–61 [CrossRef]
     [Google Scholar]
  26. Lopez-Goni I. 2001; Detection of Brucella by PCR: application limits and needs. In Brucellosis in Animals and Man . Proceedings of the Combined COST Meeting for Working Groups 1 (Epidemiology) , Pamplona, Spain June 11–12, 2001. Progress Report of Action 845: Food & Agriculture Domain Brussels: European; Cooperation in the Field of Scientific and Technical Research (COST
    [Google Scholar]
  27. Lulu A. R., Araz G. F., Khatib M. I., Mustafa M. Y., Yusuf A. R., Fenech F. F. 1988; Human brucellosis in Kuwait: a prospective study of 400 cases. Q J Med 66:39–54
     [Google Scholar]
  28. Marianelli C., Ciuchini F., Tarantino M., Pasquali P., Adone R. 2006; Molecular characterization of the rpoB gene in Brucella species: new potential molecular markers for genotyping. Microbes Infect 8:860–865 [CrossRef]
     [Google Scholar]
  29. Mayfield J. E., Bricker B. J., Godfrey H., Crosby R. M., Knight D. J., Halling S. M., Balinsky D., Tabataba L. B. 1988; The cloning, expression, and nucleotide sequence of the BCSP 31 gene from Brucella abortus . Gene 63:1–9 [CrossRef]
     [Google Scholar]
  30. Morata P., Quiepo-Ortuno M. I., Reguera J. M., Garcia-Ordonez M. A., Ca'rdenas A., Juan D., Colmenero J. D. 2003; Development and evaluation of a PCR–enzyme-linked immunosorbent assay for diagnosis of human brucellosis. J Clin Microbiol 41:144–148 [CrossRef]
     [Google Scholar]
  31. Mukherjee F., Jain J., Grillo M. J., Blasco J. M., Nair M. 2005; Evaluation of Brucella abortus S19 vaccine strains by bacteriological tests, molecular analysis of ery loci and virulence in BALB/c mice. Biologicals 33:153–160 [CrossRef]
     [Google Scholar]
  32. Navarro E., Escribano J., Fernández J. A., Solera J. 2002; Comparison of three different PCR methods for detection of Brucella spp in human blood samples. FEMS Immunol Med Microbiol 34:147–151 [CrossRef]
     [Google Scholar]
  33. Navarro E., Segura J. C., Castaño M. J., Solera J. 2006; Use of real-time quantitative polymerase chain reaction to monitor the evolution of Brucella melitensis DNA load during therapy and post-therapy follow-up in patients with brucellosis. Clin Infect Dis 42:1266–1273 [CrossRef]
     [Google Scholar]
  34. Nicoletti P. 1980; The epidemiology of bovine brucellosis. Adv Vet Sci Comp Med 24:69–89
     [Google Scholar]
  35. O'Leary S., Sheahan M., Sweeney T. 2006; Brucella abortus detection by PCR assay in blood, milk and lymph tissue of serologically positive cows. Res Vet Sci 81:170–176 [CrossRef]
     [Google Scholar]
  36. OIE 1996; WHO , Manual of Standards for Diagnostic Tests and Vaccine . , 3rd edn, chapter. 3.2.1 pp 242–255 Paris: Office International des Epizootics;
  37. 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 [CrossRef]
     [Google Scholar]
  38. Queipo-Ortuno M. I., Colmenero J. D., Reguera J. M., Garcia-Ordonez M. A., Pachon M. E., Gonzalez M., Morata P. 2005; Rapid diagnosis of human brucellosis by SYBR Green I-based real-time PCR assay and melting curve analysis in serum samples. Clin Microbiol Infect 11:713–718 [CrossRef]
     [Google Scholar]
  39. Queipo-Ortuno M. I., Morata P., Ocon P., Manchado P., de Dios Colmenero J. 2006; Rapid diagnosis of Brucella epididymo-orchitis by real-time polymerase chain reaction assay in urine samples. J Urol 176:2290–2293 [CrossRef]
     [Google Scholar]
  40. Radostits O. M., Blood D. C., Gay C. C. (editors) 1994; Veterinary Medicine. A Text Book of the Diseases of Cattle, Sheep, Pigs, Goats and Horses . , 8th edn. pp 789–812 London: Baillière Tindall;
     [Google Scholar]
  41. Redkar R., Rose S., Bricker B., DelVecchio V. 2001; Real-time detection of Brucella abortus , Brucella melitensis , and Brucella suis . Mol Cell Probes 15:43–52 [CrossRef]
     [Google Scholar]
  42. Rijpens N. P., Jannes G., Asbroeck M. V., Rossau R., Herman L. M. F. 1996; Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S–23S rRNA spacer probes. Appl Environ Microbiol 62:1683–1688
     [Google Scholar]
  43. Romero C., Gamazo C., Pardo M., López-Goñi I. 1995a; Specific detection of Brucella DNA by PCR. J Clin Microbiol 33:615–617
     [Google Scholar]
  44. Romero C. M., Padro M., Grillo M. J., Diaz R., Blasco J. M., Lopez-Goni I. 1995b; Evaluation of PCR and indirect enzyme-linked immunosorbent assay on milk samples for diagnosis of brucellosis in cattle. J Clin Microbiol 33:3198–3200
     [Google Scholar]
  45. Sen G. P., Sharma G. L. 1975; Speciation of seventy-eight Indian strains of Brucella : an epidemiological study. Indian J Anim Sci 45:537–542
     [Google Scholar]
  46. Sifuentes-Rincon A. M., Revol A., Barrera-Saldana H. A. 1997; Detection and differentiation of the six Brucella species by polymerase chain reaction. Mol Med 3:734–739
     [Google Scholar]
  47. Sreevatsan S., Bookout J. B., Ringpis F., Perumaalla V. S., Fitch T. A., Adams L. G., Haguis S. D., Elzer P. H., Bricker B. J. other authors 2000; A multiplex approach to molecular detection of Brucella abortus and/or Mycobacterium bovis infection in cattle. J Clin Microbiol 38:2602–2610
     [Google Scholar]
  48. Weynants V., Tibor A., Denoel P. A., Saegerman C., Godfroid J., Thiange P., Letesson J. J. 1996; Infection of cattle with Yersinia enterocolitica O : 9 a cause of the false positive serological reactions in bovine brucellosis diagnostic tests. Vet Microbiol 48:101–112 [CrossRef]
     [Google Scholar]
  49. Yagupsky P. 1994; Detection of Brucella melitensis by BACTEC NR660 blood culture system. J Clin Microbiol 32:1899–1901
     [Google Scholar]
/content/journal/jmm/10.1099/jmm.0.47160-0
Loading
Multiple genus-specific markers in PCR assays improve the specificity and sensitivity of diagnosis of brucellosis in field animals
J. Med. Microbiol. 56, 1309 (2007); https://doi.org/10.1099/jmm.0.47160-0
/content/journal/jmm/10.1099/jmm.0.47160-0
/content/journal/jmm/10.1099/jmm.0.47160-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error