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Veterinary Research Communications

, Volume 31, Issue 8, pp 951–965 | Cite as

Diagnosis of Canine Brucellosis: Comparison between Serological and Microbiological Tests and a PCR Based on Primers to 16S-23S rDNA Interspacer

  • L. B. Keid
  • R. M. Soares
  • N. R. Vieira
  • J. Megid
  • V. R. Salgado
  • S. A. Vasconcellos
  • M. da Costa
  • F. Gregori
  • L. J. Richtzenhain
Article

Abstract

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol–chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.

Keywords

Brucella canis canine brucellosis PCR internal transcribed spacer blood culture rapid slide agglutination test 

Abbreviations

CTAB

cetyl trimethyl ammonium bromide

ITS

interspacer

PCR

polymerase chain reaction

RBPT

Rose Bengal Plate test

R-LPS

rough lipopolysaccharide

RSAT

rapid slide agglutination test

SDS

sodium docdecyl sulphate

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References

  1. Al Dahouk, S., Tomaso, H., Nockler, K., Neubauer, H. and Frangoulidis, D., 2003. Laboratory-based diagnosis of brucellosis—a review of the literature. Part I: Techniques for direct detection and identification of Brucella spp. Clinical Laboratory, 49, 487–505.PubMedGoogle Scholar
  2. Al-Nakkas, A.F., Wright, S.G., Mustafa, A.S. and Wilson, S., 2002. Single-tube, nested PCR for the diagnosis of human brucellosis in Kuwait. Annals of Tropical Medicine and Parasitology, 96, 397–403.PubMedCrossRefGoogle Scholar
  3. Al-Nakkas, A., Mustafa, A.S. and Wright, S.G., 2005. Large-scale evaluation of a single-tube nested PCR for the laboratory diagnosis of human brucellosis in Kuwait. Journal of Medical Microbiology, 54(Pt 8), 727–730.PubMedCrossRefGoogle Scholar
  4. Alton, G.G., Jones, L.M. and Pietz, D.E., 1976. Metodos bacteriologicos. In: Las tecnicas de laboratorios en la brucelosis, 2nd edn, (WHO, Geneva), 11–67.Google Scholar
  5. Badakhsh, F.F., Carmichael, L.E. and Douglass, J.A., 1982. Improved rapid slide agglutination test for presumptive diagnosis of canine brucellosis. Clinical Microbiology, 15, 286–289.Google Scholar
  6. Baek, B.K., Lim, C.W., Rahman, M.S., Kim, C.H., Oluoch, A. and Kakoma, I., 2003. Brucella abortus infection in indigenous Korean dogs. Canadian Journal of Veterinary Research, 67, 312–314.PubMedGoogle Scholar
  7. Baily, G.G., Krahn, J.B., Drasar, B.S. and Stoker, N.G., 1992. Detection of Brucella melitensis and Brucella abortus by DNA amplification. Journal of Tropical Medicine and Hygiene, 95, 271–275.PubMedGoogle Scholar
  8. Bricker, B.J., 2002. PCR as a diagnostic tool for brucellosis. Veterinary Microbiology, 90, 435–446.PubMedCrossRefGoogle Scholar
  9. Carmichael, L.E., 1976. Canine brucellosis: an annotated review with cautionary comment. Theriogenology, 6, 105–116.CrossRefGoogle Scholar
  10. Carmichael, L.E., 1990. Brucella canis. In: K. Nielsen and J.R. Duncan (eds), Animal Brucellosis, (CRC Press, Boca Raton, FL), 335–350.Google Scholar
  11. Carmichael, L.E. and Greene, C.E., 1990. Canine brucellosis. In: C.E. Greene (ed.), Infectious Diseases of the Dog and Cat, (W.B. Sauders, Philadelphia), 573–584.Google Scholar
  12. Carmichael, L.E. and Joubert, J.C., 1987. A rapid slide agglutination test for the serodiagnosis of Brucella canis infection that employs a variant (M-) organism as antigen. Cornell Veterinarian, 77, 3–12.PubMedGoogle Scholar
  13. Carmichael, L.E. and Kenney, R.M., 1968. Canine abortion caused by Brucella canis. Journal of the American Veterinary Medical Association, 152, 605–616.PubMedGoogle Scholar
  14. Carmichael, L.E. and Kenney, R.M., 1970. Canine brucellosis: the clinical disease, pathogenesis, and immune response. Journal of the American Veterinary Medical Association, 156, 1726–1734.PubMedGoogle Scholar
  15. Carmichel, L.E. and Shin, S.J., 1996. Canine brucellosis: a diagnostician's dilemma. Seminars in Veterinary Medicine and Surgery (Small Animal), 11, 161–165.Google Scholar
  16. Carmichael, L.E., Zoha, S.J. and Flores-Castro, R., 1984. Problems in the serodiagnosis of canine brucellosis: dog responses to cell wall and internal antigens of Brucella canis. Development in Biological Standardization, 56, 371–383.Google Scholar
  17. Casanas, M.C., Queipo-Ortuno, M.I., Rodriguez-Torres, A., Orduna, A., Colmenero, J.D. and Morata, P., 2001. Specificity of a polymerase chain reaction assay of a target sequence on the 31-kilodalton Brucella antigen DNA used to diagnose human brucellosis. European Journal of Clinical Microbiology and Infectious Disease, 20, 127–131.Google Scholar
  18. Cogswell, F.B., Bantar, C.E., Hughes, T.G., Gu, Y. and Philipp, M.T., 1996. Host DNA can interfere with detection of Borrelia burgdorferi in skin biopsy specimens by PCR. Journal Clinical Microbiology, 34, 980–982.Google Scholar
  19. Elfaki, M.G., Al-Hokail, A., Nakeeb, S.M. and Al-Rabiah, F.A., 2005a. Evaluation of culture, tube agglutination, and PCR methods for the diagnosis of brucellosis in humans. Medical Science Monitor, 27, MT69–74.Google Scholar
  20. Elfaki, M.G., Uz-Zaman, T., Al-Hokail, A.A. and Nakeeb, S.M., 2005b. Detection of Brucella DNA in sera from patients with brucellosis by polymerase chain reaction. Diagnostic Microbiology and Infectious Disease, 53, 1–7.CrossRefGoogle Scholar
  21. Flores-Castro, R. and Carmichael, L.E., 1978. Canine brucellosis. Current status of methods for diagnosis. Cornell Veterinarian, 68, 76–88.PubMedGoogle Scholar
  22. Forbes, L.B., 1990. Brucella abortus infection in 14 farm dogs. Journal of American Veterinary Medical Association, 196, 911–916.Google Scholar
  23. George, L.W. and Carmichael, L.E., 1978. Development of a rose bengal stained plate-test antigen for the rapid diagnosis of Brucella canis infection. Cornell Veterinarian, 68, 530–543.PubMedGoogle Scholar
  24. Gransden, W.R. and Eykyn, S.J., 1992. Seven cases of bacteremia due to Ochrobactrum anthropi. Clinical and Infectious Diseases, 15, 1068–1069.Google Scholar
  25. Guarino, A., Serpe, L., Fusco, G., Scaramuzzo, A. and Gallo, P., 2000. Detection of Brucella species in buffalo whole blood by gene-specific PCR. Veterinary Record, 147, 634–636.PubMedGoogle Scholar
  26. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98.Google Scholar
  27. Hillis, D. M. and Dixon, M.T., 1991. Ribosomal DNA: molecular evolution and phylogenetic inference. Quarterly Review of Biology, 66, 411–452.PubMedCrossRefGoogle Scholar
  28. Johnson, C.A. and Walker, R.D., 1992. Clinical signs and diagnosis of Brucella canis infection. The Compendium on Continuing Education for the Practicing Veterinarian. Small Animal, 14, 763–772.Google Scholar
  29. Leal-Klevezas, D.S., Martinez-Vazquez, I.O., Lopez-Merino, A. and Martinez-Soriano, J.P., 1995. Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. Journal of Clinical Microbiology, 33, 3087–3090.PubMedGoogle Scholar
  30. Leal-Klevezas, D.S., Martinez-Vazquez, I.O., Garcia-Cantu, J., Lopez-Merino, A. and Martinez-Soriano, J.P., 2000. Use of polymerase chain reaction to detect Brucella abortus biovar 1 in infected goats. Veterinary Microbiology, 75, 91–97.PubMedCrossRefGoogle Scholar
  31. Matar, G.M., Khneisser, I.A. and Abdelnoor, A.M., 1996. Rapid laboratory confirmation of human brucellosis by PCR analysis of a target sequence on the 31-kilodalton Brucella antigen DNA. Journal of Clinical Microbiology, 34, 477–478.PubMedGoogle Scholar
  32. Moore, J.A. and Kakuk, T.J., 1969. Male dogs naturally infected with Brucella canis. Journal of the American Veterinary Medical Association, 155, 1352–1358.Google Scholar
  33. Morata, P., Queipo-Ortuno, M.I. and de Dios Colmenero, J., 1998. Strategy for optimizing DNA amplification in a peripheral blood PCR assay used for diagnosis of human brucellosis. Journal of Clinical Microbiology, 36, 2443–2446.PubMedGoogle Scholar
  34. Morata, P., Queipo-Ortuno, M.I., Reguera, J.M., Garcia-Ordonez, M.A., Pichardo, C. and Colmenero, J.D., 1999. Posttreatment follow-up of brucellosis by PCR assay. Journal of Clinical Microbiology, 37, 4163–4166.PubMedGoogle Scholar
  35. Morata, P., Queipo-Ortuno, M.I., Reguera, J.M., Miralles, F., Lopez-Gonzalez, J.J. and Colmenero, J.D., 2001. Diagnostic yield of a PCR assay in focal complications of brucellosis. Journal of Clinical Microbiology, 39, 3743–3746.PubMedCrossRefGoogle Scholar
  36. Navarro, E., Fernandez, J.A., Escribano, J. and Solera, J., 1999. PCR assay for diagnosis of human brucellosis. Journal of Clinical Microbiology, 37, 1654–1655.PubMedGoogle Scholar
  37. Navarro, E., Escribano, J., Fernandez, J. 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.PubMedCrossRefGoogle Scholar
  38. Navarro, E., Casao, M.A. and Solera, J., 2004. Diagnosis of human brucellosis using PCR. Expert Review of Molecular Diagnostics, 4, 115–123.PubMedCrossRefGoogle Scholar
  39. Nimri, L.F., 2003. Diagnosis of recent and relapsed cases of human brucellosis by PCR assay. BMC Infectious Disease, 28, 1–7.Google Scholar
  40. Queipo-Ortuno, M.I., Morata, P., Ocon, P., Manchado, P. and Colmenero, J.D., 1997. Rapid diagnosis of human brucellosis by peripheral-blood PCR assay. Journal of Clinical Microbiology, 35, 2927–2930.PubMedGoogle Scholar
  41. Queipo-Ortuno, M.I., Garcia-Ordonez, M.A., Colmenero, J.D. and Morata, P., 1999. Hydrogen peroxide improves the efficiency of a peripheral blood PCR assay for diagnosis of human brucellosis. Biotechniques, 27, 248–250, 252.PubMedGoogle Scholar
  42. Saah, A.J. and Hoover, D.R., 1997. ‘Sensitivity’ and ‘specificity’ reconsidered. Annals of Internal Medicine, 126, 91–94.PubMedGoogle Scholar
  43. Sambrook, J., Fritsch, E.F. and Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd edn (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).Google Scholar
  44. Taylor, D.J., Renton, J.P. and McGregor, A.B., 1975. Brucella abortus biotype 1 as a cause of abortion in a bitch. Veterinary Record, 96, 428–429.PubMedGoogle Scholar
  45. Ueda, K., Magaribuchi, T., Saegusa. J., Urano. T. and Ito. K., 1974. Spontaneous Brucella canis infection in beagles: bacteriological and serological studies. Nippon Juigaku Zasshi, 36, 381–389.PubMedGoogle Scholar
  46. Velasco, J., Romero, C., Lopez-Goni, I., Leiva, J., Diaz, R. and Moriyon, I., 1998. Evaluation of the relatedness of Brucella spp. and Ochrobactrum anthropi and description of Ochrobactrum intermedium sp. nov., a new species with a closer relationship to Brucella spp. International Journal of Systematic Bacteriology, 48, 759–768.PubMedCrossRefGoogle Scholar
  47. Wallach, J.C., Giambartolomei, G.H., Baldi, P.C. and Fossati, C.A., 2004. Human infection with M-strain of Brucella canis. Emerging Infectious Disease, 10, 146–148.Google Scholar
  48. Wanke, M.M., 2004. Canine brucellosis. Animal Reproduction Science, 82/83, 195–207.CrossRefGoogle Scholar
  49. Wilson, K., 1990. Preparation of genomic DNA from bacteria. In: F.M. Ausubel, R. Brent, R.E. Kimgston, et al. (eds), Current Protocols in Molecular Biology, (Greene Publishing Associates/John Wiley and Sons, New York), 241–245.Google Scholar
  50. Zerva, L., Bourantas, K., Mitka, S., Kansouzidou, 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.PubMedCrossRefGoogle Scholar
  51. Zoha, S.J. and Carmichael, L.E., 1982. Serological responses of dogs to cell wall and internal antigens of Brucella canis B. canis). Veterinary Microbiology, 7, 35–50.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science + Business Media, Inc. 2007

Authors and Affiliations

  • L. B. Keid
    • 1
  • R. M. Soares
    • 1
  • N. R. Vieira
    • 1
  • J. Megid
    • 2
  • V. R. Salgado
    • 2
  • S. A. Vasconcellos
    • 1
  • M. da Costa
    • 3
  • F. Gregori
    • 4
  • L. J. Richtzenhain
    • 1
  1. 1.Department of Preventive Veterinary Medicine and Animal Health, School of Veterinary MedicineUniversity of São PauloSão PauloBrazil
  2. 2.Department of Veterinary Hygiene and Public Health, School of Veterinary MedicineSão Paulo State University, BotucatuSão PauloBrazil
  3. 3.Departament of Microbiology, Health Sciences InstituteFederal University of Rio Grande do SulPorto AlegreBrazil
  4. 4.Research and Developmental Center of Animal Health, Biological Institute, São PauloSão PauloBrazil

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