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

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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.

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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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Authors and Affiliations

  1. Department of Preventive Veterinary Medicine and Animal Health, School of Veterinary Medicine, University of São Paulo, São Paulo, SP, Brazil

    L. B. Keid, R. M. Soares, N. R. Vieira, S. A. Vasconcellos & L. J. Richtzenhain

  2. Department of Veterinary Hygiene and Public Health, School of Veterinary Medicine, São Paulo State University, Botucatu, São Paulo, Brazil

    J. Megid & V. R. Salgado

  3. Departament of Microbiology, Health Sciences Institute, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil

    M. da Costa

  4. Research and Developmental Center of Animal Health, Biological Institute, São Paulo, São Paulo, Brazil

    F. Gregori

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  1. L. B. Keid
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  2. R. M. Soares
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  3. N. R. Vieira
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  4. J. Megid
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  9. L. J. Richtzenhain
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Correspondence to L. J. Richtzenhain.

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Keid, L.B., Soares, R.M., Vieira, N.R. et al. Diagnosis of Canine Brucellosis: Comparison between Serological and Microbiological Tests and a PCR Based on Primers to 16S-23S rDNA Interspacer. Vet Res Commun 31, 951–965 (2007). https://doi.org/10.1007/s11259-006-0109-6

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