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Journal of Clinical Microbiology, June 2006, p. 1982-1993, Vol. 44, No. 6
0095-1137/06/$08.00+0     doi:10.1128/JCM.02039-05

Identification and Characterization of Variable-Number Tandem-Repeat Markers for Typing of Brucella spp.{dagger}

Adrian M. Whatmore,* Stephen J. Shankster, Lorraine L. Perrett, Terry J. Murphy, Simon D. Brew, Rachel E. Thirlwall, Sally J. Cutler, and Alastair P. MacMillan

Department of Statutory and Exotic Bacterial Diseases, Veterinary Laboratories Agency, Addlestone, Surrey KT15 3NB, United Kingdom

Received 29 September 2005/ Returned for modification 2 December 2005/ Accepted 10 March 2006

Members of the genus Brucella infect many domesticated and wild animals and cause serious zoonotic infection in humans. The availability of discriminatory molecular typing tools to inform and assist conventional epidemiological approaches would be invaluable in controlling these infections, but efforts have been hampered by the genetic homogeneity of the genus. We report here on a molecular subtyping system based on 21 variable-number tandem-repeat (VNTR) loci consisting of 13 previously unreported loci and 8 loci previously reported elsewhere. This approach was applied to a collection of 121 Brucella isolates obtained worldwide and representing all six classically recognized Brucella species. The size of repeats selected for inclusion varied from 5 to 40 bp giving VNTR loci with a range of diversities. The number of alleles detected ranged from 2 to 21, and Simpson's diversity index values ranged from 0.31 to 0.92. This assay divides the 121 isolates into 119 genotypes, and clustering analysis results in groups that, with minor exceptions, correspond to conventional species designations. Reflecting this, the use of six loci in isolation was shown to be sufficient to determine species designation. On the basis of the more variable loci, the assay could also discriminate isolates originating from restricted geographical sources, indicating its potential as an epidemiological tool. Stability studies carried out in vivo and in vitro showed that VNTR profiles were sufficiently stable such that recovered strains could readily be identified as the input strain. The method described here shows great potential for further development and application to both epidemiological tracing of Brucella transmissions and in determining relationships between isolates worldwide.

* Corresponding author. Mailing address: Department of Statutory and Exotic Bacterial Diseases, Veterinary Laboratories Agency, Addlestone, Surrey KT15 3NB, United Kingdom. Phone: 44 1932 357311. Fax: 44 1932 357423. E-mail: a.whatmore{at}vla.defra.gsi.gov.uk.

{dagger} Supplemental material for this article may be found at http://jcm.asm.org/.

Journal of Clinical Microbiology, June 2006, p. 1982-1993, Vol. 44, No. 6
0095-1137/06/$08.00+0     doi:10.1128/JCM.02039-05




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