Identification and Characterization of Variable-Number Tandem-Repeat Markers for Typing of Brucella spp

Abstract

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.Brucellosis is a zoonotic disease of major public health, animal welfare, and economic significance worldwide. In humans, infection with Brucella can lead to a chronic debilitating infection; in domesticated animals, the main symptom is reproductive failure. Disease in humans usually reflects occupational exposure or the consumption of unpasteurized dairy products. Brucellosis remains a major problem in many parts of the world, particularly Mediterranean regions, western Asia, and parts of Africa and Latin America (11), although in many developed countries it has been eradicated or severely curtailed by a combination of strict veterinary hygiene measures, monitoring programs, and improved food safety measures. Brucella species have also long been considered potential biological warfare agents, and in 1954 Brucella became the first biological agent to be treated as a weapon and field tested on animals under the old U.S. offensive biological weapons program. Recent history has raised awareness in this area (28), and the organism remains on the list of Centers for Disease Control and Prevention category B potential biological warfare agents (25).Classical Brucella taxonomists developed a classification system that recognized six species based on subtle phenotypi...

Epidemiologic Relatedness between Brucella abortus Isolates from Livestock and Wildlife in South Korea

“…Some investigators reported that MLVA could classify Brucella isolates in accordance with different host origin (Groussaud et al, 2007), geographic region (Kattar et al, 2008), and species (Whatmore et al, 2006). In clustering and traceback analysis, B. abortus isolates from wild animals were included in the same group as isolates from livestock, suggesting that direct or indirect horizontal transfer may occur and that brucellosis in wildlife reservoirs may pose a threat to humans and domestic animals.…”

“…The MLVA typing assay has been recommended by many researchers to analyze the genetic diversity and to trace the origins of infection (Whatmore et al, 2006;Al Dahouk et al, 2007;Groussaud et al, 2007;Her et al, 2009;Kang et al, 2009;Abril et al, 2011). Seventeen markers (16 loci and HOOF-3) used previously were available for follow-up (Bricker et al, 2003;Her et al, 2009).…”

“…However, the Korean B. abortus isolates formed a cluster that was not well resolved by these markers, suggesting that they have been localized by clonal expansion without influence of new foreign strains . Therefore, another locus, VNTR 16, was added to increase the resolution (Whatmore et al, 2006). This marker showed two allelic types among the Korean B. abortus isolates.…”

“…Because of the high degree of sequence homology (over 90%) among Brucella species (Vizcaíno et al, 2000;Halling et al, 2005), a powerful molecular typing method was required to clarify the epidemiologic relationships among Brucella infections. Multilocus variable-number tandem repeats analysis (MLVA) is useful in tracing the epidemiology of disease outbreaks (Le Flè che et al, 2006;Whatmore et al, 2006;Groussaud et al, 2007;Kattar et al, 2008;Kang et al, 2009;Abril et al, 2011).…”

“…Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen Ltd., Seoul, Korea) according to the manufacturer's instructions and subsequently stored at 220 C until use. The number of copies of tandem repeats for 17 loci was calculated as described , and the variable-number tandem repeat (VNTR) 16 marker (Whatmore et al, 2006) was added to enhance discrimination among Korean B. abortus isolates. The amplified PCR products from 18 loci were confirmed using the Agilent 2100 Bioanalyzer with a DNA 1000 LabChip kit (Agilent Technologies, Waldbronn, Germany) following the manufacturer's protocol.…”