Journal of Microbiological Methods 78 (2009) 144149

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Journal of Microbiological Methods

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h

Molecular typing for epidemiological evaluation of Brucella abortus and Brucella canis isolated in Korea
Sung-Il Kang, Moon Her, Eun Jeong Heo, Hyang Mi Nam, Suk Chan Jung, Donghee Cho
Bacteriology and Parasitology Division, National Veterinary Research Institute, National Veterinary Research and Quarantine Service, Anyang, Gyeonggi-do 430-824, Republic of Korea

a r t i c l e

i n f o

a b s t r a c t
To investigate genotype relationships among regional groups of Brucella isolates, variable-number tandemrepeat (VNTR) analysis was conducted according to previously reported methods. Field strains of Brucella abortus and Brucella canis were isolated from 9 provinces in the Republic of Korea during the years 1996 2006 and each of the isolates was classied by eight loci of HOOF-Prints. On the basis of the alleles, the 33 B. abortus and 21 B. canis eld strains were divided into 22 and 18 distinct genotypes, respectively. Phylogenetic cluster analysis of Brucella isolates could be discriminated with geographical region in the Republic of Korea. Simpson's diversity index values of B. abortus and B. canis isolates ranged from 0 to 0.85. The stability of each locus was determined with in vivo and in vitro experiments. After twenty passages in blood agar, the VNTR numbers of loci 1 and 7 in B. abortus isolates and loci 5, 7, and 8 in B. canis isolates changed. The same change of the VNTR numbers at loci 1 and 7 was observed with B. abortus RB51 strains isolated from vaccinated cattle for the in vivo experiment. Although B. canis and B. abortus isolates were discriminated to herd levels by the HOOF-Prints, this method needs further improvement for the high variable locus. This study represents the rst epidemiological data of molecular typing of B. abortus and B. canis reported in Korea. 2009 Elsevier B.V. All rights reserved.

Article history: Received 12 December 2008 Received in revised form 17 April 2009 Accepted 7 May 2009 Available online 20 May 2009 Keywords: Brucella Diversity index Genotyping Molecular epidemiology VNTR

1. Introduction Brucella is the causative agent of brucellosis, a widespread disease of various animal species and humans. This disease is a major cause of direct economic losses in the animal industry. Brucellosis remains a major problem in many parts of the world, particularly in Mediterranean regions, western Asia, parts of Africa, and Latin America (Cobel, 1997). The genus Brucella is currently divided into six species: B. abortus, B. canis, B. suis, B. ovis, B. neotomae and B. melitensis (Cobel and Brinley-Morgan, 1984). Recently, two new species of B. ceti and B. pinnipedialis have been introduced in the marine mammals (Foster et al., 2007). Each Brucella species can infect several animal species including humans (B. melitensis, B. abortus, B. suis and B. canis), cattle (B. abortus), canines (B. canis), caprine (B. melitensis), swine (B. suis), rams (B. ovis), rats (B. neotomae), cetaceans (B. ceti), and pinnipeds (B. pinnipedialis). Conventional subtyping of Brucella strains has relied primarily on biochemical characteristics such as dye susceptibility, phage lysis and agglutination of specic sera; however most of the biological assays are time-consuming and laborious. Recently, various methods have been established for molecular subtyping of Brucella strains (Al Dahouk et al., 2003), including polymerase chain reaction-restriction fragment length polymorph-

isms (PCR-RFLP) of different genetic loci (Vizcano et al., 2000), random amplied polymorphic DNA (RAPD)-PCR (Tscherneva et al., 2000), multiplex PCR assays (Garca-Yoldi et al., 2006), and single nucleotide polymorphisms (Marianelli et al., 2006). In recent years, multilocus sequence typing, such as multilocus variable-number tandem-repeat (VNTR) analysis, has been utilized for ngerprinting microbial genomic DNA. Multilocus VNTR analysis (MLVA) has proven to be a rapid and effective technique that can discriminate many difcult-to-type bacteria, including many human pathogens (Fabre et al., 2004; Le Flche et al., 2001; Le Flche et al., 2002; Pourcel et al., 2004) such as Mycobacterium tuberculosis (Iwamoto et al., 2007), Brucella spp. (Whatmore et al., 2006), Bacillus anthracis (Keim et al., 2000), Salmonella typhimurium (Drahovsk et al., 2007), and Escherichia coli O157:H7 (Vogler et al., 2006). Our study examines multiple VNTR genetic loci in Korean Brucella eld isolates, establishes their phylogenetic relationships between herds, and provides a foundation for further molecular epidemiological investigation and phylogenetic classication of Brucella isolates in Korea. 2. Materials and methods 2.1. Brucella eld strains

Corresponding author. Tel.: +82 31 467 1829; fax: +82 31 467 1778. E-mail address: chodh@nvrqs.go.kr (D. Cho). 0167-7012/$ see front matter 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2009.05.009

The B. abortus and B. canis eld strains are listed in Table 1. Field strains of 33 B. abortus and 21 B. canis were isolated from 9 provinces

S.-I. Kang et al. / Journal of Microbiological Methods 78 (2009) 144149 Table 1 B. abortus and B. canis eld strains used in this study and their allele proles. Strainsa B. abortusb A-1 B. abortus A-2 B. abortus A-3 B. abortus A-4 B. abortus A-5 B. abortus B-1 B. abortus B-2 B. abortus B-3 B. abortus B-4 B. abortus B-5 B. abortus C-1 B. abortus C-2 B. abortus C-3 B. abortus C-4 B. abortus C-5 B. abortus D-1 B. abortus E-1 B. abortus E-2 B. abortus E-3 B. abortus E-4 B. abortus F-1 B. abortus F-2 B. abortus F-3 B. abortus G-1 B. abortus G-2 B. abortus G-3 B. abortus G-4 B. abortus G-5 B. abortus H-1 B. abortus H-2 B. abortus H-3 B. abortus H-4 B. abortus H-5 B. canis A-1 B. canis A-2 B. canis A-3 B. canis A-4 B. canis A-5 B. canis B-1 B. canis B-2 B. canis B-3 B. canis B-4 B. canis B-5 B. canis C-1 B. canis C-2 B. canis C-3 B. canis C-4 B. canis D-1 B. canis D-2 B. canis D-3 B. canis D-4 B. canis D-5 B. canis E-1 B. canis E-2
a b

145

Region Jeonbuk q q q q Jeju q q q q Chungnam q q q q Gangwon Gyeongnam q q q Jeonnam q q Gyeonggi q q q q Gyeongbuk q q q q Gyeonggi q q q q Jeonbuk q q q q Gyeongbuk q q q Jeonnam q q q q Chungbuk q

Locus Locus Locus Locus Locus Locus Locus Locus 1 2 3 4 5 6 7 8 9 9 8 9 9 9 9 9 9 9 10 9 9 9 9 12 5 10 10 10 11 11 11 11 11 12 10 11 14 10 10 10 12 4 4 4 4 4 3 3 3 3 3 6 4 4 5 3 3 3 3 3 6 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 5 5 5 5 6 7 7 7 7 5 5 5 5 5 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 4 4 4 4 5 4 4 4 4 3 3 3 3 3 3 4 4 4 4 3 3 3 3 3 3 3 3 3 3 3 3 3 14 15 15 15 15 13 13 13 13 13 13 10 10 13 13 13 13 13 13 12 12 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 7 6 6 6 6 11 10 5 11 5 8 4 4 8 7 6 7 6 6 7 7 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 17 18 17 17 17 11 11 11 10 11 16 16 16 6 16 15 16 16 15 7 8 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 8 8 8 8 8 4 4 4 4 4 6 5 6 6 4 4 4 4 4 5 5 6 6 6 6 6 8 8 8 8 8 5 5 5 6 5 10 9 9 10 11 11 12 10 11 9 12 11 11 6 10 9 9 9 7 8 7 7 7 9 8 9 8 8 12 16 15 12 17 17 18 17 17 8 10 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 13 15 15 15 15 4 4 4 4 4 7 11 11 7 8 8 8 8 8 5 5

for 2 min; followed by 94 C for 15 s, 60 C for 20 s, and 72 C for 90 s, repeated for 32 cycles. All amplication products were conrmed by electrophoresis on 4% standard agarose gels and run under 120 voltages for 55 min. 2000-bp and 25-bp ladders (25/100-bp mixed DNA ladder, Bioneer, Korea) were used as molecular size markers. Gels were stained with ethidium bromide (50 mg/ml), visualized under UV light, and photographed. The amplied samples were diluted from 1:10 to 1:100 in Hi-Di Formamide (Applied Biosystems, Cat # 4311320) depending on the estimated concentration of amplicon in each sample. A 1-ml aliquot was analyzed on an ABI 3730xl DNA capillary sequences (50-cm capillary array) (Foster City, CA, USA) with G5 dye set, running a default fragment analysis run module (GeneMapper50_POP7; injection 15 s, injection voltage 1.6 kV, run voltage 1.5 kV, and run time 1600 s). All samples were run with GeneScan 500-LIZ ROX size markers (Applied Biosystems, Cat # 4322682) as an internal standard, and the bands were sized relative to these markers by using the GeneMapper software ver. 3.7 (Applied Biosystems). Size of amplied PCR products was conrmed to triplicate. The number of repeat copies was deduced from size of amplied PCR products. 2.3. Investigation of VNTR loci stability In order to investigate the VNTR loci stability through in vitro passages, Brucella isolates were inoculated on 5% sheep blood agar (BBL, Cat # 211037) under 5% CO2 at 37 C. The cultures were transferred to fresh media 20 times at approximately 4-day intervals and subjected to VNTR analysis at each interval. Simultaneous, in vivo experiments were performed with the B. abortus RB51 strain isolated from various tissues of cattle in 46 weeks after vaccination. The isolated strains were compared with the original seed to determine the stability of tandem repeats at each of the 8 loci. 2.4. Diversity index analysis and phylogenetic construction Genetic diversity was calculated using the Simpson's diversity index (SDI) (Simpson, 1949) via the online V-DICE bioinformation tool available at the HPA website (http://www. hpa.org.uk/srmd/bioinformatics/tools/tools.htm). A number of tandem repeats of each locus were used for phylogenetic analysis. The cluster analysis was performed using the UPGMA (unweighted pair group method using arithmetic averages) of Bionumerics Version 5.0. 3. Results 3.1. Characterization of loci and cluster analysis The HOOF-Prints for the octameric tandem repeats of the 54 Brucella eld strains were characterized for the 8 loci. All 8 loci selected were specically amplied by PCR using uorescently labeled primers. Thirty-three B. abortus strains were originated from 8 different cattle herds, and 21 B. canis strains were obtained from 5 different dog breeding farms. B. abortus and B. canis isolates were divided into 22 and 18 different genotypes, respectively. In the same herd or farm, B. abortus and B. canis isolates were categorized by various genotypes. For example, the proles of 5 B. abortus isolates within a herd in Gyeonggi were categorized as G-1 (11,6,3,2,2,3,11,2), G-2 (11,7,3,2,2,3,9,2), G-3 (12,7,3,2,2,3,12,2), G-4 (10,7,3,2,2,3,11,2), and G-5 (11,7,3,2,2,3,11,2), and 4 B. canis isolates within a breeding farm in Gyeongbuk were categorized as C-1 (6,2,13,8,16,6,12,7), C-2 (4,2,10,4,16,5,16,11), C-3 (4,2,10,4,16,6,15,11), and C-4 (5,2,13,8,6,6,12,7). In particular, loci 1 and 7 of B. abortus and loci 4, 5, 7 and 8 of B. canis isolates appeared high variability within the same herd (Table 1). Whereas, B. abortus isolates showed no change of allele in loci 4, 5, 6 and 8, and B. canis strains were found to be invariant in locus 2. The genetic relationships of these isolates were determined by the UPGMA cluster analysis. B. canis isolates were relatively distinguishable

B. abortus and B. canis strains were isolated from cattle and dogs, respectively. Capital characters indicate the herd.

in Korea (Gyeongnam, Gyeongbuk, Jeonnam, Jeonbuk, Chungbuk, Chungnam, Gangwon, Gyeonggi, and Jeju) during the years 1996 2006 (Fig. 1). DNAs were extracted using a DNeasy Blood & Tissue kit (Qiagen Korea Ltd.). 2.2. PCR amplication of VNTR loci and sequencing The VNTR genotyping assay was used as in Bricker et al. (2003). PCR amplication was performed using a T3000 thermocycler (Biometra, Germany). To analyze the exact PCR product sizes, eight PCR primer sets were labeled with one of three uorescent dyes; HEX (green), NED (yellow), or 6-FAM (blue). The PCR conditions were modied to annealing temperature as follows: an initial step of 94 C

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Fig. 1. The map shows outbreak place of Brucella isolates used in this study.

B. abortus outbreak place;

B. canis outbreak place.

with some herds (Gyeonggi, Jeonbuk, Jeonnam, and Chungbuk). B. abortus isolates, also, were clustered to the same herds (Gyeonggi, Chungnam, Jeonbuk and Jeju). Hence, Brucella isolates showed a tendency to cluster by the provinces (Figs. 13). 3.2. Diversity index The Simpson's diversity index (SDI) is based on the repeat number of alleles. Different strains may have different allele sizes, and alleles with a high-repeat copy number might be more prone to rearrangement than small-repeat copy number alleles. In order to assess the diversity at individual loci, the DI was calculated using the allele sizes at all genetic loci for the collection of Brucella strains examined. The values of this index ranged from 0 to 1. Simpson's DI values for loci 1 and 7 of B. abortus isolates appeared with 0.742 and 0.843. They revealed to have higher diversity as compared with other loci. In B. canis, the DI values of loci 5, 7, and 8 were more than 0.8. But, loci 4, 5, 6 and 8 in B. abortus and locus 2 in B. canis were not detected with any diversity (Table 2). 3.3. Stability of VNTR loci In order to assess the stability of VNTR loci, changes in the original strains were examined after in vivo passage in cattle and in vitro passage in blood agar. For the in vivo study, cattle were inoculated with B. abortus RB51 vaccine, and RB51 was re-isolated from various tissues after 46 weeks vaccination. The RB51 strains recovered from various tissues showed changes in the VNTR loci 1 and 7. Locus 1

showed allele number changes in RB51 strains isolated from 4 tissues including the pre-scapular, iliac, parotid, and axillary lymph nodes. Changes in the VNTR numbers for locus 7 were observed in isolates from submandibular, iliac, and parotid lymph nodes (Fig. 4). To examine the effect of in vitro passage, the eld isolates were cultivated 20 times by serial passage over approximately 3 months. In the case of B. abortus, VNTR loci 1 and 7 were changed at the 18th and 8th passages, respectively. Thus, the diversity of loci 1 and 7 in B. abortus appeared to have similar patterns by in vivo and in vitro experiments. In case of B. canis, VNTR loci 5, 7, and 8 were changed at the 16th, 7th, and 16th passages, respectively. 4. Discussion Brucellosis is an important disease that is reported every year in Korea (Wee et al., 2008). Bovine brucellosis was rst reported in imported cattle in 1955, reached in the top of peak for 25,454 head (4498 cases) in 2006. Canine brucellosis has been reported to have 6 cases in 2002, thereafter showed up to 187 head in 2005. In 2008, bovine and canine brucellosis are continuously decreasing on 8409 and 24 head, respectively. The incidence of animal infection seems to have been declining due to the success of the national Brucella eradication program. However, to understand the sources of infection, it is necessary to study for distribution of Brucella genotypes and tools for epidemiological trace-back. In epidemiology, molecular typing can be used for trace-back analysis, which can help to identify the origin of an infection. Rapid and accurate subtyping of Brucella isolates is a key for effective outbreak tracking and the establishment of a control

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Fig. 2. The cluster analysis of 33 B. abortus isolates based on HOOF-Print.

program. Of the reported methods of subtyping, VNTR has many advantages. It is rapid, taking only a few hours compared with techniques such as pulsed eld gel electrophoresis (PFGE), which may take several days. VNTRs also appear to have greater diversity and, hence, greater discriminatory capacity as compared with other molecular typing systems (Richards and Sutherland, 1997; van Belkum et al., 1998). HOOF-Prints have been employed with entire genome sequences to identify rapidly evolving loci and are capable of differentiating Brucella isolates by the variability in the 8-bp tandem repeat (Bricker et al., 2003). The purpose of this study was to investigate the clustering of Brucella isolates by geographical origin. Furthermore, the genetic diversity of Brucella strains causing animal infection in Korea has not yet been investigated. The Brucella isolates in this study exhibited extensive variability in the range of alleles at each locus. For example, VNTR proles of loci 1 and 7 in B. abortus isolates and loci 4, 5, 7, and 8 in B. canis were highly variable. The allele numbers for each locus from B. abortus and B. canis isolates showed a similar prole with previous reports (Bricker and Ewalt, 2005; Whatmore et al., 2006). These various alleles may be due to strain mutation during replication in the host or multiple infections of different origins. So, the difference of one repeat number for each locus should consider belonging to the same allele type. In order to assess the diversity at individual locus, the DI was calculated for the population of B. abortus and B. canis isolates. Value

of this DI represents from 0 in the case of no diversity up to 1 in innite diversity. It reects the number of alleles detected and causes the individual allele frequency. When compared with the prior report (Bricker and Ewalt, 2005; Whatmore et al., 2006), we conrmed that there was a close connection between the total number of alleles and DI value. In this study, the DIs of loci 1 and 7 in B. abortus isolates appeared to be 0.74 and 0.84, which were slightly lower than DIs 0.89 and 0.92 (Whatmore et al., 2006), and DIs 0.90 and 0.87 (Bricker and Ewalt, 2005). Moreover, the diversity value of loci 4, 5, 6, and 8 in B. abortus isolates was zero, which was much lower than DI 0.590.92 reported previously (Table 2; Whatmore et al., 2006). In case of B. canis isolates, the DI values at loci 5, 6, 7, and 8 were ranged to 0.67 0.84, which were similar to DI 0.610.81 reported previously (Whatmore et al., 2006). Lower DI values for B. abortus isolates in Korea seem to deduce that they are localized by longtime steady mutation from ancestor to descendants, but further study of some more isolates may be necessary. In order to conrm the stability of isolates from each locus, we compared the number of alleles after in vivo and in vitro passages. As shown in Fig. 4, loci 1 and 7 of B. abortus were unstable through in vivo and in vitro passage as compared with other loci. These results corresponded to show high variability in 33 B. abortus eld strains. This suggested that loci 1 and 7 were unstable for use in molecular epidemiological investigations of Brucella infections. The in vitro cultivation of B. canis strains affected allele number of loci 5, 7, and 8

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Fig. 3. The cluster analysis of 21 B. canis isolates based on HOOF-Print.

by passage, and these changes are similar to 21 B. canis eld strains. Among them, particularly locus 7 showed that B. abortus and B. canis isolates rapidly mutated at seven and eight passages. Hence, locus 7 will be difcult to use as a trace-back marker. There is a necessity for some more stable loci in epidemiological applications to demonstrate relationships among farms. In conclusion, Korean isolates of B. canis and B. abortus were easily distinguished at the pack or herd levels, respectively, using VNTR analysis. However, the greater diversity and higher mutation rate showed at some loci of isolates from the same farm or of between fresh eld isolates and laboratory passaged isolates were difcult to analyze in high-resolution analysis of epidemics. Even if hypervariable loci of HOOFs need to be replaced to stabilize other loci, this

method can be used as a molecular epidemiological tool to determine relationships for Brucella infections of the Korean isolates. Acknowledgements This study was supported by a fund of the Veterinary Science Technical Development Research Project from the National Veterinary Research and Quarantine Service, Republic of Korea (Project No: CAD13-2006-09-02). References
Al Dahouk, S., Tomaso, H., Nckler, K., Neubauer, H., Frangoulidis, D., 2003. Laboratorybased diagnosis of brucellosisa review of the literature. Part1: techniques for direct detection and identication of Brucella spp. Clin. Lab. 49, 487505. Bricker, B.J., Ewalt, D.R., 2005. Evaluation of the HOOF-Print assay for typing Brucella abortus strains isolated from cattle in the United States: results with four performance criteria. BMC Microbiol. 5, 37. Bricker, B.J., Ewalt, D.R., Halling, S.M., 2003. Brucella HOOF-Prints: strain typing by multilocus analysis of variable number tandem repeats (VNTRs). BMC Microbiol. 11, 15. Cobel, M.J., 1997. Brucellosis: an overview. Emerg. Infect. Dis. 3, 213221. Cobel, M.J., Brinley-Morgan, W.J., 1984. Genus Brucella Meyer and Shaw 1920. 173AL. Bergey's Manual of Systematic Bacteriology, vol. 1. Williams & Wilkins, pp. 377388. Fabre, M., Koeck, J.L., Le Flche, P., Simon, F., Herv, V., Vergnaud, G., Pourcel, C., 2004. High genetic diversity revealed by variable-number tandem repeat genotyping and analysis of hsp65 gene polymorphism in a large collection of Mycobacterium canettii strains indicates that the M. tuberculosis complex is a recently emerged clone of M. canettii. J. Clin. Microbiol. 42, 32483255. Foster, G., Osterman, B.S., Godfroid, J., Jacques, I., Cloeckaert, A., 2007. Brucella ceti sp. nov. and Brucella pinnipedialis sp. nov. for Brucella strains with cetaceans and seals as their preferred hosts. Int. J. Syst. Evol. Microbiol. 57, 26882693. Garca-Yoldi, D., Marn, C.M., de Miguel, M.J., Muoz, P.M., Vizmanos, J.L., Lpez-Goi, I., 2006. Multiplex PCR assay for the identication and differentiation of all Brucella

Table 2 Allelic diversity of each locus in Brucella isolates. Locus Locus Locus Locus Locus Locus Locus Locus Locus
a

Diversity indexa B. abortus (n = 33) 1 2 3 4 5 6 7 8 0.742 0.360 0.512 0.000 0.000 0.000 0.843 0.000 B. canis (n = 21) 0.644 0.000 0.508 0.794 0.825 0.676 0.844 0.821

The diversity index was calculated for each locus by the method of Simpson (1949).

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Fig. 4. Allelic patterns of VNTR loci 1 and 7 after in vivo passages. The vaccinated RB51 strains were re-isolated from various tissues. Lane 1, RB 51; lane 2, pre-scapluar lymph node; lane 3, submandibular lymph node; lane 4, supramammary lymph node; lane 5, iliac lymph node; lane 6, subilial lymph node; lane 7, parotid lymph node; lane 8, axillary lymph node; lane 9, retropharyngeal lymph node.

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