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Molecular typing of Brucella melitensis endemic strains and differentiation from the vaccine strain Rev-1

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In the present study forty-four Greek endemic strains of Br. melitensis and three reference strains were genotyped by Multi locus Variable Number Tandem Repeat (ML-VNTR) analysis based on an eight-base pair tandem repeat sequence that was revealed in eight loci of Br. melitensis genome. The forty-four strains were discriminated from the vaccine strain Rev-1 by Restriction Fragment Length Polymorphism (RFLP) and Denaturant Gradient Gel Electrophoresis (DGGE). The ML-VNTR analysis revealed that endemic, reference and vaccine strains are genetically closely related, while most of the loci tested (1, 2, 4, 5 and 7) are highly polymorphic with Hunter-Gaston Genetic Diversity Index (HGDI) values in the range of 0.939 to 0.775. Analysis of ML-VNTRs loci stability through in vitro passages proved that loci 1 and 5 are non stable. Therefore, vaccine strain can be discriminated from endemic strains by allele’s clusters of loci 2, 4, 6 and 7. RFLP and DGGE were also employed to analyse omp2 gene and reveled different patterns among Rev-1 and endemic strains. In RFLP, Rev-1 revealed three fragments (282, 238 and 44 bp), while endemic strains two fragments (238 and 44 bp). As for DGGE, the electrophoretic mobility of Rev-1 is different from the endemic strains due to heterologous binding of DNA chains of omp2a and omp2b gene. Overall, our data show clearly that it is feasible to genotype endemic strains of Br. melitensis and differentiate them from vaccine strain Rev-1 with ML-VNTR, RFLP and DGGE techniques. These tools can be used for conventional investigations in brucellosis outbreaks.

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Polymerase chain reaction


Various number tandem repeats


Field strain


Tris-borate-EDTA buffer


Outer membrane protein


Restriction Fragment length polymorphism


Hypervariable octameric oligonucleotide finger-prints


European Study Group for epidemiological markers


Denaturant gradient gel electrophoresis




Hunter-Gaston genetic diversity index


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This research was fully supported by a PENED Doctoral Research Grant (project 03ED467) from the General Secretariat for Research and Technology of the Hellenic Ministry of Development and PNG Gerolymatos S.A.

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Correspondence to Dimitrios A. Kyriakidis.

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Supplementary Table 1

Size of PCR products (bp) from eight loci of Br. melitensis genome (DOC 101 kb)

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Noutsios, G.T., Papi, R.M., Ekateriniadou, L.V. et al. Molecular typing of Brucella melitensis endemic strains and differentiation from the vaccine strain Rev-1. Vet Res Commun 36, 7–20 (2012). https://doi.org/10.1007/s11259-011-9505-7

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  • Brucella melitensis
  • Rev-1
  • ML-VNTR analysis
  • omp2 polymorphism
  • Phylogenetic relationship
  • DGGE