Molecular typing of Brucella melitensis endemic strains and differentiation from the vaccine strain Rev-1 |
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Authors: | Georgios T Noutsios Rigini M Papi Loukia V Ekateriniadou Anastasios Minas Dimitrios A Kyriakidis |
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Institution: | (1) Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece;(2) National Agricultural Research Foundation, Veterinary Research Institute of Thessaloniki, NAGREF, GR-57001 Thermi, Greece;(3) Technological Educational Institute of Larissa, School of Health Sciences, Laboratory of Microbiology, GR-41110 Larissa, Greece; |
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Abstract: | 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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