Identification of Leptospira species is important because leptospirosis is an infectious disease that has reemerging in urban centers throughout the world. To date, observation of these bacteria requires dark-field microscopy, and their identification has long relied on serotyping. The development of alternative tools for identification of Leptospira serovars is required, to circumvent a major risk of contamination during sample cultivation. The need for rapid and specific characterization of leptospires has stimulated the use of new molecular biological tests. The aim of study was to investigate the possible identification of some serovars by RFLP typing

Material and Methods: DNA was extracted from 68 Leptospira serovars, belonging of 11 different pathogenic and saprophytic species. Polymerase chain reaction (PCR) assays were performed using specific primers designed to amplify a fragment of the gene coding the beta subunit of RNA polymerase (rpoB). The enzymatic digestion was performed using the enzymes TaqI, Sau3AI, MseI, MslI and EcoNI, previously selected based in Expressed Sequence Tags and nucleotide sequences available in databases. The restriction fragments were separated in polyacrylamide gels and stained with silver nitrate. Results: We were able to differentiate 58% of the serovars tested. However 29 serovars from 9 species were not identified yet, we select in database, enzymes that can identify the variations in others genes like: DNA Bgyrase gene, Translocase pre -protein gene (SecY), Isocitrate dehidrogenase gene (icdA) and used for this purpose. Conclusion: By use of restriction typing, the phylogenetic study of extremely fastidious Leptospira species can be carried out without the need to culture them. In addition, this technique is a promising tool to evaluate genetic diversity of related strains and may be useful to differentiate Leptospira serovars within the species.

Supported by: Conselho Nacional de PESQUISA (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais -FAPEMIG