Genotipagem de Mycobacterium bovis pelo multispacer sequence typing

Mycobacterium bovis, which causes bovine tuberculosis, is a species belonging to Mycobacterium tuberculosis complex. It shows 99.9% of genetic homology with other species of the complex, making the diagnosis difficult. The bovine bacilli represent a serious problem due to the direct losses in cattle, caused by such infirmity and possible sanitary barriers to exportation. Molecular typing, provide an innovative way to identify bacterial strains, trough generate while genotypes by combining different polymorphic DNA markers, contributing to their epidemiological investigation. The techniques currently used for typing of M. bovis are Spoligotyping and VNTR. However these methods are not able to recognize all genetics analyzed strains diversity such as sequencing methods based in last ones allow analyzing molecular target sequence, thereby identifying all events present in the genetic marker used. This paper aims standardize Multispacer Sequence Typing (MST) for M.bovis genotyping. Seven intergenic spacer region were analyzed on 58 M.bovis samples, coming from six brazilian's states (MG, GO, SP, MT, PR, RS), isolated between 2006 and 2010, and on four M.boviss reference strains. Four types of genetic events were observed: nucleotide mutation (SNP), insertion, deletion and tandem repeat (TR), totaling from the combination of genotypes obtained a total of 28 events. Twenty-eight type sequences (TS) have been also produced, showing a discrimination index of 93%, including the standard strain AF2122/97 [Genbank BX248333.1] used for comparison in silico. These data were used to analyze the pattern of evolutionary lineage of M. boviss isolates and correlate them with phylogeographic lineages, based on the formation of clonal complexes, generated from eBURST. This is the first study aimed identify the variability of isolates of M. bovis using MST method. Results were quite satisfactory. Method allowed us to typing and differentiate M.bovis isolate from a sequencing of few regions, even in restricted locations and in short a time as established in Bueno Brandao city.The method has the sequencing advantage and the availability of sequences analyzed in public databases which can be used by professionals around the world as an analyzes tool.