Identificação e caracterização de minicírculos de Trypanosoma vivax (Zieman, 1905) através de geração e análise de GSS (Genome Sequence Survey)

Trypanosoma vivax is a hemoparasite which causes sickness in ruminants. In Africa it is found wide-spread, mainly in tropical areas where tsetse is found, and in several South America countries, mainly in the Pantanal of Brazil and in Bolivia. T. vivax is a pathogenic parasite considered of great importance for cattle-industry of Africa and South America. Possible economic losses of 160 million dollar in the Brazilian Pantanal and lowlands of Bolivia are estimated. Despite the high economic relevance of the disease caused by T. vivax, few researches on its molecular characterization have been done up to this moment compared with human trypanosomes as T. brucei and T. cruzi, for example, a search in the NCBI-Entrez portal (24/05/05) using “Trypanosoma vivax” as a keyword showed only 439, 27, 23 and 5 entries available in the PubMed, Protein, Nucleotide and Structure sections, respectively, showing the limited knowledge about this species. The diagnosis of the infections for T. vivax remains a challenge, since parasitemias are usually very low in the majority of the infections, reinforcing the necessity to discover new markers for the development of more sensitive and species-specific assays. The technique for gene discovery used in this study was the GSS, with the objective to generate new data from the T. vivax genome, to make a preliminary innotation of these sequences and identification of minicircles. From a genomic library, 455 high quality GSS were obtained, equivalent to 0.5% of the parasite genome, that was estimated to be around 25 MB. From those 455 GSS, 331 GSS-nr were obtained, of which 108 sequences had a significant similarity with sequences deposited in the public databases and were classified in the three functional categories of GO: biological process, cellular component and molecular function. Eighteen sequences did not show any similarity with the databases used in this study, being considered orphan genes. In the present study, primers were designed to obtain minicircle sequences of different T. vivax strains, and their sensitivity was tested. Thirty-six minicircles were identified, each one showing only one conserved region and conserved sequence blocks (CSB). The estimated size of the minicircle was of 480 bp.