Caracterização genotípica de Borrelia sp e de genes de Anaplasma marginale que codificam proteínas de membrana com potencial imunogênico

Abstract

The geographic distribution of bovine borreliosis is determined by the dispersion of its vector. Borrelia theileri is the predominant species in cattle, and B. coriaceae and B. burgdorferi also been reported causing clinical disease. B. theileri cause mild disease in cattle, and still is important for its potential to be confused with the spirochete of Lyme disease, B. burgdorferi, and agents of epizootic bovine abortion, B. coriaceae. In Brazil, as well as in other South American countries, the agent of this disease has not been isolated further confusing the diagnosis. The objective of this study was to identify genotypically Borrelia sp that affects cattle in Brazil. DNA extraction, was performed from blood and ticks of cattle with positive serology by indirect ELISA with crude antigen of Borrelia burgdorferi. Primers were designed for genes of Borrelia burgdorferi and B. theileri groups: 16S, flaA, flaB, GroEL, hbb, recA, 5s-23s, p66, rrs, rpoB and glpq. After the PCR reaction, only the primers amplified rrs and rpoB sequences. The predictive amino acid sequence of RRS3 revealed 99% homology with B. hermsii and B. duttonii and predictive amino acid sequence of RPOB showed 67% homology with B. duttonii and B. recurrentis. This suggests that the species of Borrelia sp present in Brazil is not owned by group B. burgdorferi. Little is known regarding the genetic variability of genes that encode membrane proteins of Brazilian isolates of A. marginale. The products of these genes constitute an important tool, as there may be significant antigen polymorphism, which may damage cross-protection between isolates and the chances of identifying candidate immunogens. The aim of the present study was to determine the degree of conservation of sequences of these genes in a Brazilian isolate of A. marginale comparing with Saint Maries and Florida isolates. For this, primers were designed to amplify the genes omp1, omp4, omp5, omp7, omp8, omp10, omp14, omp15, sodb, opag1, opag3, virb3, am097 (VirB9-1), am956 (PepA), am254 (ef-tu), am854 by PCR. The genes were then sequenced by Sanger method and the predicted amino acid sequences aligned and homology analyzed by the program CLUSTAL W. With the exception of OMP 7 all proteins (OMP1, OMP4, OMP5, OMP8, OMP10, OMP14, OMP15, SODB, OPAG1, OPAG3, VIRB3, VIRB9-1, PepA, EF-Tu, AM854) exhibited homology greater than 92% with other A. marginale isolates. However, only OMP1, OMP5, EF-Tu, VirB3, SODB, VIRB9-1 e AM854 showed homology greater than 72% regarding to A. marginale centrale which confers cross-protection against A. marginale.