INVESTIGADORES
RUYBAL paula
congresos y reuniones científicas
Título:
MLST scheme for Anaplasma marginale reveals pathogen strong adaptation to a very specific cell type
Autor/es:
GUILLEMI ELIANA; RUYBAL PAULA; GONZALEZ SERGIO; FRUTOS ROGER; WILKOWSKY SILVINA; FARBER MARISA
Reunión:
Conferencia; TTP7 Ticks and Tick-borne pathogens international conference; 2011
Resumen:
A highly reproducible and discriminative typing system is essential for better understanding of Anaplasma marginale epidemiology, in order to analyze the population structure, design and apply strategic control measures and monitor the performance of live vaccines.The Multilocus Sequence Typing (MLST) is a highly discriminatory method for isolate characterization on the basis of 7 housekeeping genes sequenced fragments. For each gene fragment, the different sequences are assigned as distinct alleles, and each isolate is defined as a particular haplotype (the allelic profile or sequence type -ST-) by the combination of alleles at each of the seven loci. Due to the scarce knowledge status of Anaplasma marginale diversity in Argentina, the aim of this study was to develop the MLST strategy to discriminate between A. marginale isolates in order to study the genetic diversity and estimate the population structure of the pathogen among assorted enzootic regions. Fourteen annotated genes coding for highly conserved proteins were pre-selected based on reported bacterial MLST schemes. Primers were designed using Primer3 program in order to amplify an internal fragment of 500 to 700-bp.  Seven genes (dnaA, ftsz, groEl, lipA, recA, secY and sucB) were selected based on: number of single nucleotide polymorphic sites (SNPs), genome distribution, absence of selective pressure (Ka/Ks 1) and specificity of primers. PCR amplifications were performed and amplicons were sequenced using the Big Dye Terminator v3.1 kit from Applied Biosystems and analyzed on an ABI 3130XL genetic analyzer from the same supplier. Raw trace files were FASTA-converted and contigs were assembled, aligned and assigned to a defined ST using an “in-house”-developed pipeline (MLST-pipeline) available at http://bioinformatica.inta.gov.ar.  Genetic variation among these 7 gene sequences was evaluated using as references the available annotated genomes from A. marginale strains (Saint Marie, Mississippi, Puerto Rico, Florida and Virginia), 5 reference strains from Argentina (Mercedes, Salta, Quitilipi, Rosalí and Virasoro), 2 isolates from E.E.U.U ( from South Idaho and Oklahoma) and 1 from South Africa. We also analyzed 26 samples from naturally infected animals from the argentinean enzootic area. Phylogenetic analyses were performed using Maximum Likelihood (ML) method for each locus separately and for the concatenated sequences using Mega 5.05 software with 1.000 bootstrap replicates using orthologous genes from the closely related Rickettsiale Anaplasma centrale as outgroup. Phylogenetic analysis showed that in the tree from concatenated sequences the foreign reference strains were clustered separately with an external branch for the African strain. The argentinean strains clustered together in a main group. To further investigate if the topology observed for the concatenated sequences was supported by the distribution of the various haplotypes, a similar phylogenetic analysis was conducted on each locus considered separately. Tree topologies were different than those observed for the concatenated sequences, with different topologies for each locus. The locus yielding the topology resembling the most that of the concatenated genes was dnaA. DNA polymorphism analysis reflected very low number of haplotypes or alleles, with the exception of dnaA and recA which were significantly more diverse. Recombination events, accompanied by low linkage disequilibrium figures, were detected for dnA, recA and sucB using several functions from the DnaSP 5.00.02 package.The most striking features, however, were the almost complete absence of singletons and non-synonymous substitutions, indicative of negative selection and absence of population expansion; this seems to be the result of a very strong adaptation to a very specific cell type and cell environment.