MLST Scheme for Anaplasma marginale Reveals Pathogen Strong Adaptation to a Very Specific Cell Type

E. GUILLEMI; P. RUYBAL; S. GONZALEZ; R. FRUTOS; S. WILKOWSKY; M. FARBER

New Orleans

Conferencia; 11th International Conference on Molecular Epidemiology and Evolutionary Genetics of Infectious Diseases,; 2012

Elsevier

Anaplasmosis is enzootic in Argentina and is a major cause of economic losses for livestock industry. A highly reproducible and discriminative typing system is essential for better understanding the Anaplasma marginale epidemiology. 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; each isolate is defined by the alleles at each of the seven loci. This is called the sequence type (ST). Due to the scarce knowledge status of A. marginale diversity in Argentina, the aim of this study was to develop the MLST strategy to discriminate isolates to study the genetic diversity andestimate 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 finally selected. Raw trace files from 57 isolates 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. Phylogenetic analyses were performed using Maximum Likelihood method for each locus separately and for the concatenated sequences using PhyML (version 2.4.4) with 500 bootstrap replicates. 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 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.