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

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

New Orleans

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

Editorial Elsevier y Loyola University

Anaplasmosis, a tick-borne disease caused by the strictly intracellular lifestyle bacteria Anaplasma marginale, is a major cause of economic losses for livestock in Northern Argentina. Due to the scarce knowledge status of A. marginale diversity in Argentina, the aim of this study was to develop a Multilocus Sequence Typing scheme (MLST) for estimating 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. Using an ?in-house?-developed pipeline and a MySQL data base (available as a web application at http://bioinformatica.inta.gov.ar), raw trace files from 57 isolates were FASTA-converted and contigs were assembled, aligned for allele identification and assigned to a defined haplotype called sequence type (ST). Phylogenetic analyses were performed for each locus separately and for the concatenated sequences, using PhyML (version 2.4.4) with 500 bootstrap replicates. Using several functions from the DnaSP 5.00.02 package, recombination events, accompanied by low linkage disequilibrium figures, were detected for dnA, recA and sucB, linkage disequilibrium and thus lack of recombination was corroborated for  ftsZ, lipA and secY. The most striking features were the almost complete absence of geographical association among strains, very strong negative pressure on all loci, no population expansion and very limited polymorphism. A. marginale seems to be in genomic stasis as a result of a strong adaptation to a very specific cell type and cell environment as is the bovine erythrocyte.