Multi-Locus Typing Scheme for Babesia bovis and Babesia bigemina Reveals High Levels of Genetic Variability in Strains from Northern Argentina

E. GUILLEMI; P. RUYBAL; LIA V; GONZALEZ S; FARBER M; S. WILKOWSKY

New Orleans

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

Elsevier

Babesiosis, caused by the protozoans Babesia bovis and Babesia bigemina, is enzootic in north Argentina and is a cause of major economic losses to livestock industry. Current control measures are based on the use of acaricides and live attenuated vaccines. The aim of this work was to develop a MLST strategy to characterize the genetic diversity of reference and field strains of both parasites. We started by pre-selecting 23 single-copy genes from the B. bovis annotated genome. These genes were well distributed in the 4 chromosomes of the parasite. For B. bigemina the genes were identified by TBLASTN using the orthologousgenes of B. bovis as query. The final MLST scheme was based on seven genes (check, dnaJ, gpad, pkid, rcc, rip9 and rho4) for B. bovis and six (cyp, dnaJ, rcc, sbp3, sbp4 and zfc) for B. bigemina. The gene choice was supported by low dN/dS values and non- significant neutrality tests. A custom-designed bioinformatic pipeline was also developed to facilitate the automated analysis of raw sequences and further phylogenetic analysis. The open source pipeline was designed for UNIX-type Platforms and available at http://bioinformatica.inta.gov.ar We analyzed 17 isolates for B. bovis and 13 for B. bigemina. Both MLST schemes exhibited excellent discriminatory power with Simpson´s indexes of 1 for B. bovis and 0.9545 for B. bigemina. Phylogenetic analysis was conducted both on concatenated MLST loci and on each individual locus. The latter showed different phylogenies for each individual gene.  Genetic diversity was very high and mixed genotypes were frequently found for both parasites in outbreak samples. Recombination events, that could have arisen from these multiple infections, were suggested by inter and intra-loci linkage disequilibrium analysis and the lack of congruence in phylogenetic trees from individual genes. Thus, the high levels of diversity observed might be interpreted as the result of the combined contribution of mutation and intragenic recombination.