Sequence and phylogenetic analyses
In order to confirm the specificity of the amplicons yielded, positive
PCR products were subjected to direct sequencing at the Center for
Genomic Technologies of the Complutense University of Madrid (Spain).
Briefly, amplicons were sequenced in both directions with the same
internal primer pair used for PCR amplification, Big Dye™ chemistries
and an ABI 3730xl sequencer analyzer (Applied Biosystems, Foster City,
CA). Raw sequencing data in both forward and reverse directions were
manually edited by Bioedit Software, and BLAST tool
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to compare resulting
nucleotide sequences with sequences retrieved from the National Center
for Biotechnology Information (NCBI) database.
The evolutionary relationships among the identifiedBesnoitia -positive samples were inferred by a phylogenetic
analysis using the Neighbor-Joining method (Saitou & Nei, 1987) in MEGA
6. The evolutionary distances were computed using the Kimura 2-parameter
method and modelled with a gamma distribution. The reliability of the
phylogenetic analyses at each branch node was estimated by the bootstrap
method using 1000 replications. Representative sequences of differentBesnoitia spp. isolates were retrieved from the NCBI database and
included in the phylogenetic analysis for reference and comparative
purposes. Representative sequences obtained in the present study were
deposited in GenBank under the accession numbers MW035607 to MW035610.