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 identified Besnoitiasp.-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.