Molecular survey and risk factors of <i>Trypanosoma pestanai</i> in Eurasian badgers from the northern Iberian Peninsula

<i>Trypanosoma</i> (<i>Megatrypanum</i>) <i>pestanai</i> is a parasite of the Eurasian badger (<i>Meles meles</i>), reported in various European countries. However, its presence in the Iberian Peninsula had not been previously investigated. To address this knowledge gap and to assess its occurrence and potential risk factors associated with infection, we analyzed DNA from the spleens of 145 badgers sampled across 4 autonomous regions in northern Spain. Two real-time PCR assays using a reference <i>18S rRNA</i> partial sequence of <i>T. pestanai</i> (92 bp) were developed: one based on SYBR Green chemistry and the other employing a TaqMan probe. Both protocols demonstrated excellent concordance. Defining a sample as positive when at least 1 assay yielded a positive result, the overall prevalence was 35%, consistent with values previously reported in other European populations. A logistic regression model indicated a significantly higher occurrence in badgers from the Eurosiberian bioregion (42%) compared to those from the Mediterranean bioregion (19%). No significant associations were found with age or sex. A subset of positive samples was further analyzed by conventional PCR targeting approximately 900 bp of the <i>18S rRNA</i> gene and sequenced. All 9 high-quality sequences shared 99.75-100% identity with known <i>T. pestanai</i> sequences. These findings confirm that <i>T. pestanai</i> is a common parasite of Iberian badgers and suggest that more humid climatic conditions may favour its persistence, potentially through effects on host ecology or vector dynamics.