Targeted-Amplicon NGS for Blastocystis sp. in Shepherd Dogs of Portugal Discriminates Co-Colonization with Multiple Zoonotic Subtypes

Research on endoparasites in pet dogs has been growing, but shepherd dogs have largely been overlooked. These dogs frequently share close proximity not only with sheep, which are reservoirs of zoonotic subtypes of <i>Blastocystis</i>, but also with their owners. This close contact increases the potential for shepherd dogs to act as intermediates in the transmission of <i>Blastocystis</i>. To clarify the role of these dogs as reservoirs for this parasite, this study investigated the presence of <i>Blastocystis</i> in shepherd dogs. Stool samples from Portuguese shepherd dogs were analyzed using SYBR-Green-based real-time PCR and melting curve analysis followed by targeted-amplicon NGS for mixed infections detection. Our results revealed a 60% occurrence of <i>Blastocystis</i> sp. in shepherd dog stools and frequent identification of zoonotic subtypes ST1-ST4 and ST14. Additionally, we observed mixed infections and subtype diversity within individual dogs, suggesting a potential role in cross-species transmission between livestock and humans.