First report of Trypanosoma evansi A-type from the Ecuadorian Amazon: Phylogenetic and structural analyses of the VSG RoTat1.2 fragment.

Trypanosoma evansi, a protozoan parasite of the Trypanozoon subgenus, infects domestic and wild animals, and occasionally humans, worldwide. Despite its significant impact on livestock production, epidemiological studies and molecular analyses of T. evansi based on variant surface glycoproteins (VSG) remain limited in South America. We report the first molecular identification of T. evansi in two localities in the Ecuadorian Amazon and provide phylogenetic and structural analyses of the VSG RoTat 1.2 fragment. Using a retrospective biobank approach, we analyzed DNA from blood samples of dogs and horses from small farms in the Orellana and Sucumbíos provinces of the country. Three primer sets targeted the internal transcribed spacer 1 (ITS1), expression site-associated genes 6 and 7 (ESAG6/7), and VSG RoTat 1.2. Sequences were evaluated at both the nucleotide and predicted protein levels. The molecular prevalence was 19 % (5/26), including one dog from Sucumbíos and one dog plus three horses from Orellana. Although ITS and ESAG phylogenies were inconclusive, VSG analysis grouped the Ecuadorian genotypes within a distinct T. evansi clade, separate from Trypanosoma equiperdum and Trypanosoma brucei. The amino acid residues of the VSGs are similar between T. evansi isolates and different from those of the other two species. However, the overall structural conservation of the VSGs analyzed portions of the Trypanozoon species suggests a preserved membrane protein functionality. These findings reveal the silent presence of T. evansi in rural Ecuador, posing risks to animals and highlighting the need for further research on native isolates.