Epidemiological dynamics of Leishmania (Sauroleishmania) tarentolae and Trypanosoma platydactyli in reptile hosts and sand flies: from isolation to genome assembly

<i>Leishmania</i> (<i>Sauroleishmania</i>) <i>tarentolae</i> and <i>Trypanosoma platydactyli</i> are reptile-associated trypanosomatids transmitted by <i>Sergentomyia minuta</i> sand flies, posing challenges for accurate diagnosis due to the fact that they often occur in sympatry. This study aimed to isolate and characterize new strains of these parasites from reptiles and sand flies using morphological, molecular and genomic approaches. Fifty-five reptiles were captured in Apulia, Italy, and sand flies were collected and dissected under a surveillance framework. Blood samples and gut contents were cultured in Schneider's Drosophila (SC) and Tobie-Evans (TEv) media. Two positive cultures underwent whole-genome sequencing, and a new conventional PCR (cPCR) protocol targeting the <i>β-tubulin</i> gene was developed. <i>T. platydactyli</i> was isolated from 27% of <i>Tarentola mauritanica</i> geckos using SC medium and 12.5% with TE, while <i>L</i>. (<i>S</i>.) <i>tarentolae</i> was isolated from 4.15% of geckos exclusively with SC. Cytology confirmed <i>T. platydactyli</i> in 25% of gecko blood smears. cPCR revealed <i>T. platydactyli</i> in 18.75%, <i>L</i>. (<i>S</i>.) <i>tarentolae</i> in 12.5% and co-infections in 14.6%. No infections were found in <i>Podarcis siculus</i> or <i>Hemidactylus turcicus</i>. Out of 208 <i>S</i>. <i>minuta</i> sand flies tested, 19 (9.1%) were positive for <i>T. platydactyli</i>, 30 (14.4%) for <i>L</i>. (<i>S</i>.) <i>tarentolae</i>, and 15 (7.2%) were co-infected with both. The newly developed cPCR assay robustly differentiated these parasites in both reptile and sand fly samples. Monitoring of natural infections in geckos revealed persistent, low-level <i>L</i>. (<i>S</i>.) <i>tarentolae</i> infections, detectable only by molecular methods, in contrast to the intermittent parasitaemia of <i>T. platydactyli</i>, which was detectable by cytology and culture. Kimura 2-parameter (K2P) divergence profiles indicate no evidence of a recent mobilome in <i>T. platydactyli</i>, whereas <i>L</i>. (<i>S</i>.) <i>tarentolae</i> retains a small but detectable fraction of low-divergence transposable-element copies (≤5-10% K2P; <0.05% of the genome). These findings confirm the sympatric circulation of <i>L</i>. (<i>S</i>.) <i>tarentolae</i> and <i>T. platydactyli</i> in geckos and sand flies in southern Italy, highlighting <i>T. mauritanica</i> as the most common reptilian host. The successful isolation and genome assembly of these trypanosomatids, along with the newly developed molecular tool, lay a solid foundation for future epidemiological and comparative genomic investigations, emphasizing the role of reptilian hosts in maintaining trypanosomatid diversity.