First molecular evidence of Leishmania parasites in sand flies (Diptera: Phlebotominae) from Slovenia

Sand flies (Diptera: Phlebotominae) are vectors of Leishmania spp., protozoan parasites that cause leishmaniasis, a zoonosis endemic in the Mediterranean region. Although Slovenia is not considered endemic, its proximity to affected areas and the presence of competent vectors underscore the importance of entomological surveillance. As part of the CLIMOS project, we investigated sand fly species composition, seasonal abundance, and the presence of Leishmania parasites at two sites in southwestern Slovenia: Cetore and Velike Žablje.

From May to October 2023, adult sand flies were collected using Centers for Disease Control and Prevention (CDC) miniature light traps. Specimens were morphologically identified and screened for Leishmania spp. DNA using quantitative polymerase chain reaction (PCR) targeted kinetoplast DNA, followed by species confirmation with ITS-1 nested PCR and restriction fragment length polymorphism (RFLP) analysis.

A total of 274 sand flies were collected, predominantly Phlebotomus neglectus, P. perniciosus, and P. mascittii. Abundance peaked in July in Cetore and August in Velike Žablje, likely reflecting local climatic differences. Among 25 pools of female sand flies analyzed, Leishmania DNA was detected in three pools (12.0%), specifically in P. neglectus and P. mascittii. The cycle threshold values (Ct 35-37) indicated low parasite DNA loads. ITS-1 amplification was unsuccessful, likely due to low DNA concentration.

This study provides the first molecular evidence of Leishmania DNA in sand flies from Slovenia. The detection of Leishmania in P.  mascittii, a species with uncertain vector competence, is particularly noteworthy and warrants further investigation. Although species identification of the parasite was not possible, the findings suggest potential for autochthonous transmission and highlight the need for continued surveillance and research in the region.