Molecular confirmation of Leishmania infantum infection in the sand fly vector Phlebotomus neglectus ( Diptera: Phlebotominae ) in Slovenia

Leishmania infantum is the primary causative agent of zoonotic leishmaniasis in Europe and is transmitted by phlebotomine sand flies. Although Slovenia is not considered endemic, competent vectors and canine infections have been documented, suggesting conditions that may allow local transmission. During the 2023 sand fly season, 145 phlebotomine sand flies were collected at a farm site in Cetore, Slovenian Istria, and identified morphologically as Phlebotomus neglectus, P. perniciosus and P. mascittii. Female sand flies were grouped into eight monospecific pools and screened for Leishmania DNA by kinetoplast DNA real-time PCR. One pool of P. neglectus females collected in July tested positive, with a low cycle threshold value indicating a high parasite DNA load. Species-level identification using ITS-1 and hsp70 PCR-RFLP, followed by sequencing, confirmed the parasite as L. infantum. This study provides the first molecular confirmation of L. infantum in phlebotomine sand flies from Slovenia. The finding of L. infantum DNA in a proven vector species represents an early warning signal and highlights the need to implement entomological and molecular surveillance programmes in Slovenia.