Detection and isolation of Leishmania infantum and Leishmania tarentolae in sand flies from a canine leishmaniasis endemic area

Sand flies are blood feeding insects known as vectors of Leishmania spp. worldwide, with Leishmania infantum being the most relevant species in Southern Europe. The sympatric occurrence of L. infantum, which causes canine leishmaniasis (CanL), along with the reptile-associated Leishmania tarentolae, triggered the interest to investigate their transmission dynamics in sand fly vectors. Sand flies were collected over two consecutive years (2023-2024) in a CanL endemic area using CDC light traps and were morphologically identified. Female specimens were either screened for Leishmania spp. DNA by quantitative PCR (qPCR) or dissected alive for microscopic detection of flagellates. A total of 3477 sand flies (2076 males and 1401 females) were collected, and the most prevalent species were Sergentomyia minuta (n = 2379) and Phlebotomus perniciosus (n = 1020), followed by Phlebotomus neglectus (n = 44), Phlebotomus perfiliewi (n = 12), andPhlebotomus papatasi (n = 2). Promastigotes were observed microscopically in 13 S. minuta and one P. perniciosus, with L. infantum isolated from the latter. The qPCR analysis revealed that 50.0 % (n = 55/110) and 1.8 % (n = 2/110) of the S. minuta pools scored positive for L. tarentolae and L. infantum, respectively, whereas 25.0 % (n = 3/12) of P. perniciosus pools were positive for L. infantum. Among the individually tested engorged female sand flies, L. tarentolae and L. infantum were amplified from 35.8 % (n = 24/67) and 3.0 % (n = 2/67) of S. minuta, respectively, while 15.5 % (n = 9/58) of the Phlebotomus spp. (i.e., eight P. perniciosus and one Phlebotomus sp.) were positive for L. infantum. These findings confirm the co-circulation of L. infantum and L. tarentolae in sand flies, with a notably high detection rate of trypanosomatids in S. minuta through molecular and microscopic methods.