Zoonotic cutaneous leishmaniasis (ZCL) and chronic cutaneous leishmaniasis (CCL) are known to overlap in Central Tunisia. Sandflies were collected using sticky traps and CDC light-traps set in rodent burrows at the ecotones surrounding the village, in houses, and in animal shelters during July-October 2017, 2018, and 2019. A total of 17,175 sandflies were collected during the three sandfly seasons and identified morphologically to species level. Of a total of 18 sandfly species reported in Tunisia, 16 were identified in this mixed focus of ZCL and CCL. Except for the rocky mountainous areas, Phlebotomus papatasi was the most abundant sandfly species in all biotopes. In the mountainous areas, Phlebotomus sergenti is the most abundant sandfly species belonging to the genus Phlebotomus. Female sandflies were tested for the presence of Leishmania species by PCR. The overall infection prevalence of sandflies with Leishmania major and Leishmania tropica was 0.42% and 0.065%, respectively. The sequencing of PCR-amplified ITS1 products showed that L. major is the predominant species in all biotopes and transmitted mostly by P. papaptasi followed by Phlebotomus longicuspis and Sergentomyia species. Leishmania tropica was detected in Phlebotomus sergenti and in Phlebotomus longicuspis collected in bedrooms and in the ecotone of rocky mountainous areas. Our results provided strong evidence that the proximity of human settlements to biotopes of rodent reservoir hosts of L. major and of L. tropica resulted into the cocirculation of both Leishmania species leading to a mixed focus of ZCL and CCL. The epidemiology of leishmaniases in North Africa is highly complex by the high diversity of sandfly vectors and their associated Leishmania species, leading to a mixed form of cutaneous leishmaniasis. It is of major epidemiological importance to point to the risk of spillover from rural to urban areas leading to the anthroponization of cutaneous leishmaniasis. Therefore, efficient control to reduce the indoor abundance of sandfly vectors in order to reduce the incidence of leishmaniases is urgently needed.

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