LAUSR

In the Mediterranean basin, sand flies are vectors of Leishmania parasites and phleboviruses affecting humans and animals. In this study, we aimed to investigate phlebovirus and Leishmania parasites circulating in a focus of zoonotic visceral leishmaniasis (ZVL) located in a highly irrigated area within the arid Central Tunisia, known mainly to be endemic for zoonotic cutaenous leishmaniasis (ZCL) caused Leishmania major and transmitted by Phlebotomus papatasi. Sand flies were collected using CDC light traps in the village of Saddaguia, an emergent focus of ZVL located in Central Tunisia during September-October 2014, 2015, and 2016. Pools of live female sand flies were screened for phleboviruses and Leishmania by nested PCR in the polymerase gene and kinetoplast minicircle DNA, respectively. Dead sand flies were identified morphologically to species level. Sand flies of the subgenus Larroussius mainly Phlebotomus perfiliewi, Phlebotomus perniciosus, and Phlebotomus longicuspis were predominant in this ZVL focus compared to P. papatasi. A total of 1932, 1740, and 444 sand flies were tested in 2014, 2015 and 2016, respectively. Pathogen screening performed on 4116 sand flies distributed in 148 pools revealed the presence of Leishmania infantum and Toscana virus. The minimum infection rates of sand flies with TOSV in 2014, 2015, and 2016 were 0.05%, 011%, and 0.22%, respectively. The minimum infection rates of sand flies with L. infantum in 2014, 2015, and 2016 were 0.25%, 012%, and 0.79%, respectively. No L. major was detected during the 3-years investigation in this ZVL focus. Our results showed clearly the endemic co-circulation of TOSV and L. infantum in this emergent ZVL focus. However, no co-infection of TOSV and L. infantum was detected in any of the sand fly pools investigated during the three years period. TOSV was isolated from positive pools in 2015. Phylogenetic analysis showed that the Tunisian strains of TOSV belonged to the sublineage A. Based on the present findings, our results provided strong evidence that TOSV and L. infantum are transmitted by the same predominant sand fly species of the subgenus Larroussius, and subsequently, humans and dogs could be co-infected through co-infected or successive infected bites. Our results showed clearly that the development of irrigation in arid areas contributed significantly to the establishment of stable transmission cycles of L. infantum and TOSV and subsequently to the emergence of a ZVL focus within this arid bio-geographical area characterized by the presence of multiple foci of ZCL located outside the study site. Thus, more studies are needed to better understand the impact of RNA viruses shared by vectors and reservoir hosts of L. infantum on the development of zoonotic visceral leishmaniasis.