Molecular detection and identification of Leishmania DNA and blood meal analysis in Phlebotomus (Larroussius) species

Author summary Leishmania (L.) infantum is responsible for both visceral leishmaniasis and sporadic cutaneous leishmaniasis in Tunisia. The isoenzymatic typing of this taxon revealed three zymodemes and only one (L. infantum MON-1) presents the transmission cycle elucidated. In this study, we conducted an entomological survey using CDC light traps in central Tunisia wherein the three zymodemes of L. infantum coexist, to investigate the presence of L. infantum, to quantify the parasite load, and to analyze the blood meal sources in infected sand flies belonging to Larroussius (Lar.) subgenus. Our results demonstrate the role of Ph. Lar. species in L. infantum transmission and their potential role in L. major and L. killicki life cycles. The high parasite load observed in Ph. perfiliewi underline its incrimination in L. infantum transmission. Also, blood meal analysis showed that Lar. subgenus sand flies fed on cattle, goat, sheep, chicken, and human. Thus, in the light of the present results, further studies should be performed for a better understanding of L. infantum transmission cycles. Background Phlebotomus (Larroussius) perniciosus and Canis familiaris are respectively the only confirmed vector and reservoir for the transmission of Leishmania (L.) infantum MON-1 in Tunisia. However, the vector and reservoir hosts of the two other zymodemes, MON-24 and MON-80, are still unknown. The aim of this study was to analyze the L. infantum life cycle in a Tunisian leishmaniasis focus. For this purpose, we have focused on: i) the detection, quantification and identification of Leishmania among this sand fly population, and ii) the analysis of the blood meal preferences of Larroussius (Lar.) subgenus sand flies to identify the potential reservoirs. Methodology and findings A total of 3,831 sand flies were collected in seven locations from the center of Tunisia affected by human visceral leishmaniasis. The collected sand flies belonged to two genus Phlebotomus (Ph.) (five species) and Sergentomyia (four species). From the collected 1,029 Lar. subgenus female sand flies, 8.26% was positive to Leishmania by ITS1 nested PCR. Three Leishmania spp. were identified: L. infantum 28% (24/85), L. killicki 13% (11/85), and L. major 22% (19/85). To identify the blood meal sources in Ph. Lar. subgenus sand flies, engorged females were analyzed by PCR-sequencing targeting the vertebrate cytochrome b gene. Among the 177 analyzed blood-fed females, 169 samples were positive. Sequencing results showed seven blood sources: cattle, human, sheep, chicken, goat, donkey, and turkey. In addition, mixed blood meals were detected in twelve cases. Leishmania DNA was found in 21 engorged females, with a wide range of blood meal sources: cattle, chicken, goat, chicken/cattle, chicken/sheep, chicken/turkey and human/cattle. The parasite load was quantified in fed and unfed infected sand flies using a real time PCR targeting kinetoplast DNA. The average parasite load was 1,174 parasites/reaction and 90 parasites/reaction in unfed and fed flies, respectively. Conclusion Our results support the role of Ph. longicuspis, Ph. perfiliewi, and Ph. perniciosus in L. infantum transmission. Furthermore, these species could be involved in L. major and L. killicki life cycles. The combination of the parasite detection and the blood meal analysis in this study highlights the incrimination of the identified vertebrate in Leishmania transmission. In addition, we quantify for the first time the parasite load in naturally infected sand flies caught in Tunisia. These findings are relevant for a better understanding of L. infantum transmission cycle in the country. Further investigations and control measures are needed to manage L. infantum transmission and its spreading.