Background: Leishmania killicki was originally described in 1980 in southeast Tunisia. It was also recently reported in Lybia and Algeria. Nevertheless, neither vector nor reservoirs of this parasite are known. The identification of the vector and the animal reservoir host of L. killicki is critical for the establishment of an efficient control strategy. Findings: blood, popliteal lymph node, spleen, bone marrow, liver and skin were collected from 50 rodents in 2009 in south western Tunisia. Samples were smeared onto glass slides, cultured on NNN medium and tested by polymerase chain reaction for Leishmania detection. Parasites were detected by PCR from 10 Psammomys obesus and from two Ctenodactylus gundi. Parasite identification was performed simultaneously by internal transcribed spacer 1 PCR-RFLP and by PCR sequencing. Both Leishmania major and Leishmania killicki were identified from infected Psammomys and Ctenodactylus gundi respectively. Conclusion: This is the first report of Leishmania killicki identified from Ctenodactylus gundi in Tunisia. This result supports the assumption that C. gundi is a potential reservoir for Leishmania killicki.

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. 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. 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. 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: This review focuses on leishmaniasis caused by Leishmania (L.) infantum in Tunisia, a vector-borne parasitic disease transmitted through the bite of infected female sandflies. Leishmaniasis manifests as a spectrum of clinical forms, ranging from benign cutaneous lesions, to a severe and potentially fatal visceral form. In Tunisia, L. infantum is the etiological agent of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). While CL typically manifests as a single, small facial lesion, atypical forms are sometimes observed. VL primarily affects children under the age of five and immunocompromised individuals, although an increasing number of cases have been reported in immunocompetent adults in recent years. Although neglected, leishmaniasis is an emerging and growing public health concern in Tunisia, particularly due to the increasing incidence of VL among adults and potential spread of both CL and VL to previously non-endemic areas. This expansion is demonstrated by the fact that L. infantum has a geographical distribution mainly in the humid, sub-humid and semi-arid regions of the north, but gradually spreading towards the central and southern parts of the country. Methodology/principal findings: This literature review was conducted through a systematic search of PubMed, Scopus, and Google Scholar for studies published between 1904 and 2024, focusing on the clinical, epidemiological, molecular, and ecological aspects of L. infantum in Tunisia. In addition to its clinical variability, L. infantum presents a biochemical variability with three isoenzymatic variants (zymodemes) identified in Tunisia: MON-1 (predominantly associated with VL), MON-24 (predominantly associated with CL), and MON-80 (implicated in both forms). Our review found that VL remains highly endemic in northern Tunisia but has expanded southward in recent decades, while cutaneous cases due to L. infantum are increasingly recognized. Isoenzymatic and molecular studies confirm the predominance of the MON-1 zymodeme, with sporadic detection of MON-24 and MON-80. Domestic dogs remain the main reservoir, and Phlebotomus (P.) perniciosus is the principal vector for VL, though other Phlebotomus species have been implicated in CL transmission. These findings highlight the importance of integrating molecular tools alongside classical isoenzyme methods for a better understanding of parasite dynamics and epidemiological monitoring. Conclusions/significance: The transmission cycle of L. infantum is not fully elucidated, but domestic dogs and P. perniciosus are considered the primary reservoir and vector for VL, respectively, while, other potential mammalian hosts and sandflies vectors were suspected for CL. Comparative data from Algeria, Morocco, Libya, and southern Europe suggest both common patterns and local specificities in L. infantum transmission, underscoring the importance of regional collaboration. The epidemiological and clinical complexity of L. infantum , together with its expanding geographic distribution in Tunisia, underscores the need for further integrated research to clarify transmission cycles and implement effective prevention and control strategies.