Functional Analysis of Leishmania Cyclopropane Fatty Acid Synthetase

The single gene encoding cyclopropane fatty acid synthetase (CFAS) is present in Leishmania infantum, L. mexicana and L. braziliensis but absent from L. major, a causative agent of cutaneous leishmaniasis. In L. infantum, usually causative agent of visceral leishmaniasis, the CFAS gene is transcribed in both insect (extracellular) and host (intracellular) stages of the parasite life cycle. Tagged CFAS protein is stably detected in intracellular L. infantum but only during the early log phase of extracellular growth, when it shows partial localisation to the endoplasmic reticulum. Lipid analyses of L. infantum wild type, CFAS null and complemented parasites detect a low abundance CFAS-dependent C19Δ fatty acid, characteristic of a cyclopropanated species, in wild type and add-back cells. Sub-cellular fractionation studies locate the C19Δ fatty acid to both ER and plasma membrane-enriched fractions. This fatty acid is not detectable in wild type L. major, although expression of the L. infantum CFAS gene in L. major generates cyclopropanated fatty acids, indicating that the substrate for this modification is present in L. major, despite the absence of the modifying enzyme. Loss of the L. infantum CFAS gene does not affect extracellular parasite growth, phagocytosis or early survival in macrophages. However, while endocytosis is also unaffected in the extracellular CFAS nulls, membrane transporter activity is defective and the null parasites are more resistant to oxidative stress. Following infection in vivo, L. infantum CFAS nulls exhibit lower parasite burdens in both the liver and spleen of susceptible hosts but it has not been possible to complement this phenotype, suggesting that loss of C19Δ fatty acid may lead to irreversible changes in cell physiology that cannot be rescued by re-expression. Aberrant cyclopropanation in L. major decreases parasite virulence but does not influence parasite tissue tropism.     

The role of reduced pterins in resistance to reactive oxygen and nitrogen intermediates in the protozoan parasite Leishmania

During its life cycle, the protozoan parasite Leishmania experiences oxidative stress when interacting with macrophages. Reduced pterins are known scavengers of reactive oxygen and nitrogen intermediates. Leishmania has a pteridine reductase, PTR1, whose main function is to provide reduced pterins. We investigated the role of PTR1 in resistance to oxidative and nitrosative stress in Leishmania tarentolae, Leishmania infantum, and Leishmania major PTR1^?/? mutants. The PTR1^?/? cells of the three species were more sensitive to H₂O₂- and NO-induced stress. Using a fluorescent probe allowing ROI quantification, we demonstrated an increase in intracellular oxidant molecules in the PTR1^?/? mutants. The disruption of PTR1 increased metacyclogenesis in L. infantum and L. major. We purified metacyclic parasites from PTR1^?/? mutants and control cells and tested their intracellular survival in the J774 mouse cell line and in human monocyte-derived macrophages. Our results showed that PTR1^?/? null mutants survived less in both macrophage models compared to control cells and this decrease was more pronounced in macrophages activated for oxidant production. This study demonstrates that one physiological role of reduced pterins in Leishmania is to deal with oxidative and nitrosative species, and a decreased ability to provide reduced pterins leads to decreased intracellular survival.     

Vaccination with the Leishmania infantum ribosomal proteins induces protection in BALB/c mice against Leishmania chagasi and Leishmania amazonensis challenge

Leishmania chagasi and Leishmania amazonensis are the etiologic agents of different clinical forms of human leishmaniasis in South America. In an attempt to select candidate antigens for a vaccine protecting against different Leishmania species, the efficacy of vaccination using Leishmania ribosomal proteins and saponin as adjuvant was examined in BALB/c mice against challenge infection with both parasite species. Mice vaccinated with parasite ribosomal proteins purified from Leishmania infantum plus saponin showed a specific production of IFN-γ, IL-12 and GM-CSF after in vitro stimulation with L. infantum ribosomal proteins. Vaccinated mice showed a reduction in the liver and spleen parasite burdens after L. chagasi infection. After L. amazonensis challenge, vaccinated mice showed a decrease of the dermal pathology and a reduction in the parasite loads in the footpad and spleen. In both models, protection was correlated to an IL-12-dependent production of IFN-γ by CD4⁺ and CD8⁺ T cells that activate macrophages for the synthesis of NO. In the protected mice a decrease in the parasite-mediated IL-4 and IL-10 responses was also observed. In mice challenged with L. amazonensis, lower levels of anti-parasite-specific antibodies were detected. Thus, Leishmania ribosomal proteins plus saponin fits the requirements to compose a pan-Leishmania vaccine.     

UDP-galactopyranose mutases from Leishmania species that cause visceral and cutaneous leishmaniasis

Leishmaniasis is a vector-borne, neglected tropical disease caused by parasites from the genus Leishmania. Galactofuranose (Galf) is found on the cell surface of Leishmania parasites and is important for virulence. The flavoenzyme that catalyzes the isomerization of UDP-galactopyranose to UDP-Galf, UDP-galactopyranose mutase (UGM), is a validated drug target in protozoan parasites. UGMs from L. mexicana and L. infantum were recombinantly expressed, purified, and characterized. The isolated enzymes contained tightly bound flavin cofactor and were active only in the reduced form. NADPH is the preferred redox partner for both enzymes. A k_cat value of 6 ± 0.4 s⁻¹ and a K_m value of 252 ± 42 μM were determined for L. infantum UGM. For L. mexicana UGM, these values were ∼4-times lower. Binding of UDP-Galp is enhanced 10–20 fold in the reduced form of the enzymes. Changes in the spectra of the reduced flavin upon interaction with the substrate are consistent with formation of a flavin-iminium ion intermediate.     

Screening Leishmania donovani‐specific genes required for visceral infection

Comparison of the Leishmania infantum genome with Leishmania braziliensis and Leishmania major genomes has identified 25 L. infantum species‐specific genes that are absent or pseudogenes in L. major and L. braziliensis. To determine whether these L. infantum species‐specific genes are involved in visceral Leishmania infection, we cloned the orthologues of 14 L. infantum species‐specific genes from the genetically closely related Leishmania donovani and introduced them into L. major. Two of these L. donovani species‐specific genes were found to significantly increase L. major survival in visceral organs in BALB/c mice. One (orthologue of LinJ28_V3.0340; Ld2834) of these two genes was further investigated. The L. donovani Ld2834 null mutants displayed dramatically reduced virulence in BALB/c mice and were unable to survive in axenic amastigote culture conditions arguing that Ld2834 plays a crucial role in enabling L. donovani survive at the increased temperature typically associated with visceral organs. Ld2834 encodes a 50 kDa protein that is localized in the cytoplasma and has no significant sequence similarity with other known genes. This study validates the importance of comparative genomics for understanding Leishmania species pathology and argues that Leishmania species‐specific genes play important roles in tissue tropism and virulence.     

In vitro and in vivo efficacy of ether lipid edelfosine against Leishmania spp. and SbV-resistant parasites

Background

The leishmaniases are a complex of neglected tropical diseases caused by more than 20 Leishmania parasite species, for which available therapeutic arsenal is scarce and unsatisfactory. Pentavalent antimonials (SbV) are currently the first-line pharmacologic therapy for leishmaniasis worldwide, but resistance to these compounds is increasingly reported. Alkyl-lysophospoholipid analogs (ALPs) constitute a family of compounds with antileishmanial activity, and one of its members, miltefosine, has been approved as the first oral treatment for visceral and cutaneous leishmaniasis. However, its clinical use can be challenged by less impressive efficiency in patients infected with some Leishmania species, including L. braziliensis and L. mexicana, and by proneness to develop drug resistance in vitro.

Methodology/Principal Findings

We found that ALPs ranked edelfosine>perifosine>miltefosine>erucylphosphocholine for their antileishmanial activity and capacity to promote apoptosis-like parasitic cell death in promastigote and amastigote forms of distinct Leishmania spp., as assessed by proliferation and flow cytometry assays. Effective antileishmanial ALP concentrations were dependent on both the parasite species and their development stage. Edelfosine accumulated in and killed intracellular Leishmania parasites within macrophages. In vivo antileishmanial activity was demonstrated following oral treatment with edelfosine of mice and hamsters infected with L. major, L. panamensis or L. braziliensis, without any significant side-effect. Edelfosine also killed SbV-resistant Leishmania parasites in in vitro and in vivo assays, and required longer incubation times than miltefosine to generate drug resistance.

Conclusions/Significance

Our data reveal that edelfosine is the most potent ALP in killing different Leishmania spp., and it is less prone to lead to drug resistance development than miltefosine. Edelfosine is effective in killing Leishmania in culture and within macrophages, as well as in animal models infected with different Leishmania spp. and SbV-resistant parasites. Our results indicate that edelfosine is a promising orally administered antileishmanial drug for clinical evaluation.     

The Genome Sequence of Leishmania (Leishmania) amazonensis: Functional Annotation and Extended Analysis of Gene Models

We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3′-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. Additionally, we describe a hypothetical hybrid interactome of potentially secreted L. (L.) amazonensis proteins and host proteins under the assumption that parasite factors mimic their mammalian counterparts. The model predicts an interaction between an L. (L.) amazonensis heat-shock protein and mammalian Toll-like receptor 9, which is implicated in important immune responses such as cytokine and nitric oxide production. The analysis presented here represents valuable information for future studies of leishmaniasis pathogenicity and treatment.     

Leishmania infantum chagasi: A genome-based approach to identification of excreted/secreted proteins

The parasitic protozoan, Leishmania, survives in harsh environments within its mammalian and sand fly hosts. Secreted proteins likely play critical roles in the parasite’s interactions with its environment. As a preliminary identification of the spectrum of potential excreted/secreted (ES) proteins of Leishmania infantum chagasi (Lic), a causative agent of visceral leishmaniasis, we used standard algorithms to screen the annotated L. infantum genome for genes whose predicted protein products have an N-terminal signal peptide and lack transmembrane domains and membrane anchors. A suite of 181 candidate ES proteins were identified. These included several that were documented in the literature to be released by other Leishmania spp. Six candidate ES proteins were selected for further validation of their expression and release by different parasite stages. We found both amastigote-specific and promastigote-specific released proteins. The ES proteins of Lic are candidates for future studies of parasite virulence determinants and host protective immunity.     

Stage-Specific Adhesion of Leishmania Promastigotes to Sand Fly Midguts Assessed Using an Improved Comparative Binding Assay

Background

The binding of Leishmania promastigotes to the midgut epithelium is regarded as an essential part of the life-cycle in the sand fly vector, enabling the parasites to persist beyond the initial blood meal phase and establish the infection. However, the precise nature of the promastigote stage(s) that mediate binding is not fully understood.

Methodology/Principal Findings

To address this issue we have developed an in vitro gut binding assay in which two promastigote populations are labelled with different fluorescent dyes and compete for binding to dissected sand fly midguts. Binding of procyclic, nectomonad, leptomonad and metacyclic promastigotes of Leishmania infantum and L. mexicana to the midguts of blood-fed, female Lutzomyia longipalpis was investigated. The results show that procyclic and metacyclic promastigotes do not bind to the midgut epithelium in significant numbers, whereas nectomonad and leptomonad promastigotes both bind strongly and in similar numbers. The assay was then used to compare the binding of a range of different parasite species (L. infantum, L. mexicana, L. braziliensis, L. major, L. tropica) to guts dissected from various sand flies (Lu. longipalpis, Phlebotomus papatasi, P. sergenti). The results of these comparisons were in many cases in line with expectations, the natural parasite binding most effectively to its natural vector, and no examples were found where a parasite was unable to bind to its natural vector. However, there were interesting exceptions: L. major and L. tropica being able to bind to Lu. longipalpis better than L. infantum; L. braziliensis was able to bind to P. papatasi as well as L. major; and significant binding of L. major to P. sergenti and L. tropica to P. papatasi was observed.

Conclusions/Significance

The results demonstrate that Leishmania gut binding is strictly stage-dependent, is a property of those forms found in the middle phase of development (nectomonad and leptomonad forms), but is absent in the early blood meal and final stages (procyclic and metacyclic forms). Further they show that although gut binding may be necessary for parasite establishment, in several vector-parasite pairs the specificity of such in vitro binding alone is insufficient to explain overall vector specificity. Other significant barriers to development must exist in certain refractory Leishmania parasite-sand fly vector combinations. A re-appraisal of the specificity of the Leishmania-sand fly relationship is required.     

Canine antibody response to Phlebotomus perniciosus bites negatively correlates with the risk of Leishmania infantum transmission

Background

Phlebotomine sand flies are blood-sucking insects that can transmit Leishmania parasites. Hosts bitten by sand flies develop an immune response against sand fly salivary antigens. Specific anti-saliva IgG indicate the exposure to the vector and may also help to estimate the risk of Leishmania spp. transmission. In this study, we examined the canine antibody response against the saliva of Phlebotomus perniciosus, the main vector of Leishmania infantum in the Mediterranean Basin, and characterized salivary antigens of this sand fly species.

Methodology/Principal Findings

Sera of dogs bitten by P. perniciosus under experimental conditions and dogs naturally exposed to sand flies in a L. infantum focus were tested by ELISA for the presence of anti-P. perniciosus antibodies. Antibody levels positively correlated with the number of blood-fed P. perniciosus females. In naturally exposed dogs the increase of specific IgG, IgG1 and IgG2 was observed during sand fly season. Importantly, Leishmania-positive dogs revealed significantly lower anti-P. perniciosus IgG2 compared to Leishmania-negative ones. Major P. perniciosus antigens were identified by western blot and mass spectrometry as yellow proteins, apyrases and antigen 5-related proteins.

Conclusions

Results suggest that monitoring canine antibody response to sand fly saliva in endemic foci could estimate the risk of L. infantum transmission. It may also help to control canine leishmaniasis by evaluating the effectiveness of anti-vector campaigns. Data from the field study where dogs from the Italian focus of L. infantum were naturally exposed to P. perniciosus bites indicates that the levels of anti-P. perniciosus saliva IgG2 negatively correlate with the risk of Leishmania transmission. Thus, specific IgG2 response is suggested as a risk marker of L. infantum transmission for dogs.     

Constitutive mosaic aneuploidy is a unique genetic feature widespread in the Leishmania genus

Using fluorescence in situ hybridization, we determined the ploidy of four species of Leishmania: Leishmania infantum, Leishmania donovani, Leishmania tropica and Leishmania amazonensis. We found that each cell in a strain possesses a combination of mono-, di- and trisomies for all chromosomes; ploidy patterns were different among all strains/species. These results extend those we previously described in Leishmania major, demonstrating that mosaic aneuploidy is a genetic feature widespread to the Leishmania genus. In addition to the genetic consequences induced by this mosaicism, the apparent absence of alternation between haploid/diploid stages questions the modality of genetic exchange in Leishmania sp.     

Leishmania species: fatty acid composition of promastigotes

The fatty acid composition of the total lipid fractions of five different Leishmania organisms grown on Eagle's medium was determined by gas chromatography. The major fatty acids identified in the total lipid fractions of L. donovani, L. tropica major, L. tropica minor, L. tropica (England strain), and L. enriettii were C_12:0, C_13:0, C_14:0, C_15:0, C_16:0, C_17:0, C_18:0, C_18:1, C_18:2, and C_18:3. The statistical differences among the fatty acid methyl esters of different Leishmania organisms are discussed.Gas chromatographic analysis of the fatty acid methyl esters of the total lipid fractions of the original Eagle's medium and the media after harvesting of various Leishmania species revealed the presence of C_18:3 fatty acid in the total lipid fraction of the medium of L. donovani and the complete absence of 18-carbon unsaturated fatty acids in the total lipid fraction of the medium of L. enriettii. The use of such differences in the differentiation of various Leishmania species is discussed.     

Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area,the Algarve Region in Portugal

The Algarve Region (AR) in southern Portugal, which is an international tourist destination, has been considered an endemic region of zoonotic leishmaniasis caused by Leishmania infantum since the 1980s. In the present study, phlebotomine and canine surveys were conducted to identify sandfly blood meal sources and to update the occurrence of Leishmania infection in vectors and dogs. Four sandfly species were captured: Phlebotomus perniciosus, Phlebotomus ariasi, Phlebotomus sergenti and Sergentomyia minuta. In one P. perniciosus female, L. infantum DNA was detected. Blood meal tests showed that this species had no host preferences and was an opportunistic feeder. An overall canine leishmaniasis (CanL) seroprevalence of 16.06% was found; the seroprevalence was 3.88% in dogs housed in kennels and 40.63% in dogs that attended veterinary clinics. The simultaneous occurrence of dogs and P. perniciosus infected with L. infantum in the AR indicates that the region continues to be an endemic area for CanL. Our results reinforce the need for the systematic spatial distribution of phlebotomine populations and their Leishmania infection rates and the need to simultaneously perform pathogen monitoring in both invertebrate and vertebrate hosts to investigate the transmission, distribution and spreading of Leishmania infection.     

Leishmania donovani complex (Kinetoplastida, Trypanosomatidae): Comparison of deoxyribonucleic acid based techniques for typing of isolates from Ethiopia

In Ethiopia, visceral leishmaniasis (VL) is an increasing public health concern. Recently, a new outbreak of VL claimed the lives of hundreds of Ethiopians. Mapping its distribution and the identification of the causative Leishmania species is important for proper use of resources and for control planning. The choice of appropriate typing technique is the key for determining the infecting species. Here we compared three deoxyribonucleic acid (DNA) based markers. We used, for the first time, cpbE and cpbF (cpbE/F) PCR-RFLP and demonstrated that it clearly differentiates Leishmania donovani from Leishmania infantum. The cpbE/F PCR-RFLP gave identical banding pattern for all L. donovani strains irrespective of their geographic origin. With the K26 (primers) PCR-RFLP, the L. donovani strains gave a banding pattern different from L. infantum and showed variation with geographic origin. The Ethiopian isolates typed as L. donovani by the PCR-RFLP of the cpbE/F (gene) and K26 (primers) showed two types of patterns with the T2/B4 (primers) PCR-RFLP; one group with L. infantum-like and the other L. donovani-like pattern. Phylogenetic analysis using cpbE/F sequences showed variation with geographic origin of strains and the African strains of L. donovani are more distantly related to L. infantum. Moreover, the Ethiopian isolates were seen to be closely related to the Sudanese, Kenyan and Indian strains. Thus, we recommend the use of more than one marker to study the population genetics of L. donovani complex.     

Leishmania donovani lacking the Golgi GDP-Man transporter LPG2 exhibit attenuated virulence in mammalian hosts

Surface phosophoglycans such as lipophosphoglycan (LPG) or proteophosphoglycan (PPG) and glycosylinositol phospholipids (GIPLs) modulate essential interactions between Leishmania and mammalian macrophages. Phosphoglycan synthesis depends on the Golgi GDP-mannose transporter encoded by LPG2. LPG2-null (lpg2⁻) Leishmania major cannot establish macrophage infections or induce acute pathology, whereas lpg2⁻Leishmania mexicana retain virulence. lpg2⁻Leishmaniadonovani has been reported to survive poorly in cultured macrophages but in vivo survival has not been explored. Herein we discovered that, similar to lpg2⁻L. major, lpg2⁻L. donovani promastigotes exhibited diminished virulence in mice, but persisted at consistently low levels. lpg2⁻L. donovani promastigotes could not establish infection in macrophages and could not transiently inhibit phagolysosomal fusion. Furthermore, lpg2⁻ promastigotes of L. major, L. donovani and L. mexicana were highly susceptible to complement-mediated lysis. We conclude that phosphoglycan assembly and expression mediated by L. donovani LPG2 are important for promastigote and amastigote virulence, unlike L. mexicana but similar to L. major.     

In vitro leishmanicidal activity and theoretical insights into biological action of ruthenium(II) organometallic complexes containing anti-inflammatories

Leishmaniasis, a neglected tropical disease caused by protozoans of the genus Leishmania, kills around 20–30 thousand people in Africa, Asia, and Latin America annually and, despite its potential lethality, it can be treated and eventually cured. However, the current treatments are limited owing to severe side effects and resistance development by some Leishmania. These factors make it urgent to develop new leishmanicidal drugs. In the present study, three ruthenium(II) organometallic complexes containing as ligands the commercially available anti-inflammatories diclofenac (dic), ibuprofen (ibu), and naproxen (nap) were synthesized, characterized, and subjected to in vitro leishmanicidal activity. The in vitro cytotoxicity assays against Leishmania (L.) amazonensis and Leishmania (L.) infantum promastigotes have shown that complexes [RuCl(dic)(η⁶-p-cymene)] (1) and [RuCl(nap)(η⁶-p-cymene)] (3) were active against both Leishmania species. Complex [RuCl(ibu)(η⁶-p-cymene)] (2) has exhibited no activity. The IC₅₀ values for the two active complexes were respectively 7.42 and 23.55 μM, for L. (L.) amazonensis, and 8.57 and 42.25 μM, for L. (L.) infantum. Based on the toxicological results and computational analysis, we proposed a correlation between the complexes and their activity. Our results suggest both complexation to ruthenium(II) and ligands structure are key elements to leishmanicidal activity.     

Thrichomys laurentius (Rodentia; Echimyidae) as a Putative Reservoir of Leishmania infantum and L. braziliensis: Patterns of Experimental Infection

The importance of the genus Thrichomys in the retention of infection and transmission of Leishmania species is supported by previous studies that describe an ancient interaction between caviomorphs and trypanosomatids and report the natural infection of Thrichomys spp. Moreover, these rodents are widely dispersed in Brazil and recognized as important hosts of other tripanosomatids. Our main purpose was to evaluate the putative role of Thrichomys laurentius in the retention of infection and amplification of the transmission cycle of Leishmania infantum and L. braziliensis. Male and female T. laurentius (n = 24) born in captivity were evaluated for the retention of infection with these Leishmania species and followed up by parasitological, serological, hematological, biochemical, histological, and molecular assays for 3, 6, 9, or 12 months post infection (mpi). T. laurentius showed its competence as maintenance host for the two inoculated Leishmania species. Four aspects should be highlighted: (i) re-isolation of parasites 12 mpi; (ii) the low parasitic burden displayed by T. laurentius tissues; (iii) the early onset and maintenance of humoral response, and (iv) the similar pattern of infection by the two Leishmania species. Both Leishmania species demonstrated the ability to invade and maintain itself in viscera and skin of T. laurentius, and no rodent displayed any lesion, histological changes, or clinical evidence of infection. We also wish to point out the irrelevance of the adjective dermotropic or viscerotropic to qualify L. braziliensis and L. infantum, respectively, when these species are hosted by nonhuman hosts. Our data suggest that T. laurentius may act at least as a maintenance host of both tested Leishmania species since it maintained long-lasting infections. Moreover, it cannot be discarded that Leishmania spp. infection in free-ranging T. laurentius could result in higher parasite burden due the more stressing conditions in the wild. Therefore the tissular parasitism of the skin, infectiveness to the vector, and amplification of the transmission cycle of both Leishmania species could be expected.     

Molecular detection of Leishmania (Sauroleishmania) tarentolae in human blood and Leishmania (Leishmania) infantum in Sergentomyia minuta: unexpected host-parasite contacts

The detection of atypical Kinetoplastida in vertebrate hosts and vectors might suggest unexpected host-parasite contacts. Aside to major vectors of Leishmania (Leishmania) infantum in Italy (e.g. Phlebotomus perniciosus and Phlebotomus perfiliewi), the sand fly fauna also includes Sergentomyia minuta, herpetophilic and proven vector of Leishmania (Sauroleishmania) tarentolae, in which records of blood meal on mammals and detection of L. infantum DNA are increasing. This study was conducted in Central Italy aiming to molecularly detect potential atypical Leishmania host-vector contacts. Detection of Leishmania spp. DNA was performed by polymerase chain reaction (SSU rRNA, ITS1 targets) on field-collected sand fly females (N = 344), blood samples from humans (N = 185) and dogs (N = 125). Blood meal identification was also performed on engorged sand flies. Leishmania spp. DNA was found in 13.1% sand flies, 3.7% humans and 14.4% dogs. Sequence analysis identified L. infantum in S. minuta (4.4%), P. perniciosus (9.1%), humans (2.2%) and dogs (14.4%). Leishmania tarentolae was detected in S. minuta (12.6%), P. perfiliewi (6.6%) and human (1.6%) samples. Of 28 S. minuta examined for blood meal, 3.6 and 21.4% scored positive for human and lizard DNA, respectively. These results indicate the importance of one-health approach to explore new potential routes of transmission of leishmaniasis involving S. minuta.     

Leishmania (Sauroleishmania) tarentolae isolation and sympatric occurrence with Leishmania (Leishmania) infantum in geckoes,dogs and sand flies

The trypanosomatid protist Leishmania tarentolae is a saurian-associated parasite vectored by the Sergentomyia minuta sand fly. This study aimed to confirm the circulation of L. infantum and L. tarentolae in sand flies, reptiles and dogs and to isolate new strains of these protists. Reptilian and sheltered dog blood samples were collected, and sand flies were captured. Samples were tested for Leishmania spp. using duplex real-time PCR (dqPCR) and real-time PCR (qPCR); the origin of blood meal was identified in engorged sand flies by conventional PCR. The reptilian blood and intestinal content of sand fly females were cultured. Dog sera were tested by IFAT using both Leishmania species. Four Tarentola mauritanica geckoes were molecularly positive for L. infantum or L. tarentolae, with no co-infections; moreover, amastigote-like forms of L. infantum were observed in the bone marrow. 24/294 sand flies scored positive for Leishmania spp. by dqPCR, 21 S. minuta and two Phlebotomus perniciosus were positive for L. tarentolae, while only a single Ph. perniciosus was positive for L. infantum. Blood meal analysis confirmed reptile and dog in S. minuta, dog and human in Ph. perniciosus and dog in Phlebotomus neglectus. Two axenic strains of L. tarentolae were obtained. Twelve of 19 dogs scored positive for L. infantum and L. tarentolae by IFAT and three of them also for L. infantum by dqPCR, and six by qPCR. These data confirm the sympatric circulation of L. infantum and L. tarentolae in geckoes, sand flies, and dogs, and suggest that geckoes may be infected with L. infantum.