A lipophosphoglycan-independent development of Leishmania in permissive sand flies

Leishmaniases are serious parasitic diseases the etiological organisms of which are transmitted by insect vectors, phlebotominae sand flies. Two sand fly species, Phlebotomus papatasi and P. sergenti, display remarkable specificity for Leishmania parasites they transmit in nature, but many others are broadly permissive to the development of different Leishmania species. Previous studies have suggested that in 'specific' vectors the successful parasite development is mediated by parasite surface glycoconjugates and sand fly lectins, however we show here that interactions involving 'permissive' sand flies utilize another molecules. We did find that the abundant surface glycoconjugate lipophosphoglycan, essential for attachment of Leishmania major in the specific vector P. papatasi, was not required for parasite adherence or survival in the permissive vectors P. arabicus and Lutzomyia longipalpis. Attachment in several permissive sand fly species instead correlated with the presence of midgut glycoproteins bearing terminal N-acetyl-galactosamine and with the occurrence of a lectin-like activity on Leishmania surface. This new binding modality has important implications for parasite transmission and evolution. It may contribute to the successful spreading of Leishmania due to their adaptation into new vectors, namely transmission of L. infantum by Lutzomyia longipalpis; this event led to the establishment of L. infantum/chagasi in Latin America.     

Investigation of the bacterial communities associated with females of Lutzomyia sand fly species from South america

Phlebotomine sand flies are vectors of Leishmania that are acquired by the female sand fly during blood feeding on an infected mammal. Leishmania parasites develop exclusively in the gut lumen during their residence in the insect before transmission to a suitable host during the next blood feed. Female phlebotomine sand flies are blood feeding insects but their life style of visiting plants as well as animals, and the propensity for larvae to feed on detritus including animal faeces means that the insect host and parasite are exposed to a range of microorganisms. Thus, the sand fly microbiota may interact with the developing Leishmania population in the gut. The aim of the study was to investigate and identify the bacterial diversity associated with wild adult female Lutzomyia sand flies from different geographical locations in the New World. The bacterial phylotypes recovered from 16S rRNA gene clone libraries obtained from wild caught adult female Lutzomyia sand flies were estimated from direct band sequencing after denaturing gradient gel electrophoresis of bacterial 16 rRNA gene fragments. These results confirm that the Lutzomyia sand flies contain a limited array of bacterial phylotypes across several divisions. Several potential plant-related bacterial sequences were detected including Erwinia sp. and putative Ralstonia sp. from two sand fly species sampled from 3 geographically separated regions in Brazil. Identification of putative human pathogens also demonstrated the potential for sand flies to act as vectors of bacterial pathogens of medical importance in addition to their role in Leishmania transmission.     

Leishmania major survival in selective Phlebotomus papatasi sand fly vector requires a specific SCG-encoded lipophosphoglycan galactosylation pattern

Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In "selective" sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG), which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG) repeat units. For the "selective" fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal) of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated Molecular Pattern) through studies of natural isolates exhibiting a wide range of galactosylation patterns, and of a panel of isogenic L. major engineered to express similar scGal-LPG diversity by transfection of SCG-encoded β1,3-galactosyltransferases with different activities. Surprisingly, both 'poly-scGal' and 'null-scGal' lines survived poorly relative to PpapJ-sympatric L. major FV1 and other 'mono-scGal' lines. However, survival of all lines was equivalent in P. duboscqi, which naturally transmit L. major strains bearing 'null-scGal'-LPG PAMPs. We then asked whether scGal-LPG-mediated interactions were sufficient for PpapJ midgut survival by engineering Leishmania donovani, which normally express unsubstituted LPG, to express a 'PpapJ-optimal' scGal-LPG PAMP. Unexpectedly, these "L. major FV1-cloaked" L. donovani-SCG lines remained unable to survive within PpapJ flies. These studies establish that midgut survival of L. major in PpapJ flies is exquisitely sensitive to the scGal-LPG PAMP, requiring a specific 'mono-scGal' pattern. However, failure of 'mono-scGal' L. donovani-SCG lines to survive in selective PpapJ flies suggests a requirement for an additional, as yet unidentified L. major-specific parasite factor(s). The interplay of the LPG PAMP and additional factor(s) with sand fly midgut receptors may determine whether a given sand fly host is "selective" or "permissive", with important consequences to both disease transmission and the natural co-evolution of sand flies and Leishmania.     

Leishmania major Glycosylation Mutants Require Phosphoglycans (lpg2 ?) but Not Lipophosphoglycan (lpg1 ?) for Survival in Permissive Sand Fly Vectors

Background

Sand fly species able to support the survival of the protozoan parasite Leishmania have been classified as permissive or specific, based upon their ability to support a wide or limited range of strains and/or species. Studies of a limited number of fly/parasite species combinations have implicated parasite surface molecules in this process and here we provide further evidence in support of this proposal. We investigated the role of lipophosphoglycan (LPG) and other phosphoglycans (PGs) in sand fly survival, using Leishmania major mutants deficient in LPG (lpg1 ⁻), and the phosphoglycan (PG)-deficient mutant lpg2 ⁻. The sand fly species used were the permissive species Phlebotomus perniciosus and P. argentipes, and the specific vector P. duboscqi, a species resistant to L. infantum development.

Principal Findings

The lpg2 ⁻ mutants did not survive well in any of the three sand fly species, suggesting that phosphoglycans and/or other LPG2-dependent molecules are required for parasite development. In vitro, all three L. major lines were equally resistant to proteolytic activity of bovine trypsin, suggesting that sand fly-specific hydrolytic proteases or other factors are the reason for the early lpg2 ⁻ parasite killing. The lpg1 ⁻ mutants developed late-stage infections in two permissive species, P. perniciosus and P. argentipes, where their infection rates and intensities of infections were comparable to the wild type (WT) parasites. In contrast, in P. duboscqi the lpg1 ⁻ mutants developed significantly worse than the WT parasites.

Conclusions

In combination with previous studies, the data establish clearly that LPG is not required for Leishmania survival in permissive species P. perniciosus and P. argentipes but plays an important role in the specific vector P. duboscqi. With regard to PGs other than LPG, the data prove the importance of LPG2-related molecules for survival of L. major in the three sand fly species tested.     

Leishmania braziliensis: a novel mechanism in the lipophosphoglycan regulation during metacyclogenesis

During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterised by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This lipid-anchored polysaccharide is polymorphic among species with variations in sugars that branch off the conserved Gal(beta1,4)Man(alpha1)-PO4 backbone of repeat units and the oligosaccharide cap. Lipophosphoglycan has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the subgenus Leishmania. This paper describes the LPG structure for the first time in a species from the subgenus Viannia, Leishmania (Viannia) braziliensis. The LPG from the procyclic form of L. braziliensis was found to lack side chain sugar substitutions. In contrast to other species from the subgenus Leishmania, metacyclic forms of L. braziliensis makes less LPG and add 1-2 (beta1-3) glucose residues that branch off the disaccharide-phosphate repeat units of LPG. Thus, this represents a novel mechanism in the regulation of LPG structure during metacyclogenesis.     

Ecological and Control Techniques for Sand Flies (Diptera: Psychodidae) Associated with Rodent Reservoirs of Leishmaniasis

Background

Leishmaniasis remains a global health problem because of the substantial holes that remain in our understanding of sand fly ecology and the failure of traditional vector control methods. The specific larval food source is unknown for all but a few sand fly species, and this is particularly true for the vectors of Leishmania parasites. We provide methods and materials that could be used to understand, and ultimately break, the transmission cycle of zoonotic cutaneous leishmaniasis.

Methods and Findings

We demonstrated in laboratory studies that analysis of the stable carbon and nitrogen isotopes found naturally in plant and animal tissues was highly effective for linking adult sand flies with their larval diet, without having to locate or capture the sand fly larvae themselves. In a field trial, we also demonstrated using this technique that half of captured adult sand flies had fed as larvae on rodent feces. Through the identification of rodent feces as a sand fly larval habitat, we now know that rodent baits containing insecticides that have been shown in previous studies to pass into the rodents'' feces and kill sand fly larvae also could play a future role in sand fly control. In a second study we showed that rubidium incorporated into rodent baits could be used to demonstrate the level of bloodfeeding by sand flies on baited rodents, and that the elimination of sand flies that feed on rodents can be achieved using baits containing an insecticide that circulates in the blood of baited rodents.

Conclusions

Combined, the techniques described could help to identify larval food sources of other important vectors of the protozoa that cause visceral or dermal leishmaniasis. Unveiling aspects of the life cycles of sand flies that could be targeted with insecticides would guide future sand fly control programs for prevention of leishmaniasis.     

Identification of genes encoding arabinosyltransferases (SCA) mediating developmental modifications of lipophosphoglycan required for sand fly transmission of leishmania major

At key steps in the infectious cycle pathogens must adhere to target cells, but at other times detachment is required for transmission. During sand fly infections by the protozoan parasite Leishmania major, binding of replicating promastigotes is mediated by galactosyl side chain (scGal) modifications of phosphoglycan repeats of the major surface adhesin, lipophosphoglycan (LPG). Release is mediated by arabinosyl (Ara) capping of LPG scbetaGal residues upon differentiation to the infective metacyclic stage. We used intraspecific polymorphisms of LPG structure to develop a genetic strategy leading to the identification of two genes (SCA1/2) mediating scAra capping. These LPG side chain beta1,2-arabinosyltransferases (scbetaAraTs) exhibit canonical glycosyltransferase motifs, and their overexpression leads to elevated microsomal scbetaAraT activity. Although the level of scAra caps is maximal in metacyclic parasites, scbetaAraT activity is maximal in log phase cells. Because quantitative immunolocalization studies suggest this is not mediated by sequestration of SCA scbetaAraTs away from the Golgi apparatus during log phase, regulation of activated Ara precursors may control LPG arabinosylation in vivo. The SCA genes define a new family of eukaryotic betaAraTs and represent novel developmentally regulated LPG-modifying activities identified in Leishmania.     

Leishmania chitinase facilitates colonization of sand fly vectors and enhances transmission to mice

Chitinases of trypanosomatid parasites have been proposed to fulfil various roles in their blood-feeding arthropod vectors but so far none have been directly tested using a molecular approach. We characterized the ability of Leishmania mexicana episomally transfected with LmexCht1 (the L. mexicana chitinase gene) to survive and grow within the permissive sand fly vector, Lutzomyia longipalpis. Compared with control plasmid transfectants, the overexpression of chitinase was found to increase the average number of parasites per sand fly and accelerate the escape of parasites from the peritrophic matrix-enclosed blood meal as revealed by earlier arrival at the stomodeal valve. Such flies also exhibited increased damage to the structure of the stomodeal valve, which may facilitate transmission by regurgitation. When exposed individually to BALB/c mice, those flies with chitinase-overexpressing parasites spent on average 2.4-2.5 times longer in contact with their host during feeding, compared with flies with control infections. Furthermore, the lesions that resulted from these single fly bite infections were both significantly larger and with higher final parasite burdens than controls. These data show that chitinase is a multifunctional virulence factor for L. mexicana which assists its survival in Lu. longipalpis. Specifically, this enzyme enables the parasites to colonize the anterior midgut of the sand fly more quickly, modify the sand fly stomodeal valve and affect its blood feeding, all of which combine to enhance transmission.     

Leishmania infantum: Lipophosphoglycan intraspecific variation and interaction with vertebrate and invertebrate hosts

Interspecies variations in lipophosphoglycan (LPG) have been the focus of intense study over the years due its role in specificity during sand fly-Leishmania interaction. This cell surface glycoconjugate is highly polymorphic among species with variations in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO(4) backbone of repeat units. However, the degree of intraspecies polymorphism in LPG of Leishmania infantum (syn. Leishmania chagasi) is not known. In this study, intraspecific variation in the repeat units of LPG was evaluated in 16 strains of L. infantum from Brazil, France, Algeria and Tunisia. The structural polymorphism in the L. infantum LPG repeat units was relatively slight and consisted of three types: type I does not have side chains; type II has one β-glucose residue that branches off the disaccharide-phosphate repeat units and type III has up to three glucose residues (oligo-glucosylated). The significance of these modifications was investigated during in vivo interaction of L. infantum with Lutzomyia longipalpis, and in vitro interaction of the parasites and respective LPGs with murine macrophages. There were no consequential differences in the parasite densities in sand fly midguts infected with Leishmania strains exhibiting type I, II and III LPGs. However, higher nitric oxide production was observed in macrophages exposed to glucosylated type II LPG.     

Experimental transmission of Leishmania tropica to hyraxes (Procavia capensis) by the bite of Phlebotomus arabicus

The ability of the sand fly Phlebotomus (Adlerius) arabicus to transmit Leishmania tropica was studied experimentally using hyraxes (Procavia capensis), natural reservoir hosts of the parasite. Sand flies became infected with L. tropica after feeding on a lesion of needle-inoculated hyrax. Moreover, P. arabicus fed with L. tropica promastigotes transmitted the parasite to hyraxes by bite during a second bloodmeal. Although the animals remained asymptomatic after infective sand fly bite, they were PCR positive and infectious for naive sand flies. We have thus demonstrated cyclical transmission of L. tropica by P. arabicus in hyraxes. This confirms experimentally the vectorial competence of P. (Adlerius) arabicus, and demonstrates that asymptomatic reservoir hosts are infectious to appropriate vectors.     

The effect of the sugar metabolism on Leishmania infantum promastigotes inside the gut of Lutzomyia longipalpis: A sweet relationship?

It is well-known that Leishmania parasites can alter the behavior of the sand fly vector in order to increase their transmission potential. However, little is known about the contribution of the infecting host’s blood composition on subsequent sand fly infection and survival. This study focused on the host’s glucose metabolism and the insulin/insulin-like growth factor 1 (IGF-1) pathway as both metabolic processes are known to impact vector-parasite interactions of other protozoa and insect species. The focus of this study was inspired by the observation that the glycemic levels in the blood of infected Syrian golden hamsters inversely correlated to splenic and hepatic parasite burdens. To evaluate the biological impact of these findings on further transmission, Lutzomyia longipalpis sand flies were infected with blood that was artificially supplemented with different physiological concentrations of several monosaccharides, insulin or IGF-1. Normoglycemic levels resulted in transiently higher parasite loads and faster appearance of metacyclics, whereas higher carbohydrate and insulin/IGF-1 levels favored sand fly survival. Although the recorded effects were modest or transient of nature, these observations support the concept that the host blood biochemistry may affect Leishmania transmission and sand fly longevity.     

A lipophosphoglycan-independent method for isolation of infective Leishmania metacyclic promastigotes by density gradient centrifugation.

At the end of their growth in the sand fly, Leishmania parasites differentiate into the infective metacyclic promastigote stage, which is transmitted to the mammalian host. Thus, in experimental studies of parasite infectivity toward animals or macrophages, the use of purified metacyclics is generally preferred. While metacyclics of several Leishmania species can be efficiently purified with the aid of lectins or monoclonal antibodies, which differentially exploit stage-specific differences in the structure of the abundant surface glycolipid lipophosphoglycan (LPG), such reagents are unavailable for most species and they are unsuitable for studies involving LPG-deficient mutants. Here we describe a simple density gradient centrifugation method, which allows the rapid purification of infective metacyclic parasites from both wild-type and LPG-deficient Leishmania major. The purified metacyclic promastigotes are authentic, as judged by criteria such as their morphology, expression of the metacyclic-specific gene SHERP, and ability to invade and replicate within macrophages in vitro. Preliminary studies suggest that this method is applicable to other Leishmania species including L. donovani.     

Effect of sand fly saliva on Leishmania uptake by murine macrophages

Leishmania promastigotes are introduced into the skin by blood-sucking phlebotomine sand flies. In the vertebrate host, promastigotes invade macrophages, transform into amastigotes and multiply intracellularly. Sand fly saliva was shown to enhance the development of cutaneous leishmaniasis lesions by inhibiting some immune functions of the host macrophages. This study demonstrates that sand fly saliva promotes parasite survival and proliferation. First, macrophages gravitated towards increasing concentrations of sand fly saliva in vitro. Secondly, saliva increased the percentage of macrophages that became infected with Leishmania promastigotes and exacerbated the parasite load in these cells. Thus, during natural transmission, saliva probably reduces the exposure of promastigotes to the immune system by attracting macrophages to the parasite inoculation site and by accelerating the entry of promastigotes into macrophages. Saliva may also enhance lesion development by shortening the generation time of dividing intracellular amastigotes.     

Mark-release-recapture of sand flies fed on leishmanial dogs: the natural life-cycle of Leishmania infantum in Phlebotomus ariasi

Wild-caught Phlebotomus ariasi Tonnoir permitted to feed on dogs infected with Leishmania infantum Nicolle were marked with fluorescent powder and released into their natural habitat in an uninhabited area of the Cévennes in southern France. Over a period of 29 days after release, 253 females were recaptured with CDC miniature light traps or by active search at night with portable UV lamps. The ovaries and infections in the alimentary tract were then examined. The females oviposited 6 nights after in infecting blood meal. Second blood meals were never taken during the maturation of eggs. During the first ovarian cycle, midgut infections with promastigotes were only moderately heavy. The intensity of infection increased markedly during the second ovarian cycle and, in the third ovarian cycle, the first pharynx infected with paramastigotes was seen (on day 19). From day 19 to day 29, 76% of the flies had pharyngeal infections. Three out of 19 sand flies with pharyngeal infections recaptured during this period had metacyclic promastigotes in their mouthparts. The long time required for parasites to reach the proboscis in completely natural conditions suggests that their presence in the mouthparts is not a prerequisite for transmission by bite. It is more likely that transmission is most commonly by the regurgitation of metacyclic promastigotes from the thoracic midgut following damage to the stomodaeal valve by chitinase produced by the parasite during its development in the gut of the fly. Nevertheless, it is reasonable to assume that the bite of a fly with metacyclic promastigotes in the proboscis (or salivary glands) would also be infective.     

Leishmania infantum xenodiagnosis from vertically infected dogs reveals significant skin tropism

BackgroundDogs are the primary reservoir for human visceral leishmaniasis due to Leishmania infantum. Phlebotomine sand flies maintain zoonotic transmission of parasites between dogs and humans. A subset of dogs is infected transplacentally during gestation, but at what stage of the clinical spectrum vertically infected dogs contribute to the infected sand fly pool is unknown.Methodology/Principal findingsWe examined infectiousness of dogs vertically infected with L. infantum from multiple clinical states to the vector Lutzomyia longipalpis using xenodiagnosis and found that vertically infected dogs were infectious to sand flies at differing rates. Dogs with mild to moderate disease showed significantly higher transmission to the vector than dogs with subclinical or severe disease. We documented a substantial parasite burden in the skin of vertically infected dogs by RT-qPCR, despite these dogs not having received intradermal parasites via sand flies. There was a highly significant correlation between skin parasite burden at the feeding site and sand fly parasite uptake. This suggests dogs with high skin parasite burden contribute the most to the infected sand fly pool. Although skin parasite load and parasitemia correlated with one another, the average parasite number detected in skin was significantly higher compared to blood in matched subjects. Thus, dermal resident parasites were infectious to sand flies from dogs without detectable parasitemia.Conclusions/SignificanceTogether, our data implicate skin parasite burden and earlier clinical status as stronger indicators of outward transmission potential than blood parasite burden. Our studies of a population of dogs without vector transmission highlights the need to consider canine vertical transmission in surveillance and prevention strategies.     

Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies

A thorough understanding of the transmission mechanism of any infectious agent is crucial to implementing an effective intervention strategy. Here, our current understanding of the mechanisms that Leishmania parasites use to ensure their transmission from sand fly vectors by bite is reviewed. The most important mechanism is the creation of a "blocked fly" resulting from the secretion of promastigote secretory gel (PSG) by the parasites in the anterior midgut. This forces the sand fly to regurgitate PSG before it can bloodfeed, thereby depositing both PSG and infective metacyclic promastigotes in the skin of a mammalian host. Other possible factors in transmission are considered: damage to the stomodeal valve; occurrence of parasites in the salivary glands; and excretion of parasites from the anus of infected sand flies. Differences in the transmission mechanisms employed by parasites in the three subgenera, Leishmania, Viannia and Sauroleishmania are also addressed.     

Phlebotomine vectors of the leishmaniases: a review

ABSTRACT. An account is given of work published during the past 10 years incriminating species of phlebotomine sandflies as vectors of Leishmania species which infect man.
An assessment is made of the degrees of certainty of the vectorial roles of eighty-one species and subspecies of sandflies (thirty-seven Old World and forty-four New World) in the transmission of twenty-nine leishmanial parasites of mammals. At least one species of sandfly is considered to be a proven vector of each of ten parasites.
Of the eighty-one sandfly taxa, evidence is judged to be sufficient to incriminate nineteen as proven vectors (eleven Phlebotomus species and eight Lutzomyia species or subspecies) and evidence for a further fourteen (nine Phlebotomus species and five Lutzomyia species or subspecies) is considered to be strong.
The suggested criteria for incrimination of a vector are anthropophily and common infection with the same leishmanial parasite as that found in man in the same place. More weight should be given to natural infections persisting after the digestion of a bloodmeal than those in the presence of blood. Supporting evidence is a concordance in the distribution of the fly and the disease in man, proof that the fly feeds regularly on the reservoir host, a flourishing development of the parasite in infected flies and the experimental transmission of the parasite by the bite of the fly.     

First Assessment for the Presence of Phlebotomine Vectors in Bavaria,Southern Germany,by Combined Distribution Modeling and Field Surveys

Leishmaniasis is caused by protozoa of the genus Leishmania and transmitted by sand flies from mammalian reservoirs to humans. In recent years, a northward spread of L. infantum from highly endemic Mediterranean countries into previously non-endemic Central European areas has been suspected based on presumed sporadic cases of autochthonous leishmaniasis. Here, we investigated whether sand flies are prevalent in Bavaria in Southern Germany, a federal state in which autochthonous cases have previously been reported. Considering the present and future climatic conditions, we determined whether Bavaria is suitable for five sand fly species with assumed spreading tendencies towards Central Europe: Phlebotomus ariasi, P. neglectus, P. perfiliewi and P. perniciosus that are known vectors for Leishmania in Europe, and P. mascittii, a suspected but not proven vector. Within Bavaria we defined sampling regions based on their climatic suitability and their spatial distance to the sites of the autochthonous cases and/or to areas of reported sand fly detection in states adjacent to Bavaria. At 155 locations in 7 sampling regions, CDC light traps were placed during 38 nights in the summers of 2009 and 2010, resulting in 202 trap-nights. All traps were negative for sand flies. The results suggest that Bavaria is not yet endemic for sand flies, but do not exclude the possibility of sporadic cases of autochthonous human or zoonotic Leishmania infections. This study, which combined methodological approaches from different disciplines, serves as reference for future surveys and risk analyses of sand flies and leishmaniasis in so far non-endemic areas of Europe.     

Incrimination of Phlebotomus kandelakii and Phlebotomus balcanicus as vectors of Leishmania infantum in Tbilisi, Georgia

A survey of potential vector sand flies was conducted in the neighboring suburban communities of Vake and Mtatsminda districts in an active focus of visceral Leishmaniasis (VL) in Tbilisi, Georgia. Using light and sticky-paper traps, 1,266 male and 1,179 female sand flies were collected during 2006-2008. Five Phlebotomus species of three subgenera were collected: Phlebotomus balcanicus Theodor and Phlebotomus halepensis Theodor of the subgenus Adlerius; Phlebotomus kandelakii Shchurenkova and Phlebotomus wenyoni Adler and Theodor of the subgenus Larroussius; Phlebotomus sergenti Perfil'ev of the subgenus Paraphlebotomus. Phlebotomus sergenti (35.1%) predominated in Vake, followed by P. kandelakii (33.5%), P. balcanicus (18.9%), P. halepensis (12.2%), and P. wenyoni (0.3%). In Mtatsminda, P. kandelakii (76.8%) comprised over three fourths of collected sand flies, followed by P. sergenti (12.6%), P. balcanicus (5.8%), P. halepensis (3.7%), and P. wenyoni (1.1%). The sand fly season in Georgia is exceptionally short beginning in early June, peaking in July and August, then declining to zero in early September. Of 659 female sand flies examined for Leishmania, 12 (1.8%) specimens without traces of blood were infected including 10 of 535 P. kandelakii (1.9%) and two of 40 P. balcanicus (5.0%). Six isolates were successfully cultured and characterized as Leishmania by PCR. Three isolates from P. kandelakii (2) and P. balcanicus (1) were further identified as L. infantum using sequence alignment of the 70 kDa heat-shock protein gene. Importantly, the sand fly isolates showed a high percent identity (99.8%-99.9%) to human and dog isolates from the same focus, incriminating the two sand fly species as vectors. Blood meal analysis showed that P. kandelakii preferentially feeds on dogs (76%) but also feeds on humans. The abundance, infection rate and feeding behavior of P. kandelakii and the infection rate in P. balcanicus establish these species as vectors in the Tbilisi VL focus.