In Europe, sand flies (Diptera: Psychodidae: Phlebotominae) are characteristic Mediterranean fauna, though some species expand their range further north. However, the sand fly fauna of Central Europe remains underreported, particularly in Hungary where recent data is lacking due to limited and outdated entomological surveys. To address this gap, a series of sand fly surveys were conducted in Hungary, with significant findings from two trapping efforts in 2017 and 2024. In 2017, only a single female Phlebotomus papatasi was trapped in northern Hungary, which marks one of the northernmost records of the species. In 2024, a more extensive and geographically wider survey recorded 264 sand flies at 34 sites, including three species: Ph. mascittii, Ph. neglectus, and Ph. papatasi. Sand flies were found across diverse environmental settings, including urban, agricultural, and natural habitats. Particularly, the previously rare presence of Ph. mascittii at rural sites (natural rock formations) was reported. Analysis of historical and current data revealed the presence of four sand fly species in Central and South Transdanubia, with evidence suggesting potential range expansion. Blood meal analysis of engorged females identified a variety of domestic and wild host species, but no Leishmania or Phlebovirus infections were detected. Habitat modelling and linear discriminant analysis indicated substantial climate suitability across Southeast Europe, with most positive sand fly observations observed in discontinuous urban fabric CORINE Land Cover classes. This study offers important insights into the ecology, distribution, and climatic preferences of sand flies in Hungary and provides crucial baseline data to monitor potential future spread.

• Keywords
• Barcoding, Blood meal, Central Europe, Ecology, MALDI-MS, Sand flies,
• Publication type
• Journal Article MeSH

INTRODUCTION: Visceral leishmaniasis (VL), a neglected tropical disease that causes substantial morbidity and mortality, is a serious health problem in Ethiopia. Infections are caused by Leishmania (L.) donovani parasites. Most individuals remain asymptomatic, but some develop VL, which is generally fatal if not treated. We identified the area of Metema-Humera in Northwest Ethiopia as a setting in which we could follow migrant workers when they arrived in an endemic area. The demographic characteristics of this population and factors associated with their risk of asymptomatic infection are poorly characterised. METHODS: We divided our cohort into individuals who visited this area for the first time (first comers, FC) and those who had already been in this area (repeat comers, RC). We followed them from the beginning (Time 1, T1) to the end of the agricultural season (Time 2, T2), performing tests for sand fly bite exposure (anti-sand fly saliva antibody ELISA) and serology for Leishmania infection (rK39 rapid diagnostic test and the direct agglutination test) at each time point and collecting information on risk factors for infection. RESULTS: Our results show that most migrant workers come from non-endemic areas, are male, young (median age of 20 years) and are farmers or students. At T1, >80% of them had been already exposed to sand fly bites, as shown by the presence of anti-saliva antibodies. However, due to seasonality of sand flies there was no difference in exposure between FC and RC, or between T1 and T2. The serology data showed that at T1, but not at T2, a significantly higher proportion of RC were asymptomatic. Furthermore, 28.6% of FC became asymptomatic between T1 and T2. Over the duration of this study, one FC and one RC developed VL. In multivariable logistic regression of asymptomatic infection at T1, only age and the number of visits to Metema/Humera were significantly associated with asymptomatic infection. CONCLUSION: A better understanding of the dynamics of parasite transmission and the risk factors associated with the development of asymptomatic infections and potentially VL will be essential for the development of new strategies to prevent leishmaniasis.

• Keywords
• Leishmania, PCR, asymptomatic, direct agglutination test, rK39, risk factors, visceral leishmaniasis,
• Publication type
• Journal Article MeSH

Phlebotomine sand flies (Diptera: Psychodidae) are the principal vectors of phleboviruses and Leishmania spp., the causative agents of leishmaniases. The Mediterranean sand fly fauna is diverse, and leishmaniasis, mainly caused by Leishmania infantum, is endemic in the Balkan countries. Despite recent entomological surveys, only some districts of Kosovo have been sampled for sand flies, with no proof/confirmation of L. infantum. This study aimed to gain further insights into the species composition of natural sand fly populations in previously unsampled districts and areas in Kosovo without reports of leishmaniasis and to detect Leishmania DNA in sand flies. A sand fly survey was conducted in 2022 in all seven districts of Kosovo. Collected females were screened for Leishmania DNA by PCR. Positive samples were sequenced and subjected to maximum likelihood analysis with reference sequences for further molecular characterization. The trapping activities at 114 different localities resulted in 3272 caught specimens, comprising seven sand fly species of two genera, namely Phlebotomus neglectus, Ph. perfiliewi, Ph. tobbi, Ph. papatasi, Ph. simici, Ph. balcanicus and Sergentomyia minuta. Leishmania infantum DNA was detected in three individual sand flies of Ph. neglectus and Ph. perfiliewi. This study provides the most extensive sand fly survey in Kosovo and reports the first record of L. infantum DNA in sand flies, indicating autochthonous circulation of L. infantum.

• Keywords
• Balkan, Kosovo, Larroussius, PCR, Phlebotominae, leishmaniasis,
• Publication type
• Journal Article MeSH

Leishmania parasites alternate in their life cycle between promastigote stages that develop in the gut of phlebotomine sand flies and amastigotes residing inside phagocytic cells of vertebrate hosts. For experimental infections of sand flies, promastigotes are frequently used as this way of infection is technically easier although ingestion of promastigotes by sand flies is unnatural. Here we aimed to answer a critical question, to what extent do promastigote-initiated experimental infections differ from those initiated with intracellular amastigotes. We performed side-by-side comparison of Leishmania development in Phlebotomus argentipes females infected alternatively with promastigotes from log-phase cultures or amastigotes grown ex vivo in macrophages. Early stage infections showed substantial differences in parasite load and representation of morphological forms. The differences disappeared along the maturation of infections; both groups developed heavy late-stage infections with colonization of the stomodeal valve, uniform representation of infective metacyclics and equal efficiency of transmission. The results showed that studies focusing on early phase of Leishmania development in sand flies should be initiated with intracellular amastigotes. However, the use of promastigote stages for sand fly infections does not alter significantly the final outcome of Leishmania donovani development in P. argentipes and their transmissibility to the vertebrate host.

• Keywords
• Leishmania donovani, Phlebotomus, amastigotes, promastigotes, transmission,
• MeSH
• Insect Vectors parasitology   MeSH
• Host-Parasite Interactions MeSH
• Leishmania donovani growth & development   MeSH
• Leishmaniasis, Visceral parasitology   transmission   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Phlebotomus parasitology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Differentiation of extracellular Leishmania promastigotes within their sand fly vector, termed metacyclogenesis, is considered to be essential for parasites to regain mammalian host infectivity. Metacyclogenesis is accompanied by changes in the local parasite environment, including secretion of complex glycoconjugates within the promastigote secretory gel and colonization and degradation of the sand fly stomodeal valve. Deletion of the stage-regulated HASP and SHERP genes on chromosome 23 of Leishmania major is known to stall metacyclogenesis in the sand fly but not in in vitro culture. Here, parasite mutants deficient in specific genes within the HASP/SHERP chromosomal region have been used to investigate their role in metacyclogenesis, parasite transmission and establishment of infection. Metacyclogenesis was stalled in HASP/SHERP mutants in vivo and, although still capable of osmotaxis, these mutants failed to secrete promastigote secretory gel, correlating with a lack of parasite accumulation in the thoracic midgut and failure to colonise the stomodeal valve. These defects prevented parasite transmission to a new mammalian host. Sand fly midgut homogenates modulated parasite behaviour in vitro, suggesting a role for molecular interactions between parasite and vector in Leishmania development within the sand fly. For the first time, stage-regulated expression of the small HASPA proteins in Leishmania (Leishmania) has been demonstrated: HASPA2 is expressed only in extracellular promastigotes and HASPA1 only in intracellular amastigotes. Despite its lack of expression in amastigotes, replacement of HASPA2 into the null locus background delays onset of pathology in BALB/c mice. This HASPA2-dependent effect is reversed by HASPA1 gene addition, suggesting that the HASPAs may have a role in host immunomodulation.

• MeSH
• Antigens, Protozoan metabolism   MeSH
• Cell Differentiation physiology   MeSH
• Fluorescent Antibody Technique MeSH
• Insect Vectors parasitology   MeSH
• Immunoblotting MeSH
• Host-Parasite Interactions physiology   MeSH
• Leishmania major growth & development   pathogenicity   MeSH
• Leishmaniasis genetics   transmission   MeSH
• Disease Models, Animal MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Polymerase Chain Reaction MeSH
• Protozoan Proteins metabolism   MeSH
• Psychodidae parasitology   MeSH
• Virulence physiology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Antigens, Protozoan MeSH
• HASPA protein, Leishmania MeSH Browser
• Protozoan Proteins MeSH

BACKGROUND: Infection caused by parasites from L. donovani complex can manifest as a serious visceral disease or a self-healing milder cutaneous form. The different tropism and pathology in humans is caused by the interaction between parasites, host and vector determinants but the mechanisms are not well understood. In Cukurova region in Turkey we previously identified a major focus of cutaneous leishmaniasis caused by L. donovani/infantum hybrids (CUK strain) and isolated this parasite from the locally abundant sand fly, Phlebotomus tobbi. Here, we present the first experimental study with P. tobbi. We tested the susceptibility of this species to various Leishmania under laboratory conditions, characterized glycoproteins in the P. tobbi midgut putatively involved in parasite-vector interaction and compared the development of the CUK strain in the sand fly with one other dermotropic and three viscerotropic strains belonging to the L. donovani complex. METHODS: Females of laboratory reared P. tobbi, P. perniciosus and Lutzomyia longipalpis were infected using membrane feeding on rabbit blood containing promastigotes of various Leishmania species with different tropisms. The individual guts were checked microscopically for presence and localization of Leishmania parasites; the number of parasites was assessed more precisely by qPCR. In addition, glycosylation of midgut proteins of P. tobbi was studied by lectin blotting of midgut lysate with lectins specific for terminal sugars of N-type and O-type glycans. RESULTS: High infection rates, heavy parasite loads and late-stage infection with colonization of the stomodeal valve were observed in P. tobbi infected by Leishmania major or L. infantum CUK hybrid. In parallel, lectin blotting revealed the presence of O-glycosylated proteins in the P. tobbi midgut. In P. perniciosus and L. longipalpis all five Leishmania strains tested developed well. In both vectors, significantly higher parasite numbers were detected by qPCR for dermotropic L. donovani from Cyprus, however, in all other parameters studied, including localization of infection and colonization of stomodeal valve, dermotropic and viscerotropic strains were not significantly different. CONCLUSIONS: We showed high susceptibility of P. tobbi to various Leishmania spp. This, together with the presence of O-glycosylated midgut proteins in their midguts demonstrate that P. tobbi is a permissive vector. Two dermotropic and three viscerotropic strains from the L. donovani complex developed late-stage infections in natural L. infantum vectors, P. perniciosus and L. longipalpis and none of the parameters studied seem to be linked with different tropism of parasites in the vertebrate host.

• MeSH
• Gastrointestinal Tract parasitology   MeSH
• Insect Vectors parasitology   MeSH
• Rabbits MeSH
• Leishmania infantum genetics   growth & development   isolation & purification   MeSH
• Leishmania major genetics   growth & development   isolation & purification   MeSH
• Leishmaniasis, Cutaneous epidemiology   parasitology   MeSH
• Humans MeSH
• Phlebotomus parasitology   MeSH
• Psychodidae parasitology   MeSH
• Tropism MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Humans MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Geographicals
• Turkey MeSH

BACKGROUND: Despite their importance in animal and human health, the epidemiology of species of the Leishmania enriettii complex remains poorly understood, including the identity of their biological vectors. Biting midges of the genus Forcipomyia (Lasiohelea) have been implicated in the transmission of a member of the L. enriettii complex in Australia, but the far larger and more widespread genus Culicoides has not been investigated for the potential to include vectors to date. METHODOLOGY/PRINCIPAL FINDINGS: Females from colonies of the midges Culicoides nubeculosus Meigen and C. sonorensis Wirth & Jones and the sand fly Lutzomyia longipalpis Lutz & Nevia (Diptera: Psychodidae) were experimentally infected with two different species of Leishmania, originating from Australia (Leishmania sp. AM-2004) and Brazil (Leishmania enriettii). In addition, the infectivity of L. enriettii infections generated in guinea pigs and golden hamsters for Lu. longipalpis and C. sonorensis was tested by xenodiagnosis. Development of L. enriettii in Lu. longipalpis was relatively poor compared to other Leishmania species in this permissive vector. Culicoides nubeculosus was not susceptible to infection by parasites from the L. enriettii complex. In contrast, C. sonorensis developed late stage infections with colonization of the thoracic midgut and the stomodeal valve. In hamsters, experimental infection with L. enriettii led only to mild symptoms, while in guinea pigs L. enriettii grew aggressively, producing large, ulcerated, tumour-like lesions. A high proportion of C. sonorensis (up to 80%) feeding on the ears and nose of these guinea pigs became infected. CONCLUSIONS/SIGNIFICANCE: We demonstrate that L. enriettii can develop late stage infections in the biting midge Culicoides sonorensis. This midge was found to be susceptible to L. enriettii to a similar degree as Lutzomyia longipalpis, the vector of Leishmania infantum in South America. Our results support the hypothesis that some biting midges could be natural vectors of the L. enriettii complex because of their vector competence, although not Culicoides sonorensis itself, which is not sympatric, and midges should be assessed in the field while searching for vectors of related Leishmania species including L. martiniquensis and "L. siamensis".

• MeSH
• Ceratopogonidae parasitology   MeSH
• Gastrointestinal Tract parasitology   MeSH
• Insect Vectors * MeSH
• Mesocricetus MeSH
• Leishmania enriettii isolation & purification   MeSH
• Leishmaniasis parasitology   pathology   transmission   MeSH
• Guinea Pigs MeSH
• Psychodidae parasitology   MeSH
• Animals MeSH
• Check Tag
• Guinea Pigs MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The early stage of Leishmania development in sand flies is closely connected with bloodmeal digestion. Here we compared various parameters of bloodmeal digestion in sand flies that are either susceptible (Phlebotomus argentipes and P. orientalis) or refractory (P. papatasi and Sergentomyia schwetzi) to Leishmania donovani, to study the effects on vector competence. The volume of the bloodmeal ingested, time of defecation of bloodmeal remnants, timing of formation and degradation of the peritrophic matrix (PM) and dynamics of proteolytic activities were compared in four sand fly species. Both proven vectors of L. donovani showed lower trypsin activity and slower PM formation than refractory species. Interestingly, the two natural L. donovani vectors strikingly differed from each other in secretion of the PM and midgut proteases, with P. argentipes possessing fast bloodmeal digestion with a very high peak of chymotrypsin activity and rapid degradation of the PM. Experimental infections of P. argentipes did not reveal any differences in vector competence in comparison with previously studied P. orientalis; even the very low initial dose (2×103 promastigotes/ml) led to fully developed late-stage infections with colonization of the stomodeal valve in about 40% of females. We hypothesise that the period between the breakdown of the PM and defecation of the bloodmeal remnants, i.e. the time frame when Leishmania attach to the midgut in order to prevent defecation, could be one of crucial parameters responsible for the establishment of Leishmania in the sand fly midgut. In both natural L. donovani vectors this period was significantly longer than in S. schwetzi. Both vectors are equally susceptible to L. donovani; as average bloodmeal volumes taken by females of P. argentipes and P. orientalis were 0.63 μl and 0.59 μl, respectively, an infective dose corresponding to 1-2 parasites was enough to initiate mature infections.

• MeSH
• Species Specificity MeSH
• Feces parasitology   MeSH
• Insect Vectors immunology   metabolism   parasitology   physiology   MeSH
• Blood metabolism   MeSH
• Leishmania donovani growth & development   physiology   MeSH
• Leishmaniasis, Visceral immunology   MeSH
• Membranes parasitology   MeSH
• Disease Susceptibility MeSH
• Eating * MeSH
• Proteolysis MeSH
• Psychodidae immunology   metabolism   parasitology   physiology   MeSH
• Digestion * MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH

BACKGROUND: The development of pathogens transmitted by haematophagous invertebrate vectors is closely connected with the digestion of bloodmeals and is thus affected by midgut enzymatic activity. Some studies have demonstrated that avian blood inhibits Leishmania major infection in the Old World vector Phlebotomus papatasi; however, this effect has never been observed in the New World vectors of the genus Lutzomyia infected by other Leishmania species. Therefore, our study was focused on the effect of chicken blood on bloodmeal digestion and the development of Leishmania major in its natural vector Phlebotomus duboscqi, i.e. in a vector-parasite combination where the effect of blood is assumed. In addition, we tested the effect of avian blood on midgut trypsin activity and the influence of repeated feedings on the susceptibility of sand flies to Leishmania infection. METHODS: Phlebotomus duboscqi females were infected by rabbit blood containing L. major and either before or after the infection fed on chickens or mice. The individual guts were checked microscopically for presence and localization of Leishmania, parasite numbers were detected by Q-PCR. In addition, midgut trypsin activity was studied. RESULTS: Sand fly females fed on chicken blood had significantly lower midgut trypsin activity and delayed egg development compared to those fed on rabbits. On the other hand, there was no effect detected of avian blood on parasite development within the sand fly gut: similar infection rates and parasite loads were observed in P. duboscqi females infected by L. major and fed on chickens or mouse one or six days later. Similarly, previous blood feeding of sand flies on chickens or mice did not show any differences in subsequent Leishmania infections, and there was equal susceptibility of P. duboscqi to L. major infection during the first and second bloodmeals. CONCLUSION: In spite of the fact that avian blood affects trypsin activity and the oocyte development of sand flies, no effect of chicken blood was observed on the development of L. major in P. duboscqi. Our study unambiguously shows that sand fly feeding on avian hosts is not harmful to Leishmania parasites within the sand fly midgut.

• MeSH
• Gastrointestinal Tract parasitology   MeSH
• Rabbits MeSH
• Blood metabolism   MeSH
• Animal Feed MeSH
• Chickens MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Leishmania major genetics   growth & development   MeSH
• Microscopy MeSH
• Mice MeSH
• Phlebotomus parasitology   MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Phlebotomus orientalis Parrot (Diptera: Psychodidae) is the main vector of visceral leishmaniasis (VL) caused by Leishmania donovani in East Africa. Here we report on life cycle parameters and susceptibility to L. donovani of two P. orientalis colonies originating from different sites in Ethiopia: a non-endemic site in the lowlands - Melka Werer (MW), and an endemic focus of human VL in the highlands - Addis Zemen (AZ). METHODOLOGY/PRINCIPAL FINDINGS: Marked differences in life-cycle parameters between the two colonies included distinct requirements for larval food and humidity during pupation. However, analyses using Random Amplified Polymorphic DNA (RAPD) PCR and DNA sequencing of cytB and COI mitochondrial genes did not reveal any genetic differences. F1 hybrids developed successfully with higher fecundity than the parental colonies. Susceptibility of P. orientalis to L. donovani was studied by experimental infections. Even the lowest infective dose tested (2×10(3) per ml) was sufficient for successful establishment of L. donovani infections in about 50% of the P. orientalis females. Using higher infective doses, the infection rates were around 90% for both colonies. Leishmania development in P. orientalis was fast, the presence of metacyclic promastigotes in the thoracic midgut and the colonization of the stomodeal valve by haptomonads were recorded in most P. orientalis females by day five post-blood feeding. CONCLUSIONS: Both MW and AZ colonies of P. orientalis were highly susceptible to Ethiopian L. donovani strains. As the average volume of blood-meals taken by P. orientalis females are about 0.7 µl, the infective dose at the lowest concentration was one or two L. donovani promastigotes per sand fly blood-meal. The development of L. donovani was similar in both P. orientalis colonies; hence, the absence of visceral leishmaniasis in non-endemic area Melka Werer cannot be attributed to different susceptibility of local P. orientalis populations to L. donovani.

• MeSH
• Leishmania donovani pathogenicity   MeSH
• Phlebotomus genetics   parasitology   physiology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Ethiopia MeSH