BACKGROUND: Several new species of Leishmania have recently emerged in Europe, probably as the result of global changes and increased human migration from endemic areas. In this study, we tested whether two sand fly species, the Western Mediterranean Phlebotomus perniciosus and the Eastern Mediterranean P. tobbi, are competent vectors of L. donovani, L. major and L. martiniquensis. METHODOLOGY/PRINCIPAL FINDINGS: Sand flies were infected through the chick skin membrane using Leishmania species and strains of various geographical origins. Leishmania infections were evaluated by light microscopy and qPCR, and the representation of morphological forms was assessed from Giemsa-stained gut smears. Neither P. perniciosus nor P. tobbi supported the development of L. martiniquensis, but L. major and L. donovani in both species survived defecation of blood meal remnants, colonized the stomodeal valve and produced metacyclic stages. The results with L donovani have shown that infection rates in sand flies can be strain-specific; therefore, to determine vector competence or refractoriness, it is optimal to test at least two strains of Leishmania. CONCLUSIONS, SIGNIFICANCE: Both sand fly species tested are potential vectors of L. donovani and L. major in Mediterranean area. However, further studies will be needed to identify European vectors of L. martiniquensis and to test the ability of other European sand fly species to transmit L. major, L. donovani, L. tropica and L. infantum.

• MeSH
• Insect Vectors * parasitology   physiology   MeSH
• Chickens parasitology   MeSH
• Leishmania * physiology   classification   genetics   MeSH
• Leishmaniasis transmission   parasitology   MeSH
• Humans MeSH
• Phlebotomus * parasitology   physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Europe MeSH

Leishmania species, members of the kinetoplastid parasites, cause leishmaniasis, a neglected tropical disease, in millions of people worldwide. Leishmania has a complex life cycle with multiple developmental forms, as it cycles between a sand fly vector and a mammalian host; understanding their life cycle is critical to understanding disease spread. One of the key life cycle stages is the haptomonad form, which attaches to insect tissues through its flagellum. This adhesion, conserved across kinetoplastid parasites, is implicated in having an important function within their life cycles and hence in disease transmission. Here, we discover the kinetoplastid-insect adhesion proteins (KIAPs), which localise in the attached Leishmania flagellum. Deletion of these KIAPs impairs cell adhesion in vitro and prevents Leishmania from colonising the stomodeal valve in the sand fly, without affecting cell growth. Additionally, loss of parasite adhesion in the sand fly results in reduced physiological changes to the fly, with no observable damage of the stomodeal valve and reduced midgut swelling. These results provide important insights into a comprehensive understanding of the Leishmania life cycle, which will be critical for developing transmission-blocking strategies.

• MeSH
• Cell Adhesion MeSH
• Flagella * metabolism   MeSH
• Insect Vectors parasitology   MeSH
• Insect Proteins metabolism   genetics   MeSH
• Host-Parasite Interactions MeSH
• Leishmania * physiology   genetics   metabolism   MeSH
• Leishmaniasis parasitology   transmission   MeSH
• Protozoan Proteins metabolism   genetics   MeSH
• Psychodidae * parasitology   MeSH
• Life Cycle Stages MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Insect Proteins MeSH
• Protozoan Proteins MeSH

Leishmania, the dixenous trypanosomatid parasites, are the causative agents of leishmaniasis currently divided into four subgenera: Leishmania, Viannia, Sauroleishmania, and the recently described Mundinia, consisting of six species distributed sporadically all over the world infecting humans and/or animals. These parasites infect various mammalian species and also cause serious human diseases, but their reservoirs are unknown. Thus, adequate laboratory models are needed to enable proper research of Mundinia parasites. In this complex study, we compared experimental infections of five Mundinia species (L. enriettii, L. macropodum, L. chancei, L. orientalis, and four strains of L. martiniquensis) in three rodent species: BALB/c mouse, Chinese hamster (Cricetulus griseus) and steppe lemming (Lagurus lagurus). Culture-derived parasites were inoculated intradermally into the ear pinnae and progress of infection was monitored for 20 weeks, when the tissues and organs of animals were screened for the presence and quantity of Leishmania. Xenodiagnoses with Phlebotomus duboscqi were performed at weeks 5, 10, 15 and 20 post-infection to test the infectiousness of the animals throughout the experiment. BALB/c mice showed no signs of infection and were not infectious to sand flies, while Chinese hamsters and steppe lemmings proved susceptible to all five species of Mundinia tested, showing a wide spectrum of disease signs ranging from asymptomatic to visceral. Mundinia induced significantly higher infection rates in steppe lemmings compared to Chinese hamsters, and consequently steppe lemmings were more infectious to sand flies: In all groups tested, they were infectious from the 5th to the 20th week post infection. In conclusion, we identified two rodent species, Chinese hamster (Cricetulus griseus) and steppe lemming (Lagurus lagurus), as candidates for laboratory models for Mundinia allowing detailed studies of these enigmatic parasites. Furthermore, the long-term survival of all Mundinia species in steppe lemmings and their infectiousness to vectors support the hypothesis that some rodents have the potential to serve as reservoir hosts for Mundinia.

• MeSH
• Arvicolinae * parasitology   MeSH
• Cricetulus MeSH
• Cricetinae MeSH
• Leishmania * classification   MeSH
• Leishmaniasis * parasitology   MeSH
• Disease Models, Animal * MeSH
• Mice, Inbred BALB C * MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Leishmaniasis is widely regarded as a vaccine-preventable disease, but the costs required to reach pivotal Phase 3 studies and uncertainty about which candidate vaccines should be progressed into human studies significantly limits progress in vaccine development for this neglected tropical disease. Controlled human infection models (CHIMs) provide a pathway for accelerating vaccine development and to more fully understand disease pathogenesis and correlates of protection. Here, we describe the isolation, characterization and GMP manufacture of a new clinical strain of Leishmania major. Two fresh strains of L. major from Israel were initially compared by genome sequencing, in vivo infectivity and drug sensitivity in mice, and development and transmission competence in sand flies, allowing one to be selected for GMP production. This study addresses a major roadblock in the development of vaccines for leishmaniasis, providing a key resource for CHIM studies of sand fly transmitted cutaneous leishmaniasis.

• MeSH
• Phylogeny MeSH
• Insect Vectors parasitology   MeSH
• Leishmania major genetics   growth & development   physiology   MeSH
• Leishmaniasis, Cutaneous parasitology   transmission   MeSH
• Humans MeSH
• Disease Models, Animal MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Parasites genetics   MeSH
• Psychodidae parasitology   MeSH
• Whole Genome Sequencing MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Israel MeSH

BACKGROUND Leishmania major is an Old World species causing cutaneous leishmaniasis and is transmitted by Phlebotomus papatasi and Phlebotomus duboscqi. In Brazil, two isolates from patients who never left the country were characterised as L. major-like (BH49 and BH121). Using molecular techniques, these isolates were indistinguishable from the L. major reference strain (FV1). OBJECTIVES We evaluated the lipophosphoglycans (LPGs) of the strains and their behaviour in Old and New World sand fly vectors. METHODS LPGs were purified, and repeat units were qualitatively evaluated by immunoblotting. Experimental in vivo infection with L. major-like strains was performed in Lutzomyia longipalpis (New World, permissive vector) and Ph. papatasi (Old World, restrictive or specific vector). FINDINGS The LPGs of both strains were devoid of arabinosylated side chains, whereas the LPG of strain BH49 was more galactosylated than that of strain BH121. All strains with different levels of galactosylation in their LPGs were able to infect both vectors, exhibiting colonisation of the stomodeal valve and metacyclogenesis. The BH121 strain (less galactosylated) exhibited lower infection intensity compared to BH49 and FV1 in both vectors. MAIN CONCLUSIONS Intraspecific variation in the LPG of L. major-like strains occur, and the different galactosylation levels affected interactions with the invertebrate host.

• MeSH
• Species Specificity MeSH
• Galactose metabolism   MeSH
• Glycosphingolipids chemistry   metabolism   MeSH
• Insect Vectors chemistry   physiology   MeSH
• Host-Pathogen Interactions MeSH
• Leishmania major chemistry   physiology   MeSH
• Phlebotomus parasitology   MeSH
• Psychodidae parasitology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Galactose MeSH
• Glycosphingolipids MeSH
• lipophosphonoglycan MeSH Browser

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: Leishmania infantum is the most widespread etiological agent of visceral leishmaniasis (VL) in the world, with significant mortality rates in human cases. In Latin America, this parasite is primarily transmitted by Lutzomyia longipalpis, but the role of Lutzomyia migonei as a potential vector for this protozoan has been discussed. Laboratory and field investigations have contributed to this hypothesis; however, proof of the vector competence of L. migonei has not yet been provided. In this study, we evaluate for the first time the susceptibility of L. migonei to L. infantum. METHODS: Females of laboratory-reared L. migonei were fed through a chick-skin membrane on rabbit blood containing L. infantum promastigotes, dissected at 1, 5 and 8 days post-infection (PI) and checked microscopically for the presence, intensity and localisation of Leishmania infections. In addition, morphometric analysis of L. infantum promastigotes was performed. RESULTS: High infection rates of both L. infantum strains tested were observed in L. migonei, with colonisation of the stomodeal valve already on day 5 PI. At the late-stage infection, most L. migonei females had their cardia and stomodeal valve colonised by high numbers of parasites, and no significant differences were found compared to the development in L. longipalpis. Metacyclic forms were found in all parasite-vector combinations since day 5 PI. CONCLUSIONS: We propose that Lutzomyia migonei belongs to sand fly species permissive to various Leishmania spp. Here we demonstrate that L. migonei is highly susceptible to the development of L. infantum. This, together with its known anthropophily, abundance in VL foci and natural infection by L. infantum, constitute important evidence that L. migonei is another vector of this parasite in Latin America.

• Keywords
• Leishmania infantum, Lutzomyia migonei, Vector competence,
• MeSH
• Insect Vectors * MeSH
• Leishmania infantum cytology   isolation & purification   MeSH
• Microscopy MeSH
• Psychodidae growth & development   parasitology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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

We studied the development of antimony-resistant Leishmania infantum in natural vectors Lutzomyia longipalpis and Phlebotomus perniciosus to ascertain the risk of parasite transmission by sand flies. All three resistant strains produced fully mature late-stage infections in sand flies; moreover, the resistant phenotype was maintained after the passage through the vector. These results highlight the risk of circulation of resistant Leishmania strains and question the use of human drugs for treatment of dogs as Leishmania reservoirs.

• MeSH
• Antimony pharmacology   MeSH
• Insect Vectors MeSH
• Leishmania infantum pathogenicity   MeSH
• Leishmania pathogenicity   MeSH
• Phlebotomus parasitology   MeSH
• Psychodidae parasitology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antimony 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