Host infectiousness to insect vectors is a crucial parameter for understanding the transmission dynamics of insect-borne infectious diseases such as leishmaniases. Despite their importance, critical factors influencing the outwards transmission of Leishmania major, including parasite distribution within the host body and the minimum number of skin amastigotes required for vector infection, remain poorly characterized. To address these gaps, we studied these parameters in the natural North African reservoir host Meriones shawi and in BALB/c mice infected with a low parasite dose. Using qPCR, we quantified Leishmania loads in different zones (regions) of infected ear pinnae, whereas microscale infectiousness was evaluated via microbiopsies and fluorescence microscopy. The amastigote distribution within infected ears was heterogeneous, with pronounced differences between the lesion center, lesion margin, and visually unaffected surrounding skin. Phlebotomus papatasi females that fed in areas where no amastigotes were detected via microscopy did not become infected. In M. shawi, lesion margins have emerged as the most effective source of infection. The number of amastigotes at bite sites where sand fly females became infected ranged from 4--500, with as few as 2--10 amastigotes sufficient to initiate vector infection. This low infection threshold was confirmed by experiments in which P. papatasi was fed through a chick-skin membrane. In contrast, the BALB/c mouse model showed only minor differences in infectiousness between lesion centers and margins. The minimum infectious dose in BALB/c mice was approximately 100 times greater than that in M. shawi, with successful infections occurring at sites containing 1,500-10,000 amastigotes. These findings advance our understanding of Leishmania transmission by addressing critical knowledge gaps and enabling more accurate modelling of cutaneous leishmaniasis epidemiology. Moreover, this study highlights the importance of incorporating natural host models in research, as the dynamics of disease progression and transmission parameters can differ significantly between natural hosts and standard laboratory models.

• MeSH
• Gerbillinae * parasitology   MeSH
• Insect Vectors * parasitology   MeSH
• Skin parasitology   MeSH
• Leishmania major * physiology   pathogenicity   MeSH
• Leishmaniasis, Cutaneous * transmission   parasitology   MeSH
• Disease Models, Animal MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Parasite Load MeSH
• Phlebotomus * parasitology   MeSH
• Disease Reservoirs * parasitology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

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 major is responsible for zoonotic cutaneous leishmaniasis. Therapy is mainly based on the use of antimony-based drugs; however, treatment failures and illness relapses were reported. Although studies were developed to understand mechanisms of drug resistance, the interactions of resistant parasites with their reservoir hosts and vectors remain poorly understood. Here we compared the development of two L. major MON-25 trivalent antimony-resistant lines, selected by a stepwise in vitro Sb(III)-drug pressure, to their wild-type parent line in the natural vector Phlebotomus papatasi. The intensity of infection, parasite location and morphological forms were compared by microscopy. Parasite growth curves and IC50 values have been determined before and after the passage in Ph. papatasi. qPCR was used to assess the amplification rates of some antimony-resistance gene markers. In the digestive tract of sand flies, Sb(III)-resistant lines developed similar infection rates as the wild-type lines during the early-stage infections, but significant differences were observed during the late-stage of the infections. Thus, on day 7 p. i., resistant lines showed lower representation of heavy infections with colonization of the stomodeal valve and lower percentage of metacyclic promastigote forms in comparison to wild-type strains. Observed differences between both resistant lines suggest that the level of Sb(III)-resistance negatively correlates with the quality of the development in the vector. Nevertheless, both resistant lines developed mature infections with the presence of infective metacyclic forms in almost half of infected sandflies. The passage of parasites through the sand fly guts does not significantly influence their capacity to multiply in vitro. The IC50 values and molecular analysis of antimony-resistance genes showed that the resistant phenotype of Sb(III)-resistant parasites is maintained after passage through the sand fly. Sb(III)-resistant lines of L. major MON-25 were able to produce mature infections in Ph. papatasi suggesting a possible circulation in the field using this vector.

• Keywords
• Antimony resistance, Fitness, Leishmania major, Phlebotomus papatasi, experimental infection, Virulence,
• MeSH
• Gene Amplification MeSH
• Antimony * pharmacology   MeSH
• Genetic Fitness MeSH
• Leishmania major * drug effects   genetics   pathogenicity   MeSH
• Drug Resistance * genetics   MeSH
• Phlebotomus * 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

Sand fly transmitted Leishmania species are responsible for severe, wide ranging, visceral and cutaneous leishmaniases. Genetic exchange can occur among natural Leishmania populations and hybrids can now be produced experimentally, with limitations. Feeding Phlebotomus orientalis or Phlebotomus argentipes on two strains of Leishmania donovani yielded hybrid progeny, selected using double drug resistance and fluorescence markers. Fluorescence activated cell sorting of cultured clones derived from these hybrids indicated diploid progeny. Multilocus sequence typing of the clones showed hybridisation and nuclear heterozygosity, although with inheritance of single haplotypes in a kinetoplastid target. Comparative genomics showed diversity of clonal progeny between single chromosomes, and extraordinary heterozygosity across all 36 chromosomes. Diversity between progeny was seen for the HASPB antigen, which has been noted previously as having implications for design of a therapeutic vaccine. Genomic diversity seen among Leishmania strains and hybrid progeny is of great importance in understanding the epidemiology and control of leishmaniasis. As an outcome of this study we strongly recommend that wider biological archives of different Leishmania species from endemic regions should be established and made available for comparative genomics. However, in parallel, performance of genetic crosses and genomic comparisons should give fundamental insight into the specificity, diversity and limitations of candidate diagnostics, vaccines and drugs, for targeted control of leishmaniasis.

• MeSH
• Genomics MeSH
• Crosses, Genetic MeSH
• Leishmania donovani * genetics   MeSH
• Leishmaniasis, Cutaneous * MeSH
• Leishmaniasis, Visceral * diagnosis   prevention & control   epidemiology   MeSH
• Phlebotomus * genetics   MeSH
• Psychodidae * genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

Leishmaniases are neglected diseases caused by protozoans of the genus Leishmania that threaten millions of people worldwide. Cutaneous leishmaniasis (CL) caused by L. major is a typical zoonosis transmitted by phlebotomine sand flies and maintained in rodent reservoirs. The female sand fly was assumed to become infected by feeding on the skin lesion of the host, and the relative contribution of asymptomatic individuals to disease transmission was unknown. In this study, we infected 32 Meriones shawi, North African reservoirs, with a natural dose of L. major obtained from the gut of infected sand flies. Skin manifestations appeared in 90% of the animals, and xenodiagnosis with the proven vector Phlebotomus papatasi showed transmissibility in 67% of the rodents, and 45% were repeatedly infectious to sand flies. Notably, the analysis of 113 xenodiagnostic trials with 2189 sand flies showed no significant difference in the transmissibility of animals in the asymptomatic and symptomatic periods; asymptomatic animals were infectious several weeks before the appearance of skin lesions and several months after their healing. These results clearly confirm that skin lesions are not a prerequisite for vector infection in CL and that asymptomatic animals are an essential source of L. major infection. These data are important for modeling the epidemiology of CL caused by L. major.

• Keywords
• Leishmania, Meriones, Phlebotomus, asymptomatic infection, reservoir host, xenodiagnosis,
• Publication type
• Journal Article MeSH

Leishmania is the unicellular parasite transmitted by phlebotomine sand fly bite. It exists in two different forms; extracellular promastigotes, occurring in the gut of sand flies, and intracellular, round-shaped amastigotes residing mainly in vertebrate macrophages. As amastigotes originating from infected animals are often present in insufficient quality and quantity, two alternative types of amastigotes were introduced for laboratory experiments: axenic amastigotes and amastigotes from macrophages infected in vitro. Nevertheless, there is very little information about the degree of similarity/difference among these three types of amastigotes on proteomic level, whose comparison is crucial for assessing the suitability of using alternative types of amastigotes in experiments. In this study, L. mexicana amastigotes obtained from lesion of infected BALB/c mice were proteomically compared with alternatively cultivated amastigotes (axenic and macrophage-derived ones). Amastigotes of all three types were isolated, individually treated and analysed by LC-MS/MS proteomic analysis with quantification using TMT10-plex isobaric labeling. Significant differences were observed in the abundance of metabolic enzymes, virulence factors and proteins involved in translation and condensation of DNA. The most pronounced differences were observed between axenic amastigotes and lesion-derived amastigotes, macrophage-derived amastigotes were mostly intermediate between axenic and lesion-derived ones.

• Keywords
• Leishmania (L) mexicana, amastigote, axenic, lesion, macrophage, proteome, tandem mass tags labeling,
• MeSH
• Chromatography, Liquid MeSH
• Leishmania mexicana * metabolism   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Proteome metabolism   MeSH
• Proteomics MeSH
• Tandem Mass Spectrometry MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Proteome MeSH

Leishmania parasites, causative agents of leishmaniasis, are currently divided into four subgenera: Leishmania, Viannia, Sauroleishmania and Mundinia. The recently established subgenus Mundinia has a wide geographical distribution and contains five species, three of which have the potential to infect and cause disease in humans. While the other Leishmania subgenera are transmitted exclusively by phlebotomine sand flies (Diptera: Psychodidae), natural vectors of Mundinia remain uncertain. This study investigates the potential of sand flies and biting midges of the genus Culicoides (Diptera: Ceratopogonidae) to transmit Leishmania parasites of the subgenus Mundinia. Sand flies (Phlebotomus argentipes, P. duboscqi and Lutzomyia migonei) and Culicoides biting midges (Culicoides sonorensis) were exposed to five Mundinia species through a chicken skin membrane and dissected at specific time intervals post bloodmeal. Potentially infected insects were also allowed to feed on ear pinnae of anaesthetized BALB/c mice and the presence of Leishmania DNA was subsequently confirmed in the mice using polymerase chain reaction analyses. In C. sonorensis, all Mundinia species tested were able to establish infection at a high rate, successfully colonize the stomodeal valve and produce a higher proportion of metacyclic forms than in sand flies. Subsequently, three parasite species, L. martiniquensis, L. orientalis and L. sp. from Ghana, were transmitted to the host mouse ear by C. sonorensis bite. In contrast, transmission experiments entirely failed with P. argentipes, although colonisation of the stomodeal valve was observed for L. orientalis and L. martiniquensis and metacyclic forms of L. orientalis were recorded. This laboratory-based transmission of Mundinia species highlights that Culicoides are potential vectors of members of this ancestral subgenus of Leishmania and we suggest further studies in endemic areas to confirm their role in the lifecycles of neglected pathogens.

• MeSH
• Ceratopogonidae parasitology   MeSH
• Insect Vectors parasitology   MeSH
• Leishmania * MeSH
• Leishmaniasis transmission   MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Visceral leishmaniasis caused by Leishmania donovani is regarded as mostly anthroponotic, but a role for animal reservoir hosts in transmission has been suggested in East Africa. Field studies in this region have shown the presence of this parasite in several mammalian species, including rodents of the genera Arvicanthis and Mastomys. Further, the natural reservoirs of Leishmania (Mundinia) sp. causing human cutaneous disease in Ghana, West Africa, are unknown. This study assessed the potential role of the Sub-Saharan rodents Arvicanthis neumanni, A. niloticus and Mastomys natalensis as hosts of L. donovani and L. sp. from Ghana, based on experimental infections of animals and xenodiagnoses. The distribution and load of parasites were determined post mortem using qPCR from the blood, skin and viscera samples. The attractiveness of Arvicanthis and Mastomys to Phlebotomus orientalis was tested by pair-wise comparisons. None of the animals inoculated with L. donovani were infectious to P. orientalis females, although, in some animals, parasites were detected by PCR even 30 weeks post infection. Skin infections were characterized by low numbers of parasites while high parasite burdens were present in spleen, liver and lymph nodes only. Therefore, wild Arvicanthis and Mastomys found infected with L. donovani, should be considered parasite sinks rather than parasite reservoirs. This is indirectly supported also by results of host choice experiments with P. orientalis in which females preferred humans over both Arvicanthis and Mastomys, and their feeding rates on rodents ranged from 1.4 to 5.8% only. Therefore, the involvement of these rodents in transmission of L. donovani by P. orientalis is very unlikely. Similarly, poor survival of Leishmania parasites in the studied rodents and negative results of xenodiagnostic experiments do not support the involvement of Arvicanthis and Mastomys spp. in the transmission cycle of L. sp. from Ghana.

• Keywords
• Grass rats, Multimammate mice, Mundinia, Reservoir hosts, Visceral leishmaniasis, Xenodiagnosis,
• Publication type
• Journal Article MeSH

Sand flies (Diptera: Psychodidae) are natural vectors of Leishmania. For the initiation of sand fly experimental infections either Leishmania amastigotes or promastigotes can be used. In order to obtain comparable results, it is necessary to adjust and standardize procedures. During this study, we conducted promastigote- and amastigote-initiated infections of Leishmania infantum Nicolle, 1908 parasites in Phlebotomus (Larroussius) perniciosus Newstead, 1911 in two laboratories with different levels of biosafety protection. Protocol originally designed for a biosafety level 2 facility was modified for biosafety level 3 facility and infection parameters were compared. Particularly, specially designed plastic containers were used for blood feeding; feeders were placed outside the sand fly cage, on the top of the mesh; feeding was performed inside the climatic chamber; separation of engorged females was done in Petri dishes kept on ice; engorged females were kept in the cardboard containers until dissection. All experiments, conducted in both laboratories, resulted in fully developed late stage infections with high number of parasites and colonization of the stomodeal valve. We demonstrated that protocol originally designed for biosafety level 2 facilities can be successfully modified for other biosafety facilities, depending on the special requirements of the individual institution/laboratory.

• Keywords
• Leishmania, amastigote, infection, promastigote, sand fly,
• MeSH
• Laboratories * MeSH
• Leishmania infantum growth & development   physiology   MeSH
• Containment of Biohazards MeSH
• Phlebotomus parasitology   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Despite the increasing number of studies concerning insect immunity, Lutzomyia longipalpis immune responses in the presence of Leishmania infantum chagasi infection has not been widely investigated. The few available studies analyzed the role of the Toll and IMD pathways involved in response against Leishmania and microbial infections. Nevertheless, effector molecules responsible for controlling sand fly infections have not been identified. In the present study we investigated the role a signal transduction pathway, the Transforming Growth Factor-beta (TGF-β) pathway, on the interrelation between L. longipalpis and L. i. chagasi. We identified an L. longipalpis homolog belonging to the multifunctional cytokine TGF-β gene family (LlTGF-β), which is closely related to the activin/inhibin subfamily and potentially involved in responses to infections. We investigated this gene expression through the insect development and in adult flies infected with L. i. chagasi. Our results showed that LlTGF-β was expressed in all L. longipalpis developmental stages and was upregulated at the third day post L. i. chagasi infection, when protein levels were also higher as compared to uninfected insects. At this point blood digestion is finished and parasites are in close contact with the insect gut. In addition, we investigated the role of LlTGF-β on L. longipalpis infection by L. i. chagasi using either gene silencing by RNAi or pathway inactivation by addition of the TGF-β receptor inhibitor SB431542. The blockage of the LlTGF-β pathway increased significantly antimicrobial peptides expression and nitric oxide levels in the insect gut, as expected. Both methods led to a decreased L. i. chagasi infection. Our results show that inactivation of the L. longipalpis TGF-β signal transduction pathway reduce L. i. chagasi survival, therefore suggesting that under natural conditions the parasite benefits from the insect LlTGF-β pathway, as already seen in Plamodium infection of mosquitoes.

• Keywords
• Leishmania, Lutzomyia longipalpis, TGF-β, activin, innate immunity, vector-parasite interaction,
• MeSH
• Survival Analysis MeSH
• Insect Vectors immunology   parasitology   MeSH
• Host-Pathogen Interactions * MeSH
• Leishmania infantum growth & development   MeSH
• Immunity, Innate MeSH
• Psychodidae immunology   parasitology   MeSH
• Signal Transduction MeSH
• Gene Expression Profiling MeSH
• Transforming Growth Factor beta metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Transforming Growth Factor beta MeSH