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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.
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Flagelos , Leishmania , Psychodidae , Animales , Leishmania/fisiología , Leishmania/genética , Leishmania/metabolismo , Psychodidae/parasitología , Flagelos/metabolismo , Adhesión Celular , Insectos Vectores/parasitología , Interacciones Huésped-Parásitos , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Estadios del Ciclo de Vida , Leishmaniasis/parasitología , Leishmaniasis/transmisión , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , FemeninoRESUMEN
The leishmaniases are globally important parasitic diseases for which no human vaccines are currently available. To facilitate vaccine development, we conducted an open-label observational study to establish a controlled human infection model (CHIM) of sand fly-transmitted cutaneous leishmaniasis (CL) caused by Leishmania major. Between 24 January and 12 August 2022, we exposed 14 participants to L. major-infected Phlebotomus duboscqi. The primary objective was to demonstrate effectiveness of lesion development (take rate) and safety (absence of CL lesion at 12 months). Secondary and exploratory objectives included rate of lesion development, parasite load and analysis of local immune responses by immunohistology and spatial transcriptomics. Lesion development was terminated by therapeutic biopsy (between days 14 and 42 after bite) in ten participants with clinically compatible lesions, one of which was not confirmed by parasite detection. We estimated an overall take rate for CL development of 64% (9/14). Two of ten participants had one and one of ten participants had two lesion recurrences 4-8 months after biopsy that were treated successfully with cryotherapy. No severe or serious adverse events were recorded, but as expected, scarring due to a combination of CL and the biopsy procedure was evident. All participants were lesion free at >12-month follow-up. We provide the first comprehensive map of immune cell distribution and cytokine/chemokine expression in human CL lesions, revealing discrete immune niches. This CHIM offers opportunities for vaccine candidate selection based on human efficacy data and for a greater understanding of immune-mediated pathology. ClinicalTrials.gov identifier: NCT04512742 .
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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.
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Antimonio , Resistencia a Medicamentos , Leishmania major , Leishmaniasis Cutánea , Phlebotomus , Phlebotomus/parasitología , Phlebotomus/efectos de los fármacos , Leishmania major/efectos de los fármacos , Leishmania major/genética , Animales , Antimonio/farmacología , Resistencia a Medicamentos/genética , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/transmisión , Insectos Vectores/parasitología , Insectos Vectores/efectos de los fármacos , Fenotipo , Antiprotozoarios/farmacología , Concentración 50 Inhibidora , FemeninoRESUMEN
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.
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Arvicolinae , Modelos Animales de Enfermedad , Leishmania , Leishmaniasis , Ratones Endogámicos BALB C , Animales , Leishmania/clasificación , Leishmaniasis/parasitología , Ratones , Cricetinae , Arvicolinae/parasitología , Cricetulus , FemeninoRESUMEN
Nearly all aerobic organisms are equipped with catalases, powerful enzymes scavenging hydrogen peroxide and facilitating defense against harmful reactive oxygen species. In trypanosomatids, this enzyme was not present in the common ancestor, yet it had been independently acquired by different lineages of monoxenous trypanosomatids from different bacteria at least three times. This observation posited an obvious question: why was catalase so "sought after" if many trypanosomatid groups do just fine without it? In this work, we analyzed subcellular localization and function of catalase in Leptomonas seymouri. We demonstrated that this enzyme is present in the cytoplasm and a subset of glycosomes, and that its cytoplasmic retention is H2O2-dependent. The ablation of catalase in this parasite is not detrimental in vivo, while its overexpression resulted in a substantially higher parasite load in the experimental infection of Dysdercus peruvianus. We propose that the capacity of studied flagellates to modulate the catalase activity in the midgut of its insect host facilitates their development and protects them from oxidative damage at elevated temperatures.
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Catalasa , Peróxido de Hidrógeno , Trypanosomatina , Catalasa/metabolismo , Animales , Trypanosomatina/enzimología , Trypanosomatina/genética , Peróxido de Hidrógeno/metabolismo , Citoplasma , Microcuerpos/metabolismoRESUMEN
Attachment to a substrate to maintain position in a specific ecological niche is a common strategy across biology, especially for eukaryotic parasites. During development in the sand fly vector, the eukaryotic parasite Leishmania adheres to the stomodeal valve, as the specialised haptomonad form. Dissection of haptomonad adhesion is a critical step for understanding the complete life cycle of Leishmania. Nevertheless, haptomonad studies are limited, as this is a technically challenging life cycle form to investigate. Here, we have combined three-dimensional electron microscopy approaches, including serial block face scanning electron microscopy (SBFSEM) and serial tomography to dissect the organisation and architecture of haptomonads in the sand fly. We showed that the attachment plaque contains distinct structural elements. Using time-lapse light microscopy of in vitro haptomonad-like cells, we identified five stages of haptomonad-like cell differentiation, and showed that calcium is necessary for Leishmania adhesion to the surface in vitro. This study provides the structural and regulatory foundations of Leishmania adhesion, which are critical for a holistic understanding of the Leishmania life cycle.
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Leishmania , Psychodidae , Animales , Microscopía ElectrónicaRESUMEN
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.
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Trypanosomatid pathogens are transmitted by blood-feeding insects, causing devastating human infections. These parasites show important phenotypic shifts that often impact parasite pathogenicity, tissue tropism, or drug susceptibility. The evolutionary mechanisms that allow for the selection of such adaptive phenotypes remain only poorly investigated. Here, we use Leishmania donovani as a trypanosomatid model pathogen to assess parasite evolutionary adaptation during experimental sand fly infection. Comparing the genome of the parasites before and after sand fly infection revealed a strong population bottleneck effect as judged by allele frequency analysis. Apart from random genetic drift caused by the bottleneck effect, our analyses revealed haplotype and allelic changes during sand fly infection that seem under natural selection given their convergence between independent biological replicates. Our analyses further uncovered signature mutations of oxidative DNA damage in the parasite genomes after sand fly infection, suggesting that Leishmania suffers from oxidative stress inside the insect digestive tract. Our results propose a model of Leishmania genomic adaptation during sand fly infection, with oxidative DNA damage and DNA repair processes likely driving haplotype and allelic selection. The experimental and computational framework presented here provides a useful blueprint to assess evolutionary adaptation of other eukaryotic pathogens inside their insect vectors, such as Plasmodium spp, Trypanosoma brucei, and Trypanosoma cruzi.
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Leishmania donovani , Psychodidae , Humanos , Animales , Estrés Oxidativo/genética , Reparación del ADN/genética , MutaciónRESUMEN
Parasites of the genus Porcisia, together with the genus Endotrypanum, form a sister clade to the species-rich and medically important genus Leishmania. Both Porcisia species, P. hertigi and P. deanei, are dixenous parasites of Neotropical porcupines. Almost 50 years after their first discovery, knowledge of their life cycle remains poor and their insect vectors are unknown. Because competent vectors of their closest phylogenetic relatives, genera Endotrypanum and Leishmania, are phlebotomine sand flies (Diptera: Psychodidae) and/or biting midges (Diptera: Ceratopogonidae), we examined here the potential of both sand flies and biting midges to transmit Porcisia parasites. The insects (Lutzomyia longipalpis, L. migonei and Culicoides sonorensis) were exposed to parasites through the chicken skin membrane and dissected at various time intervals post bloodmeal. Potentially infected females were also allowed to feed on the ears of anaesthetized BALB/c mice and the presence of parasite DNA was subsequently confirmed in the mice by PCR. Porcisia hertigi did not survive defecation in L. longipalpis or L. migonei, suggesting that these sand fly species are unlikely to serve as natural vectors of this parasite. Similarly, P. hertigi infections were lost in Culicoides midges. In contrast, mature P. deanei infections developed in 51-61% of L. longipalpis females, 7.3% of L. migonei females and 7.7% of Culicoides sonorensis females. In all three vector species, P. deanei colonized predominantly Malpighian tubules and produced metacyclic infective forms. Transmission of P. daenei to BALB/c mice was demonstrated via the prediuresis of L. longipalpis females. This mode of transmission, as well the colonization of Malpighian tubules as the dominant tissue of the vector, is unique among trypanosomatids. In conclusion, we demonstrated the vector competence of L. longipalpis for P. deanei but not for P. hertigi, and further studies are needed to evaluate competence of other Neotropical vectors for these neglected parasites.
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Leishmania , Psychodidae , Animales , Femenino , Insectos Vectores , Estadios del Ciclo de Vida , Ratones , FilogeniaRESUMEN
The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network associated with parasite fitness gain, with genome instability causing highly reproducible, gene dosage-independent and -dependent changes. Reduction of flagellar transcripts and increase in coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation were not correlated to dosage changes of the corresponding genes, revealing a gene dosage-independent, post-transcriptional mechanism of regulation. In contrast, abundance of gene products implicated in post-transcriptional regulation itself correlated to corresponding gene dosage changes. Thus, RNA abundance during parasite adaptation is controled by direct and indirect gene dosage changes. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of modified ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.
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Leishmania donovani , Leishmaniasis Visceral , Regulación de la Expresión Génica , Inestabilidad Genómica , Humanos , Leishmania donovani/genética , Leishmaniasis Visceral/parasitología , ProteómicaRESUMEN
Background: Leishmaniasis is a globally important yet neglected parasitic disease transmitted by phlebotomine sand flies. With new candidate vaccines in or near the clinic, a controlled human challenge model (CHIM) using natural sand fly challenge would provide a method for early evaluation of prophylactic efficacy. Methods : We evaluated the biting frequency and adverse effects resulting from exposure of human volunteers to bites of either Phlebotomus papatasi or P. duboscqi, two natural vectors of Leishmania major. 12 healthy participants were recruited (mean age 40.2 ± 11.8 years) with no history of significant travel to regions where L. major-transmitting sand flies are prevalent. Participants were assigned to either vector by 1:1 allocation and exposed to five female sand flies for 30 minutes in a custom biting chamber. Bite frequency was recorded to confirm a bloodmeal was taken. Participant responses and safety outcomes were monitored using a visual analogue scale (VAS), clinical examination, and blood biochemistry. Focus groups were subsequently conducted to explore participant acceptability. Results: All participants had at least one successful sand fly bite with none reporting any serious adverse events, with median VAS scores of 0-1/10 out to day 21 post-sand fly bite. Corresponding assessment of sand flies confirmed that for each participant at least 1/5 sand flies had successfully taken a bloodmeal (overall mean 3.67±1.03 bites per participant). There was no significant difference between P. papatasi and P. duboscqi in the number of bites resulting from 5 sand flies applied to human participants (3.3±0.81 vs 3.00±1.27 bites per participant; p=0.56) . In the two focus groups (n=5 per group), themes relating to positive participant-reported experiences of being bitten and the overall study, were identified. Conclusions: These results validate a protocol for achieving successful sand fly bites in humans that is safe, well-tolerated and acceptable for participants. Clinicaltrials.gov registration: NCT03999970 (27/06/2019).
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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.
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Ceratopogonidae/parasitología , Insectos Vectores/parasitología , Leishmania , Leishmaniasis/transmisión , Animales , RatonesRESUMEN
Catalase is one of the most abundant enzymes on Earth. It decomposes hydrogen peroxide, thus protecting cells from dangerous reactive oxygen species. The catalase-encoding gene is conspicuously absent from the genome of most representatives of the family Trypanosomatidae. Here, we expressed this protein from the Leishmania mexicana Β-TUBULIN locus using a novel bicistronic expression system, which relies on the 2A peptide of Teschovirus A. We demonstrated that catalase-expressing parasites are severely compromised in their ability to develop in insects, to be transmitted and to infect mice, and to cause clinical manifestation in their mammalian host. Taken together, our data support the hypothesis that the presence of catalase is not compatible with the dixenous life cycle of Leishmania, resulting in loss of this gene from the genome during the evolution of these parasites.
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Catalasa/genética , Leishmania mexicana/crecimiento & desarrollo , Leishmania mexicana/patogenicidad , Estadios del Ciclo de Vida/genética , Proteínas Protozoarias/genética , Factores de Virulencia/genética , Animales , Catalasa/metabolismo , Células Cultivadas , Femenino , Leishmania mexicana/genética , Ratones , Ratones Endogámicos BALB C , Psychodidae/parasitología , Teschovirus/genética , Virulencia , Factores de Virulencia/metabolismoRESUMEN
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.
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Leishmania major/fisiología , Leishmaniasis Cutánea/parasitología , Animales , Modelos Animales de Enfermedad , Humanos , Insectos Vectores/parasitología , Israel , Leishmania major/genética , Leishmania major/crecimiento & desarrollo , Leishmaniasis Cutánea/transmisión , Ratones Endogámicos BALB C , Parásitos/genética , Filogenia , Psychodidae/parasitología , Secuenciación Completa del GenomaRESUMEN
The clinical manifestation of leishmaniases depends on parasite species, host genetic background, and immune response. Manifestations of human leishmaniases are highly variable, ranging from self-healing skin lesions to fatal visceral disease. The scope of standard model hosts is insufficient to mimic well the wide disease spectrum, which compels the introduction of new model animals for leishmaniasis research. In this article, we study the susceptibility of three Asian rodent species (Cricetulus griseus, Lagurus lagurus, and Phodopus sungorus) to Leishmania major and L. donovani. The external manifestation of the disease, distribution, as well as load of parasites and infectiousness to natural sand fly vectors, were compared with standard models, BALB/c mice and Mesocricetus auratus. No significant differences were found in disease outcomes in animals inoculated with sand fly- or culture-derived parasites. All Asian rodent species were highly susceptible to L. major. Phodopus sungorus showed the non-healing phenotype with the progressive growth of ulcerative lesions and massive parasite loads. Lagurus lagurus and C. griseus represented the healing phenotype, the latter with high infectiousness to vectors, mimicking best the character of natural reservoir hosts. Both, L. lagurus and C. griseus were also highly susceptible to L. donovani, having wider parasite distribution and higher parasite loads and infectiousness than standard model animals.
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Leishmaniases are neglected tropical diseases and Leishmania (Leishmania) infantum and Leishmania (Viannia) braziliensis are the most important causative agents of leishmaniases in the New World. These two parasite species may co-circulate in a given endemic area but their interactions in the vector have not been studied yet. We conducted experimental infections using both single infections and co-infections to compare the development of L. (L.) infantum (OGVL/mCherry) and L. (V.) braziliensis (XB29/GFP) in Lutzomyia longipalpis and Lutzomyia migonei. Parasite labelling by different fluorescein proteins enabled studying interspecific competition and localization of different parasite species during co-infections. Both Leishmania species completed their life cycle, producing infective forms in both sand fly species studied. The same happens in the co infections, demonstrating that the two parasites conclude their development and do not compete with each other. However, infections produced by L. (L.) infantum reached higher rates and grew more vigorously, as compared to L. (V.) braziliensis. In late-stage infections, L. (L.) infantum was present in all midgut regions, showing typical suprapylarian type of development, whereas L. (V.) braziliensis was concentrated in the hindgut and the abdominal midgut (peripylarian development). We concluded that both Lu. migonei and Lu. longipalpis are equally susceptible vectors for L. (L.) infantum, in laboratory colonies. In relation to L. (V.) braziliensis, Lu. migonei appears to be more susceptible to this parasite than Lu. longipalpis.
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Insectos Vectores/parasitología , Leishmania braziliensis/fisiología , Leishmania infantum/fisiología , Psychodidae/parasitología , Animales , Sistema Digestivo/parasitología , Femenino , Leishmania braziliensis/crecimiento & desarrollo , Leishmania infantum/crecimiento & desarrollo , Estadios del Ciclo de VidaRESUMEN
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.
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Cutaneous leishmaniasis caused by Leishmania major is a typical zoonosis circulating in rodents. In Sub-Saharan Africa the reservoirs remain to be identified, although L. major has been detected in several rodent species including members of the genera Arvicanthis and Mastomys. However, differentiation of true reservoir hosts from incidental hosts requires in-depth studies both in the field and in the laboratory, with the best method for testing the infectiousness of hosts to biting vectors being xenodiagnosis. Here we studied experimental infections of three L. major strains in Arvicanthis neumanni, A. niloticus and Mastomys natalensis; the infections were initiated either with sand fly-derived or with culture-derived Leishmania promastigotes. Inoculated rodents were monitored for several months and tested by xenodiagnoses for their infectiousness to Phlebotomus duboscqi, the natural vector of L. major in Sub-Saharan Africa. 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 P. duboscqi was tested by pair-wise comparisons. Three L. major strains used significantly differed in infectivity: the Middle Eastern strain infected a low proportion of rodents, while two Sub-Saharan isolates (LV109, LV110) infected a high percentage of animals and LV110 also produced higher parasite loads in all host species. All three rodent species maintained parasites of the LV109 strain for 20-25 weeks and were able to infect P. duboscqi without apparent health complications: infected animals showed only temporary swellings or changes of pigmentation at the site of inoculation. However, the higher infection rates, more generalized distribution of parasites and longer infectiousness period to sand flies in M. natalensis suggest that this species plays the more important reservoir role in the life cycle of L. major in Sub-Saharan Africa. Arvicanthis species may serve as potential reservoirs in seasons/periods of low abundance of Mastomys.
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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".