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 * parazitologie   MeSH
• hmyz - vektory * parazitologie   MeSH
• kůže parazitologie   MeSH
• Leishmania major * fyziologie   patogenita   MeSH
• leishmanióza kožní * přenos   parazitologie   MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• parazitární zátěž MeSH
• Phlebotomus * parazitologie   MeSH
• zdroje nemoci * parazitologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články 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 * parazitologie   MeSH
• Cricetulus MeSH
• křečci praví MeSH
• Leishmania * klasifikace   MeSH
• leishmanióza * parazitologie   MeSH
• modely nemocí na zvířatech * MeSH
• myši inbrední BALB C * MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• Klíčová slova
• Leishmania, Meriones, Phlebotomus, asymptomatic infection, reservoir host, xenodiagnosis,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Species belonging to the subgenus Sauroleishmania are parasites of reptiles, and traditionally considered to be non-pathogenic to mammals. Knowledge of the development of these parasites in sand flies and their mechanism of transmission is currently lacking. The main aim of this study was to test the susceptibility of various sand fly species to infection by two Sauroleishmania species, focusing on the localization of parasites in the sand fly intestinal tract. METHODS: The development of Leishmania (Sauroleishmania [S.]) adleri and Leishmania (S.) hoogstraali was studied in six sand fly species (Phlebotomus orientalis, P. argentipes, P. sergenti, P. papatasi, P. duboscqi, Sergentomyia schwetzi). Sand flies were fed through a chick-skin membrane on blood containing Sauroleishmania promastigotes, and they were dissected at various time intervals post blood meal (PBM). Guts were examined microscopically for the presence of parasites, and the intensity and localizations of infections were recorded. Morphological forms of both Sauroleishmania species developing in P. orientalis were analyzed. Experimental infections of geckos using sand fly-derived promastigotes were also performed, and the reptiles were repeatedly examined for Sauroleishmania infection by xenodiagnosis and PCR analysis. RESULTS: High infection rates for both Sauroleishmania species were observed in P. orientalis and P. argentipes, with the parasites migrating anteriorly and undergoing a peripylarian type of development, including colonization of the stomodeal valve. Conversely, the development of L. (S.) adleri in P. sergenti, P. papatasi and Se. schwetzi was restricted to the sand fly hindgut (hypopylarian type of development). Five morphological forms were distinguished for both Sauroleishmania species developing in P. orientalis. All experimentally infected geckos scored negative for Sauroleishmania based on xenodiagnosis and molecular analysis. CONCLUSIONS: The results showed that Sauroleishmania promastigotes can undergo either a peripylarian or hypopylarian type of development in the sand fly intestinal tract, depending on the sand fly species infected. We demonstrated that P. argentipes and P. orientalis, two sand fly species known as permissive vectors for mammalian parasites of subgenus Leishmania, are also highly susceptible to Sauroleishmania as the parasites developed mature late-stage infections, including colonization of the sand fly stomodeal valve. Thus, the role of Phlebotomus sand flies in transmission of Sauroleishmania should be reconsidered and further investigated.

• Klíčová slova
• Geckos, Leishmaniasis, Phlebotomus, Sand flies, Sauroleishmania, Sergentomyia,
• MeSH
• ještěři * MeSH
• Leishmania * MeSH
• Phlebotomus * parazitologie   MeSH
• Psychodidae * parazitologie   MeSH
• savci MeSH
• xenodiagnóza MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• Cricetulus griseus, Lagurus lagurus, Phlebotomus, Phodopus sungorus, cutaneous leishmaniasis, infectiousness, model animals, sand fly, visceral leishmaniasis, xenodiagnosis,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• Grass rats, Multimammate mice, Mundinia, Reservoir hosts, Visceral leishmaniasis, Xenodiagnosis,
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• Arvicanthis, Grass rats, Leishmaniases, Mastomys, Multimammate mice, Wild reservoir, Xenodiagnosis,
• Publikační typ
• časopisecké články MeSH

Leishmania parasites cause human cutaneous, mucocutaneous and visceral leishmaniasis. Several studies proposed involvement of certain genes in infectivity of these parasites based on differential mRNA expression data. Due to unusual gene expression mechanism, functions of such genes must be further validated experimentally. Here, we investigated a role of one of the putative virulence factors, LmxM.22.0010-encoded BTN1 (a protein involved in Batten disease in humans), in L. mexicana infectivity. Due to the incredible plasticity of the L. mexicana genome, we failed to obtain a complete knock-out of LmxM.22.0010 using conventional recombination-based approach even after ablating four alleles of this gene. To overcome this, we established a modified CRISPR-Cas9 system with genomic expression of Cas9 nuclease and gRNA. Application of this system allowed us to establish a complete BTN1 KO strain of L. mexicana. The mutant strain did not show any difference in growth kinetics and differentiation in vitro, as well as in the infectivity for insect vectors and mice hosts. Based on the whole-transcriptome profiling, LmxM.22.0010-encoded BTN1 was considered a putative factor of virulence in Leishmania. Our study suggests that ablation of LmxM.22.0010 does not influence L. mexicana infectivity and further illustrates importance of experimental validation of in silico-predicted virulence factors. Here we also describe the whole genome sequencing of the widely used model isolate L. mexicana M379 and report a modified CRISPR/Cas9 system suitable for complete KO of multi-copy genes in organisms with flexible genomes.

• MeSH
• CRISPR-Cas systémy * MeSH
• genový knockout metody   MeSH
• hmyz - vektory parazitologie   MeSH
• Leishmania mexicana genetika   patogenita   MeSH
• leishmanióza kožní parazitologie   MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• počítačová simulace MeSH
• protozoální geny * MeSH
• Psychodidae parazitologie   MeSH
• stanovení celkové genové exprese MeSH
• virulence genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• validační studie MeSH

Aneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania, a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro-cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro, aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness.IMPORTANCE Aneuploidy is usually detrimental in multicellular organisms, but in several microorganisms, it can be tolerated and even beneficial. Leishmania-a protozoan parasite that kills more than 30,000 people each year-is emerging as a new model for aneuploidy studies, as unexpectedly high levels of aneuploidy are found in clinical isolates. Leishmania lacks classical regulation of transcription at initiation through promoters, so aneuploidy could represent a major adaptive strategy of this parasite to modulate gene dosage in response to stressful environments. For the first time, we document the dynamics of aneuploidy throughout the life cycle of the parasite, in vitro and in vivo We show its adaptive impact on transcription and its interaction with regulation. Besides offering a new model for aneuploidy studies, we show that further genomic studies should be done directly in clinical samples without parasite isolation and that adequate methods should be developed for this.

• Klíčová slova
• Leishmania, aneuploidy, gene dosage, genomics, life cycle,
• MeSH
• aneuploidie * MeSH
• biologická adaptace * MeSH
• exprese genu * MeSH
• genom protozoální MeSH
• křečci praví MeSH
• Leishmania donovani genetika   MeSH
• lidé MeSH
• Psychodidae MeSH
• sekvenční analýza DNA MeSH
• stanovení celkové genové exprese MeSH
• životní prostředí MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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 parazitologie   MeSH
• gastrointestinální trakt parazitologie   MeSH
• hmyz - vektory * MeSH
• křeček rodu Mesocricetus MeSH
• Leishmania enriettii izolace a purifikace   MeSH
• leishmanióza parazitologie   patologie   přenos   MeSH
• morčata MeSH
• Psychodidae parazitologie   MeSH
• zvířata MeSH
• Check Tag
• morčata MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH