Publications by authors named "J Sadlova"

48 Publications

Genome Analysis of and spp., Closest Phylogenetic Relatives of , Highlights the Role of Amastins in Shaping Pathogenicity.

Genes (Basel) 2021 Mar 20;12(3). Epub 2021 Mar 20.

Life Science Research Centre, Faculty of Science, University of Ostrava, 71000 Ostrava, Czech Republic.

While numerous genomes of spp. have been sequenced and analyzed, an understanding of the evolutionary history of these organisms remains limited due to the unavailability of the sequence data for their closest known relatives, and spp., infecting sloths and porcupines. We have sequenced and analyzed genomes of three members of this clade in order to fill this gap. Their comparative analyses revealed only minute differences from genome in terms of metabolic capacities. We also documented that the number of genes under positive selection on the / branch is rather small, with the flagellum-related group of genes being over-represented. Most significantly, the analysis of gene family evolution revealed a substantially reduced repertoire of surface proteins, such as amastins and biopterin transporters BT1 in the species when compared to amastigote-dwelling . This reduction was especially pronounced for δ-amastins, a subfamily of cell surface proteins crucial in the propagation of amastigotes inside vertebrate macrophages and, apparently, dispensable for , which do not infect such cells.
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http://dx.doi.org/10.3390/genes12030444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004069PMC
March 2021

Catalase impairs development and virulence.

Virulence 2021 12;12(1):852-867

Life Science Research Centre, Faculty of Science, University of Ostrava, Ostrava, Czech Republic.

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 locus using a novel bicistronic expression system, which relies on the 2A peptide of . 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 , resulting in loss of this gene from the genome during the evolution of these parasites.
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http://dx.doi.org/10.1080/21505594.2021.1896830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7971327PMC
December 2021

Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation or survival.

Nat Commun 2021 02 23;12(1):1244. Epub 2021 Feb 23.

York Biomedical Research Institute, University of York, York, UK.

Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here, we apply kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 are dispensable, 44 protein kinase genes are refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering reveal functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism.
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http://dx.doi.org/10.1038/s41467-021-21360-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902614PMC
February 2021

Characterization of a new Leishmania major strain for use in a controlled human infection model.

Nat Commun 2021 01 11;12(1):215. Epub 2021 Jan 11.

York Biomedical Research Institute, Hull York Medical School, University of York, York, UK.

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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http://dx.doi.org/10.1038/s41467-020-20569-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801518PMC
January 2021

The Phlebotomus papatasi systemic transcriptional response to trypanosomatid-contaminated blood does not differ from the non-infected blood meal.

Parasit Vectors 2021 Jan 6;14(1):15. Epub 2021 Jan 6.

Department of Biochemistry, University of Oxford, South Parks Rd, Oxford, OX1 3QU, UK.

Background: Leishmaniasis, caused by parasites of the genus Leishmania, is a disease that affects up to 8 million people worldwide. Parasites are transmitted to human and animal hosts through the bite of an infected sand fly. Novel strategies for disease control require a better understanding of the key step for transmission, namely the establishment of infection inside the fly.

Methods: The aim of this work was to identify sand fly systemic transcriptomic signatures associated with Leishmania infection. We used next generation sequencing to describe the transcriptome of whole Phlebotomus papatasi sand flies when fed with blood alone (control) or with blood containing one of three trypanosomatids: Leishmania major, L. donovani and Herpetomonas muscarum, the latter being a parasite not transmitted to humans.

Results: Of the trypanosomatids studied, only L. major was able to successfully establish an infection in the host P. papatasi. However, the transcriptional signatures observed after each parasite-contaminated blood meal were not specific to success or failure of a specific infection and they did not differ from each other. The transcriptional signatures were also indistinguishable after a non-contaminated blood meal.

Conclusions: The results imply that sand flies perceive Leishmania as just one feature of their microbiome landscape and that any strategy to tackle transmission should focus on the response towards the blood meal rather than parasite establishment. Alternatively, Leishmania could suppress host responses. These results will generate new thinking around the concept of stopping transmission by controlling the parasite inside the insect.
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http://dx.doi.org/10.1186/s13071-020-04498-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789365PMC
January 2021

Central Asian Rodents as Model Animals for and Research.

Microorganisms 2020 Sep 20;8(9). Epub 2020 Sep 20.

Department of Parasitology, Faculty of Science, Charles University, 12844 Prague, Czech Republic.

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 ( and ) to and 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 . 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 . showed the non-healing phenotype with the progressive growth of ulcerative lesions and massive parasite loads. and represented the healing phenotype, the latter with high infectiousness to vectors, mimicking best the character of natural reservoir hosts. Both, and were also highly susceptible to . having wider parasite distribution and higher parasite loads and infectiousness than standard model animals.
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http://dx.doi.org/10.3390/microorganisms8091440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563294PMC
September 2020

Impact of clinically acquired miltefosine resistance by Leishmania infantum on mouse and sand fly infection.

Int J Parasitol Drugs Drug Resist 2020 08 1;13:16-21. Epub 2020 May 1.

Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Belgium. Electronic address:

Objectives: This study evaluated the implications of clinically acquired miltefosine resistance (MIL-R) by assessing virulence in mice and sand flies to reveal the potential of MIL-R strains to circulate.

Methods: Experimental infections with the MIL-R clinical Leishmania infantum isolate MHOM/FR/2005/LEM5159, having a defect in the LiROS3 subunit of the MIL-transporter, and its syngeneic experimentally reconstituted MIL-S counterpart (LEM5159) were performed in BALB/c mice and Lutzomyia longipalpis and Phlebotomus perniciosus sand flies. In mice, the amastigote burdens in liver and spleen were compared microscopically using Giemsa smears and by bioluminescent imaging. During the sand fly infections, the percentage of infected flies, parasite load, colonization of the stomodeal valve and metacyclogenesis were evaluated. The stability of the MIL-R phenotype after sand fly and mouse passage was determined as well.

Results: The fitness of the MIL-R strain differed between the mouse and sand fly infection model. In mice, a clear fitness loss was observed compared to the LiROS3-reconstituted susceptible strain. This defect could be rescued by episomal reconstitution with a wildtype LiROS3 copy. However, this fitness loss was not apparent in the sand fly vector, resulting in metacyclogenesis and efficient colonization of the stomodeal valve. Resistance was stable after passage in both sand fly and mouse.

Conclusion: The natural MIL-R strain is significantly hampered in its ability to multiply and cause a typical visceral infection pattern in BALB/c mice. However, this LiROS3-deficient strain efficiently produced mature infections and metacyclic promastigotes in the sand fly vector highlighting the transmission potential of this particular MIL-R clinical Leishmania strain.
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http://dx.doi.org/10.1016/j.ijpddr.2020.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215113PMC
August 2020

Development of Leishmania (Mundinia) in guinea pigs.

Parasit Vectors 2020 Apr 8;13(1):181. Epub 2020 Apr 8.

Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic.

Background: Leishmaniasis is a human and animal disease caused by parasites of the genus Leishmania, which is now divided into four subgenera, Leishmania, Viannia, Sauroleishmania and Mundinia. Subgenus Mundinia, established in 2016, is geographically widely dispersed, its distribution covers all continents, except Antarctica. It consists of 5 species; L. enriettii and L. macropodum are parasites of wild mammals while L. martiniquensis, L. orientalis and an unnamed Leishmania sp. from Ghana are infectious to humans. There is very little information on natural reservoir hosts and vectors for any Mundinia species.

Methods: Experimental infections of guinea pigs with all five Mundinia species were performed. Animals were injected intradermally with 10 culture-derived promastigotes into both ear pinnae. The courses of infections were monitored weekly; xenodiagnoses were performed at weeks 4 and 8 post-infection using Lutzomyia migonei. The distribution of parasites in different tissues was determined post-mortem by conventional PCR.

Results: No significant differences in weight were observed between infected animals and the control group. Animals infected with L. enriettii developed temporary lesions at the site of inoculation and were infectious to Lu. migonei in xenodiagnoses. Animals infected with L. martiniquensis and L. orientalis developed temporary erythema and dry lesions at the site of inoculation, respectively, but were not infectious to sand flies. Guinea pigs infected by L. macropodum and Leishmania sp. from Ghana showed no signs of infection during experiments, were not infectious to sand flies and leishmanial DNA was not detected in their tissue samples at the end of experiments at week 12 post-inoculation.

Conclusions: According to our results, guinea pigs are not an appropriate model organism for studying Mundinia species other than L. enriettii. We suggest that for better understanding of L. (Mundinia) biology it is necessary to focus on other model organisms.
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http://dx.doi.org/10.1186/s13071-020-04039-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140393PMC
April 2020

Experimental infections and co-infections with Leishmania braziliensis and Leishmania infantum in two sand fly species, Lutzomyia migonei and Lutzomyia longipalpis.

Sci Rep 2020 02 27;10(1):3566. Epub 2020 Feb 27.

Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic.

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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http://dx.doi.org/10.1038/s41598-020-60600-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046706PMC
February 2020

Transmission potential of paromomycin-resistant Leishmania infantum and Leishmania donovani.

J Antimicrob Chemother 2020 04;75(4):951-957

Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Antwerp, Belgium.

Objectives: Former studies demonstrated quick selection of paromomycin resistance for Leishmania infantum and Leishmania donovani accompanied by increased fitness. The present study aimed to interpret these findings in an epidemiological context by comparing infection of WT and experimentally derived paromomycin-resistant strains in the sand fly vector.

Methods: Depending on the Leishmania species, Lutzomyia longipalpis and Phlebotomus perniciosus or Phlebotomus argentipes sand flies were artificially infected with procyclic promastigotes of WT and paromomycin-resistant L. infantum (MHOM/FR/96/LEM3323-cl4) or L. donovani (MHOM/NP/03/BPK275/0-cl18). The infection rate and gut/stomodeal valve colonization were determined to monitor parasite phenotypic behaviour within the vector. The impact of the previously described gain of fitness in the vertebrate host on infectivity for the vector was assessed by feeding L. longipalpis on Syrian golden hamsters heavily infected with either WT or paromomycin-resistant parasites.

Results: WT and paromomycin-resistant Leishmania of both species behaved similarly in terms of infection and parasite location within the studied sand fly species. Blood feeding on infected hamsters did not reveal differences in acquisition of WT and paromomycin-resistant parasites, despite the higher organ burdens observed for the paromomycin-resistant strain. Strains remained resistant after passage in the vector.

Conclusions: Although paromomycin-resistant parasites show an increased parasite fitness in vitro and in laboratory rodents, the intrinsic infection potential of paromomycin-resistant parasites remains unaltered in the sand fly. Of importance is the fact that paromomycin-resistant Leishmania are able to complete development in the natural vectors and produce stomodeal infection with metacyclic forms, which clearly suggests their potential to spread and circulate in nature.
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http://dx.doi.org/10.1093/jac/dkz517DOI Listing
April 2020

Host competence of the African rodents and for from Ethiopia and . () sp. from Ghana.

Int J Parasitol Parasites Wildl 2020 Apr 6;11:40-45. Epub 2019 Dec 6.

Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic.

Visceral leishmaniasis caused by 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 and . Further, the natural reservoirs of ( sp. causing human cutaneous disease in Ghana, West Africa, are unknown. This study assessed the potential role of the Sub-Saharan rodents , and as hosts of and . from Ghana, based on experimental infections of animals and xenodiagnoses. The distribution and load of parasites were determined using qPCR from the blood, skin and viscera samples. The attractiveness of and to was tested by pair-wise comparisons. None of the animals inoculated with were infectious to 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 and found infected with should be considered parasite sinks rather than parasite reservoirs. This is indirectly supported also by results of host choice experiments with in which females preferred humans over both and , and their feeding rates on rodents ranged from 1.4 to 5.8% only. Therefore, the involvement of these rodents in transmission of by is very unlikely. Similarly, poor survival of parasites in the studied rodents and negative results of xenodiagnostic experiments do not support the involvement of and spp. in the transmission cycle of from Ghana.
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http://dx.doi.org/10.1016/j.ijppaw.2019.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920283PMC
April 2020

Comparative genomics of Leishmania (Mundinia).

BMC Genomics 2019 Oct 11;20(1):726. Epub 2019 Oct 11.

Life Science Research Centre, Faculty of Science, University of Ostrava, Ostrava, Czech Republic.

Background: Trypanosomatids of the genus Leishmania are parasites of mammals or reptiles transmitted by bloodsucking dipterans. Many species of these flagellates cause important human diseases with clinical symptoms ranging from skin sores to life-threatening damage of visceral organs. The genus Leishmania contains four subgenera: Leishmania, Sauroleishmania, Viannia, and Mundinia. The last subgenus has been established recently and remains understudied, although Mundinia contains human-infecting species. In addition, it is interesting from the evolutionary viewpoint, representing the earliest branch within the genus and possibly with a different type of vector. Here we analyzed the genomes of L. (M.) martiniquensis, L. (M.) enriettii and L. (M.) macropodum to better understand the biology and evolution of these parasites.

Results: All three genomes analyzed were approximately of the same size (~ 30 Mb) and similar to that of L. (Sauroleishmania) tarentolae, but smaller than those of the members of subgenera Leishmania and Viannia, or the genus Endotrypanum (~ 32 Mb). This difference was explained by domination of gene losses over gains and contractions over expansions at the Mundinia node, although only a few of these genes could be identified. The analysis predicts significant changes in the Mundinia cell surface architecture, with the most important ones relating to losses of LPG-modifying side chain galactosyltransferases and arabinosyltransferases, as well as β-amastins. Among other important changes were gene family contractions for the oxygen-sensing adenylate cyclases and FYVE zinc finger-containing proteins.

Conclusions: We suggest that adaptation of Mundinia to different vectors and hosts has led to alternative host-parasite relationships and, thereby, made some proteins redundant. Thus, the evolution of genomes in the genus Leishmania and, in particular, in the subgenus Mundinia was mainly shaped by host (or vector) switches.
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http://dx.doi.org/10.1186/s12864-019-6126-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6787982PMC
October 2019

Impaired development of a miltefosine-resistant Leishmania infantum strain in the sand fly vectors Phlebotomus perniciosus and Lutzomyia longipalpis.

Int J Parasitol Drugs Drug Resist 2019 12 10;11:1-7. Epub 2019 Sep 10.

Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Universiteitsplein 1, B-2610, Wilrijk, Belgium. Electronic address:

Objectives: To gain insight into the propagation of miltefosine (MIL) resistance in visceral leishmaniasis, this laboratory study explored development of resistant parasites with a defective miltefosine transporter (MT) in sand flies.

Methods: Infectivity, colonization of stomodeal valve and metacyclogenesis of a MIL-resistant (MIL-R) Leishmania infantum LEM3323 line with a defective MT were assessed in the natural sand fly vectors Phlebotomus perniciosus and Lutzomyia longipalpis. Given our recent description of partial drug dependency of the MT-deficient line, the impact of MIL pre-exposure on sand fly infectivity was explored as well.

Results: A significant reduction in sand fly infection, stomodeal valve colonization and differentiation into metacyclics (determined by a lower flagellum/cell body length ratio) was observed in both vectors for MIL-R as compared to the isogenic parent MIL-susceptible line. Re-introduction of the wildtype MT gene into MIL-R tended to partially rescue the capacity to infect sand flies. Pre-exposure to MIL did not alter infectivity of the MIL-R line.

Conclusion: The MIL resistant L. infantum LEM3323 line is significantly hampered in its development and transmissibility potential in two sand fly vector species. Additional studies are warranted to evaluate whether this applies to other visceral Leishmania parasites with acquired MIL-resistance.
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http://dx.doi.org/10.1016/j.ijpddr.2019.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804374PMC
December 2019

Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections.

PLoS Pathog 2019 06 26;15(6):e1007828. Epub 2019 Jun 26.

Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

The protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite's life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single paralysed mutant lacking the central pair protein PF16 or an uncoordinated swimmer lacking the axonemal protein MBO2 showed that these promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L. mexicana need directional motility for successful colonisation of sand flies.
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http://dx.doi.org/10.1371/journal.ppat.1007828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6615630PMC
June 2019

Multiple radiations of spiny mice (Rodentia: Acomys) in dry open habitats of Afro-Arabia: evidence from a multi-locus phylogeny.

BMC Evol Biol 2019 03 4;19(1):69. Epub 2019 Mar 4.

Institute of Vertebrate Biology of the Czech Academy of Sciences, 603 65, Brno, Czech Republic.

Background: Spiny mice of the genus Acomys are distributed mainly in dry open habitats in Africa and the Middle East, and they are widely used as model taxa for various biological disciplines (e.g. ecology, physiology and evolutionary biology). Despite their importance, large distribution and abundance in local communities, the phylogeny and the species limits in the genus are poorly resolved, and this is especially true for sub-Saharan taxa. The main aims of this study are (1) to reconstruct phylogenetic relationships of Acomys based on the largest available multilocus dataset (700 genotyped individuals from 282 localities), (2) to identify the main biogeographical divides in the distribution of Acomys diversity in dry open habitats in Afro-Arabia, (3) to reconstruct the historical biogeography of the genus, and finally (4) to estimate the species richness of the genus by application of the phylogenetic species concept.

Results: The multilocus phylogeny based on four genetic markers shows presence of five major groups of Acomys called here subspinosus, spinosissimus, russatus, wilsoni and cahirinus groups. Three of these major groups (spinosissimus, wilsoni and cahirinus) are further sub-structured to phylogenetic lineages with predominantly parapatric distributions. Combination of alternative species delimitation methods suggests the existence of 26 molecular operational taxonomic units (MOTUs), potentially corresponding to separate species. The highest genetic diversity was found in Eastern Africa. The origin of the genus Acomys is dated to late Miocene (ca. 8.7 Ma), when the first split occurred between spiny mice of eastern (Somali-Masai) and south-eastern (Zambezian) savannas. Further diversification, mostly in Plio-Pleistocene, and the current distribution of Acomys were influenced by the interplay of global climatic factors (e.g., Messinian salinity crisis, intensification of Northern Hemisphere glaciation) with local geomorphology (mountain chains, aridity belts, water bodies). Combination of divergence dating, species distribution modelling and historical biogeography analysis suggests repeated "out-of-East-Africa" dispersal events into western Africa, the Mediterranean region and Arabia.

Conclusions: The genus Acomys is very suitable model for historical phylogeographic and biogeographic reconstructions of dry non-forested environments in Afro-Arabia. We provide the most thorough phylogenetic reconstruction of the genus and identify major factors that influenced its evolutionary history since the late Miocene. We also highlight the urgent need of integrative taxonomic revision of east African taxa.
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http://dx.doi.org/10.1186/s12862-019-1380-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6399835PMC
March 2019

Host competence of African rodents and for .

Int J Parasitol Parasites Wildl 2019 Apr 24;8:118-126. Epub 2019 Jan 24.

Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic.

Cutaneous leishmaniasis caused by is a typical zoonosis circulating in rodents. In Sub-Saharan Africa the reservoirs remain to be identified, although has been detected in several rodent species including members of the genera and . 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 strains in , and the infections were initiated either with sand fly-derived or with culture-derived promastigotes. Inoculated rodents were monitored for several months and tested by xenodiagnoses for their infectiousness to the natural vector of in Sub-Saharan Africa. The distribution and load of parasites were determined using qPCR from the blood, skin and viscera samples. The attractiveness of and to was tested by pair-wise comparisons. Three 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 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 suggest that this species plays the more important reservoir role in the life cycle of in Sub-Saharan Africa. species may serve as potential reservoirs in seasons/periods of low abundance of .
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http://dx.doi.org/10.1016/j.ijppaw.2019.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356118PMC
April 2019

Behavioural strategies of three wild-derived populations of the house mouse (Mus m. musculus and M. m. domesticus) in five standard tests of exploration and boldness: Searching for differences attributable to subspecies and commensalism.

Behav Processes 2018 Dec 26;157:133-141. Epub 2018 Sep 26.

Department of Zoology, Faculty of Science, Charles University, Vinicna 7 128 44 Prague 2, Czech Republic; National Institute of Mental Health, Topolova 748, 250 67, Klecany, Czech Republic. Electronic address:

Animal populations adopting a commensal way of life, e. g. house mice in buildings and stores, are subject to different selection pressures than those living in a non-commensal environment. This may radically influence their behaviour. This study investigated the effects of a commensal way of life on exploratory behaviour in mice. The focal population was non-commensal Mus musculus musculus from Northern Iran. To assess the effect of commensal way of life on exploratory behaviour, it was compared with commensal M. m. musculus from the Czech Republic and to assess the effect of subspecies, it was compared to non-commensal M. m. domesticus from Eastern Syria. We compared their behaviour in five tests of exploratory behaviour and boldness: an open field test with 1) free exploration and 2) forced exploration, 3) hole-board test, 4) test of vertical activity and 5) elevated plus maze. We detected a significant effect of population on behaviour in all five tests. M. m. domesticus was generally bolder and more active than M. m. musculus. Commensal mice were characterized by a higher level of vertical activity (climbing, rearing, jumping). These results suggest that the specific selection pressures of the commensal lifestyle select mice for higher affinity towards elevated places.
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http://dx.doi.org/10.1016/j.beproc.2018.09.008DOI Listing
December 2018

Refractoriness of Sergentomyia schwetzi to Leishmania spp. is mediated by the peritrophic matrix.

PLoS Negl Trop Dis 2018 04 4;12(4):e0006382. Epub 2018 Apr 4.

Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic.

Background: The peritrophic matrix (PM) is an acellular chitin-containing envelope which in most blood sucking insects encloses the ingested blood meal and protects the midgut epithelium. Type I PM present in sand flies and other blood sucking batch feeders is secreted around the meal by the entire midgut in response to feeding. Here we tested the hypothesis that in Sergentomyia schwetzi the PM creates a physical barrier that prevents escape of Leishmania parasites from the endoperitrophic space.

Methodology/principal Findings: Morphology and ultrastructure of the PM as well the production of endogenous chitinase in S. schwetzi were compared with three sand fly species, which are natural vectors of Leishmania. Long persistence of the PM in S. schwetzi was not accompanied by different morphology or decreased production of chitinase. To confirm the role of the PM in refractoriness of S. schwetzi to Leishmania parasites, culture supernatant from the fungus Beauveria bassiana containing chitinase was added to the infective bloodmeal to disintegrate the PM artificially. In females treated with B. bassiana culture supernatants the PM was weakened and permeable, lacking multilayered inner structure; Leishmania colonized the midgut and the stomodeal valve and produced metacyclic forms. In control females Leishmania infections were lost during defecation.

Conclusions/significance: Persistence of the PM till defecation of the bloodmeal represents an important factor responsible for refractoriness of S. schwetzi to Leishmania development. Leishmania major as well as L. donovani promastigotes survived defecation and developed late-stage infections only in females with PM disintegrated artificially by B. bassiana culture supernatants containing exogenous chitinase.
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http://dx.doi.org/10.1371/journal.pntd.0006382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902042PMC
April 2018

Lower galactosylation levels of the Lipophosphoglycan from Leishmania (Leishmania) major-like strains affect interaction with Phlebotomus papatasi and Lutzomyia longipalpis.

Mem Inst Oswaldo Cruz 2018 19;113(5):e170333. Epub 2018 Feb 19.

Instituto René Rachou, Fundação Oswaldo Cruz-Fiocruz, Belo Horizonte, MG, Brasil.

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.
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http://dx.doi.org/10.1590/0074-02760170333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5853761PMC
April 2018

Leishmania mortality in sand fly blood meal is not species-specific and does not result from direct effect of proteinases.

Parasit Vectors 2018 01 15;11(1):37. Epub 2018 Jan 15.

Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic.

Background: Leishmania development in sand flies is confined to the alimentary tract and is closely connected with blood meal digestion. Previously, it has been published that activities of sand fly midgut proteases are harmful to Leishmania, especially to amastigote-promastigote transition forms. However, our experiments with various Leishmania-sand fly pairs gave quite opposite results.

Methods: We evaluated the effect of semi-digested midgut content on different life stages of Leishmania donovani and Leishmania major in vitro. Various morphological forms of parasites, including macrophage-derived amastigotes and transition forms, were incubated 2 h with midguts dissected at various intervals (6-72 h) post-blood meal or with commercially available proteinase, and their viability was determined using flow cytometry. In parallel, using amastigote-initiated experimental infections, we compared development of L. donovani in sand flies that are either susceptible (Phlebotomus argentipes and P. orientalis) or refractory (P. papatasi and Sergentomyia schwetzi) to this parasite.

Results: In vitro, sand fly midgut homogenates affected L. major and L. donovani in a similar way; in all sand fly species, the most significant mortality effect was observed by the end of the blood meal digestion process. Surprisingly, the most susceptible Leishmania stages were promastigotes, while mortality of transforming parasites and amastigotes was significantly lower. Parasites were also susceptible to killing by rabbit blood in combination with proteinase, but resistant to proteinase itself. In vivo, L. donovani developed late-stage infections in both natural vectors; in P. argentipes the development was much faster than in P. orientalis. On the other hand, in refractory species P. papatasi and S. schwetzi, promastigotes survived activity of digestive enzymes but were lost during defecation.

Conclusions: We demonstrated that Leishmania transition forms are more resistant to the killing effect of semi-digested blood meal than 24 h-old promastigotes. Data suggest that Leishmania mortality is not caused directly by sand fly proteases, we assume that this mortality results from toxic products of blood meal digestion. Survival of L. donovani promastigotes in refractory sand flies until blood meal defecation, together with similar mortality of Leishmania parasites incubated in vitro with midgut homogenates of susceptible as well as refractory species, contradict the previously raised hypotheses about the role of midgut proteases in sand fly vector competence to Leishmania.
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http://dx.doi.org/10.1186/s13071-018-2613-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5769529PMC
January 2018

Lipophosphoglycan polymorphisms do not affect Leishmania amazonensis development in the permissive vectors Lutzomyia migonei and Lutzomyia longipalpis.

Parasit Vectors 2017 Dec 16;10(1):608. Epub 2017 Dec 16.

Instituto René Rachou/FIOCRUZ, Belo Horizonte, MG, Brazil.

Background: Lipophosphoglycan (LPG) is a dominant surface molecule of Leishmania promastigotes. Its species-specific polymorphisms are found mainly in the sugars that branch off the conserved Gal(β1,4)Man(α1)-PO backbone of repeat units. Leishmania amazonensis is one of the most important species causing human cutaneous leishmaniasis in the New World. Here, we describe LPG intraspecific polymorphisms in two Le. amazonensis reference strains and their role during the development in three sand fly species.

Results: Strains isolated from Lutzomyia flaviscutellata (PH8) and from a human patient (Josefa) displayed structural polymorphism in the LPG repeat units, possessing side chains with 1 and 2 β-glucose or 1 to 3 β-galactose, respectively. Both strains successfully infected permissive vectors Lutzomyia longipalpis and Lutzomyia migonei and could colonize their stomodeal valve and differentiate into metacyclic forms. Despite bearing terminal galactose residues on LPG, Josefa could not sustain infection in the restrictive vector Phlebotomus papatasi.

Conclusions: LPG polymorphisms did not affect the ability of Le. amazonensis to develop late-stage infections in permissive vectors. However, the non-establishment of infection in Ph. papatasi by Josefa strain suggested other LPG-independent factors in this restrictive vector.
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http://dx.doi.org/10.1186/s13071-017-2568-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5732482PMC
December 2017

Leishmania HASP and SHERP Genes Are Required for In Vivo Differentiation, Parasite Transmission and Virulence Attenuation in the Host.

PLoS Pathog 2017 01 17;13(1):e1006130. Epub 2017 Jan 17.

Centre for Immunology and Infection, Department of Biology, University of York, York, United Kingdom.

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.
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http://dx.doi.org/10.1371/journal.ppat.1006130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5271408PMC
January 2017

Leishmania donovani development in Phlebotomus argentipes: comparison of promastigote- and amastigote-initiated infections.

Parasitology 2017 04 23;144(4):403-410. Epub 2016 Nov 23.

Department of Parasitology,Faculty of Science,Charles University,Vinicna 7, 128 44 Prague 2,Czech Republic.

Leishmania parasites alternate in their life cycle between promastigote stages that develop in the gut of phlebotomine sand flies and amastigotes residing inside phagocytic cells of vertebrate hosts. For experimental infections of sand flies, promastigotes are frequently used as this way of infection is technically easier although ingestion of promastigotes by sand flies is unnatural. Here we aimed to answer a critical question, to what extent do promastigote-initiated experimental infections differ from those initiated with intracellular amastigotes. We performed side-by-side comparison of Leishmania development in Phlebotomus argentipes females infected alternatively with promastigotes from log-phase cultures or amastigotes grown ex vivo in macrophages. Early stage infections showed substantial differences in parasite load and representation of morphological forms. The differences disappeared along the maturation of infections; both groups developed heavy late-stage infections with colonization of the stomodeal valve, uniform representation of infective metacyclics and equal efficiency of transmission. The results showed that studies focusing on early phase of Leishmania development in sand flies should be initiated with intracellular amastigotes. However, the use of promastigote stages for sand fly infections does not alter significantly the final outcome of Leishmania donovani development in P. argentipes and their transmissibility to the vertebrate host.
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http://dx.doi.org/10.1017/S0031182016002067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5368687PMC
April 2017

Genome of Leptomonas pyrrhocoris: a high-quality reference for monoxenous trypanosomatids and new insights into evolution of Leishmania.

Sci Rep 2016 Mar 29;6:23704. Epub 2016 Mar 29.

Biology Centre, Institute of Parasitology, Czech Academy of Sciences, 370 05 České Budějovice (Budweis), Czech Republic.

Many high-quality genomes are available for dixenous (two hosts) trypanosomatid species of the genera Trypanosoma, Leishmania, and Phytomonas, but only fragmentary information is available for monoxenous (single-host) trypanosomatids. In trypanosomatids, monoxeny is ancestral to dixeny, thus it is anticipated that the genome sequences of the key monoxenous parasites will be instrumental for both understanding the origin of parasitism and the evolution of dixeny. Here, we present a high-quality genome for Leptomonas pyrrhocoris, which is closely related to the dixenous genus Leishmania. The L. pyrrhocoris genome (30.4 Mbp in 60 scaffolds) encodes 10,148 genes. Using the L. pyrrhocoris genome, we pinpointed genes gained in Leishmania. Among those genes, 20 genes with unknown function had expression patterns in the Leishmania mexicana life cycle suggesting their involvement in virulence. By combining differential expression data for L. mexicana, L. major and Leptomonas seymouri, we have identified several additional proteins potentially involved in virulence, including SpoU methylase and U3 small nucleolar ribonucleoprotein IMP3. The population genetics of L. pyrrhocoris was also addressed by sequencing thirteen strains of different geographic origin, allowing the identification of 1,318 genes under positive selection. This set of genes was significantly enriched in components of the cytoskeleton and the flagellum.
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http://dx.doi.org/10.1038/srep23704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810370PMC
March 2016

Lutzomyia migonei is a permissive vector competent for Leishmania infantum.

Parasit Vectors 2016 Mar 17;9:159. Epub 2016 Mar 17.

Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic.

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.
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http://dx.doi.org/10.1186/s13071-016-1444-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797322PMC
March 2016

The Biting Midge Culicoides sonorensis (Diptera: Ceratopogonidae) Is Capable of Developing Late Stage Infections of Leishmania enriettii.

PLoS Negl Trop Dis 2015 14;9(9):e0004060. Epub 2015 Sep 14.

Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic.

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".
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http://dx.doi.org/10.1371/journal.pntd.0004060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4569557PMC
March 2016

Natural infection of bats with Leishmania in Ethiopia.

Acta Trop 2015 Oct 29;150:166-70. Epub 2015 Jul 29.

Department of Parasitology, Faculty of Science, Charles University in Prague, Vinicna 7, 128 44 Prague 2, Czech Republic. Electronic address:

The leishmaniases, a group of diseases with a worldwide-distribution, are caused by different species of Leishmania parasites. Both cutaneous and visceral leishmaniasis remain important public health problems in Ethiopia. Epidemiological cycles of these protozoans involve various sand fly (Diptera: Psychodidae) vectors and mammalian hosts, including humans. In recent years, Leishmania infections in bats have been reported in the New World countries endemic to leishmaniasis. The aim of this study was to survey natural Leishmania infection in bats collected from various regions of Ethiopia. Total DNA was isolated from spleens of 163 bats belonging to 23 species and 18 genera. Leishmania infection was detected by real-time (RT) PCR targeting a kinetoplast (k) DNA and internal transcribed spacer one (ITS1) gene of the parasite. Detection was confirmed by sequencing of the PCR products. Leishmania kDNA was detected in eight (4.9%) bats; four of them had been captured in the Aba-Roba and Awash-Methara regions that are endemic for leishmaniasis, while the other four specimens originated from non-endemic localities of Metu, Bedele and Masha. Leishmania isolates from two bats were confirmed by ITS1 PCR to be Leishmania tropica and Leishmania major, isolated from two individual bats, Cardioderma cor and Nycteris hispida, respectively. These results represent the first confirmed observation of natural infection of bats with the Old World Leishmania. Hence, bats should be considered putative hosts of Leishmania spp. affecting humans with a significant role in the transmission.
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http://dx.doi.org/10.1016/j.actatropica.2015.07.024DOI Listing
October 2015

Comparison of Bloodmeal Digestion and the Peritrophic Matrix in Four Sand Fly Species Differing in Susceptibility to Leishmania donovani.

PLoS One 2015 1;10(6):e0128203. Epub 2015 Jun 1.

Department of Parasitology, Faculty of Science Charles University, Prague, Czech Republic.

The early stage of Leishmania development in sand flies is closely connected with bloodmeal digestion. Here we compared various parameters of bloodmeal digestion in sand flies that are either susceptible (Phlebotomus argentipes and P. orientalis) or refractory (P. papatasi and Sergentomyia schwetzi) to Leishmania donovani, to study the effects on vector competence. The volume of the bloodmeal ingested, time of defecation of bloodmeal remnants, timing of formation and degradation of the peritrophic matrix (PM) and dynamics of proteolytic activities were compared in four sand fly species. Both proven vectors of L. donovani showed lower trypsin activity and slower PM formation than refractory species. Interestingly, the two natural L. donovani vectors strikingly differed from each other in secretion of the PM and midgut proteases, with P. argentipes possessing fast bloodmeal digestion with a very high peak of chymotrypsin activity and rapid degradation of the PM. Experimental infections of P. argentipes did not reveal any differences in vector competence in comparison with previously studied P. orientalis; even the very low initial dose (2×103 promastigotes/ml) led to fully developed late-stage infections with colonization of the stomodeal valve in about 40% of females. We hypothesise that the period between the breakdown of the PM and defecation of the bloodmeal remnants, i.e. the time frame when Leishmania attach to the midgut in order to prevent defecation, could be one of crucial parameters responsible for the establishment of Leishmania in the sand fly midgut. In both natural L. donovani vectors this period was significantly longer than in S. schwetzi. Both vectors are equally susceptible to L. donovani; as average bloodmeal volumes taken by females of P. argentipes and P. orientalis were 0.63 μl and 0.59 μl, respectively, an infective dose corresponding to 1-2 parasites was enough to initiate mature infections.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0128203PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4452187PMC
April 2016

Xenodiagnosis of Leishmania donovani in BALB/c mice using Phlebotomus orientalis: a new laboratory model.

Parasit Vectors 2015 Mar 14;8:158. Epub 2015 Mar 14.

Department of Parasitology, Faculty of Science, Charles University in Prague, Prague 2, Vinicna 7, 128 44, Czech Republic.

Background: In areas endemic for visceral leishmaniasis (VL), the majority of infected hosts remain asymptomatic but potentially infectious to biting sand flies. Their infectiousness for sand fly vectors is crucial for the transmission of the disease and can be quantified only by xenodiagnosis. However, in the case of human hosts, xenodiagnosis can be problematic for ethical and logistic reasons. The BALB/c mouse model described in this paper was designed to enable xenodiagnostic studies on VL hosts circumventing the need for human volunteers, it permits xenodiagnosis using the same individual host repeatedly, over several months.

Methods: BALB/c mice were intradermally inoculated in the ear pinnae with Leishmania donovani, primarily metacyclic stages isolated from the thoracic midguts of experimentally-infected Phlebotomus orientalis females. Naïve sand flies were allowed to feed on anaesthetized mice in 1-3-weeks- interval, firstly on the site of inoculation of L. donovani (weeks 2-8 post infection, p.i.), later on the whole body of mice (weeks 9 - 15 p.i.). Infections of sand flies were evaluated microscopically or by PCR analysis.

Results: Although infected mice did not show any signs of disease, 19% (N = 876) of the P. orientalis females that fed at the site of inoculation, became infected. The majority of L. donovani-positive females (76%) had heavy infections with their stomodeal valves colonized by attached parasites. Inoculated mouse ears remained infective for sand flies until week 15 p.i. Females feeding on other parts of the body remained negative with exception of two groups feeding on contralateral ears by week 12 p.i. On week 15, however, these two mice returned negative at xenodiagnosis of the contralateral ears. In sacrificed mice, the highest parasite numbers were found in inoculated ears and their draining lymph nodes. Infections were detected also in the spleen, liver, blood and rarely in the contralateral ear.

Conclusions: The study showed that BALB/c mice harbored parasites in sufficient numbers to promote heavy infections in P. orientalis and thus comprised a suitable laboratory model for xenodiagnoses of L. donovani. Parasites persisted in the inoculation site and were found transmissible for months to sand flies biting on the same site.
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http://dx.doi.org/10.1186/s13071-015-0765-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4364506PMC
March 2015

Detection of Leishmania donovani and L. tropica in Ethiopian wild rodents.

Acta Trop 2015 May 18;145:39-44. Epub 2015 Feb 18.

Department of Parasitology, Faculty of Science, Charles University in Prague, Vinicna 7, 128 44 Prague 2, Czech Republic. Electronic address:

Human visceral (VL, also known as Kala-azar) and cutaneous (CL) leishmaniasis are important infectious diseases affecting countries in East Africa that remain endemic in several regions of Ethiopia. The transmission and epidemiology of the disease is complicated due to the complex life cycle of the parasites and the involvement of various Leishmania spp., sand fly vectors, and reservoir animals besides human hosts. Particularly in East Africa, the role of animals as reservoirs for human VL remains unclear. Isolation of Leishmania donovani parasites from naturally infected rodents has been reported in several endemic countries; however, the status of rodents as reservoirs in Ethiopia remains unclear. Here, we demonstrated natural Leishmania infections in rodents. Animals were trapped in 41 localities of endemic and non-endemic areas in eight geographical regions of Ethiopia and DNA was isolated from spleens of 586 rodents belonging to 21 genera and 38 species. Leishmania infection was evaluated by real-time PCR of kinetoplast (k)DNA and confirmed by sequencing of the PCR products. Subsequently, parasite species identification was confirmed by PCR and DNA sequencing of the 18S ribosomal RNA internal transcribed spacer one (ITS1) gene. Out of fifty (8.2%) rodent specimens positive for Leishmania kDNA-PCR and sequencing, 10 were subsequently identified by sequencing of the ITS1 showing that five belonged to the L. donovani complex and five to L. tropica. Forty nine kDNA-positive rodents were found in the endemic localities of southern and eastern Ethiopia while only one was identified from northwestern Ethiopia. Moreover, all the ten ITS1-positive rodents were captured in areas where human leishmaniasis cases have been reported and potential sand fly vectors occur. Our findings suggest the eco-epidemiological importance of rodents in these foci of leishmaniasis and indicate that rodents are likely to play a role in the transmission of leishmaniasis in Ethiopia, possibly as reservoir hosts.
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http://dx.doi.org/10.1016/j.actatropica.2015.02.006DOI Listing
May 2015