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.

• MeSH
• biologické modely MeSH
• buněčná diferenciace * MeSH
• CRISPR-Cas systémy genetika   MeSH
• delece genu MeSH
• flagella enzymologie   MeSH
• Leishmania mexicana cytologie   enzymologie   MeSH
• leishmanióza parazitologie   patologie   MeSH
• mutace genetika   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• protein Cas9 metabolismus   MeSH
• proteinkinasy genetika   metabolismus   MeSH
• proteom metabolismus   MeSH
• Psychodidae parazitologie   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protein Cas9 MeSH
• proteinkinasy MeSH
• proteom MeSH

OBJECTIVES: This study aimed to assess vestibular function in 39 patients who underwent neurectomy for vestibular schwannoma. METHOD: Semicircular canal reactivity was measured by video head-impulse test using high-frequency passive head acceleration. Response gain was calculated as a ratio between the areas under the eye-velocity curve and the head-velocity curve. STATISTICAL ANALYSIS: Student t-test was used for to compare quantitative variables. ANOVA was used to test inter-group differences in categoric variables. RESULTS: In all cases, surgery-side gain on head impulse test was low, with increased gain asymmetry. A subgroup of 7 patients (18%) showed relatively high gain in vestibulo-ocular reflex on the surgery side. Caloric reaction was absent in all cases. These findings indicate that residual vestibular function can be conserved following vestibular schwannoma extirpation. CONCLUSION: Cases with moderate vestibulo-ocular reflex gain were a subgroup with partial conservation of vestibular nerve fibers. Whether this is a predictor of better functional prognosis remains to be elucidated. Higher gain correlated with less extensive surgery and sparing of the inferior vestibular nerve. Low gain correlated with complete vestibular neurectomy. This information may guide rehabilitation strategy following surgery.

• Klíčová slova
• Head-impulse test, Neurectomy, Vestibular schwannoma, Vestibulo-ocular reflex,
• MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• nervová vlákna MeSH
• neurochirurgické výkony metody   MeSH
• pooperační komplikace patofyziologie   MeSH
• pooperační období MeSH
• prognóza MeSH
• pulsní rotační test MeSH
• senioři MeSH
• vestibulární aparát patofyziologie   MeSH
• vestibulární funkční testy MeSH
• vestibulární schwannom patofyziologie   chirurgie   MeSH
• vestibulookulární reflex MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• křečci praví MeSH
• Leishmania donovani * MeSH
• Leishmania infantum * MeSH
• paromomycin farmakologie   MeSH
• Phlebotomus * MeSH
• Psychodidae * MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• paromomycin MeSH

Leishmania rely heavily on glycans to complete their digenetic life cycle in both mammalian and phlebotomine sand fly hosts. Leishmania promastigotes secrete a proteophosphoglycan-rich gel (Promastigote Secretory Gel, PSG) that is regurgitated during transmission and can exacerbate infection in the skin. Here we explored the role of PSG from natural Leishmania-sand fly vector combinations by obtaining PSG from Leishmania (L.) major-infected Phlebotomus (P.) papatasi and P. duboscqi and L. tropica-infected P. arabicus. We found that, in addition to the vector's saliva, the PSG from L. major and L. tropica potently exacerbated cutaneous infection in BALB/c mice, improved the probability of developing a patent cutaneous lesion, parasite growth and the evolution of the lesion. Of note, the presence of PSG in the inoculum more than halved the prepatent period of cutaneous L. tropica infection from an average of 32 weeks to 13 weeks. In addition, L. major and L. tropica PSG extracted from the permissive experimental vector, Lutzomyia (Lu.) longipalpis, also exacerbated infections in mice. These results reinforce and extend the hypothesis that PSG is an important and evolutionarily conserved component of Leishmania infection that can be used to facilitate experimental infection for drug and vaccine screening.

• Klíčová slova
• Cutaneous leishmaniasis, Leishmania, Leishmania major, Leishmania mexicana, Leishmania tropica, PSG, sand fly, transmission, zoonoses,
• MeSH
• kůže účinky léků   parazitologie   patologie   MeSH
• Leishmania major chemie   MeSH
• Leishmania tropica chemie   MeSH
• leishmanióza kožní parazitologie   patologie   MeSH
• membránové proteiny aplikace a dávkování   chemie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• parazitární zátěž MeSH
• Phlebotomus parazitologie   MeSH
• proteoglykany aplikace a dávkování   chemie   MeSH
• protozoální proteiny aplikace a dávkování   chemie   MeSH
• sliny MeSH
• syndrom vzplanutí nemoci MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH
• Ppg1 protein, Leishmania MeSH Prohlížeč
• proteoglykany MeSH
• protozoální proteiny MeSH

A 2-year longitudinal study of enzyme-linked immunosorbent assay (ELISA) antibodies against Phlebotomus perniciosus and Phlebotomus papatasi (Diptera: Psychodidae) sandfly saliva was performed in 32 Beagle dogs treated preventively with an imidacloprid-permethrin topical insecticide in an endemic area in Spain. Dogs were grouped into three sandfly exposure groups according to the time of inclusion in the study. Assays analysed immunoglobulin G (IgG) against salivary gland homogenates (SGH) of both species and recombinant P. papatasi rSP32 and P. perniciosus rSP03B proteins in serum. The dogs were participating in a Leishmania infantum (Kinetoplastida: Trypanosomatidae) vaccine trial and were experimentally infected with the parasite in the second year. No dog acquired natural L. infantum infections during the first year, but most developed anti-saliva antibodies, and median log-transformed optical densities (LODs) were seasonal, mimicking those of local sandflies. This indicates that the repellent efficacy of the insecticide used is below 100%. Multi-level modelling of LODs revealed variability among dogs, autocorrelation and differences according to the salivary antigen and the dog's age. However, dog seroprevalence, estimated using pre-exposure LODs as cut-offs, was relatively low. This, and the fact that dogs did not become naturally infected with L. infantum, would support the efficacy and usefulness of this imidacloprid-permethrin topical insecticide in canine leishmaniasis control.

• Klíčová slova
• Antibodies, antigens, dogs, insecticides, saliva, sandfly,
• MeSH
• biologické markery krev   MeSH
• dusíkaté sloučeniny aplikace a dávkování   farmakologie   MeSH
• kousnutí a bodnutí hmyzem prevence a kontrola   MeSH
• longitudinální studie MeSH
• neonikotinoidy aplikace a dávkování   farmakologie   MeSH
• permethrin aplikace a dávkování   farmakologie   MeSH
• Phlebotomus účinky léků   MeSH
• protilátky krev   účinky léků   MeSH
• psi imunologie   MeSH
• repelenty proti hmyzu aplikace a dávkování   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• psi imunologie   MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Španělsko MeSH
• Názvy látek
• biologické markery MeSH
• dusíkaté sloučeniny MeSH
• imidacloprid MeSH Prohlížeč
• neonikotinoidy MeSH
• permethrin MeSH
• protilátky MeSH
• repelenty proti hmyzu MeSH

Sandflies (Diptera: Psychodidae) (Newstead, 1911) are blood-feeding insects that transmit human pathogens including Leishmania (Trypanosomatida: Trypanosomatidae) parasites, causative agents of the leishmaniases. To elucidate Leishmania transmission cycles, conclusive identification of vector species is essential. Molecular approaches including matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) protein profiling have recently emerged to complement morphological identification. The aim of this study was to evaluate the effect of the trap type used to collect sandflies, specifically Centers for Disease Control (CDC) light or sticky traps, the two most commonly used in sandfly surveys, on subsequent MALDI-TOF MS protein profiling. Specimens of five species (Phlebotomus ariasi, Phlebotomus papatasi, Phlebotomus perniciosus, Phlebotomus sergenti, Sergentomyia minuta) collected in periurban and agricultural habitats in southeast Spain were subjected to protein profiling. Acquired protein spectra were queried against an in-house reference database and their quality assessed to evaluate the trap type effect. The results indicate that trap choice can substantially affect the quality of protein spectra in collected sandflies. Whereas specimens retrieved from light traps produced intense and reproducible spectra that allowed reliable species determination, profiles of specimens from sticky traps were compromised and often did not enable correct identification. Sticky traps should therefore not be used in surveys that deploy MALDI-TOF MS protein profiling for species identification.

• Klíčová slova
• MALDI-TOF MS protein profiling, Phlebotomus, light traps, species identification, sticky traps, trapping methods,
• MeSH
• odběr biologického vzorku metody   MeSH
• Psychodidae klasifikace   genetika   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice * MeSH
• stanovení celkové genové exprese * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Španělsko 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

The frequency of sandfly-host contacts can be measured by host antibody levels against sandfly salivary proteins. Recombinant salivary proteins are suggested to represent a valid replacement for salivary gland homogenate (SGH); however, it is necessary to prove that such antigens are recognized by antibodies against various populations of the same species. Phlebotomus perniciosus (Diptera: Psychodidae) is the main vector of Leishmania infantum (Trypanosomatida: Trypanosomatidae) in southwest Europe and is widespread from Portugal to Italy. In this study, sera were sampled from naturally exposed dogs from distant regions, including Campania (southern Italy), Umbria (central Italy) and the metropolitan Lisbon region (Portugal), where P. perniciosus is the unique or principal vector species. Sera were screened for anti-P. perniciosus antibodies using SGH and 43-kDa yellow-related recombinant protein (rSP03B). A robust correlation between antibodies recognizing SGH and rSP03B was detected in all regions, suggesting substantial antigenic cross-reactivity among different P. perniciosus populations. No significant differences in this relationship were detected between regions. Moreover, rSP03B and the native yellow-related protein were shown to share similar antigenic epitopes, as canine immunoglobulin G (IgG) binding to the native protein was inhibited by pre-incubation with the recombinant form. These findings suggest that rSP03B should be regarded as a universal marker of sandfly exposure throughout the geographical distribution of P. perniciosus.

• Klíčová slova
• Leishmania infantum, Mediterranean region, Phlebotomus spp., antibody response, dog, markers of exposure, salivary proteins, sandflies,
• MeSH
• antigeny analýza   MeSH
• hmyz - vektory parazitologie   fyziologie   MeSH
• hmyzí proteiny analýza   MeSH
• kousnutí a bodnutí hmyzem epidemiologie   etiologie   MeSH
• Leishmania infantum fyziologie   MeSH
• leishmanióza parazitologie   veterinární   MeSH
• nemoci psů epidemiologie   etiologie   MeSH
• Phlebotomus parazitologie   fyziologie   MeSH
• psi MeSH
• rekombinantní proteiny analýza   MeSH
• slinné proteiny a peptidy analýza   MeSH
• zvířata MeSH
• Check Tag
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Itálie epidemiologie   MeSH
• Portugalsko epidemiologie   MeSH
• Názvy látek
• antigeny MeSH
• hmyzí proteiny MeSH
• rekombinantní proteiny MeSH
• slinné proteiny a peptidy MeSH

Trypanosomes are known as widespread blood parasites of birds; however, knowledge of their prevalences in vectors and their overall biodiversity is rather limited. To assess the prevalences in potential vectors, we have microscopically examined ornithophilic bloodsucking Diptera (Culicidae, Simuliidae and Hippoboscidae) for the presence of trypanosomatids in their guts. In total, 3270 specimens were dissected, namely Culex pipiens Linnaeus, 1758 (n = 898), C. modestus Ficalbi, 1890 (136), Simulium vernum (Macquart, 1838) (1455), S. angustipes Edwards, 1915 (221) and Ornithomyia avicularia (Linnaeus, 1758) (560). All insect species were found to be infected with trypanosomatids, and the prevalence ranged from 4 to 8% but reached 60% in S. vernum. Blackflies and hippoboscids exclusively harboured trypanosomes (both T. cf. avium s.s. Danilewsky, 1885; T. corvi/culicavium group in hippoboscids). Mosquitoes were infected with T. culicavium Votypka, 2012 and T. avium s. l. but also with monoxenous parasites, namely Crithidia brevicula Frolov and Malysheva, 1989, and Paratrypanosoma confusum Votypka and Lukes, 2013. Only 4% of the isolated parasite strains were monoxenous whereas the majority were avian trypanosomes, confirming the vectorial status of the studied insects.

• Klíčová slova
• Crithidia, Culex, Ornithomyia, Paratrypanosoma, Simulium, Trypanosoma, kinetoplastida, prevalence,
• MeSH
• Culicidae parazitologie   MeSH
• Diptera parazitologie   MeSH
• dravci parazitologie   MeSH
• hmyz - vektory parazitologie   MeSH
• Simuliidae parazitologie   MeSH
• Trypanosomatina izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

Phlebotomine sandflies, vectors of Leishmania (Kinetoplastida: Trypanosomatidae) parasites that affect millions of people worldwide, breed in terrestrial biotopes. As immature stages are rarely accessible, the detection of their natural breeding sites is primarily based on findings of juvenile males with unrotated external genitalia. In males, permanent 180° rotation on the longitudinal body axis occurs soon after eclosion; however, no study has as yet addressed this aspect in detail. The present study describes the timing and duration of the rotation of male external genitalia in eight highly medically important sandfly species belonging to the genera Sergentomyia, Lutzomyia and Phlebotomus (all: Diptera: Psychodidae), kept under controlled laboratory conditions. The average duration of rotation was species-specific and varied from 12 h in Sergentomyia schwetzi to 33 h in Phlebotomus sergenti. Significant differences in rotation times were found among species, even between two closely related species of the subgenus Larroussius, Phlebotomus orientalis and Phlebotomus tobbi. The rotation of genitalia in all three studied genera was randomly oriented and similar numbers of clockwise and counter-clockwise events were observed. The study also addresses the effects of some external factors. In all species studied, rotation was not affected by the time of day of eclosion. Similarly, no differences in total rotation time were found between Phlebotomus papatasi males maintained at 25 and 20 °C, respectively. The present findings will assist in the search for natural breeding sites and in studies aimed at elucidating strategies for integrated sandfly and leishmaniasis control.

• Klíčová slova
• Lutzomyia, Phlebotomus, Sergentomyia, biology and behaviour, laboratory colony, leishmaniasis,
• MeSH
• kukla anatomie a histologie   růst a vývoj   MeSH
• mužské pohlavní orgány anatomie a histologie   růst a vývoj   MeSH
• Phlebotomus anatomie a histologie   růst a vývoj   MeSH
• Psychodidae anatomie a histologie   růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH