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

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

Následující kazuistické sdělení popisuje případ 69letého muže s viscerální leishmaniózou. Progresivně se horšící klinický stav nemocného spolu s chudým nálezem na zobrazovacích metodách a nespecifickými laboratorní nálezy vedly k diagnostickým úskalím tohoto v České republice raritně zachyceného infekčního onemocnění. Práce je doplněna o přehled recentních epidemiologických dat, diagnostických postupů a léčbu leishmaniózy.

This case report describes a 69-year-old man with visceral Leishmaniasis. The progressively worsening clinical condition of the patient, combined with nonspecific imaging findings and laboratory results, led to diagnostic difficulties in identifying this rarely encountered infectious disease in the Czech Republic. The paper is supplemented with an overview of recent epidemiological data, diagnostic procedures, and treatment of Leishmaniasis.

• MeSH
• Fever of Unknown Origin etiology   MeSH
• Leishmania infantum isolation & purification   MeSH
• Leishmaniasis, Visceral * diagnostic imaging   drug therapy   MeSH
• Humans MeSH
• Positron Emission Tomography Computed Tomography MeSH
• Renal Insufficiency etiology   MeSH
• Aged MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Aged MeSH
• Publication type
• Case Reports MeSH

Útlum kostní dřeně v sobě skrývá nepřeberné množství etiopatogenetických příčin, ve kterých se lékař snaží vyznat a porozumět jim. Kazuistika naší pacientky byla o to záludnější, že se jednalo o imunokompromitovanou osobu, jejíž klinický obraz a laboratorní výsledky byly zkresleny imunosupresivní terapií. V následujícím článku se snažíme upozornit na důležitost odebrání základních anamnestických údajů, které by neměly býti opomíjeny, a důkladné fyzikální a laboratorní vyšetřovací metody. Nemalou, ne-li nejdůležitější úlohu hraje vysoce erudované hematologické pracoviště, které nám s diagnózou a následným léčebným řešením velmi pomohlo a patří mu nemalé poděkování. Věřím, že následující článek obohatí každého lékaře různé odbornosti o velmi zajímavou a raritní záležitost.

Bone marrow decline hides a plethora of etiopathogenetic causes, which the physician tries to understand. Our patient's case was all the more tricky because she was an immunocompromised person whose clinical picture and laboratory results were distorted by immunosuppressive therapy. In the following article, we try to highlight the importance of taking basic anamnestic data that should not be neglected and thorough physical and laboratory investigations. The highly erudite haematology department played a major, if not the most important role and is greatly helped with the diagnosis and subsequent treatment and is to be thanked. I believe that the following article will enrich every physician of different specialties with a very interesting and rare matter.

• MeSH
• Amphotericin B pharmacology   therapeutic use   MeSH
• Immunosuppression Therapy MeSH
• Bone Marrow pathology   MeSH
• Leishmania isolation & purification   MeSH
• Leishmaniasis * diagnosis   drug therapy   complications   transmission   MeSH
• Humans MeSH
• Pancytopenia etiology   MeSH
• Aged MeSH
• Check Tag
• Humans MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Case Reports MeSH

African trypanosomes are medically important parasites that cause sleeping sickness in humans and nagana in animals. In addition to their pathogenic role, they have emerged as valuable model organisms for studying fundamental biological processes. Protein tagging is a powerful tool for investigating protein localization and function. In a previous study, we developed two plasmids for rapid and reproducible polymerase chain reaction-based protein tagging in trypanosomes, which enabled the subcellular mapping of 89% of the trypanosome proteome. However, the limited selection of fluorescent protein tags and selectable markers restricted the flexibility of this approach. Here, we present an extended set of >100 plasmids that incorporate universal primer annealing sequences, enabling protein tagging with a range of fluorescent, biochemical and epitope tags, using five different selection markers. We evaluated the suitability of various fluorescent proteins for live and fixed cell imaging, fluorescent movies, and we demonstrate the use of tagging plasmids encoding tandem epitope tags to support expansion microscopy approaches. We show that this series of plasmids is functional in other trypanosomatid parasites, significantly increasing its value. Finally, we developed a new plasmid for tagging glycosylphosphatidylinositol-anchored proteins. We anticipate that this will be an important toolset for investigating trypanosomatid protein localization and function.

• MeSH
• Humans MeSH
• Plasmids genetics   MeSH
• Protozoan Proteins * metabolism   genetics   MeSH
• Protein Transport MeSH
• Trypanosoma brucei brucei metabolism   genetics   MeSH
• Trypanosomatina * metabolism   genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

The exon junction complex (EJC) is a key player in metazoan mRNA quality control and is placed upstream of the exon-exon junction after splicing. Its inner core is composed of Magoh, Y14, eIF4AIII and BTZ and the outer core of proteins involved in mRNA splicing (CWC22), export (Yra1), translation (PYM) and nonsense mediated decay (NMD, UPF1/2/3). Trypanosoma brucei encodes only two genes with introns, but all mRNAs are processed by trans-splicing. The presence of three core EJC proteins and a potential BTZ homologue (Rbp25) in trypanosomes has been suggested to adapt of the EJC function to mark trans-spliced mRNAs. We analysed trypanosome EJC components and noticed major differences between eIF4AIII and Magoh/Y14: (i) whilst eIF4AIII is essential, knocking out both Magoh and Y14 elicits only a mild growth phenotype (ii) eIF4AIII localization is mostly nucleolar, while Magoh and Y14 are nucleolar and nucleoplasmic but excluded from the cytoplasm (iii) eIF4AIII associates with nucleolar proteins and the splicing factor CWC22, but not with Y14 or Magoh, while Magoh and Y14 associate with each other, but not with eIF4AIII, CWC22 or nucleolar proteins. Our data argue against the presence of a functional EJC in trypanosomes, but indicate that eIF4AIII adopted non-EJC related, essential functions, while Magoh and Y14 became redundant. Trypanosomes also possess homologues to the NMD proteins UPF1 and UPF2. Depletion of UPF1 causes only a minor reduction in growth and phylogenetic analyses show several independent losses of UPF1 and UPF2, as well as complete loss of UPF3 in the Kinetoplastida group, indicating that UPF1-dependent NMD is not essential. Regardless, we demonstrate that UPF1 depletion restores the mRNA levels of a PTC reporter. Altogether, we show that the almost intron-less trypanosomes are in the process of losing the canonical EJC/NMD pathways: Y14 and Magoh have become redundant and the still-functional UPF1-dependent NMD pathway is not essential.

• MeSH
• Eukaryotic Initiation Factor-4A metabolism   genetics   MeSH
• Exons genetics   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Nonsense Mediated mRNA Decay * MeSH
• Protozoan Proteins * metabolism   genetics   MeSH
• RNA Splicing MeSH
• Trypanosoma brucei brucei * metabolism   genetics   MeSH
• Publication type
• Journal Article MeSH

The olfactory response of insect vectors such as phlebotomine sand flies is a key facet for investigating their interactions with vertebrate hosts and associated vector-borne pathogens. Such studies are mainly performed by assessing the electrophysiological response and the olfactory behaviour of these arthropods towards volatile organic compounds (VOCs) produced by hosts. Nonetheless, few studies are available for species of the subgenera Lutzomyia and Nyssomyia in South America, leaving a void for Old World sand fly species of the genus Phlebotomus. In this study, we evaluated the olfactory responses of Phlebotomus perniciosus, one of the most important vectors of Leishmania infantum in the Old World. To test the P. perniciosus behavioural response to VOCs, 28 compounds isolated from humans and dogs were assessed using electrophysiological (i.e., electroantennogram, EAG) and behavioural assays (i.e., Y-tube olfactometer). In the EAG trials, 14 compounds (i.e., acetic acid, nonanoic acid, 2-propanol, 2-butanol, pentanal, hexanal, nonanal, trans-2-nonenal, decanal, myrcene, p-cymene, verbenone, 2-ethyl-1-hexanol, and acetonitrile) elicited high antennal responses (i.e., ≥ 0.30 mV) in female sand flies, being those VOCs selected for the behavioural assays. From the 14 compounds tested in the Y-tube olfactometer, nonanal was significantly attractive for P. perniciosus females, whereas myrcene and p-cymene were significantly repellents (p < 0.05). The attraction indexes varied from 0.53 for nonanal (i.e., most attractive) to -0.47 to p-cymene (i.e., most repellent). Overall, our results shed light on the role of olfactory cues routing host seeking behaviour in P. perniciosus, with implications to develop sustainable sand fly monitoring as well as control in leishmaniasis endemic areas.

• MeSH
• Behavior, Animal drug effects   MeSH
• Insect Vectors physiology   drug effects   MeSH
• Leishmania infantum drug effects   physiology   MeSH
• Humans MeSH
• Phlebotomus * physiology   drug effects   MeSH
• Dogs MeSH
• Volatile Organic Compounds * pharmacology   chemistry   analysis   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Dogs MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Leishmania is a genus of the family Trypanosomatidae that unites obligatory parasitic flagellates causing a variety of vector-borne diseases collectively called leishmaniasis. The symptoms range from relatively innocuous skin lesions to complete failures of visceral organs. The disease is exacerbated if a parasite harbors Leishmania RNA viruses (LRVs) of the family Pseudototiviridae. Screening a novel isolate of L. braziliensis, we revealed that it possesses not a toti-, but a bunyavirus of the family Leishbuviridae. To the best of our knowledge, this is a very first discovery of a bunyavirus infecting a representative of the Leishmania subgenus Viannia. We suggest that these viruses may serve as potential factors of virulence in American leishmaniasis and encourage researchers to test leishmanial strains for the presence of not only LRVs, but also other RNA viruses.

• MeSH
• Bunyaviridae classification   genetics   isolation & purification   MeSH
• Phylogeny MeSH
• Leishmania braziliensis * genetics   isolation & purification   MeSH
• Humans MeSH
• Orthobunyavirus genetics   classification   isolation & purification   physiology   MeSH
• RNA Viruses genetics   classification   isolation & purification   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Several new species of Leishmania have recently emerged in Europe, probably as the result of global changes and increased human migration from endemic areas. In this study, we tested whether two sand fly species, the Western Mediterranean Phlebotomus perniciosus and the Eastern Mediterranean P. tobbi, are competent vectors of L. donovani, L. major and L. martiniquensis. METHODOLOGY/PRINCIPAL FINDINGS: Sand flies were infected through the chick skin membrane using Leishmania species and strains of various geographical origins. Leishmania infections were evaluated by light microscopy and qPCR, and the representation of morphological forms was assessed from Giemsa-stained gut smears. Neither P. perniciosus nor P. tobbi supported the development of L. martiniquensis, but L. major and L. donovani in both species survived defecation of blood meal remnants, colonized the stomodeal valve and produced metacyclic stages. The results with L donovani have shown that infection rates in sand flies can be strain-specific; therefore, to determine vector competence or refractoriness, it is optimal to test at least two strains of Leishmania. CONCLUSIONS, SIGNIFICANCE: Both sand fly species tested are potential vectors of L. donovani and L. major in Mediterranean area. However, further studies will be needed to identify European vectors of L. martiniquensis and to test the ability of other European sand fly species to transmit L. major, L. donovani, L. tropica and L. infantum.

• MeSH
• Insect Vectors * parasitology   physiology   MeSH
• Chickens parasitology   MeSH
• Leishmania * physiology   classification   genetics   MeSH
• Leishmaniasis transmission   parasitology   MeSH
• Humans MeSH
• Phlebotomus * parasitology   physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Europe MeSH

We investigated gene expression patterns in Lutzomyia and Phlebotomus sand fly vectors of leishmaniases. Using quantitative PCR, we assessed the expression stability of potential endogenous control genes commonly used in dipterans. We analyzed Lutzomyia longipalpis and Phlebotomus papatasi samples from L3 and L4 larval stages, adult sand flies of different sexes, diets, dsRNA injection, and Leishmania infection. Six genes were evaluated: actin, α-tubulin, GAPDH, 60 S ribosomal proteins L8 and L32 (RiboL8 and RiboL32), and elongation factor 1-α (EF1-α). EF1-α was among the most stably expressed along with RiboL8 in L. longipalpis larvae and RiboL32 in adults. In P. papatasi, EF1-α and RiboL32 were the top in larvae, while EF1-α and actin were the most stable in adults. RiboL8 and actin were the most stable genes in dissected tissues and infected guts. Additionally, five primer pairs designed for L. longipalpis or P. papatasi were effective in PCR with Lutzomyia migonei, Phlebotomus duboscqi, Phlebotomus perniciosus, and Sergentomyia schwetzi cDNA. Furthermore, L. longipalpis RiboL32 and P. papatasi α-tubulin primers were suitable for qPCR with cDNA from the other four species. Our research provides tools to enhance relative gene expression studies in sand flies, facilitating the selection of endogenous control for qPCR.

• MeSH
• Genes, Essential * MeSH
• Insect Vectors genetics   MeSH
• Genes, Insect MeSH
• Larva genetics   MeSH
• Leishmania genetics   MeSH
• Phlebotomus * genetics   MeSH
• Psychodidae genetics   MeSH
• Gene Expression Profiling methods   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Sand flies (Diptera: Psychodidae: Phlebotominae) are blood-feeding insects that transmit the protozoan parasites Leishmania spp. and various arboviruses. The Balkan region, including the Republic of Kosovo, harbours a diverse sand fly fauna. Vector species of Leishmania infantum as well as phleboviruses are endemic; however, recent data are scarce. We performed a cross-sectional study to update the current sand fly distribution in Kosovo and assess biological as well as environmental factors associated with sand fly presence. CDC light trapping was conducted at 46 locations in 2022 and 2023, specifically targeting understudied regions in Kosovo. Individual morphological species identification was supported by molecular barcoding. The occurrence data of sand flies was used to create distribution maps and perform environmental analyses, taking elevation, wind speed and climate-related factors into account. In addition, PCR-based blood meal analysis and pathogen screening were conducted. Overall, 303 specimens of six sand fly species were trapped, predominated by Phlebotomus neglectus (97%). Barcodes from eight of nine known endemic sand fly species were obtained. Combining our data with previous surveys, we mapped the currently known sand fly distribution based on more than 4000 specimens at 177 data points, identifying Ph. neglectus and Ph. perfiliewi as the predominant species. Environmental analyses depicted two geographical groups of sand flies in Kosovo, with notable differences between the species. In total, 223 blood meals of five sand fly species were analysed. Of seven identified host species, the predominant blood meal source was observed to be cattle, but the DNA of dogs and humans, among others, was also detected. This study assessed biological as well as ecological factors of sand fly occurrence, which should help better understand and evaluate potential hot spots of disease transmission in Kosovo.

• MeSH
• Insect Vectors * physiology   parasitology   MeSH
• Leishmania infantum physiology   MeSH
• Phlebotomus * classification   physiology   parasitology   MeSH
• Cross-Sectional Studies MeSH
• Dogs MeSH
• Psychodidae physiology   parasitology   MeSH
• Animal Distribution * MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Dogs MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Kosovo MeSH