BACKGROUND: We evaluated various membranes for blood-feeding in nine sand fly species from different genera and subgenera. Most of these species are vectors of human-pathogenic Leishmania, whereas Sergentomyia minuta is a herpetophilic sand fly species and a proven vector of Leishmania (Sauroleishmania) tarentolae. METHODS: Female sand flies were offered blood through a range of membranes (chicken, reptilian, and frog skin; synthetic collagen; pig intestine; and duck foot webbing). Two feeding systems (glass feeder, Hemotek) and different blood sources (human, ovine, avian, and reptilian) were used. Feeding trials were conducted under varying thermal and light conditions to determine the optimal parameters. RESULTS: Among the 4950 female S. minuta tested, only a negligible fraction took a blood meal: 2% of the females fed on avian blood, and 0.2% of the females fed on human blood. In eight other species, the chicken membrane was generally more effective than synthetic membranes or pig intestines. For example, Phlebotomus duboscqi refused synthetic membranes, while Lutzomyia longipalpis and P. perniciosus avoided both synthetic membranes and pig intestines. The most effective membrane was duck foot webbing, with four species feeding more readily through it than through the chicken membrane. Additionally, applying coagulated blood plasma to the outer surface of chicken or synthetic membranes significantly increased feeding rates. CONCLUSIONS: Female S. minuta did not reliably feed on blood through the tested membranes, preventing laboratory infection experiments from confirming their vector competence for human-pathogenic Leishmania. However, for future experimental infections of other sand fly species, duck foot webbing has emerged as an effective membrane, and the application of blood plasma to the exterior of membranes may increase the feeding rates.

• Klíčová slova
• Leishmania, Lutzomyia, Phlebotomus, Sergentomyia minuta, Artificial feeding, Vector competence,
• MeSH
• krev * MeSH
• lidé MeSH
• membrány MeSH
• prasata MeSH
• Psychodidae * fyziologie   MeSH
• stravovací zvyklosti * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Equids may be infected by zoonotic Leishmania spp., including Leishmania infantum, in regions where canine leishmaniasis (CanL) is endemic, and Leishmania martiniquensis, which has been reported in horses from Central Europe. This study was designed to evaluate the occurrence of both Leishmania spp. among equids living in CanL endemic areas of Italy, as well as to identify dipteran vectors from the same habitats. From March to October 2023, blood, serum and tissue samples from skin lesions were collected from equids (n = 98; n = 56 donkeys and n = 42 horses) living in Italy, as well as sand flies and biting midges. Blood samples (n = 98) and skin lesions (n = 56) were tested for Leishmania spp. by conventional and real time PCRs and sera were tested by immunofluorescence antibody tests (IFAT) for both L. infantum and L. martiniquensis. Insects were morphologically identified, and female specimens (n = 268 sand flies, n = 7 biting midges) analyzed for Leishmania DNA, as well as engorged sand flies (n = 16) for blood-meal detection. Two animals with skin lesions (i.e., one donkey and one horse) scored positive for Leishmania spp. DNA, and 19 animals (i.e., 19.4%; n = 13 donkeys and n = 6 horses) were seropositive for L. infantum, with five of them also for L. martiniquensis. Most seropositive animals had no dermatological lesions (i.e., 68.4%) while both animals molecularly positive for Leishmania spp. scored seronegative. Of the 356 sand flies collected, 12 females (i.e., n = 8 Sergentomyia minuta; n = 3 Phlebotomus perniciosus, n = 1 Phlebotomus perfiliewi) were positive for Leishmania spp. DNA, and one out of seven biting midges collected was DNA-positive for L. infantum. Moreover, engorged sand flies scored positive for human and equine DNA. Data suggest that equids living in CanL endemic areas are exposed to Leishmania spp., but their role in the circulation of the parasite needs further investigations.

• MeSH
• Ceratopogonidae parazitologie   MeSH
• endemické nemoci veterinární   MeSH
• Equidae * parazitologie   MeSH
• hmyz - vektory * parazitologie   MeSH
• koně parazitologie   MeSH
• Leishmania infantum izolace a purifikace   genetika   MeSH
• Leishmania * izolace a purifikace   genetika   klasifikace   MeSH
• leishmanióza * veterinární   epidemiologie   parazitologie   přenos   MeSH
• nemoci koní parazitologie   epidemiologie   MeSH
• nemoci psů * parazitologie   epidemiologie   přenos   MeSH
• psi MeSH
• Psychodidae parazitologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• psi MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Itálie epidemiologie   MeSH

Leishmania parasites, causative agents of leishmaniasis, are currently divided into four subgenera: Leishmania, Viannia, Sauroleishmania and Mundinia. The recently established subgenus Mundinia has a wide geographical distribution and contains five species, three of which have the potential to infect and cause disease in humans. While the other Leishmania subgenera are transmitted exclusively by phlebotomine sand flies (Diptera: Psychodidae), natural vectors of Mundinia remain uncertain. This study investigates the potential of sand flies and biting midges of the genus Culicoides (Diptera: Ceratopogonidae) to transmit Leishmania parasites of the subgenus Mundinia. Sand flies (Phlebotomus argentipes, P. duboscqi and Lutzomyia migonei) and Culicoides biting midges (Culicoides sonorensis) were exposed to five Mundinia species through a chicken skin membrane and dissected at specific time intervals post bloodmeal. Potentially infected insects were also allowed to feed on ear pinnae of anaesthetized BALB/c mice and the presence of Leishmania DNA was subsequently confirmed in the mice using polymerase chain reaction analyses. In C. sonorensis, all Mundinia species tested were able to establish infection at a high rate, successfully colonize the stomodeal valve and produce a higher proportion of metacyclic forms than in sand flies. Subsequently, three parasite species, L. martiniquensis, L. orientalis and L. sp. from Ghana, were transmitted to the host mouse ear by C. sonorensis bite. In contrast, transmission experiments entirely failed with P. argentipes, although colonisation of the stomodeal valve was observed for L. orientalis and L. martiniquensis and metacyclic forms of L. orientalis were recorded. This laboratory-based transmission of Mundinia species highlights that Culicoides are potential vectors of members of this ancestral subgenus of Leishmania and we suggest further studies in endemic areas to confirm their role in the lifecycles of neglected pathogens.

• MeSH
• Ceratopogonidae parazitologie   MeSH
• hmyz - vektory parazitologie   MeSH
• Leishmania * MeSH
• leishmanióza přenos   MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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

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

BACKGROUND: Leishmania virulence factors responsible for the complicated epidemiology of the various leishmaniases remain mainly unidentified. This study is a characterization of a gene previously identified as upregulated in two of three overlapping datasets containing putative factors important for Leishmania's ability to establish mammalian intracellular infection and to colonize the gut of an insect vector. METHODOLOGY/PRINCIPAL FINDINGS: The investigated gene encodes ATP/GTP binding motif-containing protein related to Leishmania development 1 (ALD1), a cytosolic protein that contains a cryptic ATP/GTP binding P-loop. We compared differentiation, growth rates, and infective abilities of wild-type and ALD1 null mutant cell lines of L. mexicana. Loss of ALD1 results in retarded growth kinetics but not defects in differentiation in axenic culture. Similarly, when mice and the sand fly vector were infected with the ALD1 null mutant, the primary difference in infection and colonization phenotype relative to wild type was an inability to achieve maximal host pathogenicity. While ability of the ALD1 null mutant cells to infect macrophages in vitro was not affected, replication within macrophages was clearly curtailed. CONCLUSIONS/SIGNIFICANCE: L. mexicana ALD1, encoding a protein with no assigned functional domains or motifs, was identified utilizing multiple comparative analyses with the related and often experimentally overlooked monoxenous flagellates. We found that it plays a role in Leishmania infection and colonization in vitro and in vivo. Results suggest that ALD1 functions in L. mexicana's general metabolic network, rather than function in specific aspect of virulence as anticipated from the compared datasets. This result validates our comparative genomics approach for finding relevant factors, yet highlights the importance of quality laboratory-based analysis of genes tagged by these methods.

• MeSH
• hmyz - vektory parazitologie   MeSH
• Leishmania mexicana genetika   patogenita   MeSH
• leishmanióza kožní parazitologie   MeSH
• makrofágy parazitologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• proteiny vázající GTP genetika   metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• Psychodidae parazitologie   MeSH
• virulence MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny vázající GTP MeSH
• protozoální proteiny MeSH