BACKGROUND: Ixodes inopinatus was described from Spain on the basis of morphology and partial sequencing of 16S ribosomal DNA. However, several studies suggested that morphological differences between I. inopinatus and Ixodes ricinus are minimal and that 16S rDNA lacks the power to distinguish the two species. Furthermore, nuclear and mitochondrial markers indicated evidence of hybridization between I. inopinatus and I. ricinus. In this study, we tested our hypothesis on tick dispersal from North Africa to Southern Europe and determined the prevalence of selected tick-borne pathogens (TBPs) in I. inopinatus, I. ricinus, and their hybrids. METHODS: Ticks were collected in Italy and Algeria by flagging, identified by sequencing of partial TROSPA and COI genes, and screened for Borrelia burgdorferi s.l., B. miyamotoi, Rickettsia spp., and Anaplasma phagocytophilum by polymerase chain reaction and sequencing of specific markers. RESULTS: Out of the 380 ticks, in Italy, 92 were I. ricinus, 3 were I. inopinatus, and 136 were hybrids of the two species. All 149 ticks from Algeria were I. inopinatus. Overall, 60% of ticks were positive for at least one TBP. Borrelia burgdorferi s.l. was detected in 19.5% of ticks, and it was significantly more prevalent in Ixodes ticks from Algeria than in ticks from Italy. Prevalence of Rickettsia spotted fever group (SFG) was 51.1%, with significantly greater prevalence in ticks from Algeria than in ticks from Italy. Borrelia miyamotoi and A. phagocytophilum were detected in low prevalence (0.9% and 5.2%, respectively) and only in ticks from Italy. CONCLUSIONS: This study indicates that I. inopinatus is a dominant species in Algeria, while I. ricinus and hybrids were common in Italy. The higher prevalence of B. burgdorferi s.l. and Rickettsia SFG in I. inopinatus compared with that in I. ricinus might be due to geographical and ecological differences between these two tick species. The role of I. inopinatus in the epidemiology of TBPs needs further investigation in the Mediterranean Basin.

• Klíčová slova
• Anaplasma phagocytophilum, B. miyamotoi, Borrelia burgdorferi s.l., Ixodes inopinatus, Ixodes ricinus, Rickettsia SFG, Algeria, Hybrids, Italy,
• MeSH
• Anaplasma phagocytophilum genetika   izolace a purifikace   klasifikace   MeSH
• Borrelia burgdorferi genetika   izolace a purifikace   klasifikace   MeSH
• Borrelia genetika   izolace a purifikace   klasifikace   MeSH
• hybridizace genetická MeSH
• klíště * mikrobiologie   MeSH
• nemoci přenášené klíšťaty epidemiologie   mikrobiologie   MeSH
• prevalence MeSH
• Rickettsia * izolace a purifikace   genetika   klasifikace   MeSH
• RNA ribozomální 16S genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Alžírsko epidemiologie   MeSH
• Itálie epidemiologie   MeSH
• Názvy látek
• RNA ribozomální 16S MeSH

BACKGROUND: Previous studies on the tick infestation of birds in the Carpathian Basin focused on songbirds (Passeriformes). Thus, the primary aim of the present work was to extend the scope of previous studies, i.e. to include aquatic (water-associated) bird species in a similar context, especially considering that these birds are usually long-distance migrants. METHODS: Between March 2021 and August 2023, 11,919 birds representing 126 species were checked for the presence of ticks. From 352 birds belonging to 40 species, 905 ixodid ticks were collected. Tick species were identified morphologically and/or molecularly. RESULTS: Ticks from avian hosts belonged to seven species: Ixodes ricinus (n = 448), I. frontalis (n = 31), I. festai (n = 2), I. arboricola (n = 36), I. lividus (n = 4), Haemaphysalis concinna (n = 382) and Dermacentor reticulatus (n = 2). Nymphs of I. ricinus occurred with a single activity peak around March-May, whereas its larvae typically infested birds in May, June or July. By contrast, H. concinna usually had its activity maximum during the summer (nymphs in June-July, larvae later in July-August). Interestingly, two ornithophilic species, I. frontalis and I. arboricola, were most active around winter months (between October and April). A significantly lower ratio of aquatic birds was found tick-infested than songbirds. Several new tick-host associations were revealed, including I. ricinus from Greylag Goose (Anser anser) and D. reticulatus from Great Egret (Ardea alba) and Sedge Warbler (Acrocephalus schoenobaenus). Ticks were collected for the first time in Europe from two species of predatory birds as well as from Little Bittern (Ixobrychus minutus). Bird species typically inhabiting reedbeds were most frequently infested with H. concinna, and most ticks localized at their throat, as opposed to forest-dwelling avian hosts, on which I. ricinus predominated and ticks were more evenly distributed. CONCLUSIONS: In the evaluated region, aquatic birds appear to be less important in tick dispersal than songbirds. However, newly revealed tick-host associations in this category attest to their hitherto neglected contribution. The results suggest that the habitat type will have significant impact not only on the species composition but also on the feeding location of ticks on birds.

• Klíčová slova
• Accipitriformes, Ixodidae, Passeriformes, Pelecaniformes,
• MeSH
• ekosystém MeSH
• husy MeSH
• infestace klíšťaty * epidemiologie   veterinární   MeSH
• Ixodidae * MeSH
• klíště * MeSH
• larva MeSH
• nymfa MeSH
• Passeriformes * MeSH
• zpěvní ptáci * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Ticks (Family Ixodidae) spend most of their life cycle as immature stages in the soil and litter, and as any other soil invertebrates, are likely to be controlled top-down by soil-dwelling predators. To date, the ability of soil invertebrate predators to control ixodid tick population remains little known, partly due to methodological difficulties. In the current study, we developed and successfully tested a novel method of labeling live Ixodes ricinus (L., 1758) (Ixodida: Ixodidae) nymphs with a 15N isotope label. Labeled ticks were used in a small-scale 8-day-long microcosm experiment to reveal soil predators attacking nymphs. Only a small fraction (4.1% of all samples) of soil generalist predators preyed upon nymphs. A strong 15N label was found in 5 predator species, namely 2 spiders (Pachygnatha listeri Sundevall, 1830, Tetragnathidae and Ozyptila sp., Theridiidae), 2 gamasid mites (Pergamasus beklemischevi Sellnick, 1929 and Pergamasus quisquiliarum [Canestrini, 1882], Parasitidae), and 1 staphylinid beetle (Geostiba circellaris [Gravenhorst, 1806], Staphylinidae). The isotopic labeling can be a useful tool in revealing a range of invertebrate predators that can control tick populations in soil.

• Klíčová slova
• Ixodes ricinus, laboratory experiment, natural enemies, soil food webs, tick ecology,
• MeSH
• brouci * MeSH
• Ixodidae * MeSH
• izotopové značení MeSH
• klíště * MeSH
• nymfa MeSH
• půda MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• půda MeSH

The structure and biochemical properties of protease inhibitors from the thyropin family are poorly understood in parasites and pathogens. Here, we introduce a novel family member, Ir-thyropin (IrThy), which is secreted in the saliva of Ixodes ricinus ticks, vectors of Lyme borreliosis and tick-borne encephalitis. The IrThy molecule consists of two consecutive thyroglobulin type-1 (Tg1) domains with an unusual disulfide pattern. Recombinant IrThy was found to inhibit human host-derived cathepsin proteases with a high specificity for cathepsins V, K, and L among a wide range of screened cathepsins exhibiting diverse endo- and exopeptidase activities. Both Tg1 domains displayed inhibitory activities, but with distinct specificity profiles. We determined the spatial structure of one of the Tg1 domains by solution NMR spectroscopy and described its reactive center to elucidate the unique inhibitory specificity. Furthermore, we found that the inhibitory potency of IrThy was modulated in a complex manner by various glycosaminoglycans from host tissues. IrThy was additionally regulated by pH and proteolytic degradation. This study provides a comprehensive structure-function characterization of IrThy-the first investigated thyropin of parasite origin-and suggests its potential role in host-parasite interactions at the tick bite site.

• Klíčová slova
• cathepsin, cysteine protease, parasite, protease inhibitor, protein structure, saliva, thyropin, tick,
• MeSH
• cystein MeSH
• glykosaminoglykany MeSH
• kathepsiny metabolismus   MeSH
• klíště * metabolismus   MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• sliny * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cystein MeSH
• glykosaminoglykany MeSH
• kathepsiny MeSH

Ticks are blood-feeding arachnids that are known to transmit various pathogenic microorganisms to their hosts. During blood feeding, ticks activate their metabolism and immune system to efficiently utilise nutrients from the host's blood and complete the feeding process. In contrast to insects, in which the fat body is known to be a central organ that controls essential metabolic processes and immune defense mechanisms, the function of the fat body in tick physiology is still relatively unexplored. To fill this gap, we sought to uncover the repertoire of genes expressed in the fat body associated with trachea (FB/Tr) by analyzing the transcriptome of individual, partially fed (previtellogenic) Ixodes ricinus females. The resulting catalog of individual mRNA sequences reveals a broad repertoire of transcripts encoding proteins involved in nutrient storage and distribution, as well as components of the tick immune system. To gain a detailed insight into the secretory products of FB/Tr specifically involved in inter-tissue transport and humoral immunity, the transcriptomic data were complemented with the proteome of soluble proteins in the hemolymph of partially fed female ticks. Among these proteins, the hemolipoglyco-carrier proteins were predominant. When comparing immune peptides and proteins from the fat body with those produced by hemocytes, we found that the fat body serves as a unique producer of certain immune components. Finally, time-resolved transcriptional regulation of selected immune transcripts from the FB/Tr was examined in response to experimental challenges with model microbes and analyzed by RT-qPCR. Overall, our data show that the fat body of ticks, similar to insects, is an important metabolic tissue that also plays a remarkable role in immune defense against invading microbes. These findings improve our understanding of tick biology and its impact on the transmission of tick-borne pathogens.

• MeSH
• hemolymfa * MeSH
• klíště * genetika   metabolismus   MeSH
• proteiny členovců genetika   metabolismus   MeSH
• proteomika MeSH
• stanovení celkové genové exprese MeSH
• tukové těleso metabolismus   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny členovců MeSH

Ticks are hematophagous arthropods that use a complex mixture of salivary proteins to evade host defenses while taking a blood meal. Little is known about the immunological and physiological consequences of tick feeding on humans. Here, we performed the first bulk and single-nucleus RNA sequencing (snRNA-seq) of skin and blood of four persons presenting with naturally acquired, attached Ixodes scapularis ticks. Pathways and individual genes associated with innate and adaptive immunity were identified based on bulk RNA sequencing, including interleukin-17 signaling and platelet activation pathways at the site of tick attachment or in peripheral blood. snRNA-seq further revealed that the Hippo signaling, cell adhesion, and axon guidance pathways were involved in the response to an I. scapularis bite in humans. Features of the host response in these individuals also overlapped with that of laboratory guinea pigs exposed to I. scapularis and which acquired resistance to ticks. These findings offer novel insights for the development of new biomarkers for I. scapularis exposure and anti-tick vaccines for human use.

• Klíčová slova
• RNA-seq, acquired resistance, human, immune pathways, tick, tick-borne pathogens,
• MeSH
• klíště * genetika   MeSH
• kousnutí klíštětem * MeSH
• lidé MeSH
• morčata MeSH
• RNA malá jaderná MeSH
• sekvence nukleotidů MeSH
• stravovací zvyklosti fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• morčata MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA malá jaderná MeSH

Ticks are ectoparasites that feed on blood and have an impressive ability to consume and process enormous amounts of host blood, allowing extremely long periods of starvation between blood meals. The central role in the parasitic lifestyle of ticks is played by the midgut. This organ efficiently stores and digests ingested blood and serves as the primary interface for the transmission of tick-borne pathogens. In this study, we used a label-free quantitative approach to perform a novel dynamic proteomic analysis of the midgut of Ixodesricinus nymphs, covering their development from unfed to pre-molt stages. We identified 1534 I. ricinus-specific proteins with a relatively low proportion of host proteins. This proteome dataset, which was carefully examined by manual scrutiny, allowed precise annotation of proteins important for blood meal processing and their dynamic changes during nymphal ontogeny. We focused on midgut molecules related to lipid hydrolysis, storage, and transport, opening a yet unexplored avenue for studying lipid metabolism in ticks. Further dynamic profiling of the tick's multi-enzyme digestive network, protease inhibitors, enzymes involved in redox homeostasis and detoxification, antimicrobial peptides, and proteins responsible for midgut colonization by Borrelia spirochetes promises to uncover new targets for targeting tick nymphs, the most critical life stage for transmission the pathogens that cause tick-borne diseases.

• Klíčová slova
• Borrelia, Ixodes, antimicrobial peptides, label-free quantification, lipid metabolism, midgut, protease inhibitors, proteases, proteome, ticks,
• MeSH
• klíště * parazitologie   MeSH
• proteom MeSH
• proteomika MeSH
• trávicí systém MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteom MeSH

Tick saliva injected into the vertebrate host contains bioactive anti-proteolytic proteins from the cystatin family; however, the molecular basis of their unusual biochemical and physiological properties, distinct from those of host homologs, is unknown. Here, we present Ricistatin, a novel secreted cystatin identified in the salivary gland transcriptome of Ixodes ricinus ticks. Recombinant Ricistatin inhibited host-derived cysteine cathepsins and preferentially targeted endopeptidases, while having only limited impact on proteolysis driven by exopeptidases. Determination of the crystal structure of Ricistatin in complex with a cysteine cathepsin together with characterization of structural determinants in the Ricistatin binding site explained its restricted specificity. Furthermore, Ricistatin was potently immunosuppressive and anti-inflammatory, reducing levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α and nitric oxide in macrophages; IL-2 and IL-9 levels in Th9 cells; and OVA antigen-induced CD4+ T cell proliferation and neutrophil migration. This work highlights the immunotherapeutic potential of Ricistatin and, for the first time, provides structural insights into the unique narrow selectivity of tick salivary cystatins determining their bioactivity.

• Klíčová slova
• Cystatins, Host–parasite interactions, Ixodes ricinus, Protease inhibition, Protein structure, Tick saliva,
• MeSH
• cystatiny * farmakologie   MeSH
• cystein metabolismus   MeSH
• endopeptidasy metabolismus   MeSH
• kathepsiny metabolismus   MeSH
• klíště * chemie   MeSH
• obratlovci MeSH
• proteasy metabolismus   MeSH
• slinné cystatiny chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cystatiny * MeSH
• cystein MeSH
• endopeptidasy MeSH
• kathepsiny MeSH
• proteasy MeSH
• slinné cystatiny MeSH

BACKGROUND: Ixodes ricinus is an important vector of several pathogens, primarily in Europe. Recently, Ixodes inopinatus was described from Spain, Portugal, and North Africa and then reported from several European countries. In this study, a multiplex polymerase chain reaction (PCR) was developed to distinguish I. ricinus from I. inopinatus and used in the surveillance of I. inopinatus in Algeria (ALG) and three regions in the Czech Republic (CZ). METHODS: A multiplex PCR on TROSPA and sequencing of several mitochondrial (16S rDNA, COI) and nuclear markers (TROSPA, ITS2, calreticulin) were used to differentiate these two species and for a subsequent phylogenetic analysis. RESULTS: Sequencing of TROSPA, COI, and ITS2 separated these two species into two subclades, while 16S rDNA and calreticulin could not distinguish I. ricinus from I. inopinatus. Interestingly, 23 nucleotide positions in the TROSPA gene had consistently double peaks in a subset of ticks from CZ. Cloning of these PCR products led to a clear separation of I. ricinus and I. inopinatus indicating hybridization and introgression between these two tick taxa. Based on a multiplex PCR of TROSPA and analysis of sequences of TROSPA, COI, and ITS2, the majority of ticks in CZ were I. ricinus, no I. inopinatus ticks were found, and 10 specimens showed signs of hybridization. In contrast, most ticks in ALG were I. inopinatus, four ticks were I. ricinus, and no signs of hybridization and introgression were detected. CONCLUSIONS: We developed a multiplex PCR method based on the TROSPA gene to differentiate I. ricinus and I. inopinatus. We demonstrate the lack of evidence for the presence of I. inopinatus in Central Europe and propose that previous studies be re-examined. Mitochondrial markers are not suitable for distinguishing I. inopinatus from I. ricinus. Furthermore, our data indicate that I. inopinatus and I. ricinus can hybridize, and the hybrids can survive in Europe.

• Klíčová slova
• 16S rDNA, Algeria, COI, Czech Republic, ITS2, Ixodes inopinatus, Ixodes ricinus, TROSPA, Tick,
• MeSH
• fylogeneze MeSH
• klíště * MeSH
• multiplexová polymerázová řetězová reakce MeSH
• ribozomální DNA genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Evropa MeSH
• Názvy látek
• ribozomální DNA MeSH

BACKGROUND: Vector-borne zoonotic diseases are a concerning issue in Europe. Lyme disease and tick-borne encephalitis virus (TBEV) have been reported in several countries with a large impact on public health; other emerging pathogens, such as Rickettsiales, and mosquito-borne flaviviruses have been increasingly reported. All these pathogens are linked to wild ungulates playing roles as tick feeders, spreaders, and sentinels for pathogen circulation. This study evaluated the prevalence of TBEV, Borrelia burgdorferi sensu lato, Rickettsia spp., Ehrlichia spp., and Coxiella spp. by biomolecular screening of blood samples and ticks collected from wild ungulates. Ungulates were also screened by ELISA and virus neutralization tests for flaviviral antibody detection. RESULTS: A total of 274 blood samples were collected from several wild ungulate species, as well as 406 Ixodes ricinus, which were feeding on them. Blood samples tested positive for B. burgdorferi s.l. (1.1%; 0-2.3%) and Rickettsia spp. (1.1%; 0-2.3%) and showed an overall flaviviral seroprevalence of 30.6% (22.1-39.2%): 26.1% (17.9-34.3%) for TBEV, 3.6% (0.1-7.1%) for Usutu virus and 0.9% (0-2.7%) for West Nile virus. Ticks were pooled when possible and yielded 331 tick samples that tested positive for B. burgdorferi s.l. (8.8%; 5.8-11.8%), Rickettsia spp. (26.6%; 21.8-31.2%) and Neoehrlichia mikurensis (1.2%; 0-2.4%). TBEV and Coxiella spp. were not detected in either blood or tick samples. CONCLUSIONS: This research highlighted a high prevalence of several tick-borne zoonotic pathogens and high seroprevalence for flaviviruses in both hilly and alpine areas. For the first time, an alpine chamois tested positive for anti-TBEV antibodies. Ungulate species are of particular interest due to their sentinel role in flavivirus circulation and their indirect role in tick-borne diseases and maintenance as Ixodes feeders and spreaders.

• Klíčová slova
• Flavivirus, Ixodes ricinus, Molecular biology, Vector-borne zoonotic pathogens, Virus neutralization test, Wild ungulates,
• MeSH
• Coxiella MeSH
• klíště * MeSH
• komáří přenašeči MeSH
• Rickettsia * MeSH
• savci MeSH
• séroepidemiologické studie MeSH
• viry klíšťové encefalitidy * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Evropa MeSH