Avian louse flies (Hippoboscidae: Ornithomyinae) are blood-feeding parasites with largely unexplored vector potential, although they have been implicated in the transmission of pathogens of medical and veterinary relevance, such as West-Nile virus, Babesia and Trypanosoma species. We screened 253 specimens of nine Ornithomyinae species for selected bacteria (Anaplasma, Rickettsia, Bartonella, Borrelia, and Ehrlichia spp.) as well as for trypanosomes and piroplasms. Anaplasma phagocytophilum was detected in a single Ornithomya avicularia specimen, and Haematospirillum jordaniae was identified in three Ornithomya fringillina individuals from the same location, representing the first report of these bacteria in avian louse flies. In addition, eight different Trypanosoma sequences were obtained, belonging to the T. corvi/culicavium and T. bennetti groups. Unexpectedly, a sequence resembling T. theileri was recovered from an Ornithoica turdi specimen. Our findings suggest that the role of louse flies in transmitting the investigated bacterial pathogens is likely minimal, but their involvement in the ecology of Trypanosoma species warrants further study. Consistent with previous work, we propose that louse flies could serve as valuable sentinels for monitoring pathogens in wild bird populations.

Background Potentially zoonotic pathogens have been previously detected in bat-associated ticks, but their role in disease transmission and their frequency of feeding on non-bat hosts is poorly known. Methodology/Principal findings We used molecular blood meal analysis to reveal feeding patterns of the bat-associated tick species Ixodes ariadnae, I. simplex, and I. vespertilionis collected from cave and mine walls in Central and Southeastern Europe. Vertebrate DNA, predominantly from bats, was detected in 43.5% of the samples (70 of 161 ticks) but in these ticks we also detected the DNA of non-chiropteran hosts, such as dog, Canis lupus familiaris, wild boar, Sus scrofa, and horse, Equus caballus, suggesting that bat-associated ticks may exhibit a much broader host range than previously thought, including domestic and wild mammals. Furthermore, we detected the zoonotic bacteria Neoehrlichia mikurensis in bat ticks for the first time, and other bacteria, such as Bartonella and Wolbachia. Conclusions/Significance In the light of these findings, the role of bat ticks as disease vectors should be urgently re-evaluated in more diverse host systems, as they may contribute to pathogen transmission between bats and non-chiropteran hosts.

Birds play a crucial role in disseminating ticks that carry pathogens of high veterinary-medical importance. The aim of this study was to analyze data of a long-term tick collection from birds at a single stop-over site in Central Europe, Hungary. Over eight years (2015–2022) 5833 ticks (ten species) were collected from 2395 tick-infested birds. The most abundant species were Ixodes ricinus (n = 3971) and Haemaphysalis concinna (n = 1706). Ixodes ricinus nymphs and larvae were the most frequently occurring on resident and short-distance migratory birds with forest habitat but Ha. concinna was the most abundant species on reed-associated, long-distance migrants. Haemaphysalis concinna occurred mostly on birds feeding above the ground level, while I. ricinus predominated on ground feeding birds. Infestation with I. ricinus nymphs always peaked in the first half of the year, in contrast to larvae which were more abundant on avian hosts in the autumn. At the same time, Ha. concinna larvae and nymphs had their peak numbers in the summer. This is the first long-term study on the tick infestation of birds in Central Europe. The study shows that, migration distance, habitat type, and typical feeding level of birds, as well as characteristics of tick life cycle are all key factors in the role of birds as tick disseminators. It was revealed that Savi’s Warbler ( Locustella luscinioides ) is the most frequent hosts of Ha. concinna in Central Hungary.

Background Louse flies (Diptera, Hippoboscidae) are important blood-sucking parasites of birds and mammals with a worldwide distribution. The aim of our study was to collect louse flies from birds across multiple sites in Hungary and evaluate the effects of avian traits on louse fly-host relationships. Methods Between 2015 and 2022, 237 louse flies were collected from birds at multiple locations in Hungary. The louse flies were identified to species level by morphological and molecular methods. Louse fly species and their seasonal dynamics were analyzed. Results Six louse fly species were identified: Ornithomya avicularia, Ornithomya fringillina, Ornithomya biloba, Ornithomya chloropus, Ornithoica turdi and Ornithoctona laticornis. Results of statistical analyses indicated that habitat, migration habits and the feeding places of birds have significant effects on their possible role as hosts of O. avicularia, O. fringillina and O. turdi. Analysis of the temporal distribution of avian louse flies showed different seasonal patterns according to species. Phylogenetic analyses highlighted that O. turdi clustered separately from other members of the subfamily Ornithomyinae which thus did not form a monophyletic group. Conclusions This study presents one of the longest continuous collections of ornithophilic louse fly species in Europe so far. Avian traits were shown to influence louse-fly infestation. To our best knowledge, this is the first report on O. laticornis in Europe. The ability of this African louse fly species to survive in Europe, as demonstrated in the present study, may be an indication of its future establishment. Our findings, in accordance with previous reports, also indicated that the subfamily Ornithomyinae should be taxonomically revised.

AbstractBackgroundReports on adultHyalommaticks in certain regions of the Carpathian Basin date back to the 19thcentury. These ticks were thought to emerge from nymphs dropping from birds, then molting to adults. Although the role of migratory birds in carrying ticks of this genus is known from all parts of Europe, in most countries no contemporaneous multiregional surveillance of bird-associated ticks was reported which could allow the recognition of hotspots in this context.MethodsIxodid ticks were collected from birds at seven ringing stations in Hungary, including both the spring and autumn migration period in 2022.IxodesandHaemaphysalisspecies were identified morphologically, whereasHyalommaspecies molecularly.ResultsFrom 38 passeriform bird species 957 ixodid ticks were collected. The majority of developmental stages were nymphs (n=588), but 353 larvae and 16 females were also present. On most birds (n=381) only a single tick was found and the maximum number of ticks removed from the same bird was 30. Tick species were identified asIxodes ricinus(n=598),Ixodes frontalis(n=18),Ixodes lividus(n=6),Haemaphysalis concinna(n=322), andD. reticulatus(n=1). All twelveHyalommasp. ticks (11 engorged nymphs and an unengorged larva) were identified asHyalomma rufipesbased on three mitochondrial markers. This species was only found in the Transdanubian region and along its southeastern border. The Common Blackbird (Turdus merula) and the European Robin (Erithacus rubecula) were the two main hosts ofI. ricinusandI. frontalis, whereasH. concinnawas almost exclusively collected form long-distance migrants. The predominant hosts ofH. rufipeswere reed-associated bird species, the Sedge Warbler (Acrocephalus schoenobaenus) and the Bearded Reedling (Panurus biarmicus), both harboring these ticks at the end of June (i.e., the nesting period) in southwestern Hungary.ConclusionsThis study provides ornithological explanation for the regional, century-long presence of adultHyalommaticks under continental climate in the Transdanubian Region of the Carpathian Basin. More importantly, the autochthonous occurrence of aH. rufipespopulation was revealed for the first time in Europe, based on the following observations: (1) the bird species infested withH. rufipesare not known to migrate during their nesting period; (2) one larva was not yet engorged; (3) the larva and the nymphs must have belonged to different local generations; and (4) allH. rufipesfound in the relevant location were identical in their haplotypes based on three maternally inherited mitochondrial markers, probably reflecting founder effect. This study also demonstrated that the species of ticks carried by birds were significantly different between collection sites even within a geographically short distance (200 km). Therefore, within a country multiregional monitoring is inevitable to assess the overall epidemiological significance of migratory birds in importing exotic ticks, and also in maintaining newly established tick species.

In Eurasia, the geographically most widespread ixodid tick species of the bat families Rhinolophidae Gray, Vespertilionidae Gray, and Miniopteridae Dobson were considered to belong to four species, Ixodes vespertilionis Koch, I. collaris Hornok, I. ariadnae Hornok, and I. simplex Neumann. Previous data attest that bat-associated tick species from Eastern Asia show remarkable genetic difference from the above four tick species, but in the absence of detailed morphological comparison these were regarded as conspecific. In this study we compensate for this lack of data on three bat-associated tick species, reporting their morphological comparison, as well as molecular and phylogenetic relationships. According to the results we describe the females of three tick species new to science, i.e., I. nipponrhinolophi Hornok & Takano, sp. nov., I. fuliginosus Hornok & Takano, sp. nov., and I. fujitai Hornok & Takano, sp. nov. In case of all three new tick species the cytochrome c oxidase subunit (coxI) gene showed remarkably high sequence differences from the species that they previously were thought to belong to, well exceeding the average limit delineating ixodid tick species. This, as well as observed morphological differences fully justify their taxonomical status as new species.

Reports on adult Hyalomma ticks in certain regions of the Carpathian Basin date back to the 19th century. These ticks were thought to emerge from nymphs dropping from birds, then molting to adults. Although the role of migratory birds in carrying ticks of this genus is known from all parts of Europe, in most countries no contemporaneous multiregional surveillance of bird-associated ticks was reported which could allow the recognition of hotspots in this context. From 38 passeriform bird species 956 ixodid ticks were collected at seven locations in Hungary. Tick species were identified as Ixodes ricinus (n = 598), Ixodes frontalis (n = 18), Ixodes lividus (n = 6), Haemaphysalis concinna (n = 321), and D. reticulatus (n = 1). All 12 Hyalomma sp. ticks (11 engorged nymphs and 1 unengorged larva) were identified as H. rufipes based on three mitochondrial markers. This species was only found in the Transdanubian region and along its southeastern border. The Common Blackbird and the European Robin were the two main hosts of I. ricinus and I. frontalis, whereas H. concinna was almost exclusively collected form long-distance migrants. The predominant hosts of H. rufipes were reed-associated bird species, the Sedge Warbler and the Bearded Reedling, both in their nesting period. This study provides ornithological explanation for the regional, century-long presence of adult Hyalomma ticks under continental climate in the Transdanubian Region of the Carpathian Basin. More importantly, the autochthonous occurrence of a H. rufipes population was revealed for the first time in Europe, based on the following observations: (1) the bird species infested with H. rufipes are not known to migrate during their nesting period; (2) one larva was not yet engorged; (3) the larva and the nymphs must have belonged to different local generations; and (4) all H. rufipes found in the relevant location were identical in their haplotypes based on three maternally inherited mitochondrial markers, probably reflecting founder effect. This study also demonstrated regional and temporal differences in tick species carried by birds.

Hungary is situated in the southern part of Central Europe, next to the northern boundary of the Mediterranean region. This geographical position may allow the northward expansion of Mediterranean ixodid tick species into Hungary, particularly in the era of warming climate. During the past 14 years numerous surveys have been published on the species and activity of hard ticks occurring in the country. However, it was 60 years ago that the last comprehensive review of ixodid ticks of Hungary was published, and only in Hungarian language. The purpose of the present checklist is to provide a comprehensive and complete overview of the ixodid fauna of Hungary, based on tick reports published so far in Hungarian or English, also including hitherto unpublished data. Altogether 27 hard tick species were identified in Hungary, of which 21 can be regarded as indigenous. Most importantly, the autochthonous occurrence of Rhipicephalus sanguineus sensu lato was not known prior to 2005, but during the last 14 years increasing numbers of cases have been reported, attesting the emergence of this tick species in Hungary. Whereas R. sanguineus sensu lato was always associated with dogs and cats in Hungary, other tick species show differences in host associations according to habitat type, seasonal activity and questing height. Changes in the distribution, abundance and seasonality of a few tick species were also noted.

Birds are hosts for several zoonotic pathogens. Because of their high mobility, especially of longdistance migrants, birds can disperse these pathogens, affecting their distribution and phylogeography. We focused on Borrelia burgdorferi sensu lato, which includes the causative agents of Lyme borreliosis, as an example for tick-borne pathogens, to address the role of birds as propagation hosts of zoonotic agents at a large geographical scale. We collected ticks from passerine birds in 11 European countries. B. burgdorferi s.l. prevalence in Ixodes spp. was 37% and increased with latitude. The fieldfare Turdus pilaris and the blackbird T. merula carried ticks with the highest Borrelia prevalence (92 and 58%, respectively), whereas robin Erithacus rubecula ticks were the least infected (3.8%). Borrelia garinii was the most prevalent genospecies (61%), followed by B. valaisiana (24%), B. afzelii (9%), B. turdi (5%) and B. lusitaniae (0.5%). A novel Borrelia genospecies "Candidatus Borrelia aligera" was also detected. Multilocus sequence typing (MLST) analysis of B. garinii isolates together with the global collection of B. garinii genotypes obtained from the Borrelia MLST public database revealed that: (a) there was little overlap among genotypes from different continents, (b) there was no geographical structuring within Europe, and (c) there was no evident association pattern detectable among B. garinii genotypes from ticks feeding on birds, questing ticks or human isolates. These findings strengthen the hypothesis that the population structure and evolutionary biology of tick-borne pathogens are shaped by their host associations and the movement patterns of these hosts.

BACKGROUND:Despite the increasingly recognized eco-epidemiological significance of bats, data from molecular analyses of vector-borne bacteria in bat ectoparasites are lacking from several regions of the Old and New Worlds.METHODS:During this study, six species of ticks (630 specimens) were collected from bats in Hungary, Romania, Italy, Kenya, South Africa, China, Vietnam and Mexico. DNA was extracted from these ticks and analyzed for vector-borne bacteria with real-time PCRs (screening), as well as conventional PCRs and sequencing (for pathogen identification), based on the amplification of various genetic markers.RESULTS:In the screening assays, Rickettsia DNA was only detected in bat soft ticks, whereas Anaplasma phagocytophilum and haemoplasma DNA were present exclusively in hard ticks. Bartonella DNA was significantly more frequently amplified from hard ticks than from soft ticks of bats. In addition to Rickettsia helvetica detected by a species-specific PCR, sequencing identified four Rickettsia species in soft ticks, including a Rickettsia africae-like genotype (in association with a bat species, which is not known to migrate to Africa), three haemotropic Mycoplasma genotypes in Ixodes simplex, and Bartonella genotypes in I. ariadnae and I. vespertilionis.CONCLUSIONS:Rickettsiae (from both the spotted fever and the R. felis groups) appear to be associated with soft rather than hard ticks of bats, as opposed to bartonellae. Two tick-borne zoonotic pathogens (R. helvetica and A. phagocytophilum) have been detected for the first time in bat ticks. The present findings add Asia (China) to the geographical range of R. lusitaniae, as well as indicate the occurrence of R. hoogstraalii in South Africa. This is also the first molecular evidence for the autochthonous occurrence of a R. africae-like genotype in Europe. Bat haemoplasmas, which are closely related to haemoplasmas previously identified in bats in Spain and to "Candidatus Mycoplasma haemohominis", are reported here for the first time from Central Europe and from any bat tick.

Ecdysteroids (arthropod molting hormones) play an important role in the development and sexual maturation of arthropods, and they have been shown to have anabolic and "energizing" effect in higher vertebrates. The aim of this study was to assess ecdysteroid diversity, levels according to bird species and months, as well as to observe the molting status of hard ticks (Acari: Ixodidae) infesting the birds. Therefore, blood samples and ticks were collected from 245 birds (244 songbirds and a quail). Mass spectrometric analyses showed that 15 ecdysteroids were regularly present in the blood samples. Molting hormones biologically most active in insects (including 20-hydroxyecdysone [20E], 2deoxy-20E, ajugasterone C and dacryhainansterone) reached different levels of concentration according to bird species and season. Similarly to ecdysteroids, the seasonal presence of affected, apolytic ticks peaked in July and August. In conclusion, this study demonstrates the presence of a broad range and high concentrations of ecdysteroids in the blood stream of wild-living passerine birds. These biologically active, anabolic compounds might possibly contribute to the known high metabolic rate of songbirds.

Bats are important zoonotic reservoirs for many pathogens worldwide. Although their highly specialized ectoparasites, bat flies (Diptera: Hippoboscoidea), can transmit Bartonella bacteria including human pathogens, their eco-epidemiology is unexplored. Here, we analyzed the prevalence and diversity of Bartonella strains sampled from 10 bat fly species from 14 European bat species. We found high prevalence of Bartonella spp. in most bat fly species with wide geographical distribution. Bat species explained most of the variance in Bartonella distribution with the highest prevalence of infected flies recorded in species living in dense groups exclusively in caves. Bat gender but not bat fly gender was also an important factor with the more mobile male bats giving more opportunity for the ectoparasites to access several host individuals. We detected high diversity of Bartonella strains (18 sequences, 7 genotypes, in 9 bat fly species) comparable with tropical assemblages of bat-bat fly association. Most genotypes are novel (15 out of 18 recorded strains have a similarity of 92–99%, with three sequences having 100% similarity to Bartonella spp. sequences deposited in GenBank) with currently unknown pathogenicity; however, 4 of these sequences are similar (up to 92% sequence similarity) to Bartonella spp. with known zoonotic potential. The high prevalence and diversity of Bartonella spp. suggests a long shared evolution of these bacteria with bat flies and bats providing excellent study targets for the eco-epidemiology of host-vector-pathogen cycles.

In Europe, several species of bats, owls and kestrels exemplify highly urbanised, flying vertebrates, which may get close to humans or domestic animals. Bat droppings and bird pellets may have epidemiological, as well as diagnostic significance from the point of view of pathogens. In this work 221 bat faecal and 118 bird pellet samples were screened for a broad range of vector-borne bacteria using PCR-based methods. Rickettsia DNA was detected in 13 bat faecal DNA extracts, including the sequence of a rickettsial insect endosymbiont, a novel Rickettsia genotype and Rickettsia helvetica. Faecal samples of the pond bat (Myotis dasycneme) were positive for a Neorickettsia sp. and for haemoplasmas of the haemofelis group. In addition, two bird pellets (collected from a Long-eared Owl, Asio otus, and from a Common Kestrel, Falco tinnunculus) contained the DNA of a Rickettsia sp. and Anaplasma phagocytophilum, respectively. In both of these bird pellets the bones of Microtus arvalis were identified. All samples were negative for Borrelia burgdorferi s.l., Francisella tularensis, Coxiella burnetii and Chlamydiales. In conclusion, bats were shown to pass rickettsia and haemoplasma DNA in their faeces. Molecular evidence is provided for the presence of Neorickettsia sp. in bat faeces in Europe. In the evaluated regions bat faeces and owl/kestrel pellets do not appear to pose epidemiological risk from the point of view of F. tularensis, C. burnetii and Chlamydiales. Testing of bird pellets may provide an alternative approach to trapping for assessing the local occurrence of vector-borne bacteria in small mammals.

Kinetoplastids are flagellated protozoa, including principally free-living bodonids and exclusively parasitic trypanosomatids. In the most species-rich genus, Trypanosoma , more than thirty species were found to infect bats worldwide. Bat trypanosomes are also known to have played a significant role in the evolution of T. cruzi , a species with high veterinary medical significance. Although preliminary data attested the occurrence of bat trypanosomes in Hungary, these were never sought for with molecular methods. Therefore, amplification of an approx. 900-bp fragment of the 18S rRNA gene of kinetoplastids was attempted from 307 ixodid and 299 argasid ticks collected from bats, and from 207 cimicid bugs collected from or near bats in Hungary and Romania. Three samples, one per each bat ectoparasite group, were PCR positive. Sequencing revealed the presence of DNA from free-living bodonids ( Bodo saltans and neobodonids), but no trypanosomes were detected. The most likely source of bodonid DNA detected here in engorged bat ectoparasites is the blood of their bat hosts. However, how bodonids were acquired by bats, can only be speculated. Bats are known to drink from freshwater bodies, i.e. the natural habitats of B. saltans and related species, allowing bats to ingest bodonids. Consequently, these results suggest that at least the DNA of bodonids might pass through the alimentary mucosa of bats into their circulation. The above findings highlight the importance of studying bats and other mammals for the occurrence of bodonids in their blood and excreta, with potential relevance to the evolution of free-living kinetoplastids towards parasitism.

Haemaphysalis concinna is the second most common tick species attaching to birds in Hungary. Recently, Babesia genotypes, found in Siberia and the Far East, have been detected in this tick species collected from the vegetation in Hungary and Slovakia. The aim of this study was to molecularly investigate if these piroplasms also occur in H. concinna carried by migratory birds, which might explain their occurrence in the western Palaearctic. During a 2-year period, 321 H. concinna larvae and nymphs were collected from 121 passerine birds (of 19 species) in Hungary. These were molecularly investigated for the presence of piroplasm DNA with PCR and sequencing. The prevalence of PCR positive ticks was 15.9% (51 out of 321). Piroplasm PCR positivity of H. concinna ticks was significantly more frequent during the summer and autumn compared to spring, suggesting that migratory birds arriving in Hungary from the north or north east are the most important in the dispersal of H. concinna-associated piroplasms. Three genotypes, i.e. Babesia sp. "Irk-Hc133", "Irk-Hc130" (originally found in Irkutsk, Siberia) and "Kh-Hc222" (originally found in Khabarovsk, Far East) were detected. Phylogenetically all these belonged to the group formed by Babesia spp. of ruminants. Four bird species, which had 14-60% prevalence of PCR positive ticks, are known to be associated with northeast to southwest autumn migration. In conclusion, the presence of Central and East Asian Babesia genotypes in Central Europe are most likely related to bird species with known eastern migratory habit and/or phylogenetically substantiated connections between their eastern and western Eurasian populations.

In this study 308 ticks (Ixodes ariadnae: 26 larvae, 14 nymphs, five females; I. vespertilionis: 89 larvae, 27 nymphs, eight females; I. simplex: 80 larvae, 50 nymphs, nine females) have been collected from 200 individuals of 17 bat species in two countries, Hungary and Romania. After DNA extraction these ticks were molecularly analysed for the presence of piroplasm DNA. In Hungary I. ariadnae was most frequently identified from bat species in the family Vespertilionidae, whereas I. vespertilionis was associated with Rhinolophidae. Ixodes ariadnae was not found in Romania. Four, four and one new bat host species of I. ariadnae, I. vespertilionis and I. simplex were identified, respectively. DNA sequences of piroplasms were detected in 20 bat ticks (15 larvae, four nymphs and one female). I. simplex carried piroplasm DNA sequences significantly more frequently than I. vespertilionis. In I. ariadnae only Babesia vesperuginis DNA was detected, whereas in I. vespertilionis sequences of both B. vesperuginis and B. crassa. From I. simplex the DNA of B. canis, Theileria capreoli, T. orientalis and Theileria sp. OT3 were amplified, as well as a shorter sequence of the zoonotic B. venatorum. Bat ticks are not known to infest dogs or ruminants, i.e. typical hosts and reservoirs of piroplasms molecularly identified in I. vespertilionis and I. simplex. Therefore, DNA sequences of piroplasms detected in these bat ticks most likely originated from the blood of their respective bat hosts. This may indicate either that bats are susceptible to a broader range of piroplasms than previously thought, or at least the DNA of piroplasms may pass through the gut barrier of bats during digestion of relevant arthropod vectors. In light of these findings, the role of bats in the epidemiology of piroplasmoses deserves further investigation.

Ixodes ariadnae is a tick species of bats so far reported only in Central Europe, with its description based on the female and nymph. This study describes the male and larva in order to complete the description of the species. Male ticks collected from cave walls in Hungary showed a different morphology from those of I. vespertilionis and I. simplex. Molecular analysis of the cytochrome oxidase subunit I (COI) gene of these ticks verified them as conspecific to I. ariadnae. In addition, a larva of I. ariadnae was removed from a Daubenton's bat (Myotis daubentonii Kuhl, 1817). The male of I. ariadnae is characterized by long legs (7-8mm; I. vespertilionis: 8-10mm; I. simplex: 2-2.2mm), relatively short palpal setae (30-100μm; I. vespertilionis: 100-200μm; I. simplex: 20-50μm) and straight lateral edge of palps, the genital aperture (enclosed by a line of sclerotization) situated anteriorly to second intercoxal space and rounded coxae. The larva of I. ariadnae has long legs (2-2.2mm; I. vespertilionis: 1.6-1.8mm; I. simplex: 1mm), broad palps (length×width: 200×90μm; I. vespertilionis: 200×70μm; I. simplex: 140×60μm), pentagonal and posteriorly reverse bell-shaped scutum. These features allow to distinguish the male and the larva of I. ariadnae from those of I. vespertilionis (of which the male has longer palpal setae and curved lateral edge of palps, the genital aperture is situated posterior to the second intercoxal space, and the second coxae are squared; the larva of I. vespertilionis has narrower palps and posteriorly triangular scutum) and I. simplex (of which the male and the larva have considerably shorter legs, palps).