Genomic signatures of climate adaptation in bank voles.

Evidence for divergent selection and adaptive variation across the landscape can provide insight into a species' ability to adapt to different environments. However, despite recent advances in genomics, it remains difficult to detect the footprints of climate-mediated selection in natural populations. Here, we analysed ddRAD sequencing data (21,892 SNPs) in conjunction with geographic climate variation to search for signatures of adaptive differentiation in twelve populations of the bank vole (Clethrionomys glareolus) distributed across Europe. To identify the loci subject to selection associated with climate variation, we applied multiple genotype-environment association methods, two univariate and one multivariate, and controlled for the effect of population structure. In total, we identified 213 candidate loci for adaptation, 74 of which were located within genes. In particular, we identified signatures of selection in candidate genes with functions related to lipid metabolism and the immune system. Using the results of redundancy analysis, we demonstrated that population history and climate have joint effects on the genetic variation in the pan-European metapopulation. Furthermore, by examining only candidate loci, we found that annual mean temperature is an important factor shaping adaptive genetic variation in the bank vole. By combining landscape genomic approaches, our study sheds light on genome-wide adaptive differentiation and the spatial distribution of variants underlying adaptive variation influenced by local climate in bank voles.

Season and host-community composition inside roosts may affect host-specificity of bat flies.

Bat flies are one of the most abundant ectoparasites of bats, showing remarkable morphological adaptations to the parasitic habit, while the relationship with their hosts is characterized by a high level of specificity. By collecting bat flies from live hosts, our intention was to elucidate the seasonal differences in bat fly occurrence and to describe factors regulating the level of incipient host specificity. Our results indicate that the prevalence and the intensity of infestation is increasing from spring to autumn for most host species, with significant differences among different fly species. Males showed higher infestation levels than females in autumn, suggesting a non-random host choice by flies, targeting the most active host sex. Bat-bat fly host specificity shows seasonal changes and host choice of bat flies are affected by the seasonal differences in hosts' behavior and ecology, the intensity of infestation and the species composition of the local host community. Nycteribiid bat flies showed lower host specificity in the swarming (boreal autumn) period, with higher prevalence recorded on non-primary hosts. Choosing a non-primary bat host may be an adaptive choice for bat flies in the host's mating period, thus increasing their dispersive ability in a high activity phase of their hosts.

First record of Ixodes (Scaphixodes) caledonicus in the Carpathian Basin and first time molecular-phylogenetic analysis of this tick species with updated host records and geographical range.

Four Ixodes species represent the subgenus Scaphixodes Schulze, 1941 in Europe, but none of them were reported to be compared in a molecular-phylogenetic context. This study compensates for this lack of data. A tick larva, morphologically identified as Ixodes (Scaphixodes) caledonicus Nuttall, 1910, was collected from an Alpine swift (Tachymarptis melba) during its nesting period in Transylvania, Romania. Following DNA extraction, PCR analyses and sequencing in part with newly designed primers, three genetic markers of this specimen were amplified and compared to GenBank data, and two were analyzed phylogenetically. Based on sequence comparisons of its mitochondrial cytochrome c oxidase subunit I (cox1) and nuclear 28S rRNA genes I. caledonicus appeared to be closely related to members of the subgenus Pholeoixodes. However, the topology of the concatenated cox1 and 16S rRNA gene phylogenetic tree clearly showed its clustering with Ixodes (Scaphixodes) philipi. In conclusion, I. caledonicus is part of the tick fauna of Romania and is expected to occur also in other countries of the Carpathian Basin where rocky cliffs are available for nesting of swifts and other birds. This is the first species of the subgenus Scaphixodes in Europe, for which the traditional (morphology-based) taxonomic assignment is confirmed by molecular-phylogenetic analyses.

Bat Ectoparasites (Acari, Diptera, Hemiptera, Siphonaptera) in the Grand Maghreb (Algeria, Libya, Mauritania, Morocco and Tunisia): A Literature Review and New Data.

BACKGROUND: Arthropods parasites of bats play a crucial role in both ecological and public health contexts, as they have the potential to transmit zoonotic agents. The study aims to identify the distribution, and host-parasite associations of bat ectoparasites in the Grand Maghreb region (Algeria, Libya, Mauritania, Morocco and Tunisia), which has been largely understudied. METHODS: A thorough analysis of published records was conducted and we included our own field data. RESULTS: The checklist reveals a total of 43 ectoparasite species, encompassing a range of taxa. The list comprises 9 tick species, 11 mite species (including a chigger-mite), 11 bat fly species, 3 species of bugs, and 9 species of fleas. Extensive research efforts uncovered 141 host-parasite associations. Our data presents several new country records, documenting for the first time the presence of Carios vespertilionis and Raymondia huberi in Tunisia, Ixodes simplex and Spinturnix plecotinus in Algeria. CONCLUSION: By compiling and analysing available information, we have provided for the first time an up-to-date checklist of bat ectoparasites and their host associations in the region. This knowledge contributes to a better understanding of the epidemiological implications associated with bat ectoparasites, emphasizing their ecological and public health importance. The study's findings call for continued investigations and monitoring of bat ectoparasites to mitigate potential risks and safeguard both human and animal populations.

Toxoplasma gondii in small mammals in Romania: the influence of host, season and sampling location.

BACKGROUND: Toxoplasma gondii is a protozoan parasite that infects a large spectrum of warm-blooded animals, including humans. Small rodents and insectivores play an important role in the epidemiology of T. gondii and may serve as a source of infection for both, domestic and wild definitive felid hosts. Factors influencing the occurrence of T. gondii in wild small mammals are unknown, despite the fact that many intermediate host species are identified. We have used small mammals (Rodentia and Lipotyphla) captured over two years in various habitats, both in urbanised and in natural landscapes. We assessed the importance of land-use, season and host ecology on T. gondii infection. RESULTS: We examined 471 individuals belonging to 20 small mammal species, collected at 63 locations spread over wide altitude, habitat and land-use ranges from Romania. Heart tissue samples were individually analysed by PCR targeting the 529 bp repetitive DNA fragment of T. gondii. The overall prevalence of infection was 7.3%, with nine species of rodents and two species of shrews being found to carry T. gondii DNA. Five species showed high frequency of infection, with the highest prevalence found in Myodes glareolus (35.5%), followed by Spermophilus citellus (33.3%), Sorex minutus (23.1%), S. araneus (21.7%) and Micromys minutus (11.1%). Adults seemed more often infected than young, however when controlling for season, the difference was not significant, as in spring both adults and young showed higher infection rates, but more adults were sampled. Contrary to our expectations, urban/rural areas (with their implicit high density of domestic feline presence) had no effect on infection prevalence. In addition, neither habitat, nor land-use at sampling sites was important as only geographical location and host species were contributing factors to the infection risk. CONCLUSIONS: High prevalence of T. gondii infection showed a highly localised, patchy occurrence, with long living and higher mobility host species being the most common carriers, especially during autumn.

Detection of bacterial and protozoan pathogens in individual bats and their ectoparasites using high-throughput microfluidic real-time PCR.

Among the most studied mammals in terms of their role in the spread of various pathogens with possible zoonotic effects are bats. These are animals with a very complex lifestyle, diet, and behavior. They are able to fly long distances, thus maintaining and spreading the pathogens they may be carrying. These pathogens also include vector-borne parasites and bacteria that can be spread by ectoparasites such as ticks and bat flies. In the present study, high-throughput screening was performed and we detected three bacterial pathogens: Bartonella spp., Neoehrlichia mikurensis and Mycoplasma spp., and a protozoan parasite: Theileria spp. in paired samples from bats (blood and ectoparasites). In the samples from the bat-arthropod pairs, we were able to detect Bartonella spp. and Mycoplasma spp. which also showed a high phylogenetic diversity, demonstrating the importance of these mammals and the arthropods associated with them in maintaining the spread of pathogens. Previous studies have also reported the presence of these pathogens, with one exception, Neoehrlichia mikurensis, for which phylogenetic analysis revealed less genetic divergence. High-throughput screening can detect more bacteria and parasites at once, reduce screening costs, and improve knowledge of bats as reservoirs of vector-borne pathogens. IMPORTANCE The increasing number of zoonotic pathogens is evident through extensive studies and expanded animal research. Bats, known for their role as reservoirs for various viruses, continue to be significant. However, new findings highlight the emergence of Bartonella spp., such as the human-infecting B. mayotimonensis from bats. Other pathogens like N. mikurensis, Mycoplasma spp., and Theileria spp. found in bat blood and ectoparasites raise concerns, as their impact remains uncertain. These discoveries underscore the urgency for heightened vigilance and proactive measures to understand and monitor zoonotic pathogens. By deepening our knowledge and collaboration, we can mitigate these risks, safeguarding human and animal well-being.

The Role of Wild Boars in the Circulation of Tick-Borne Pathogens: The First Evidence of Rickettsia monacensis Presence.

Most wild mammals can serve as hosts both for tick-borne pathogens (TBPs) and for the ticks themselves. Among these, wild boars, due to their large body size, habitat and life span, show high exposure to ticks and TBPs. These species are now one of the widest-ranging mammals in the world, as well as the most widespread suid. Despite the fact that certain local populations have been decimated by African swine fever (ASF), wild boars are still considered overabundant in most parts of the world, including Europe. Altogether, their long-life expectancy, large home ranges including migration, feeding and social behaviors, wide distribution, overabundance and increased chances of interactions with livestock or humans make them suitable sentinel species for general health threats, such as antimicrobial-resistant microorganisms, pollution and ASF geographical distribution, as well as for the distribution and abundance of hard ticks and also for certain TBPs, such as Anaplasma phagocytophilum. The aim of this study was to evaluate the presence of rickettsial agents in wild boars from two counties in Romania. Among 203 blood samples of wild boars (Sus scrofa ssp. attila) collected during 3 (2019-2022) hunting seasons (September-February), 15 were found positive for tick-borne pathogen DNA. Six wild boars were positive for A. phagocytophilum DNA presence and nine for Rickettsia spp. The identified rickettsial species were R. monacensis (six) and R. helvetica (three). No animal was positive either for Borrelia spp., Ehrlichia spp. or Babesia spp. To the best of our knowledge, this is the first report of R. monacensis in European wild boars, thus adding the third species from the SFG Rickettsia, in the epidemiology of which this wild species may have a role as a reservoir host.

Ornithological and molecular evidence of a reproducing Hyalomma rufipes population under continental climate in Europe.

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.

Structural differences in the gut microbiome of bats using terrestrial vs. aquatic feeding resources.

Bat gut microbiomes are adapted to the specific diets of their hosts. Despite diet variation has been associated with differences in bat microbiome diversity, the influence of diet on microbial community assembly have not been fully elucidated. In the present study, we used available data on bat gut microbiome to characterize the microbial community assembly of five selected bat species (i.e., Miniopterus schreibersii, Myotis capaccinii, Myotis myotis, Myotis pilosus, and Myotis vivesi), using network analysis. These bat species with contrasting habitat and food preferences (i.e., My. capaccinii and My. pilosus can be piscivorous and/or insectivorous; Mi. schreibersii and My. myotis are exclusively insectivorous; while My. vivesi is a marine predator) offer an invaluable opportunity to test the impact of diet on bat gut microbiome assembly. The results showed that My. myotis showed the most complex network, with the highest number of nodes, while My. vivesi has the least complex structured microbiome, with lowest number of nodes in its network. No common nodes were observed in the networks of the five bat species, with My. myotis possessing the highest number of unique nodes. Only three bat species, My. myotis, My. pilosus and My. vivesi, presented a core microbiome and the distribution of local centrality measures of nodes was different in the five networks. Taxa removal followed by measurement of network connectivity revealed that My. myotis had the most robust network, while the network of My. vivesi presented the lowest tolerance to taxa removal. Prediction of metabolic pathways using PICRUSt2 revealed that Mi. schreibersii had significantly higher functional pathway's richness compared to the other bat species. Most of predicted pathways (82%, total 435) were shared between all bat species, while My. capaccinii, My. myotis and My. vivesi, but no Mi. schreibersii or My. pilosus, showed specific pathways. We concluded that despite similar feeding habits, microbial community assembly can differ between bat species. Other factors beyond diet may play a major role in bat microbial community assembly, with host ecology, sociality and overlap in roosts likely providing additional predictors governing gut microbiome of insectivorous bats.

An update on the occurrence of flies (Diptera: Muscidae, Calliphoridae) and sucking lice (Phthiraptera: Anoplura) of veterinary importance in Malta: First record of Lucilia cuprina and Linognathus africanus.

To obtain new data on the species diversity, host associations and spatiotemporal occurrence of flies and blood-sucking lice of veterinary importance in Malta, ectoparasites were collected at cattle, sheep and goat, pig farms, as well as dog shelters, and in two places in the absence of domestic animals. The species were identified morphologically, but voucher specimens were also analyzed with molecular-phylogenetic methods following DNA extraction. Altogether 3,095 flies (Diptera: Muscidae, Calliphoridae) were collected at farms and kennels near domestic animals, as well as 37 blowflies (Calliphoridae) in rural and urban areas without animals nearby. Regarding Muscidae, the great majority of flies (n = 3,084) were identified as the common housefly (Musca domestica). Eight flies represented the stable fly (Stomoxys calcitrans). Three blowflies associated with dogs and small ruminants belonged to Lucilia cuprina. By contrast, all 37 blowflies collected without domestic animals nearby, were identified as Lucilia sericata. In addition, 22 sucking lice were collected from goats, and all belonged to Linognathus africanus. Molecular identification of 28 flies and four lice confirmed the above species. Considering the sex ratio of M. domestica among samples collected randomly at cattle farms, females predominated in the whole study period, but the abundance of males increased significantly toward the autumn. Stomoxys calcitrans was associated with cattle and dogs, whereas L. cuprina was found near small ruminants and dogs. To our knowledge, this is the first study including the molecular analysis of flies and lice of veterinary-medical importance from Malta. The most important finding of this study is the first evidence for the autochthonous occurrence of L. cuprina in Malta. The exclusive presence of L. cuprina at animal-keeping facilities in rural areas and association of L. sericata with urban areas void of livestock might reflect similar habitat preference of these species in Malta to what was reported in South Africa. Based on the sucking-louse burden in the examined goat herds, the situation in Malta was similar to northern Africa where the exclusive presence of L. africanus was reported, unlike toward the north in the Mediterranean Basin where populations of this species are mixed with Linognathus stenopsis.

Phylogenetic analyses of Ixodes rugicollis with notes on its morphology in comparison with Ixodes cornutus.

BACKGROUND: The subgenus Pholeoixodes contains Ixodes species typically associated with birds that nest in cavities or with carnivorous mammals that are burrow-dwelling. Among ticks infesting the latter, Ixodes rugicollis is regarded as the rarest species in the western Palearctic. Despite the unique morphology of this species, its identification (especially of subadult stages) is difficult, and molecular-phylogenetic data to offer other diagnostic methods and a better understanding of its taxonomy are not available. METHODS: In this study, a female and a male of I. rugicollis were collected in Romania. The female was compared morphologically to another female of this species collected in France and to the lectotype of Ixodes cornutus (from Tajikistan), which has similar morphology and host association. Following DNA extraction, two mitochondrial (cytochrome c oxidase subunit I: cox1 and the 16S rRNA gene) and two nuclear genetic markers (18S and 28S rRNA genes) of I. rugicollis were amplified and analyzed in a phylogenetic context. RESULTS: Females of I. rugicollis and I. cornutus differed in the shape of their palps, scutum and areae porosae and the size of peritremes, but they were similar in palpal setal length, dental formula and arrangement of anal setae. Measurements of two I. rugicollis females examined were not less different from each other than from I. cornutus. Phylogenetically, I. rugicollis clustered with other members of its subgenus. The topology of all trees showed the position of bat-associated tick species of the subgenus Eschatocephalus among Pholeoixodes species. CONCLUSIONS: For the first time to our knowledge, this study provides high-resolution digital pictures of male and female I. rugicollis as well as corresponding molecular data. Morphological comparison of this species with I. cornutus could not resolve uncertainties in the validity of the latter species, which can only be accomplished after collecting new specimens of I. cornutus and consequent molecular comparisons. This study includes the first comprehensive molecular-phylogenetic analysis of western Palearctic Pholeoixodes species based on both nuclear and mitochondrial genetic markers and including I. rugicollis. The results of these confirm the phylogenetic position of subgenus Eschatocephalus within Pholeoixodes, justifying the need to merge them to comply with the taxonomic criterion of monophyly.

Shift in the seasonality of ixodid ticks after a warm winter in an urban habitat with notes on morphotypes of Ixodes ricinus and data in support of cryptic species within Ixodes frontalis.

This study was initiated to assess the seasonality and to investigate the morphology of questing ixodid ticks in an urban habitat in Central Europe, Hungary. A neglected part of a large cemetery, with sparse tree covering and dense lower vegetation, was sampled monthly from February 2019 to May 2021. All ticks were analyzed morphologically, and selected specimens by amplifying and sequencing two genetic markers. During the study 3818 ticks were collected, including Ixodes ricinus (n = 2772), Ixodes frontalis (n = 350) and Haemaphysalis concinna (n = 696). Ixodes ricinus adults and nymphs showed year-round activity, whereas H. concinna was not active during winter months and early spring. Most I. frontalis nymphs were collected in late winter and early spring, whereas the peak activity of larvae was during late autumn. Interestingly, during the spring, the peak activity of I. ricinus adults and nymphs was later (in May) when preceded by a warm winter in 2020. In contrast, the 2019 and 2021 spring activity peaks occurred in March and April after sharply rising temperatures in February. This shift in the peak activity of I. ricinus coincided with the initiation of questing activity of H. concinna. Three notably different morphotypes and four malformed specimens of I. ricinus were found. However, these were not significantly different in their mitochondrial haplotypes and phylogenetic clustering from typical specimens of this species. On the other hand, I. frontalis was represented by two remarkably different haplogroups, between which in the nymph stage there were no recognizable morphological differences, suggesting the status of these as cryptic species.

Hard ticks (Acari: Ixodidae) associated with birds in Europe: Review of literature data.

Hard ticks (Acari: Ixodidae) are considered the most important transmitters of pathogens in the temperate zone that covers most of Europe. In the era of climate change tick-borne diseases are predicted to undergo geographical range expansion toward the north through regions that are connected to southern areas of the continent by bird migration. This alone would justify the importance of synthesized knowledge on the association of tick species with avian hosts, yet birds also represent the most taxonomically and ecologically diverse part of urban vertebrate fauna. Birds frequently occur in gardens and near animal keeping facilities, thus playing a significant role in the dispersal of ticks and tick-borne pathogens in synanthropic environments. The primary aim of this review is to provide a comprehensive reference source (baseline data) for future studies, particularly in the context of discovering new tick-host associations after comparison with already published data. The records on the ixodid tick infestations of birds were assessed from nearly 200 papers published since 1952. In this period, 37 hard tick species were reported from 16 orders of avian hosts in Europe. Here we compile a list of these tick species, followed by the English and Latin name of all reported infested bird species, as well as the tick developmental stage and country of origin whenever this information was available. These data allowed a first-hand analysis of general trends regarding how and at which developmental stage of ticks tend to infest avian hosts. Five tick species that were frequently reported from birds and show a broad geographical distribution in the Western Palearctic (Ixodes arboricola, I. frontalis, I. ricinus, Haemaphysalis concinna and Hyalomma marginatum) were also selected for statistical comparisons. Differences were demonstrated between these tick species regarding their association with bird species that typically feed from the ground and those that rarely occur at the soil level. The ecology of these five bird-infesting tick species is also illustrated here according to avian orders, taking into account the ecology (habitat type) and activity (circadian rhythm and feeding level) of most bird species that represent a certain order.

Zoonotic ecotype-I of Anaplasma phagocytophilum in sympatric wildcat, pine marten and red squirrel - Short communication.

Anaplasma phagocytophilum is the causative agent of granulocytic anaplasmosis in humans, dogs, cats, horses and tick-borne fever in ruminants. In Europe, its main vector is the tick species Ixodes ricinus. In this study, spleen and liver samples, as well as ticks from 18 wild-living mammals (belonging to seven species) were analysed for the presence of A. phagocytophilum with molecular methods. The zoonotic ecotype-I of A. phagocytophilum was identified in a European wildcat (Felis silvestris) and its tick, a European pine marten (Martes martes) and a Eurasian red squirrel (Sciurus vulgaris). All PCR-positive samples were collected in 2019 and originated in the same geographic area. These results indicate that taxonomically diverse mammalian species can maintain the local enzootic cycle of the same genotype of A. phagocytophilum. To the best of our knowledge, this is the first report of the zoonotic variant of A. phagocytophilum in the wildcat and in the European pine marten in a broad geographical context, as well as in the red squirrel in Hungary. Since all these host species are well known for their urban and peri-urban presence, the results of this study verify their role in the synanthropic enzootic cycle of granulocytic anaplasmosis and tick-borne fever.

On the way between Africa and Europe: Molecular taxonomy of ticks collected from birds in Malta.

The Maltese Archipelago is situated in the middle of the Mediterranean Basin, between Europe and Africa, therefore representing an important stopover site for migratory birds between these two continents. Despite this, up-to-date information is not available on tick species associated with birds in Malta. Therefore, in this study, birds mist-netted for ringing by BirdLife Malta were examined for the presence of ticks between September, 2019 and May, 2021. Ticks were identified morphologically and molecularly, using three genetic markers. During the study period, 57 individuals of 22 bird species were found tick-infested, from which altogether 113 ixodid ticks were collected. The majority of developmental stages were nymphs, but 13 larvae and one female were also found. These ticks belonged to nine species: Ixodes cumulatimpunctatus (n=1), Ixodes ricinus (n=2), Ixodes acuminatus (n=2), Ixodes frontalis (n=5), Ixodes festai (n=1), one species of the Amblyomma marmoreum complex (n=8), Hyalomma rufipes (n=78), Hyalomma marginatum (n=7) and Hyalomma lusitanicum (n=1). Eight Hyalomma sp. ticks could only be identified on the genus level. Regarding seasonality, all Palearctic Ixodes species were carried by birds exclusively in the autumn (i.e., north to south), whereas H. rufipes (with predominantly Afrotropical distribution) was exclusively collected in the spring (i.e., carried south to north). Two tick species that occurred on birds in Malta, i.e., a species of the A. marmoreum complex and I. cumulatimpunctatus are only indigenous in the Afrotropical zoogeographic region. This is the first finding of the latter tick species in Europe, and four tick species were identified for the first time in Malta. In conclusion, the diversity of tick species regularly arriving in Europe from Africa is most likely higher than reflected by data obtained in Mediterranean countries of mainland Europe. Most notably, ticks of the genus Amblyomma appear to be underrepresented in previous datasets. Ticks of the subgenus Afrixodes (represented by I. cumulatimpunctatus) might also be imported into Europe by migratory birds.

Molecular epidemiological study on ticks and tick-borne protozoan parasites (Apicomplexa: Cytauxzoon and Hepatozoon spp.) from wild cats (Felis silvestris), Mustelidae and red squirrels (Sciurus vulgaris) in central Europe, Hungary.

BACKGROUND: Among live wild mammals adapted to urban and peri-urban habitats in Europe, members of the families Felidae, Mustelidae and Sciuridae deserve special attention as pathogen reservoirs because all of these families include members that are kept as pets. We report here the results of our study on two important groups of tick-borne protozoan parasites in ticks and tissues of wild cats, mustelids and red squirrels. METHODS: DNA was extracted from the tissues of carnivores (wild cats, mustelids; n = 16) and red squirrels (n = 4), as well as from ixodid ticks (n = 89) collected from these hosts. These DNA extracts were screened for piroplasms and Hepatozoon spp. using conventional PCR analysis and sequencing. In addition, 53 pooled samples of 259 questing Haemaphysalis concinna ticks were evaluated for the presence of Hepatozoon DNA, followed by phylogenetic analyses. RESULTS: One wild cat was found to be coinfected with Cytauxzoon europaeus and a new genotype of Hepatozoon felis, and two additional wild cats were infected with H. felis from a different phylogenetic group. In mustelids, Hepatozoon martis and two further Hepatozoon genotypes were detected. The latter clustered separately, close to others reported from eastern Asia. In addition, Hepatozoon sciuri was detected in red squirrels. Morphologic and molecular analyses verified eight tick species. One wild cat was infected with a H. felis genotype that was significantly different from that in Ixodes ricinus females infesting this cat. Only three pools of questing H. concinna nymphs tested positive for Hepatozoon, one of which contained H. martis. CONCLUSIONS: This study provides the first evidence of the occurrence of any Cytauxzoon species and of three Hepatozoon species in Hungary. In addition to H. martis, two further mustelid-associated Hepatozoon genotypes were detected, one of which was new in terms of phylogenetic and broader geographical contexts. This may be the first indication that H. felis genotypes from both of its phylogenetic groups occur in Europe. This also appears to be the first evidence of H. felis and C. europaeus coinfection in felids in Europe, and of autochthonous H. felis infection in wild cats north of the Mediterranean Basin. New tick-host associations were also observed in this study. Based on the results, H. felis and H. martis might survive transstadially in I. ricinus and H. concinna, respectively.

Diversity of tick species and associated pathogens on peri-urban wild boars - First report of the zoonotic Babesia cf. crassa from Hungary.

Wild boars show increasing numbers and population densities throughout Europe, including Hungary. While their presence is appreciated as game animals, they are also responsible for significant agricultural damage, habitat degradation and water quality issues. In addition, wild boars may harbor ticks and can act as reservoirs of tick-borne pathogens, thus posing a risk of transmission towards humans and domestic animals. This latter aspect of their veterinary-medical and epidemiological significance has become especially important in recent years, because increasing numbers of wild boars are reported to enter urban areas. Despite of this, reports on tick infestations of wild boars are scarce in Europe. For this study, 333 ixodid ticks were collected from 51 wild boars at 32 peri-urban locations in 14 counties of Hungary, during 2005-2008 (older samples) and 2019-2020 (new samples). Five species of ticks were identified: Dermacentor reticulatus (n = 165), Ixodes ricinus (n = 90) and Haemaphysalis concinna (n = 29) in both sample groups, while H. inermis (n = 29) and D. marginatus (n = 20) were only found among the old samples. The seasonality of collected ticks corresponded to their known activities. After DNA extraction, ticks were screened for three groups of tick-borne pathogens. All samples were negative for brucellae, recently reported to be carried and transmitted transovarially by D. marginatus. Four D. reticulatus contained Babesia canis DNA, while in one H. concinna nymph the recently discovered zoonotic B. cf. crassa (reported in Slovenia within 80 km of our sampling site) was detected. In addition, Anaplasma phagocytophilum was identified in D. reticulatus (n = 1), H. concinna (n = 3) and in its known vector, I. ricinus (n = 15). Phylogenetically, three out of four A. phagocytophilum genotypes clustered with zoonotic ones. In conclusion, despite of the high prevalence of Brucella suis in wild boars in Hungary, no evidence was found in support of the epidemiological role of ticks in transmitting brucellae. On the other hand, wild boars might introduce B. canis-carrier D. reticulatus into urban areas, unlike birds (which are not known to carry this tick species in the country). Most importantly, tick-infested wild boars can contribute to the spread of a novel zoonotic Babesia sp. and of the zoonotic variants of A. phagocytophilum.

Rickettsiaceae in two reptile-associated tick species, Amblyomma exornatum and Africaniella transversale: First evidence of Occidentia massiliensis in hard ticks (Acari: Ixodidae).

All species of hard ticks associated with reptiles as hosts throughout their life cycle, are currently assigned to genera including Amblyomma and Africaniella. Among these species, based on literature data, Africaniella transversale has never been investigated for the presence of tick-borne pathogens. In this study, seven DNA extracts (two from A. transversale and five from Amblyomma exornatum) were screened for the presence of important tick-borne protozoa (piroplasms) and bacteria (Anaplasmataceae and Rickettsiaceae) with conventional PCRs and sequencing. A new heat shock protein chaperonin (groEL) gene-specific PCR was also developed to identify Occidentia spp. in these samples. In A. transversale, Occidentia massiliensis (previously detected in rodent-associated soft ticks) and Rickettsia hoogstraalii were present. While the latter was molecularly identical with formerly reported sequences of this rickettsia, the genotype of O. massiliensis was new based on sequence and phylogenetic analyses of its groEL gene. In A. exornatum, a Rickettsia genotype closely related to R. tamurae and R. monacensis, was detected. The ompA sequence of this genotype was identical to that of Rickettsia sp. Ae-8 reported from A. exornatum in a reptile breeding facility in the USA. These results show that A. transversale might carry O. massiliensis which (unless having a symbiotic nature in ticks) may originate either from the reptile host of this hard tick species or the rodent prey of reptiles. This is also the first detection of the reptile tick-associated Rickettsia sp. Ae-8 (phylogenetically aligning with R. tamurae, R. monacensis) in Africa, i.e. within the original geographical range of A. exornatum.

Investigation on haplotypes of ixodid ticks and retrospective finding of Borrelia miyamotoi in bank vole (Myodes glareolus) in Switzerland.

The current status of tick species, important tick-borne bacteria and protozoan parasites is well-documented in Switzerland. However, reports on the genetic diversity and geographical relationships of tick species in this country appear to be in part lacking or outdated. Thus, the aim of this study was to collect ticks from various host species in southern Switzerland, to compare them in a geographical context and to screen in these samples rare tick-borne pathogens hitherto not reported or having low prevalence in Switzerland. In 2019-2020 altogether 177 ixodid ticks were collected from the vegetation, as well as from humans (n = 17), dogs (n = 23), cats (n = 41), red deer (n = 8), a European rabbit and a European hedgehog at 25 locations in three cantons of south Switzerland. Tick species were identified morphologically, followed by DNA extraction and comparison of mitochondrial haplotypes with molecular-phylogenetic methods. Tick DNA extracts, as well as sixty-two rodent liver or spleen tissue DNA extracts (representing six species) available from 2005 to 2006 were screened for trypanosomes, Occidentia massiliensis and Borrelia miyamotoi. Morphologically, three tick species were identified: Ixodes ricinus (n = 170), Rhipicephalus sanguineus sensu lato (n = 6) and I. hexagonus (n = 1). In contrast to companion animals (dogs, cats) immature ticks (larvae and nymphs) predominated on humans, which was a highly significant association (P < 0.0001). Molecular comparison of the cytochrome c oxidase subunit I (cox1) gene with GenBank data established the species as R. sanguineus sensu stricto and confirmed I. hexagonus, both showing 99.8-100% sequence identity to conspecific ticks from northern Italy. Seventy-nine specimens morphologically identified as I. ricinus revealed high 16S rRNA gene haplotype diversity and represented two phylogenetic groups. Two I. ricinus haplotypes from Switzerland belonged to the same haplogroup with I. inopinatus from Spain, Germany and Austria as well as with I. ricinus reported from a broad geographical range of Europe (including Italy, the Netherlands, Poland, Latvia and Sweden). All 141 tick DNA extracts (from five R. sanguineus s.l., 135 I. ricinus and one I. hexagonus) and 62 rodent tissue DNA extracts were negative for trypanosomes and O. massiliensis. However, B. miyamotoi was identified in a bank vole (Myodes glareolus) and three ticks by sequencing. From Switzerland, this is the first report of tick haplotypes that are phylogenetically closely related to I. inopinatus. However, based on their morphology, both specimens are considered as I. ricinus. These results highlight the importance that the identification of I. inopinatus should be based on coherent morphologic and molecular properties. This is also the first report of rodent-borne B. miyamotoi in Switzerland. Taking into account the year of collection (2005), in a chronological order this might be the first indication of B. miyamotoi in any rodent species in Europe.

Molecular epidemiological study of Trichomonas gallinae focusing on central and southeastern Europe.

Trichomonas gallinae is a geographically widespread protozoan parasite of birds. In this study, oropharyngeal swab samples were collected in Hungary and Romania from 99 columbiform birds, including 76 feral pigeons (Columba livia domestica: 42 kept for racing, 32 with urban and two with rural habitat), four common wood pigeons (C. palumbus), 16 ring doves (Streptopelia risoria) and three Eurasian collared doves (S. decaocto). These samples were analyzed for the presence of T. gallinae using molecular methods. Racing feral pigeons had significantly higher prevalence of T. gallinae infection than urban feral pigeons. The rate of PCR-positivity was the highest among wood pigeons and ring doves. Based on 18S rRNA gene, T. gallinae was the most heterogenous among racing feral pigeons sampled in a trading-breeding place. Clinical signs were associated with only one 18S rRNA gene subtype. The most divergent 18S rRNA gene subtype, Trichomonas sp. Hu-TG37 clustered with T. canistomae and T. tenax and represents probably a new species. To our knowledge, this is the first report on the genetic diversity of T. gallinae in the southeastern European region. The results suggest that most detected T. gallinae 18S rRNA gene subtypes are not host-specific and do not cause clinical signs. The highest number of 18S rRNA gene subtypes was demonstrated among racing feral pigeons. Significantly more captive than free-living columbiform birds had T. gallinae infection. These data highlight the importance of epizootic monitoring of the genetic diversity and presence of T. gallinae in trading-breeding places of pigeons and doves.