Parasitological and molecular investigation of consequences of raw meat feeding (BARF) in dogs and cats: implications for other pets living nearby.

Considering the original nature of carnivorism among dogs and cats, nowadays these pets are increasingly fed BARF (Biologically Appropriate Raw Food/Bone And Raw Food) diet to improve their health conditions. However, this diet may also carry health risks, such as vitamin or mineral deficiencies, and infection with pathogens including parasites. In our study, fecal samples of 89 pet animals kept on BARF diet were subjected to coprological examination followed by molecular analyses. Six of them shed eggs of Dicrocoelium dendriticum. This result was confirmed with PCR and sequencing, and in one case, the DNA of Fasciola hepatica was also demonstrated. In addition, oocysts of Cystoisospora canis, a Cystoisospora ohioensis-like sp. and Eimeria stiedai, as well as sporocysts of a Sarcocystis sp. were also detected. All samples were negative for Neospora caninum and Toxoplasma gondii. In conclusion, no evidence was found for the infection of BARF-fed dogs and cats with parasites that are usually associated with this diet and considered as clinico-pathological risk factors for these pets themselves (e.g., N. caninum, T. gondii). However, fluke eggs (probably originating from ruminants) and oocysts of E. stiedai (from rabbit liver in the food) were demonstrated as pseudoparasites. These species are usually not considered among parasite-associated risks of BARF-feeding, implying that other animals living near BARF-fed pets are neglected in this context. However, where intermediate hosts of D. dendriticum occur in urban areas, BARF-feeding may indirectly affect later other dogs and cats. It was also shown here that BARF-feeding may contribute to the contamination of the environment with E. stiedai oocysts, thus increasing the risks of biliary coccidiosis in nearby living pet rabbits that would otherwise not have access to oocysts of E. stiedai.

Molecular evidence of Monocercomonas and Acanthamoeba in the feces of captive reptiles.

Reptiles are frequently kept as pet animals. They are considered as important reservoirs of protozoa with veterinary-medical significance. At a reptile farm in Ireland, fecal samples were collected from 98 captive reptiles, representing 43 species of three orders (Squamata, Testudines, and Crocodylia). After DNA extraction, all samples were screened by conventional PCRs, targeting the ribosomal small subunit (SSU) RNA and alpha-tubulin genes of trichomonads and SSU RNA gene of Acanthamoeba spp. One leopard gecko (Eublepharis macularius) was positive for a not yet reported species/genotype of the genus Monocercomonas, different from M. colubrorum. Various Acanthamoeba genotypes were detected in six reptilian species, i.e., Acanthamoeba genotype T11 in Eunectes notaeus and Heloderma suspectum/horridum; genotype T4 in Varanus exanthematicus, Chlamydosaurus kingii, and Macrochelys temminckii; and the genotype T13 in Iguana iguana. Some of these amoeba species might have clinicopathological significance in both humans and animals. Our findings highlight the importance to monitor pathogenic protozoa in pet as well as wildlife reptiles, as a source of possible infection for animals and humans living nearby.

First report of molecular taxonomic analyses of European beaver metazoan parasites from Hungary.

European beaver (Castor fiber L. 1758) is the biggest rodent species living in Europe. Beavers are semi-aquatic animals; they are defecating directly into the water; thus, they have an important role in spreading parasites related to water (e.g., protozoa and flukes). The first specimens of this once extinct rodent species in Hungary turned up in Szigetköz (upper flow of the Hungarian Danube) in 1991 dispersed from Austria. The reintroduction to Hungary started in 1996, and the population slowly increased in number up to around 4000 individuals, but the knowledge about their parasites is lacking. This is the first report on the metazoan parasites of beavers in Hungary and their molecular taxonomy. In the 5-year study, 47 beavers were trapped in four locations and euthanized with permission. Three different metazoan parasites were collected: larvae and adults of Platypsyllus castoris beetles, nymphs and adults of Schizocarpus sp. mites and eggs and adults of Stichorchis subtriquetrus flukes. From these three parasite species, molecular taxonomic studies were also carried out. The low number of metazoan parasites species detected in Hungarian beavers compared to other European countries (e.g., Poland) might be attributed to host population bottleneck effect during reintroduction. As parasites represent a significant component of the biodiversity and ecosystem, the conservation efforts should focus not only on host species but also on their parasites.

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.

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.

Dogs are final hosts of Sarcocystis morae (Apicomplexa: Sarcocystidae): First report of this species in Hungary and its region - Short communication.

In this study, faecal samples of four American Staffordshire terrier dogs (used for illegal fighting) were analysed by DNA extraction, molecular-phylogenetic and parasitological methods, in order to examine the occurrence of protozoan, apicomplexan parasites. In one sample, the DNA of Sarcocystis morae was shown to be present. This species was identified based on 100% identity with already reported sequences of S. morae from cervids in Lithuania and Spain. The result was also confirmed by phylogenetic analysis. The sporocysts of the canine S. morae isolate measured 14.95 × 9.75 µm on average. This is the first molecular evidence in support of the final host role of domestic dogs in the life cycle of S. morae. The most likely source of the infection was raw meat given to the examined dog to increase its physical achievement. In conclusion, under similar circumstances dogs may participate in the life cycle of S. morae in a 'natural way', shedding sporocysts/oocysts when used for hunting or taken to walks in forested areas.

Anaplasmataceae closely related to Ehrlichia chaffeensis and Neorickettsia helminthoeca from birds in Central Europe, Hungary.

Increasing amount of data attest that (in the context of vector-borne infections) birds are not only important as hosts of blood-sucking arthropod vectors, but also as reservoirs of vector-borne pathogens. From 2015 to 2019 cadavers of 100 birds (from 45 species, nine orders) were collected in Hungary, and their organs were screened for DNA from a broad range of vector-borne bacteria with PCR and sequencing. Molecular analyses revealed the presence of Anaplasmataceae, and sequencing identified bacteria closely related to Neorickettsia helminthoeca and Ehrlichia chaffeensis in a Eurasian teal (Anas crecca) and a song thrush (Turdus philomelos), respectively. All samples were PCR negative for rickettsiae, borreliae, Francisella and Coxiella spp., as well as for piroplasms. To our knowledge, this is the first report of a Neorickettsia and an Ehrlichia sp., which belong to the phylogenetic groups of N. helminthoeca and E. chaffeensis, respectively, from Europe. The potential presence of these two vector-borne bacteria needs to be taken into account during future studies on the eco-epidemiology of Anaplasmataceae in Europe.

The current situation of canine dirofilariosis in Hungary.

Between April and September 2017, blood samples were collected from 344 randomly selected dogs older than 1 year in 180 settlements of 19 counties in Hungary. The dogs lived exclusively outdoors, had never travelled and had neither been examined for Dirofilaria infection nor treated against mosquitoes with insecticides or/and filarioid worms with macrocyclic lactones. Dirofilaria infection was examined with a modified Knott's test for microfilariae, DiroCHEK®, for the presence of D. immitis antigen, as well as by multiplex and conventional PCR. Altogether, 77 (22.4%) dogs living in 58 settlements of 17 counties were found to be infected with one or both Dirofilaria species based on the PCR techniques. Twenty-eight (8.1%) and 38 (11.1%) dogs were infected with D. immitis and D. repens, respectively. Coinfections were recorded in 11 samples (3.2%) collected in 11 locations of 8 counties. The results confirmed that both dirofilarioses are endemic in dogs and the eastern areas of the country are hyperendemic for heartworm disease. Temperature showed a significant association with the prevalence of D. immitis (OR 2.41, 95% CI 1.24-4.86, p = 0.012) but not with that of D. repens (OR 1.37, 95% CI 0.78-2.47, p = 0.286). The prevalence of neither D. immitis (OR 0.99, 95% CI 0.98-1.00, p = 0.213) nor D. repens (OR 1.01, 95% CI 0.99-1.01, p = 0.094) showed a significant correlation with precipitation. The number of yearly growing degree days (GDD) based on the lifecycle of Dirofilaria in mosquitoes ranged between 3.73 and 7.57 for the Hungarian districts. The GDD showed a significant positive association with the prevalence of D. immitis (OR 2.38, 95% CI 1.43-4.15, p = 0.001) and a non-significant positive relationship with that of D. repens (OR 1.25, 95% CI 0.83-1.95, p = 0.291).

First broad-range molecular screening of tick-borne pathogens in Ixodes (Pholeoixodes) kaiseri, with special emphasis on piroplasms.

Recently, the occurrence of Ixodes (Pholeoixodes) kaiseri has been reported for the first time in several European countries, but data on the molecular analysis of this hard tick species are still lacking. Therefore, in this study DNA extracts of 28 I. kaiseri (collected from dogs and red foxes in Germany, Hungary and Romania) were screened with reverse line blot hybridisation (RLB), PCR and sequencing for the presence of 43 tick-borne pathogens or other members of their families from the categories of Anaplasmataceae, piroplasms, rickettsiae and borreliae. Rickettsia helvetica DNA was detected in one I. kaiseri female (from a red fox, Romania), for the first time in this tick species. Six ticks (from red foxes, Romania) contained the DNA of Babesia vulpes, also for the first time in the case of I. kaiseri. Molecular evidence of R. helvetica and B. vulpes in engorged I. kaiseri does not prove that this tick species is a vector of the above two pathogens, because they might have been taken up by the ticks from the blood of foxes. In addition, one I. kaiseri female (from a dog, Hungary) harboured Babesia sp. badger type-B, identified for the first time in Hungary and Central Europe (i.e. it has been reported previously from Western Europe and China). The latter finding can be explained by either the susceptibility of dogs to Babesia sp. badger type-B, or by transstadial survival of this piroplasm in I. kaiseri.

Genetic diversity, piroplasms and trypanosomes in Rhipicephalus microplus and Hyalomma anatolicum collected from cattle in northern Pakistan.

The two most important tick species in Pakistan are Rhipicephalus microplus and Hyalomma anatolicum. When associated with cattle, these have one or three host life cycles, respectively, with potential implications for their population genetics and for their vector role in the transmission of pathogens. To compare the two tick species in this context with molecular-phylogenetic methods, during the present study 123 ticks were collected from cattle in northern Pakistan. Two mitochondrial markers of 36 ticks were molecularly analyzed. All 11 R. microplus specimens had identical cox1 haplotypes, whereas the 25 H. anatolicum specimens had nine cox1 haplotypes. The latter belonged to two distinct phylogenetic lineages with high support. However, in the 16S rRNA gene these differences were less evident. Among the 113 ticks molecularly analyzed for tick-borne protozoa, the sequence of Babesia occultans was successfully amplified from two specimens of H. anatolicum. Theileria annulata was present in both R. microplus (10.4%) and H. anatolicum (27.3%), with significantly higher prevalence rate in the latter species. Only one tick, a H. anatolicum female, was positive in the PCR detecting Trypanosoma spp. Sequencing revealed the presence of a new genotype, with the closest phylogenetic relationship to stercorarian trypanosomes (in particular, to a tick-associated Trypanosoma sp. from Japan). In conclusion, the above differences between R. microplus and H. anatolicum may be partly related to their life cycles involving one host or three hosts, respectively. Among the others, host switching (reducing chances of inbreeding) and shorter periods spent on-host (reducing gene flow between cattle herds) are supposed to be important drivers of cox1 gene diversification in case of H. anatolicum as a three host tick species. These results highlight the importance of studying differences in intraspecific genetic diversity and piroplasm burdens between one host and three host ticks in the local scale. In addition, a Trypanosoma sp. molecularly identified in H. anatolicum is reported here for the first time from South Asia, deserving further evaluation concerning its host and vector species.

Sarcocystis rileyi emerging in Hungary: is rice breast disease underreported in the region?

Reports of Sarcocystis rileyi-like protozoa ('rice breast disease') from anseriform birds had been rare in Europe until the last two decades, when S. rileyi was identified in northern Europe and the UK. However, despite the economic losses resulting from S. rileyi infection, no recent accounts are available on its presence (which can be suspected) in most parts of central, western, southern and eastern Europe. Between 2014 and 2019, twelve mallards (Anas platyrhynchos) were observed to have rice breast disease in Hungary, and the last one of these 12 cases allowed molecular identification of S. rileyi, as reported here. In addition, S. rileyi was molecularly identified in the faeces of one red fox (Vulpes vulpes). The hunting season for mallards in Hungary lasts from mid-August to January, which in Europe coincides with the wintering migration of anseriform birds towards the south. Based on this, as well as bird ringing data, it is reasonable to suppose that the first S. rileyi-infected mallards arrived in Hungary from the north. on the other hand, red foxes (Vulpes vulpes), which are final hosts of S. rileyi, are ubiquitous in Hungary, and our molecular finding confirms an already established autochthonous life cycle of S. rileyi in the region. Taken together, this is the first evidence for the occurrence of S. rileyi in Hungary and its region.

Tick- and fly-borne bacteria in ungulates: the prevalence of Anaplasma phagocytophilum, haemoplasmas and rickettsiae in water buffalo and deer species in Central Europe, Hungary.

BACKGROUND: Hunting constitutes an important industry in Europe. However, data on the prevalence of vector-borne bacteria in large game animal species are lacking from several countries. Blood or spleen samples (239 and 270, respectively) were taken from red, fallow and roe deer, as well as from water buffaloes, mouflons and wild boars in Hungary, followed by DNA extraction and molecular analyses for Anaplasma phagocytophilum, haemoplasmas and rickettsiae. RESULTS: Based on blood samples, the prevalence rate of A. phagocytophilum infection was significantly higher in red deer (97.9%) than in fallow deer (72.7%) and roe deer (60%), and in all these compared to mouflons (6.3%). In addition, 39.2% of the spleen samples from wild boars were PCR positive for A. phagocytophilum, but none of the buffalos. Based on blood samples, the prevalence rates of both Mycoplasma wenyonii (Mw) and 'Candidatus M. haemobos' (CMh) infections were significantly higher in buffaloes (Mw: 91.2%; CMh: 73.3%) than in red deer (Mw: 64.6%; CMh: 45.8%), and in both of them compared to fallow deer (Mw: 30.3%; CMh: 9.1%) and roe deer (Mw: 20%; CMh: 1.5%). The prevalence of Mw and CMh infection significantly correlated with the body sizes of these hosts. Furthermore, Mw was significantly more prevalent than CMh in buffaloes, red and roe deer. Mycoplasma ovis was detected in mouflons, M. suis in wild boars, R. helvetica in one fallow deer and one mouflon, and an unidentified Rickettsia sp. in a fallow deer. CONCLUSIONS: Forest-dwelling game animal species were found to be important carriers of A. phagocytophilum. In contrast, animals grazing grassland (i.e. buffaloes) were less likely to get infected with this Ixodes ricinus-borne pathogen. Water buffaloes, deer species, mouflons and wild boars harbored haemoplasmas that may affect domestic ungulates. Evaluated animals with larger body size had significantly higher prevalence of infection with haemoplasmas compared to smaller deer species. The above host species rarely carried rickettsiae.

Haematospirillum and insect Wolbachia DNA in avian blood.

In this study, blood samples of 259 Acrocephalus sp. warblers were molecularly analysed for Anaplasmataceae and Rhodospirillaceae based on PCR amplification of 16S rRNA gene fragments. One bird blood sample (from Reed Warbler, Acrocephalus scirpaceus) yielded a sequence with 99.8% identity to Haematospirillum jordaniae. This is the first molecular evidence for the occurrence of this species in the blood of any vertebrate other than human. Another bird blood sample (from Marsh Warbler: Acrocephalus palustris) yielded a Wolbachia sequence, closely related to a moth endosymbiont with 99.8% identity. A nematode origin of Wolbachia DNA detected here in avian blood can be excluded, because results of phylogenetic analysis showed its closest alignment with insect wolbachiae. This is the first finding of insect Wolbachia DNA in the circulatory system of birds, which can be explained either by the inoculation of wolbachiae by blood-sucking vectors, or passing of Wolbachia DNA from the gut into the blood of this insectivorous bird species.

Assessing bat droppings and predatory bird pellets for vector-borne bacteria: molecular evidence of bat-associated Neorickettsia sp. in Europe.

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.

Molecular investigations of the bat tick Argas vespertilionis (Ixodida: Argasidae) and Babesia vesperuginis (Apicomplexa: Piroplasmida) reflect "bat connection" between Central Europe and Central Asia.

Argas vespertilionis is a geographically widespread haematophagous ectoparasite species of bats in the Old World, with a suspected role in the transmission of Babesia vesperuginis. The aims of the present study were (1) to molecularly screen A. vespertilionis larvae (collected in Europe, Africa and Asia) for the presence of piroplasms, and (2) to analyze mitochondrial markers of A. vespertilionis larvae from Central Asia (Xinjiang Province, Northwestern China) in a phylogeographical context. Out of the 193 DNA extracts from 321 A. vespertilionis larvae, 12 contained piroplasm DNA (10 from Hungary, two from China). Sequencing showed the exclusive presence of B. vesperuginis, with 100% sequence identity between samples from Hungary and China. In addition, A. vespertilionis cytochrome oxidase c subunit 1 (cox1) and 16S rRNA gene sequences had 99.1-99.2 and 99.5-100% similarities, respectively, between Hungary and China. Accordingly, in the phylogenetic analyses A. vespertilionis from China clustered with haplotypes from Europe, and (with high support) outside the group formed by haplotypes from Southeast Asia. This is the first molecular evidence on the occurrence of B. vesperuginis in Asia. Bat ticks from hosts in Vespertilionidae contained only the DNA of B. vesperuginis (in contrast with what was reported on bat ticks from Rhinolophidae and Miniopteridae). Molecular taxonomic analyses of A. vespertilionis and B. vesperuginis suggest a genetic link of bat parasites between Central Europe and Central Asia, which is epidemiologically relevant in the context of any pathogens associated with bats.

DNA of free-living bodonids (Euglenozoa: Kinetoplastea) in bat ectoparasites: potential relevance to the evolution of parasitic trypanosomatids.

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.

Molecular screening for Anaplasmataceae in ticks and tsetse flies from Ethiopia.

Hard ticks and tsetse flies are regarded as the most important vectors of disease agents in Sub-Saharan Africa. With the aim of screening these blood-sucking arthropods for vector-borne pathogens belonging to the family Anaplasmataceae in South-Western Ethiopia, four species of tsetse flies (collected by traps) and seven species of ixodid ticks (removed from cattle) were molecularly analysed. DNA was extracted from 296 individual ticks and from 162 individuals or pools of tsetse flies. Besides known vector-pathogen associations, in Amblyomma cohaerens ticks sequences of Anaplasma marginale and A. phagocytophilum were detected, the latter for the first time in any ticks from cattle in Africa. In addition, part of the gltA gene of Ehrlichia ruminantium was successfully amplified from tsetse flies (Glossina pallidipes). First-time identification of sequences of the above pathogens in certain tick or tsetse fly species may serve as the basis of further epidemiological and transmission studies.

First record of Ixodes ariadnae in Germany - Short communication.

A long-legged tick was collected from a hibernating greater mouse-eared bat (Myotis myotis) in Baden-Württemberg, Germany. Based on morphological characteristics as well as on partial COI and 16S rDNA gene sequences the tick was identified as an engorged female of Ixodes ariadnae. The greater mouseeared bat is a new host record for this tick species. Taking into account the geographical position of the collection site and the known migration distance of the greater mouse-eared bat, the present data suggest the autochthonous occurrence of I. ariadnae in Germany. This is the first record of I. ariadnae in Germany, and in any country other than Hungary, where this species has been recently discovered.

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.

Investigations of the tick burden on passeriform, water-associated and predatory birds reveal new tick-host associations and habitat-related factors of tick infestation.

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.