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Babesia vesperuginis was molecularly detected in 10% (5/48) of common pipistrelle bats ( Pipistrellus pipistrellus) in Shihezi City, Northwestern China. Interestingly, four bat ticks ( Argas vespertilionis), from Babesia DNA-positive common pipistrelle bats, were also positive for B. vesperuginis. Our findings extend the geographic range of the common pipistrelle bat as a reservoir of B. vesperuginis in Asia.

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.

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.

Babesia vesperuginis is the only piroplasm known to infect bats. Unlike most members of the genus Babesia, it is probably transmitted by a soft tick species (i.e. Argas vespertilionis). Recently, two studies have been conducted to clarify the phylogenetic status of this species, and both agreed on placing it into a basal position among Babesia sensu stricto (s.s.). However, several important groups of piroplasms were not included in the already reported phylogenetic trees of B. vesperuginis isolates. Therefore, the aim of the present study was to amplify an approx. 950-bp fragment of the cytochrome c oxidase subunit 1 (cox1) gene of B. vesperuginis from A. vespertilionis specimens, and to compare its sequences with those from other piroplasmid groups in a broader phylogenetic context. Sequence comparisons focusing on either 18S rRNA or cox1 genes, as well as phylogenetic analyses involving separate and concatenated 18S rRNA and cox1 sequences indicate that B. vesperuginis is more closely related to the phylogenetic group of Theileriidae than to Babesia s.s. In particular, B. vesperuginis clustered closest to Cytauxzoon felis and the 'prototheilerid' B. conradae. The results of this study highlight that B. vesperuginis is a unique and taxonomically important species, which should be included in future studies aimed at resolving the comprehensive phylogeny of Piroplasmida.

Background: Babesia spp. are hemoparasites which infect the red blood cells of a large variety of mammals. In bats, the only known species of the genus is Babesia vesperuginis. However, except a few old reports, the host range and geographical distribution of this bat parasite have been poorly studied. This study aimed to investigate the presence of piroplasms in tissues of bats collected in four different countries from eastern and central Europe: Austria, Czech Republic, Hungary and Romania., Methods: A total of 461 bat carcasses (24 species) were collected between 2001 and 2016 from caves, mines and buildings. PCR was performed using specific primers targeting a portion of the 18S rDNA nuclear gene and cytochrome c oxidase subunit 1 mitochondrial gene, followed by sequencing., Results: The results of this study show for the first time the presence of B. vesperuginis in bats in central and eastern Europe. The phylogenetic analysis of the 18S rDNA nuclear gene revealed no variability between the sequences and the phylogenetic analysis of the cox1 mitochondrial gene proved that B. vesperuginis could be divided into two subclades., Conclusion: Our study showed a broad geographical distribution of B. vesperuginis in European bats, reporting its presence in five new host species (M. cf. alcathoe, M. bechsteinii, M. myotis, Pi. nathusii and V. murinus) and three new countries.

Rhipicephalus turanicus is an important tick species potentially carrying tick-borne pathogens. Several tick species have obvious subspecies divergence. However few studies aimed to examine the existence of divergence within R. turanicus. Therefore, a detailed morphological and molecular analysis was conducted for comparing R. turanicus from the Mediterranean Basin (represented by Albania) and Central Asia (Northwestern China). Altogether 315 adult ticks of R. turanicus (103 from Albania and 212 from China) were morphologically and molecularly analysed. DNA samples were used for mitochondrial 16S rRNA and cox1 gene sequences analysis. In addition, as potentially genetic markers, three fragments including partial nad1-16S rRNA, nad2-cox1, cox1-tRNA-Lys, were designed and then phylogenetically analyzed. Based on detailed morphological observations, only basis capituli length:width ratio (females), the length, the width and the length:width ratio of the scutum (males) had differences between R. turanicus from China and Albania. Gene divergences of 16S rRNA, cox1, partial nad1-16S rRNA, nad2-cox1 and cox1-tRNA-Lys from China and Albania ticks were 3.53-4.84, 3.57-4.92, 3.57-4.07, 3.57-4.39 and 3.18-4.69%, respectively. The evaluated five genetic markers revealed two phylogenetic branches in R. turanicus. Obvious differences exist within R. turanicus based on morphological and genetic analysis. Three newly designed genetic markers (partial nad1-16S rRNA, nad2-cox1 and cox1-tRNA-Lys) in this study may be suitable genetic tools for identification and analysis in R. turanicus. Subspecies analysis of R. turanicus from other regions of the world should be initiated in the future.

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.

Background: In Europe, hard ticks of the subgenus Pholeoixodes (Ixodidae: Ixodes) are usually associated with burrow-dwelling mammals and terrestrial birds. Reports of Pholeoixodes spp. from carnivores are frequently contradictory, and their identification is not based on key diagnostic characters. Therefore, the aims of the present study were to identify ticks collected from dogs, foxes and badgers in several European countries, and to reassess their systematic status with molecular analyses using two mitochondrial markers., Results: Between 2003 and 2017, 144 Pholeoixodes spp. ticks were collected in nine European countries. From accurate descriptions and comparison with type-materials, a simple illustrated identification key was compiled for adult females, by focusing on the shape of the anterior surface of basis capituli. Based on this key, 71 female ticks were identified as I. canisuga, 21 as I. kaiseri and 21 as I. hexagonus. DNA was extracted from these 113 female ticks, and from further 31 specimens. Fragments of two mitochondrial genes, cox1 (cytochrome c oxidase subunit 1) and 16S rRNA, were amplified and sequenced. Ixodes kaiseri had nine unique cox1 haplotypes, which showed 99.2-100% sequence identity, whereas I. canisuga and I. hexagonus had eleven and five cox1 haplotypes, respectively, with 99.5-100% sequence identity. The distribution of cox1 haplotypes reflected a geographical pattern. Pholeoixodes spp. ticks had fewer 16S rRNA haplotypes, with a lower degree of intraspecific divergence (99.5-100% sequence identity) and no geographical clustering. Phylogenetic analyses were in agreement with morphology: I. kaiseri and I. hexagonus (with the similar shape of the anterior surface of basis capituli) were genetically more closely related to each other than to I. canisuga. Phylogenetic analyses also showed that the subgenus Eschatocephalus (bat ticks) clustered within the subgenus Pholeoixodes., Conclusions: A simple, illustrated identification key is provided for female Pholeoixodes ticks of carnivores (including I. hexagonus and I. rugicollis) to prevent future misidentification of these species. It is also shown that I. kaiseri is more widespread in Europe than previously thought. Phylogenetic analyses suggest that the subgenus Pholeoixodes is not monophyletic: either the subgenus Eschatocephalus should be included in Pholeoixodes, or the latter subgenus should be divided, which is a task for future studies.

Background: Bats are regarded as the primary (ancestral) hosts of bugs of the family Cimicidae. The historically and economically most important species in the family is the common bedbug (Cimex lectularius), because of its worldwide occurrence and association with humans. This molecular-phylogenetic study was initiated in order to expand the knowledge on the phylogeny of cimicid bugs of bats, by investigating samples from Hungary, Romania (representing central-eastern Europe) and two further countries (South Africa and Vietnam)., Results: Altogether 216 cimicid bugs were collected (73 Ci. lectularius, 133 Ci. pipistrelli, nine Cacodmus ignotus and one Ca. sparsilis). Members of the Cimex lectularius species group were found both in the environment of bats (only Myotis emarginatus, which is a cave/attic-dwelling species) and on three crevice-dwelling bat species (two pipistrelloid bats and M. bechsteinii). On the other hand, Ci. pipistrelli always occurred off-host (near M. myotis/blythii, which are cave/attic-dwelling species). In addition, two Cacodmus spp. were collected from Pipistrellus hesperidus. The morphological characters of these specimens are illustrated with high resolution pictures. Analysis of cytochrome c oxidase subunit 1 (cox1) sequences generated from 38 samples indicated relative genetic homogeneity of Ci. pipistrelli, while the Ci. lectularius group had two haplotypes (collected from pipistrelloid bats in Hungary and Vietnam) highly divergent from other members of this species group. These results were confirmed with molecular and phylogenetic analyses based on the internal transcribed spacer 2 (ITS2). Bat-associated bugs morphologically identified as Ca. ignotus and Ca. sparsilis were different in their cox1, but identical in their ITS2 sequences., Conclusions: Molecular evidence is provided here on the existence of two new genotypes, most likely new species, within the Ci. lectularius species group. The relevant specimens (unlike the others) were collected from pipistrelloid bats, therefore the association of Ci. lectularius with different bat host species (pipistrelloid vs myotine bats) should be evaluated further as a possible background factor of this genetic divergence. In addition, Ca. ignotus is reported for the first time in South Africa.

Among true bugs (Insecta: Hemiptera), only hematophagous species (families Reduviidae, Cimicidae) have high veterinary and medical significance. In addition, several predatory and plant-feeding bug species, which also have piercing-sucking mouthparts, are known to occasionally bite humans. The majority of such examples are known from the New World. Here, we report the first case concerning the human-biting potential of the western conifer seed bug, Leptoglossus occidentalis Heidemann (Hemiptera: Coreidae). This is a phytophagous bug species, which has become widespread in North America, and has also been introduced into Europe where it shows a rapidly expanding geographical range., (© The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Man-made barriers are well known for their effects on ecosystems. Habitat fragmentation, for instance, is a recognised consequence of modern-day infrastructure. The aim of the present study was to investigate the diversity and abundance of tick species, as well as the risks of acquiring tick-borne infections in habitats adjacent to a freeway. Therefore, ixodid ticks were collected from the vegetation at two-week intervals (in the main tick season, from March to June) in eight habitats of different types (forest, grove, grassland) along both sides of a freeway. Ixodes ricinus females were molecularly screened for three species of tick-borne bacteria. In the study period, 887 ixodid ticks were collected. These included 704 I. ricinus (79.4%), 51 Dermacentor reticulatus (5.7%), 78 D. marginatus (8.8%), 35 Haemaphysalis inermis (3.9%) and 19 H. concinna (2.1%). There was no significant difference in the abundance of tick species between similar habitats separated by the freeway, except for the absence of Dermacentor spp. on one side. In I. ricinus females, the overall prevalence of Anaplasma phagocytophilum was low, and (in part due to this low rate) did not show significant difference between the two sides of the freeway. Rickettsia helvetica had significantly different overall prevalence between two distant habitats along the same side of the freeway (12.3% vs. 31.4%), but not between habitats on the opposite sides. Borrelia burgdorferi s.l. showed significantly different overall prevalence between habitats both on the same and on the opposite sides of the freeway (8.6-35.9%), and the difference was higher if relevant habitats were also separated by the freeway. Importantly, the prevalence rate of the Lyme disease agent was highest in a forested resting area of the freeway, and was significantly inversely proportional to the prevalence of A. phagocytophilum (taking into account all evaluated habitats), apparently related to deer population density. Prevalence rates of these bacteria also differed significantly on single sampling occasions between: (1) closely situated habitats of different types; (2) distant and either similar or different habitat types; and (3) habitats on the opposite sides of the freeway. In conclusion, the findings of the present study show that a fenced freeway may contribute to differences in tick species diversity and tick-borne pathogen prevalence along its two sides, and this effect is most likely a consequence of its barrier role preventing deer movements.

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., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

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.

Dermacentor reticulatus is a tick species of high medical and veterinary importance, emerging in several parts of Europe. Up to now most studies focusing on zoonotic rickettsiae in D. reticulatus were based on ticks collected in a limited part of the questing period, and did not take into account the potential seasonal variations in the rate of infection with tick-borne rickettsiae. The aim of the present study was to investigate the latter phenomenon, i.e. to screen D. reticulatus adults, collected monthly in two urban habitats of Budapest, for the presence of three zoonotic Rickettsia spp. Altogether 852 D. reticulatus adults were collected, which showed significantly similar seasonal activity in the two evaluated habitats. Among the 413 molecularly analysed ticks, R. helvetica-infected D. reticulatus were only collected during autumn in habitat-1, in contrast to habitat-2. The overall prevalence of R. raoultii in D. reticulatus adults was significantly higher in habitat-1 than in habitat-2. In addition, the seasonal distribution of R. raoultii-infected ticks was different between the two habitats (in habitat-2 significantly more R. raoultii-infected ticks were collected in the autumn, in comparison with winter and spring). Rickettsia slovaca was not detected in any of the molecularly analysed ticks. The results clearly indicate that a single-time or seasonally biased collection of D. reticulatus adults and their subsequent molecular analysis may not be informative on the real prevalence of rickettsiae. This is because the availability/ activity of infected ticks shows significant seasonal fluctuations, both within and between habitats. Instead, for screening D. reticulatus-borne rickettsiae, it is important to collect monthly samples and then to assess seasonal prevalence and actual habitat-associated eco-epidemiological risks.

Background: Recently, a high degree of mitochondrial gene heterogeneity was demonstrated between conspecific ixodid ticks of bats in Eurasia. Argas vespertilionis is a soft tick species of mainly vespertilionid bats, also with a wide distribution in the Old World. The aim of this study was to investigate the morphology, mitochondrial gene heterogeneity and host range of A. vespertilionis in the Old World., Results: Altogether 318 soft tick larvae were collected from 17 bat species (belonging to six genera) in seven countries. Based on the general morphology (setal arrangement) of 314 A. vespertilionis larvae, and the detailed measurements of fifteen larvae, only minor morphological differences (in dorsal plate size and the type of serrate setae) were observed between specimens from Europe and Vietnam. On the other hand, cytochrome c oxidase subunit 1 (cox1) and 16S rRNA gene sequence analyses of 17 specimens showed that A. vespertilionis from Europe is genetically different (with up to 7.5% cox1 and 5.7% 16S rRNA gene sequence divergence) from specimens collected in Vietnam, and their phylogenetic separation is well supported., Conclusion: In its evaluated geographical range, no larval phenotypic differences justify the existence of separate species under the name A. vespertilionis. However, phylogenetic analyses based on two mitochondrial markers suggest that it represents a complex of at least two putative cryptic species. The broad host range of A. vespertilionis might partly explain its lower degree of mitochondrial gene heterogeneity in comparison with ixodid bat tick species over the same geographical region of Eurasia.

Background: Rhipicephalus sanguineus belongs to a complex of hard tick species with high veterinary-medical significance. Recently, new phylogenetic units have been discovered within R. sanguineus, which therefore needs taxonomic revision. The present study was initiated to provide new information on the phylogeography of relevant haplotypes from less studied regions of Europe and Africa. With this aim, molecular-phylogenetic analyses of two mitochondrial markers were performed on 50 ticks collected in Hungary, the Balkans, countries along the Mediterranean Sea, Kenya and Ivory Coast., Results: In the "temperate lineage" of R. sanguineus, based on cytochrome c oxidase subunit 1 (cox1) and 16S rRNA genes, Rhipicephalus sp. I was only found in the eastern part of the Mediterranean Basin (with relatively homogenous haplotypes), whereas Rhipicephalus sp. II occurred in the middle-to-western part of this region (with phylogenetically dichotomous haplotypes). Ticks identified as R. leporis (based on morphology and cox1 gene) were found in Kenya and Ivory Coast. These clustered phylogenetically within R. sanguineus (s.l.) ("tropical lineage")., Conclusions: In the Mediterranean Basin two mitochondrial lineages of R. sanguineus, i.e. Rhipicephalus sp. I and Rhipicephalus sp. II exist, which show different geographical distribution. Therefore, data from this study confirm limited gene flow between Rhipicephalus sp. I and Rhipicephalus sp. II, but more evidence (analyses of nuclear markers, extensive morphological and biological comparison etc.) are necessary to infer if they belong to different species or not. The phylogenetic relationships of eastern and western African ticks, which align with R. leporis, need to be studied further within R. sanguineus (s.l.) ("tropical lineage").

The European badger (Meles meles) is a widespread mammal in most countries of the European continent, with increasingly recognized veterinary/medical importance owing to its preferred habitats (including pastures and urban environments), broad spectrum of food items, and role as a game hunting target. However, ticks and tick-borne pathogens associated with badgers are only partly known, and most of them have not yet been analysed with molecular biological methods The aim of this study was to perform molecular taxonomic analysis of ticks collected from a road-killed European badger, as well as to molecularly investigate its ticks and blood sample for the presence of Anaplasmataceae and piroplasms. Ticks from the badger were morphologically identified as females of Ixodes rugicollis. Based on its cytochrome oxidase subunit I (COI) and 16S rRNA sequences, I. rugicollis phylogenetically clustered together with I. lividus and I. arboricola, i.e. other members of the subgenus Pholeoixodes. The blood sample of the badger contained the DNA of Candidatus Neoehrlichia sp. (FU98) recently identified in red fox in Austria and the Czech Republic. This genotype is most closely related to Ca. N. lotoris (from raccoons in North America), and has lower sequence identity with the I. ricinus-transmitted zoonotic agent, Ca. N. mikurensis found in Eurasia. In the blood of the badger and in one female I. rugicollis, the DNA of a new Babesia genotype was also present, which differed from a piroplasm detected in M. meles in Spain, and clustered phylogenetically in the B. microti clade. Phylogenetic analysis of I. rugicollis (based on two genetic markers) confirms its status in subgenus Pholeoixodes. Ca. Neoehrlichia sp. (FU98) was identified for the first time in M. meles and in Hungary. In addition, a molecularly previously not yet characterized Babesia genotype occurs in badgers in Central Europe., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Background: Haemaphysalis erinacei is one of the few ixodid tick species for which valid names of subspecies exist. Despite their disputed taxonomic status in the literature, these subspecies have not yet been compared with molecular methods. The aim of the present study was to investigate the phylogenetic relationships of H. erinacei subspecies, in the context of the first finding of this tick species in Romania., Results: After morphological identification, DNA was extracted from five adults of H. e. taurica (from Romania and Turkey), four adults of H. e. erinacei (from Italy) and 17 adults of H. e. turanica (from China). From these samples fragments of the cytochrome c oxidase subunit 1 (cox1) and 16S rRNA genes were amplified via PCR and sequenced. Results showed that cox1 and 16S rRNA gene sequence divergences between H. e. taurica from Romania and H. e. erinacei from Italy were below 2%. However, the sequence divergences between H. e. taurica from Romania and H. e. turanica from China were high (up to 7.3% difference for the 16S rRNA gene), exceeding the reported level of sequence divergence between closely related tick species. At the same time, two adults of H. e. taurica from Turkey had higher 16S rRNA gene similarity to H. e. turanica from China (up to 97.5%) than to H. e. taurica from Romania (96.3%), but phylogenetically clustered more closely to H. e. taurica than to H. e. turanica., Conclusions: This is the first finding of H. erinacei in Romania, and the first (although preliminary) phylogenetic comparison of H. erinacei subspecies. Phylogenetic analyses did not support that the three H. erinacei subspecies evaluated here are of equal taxonomic rank, because the genetic divergence between H. e. turanica from China and H. e. taurica from Romania exceeded the usual level of sequence divergence between closely related tick species, suggesting that they might represent different species. Therefore, the taxonomic status of the subspecies of H. erinacei needs to be revised based on a larger number of specimens collected throughout its geographical range.

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., Competing Interests: The authors have declared that no competing interests exist.

Fourteen long-legged ixodid ticks (6 nymphs and 8 larvae) were collected from Bechstein's bat (Myotis bechsteinii) in Rochefort, Belgium. All ticks were morphologically identified as Ixodes ariadnae, based on their long legs (Haller's organ longer than maximum diameter of tarsus I), broad palps and posteriorly reverse bell-shaped scutum with wavy surface. The DNA was extracted from these ticks, followed by PCR amplification of part of their cytochrome oxidase subunit I (COI) gene. All obtained sequences were 100% identical with each other, and with the COI sequence of I. ariadnae reported previously from Hungary and Germany. Taking into account that the collection site in the present study is close to the French border of Belgium, and migration of Bechstein's bat is known between Belgium and France, it is reasonable to suppose that I. ariadnae also occurs in France. This is the first record of I. ariadnae in Western Europe, outside its formerly known geographical range (Central Europe).

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)., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

It has been reported from cities in Central Europe that clinical cases of canine babesiosis are most frequent in spring time, despite the fact that the peak activity of Dermacentor reticulatus (the vector of Babesia canis) is during autumn. The present study was initiated to evaluate the seasonal distribution of B. canis-infected D. reticulatus ticks in this context. In two habitats of Budapest 852 D. reticulatus adults were collected between August, 2014 and June, 2015. Among the molecularly analysed 413 ticks 8.2% were PCR positive for piroplasms. Both formerly reported 18S rDNA genotypes of B. canis: ("A" and "B") were identified. In habitat-1 B. canis-infected ticks were detected only in spring. Similarly, in habitat-2 B. canis-infected ticks occurred significantly more frequently during winter and spring than in the autumn (24.6% vs. 1.4%), and their monthly distribution showed significant negative correlation with tick size. The prevalence of infected ticks was the highest (43.5%) in late February. In addition, a month-dependent time-shift was noted in the appearance of the two B. canis 18S rDNA genotypes: the less pathogenic "A" predominating earlier, and the more pathogenic "B" later. It is known from literature that D. reticulatus individuals that moult to adult in the spring are smaller in size. Thus, the above results suggest that in urban habitats the occurrence of B. canis-infected ticks (or their questing activity) is more likely, when there are freshly emerged adults in the population, i.e. early in the questing season. It was also observed that the temporal distribution of D. reticulatus ticks carrying different B. canis genotypes was not random., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Background: In a recent study on ixodid bat ticks from Eurasia, a high genetic difference was found between Ixodes vespertilionis from Europe and Vietnam. Accordingly, it was proposed that I. vespertilionis is a species complex, with at least one additional, hitherto undescribed species. The aim of the present study was to investigate the morphology of bat ticks from Vietnam and to assess their taxonomic status in comparison with those collected in Europe., Findings: Ixodid bat ticks (two females and two nymphs) collected from the pomona leaf-nosed bat (Hipposideros pomona) (Hipposideridae) and intermediate horseshoe bat (Rhinolophus affinis) (Rhinolophidae) in Vietnam showed major morphological differences from European isolates of I. vespertilionis, including the shape of the scutum, the enclosure and shape of porose areas, the presence of a caudo-lateral collar-like ridge ventrally on the basis capituli, polytrich coxae with short setae, and grouped (non-linear) arrangement of anterior pit sensillae in Haller's organ., Conclusions: In this study the female and the nymph of an ixodid bat tick species from Vietnam are described for the first time. The genetic and morphological differences between I. vespertilionis Koch, 1844 and these bat ticks from Vietnam justify the status of the latter as a distinct species, Ixodes collaris Hornok n. sp.

Ecdysteroids are important hormones that regulate moulting in arthropods. Three-host ixodid ticks normally moult to the next stage after finishing their blood meal, in the off-host environment. Presumably, three-host ticks that feed on the blood of insectivorous vertebrate hosts can be exposed to high levels of exogenous ecdysteroids causing them to initiate apolysis (the first step of moulting) on the vertebrate host. The aim of the present study was to investigate whether ticks undergo apolysis on insectivorous song birds, and if this phenomenon is associated with the seasonal variation in the availability of moths and with the presence of naturally acquired ecdysteroids in avian blood. During a triannual survey, 3330 hard tick larvae and nymphs were collected from 1164 insectivorous song birds of 46 species. A noteworthy proportion of ticks, 20.5%, showed apolysis. The occurrence of apolytic ticks on birds was correlated with the known seasonality of lepidopteran caterpillars. In addition, 18 blood samples of tick-infested birds were analysed with liquid chromatography - tandem mass spectrometry. Eight samples contained ecdysteroids or their derivatives, frequently in high concentrations, and the presence of these was associated with tick apolysis. In conclusion, naturally acquired ecdysteroids may reach high levels in the blood of insectivorous passerine birds, and will affect ticks (feeding on such blood) by shortening their parasitism.

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.

Background: Increasing numbers of genetic variants are being recognized among piroplasms, but the precise taxonomical status, the tick vector and the geographical range of several species or genotypes are still unknown. Bovine piroplasmosis was reported to re-emerge in north-east Hungary. Because Theileria-infection was newly diagnosed in one cattle herd in the same region of the country, the aim of this study was to molecularly identify the relevant agent, to find its local vector tick species, and to examine the range of Babesia/Theileria spp. of ruminants in Haemaphysalis sp. ticks collected previously in Hungary., Findings: Blood samples were drawn on two occasions from 90 dairy cattle in northern Hungary, and ticks were collected on their pastures. In addition, questing ticks (315 Haemaphysalis inermis, 259 H. concinna and 22 H. punctata), which originated mainly in the same region of the country from 2007, were included in the study. DNA was extracted from these samples, followed by molecular analysis for piroplasms. In the cattle Theileria orientalis was identified, with 100 % sequence homology to isolates from Japan, China, South-Africa and Australia. Based on GenBank data this genotype has not been previously reported in Europe. The prevalence of infection in the herd remained almost constant in the main tick season, suggesting exposure in previous years. Retrospective analysis of ticks revealed the presence of Babesia crassa in H. inermis, for the first time in Europe and in this tick species. On the other hand, H. concinna carried five different piroplasms, including B. motasi that was also newly detected in Central-Eastern Europe and in this tick species; whereas H. punctata harboured Theileria sp. OT3, hitherto known to occur in the Mediterranean region., Conclusions: Results of this study broaden the range of piroplasms that are infective for ruminants in Central-Eastern Europe. Although bovine babesiosis and theileriosis was known to occur in Hungary, molecular evidence is provided here for the first time on the presence of Babesia and/or Theileria spp. of sheep, goats and cervids in Hungary.

Background: Phylogeographical studies allow precise genetic comparison of specimens, which were collected over large geographical ranges and belong to the same or closely related animal species. These methods have also been used to compare ticks of veterinary-medical importance. However, relevant data are missing in the case of ixodid ticks of bats, despite (1) the vast geographical range of both Ixodes vespertilionis and Ixodes simplex, and (2) the considerable uncertainty in their taxonomy, which is currently unresolvable by morphological clues., Methods: In the present study 21 ticks were selected from collections or were freshly removed from bats or cave walls in six European and four Asian countries. The DNA was extracted and PCRs were performed to amplify part of the cytochrome oxidase I (COI), 16S and 12S rDNA genes, followed by sequencing for identification and molecular-phylogenetic comparison., Results: No morphological differences were observed between Ixodes vespertilionis specimens from Spain and from other parts of Europe, but corresponding genotypes had only 94.6 % COI sequence identity. An I. vespertilionis specimen collected in Vietnam was different both morphologically and genetically (i.e. with only 84.1 % COI sequence identity in comparison with I. vespertilionis from Europe). Two ticks (collected in Vietnam and in Japan) formed a monophyletic clade and shared morphological features with I. ariadnae, recently described and hitherto only reported in Europe. In addition, two Asiatic specimens of I. simplex were shown to differ markedly from European genotypes of the same species. Phylogenetic relationships of ticks showed similar clustering patterns with those of their associated bat host species., Conclusions: Although all three ixodid bat tick species evaluated in the present study appear to be widespread in Eurasia, they exhibit pronounced genetic differences. Data of this study also reflect that I. vespertilionis may represent a species complex.

Background: Bovine besnoitiosis has been recently diagnosed in a three-parted herd of 796 Aubrac and Charolais beef cattle in Hungary. A large scale serological, histological and molecular survey was initiated in order to uncover important factors in the local epidemiology of the disease., Findings: Blood samples were collected (three times from the whole herd, and repeatedly from selected animals) for serological screening by ELISA. In addition, various organs from aborted fetuses and newborn calves, skin and colostrum samples of seropositive heifers/cows, and ticks collected from the cattle were histologically and/or molecularly analysed for the presence of Besnoitia besnoiti. All fetal and calf tissues, as well as colostrum and tick samples from cows were PCR negative. Based on ELISA results, only very few local cows seroconverted after mating with imported, infected bulls, and not necessarily as a consequence of this event. Among calves that were born to seropositive, imported cows and stayed with their mother until weaning at seven months of age, seroprevalence decreased significantly, but remained high. At the same time, 28 calves born from seropositive cows, but separated from their dams immediately after receiving colostrum, were successfully reared and remained uninfected. Following a second herd-level screening, all Aubrac cattle (except for heifer calves) and all seropositive Charolais cows and bulls were culled. Manifestation of the disease is currently sporadic. Among chronically affected heifers two types of skin lesions were noted, and histological evaluation indicated marked distension of sweat gland ducts with membrane-bound structures (resembling cystozoites) in their contents., Conclusions: Transmission through natural mating, as well as transplacental, colostral and tick-borne transmission of B. besnoiti was either unlikely or did not occur. However, the risk for spreading of the infection was high, when calves stayed with their mother during suckling, and if animals were kept in the same stable (although physically separated) during the main fly season. Herd replacement and generation exchange (i.e. early weaning and artificial feeding) appear to be the successful strategies for the local eradication of bovine besnoitiosis. Adding to the already known mechanical transmission of B. besnoiti by blood-sucking flies, results of the present study suggest that secretophagous flies should also be evaluated as potential vectors of this coccidium species.

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.

Background: Bats are among the most eco-epidemiologically important mammals, owing to their presence in human settlements and animal keeping facilities. Roosting of bats in buildings may bring pathogens of veterinary-medical importance into the environment of domestic animals and humans. In this context bats have long been studied as carriers of various pathogen groups. However, despite their close association with arthropods (both in their food and as their ectoparasites), only a few molecular surveys have been published on their role as carriers of vector-borne protozoa. The aim of the present study was to compensate for this scarcity of information., Findings: Altogether 221 (mostly individual) bat faecal samples were collected in Hungary and the Netherlands. The DNA was extracted, and analysed with PCR and sequencing for the presence of arthropod-borne apicomplexan protozoa. Babesia canis canis (with 99-100% homology) was identified in five samples, all from Hungary. Because it was excluded with an Ixodidae-specific PCR that the relevant bats consumed ticks, these sequences derive either from insect carriers of Ba. canis, or from the infection of bats. In one bat faecal sample from the Netherlands a sequence having the highest (99%) homology to Besnoitia besnoiti was amplified., Conclusions: These findings suggest that some aspects of the epidemiology of canine babesiosis are underestimated or unknown, i.e. the potential role of insect-borne mechanical transmission and/or the susceptibility of bats to Ba. canis. In addition, bats need to be added to future studies in the quest for the final host of Be. besnoiti.

The occurrence of the nematode Angiostrongylus vasorum, also known as the French heartworm, is increasingly being reported from various European countries. The adults of this parasite species live in the pulmonary arteries and right cardiac ventricle of wild canids and domestic dogs. Larval stages and eggs in the lungs induce inflammatory verminous pneumonia, causing severe respiratory disease in dogs. Furthermore, haematological and neurological signs and even death may occur. In Hungary, A. vasorum has been identified in red foxes, golden jackals and in two dogs and some slugs. In this first large-scale survey, 1247 sera from pet dogs were collected and tested by an ELISA for the detection of circulating antigen of A. vasorum and by a separate ELISA to detect specific antibodies against the parasite. A total of 1.36% (n = 17, 95 % confidence intervals, CI: 0.80 - 2.17 %) of the animals were positive in both ELISAs, while 1.76 % (n = 22, CI: 1.11 - 2.66 %) of the tested dogs were antigen-positive only and 2.73 % (n = 34, CI: 1.90 - 3.79 %) were positive for specific antibodies only. Regions with antigen- and antibody-positive animals overlapped and were distributed over nearly the whole sampled areas of the country. A considerable number of cases was observed in Budapest and also in the southern part of the country bordering Croatia, while in the most eastern part bordering Ukraine no positive samples were detected. These results confirm the endemic occurrence of A. vasorum in dogs originating from different parts of Hungary and the significant advantages of A. vasorum serology in epidemiological studies.

Background: Reports on Sarcocystis-infection of cattle are outdated or lacking in many European countries, including those in the Central-Eastern part of the continent. Therefore, to assess the prevalence of Sarcocystis spp. among bovids in Hungary, a countrywide survey was initiated. In addition, fulminant deaths of four cattle, that showed clinical signs and post mortem lesions resembling acute sarcocystiosis ("Dalmeny disease"), were investigated., Methods: During the countrywide survey individual heart and oesophagus samples were collected at slaughterhouses from 151 beef cattle and from 15 buffalo, kept in 31 places of Hungary. Analysis for Sarcocystis spp. was carried out with conventional PCRs for the 18S rDNA gene and gel electrophoresis, followed by sequencing of 36 strongly positive samples. Mortality cases were evaluated by histological, molecular, bacteriological and virological analyses of samples from various organs., Results: Among slaughtered cattle the rate of Sarcocystis-infection was 66%. S. cruzi was identified as the most prevalent species in aurochs-like breed, and the zoonotic S. hominis in Hungarian grey cattle. Concerning the sudden deaths of cattle, Sarcocystis-infection could not be demonstrated in organs showing haemorrhages, but S. cruzi cysts were present in the muscles. In one case "S. sinensis" was molecularly identified in the blood (indicating sarcocystaemia). Results of analyses for bacterial/viral pathogens were negative., Conclusions: S. cruzi appears to be the most prevalent Sarcocystis sp. in cattle in Hungary, followed by the zoonotic S. hominis. However, the rate of infection with both species was shown to differ between cattle breeds. The suspected role of Sarcocystis spp. as causative agents of the fatal cases could not be confirmed.

Background: To date, only one report of a small Babesia infection based on microscopic observation which caused babesiosis in two dogs in Hungary has been published. Babesiosis due to Babesia canis - which is endemic in the local dogs - has only been detected in captive grey wolves. No information is available on babesial/theilerial infections in red foxes in Hungary. The aim of the study was to screen red foxes in Hungary for babesial parasites by PCR and to compare their partial 18S rRNA gene sequences to those parasites of domestic dogs and wild canids from other countries., Methods: Blood samples of 404 red foxes originating from 316 locations representing all 19 Hungarian counties were screened in Hungary for babesial parasites by PCR and the partial 18S rRNA gene sequences were compared to those parasites of domestic dogs and wild canids from other countries., Results: Altogether 81 red foxes out of 404 (20.0%; 95% CI: 16.4-24.2%) shot in 74 locations and in 17 of the 19 Hungarian counties were found to be infected with Babesia cf. microti by PCR., Conclusions: This is the first report to demonstrate the occurrence of Babesia cf. microti in Hungary, and its widespread presence in the fox population throughout the country. Further studies are needed to identify the tick species involved in its transmission, and whether other mechanisms of transmission are involved in its spread in fox populations.

Background: Recently a new hard tick species, Ixodes ariadnae has been discovered, adding to the two known ixodid tick species (I. vespertilionis and I. simplex) of bats in Europe., Findings: Scanning electron microscopic comparison of adult females of these species shows morphological differences concerning the palps, the scutum, the Haller's organ, the coxae, as well as the arrangement and fine structure of setae. Molecular analysis of 10 geographically different isolates revealed 90-95% sequence homology in the 12S and 16S rDNA genes of bat tick species. Based on 12S rDNA sequences, genotypes of I. ariadnae clustered closest to I. simplex, whereas according to their 16S rDNA gene they were closest to I. vespertilionis. The subolesin gene of I. ariadnae had only 91% sequence homology with that of I. ricinus, and is the longest known among hard tick species., Conclusions: The present study illustrates the morphology and clarifies the phylogenetic relationships of the three known bat tick species that occur in Europe. According to its subolesin gene I. ariadnae may have a long evolutionary history.

Ticks, as vectors of several notorious zoonotic pathogens, represent an important and increasing threat for human and animal health in Europe. Recent applications of new technology revealed the complexity of the tick microbiome, which may affect its vectorial capacity. Appreciation of these complex systems is expanding our understanding of tick-borne pathogens, leading us to evolve a more integrated view that embraces the 'pathobiome'; the pathogenic agent integrated within its abiotic and biotic environments. In this review, we will explore how this new vision will revolutionize our understanding of tick-borne diseases. We will discuss the implications in terms of future research approaches that will enable us to efficiently prevent and control the threat posed by ticks., Competing Interests: Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Background: Coxiella burnetii, the etiologic agent of Q fever, is a highly infectious zoonotic bacterium. Genetic information about the strains of this worldwide distributed agent circulating on the African continent is limited. The aim of the present study was the genetic characterization of C. burnetii DNA samples detected in ticks collected from Ethiopian cattle and their comparison with other genotypes found previously in other parts of the world., Methodology/principal Findings: A total of 296 tick samples were screened by real-time PCR targeting the IS1111 region of C. burnetii genome and from the 32 positive samples, 8 cases with sufficient C. burnetii DNA load (Amblyomma cohaerens, n = 6; A. variegatum, n = 2) were characterized by multispacer sequence typing (MST) and multiple-locus variable-number tandem repeat analysis (MLVA). One novel sequence type (ST), the proposed ST52, was identified by MST. The MLVA-6 discriminated the proposed ST52 into two newly identified MLVA genotypes: type 24 or AH was detected in both Amblyomma species while type 26 or AI was found only in A. cohaerens., Conclusions/significance: Both the MST and MLVA genotypes of the present work are closely related to previously described genotypes found primarily in cattle samples from different parts of the globe. This finding is congruent with the source hosts of the analyzed Ethiopian ticks, as these were also collected from cattle. The present study provides genotype information of C. burnetii from this seldom studied East-African region as well as further evidence for the presumed host-specific adaptation of this agent.

The aim of the present study was to compare different urban biotopes for the occurrence of ixodid tick species, for the population density of Ixodes ricinus and for the prevalence rates of two emerging, zoonotic pathogens. Altogether 2455 ticks were collected from the vegetation on 30 places (forests, parks, cemeteries) of Budapest, Hungary. I. ricinus and Haemaphysalis concinna were collected in all three biotope types, but Dermacentor reticulatus only in parks and forests, and D. marginatus only in a forest. Highest population density of I. ricinus was observed in neglected parts of cemeteries. In females of this tick species the prevalence rates of Anaplasma phagocytophilum and Borrelia burgdorferi s.l. were significantly lower in cemeteries, than in parks or forests. In conclusion, risks associated with the presence of ticks and tick-borne pathogens may be high in a city, but this depends on biotope types, due to habitat-related differences in the vegetation, as well as in the availability of tick hosts and pathogen reservoirs., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The expanding family of Francisellaceae includes the genus Francisella, where several pathogen bacteria, e.g. the zoonotic F. tularensis, and different Francisella-like agents belong to. Francisella-like endosymbionts (FLEs) are widespread in hard and soft ticks and their pathogenicity is unknown. The examination of 296 ticks collected in Ethiopia was performed for the detection of F. tularensis and FLEs using polymerase chain reaction (PCR) assays based on the amplification of 16S rRNA, sdhA and tul4 gene fragments. FLE was described in one Hyalomma rufipes tick based on the 16S rRNA and sdhA gene sequences. The 16S rRNA gene fragment was identical with the ones detected previously in Rhipicephalus sanguineus and Hyalomma marginatum marginatum in Bulgaria. The presence of endosymbionts with identical 16S rRNA gene sequence in both Rhipicephalus and Hyalomma species further supports the hypotheses, that certain FLEs had independent evolution from their tick hosts., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Background: The majority of vector-borne infections occur in the tropics, including Africa, but molecular eco-epidemiological studies are seldom reported from these regions. In particular, most previously published data on ticks in Ethiopia focus on species distribution, and only a few molecular studies on the occurrence of tick-borne pathogens or on ecological factors influencing these. The present study was undertaken to evaluate, if ticks collected from cattle in different Ethiopian biotopes harbour (had access to) different pathogens., Methods: In South-Western Ethiopia 1032 hard ticks were removed from cattle grazing in three kinds of tick biotopes. DNA was individually extracted from one specimen of both sexes of each tick species per cattle. These samples were molecularly analysed for the presence of tick-borne pathogens., Results: Amblyomma variegatum was significantly more abundant on mid highland, than on moist highland. Rhipicephalus decoloratus was absent from savannah lowland, where virtually only A. cohaerens was found. In the ticks Coxiella burnetii had the highest prevalence on savannah lowland. PCR positivity to Theileria spp. did not appear to depend on the biotope, but some genotypes were unique to certain tick species. Significantly more A. variegatum specimens were rickettsia-positive, than those of other tick species. The presence of rickettsiae (R. africae) appeared to be associated with mid highland in case of A. variegatum and A. cohaerens. The low level of haemoplasma positivity seemed to be equally distributed among the tick species, but was restricted to one biotope type., Conclusions: The tick biotope, in which cattle are grazed, will influence not only the tick burden of these hosts, but also the spectrum of pathogens in their ticks. Thus, the presence of pathogens with alternative (non-tick-borne) transmission routes, with transstadial or with transovarial transmission by ticks appeared to be associated with the biotope type, with the tick species, or both, respectively.

Background: The prevalence of bovine babesiosis caused by Babesia divergens has been declining during the past decades in northeastern Hungary, and no cases have been observed since 2008. Infections of cattle with B. major and Theileria buffeli were hitherto reported in southern and western Europe. In other parts of the globe, there is evidence of emergence and a growing clinical importance of T. buffeli and closely related genotypes of the T. orientalis complex., Findings: In a herd of 88 beef cattle kept in northeastern Hungary, bovine piroplasmosis was diagnosed in nine animals through the examination of blood smears or by molecular methods. B. major was identified in five animals, two of which died. In addition, four cattle harboured T. buffeli, and one of these animals was anaemic. Despite their presence, a contributory role of Anaplasma marginale and A. phagocytophilum could not be established in the disease cases., Conclusions: In this study B. major and bovine theileriosis is reported for the first time in central-eastern Europe, where clinical cases were associated with a mild winter.

Background: Recently, Hepatozoon canis infection has been detected among shepherd, hunting and stray dogs in the southern part of Hungary, which is considered to be free of Rhipicephalus sanguineus sensu lato and close to the border with Croatia. The aim of this study was to acquire information on the possibility that red foxes and/or golden jackals could play a role in the appearance and spread of H. canis in Hungary., Methods: A conventional PCR was used to amplify a 666 bp long fragment of the Hepatozoon 18S rRNA gene from blood samples collected from 334 foxes shot in 231 locations in 16 counties and 15 golden jackals shot in 9 locations in two southwestern counties close to Croatia. A second PCR assay was performed in some of the samples positive by the first PCR to amplify a larger segment (approximately 1500 bp) of the 18S rRNA gene of Hepatozoon spp. for further phylogenetic analysis., Results: Hepatozoon infection was detected in canids shot in 30 locations and 9 counties. Altogether 26 foxes (8.0%, 95% CI: 5-11%) and 9 jackals (60%, 95% CI: 33-81%) were PCR positive. Hepatozoon canis sequences were obtained from 12 foxes and 7 jackals. DNA sequences from 16 animals were 99-100% similar to H. canis from Croatian foxes or dogs while two of the sequences were 99% similar to an Italian fox. Half (13/26) of the infected red foxes and all golden jackals were shot in the two southwestern counties., Conclusions: This is the first report on molecular evidence of H. canis in red foxes (Vulpes vulpes) and golden jackals (Canis aureus) from Hungary, which is considered free from the tick vector of H. canis, R. sanguineus. Although no R. sanguineus sensu lato had been found on infected or non-infected wild canids, the detection of authochnous canine hepatozoonosis in Hungary might imply that the range of R. sanguineus sensu lato has reached this country.

Background: In Europe two ixodid bat tick species, Ixodes vespertilionis and I. simplex were hitherto known to occur., Methods: Bat ticks were collected from cave walls and bats in Hungary. Their morphology and genotypes were compared with microscopy and conventional PCR (followed by sequencing), respectively., Results: A year-round activity of I. vespertilionis was observed. Molecular analysis of the cytochrome oxidase subunit I (COI) gene of twenty ticks from different caves showed that the occurrence of the most common genotype was associated with the caves close to each other. A few specimens of a morphologically different tick variant were also found and their COI analysis revealed only 86-88% sequence homology with I. simplex and I. vespertilionis, respectively., Conclusions: The microenvironment of caves (well separated from each other) appears to support the existence of allopatric I. vespertilionis COI genotypes, most likely related to the distance between caves and to bat migration over-bridging certain caves. The name I. ariadnae sp. nov. is given to the new tick species described here for the first time.

Background: Birds have long been known as carriers of ticks, but data from the literature are lacking on their role as a reservoir in the epidemiology of certain tick-borne disease-causing agents. Therefore, the aim of this study was to evaluate the presence of three emerging, zoonotic tick-borne pathogens in blood samples and ticks of birds and to assess the impact of feeding location preference and migration distance of bird species on their tick infestation., Methods: Blood samples and ticks of birds were analysed with TaqMan real-time PCRs and conventional PCR followed by sequencing., Results: During the spring and autumn bird migrations, 128 blood samples and 140 ticks (Ixodes ricinus, Haemaphysalis concinna and a Hyalomma specimen) were collected from birds belonging to 16 species. The prevalence of tick infestation and the presence of tick species were related to the feeding and migration habits of avian hosts. Birds were shown to be bacteraemic with Rickettsia helvetica and Anaplasma phagocytophilum, but not with Candidatus Neoehrlichia mikurensis. The prevalence of rickettsiae was high (51.4%) in ticks, suggesting that some of them may have acquired their infection from their avian host., Conclusion: Based on the present results birds are potential reservoirs of both I. ricinus transmitted zoonotic pathogens, R. helvetica and A. phagocytophilum, but their epidemiological role appears to be less important concerning the latter, at least in Central Europe.

Background: Besnoitia besnoiti, the cause of bovine besnoitiosis, is a cyst-forming coccidian parasite that has recently been shown to be spreading in several Western and Southern European countries., Findings: Clinical cases of bovine besnoitiosis were confirmed for the first time in Hungary, by histological, serological and PCR analyses., Conclusions: This is the first report of autochthonous bovine besnoitiosis in Central-Eastern Europe. The emergence of bovine besnoitiosis in this region represents a further example, when human activity (i.e. cattle trading) is the main factor involved in the geographical spread of an infectious disease.

This is the first large-scale molecular investigation of fleas from a geographically widespread and highly urbanized species, the northern white-breasted hedgehog. In this study, 759 fleas (the majority were Archaeopsylla erinacei) collected from 134 hedgehogs were molecularly analyzed individually or in pools for the presence of three groups of vector-borne pathogens. All flea samples were positive for rickettsiae: In two samples (1.5%) Rickettsia helvetica and in 10% of the others a novel rickettsia genotype were identified. Additionally, Bartonella henselae (the causative agent of cat scratch disease in humans) was demonstrated in one flea (0.7%), and hemoplasmas of the hemofelis group were identified in seven other samples (5.2%). The findings of vector-borne agents not detected before in A. erinacei fleas broaden the range of those diseases of veterinary-medical importance, of which hedgehogs may play a role in the epidemiology.

Blood samples were collected from 100 shepherd dogs, 12 hunting dogs and 14 stray dogs (apparently healthy) in southern Hungary to screen for the presence of emerging tick-borne pathogens. Based on real-time PCR results, 14 dogs (11%) had single or dual haemoplasma infection, and a same number of samples were positive for Anaplasma phagocytophilum. In one sample Coxiella burnetii was molecularly identified, and 20.3% of dogs seroconverted to the Q fever agent. Rickettsaemia (sensu stricto) was also detected in one animal. This is the first molecular evidence of autochthonous infection of dogs with the above pathogens in Hungary. The relatively high prevalence of haemoplasma and anaplasma infection among non-pet dogs is suggestive of a prolonged carrier status and bacteraemia of these animals rendering them epidemiologically significant as potential reservoirs and sentinels for tick-borne infections., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Blood samples and ticks were collected from 100 shepherd dogs, 12 hunting dogs and 14 stray dogs in southern Hungary, in order to screen them for the presence of Hepatozoon spp. by PCR. Out of 126 blood samples, 33 were positive (26%). Significantly more shepherd dogs (31%) were infected, than hunting (8%) and stray dogs (7%). Three genotypes of Hepatozoon canis were identified by sequencing, differing from each other in up to six nucleotides in the amplified portion of their 18S rRNA gene. In Dermacentor marginatus larvae/nymphs and Dermacentor reticulatus nymphs, H. canis was present only if they had been collected from PCR-positive dogs, and the genotypes were identical in the ticks and their hosts. However, two Haemaphysalis concinna nymphs removed from a PCR-negative dog were found positive for H. canis, and the genotype detected in specimens of this tick species differed from that in the blood of their respective hosts. These results indicate that canine hepatozoonosis may be highly prevalent in regions where Rhipicephalus sanguineus is considered to be non-endemic. In addition, H. canis was identified for the first time in Hungary, as well as in D. marginatus, D. reticulatus and Ha. concinna ticks. Canine hepatozoonosis was significantly more prevalent west of the Danube river (where higher densities of red fox and golden jackal populations occur), suggesting a role of wild carnivores in its epidemiology., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Several new taxa belonging to the genus Francisella have been described recently. The present study describes the prevalence of Francisella tularensis and Francisella-like endosymbionts (FLE) in ticks collected from Hungary from 2007 to 2009 and characterizes the genetic variability of FLEs. A total of 5402 Ixodid ticks (Ixodes ricinus, I. acuminatus, Dermacentor marginatus, D. reticulatus, Haemaphysalis inermis, H. concinna, H. punctata) were collected from vegetation and animal hosts and tested with conventional PCR, detecting the 16S rRNA and tul4 genes. F. tularensis ssp. holarctica was found in 2 pools of H. concinna and 1 pool of D. reticulatus, both representing minimum prevalence (calculated with 1 infected tick per pool) of 0.27% whereas the sequences of a FLE were detected in 11 pools of D. reticulatus showing a minimum prevalence of 3%. Although the tul4 gene sequence of this FLE was identical to all Hungarian and Portuguese FLEs found earlier, and the 16S rRNA sequence was also identical to the sequence of the endosymbiont of D. reticulatus described in Bulgaria, these 16S rRNA gene coding sequences differed in 2 nucleotides from the one found earlier in this tick species in Hungary. This divergence may appear to be a minor difference between the sequences, potentially even resulting from a technical failure, but it could also indicate a significant difference stemming from the conservative genetic character of Francisellaceae. Thus, it raises a question about the number of FLE variants circulating in D. reticulatus in Europe and indicates the need for further data about the FLEs described in other parts of the continent and new FLE genotyping markers.