Krisztina Szőke

DNA of Piroplasms of Ruminants and Dogs in Ixodid Bat Ticks.

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.

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.

Mitochondrial gene heterogeneity of the bat soft tick Argas vespertilionis (Ixodida: Argasidae) in the Palaearctic

BackgroundRecently, 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.ResultsAltogether 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.ConclusionIn 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.

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.

Molecular identification of badger-associated Babesia sp. DNA in dogs: Updated phylogeny of piroplasms infecting Caniformia

BackgroundPiroplasms are unicellular, tick-borne parasites. Among them, during the past decade, an increasing diversity of Babesia spp. has been reported from wild carnivores. On the other hand, despite the known contact of domestic and wild carnivores (e.g. during hunting), and a number of ixodid tick species they share, data on the infection of dogs with babesiae from other families of carnivores are rare.MethodsIn this study blood samples were collected from 90 dogs and five road-killed badgers. Ticks were also removed from these animals. The DNA was extracted from all blood samples, and from 33 ticks of badgers, followed by molecular analysis for piroplasms with PCR and sequencing, as well as by phylogenetic comparison of detected genotypes with piroplasms infecting carnivores.ResultsEleven of 90 blood DNA extracts from dogs, and all five samples from badgers were PCR-positive for piroplasms. In addition to the presence of B. canis DNA in five dogs, sequencing identified the DNA of badger-associated “Babesia sp. Meles-Hu1” in six dogs and in all five badgers. The DNA of “Babesia sp. Meles-Hu1” occurred significantly more frequently in dogs often taken to forests (i.e. the preferred habitat of badgers in Hungary), than in dogs without this characteristic. Moreover, detection of DNA from this Babesia sp. was significantly associated with hunting dogs in comparison with dogs not used for hunting. Two PCR-positive dogs (in one of which the DNA of the badger-associated Babesia sp. was identified, whereas in the other the DNA of B. canis was present) showed clinical signs of babesiosis. Engorged specimens of both I. canisuga and I. hexagonus were collected from badgers with parasitaemia, but only I. canisuga contained the DNA of “Babesia sp. Meles-Hu1”. This means a significant association of the DNA from “Babesia sp. Meles-Hu1” with I. canisuga. Phylogenetically, “Babesia sp. Meles-Hu1” belonged to the “B. microti” group.ConclusionsThis is the first detection of the DNA from a badger-associated Babesia sp. in dogs, one of which also showed relevant clinical signs. Based on the number of dogs with blood samples containing the DNA of “Babesia sp. Meles-Hu1” in this study (i.e. exceeding the number of B. canis-positives), these findings should not be regarded as isolated cases. It is assumed that dogs, which are used for hunting or frequently visit forests, are more likely to be exposed to this piroplasm, probably as a consequence of infestation with I. canisuga from badgers or from the burrows of badgers. The above results suggest that “Babesia sp. Meles-Hu1” should be added to the range of piroplasms, which are naturally capable of infecting hosts from different families of Caniformia.

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.

Impact of a freeway on the dispersal of ticks and Ixodes ricinus-borne pathogens: forested resting areas may become Lyme disease hotspots

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.

Molecular evidence of a badger-associated Ehrlichia sp., a Candidatus Neoehrlichia lotoris-like genotype and Anaplasma marginale in dogs

The family Anaplasmataceae contains pathogenic and endosymbiotic bacteria of veterinary-medical importance. In this study, 90 blood samples from rural dogs, five blood samples from road-killed European badgers and 34 ticks, i.e. 27 Ixodes (Pholeoixodes) canisuga, six I. (Ph.) hexagonus and one Haemaphysalis concinna collected from the badgers were molecularly analysed for members of Anaplasmataceae. Apart from the molecular evidence of Anaplasma phagocytophilum in one dog and Wolbachia sp. associated with Dirofilaria repens in five dogs, four species/genotypes not yet known to occur in canine hosts have also been found. These included A. marginale in two dogs, a badger-associated Ehrlichia sp. in one dog, a Candidatus Neoehrlichia lotoris-like genotype in six dogs and the DNA of arthropod-associated wolbachiae in three dogs. In two badgers the DNA from the Candidatus N. lotoris-like genotype was identified. Among ticks, four I. canisuga carried the DNA of the above badger-associated Ehrlichia sp., one I. canisuga contained the Candidatus N. lotoris-like genotype, and in H. concinna Wolbachia DNA was present. In conclusion, results shown here should be interpreted as the first molecular evidence for exposure of dogs to three members of Anaplasmataceae, i.e. A. marginale, a badger-associated Ehrlichia sp. and a Candidatus N. lotoris-like agent. The presence of DNA in the blood of relevant animals may also indicate susceptibility to these bacteria, but in support of this, further studies are needed.

High mitochondrial sequence divergence in synanthropic flea species (Insecta: Siphonaptera) from Europe and the Mediterranean

BackgroundAdult fleas are haematophagous ectoparasites of warm-blooded vertebrates, particularly mammals. Among them, the cat flea (Ctenocephalides felis) and the human flea (Pulex irritans) have high veterinary-medical significance, owing to their cosmopolitan distribution and role in the transmission of important vector-borne pathogens. While the taxonomy of Ct. felis has been investigated on a morphological basis during the past decades, its molecular-phylogenetic analyses have been only recently conducted. This study expands the knowledge on Ct. felis from hitherto less studied geographical regions, and includes representatives from additional flea families, less investigated with molecular approaches.MethodsFleas were collected in four countries of the Mediterranean Basin (Croatia, Italy, Malta and Israel), as well as in Hungary, from domestic and wild carnivores, rodents and humans. The DNA extracts of representative fleas (n = 148), belonging to ten species of eight genera, were used for PCR amplification of part of their cytochrome c oxidase subunits 1, 2 (cox1, cox2) and 18S rRNA genes, followed by sequencing and phylogenetic analyses.ResultsThe majority (65.6%) of Ct. felis felis cox2 sequences showed 99.4–100% similarity to each other (haplogroup A), whereas those from Malta and Israel had 98.1–98.7% sequence similarity (haplogroup B), and a third sequence from Israel (haplotype C) had as low as 96.3% sequence similarity in comparison with a reference sequence from group “A”. Except for the shape of the head, no consistent morphological differences (e.g. in chaetotaxy) were found between haplogroups “A” and “C”. Haplotypes of Ct. canis were genetically more homogenous, with 99.6–100% sequence similarity to each other. However, when P. irritans collected from humans was compared to those from three species of wild carnivores, these only had 96.6% cox2 similarity. The mouse flea, Leptopsylla segnis and the northern rat flea, Nosopsyllus fasciatus were both shown to have haplotypes with low intraspecific cox2 similarities (96.2 and 94.4%, respectively). Taken together, differences between mitochondrial lineages within four flea species exceeded that observed between two Chaetopsylla spp. (which had 97.3% cox2 similarity). The topologies of cox1 and cox2 phylogenetic trees were in line with relevant sequence comparisons. Conversely, 18S rRNA gene analyses only resolved differences above the species level.ConclusionsCtenocephalides felis felis, P. irritans, L. segnis and N. fasciatus were shown to have such a high level of mitochondrial gene heterogeneity, that the uniformity of these flea taxa should be reconsidered. Although the present results are limited (especially in the case of L. segnis and N. fasciatus), there appears to be no geographical or host restriction, which could explain the divergence of these genetic lineages.