Nóra Takács

Birds as potential reservoirs of tick-borne pathogens: First evidence of bacteraemia with Rickettsia helvetica

BackgroundBirds 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.MethodsBlood samples and ticks of birds were analysed with TaqMan real-time PCRs and conventional PCR followed by sequencing.ResultsDuring 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.ConclusionBased 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.

First report on Babesia cf. microti infection of red foxes (Vulpes vulpes) from Hungary

BackgroundTo 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.MethodsBlood 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.ResultsAltogether 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.ConclusionsThis 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.

Occurrence of ticks and prevalence of Anaplasma phagocytophilum and Borrelia burgdorferi s.l. in three types of urban biotopes: forests, parks and cemeteries.

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.

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.

Re-emergence of bovine piroplasmosis in Hungary: has the etiological role of Babesia divergens been taken over by B. major and Theileria buffeli?

BackgroundThe 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.FindingsIn 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.ConclusionsIn 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.

Sarcocystis-infection of cattle in Hungary

BackgroundReports 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.MethodsDuring 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.ResultsAmong 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.ConclusionsS. 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.

Molecular analysis of Ixodes rugicollis, Candidatus Neoehrlichia sp. (FU98) and a novel Babesia genotype from a European badger (Meles meles).

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.

East and west separation of Rhipicephalus sanguineus mitochondrial lineages in the Mediterranean Basin

BackgroundRhipicephalus 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.ResultsIn 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”).ConclusionsIn 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”).

Diversity of Haemaphysalis-associated piroplasms of ruminants in Central-Eastern Europe, Hungary.

BackgroundIncreasing 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.FindingsBlood 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.ConclusionsResults 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.

Contributions to the phylogeny of Ixodes (Pholeoixodes) canisuga, I. (Ph.) kaiseri, I. (Ph.) hexagonus and a simple pictorial key for the identification of their females

BackgroundIn 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.ResultsBetween 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.ConclusionsA 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.