Diverse tick-borne microorganisms identified in free-living ungulates in Slovakia

Background: Free-living ungulates are hosts of ixodid ticks and reservoirs of tick-borne microorganisms in central Europe and many regions around the world. Tissue samples and engorged ticks were obtained from roe deer, red deer, fallow deer, mouflon, and wild boar hunted in deciduous forests of south-western Slovakia. DNA isolated from these samples was screened for the presence of tick-borne microorganisms by PCR-based methods. Results: Ticks were found to infest all examined ungulate species. The principal infesting tick was Ixodes ricinus, identified on 90.4% of wildlife, and included all developmental stages. Larvae and nymphs of Haemaphysalis concinna were feeding on 9.6% of wildlife. Two specimens of Dermacentor reticulatus were also identified. Ungulates were positive for A. phagocytophilum and Theileria spp. Anaplasma phagocytophilum was found to infect 96.1% of cervids, 88.9% of mouflon, and 28.2% of wild boar, whereas Theileria spp. was detected only in cervids (94.6%). Importantly, a high rate of cervids (89%) showed mixed infections with both these microorganisms. In addition to A. phagocytophilum and Theileria spp., Rickettsia helvetica, R. monacensis, unidentified Rickettsia sp., Coxiella burnetii, "Candidatus Neoehrlichia mikurensis", Borrelia burgdorferi (s.l.) and Babesia venatorum were identified in engorged I. ricinus. Furthermore, A. phagocytophilum, Babesia spp. and Theileria spp. were detected in engorged H. concinna. Analysis of 16S rRNA and groEL gene sequences revealed the presence of five and two A. phagocytophilum variants, respectively, among which sequences identified in wild boar showed identity to the sequence of the causative agent of human granulocytic anaplasmosis (HGA). Phylogenetic analysis of Theileria 18S rRNA gene sequences amplified from cervids and engorged I. ricinus ticks segregated jointly with sequences of T. capreoli isolates into a moderately supported monophyletic clade. Conclusions: The findings indicate that free-living ungulates are reservoirs for A. phagocytophilum and Theileria spp. and engorged ixodid ticks attached to ungulates are good sentinels for the presence of agents of public and veterinary concern. Further analyses of the A. phagocytophilum genetic variants and Theileria species and their associations with vector ticks and free-living ungulates are required.Fil: Kazimírová, Mária. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Hamšíková, Zuzana. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Spitalská, Eva. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; EslovaquiaFil: Minichová, Lenka. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; EslovaquiaFil: Mahríková, Lenka. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Caban, Radoslav. Široká ; EslovaquiaFil: Sprong, Hein. National Institute for Public Health and Environment.Laboratory for Zoonoses and Environmental Microbiology; Países BajosFil: Fonville, Manoj. National Institute for Public Health and Environment.Laboratory for Zoonoses and Environmental Microbiology; Países BajosFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kocianová, Elena. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; Eslovaqui

Babesia spp. in ticks and wildlife in different habitat types of Slovakia

Background: Babesiosis is an emerging and potentially zoonotic disease caused by tick-borne piroplasmids of the Babesia genus. New genetic variants of piroplasmids with unknown associations to vectors and hosts are recognized. Data on the occurrence of Babesia spp. in ticks and wildlife widen the knowledge on the geographical distribution and circulation of piroplasmids in natural foci. Questing and rodent-attached ticks, rodents, and birds were screened for the presence of Babesia-specific DNA using molecular methods. Spatial and temporal differences of Babesia spp. prevalence in ticks and rodents from two contrasting habitats of Slovakia with sympatric occurrence of Ixodes ricinus and Haemaphysalis concinna ticks and co-infections of Candidatus N. mikurensis and Anaplasma phagocytophilum were investigated. Results: Babesia spp. were detected in 1.5 % and 6.6 % of questing I. ricinus and H. concinna, respectively. Prevalence of Babesia-infected I. ricinus was higher in a natural than an urban/suburban habitat. Phylogenetic analysis showed that Babesia spp. from I. ricinus clustered with Babesia microti, Babesia venatorum, Babesia canis, Babesia capreoli/Babesia divergens, and Babesia odocoilei. Babesia spp. amplified from H. concinna segregated into two monophyletic clades, designated Babesia sp. 1 (Eurasia) and Babesia sp. 2 (Eurasia), each of which represents a yet undescribed novel species. The prevalence of infection in rodents (with Apodemus flavicollis and Myodes glareolus prevailing) with B. microti was 1.3 % in an urban/suburban and 4.2 % in a natural habitat. The majority of infected rodents (81.3 %) were positive for spleen and blood and the remaining for lungs and/or skin. Rodent-attached I. ricinus (accounting for 96.3 %) and H. concinna were infected with B. microti, B. venatorum, B. capreoli/B. divergens, Babesia sp. 1 (Eurasia), and Babesia sp. 2 (Eurasia). All B. microti and B. venatorum isolates were identical to known zoonotic strains from Europe. Less than 1.0 % of Babesia-positive ticks and rodents carried Candidatus N. mikurensis or A. phagocytophilum.Inst. de PatobiologíaFil: Hamsikova, Zuzana. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Kazimirová, Mária. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Harustiakova, Danka. Masaryk University. Faculty of Medicine and Faculty of Science, Institute of Biostatistics and Analyses; República ChecaFil: Mahrikova, Lenka. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Slovak, Mirko. Slovak Academy of Sciences. Institute of Zoology; EslovaquiaFil: Berthova, Lenka. Slovak Academy of Sciences. Biomedical Research Center. Institute of Virology; EslovaquiaFil: Kocianova, Elena. Slovak Academy of Sciences. Biomedical Research Center. Institute of Virology; EslovaquiaFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Molecular detection and phylogenetic analysis of Hepatozoon spp. in questing Ixodes ricinus ticks and rodents from Slovakia and Czech Republic

By amplification and sequencing of 18S rRNA gene fragments, Hepatozoon spp. DNA was detected in 0.08 % (4/5057) and 0.04 % (1/2473) of questing Ixodes ricinus ticks from Slovakia and Czech Republic, respectively. Hepatozoon spp. DNA was also detected in spleen and/or lungs of 4.45 % (27/606) of rodents from Slovakia. Prevalence of infection was significantly higher in Myodes glareolus (11.45 %) than in Apodemus spp. (0.28 %) (P < 0.001). Sequencing of 18S rRNA Hepatozoon spp. gene amplicons from I. ricinus showed 100 % identity with Hepatozoon canis isolates from red foxes or dogs in Europe. Phylogenetic analysis showed that at least two H. canis 18S rRNA genotypes exist in Slovakia of which one was identified also in the Czech Republic. The finding of H. canis in questing I. ricinus suggests the geographical spread of the parasite and a potential role of other ticks as its vectors in areas where Rhipicephalus sanguineus is not endemic. Sequencing of 18S rRNA gene amplicons from M. glareolus revealed the presence of two closely related genetic variants, Hepatozoon sp. SK1 and Hepatozoon sp. SK2, showing 99–100 % identity with isolates from M. glareolus from other European countries. Phylogenetic analysis demonstrates that 18S rRNA variants SK1 and SK2 correspond to previously described genotypes UR1 and UR2 of H. erhardovae, respectively. The isolate from Apodemus flavicollis (Hepatozoon sp. SK3b) was 99 % identical with isolates from reptiles in Africa and Asia. Further studies are necessary to identify the taxonomic status of Hepatozoon spp. parasitizing rodents in Europe and the host-parasite interactions in natural foci.Fil: Hamšíková, Zuzana. Slovak Academy of Sciences; EslovaquiaFil: Silaghi, Cornelia. Universitat Zurich; Suiza. Ludwig Maximilians Universitat; AlemaniaFil: Rudolf, Ivo. Academy of Sciences of the Czech Republic; República ChecaFil: Venclíková, Kristýna. Academy of Sciences of the Czech Republic; República ChecaFil: Mahríková, Lenka. Slovak Academy of Sciences; EslovaquiaFil: Slovák, Mirko. Slovak Academy of Sciences; EslovaquiaFil: Mendel, Jan. Academy of Sciences of the Czech Republic; República ChecaFil: Blažejová, Hana. Academy of Sciences of the Czech Republic; República ChecaFil: Berthová, Lenka. Academy of Sciences of the Czech Republic; República ChecaFil: Kocianová, Elena. Academy of Sciences of the Czech Republic; República ChecaFil: Hubálek, Zdeněk. Academy of Sciences of the Czech Republic; República ChecaFil: Schnittger, Leonhard. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Kazimírová, Mária. Slovak Academy of Sciences; Eslovaqui

Additional file 1: Table S1. of Babesia spp. in ticks and wildlife in different habitat types of Slovakia

Prevalence of Babesia spp. in questing Haemaphysalis concinna per site in 2011–2013. Table S2. Occurence of Babesia spp. in questing Ixodes ricinus in Bratislava and Fúgelka. Table S3. Occurence of Babesia spp. in questing Ixodes ricinus in 2011–2013. Table S4. Occurence of Babesia spp. in questing Ixodes ricinus males, females and nymphs. Table S5. Variables remaining in the best selected model for Babesia microti prevalence in rodents. Table S6. Occurence of Babesia, Candidatus N. mikurensis and Anaplasma phagocytophilum in questing Ixodes ricinus. Table S7. Accession numbers of Apicomplexa 18S rRNA gene sequences (PDF 238 kb

Additional file 1: of Molecular evidence of Rickettsia spp. in ixodid ticks and rodents in suburban, natural and rural habitats in Slovakia

PCR protocols for the detection of Rickettsia spp. and Coxiella burnetii. (DOCX 21 kb

Relative density of host-seeking ticks in different habitat types of south-western Slovakia

Ixodes ricinus is a vector of microbial pathogens of medical and veterinary importance in Europe. Recently, increasing abundance of ticks has been observed in urban and suburban areas. The aim of this study was to investigate the tick species composition and examine correlations between local environmental variables and the relative density of host-seeking I. ricinus in two habitat types. Questing ticks were collected along six 100 m2 transects in urban/suburban locations of Bratislava town, and in a non-fragmented deciduous forest in the Small Carpathians Mountains (south-western Slovakia) during 2011–2013. In total, 6015 I. ricinus were collected (3435 and 2580 in the urban/suburban and natural habitat, respectively), out of which over 80 % were nymphs. Haemaphysalis concinna comprised 1.3 % of the tick collections. Peak I. ricinus nymph and adult host-seeking activities were registered in April–June. Spatial and temporal variation in tick relative density and differences in the subadult/adult ratio were observed between habitats and between locations within the same habitat type. The relative density of questing I. ricinus nymphs correlated negatively with altitude, geographical aspect and saturation deficit in a 64-day period comprising the 8-day period including the date of tick sampling and previous 56 days. No significant correlation was found between roe deer density and questing nymph density. The study revealed the presence of abundant I. ricinus populations in green areas of Bratislava, suggesting a risk of exposure of town dwellers and domestic and companion animals to potentially infected tick