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Wild-living rodents are important hosts for zoonotic pathogens. Bartonella infections are widespread in rodents; however, in Slovakia, knowledge on the prevalence of these bacteria in small mammals is limited. We investigated the prevalence and diversity of Bartonella species in the spleens of 640 rodents of six species (Apodemus flavicollis, Apodemus sylvaticus, Myodes glareolus, Microtus arvalis, Microtus subterraneus, and Micromys minutus) and in the European mole (Talpa europaea) from three different habitat types in south-western and central Slovakia. Overall, the prevalence of Bartonella spp. in rodents was 64.8%; a rate of 73.8% was found in natural habitat (deciduous forest), 56.0% in suburban forest park and 64.9% in rural habitat. Bartonella spp. were detected in 63.0% of A. flavicollis, 69% of My. glareolus and 61.1% of M. arvalis and in T. europaea. However, Bartonella were not found in the other examined rodents. Molecular analyses of the 16S-23S rRNA intergenic spacer region revealed the presence of four different Bartonella spp. clusters. We identified B. taylorii, B. rochalimae, B. elizabethae, B. grahamii and Bartonella sp. wbs11 in A. flavicollis and My. glareolus. Bartonella genotypes ascribed to B. taylorii and B. rochalimae were found in M. arvalis. B. taylorii was identified in T. europaea. Questing Ixodes ricinus ticks that were collected at the study sites were not infected with Bartonella. This study improves our understanding of the ecoepidemiology of Bartonella spp. in Europe and underlines the necessity for further research on Bartonella-host-vector associations and their consequences on animal and human health in Slovakia.

A total of 750 ticks feeding on humans were collected during the years 2008-2018. The majority of ticks (94.8 %) came from Slovakia, with 3.5 % from the Czech Republic, 0.9 % from Austria, and 0.3 % from Hungary. Travellers from Ukraine, Croatia, France, and Cuba also brought one tick from each of these countries. The majority of the analysed ticks were identified as Ixodes ricinus (94.3 %). Dermacentor reticulatus (0.93 %), Haemaphysalis concinna (0.1 %), Haemaphysalis sp. (0.1 %), Ixodes arboricola (0.1 %), and Rhipicephalus sp. (0.1 %) were also encountered. The most frequently found stage of I. ricinus was the nymph (69.9 %) followed by adult females (20.4 %) and larvae (8.3 %). Ticks were predominantly found on children younger than 10 years (46.3 %) and adults between 30-39 years (21.4 %). In children younger than 10 years, the ticks were usually found on the head, while in other age categories, the ticks were predominantly attached to legs. Ticks were further individually analysed for the presence of Rickettsia spp., Coxiella burnetii, Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Neoehrlichia mikurensis, Bartonella spp. and Babesia spp. The overall prevalences of tick-borne bacteria assessed in I. ricinus ticks acquired in Slovakia were: rickettsiae 25.0 % (95 % CI: 21.7-28.2), B. burgdorferi s.l. 20.5 % (95 % CI: 17.4-23.5), A. phagocytophilum 13.5 % (95 % CI: 10.9-16.0), Babesia spp. 5.2 % (95 % CI: 3.5-6.9), C. burnetii 3.0 % (95 % CI: 1.5-4.6), and N. mikurensis 4.4 % (95 % CI: 2.0-6.8). Pathogenic species Rickettsia raoultii, Rickettsia helvetica, Rickettsia monacensis, A. phagocytophilum, Borrelia garinii, Borrelia afzelii, Borrelia valaisiana, Babesia microti, and Babesia divergens were identified in D. reticulatus and I. ricinus ticks.

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; Eslovaquia Fil: Hamšíková, Zuzana. Slovak Academy of Sciences. Institute of Zoology; Eslovaquia Fil: Spitalská, Eva. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; Eslovaquia Fil: Minichová, Lenka. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; Eslovaquia Fil: Mahríková, Lenka. Slovak Academy of Sciences. Institute of Zoology; Eslovaquia Fil: Caban, Radoslav. Široká ; Eslovaquia Fil: Sprong, Hein. National Institute for Public Health and Environment.Laboratory for Zoonoses and Environmental Microbiology; Países Bajos Fil: Fonville, Manoj. National Institute for Public Health and Environment.Laboratory for Zoonoses and Environmental Microbiology; Países Bajos Fil: 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; Argentina Fil: Kocianová, Elena. Slovak Academy of Sciences. Institute of Virology. Biomedical Research Center,; Eslovaquia

The incidence of tick-borne diseases caused by Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Rickettsia spp. has been rising in Europe in recent decades. Early pre-assessment of acarological hazard still represents a complex challenge. The aim of this study was to model Ixodes ricinus questing nymph density and its infection rate with B. burgdorferi s.l., A. phagocytophilum and Rickettsia spp. in five European countries (Italy, Germany, Czech Republic, Slovakia, Hungary) in various land cover types differing in use and anthropisation (agricultural, urban and natural) with climatic and environmental factors (Normalized Difference Vegetation Index (NDVI), Normalized Difference Water Index (NDWI), Land Surface Temperature (LST) and precipitation). We show that the relative abundance of questing nymphs was significantly associated with climatic conditions, such as higher values of NDVI recorded in the sampling period, while no differences were observed among land use categories. However, the density of infected nymphs (DIN) also depended on the pathogen considered and land use. These results contribute to a better understanding of the variation in acarological hazard for Ixodes ricinus transmitted pathogens in Central Europe and provide the basis for more focused ecological studies aimed at assessing the effect of land use in different sites on tick–host pathogens interaction.

Hyalomma ticks are well-known vectors transmitting infectious agents, which can result in severe and potentially fatal diseases in humans. Migratory birds may carry infected ticks over long distances. Here, we report on records of ticks of the H. marginatum complex in birds from Central Europe during the spring migration in 2008-2012. A total of 1172 birds belonging to 32 species, 16 families, and 3 orders was examined for ticks. Sixteen individuals of 6 passerine species were found to transport 30 ticks, identified as individuals belonging to the H. marginatum species complex (consisting of H. isaaci, H. marginatum sensu stricto, H. rufipes, H. turanicum, and H. glabrum) during 5 spring seasons. Infested bird species included the great reed warbler Acrocephalus arundinaceus, the Eurasian reed warbler A. scirpaceus, the marsh warbler A. palustris, the sedge warbler A. schoenobaenus, Savi's warbler Locustella luscinioides, and the common nightingale Luscinia megarhynchos. All of these Central European breeders are migratory species wintering in Africa. To our knowledge, this is the first study to record ticks of the H. marginatum complex on the great reed warbler and Savi's warbler.

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; Eslovaquia Fil: Silaghi, Cornelia. Universitat Zurich; Suiza. Ludwig Maximilians Universitat; Alemania Fil: Rudolf, Ivo. Academy of Sciences of the Czech Republic; República Checa Fil: Venclíková, Kristýna. Academy of Sciences of the Czech Republic; República Checa Fil: Mahríková, Lenka. Slovak Academy of Sciences; Eslovaquia Fil: Slovák, Mirko. Slovak Academy of Sciences; Eslovaquia Fil: Mendel, Jan. Academy of Sciences of the Czech Republic; República Checa Fil: Blažejová, Hana. Academy of Sciences of the Czech Republic; República Checa Fil: Berthová, Lenka. Academy of Sciences of the Czech Republic; República Checa Fil: Kocianová, Elena. Academy of Sciences of the Czech Republic; República Checa Fil: Hubálek, Zdeněk. Academy of Sciences of the Czech Republic; República Checa Fil: 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; Argentina Fil: Kazimírová, Mária. Slovak Academy of Sciences; Eslovaquia

Background Candidatus Neoehrlichia mikurensis (CNM) is an emerging tick-borne pathogen causing severe disease in immunocompromised patients. In Europe, Ixodes ricinus is the primary vector and rodents act as reservoir hosts. New data on the prevalence of CNM in ticks and rodents contribute to the knowledge on the distribution of endemic areas and circulation of the bacterium in natural foci. Methods Questing ticks were collected and rodents were trapped in urban/suburban and natural habitats in South-Western Slovakia from 2011 to 2014. DNA from questing and rodent-attached ticks and rodent tissues were screened for CNM by real-time PCR. Rodent spleen samples positive for CNM were characterised at the groEL gene locus. Spatial and temporal differences in CNM prevalence in ticks and rodents and co-infections of ticks with CNM and Anaplasma phagocytophilum were analysed. Results The presence of CNM was confirmed in questing and rodent-attached I. ricinus ticks and in rodents. Total prevalence in both ticks and rodents was significantly higher in the natural habitat (2.3 % and 10.1 %, respectively) than in the urban/suburban habitat (1.0 % and 3.3 %, respectively). No seasonal pattern in CNM prevalence in ticks was observed, but prevalence in rodents was higher in autumn than in spring. CNM was detected in Apodemus flavicollis, Myodes glareolus, Microtus arvalis and Micromys minutus, with the highest prevalence in M. arvalis (30 %). By screening CNM dissemination in rodent tissues, infection was detected in lungs of all specimens with positive spleens and in blood, kidney, liver and skin of part of those individuals. Infection with CNM was detected in 1.3 % of rodent attached I. ricinus ticks. Sequences of a fragment of the groEL gene from CNM-positive rodents showed a high degree of identity with sequences of the gene amplified from ticks and infected human blood from Europe. Only 0.1 % of CNM-positive questing ticks carried A. phagocytophilum. Ticks infected with CNM prevailed in the natural habitat (67.2 %), whereas ticks infected with A. phagocytophilum prevailed in the urban/suburban habitat (75.0 %). Conclusion The study confirmed the circulation of CNM between I. ricinus ticks and rodents in South-Western Slovakia, and indicates a potential risk of contracting human infections.

Rickettsiae, obligate intracellular Gram-negative bacteria, responsible for mild to severe diseases in humans are associated with arthropod vectors. Dermacentor marginatus and Dermacentor reticulatus are known vectors of Rickettsia slovaca and Rickettsia raoultii distributed across Europe. A total of 794 D. marginatus, D. reticulatus and Ixodes ricinus adult ticks were collected from the vegetation, removed from horses, sheep, goats and dogs in Slovakia. The DNA of Rickettsia sp. was found in 229 ticks by PCR amplifying parts of gltA, ompA and sca4 genes. Next analyses of Rickettsia-positive samples by PCR-RFLP and/or sequencing showed D. reticulatus ticks were more infected with R. raoultii and D. marginatus were more infected with R. slovaca. The prevalence of R. raoultii was 8.1-8.6% and 22.3-27% in D. marginatus and D. reticulatus, respectively. The prevalence of R. slovaca was 20.6-24.3% in D. marginatus and 1.7-3.4% in D. reticulatus. Intracellular growth of R. raoultii isolate from D. marginatus tick was evaluated by rOmpA-based quantitative SybrGreen PCR assay. The highest point of multiplication was recorded on the 7th and 8th day postinfection in Vero and L929 cells, respectively. R. raoultii was transmitted during feeding of R. raoultii-positive ticks to guinea pigs and subsequently rickettsial infection was recorded in all organs, the highest infection was in spleen, liver and heart. Our study describes the detection and isolation of tick-borne pathogens R. raoultii and R. slovaca, show that they are spread in Slovakia and highlight their risk for humans.

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 m(2) 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 ticks.

Tick-borne bacterial zoonoses of livestock and free-ranging ungulates caused by Anaplasma spp. are common in Central Europe. The aim of this study was to analyze the prevalence of Anaplasma phagocytophilum and other tick-borne bacteria in wild animals from western Slovakia. Infection with A. phagocytophilum was recorded in 62.86% of analyzed roe deer (Capreolus capreolus), in two red deer (Cervus elaphus) and two wild boars (Sus scrofa). Dermacentor reticulatus and Ixodes ricinus ticks collected on red deer were not A. phagocytophilum-infected. However, spotted fever group rickettsiae were detected in ticks collected from red deer. High prevalence of A. phagocytophilum in roe deer as well as positive red deer and wild boars suggest the occurrence of natural foci in western Slovakia.

Rickettsia slovaca, a member of spotted fever rickettsiae, is an agent of a mild human disease known as Tibola or Debonel. Using quantitative real-time PCR we identified the highest point of multiplication of wild and standard type (strain B) of R. slovaca on the second vs. the fourth day postinfection. Comparing both types of R. slovaca by transmission electron microscopy substantiated different cytopathological and morphological changes in infected cells and other differences, for example a slight shift of stages during the life cycle that presented in a variety of forms and localization of the studied Rickettsia within the infected cells. Our study provides a valuable insight into the pathogenicity and virulence of R. slovaca.

Ultrastructural changes induced by Rickettsia slovaca standard type (ST) and wild type (WT) were examined during their life cycle in L929 and Vero cells. R. slovaca invaded the cytoplasm of the host cell by phagocytosis on the 1st d p.i. Rickettsiae adhering to the cytoplasmic membrane were engulfed by cellular extensions and occurred in phagocytic vacuoles. Binary fission of rickettsia was observed. The nuclear chromatin of eukaryotic cells was rearranged and condensed during 3rd and 6th d p.i. Finally, loss of the plasma membrane integrity, destruction of cytoplasm and nucleus resulted in cell lysis. Degeneration of the host cell caused by WT and ST was observed after 4 and 5 d p.i. in L929 cells and after 3 and 6 d p.i. in Vero cells, respectively. WT type was able to penetrate into the nucleus of the host cell and was responsible for dilatation of the perinuclear space and endoplasmic reticulum, causing more pronounced and different cytopathological changes than the ST.

Borrelia spirochetes in bird-feeding ticks were studied in the Czech Republic. During the postbreeding period (July to September 2005), 1,080 passerine birds infested by 2,240 Ixodes ricinus subadult ticks were examined. Borrelia garinii was detected in 22.2% of the ticks, Borrelia valaisiana was detected in 12.8% of the ticks, Borrelia afzelii was detected in 1.6% of the ticks, and Borrelia burgdorferi sensu stricto was detected in 0.3% of the ticks. After analysis of infections in which the blood meal volume and the stage of the ticks were considered, we concluded that Eurasian blackbirds ( Turdus merula ), song thrushes ( Turdus philomelos ), and great tits ( Parus major ) are capable of transmitting B. garinii ; that juvenile blackbirds and song thrushes are prominent reservoirs for B. garinii spirochetes; that some other passerine birds investigated play minor roles in transmitting B. garinii ; and that the presence B. afzelii in ticks results from infection in a former stage. Thus, while B. garinii transmission is associated with only a few passerine bird species, these birds have the potential to distribute millions of Lyme disease spirochetes between urban areas.

A total of 80 adult ticks (55 Haemaphysalis inermis, 12 Dermacentor reticulatus, 11 D. marginatus, 2 Ixodes ricinus) were collected from vegetation in three areas of Slovakia (forest and pasture habitat) in central Europe. Forty-six (46 ticks) (57.5%) of all species tested were positive by the hemocyte test, PCR assays based on the gltA and ompA genes showed a Rickettsiaceae infection in 77.5% of the ticks, whereas only one H. inermis tick was positive for Anaplasmataceae on a 16S rRNA-based PCR. Isolation of rickettsiae was attempted on all collected ticks by means of the shell vial technique, 52 isolates of which were inoculated into Vero cells and 28 into L929 cells. Rickettsiae were detected in 50% (40/80) of the cell lines using the Gimenez staining method, whereas 33.8% (27/80) of the cell lines were PCR-positive for Rickettsia species. The presence of rickettsiae was shown by PCR to be around 30.8% (16/52) in Vero and 39.3% (11/28) in L929 cell lines. Sequencing results showed that detected infections were Rickettsia sp., R. raoultii, and Anaplasma phagocytophilum in ticks, and R. slovaca in cell lines. This is the first report of R. raoultii in Slovakia. Observations by electron microscopy of the R. slovaca isolate from Vero cell lines showed a microcapsular layer, typical Gram-negative cell wall, and a cytoplasmic membrane.

Blackbirds ( Turdus merula ) and song thrushes ( Turdus philomelos ) were found to carry 95% of all spirochete-infected tick larvae among 40 bird species captured in Central Europe. More than 90% of the infections were typed as Borrelia garinii and Borrelia valaisiana . We conclude that thrushes are key players in the maintenance of these spirochete species in this region of Central Europe.

Ticks are well-known vectors for a wide range of pathogenic microorganisms. We examined the presence of Rickettsia spp., Anaplasma spp., Borrelia spp., Coxiella burnetii and Francisella tularensis in Ixodes ricinus ticks collected in central Slovakia using oligo-chip based assay. Rickettsiae were detected in 5.6% of examined ticks. Borreliae and anaplasmae were identified in 2.1% and 2.8% ticks, respectively. All tested samples were negative for presence of Coxiella burnetii and Francisella tularensis. All these results were compared with those obtained by PCR analysis, and a close correlation between them was found. In addition, rickettsiae of spotted fever group (SFG), Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato were found in ticks using genera or species-specific PCR methods. They are circulating in 10 out of 18 studied localities.

The purpose of this study was to investigate the role of wild animals for Anaplasma phagocytophilum, other ehrlichiae/anaplasmae, Rickettsia helvetica and other rickettsiae and whether different genetic variants of A. phagocytophilum in central Slovakia exist. A total of 109 spleen samples from 49 red deer (Cervus elaphus), 30 roe deer (Capreolus capreolus), 28 wild boar (Sus scrofa) and two mouflon (Ovis musimon) were collected from June 2005 to December 2006. Polymerase chain reaction (PCR) amplification of the16S rRNA gene was used for detection of ehrlichiae/anaplasmae. A nested PCR targeting part (392 bp) of groESL gene was applied for the specific detection of A. phagocytophilum. Fragments of the gltA and ompA genes (381 bp and 632 bp, respectively) were amplified to detect rickettsiae, followed by sequencing. A. phagocytophilum and R. helvetica were detected in wild animals. The prevalence of A. phagocytophilum was 50.0 ± 18.2% in roe deer and 53.1 ± 14.1% in red deer. None of the 28 wild boar was PCR positive for ehrlichiae/anaplasmae. A. phagocytophilum was detected in one mouflon. R. helvetica was found in one roe deer. Our study suggests a role of cervids as a natural reservoir of A. phagocytophilum in Slovakia. However, the role of cervids and wild boars in the circulation of R. helvetica remains unknown. The analysis of sequence variation in the msp4 coding region of A. phagocytophilum showed the presence of different variants previously described in ruminants.

This study assessed the parasitization of cavity-nesting birds and ground-nesting/foraging birds with larvae and nymphs of two Ixodes species, Ixodes ricinus and Ixodes arboricola. Totals of 679 (52.3%) I. ricinus and 619 (47.7%) I. arboricola ticks were collected from 15 species of passerine birds which were caught during the nesting and non-nesting periods of 2003-2006, in the south-eastern part of the Czech Republic, the Drahanska Vrchovina Uplands. In the non-nesting period from October to March, 6.8% (101/1492) of birds were infested with ticks, mainly with I. arboricola larvae. In the non-nesting period, the average intensity of infestation by I. arboricola and I. ricinus was 8.5 and 1.5 individuals per infested bird, respectively. In the nesting period from April to June, 21.6% (50/232) of birds were infested by both tick species but mainly with I. ricinus nymphs. The average intensity of infestation by I. ricinus and I. arboricola was 13.3 and 10.8 individuals per infested bird, respectively. Altogether, 23.2% of the infested birds were parasitized by both immature life stages of one or both tick species. From an enzootic perspective, co-feeding and co-infestation of I. ricinus and I. arboricola subadults on passerine birds might happen and may be important for the dissemination of tick-borne agents.

Background Ixodes ricinus is the principal vector of Anaplasma phagocytophilum, the ethiological agent of granulocytic anaplasmosis in Europe. Anaplasmosis is an emerging zoonotic disease with a natural enzootic cycle. The reservoir competence of rodents is unclear. Monitoring of A. phagocytophilum prevalence in I. ricinus and rodents in various habitat types of Slovakia may contribute to the knowledge about the epidemiology of anaplasmosis in Central Europe. Methods Over 4400 questing ixodid ticks, 1000 rodent-attached ticks and tissue samples of 606 rodents were screened for A. phagocytophilum DNA by real-time PCR targeting the msp2 gene. Ticks and rodents were captured along six transects in an urban/suburban and natural habitat in south-western Slovakia during 2011–2014. Estimates of wildlife (roe deer, red deer, fallow deer, mouflon, wild boar) densities in the study area were taken from hunter’s yearly reports. Spatial and temporal differences in A. phagocytophilum prevalence in questing I. ricinus and relationships with relative abundance of ticks and wildlife were analysed. Results Overall prevalence of A. phagocytophilum in questing I. ricinus was significantly higher in the urban/suburban habitat (7.2 %; 95 % CI: 6.1–8.3 %) compared to the natural habitat (3.1 %; 95 % CI: 2.5–3.9 %) (χ2 = 37.451; P

Information on circulation of rickettsiae between small mammals and their ectoparasitic mites is scarce. In this study, we investigated infestation rates of rodents with mites in some areas of SW Slovakia and the role of mites as possible vectors of pathogenic rickettsiae. A total of 615 rodents of five species were caught during 2010–2012. All individuals were examined for ectoparasites which resulted in 2821 mites belonging to three species of Laelapidae and six species of Trombiculidae. The most common Laelapidae species was Laelaps agilis (81.25 %), followed by Haemogamasus nidi, and Eulaelaps stabularis. Hirsutiella zachvatkini (16.52 %) was the dominant species of the family Trombiculidae. DNA extracted from rodent blood and ectoparasitic mites was examined for the presence of rickettsiae by PCR. By pooling mites, 345 pool samples were created, of which 112 (32.46 %) were found to be positive for the rickettsial DNA. From 487 examined rodent blood samples, rickettsial DNA was found in 46 (9.44 %). Sequencing DNA from the positive blood samples and mites revealed the identity of Rickettsia helvetica and Rickettsia monacensis. The results of the study suggest that ectoparasitic mites may be reservoirs as well as vectors of some pathogenic rickettsiae.

In this study, three tick species (Ixodes ricinus, Dermacentor marginatus, and D. reticulatus), small terrestrial mammals, and game were examined by PCR for the presence of tick-borne pathogens Anaplasma phagocytophilum, Coxiella burnetii, Rickettsia spp., and Borrelia burgdorferi Sensu lato.

A total of 235 adult ticks collected from vegetation in Slovakia and Hungary in 1998-2000 were tested for Coxiella burnetii by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). C. burnetii was identified in six ticks of Ixodes ricinus, Dermacentor marginatus, and Haemaphysalis concinna species.

Ixodes ricinus ticks collected from 835 birds and from vegetation in the Czech Republic were analyzed. Host-seeking ticks ( n = 427) were infected predominantly by Borrelia afzelii (25%). Ticks ( n = 1,012) from songbirds (Passeriformes) were infected commonly by Borrelia garinii (12.1%) and Borrelia valaisiana (13.4%). Juveniles of synanthropic birds, Eurasian blackbirds ( Turdus merula ) and song thrushes ( Turdus philomelos ), were major reservoir hosts of B. garinii.

In Europe, Borrelia burgdorferi sensu lato is diverse, including B.burgdorferi s.s., B.garinii, B.afzelii, B.valaisiana and B.lusitaniae. In this study, we focused on the distribution of the different B.burgdorferi species among Ixodes ricinus adult ticks collected in an endemic area within Slovakia. We compared results of prevalence of B.burgdorferi infection in ticks obtained by immunofluorescence (IF) and by isolation. Isolates were characterized by restriction fragment length polymorphism (RFLP) of the rrf-rrl intergenic spacer genes using MseI. Using immunofluorescence we observed that 56/114 (49%) ticks were infected by B.burgdorferi s.l. Males were found to be more often infected (32/57, 56%) than females (24/57, 42%) but the difference was not significant (p = 0.1895). From the same 114 ticks a total of 37 isolates were obtained: 19 from males (33%) and 18 from females (32%). The RFLP identification revealed 25 B.afzelii (68%), 5 B.garinii (14%), 5 B.valaisiana (14%) and 2 B.lusitaniae (5%). The infection in ticks was more often detected by IF than by isolation (p = 0.0153) and isolation success was higher when the infection degree in ticks was high (p = 0.0397). The infection prevalence observed in this area is among the highest observed in Europe.

The reported incidence of vector-borne diseases including various cases of Rickettsioses in humans is increasing due to a combination of climatic and social factors, escalating the opportunities for contact between people and ticks, fleas or lice. Many of the emerging infectious diseases currently challenging human health in Europe are transmitted by ticks which normally feed on domestic or wild animals. Each Rickettsia spp. has one or several tick vectors, and their geographical distribution varies according to geographical conditions; e.g.; altitude or temperature, which is gradually changing due to a global warming. Evidence of Rickettsia spp. particularly of a newly discovered species is a strong indication that a great number of diseases may be caused by so far undetected or unrecognized organisms. Their diagnosis relies mostly on rare "spot like" cooperation of clinicians with scientists, the members of the working groups that are devoted to the scientific studies of the corresponding research areas. The clinical picture of the disease caused by rickettsiae varies significantly from flu like symptoms to severe fatal outcomes, reflecting the various factors, e.g. a variability of virulence of rickettsial species due to cell invasion, dissemination of rickettsiae, genomics, immune response of an infected organism, or a tricky impact of a treatment. Several major reviews on rickettsioses have been previously published, e.g. in 1997 (Raoult and Roux, 1997a), in 2005 (Parola et al., 2005), and in 2011 (Botelho-Nevers and Raoult, 2011). In this work we intend to present a short historical overview and to describe new trends in research studies of rickettsiology. The main focus will be on rickettsioses affecting Europe΄s population.

Two strains of Coxiella burnetii and two strains of an unidentified rickettsial organism were isolated for the first time from Ixodes ricinus ticks collected in the Alpine region of Tirol, Austria. The C. burnetii strains belong to the group of agents causing acute forms of Q fever. The other two strains of isolated rickettsial agent share some antigenic epitopes with C. burnetii and R. prowazekii but they differ from them by their high sensitivity to freezing and refreezing and by poor multiplication in yolk sacs of chick embryos. There is at present no evidence that these organisms cause human illness and no ecological information is available. We suggest they may be some new species of rickettsiae or rickettsia-like organisms.

Partially engorged female ticks were used as laboratory animals in microbiological research. The ticks, which were inoculated intracoelomally, became a convenient substrate for the detection of viruses, rickettsiae and protozoal parasites. This research concerned the isolation of newly recovered micro-organisms, the study of development, structure and distribution of microbial agents in ticks, and the study of their interaction with other pathogens or symbionts during mixed infection in a tick body. The isolation and maintenance of Rickettsiella phytoseiuli, the organism not of tick-borne origin, was achieved. For use in Central Europe the tick Dermacentor reticulatus is recommended for the above investigations.

SDS-PAGE, immunoblotting and serological methods such as microimmunofluorescence (MIF) test and ELISA were used to compare protein and lipopolysaccharide (LPS) profiles and antigenicity of 12 Coxiella burnetii strains isolated mostly from ticks in Europe and Mongolia with three reference C. burnetii strains originating from USA, namely Nine Mile from tick, Priscilla from goat placenta and S from human heart valve. Among strains from Europe and Mongolia, no significant differences in protein and LPS profiles were observed, irrespective of their origin, i.e. the country and source of isolation. The LPS profiles of these strains appeared to be more related to those of Nine Mile strain associated with acute Q fever, than to those of strains S and Priscilla associated with chronic Q fever. In immunoblots all strains isolated from Slovakia and Poland reacted more expressively with rabbit serum against Nine Mile than with serum against Priscilla strain. In the MIF test and ELISA there were no substantial differences in antibody-binding capacity between the reference and newly isolated C. burnetii strains, except for strain Priscilla reacting with homologous serum in lower antigenic concentration than other strains under study. However, in the MIF test much higher antigenic concentrations of each C. burnetii strain was required to detect antibodies in the Priscilla serum than in the Nine Mile, Luga and S sera.

Wild birds are known to be a reservoir of infectious disease agents and disseminatory hosts of ticks. The purpose of this work was to obtain information about the occurrence of rickettsial, anaplasmal, and borrelial infections in some ticks that parasitize wild birds in the Czech Republic. A total of 549 subadult ticks of three species Ixodes arboricola (75.0%), Ixodes ricinus (23.1%), and Haemaphysalis concinna (1.8%) were collected from 20 species of birds (Passeriformes). Rickettsiae were detected in 44.0% larvae and 24.5% nymphs of I. arboricola collected from Parus major, Poecile palustris, and Sitta europaea. Rickettsiae-positive I. ricinus larvae (13.7%) were collected from P. major, Cyanistes caeruleus, and S. europaea, and 2.6% of nymphs from Erithacus rubecula and Prunella modularis. Comparison of sequences of a gltA gene fragment with data available in GenBank identified Rickettsia helvetica, a spotted fever rickettsia associated with human infections, and other Rickettsia spp. Anaplasma phagocytophilum was found only in two I. ricinus nymphs collected from E. rubecula and P. major. Infections with Borrelia burgdorferi sensu lato were recorded in 1.3% larvae of I. arboricola acquired from P. palustris and P. major and in 11.8% larvae and 25.0% nymphs of I. ricinus collected from P. major, P. palustris, C. caeruleus, Acrocephalus schoenobaenus, Turdus merula, Carpodacus erythrinus, Sylvia atricapilla, P. modularis, and Phylloscopus collybita. Reverse-line blot hybridization showed infections with Borrelia garinii and Borrelia valaisiana and mixed infections with these two genospecies. This is the first record of a high rate of rickettsial infection in I. arboricola subadult ticks acquired from birds in the Czech Republic and in central Europe. Our study suggests that I. arboricola, P. major, and P. palustris play important roles in circulating rickettsiae.

Summary In studies carried out in 1991 in the foothills of the southern part of the B~varian Forest, in the district of Freyung/Grafenau, ticks and small mammals were collected and examined for the presence of Coxiella (C.) burnetii and sera of small mammals and cattle investigated for antibodies against this rickettsia. A total of 1716 imagines and nymphs of Ixodes ricinus were collected by flagging and 892 larvae and nymphs of the same tick species removed from small mammals. In addition to 1095 serum samples from cattle, 326 specimens of nine species of small terrestrian mammals were examined. Neither in ticks nor in rodents, C. burnetii was detected, however, in 17 of 21 localities, seropositive cattle were found. Altogether, 12% of all 1095 heads of cattle tested were seropositive for C. burnetii antibodies. These serological results indicated a wide dissemination of C. burnetii in cattle of the region investigated, but there was no indication of a natural focus. As in other areas of Europe, an independent natural cycle of the agent involving cattle only is assumed to occur in this region.

The experimental study investigated the ability of tortoise tick Hyalomma aegyptium to play a role in forming and maintaining natural foci of Q fever. We tested the competence of H. aegyptium larvae to acquire Coxiella burnetii infection from mammals, serve as a C. burnetii vector between mammalian hosts, and be a long-term carrier of C. burnetii, including interstadial transmission. H. aegyptium larvae were allowed to feed on guinea pigs experimentally infected with C. burnetii. Engorged larvae molted to nymphs, some of which were preserved in 96% ethanol and later examined by polymerase chain reaction (PCR) using C. burnetii-specific primers (CBCOS, CBCOE). Prevalence of C. burnetii among these nymphs was 5.6% (n = 235). Remaining nymphs then fed on other, C. burnetii-negative guinea pigs; and according to results of both, micro-agglutination reaction, and ELISA, they successfully transmitted C. burnetii to those new hosts. Detached engorged nymphs molted to adults, which were kept alive long term and then placed in 96% ethanol 383 days post-infection. Thereafter, they were examined by PCR in the same manner as were the nymphs. Prevalence of C. burnetii among adult H. aegyptium was 28.9% (n = 90). According to our results, tortoise-specific ticks have indisputable potential in the epidemiology of Q fever natural foci.

Lyme-Borreliose ist eine der häufigsten von Zecken vermittelten Erkrankungen, die in der Slowakei auftreten. In dieser Studie wurde Borrelia burgdorferi sensu lato in Wirt-suchenden Zecken, die aus drei Regionen der Slowakei gesammelt worden sind, nachgewiesen und kultiviert. Zur Identifizierung der Genospezies wurden zwei Methoden eingesetzt, die RFLP (restriction fragment length polymorphism) und der "reverse line blot". Ebenso wurde die Prävalenz von B. burgdorferi s.l. in den Zecken ermittelt, welche rund 32% betrug. Von den vier identifizierten Genospezies, B. afzelii, B. burgdorferi sensu stricto, B. garinii und B. valaisiana, wurde B. garinii am häufigsten nachgewiesen. Lyme borreliosis is one of the most common tick-borne diseases that occur in Slovakia. In this study, Borrelia burgdorferi sensu lato was detected and cultivated from questing ticks collected in three areas of Slovakia. Two methods, restriction fragment length polymorphism and reverse line blot, were used for identification of isolates and determination of the prevalence of B. burgdorferi s.l. in the ticks. The prevalence of B. burgdorferi s.l. in I. ricinus detected by reverse line blot was 31.9%. Four genospecies, namely B. garinii, B. valaisiana, B. afzelii and B. burgdorferi sensu stricto were found. B. garinii was the most prevalent genospecies.

The aim of this study was to determine the risks of human anaplasmosis in an area of central Slovakia endemic for Lyme borreliosis. The circulation of Anaplasma phagocytophilum in ticks and wild animals has been observed in natural foci in this area for several years. Samples of human sera from patients with Lyme borreliosis and persons with a history of recent tick bite and clinical symptoms indicating Lyme borreliosis were collected in central Slovakia. A total of 76 human sera were analyzed using an indirect HGE IgG immunofluorescent assay kit. IgG antibodies against A. phagocytophilum were found in 19 (25%) sera (15 female, 4 male patients). A. phagocytophilum infection was serologically confirmed in one (3.8%) child, 12 (38.7%) persons aged 22–56 and six (31.6%) persons older than 56. A statistically significant difference in seroprevalence (P < 0.01) was observed between children (3.8%, 1/26) and adults (36%, 18/50). Antibodies against A. phagocytophilum were detected in seven patients with clinically diagnosed Lyme borreliosis and in another seven individuals with assessed antiborrelia antibodies. IgG antibodies against A. phagocytophilum were detected in five persons seronegative for borrelia. The most frequent clinical symptoms in patients with positive A. phagocytophilum serology were cephalalgia, arthralgia, myalgia, fever, exanthema, neurological symptoms and lymphadenopathy. Positive sera were obtained from patients living in villages and towns in the orographic entities Vtacnik (5/19), Stiavnicke vrchy (1/19), Kremnicke vrchy (10/19) and Žiarska kotlina (3/19). Our results demonstrate the risk of acquiring A. phagocytophilum infection in natural foci in central Slovakia. Human anaplasmosis should be considered in the differential diagnosis, especially in cases of acute febrile illness with tick-bite history.

In winter months during 2003-2006, wild birds were captured and examined for ticks and chiggers at two sites near Brno, Czech Republic. In total, 1,362 birds, mostly passerines, were examined. The tick Ixodes arboricola Schulze et Schlottke, 1929 was found on 47 (3%) birds of six species. Ixodes ricinus Linnaeus, 1758 was found on 11 (1%) birds of five species. Larvae of chiggers Ascoschoengastia latyshevi (Schluger 1955) were found on 13 (1%) birds of six species. I. arboricola and A. latyshevi associated with hole-nesting birds can appear on birds rather frequently even during winter months. I. ricinus occurs on birds in winter sporadically.

Birds have been found to be a reservoir host of borrelia. In order to assess the situation in Slovakia ticks were collected from a total of 3057 mist-netted, ringed and released passerine birds in two locations at 500 m (in 2001) and 1000 m (in 2003) above sea level in the Bukovske Vrchy Hills, part of the Carpathian region in the north-east of Slovakia. A total of 75 birds of 16 species were infested with subadult ticks of Ixodes ricinus species (prevalence of parasitization 5%). Sixty-two larvae from 31 birds of 9 species and 80 nymphs from 52 birds of 15 species were found. The highest intensity of parasitization was observed on blackbirds Turdus merula, song thrushes T. philomelos and dunnocks Prunella modularis. Six Ixodes ricinus adult ticks were found on humans working with birds, and one I. ricinus female tick on their dog. In ticks, the presence of Rickettsia sp., Coxiella burnetii, Borrelia burgdorferi sensu lato and members of the Anaplasmataceae and Piroplasmidae, were investigated by polymerase chain reaction, followed by sequence analysis. Rickettsia sp. was found in 1 nymph from the European robin Erithacus rubecula, in 3 adult ticks (1 male, 2 females) from humans and in the tick from the dog. The closely related Ehrlichia- like species "Schotti variant" was detected in 1 nymph from the song thrush. Borrelia afzelii was identified in 1 male and B. garinii in 1 female tick collected on humans. Ixodes ricinus was found to be the vector of a wide spectrum of tick-borne pathogens in a mountainous area of the Carpathians. Because of the low yield of ticks and pathogens the importance of birds as reservoir hosts is still poorly understood.

Knowledge of the duration of tick feeding can contribute to assessment of disease transmission risk, and may be estimated from changes in body dimensions during engorgement. Application of this approach to the European tick, Ixodes ricinus, is described here. Nymphal and adult female I. ricinus ticks were detached at predetermined intervals from laboratory rabbits and their state of engorgement estimated by calculating either the scutal index (the ratio of the width of the scutum to the length of the idiosoma) or the coxal index (the ratio of the width of the scutum to the distance between the basal coxae of the fourth pair of legs – the coxal gap). The coxal index proved to be more accurate in estimating duration of feeding over the first 24 h, but the scutal index was more accurate later in feeding. The coxal index may be used in combination with the scutal index to estimate the period of time that ticks removed from patients have been feeding, thus assisting with the assessment of pathogen transmission risk.

Studies on Lyme borreliosis and other tick-borne zoonoses in the Austrian and Slovakian borderland, a region endemic for tularemia, revealed a relatively high prevalence of infection with Borrelia burgdorferi s.l. and Francisella tularensis in small terrestrial mammals, as well as in the ticks, during a one-year survey. The occurrence of coinfection with the agents of Lyme borreliosis and tularemia was assessed in different species of rodents.Organs of small mammals, live-trapped mostly in six-week intervals from May 1994 to April 1995, were cultured on appropriate media in order to grow borreliae and F. tularensis.Infection with B. burgdorferi s.l. and also with F. tularensis was found in all the most abundant rodent species. A significant difference was observed in the time period of isolation of these agents. Borrelia was cultured from May to January (PCR detected borrelia up to April), while F. tularensis was isolated from August to December. Coinfection was seen in two species of voles, Clethrionomys glareolus trapped in August and Microtus arvalis in October. The Borrelia strains isolated from these animals were identified as B. garinii. Isolates of F. tularensis belonged to the subspecies holarctica, biovar II.Results obtained indicate that in endemic regions for tularemia the prevalence of infection with borreliae could be modified in different animal species mainly during epizootic outbreaks of tularemia.

Laboratory animals (mice and guinea pigs) were infected with the isolates of Coxiella burnetii (Derrick, 1939) obtained from bovine milk (M18 and M35) and the ticks Ixodes ricinus (Linnaeus, 1758) and Dermacentor marginatus (Sulzer, 1776) (Kl3 and Kl6, respectively), and with the reference strain Nine Mile. Neither mortality nor lethality occurred with the mice. Antibody response in mice infected with isolates from milk was lower (1:16-512) than that from ticks (1:32-4096). Onset of seropositivity also occurred later - on the 10th day post-infection (p.i.) for M18 and M35 in comparison with the 7th day for Kl3 and Kl6. In guinea pigs, infection manifested by fever. The fever was less evident in guinea pigs infected with isolates from milk (39.5-40.1 degrees C) than in guinea pigs infected with isolates from ticks (39.5-40.6 degrees C). Partially engorged females of Dermacentor reticulatus (Fabricius, 1794) were inoculated with isolates M18 and Kl3. No differences in the multiplication of C. burnetii in haemocytes between these two isolates were ascertained.

Young semi-domesticated pigeons captured or hatched from eggs gathered in Bratislava during 1989–1991 were examined for complement fixing antibodies to Chlamydia psittaci and agglutinating antibodies to Coxiella burnetii. Antibodies to Ch. psittaci were present in 76% of birds younger than 24 h, in 47.7% between 1 and 10 days of age and in 12% of nestlings over 10 days old. Antibodies to Ch. psittaci were also detected in “crop milk” of 4.1% of 1 to 10 day old birds and in 4.5% of specimens older than 10 days. Antibodies to C. burnetii were not found in juvenile birds under 24 h old, but antibodies against this agent were present in 16.4% birds between 1 and 10 days old and in 18% over 10 days old. Antibodies to C. burnetii were also detected in “crop milk” collected from crops of 2% of the young birds between 1 and 10 days.

Between 1987-1989 almost 7000 adult Ixodes ricinus, Dermacentor reticulatus, Dermacentor marginatus, Haemaphysalis concinna, Haemaphysalis punctata and Haemaphysalis inermis ticks collected in all 38 districts of Slovakia were screened for the presence of Coxiella burnettii. The proportion of ticks containing C. burnetii as indicated by the haemocyte test was less than 3%. Attempts to recover C. burnetii by inoculation of yolk sacs of embryonated hen eggs from pools of 1-6 specimens of haemocyte test positive ticks resulted in the isolation of 10 rickettsial strains. Six strains were recovered from I. ricinus, the remaining ones from single pools of D. reticulatus, D. marginatus. H. concinna and H. inermis ticks. In addition to the previous recovery of C. burnetii from H. punctata ticks, the agent was thus isolated from all important ticks living in Slovakia. The agent was found in tick habitats regardless of the latitude and altitude in the entire country. These results are not consistent with the negligible number of Q fever cases occurring in past years in Slovakia.

Background: Natural foci of tick-borne spotted fever group (SFG) rickettsiae of public health concern have been found in Slovakia, but the role of rodents in their circulation is unclear. Ticks (Ixodes ricinus, Ixodes trianguliceps, Dermacentor marginatus, Dermacentor reticulatus, Haemaphysalis concinna and Haemaphysalis inermis) and tissues of rodents (Apodemus flavicollis, Apodemus sylvaticus, Myodes glareolus, Microtus arvalis, Microtus subterraneus and Micromys minutus) were examined for the presence of SFG rickettsiae and Coxiella burnetii by molecular methods. Suburban, natural and rural habitats were monitored to acquire information on the role of ticks and rodents in the agents' maintenance in various habitat types of Slovakia.Results: The overall prevalence of rickettsial infection in questing I. ricinus and D. marginatus was 6.6% and 21.4%, respectively. Rickettsia helvetica, R. monacensis and non-identified rickettsial species were detected in I. ricinus, whereas R. slovaca and R. raoultii were identified in D. marginatus. Rickettsia spp.-infected I. ricinus occurred during the whole tick questing period. Rickettsia helvetica dominated (80.5%) followed by R. monacensis (6.5%). The species were present in all studied habitats. Rickettsia slovaca (66.7%) and R. raoultii (33.3%) were identified in D. marginatus from the rural habitat. Apodemus flavicollis was the most infested rodent species with I. ricinus, but My. glareolus carried the highest proportion of Rickettsia-positive I. ricinus larvae. Only 0.5% of rodents (A. flavicollis) and 5.2% of engorged I. ricinus removed from My. glareolus, A. flavicollis and M. arvalis were R. helvetica- and R. monacensis-positive. Coxiella burnetii was not detected in any of the tested samples. We hypothesize that rodents could play a role as carriers of infected ticks and contribute to the maintenance of rickettsial pathogens in natural foci.Conclusions: Long-term presence of SFG Rickettsia spp. was confirmed in questing ticks from different habitat types of Slovakia. The results suggest a human risk for infection with the pathogenic R. helvetica, R. monacensis, R. slovaca and R. raoultii.