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Background: Increased glucose uptake and utilization via aerobic glycolysis are among the most prominent hallmarks of tumor cell metabolism. Accumulating evidence suggests that similar metabolic changes are also triggered in many virus-infected cells. Viral propagation, like highly proliferative tumor cells, increases the demand for energy and macromolecular synthesis, leading to high bioenergetic and biosynthetic requirements. Although significant progress has been made in understanding the metabolic changes induced by viruses, the interaction between host cell metabolism and arenavirus infection remains unclear. Our study sheds light on these processes during lymphocytic choriomeningitis virus (LCMV) infection, a model representative of the Arenaviridae family., Methods: The impact of LCMV on glucose metabolism in MRC-5 cells was studied using reverse transcription-quantitative PCR and biochemical assays. A focus-forming assay and western blot analysis were used to determine the effects of glucose deficiency and glycolysis inhibition on the production of infectious LCMV particles., Results: Despite changes in the expression of glucose transporters and glycolytic enzymes, LCMV infection did not result in increased glucose uptake or lactate excretion. Accordingly, depriving LCMV-infected cells of extracellular glucose or inhibiting lactate production had no impact on viral propagation. However, treatment with the commonly used glycolytic inhibitor 2-deoxy-D-glucose (2-DG) profoundly reduced the production of infectious LCMV particles. This effect of 2-DG was further shown to be the result of suppressed N-linked glycosylation of the viral glycoprotein., Conclusions: Although our results showed that the LCMV life cycle is not dependent on glucose supply or utilization, they did confirm the importance of N-glycosylation of LCMV GP-C. 2-DG potently reduces LCMV propagation not by disrupting glycolytic flux but by inhibiting N-linked protein glycosylation. These findings highlight the potential for developing new, targeted antiviral therapies that could be relevant to a wider range of arenaviruses., (© 2023. The Author(s).)

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., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

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.

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., Competing Interests: The authors declare no conflict of interest.

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.

This paper presents a method for the determination of acebutolol, betaxolol, bisoprolol, metoprolol, nebivolol and sotalol in human serum by liquid-liquid extraction and ultra-high-performance liquid chromatography coupled with ultra-high-resolution TOF mass spectrometry. After liquid-liquid extraction, beta blockers were separated on a reverse-phase analytical column (Acclaim RS 120; 100 × 2.1 mm, 2.2 μm). The total run time was 6 min for each sample. Linearity, limit of detection, limit of quantification, matrix effects, specificity, precision, accuracy, recovery and sample stability were evaluated. The method was successfully applied to the therapeutic drug monitoring of 108 patients with hypertension. This method was also used for determination of beta blockers in 33 intoxicated patients., (Copyright © 2016 John Wiley & Sons, Ltd.)

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.

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

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.

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.

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., Conclusion: Our findings suggest that I. ricinus and rodents play important roles in the epidemiology of zoonotic Babesia spp. in south-western Slovakia. Associations with vertebrate hosts and the pathogenicity of Babesia spp. infecting H. concinna ticks need to be further explored.

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.

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.

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%) (χ(2) = 37.451; P < 0.001). Significant local differences in prevalence of infected questing ticks were found among transects within each habitat as well as among years and between seasons. The trapped rodents belonged to six species. Apodemus flavicollis and Myodes glareolus prevailed in both habitats, Microtus arvalis was present only in the natural habitat. I. ricinus comprised 96.3% of the rodent-attached ticks, the rest were Haemaphysalis concinna, Ixodes trianguliceps and Dermacentor reticulatus. Only 0.5% of rodent skin and 0.6% of rodent-attached ticks (only I. ricinus) were infected with A. phagocytophilum. Prevalence of A. phagocytophilum in questing I. ricinus did not correlate significantly with relative abundance of ticks or with abundance of wildlife in the area., Conclusion: The study confirms that urban I. ricinus populations are infected with A. phagocytophilum at a higher rate than in a natural habitat of south-western Slovakia and suggests that rodents are not the main reservoirs of the bacterium in the investigated area.

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.

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.

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.

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.

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.

In this study, we detected Rickettsia helvetica, Candidatus Midichloria mitochondrii, Anaplasma phagocytophilum, Ehrlichia muris, Candidatus Neoehrlichia mikurensis, and Bartonella sp. infections in wild rodents and ticks collected from the vegetation of central Slovakia. The microorganisms were identified by PCR and sequencing. Yellow-necked mice (Apodemus flavicollis) were infected with E. muris and Bartonella sp., while ticks Ixodes ricinus collected from the vegetation were infected with R. helvetica, Candidatus M. mitochondrii, Candidatus N. mikurensis, A. phagocytophilum, and E. muris.

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 Vtácnik (5/19), Stiavnické vrchy (1/19), Kremnické vrchy (10/19) and Ziarska 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.

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.

Austria is well known as an endemic area of Lyme borreliosis. To assess the annual variation of rodent populations that may host agents of Lyme borreliosis we collected rodents in northeastern Austria. Life traps were set out every six weeks during a year consecutively in one each of the three different zones (Hohenau, Ernstbrunn, Vienna Woods) that cover the main habitat characteristics of small mammals in northeastern Austria. Rodents were collected and identified. Samples of heart, urine bladder and brain were removed under aseptic conditions for cultivation of borrelia. Samples of heart muscle were additionally used for molecular detection of borrelia by Real-Time polymerase chain reaction. PCR was performed with borrelia universal primers and with species-specific primers. 938 mice were caught, most frequently Apodemus flavicollis (44%), followed by Clethrionomys glareolus (35%), Microtus arvalis (9%), A. sylvaticus (7%) and Mus musculus (6%). Significant differences were seen in the total number of catch per area (Hohenau, Ernstbrunn, Vienna Woods equal 10:9:2) and in the distribution of the various rodent species in the respective areas. Borrelia strains were grown from only 65 (7%) animals, and more frequently isolated from bladder wall than from heart muscle, and only once from brain. Heart specimens of 223 animals were positive by borrelia PCR (24%), most frequently of the rodent species A. flavicollis (43%) and C. glareolus (38%). Borrelia afzelii was most frequently identified, followed by B. burgdorferi sensu stricto, B. garinii and by mixed infection of B. afzelii with B. burgdorferi sensu stricto. B. garinii was most frequently detected in heart samples of A. sylvaticus (about 20%). In about 3% of PCR positive samples the identification of one of the three mentioned genospecies of borrelia could not be ascertained with the test panel used. The results confirm the rodent species A. flavicollis, A. sylvaticus, M. arvalis and C. glareolus as reservoir animals for B. afzelii, B. garinii and B. burgdorferi sensu stricto, agents of Lyme borreliosis. Notable is the salient presence of B. garinii in heart specimens of A. sylvaticus.

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 Bukovské 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.

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.