Diversity and prevalence of Bartonella species in small mammals from Slovakia, Central Europe.

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.

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

By amplification and sequencing of 18S rRNA gene fragments, Hepatozoon spp. DNA was detected in 0.08 % (4/5057) and 0.04 % (1/2473) of questing Ixodes ricinus ticks from Slovakia and Czech Republic, respectively. Hepatozoon spp. DNA was also detected in spleen and/or lungs of 4.45 % (27/606) of rodents from Slovakia. Prevalence of infection was significantly higher in Myodes glareolus (11.45 %) than in Apodemus spp. (0.28 %) (P < 0.001). Sequencing of 18S rRNA Hepatozoon spp. gene amplicons from I. ricinus showed 100 % identity with Hepatozoon canis isolates from red foxes or dogs in Europe. Phylogenetic analysis showed that at least two H. canis 18S rRNA genotypes exist in Slovakia of which one was identified also in the Czech Republic. The finding of H. canis in questing I. ricinus suggests the geographical spread of the parasite and a potential role of other ticks as its vectors in areas where Rhipicephalus sanguineus is not endemic. Sequencing of 18S rRNA gene amplicons from M. glareolus revealed the presence of two closely related genetic variants, Hepatozoon sp. SK1 and Hepatozoon sp. SK2, showing 99-100 % identity with isolates from M. glareolus from other European countries. Phylogenetic analysis demonstrates that 18S rRNA variants SK1 and SK2 correspond to previously described genotypes UR1 and UR2 of H. erhardovae, respectively. The isolate from Apodemus flavicollis (Hepatozoon sp. SK3b) was 99 % identical with isolates from reptiles in Africa and Asia. Further studies are necessary to identify the taxonomic status of Hepatozoon spp. parasitizing rodents in Europe and the host-parasite interactions in natural foci.

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

Ixodes ricinus is a vector of microbial pathogens of medical and veterinary importance in Europe. Recently, increasing abundance of ticks has been observed in urban and suburban areas. The aim of this study was to investigate the tick species composition and examine correlations between local environmental variables and the relative density of host-seeking I. ricinus in two habitat types. Questing ticks were collected along six 100 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.

First detections of Rickettsia helvetica and R. monacensis in ectoparasitic mites (Laelapidae and Trombiculidae) infesting rodents in south-western Slovakia.

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.

Rickettsia slovaca and Rickettsia raoultii in Dermacentor marginatus and Dermacentor reticulatus ticks from Slovak Republic.

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.

Synanthropic birds influence the distribution of Borrelia species: analysis of Ixodes ricinus ticks feeding on passerine birds.

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.

Pathogenic microorganisms in ticks removed from Slovakian residents over the years 2008-2018.

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.

Tortoise tick Hyalomma aegyptium as long term carrier of Q fever agent Coxiella burnetii--evidence from experimental infection.

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.

Differential role of passerine birds in distribution of Borrelia spirochetes, based on data from ticks collected from birds during the postbreeding migration period in Central Europe.

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.

Blackbirds and song thrushes constitute a key reservoir of Borrelia garinii, the causative agent of borreliosis in Central Europe.

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.

Serologic evidence of Anaplasma phagocytophilum infections in patients with a history of tick bite in central Slovakia.

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.

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

BACKGROUND: Free-living ungulates are hosts of ixodid ticks and reservoirs of tick-borne microorganisms in central Europe and many regions around the world. Tissue samples and engorged ticks were obtained from roe deer, red deer, fallow deer, mouflon, and wild boar hunted in deciduous forests of south-western Slovakia. DNA isolated from these samples was screened for the presence of tick-borne microorganisms by PCR-based methods. RESULTS: Ticks were found to infest all examined ungulate species. The principal infesting tick was Ixodes ricinus, identified on 90.4% of wildlife, and included all developmental stages. Larvae and nymphs of Haemaphysalis concinna were feeding on 9.6% of wildlife. Two specimens of Dermacentor reticulatus were also identified. Ungulates were positive for A. phagocytophilum and Theileria spp. Anaplasma phagocytophilum was found to infect 96.1% of cervids, 88.9% of mouflon, and 28.2% of wild boar, whereas Theileria spp. was detected only in cervids (94.6%). Importantly, a high rate of cervids (89%) showed mixed infections with both these microorganisms. In addition to A. phagocytophilum and Theileria spp., Rickettsia helvetica, R. monacensis, unidentified Rickettsia sp., Coxiella burnetii, "Candidatus Neoehrlichia mikurensis", Borrelia burgdorferi (s.l.) and Babesia venatorum were identified in engorged I. ricinus. Furthermore, A. phagocytophilum, Babesia spp. and Theileria spp. were detected in engorged H. concinna. Analysis of 16S rRNA and groEL gene sequences revealed the presence of five and two A. phagocytophilum variants, respectively, among which sequences identified in wild boar showed identity to the sequence of the causative agent of human granulocytic anaplasmosis (HGA). Phylogenetic analysis of Theileria 18S rRNA gene sequences amplified from cervids and engorged I. ricinus ticks segregated jointly with sequences of T. capreoli isolates into a moderately supported monophyletic clade. CONCLUSIONS: The findings indicate that free-living ungulates are reservoirs for A. phagocytophilum and Theileria spp. and engorged ixodid ticks attached to ungulates are good sentinels for the presence of agents of public and veterinary concern. Further analyses of the A. phagocytophilum genetic variants and Theileria species and their associations with vector ticks and free-living ungulates are required.

Effect of Climate and Land Use on the Spatio-Temporal Variability of Tick-Borne Bacteria in Europe.

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.

Detection and identification of Borrelia burgdorferi sensu lato genospecies in ticks from three different regions in Slovakia.

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.

Seasonal infestation of birds with immature stages of Ixodes ricinus and Ixodes arboricola.

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.

Molecular evidence of Rickettsia spp. in ixodid ticks and rodents in suburban, natural and rural habitats 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.

Detection of Anaplasma phagocytophilum, Coxiella burnetii, Rickettsia spp., and Borrelia burgdorferi s. l. in Ticks, and wild-living animals in western and middle Slovakia.

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.

Seasonal variations in detecting Borrelia burgdorferi sensu lato in rodents from north eastern Austria.

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.

Ticks (Ixodidae) from passerine birds in the Carpathian region.

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.

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

BACKGROUND: Babesiosis is an emerging and potentially zoonotic disease caused by tick-borne piroplasmids of the Babesia genus. New genetic variants of piroplasmids with unknown associations to vectors and hosts are recognized. Data on the occurrence of Babesia spp. in ticks and wildlife widen the knowledge on the geographical distribution and circulation of piroplasmids in natural foci. Questing and rodent-attached ticks, rodents, and birds were screened for the presence of Babesia-specific DNA using molecular methods. Spatial and temporal differences of Babesia spp. prevalence in ticks and rodents from two contrasting habitats of Slovakia with sympatric occurrence of Ixodes ricinus and Haemaphysalis concinna ticks and co-infections of Candidatus N. mikurensis and Anaplasma phagocytophilum were investigated. RESULTS: Babesia spp. were detected in 1.5 % and 6.6 % of questing I. ricinus and H. concinna, respectively. Prevalence of Babesia-infected I. ricinus was higher in a natural than an urban/suburban habitat. Phylogenetic analysis showed that Babesia spp. from I. ricinus clustered with Babesia microti, Babesia venatorum, Babesia canis, Babesia capreoli/Babesia divergens, and Babesia odocoilei. Babesia spp. amplified from H. concinna segregated into two monophyletic clades, designated Babesia sp. 1 (Eurasia) and Babesia sp. 2 (Eurasia), each of which represents a yet undescribed novel species. The prevalence of infection in rodents (with Apodemus flavicollis and Myodes glareolus prevailing) with B. microti was 1.3 % in an urban/suburban and 4.2 % in a natural habitat. The majority of infected rodents (81.3 %) were positive for spleen and blood and the remaining for lungs and/or skin. Rodent-attached I. ricinus (accounting for 96.3 %) and H. concinna were infected with B. microti, B. venatorum, B. capreoli/B. divergens, Babesia sp. 1 (Eurasia), and Babesia sp. 2 (Eurasia). All B. microti and B. venatorum isolates were identical to known zoonotic strains from Europe. Less than 1.0 % of Babesia-positive ticks and rodents carried Candidatus N. mikurensis or A. phagocytophilum. 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.