Circulation of Rickettsia species and rickettsial endosymbionts among small mammals and their ectoparasites in Eastern Slovakia.

Bacteria belonging to the genus Rickettsia are known as causative agents of vector-borne zoonotic diseases, such as spotted fevers, epidemic typhus and endemic typhus. Different species of ticks, mites and fleas could act as reservoirs and arthropod vectors of different pathogenic Rickettsia species. The aim of this work was to establish active surveillance of Rickettsia spp. in mites, ticks and fleas collected from small mammals (rodents and shrews) in Eastern Slovakia. A total of 964 animal ear biopsies, 871 mites, 667 ticks and 743 fleas were collected from small mammals in the Kosice region, Eastern Slovakia. All specimens were identified using specialized taxonomic keys, and were conserved in ethanol until DNA extraction was performed. After DNA extraction, identification of Rickettsia species was performed by PCR-based methods. The total prevalence of rickettsiae from ear biopsies was 4.6% (95% CI, 3.2-5.9), in tested mites 9.3% (95% CI, 7.4-11.2), 17.2% (95% CI, 14.3-20.1) in I. ricinus ticks and 3.5% (95% CI, 2.2-4.8) in fleas. Sequence analysis of the partial gltA gene and Rickettsia helvetica-, Rickettsia slovaca-, Rickettsia raoultii- species specific real-time PCR tests revealed the presence of R. helvetica, R. slovaca, unidentified Rickettsia and rickettsial endosymbionts. These pathogenic and symbiotic species were confirmed in the following ectoparasite species-Laelaps jettmari, Haemogamasus nidi, Laelaps agilis and Eulaelaps stabularis mites, Ixodes ricinus ticks, Ctenophthalmus solutus, C. assimilis and Megabothris turbidus fleas infesting host-Apodemus agrarius, A. flavicollis, Microtus arvalis and Myodes glareolus small mammals. These results confirm the circulation of R. helvetica, R. slovaca, unidentified Rickettsia and rickettsial endosymbionts in mites, ticks and fleas collected on small mammals in the Kosice region, Eastern Slovakia.

Molecular evidence and diversity of the spotted-fever group Rickettsia spp. in small mammals from natural, suburban and urban areas of Eastern Slovakia.

Rickettsiae of the spotted fever group are considered as emerging pathogens; ticks, fleas and mites are known to be their vectors. However, the prevalence and species diversity of rickettsiae in small mammals and the role of these hosts in the circulation of bacteria are much less studied. During 2014-2016, a total of 250 small mammals (Apodemus agrarius, Apodemus flavicollis, Apodemus uralensis, Myodes glareolus, Crocidura leucodon, Crocidura suaveolens, Micromys minutus, Microtus arvalis, Microtus subterraneus, Neomys fodiens and Sorex minutus) were captured in natural, suburban and urban habitats of eastern Slovakia. Ear biopsies of 245 individuals were examined for the presence of SFG rickettsiae by molecular methods. The overall prevalence of Rickettsia spp. in small mammals was 11%. The predominant species, Rickettsia helvetica was confirmed in the striped field mouse (A. agrarius), the yellow-necked mouse (A. flavicollis), the bank vole (M. glareolus) and the common vole (M. arvalis) in natural and suburban habitats, followed by Rickettsia sp. closely related to R. felis identified in A. flavicollis captured in a suburban habitat. Finally, R. slovaca was found in only one yellow-necked mouse (A. flavicollis) captured in a natural habitat, with the sympatric occurrence of Dermacentor marginatus and Dermacentor reticulatus ticks. We assume the presence of R. slovaca especially in sites with the occurrence of Dermacentor spp. All small mammals captured in the urban habitat tested were negative for the presence of rickettsiae. This study brings the first molecular evidence of R. slovaca in a rodent captured in Slovakia. Rickettsia sp. closely related to R. felis was first time detected in A. flavicollis in suburban site of Slovakia. The highest species diversity of rickettsiae was observed in A. flavicollis, and the highest prevalence of bacteria was recorded in M. glareolus. The highest occurrence of rickettsiae-positive small mammals was recorded during the spring and autumn months, May, June and September, respectively. This may be related with the seasonal activity of the tick vectors. This study confirms the long-term persistence of Rickettsia spp. in small mammals in natural and suburban habitats of Slovakia. Some rodent species that have a wider ecological valency may contribute to the maintenance, circulation and dissemination of rickettsiae within and out the natural foci more significantly than those species that have narrower relation to the certain type of habitat.

Fleas and Ticks of Red Foxes as Vectors of Canine Bacterial and Parasitic Pathogens, in Slovakia, Central Europe.

The present survey aimed to investigate flea and tick fauna parasitizing Slovak red fox populations with special emphasis on canine pathogens they transmit. A total of 407 fleas and 105 ticks were collected from 90 red foxes from two geographically distant regions. Seven flea species (Chaetopsylla globiceps, Pulex irritans, Archaeopsylla erinacei, Chaetopsylla rothschildi, Chaetopsylla trichosa, Ctenocephalides canis, and Ctenopthalmus assimilis) and three species of hard ticks (Ixodes ricinus, Ixodes hexagonus, Haemaphysalis concinna) were recorded on sampled animals. Consequently, the DNA of five different pathogen taxa was confirmed in collected arthropod vectors: Bartonella spp. (in P. irritans, Ch. globiceps, and Ct. assimilis), Rickettsia spp. (in A. erinacei, I. ricinus, I. hexagonus, and H. concinna), Anaplasma phagocytophilum (in I. ricinus), Theileria sp. (in Ch. globiceps and H. concinna), and Hepatozoon canis (in I. ricinus and I. hexagonus). Mycoplasma spp., Dipylidium caninum, and Acanthocheilonema reconditum were not found in fleas or ticks in this study.

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.

Genetic diversity of Bartonella genotypes found in the striped field mouse (Apodemus agrarius) in Central Europe.

We investigated the diversity of Bartonella in Apodemus agrarius, an important rodent of peri-domestic habitats, which has spread into Europe in the past 1000 years. Spleen samples of 344 A. agrarius from Eastern Slovakia were screened for the presence of Bartonella spp. using 16S-23S rRNA internal transcribed spacer region and bacteria were detected in 9% of rodents. Based on sequencing of three housekeeping genes (gltA, rpoB and groEL) Bartonella genotypes were ascribed to the species typical for mice and voles: B. grahamii, B. taylorii and B. birtlesii. However, the study also confirmed presence of genotypes belonging to the B. clarridgeiae/B. rochalimae clade, and the B. elizabethae/B. tribocorum clade, which are not commonly found in woodland rodents. In addition, a potential recombination event between these two genotypes was noted, which highlights an important role of A. agrarius in shaping Bartonella diversity and evolution.

Presence of Candidatus Neoehrlichia mikurensis and Babesia microti in rodents and two tick species (Ixodes ricinus and Ixodes trianguliceps) in Slovakia.

Rodents are important reservoir hosts of many tick-borne pathogens. Their importance in the circulation of the emerging bacterial agent, Candidatus Neoehrlichia mikurensis and the intraerythrocytic protozoan parasite, Babesia microti has been recently proposed. The aim of the present study was to identify the presence and genetic diversity of Candidatus N. mikurensis and B. microti circulating in the natural foci among rodents and two species of ixodid ticks (Ixodes ricinus and Ixodes trianguliceps). In 2011-2013, rodents were captured at sampling sites in Eastern Slovakia. A total of 997 rodents (324 Apodemus agrarius, 350 Apodemus flavicollis, 271 Myodes glareolus, and 52 other rodent species), 788 feeding ticks from rodents, and 1375 questing ticks were investigated for the presence of pathogens by molecular methods followed by DNA sequencing. Candidatus N. mikurensis was detected in 2.4% of questing I. ricinus nymphs and 2.6% of questing adult I. ricinus ticks, spleens of rodents (1.6%), as well as in feeding larval I. ricinus (0.3%) and feeding larval I. trianguliceps ticks (3.3%). The 16S rRNA and gltA gene sequences of Candidatus N. mikurensis obtained in this study confirmed a high degree of genetic identity of this bacterium in Europe. DNA of B. microti was found in ear (0.6%) and spleen biopsies of rodents (1.9%), in rodent foetus (3.8%) and feeding larval (5.2%) and nymphal (8.7%) I. ricinus, in questing nymphal I. ricinus (0.5%) and questing adult I. ricinus ticks (0.3%). None of the 112 I. trianguliceps ticks were infected. B. microti was represented by two different genotypes: 92% of the positive samples belonged to the zoonotic type strain from Jena (Germany). The results of this study underline the importance of rodents in the circulation of both emerging pathogens in natural foci.

Seasonal analysis of Rickettsia species in ticks in an agricultural site of Slovakia.

Many rickettsiae of the spotted fever group are emerging pathogens causing serious diseases associated with vertebrate hosts. Ixodidae ticks are known as their vectors. Investigation of the relative abundance of questing Ixodes ricinus and their infection with Rickettsia spp. in an agricultural site comprising a game reserve in Slovakia was the aim of this study. In total, 2198 I. ricinus (492 larvae, 1503 nymphs and 203 adults) were collected by flagging the vegetation along 100 m(2) transects in Rozhanovce (eastern Slovakia): 334, 595 and 1269 in 2011, 2012 and 2013, respectively. Considering questing nymphs and adults, the highest relative density of 81 individuals/100 m(2) was observed in May 2013, the lowest of 0.3 individuals/100 m(2) in March 2012. A total of 1056 ticks (853 nymphs, 100 females and 103 males; 2011: n = 329, 2012: n = 509 and 2013: n = 218) were individually screened by PCR-based methods for the presence of Rickettsia spp. The overall prevalences were 7.3% for nymphs, 15% for females, 7.8% for males; 7.0% in 2011, 8.4% in 2012, and 8.7% in 2013. The maximum prevalences were observed in July in nymphs and in May in adults. Sequencing showed infection with R. helvetica in 73 ticks (72.6% nymphs, 16.4% females, 11% males) and with R. monacensis in 11 ticks (8 nymphs, 3 females). The results showed the circulation of pathogenic Rickettsia species in the agricultural site and a potential risk for humans to encounter infected ticks.