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.

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.

Rickettsia species in fleas collected from small mammals in Slovakia.

Epidemiological and epizootiological studies of Rickettsia felis and other Rickettsia spp. are very important, because their natural cycle has not yet been established completely. In total, 315 fleas (Siphonaptera) of 11 species of Ceratophyllidae, Hystrichopsyllidae and Leptopsyllidae families were tested for the presence of Rickettsia species and Coxiella burnetii with conventional and specific quantitative real-time PCR assays. Fleas were collected from five rodent hosts (Myodes glareolus, Apodemus flavicollis, Apodemus agrarius, Microtus subterraneus, Microtus arvalis) and three shrew species (Sorex araneus, Neomys fodiens, Crocidura suaveolens) captured in Eastern and Southern Slovakia. Overall, Rickettsia spp. was found in 10.8% (34/315) of the tested fleas of Ctenophthalmus agyrtes, Ctenophthalmus solutus, Ctenophthalmus uncinatus and Nosopsyllus fasciatus species. Infected fleas were coming from A. flavicollis, A. agrarius, and M. glareolus captured in Eastern Slovakia. C. burnetii was not found in any fleas. R. felis, Rickettsia helvetica, unidentified Rickettsia, and rickettsial endosymbionts were identified in fleas infesting small mammals in the Kosice region, Eastern Slovakia. This study is the first report of R. felis infection in C. solutus male flea collected from A. agrarius in Slovakia.

Distinct Anaplasma phagocytophilum genotypes associated with Ixodes trianguliceps ticks and rodents in Central Europe.

Rodents are important reservoir hosts of tick-borne pathogens. Anaplasma phagocytophilum is the causative agent of granulocytic anaplasmosis of both medical and veterinary importance. In Europe, this pathogen is primarily transmitted by the Ixodes ricinus tick among a wide range of vertebrate hosts. However, to what degree A. phagocytophilum exhibits host specificity and vector association is poorly understood. To assess the extent of vector association of this pathogen and to clarify its ecology in Central Europe we have analyzed and compared the genetic variability of A. phagocytophilum strains from questing and feeding I. ricinus and Ixodes trianguliceps ticks, as well as from rodent' tissue samples. Tick collection and rodent trapping were performed during a 2-year study (2011-2012) in ecologically contrasting setting at four sites in Eastern Slovakia. Genetic variability of this pathogen was studied from the collected samples by DNA amplification and sequencing of four loci followed by Bayesian phylogenetic analyses. A. phagocytophilum was detected in questing I. ricinus ticks (0.7%) from all studied sites and in host feeding I. trianguliceps ticks (15.2%), as well as in rodent biopsies (ear - 1.6%, spleen - 2.2%), whereas A. phagocytophilum was not detected in rodents from those sites where I. trianguliceps ticks were absent. Moreover, Bayesian phylogenetic analyses have shown the presence of two distinct clades, and tree topologies were concordant for all four investigated loci. Importantly, the first clade contained A. phagocytophilum genotypes from questing I. ricinus and feeding I. ricinus from a broad array of hosts (i.e.,: humans, ungulates, birds and dogs). The second clade comprised solely genotypes found in rodents and feeding I. trianguliceps. In this study we have confirmed that A. phagocytophilum strains display specific host and vector associations also in Central Europe similarly to A. phagocytophilum' molecular ecology in United Kingdom. This study suggests that A. phagocytophilum genotypes associated with rodents are probably transmitted solely by I. trianguliceps ticks, thus implying that rodent-associated A. phagocytophilum strains may not pose a risk for humans.

Detection of shrew-borne hantavirus in Eurasian pygmy shrew (Sorex minutus) in Central Europe.

Recently, it was found that not only rodents but also shrews are reservoir hosts of hantaviruses. In Central Europe, only Seewis virus, associated with the Eurasian common shrew (Sorex araneus), has been recognized until now. In the present report, tissue samples from shrews belonging to Crocidurinae and Soricinae subfamilies, trapped in Czech Republic, Germany, and Slovakia, were screened for the presence of novel hantaviruses. Three new hantavirus partial L-segment sequences were obtained from pygmy shrews (Sorex minutus) trapped in Czech Republic and Germany. Complete nucleocapsid protein- and glycoprotein precursor-coding S- and M-segment sequences were then determined for the newly recognized hantavirus strains, CZ/Beskydy/412/2010/Sm, CZ/Drahany/420/2010/Sm, and DE/Dürrbach/1912/2009/Sm. Phylogenetic analyses showed that they represent strains of Asikkala virus (ASIV), a novel hantavirus also found in pygmy shrews from Finland. Our study reveals a broad geographic distribution of ASIV across Europe and indicates pygmy shrew as the primary reservoir host. Future studies will have to determine the pathogenic relevance of ASIV.

Broad geographical distribution and high genetic diversity of shrew-borne Seewis hantavirus in Central Europe.

For a long time hantaviruses were believed to be exclusively rodent-borne pathogens. Recent findings of numerous shrew- and mole-borne hantaviruses raise important questions on their phylogenetic origin. The objective of our study was to prove the presence and distribution of shrew-associated Seewis virus (SWSV) in different Sorex species in Central Europe. Therefore, a total of 353 Sorex araneus, 59 S. minutus, 27 S. coronatus, and one S. alpinus were collected in Germany, the Czech Republic, and Slovakia. Screening by hantavirus-specific L-segment RT-PCR revealed specific amplification products in tissues of 49 out of 353 S. araneus and four out of 59 S. minutus. S-segment sequences were obtained for 45 of the L-segment positive S. araneus and all four L-segment positive S. minutus. Phylogenetic investigation of these sequences from Germany, the Czech Republic, and Slovakia demonstrated their similarity to SWSV sequences from Hungary, Finland, Austria, and other sites in Germany. The low intra-cluster sequence variability and the high inter-cluster divergence suggest a long-term SWSV evolution in isolated Sorex populations. In 28 of the 49 SWSV S-segment sequences, an additional putative open reading frame (ORF) on the opposite strand to the nucleocapsid protein-encoding ORF was identified. This is the first comprehensive sequence analysis of SWSV strains from Germany, the Czech Republic, and Slovakia, indicating its broad geographical distribution and high genetic divergence. Future studies have to prove whether both S. araneus and S. minutus represent SWSV reservoir hosts or spillover infections are responsible for the parallel molecular detection of SWSV in both species.

Small mammals (Insectivora, Rodentia) as a potential source of chlamydial infection in East Slovakia.

The presence of antibodies against Chlamydophila (Chlamydia) psittaci in small mammals (Insectivora, Rodentia) in the region of East Slovakia are presented. The hosts were caught in several areas of Slovakia in habitats with different levels of anthropogenic disturbance. Research was carried out during 2000-2002. The authors examined 1,947 sera coming from 4 insectivore and 10 rodent species. Each serum was examined by micromethod of complement binding reactions using antigen Chlamydophila (Chlamydia) psittaci. Chlamydial infections were found in 251 individuals (prevalence 12.9 %) of 8 mammal species. The antichlamydial antibodies were proved at levels ranging from 1:32-1:1024. The highest prevalence of antibodies was detected in the most abundant rodent species Apodemus microps (14.8 %), Apodemus agrarius (13.9 %), Apodemus flavicolis (12.4 %), Microtus arvalis (12 %), and Clethrionomys glareolus (10.9 %). Positive hosts were registered in all studied localities. Testing of prevalence values in the individual research years confirmed significant changes. Our results showed that small mammals probably play an important role in the circulation of chlamydiae in nature.

Anti-Borrelia antibodies in rodents: important hosts in ecology of Lyme disease.

Data presented in this study focuses on the presence of anti-Borrelia antibodies in small mammals from Eastern Slovakia during 2000-2003. The total seropositivity observed was 18.78% in rodents. Amongst all species, the total seroprevalence in Apodemus flavicolis was the highest (20.87%), followed by Apodemus agrarius (19.58%) and Clethrionomys glareolus (11.11%). However, the prevalence in Apodemus flavicolis during the year 2000-2001 was higher (26.72%), which reduced to 10.60% in 2002-2003. To compare the year range of seroprevalence in other small mammals was not feasible due to the small sample number. Area-wise distribution of anti-Borrelia antibodies was even (18.75% to 20%) in this study, except in the Bot'any province (0%). This confirms the equal distribution of Borrelia spirochetes in the other 3 localities. Prevalence of anti-Borrelia antibodies during summer was significantly higher than during autumn and early spring. The overall study also reviews the importance of small mammals in Lyme disease ecology.