Ticks and their epidemiological role in Slovakia: from the past till present.

In Slovakia, 22 tick species have been found to occur to date. Among them, Ixodes ricinus, Dermacentor reticulatus, D. marginatus and marginally Haemaphysalis concinna, H. inermis and H. punctata have been identified as the species of public health relevance. Ticks in Slovakia were found to harbour and transmit zoonotic and/or potentially zoonotic agents such as tick-borne encephalitis virus (TBEV), spirochaetes of the Borrelia burgdorferi sensu lato (s.l.) complex, the relapsing fever sprirochaete Borrelia miyamotoi, bacteria belonging to the orders Rickettsiales (Rickettsia spp., Anaplasma phagocytophilum, Neoehrlichia mikurensis), Legionellales (Coxiella burnetii), and Thiotrichales (Francisella tularensis), and Babesia spp. parasites (order Piroplasmida). Ixodes ricinus is the principal vector of the largest variety of microorganisms including viruses, bacteria and piroplasms. TBEV, B. burgdorferi s.l., rickettsiae of the spotted fever group, C. burnetii and F. tularensis have been found to cause serious diseases in humans, whereas B. miyamotoi, A. phagocytophilum, N. mikurensis, Babesia microti, and B. venatorum pose lower or potential risk to humans. Distribution of TBEV has a focal character. During the last few decades, new tick-borne encephalitis (TBE) foci and their spread to new areas have been registered and TBE incidence rates have increased. Moreover, Slovakia reports the highest rates of alimentary TBE infections among the European countries. Lyme borreliosis (LB) spirochaetes are spread throughout the distribution range of I. ricinus. Incidence rates of LB have shown a slightly increasing trend since 2010. Only a few sporadic cases of human rickettsiosis, anaplasmosis and babesiosis have been confirmed thus far in Slovakia. The latest large outbreaks of Q fever and tularaemia were recorded in 1993 and 1967, respectively. Since then, a few human cases of Q fever have been reported almost each year. Changes in the epidemiological characteristics and clinical forms of tularaemia have been observed during the last few decades. Global changes and development of modern molecular tools led to the discovery and identification of emerging or new tick-borne microorganisms and symbionts with unknown zoonotic potential. In this review, we provide a historical overview of research on ticks and tick-borne pathogens in Slovakia with the most important milestones and recent findings, and outline future directions in the investigation of ticks as ectoparasites and vectors of zoonotic agents and in the study of tick-borne diseases.

The Population Structure of Borrelia lusitaniae Is Reflected by a Population Division of Its Ixodes Vector.

Populations of vector-borne pathogens are shaped by the distribution and movement of vector and reservoir hosts. To study what impact host and vector association have on tick-borne pathogens, we investigated the population structure of Borrelia lusitaniae using multilocus sequence typing (MLST). Novel sequences were acquired from questing ticks collected in multiple North African and European locations and were supplemented by publicly available sequences at the Borrelia Pubmlst database (accessed on 11 February 2020). Population structure of B. lusitaniae was inferred using clustering and network analyses. Maximum likelihood phylogenies for two molecular tick markers (the mitochondrial 16S rRNA locus and a nuclear locus, Tick-receptor of outer surface protein A, trospA) were used to confirm the morphological species identification of collected ticks. Our results confirmed that B. lusitaniae does indeed form two distinguishable populations: one containing mostly European samples and the other mostly Portuguese and North African samples. Of interest, Portuguese samples clustered largely based on being from north (European) or south (North African) of the river Targus. As two different Ixodes species (i.e., I. ricinus and I. inopinatus) may vector Borrelia in these regions, reference samples were included for I. inopinatus but did not form monophyletic clades in either tree, suggesting some misidentification. Even so, the trospA phylogeny showed a monophyletic clade containing tick samples from Northern Africa and Portugal south of the river Tagus suggesting a population division in Ixodes on this locus. The pattern mirrored the clustering of B. lusitaniae samples, suggesting a potential co-evolution between tick and Borrelia populations that deserve further investigation.

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.

Local Population Structure and Seasonal Variability of Borrelia garinii Genotypes in Ixodes ricinus Ticks, Slovakia.

Lyme disease (LD) is the most common tick-borne human disease in Europe, and Borrelia garinii, which is associated with avian reservoirs, is one of the most genetically diverse and widespread human pathogenic genospecies from the B. burgdorferi sensu lato (s.l.) complex. The clinical manifestations of LD are known to vary between regions and depend on the genetic strain even within Borrelia genospecies. It is thus of importance to explore the genetic diversity of such pathogenic borreliae for the wide range of host and ecological contexts. In this study, multilocus sequence typing (MLST) was employed to investigate the local population structure of B. garinii in Ixodes ricinus ticks. The study took place in a natural wetland in Slovakia, temporally encompassing spring and autumn bird migration periods as well as the breeding period of resident birds. In total, we examined 369 and 255 ticks collected from 78 birds and local vegetation, respectively. B. burgdorferi s.l. was detected in 43.4% (160/369) of ticks recovered from birds and in 26.3% (67/255) of questing ticks, respectively. Considering the ticks from bird hosts, the highest prevalence was found for single infections with B. garinii (22.5%). Infection intensity of B. garinii in bird-feeding ticks was significantly higher than that in questing ticks. We identified ten B. garinii sequence types (STs) occurring exclusively in bird-feeding ticks, two STs occurring exclusively in questing ticks, and one ST (ST 244) occurring in both ticks from birds and questing ticks. Four B. garinii STs were detected for the first time herein. With the exception of ST 93, we detected different STs in spring and summer for bird-feeding ticks. Our results are consistent with previous studies of the low geographic structuring of B. garinii genotypes. However, our study reveals some consistency in local ST occurrence and a geographic signal for one of the clonal complexes.

Simultaneous Occurrence of Borrelia miyamotoi, Borrelia burgdorferi Sensu Lato, Anaplasma phagocytophilum and Rickettsia helvetica in Ixodes ricinus Ticks in Urban Foci in Bratislava, Slovakia.

BACKGROUND: Questing Ixodes ricinus ticks were collected in two urban parks (Zelezná studienka and Horský park) of the capital city of Slovakia, Bratislava, during two consecutive years in 2011 and 2012. A total of 932 ticks were analyzed for the presence of tick-borne agents: B. miyamotoi, B. burgdorferi s.l., A. phagocytophilum and R. helvetica. RESULTS: PCR analysis confirmed the presence of all pathogens at both localities. The overall infection prevalence of B. miyamotoi, B. burgdorferi s.l., A. phagocytophilum and R. helvetica was 0.75, 13.2, 5.6 and 8.9%, respectively. B. burgdorferi s.l. positive samples were represented by six genospecies. The most frequent one was B. afzelii followed by B. garinii and B. valaisiana. CONCLUSION: Our study confirms the presence of I. ricinus ticks and at least nine tick-borne bacterial agents in city forest parks, which are used for recreational purposes. Ordination analysis revealed significant differences in the composition of pathogens with respect to study site location, time of season and ambient temperature, despite the fact that both sites are located relatively close to one another within the city.

Seasonal Patterns in the Prevalence and Diversity of Tick-Borne Borrelia burgdorferi Sensu Lato, Anaplasma phagocytophilum and Rickettsia spp. in an Urban Temperate Forest in South Western Slovakia.

In Europe, Ixodes ricinus is the most important vector of tick-borne zoonotic bacteria. It transmits spirochaetes from the Borrelia burgdorferi sensu lato complex, Anaplasma phagocytophilum and Rickettsia spp. Although spatial differences in the prevalence of tick-borne pathogens have been intensively studied, seasonal (within-year) fluctuations in the prevalence of these pathogens within sites are often overlooked. We analyzed the occurrence and seasonal dynamics of Ixodes ricinus in an urban forest in Bratislava, Slovakia. Furthemore, we examined temporal trends in the community structure of B. burgdorferi s.l., A. phagocytophilum and Rickettsia spp. in questing and bird-feeding ticks. The total prevalence for B. burgdorferi s.l. in questing I. ricinus was 6.8%, involving six genospecies with the dominance of bird-associated B. garinii and B. valaisiana.A. phagocytophilum, R. helvetica and R. monacensis occurred in 5.9%, 5.0% and 0.2% of questing ticks, respectively. In total, 12.5% and 4.4% of bird-feeding I. ricinus ticks carried B. burgdorferi s.l. and R. helvetica. The total prevalence of B. burgdorferi s.l. in our study site was two times lower than the mean prevalence for Europe. In contrast, A. phagocytophilum prevalence was significantly higher compared to those in other habitats of Slovakia. Our results imply that tick propagation and the transmission, suppression and seasonal dynamics of tick-borne pathogens at the study site were primarily shaped by abundance and temporal population fluctuations in ruminant and bird hosts.

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.

Tick-borne pathogens and their reservoir hosts in northern Italy.

The aim of this study was to determine the occurrence of Anaplasma phagocytophilum, Rickettsia spp., Babesia spp., and Candidatus Neoehrlichia mikurensis in Ixodes spp. ticks removed from wildlife, domestic animals and humans in the Province of Trento (northern Italy) in order to better understand their ecology and provide public health professionals with an updated list of pathogens which should be considered during their diagnostic procedures after a tick bite. During 2011-2012, 848 feeding ticks at all life stages (adults, nymphs and larvae) from various hosts (wild ungulates, birds and rodents; domestic sheep, dogs and humans) were collected. The highest prevalences of A. phagocytophilum and Rickettsia spp. were detected in adult and nymphal tick stages feeding on wild ungulates (11.4% prevalence for both pathogens), while the Babesia spp. prevailed in nymphal and larval ticks feeding on wild birds (7.7%). A wide spectrum of tick-borne agents was present in larval ticks: those detached from wild ungulates were positive for A. phagocytophilum, B. venatorum, R. helvetica, R. monacensis and R. raoultii, while those removed from wild rodents were positive for B. venatorum, R. helvetica, R. monacensis and Ca. N. mikurensis, and ticks from wild birds carried A. phagocytophilum, B. venatorum, B. capreoli and R. helvetica. This study provides evidence of circulation of five tick-borne pathogens not reported in this region before, specifically R. raoultii, R. monacensis, B. venatorum, B. capreoli and B. microti. Furthermore, it discusses the epidemiological role of the animal species from which the ticks were collected highlighting the needs for more experimental studies especially for those pathogens where transovarial transmission in ticks has been demonstrated.

Prevalence and genetic variability of Anaplasma phagocytophilum in wild rodents from the Italian alps.

BACKGROUND: Human granulocytic anaplasmosis is a zoonotic bacterial disease with increasing relevance for public health in Europe. The understanding of its sylvatic cycle and identification of competent reservoir hosts are essential for improving disease risk models and planning preventative measures. RESULTS: In 2012 we collected single ear biopsy punches from 964 live-trapped rodents in the Province of Trento, Italy. Genetic screening for Anaplasma phagocytophilum (AP) was carried out by PCR amplification of a fragment of the 16S rRNA gene. Fifty-two (5.4%) samples tested positive: 49/245 (20%) from the bank vole (Myodes glareolus) and 3/685 (0.4%) samples collected from the yellow-necked mouse (Apodemus flavicollis). From these 52 positive samples, we generated 38 groEL and 39 msp4 sequences. Phylogenetic analysis confirmed the existence of a distinct rodent strain of AP. CONCLUSIONS: Our results confirm the circulation of a specific strain of AP in rodents in our study area; moreover, they provide further evidence of the marginal role of A. flavicollis compared to M. glareolus as a reservoir host for this pathogen.

Wild boar (Sus scrofa) - reservoir host of Toxoplasma gondii, Neospora caninum and Anaplasma phagocytophilum in Slovakia.

In Central Europe the wild boar population is permanently growing and consequently Cf foodborne infections. In this study serological and molecular detection of Toxoplasma gondii and Neospora caninum in wild boars was evaluated. Moreover, same samples were screened for the presence and genetic variability of tick-borne bacterium Anaplasma phagocytophilum. Blood samples collected from 113 wild boars from Southern Slovakia were examined for antibodies to T. gondii by indirect and to N. caninum by competitive ELISA. The presence of parasitic DNA in blood samples was determined by standard or real time PCR techniques. Antibodies against T. gondii and N. caninum were detected in 45 (39.8%) and 38 (33.6%) animals, respectively. Females were more frequently infected for both pathogens than males. The high seropositivity against both coccidia indicates a permanent occurrence of these pathogens in the studied locality. T. gondii DNA was confirmed in five seropositive boars (4.4%) and N. caninum in 23 blood samples (20.4%). Three out of 23 N. caninum PCR positive animals did not show seropositivity. Three out of 113 blood samples of wild boars were positive for A. phagocytophilum (2.7%). The obtained A. phagocytophilum sequences were 100% identical with GenBankTM isolates from Slovak dog (KC985242); German horse (JF893938) or wild boar (EF143810) and red deer (EF143808) from Poland. Coinfections of T. gondii with N. caninum and N. caninum with A. phagocytophilum were detected in single cases. Results suggest a potential zoonotic risk of toxoplasmosis transmission to humans and the spread of neosporosis to farm animals.

Natural foci of Borrelia lusitaniae in a mountain region of Central Europe.

Lyme borreliosis is the most prevalent tick-borne disease in Europe. It is caused by spirochaetes of the Borrelia burgdorferi sensu lato (s.l.) complex and transmitted to humans by ticks of the genus Ixodes. Borrelia afzelii, Borrelia garinii, and Borrelia valaisiana are the most common genospecies in Central Europe. In contrast, Borrelia lusitaniae predominates in Mediterranean countries such as Portugal, Morocco, and Tunisia. In Slovakia, its prevalence is low and restricted to only a few sites. The aim of our research was to study the expansion of ticks into higher altitudes in the ecosystem of the Malá Fatra mountains (north Slovakia) and their infection with B. burgdorferi s.l. pathogens. Questing ticks were collected by flagging in seven years (2004, 2006-2011) at three different altitudes: low (630-660 m above sea level (ASL)), intermediate (720-750 m ASL), and high (1040-1070 m ASL). Tick abundance was highest at the lowest altitude and lowest at the highest altitude. The average infection prevalence of B. burgdorferi s.l. in nymphs and adults was 16.8% and 36.2%, respectively. The number of infected ticks decreased from 38.5% at the lowest altitude to 4.4% at the highest altitude. B. lusitaniae was the most frequently found genospecies (>60% of the ticks found positive for B. burgdorferi s.l.) in all sites in all the studied years with the exception of 2008 when B. afzelii predominated (62%). Our study confirms the spread of Ixodes ricinus ticks to higher altitudes in Slovakia. The discovery that our mountain study sites were a natural foci of B. lusitaniae was unexpected because this genospecies is usually associated with lizards and xerothermic habitats.

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.

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.

Candidatus Neoehrlichia mikurensis and its co-circulation with Anaplasma phagocytophilum in Ixodes ricinus ticks across ecologically different habitats of Central Europe.

BACKGROUND: Candidatus Neoehrlichia mikurensis is a newly emerging tick-borne bacterium from the family Anaplasmataceae. Its presence in Ixodes ricinus ticks was reported from various European countries, however, it's ecology and co-circulation with another member of the same family, Anaplasma phagocytophilum has not been rigorously studied yet. FINDINGS: Candidatus N. mikurensis was detected in all sampling sites. In total, 4.5% of ticks were positive including larvae. The highest positivity was detected in Austria with a prevalence of 23.5%. The probability of Candidatus N. mikurensis occurrence increased with the proportion of ticks infected with Anaplasma phagocytophilum. CONCLUSION: A positive association between the occurrences of Candidatus N. mikurensis and A. phagocytophilum indicates that both bacteria share similar ecology for their natural foci in Central Europe.

Rickettsial infection in Ixodes ricinus ticks in urban and natural habitats of Slovakia.

A total of 1810 Ixodes ricinus ticks was collected from the vegetation from 2 different habitat types: urban and natural. Urban habitats were represented by cemeteries and public parks in the following towns: Bratislava, Malacky, and Martin at 150 m and 400 m above sea level. Natural habitats were selected in the mountain forest of the Martinské hole Mts. in Central Slovakia at 3 different altitudinal levels, i.e. 600 m, 800 m and 1000 ma.s.l. All ticks were tested for the presence of spotted fever group rickettsiae. The DNA of Rickettsia spp. was identified in 9% of all tested ticks. Rickettsia-infected ticks were present in both, urban and sylvatic sites at all studied altitudes. Four different species of Rickettsia were present in positive I. ricinus ticks. Rickettsia helvetica was identified in 77 out of 87 Rickettsia-positive I. ricinus ticks, followed by 8 samples that belonged to Rickettsia monacensis and 2 of the positive ticks were infected with different unidentified Rickettsia spp. Due to the association of R. helvetica and R. monacensis with human infections, it is essential to understand which species of Rickettsia circulate in the natural foci of Slovakia. Circulation of pathogenic rickettsiae in urban as well as natural habitats at different altitudinal levels in Slovakia emphasizes that infection risk is very common throughout this Central European country.

Toxoplasma gondii, Neospora caninum and tick-transmitted bacterium Anaplasma phagocytophilum infections in one selected goat farm in Slovakia.

Parasitic diseases of livestock together with poor welfare conditions can negatively affect the health status and production of small ruminants. Protozoan parasites and tick-borne infectious agents are common threat of livestock including small ruminants mostly during the pasture season. Therefore the priority of the study was to analyse the circulation and presence of two protozoan parasites Toxoplasma gondii and Neospora caninum as well as tick-transmitted bacterium Anaplasma phagocytophilum in one selected goat farm in Eastern Slovakia. Throughout a three-year study period we have repeatedly screened the sera and blood of goats and dogs from monitored farm. In total, 343 blood serum samples from 116 goats were examined by ELISA. The mean seropositivity for T. gondii was 56.9% (66/116, CI (95%) = 48-66.0) and 15.5% (18/116, CI (95%) = 9.3-22.7) for N. caninum. The permanent occurrence of anti-Toxoplasma and anti-Neospora antibodies was detected in repeatedly examined goats during the whole monitored period. The presence of both parasites in the flock was analysed by PCR. DNA of T. gondii was confirmed in 12 out of 25 Toxoplasma-seropositive goats and N. caninum in 14 samples out of 18 Neospora-seropositive animals; four goats were co-infected with both pathogens. The risk of endogenous transmission of both parasites was pursued by examination of 41 kid's sera, where seropositivity for toxoplasmosis was 31.7% and for neosporosis 14.6%. In dogs 61.1% seropositivity for T. gondii and 38.9% for N. caninum was found, however, their faeces were negative for coccidian oocysts. Eight out of 108 tested animals were infected with A. phagocytophilum, the causative agent of tick-borne fever. Seven of them were simultaneously infected with T. gondii and A. phagocytophilum, out of which four goats were concurrently infected with all three pathogens.

A system to simultaneously detect tick-borne pathogens based on the variability of the 16S ribosomal genes.

BACKGROUND: DNA microarrays can be used to quickly and sensitively identify several different pathogens in one step. Our previously developed DNA microarray, based on the detection of variable regions in the 16S rDNA gene (rrs), which are specific for each selected bacterial genus, allowed the concurrent detection of Borrelia spp., Anaplasma spp., Francisella spp., Rickettsia spp. and Coxiella spp. METHODS: In this study, we developed a comprehensive detection system consisting of a second generation DNA microarray and quantitative PCRs. New oligonucleotide capture probes specific for Borrelia burgdorferi s.l. genospecies and Candidatus Neoehrlichia mikurensis were included. This new DNA microarray system required substantial changes in solution composition, hybridization conditions and post-hybridization washes. RESULTS: This second generation chip displayed high specificity and sensitivity. The specificity of the capture probes was tested by hybridizing the DNA microarrays with Cy5-labeled, PCR-generated amplicons encoding the rrs genes of both target and non-target bacteria. The detection limit was determined to be 10(3) genome copies, which corresponds to 1-2 pg of DNA. A given sample was evaluated as positive if its mean fluorescence was at least 10% of the mean fluorescence of a positive control. Those samples with fluorescence close to the threshold were further analyzed using quantitative PCRs, developed to identify Francisella spp., Rickettsia spp. and Coxiella spp. Like the DNA microarray, the qPCRs were based on the genus specific variable regions of the rrs gene. No unspecific cross-reactions were detected. The detection limit for Francisella spp. was determined to be only 1 genome copy, for Coxiella spp. 10 copies, and for Rickettsia spp., 100 copies. CONCLUSIONS: Our detection system offers a rapid method for the comprehensive identification of tick-borne bacteria, which is applicable to clinical samples. It can also be used to identify both pathogenic and endosymbiontic bacteria in ticks for eco-epidemiological studies, tick laboratory colony testing, and many other applications.

Ixodes ricinus abundance and its infection with the tick-borne pathogens in urban and suburban areas of Eastern Slovakia.

BACKGROUND: Raising abundance of ticks and tick-borne diseases in Europe is the result of multiple factors including climate changes and human activities. Herein, we investigated the presence and seasonal activity of Ixodes ricinus ticks from 10 urban and suburban sites in two different geographical areas of southeastern and northeastern Slovakia during 2008-2010. Our aim was to study the abundance of ticks in correlation with the environmental factors and their infection with Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Neoehrlichia mikurensis. METHODS: Questing I. ricinus ticks were collected from ten urban and suburban sites in Eastern Slovakia. A total of 670 ticks were further analysed for the presence of B. burgdorferi s.l., A. phagocytophilum and N. mikurensis by molecular methods. Tick site and environmental relations were analysed using General Linear Models (LM). The differences between the number of Lyme borreliosis cases between the Kosice and Bardejov regions during a ten-year period were tested by Wilcoxon matched pairs test. RESULTS: In total, 2921 (1913 nymphs, 1008 adults) I. ricinus ticks were collected from 10 study sites during the main questing season. Tick activity and relative abundance differed between locations and months. Temperature and humidity were the main factors affecting the tick abundance and questing activity. Out of 670 examined ticks, 10.15% were infected with spirochetes from B. burgdorferi s.l. complex (represented by B. afzelii, B. garinii, B.valaisiana and B. burgdorferi s.s.), 2.69% with the A. phagocytophilum and 2.39% with N. mikurensis. The number of Lyme borreliosis cases per 100,000 inhabitants in the Bardejov region was significantly higher than in the Kosice region. CONCLUSIONS: Our data indicate that the risk of infection with tick-borne pathogens in Eastern Slovakia is common since 15.2% of ticks were infected at least with one of the tested microorganisms. Even though the abundance of ticks was affected by the microclimatic conditions and the prevalence of pathogens differed between the habitats, the infection risk for humans is also affected by human activities leading to an increased contact with infected ticks.

Driving forces for changes in geographical distribution of Ixodes ricinus ticks in Europe.

Many factors are involved in determining the latitudinal and altitudinal spread of the important tick vector Ixodes ricinus (Acari: Ixodidae) in Europe, as well as in changes in the distribution within its prior endemic zones. This paper builds on published literature and unpublished expert opinion from the VBORNET network with the aim of reviewing the evidence for these changes in Europe and discusses the many climatic, ecological, landscape and anthropogenic drivers. These can be divided into those directly related to climatic change, contributing to an expansion in the tick's geographic range at extremes of altitude in central Europe, and at extremes of latitude in Scandinavia; those related to changes in the distribution of tick hosts, particularly roe deer and other cervids; other ecological changes such as habitat connectivity and changes in land management; and finally, anthropogenically induced changes. These factors are strongly interlinked and often not well quantified. Although a change in climate plays an important role in certain geographic regions, for much of Europe it is non-climatic factors that are becoming increasingly important. How we manage habitats on a landscape scale, and the changes in the distribution and abundance of tick hosts are important considerations during our assessment and management of the public health risks associated with ticks and tick-borne disease issues in 21(st) century Europe. Better understanding and mapping of the spread of I. ricinus (and changes in its abundance) is, however, essential to assess the risk of the spread of infections transmitted by this vector species. Enhanced tick surveillance with harmonized approaches for comparison of data enabling the follow-up of trends at EU level will improve the messages on risk related to tick-borne diseases to policy makers, other stake holders and to the general public.