Spatial and temporal variation of five different pathogens and symbionts in Ixodes ricinus nymphs in the Netherlands.

The incidence of diseases caused by pathogens transmitted by the tick Ixodes ricinus vary over time and space through incompletely understood mechanisms. An important determinant of the disease risk is the density of infected ticks, which is the infection prevalence times the density of questing ticks. We therefore investigated the spatial and temporal variation of four pathogens and one of the most abundant symbionts in Ixodes ricinus in questing nymphs over four years of monthly collections in 12 locations in the Netherlands. The infection prevalence of all microbes showed markedly different patterns with significant spatial variation for Borrelia burgdorferi (s.l.), Neoehrlichia mikurensis, Rickettsia helvetica, and Midichloria mitochondrii, significant seasonal variation of B. burgdorferi (s.l.), N. mikurensis, and M. mitochondrii and a significant interannual variation of R. helvetica. Despite its ubiquitous presence, no spatio-temporal variation was observed for the infection prevalence of B. miyamotoi. The variation in infection prevalence was generally smaller than the variation in the density of nymphs, which fluctuated substantially both seasonally and between locations. This means that the variation in the densities of infected nymphs for all pathogens was mostly the result of the variation in densities of nymphs. We also investigated whether there were positive or negative associations between the symbionts, and more specifically whether ticks infected with vertically transmitted symbionts like M. mitochondrii and R. helvetica, have a higher prevalence of horizontally transmitted symbionts, such as B. burgdorferi (s.l.) and N. mikurensis. We indeed found a clear positive association between M. mitochondrii and B. burgdorferi (s.l.). The positive association between R. helvetica and B. burgdorferi (s.l.) was less clear and was only shown in two locations. Additionally, we found a clear positive association between B. burgdorferi (s.l.) and N. mikurensis, which are both transmitted by rodents. Our longitudinal study indicated strong between-location variation, some seasonal patterns and hardly any differences between years for most symbionts. Positive associations between symbionts were observed, suggesting that infection with a (vertically transmitted) symbiont may influence the probability of infection with other symbionts, or that there is a common underlying mechanism (e.g. feeding on rodents).

The prevalence of pathogens in ticks collected from humans in Belgium, 2021, versus 2017.

BACKGROUND: Ticks carry a variety of microorganisms, some of which are pathogenic to humans. The human risk of tick-borne diseases depends on, among others, the prevalence of pathogens in ticks biting humans. To follow-up on this prevalence over time, a Belgian study from 2017 was repeated in 2021. METHODS: During the tick season 2021, citizens were invited to have ticks removed from their skin, send them and fill in a short questionnaire on an existing citizen science platform for the notification of tick bites (TekenNet). Ticks were morphologically identified to species and life stage level and screened using multiplex qPCR targeting, among others, Borrelia burgdorferi (sensu lato), Anaplasma phagocytophilum, Borrelia miyamotoi, Neoehrlichia mikurensis, Babesia spp., Rickettsia helvetica and tick-borne encephalitis virus (TBEV). The same methodology as in 2017 was used. RESULTS: In 2021, the same tick species as in 2017 were identified in similar proportions; of 1094 ticks, 98.7% were Ixodes ricinus, 0.8% Ixodes hexagonus and 0.5% Dermacentor reticulatus. A total of 928 nymphs and adults could be screened for the presence of pathogens. Borrelia burgdorferi (s.l.) was detected in 9.9% (95% CI 8.2-12.0%), which is significantly lower than the prevalence of 13.9% (95% CI 12.2-15.7%) in 2017 (P = 0.004). The prevalences of A. phagocytophilum (4.7%; 95% CI 3.5-6.3%) and R. helvetica (13.3%; 95% CI 11.2-15.6%) in 2021 were significantly higher compared to 2017 (1.8%; 95% CI 1.3-2.7% and 6.8%; 95% CI 5.6-8.2% respectively) (P < 0.001 for both). For the other pathogens tested, no statistical differences compared to 2017 were found, with prevalences ranging between 1.5 and 2.9% in 2021. Rickettsia raoultii was again found in D. reticulatus ticks (n = 3/5 in 2021). Similar to 2017, no TBEV was detected in the ticks. Co-infections were found in 5.1% of ticks. When combining co-infection occurrence in 2017 and 2021, a positive correlation was observed between B. burgdorferi (s.l.) and N. mikurensis and B. burgdorferi (s.l.) and B. miyamotoi (P < 0.001 for both). CONCLUSIONS: Although the 2021 prevalences fell within expectations, differences were found compared to 2017. Further research to understand the explanations behind these differences is needed.

Xenodiagnosis in the wild: A methodology to investigate infectiousness for tick-borne bacteria in a songbird reservoir.

A crucial factor to predict the persistence and spread of infections in natural systems is the capacity of reservoir hosts to maintain the infection and transmit it to others. This is known to greatly vary within and between species and through time, although the latter part of the variation is often less well understood in the wild. Borrelia garinii is one of the causal agents of Lyme disease in humans and is transmitted among avian hosts by the hard tick Ixodes ricinus. Great tits are known to be a reservoir in Europe for B. garinii. For tick-borne pathogens like B. garinii, infectiousness or host-to-vector transmission can be measured using xenodiagnosis where pathogen-free vectors are fed on a host, and the blood-fed vectors are subsequently tested for the pathogen. Here we describe and evaluate a methodology to quantify infectiousness for tick-borne pathogens in individual wild great tits (Parus major), involving captures and recaptures of targeted individuals. The methodology can potentially be applied to other species where recapturing is sufficiently guaranteed. We successfully recaptured most of the infested great tits two to three days after initial infestation (i.e. just before ticks have fully fed) with sufficient numbers of I. ricinus larval ticks, which were subsequently screened for B. garinii using a newly developed B. garinii-specific real-time PCR assay. Higher larval tick numbers were recovered from birds during the breeding seasons than during the winter months. Our novel B. garinii-qPCR performed well, and greatly reduced the amount of Sanger sequencing needed. Preliminary results suggest both seasonal and individual variation in infectiousness; heterogeneity that needs to be unravelled to further understand the contribution of resident birds to the epidemiology of B. garinii.

T(r)icky Environments: Higher Prevalence of Tick-Borne Zoonotic Pathogens in Rodents from Natural Areas Compared with Urban Areas.

Background: Urban areas are unique ecosystems with stark differences in species abundance and composition compared with natural ecosystems. These differences can affect pathogen transmission dynamics, thereby altering zoonotic pathogen prevalence and diversity. In this study, we screened small mammals from natural and urban areas in the Netherlands for up to 19 zoonotic pathogens, including viruses, bacteria, and protozoan parasites. Materials and Methods: In total, 578 small mammals were captured, including wood mice (Apodemus sylvaticus), bank voles (Myodes glareolus), yellow-necked mice (Apodemus flavicollis), house mice (Mus musculus), common voles (Microtus arvalis), and greater white-toothed shrews (Crocidura russula). We detected a wide variety of zoonotic pathogens in small mammals from both urban and natural areas. For a subset of these pathogens, in wood mice and bank voles, we then tested whether pathogen prevalence and diversity were associated with habitat type (i.e., natural versus urban), degree of greenness, and various host characteristics. Results: The prevalence of tick-borne zoonotic pathogens (Borrelia spp. and Neoehrlichia mikurensis) was significantly higher in wood mice from natural areas. In contrast, the prevalence of Bartonella spp. was higher in wood mice from urban areas, but this difference was not statistically significant. Pathogen diversity was higher in bank voles from natural habitats and increased with body weight for both rodent species, although this relationship depended on sex for bank voles. In addition, we detected methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamase/AmpC-producing Escherichia coli, and lymphocytic choriomeningitis virus for the first time in rodents in the Netherlands. Discussion: The differences between natural and urban areas are likely related to differences in the abundance and diversity of arthropod vectors and vertebrate community composition. With increasing environmental encroachment and changes in urban land use (e.g., urban greening), it is important to better understand transmission dynamics of zoonotic pathogens in urban environments to reduce potential disease risks for public health.

Recreational hazard: Vegetation and host habitat use correlate with changes in tick-borne disease hazard at infrastructure within forest stands.

Studies on density and pathogen prevalence of Ixodes ricinus indicate that vegetation and local host community drive much of their variation between green spaces. Contrarily, micro-geographic variation is understudied, although its understanding could reduce disease risk. We studied the density of infectious nymphal Ixodes sp. ("DIN", proxy for disease hazard), density of questing nymphs ("DON") and nymphal infection prevalence ("NIP") near recreational forest infrastructure. Drag sampling within forest stands and at adjacent benches and trails was combined with vegetation surveys, camera trapping hosts and pathogen screening of ticks. We analysed Borrelia burgdorferi s.l. and its genospecies, with complementary analyses on Rickettsia sp., Anaplasma phagocytophilum, Neoehrlichia mikurensis and Borrelia miyamotoi. DIN was highest in forest interior and at trails enclosed by forest. Lower disease hazard was observed at benches and trails at forest edges. This infrastructure effect can be attributed to variation in vegetation characteristics and the habitat use of tick hosts, specifically roe deer, rodents and songbirds. DON is the main driver of DIN at micro-geographic scale and negatively affected by infrastructure and forest edges. A positive association with vegetation cover in understorey and canopy was observed, as were positive trends for local rodent and songbird abundance. NIP of different pathogens was affected by different drivers. Lower B. burgdorferi s.l. prevalence in the interior of forest stands, driven by its most prevalent genospecies B. afzelii, points towards higher density of uninfected hosts there. B. afzelii was positively associated with understorey containing tall species and with high canopy cover, whereas local bird community composition predicts B. garinii prevalence. A positive effect of songbird abundance and a negative effect of pigeons were observed. Our findings support amplification and inhibition mechanisms within forest stands and highlight that the effect of established drivers of DIN may differ based on the considered spatial scale.

Differential associations of horizontally and vertically transmitted symbionts on Ixodes ricinus behaviour and physiology.

BACKGROUND: Ixodes ricinus ticks are infected with a large diversity of vertically and horizontally transmitted symbionts. While horizontally transmitted symbionts rely on a vertebrate host for their transmission, vertically transmitted symbionts rely more on the survival of their invertebrate host for transmission. We therefore hypothesized horizontally transmitted symbionts to be associated with increased tick activity to increase host contact rate and vertically transmitted symbionts to be associated with higher tick weight and lipid fraction to promote tick survival. METHODS: We used a behavioural assay to record the questing activity of I. ricinus ticks. In addition, we measured weight and lipid fraction and determined the presence of ten symbiont species in these ticks using qPCR, of which six were vertically transmitted and four horizontally transmitted. RESULTS: Vertically transmitted symbionts (e.g. Midichloria mitochondrii) were associated with an increase in tick weight, whereas horizontally transmitted symbionts (e.g. Borrelia burgdorferi sensu lato) were often associated with lower weight and lipid fraction of ticks. Moreover, horizontally transmitted symbionts (e.g. B. burgdorferi s.l.) were associated with increased tick activity, which may benefit pathogen transmission and increases tick-borne disease hazard. CONCLUSIONS: Our study shows that horizontally and vertically transmitted symbionts differentially influence the behaviour and physiology of I. ricinus and warrants future research to study the underlying mechanisms and effects on transmission dynamics of tick-borne pathogens.

Increased rat-borne zoonotic disease hazard in greener urban areas.

Urban greening has benefits for both human and environmental health. However, urban greening might also have negative effects as the abundance of wild rats, which can host and spread a great diversity of zoonotic pathogens, increases with urban greenness. Studies on the effect of urban greening on rat-borne zoonotic pathogens are currently unavailable. Therefore, we investigated how urban greenness is associated with rat-borne zoonotic pathogen prevalence and diversity, and translated this to human disease hazard. We screened 412 wild rats (Rattus norvegicus and Rattus rattus) from three cities in the Netherlands for 18 different zoonotic pathogens: Bartonella spp., Leptospira spp., Borrelia spp., Rickettsia spp., Anaplasma phagocytophilum, Neoehrlichia mikurensis, Spiroplasma spp., Streptobacillus moniliformis, Coxiella burnetii, Salmonella spp., methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase (ESBL)/AmpC-producing Escherichia coli, rat hepatitis E virus (ratHEV), Seoul orthohantavirus, Cowpox virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Toxoplasma gondii and Babesia spp. We modelled the relationships between pathogen prevalence and diversity and urban greenness. We detected 13 different zoonotic pathogens. Rats from greener urban areas had a significantly higher prevalence of Bartonella spp. and Borrelia spp., and a significantly lower prevalence of ESBL/AmpC-producing E. coli and ratHEV. Rat age was positively correlated with pathogen diversity while greenness was not related to pathogen diversity. Additionally, Bartonella spp. occurrence was positively correlated with that of Leptospira spp., Borrelia spp. and Rickettsia spp., and Borrelia spp. occurrence was also positively correlated with that of Rickettsia spp. Our results show an increased rat-borne zoonotic disease hazard in greener urban areas, which for most pathogens was driven by the increase in rat abundance rather than pathogen prevalence. This highlights the importance of keeping rat densities low and investigating the effects of urban greening on the exposure to zoonotic pathogens in order to make informed decisions and to take appropriate countermeasures preventing zoonotic diseases.

Dermacentor reticulatus - a tick on its way from glacial refugia to a panmictic Eurasian population.

The ornate dog tick (Dermacentor reticulatus) shows a recently expanding geographic distribution. Knowledge on its intraspecific variability, population structure, rate of genetic diversity and divergence, including its evolution and geographic distribution, is crucial to understand its dispersal capacity. All such information would help to evaluate the potential risk of future spread of associated pathogens of medical and veterinary concern. A set of 865 D. reticulatus ticks was collected from 65 localities across 21 countries, from Portugal in the west to Kazakhstan and southern Russia in the east. Cluster analyses of 16 microsatellite loci were combined with nuclear (ITS2, 18S) and mitochondrial (12S, 16S, COI) sequence data to uncover the ticks' population structures and geographical patterns. Approximate Bayesian computation was applied to model evolutionary relationships among the found clusters. Low variability and a weak phylogenetic signal showing an east-west cline were detected both for mitochondrial and nuclear sequence markers. Microsatellite analyses revealed three genetic clusters, where the eastern and western cluster gradient was supplemented by a third, northern cluster. Alternative scenarios could explain such a tripartite population structure by independent formation of clusters in separate refugia, limited gene flow connected with isolation by distance causing a "bipolar pattern", and the northern cluster deriving from admixture between the eastern and western populations. The best supported demographic scenario of this tick species indicates that the northern cluster derived from admixture between the eastern and western populations 441 (median) to 224 (mode) generations ago, suggesting a possible link with the end of the Little Ice Age in Europe.

Screen the unforeseen: Microbiome-profiling for detection of zoonotic pathogens in wild rats.

Wild rats can host various zoonotic pathogens. Detection of these pathogens is commonly performed using molecular techniques targeting one or a few specific pathogens. However, this specific way of surveillance could lead to (emerging) zoonotic pathogens staying unnoticed. This problem may be overcome by using broader microbiome-profiling techniques, which enable broad screening of a sample's bacterial or viral composition. In this study, we investigated if 16S rRNA gene amplicon sequencing would be a suitable tool for the detection of zoonotic bacteria in wild rats. Moreover, we used virome-enriched (VirCapSeq) sequencing to detect zoonotic viruses. DNA from kidney samples of 147 wild brown rats (Rattus norvegicus) and 42 black rats (Rattus rattus) was used for 16S rRNA gene amplicon sequencing of the V3-V4 hypervariable region. Blocking primers were developed to reduce the amplification of rat host DNA. The kidney bacterial composition was studied using alpha- and beta-diversity metrics and statistically assessed using PERMANOVA and SIMPER analyses. From the sequencing data, 14 potentially zoonotic bacterial genera were identified from which the presence of zoonotic Leptospira spp. and Bartonella tribocorum was confirmed by (q)PCR or Sanger sequencing. In addition, more than 65% of all samples were dominated (>50% reads) by one of three bacterial taxa: Streptococcus (n = 59), Mycoplasma (n = 39) and Leptospira (n = 25). These taxa also showed the highest contribution to the observed differences in beta diversity. VirCapSeq sequencing in rat liver samples detected the potentially zoonotic rat hepatitis E virus in three rats. Although 16S rRNA gene amplicon sequencing was limited in its capacity for species level identifications and can be more difficult to interpret due to the influence of contaminating sequences in these low microbial biomass samples, we believe it has potential to be a suitable pre-screening method in the future to get a better overview of potentially zoonotic bacteria that are circulating in wildlife.

Tick microbial associations at the crossroad of horizontal and vertical transmission pathways.

BACKGROUND: Microbial communities can affect disease risk by interfering with the transmission or maintenance of pathogens in blood-feeding arthropods. Here, we investigated whether bacterial communities vary between Ixodes ricinus nymphs which were or were not infected with horizontally transmitted human pathogens. METHODS: Ticks from eight forest sites were tested for the presence of Borrelia burgdorferi sensu lato, Babesia spp., Anaplasma phagocytophilum, and Neoehrlichia mikurensis by quantitative polymerase chain reaction (qPCR), and their microbiomes were determined by 16S rRNA amplicon sequencing. Tick bacterial communities clustered poorly by pathogen infection status but better by geography. As a second approach, we analysed variation in tick microorganism community structure (in terms of species co-infection) across space using hierarchical modelling of species communities. For that, we analysed almost 14,000 nymphs, which were tested for the presence of horizontally transmitted pathogens B. burgdorferi s.l., A. phagocytophilum, and N. mikurensis, and the vertically transmitted tick symbionts Rickettsia helvetica, Rickettsiella spp., Spiroplasma ixodetis, and Candidatus Midichloria mitochondrii. RESULTS: With the exception of Rickettsiella spp., all microorganisms had either significant negative (R. helvetica and A. phagocytophilum) or positive (S. ixodetis, N. mikurensis, and B. burgdorferi s.l.) associations with M. mitochondrii. Two tick symbionts, R. helvetica and S. ixodetis, were negatively associated with each other. As expected, both B. burgdorferi s.l. and N. mikurensis had a significant positive association with each other and a negative association with A. phagocytophilum. Although these few specific associations do not appear to have a large effect on the entire microbiome composition, they can still be relevant for tick-borne pathogen dynamics. CONCLUSIONS: Based on our results, we propose that M. mitochondrii alters the propensity of ticks to acquire or maintain horizontally acquired pathogens. The underlying mechanisms for some of these remarkable interactions are discussed herein and merit further investigation. Positive and negative associations between and within horizontally and vertically transmitted symbionts.

Quantitative microbial population study reveals geographical differences in bacterial symbionts of Ixodes ricinus.

BACKGROUND: Ixodes ricinus ticks vector pathogens that cause serious health concerns. Like in other arthropods, the microbiome may affect the tick's biology, with consequences for pathogen transmission. Here, we explored the bacterial communities of I. ricinus across its developmental stages and six geographic locations by the 16S rRNA amplicon sequencing, combined with quantification of the bacterial load. RESULTS: A wide range of bacterial loads was found. Accurate quantification of low microbial biomass samples permitted comparisons to high biomass samples, despite the presence of contaminating DNA. The bacterial communities of ticks were associated with geographical location rather than life stage, and differences in Rickettsia abundance determined this association. Subsequently, we explored the geographical distribution of four vertically transmitted symbionts identified in the microbiome analysis. For that, we screened 16,555 nymphs from 19 forest sites for R. helvetica, Rickettsiella spp., Midichloria mitochondrii, and Spiroplasma ixodetis. Also, the infection rates and distributions of these symbionts were compared to the horizontally transmitted pathogens Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and Neoehrlichia mikurensis. The infection rates of all vertically transmitted symbionts differed between the study sites, and none of the symbionts was present in all tested ticks suggesting a facultative association with I. ricinus. The proportions in which symbionts occurred in populations of I. ricinus were highly variable, but geographically close study sites expressed similar proportions. These patterns were in contrast to what we observed for horizontally transmitted pathogens. Lastly, nearly 12% of tested nymphs were free of any targeted microorganisms, which is in line with the microbiome analyses. CONCLUSIONS: Our results show that the microbiome of I. ricinus is highly variable, but changes gradually and ticks originating from geographically close forest sites express similar bacterial communities. This suggests that geography-related factors affect the infection rates of vertically transmitted symbionts in I. ricinus. Since some symbionts, such as R. helvetica can cause disease in humans, we propose that public health investigations consider geographical differences in its infection rates.

Seasonal dynamics of tick burden and associated Borrelia burgdorferi s.l. and Borrelia miyamotoi infections in rodents in a Dutch forest ecosystem.

Ixodes ricinus ticks transmit Borrelia burgdorferi sensu lato (s.l.) as well as Borrelia miyamotoi. Larvae become infected when feeding on infected rodents, with horizontal transmission of B. burgdorferi and horizontal and vertical transmission of B. miyamotoi. We studied seasonal dynamics of infection rates of I. ricinus and their rodent hosts, and hence transmission risk of these two distinctly different Borrelia species. Rodents were live-trapped and inspected for ticks from May to November in 2013 and 2014 in a forest in The Netherlands. Trapped rodents were temporarily housed in the laboratory and detached ticks were collected. Borrelia infections were determined from the trapped rodents and collected ticks. Borrelia burgdorferi s.l. and B. miyamotoi were found in ticks as well as in rodents. Rodent density was higher in 2014, whereas tick burden as well as the Borrelia infection rates in rodents were higher in 2013. The density of B. miyamotoi-infected nymphs did not differ between the years. Tick burdens were higher on Apodemus sylvaticus than on Myodes glareolus, and higher on males than on females. Borrelia-infection rate of rodents varied strongly seasonally, peaking in summer. As the larval tick burden also peaked in summer, the generation of infected nymphs was highest in summer. We conclude that the heterogeneity of environmental and host-specific factors affects the seasonal transmission of Borrelia spp., and that these effects act more strongly on horizontally transmitted B. burgdorferi spp. than on the vertically transmitted B. miyamotoi.

Prevalence of Anaplasma phagocytophilum in questing Ixodes ricinus nymphs across twenty recreational areas in England and Wales.

Human granulocytic anaplasmosis and tick-borne fever, affecting livestock, are diseases caused by an infection with the bacterium Anaplasma phagocytophilum. Its transmission dynamics between vertebrate hosts and ticks remain largely unknown and the potential impact on public health in the United Kingdom is unclear. This study aimed to assess the distribution and estimate the prevalence of A. phagocytophilum in questing Ixodes ricinus at recreational locations across England and Wales over six years. An additional objective was to investigate possible associations between prevalence, habitat and presence of ruminant hosts. Ixodes ricinus ticks were collected each spring at 20 recreational locations across England and Wales between 2014 and 2019. Nymphs were tested for infection with A. phagocytophilum by detection of bacterial genome in DNA extracts, targeting the msp2 gene locus. Positive samples were further investigated for the presence of different ecotypes based on the GroEL region. Of 3,919 nymphs tested, the mean infection prevalence was 3.6% [95%CI: 3.1-4.3] and ranged from 0 to 20.4%. Northern England had a higher overall prevalence (4.7% [95%CI: 3.4-6.4]) compared to Southern England (1.8% [95%CI: 1.3-2.5]) and the presence of sheep was associated with higher A. phagocytophilum prevalence (8.4% [95%CI: 6.9-10.1] vs 1.2% [95%CI: 0.8-1.7] when absent). There was also a negative correlation with the prevalence of Borrelia burgdorferi s.l. (causing Lyme borreliosis). When investigating the diversity of A. phagocytophilum, ecotype I accounted for 86.8% of samples and ecotype II for 13.2%. Our study presents an overview of A. phagocytophilum prevalence in questing I. ricinus in recreational areas across England and Wales and discusses the potential public and veterinary health relevance.

Self-reported symptoms and health complaints associated with exposure to Ixodes ricinus-borne pathogens.

BACKGROUND: The impact of infections with tick-borne pathogens (TBPs) other than Borrelia burgdorferi (s.l.) and tick-borne encephalitis virus (TBEV) on public health in Europe remains unclear. Our goal is to evaluate whether the presence of these TBPs in ticks can be associated with self-reported health complaints. METHODS: We enrolled individuals who were bitten by I. ricinus between 2012 and 2015 and collected their relevant demographic and clinical information using a self-administered online questionnaire. A total of 4163 I. ricinus ticks sent by the participants were subject to molecular analyses for detection of specific TBPs. Associations between the presence of TBPs in ticks and self-reported complaints and symptoms were evaluated by means of a stepwise approach using a generalized linear model (GLM). RESULTS: Of 17 self-reported complaints and symptoms significant in the univariate analyses, 3 had a highly significant association (P < 0.01) with at least one TBP in the multivariate analysis. Self-reported Lyme borreliosis was significantly associated (P < 0.001) with B. burgdorferi (s.l.) infection. Facial paralysis was associated (P < 0.01) with infection with B. miyamotoi, N. mikurensis and R. helvetica. Finally, a significant association (P < 0.001) was found between nocturnal sweating and A. phagocytophilum. CONCLUSIONS: We found associations between the presence of TBPs in ticks feeding on humans and self-reported symptoms. Due to the subjective nature of such reports and the fact that infection was determined in the ticks and not in the patient samples, further prospective studies utilizing diagnostic modalities should be performed before any clinical outcome can be causally linked to infection with TBPs.

Prevalence and predictors of vector-borne pathogens in Dutch roe deer.

BACKGROUND: The main objective of this study was to determine the prevalence of nine vector-borne pathogens or pathogen genera in roe deer (Capreolus capreolus) in the Netherlands, and to identify which host variables predict vector-borne pathogen presence in roe deer. The host variables examined were the four host factors 'age category', 'sex', 'nutritional condition' and 'health status', as well as 'roe deer density'. METHODS: From December 2009 to September 2010, blood samples of 461 roe deer were collected and analysed by polymerase chain reaction (PCR) for the presence of genetic material from Anaplasma phagocytophilum, Bartonella spp., Babesia spp., Borrelia burgdorferi sensu lato (s.l.), Borrelia miyamotoi, Neoehrlichia mikurensis, Rickettsia spp., and epizootic haemorrhagic disease virus (EHDV), and by commercial enzyme-linked immunosorbent assay (ELISA) for antibodies against bluetongue virus (BTV). The possible associations of host factors and density with pathogen prevalence and co-infection, and in the case of A. phagocytophilum with bacterial load, were assessed using generalized linear modelling. RESULTS AND CONCLUSION: Analysis revealed the following prevalence in roe deer: A. phagocytophilum 77.9%, Bartonella spp. 77.7%, Babesia spp. 17.4%, Rickettsia spp. 3.3%, B. burgdorferi sensu lato 0.2%. Various co-infections were found, of which A. phagocytophilum and Bartonella spp. (49.7% of infected roe deer) and A. phagocytophilum, Bartonella spp. and Babesia spp. (12.2% of infected roe deer) were the most common. Anaplasma phagocytophilum, Babesia spp., and co-infection prevalence were significantly higher in calves than in adult roe deer, whereas the prevalence of Bartonella spp. was lower in roe deer in good nutritional condition than in deer in poor nutritional condition. Local roe deer density was not associated with pathogen presence. The high prevalence of A. phagocytophilum, Bartonella spp., and Babesia spp. is evidence for the role of roe deer as reservoirs for these pathogens. Additionally, the results suggest a supportive role of roe deer in the life-cycle of Rickettsia spp. in the Netherlands.

The Genetic Diversity of Rickettsiella Symbionts in Ixodes ricinus Throughout Europe.

Rickettsiella species are bacterial symbionts that are present in a great variety of arthropod species, including ixodid ticks. However, little is known about their genetic diversity and distribution in Ixodes ricinus, as well as their relationship with other tick-associated bacteria. In this study, we investigated the occurrence and the genetic diversity of Rickettsiella spp. in I. ricinus throughout Europe and evaluated any preferential and antagonistic associations with Candidatus Midichloria mitochondrii and the pathogens Borrelia burgdorferi sensu lato and Borrelia miyamotoi. Rickettsiella spp. were detected in most I. ricinus populations investigated, encompassing a wide array of climate types and environments. The infection prevalence significantly differed between geographic locations and was significantly higher in adults than in immature life stages. Phylogenetic investigations and protein characterization disclosed four Rickettsiella clades (I-IV). Close phylogenetic relations were observed between Rickettsiella strains of I. ricinus and other arthropod species. Isolation patterns were detected for Clades II and IV, which were restricted to specific geographic areas. Lastly, although coinfections occurred, we did not detect significant associations between Rickettsiella spp. and the other tick-associated bacteria investigated. Our results suggest that Rickettsiella spp. are a genetically and biologically diverse facultative symbiont of I. ricinus and that their distribution among tick populations could be influenced by environmental components.

Occurrence of tick-borne pathogens in questing Ixodes ricinus ticks from Wester Ross, Northwest Scotland.

BACKGROUND: Lyme borreliosis and other tick-borne diseases emerge from increased interactions between humans, other animals, and infected ticks. The risk of acquiring a tick-borne infection varies across space and time, so knowledge of the occurrence and prevalence of pathogens in ticks can facilitate disease diagnosis in a specific area and the implementation of mitigation measures and awareness campaigns. Here we identify the occurrence and prevalence of several pathogens in Ixodes ricinus ticks in Wester Ross, Northwest Scotland, a region of high tourism and tick exposure, yet data-poor in terms of tick-borne pathogens. METHODS: Questing I. ricinus nymphs (n = 2828) were collected from 26 sites in 2018 and 2019 and tested for the presence of tick-borne pathogens using PCR-based methods. Prevalence was compared with other regions of Scotland, England, Wales, and the Netherlands. RESULTS: Anaplasma phagocytophilum (4.7% prevalence), Borrelia burgdorferi sensu lato (s.l.) (2.2%), Babesia from clade X (0.2%), Rickettsia helvetica (0.04%), and Spiroplasma ixodetis (0.4%) were detected, but no Neoehrlichia mikurensis, Borrelia miyamotoi, or Babesia microti. Typing of A. phagocytophilum using a fragment of the GroEL gene identified the presence of both ecotype I and ecotype II. Genospecies identification of Borrelia burgdorferi s.l. revealed B. afzelii (53% of infected nymphs), B. garinii (9%), B. burgdorferi sensu stricto (7%), and B. valaisiana (31%). We found similar prevalence of A. phagocytophilum in Wester Ross as in the Netherlands, but higher than in other parts of Great Britain. We found lower B. burgdorferi s.l. prevalence than in England or the Netherlands, and similar to some other Scottish studies. We found higher prevalence of B. valaisiana and lower prevalence of B. garinii than in other Scottish studies. We found S. ixodetis at much lower prevalence than in the Netherlands, and R. helvetica at much lower prevalence than in England and the Netherlands. CONCLUSIONS: As far as we know, this is the first description of S. ixodetis in Great Britain. The results are relevant for disease surveillance and management for public and veterinary health. The findings can also aid in designing targeted public health campaigns and in raising awareness among outdoor recreationists and professionals.

Detection of Anaplasma phagocytophilum in European brown hares (Lepus europaeus) using three different methods.

European brown hare (Lepus europaeus Pallas 1778) is a broadly distributed lagomorph species in Europe, recognized as a host for Ixodes ricinus and reservoir of a wide range of pathogens with zoonotic potential. Even though Lepus europaeus represents an important game animal in Central Europe, the data available on Anaplasma phagocytophilum in this lagomorph are scarce. In this study, three populations of brown hare from distinct localities in the Czech Republic were analysed for the presence of Anaplasma phagocytophilum DNA. We used standard qPCR, targeting the msp2 gene and adapted the same assay also for digital droplet PCR. Out of 91 samples, these two methods identified 9 and 12 as positive, respectively. For taxonomic analysis, we amplified the groEL gene from five of six samples that were found positive by both methods. In phylogenetic analyses, this haplotype belongs to ecotype 1, and to the subclade with isolates from cervids and I. ricinus. Our findings underline the importance of correct result interpretation and positivity cut-off set-up for different detection methods of A. phagocytophilum. This bacterium is characterized by a high intraspecific variability and highly sensitive detection itself, is not enough. Detailed molecular typing is necessary to define the zoonotic potential of different strains and their natural reservoirs.

Hedgehogs and Squirrels as Hosts of Zoonotic Bartonella Species.

Free-living animals frequently play a key role in the circulation of various zoonotic vector-borne pathogens. Bacteria of the genus Bartonella are transmitted by blood-feeding arthropods and infect a large range of mammals. Although only several species have been identified as causative agents of human disease, it has been proposed that any Bartonella species found in animals may be capable of infecting humans. Within a wide-ranging survey in various geographical regions of the Czech Republic, cadavers of accidentally killed synurbic mammalian species, namely Eurasian red squirrel (Sciurus vulgaris), European hedgehog (Erinaceus europaeus) and Northern white-breasted hedgehog (Erinaceus roumanicus), were sampled and tested for Bartonella presence using multiple PCR reaction approach targeting several DNA loci. We demonstrate that cadavers constitute an available and highly useful source of biological material for pathogen screening. High infection rates of Bartonella spp., ranging from 24% to 76%, were confirmed for all three tested mammalian species, and spleen, ear, lung and liver tissues were demonstrated as the most suitable for Bartonella DNA detection. The wide spectrum of Bartonella spp. that were identified includes three species with previously validated zoonotic potential, B. grahamii, B. melophagi and B. washoensis, accompanied by 'Candidatus B. rudakovii' and two putative novel species, Bartonella sp. ERIN and Bartonella sp. SCIER.

Seoul Virus in Pet and Feeder Rats in The Netherlands.

Seoul virus (SEOV) is a zoonotic orthohantavirus carried by rats. In humans, SEOV can cause hemorrhagic fever with renal syndrome. Recent human SEOV cases described in the USA, United Kingdom, France and the Netherlands were associated with contact with pet or feeder rats. The prevalence of SEOV in these types of rats is unknown. We collected 175 pet and feeder rats (Rattus norvegicus) from private owners, ratteries and commercial breeders/traders in the Netherlands. Lung tissue of the rats was tested using a SEOV real-time RT-qPCR and heart fluid was tested for the presence of antibodies against SEOV. In all three investigated groups, RT-qPCR-positive rats were found: in 1/29 rats from private owners (3.6%), 2/56 rats from ratteries (3.4%) and 11/90 rats from commercial breeders (12.2%). The seroprevalence was largely similar to the prevalence calculated from RT-qPCR-positive rats. The SEOV sequences found were highly similar to sequences previously found in domesticated rats in Europe. In conclusion, SEOV is spread throughout different populations of domesticated rats.