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.

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.

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.

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.

Circulation of Babesia Species and Their Exposure to Humans through Ixodes Ricinus.

Human babesiosis in Europe has been attributed to infection with Babesia divergens and, to a lesser extent, with Babesia venatorum and Babesia microti, which are all transmitted to humans through a bite of Ixodes ricinus. These Babesia species circulate in the Netherlands, but autochthonous human babesiosis cases have not been reported so far. To gain more insight into the natural sources of these Babesia species, their presence in reservoir hosts and in I. ricinus was examined. Moreover, part of the ticks were tested for co-infections with other tick borne pathogens. In a cross-sectional study, qPCR-detection was used to determine the presence of Babesia species in 4611 tissue samples from 27 mammalian species and 13 bird species. Reverse line blotting (RLB) and qPCR detection of Babesia species were used to test 25,849 questing I. ricinus. Fragments of the 18S rDNA and cytochrome c oxidase subunit I (COI) gene from PCR-positive isolates were sequenced for confirmation and species identification and species-specific PCR reactions were performed on samples with suspected mixed infections. Babesia microti was found in two widespread rodent species: Myodes glareolus and Apodemus sylvaticus, whereas B. divergens was detected in the geographically restricted Cervus elaphus and Bison bonasus, and occasionally in free-ranging Ovis aries. B. venatorum was detected in the ubiquitous Capreolus capreolus, and occasionally in free-ranging O. aries. Species-specific PCR revealed co-infections in C. capreolus and C. elaphus, resulting in higher prevalence of B. venatorum and B. divergens than disclosed by qPCR detection, followed by 18S rDNA and COI sequencing. The non-zoonotic Babesia species found were Babesia capreoli, Babesia vulpes, Babesia sp. deer clade, and badger-associated Babesia species. The infection rate of zoonotic Babesia species in questing I. ricinus ticks was higher for Babesia clade I (2.6%) than Babesia clade X (1.9%). Co-infection of B. microti with Borrelia burgdorferi sensu lato and Neoehrlichia mikurensis in questing nymphs occurred more than expected, which reflects their mutual reservoir hosts, and suggests the possibility of co-transmission of these three pathogens to humans during a tick bite. The ubiquitous spread and abundance of B. microti and B. venatorum in their reservoir hosts and questing ticks imply some level of human exposure through tick bites. The restricted distribution of the wild reservoir hosts for B. divergens and its low infection rate in ticks might contribute to the absence of reported autochthonous cases of human babesiosis in the Netherlands.

Seasonal patterns and spatial variation of Borrelia burgdorferi (sensu lato) infections in Ixodes ricinus in the Netherlands.

BACKGROUND: The incidence of Lyme borreliosis varies over time and space through as yet incompletely understood mechanisms. In Europe, Lyme borreliosis is caused by infection with a Borrelia burgdorferi (s.l.) genospecies, which is primarily transmitted by a bite of Ixodes ricinus nymphs. The aim of this study was to investigate the spatial and temporal variation in nymphal infection prevalence of B. burgdorferi (s.l.) (NIP), density of questing nymphs (DON) and the resulting density of infected nymphs (DIN). METHODS: We investigated the infection rates in I. ricinus nymphs that were collected monthly between 2009 and 2016 in 12 locations in the Netherlands. Using generalized linear mixed models, we explored how the NIP, DON and DIN varied during the seasons, between years and between locations. We also determined the genospecies of the Borrelia infections and investigated whether the genospecies composition differed between locations. RESULTS: The overall NIP was 14.7%. A seasonal pattern in infection prevalence was observed, with higher estimated prevalences in the summer than in the spring and autumn. This, combined with higher nymphal densities in summer, resulted in a pronounced summer peak in the estimated DIN. Over the 7.5-year study period, a significant decrease in infection prevalence was found, as well as a significant increase in nymphal density. These two effects appear to cancel each other out; the density of infected nymphs, which is the product of NIP × DON, showed no significant trend over years. Mean infection prevalence (NIP, averaged over all years and all months) varied considerably between locations, ranging from 5 to 26%. Borrelia genospecies composition differed between locations: in some locations almost all infections consisted of B. afzelii, whereas other locations had more diverse genospecies compositions. CONCLUSION: In the Netherlands, the summer peak in DIN is a result of peaks in both NIP and DON. No significant trend in DIN was observed over the years of the study, and variations in DIN between locations were mostly a result of the variation in DON. There were considerable differences in acarological risk between areas in terms of infection prevalence and densities of ticks as well as in Borrelia genospecies composition.

The Role of Peridomestic Animals in the Eco-Epidemiology of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is an important tick-borne zoonotic agent of human granulocytic anaplasmosis (HGA). In Europe, the Ixodes ticks are the main vector responsible for A. phagocytophilum transmission. A wide range of wild animals is involved in the circulation of this pathogen in the environment. Changes in populations of vertebrates living in different ecosystems impact the ecology of ticks and the epidemiology of tick-borne diseases. In this study, we investigated four species, Western European hedgehog (Erinaceus europaeus), northern white-breasted hedgehog (Erinaceus roumanicus), Eurasian red squirrel (Sciurus vulgaris), and the common blackbird (Turdus merula), to describe their role in the circulation of A. phagocytophilum in urban and periurban ecosystems. Ten different tissues were collected from cadavers of the four species, and blood and ear/skin samples from live blackbirds and hedgehogs. Using qPCR, we detected a high rate of A. phagocytophilum: Western European hedgehogs (96.4%), northern white-breasted hedgehogs (92.9%), Eurasian red squirrels (60%), and common blackbirds (33.8%). In the groEL gene, we found nine genotypes belonging to three ecotypes; seven of the genotypes are associated with HGA symptoms. Our findings underline the role of peridomestic animals in the ecology of A. phagocytophilum and indicate that cadavers are an important source of material for monitoring zoonotic pathogens. Concerning the high prevalence rate, all investigated species play an important role in the circulation of A. phagocytophilum in municipal areas; however, hedgehogs present the greatest anaplasmosis risk for humans. Common blackbirds and squirrels carry different A. phagocytophilum variants some of which are responsible for HGA.

Hedgehogs, Squirrels, and Blackbirds as Sentinel Hosts for Active Surveillance of Borrelia miyamotoi and Borrelia burgdorferi Complex in Urban and Rural Environments.

Lyme borreliosis (LB), caused by spirochetes of the Borrelia burgdorferi sensu lato (s.l.) complex, is one of the most common vector-borne zoonotic diseases in Europe. Knowledge about the enzootic circulation of Borrelia pathogens between ticks and their vertebrate hosts is epidemiologically important and enables assessment of the health risk for the human population. In our project, we focused on the following vertebrate species: European hedgehog (Erinaceus europaeus), Northern white-breasted hedgehog (E. roumanicus), Eurasian red squirrel (Sciurus vulgaris), and Common blackbird (Turdus merula). The cadavers of accidentally killed animals used in this study constitute an available source of biological material, and we have confirmed its potential for wide monitoring of B. burgdorferi s.l. presence and genospecies diversity in the urban environment. High infection rates (90% for E. erinaceus, 73% for E. roumanicus, 91% for S. vulgaris, and 68% for T. merula) were observed in all four target host species; mixed infections by several genospecies were detected on the level of individuals, as well as in particular tissue samples. These findings show the usefulness of multiple tissue sampling as tool for revealing the occurrence of several genospecies within one animal and the risk of missing particular B. burgdorferi s.l. genospecies when looking in one organ alone.