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In large parts of the northern hemisphere, multiple deer species coexist, and management actions can strongly influence wild deer communities. Such changes may also indirectly influence other species in the community, such as small mammals and birds, because deer can have strong effects on their habitats and resources. Deer, small mammals and birds play an important role in the dynamics of tick-borne zoonotic diseases. It is, however, relatively underexplored how the abundance and composition of vertebrate communities may affect the outbreak potential, maintenance and circulation of tick-borne pathogens. In this study we focus on the outbreak potential by exploring how the basic reproduction number R 0 for different tick-borne pathogens depends on host community composition. We used published data on co-varying roe deer (Capreolus capreolus) and fallow deer (Dama dama) densities following a hunting ban, and different small mammal and bird densities, to investigate how the change in host community influences the R 0 of four tick-borne pathogens: one non-zoonotic, namely Anaplasma phagocytophilum ecotype 2, and three zoonotic, namely A. phagocytophilum ecotype 1, Borrelia afzelii and Borrelia garinii. We calculated R 0 using a next generation matrix approach, and used elasticities to quantify the contributions to R 0 of the different groups of host species. The value of R 0 for A. phagocytophilum ecotype 1 was higher with high fallow deer density and low roe deer density, while it was the other way round for A. phagocytophilum ecotype 2. For B. afzelii, R 0 was mostly related to the density of small mammals and for B. garinii it was mostly determined by bird density. Our results show that the effect of species composition is substantial in the outbreak potential of tick-borne pathogens. This implies that also management actions that change this composition, can (indirectly and unintentionally) affect the outbreak potential of tick-borne diseases., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier GmbH.)

The distribution of tick-borne encephalitis virus (TBEV) is expanding to Western European countries, including the Netherlands, but the contribution of different rodent species to the transmission of TBEV is poorly understood. We investigated whether two species of wild rodents native to the Netherlands, the wood mouse Apodemus sylvaticus and the yellow-necked mouse Apodemus flavicollis, differ in their relative susceptibility to experimental infection with TBEV. Wild-caught individuals were inoculated subcutaneously with the classical European subtype of TBEV (Neudoerfl) or with TBEV-NL, a genetically divergent TBEV strain from the Netherlands. Mice were euthanised and necropsied between 3 and 21 days post-inoculation. None of the mice showed clinical signs or died during the experimental period. Nevertheless, TBEV RNA was detected up to 21 days in the blood of both mouse species and TBEV was also isolated from the brain of some mice. Moreover, no differences in infection rates between virus strains and mouse species were found in blood, spleen, or liver samples. Our results suggest that the wood mouse and the yellow-necked mouse may equally contribute to the transmission cycle of TBEV in the Netherlands. Future experimental infection studies that include feeding ticks will help elucidate the relative importance of viraemic transmission in the epidemiology of TBEV., (© 2023. The Author(s).)

Background: Patients treated for Lyme borreliosis (LB) frequently report persistent symptoms. Little is known about risk factors and etiology., Methods: In a prospective observational cohort study with a follow-up of one year, we assessed a range of microbiological, immunological, genetic, clinical, functional, epidemiological, psychosocial and cognitive-behavioral variables as determinants of persistent symptoms after treatment for LB. Between 2015 and 2018 we included 1135 physician-confirmed LB patients at initiation of antibiotic therapy, through clinical LB centers and online self-registration. Two reference cohorts of individuals without LB (n = 4000 and n = 2405) served as a control. Prediction analyses and association studies were used to identify determinants, as collected from online questionnaires (three-monthly) and laboratory tests (twice)., Findings: Main predictors of persistent symptoms were baseline poorer physical and social functioning, higher depression and anxiety scores, more negative illness perceptions, comorbidity, as well as fatigue, cognitive impairment, and pain in 295 patients with persistent symptoms. The primary prediction model correctly indicated persistent symptoms in 71.0% of predictions (AUC 0.79). In patients with symptoms at baseline, cognitive-behavioral responses to symptoms predicted symptom persistence. Of various microbiological, immunological and genetic factors, only lower IL-10 concentrations in ex vivo stimulation experiments were associated with persistent symptoms. Clinical LB characteristics did not contribute to the prediction of persistent symptoms., Interpretation: Determinants of persistent symptoms after LB were mainly generic, including baseline functioning, symptoms and cognitive-behavioral responses. A potential role of host immune responses remains to be investigated., Funding: Netherlands Organisation for Health Research and Development (ZonMw); the Dutch Ministry of Health, Welfare and Sport (VWS)., Competing Interests: Declaration of interests MEB, FRvdS and JWH report collaborations on LB diagnostics with Pfizer, Antigen Discovery Inc., Oxford Immunotec, AID/GenID, and InVitaLab; funding to institution and/or material supplied for research free of charge, no personal financial compensation. MEB reports support for travel expenses related to LB symposium at Massachusetts General Hospital. LABJ and BJK are co-inventor of Spirofind, an experimental in-house assay for Lyme borreliosis, which is owned by Radboudumc and will not be developed further as potential diagnostic test. CCvdW is a consortium partner and partner in sub-project in the NMCB consortium (The Dutch ME/CFS Cohort and Biobank Consortium) funded by Netherlands Organization for Health Research and Development (ZonMw, project numbers 10091012110027 and 10091012110018). The other authors report no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (© 2023. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

BackgroundArthropod vectors such as ticks, mosquitoes, sandflies and biting midges are of public and veterinary health significance because of the pathogens they can transmit. Understanding their distributions is a key means of assessing risk. VectorNet maps their distribution in the EU and surrounding areas.AimWe aim to describe the methodology underlying VectorNet maps, encourage standardisation and evaluate output.Methods: Vector distribution and surveillance activity data have been collected since 2010 from a combination of literature searches, field-survey data by entomologist volunteers via a network facilitated for each participating country and expert validation. Data were collated by VectorNet members and extensively validated during data entry and mapping processes.ResultsAs of 2021, the VectorNet archive consisted of ca 475,000 records relating to > 330 species. Maps for 42 species are routinely produced online at subnational administrative unit resolution. On VectorNet maps, there are relatively few areas where surveillance has been recorded but there are no distribution data. Comparison with other continental databases, namely the Global Biodiversity Information Facility and VectorBase show that VectorNet has 5-10 times as many records overall, although three species are better represented in the other databases. In addition, VectorNet maps show where species are absent. VectorNet's impact as assessed by citations (ca 60 per year) and web statistics (58,000 views) is substantial and its maps are widely used as reference material by professionals and the public.ConclusionVectorNet maps are the pre-eminent source of rigorously validated arthropod vector maps for Europe and its surrounding areas.

Biodiversity loss and the emergence of zoonotic diseases are two major global challenges. An urgent question is how ecosystems and wildlife communities can be restored whilst minimizing the risk of zoonotic diseases carried by wildlife. Here, we evaluate how current ambitions to restore Europe's natural ecosystems may affect the hazard of diseases vectored by the tick Ixodes ricinus at different scales. We find that effects of restoration efforts on tick abundance are relatively straightforward but that the interacting effects of vertebrate diversity and abundance on pathogen transmission are insufficiently known. Long-term integrated surveillance of wildlife communities, ticks, and their pathogens is needed to understand their interactions and to prevent nature restoration from increasing tick-borne disease (TBD) hazard., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Tick-borne encephalitis (TBE) is a viral disease endemic in Eurasia. The virus is mainly transmitted to humans via ticks and occasionally via the consumption of unpasteurized milk products. The European Centre for Disease Prevention and Control reported an increase in TBE incidence over the past years in Europe as well as the emergence of the disease in new areas. To better understand this phenomenon, we investigated the drivers of TBE emergence and increase in incidence in humans through an expert knowledge elicitation. We listed 59 possible drivers grouped in eight domains and elicited forty European experts to: (i) allocate a score per driver, (ii) weight this score within each domain, and (iii) weight the different domains and attribute an uncertainty level per domain. An overall weighted score per driver was calculated, and drivers with comparable scores were grouped into three terminal nodes using a regression tree analysis. The drivers with the highest scores were: (i) changes in human behavior/activities; (ii) changes in eating habits or consumer demand; (iii) changes in the landscape; (iv) influence of humidity on the survival and transmission of the pathogen; (v) difficulty to control reservoir(s) and/or vector(s); (vi) influence of temperature on virus survival and transmission; (vii) number of wildlife compartments/groups acting as reservoirs or amplifying hosts; (viii) increase of autochthonous wild mammals; and (ix) number of tick species vectors and their distribution. Our results support researchers in prioritizing studies targeting the most relevant drivers of emergence and increasing TBE incidence.

Background: Tick-borne encephalitis virus (TBEV) can cause severe neurological disease in humans. Its geographical distribution is expanding in Western Europe with unresolved causes and spatial patterns, necessitating enhanced surveillance. Monitoring the virus in the environment is complicated, as it usually relies on destructive sampling of small rodents to test organs for TBEV, which in addition to ethical considerations also raises issues for long-term monitoring or longitudinal studies. Moreover, even when the virus is not detected in the blood or organs of the rodent, TBEV can still be transmitted from an infected tick to uninfected ticks feeding nearby. This is due to the ability of TBEV to replicate and migrate locally within the epidermis of small mammals, including those that do not appear to have systemic infection. This suggests that the virus may be detectable in skin biopsies, which has been confirmed in experimentally infected laboratory rodents, but it remains unknown if this sample type may be a viable alternative to destructively obtained samples in the monitoring of natural TBEV infection. Here we test ear tissue and dried blood spot (DBS) samples from rodents to determine whether TBEV-RNA can be detected in biological samples obtained non-destructively., Methods: Rodents were live-trapped and sampled at three woodland areas in The Netherlands where presence of TBEV has previously been recorded. Ear tissue (n = 79) and DBSs (n = 112) were collected from a total of 117 individuals and were tested for TBEV-RNA by real-time RT-PCR., Results: TBEV-RNA was detected in five rodents (4.3% of tested individuals), all of which had a TBEV-positive ear sample, while only two out of four of these individuals (for which a DBS was available) had a positive DBS. This equated to 6.3% of ear samples and 1.8% of DBSs testing positive for TBEV-RNA., Conclusions: We provide the first evidence to our knowledge that TBEV-RNA can be detected in samples obtained non-destructively from naturally infected wild rodents, providing a viable sampling alternative suitable for longitudinal surveillance of the virus., (© 2023. The Author(s).)

Tick-borne encephalitis virus (TBEV) may cause tick-borne encephalitis (TBE), a potential life-threatening infection of the central nervous system in humans. Phylogenetically, TBEVs can be subdivided into three main subtypes, which differ in endemic region and pathogenic potential. In 2016, TBEV was first detected in the Netherlands. One of two detected strains, referred to as Salland, belonged to the TBEV-Eu subtype, yet diverged ≥ 2% on amino acid level from other members of this subtype. Here, we report the successful rescue of this strain using infectious subgenomic amplicons and its subsequent in vitro characterization by comparison to two well-characterized TBEV-Eu strains; Neudoerfl and Hypr. In the human alveolar epithelial cell line A549, growth kinetics of Salland were comparable to the high pathogenicity TBEV-Eu strain Hypr, and both strains grew considerably faster than the mildly pathogenic strain Neudoerfl. In the human neuroblastoma cell line SK-N-SH, Salland replicated faster and to higher infectious titers than both reference strains. All three TBEV strains infected primary human monocyte-derived dendritic cells to a similar extent and interacted with the type I interferon system in a similar manner. The current study serves as the first in vitro characterization of the novel, divergent TBEV-Eu strain Salland., (© 2023. The Author(s).)

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., (Copyright © 2023 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Background: Hepatozoon spp. are tick-borne parasites causing subclinical to clinical disease in wild and domestic animals. Aim of this study was to determine Hepatozoon prevalence and species distribution among wild mammals and ticks in Europe., Methods: Samples of wild mammals and ticks, originating from Austria, Bosnia and Herzegovina, Croatia, Belgium and the Netherlands, were tested with PCR to amplify a ~ 670-bp fragment of the small subunit ribosomal RNA gene., Results: Of the 2801 mammal samples that were used for this study, 370 (13.2%) tested positive. Hepatozoon canis was detected in samples of 178 animals (3 Artiodactyla, 173 Carnivora, 1 Eulipotyphia, 1 Lagomorpha), H. martis in 125 (3 Artiodactyla, 122 Carnivora), H. sciuri in 13 (all Rodentia), Hepatozoon sp. in 47 (among which Hepatozoon sp. Vole isolate, all Rodentia) and H. ayorgbor in 4 (all Rodentia). Regarding origin, 2.9% (6/208) tested positive from Austria, 2.8% (1/36) from Bosnia and Herzegovina, 14.6% (173/1186) from Croatia and 13.9% (190/1371) from Belgium/the Netherlands. Of the 754 ticks collected, 0.0% (0/35) Hyalomma sp., 16.0% (4/25) Dermacentor spp., 0.0% (0/23) Haemaphysalis spp., 5.3% (24/50) Ixodes and 1.4% (3/221) Rhipicephalus spp. tested positive for Hepatozoon (4.2%; 32/754), most often H. canis (n = 22)., Conclusions: Hepatozoon canis is most present in mammals (especially in Carnivora such as gray wolves and golden jackals) and ticks, followed by H. martis, which was found merely in stone martens and pine martens. None of the rodent-associated Hepatozoon spp. were detected in the ticks, suggesting the possible implication of other arthropod species or non-vectorial routes in the transmission cycle of the hemoprotozoans in rodents. Our findings of H. canis in ticks other than R. sanguineus add to the observation that other ticks are also involved in the life cycle of Hepatozoon. Now that presence of Hepatozoon has been demonstrated in red foxes, gray wolves, mustelids and rodents from the Netherlands and/or Belgium, veterinary clinicians should be aware of the possibility of spill-over to domestic animals, such as dogs., (© 2023. The Author(s).)

An outbreak of disease in a Swedish beef cattle herd initiated an in-depth study to investigate the presence of bacteria and viruses in the blood of clinically healthy ( n = 10) and clinically diseased cattle ( n = 20) using whole-genome shotgun sequencing (WGSS). The occurrence of infectious agents was also investigated in ticks found attached to healthy cattle ( n = 61) and wild deer (n = 23), and in spleen samples from wild deer ( n = 30) and wild boars ( n = 10). Moreover, blood samples from 84 clinically healthy young stock were analysed for antibodies against Anaplasma phagocytophilum and Babesia divergens. The WGSS revealed the presence of at least three distinct Mycoplasma variants that were most closely related to Mycoplasma wenyonii . Two of these were very similar to a divergent M. wenyonii variant previously only detected in Mexico. These variants tended to be more common in the diseased cattle than in the healthy cattle but were not detected in the ticks or wild animals. The DNA of A. phagocytophilum was detected in similar proportions in diseased (33%) and healthy (40%) cattle, while 70% of the deer, 8% of ticks collected from the cattle and 19% of the ticks collected from deer were positive. Almost all the isolates from the cattle, deer and ticks belonged to Ecotype 1. Based on sequencing of the groEL-gene, most isolates of A. phagocytophilum from cattle were similar and belonged to a different cluster than the isolates from wild deer. Antibodies against A. phagocytophilum were detected in all the analysed samples. In conclusion, uncommon variants of Mycoplasma were detected, probably associated with the disease outbreak in combination with immune suppression due to granulocytic anaplasmosis. Moreover, A. phagocytophilum was found to be circulating within this cattle population, while circulation between cattle and deer occurred infrequently.

Background: Trypanosomes are protozoan parasites of vertebrates that are of medical and veterinary concern. A variety of blood-feeding invertebrates have been identified as vectors, but the role of ticks in trypanosome transmission remains unclear., Methods: In this study, we undertook extensive molecular screening for the presence and genetic diversity of trypanosomes in field ticks., Results: Examination of 1089 specimens belonging to 28 tick species from Europe and South America led to the identification of two new trypanosome strains. The prevalence may be as high as 4% in tick species such as the castor bean tick Ixodes ricinus, but we found no evidence of transovarial transmission. Further phylogenetic analyses based on 18S rRNA, EF1-α, hsp60 and hsp85 gene sequences revealed that different tick species, originating from different continents, often harbour phylogenetically related trypanosome strains and species. Most tick-associated trypanosomes cluster in a monophyletic clade, the Trypanosoma pestanai clade, distinct from clades of trypanosomes associated with transmission by other blood-feeding invertebrates., Conclusions: These observations suggest that ticks may be specific arthropod hosts for trypanosomes of the T. pestanai clade. Phylogenetic analyses provide further evidence that ticks may transmit these trypanosomes to a diversity of mammal species (including placental and marsupial species) on most continents., (© 2023. The Author(s).)

The European mole ( Talpa europaea ) has a widespread distribution throughout Europe. However, little is known about the presence of zoonotic pathogens in European moles. We therefore tested 180 moles from the middle and the south of the Netherlands by (q)PCR for the presence of multiple (tick-borne) zoonotic pathogens. Spotted fever Rickettsia was found in one (0.6%), Leptospira spp. in three (1.7%), Bartonella spp. in 69 (38.3%) and Hantaviridae in 89 (49.4%) of the 180 moles. Infections with Anaplasma phagocytophilum , Babesia spp., Neoehrlichia mikurensis , Borrelia spp., Spiroplasma spp. and Francisella tularensis were not found. In addition, in a subset of 35 moles no antibodies against Tick-borne encephalitis virus were found. The obtained sequences of Bartonella spp. were closely related to Bartonella spp. sequences from moles in Spain and Hungary. The Hantaviridae were identified as the mole-borne Nova virus, with high sequence similarity to sequences from other European countries, and Bruges virus. Though the zoonotic risk from moles appears limited, our results indicate that these animals do play a role in multiple host-pathogen cycles.

Tick-borne encephalitis virus (TBEV) is an emerging pathogen that was first detected in ticks and humans in the Netherlands in 2015 (ticks) and 2016 (humans). To learn more about its distribution and prevalence in the Netherlands, we conducted large-scale surveillance in ticks and rodents during August 2018-September 2020. We tested 320 wild rodents and >46,000 ticks from 48 locations considered to be at high risk for TBEV circulation. We found TBEV RNA in 3 rodents (0.9%) and 7 tick pools (minimum infection rate 0.02%) from 5 geographically distinct foci. Phylogenetic analyses indicated that 3 different variants of the TBEV-Eu subtype circulate in the Netherlands, suggesting multiple independent introductions. Combined with recent human cases outside known TBEV hotspots, our data demonstrate that the distribution of TBEV in the Netherlands is more widespread than previously thought.

Wild and domestic animals can be usefully employed as sentinels for the surveillance of diseases with an impact on public health. In the case of tick-borne encephalitis virus (TBEV), the detection of antibodies in animals can be more effective than screening ticks for detecting TBEV foci, due to the patchy distribution of the virus. In the Piedmont region, northwestern Italy, TBEV is considered absent, but an increase in tick densities, of Ixodes ricinus in particular, has been observed, and TBEV is spreading in bordering countries, e.g., Switzerland. Therefore, we collected sera from wild ungulates during the hunting season (October-December) from 2017 to 2019 in the Susa Valley, Italian western Alps, and screened them for TBEV antibodies by a commercial competitive ELISA test. We collected 267 serum samples by endocranial venous sinuses puncture from red deer, roe deer and northern chamois carcasses. The animals were hunted in 13 different municipalities, at altitudes ranging between 750 and 2800 m a.s.l. The serological survey for TBEV yielded negative results. Borderline results for five serum samples were further confirmed as negative for TBEV by a plaque reduction neutralisation test. To date, our results indicate that TBEV is not circulating in western Piedmont. However, monitoring of TBEV should continue since TBEV and its vector are spreading in Europe. The wide-range distribution of wild ungulates and their role as feeding hosts, make them useful indicators of the health threats posed by Ixodid ticks.

Background: The extent to which infections with Ixodes ricinus -borne pathogens (TBPs), other than Borrelia burgdorferi s. l. and tick-borne encephalitis virus (TBEV), cause disease in humans remains unclear. One of the reasons is that adequate diagnostic modalities are lacking in routine or research settings., Methods: We evaluated the analytical specificity, sensitivity and robustness of qPCR assays for the detection of Anaplasma phagocytophilum , Neoehrlichia mikurensis , Spiroplasma ixodetis , several Babesia species and Spotted Fever Rickettsia species as well as Bartonella species in human samples., Results: The qPCRs were found to perform well, given the difficulties of dealing with microorganisms for which confirmed patient materials are scarce or non-existent, a hurdle that was partially overcome by using synthetic controls. Spiking blood samples with the tested microorganisms showed that the detection of the TBPs was not inhibited by the presence of blood. The acceptable sensitivity when multiplexing the different pathogens, the good inter-assay variability and the absence of cross-reactivity make them potentially suitable as human diagnostics., Conclusions: The qPCRs evaluated in this study are technically suitable for the laboratory diagnostic assessment of clinical samples for infection with tick-borne pathogens. However, clinical validation and independent confirmation are still needed, pending the availability of sufficient human samples for testing in different laboratories.

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., (© 2022 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

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., (© 2022. The Author(s).)

Background: Various studies have evaluated the infection of Ixodes ticks and humans with the relapsing fever spirochaete Borrelia miyamotoi. However, to our knowledge, the prevalence of infection and disease has not been assessed systematically. We aimed to examine the prevalence of B miyamotoi in Ixodes ticks and humans, and the disease it can cause, in the northern hemisphere., Methods: For this systematic review and meta-analysis, we searched PubMed and Web of Science up to March 1, 2021. Studies assessing Ixodes tick infection published since Jan 1, 2011 were eligible, whereas no time limitation was placed on reports of human infection and disease. We extracted B miyamotoi test positivity ratios and used a random-effects model to calculate estimated proportions of infected ticks, infected humans, and human disease with 95% CI. This study was registered with PROSPERO, CRD42021268996., Findings: We identified 730 studies through database searches and 316 additional studies that referenced two seminal articles on B miyamotoi. Of these 1046 studies, 157 were included in the review, reporting on 165 637 questing ticks, 45 608 unique individuals, and 504 well described cases of B miyamotoi disease in humans. In ticks, the highest prevalence of B miyamotoi was observed in Ixodes persulcatus (2·8%, 95% CI 2·4-3·1) and the lowest in Ixodes pacificus (0·7%, 0·6-0·8). The overall seroprevalence in humans was 4·4% (2·8-6·3), with significantly (p<0·0001) higher seroprevalences in the high-risk group (4·6%, 2·6-7·1), participants with confirmed or suspected Lyme borreliosis (4·8%, 1·8-8·8), and individuals suspected of having a different tick-borne disease (11·9%, 5·6-19·9) than in healthy controls (1·3%, 0·4-2·8). Participants suspected of having a different tick-borne disease tested positive for B miyamotoi by PCR significantly more often than did the high-risk group (p=0·025), with individuals in Asia more likely to test positive than those in the USA (odds ratio 14·63 [95% CI 2·80-76·41])., Interpretation: B miyamotoi disease should be considered an emerging infectious disease, especially in North America and Asia. Prospective studies and increased awareness are required to obtain further insights into the burden of disease., Funding: ZonMW and the European Regional Development Fund (Interreg)., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

To identify ideal elements for the monitoring and prevention of tick-borne diseases (TBD), we analysed the surveillance systems in place in the Netherlands, Spain and Italy. We applied a semi-quantitative evaluation to identify outcomes and assess the degree of One Health implementation. Differences emerged in the surveillance initiatives, as well as the One Health scores. The Dutch surveillance is dominated by a high level of transdisciplinary and trans-sectoral collaboration, enabling communication and data sharing among actors. Different project-based monitoring, research and educational activities are centrally coordinated and the non-scientific community is actively involved. All this yielded measurable health outcomes. In Italy and Spain, TBD surveillance and reporting systems are based on compulsory notification. Law enforcement, alongside dedicated time and availability of economic resources, is fragmented and limited to the most severe health issues. Veterinary and human medicine are the most involved disciplines, with the first prevailing in some contexts. Stakeholders are marginally considered and collaborations limited to local initiatives. Research activities have mostly contributed to gaining knowledge on the distribution of tick vectors and discovery of new pathogens. Although all TBD surveillance plans comply with EU regulations, initiatives characterised by transdisciplinary collaboration may be more effective for the surveillance and prevention of TBD.

Following findings in Northern America of SARS-CoV-2 infections in white-tailed deer, there is concern of similar infections in European deer and their potential as reservoirs of SARS-CoV-2 including opportunities for the emergence of new variants. UK deer sera were collected in 2020-2021 from 6 species and a hybrid with 1748 tested using anti-spike and anti-nucleocapsid serology assays. No samples were positive on both assays nor by surrogate neutralization testing. There is no evidence that spill-over infections of SARS-CoV-2 occurred from the human population to UK deer or that SARS-CoV-2 has been circulating in UK deer (over the study period). Although it cannot be ruled out, study results indicate that spill-over infections followed by circulation of SARS-CoV-2 to the most common European deer species is small., (© 2022 Crown copyright. Transboundary and Emerging Diseases published by Wiley-VCH GmbH. This article is published with the permission of the Controller of HMSO and the Queen's Printer for Scotland.)

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., (© 2022. The Author(s).)

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., (© 2021. The Author(s).)

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., (Copyright © 2022. Published by Elsevier GmbH.)

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., (© 2022. The Author(s).)

Ca. Neoehrlichia mikurensis is widely prevalent in I. ricinus across Europe and has been associated with human disease. However, diagnostic modalities are limited, and much is still unknown about its biology. Here, we present the first complete Ca. Neoehrlichia mikurensis genomes directly derived from wildlife reservoir host tissues, using both long- and short-read sequencing technologies. This pragmatic approach provides an alternative to obtaining sufficient material from clinical cases, a difficult task for emerging infectious diseases, and to expensive and challenging bacterial isolation and culture methods. Both genomes exhibit a larger chromosome than the currently available Ca. Neoehrlichia mikurensis genomes and expand the ability to find new targets for the development of supportive laboratory diagnostics in the future. Moreover, this method could be utilized for other tick-borne pathogens that are difficult to culture.

Public and animal health authorities face many challenges in surveillance and control of vector-borne diseases. Those challenges are principally due to the multitude of interactions between vertebrate hosts, pathogens, and vectors in continuously changing environments. VectorNet, a joint project of the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) facilitates risk assessments of VBD threats through the collection, mapping and sharing of distribution data for ticks, mosquitoes, sand flies, and biting midges that are vectors of pathogens of importance to animal and/or human health in Europe. We describe the development and maintenance of this One Health network that celebrated its 10th anniversary in 2020 and the value of its most tangible outputs, the vector distribution maps, that are freely available online and its raw data on request. VectorNet encourages usage of these maps by health professionals and participation, sharing and usage of the raw data by the network and other experts in the science community. For the latter, a more complete technical description of the mapping procedure will be submitted elsewhere., Competing Interests: GH and CM are employed by AviaGis and FS by Francis Schaffner Consultancy/Mabritec AG, Riehen. RS is employed by Wageningen Academic Publishers. WW was employed by Environmental Research Group Oxford Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Braks, Schaffner, Medlock, Berriatua, Balenghien, Mihalca, Hendrickx, Marsboom, Van Bortel, Smallegange, Sprong, Gossner, Czwienczek, Dhollander, Briët and Wint.)

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., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

Background: A number of tick-borne pathogens circulate in the Belgian tick population in addition to the causative agent of Lyme borreliosis. However, so far, only a few patients with tick-borne diseases other than Lyme borreliosis have been reported in Belgium. The aim of this study was to investigate the occurrence of other human tick-borne infections in Belgium and their possible clinical manifestation., Methods: Patients with fever (> 37.5 °C) after a tick bite or those with erythema migrans (EM) were included in the study. EDTA-blood samples were screened for the presence of DNA from Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Anaplasma phagocytophilum, Neoehrlichia mikurensis, spotted fever group rickettsiae (genus Rickettsia), Babesia spp., Bartonella spp., Spiroplasma ixodetis and tick-borne encephalitis virus, using multiplex PCR methods. A questionnaire on, among others, demographics and clinical symptoms, was also filled in., Results: Over a period of 3 years, 119 patients with EM and 14 patients with fever after a recent tick bite were enrolled in the study. Three samples initially tested positive for N. mikurensis by quantitative PCR (qPCR), but the results could not be confirmed by other PCR methods, and repetition of the DNA extraction procedure and qPCR test was not successful. The qPCR test results for the other tick-borne pathogens were negative., Conclusions: In general, only a few patients with fever after a tick bite could be identified. Although no tick-borne pathogens were detected, their occurrence cannot be excluded based on the limited number of patients and the limitations inherent to current methodologies. This study underscores the possibility of false-positive PCR results and the necessity for the development of multiple independent tools for the sensitive and specific detection of emerging tick-borne pathogens., (© 2022. The Author(s).)

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., (© 2021 Wiley-VCH GmbH.)

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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

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., (© 2021. The Author(s).)

Background: Several ungulate species are feeding and propagation hosts for the tick Ixodes ricinus as well as hosts to a wide range of zoonotic pathogens. Here, we focus on Anaplasma phagocytophilum and Borrelia burgdorferi (s.l.), two important pathogens for which ungulates are amplifying and dilution hosts, respectively. Ungulate management is one of the main tools to mitigate human health risks associated with these tick-borne pathogens. Across Europe, different species of ungulates are expanding their ranges and increasing in numbers. It is currently unclear if and how the relative contribution to the life-cycle of I. ricinus and the transmission cycles of tick-borne pathogens differ among these species. In this study, we aimed to identify these relative contributions for five European ungulate species., Methods: We quantified the tick load and collected ticks and spleen samples from hunted fallow deer (Dama dama, n = 131), moose (Alces alces, n = 15), red deer (Cervus elaphus, n = 61), roe deer (Capreolus capreolus, n = 30) and wild boar (Sus scrofa, n = 87) in south-central Sweden. We investigated the presence of tick-borne pathogens in ticks and spleen samples using real-time PCR. We determined if ungulate species differed in tick load (prevalence and intensity) and in infection prevalence in their tissue as well as in the ticks feeding on them., Results: Wild boar hosted fewer adult female ticks than any of the deer species, indicating that deer are more important as propagation hosts. Among the deer species, moose had the lowest number of female ticks, while there was no difference among the other deer species. Given the low number of infected nymphs, the relative contribution of all ungulate species to the transmission of B. burgdorferi (s.l.) was low. Fallow deer, red deer and roe deer contributed more to the transmission of A. phagocytophilum than wild boar., Conclusions: The ungulate species clearly differed in their role as a propagation host and in the transmission of B. burgdorferi and A. phagocytophilum. This study provides crucial information for ungulate management as a tool to mitigate zoonotic disease risk and argues for adapting management approaches to the local ungulate species composition and the pathogen(s) of concern., (© 2021. The Author(s).)

An understanding of the relationships of ticks and tick-borne pathogens can only be achieved by integrating data from multiple studies. The publication of raw material is a necessary step for wide-area meta-analyses and study design, data collection and reporting require harmonization. This is an opinion paper, not a consensus position, and is open to debate. This work reflects our view about how data should be communicated in mainstream journals. We indicate rules that should be observed in recording weather data, to avoid serendipitous correlations between the density of ticks and climate variables and recommend the inclusion of raw data in reports. We stress the need for standardized methods to collect ticks that cannot be obtained by standard flagging. The reporting of infection rates of pathogens in ticks should avoid conclusions based on pure molecular findings in feeding ticks. Studies demonstrating the vectorial capacity of ticks should not be supported only by molecular surveys of feeding ticks. Vacuous conclusions about vectorial or reservoir status based solely on the finding of genomic material of a pathogen should be discouraged. We stress that phylogenetic studies based on random selection of sequences from GenBank are unsuitable. We firmly support the development of a dedicated server of curated sequences of ticks and pathogens as a standard for future studies.

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.

Background: Tick-borne pathogens other than Borrelia burgdorferi sensu lato - the causative agent of Lyme borreliosis - are common in Ixodes ricinus ticks. How often these pathogens cause human disease is unknown. In addition, diagnostic tools to identify such diseases are lacking or reserved to research laboratories. To elucidate their prevalence and disease burden, the study 'Ticking on Pandora's Box' has been initiated, a collaborative effort between Amsterdam University Medical Center and the National Institute for Public Health and the Environment., Methods: The study investigates how often the tick-borne pathogens Anaplasma phagocytophilum, Babesia species, Borrelia miyamotoi, Neoehrlichia mikurensis, spotted fever group Rickettsia species and/or tick-borne encephalitis virus cause an acute febrile illness after tick-bite. We aim to determine the impact and severity of these tick-borne diseases in the Netherlands by measuring their prevalence and describing their clinical picture and course of disease. The study is designed as a prospective case-control study. We aim to include 150 cases - individuals clinically suspected of a tick-borne disease - and 3 matched healthy control groups of 200 persons each. The controls consist respectively of a group of individuals with either a tick-bite without complaints, the general population and of healthy blood donors. During a one-year follow-up we will acquire blood, urine and skin biopsy samples and ticks at baseline, 4 and 12 weeks. Additionally, participants answer modified versions of validated questionnaires to assess self-reported symptoms, among which the SF-36, on a 3 monthly basis., Discussion: This article describes the background and design of the study protocol of 'Ticking on Pandora's Box'. With our study we hope to provide insight into the prevalence, clinical presentation and disease burden of the tick-borne diseases anaplasmosis, babesiosis, B. miyamotoi disease, neoehrlichiosis, rickettsiosis and tick-borne encephalitis and to assist in test development as well as provide recommendations for national guidelines., Trial Registration: NL9258 (retrospectively registered at Netherlands Trial Register, trialregister.nl in in February 2021).

Background: Ticks of the genus Hyalomma, which are vectors for several tick-borne diseases, are occasionally found in areas outside their endemic range including northern parts of Europe. The objective of this study was to analyse adult Hyalomma ticks that were recently found in the Netherlands., Methods: Hyalomma ticks were morphologically identified. Cluster analysis, based upon sequence data (cox1 barcoding) for molecular identification, and pathogen detection were performed. Additionally, a cross-sectional survey of horses was conducted to actively search for Hyalomma ticks in summer 2019. Analysis of temperature was done to assess the possibility of (i) introduced engorged nymphs moulting to adults and (ii) establishment of populations in the Netherlands., Results: Seventeen adult Hyalomma ticks (one in 2018, eleven in 2019, five in 2020) were found by citizens and reported. Fifteen ticks were detected on horses and two on humans. Twelve were identified as H. marginatum, one as H. rufipes and four, of which only photographic images were available, as Hyalomma sp. No Crimean-Congo haemorrhagic fever virus or Babesia/Theileria parasites were detected. One adult tick tested positive for Rickettsia aeschlimannii. In the cross-sectional horse survey, no Hyalomma ticks were found. Analysis of temperatures showed that engorged nymphs arriving on migratory birds in spring were able to moult to adults in 2019 and 2020, and that cumulative daily temperatures in the Netherlands were lower than in areas with established H. marginatum populations., Conclusions: Our results show that Hyalomma ticks are regularly introduced in the Netherlands as nymphs. Under the Dutch weather conditions, these nymphs are able to develop to the adult stage, which can be sighted by vigilant citizens. Only one human pathogen, Rickettsia aeschlimannii, was found in one of the ticks. The risk of introduction of tick-borne diseases via Hyalomma ticks on migratory birds is considered to be low. Establishment of permanent Hyalomma populations is considered unlikely under the current Dutch climatic conditions.

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 . S pecies-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.

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.

Background: The density of Ixodes ricinus nymphs infected with Anaplasma phagocytophilum is one of the parameters that determines the risk for humans and domesticated animals to contract anaplasmosis. For this, I. ricinus larvae need to take a bloodmeal from free-ranging ungulates, which are competent hosts for A. phagocytophilum., Methods: Here, we compared the contribution of four free-ranging ungulate species, red deer (Cervus elaphus), fallow deer (Dama dama), roe deer (Capreolus capreolus), and wild boar (Sus scrofa), to A. phagocytophilum infections in nymphs. We used a combination of camera and live trapping to quantify the relative availability of vertebrate hosts to questing ticks in 19 Dutch forest sites. Additionally, we collected questing I. ricinus nymphs and tested these for the presence of A. phagocytophilum. Furthermore, we explored two potential mechanisms that could explain differences between species: (i) differences in larval burden, which we based on data from published studies, and (ii) differences in associations with other, non-competent hosts., Results: Principal component analysis indicated that the density of A. phagocytophilum-infected nymphs (DIN) was higher in forest sites with high availability of red and fallow deer, and to a lesser degree roe deer. Initial results suggest that these differences are not a result of differences in larval burden, but rather differences in associations with other species or other ecological factors., Conclusions: These results indicate that the risk for contracting anaplasmosis in The Netherlands is likely highest in the few areas where red and fallow deer are present. Future studies are needed to explore the mechanisms behind this association.

Objectives: A single dose of doxycycline after a tick bite can prevent the development of Lyme borreliosis in North America, but extrapolation to Europe is hampered by differences in Borrelia burgdorferi sensu lato genospecies and tick species. We assessed the efficacy of prophylaxis after a tick bite in Europe., Methods: We conducted an open-label randomized controlled trial, administering a single dose of 200 mg doxycycline within 72 h after removing an attached tick from the skin, compared to no treatment. Potential participants ≥ 8 years of age who reported a recent tick bite online were invited for the study. After informed consent, they were randomly assigned to either the prophylaxis or the no-treatment group. Participants in the prophylaxis group were asked to visit their general practitioner to administer the antibiotics. All participants were followed up by online questionnaires. Our primary outcome was the development of physician-confirmed Lyme borreliosis in a modified-intention-to-treat analysis. This study is registered in the Netherlands Trial Register (NTR3953) and is closed., Results: Between April 11, 2013, and June 10, 2015, 3538 potential participants were randomized, of whom 1689 were included in the modified-intention-to-treat analysis. 10 cases of Lyme borreliosis were reported out of 1041 participants (0.96%) in the prophylaxis group, and 19 cases out of 648 no-treatment participants (2.9%), resulting in a relative risk reduction of 67% (95% CI 31 - 84%), and a number-needed-to-treat of 51 (95% CI 29 - 180). No serious adverse events were reported., Conclusions: This primary care-based trial provides evidence that a single dose of doxycycline can prevent the development of Lyme borreliosis after an Ixodes ricinus tick bite., Competing Interests: Declaration of Competing Interest We declare no competing interests., (Copyright © 2020 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

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.

Ixodes ricinus is the vector for Borrelia afzelii, the predominant cause of Lyme borreliosis in Europe, whereas Ixodes scapularis is the vector for Borrelia burgdorferi in the USA. Transcription of several I. scapularis genes changes in the presence of B. burgdorferi and contributes to successful infection. To what extend B. afzelii influences gene expression in I. ricinus salivary glands is largely unknown. Therefore, we measured expression of uninfected vs. infected tick salivary gland genes during tick feeding using Massive Analysis of cDNA Ends (MACE) and RNAseq, quantifying 26.179 unique transcripts. While tick feeding was the main differentiator, B. afzelii infection significantly affected expression of hundreds of transcripts, including 465 transcripts after 24 h of tick feeding. Validation of the top-20 B. afzelii-upregulated transcripts at 24 h of tick feeding in ten biological genetic distinct replicates showed that expression varied extensively. Three transcripts could be validated, a basic tail protein, a lipocalin and an ixodegrin, and might be involved in B. afzelii transmission. However, vaccination with recombinant forms of these proteins only marginally altered B. afzelii infection in I. ricinus-challenged mice for one of the proteins. Collectively, our data show that identification of tick salivary genes upregulated in the presence of pathogens could serve to identify potential pathogen-blocking vaccine candidates.

In mountain areas of northwestern Italy, ticks were rarely collected in the past. In recent years, a marked increase in tick abundance has been observed in several Alpine valleys, together with more frequent reports of Lyme borreliosis. We then carried out a four-year study to assess the distribution and abundance of ticks and transmitted pathogens and determine their altitudinal limit in a natural park area in Piedmont region. Ixodes ricinus and Dermacentor marginatus were collected from both the vegetation and hunted wild ungulates. Tick abundance was significantly associated with altitude, habitat type and signs of animal presence, roe deer's in particular. Ixodes ricinus prevailed in distribution and abundance and, although their numbers decreased with increasing altitude, we recorded the presence of all active life stages of up to around 1700 m a.s.l., with conifers as the second most infested habitat after deciduous woods. Molecular analyses demonstrated the infection of questing I. ricinus nymphs with B. burgdorferi sensu lato (15.5 %), Rickettsia helvetica and R. monacensis (20.7 %), Anaplasma phagocytophilum (1.9 %), Borrelia miyamotoi (0.5 %) and Neoehrlichia mikurensis (0.5 %). One third of the questing D. marginatus were infected with R. slovaca. We observed a spatial aggregation of study sites infested by B. burgdorferi s.l. infected ticks below 1400 m. Borrelia-infected nymphs prevailed in open areas, while SFG rickettsiae prevalence was higher in coniferous and deciduous woods. Interestingly, prevalence of SFG rickettsiae in ticks doubled above 1400 m, and R. helvetica was the only pathogen detected above 1800 m a.s.l. Tick infestation on hunted wild ungulates indicated the persistence of tick activity during winter months and, when compared to past studies, confirmed the recent spread of I. ricinus in the area. Our study provides new insights into the population dynamics of ticks in the Alps and confirms a further expansion of ticks to higher altitudes in Europe. We underline the importance of adopting a multidisciplinary approach in order to develop effective strategies for the surveillance of tick-borne diseases, and inform the public about the hazard posed by ticks, especially in recently invaded areas., Competing Interests: Declaration of Competing Interest All authors declare the absence of any financial or personal interests that could inappropriately influence the current work. The final article has been approved by all authors., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Rhipicephalus ticks are competent vectors of several pathogens, such as Spotted Fever Group Rickettsiae (SFGR) and many Babesia species. Within this genus, different R. sanguineus s.l. lineages show an unequal vector competence and resistance regarding some pathogenic strains. Current literature supports that tick endosymbionts may play an essential role in the transmission ability of a vector. Indeed, the microbial community of Rhipicephalus seems to be dominated by Coxiella-like endosymbionts (CLE). Still, their co-evolutionary associations with the complicated phylogeny of Rhipicephalus lineages and their transmissible pathogens remain unclear. We performed a phylogenetic congruence analysis to address whether divergent R. sanguineus s.l. lineages had a different symbiont composition. For that, we applied a PCR based approach to screen part of the microbial community present in 279 Rhipicephalus ticks from the Iberian Peninsula and Africa. Our analyses detected several qPCR-positive signals for both SFGR and Babesia species, of which we suggest R. sanguineus-tropical lineage as a natural vector of Babesia vogeli and R. sanguineus-temperate lineage of SFGR. The acquisition of 190 CLE sequences allowed to evaluate co-phylogenetic associations between the tick and the symbiont. With this data, we observed a strong but incomplete co-cladogenesis between CLE strains and their Rhipicephalus tick lineages hosts., (© FEMS 2020.)

For the development of sustainable control of tick-borne diseases, insight is needed in biological factors that affect tick populations. Here, the ecological interactions among Ixodiphagus hookeri , Ixodes ricinus , and two vertebrate species groups were investigated in relation to their effects on tick-borne disease risk. In 1129 questing ticks, I. hookeri DNA was detected more often in I. ricinus nymphs (4.4%) than in larvae (0.5%) and not in adults. Therefore, we determined the infestation rate of I. hookeri in nymphs from 19 forest sites, where vertebrate, tick, and tick-borne pathogen communities had been previously quantified. We found higher than expected co-occurrence rates of I. hookeri with deer-associated Anaplasma phagocytophilum , and lower than expected rates with rodent-associated Borrelia afzelii and Neoehrlichia mikurensis . The prevalence of I. hookeri in nymphs varied between 0% and 16% and was positively correlated with the encounter probability of ungulates and the densities of all life stages of I. ricinus . Lastly, we investigated the emergence of I. hookeri from artificially fed, field-collected nymphs. Adult wasps emerged from seven of the 172 fed nymphs. From these observations, we inferred that I. hookeri is parasitizing I. ricinus larvae that are feeding on deer, rather than on rodents or in the vegetation. Since I. hookeri populations depend on deer abundance, the main propagation host of I. ricinus , these wasps have no apparent effect on tick populations. The presence of I. hookeri may directly interfere with the transmission cycle of A. phagocytophilum , but not with that of B. afzelii or N. mikurensis .