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Abstract 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).

Substantial exposure to Borrelia miyamotoi occurs through bites from Ixodes ricinus ticks in the Netherlands, which also transmit Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum. Direct evidence for B. miyamotoi infection in European populations is scarce. A flu-like illness with high fever, resembling human granulocytic anaplasmosis, has been attributed to B. miyamotoi infections in relatively small groups. Borrelia miyamotoi infections associated with chronic meningoencephalitis have also been described in case reports. Assuming that an IgG antibody response against B. miyamotoi antigens reflects (endured) infection, the seroprevalence in different risk groups was examined. Sera from nine out of ten confirmed B. miyamotoi infections from Russia were found to be positive with the recombinant antigen used, and no significant cross-reactivity was observed in secondary syphilis patients. The seroprevalence in blood donors was set at 2.0% (95% CI 0.4–5.7%). Elevated seroprevalences in individuals with serologically confirmed, 7.4% (2.0–17.9%), or unconfirmed, 8.6% (1.8–23%), Lyme neuroborreliosis were not significantly different from those in blood donors. The prevalence of anti-B. miyamotoi antibodies among forestry workers was 10% (5.3–16.8%) and in patients with serologically unconfirmed but suspected human granulocytic anaplasmosis was 14.6% (9.0–21.8%); these were significantly higher compared with the seroprevalence in blood donors. Our findings indicate that infections with B. miyamotoi occur in tick-exposed individuals in the Netherlands. In addition, B. miyamotoi infections should be considered in patients reporting tick bites and febrile illness with unresolved aetiology in the Netherlands, and other countries where I. ricinus ticks are endemic.

We report here one new, hospitalized case of Anaplasma phagocytophilum in Belgium. The clinical presentation of anaplasmosis, its treatment and the molecular and serological relevant laboratory methods are briefly developed.

Background . In the northern hemisphere, ticks of the Ixodidae family are vectors of diseases such as Lyme borreliosis, Rocky Mountain spotted fever and tick-borne encephalitis. Most of these ticks are generalists and have a three-host life cycle for which they are dependent on three different hosts for their blood meal. Finding out which host species contribute most in maintaining ticks and the pathogens they transmit, is imperative in understanding the drivers behind the dynamics of a disease. Methods. We performed a systematic review to identify the most important vertebrate host species for Ixodes ricinus and Borrelia burgdorferi s.l. as a well-studied model system for tick-borne diseases. We analyzed data from 66 publications and quantified the relative contribution for 15 host species. Review results. We found a positive correlation between host body mass and tick burdens for the different stages of I. ricinus . We show that nymphal burdens of host species are positively correlated with infection prevalence with B. burgdorferi s.l., which is again positively correlated with the realized reservoir competence of a host species for B. burgdorferi s.l. Our quantification method suggests that only a few host species, which are amongst the most widespread species in the environment (rodents, thrushes and deer), feed the majority of I. ricinus individuals and that rodents infect the majority of I. ricinus larvae with B. burgdorferi s.l. Discussion. We argue that small mammal-transmitted Borrelia spp. are maintained due to the high density of their reservoir hosts, while bird-transmitted Borrelia spp. are maintained due to the high infection prevalence of their reservoir hosts. Our findings suggest that Ixodes ricinus and Borrelia burgdorferi s.l. populations are maintained by a few widespread host species. The increase in distribution and abundance of these species, could be the cause for the increase in Lyme borreliosis incidence in Europe in recent decades.

Rab4 regulates recycling from early endosomes. We investigated the role of the rab4 effector rabaptin-5α and its putative partner γ1-adaptin in membrane recycling. We found that rabaptin-5α forms a ternary complex with the γ1-σ1 subcomplex of AP-1, via a direct interaction with the γ1-subunit. The binding site for γ1-adaptin is in the hinge region of rabaptin-5α, which is distinct from rab4- and rab5-binding domains. Endogenous or ectopically expressed γ1- adaptin localized to both the trans-Golgi network and endosomes. Co-expressed rabaptin-5α and γ1-adaptin, however, co-localized in a rab4-dependent manner on recycling endosomes. Transfection of rabaptin-5α caused enlarged endosomes and delayed recycling of transferrin. RNAi of rab4 had an opposing effect on transferrin recycling. Collectively, our data show that rab4-GTP acts as a scaffold for a rabaptin-5α- γ1-adaptin complex on recycling endosomes and that interactions between rab4, rabaptin-5α and γ1-adaptin regulate membrane recycling. [ABSTRACT FROM AUTHOR]

Tick-borne encephalitis virus (TBEV) is an emerging pathogen in the Netherlands. Multiple divergent viral strains are circulating and the focal distribution of TBEV remains poorly understood. This may, however, be explained by differences in the susceptibility of tick populations for specific viruses and viral strains, and by viral strains having higher infection success in their local tick population. We investigated this hypothesis by exposing Dutch Ixodes ricinus ticks to two different TBEV strains: TBEV-NL from the Netherlands and TBEV-Neudoerfl from Austria. In addition, we exposed ticks to louping Ill virus (LIV), which is endemic to large parts of the United Kingdom and Ireland, but has not been reported in the Netherlands. Ticks were collected from two locations in the Netherlands: one location without evidence of TBEV circulation and one location endemic for the TBEV-NL strain. Ticks were infected in a biosafety level 3 laboratory using an artificial membrane feeding system. Ticks collected from the region without evidence of TBEV circulation had lower infection rates for TBEV-NL as compared to TBEV-Neudoerfl. Vice versa , ticks collected from the TBEV-NL endemic region had higher infection rates for TBEV-NL compared to TBEV-Neudoerfl. In addition, LIV infection rates were much lower in Dutch ticks compared to TBEV, which may explain why LIV is not present in the Netherlands. Our findings show that ticks from two distinct geographical populations differ in their susceptibility to TBEV strains, which could be the result of differences in the genetic background of the tick populations.

Endosymbiosis-where a microbe lives and replicates within a host-is an important contributor to organismal function that has accelerated evolutionary innovations and catalyzed the evolution of complex life. The evolutionary processes associated with transitions to endosymbiosis, however, are poorly understood. Here, we leverage the wide diversity of host-associated lifestyles of the genus Arsenophonus to reveal the complex evolutionary processes that occur during the transition to a vertically transmitted endosymbiotic lifestyle from strains maintained solely by horizontal (infectious) transmission. We compared the genomes of 38 strains spanning diverse lifestyles from horizontally transmitted pathogens to obligate interdependent endosymbionts. Among culturable strains, we observed those with vertical transmission had larger genome sizes than closely related horizontally transmitting counterparts, consistent with evolutionary innovation and the rapid gain of new functions. Increased genome size was a consequence of prophage and plasmid acquisition, including a cargo of type III effectors, alongside the concomitant loss of CRISPR-Cas genome defense systems, enabling mobile genetic element expansion. Persistent endosymbiosis was also associated with loss of type VI secretion, which we hypothesize to be a consequence of reduced microbe-microbe competition. Thereafter, the transition to endosymbiosis with strict vertical inheritance was associated with the expected relaxation of purifying selection, gene pseudogenization, metabolic degradation, and genome reduction. We argue that reduced phage predation in endosymbiotic niches drives the loss of genome defense systems driving rapid genome expansion upon the adoption of endosymbiosis and vertical transmission. This remodeling enables rapid horizontal gene transfer-mediated evolutionary innovation and precedes the reductive evolution traditionally associated with adaptation to endosymbiosis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Crimean-Congo haemorrhagic fever (CCHF) is a widely distributed and potentially fatal tick-borne viral disease with no licensed specific treatments or vaccines. In 2019, WHO published an advanced draft of a research and development roadmap for CCHF that prioritised the development and deployment of the medical countermeasures most needed by CCHF-affected countries. This Personal View presents updated CCHF research and development priorities and is the product of broad consultation with a working group of 20 leading experts in 2023-24. The strategic goals, milestones, and timelines have been revised and expanded to reflect scientific advances since 2019, including the identification of antibodies with therapeutic potential and the progression of four vaccine candidates through phase 1 clinical trials. This update emphasises the need for a One Health approach to manage CCHF, from integrated cross-sectoral surveillance to novel interventions that target ticks and their vertebrate hosts to reduce CCHF virus transmission to humans. The overarching vision for rapid diagnostics and specific therapeutics by 2028, followed by options to limit CCHF virus transmission and control disease by 2030, is deliberately ambitious and will only be achieved through coordinated international action from affected countries, funders, scientists, product developers, manufacturers, regulators, national authorities, and policy makers., Competing Interests: Declaration of interests JF declares a grant from the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación, Spain (grant number MCIN/AEI/10.13039/501100011033) and the EU–European Regional Development Fund (grant number BIOGAL PID2020–116761GB-I00). JF has also received support from the Federal University of Uberlândia and the Brazilian Federal Foundation for Support and Evaluation of Graduate Education (grant number 88887.936876/2024–00) to attend the 2024 Annual Scientific Meeting of the European Veterinary Parasitology College at the University of Agronomic Sciences and Veterinary Medicine, Bucharest, Romania. TF declares funding (paid to his institution) from GuardRX (not-for-profit) and the Pandemic Institute (Liverpool) for the UMIT-1 phase 1b Crimean–Congo haemorrhagic fever (CCHF) therapeutics trial in Türkiye; from the Medical Research Council (MRC) for the UMIT-2 phase 2b CCHF therapeutics trial in Türkiye and Iraq; from the MRC for the GeneDrive PCR platform and for development of a CCHF lateral flow test. SG has received grants (paid to his institution) from the German Research Foundation, European Commission, German Government, Coalition for Epidemic Preparedness Innovations (CEPI), and Kirmser Foundation; and has received speaker honoraria and support for attending meetings from WHO and CEPI. RH is in receipt of grant funding from the Biotechnology and Biological Sciences Research Council, the Defense Threat Reduction Agency, and the MRC to investigate CCHF virus genome reassortment, sequence CCHF virus variants, and develop diagnostics for low-resource settings; and is an investigator in the EU H2020-funded CCHFVaccine project. RH is a named inventor on a patent (PCT/GB2013/0503174; WO 2014/132013) for a vectored CCHF vaccine. GKob received federal funding from the Canadian Institute for Health Research to develop immunogens based on CCHF glycoproteins (2016–21), is an inventor on a patent for CCHF virus immunogenic compositions, and received funding from Université Laval and GUARDRx (not-for-profit) to attend meetings related to the original CCHF Research and Development (R&D) Roadmap (2018–20). AC, PCF, PJH, and JPG worked at the Wellcome Trust during the time that the CCHF Roadmap was being revised and this Personal View was written. TJGB, AES, and JW received support from WHO to attend meetings related to the original CCHF R&D Roadmap (2018–20). TJGB, AES, JW, and JO received funding from the Wellcome Trust (grant number 226729/Z/22/Z) to coordinate the 2023–24 update to the CCHF R&D Roadmap and to complete this Personal View. In addition, the Wellcome Trust covered convening costs for TJGB, AES, JW, VB, M-PP, LHB, TF, PBHF, SG, RH, BJ, JF, GP, AP, and HS to attend a meeting of the CCHF working group in London, UK (Feb 13–14, 2023). All other authors declare no competing interests. The authors alone are responsible for the views expressed in this Personal View; they do not necessarily represent the views, decisions, or policies of the institutions with which the authors are affiliated. Editorial note: The Lancet Group takes a neutral position with respect to territorial claims in published maps and institutional affiliations., (Crown Copyright © 2024 Published by Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

The taxa Ixodes ricinus and Ixodes inopinatus are sympatric in Tunisia. The genetics underlying their morphological differences are unresolved. In this study, ticks collected in Jouza-Amdoun, Tunisia, were morphologically identified and sequenced using Oxford Nanopore Technologies. Three complete genome assemblies of I. inopinatus and three of I. ricinus with BUSCO scores of ∼98% were generated, including the reconstruction of mitochondrial genomes and separation of both alleles of the TRPA1, TROSPA and calreticulin genes. Deep sequencing allowed the first descriptions of complete bacterial genomes for " Candidatus Midichloria mitochondrii", Rickettsia helvetica and R. monacensis from North Africa, and the discovery of extensive integration of parts of the Spiroplasma ixodetis and " Ca . M. mitochondrii" into the nuclear genome of these ticks. Phylogenetic analyses of the mitochondrial genome, the nuclear genes, and symbionts showed differentiation between Tunisian and Dutch ticks, but high genetic similarities between Tunisian I. ricinus and I. inopinatus . Subtraction of the genome assemblies identified the presence of some unique sequences, which could not be confirmed when screening a larger batch of I. ricinus and I. inopinatus ticks using PCR. Our findings yield compelling evidence that I. inopinatus is genetically highly similar, if not identical, to sympatric I. ricinus. Defined morphological differences might be caused by extrinsic factors such as micro-climatic conditions or bloodmeal composition. Our findings support the existence of different lineages of I. ricinus as well of its symbionts/pathogens from geographically dispersed locations., Competing Interests: 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., (© 2024 The Authors.)

Ixodes ricinus is the main vector of the causative agents of Lyme neuroborreliosis. This tick species can also transmit tick-borne encephalitis virus (TBEV), spotted fever group (SFG) Rickettsia and Borrelia miyamotoi to humans. These tick-borne pathogens are present in Dutch ticks and have also been associated with human neurological infections, but well characterized disease cases are seldom reported. We therefore assessed the evidence for TBEV, SFG Rickettsia or B. miyamotoi infection in clinically well-described patients suspected of Lyme neuroborreliosis. We retrospectively included patients with specific predefined clinical criteria from patients that were tested for Lyme neuroborreliosis between 2010 and 2014 at an academic Lyme borreliosis Center. Serology was performed on available serum samples, and cerebrospinal fluid (CSF) was tested by molecular methods. Out of 514 potentially eligible patients, 176 individual patients were included. None of CSF samples was positive for the tested tick-borne pathogens, except for one previously described patient with Borrelia miyamotoi disease (BMD). Serology revealed 27, 14 and three patients with antibodies against SFG Rickettsia, B. miyamotoi and TBEV, respectively. No distinctive clinical symptoms or signs could be associated with seropositivity against any of these tick-borne pathogens. Apart from the previously published BMD case, we were unable to find convincing evidence of new cases of tick-borne encephalitis, spotted fever rickettsiosis or BMD in a cohort of patients suspected of Lyme neuroborreliosis. While antibodies against these tick-borne pathogens were detected, we could not associate these findings to clinical symptoms or signs. Therefore, prospective studies on humans with tick exposure are necessary to describe the prevalence, etiology and clinical symptoms of these tick-borne diseases other than Lyme borreliosis and tick-borne encephalitis., 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 © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic disease associated with its principal tick vector, Hyalomma spp. with increasing fatal incidence worldwide. Accordingly, CCHF is a World Health Organization-prioritized disease with the absence of effective preventive interventions and approved vaccines or effective treatments. This perspective raised from a multidisciplinary gap analysis considering a One Health approach beneficial for human and animal health and the environment exploring international collaborations, gaps and recommendations., (© 2024. The Author(s).)

An infection with the tick-borne Rickettsia helvetica has been associated with a broad spectrum of clinical manifestations in humans, but patients are only seldomly reported. Understanding its disease etiology necessitates well-stablished infection models, improving to recognize and diagnose patients with R. helvetica infection and facilitating the development of effective control strategies. In this study, we used C3H/HeN mice as a model to establish R. helvetica infection, achieving a high infection prevalence (89-100 %). While the liver and the spleen DNA consistently tested positive for infection in all challenged mice, additional infected organs included the kidneys, heart, and the lungs. Notably, a low prevalence of infection was observed in I. ricinus nymphs fed on R. helvetica -challenged mice. In addition, larvae were refractory to infection, suggesting that ticks exhibit low susceptibility to the pathogen. To the best of our knowledge, this is the first study of an animal model for R. helvetica infection. It serves as a valuable tool for advancing research on the interactions among the bacterium and its vertebrate host., Competing Interests: 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., (© 2024 The Authors.)

Background: Ticks carry a variety of microorganisms, some of which are pathogenic to humans. The human risk of tick-borne diseases depends on, among others, the prevalence of pathogens in ticks biting humans. To follow-up on this prevalence over time, a Belgian study from 2017 was repeated in 2021., Methods: During the tick season 2021, citizens were invited to have ticks removed from their skin, send them and fill in a short questionnaire on an existing citizen science platform for the notification of tick bites (TekenNet). Ticks were morphologically identified to species and life stage level and screened using multiplex qPCR targeting, among others, Borrelia burgdorferi (sensu lato), Anaplasma phagocytophilum, Borrelia miyamotoi, Neoehrlichia mikurensis, Babesia spp., Rickettsia helvetica and tick-borne encephalitis virus (TBEV). The same methodology as in 2017 was used., Results: In 2021, the same tick species as in 2017 were identified in similar proportions; of 1094 ticks, 98.7% were Ixodes ricinus, 0.8% Ixodes hexagonus and 0.5% Dermacentor reticulatus. A total of 928 nymphs and adults could be screened for the presence of pathogens. Borrelia burgdorferi (s.l.) was detected in 9.9% (95% CI 8.2-12.0%), which is significantly lower than the prevalence of 13.9% (95% CI 12.2-15.7%) in 2017 (P = 0.004). The prevalences of A. phagocytophilum (4.7%; 95% CI 3.5-6.3%) and R. helvetica (13.3%; 95% CI 11.2-15.6%) in 2021 were significantly higher compared to 2017 (1.8%; 95% CI 1.3-2.7% and 6.8%; 95% CI 5.6-8.2% respectively) (P < 0.001 for both). For the other pathogens tested, no statistical differences compared to 2017 were found, with prevalences ranging between 1.5 and 2.9% in 2021. Rickettsia raoultii was again found in D. reticulatus ticks (n = 3/5 in 2021). Similar to 2017, no TBEV was detected in the ticks. Co-infections were found in 5.1% of ticks. When combining co-infection occurrence in 2017 and 2021, a positive correlation was observed between B. burgdorferi (s.l.) and N. mikurensis and B. burgdorferi (s.l.) and B. miyamotoi (P < 0.001 for both)., Conclusions: Although the 2021 prevalences fell within expectations, differences were found compared to 2017. Further research to understand the explanations behind these differences is needed., (© 2024. The Author(s).)

Severe babesiosis with 9.8% parasitemia was diagnosed in a patient in the Netherlands who had previously undergone splenectomy. We confirmed Babesia venatorum using PCR and sequencing. B. venatorum was also the most prevalent species in Ixodes ricinus ticks collected around the patient's home. Our findings warrant awareness for severe babesiosis in similar patients.

A crucial factor to predict the persistence and spread of infections in natural systems is the capacity of reservoir hosts to maintain the infection and transmit it to others. This is known to greatly vary within and between species and through time, although the latter part of the variation is often less well understood in the wild. Borrelia garinii is one of the causal agents of Lyme disease in humans and is transmitted among avian hosts by the hard tick Ixodes ricinus . Great tits are known to be a reservoir in Europe for B. garinii . For tick-borne pathogens like B. garinii , infectiousness or host-to-vector transmission can be measured using xenodiagnosis where pathogen-free vectors are fed on a host, and the blood-fed vectors are subsequently tested for the pathogen. Here we describe and evaluate a methodology to quantify infectiousness for tick-borne pathogens in individual wild great tits ( Parus major ), involving captures and recaptures of targeted individuals. The methodology can potentially be applied to other species where recapturing is sufficiently guaranteed. We successfully recaptured most of the infested great tits two to three days after initial infestation (i.e. just before ticks have fully fed) with sufficient numbers of I. ricinus larval ticks, which were subsequently screened for B. garinii using a newly developed B. garinii- specific real-time PCR assay. Higher larval tick numbers were recovered from birds during the breeding seasons than during the winter months. Our novel B. garinii- qPCR performed well, and greatly reduced the amount of Sanger sequencing needed. Preliminary results suggest both seasonal and individual variation in infectiousness; heterogeneity that needs to be unravelled to further understand the contribution of resident birds to the epidemiology of B. garinii ., Competing Interests: 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., (© 2024 The Authors.)

The incidence of diseases caused by pathogens transmitted by the tick Ixodes ricinus vary over time and space through incompletely understood mechanisms. An important determinant of the disease risk is the density of infected ticks, which is the infection prevalence times the density of questing ticks. We therefore investigated the spatial and temporal variation of four pathogens and one of the most abundant symbionts in Ixodes ricinus in questing nymphs over four years of monthly collections in 12 locations in the Netherlands. The infection prevalence of all microbes showed markedly different patterns with significant spatial variation for Borrelia burgdorferi ( s.l. ), Neoehrlichia mikurensis , Rickettsia helvetica , and Midichloria mitochondrii , significant seasonal variation of B. burgdorferi ( s.l. ), N. mikurensis , and M. mitochondrii and a significant interannual variation of R. helvetica . Despite its ubiquitous presence, no spatio-temporal variation was observed for the infection prevalence of B. miyamotoi . The variation in infection prevalence was generally smaller than the variation in the density of nymphs, which fluctuated substantially both seasonally and between locations. This means that the variation in the densities of infected nymphs for all pathogens was mostly the result of the variation in densities of nymphs. We also investigated whether there were positive or negative associations between the symbionts, and more specifically whether ticks infected with vertically transmitted symbionts like M. mitochondrii and R. helvetica , have a higher prevalence of horizontally transmitted symbionts, such as B. burgdorferi ( s.l. ) and N. mikurensis . We indeed found a clear positive association between M. mitochondrii and B. burgdorferi ( s.l. ). The positive association between R. helvetica and B. burgdorferi ( s.l. ) was less clear and was only shown in two locations. Additionally, we found a clear positive association between B. burgdorferi ( s.l. ) and N. mikurensis , which are both transmitted by rodents. Our longitudinal study indicated strong between-location variation, some seasonal patterns and hardly any differences between years for most symbionts. Positive associations between symbionts were observed, suggesting that infection with a (vertically transmitted) symbiont may influence the probability of infection with other symbionts, or that there is a common underlying mechanism (e.g. feeding on rodents)., Competing Interests: 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., (© 2024 The Authors.)

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

Species composition and densities of wild ungulate communities in Europe have changed over the last decades. As ungulates play an important role in the life-cycle of the tick species Ixodes ricinus , these changes could affect both the life-cycle of I. ricinus and the transmission of tick-borne pathogens like Borrelia burgdorferi ( s.l. ) and Anaplasma phagocytophilum . Due to morphological and behavioural differences among the ungulate species, these species might have different effects on the densities of questing I. ricinus , either directly through a bloodmeal or indirectly via the impact of ungulates on rodent numbers via the vegetation. In this study, we aimed to investigate these direct and indirect effects of five different ungulate species, fallow deer ( Dama dama ), roe deer ( Capreolus capreolus ), red deer ( Cervus elaphus ), moose ( Alces alces ), and wild boar ( Sus scrofa ), on the presence and abundance of I. ricinus ticks. In the summer of 2019, on 20 1 × 1 km transects in south-central Sweden that differed in ungulate community composition, we collected data on tick presence and abundance (by dragging a cloth), ungulate community composition (using camera traps), vegetation height (using the drop-disc method), temperature above field layer and rodent abundance (by snap-trapping). Using generalized linear mixed models we did not find any associations between vegetation height and tick presence/abundance or ungulate visitation frequencies, or between ungulate visitation frequencies and the presence/abundance of questing I. ricinus . The power of our analyses was, however, low due to very low tick and rodent numbers. We did find a negative association between adult ticks and air temperature, where we were more likely to find adult ticks if temperature in the field layer was lower. We conclude that more elaborate long-term studies are needed to elucidate the investigated associations. Such future studies should differentiate among the potential impacts of different ungulate species instead of treating all ungulate species as one group., Competing Interests: 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., (© 2024 The Authors. Published by Elsevier B.V.)

Borrelia miyamotoi is an emerging Ixodes tick-borne human pathogen in the Northern hemisphere. The aim of the current study was to compare whole genome sequences of B. miyamotoi isolates from different continents. Using a combination of Illumina and PacBio platforms and a novel genome assembly and plasmid typing pipeline, we reveal that the 21 sequenced B. miyamotoi isolates and publically available B. miyamotoi genomes from North America, Asia, and Europe form genetically distinct populations and cluster according to their geographical origin, where distinct Ixodes species are endemic. We identified 20 linear and 17 circular plasmid types and the presence of specific plasmids for isolates originating from different continents. Linear plasmids lp12, lp23, lp41, and lp72 were core plasmids found in all isolates, with lp41 consistently containing the vmp expression site. Our data provide insights into the genetic basis of vector competence, virulence, and pathogenesis of B. miyamotoi ., Competing Interests: All authors declare no competing interests., (© 2024 The Authors.)

Usutu virus (USUV) is an emerging flavivirus that is maintained in an enzootic cycle with mosquitoes as vectors and birds as amplifying hosts. In Europe, the virus has caused mass mortality of wild birds, mainly among Common Blackbird (Turdus merula) populations. While mosquitoes are the primary vectors for USUV, Common Blackbirds and other avian species are exposed to other arthropod ectoparasites, such as ticks. It is unknown, however, if ticks can maintain and transmit USUV. We addressed this question using in vitro and in vivo experiments and field collected data. USUV replicated in IRE/CTVM19 Ixodes ricinus tick cells and in injected ticks. Moreover, I. ricinus nymphs acquired the virus via artificial membrane blood-feeding and maintained the virus for at least 70 days. Transstadial transmission of USUV from nymphs to adults was confirmed in 4.9% of the ticks. USUV disseminated from the midgut to the haemocoel, and was transmitted via the saliva of the tick during artificial membrane blood-feeding. We further explored the role of ticks by monitoring USUV in questing ticks and in ticks feeding on wild birds in the Netherlands between 2016 and 2019. In total, 622 wild birds and the Ixodes ticks they carried were tested for USUV RNA. Of these birds, 48 (7.7%) carried USUV-positive ticks. The presence of negative-sense USUV RNA in ticks, as confirmed via small RNA-sequencing, showed active virus replication. In contrast, we did not detect USUV in 15,381 questing ticks collected in 2017 and 2019. We conclude that I. ricinus can be infected with USUV and can transstadially and horizontally transmit USUV. However, in comparison to mosquito-borne transmission, the role of I. ricinus ticks in the epidemiology of USUV is expected to be minor., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bakker et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The tick Ixodes ricinus parasitizes a wide range of vertebrates. These hosts vary in the relative contribution to the feeding of the different tick life stages, and their interplay is pivotal in the transmission dynamics of tick-borne pathogens. We aimed to know if there is a phylogenetic signal in the feeding and propagation hosts of I. ricinus , independently of other traits, as well as in the amplification of Borrelia burgdorferi ( s.l. ) in feeding larvae. We used a compilation of 1127 published field surveys in Europe, providing data for 96,586 hosts, resulting in 265,124 larvae, 72,080 nymphs and 37,726 adults. The load of immature ticks on hosts showed a significant phylogenetic signal towards the genera Psammodromus , Podarcis, and Lacerta (nymphs only). We hypothesize that such signal is the background hallmark of the primitive hosts associations of I. ricinus , probably in the glaciation refugia. A secondary phylogenetic signal for tick immatures appeared for some genera of Rodentia and Eulipotyphla. Results suggest the notion that the tick gained these hosts after spread from glaciation refugia. Analyses support a phylogenetic signal in the tick adults, firmly linked to Cetartiodactyla, but not to Carnivora or Aves. This study provides the first demonstration of host preferences in the generalist tick I. ricinus . We further demonstrate that combinations of vertebrates contribute in different proportions supporting the tick life-cycle in biogeographical regions of the Western Palaearctic as each region has unique combinations of dominant hosts. Analysis of the amplification of B. burgdorferi ( s.l. ) demonstrated that each genospecies is better amplified by competent reservoirs with which a strong phylogenetic signal exists. These vertebrates are the same along the spatial range: environmental traits do not change the reservoirs along the large territory studied. The transmission of B. burgdorferi ( s.l. ) is amplified by a few species of vertebrates, that share biogeographical regions with the tick vector in variable proportions., Competing Interests: 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. Given their role as Co-Editor, Agustín Estrada-Peña had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Professor Aneta Kostadinova (Editor-in-Chief)., (© 2024 Published by Elsevier B.V.)

BackgroundTo be better prepared for emerging wildlife-borne zoonoses, we need to strengthen wildlife disease surveillance.AimThe aim of this study was to create a topical overview of zoonotic pathogens in wildlife species to identify knowledge gaps and opportunities for improvement of wildlife disease surveillance.MethodsWe created a database, which is based on a systematic literature review in Embase focused on zoonotic pathogens in 10 common urban wildlife mammals in Europe, namely brown rats, house mice, wood mice, common voles, red squirrels, European rabbits, European hedgehogs, European moles, stone martens and red foxes. In total, we retrieved 6,305 unique articles of which 882 were included.ResultsIn total, 186 zoonotic pathogen species were described, including 90 bacteria, 42 helminths, 19 protozoa, 22 viruses and 15 fungi. Most of these pathogens were only studied in one single animal species. Even considering that some pathogens are relatively species-specific, many European countries have no (accessible) data on zoonotic pathogens in these relevant animal species. We used the Netherlands as an example to show how this database can be used by other countries to identify wildlife disease surveillance gaps on a national level. Only 4% of all potential host-pathogen combinations have been studied in the Netherlands.ConclusionsThis database comprises a comprehensive overview that can guide future research on wildlife-borne zoonotic diseases both on a European and national scale. Sharing and expanding this database provides a solid starting point for future European-wide collaborations to improve wildlife disease surveillance.

Vector-borne diseases (VBDs) are considered as (re-)emerging, but information on the transmission cycles and wildlife reservoirs is often incomplete, particularly with regard to urban areas. The present study investigated blood samples from European hedgehogs ( Erinaceus europaeus ) presented at wildlife rehabilitation centres in the region of Hanover. Past exposure to B. burgdorferi sensu lato (s.l.) and tick-borne encephalitis virus (TBEV) was assessed by serological detection of antibodies, while current infections with Borrelia spp., Anaplasma phagocytophilum , Rickettsia spp., Neoehrlichia mikurensis , Bartonella spp., Babesia spp. and Spiroplasma ixodetis were investigated by (q)PCR. Of 539 hedgehogs tested for anti- Borrelia antibodies, 84.8% (457/539) were seropositive, with a higher seropositivity rate in adult than subadult animals, while anti-TBEV antibodies were detected in one animal only (0.2%; 1/526). By qPCR, 31.2% (168/539) of hedgehog blood samples were positive for Borrelia spp., 49.7% (261/525) for A. phagocytophilum , 13.0% (68/525) for Bartonella spp., 8.2% for S. ixodetis (43/525), 8.0% (42/525) for Rickettsia spp. and 1.3% (7/525) for Babesia spp., while N. mikurensis was not detected. While further differentiation of Borrelia spp. infections was not successful, 63.2% of the A. phagocytophilum infections were assigned to the zoonotic ecotype I and among Rickettsia spp. infections, 50.0% to R. helvetica by ecotype- or species-specific qPCR, respectively. Sequencing revealed the presence of a Rickettsia sp. closely related to Rickettsia felis in addition to a Bartonella sp. previously described from hedgehogs, as well as Babesia microti and Babesia venatorum . These findings show that hedgehogs from rehabilitation centres are valuable sources to identify One Health pathogens in urban areas. The hedgehogs are not only exposed to pathogens from fleas and ticks in urban areas, but they also act as potent amplifiers for these vectors and their pathogens, relevant for citizens and their pets., Competing Interests: The authors declare that they have no conflicts of interest., (© 2024 The Authors.)

Ticks are involved in the transmission a plethora of pathogens. To effectively control ticks and mitigate the risks associated with tick-borne diseases, it is important to implement tick control measures. These may include the use of acaricides as well as the development and implementation of an alternative, environmentally friendly tick management program that include practices such as habitat modification or establishing biological control. Ixodiphagus hookeri Howard is a tick-specific parasitoid wasp that predates on several species of ixodid ticks and could contribute to the control of the tick population. This work aimed to detect the presence of parasitoid wasps in ticks ( Ixodidae ) using genetic approaches. Several tick species of the genera Ixodes , Haemaphysalis, and Dermacentor , with a sympatric occurrence in the Slovak Karst National Park in southeastern Slovakia, were screened for the presence of wasps of the genus Ixodiphagus. The DNA of the parasitoids was detected in four tick species from three genera. This work presents the first molecular detection of parasitoids in two Dermacentor tick species, as well as the first molecular identification of Ixodiphagus wasps in Ixodes ricinus and Haemaphysalis concinna ticks from the Karst area. In the given area, it was observed that I. ricinus and H. concinna ticks are hyper-parasitized by wasps. Moreover, it was observed that wasps here can parasitize several tick species, some of which are of less significance for human and animal health (as they transmit fewer pathogens).

Background: Hippoboscid flies are bloodsucking arthropods that can transmit pathogenic microorganisms and are therefore potential vectors for pathogens such as Bartonella spp. These Gram-negative bacteria can cause mild-to-severe clinical signs in humans and animals; therefore, monitoring Bartonella spp. prevalence in louse fly populations appears to be a useful prerequisite for zoonotic risk assessment., Methods: Using convenience sampling, we collected 103 adult louse flies from four ked species (Lipoptena cervi, n = 22; Lipoptena fortisetosa, n = 61; Melophagus ovinus, n = 12; Hippobosca equina, n = 8) and the pupae of M. ovinus (n = 10) in the federal state of Saxony, Germany. All the samples were screened by polymerase chain reaction (PCR) for Bartonella spp. DNA, targeting the citrate synthase gene (gltA). Subsequently, PCRs targeting five more genes (16S, ftsZ, nuoG, ribC and rpoB) were performed for representatives of revealed gltA genotypes, and all the PCR products were sequenced to identify the Bartonella (sub)species accurately., Results and Conclusions: The overall detection rates for Bartonella spp. were 100.0%, 59.1%, 24.6% and 75.0% in M. ovinus, L. cervi, L. fortisetosa and H. equina, respectively. All the identified bartonellae belong to the Bartonella schoenbuchensis complex. Our data support the proposed reclassification of the (sub)species status of this group, and thus we conclude that several genotypes of B. schoenbuchensis were detected, including Bartonella schoenbuchensis subsp. melophagi and Bartonella schoenbuchensis subsp. schoenbuchensis, both of which have previously validated zoonotic potential. The extensive PCR analysis revealed the necessity of multiple PCR approach for proper identification of the ruminant-associated bartonellae., (© 2024 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

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., 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 © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)