Two Anaplasma phagocytophilum Strains in Ixodes scapularis Ticks,Canada

We developed PCR-based assays to distinguish a human pathogenic strain of Anaplasma phagocytophilum, Ap-ha, from Ap-variant 1, a strain not associated with human infection. The assays were validated on A. phagocytophilum-infected blacklegged ticks (Ixodes scapularis) collected in Canada. The relative prevalence of these 2 strains in I. scapularis ticks differed among geographic regions.     

A bioinformatics pipeline for a tick pathogen surveillance multiplex amplicon sequencing assay

The Centers for Disease Control and Prevention's national tick and tick-borne pathogen surveillance program collects information to better understand the regional distribution, prevalence, and exposure risk of host-seeking medically important ticks in the United States. A recently developed next generation sequencing (NGS) targeted multiplex PCR amplicon sequencing (MPAS) assay has enhanced the detection capabilities for Ixodes-associated human pathogens found in Ixodes scapularis and Ixodes pacificus ticks compared to the routinely used real-time PCR assay. To operationalize the MPAS assay for the large number of tick surveillance submissions processed each year, a reproducible high throughput bioinformatics pipeline is needed. We describe the development and validation of the MPAS pipeline, a bioinformatics pipeline that identifies and summarizes amplicon sequences produced by the MPAS assay. This pipeline is portable and reproducible across different computing environments, and flexible by allowing modifications to input parameters, assay primer and reference sequences. The automation of the summary report, BLAST report, and phylogenetic analysis reduces the amount of time needed for downstream analysis. To validate this pipeline, we compared the analysis of a MPAS assay dataset consisting of 175 I. scapularis nymphs with the MPAS pipeline and previously published results analyzed with a CLC Genomic Workbench workflow. The MPAS pipeline identified the same number of positive ticks for Anaplasma phagocytophilum and Babesia species as the original analysis, but the MPAS pipeline provided enhanced sequencing resolution of Borrelia burgdorferi sensu lato co-infected samples. The reproducibility, flexibility, analysis automation, and improved sequence resolution of the MPAS pipeline make it well suited for a high throughput tick pathogen surveillance program.     

The European hedgehog (Erinaceus europaeus) – A suitable reservoir for variants of Anaplasma phagocytophilum?

The European hedgehog (Erinaceus europaeus) is a common insectivore in most parts of Europe and is frequently infested by the ticks Ixodes ricinus and I. hexagonus. I. ricinus ticks have been found infected with Anaplasma phagocytophilum, an obligate intracellular bacterium, but little is known about the potential of the hedgehog as a reservoir host. In this study, the infection with A. phagocytophilum and the genetic variants involved were investigated in a captive hedgehog population which was kept in a fenced, natural grass and bush garden habitat, and also in its ticks. Additionally hedgehogs from hedgehog caretaking stations were investigated. EDTA blood and ticks were collected from the captive hedgehog population once a month from March to October 2007 and in March and April 2008. All 3 developmental stages of I. ricinus and I. hexagonus occurred on the hedgehogs. After DNA extraction, the samples were screened for A. phagocytophilum with a real-time PCR, and selected samples were further investigated with a nested PCR targeting the partial 16S rRNA gene, followed by sequencing. One hundred thirty-six out of 220 hedgehog blood samples (61.8%) from altogether 48 individuals, 413 out of 563 I. ricinus samples and 90 out of 338 I. hexagonus samples were PCR-positive. Thirty-two hedgehogs were positive more than once, most frequently twice or 3 times, but also up to 9 times. Sequencing of the partial 16S rRNA gene resulted in 6 variants, but one variant (‘A’) was the most frequent which appeared in 93.8% of the positive hedgehogs. This variant (equaling Frankonia II, GenBank AF136712) has recently been reported from human, equine, and canine granulocytic anaplasmosis cases and thus, its specific association with hedgehogs is an important finding in the epidemiology of A. phagocytophilum in Europe.The high infection rate of both hedgehogs and ticks with A. phagocytophilum and the simultaneous infestation with 2 tick species of all developmental stages suggest that the hedgehog may be a suitable reservoir for at least some variants of A. phagocytophilum.     

The prevalence and distribution of Anaplasma phagocytophilum genotypes in Ixodes ricinus nymphs collected from farm- and woodland sites in Ireland

Despite the economic importance of grass-based livestock production in Ireland and the fact that many veterinarians and farmers regard tickborne fever (TBF) as an increasingly important disease, especially in sheep, little is currently known about the prevalence and genetic diversity of its causative agent, Anaplasma phagocytophilum. In the present study, 1376 nymphal Ixodes ricinus ticks collected from woodland, farmland, bog and limestone pavement habitats were screened for A. phagocytophilum using TaqMan PCR. Positive samples were further analysed by conventional nested PCR targeting the 16S rRNA, msp4 and groEL loci.Overall 4.5% I. ricinus nymphs were found to be infected. The genetic heterogeneity was comparable to that reported elsewhere in Europe, with greater genetic diversity of 16S variants in ticks collected from farmland than from woodland. All isolates belonged to groEL ecotype I indicating that rodents and birds do not contribute to the epidemiology of tickborne fever in Ireland. In the 16S and groEL loci, a number of the Irish isolates matched European sequences from humans, horses and dogs. The 16S sequences that were identical to human isolates from Europe also matched Ap-ha, the most common human pathogenic strain in the USA. Three isolates also matched published sequences from horses in the msp4 locus. No isolate matched human, equine and canine sequences in all 3 loci. Our results represent the first molecular characterization of Irish A. phagocytophilum isolates.     

Evaluation of a novel multiplex PCR amplicon sequencing assay for detection of human pathogens in Ixodes ticks

Tickborne diseases are an increasing public health concern in the United States, where the majority of notifiable cases are caused by pathogens vectored by Ixodes ticks. To better monitor changes in acarological risk of human encounters with these ticks and their associated pathogens, the Centers for Disease Control and Prevention (CDC) recently established a national tick and tickborne pathogen surveillance program. Here, we describe and evaluate a new Multiplex PCR Amplicon Sequencing (MPAS) assay for potential use in surveillance programs targeting two common human-biting vector ticks, Ixodes scapularis and Ixodes pacificus. The ability of the MPAS assay to detect five Ixodes-associated human pathogens (Borrelia burgdorferi sensu stricto, Borrelia mayonii, Borrelia miyamotoi, Anaplasma phagocytophilum and Babesia microti) was compared to that of a previously published and routinely used probe-based (TaqMan) PCR testing algorithm for pathogen detection in Ixodes ticks. Assay performance comparisons included a set of 175 host-seeking Ixodes nymphs collected in Connecticut as well as DNA from our pathogen reference collection. The MPAS assay and the CDC standard TaqMan PCR pathogen testing algorithm were found to have equivalent detection sensitivity for Ixodes-associated human pathogens. However, the MPAS assay was able to detect a broader range of tick-associated microorganisms, more effectively detected co-infections of multiple pathogens in a single tick (including different species within the Borrelia burgdorferi sensu lato complex), and required a smaller volume of test sample (thus preserving more sample for future testing).     

Inter-annual variation in prevalence of Borrelia burgdorferi sensu stricto and Anaplasma phagocytophilum in host-seeking Ixodes scapularis (Acari: Ixodidae) at long-term surveillance sites in the upper midwestern United States: Implications for public health practice

The geographic range of the blacklegged tick, Ixodes scapularis, and its associated human pathogens have expanded substantially over the past 20 years putting an increasing number of persons at risk for tick-borne diseases, particularly in the upper midwestern and northeastern United States. Prevention and diagnosis of tick-borne diseases rely on an accurate understanding by the public and health care providers of when and where persons may be exposed to infected ticks. While tracking changes in the distribution of ticks and tick-borne pathogens provides fundamental information on risk for tick-borne diseases, metrics that incorporate prevalence of infection in ticks better characterize acarological risk. However, assessments of infection prevalence are more labor intensive and costly than simple measurements of tick or pathogen presence. Our objective was to examine whether data derived from repeated sampling at longitudinal sites substantially influences public health recommendations for Lyme disease and anaplasmosis prevention, or if more constrained sampling is sufficient. Here, we summarize inter-annual variability in prevalence of the agents of Lyme disease (Borrelia burgdorferi s.s.) and anaplasmosis (Anaplasma phagocytophilum) in host-seeking I. scapularis nymphs and adults at 28 longitudinal sampling sites in the Upper Midwestern US (Michigan, Minnesota, and Wisconsin). Infection prevalence was highly variable among sites and among years within sites. We conclude that monitoring infection prevalence in ticks aids in describing coarse acarological risk trends, but setting a fixed prevalence threshold for prevention or diagnostic decisions is not feasible given the observed variability and lack of temporal trends. Reducing repeated sampling of the same sites had minimal impact on regional (Upper Midwest) estimates of average infection prevalence; this information should be useful in allocating scarce public health resources for tick and tick-borne pathogen surveillance, prevention, and control activities.     

Potential Role of Deer Tick Virus in Powassan Encephalitis Cases in Lyme Disease–endemic Areas of New York,USA

Powassan virus, a member of the tick-borne encephalitis group of flaviviruses, encompasses 2 lineages with separate enzootic cycles. The prototype lineage of Powassan virus (POWV) is principally maintained between Ixodes cookei ticks and the groundhog (Marmota momax) or striped skunk (Mephitis mephitis), whereas the deer tick virus (DTV) lineage is believed to be maintained between Ixodes scapularis ticks and the white-footed mouse (Peromyscus leucopus). We report 14 cases of Powassan encephalitis from New York during 2004–2012. Ten (72%) of the patients were residents of the Lower Hudson Valley, a Lyme disease–endemic area in which I. scapularis ticks account for most human tick bites. This finding suggests that many of these cases were caused by DTV rather than POWV. In 2 patients, DTV infection was confirmed by genetic sequencing. As molecular testing becomes increasingly available, more cases of Powassan encephalitis may be determined to be attributable to the DTV lineage.     

Sheep experimentally infected with a human isolate of Anaplasma phagocytophilum serve as a host for infection of Ixodes scapularis ticks

Anaplasma phagocytophilum, first identified as a pathogen of ruminants in Europe, has more recently been recognized as an emerging tick-borne pathogen of humans in the U.S. and Europe. A. phagocytophilum is transmitted by Ixodes spp., but the tick developmental cycle and pathogen/vector interactions have not been fully described. In this research, we report on the experimental infection of sheep with the human NY-18 isolate of A. phagocytophilum which then served as a host for infection of I. scapularis nymphs and adults. A. phagocytophilum was propagated in the human promyelocytic cell line, HL-60, and the infected cell cultures were then used to infect sheep by intravenous inoculation. Infections in sheep were confirmed by PCR and an Anaplasma-competitive ELISA. Clinical signs were not apparent in any of the infected sheep, and only limited hematologic and mild serum biochemical abnormalities were identified. While A. phagocytophilum morulae were rarely seen in neutrophils, blood film evaluation revealed prominent large granular lymphocytes, occasional plasma cells, and rare macrophages. Upon necropsy, gross lesions were restricted to the lymphoid system. Mild splenomegaly and lymphadenomegaly with microscopic evidence of lymphoid hyperplasia was observed in all infected sheep. Female I. scapularis that were allowed to feed and acquire infection on each of the 3 experimentally infected sheep became infected with A. phagocytophilum as determined by PCR of guts (80–87%) and salivary glands (67–100%). Female I. scapularis that acquired infection as nymphs on an experimentally infected sheep transmitted A. phagocytophilum to a susceptible sheep, thus confirming transstadial transmission. Sheep proved to be a good host for the production of I. scapularis infected with this human isolate of A. phagocytophilum, which can be used as a model for future studies of the tick/pathogen interface.     

Lyme Disease,Virginia, USA, 2000–2011

Lyme disease, caused by the bacterium Borrelia burgdorferi and transmitted in the eastern United States by the black-legged tick (Ixodes scapularis), is increasing in incidence and expanding geographically. Recent environmental modeling based on extensive field collections of host-seeking I. scapularis ticks predicted a coastal distribution of ticks in mid-Atlantic states and an elevational limit of 510 m. However, human Lyme disease cases are increasing most dramatically at higher elevations in Virginia, a state where Lyme disease is rapidly emerging. Our goal was to explore the apparent incongruity, during 2000–2011, between human Lyme disease data and predicted and observed I. scapularis distribution. We found significantly higher densities of infected ticks at our highest elevation site than at lower elevation sites. We also found that I. scapularis ticks in Virginia are more closely related to northern than to southern tick populations. Clinicians and epidemiologists should be vigilant in light of the changing spatial distributions of risk.     

Anaplasma phagocytophilum in European bison (Bison bonasus) and their ticks from Lithuania and Poland

The increasing population of European bison (Bison bonasus) can contribute to the prevalence of zoonotic pathogens. The aim of the present study was to assess the presence of A. phagocytophilum infection in European bison tissues as well as ticks removed from European bison in Lithuania and Poland. A further objective of this work was to compare the detected A. phagocytophilum strains. A total of 85 tissue samples (spleen) of European bison and 560 ticks belonging to two species, Ixodes ricinus (n = 408) and Dermacentor reticulatus (n = 152) were tested. DNA of A. phagocytophilum was detected based on RT-PCR in 40% of the European bison samples, 8.8% of the I. ricinus and 5.9% of the D. reticulatus ticks. Analysis of the obtained partial 16S rRNA gene sequences of A. phagocytophilum revealed the presence of three variants with two polymorphic sites. Furthermore, phylogenetic analysis with partial msp4 gene sequences grouped A. phagocytophilum variants into three clusters. This study revealed that the groEL gene sequences of A. phagocytophilum from European bison and their ticks grouped into ecotype I and only one sequence from Lithuanian European bison belonged to ecotype II. The results of the present study indicated that European bison may play a role as a natural reservoir of A. phagocytophilum.     

Prevalence of Rickettsia spp., Anaplasma phagocytophilum,and Coxiella burnetii in adult Ixodes ricinus ticks from 29 study areas in central and southern Sweden

A total of 887 adult Ixodes ricinus ticks (469 females and 418 males) from 29 different localities in Sweden were screened for Rickettsia, Anaplasma, and Coxiella DNA using PCR and then subjected to gene sequencing. Rickettsial DNA was detected in 9.5–9.6% of the ticks. Most of the positive ticks were infected with Rickettsia helvetica. One tick harbored another spotted fever rickettsia, closely related to or identical with R. sibirica not previously found in I. ricinus nor in Sweden. Six of the ticks (0.7%) were infected with an Anaplasma sp., presumably A. phagocytophilum. Coxiella burnetii DNA was not detected in any of the ticks. The detection of R. helvetica and A. phagocytophilum in several of the localities sampled suggests that these potentially human-pathogenic agents are common in Sweden.     

Increased diversity of zoonotic pathogens and Borrelia burgdorferi strains in established versus incipient Ixodes scapularis populations across the Midwestern United States

The center of origin theory predicts that genetic diversity will be greatest near a specie’s geographic origin because of the length of time for evolution. By corollary, diversity will decrease with distance from the origin; furthermore, invasion and colonization are frequently associated with founder effects that reduce genetic variation in incipient populations. The blacklegged tick, Ixodes scapularis, harbors a suite of zoonotic pathogens, and the geographic range of the tick is expanding in the upper Midwestern United States. Therefore, we posited that diversity of I. scapularis-borne pathogens across its Midwestern range should correlate with the rate of the range expansion of this tick as well as subsequent disease emergence. Analysis of 1565 adult I. scapularis ticks from 13 sites across five Midwestern states revealed that tick infection prevalence with multiple microbial agents (Borrelia burgdorferi, Borrelia miyamotoi, Babesia odocoilei, Babesia microti, and Anaplasma phagocytophilum), coinfections, and molecular genetic diversity of B. burgdorferi all were positively correlated with the duration of establishment of tick populations, and therefore generally support the center of origin – pathogen diversity hypothesis. The observed differences across the gradient of establishment, however, were not strong and were nuanced by the high frequency of coinfections in tick populations at both established and recently-invaded tick populations. These results suggest that the invasion of ticks and their associated pathogens likely involve multiple means of pathogen introduction, rather than the conventionally presented scenario whereby infected, invading ticks are solely responsible for introducing pathogens to naïve host populations.     

High Prevalence of Borrelia miyamotoi among Adult Blacklegged Ticks from White-Tailed Deer

We compared the prevalence of Borrelia miyamotoi infection in questing and deer-associated adult Ixodes scapularis ticks in Wisconsin, USA. Prevalence among deer-associated ticks (4.5% overall, 7.1% in females) was significantly higher than among questing ticks (1.0% overall, 0.6% in females). Deer may be a sylvatic reservoir for this newly recognized zoonotic pathogen.     

Molecular detection and phylogeny of Anaplasma spp. closely related to Anaplasma phagocytophilum in small ruminants from China

The genus Anaplasma comprises eight bacterial species that are obligate intracellular pathogens that affect human and animal health. The zoonotic species A. phagocytophilum is the causative agent of tick-borne fever in ruminants, and of granulocytic anaplasmosis in horses, dogs, and humans. Recently, novel strains related to A. phagocytophilum (A. phagocytophilum-like 1/Japanese variant and A. phagocytophilum-like 2/Chinese variant) have been identified. The aim of this study was to reveal the prevalence and phylogeny of A. phagocytophilum and related stains in small ruminants and ticks in China based on sequences of the 16S rRNA combined restriction fragment length polymorphism (RFLP) and groEL genes. PCR–RFLP and phylogenetic analyses based on the 16S rRNA gene showed the presence of A. phagocytophilum-like 1 and 2 variants in sampled animals from China, with prevalence rates of 22.6% (303/1338) and 0.7% (10/1338), respectively. Only A. phagocytophilum-like 1 DNA was found in Haemaphysalis longicornis. The phylogeny based on the groEL gene showed inclusion of A. phagocytophilum-like 1 and some A. phagocytophilum-like 2 strains in two unique clades distinct from, but related to, Japanese and Chinese strains of related A. phagocytophilum, respectively. One noteworthy result was that the SSAP2f/SSAP2r primers detected Ehrlichia spp. strains. Moreover, the A. phagocytophilum-like 1 and 2 strains should be considered in the differential diagnosis of caprine and ovine anaplasmosis. Further investigations should be conducted to provide additional epidemiological information about A. phagocytophilum and A. phagocytophilum-like variants in animals and ticks.     

Human Babesiosis,Maine, USA, 1995–2011

We observed an increase in the ratio of pathogenic Babesia microti to B. odocoilei in adult Ixodes scapularis ticks in Maine. Risk for babesiosis was associated with adult tick abundance, Borrelia burgdorferi infection prevalence, and Lyme disease incidence. Our findings may help track risk and increase the focus on blood supply screening.     

Comparison of acarological risk metrics derived from active and passive surveillance and their concordance with tick-borne disease incidence

Tick-borne diseases continue to threaten human health across the United States. Both active and passive tick surveillance can complement human case surveillance, providing spatio-temporal information on when and where humans are at risk for encounters with ticks and tick-borne pathogens. However, little work has been done to assess the concordance of the acarological risk metrics from each surveillance method. We used data on Ixodes scapularis and its associated human pathogens from Connecticut (2019–2021) collected through active collections (drag sampling) or passive submissions from the public to compare county estimates of tick and pathogen presence, infection prevalence, and tick abundance by life stage. Between the surveillance strategies, we found complete agreement in estimates of tick and pathogen presence, high concordance in infection prevalence estimates for Anaplasma phagocytophilum, Borrelia burgdorferi sensu stricto, and Babesia microti, but no consistent relationships between actively and passively derived estimates of tick abundance or abundance of infected ticks by life stage. We also compared nymphal metrics (i.e., pathogen prevalence in nymphs, nymphal abundance, and abundance of infected nymphs) with reported incidence of Lyme disease, anaplasmosis, and babesiosis, but did not find any consistent relationships with any of these metrics. The small spatial and temporal scale for which we had consistently collected active and passive data limited our ability to find significant relationships. Findings are likely to differ if examined across a broader spatial or temporal coverage with greater variation in acarological and epidemiological outcomes. Our results indicate similar outcomes between some actively and passively derived tick surveillance metrics (tick and pathogen presence, pathogen prevalence), but comparisons were variable for abundance estimates.     

Genetic diversity of Anaplasma and Ehrlichia bacteria found in Dermacentor and Ixodes ticks in Mongolia

Anaplasma and Ehrlichia are tick-borne bacterial pathogens that cause human granulocytic anaplasmosis, human monocytic ehrlichiosis, and are severe threats to livestock economies like Mongolia. In this study, ticks were collected, identified, and pooled (n = 299) from three distinct environments across central Mongolia. Each pool was initially tested for Anaplasma/Ehrlichia using a 16S rRNA PCR assay that detects both genera, and specific PCR testing was done to identify those positive samples. Maximum likelihood estimation (MLE) of infection rates of ticks collected from the environment in Selenge aimag (province) found infection rates of Ixodes persulcatus ticks to be 2.0% (95% CI: 0.7, 4.3%) for A. phagocytophilum and 0.8% (95% CI: 0.1, 2.5%) for both nonspecific Ehrlichia and Anaplasma. Ehrlichia muris was only detected in I. persulcatus ticks collected from the Selenge aimag, where the MLE was 1.2% (95% CI: 0.1, 2.5%). The calculated MLE infection rate of Anaplasma spp. in questing Dermacentor nuttalli ticks ranged from 1.9% (95% CI: 1.1, 9.1%) in the Tov aimag to 2.3% (95% CI: 1.3, 10.8%) in the Selenge aimag. However, when examining MLE in ticks removed from livestock, estimates increase substantially, ranging from 7.8% (95% CI: 4.2, 13.3%) in Dornogovi to 22.5% (95% CI: 14.3, 34.3%) in Selenge, suggesting that livestock play a key role in disease maintenance. Considering the collective economic losses that can result from these pathogens and the potential for illness in nomadic herdsmen, these results highlight the need for enhanced TBD surveillance and prevention measures within Mongolia.     

Synchronous phenology of juvenile Ixodes scapularis,vertebrate host relationships,and associated patterns of Borrelia burgdorferi ribotypes in the midwestern United States

To elucidate features of enzootic maintenance of the Lyme disease bacterium that affect human risk of infection, we conducted a longitudinal study of the phenology of the vector tick, Ixodes scapularis, at a newly invaded site in the north-central United States. Surveys for questing ticks and ticks parasitizing white-footed mice and eastern chipmunks revealed that I. scapularis nymphal and larval activity peaked synchronously in June and exhibited an atypical, unimodal seasonality. Adult seasonal activity was bimodal and distributed evenly in spring and fall. We discuss implications of these phenology data for the duration of the I. scapularis life cycle. Densities of Borrelia burgdorferi-infected, questing nymphs were comparable to those found in endemic areas elsewhere in the midwestern and northeastern U.S. Molecular genetic diversity of B. burgdorferi infecting these ticks and rodents was assessed by analysis of the ribosomal spacer types (RSTs). RST 1, a clade that includes strains with highly pathogenic properties, was relatively uncommon (3.4%) in contrast to the northeastern U.S., whereas less pathogenic ribotypes of the RST 2 and 3 clades were more common. These features of the ecology of this midwestern Lyme disease system likely contribute to the lower incidence of Lyme disease in humans in the Upper Midwest compared with that of the Northeast owing to reduced exposure to pathogenic strains of B. burgdorferi.     

Bartonella spp. Transmission by Ticks Not Established

Bartonella spp. infect humans and many animal species. Mainly because PCR studies have demonstrated Bartonella DNA in ticks, some healthcare providers believe that these microorganisms are transmitted by ticks. B. henselae, in particular, is regarded as being present in and transmissible by the Ixodes scapularis tick. The presence of a microbial agent within a tick, however, does not imply that the tick might transmit it during the course of blood feeding and does not confer epidemiologic importance. After a critical review of the evidence for and against tick transmission, we conclude that transmission of any Bartonella spp. by ticks, to animals or humans, has not been established. We are unaware of any well-documented case of B. henselae transmission by I. scapularis ticks.     

Woodchip borders at the forest ecotone as an environmental control measure to reduce questing tick density along recreational trails in Ottawa,Canada

Recent studies have highlighted the occurrence and distribution of Ixodes scapularis ticks infected with Borrelia burgdorferi sensu stricto (s.s.) around the city of Ottawa, Ontario, Canada, and the need for strategies to mitigate the risk of human exposure and infection. We conducted a field study from July to October 2018 to examine the effectiveness of ecotonal woodchip borders as an environmental control method to suppress the density of host-seeking ticks along recreational trails in Ottawa. We used an experimental design with ten 100-m trail replicates randomized to intervention or control groups, and monitored questing tick density at weekly intervals in mid-summer and early fall. We compared questing tick density between woodchip-treated and untreated trails using a mixed-effects Poisson regression model. Of the 138 I. scapularis ticks collected, there were 86 adult and nymphal ticks, 37 (43 %) of which were positive for B. burgdorferi s.s. A total of 58 Haemaphysalis leporispalustris were also collected. Mean combined adult and nymphal I. scapularis density was 1.15 (1.40 standard deviation; SD) per 100 m in the control group compared to 0.28 (0.56 SD) per 100 m in the intervention group, reflecting a 75 % reduction in questing tick density on trail replicates treated with woodchip borders (p < 0.001). An effect of the intervention was observed in both sampling periods. This study indicates that woodchip borders may be an effective strategy to suppress questing tick density along trail margins where recreational trail users are more active, thereby reducing the likelihood of tick encounters.