Ticks and Tick-borne Diseases

Tick-borne relapsing fever (TBRF) spirochetes are maintained in nature through vector-associated transmission routes primarily in argasid ticks. However, the extent to which strain-level differences influence the maintenance of TBRF spirochetes remains unclear. We evaluated two Borrelia turicatae strains, Bt-SSK1 and Bt-FCB, in Ornithodoros turicata. Both strains were acquired by female ticks, and after mating we unexpectedly observed that they were hyperparasitized by uninfected and unfed male O. turicata. Bt- SSK1 was maintained in males while Bt-FCB was not. Transovarial transmission (TOT) of Bt-SSK1 and Bt-FCB was also evaluated and striking differences were observed between strains. Bt-SSK1 was vertically maintained in F1 progeny but we failed to detect TOT of Bt-FCB. TOT of Bt-SSK1 occurred inefficiently in early ovipositions but increasing in later or delayed reproductive events, indicating a timing-dependent barrier. Using a gfp-expressing Bt-FCB strain, we show that failure of vertical transmission is associated with a lack of persistent oocyte colonization despite dissemination to ovarian tissues. These findings demonstrate strain-dependent differences in oocyte colonization and represent a critical bottleneck governing vertical transmission and persistence of relapsing fever spirochetes in tick populations. ImportanceVector-borne pathogens rely on diverse transmission strategies to persist in nature, yet the biological factors that govern these processes remain poorly understood. In this study, we demonstrated that strains of Borrelia turicatae differ markedly in their ability to persist within Ornithodoros turicata ticks. We also discovered that male ticks frequently hyperparasitized engorged females, creating an unrecognized transmission route in which previously uninfected ticks can acquire, maintain, and transmitted the B. turicatae. We further showed phenotypic differences in vertical transmission and that it is temporally regulated and linked to successful colonization of developing oocytes, identifying a critical bottleneck in pathogen maintenance. Together, these findings revealed that strain-specific traits influence both horizontal maintenance within tick populations and vertical transmission to progeny. This work provides insight into the ecological and evolutionary processes that enable relapsing fever spirochetes to persist in nature.

Ticks are obligate hematophagous arthropods and feed exclusively on blood. As blood is nutrient-poor, ticks rely on bacterial endosymbionts to synthesise nutrients, yet the diversity and functional roles of these symbionts in Australian ticks remain largely uncharacterised. This is critical to address as these ticks are of high medical importance in Australia. In this study, shotgun metagenomic sequencing was performed on Bothriocroton concolor, Bothriocroton hydrosauri, Haemaphysalis longicornis and Ixodes holocyclus, enabling the recovery of six complete or partial metagenome-assembled genomes (MAGs). These comprised Coxiella-like endosymbionts (CLE), a facultative Rickettsia symbiont, and two Midichloria mitochondrii strains (Ixholo1 and Ixholo2). Functional annotation of these taxon-specific symbionts revealed the absence of virulence factors and the presence of B-vitamin and/or heme biosynthesis genes, indicative of nutritional mutualism, which is essential for tick hematophagy. The CLEs additionally harbour genes of the shikimate pathway, which modulate blood feeding in ticks by regulating serotonin biosynthesis. Furthermore, functional annotation and pangenomic analysis of Midichloria spp. found evidence that the genus may encompass multiple species, as well as the retention of genes potentially associated with an intramitochondrial lifestyle in M. mitochondrii Ixholo2. Tick microbiomes are dominated by non-pathogenic microorganisms, which are often overshadowed by pathogens. These include the endosymbionts, which can influence host biology and pathogen transmission, and are fundamental for the development of diagnostic tools and taxon-specific tick biocontrols.

Background Trypanosoma cruzi, the causative agent of Chagas disease, is subdivided into distinct genetic groups known as Discrete Typing Units (DTUs), each with distinct genetic traits that influence epidemiology and transmission dynamics. Several triatomine species serve as potential vectors of T. cruzi in the United States. However, despite the growing number of Chagas disease cases in the country, little is known about the genetic diversity and population structure of T. cruzi in natural vector populations. Methodology/Principal Findings We applied a multilocus metabarcoding approach to improve DTU resolution and characterize the genetic diversity and structure of T. cruzi in triatomines collected across five states of the southern United States. Five single-copy nuclear markers and one mitochondrial marker were amplified and processed by high-throughput sequencing to assess genetic diversity. We recovered 35 nuclear and 15 mitochondrial haplotypes from 70 infected specimens. Overall, genetic diversity was low ({pi} < 0.01 at all nuclear loci), with DTUs TcI and the North American lineage of TcIV detected, TcI being the most prevalent. Geographic structuring was particularly evident in TcI strains, which exhibited a distinctive haplotype profile in Florida populations, potentially linked to the recently revalidated vector species Triatoma ambigua. Mitochondrial introgression from TcIV into TcI suggests inter-DTU genetic exchange in these populations. Multiple haplotypes within individual insects detected across single-copy nuclear markers, support multiclonal infection as common feature of T. cruzi in natural vectors. Conclusions/Significance These findings provide new insights into the genetic landscape and evolution of T. cruzi in the United States. Evolutionary connectivity through mitochondrial introgression and frequent multiclonality highlights the importance of deep sequencing approaches for resolving T. cruzi genetic diversity, with direct implications for understanding for transmission dynamics, disease monitoring and control.

BackgroundDog-mediated rabies remains a major neglected tropical disease and an important cause of preventable human mortality in sub-Saharan Africa. Despite ongoing control efforts, limited laboratory-confirmed surveillance data hinder accurate assessment of the disease burden and the development of evidence-based interventions in Ghana. This study investigated the epidemiological characteristics of laboratory-confirmed rabies cases among suspected animal submissions and evaluated the contribution of routine diagnostic surveillance to rabies control. Methodology/Principal FindingsA retrospective analysis was conducted using laboratory surveillance records of suspected rabies cases submitted to a regional veterinary laboratory in Ghana between 2020 and 2025. Brain tissue samples were examined using Sellers staining, the direct fluorescent antibody test (FAT), and polymerase chain reaction (PCR). Epidemiological information, including animal species, vaccination history, ownership status, bite history, and diagnostic outcomes, was extracted and analysed descriptively. Of the 51 suspected cases examined, 46 (90.2%) were laboratory confirmed as rabies. Domestic dogs accounted for 96.1% of submissions, while cats represented 3.9%. Most suspected animals were either unvaccinated (64.7%) or had unknown vaccination status (33.3%), and 90.2% had reportedly bitten humans before submission. Confirmed cases were detected throughout the study period, indicating sustained viral circulation and continued zoonotic risk within the study area. Conclusions/SignificanceThe consistently high proportion of laboratory-confirmed rabies cases demonstrates persistent transmission of rabies virus among domestic animals and highlights substantial risks to human health. The integration of conventional and advanced laboratory diagnostic methods strengthened surveillance and improved case confirmation. These findings underscore the urgent need to expand dog vaccination coverage, strengthen laboratory-based surveillance, improve bite reporting systems, and enhance collaboration between veterinary and public health sectors. Reinforcing integrated One Health strategies will be essential to accelerate progress towards the global goal of eliminating dog-mediated human rabies by 2030. Author SummaryRabies remains one of the worlds oldest and most devastating zoonotic diseases, which is fatal yet preventable, causing thousands of human deaths each year, particularly in Africa and Asia. In Ghana, domestic dogs are the principal reservoir of the rabies virus, but information on the epidemiology of animal rabies from routine laboratory surveillance is limited in many regions. We reviewed six years (2020 - 2025) of laboratory records from the Regional Veterinary Laboratory in Techiman, Bono East Region, to describe the epidemiological characteristics of suspected animal rabies cases and laboratory-confirmed infections. Of the 51 brain samples submitted for diagnosis, 46 (90.2%) were confirmed positive for rabies. Domestic dogs accounted for almost all submissions, and most confirmed cases involved animals that were either unvaccinated or had an unknown vaccination history. Furthermore, most suspected animals had reportedly bitten humans before laboratory investigation, highlighting the continuing public health risk posed by canine rabies. The study also documents how regional diagnostic capacity was strengthened through collaboration with national reference laboratories, enabling the routine use of internationally recommended diagnostic methods such as the fluorescent antibody test and polymerase chain reaction. These findings provide valuable evidence to support enhanced rabies surveillance, increased mass dog vaccination, improved public awareness, and strengthened One Health collaboration. Such measures are essential for reducing human rabies deaths and supporting Ghanas contribution to the global goal of eliminating dog-mediated human rabies by 2030.

Tick-borne pathogen (TBP) surveillance strategies that rely exclusively on targeted methods like PCR (PCR) or immunoblots do not benefit from strain-level sequence variation. Bacterial 16S rRNA metabarcoding offers more agnostic detection but is constrained in I. scapularis by the dominance of a maternally inherited endosymbiont, Rickettsia buchneri. Here we report the design and evaluation of three R. buchneri-specific blocking primers to suppress endosymbiont amplification during full-length 16S rRNA library preparation. Of these, primer 18F-Rb-C3 reduced R. buchneri relative abundance approximately 32-fold. We applied 18F-Rb-C3 with V4-16S metabarcode sequencing on 67 ticks collected from farm animals in Eastern Ontario and compared Borrelia species detection against qPCR. The V4-16S rRNA metabarcoding identified Borrelia species in 21 samples, whereas qPCR detected Borrelia in 24 samples and 11 samples were detected by both methods. Additionally, metabarcoding detected Anaplasma phagocytophilum in 12 samples, including seven samples coinfected with Borrelia, in the same assay. Variation relevant to strain surveillance was also detected by sequencing, though V4-16S was not sufficient to resolve closely related Borrelia genospecies or A. phagocytophilum variants. These findings demonstrate that blocking primer 18F-Rb-C3 enhances sensitivity of amplicon sequencing to the level of qPCR while also detecting other pathogens and sequence variants in a single assay.

Background: Alpha-gal syndrome (AGS) is an emerging disease, increasingly recognized as a public health concern in the United States. The primary cause of AGS in the United States is the bite of Amblyomma americanum ticks. White-tailed deer serve both as a preferred food source and as transport for A. americanum. In this work, we aim to quantify the effect of white-tailed deer abundance on number of AGS cases in United States counties. Methods: To mitigate concerns about confounding, we used the front-door formula, leveraging biological knowledge about the causal process. Due to lack of official data, our analysis relied on data made available by citizen science efforts. Results: We found that a higher number of reported white-tailed deer sightings in 2020 was associated with the county-level presence of A. americanum in 2024. In turn, county-level presence of A. americanum was associated with a higher number of self-reported AGS cases. We estimated that if white-tailed deer abundance had increased by 50%, 75%, or 100% in 2020, there would have been 89 (95%CI: 12, 252), 126 (12, 354) or 159 (6, 448) additional AGS self-reported cases in the US in 2025. Conclusions: The estimated associations are compatible with an effect of white-tailed deer abundance on AGS in the country. Due to measurement error, the low granularity of the available data, the ecological nature of the design, and the modelling choices, our effect estimates should be interpreted cautiously. Further studies are needed to quantify the population-level effect of white-tailed deer on AGS.

This data paper presents a curated, georeferenced dataset of the frequencies of the two main target site mutations (V1016G and F1534C) associated with resistance to pyrethroid insecticides in Aedes albopictus in Italy. Populations were collected in 102 out 107 Italian provinces between 2023 and 2025. Specimens were sampled by members of the Mosquito Insecticide Resistance Italian Network (MosqIRIT) as part of RN2 activities within the INF-ACT project. Genotyping was performed on 3,517 individuals by specific allele-specific PCR assays. Each record includes metadata on sampling site, administrative location, developmental stage, collection method, and mutation-specific genotype frequencies. To support spatial analysis modelling effort, the dataset integrates geographic, eco-climatic, and demographic data. This resource will support mosquito control programs, pyrethroid resistance monitoring and managing, as well as ecological modelling, and is compliant with the FAIR data program.

Tick-borne encephalitis virus (TBEV) is a zoonotic flavivirus maintained in complex enzootic cycles involving ticks and vertebrate hosts. While vector-mediated transmission has been extensively studied, host-associated mechanisms, including vertical transmission and prolonged viral carriage in natural reservoir populations, remain poorly understood. Here, we investigated TBEV circulation, genetic diversity, and evidence for vertical transmission in bank voles (Clethrionomys glareolus) from a highly endemic region of North-eastern Poland. A total of 258 wild rodents were screened using molecular and serological approaches. TBEV RNA was detected in 14.3% of individuals, whereas seroprevalence was substantially lower (6.6%), revealing limited concordance between viral presence and humoral response under natural conditions. Near-complete genome sequences were obtained from 10 TBEV-positive individuals, representing the first near-complete TBEV genomes generated from wild rodents in Poland. All isolates belonged to the European subtype (TBEV-Eu), and phylogenetic analysis revealed two genetically distinct viral clades co-circulating within a narrow spatiotemporal window. Importantly, TBEV RNA and/or envelope protein were detected in embryos from naturally infected females, providing evidence consistent with vertical transmission in a wild reservoir host. Together, these findings suggest that vertical transmission and complex host infection dynamics may be underappreciated components of TBEV maintenance in natural reservoir populations. Our results highlight the need to integrate vertebrate host dynamics into models of TBEV ecology and support expanded wildlife-based surveillance to better understand and predict zoonotic risk.

Traditional surveillance underestimates Lyme disease (LD) incidence in the United States (US). We aimed to estimate national LD incidence using validated algorithms to identify LD cases in administrative claims data. We identified potential LD cases in commercial and Medicare claims, classified cases by disease stage, adjusted case counts using algorithm-specific positive predictive values, and standardized the adjusted counts to the US population. The study population included >66 million individuals per year. After adjustment and standardization, we estimated 191.7, 26.8, and 16.6 new cases per 100,000 population in high-incidence, neighboring, and low-incidence states, respectively, with 24% of cases diagnosed with disseminated disease. The relative burden of disseminated LD was highest in low-incidence states (28.6%) and increased with age. This study corroborates published estimates of national LD incidence and elucidates patterns of disease stage at diagnosis. The substantial burden of disseminated LD underscores the need for earlier detection and treatment of LD.

Ticks transmit pathogens across wildlife, domestic animals, and humans, and are therefore considered important vectors under the One Health framework. In India, the burden of tick-borne diseases, such as Kyasanur Forest Disease and Crimean-Congo Hemorrhagic Fever, remains poorly quantified due to fragmented surveillance and limited spatial data. Here, we present TickMapKB, a curated spatial knowledgebase documenting 72 tick species across more than 600 georeferenced locations in India. The majority of the species belonged to the genera Haemaphysalis, Rhipicephalus, and Hyalomma. Additionally, the resource integrates host associations, pathogen and disease information, morphological identification keys, acaricide resistance profiles, and protein annotations into a single, interactive platform accessible at https://cb.imsc.res.in/tickmapkb/. Specifically, the resource captures 53 pathogens, over 3000 protein annotations, and morphological keys from 26 published resources. TickMapKB thus provides researchers, clinicians, and other stakeholders with integrated spatial, biological, and resistance information to support surveillance planning, resistance management, and tick-borne disease risk assessment.

Alpha-gal syndrome (AGS) is a tick bite-associated allergic condition induced by the lone star tick (Amblyomma americanum). Illinois had no confirmed AGS case data as of January 2026, when mandatory reporting began under the state's TICK Act, leaving practitioners without data to guide screening or resource allocation. We constructed a county-level proxy risk score using a Bayesian conditional autoregressive spatial model applied to three Illinois surveillance sources from 2019-2022: tick abundance, ehrlichiosis cases, and tick establishment status, all linked by a shared vector. Ehrlichiosis was modeled as a population-adjusted rate, ticks as a relative-intensity index, and establishment status as a fixed ecological component. The combined risk score identified a high-risk cluster in far southern Illinois that remained stable across alternative weighting scenarios. This approach is transferable to jurisdictions lacking direct AGS surveillance and offers a starting point for clinician education pending validation against confirmed case data.

BackgroundAedes albopictus is among the worlds most invasive mosquito species and an important vector of dengue, Zika, and chikungunya viruses. Its global spread has been strongly associated with human-mediated transport and international trade, particularly through commodities such as used tires and ornamental plants. However, integrative studies combining population genetics, microbial symbiosis, and trade connectivity remain limited in Latin America, constraining understanding of invasion dynamics and dispersal processes. MethodsAedes albopictus populations from five Colombian departments sampled between 2019 and 2023 were analyzed using eight microsatellite loci and a [~]1.3-kb mitochondrial COI fragment. Wolbachia infection and lineage composition (wAlbA/wAlbB) were evaluated by PCR, and arbovirus detection (DENV, CHIKV, ZIKV) was performed using multiplex RT-PCR. Nuclear and mitochondrial differentiation (FST, {Phi}ST), mito-nuclear discordance, and trade-related connectivity were evaluated in relation to geographic, national transport, and international trade variables derived from customs databases. ResultsMicrosatellite analyses revealed admixed but structured populations consistent with multiple introductions and contemporary gene flow. Colombian populations showed nuclear genetic affinities with Asian, European, and North American populations, supporting a complex invasion history involving multiple geographically distributed lineages. In contrast, mitochondrial COI data showed evidence of regional genetic structure and heterogeneous mito-nuclear discordance among several population pairs. Notably, nuclear and mitochondrial markers captured contrasting dimensions of the invasion process: nuclear differentiation was positively associated with international trade intensity, particularly shipment frequency and used tire importation, whereas mitochondrial differentiation retained stronger regional structure and showed no detectable association with trade-related variables. Wolbachia prevalence ranged from 34% to 100% across departments and showed exploratory patterns consistent with localized mitochondrial differentiation. Natural detection of DENV and CHIKV RNA in larvae provided evidence of local arbovirus circulation. ConclusionsIntegrating population genetics, trade connectivity, and Wolbachia screening supports a scenario in which the Colombian invasion of Ae. albopictus has been shaped by multiple introductions, contemporary human-mediated connectivity, and partially discordant mito-nuclear histories. These findings highlight how different genomic compartments retain complementary signatures of invasion dynamics, with contemporary trade-associated connectivity primarily reflected in nuclear structure and regional lineage persistence retained in mitochondrial variation. Author SummaryThe Asian tiger mosquito, Aedes albopictus, is one of the worlds most invasive mosquito vectors and continues to expand across Latin America through human transportation and trade networks. However, the processes shaping its spread in the region remain poorly understood. We combined population genetics, international trade data, Wolbachia screening, and arbovirus surveillance to investigate the invasion dynamics of Ae. albopictus in Colombia. Our results revealed evidence of multiple introductions and ongoing genetic admixture, with international trade connectivity emerging as an important predictor of contemporary nuclear genetic structure. In contrast, mitochondrial DNA retained stronger regional patterns, generating heterogeneous mito-nuclear discordance among populations. These findings suggest that different genomic compartments retain distinct signatures of the invasion process, with trade-associated connectivity reflected primarily in nuclear variation and stronger regional structure preserved in mitochondrial lineages. More broadly, our study highlights the complex invasion dynamics of Ae. albopictus in Latin America illustrates how integrating genetics and human connectivity data can improve understanding of invasive vector spread.

Accurately determining the mating status of the agricultural fruit fly pest Ceratitis capitata, commonly known as Medfly, is essential for timely and effective eradication efforts. To overcome the limitations of subjective DAPI-based staining assessments of females captured in Jackson dry traps and Multilure liquid traps, we developed a multi-tier molecular diagnostic method that unequivocally detects mating status using DNA probes targeting the male-specific Y114 locus on the Y-chromosome of the species. Our protocol integrates morphological evaluation with increasingly sensitive molecular assays through the following steps: 1) A preliminary quality assessment of the specimens physical condition, DNA preservation, and mating status using conventional PCR followed by agarose electrophoresis (cPCR); 2) Quantification and real-time detection of sperm presence via quantitative PCR (qPCR); and 3) Detection of trace sperm amounts through droplet digital PCR (ddPCR). This PCR-based framework is designed for samples collected in the field, enabling accurate analysis of specimens exposed to adverse environmental conditions and varying levels of preservation after 2- and 3-weeks weathering times in traps. It allows quantitative determination of mating status even when sperm concentrations are extremely low, such as during transient copulation, and achieves detection limits down to approximately 14 spermatozoa in a mated female. By accounting for variable specimen quality and the performance characteristics of each molecular platform, this tiered approach ensures highly sensitive and unequivocal detection of mated females. The methodology can be used to assist eradication efforts across the C. capitata geographic range through the timely detection of mated females, halting their expansion and establishment into novel regions reducing control and eradication costs.

While emerging diseases pose a global threat to amphibians, the dynamics of understudied vector-borne blood pathogens remain poorly understood. Pathogen occurrence is driven by a combination of environmental, ecological, and phylogenetic factors, yet how these drivers shape blood pathogen communities in tropical amphibians is largely unknown. In this study, we used molecular screening and phylogenetic linear models (PGLMMs) to evaluate how climate and ecomorphology influence the incidence of three blood pathogen groups (Trypanosomatidae, Hepatozoon, and Rickettsia) in wild anurans from a protected area in the Brazilian Atlantic Forest. Among 93 individuals sampled, over 93% were infected with at least one pathogen. Trypanosomatidae was the most common (76.3%), followed by Rickettsia (69.9%) and Hepatozoon (16.1%). Pathogen responses to temperature were contrasting: Hepatozoon occurrence increased in warmer periods, while Trypanosomatidae declined. Furthermore, rheophilic species showed a lower probability of Rickettsia infection, providing the first evidence that microhabitat use influences blood pathogen dynamics in amphibians. Our findings demonstrate that hemoparasites prevalence is driven by a multifaceted interplay of variables, highlighting that conservation strategies must account for these pathogen-specific responses to habitat use and environmental change, even within protected areas.

BackgroundMalaria caused by Plasmodium knowlesi and lymphatic filariasis caused by Brugia malayi are mosquito-borne infections with non-human primates as reservoirs. P. knowlesi has emerged as a significant cause of human malaria in Southeast Asia over the past two decades. Belitung district, Indonesia, was until recently assumed to have eliminated B. malayi until infections were detected in humans and long-tailed macaques. To investigate whether the local reservoir of B. malayi is also a reservoir for malaria we screened macaques from 4 areas in Belitung for malaria parasites. Methods and findingsBlood samples from 163 long-tailed macaques (Macaca fascicularis) that had been tested for B. malayi were examined by quantitative PCR assays specific for Plasmodium spp., P. knowlesi, P. inui, P. coatneyi and P. cynomolgi. A total of 130 macaques (79.8%) tested positive in the pan-Plasmodium qPCR assay. Plasmodium inui was most prevalent (41.7%), followed by P. knowlesi (38.7%), P. coatneyi (24.5%) and P. cynomolgi (13.5%). Multiple species infections, with 2-3 Plasmodium species were detected in 37% of macaques. Notably, 20 (91%) of 22 B. malayi-positive macaques were co-infected with at least one Plasmodium species. We sequenced the complete mitochondrion from 9 samples diagnosed by qPCR as mono-infections. Phylogenetic analysis confirmed 7 as P. knowlesi, and the other two as P. inui and P. coatneyi. Phylogenetic and pairwise analysis revealed that P. knowlesi isolates from Belitung were closely related to each other and to P. knowlesi from humans and monkeys from Thailand, Malaysia and Indonesia. ConclusionsMolecular evidence shows high prevalence of zoonotic malaria parasites in macaques from Belitung, emphasizing the risk of human transmission. Increased surveillance, improved diagnostics, and targeted interventions are needed to prevent zoonotic spillover of P. knowlesi as it has been observed for B. malayi in Belitung. Author summaryWe examined macaques for Plasmodium parasites in Belitung Island, Indonesia, a region classified as free of locally transmitted human malaria. Plasmodium knowlesi is a monkey parasite that commonly infects humans and has emerged as a concern in Southwest Asia over the last 20 years. In Belitung, macaques are infected with Brugia malayi, a filarial nematode, that causes lymphatic filariasis in humans. Blood samples from 163 macaques were screened for Plasmodium DNA and 80% carried malaria parasites. Plasmodium inui was most common, followed by P. knowlesi, P. coatneyi, and P. cynomolgi. Many macaques were infected with multiple parasites simultaneously, but no host was infected with all four Plasmodium species. Ninety one percent of B. malayi-positive macaques were co-infected with malaria parasites, including P. knowlesi, indicating multi-parasite infections that could potentially spread to humans. Analysis of the mitochondrial genome of 7 P. knowlesi isolates from Belitung showed that they were most similar to each other and both human and monkey samples from Malaysia, Thailand and Indonesia. The study highlights the need for enhanced malaria monitoring and prevention, given the complex epidemiology of co-infecting parasites and risk to humans and animals.

The black-legged tick (Ixodes scapularis), a key vector of Lyme disease, anaplasmosis, and babesiosis, exhibits regionally distinct patterns of seasonal activity driven by climate. Consequently, the relative timing of larval and nymphal activity varies across geographic locations, influencing pathogen transmission dynamics. Early-emerging nymphs may increase pathogen transmission, whereas early-emerging larvae may reduce transmission. In addition, synchrony between the two life-stages facilitates co-feeding transmission, which contributes to pathogen maintenance and coinfection risk. Temperature is thought to be an important driver of tick phenology, but existing mechanistic models that incorporate temperature fail to accurately capture the timing of larval and nymphal tick activity. To address this limitation, we developed a mechanistic model that includes two additional factors: humidity-dependent questing and low rates of overwinter development. To assess the value of these factors for explaining real-world patterns, we fitted alternative models to tick collection data from the National Ecological Observatory Network. In doing so, we found that explicitly incorporating humidity is necessary to reproduce observed tick phenology, with larval ticks being especially sensitive to relative humidity compared to other life stages. In addition, we found that accounting for humidity had a larger effect at Mid-Atlantic sites than at Northeastern sites, underscoring the importance of region-specific interactions between temperature and humidity in shaping I. scapularis phenology. By more accurately capturing tick seasonality compared to existing mechanistic models, our model illustrates the importance of accounting for factors beyond temperature for investigating how climate variability influences seasonal tick activity and pathogen transmission.

Heartland virus (HRTV) and Bourbon virus (BRBV) are emerging tick-borne arboviruses transmitted by the lone star tick (Amblyomma americanum) that have caused dozens of cases of human disease in the United States, including multiple fatalities. Despite their significance, entomological, clinical, and molecular surveillance remains sparse, limiting our understanding of HRTV and BRBV distribution and risk. The Nebraska Department of Health and Human Services and the Nebraska Public Health Laboratory expanded tick-borne pathogen surveillance to include HRTV and BRBV in A. americanum ticks beginning in 2024. Here, we report the first detections of HRTV and BRBV in Nebraska and present a multi-segment phylogenetic analysis of complete virus genomes. Using a newly developed amplicon-based whole genome sequencing strategy, we generated complete HRTV genomes from three PCR-positive A. americanum pools collected in two counties in eastern Nebraska. Additionally, we generated a complete BRBV genome from a single PCR-positive A. americanum pool. A time-calibrated phylogenetic analysis of the L segment containing all publicly available HRTV sequences determined that the 3 genomes from Nebraska form a monophyletic cluster that initially diverged from viruses isolated from Missouri in the early 2000s, corresponding with the expansion of A. americanum into Nebraska. A phylogenetic analysis of BRBV segment 2 indicates that the genome from Nebraska sits on a long branch and likely diverged from other genomes sequenced in the early 2010s. Topological concordance across each segment suggests minimal occurrences of reassortment among the HRTV and BRBV genome sequences. These findings document the expansion of HRTV and BRBV to the western margin of the A. americanum range and demonstrate the utility of enhanced surveillance and whole genome sequencing for characterizing the spread of tick-borne arboviruses.

Rabies remains a major zoonotic public health threat, associated with approximately 59,000 annual deaths globally, with Africa bearing 36% of this burden. In Uganda, an estimated 36 human deaths and 1.8 million dog bite exposures occur annually, yet national dog vaccination coverage remains critically low at approximately 10%. This study investigated factors associated with rabies vaccination uptake among dog owners in Butaleja Town Council, Eastern Uganda, a high-risk setting. A cross-sectional survey was conducted among 173 dog owners between May 18 and June 28, 2024, using semi-structured questionnaires. The primary outcome was vaccination uptake ([&ge;]1 dose in the past year). Modified Poisson regression with robust variance estimated adjusted prevalence ratios for all socio-demographic, veterinary system, health system, dog-related, and knowledge factors. Among 173 dog owners, 57.2% vaccinated at least one dog, corresponding to 54.6% (166/304) of all dogs vaccinated. Factors associated with vaccination uptake included older age (21-40 years: aPR 1.43, 95% CI: 1.15-1.71; 41-60 years: aPR 1.53, 95% CI: 1.22-1.82; [&ge;]61 years: aPR 1.56, 95% CI: 1.33-2.23), higher education (primary: aPR 1.20, 95% CI: 1.00-1.45; secondary: aPR 1.55, 95% CI: 1.20-2.00), higher income, access to veterinary clinics (aPR 2.50, 95% CI: 1.52-3.75), and participation in community education sessions (aPR 1.71, 95% CI: 1.34-2.15). While household-level uptake showed moderate engagement, the resulting dog population vaccination coverage of 48.4% remained substantially below the 70% World Health Organization threshold required for herd immunity. Achieving the "Zero by 30" elimination dog-bite mediated Rabies target in this setting requires targeted interventions addressing the identified associated factors including; enhanced community education, improved veterinary service accessibility, reminder systems, and strategies to convert partial household vaccination into complete coverage particularly among younger, less educated, and lower-income dog owners.

Outer surface protein A (OspA)-based Lyme disease vaccines elicit antibodies that inhibit transmission of infectious Borrelia burgdorferi spirochetes from ticks to humans, although the mechanism by which this occurs is unclear. Here we demonstrate using high-resolution, single-cell fluorescence video microscopy that B. burgdorferi motility is arrested within minutes by human OspA-specific monoclonal antibodies and polyclonal OspA antisera from mice immunized with a candidate OspA mRNA lipid nanoparticle vaccine.

BackgroundThe ecological dynamics between Trypanosoma parasites and their wild mammalian hosts, such as bats and armadillos, are complex. Recent 18S rRNA metabarcoding studies have reported extraordinary levels of hidden parasite diversity and frequent multi-lineage coinfections within individual wild hosts. However, the boundary between genuine biological coinfection and methodological artifact remains difficult to establish. Based on Gauses principle of competitive exclusion, the mammalian bloodstream represents a highly constrained niche where stable coexistence of identical ecological competitors is theoretically rare. We hypothesize that previously reported hyper-diverse Trypanosoma coinfections are largely bioinformatic artifacts, and that true intra-host dynamics instead favor single-lineage dominance. MethodsTo test this hypothesis, we sequenced samples from 27 wild armadillos (Dasypus novemcinctus) and 26 bats from Ecuador. The 18S rRNA gene was amplified via nested PCR and sequenced using an Oxford Nanopore Technologies MinION platform. We developed a progressively stringent bioinformatics pipeline to evaluate coinfection hypotheses. Raw reads were processed through three alignment scenarios: Lenient, Moderate, and Conservative. These scenarios modulate sequence identity, mapping quality (MAPQ), and coverage thresholds to effectively isolate true biological signals from alignment ambiguity. ResultsUnder lenient alignment parameters, the resulting profiles mirrored previous literature, exhibiting massive apparent intra-host multi-lineage diversity. However, as bioinformatic stringency increased to conservative thresholds ([&ge;] 98% sequence identity, [&ge;] 99% coverage, and MAPQ [&ge;] 30), artifactual pseudo-coinfections collapsed. The highly restricted dataset demonstrated overwhelming single-lineage dominance, validating only three active mixed infections out of the retained samples. Furthermore, our rigorous pipeline isolated rare but genuine biological signals, including the detection of Trypanosoma cruzi marinkellei--historically considered a bat-restricted subgenus--within the terrestrial armadillo cohort. We also confirmed the presence of T. cruzi DTU III (TcIII) in Ecuadorian armadillos, representing a significant biogeographical record for the region. ConclusionsOnce methodological noise is computationally stripped away, active multi-strain Trypanosoma coinfections in the host bloodstream are revealed to be ecologically anomalous. Our findings strongly support the principle of competitive exclusion, suggesting established lineages actively suppress competitors. While Oxford Nanopore sequencing offers necessary resolution for wildlife parasitology, fine-tuning algorithmic parameters is critical to accurately represent host-parasite networks and prevent the artificial inflation of intra-host diversity metrics. Author summaryPrevious studies using DNA metabarcoding have reported that wild mammals, such as bats, frequently harbor complex communities of multiple Trypanosoma parasite lineages simultaneously. However, ecological principles suggest that identical competitors struggle to coexist stably within a constrained environment like the host bloodstream. To investigate whether these reported high coinfection rates reflect true biology or methodological artifacts, we sequenced the 18S rRNA gene of Trypanosoma from 26 bats and 27 armadillos in Ecuador. We processed the sequencing data through computational pipelines with progressively stricter filtering parameters. We observed that under lenient filtering, animals appeared to have highly diverse, mixed infections. Conversely, when strict parameters were applied to remove potential analytical noise, the artificial complexity collapsed, revealing that the vast majority of hosts were dominated by a single parasite lineage. We confirmed only three active mixed infections in our highly restricted dataset. Our findings indicate that active multi-strain Trypanosoma coinfections are rare, aligning with the principle of competitive exclusion. These results highlight the necessity of applying rigorous bioinformatic filters to accurately evaluate host-parasite interactions and avoid overestimating diversity metrics.