Ticks and Tick-borne Diseases

Tickborne diseases are a significant burden in many parts of the world. In the upper Midwestern United States, Lyme disease is the most common tickborne disease. It is carried by Ixodes scapularis. This vector can also transmit the pathogens causing anaplasmosis, babesiosis, ehrlichiosis, and several more tickborne diseases in this region. There is also concern for other tick species, such as Amblyomma americanum, that are expanding their ranges northward. We launched a citizen science passive tick surveillance program in 2024 to investigate tick species ranges in the upper Midwest, as well as the pathogens carried by I. scapularis. We received over 12,000 ticks in the first two years of this program, primarily from Wisconsin. While we received submissions of adult A. americanum outside of their endemic range, we did not see evidence of establishment in our study area. We measured pathogen prevalence in adult female I. scapularis (n=707) and observed 51% positivity for Borrelia burgdorferi, 9% for Babesia microti, 9% for Anaplasma phagocytophilum, and 3% for Ehrlichia muris eauclairensis. Multiple pathogens were identified in 14% of tested specimens, and significant associations were observed between B. burgdorferi and B. microti, and B. burgdorferi and E. muris eauclairensis. Pathogen prevalences varied across time and geography. Our results can begin to inform risk assessment for tickborne diseases in our region. A non-technical version of this document with interactive maps is available here: https://storymaps.arcgis.com/stories/8008c9d710b5400599f3c6cf88b2c546 Our online data dashboard is available here: redcap.link/TICS

Alongshan virus (ALSV) is an emerging tick-borne segmented RNA virus belonging to the Jingmenvirus group and has been reported in humans, ticks, and vertebrates across Asia and Europe. Despite its potential public health relevance, its distribution and genetic diversity remain poorly characterized in several European regions where tick-borne pathogens are endemic. In this study, we developed a specific TaqMan-based real-time RT-PCR assay targeting a conserved region of ALSV segment 2 and used it to investigate the presence of ALSV RNA in Ixodes ricinus ticks collected in northeastern Italy. The assay showed high linearity over a broad dynamic range and no cross-reactivity with related flaviviruses. A total of 212 archival tick samples collected between March 2021 and November 2022 were screened, and 28 samples (13.2%) tested positive for ALSV RNA. Positive ticks were detected in the provinces of Belluno and Vicenza and included individual adult males and nymph pools. A subset of positive samples was further characterized by nested PCR and Sanger sequencing of all four genomic segments. Phylogenetic analyses showed that Italian ALSV sequences clustered within the broader European ALSV diversity and were closely related to strains from Central and Northern Europe, without forming a distinct country-specific lineage. Sequence comparisons suggested purifying selection and revealed differences in predicted structural proteins between European and Chinese strains. These findings provide the first molecular evidence of ALSV circulation in Italy and support further studies to clarify its epidemiology, host range, genetic diversity, and potential clinical relevance. IMPORTANCEAlongshan virus (ALSV) is an emerging tick-borne virus identified in febrile patients in China and subsequently detected in ticks in Russian Federation and several European countries. Although severe disease has not yet been reported in humans, surveillance and elucidation of the virus distribution are essential to assess its pathogenicity and potential public health impact. We developed a specific real-time RT-PCR protocol and detected ALSV in Ixodes ricinus ticks collected in northeastern Italy. Sequence analyses suggested multiple introductions and revealed differences in structural proteins between European and Chinese strains, suggesting potential adaptation and differences in pathogenicity. Since the clinical signs of ALSV infection in humans may overlap with those of tick-borne encephalitis (TBE), differential diagnostic procedures should be developed to improve patient management, particularly in TBE-endemic regions such as northeastern of Italy.

Fasciolosis caused by Fasciola hepatica is an economically important disease in sheep and cattle. Knowledge of the population genetic structure of F. hepatica is important for understanding gene flow and informing disease control. In the present study, we designed, developed, and validated a multilocus sequence typing (MLST) scheme based on six markers. These markers were selected by aligning newly sequenced whole-genome sequence (WGS) data with available reference genomes and selecting variable regions with five or more single-nucleotide polymorphisms SNPs from different scaffolds of the F. hepatica reference genome Fasciola 10x pilon (GCA_900302435.1). Twenty markers were initially identified, of which 12 were multiplexed for deep amplicon sequencing after validation on worm and faecal eggs DNA; six markers were ultimately retained for downstream population genetics analysis. These markers were used to investigate population genetic structure in 15 cattle- and 27 sheep-derived F. hepatica populations in UK. A total of 53 unique alleles from six MLST markers were identified from 30 faecal (cattle = 13, sheep = 17) and 12 adult worm (cattle = 2, sheep = 10) populations. Shared alleles were observed in sheep- and cattle-derived populations. The highest allelic variation was observed in the Scottish Borders, Southern Scotland, and South-West England, and the lowest in North-West England. Minimal genetic differentiation was observed between cattle- and sheep-derived populations, with most genetic structuring within rather than between populations. Five markers showed high allelic polymorphism, whereas one marker showed low levels of allelic polymorphism, highlighting the importance of multilocus approaches. Overall, this six MLST-marker panel provides a tool for population genetic studies, revealing high gene flow and clonal expansion of F. hepatica across hosts and regions in the UK.

Puumala hantavirus (PUUV, Orthohantavirus puumalaense) is one of the primary causative agents of haemorrhagic fever with renal syndrome in Europe and is maintained in natural populations of the bank vole (Clethrionomys glareolus, also known as Myodes glareolus). Despite public health relevance, we are only starting to understand the molecular properties and interplay between environmental and ecological factors of the pathogen that explain PUUV infection in bank voles. Here, we investigated PUUV occurrence, genetic structure, and environmental associations in bank voles sampled from two boreal forest areas in northern Sweden, during a complete vole population cycle (2020-2023). In total, 519 voles were screened for PUUV RNA using targeted reverse transcription PCR (RT-PCR). PUUV small (S-) segment RNA was detected in both study areas and observed infection patterns varied with sex, body weight, season and year. Specifically, we detected significant interactions between season and area and between season and body weight, with males showing consistently higher infection probabilities. Infection probability was also higher during periods of increased vole abundance and peaked in 2022. Phylogenetic analysis of partial S segment sequences demonstrated that all detected sequences clustered within the North-Scandinavian PUUV lineage, with no apparent spatial differentiation, indicating limited genetic structuring between the sampling areas. Habitat analyses at multiple spatial scales did not identify significant associations between PUUV occurrence and land-use variables, suggesting that infection dynamics were driven primarily by host demographic and temporal factors rather than broad-scale habitat composition. These findings highlight the importance of host demographics and temporal dynamics in shaping PUUV epidemiology in its reservoir, and provide additional insight into the molecular ecology of PUUV in northern Europe.

Rickettsia senegalensis is a novel Rickettsia species isolated from cat fleas, Ctenocephalides felis, in Senegal. Genomic analysis confirmed its status as a distinct species, placing it within the transitional Rickettsia group, within a R. felis cluster. Furthermore, rickettsial genes identical to those of Rickettsia senegalensis had been already identified in several hematophagous arthropods, including fleas and ticks parasitizing various hosts such as cats, dogs, opossums, and rodents in tropical and subtropical regions all over the world. It has also been detected in cat tissues, suggesting a potential host-pathogen association. Here we formally propose Rickettsia senegalensis sp. nov. as a new species. The type strain of this species is strain PU01-02T (= CSUR R184T = DSM 28250T). Strain PU01-02T grows aerobically in XTC-2, SF9, and LD652 cell lines at 28 {degrees}C in a CO2-free atmosphere. The genome of strain PU01-02T has a size of 1.62 Mb and a G+C content of 33.2%. RepositoriesThe genome sequence of Rickettsia senegalensis sp. nov. strain PU01-02T has been deposited in GenBank under accession number JBVYTQ000000000, and the rrs, gltA, ompB and sca4 gene sequences under accession numbers KF666476, KF666472, KF666470, KF666474, respectively. The plasmid accession numbers are PZ272915, PZ272916, and PZ272917, for pRS01, pRS02 and pRS03, respectively.

Objectives: To characterise the national epidemiology of tick-induced mammalian meat allergy (MMA) in Australia, including temporal trends in Gal specific IgE testing and case detection, geographic distribution, demographic risk factors, and longitudinal antibody dynamics. Design: Retrospective analysis of Gal sIgE ImmunoCAP test results from 1 January 2014 to 31 December 2024. Setting, Participants: Deidentified laboratory records from 14,075 individuals tested across Australia, with residential postcodes mapped to Statistical Areas Level 3 (SA3). A subset of 1,515 individuals with repeat testing contributed to longitudinal analyses. Main outcome measures: Test volumes, suspected MMA case counts, positivity rates, spatial clustering metrics, demographic risk ratios, and longitudinal Gal sIgE trajectories. Results: Overall, 35.7% (5,025) of individuals tested positive. Testing volume increased 331% over the study period, with case detection accelerating sharply from 2020. Decomposition analysis attributed 59-81% of case growth to expanded testing, with the remainder unexplained by surveillance expansion alone. Cases were concentrated along the eastern seaboard within the range of Ixodes holocyclus, with extreme spatial clustering: three SA3 regions accounted for over one-quarter of national cases. Females comprised the majority of positive cases, and MMA risk increased with age, peaking at 45-74 years. Among 1,515 individuals with serial testing, Gal sIgE levels antibodies declined predictably in 93% of people. Conclusions: This first national assessment of MMA in Australia reveals a substantial, geographically concentrated, and growing burden. The extreme spatial concentration of cases suggests that targeted public health interventions could efficiently address a large proportion of national disease burden. While testing expansion is the dominant driver of rising case numbers, it does not fully account for observed trends, and prospective studies are needed to disentangle surveillance effects from genuine disease emergence. Declining antibody levels support the utility of serial testing for clinical monitoring, though persistent sensitisation underscores the need for sustained risk mitigation and continued surveillance.

Black-footed ferrets (Mustela nigripes) are highly susceptible to sylvatic plague caused by Yersinia pestis, but the genetic basis of this vulnerability remains poorly understood. Here, comparative immunogenomic analyses across Carnivora species identified a conserved class of immunoglobulin lambda variable (IGLV) genes with unusually long antigen-binding sites (CDRL1) that are common among Caniformia species but absent in Feliformia species. First discovered in the domestic ferret (Mustela putorius furo), these genes encode tyrosine-rich and anionic motifs resembling the chemokine receptor CCR5 and contain experimentally validated sulfotyrosines previously associated with pathogen-interacting interfaces. Evolutionary analyses revealed distinct selective pressures across Caniformia lineages and showed strong purifying selection acting on long-CDRL1 IGLV genes in mustelids and bears. Antibody repertoire sequencing demonstrated that these genes are actively utilized in expressed repertoires and that their usage correlates with evolutionary conservation. Functional analyses of monoclonal antibodies derived from the long-CDRL1 IGLV gene identified an antibody that significantly reduced intracellular Y. pestis survival in macrophages and revealed a positive correlation between anti-plague activity and sulfotyrosine signal. Notably, all analyzed black-footed ferrets carried a frameshifting deletion in the long-CDRL1 IGLV gene resulting in loss of its expression in antibody repertoires. Together, these findings uncover a germline-encoded immunoglobulin feature conserved across dog-like carnivores and suggest a potential link between antibody germline variation and immune responses to plague.

Wildlife hosts play important roles in the ecology and transmission of vector-borne parasites, yet information on host associations remains scarce in many biodiverse tropical regions. Within a One Health framework, characterizing parasite diversity in wildlife can improve understanding of ecosystem health and disease emergence. Road-killed animals provide a non-invasive opportunity to investigate host-parasite interactions while minimizing disturbance to natural habitats. We screened 127 liver and intestinal tissue samples obtained from 76 road-killed vertebrates collected near protected areas in two Ecuadorian biodiversity hotspots, the Tropical Andes and Choco-Darien, for trypanosomatids and other vector-borne microorganisms. Molecular analyses targeted the 18S rRNA and cytochrome b genes of trypanosomatids and included additional screening for Trypanosoma cruzi, Trypanosoma rangeli, Rickettsia spp., and piroplasmids. Twenty-nine samples were positive for kinetoplastids. We detected diverse trypanosomatids representing the genera Leishmania, Porcisia, Trypanosoma, Phytomonas, Blastocrithidia, and Obscuromonas, as well as free-living kinetoplastids of the order Neobodonida. The most frequently detected species was Leishmania amazonensis, identified in 17 samples from at least 13 species of birds, reptiles, and caecilians, predominantly in liver tissue, suggesting previously unrecognized host associations. We also identified a putatively novel species of Porcisia and three potentially undescribed avian trypanosomes belonging to the subgenus Ornithotrypanum. No evidence of T. cruzi, T. rangeli, Rickettsia spp., or piroplasmids was found. Our findings identify birds, reptiles, and caecilians as potential reservoir hosts of L. amazonensis. In addition, we substantially expanded current knowledge of kinetoplastid diversity in Ecuadorian wildlife. This study demonstrates the value of road-killed animals as a practical, non-invasive resource for wildlife pathogen surveillance and highlights the importance of integrating biodiversity research into One Health approaches to better understand parasite transmission dynamics in rapidly changing tropical ecosystems. Author summaryMany parasites that affect humans circulate naturally in wildlife, but identifying their animal hosts is often difficult in remote, biodiverse regions. We used road-killed animals as a non-invasive source of biological material to investigate parasites in wildlife from two biodiversity hotspots in Ecuador. By analyzing tissues from birds, reptiles, amphibians, and mammals, we found a remarkable diversity of kinetoplastid flagellates, a group that includes the agents of Chagas disease and leishmaniasis. Although we did not detect human-infective trypanosomes, we repeatedly identified Leishmania amazonensis (a species causing human disease) in birds, reptiles, and caecilians. These vertebrate groups have not previously been recognized as potential hosts of this parasite. We also discovered several undescribed trypanosomatid species, emphasizing how little is known about parasite diversity in tropical wildlife. Our results show that road-killed animals can provide valuable information on host-parasite interactions without disturbing living populations. Such surveillance contributes to One Health efforts by improving our understanding of how environmental change, wildlife, and human health are interconnected.

Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus, remains a significant public health and veterinary problem in endemic regions. Although chemotherapy and control programs exist, the development of complementary immunotherapeutic tools is increasingly needed. This study evaluated the generation and functional activity of hyperimmune serum (HIS) produced in three adult male castrated llamas (Lama glama) immunized with antigenic material derived from protoscoleces (PSCs) of the parasite. Sera collected after each of the first six immunizations were assessed by ELISA to quantify antigen-specific IgG responses, and their biological effects were tested in vitro using viable PSCs. Motility was measured using video-assisted paired-image scoring across serial serum dilutions (1:2-1:2048), and the methylene blue exclusion assay was used to assess viability. Hyperimmune serum produced a clear, reproducible, dose-dependent inhibition of PSC motility and viability. Higher titers of early inoculations reduced motility by 70-85%, while sera from the fifth and sixth inoculations achieved complete suppression. Naive serum and PBS controls showed no inhibitory effect. ELISA titers strongly correlated with biological activity, indicating that higher humoral responses predicted functional inhibition. These findings demonstrate the feasibility of generating potent anti-Echinococcus granulosus polyclonal antibodies in camelids and support their potential application in passive immunization strategies. The study establishes a foundation for future development of llama-derived immunobiological reagents, including nanobody-based tools, for the control of cystic echinococcosis.

O_LIStudying tick behavior is crucial for understanding how climate, disturbances, and land-use changes shape tick populations and tick-borne disease risk. Mark-release-recapture studies can provide valuable answers to questions regarding tick movement and behavior, population sizes, and survivorship. C_LIO_LIStandard tick mark-release-recapture provides limited resolution to understanding individual behaviors, limiting our ability to answer questions that require repeated observations of the same individuals. We developed a new, operationally simple method to track large populations of individual ticks over space and time. C_LIO_LIWe found non-random movement patterns, including directed movement towards grass, vegetation-dependent dispersal distances and rate, and sex-based differences in movement. C_LIO_LIPractical implication: This method can be applied to other tick species to assess tick longevity, determine dispersal ranges and rate, and analyze questing behavior and success. C_LI

Ixodes scapularis ticks transmit a number of pathogens important to human health, including Borrelia burgdorferi, the causative agent of Lyme disease. While I. scapularis is found across the Eastern United States, Lyme disease transmission is largely limited to the Northeast and Upper Midwest and is nearly absent in the South. This indicates that differences in northern versus southern clades of I. scapularis are associated with differences in Lyme disease transmission risk. I. scapularis is undergoing range expansion, including into the Great Plains region of the United States. Determining where I. scapularis populations in the Great Plains originated can inform future risk of Lyme disease transmission in this region. In this study, we use a population genomics framework to characterize diversity, structure, and B. burgdorferi infection rates of I. scapularis populations in the Great Plains region. We generated whole genome sequence data and single nucleotide polymorphism (SNP) datasets from I. scapularis ticks collected in Iowa, Kansas, Nebraska, and South Dakota to compare to publicly available data from across the species range. Our analysis of 200 I. scapularis SNP datasets indicated geographically well-defined populations that correspond to historical northern and southern ancestral clades of I. scapularis. Ticks from South Dakota, Iowa, and northeastern Nebraska are genetically similar populations from the Upper Midwest, while ticks from Kansas are more genetically similar to ticks from the Southeast. Ticks collected in the central eastern region of Nebraska, however, represent an evenly admixed population of both northern and southern genomic backgrounds. Analysis of B. burgdorferi reads from genomic datasets shows [~]50% infection rate in ticks from Iowa and northeastern Nebraska, whereas ticks from Kansas show no evidence of B. burgdorferi infection. Of note, evenly admixed ticks from the central eastern region of Nebraska also show no evidence of B. burgdorferi infection. These results provide further evidence that tick genomics may influence traits associated with B. burgdorferi infection status, and thus the potential for Lyme disease transmission. As the range of I. scapularis continues to expand, bringing historically isolated populations into contact, there is a clear need to understand the consequence of genomic admixture for B. burgdorferi transmission potential to inform future risk of Lyme disease in the United States.

IntroductionWith rising tick-borne disease (TBD) cases and the geographical expansion of tick populations, the need for effective surveillance and public education regarding local risk is crucial. This study assessed the effectiveness of tick-check stations as a tool for tick surveillance, and their impact on community knowledge, attitude, and practices (KAP) related to ticks and TBD. MethodsTo assess the effectiveness of tick-check stations for surveillance, we evaluated station engagement and compared tick density estimates, species composition, and life-stage distributions with those obtained through concurrent active surveillance. In addition, we compared submission numbers and tick species and life stages to those collected through a mail-in submission system conducted by the Colorado Department of Public Health and Environment (CDPHE). To quantify feasibility, we estimated effort per tick and compared effort across simulated sampling scenarios. Finally, in-person surveys were conducted at trailheads to assess baseline tick KAP and to evaluate differences between sites with and without tick-check stations ResultsEngagement with tick-check stations was sustained throughout the study. Temporal tick densities estimated from tick-check station submissions were correlated with density estimates from active surveillance (R = 0.534), and species composition and life-stage distributions did not significantly differ between methods. Tick-check stations required less effort per tick than active surveillance when sampling sites were nearby or tick densities were low, whereas sites that were farther away or had higher tick densities required less effort per tick under a hybrid surveillance approach. When asked to list tick-borne pathogens in Colorado, 47% of survey participants who had read tick-check station signage identified Rocky Mountain spotted fever compared with 20% of participants in the control group (p = 0.007; odds ratio). Notably, a low proportion of survey participants (24%) reported performing tick-checks to prevent tick bites. ConclusionTick-check stations can provide tick density estimates comparable to active surveillance while requiring less effort in many scenarios, particularly in low-density settings. Our findings also highlight opportunities for targeted outreach to address gaps in TBD knowledge. As both a surveillance and educational tool, tick-check stations offer a sustainable approach for expanding tick monitoring in resource-limited settings.

Orientia tsutsugamushi is a mite-transmitted obligate intracellular bacterium that causes the potentially deadly zoonosis, scrub typhus. The absence of genetic tools for Orientia have limited studies of the microbe-host interactions that underlie scrub typhus. To address this gap, we developed a protocol for transforming and achieving allelic exchange in O. tsutsugamushi str. Ikeda. From evaluating multiple cell lines and antibiotics, we found that contact-inhibited EA.hy926 human endothelial-like cells best supported Orientia replication and that chloramphenicol was an effective selection marker. We engineered a homologous recombination cassette encoding a codon-modified version of the O. tsutsugamushi ank13 gene (OTT_RS04140) (CMank13) and its promoter alongside genes for mScarlet-I and chloramphenicol acetyltransferase under control of the O. tsutsugamushi tsa22-up and tsa56-down promoters, respectively. A PCR product encompassing the cassette and chromosomal flanking regions was transformed into O. tsutsugamushi via electroporation or CaCl2, the latter of which better preserved bacterial and host cell viability. EA.hy926 cells inoculated with transformed O. tsutsugamushi were grown in glass-bottom plates in the presence of chloramphenicol and imaged by live-cell microscopy to identify cultures containing mScarlet-I positive bacteria, which could be maintained in perpetuity. Chromosomal integration of the CMank13 cassette and loss of wild-type ank13 were verified by PCR and nanopore sequencing. This report establishes platforms for genetically manipulating O. tsutsugamushi and building additional genetic tools to investigate this globally significant pathogen. IMPORTANCEOrientia tsutsugamushi causes scrub typhus, a globally emerging rickettsiosis that can have a high mortality rate and has been a known human disease since the fourth century. Of the genera of obligate intracellular bacterial pathogens that cause human disease, Orientia is the only one for which genetic tools have not been developed. This has limited understanding of O. tsutsugamushi-host dynamics that drive the bacteriums pathobiology and hindered development of novel treatment or protection strategies against scrub typhus. Here, we successfully transformed and achieved allelic exchange in O. tsutsugamushi. Transgenic bacteria were selected via antibiotic resistance, validated by PCR and nanopore sequencing, and visualized by immunofluorescence and live-cell fluorescence imaging. Our report includes detailed descriptions of empirically determined host cell cultivation, multiplicity of infection, transformation, and selection conditions to provide a foundation on which other researchers can build. Overall, this work begins to establish a genetic toolbox for O. tsutsugamushi.

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.

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.

The black legged tick, Ixodes scapularis, is a vector of the bacterium that causes Lyme disease and several other illnesses, including anaplasmosis, babesiosis, and tick-borne encephalitis. Although high-quality genome annotations are available for I. scapularis, functional understanding of I. scapularis genes is limited. To address this, we developed a platform for genome-wide CRISPR-Cas9 knockout screening in I. scapularis cells. To evaluate the platform, we performed a screen to identify genes associated with cellular fitness, and screens for resistance to treatment with copper chloride, Antimycin A, or Destruxin A (DA), a cyclic hexadepsipeptide produced by the pathogenic fungus Metarhizium anisopliae. In each case, the screens implicate specific sets of conserved and non-conserved I. scapularis genes in relevant cellular functions, providing the first experimental evidence of function for a large set of I. scapularis genes. Altogether, in this first-of-its-kind effort for the arthropod subclass Acari, we present an unbiased genome-wide CRISPR-Cas9 knockout cell screening platform, related resources, and datasets that will be broadly useful to efficiently uncover cellular functions of I. scapularis genes.

Lyme disease is a tick-borne illness caused by the spirochaete bacterium Borrelia (Borreliella) burgdorferi. The black-legged tick Ixodes scapularis, which transmits B. burgdorferi and several other human pathogens, is endemic to the eastern United States and, due to climate change, is rapidly expanding into central and eastern Canada. Amplification and sequencing of bacterial DNA from I. scapularis is increasingly used to monitor the presence and abundance of B. burgdorferi and associated bacteria. However, variation in the nature of molecular data collected across studies presents challenges for analysis and interpretation. Here we use full-length Oxford Nanopore 16S ribosomal RNA gene amplicon sequencing to characterize the microbiome of I. scapularis, with an explicit focus on distinguishing between tick-adapted bacteria (endosymbionts and pathogens) and environmentally acquired bacteria (external sources, including soil, vegetation or vertebrate hosts). We show that environmental dominance strength differs between these two ecological classes of bacteria, and that environmental dominance does not appear to represent stochastic background alone; environmentally derived bacterial taxa detected in tick microbiomes are not mere contaminants. Paired soil microbiome profiling from tick collection sites will be required to test whether environmental dominance and associated co-occurrence structure track with seasonal changes in exposure and environmental microbial populations.

West Nile virus (WNV) and Usutu virus (USUV) are neurotropic Orthoflaviviruses sharing a similar enzootic transmission cycle primarily involving Culex pipiens mosquitoes as vectors and birds as amplifying hosts. First identified in Africa, both viruses established endemicity across Europe over the past two decades, most likely introduced and spread by migratory bird species along Mediterranean flyways. In avian species, infection outcomes range from subclinical to fatal neuroinvasive disease, varying by viral strain, host immunity, and species susceptibility. Southern France emerges as a key hotspot for the circulation of these viruses, supported by diverse avian habitats conducive to year-round viral maintenance. This study investigated the prevalence of WNV and USUV in more than 2500 sedentary and migratory wild birds from these regions during 2024-2025 using molecular surveillance. Samples were collected using mist net and bird boxes, across multiple passerine and non-passerine taxa, spanning wetlands, urban fringes, and agricultural zones. Our analyses revealed widespread viral circulation across diverse species, mainly among passerines such as great tits, house sparrows, and barn swallows with USUV detected at higher rates than WNV in both study years. Overall prevalence was markedly higher in 2024 than in 2025, potentially reflecting climatic or ecological drivers. Migratory individuals likely seed viral introductions during seasonal passages, whereas resident populations sustain local enzootic cycles, facilitating overwintering persistence. These results highlight the pivotal role of mixed avifauna in arbovirus dynamics within Mediterranean Europe and emphasize the necessity for integrated, year-round surveillance targeting high-risk species and habitats. Enhanced monitoring will aid in predicting spillover risks and informing vector control strategies to mitigate zoonotic threats.

Naturally occurring variation in the bovine mannose receptor C-type 1 gene (MRC1) may shape macrophage responses to Mycobacterium (M.) bovis, a key driver of bovine tuberculosis (bTB). We identified four coding region SNPs in MRC1 across Bos taurus (Holstein Friesian, Brown Swiss) and Bos indicus (Boran, Sahiwal) cattle breeds, including a non-synonymous variant, rs380943118 (c.2963G>A; Ser988Asn) in C-type lectin-like domain (CTLD) 6, most prevalent in Sahiwal cattle. Structural modelling suggested that the S988N substitution, which is spatially separated from the monosaccharide binding site of CTLD4, might indirectly affect glycan binding, perhaps through a conformational change in the receptor. Monocyte-derived macrophages upregulated MR expression during differentiation, with heterozygous (G/A) animals showing higher MR expression and increased uptake of GFP-M. bovis BCG, although differences were not statistically significant. Anti-CD206 blockade did not inhibit BCG internalization, either indicating that this specific antibody did not bind to a CTLD involved in ligand binding or that MR is not the sole entry receptor. These results highlight naturally occurring MRC1 polymorphisms that may influence MR structure and macrophage function, providing a foundation for future studies to assess their role in bTB susceptibility.

Tick-borne pathogens can reshape vector microbiomes in ways that influence pathogen colonisation and transmission, yet the interplay between Ehrlichia ruminantium and the microbiota of its tick vectors remains uncharacterised. We profiled bacterial communities in haemolymph, midgut, and salivary glands of infected (n = 11) and uninfected (n = 12) Am. gemma ticks, a vector of E. ruminantium in East Africa, collected from cattle in Kajiado County, Kenya, using near-full-length 16S rRNA gene amplicon sequencing on the Oxford Nanopore platform. Community composition, alpha and beta diversity, co-occurrence networks, keystone taxa, and PICRUSt2-inferred functional profiles were compared across tissue-infection status groups. We identified 226 bacterial genera dominated by Coxiella, Pseudomonas, Acinetobacter, Proteus, and Rickettsia. Infection was associated with tissue-specific shifts in community composition (PERMANOVA R{superscript 2} = 0.14, p < 0.001) and co-occurrence network structure, with midgut networks showing complete hub taxon turnover (Jaccard = 0.000, p = 0.043). Haemolymph communities converged around Luteimonas as a keystone taxon, while opportunistic Proteobacteria, including Acinetobacter and Serratia, emerged as keystones in infected midgut. Endosymbiotic Rickettsia was near-absent in infected tissues (0.3% vs 9.3% mean relative abundance in midgut), consistent with competitive exclusion. Functional inference identified FDR-significant enrichment of predicted aerobactin siderophore biosynthesis, antimicrobial efflux, and oxidative stress response gene families in infected microbiota. These findings show tissue-specific restructuring of the Am. gemma microbiome associated with E. ruminantium infection and point to candidate targets for microbiome-based interventions against heartwater. ImportanceHeartwater, caused by the bacterium Ehrlichia ruminantium and transmitted by Amblyomma ticks, kills up to 90% of susceptible ruminants and is one of the most devastating tick-borne diseases in sub-Saharan Africa. Controlling heartwater requires understanding how the pathogen interacts with the microbial communities living inside its tick vector. In this exploratory study, we show that E. ruminantium infection is associated with tissue-specific shifts in the Amblyomma tick microbiome, including reduced abundance of beneficial symbionts, elevated representation of opportunistic bacteria among community hubs, and enrichment of iron acquisition and antimicrobial resistance functions. The midgut, the first tissue colonised during infection, showed the most marked structural reorganisation. These tissue-resolved microbiome signatures point to potential targets for novel control strategies, such as anti-microbiota vaccines or approaches that reinforce natural colonisation resistance, offering new strategies to reduce heartwater transmission and protect livestock livelihoods across Africa.