Ticks and Tick-borne Diseases

Neoehrlichia mikurensis is widely detected in tick populations across Europe, yet its host range beyond small rodents remains poorly understood. Large wild mammals are frequently exposed to tick vectors, but their role in the transmission cycle of this bacterium is unclear. Wild boars (Sus scrofa) are among the most abundant large mammals in southern Sweden and commonly inhabit tick-infested environments, making them a potentially relevant host species. In this study, spleen samples from 87 wild boars collected between 2023 and 2025 across southern and central Sweden were analysed for the presence of N. mikurensis DNA using quantitative polymerase chain reaction. N. mikurensis DNA was not detected in any of the analysed samples, despite sampling in regions where the bacterium is known to be prevalent in ticks. These results indicate that wild boars may have a limited role in the maintenance of N. mikurensis in Sweden, although the possibility of rare or transient infection cannot be excluded. Despite being based on negative findings, this study contributes to a more comprehensive understanding of the host range of N. mikurensis and highlights the importance of continued evaluation of wildlife species in the transmission dynamics of tick-borne pathogens.

Tick-borne disease risk reflects the combined influence of vector abundance, pathogen prevalence, human exposure, and seasonal activity, yet these components are rarely integrated at the county level. We developed species-specific ecological risk scores for three medically important tick species (Acari: Ixodidae): Amblyomma americanum (L.), Dermacentor variabilis (Say), and Ixodes scapularis Say, across all 102 Illinois counties using data from 2019 to 2022. County-level predicted tick abundance and pathogen prevalence were scaled and combined using equal weights to generate primary vector-level risk scores. Robustness to the equal-weight assumption was evaluated by systematically varying the relative weights assigned to abundance and pathogen prevalence, while a separate sensitivity analysis incorporated reported tick-bite incidence where available. Associations with human disease were evaluated using negative binomial regression of tick-borne disease case counts on the corresponding risk scores, with county population included as an offset. Monthly phenology indicators were constructed by integrating human disease cases, reported tick bites, and sampled tick abundance and were evaluated in relation to temperature and precipitation. Seasonal risk was estimated by combining county-level vector risk with species-specific monthly phenology, generating monthly risk surfaces that were deployed through an interactive R Shiny application. Higher risk scores were significantly associated with greater disease case rates for each tick species. County rankings were highly stable under moderate changes in abundance and prevalence weighting. Seasonal activity differed among species, with A. americanum and D. variabilis showing concentrated warm-season risk and I. scapularis exhibiting a broader seasonal profile. These county-level seasonal risk maps identify where and when relative tick-borne disease risk is elevated in Illinois, supporting targeted prevention, surveillance, and clinical awareness.

Vegetation plays a critical role in shaping fine-scale tick density by structuring local microclimates, habitat resources, and host interactions that largely determine tick survival and activity, with implications for tick-borne disease transmission. Tick burden in the United States continues to increase, with annual reported tick-borne disease cases more than doubling from 2004 through 2019. These trends call for a broader scope of tick-mitigation strategies that go beyond traditional prevention measures that focus on personal protection; and there is an opportunity to inform habitat-level interventions. Here, we quantify fine-scale vegetation structure across 48 subplots at tick surveillance sites in Western Massachusetts to assess local habitat features associated with Ixodes scapularis density and potential for human-tick contact risk. In assessing understory and overstory vegetation attributes, this study identified life stage-specific differences in predicted tick density, with higher adult densities in areas with deeper leaf litter and presence of invasive plant species and nymphs associated with high presence of ferns. Both adult and nymphal Ix. scapularis were predicted at higher densities in relatively low basal area forests (18.9 to 26.9 m2/hectare). This study design provides a simple, low-cost framework for organizations looking to incorporate tick-informed forest measurement ecology into their public health tick surveillance protocols with minimal training.

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.

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.

Mosquito-borne orthoflaviviruses pose a significant threat to human and animal health worldwide. In Europe, West Nile (WNV), Usutu (USUV) and Bagaza (BAGV) viruses have expanded their geographic distribution and are detected with increasing frequency. Following the emergence of BAGV in 2021, we investigated the circulation dynamics and genomic diversity of orthoflaviviruses in southern Portugal, where their epidemiology remains poorly understood. Active Orthoflavivirus surveillance was conducted in birds and mosquitoes during 2021-2023 using serological and molecular assays, complemented by viral genome sequencing and phylogenomic analyses. Viral RNA was detected in 3.5% (41/1,171) of birds, while seroneutralization assays identified an overall seroprevalence of 55.3% (503/910). WNV RNA (lineage-1, strains WMed-1.2 and WMed-1.3) was detected annually in resident birds, including Alectoris rufa, Cyanopica cooki and Garrulus glandarius (1.5%, 18/1,171), and in Culex univittatus mosquitoes (0.8%, 2/255). Together with a WNV-specific seroprevalence of 28.5% (259/910) in birds, these findings support sustained local circulation. USUV RNA (Africa 3.1 sub-lineage) was detected in A. rufa in 2021 and 2023 (0.2%, 2/1,171), but seroprevalence was low (1.3%, 8/623), consistent with intermittent circulation. BAGV RNA (genotype-2) was detected only during the 2021 outbreak in A. rufa and Emberiza calandra (1.8%, 21/1,171), although BAGV-specific antibodies (6.6%, 60/910) were detected for up to two years after the outbreak, suggesting continued exposure or low-level circulation. Overall, our findings reveal contrasting circulation dynamics of orthoflaviviruses in southern Portugal. Sustained WNV circulation implies the need to strengthen avian and human surveillance in Portugal to better assess its public health risk, while the sporadic detection of USUV and BAGV underscores the value of long-term, integrated One Health surveillance for improving early warning and preparedness for emerging mosquito-borne diseases.

Background: Treponema pallidum, the causative agent of yaws, bejel, and syphilis, is a significant pathogen affecting both humans and nonhuman primates (NHPs). While its presence is documented in several African NHP species, the continental-scale distribution, ecological drivers, and status in North African populations remain poorly understood. We conducted a large-scale, non-invasive survey to investigate the circulation of T. pallidum across African NHP populations and evaluate geographical and ecological patterns of exposure. Results: A total of 1,696 fecal samples from 11 wild NHP species collected across five African countries were screened for anti-T. pallidum antibodies using an automated chemiluminescent Simple Western assay, and fecal and dipteran samples were analyzed by molecular methods. Widespread seropositivity was observed across all African sites and multiple primate taxa, including great apes and cercopithecids. We provide the first evidence of T. pallidum antibodies in Barbary macaques (Macaca sylvanus) from North Africa, extending the known distribution of the pathogen to the Mediterranean basin. In West Africa, chimpanzees (Pan troglodytes verus) from Senegal and Guinea exhibited particularly high seroprevalence, confirming this region as a major endemic hotspot. In Central Africa, substantial circulation was documented in Cercopithecus wolfi, Cercopithecus ascanius, and western lowland gorillas (Gorilla gorilla gorilla). Arid/savanna biomes showed significantly higher exposure than Mediterranean and tropical rainforest biomes. No T. pallidum DNA was detected in fecal or dipteran samples. Conclusion: Our findings demonstrate widespread circulation of T. pallidum among African NHPs, reveal its presence in North African primate populations for the first time. The observed high seroprevalence, coupled with significant variations across species and geographical regions, suggests the existence of multiple transmission pathways adapted to specific host behaviors and environmental constraints. These results support the existence of a broad multihost sylvatic reservoir and provide new insights into the ecology of T. pallidum, with important implications for wildlife health, One Health surveillance, and yaws eradication efforts.

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.

Climate change is increasingly invoked to explain long-term shifts in vector-borne disease, yet observed incidence reflects both ecological processes and the systems used to detect disease. Distinguishing environmental signals from changes in surveillance is therefore essential for attributing disease trends to global change. We used Lyme borreliosis in Poland as a national case study, analysing 259,003 notifications across 16 administrative regions from 2009 to 2024 (1,024 region-quarters). Multivariable negative binomial models estimated associations with lagged climatic conditions while accounting for temporal trend, a post-2016 structural step, COVID-19 healthcare disruption, seasonality and regional heterogeneity. Hospitalisation proportion provided an independent population-level indicator of changing observed case mix. Reported incidence increased 2.93-fold, whereas hospitalisation proportion declined from 26.4% to 3.5%. Temperature at a three-quarter lag (incidence rate ratio [IRR] 1.069 per 1'C, 95% CI 1.045 - 1.093) and cloud cover at a one-quarter lag (IRR 1.127 per okta, 1.063 - 1.195) were independently associated with reported incidence. However, changes in the observation process were of greater magnitude: the post-2016 structural step was associated with a 57% increase in notifications (IRR 1.571, 1.465 - 1.684), whereas the COVID-19 period was associated with a 48% reduction (IRR 0.524, 0.482 - 0.568). The divergence between increasing notifications and declining hospitalisation indicates substantial expansion in ascertainment of milder disease, such that notification trends cannot be interpreted as proportional changes in transmission. Our findings show that climatic signals can coexist with, and be obscured or amplified by, changes in disease observation. Robust attribution of vector-borne disease responses to global environmental change therefore requires explicit modelling of surveillance processes and integration of epidemiological, healthcare and ecological data.

Dirofilariasis, or heartworm disease, is a life-threatening parasitic infection caused by the nematode parasite Dirofilaria immitis. Mosquito vectors in tropical and subtropical regions transmit the infection. Current treatment options are expensive, have a short-term effect, and depend on anthelminthic therapy. There are no licensed vaccines available for preventing heartworm infections. The primary goal of this study was to identify potential vaccine candidates, develop a tetravalent fusion protein (rDiHAVO) vaccine antigen, and evaluate its immunogenicity and efficacy in a mouse model of D. immitis infection. Results show that the rDiHAVO is highly immunogenic in mice and confers over 95% protection against a challenge infection. These findings suggest that rDiHAVO is a potential vaccine candidate against Dirofilariasis. Additional testing in dogs can advance this vaccine candidate to clinical trials.

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.

Orientia tsutsugamushi is the causative agent of scrub typhus infection in humans. This pathogen is transmitted into mammalian hosts including humans through Leptotrombidium mites. Till date, detection of O. tsutsugamushi in human was performed using PCR assay with 56kDa type specific antigen (TSA56), 47kDa outer membrane protein (47kDa-OMP) and groEL chaperonin gene. To understand the efficiency of different genes for the detection of O. tsutsugamushi in Leptotrombidium mites, we performed a comparative PCR assay with various universal primers targeting different genes including TSA56, 47kDa-OMP and groEL gene. We have collected 500 Leptotrombidium mites from Vellore, Tamil Nadu and, the mites were segregated into 50 pools with 10 mites in each pool were used for DNA extraction. The molecular detection of O. tsutsugamushi was performed using PCR assays (including conventional, nested and duplex PCR). The comparative PCR analysis showed nested PCR of groEL gene has high efficiency in detecting the presence of O. tsutsugamushi in chigger samples compared to other genes. Interestingly, groEL target demonstrated high efficiency for detection of O. tsutsugamushi in chigger samples and, when used with nested primers, can facilitate differentiation of O. tsutsugamushi from other rickettsial pathogens. Thus, we conclude groEL gene nested PCR assay can be used routinely to detect O. tsutsugamushi in trombiculid mites.

Background Following the emergence of a novel Shamonda-like virus (SHAV) in Europe, suspected clinical cases in dairy cattle were reported in Northeastern Italy. Investigation was required in dairy cattle in Northeastern Italy. Methods Samples were collected from 28 farms displaying fever and reduced milk yield and were screened using molecular methods (PanSimbu real-time RT-PCR followed by Sanger sequencing and differential PCRs for SBV/BTV). Positive samples were subsequently confirmed by Sanger sequencing and selected samples underwent whole-genome sequencing using a metagenomic approach. Results Twenty-four among sera and blood samples from 12 farms tested positive for SHAV. WGS confirmed that Italian strains share maximum identity with the European SHAV-EU1 clade. Limitations This study concerns the results of clinical investigations on suspected cases in the two autonomous provinces of Trento and Bolzano. At present, it is not possible to infer on the circulation of the virus in other Italian provinces. Conclusions This study documents the first detection and genetic characterization of SHAV-EU1 in Italian cattle, indicating active circulation of this virus across Europe. Its identification in two north-eastern Italian provinces does not rule out wider, undetected circulation. In this regard, assessing its geographical extent requires careful evaluation, just as much remains to be discovered about its host range and the severity of symptoms.

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.

Official Lyme disease incidence figures for England are based on the number of cases with a laboratory-confirmed diagnosis from the national reference laboratory. However, these figures underestimate the true incidence of acute cases since patients can also be diagnosed clinically without the need for laboratory testing. The use of a multiplication factor has been proposed to estimate total Lyme disease incidence from the number of laboratory-confirmed cases. The current multiplication factor is derived from data spanning 1998 to 2016 and is thought to be outdated, in part due to the introduction of new national Lyme disease management guidance in 2018. The aim of this study was to analyse more recent laboratory and primary care data to identify trends in case numbers and to derive updated estimates of the total annual number of acute Lyme disease cases in England. Data spanning 2013 to 2024 from the national reference laboratory and a primary care data source were analysed using seasonal time series models. The presence of structural breaks was investigated using the PELT or BinSeg methods, and annual multiplication factors were estimated using a Poisson regression model. No substantial change was found in the number of laboratory-confirmed Lyme cases from 2013 to 2024 in England, whereas the number of cases diagnosed in primary care increased significantly over the same period, with an average of an additional 498 cases per year. This has increased our best estimate of the multiplication factor from 2.35 (95% confidence interval, 1.81 - 2.88) to 6.20 (6.17 - 6.24), which is the average for the years 2020 - 2024. Use of this updated multiplication factor should improve the accuracy of annual symptomatic Lyme disease case estimates for England.

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.

Understanding the vertebrate hosts exploited by Culicoides biting midges is fundamental to assessing the transmission risk of livestock arboviruses. Blood meal analysis provides direct evidence of host-vector interactions under natural conditions; however, molecular data on Culicoides host feeding are lacking in Ireland. This study aimed to identify the vertebrate blood meal hosts of Culicoides species of veterinary importance and establish host-vector associations relevant to arbovirus transmission. A total of 115 morphologically blood-fed Culicoides females collected from 13 sites across Ireland were analysed using a sequential molecular workflow. Species-specific real-time PCR assays were used to screen for vertebrate hosts, with positive detections confirmed by mitochondrial DNA sequencing. Blood-fed Culicoides were subsequently identified to species level using DNA barcoding, enabling direct linkage between vector species and their vertebrate hosts. Vertebrate DNA was detected in 50 specimens, with cattle and horses accounting for the majority of identified blood meals. Culicoides obsoletus accounted for most cattle (n = 12) and horse (n = 11) blood meals. Culicoides scoticus fed on cattle (n = 6), horses (n = 4) and sheep (n = 1), while C. dewulfi and C. chiopterus fed predominantly on cattle, with occasional horse blood meals. Four cattle blood meals were identified from C. punctatus. No cervid-derived blood meals were detected. This study provides the first molecular characterisation of Culicoides blood meal hosts in Ireland, confirming host-vector associations between major vector species and livestock hosts of veterinary importance. Although the number of successfully characterised blood meals was modest, reflecting the inherent difficulty of recovering recently blood-fed Culicoides from field collections, the findings establish an important baseline for Ireland. The sequential molecular workflow provides a practical framework for future blood meal investigations and will support improved understanding of host-vector interactions relevant to arbovirus surveillance and risk assessment.