Pathogen surveillance contributes to the prediction of outbreaks and implementation of strategies to minimize disease transmission. White-tailed deer (Odocoileus virginianus) host important disease vectors and often support the proliferation of associated pathogens. Such vectors include ticks: hematophagous acarids that facilitate pathogen transmission between animals through blood feeding. Piroplasmida, an order of blood-borne protozoa including Babesia and Theileria, exploits this feeding to infect definitive hosts. Accordingly, we investigated the prevalence of Theileria cervi infections in Massachusetts white-tailed deer. Conventional polymerase chain reaction (PCR) was used to detect parasite gene fragments encoding the small ribosomal subunit (18S rRNA). Confirmation of species identity was determined by Oxford nanopore sequencing and Sequencher analysis. This revealed a 32.6% prevalence of T. cervi across Massachusetts. Whilst surveillance of deer blood uncovers parasite abundance and distribution, inversely, this allows us to also monitor vector populations. As Amblyomma americanum is the only vector implicated in the transmission of T. cervi, the detection of T. cervi in regions without anticipated Amblyomma presence may indicate expansion of vector populations. These findings provide valuable insights into the prevalence and ecology of this piroplasm and its vector, with possible implications for veterinary and public health.

Anaplasma phagocytophilum is a tick-borne bacterium and the causative agent of human granulocytic anaplasmosis. Two major variants have been identified in North America: a human pathogenic variant (Ap-ha) that is maintained by small mammals and a nonpathogenic variant (Ap-v1) that is maintained by white-tailed deer (Odocoileus virginianus). Despite white-tailed deer being recognized as the main reservoir of Ap-v1, few studies have directly demonstrated transmission of Ap-v1 from white-tailed deer to feeding I. scapularis ticks, and transmission to feeding larvae remains undescribed. In this study, we collected samples from an outdoor white-tailed deer research facility during an I. scapularis larval questing season in the eastern United States to investigate the variants infecting white-tailed deer and acquisition by feeding larvae. Although both variants were present in questing ticks onsite, white-tailed deer were persistently infected with only Ap-v1 and transferred this variant to feeding I. scapularis larvae with high efficiency. These results support the concept that white-tailed deer play a role in the maintenance of Ap-v1 in nature and highlight the utility of captive white-tailed deer research facilities for studying vector–pathogen–host biology.

Deer keds (Lipoptena spp. and Neolipoptena ferrisi) are hematophagous ectoparasites that primarily infest white-tailed deer (Odocoileus virginianus) and other cervids in the United States. The distribution of deer keds in the northeastern United States and the pathogens they harbor remains relatively unexplored. In this study, we examined the geographical distribution and pathogen prevalence of deer keds in Massachusetts by collecting samples from white-tailed deer and testing for tick-borne pathogens. Deer keds were collected across the state, including in four previously unrecorded counties, indicating a wide distribution. Pathogen screening revealed the presence of Anaplasma phagocytophilum DNA in 30% of the keds, but no other pathogens were detected. The medical and biological significance of detecting A. phagocytophilum DNA in deer keds requires future studies. This research provides a baseline for the distribution and pathogen prevalence of deer keds in Massachusetts and highlights the potential of deer keds as sentinels for monitoring deer-associated microbes.

Spotted fever group (SFG) rickettsioses are increasingly recognized worldwide as threats to public health. Rickettsia rickettsii, Rickettsia parkeri, and Rickettsia rickettsii subspecies californica cause spotted fever rickettsioses, including Rocky Mountain spotted fever. These disease agents are transmitted to humans by various tick vectors in the United States. There is growing concern that other tick species, such as Haemaphysalis leporispalustris, may also transmit new and potentially unrecognized SFG rickettsial pathogens. In this study, we found that 6.1 % of 296 questing H. leporispalustris ticks (21 larvae, 260 nymphs, 9 males, and 6 females) collected from 38 towns across nine counties in Maine, USA, were positive for Rickettsia spp. Further multilocus sequence typing and phylogenetic analysis revealed that this is a new Rickettsia genotype (Rickettsia sp. ME2023) belonging to the SFG group and close to Candidatus Rickettsia lanei. Tick vectors and rickettsial species associated with SFG rickettsioses in New England warrant further investigation. Additionally, the role of H. leporispalustris in pathogen enzootic cycles and transmission requires further study.

Borrelia burgdorferi’s inosine-5′-monophosphate dehydrogenase (IMPDH, GuaB encoded by the guaB gene) is a potential therapeutic target. GuaB is necessary for B. burgdorferi replication in mammalian hosts but not in standard laboratory culture conditions. Therefore, we cannot test novel GuaB inhibitors against B. burgdorferi without utilizing mammalian infection models. This study aimed to evaluate modifications to a standard growth medium that may mimic mammalian conditions and induce the requirement of GuaB usage for replication. The effects of two GuaB inhibitors (mycophenolic acid, 6-chloropurine riboside at 125 μM and 250 μM) were assessed against B. burgdorferi (guaB+) grown in standard Barbour–Stoenner–Kelly-II (BSK-II) medium (6% rabbit serum) and BSK-II modified to 60% concentration rabbit serum (BSK-II/60% serum). BSK-II directly supplemented with adenine, hypoxanthine, and nicotinamide (75 μM each, BSK-II/AHN) was also considered as a comparison group. In standard BSK-II, neither mycophenolic acid nor 6-chloropurine riboside affected B. burgdorferi growth. Based on an ANOVA, a dose-dependent increase in drug effects was observed in the modified growth conditions (F = 4.471, p = 0.001). Considering higher drug concentrations at exponential growth, mycophenolic acid at 250 μM reduced spirochete replication by 48% in BSK-II/60% serum and by 50% in BSK-II/AHN (p < 0.001 each). 6-chloropurine riboside was more effective in both mediums than mycophenolic acid, reducing replication by 64% in BSK-II/60% serum and 65% in BSK-II/AHN (p < 0.001 each). These results demonstrate that modifying BSK-II medium with physiologically relevant levels of mammalian serum supports replication and induces the effects of GuaB inhibitors. This represents the first use of GuaB inhibitors against Borrelia burgdorferi, building on tests against purified B. burgdorferi GuaB. The strong effects of 6-chloropurine riboside indicate that B. burgdorferi can salvage and phosphorylate these purine derivative analogs. Therefore, this type of molecule may be considered for future drug development. Optimization of this culture system will allow for better assessment of novel Borrelia-specific GuaB inhibitor molecules for Lyme disease interventions. The use of GuaB inhibitors as broadcast sprays or feed baits should also be evaluated to reduce spirochete load in competent reservoir hosts.

Ticks are vectors of many human and animal zoonotic disease-causing agents causing significant global health and economic strain. Repellents and acaricides are integral to the human capacity for personal protection from tick bites. Nootkatone, a naturally occurring sesquiterpene found in the Alaskan cedar tree, grapefruit, and other sources, has been documented to be a potent acaricide. Research has also noted repellent effects against some tick species. In this study, our aim was to investigate the effect of synthetic, high-purity (+)-nootkatone on adult Ixodes scapularis, Dermacentor variabilis, and Amblyomma americanum ticks in an in vitro, vertical filter paper bioassay. (+)-nootkatone showed compelling tick repellency, but median effective concentrations (EC50) significantly differed among species. Ixodes scapularis were repelled at very low concentrations (EC50 = 0.87 ± 0.05 µg/cm2). Higher concentrations were required to repel D. variabilis (EC50 = 252 ± 12 µg/cm2) and A. americanum (EC50 = 2313 ± 179 µg/cm2). Significant post-exposure mortality, assessed 24 h after repellency trials, was also observed in I. scapularis but was absent entirely in D. variabilis and A. americanum. These tests demonstrate that nootkatone has a promising dual-action personal protection capacity against adult I. scapularis ticks, warranting further investigation in more natural environments and in the presence of host cues.

Recent changes in climate and human land-use have resulted in alterations of the geographic range of many species, including human pathogens. Geographic range expansion and population growth of human pathogens increase human disease risk. Relatively little empirical work has investigated the impact of range changes on within-population variability, a contributor to both colonization success and adaptive potential, during the precise time in which populations are colonized. This is likely due to the difficulties of collecting appropriate natural samples during the dynamic phase of migration and colonization. We systematically collected blacklegged ticks ( Ixodes scapularis ) across New York State (NY), USA, between 2006 and 2019, a time period coinciding with a rapid range expansion of ticks and their associated pathogens including Borrelia burgdorferi , the etiological agent of Lyme disease. These samples provide a unique opportunity to investigate the population dynamics of human pathogens as they expand into novel territory. We observed that founder effects were short-lived, as gene flow from long-established populations brought almost all B. burgdorferi lineages to newly colonized populations within just a few years of colonization. By 7 years post-colonization, B. burgdorferi lineage frequency distributions were indistinguishable from long-established sites, indicating that local B. burgdorferi populations experience similar selective pressures despite geographic separation. The B. burgdorferi lineage dynamics elucidate the processes underlying the range expansion and demonstrate that migration into, and selection within, newly colonized sites operate on different time scales.

Tick-borne diseases and a tick-induced red meat allergy have become increasingly common in the northeastern USA and elsewhere. At the scale of local communities, few studies have documented tick densities or infection levels to characterize current conditions and provide a baseline for further monitoring. Using the town of Nantucket, MA, as a case study, we recorded tick densities by drag sampling along hiking trails in nature preserves on two islands. Nymphal blacklegged ticks (Ixodes scapularis Say) were most abundant at shadier sites and least common in grasslands and scrub oak thickets (Quercus ilicifolia). Lone star ticks (Amblyomma americanum L.) were common on Tuckernuck Island and rare on Nantucket Island, while both tick species were more numerous in 2021 compared to 2020 and 2022. We tested for pathogens in blacklegged nymphs at five sites over two years. In 2020 and 2021, infection levels among the four Nantucket Island sites averaged 10% vs. 19% for Borrelia burgdorferi, 11% vs. 15% for Babesia microti, and 17% (both years) for Anaplasma phagocytophilum, while corresponding levels were significantly greater on Tuckernuck in 2021. Our site-specific, quantitative approach represents a practical example of how potential exposure to tick-borne diseases can be monitored on a local scale.

Borrelia burgdorferi is a human pathogen vectored by Ixodes ticks and maintained in nature by a suite of competent vertebrate reservoirs. White-tailed deer (WTD) are considered to be noncompetent reservoirs for B. burgdorferi. Sera from other deer species have been found to be borreliacidal, and similar mechanisms could explain the lack of reservoir competence of WTD. Therefore, we determined whether WTD serum can kill B. burgdorferi. Using an in vitro serum sensitivity assay and subculturing of spirochetes, we demonstrated that WTD serum effectively kills B. burgdorferi. The borreliacidal activity of WTD serum likely contributes to the inability of WTD to efficiently harbor and transmit B. burgdorferi.

Borrelia burgdorferi is an important tickborne human pathogen and can be grouped into separate strains based on the outer surface protein C (ospC) gene. The detection and characterization of different ospC genotypes is vital for research on B. burgdorferi and the risk it poses to humans. Here we present a novel, multiplex assay based on Luminex xMAP technology for the detection of B. burgdorferi ospC genotypes. The assay has 5 major steps: amplification of the ospC gene, enzymatic purification, incorporation of biotinylated nucleotides into the template DNA, hybridization to Luminex microspheres, and detection of fluorescent signals corresponding to each ospC genotype. This protocol can be used for the characterization of ospC genotypes in B. burgdorferi infected ticks, reservoir hosts, and/or clinical samples. (The last step in this version contains a supplemental video with extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.)

Reservoir host associations have been observed among and within Borrelia genospecies, and host complement-mediated killing is a major determinant in these interactions. In North America, only a subset of Borrelia burgdorferi lineages cause the majority of disseminated infections in humans. We hypothesize that differential resistance to human complement-mediated killing may be a major phenotypic determinant of whether a lineage can establish systemic infection. As a corollary, we hypothesize that borreliacidal action may differ among human subjects. To test these hypotheses, we isolated primary B. burgdorferi clones from field-collected ticks and determined whether the killing effects of human serum differed among those clones in vitro and/or whether these effects were consistent among human sera. Clones associated with human invasiveness did not show higher survival in human serum compared to noninvasive clones. These results indicate that differential complement-mediated killing of B. burgdorferi lineages is not a determinant of invasiveness in humans. Only one significant difference in the survivorship of individual clones incubated in different human sera was detected, suggesting that complement-mediated killing of B. burgdorferi is usually similar among humans. Mechanisms other than differential human complement-mediated killing of B. burgdorferi lineages likely explain why only certain lineages cause the majority of disseminated human infections.

Borrelia burgdorferi, the causative agent of Lyme disease, has a highly reduced genome and relies heavily on glycolysis for carbon metabolism. As such, established inhibitors of lactate dehydrogenase (LDH) were evaluated in cultures to determine the extent of their impacts on B. burgdorferi growth. Both racemic and enantiopure (AT-101) gossypol, as well as oxamate, galloflavin, and stiripentol, caused the dose-dependent suppression of B. burgdorferi growth in vitro. Racemic gossypol and AT-101 were shown to fully inhibit spirochetal growth at concentrations of 70.5 and 187.5 μM, respectively. Differences between racemic gossypol and AT-101 efficacy may indicate that the dextrorotatory enantiomer of gossypol is a more effective inhibitor of B. burgdorferi growth than the levorotatory enantiomer. As a whole, LDH inhibition appears to be a promising mechanism for suppressing Borrelia growth, particularly with bulky LDH inhibitors like gossypol.

A wide range of pathogens, such as bacteria, viruses, and parasites can be transmitted by ticks and can cause diseases, such as Lyme disease, anaplasmosis, or Rocky Mountain spotted fever. Landscape and climate changes are driving the geographic range expansion of important tick species. The morphological identification of ticks is critical for the assessment of disease risk; however, this process is time-consuming, costly, and requires qualified taxonomic specialists. To address this issue, we constructed a tick identification tool that can differentiate the most encountered human-biting ticks, Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis, by implementing artificial intelligence methods with deep learning algorithms. Many convolutional neural network (CNN) models (such as VGG, ResNet, or Inception) have been used for image recognition purposes but it is still a very limited application in the use of tick identification. Here, we describe the modified CNN-based models which were trained using a large-scale molecularly verified dataset to identify tick species. The best CNN model achieved a 99.5% accuracy on the test set. These results demonstrate that a computer vision system is a potential alternative tool to help in prescreening ticks for identification, an earlier diagnosis of disease risk, and, as such, could be a valuable resource for health professionals.

Borrelia burgdorferi is an important tickborne human pathogen and can be grouped into separate strains based on the outer surface protein C (ospC) gene. The detection and characterization of different ospC genotypes is vital for research on B. burgdorferi and the risk it poses to humans. Here we present a novel, multiplex assay based on Luminex xMAP technology for the detection of B. burgdorferi ospC genotypes. The assay has 5 major steps: amplification of the ospC gene, enzymatic purification, incorporation of biotinylated nucleotides into the template DNA, hybridization to Luminex microspheres, and detection of fluorescent signals corresponding to each ospC genotype. This protocol can be used for the characterization of ospC genotypes in B. burgdorferi infected ticks, reservoir hosts, and/or clinical samples.

Borrelia burgdorferi is an important tickborne human pathogen comprising several strains based on nucleotide sequence of the outer surface protein C (ospC) gene. Detection and characterization of different ospC genotypes is vital for research on B. burgdorferi and the risk it poses to humans. Here we present a novel, multiplex assay based on Luminex xMAP technology for the detection of B. burgdorferi ospC genotypes. The assay has five major steps: amplification of the ospC gene, hydrolyzation of surplus primers and nucleotides, incorporation of biotinylated nucleotides into the template DNA, hybridization to Luminex microspheres, and detection of fluorescent signals corresponding to each ospC genotype. We validated the protocol by comparing results obtained from our method against results from an established ospC genotyping method. This protocol can be used for the characterization of ospC genotypes in B. burgdorferi infected ticks, reservoir hosts, and/or clinical samples.

During 2013-2019, Borrelia miyamotoi infection was detected in 19 US states. Infection rate was 0.5%-3.2%; of B. miyamotoi-positive ticks, 59.09% had concurrent infections. B. miyamotoi is homogeneous with 1 genotype from Ixodes scapularis ticks in northeastern and midwestern states and 1 from I. pacificus in western states.

Tick-borne diseases such as Lyme, babesiosis, and anaplasmosis are a continuing threat to public health in New England due to the abundance of infected blacklegged ticks (Ixodes scapularis). The goals of this project are to monitor blacklegged and invading lone star ticks (Amblyomma americanum) at sentinel sites on the islands of Nantucket and Tuckernuck to document current conditions and provide a baseline for detecting changes over time. Results from three field seasons (2020-2022) will be used to inform the public and develop cost-effective procedures for monitoring abundances of these ticks and the disease agents that infect them. In June 2020, we selected ten sentinel sites where it is feasible to drag-sample for ticks along established trails, nine on Nantucket and one on Tuckernuck. At each site, we characterized the vegetation and sampled ticks on four days to report mean numbers of ticks per kilometer of trail. Densities of blacklegged nymphs were greatest at sites with the most tree canopy and shade, and lowest at sites with the least shade, such as open grasslands. Nymph densities were high at sites

From July 2006 through August 2017, a passive surveillance study of Ixodes ticks submitted from California, Oregon, and Washington was conducted by the TickReport program at the University of Massachusetts, Amherst. In total, 549 human-biting Ixodes ticks were submitted comprising both endemic and nonendemic species. We found that 430 endemic ticks were from 3 Ixodes species: Ixodes pacificus, Ixodes spinipalpis, and Ixodes angustus, whereas Ixodes scapularis (n = 111) was the most common species among the 119 nonendemic ticks. The submission peak for nymphal I. pacificus and I. spinipalpis was June, while submission peak for adult I. pacificus and nymphal I. angustus was April and September, respectively. Endemic ticks commonly attached to the lower extremities of their victims, and individuals younger than 9 years old were frequently bitten. The infection prevalence of Borrelia burgdorferi sensu lato, Borrelia miyamotoi, and Anaplasma phagocytophilum in I. pacificus ticks was 1.31%, 1.05%, and 0.52%, respectively, and the prevalence of B. burgdorferi s. l. and A. phagocytophilum in I. spinipalpis ticks was 14.29% and 10.71%, respectively. Furthermore, two species within the B. burgdorferi s. l. complex were detected in West Coast ticks: B. burgdorferi sensu stricto and Borrelia lanei. I. spinipalpis had the highest Borrelia prevalence among endemic ticks, and it was caused exclusively by B. lanei. Borrelia mayonii, Babesia microti, and Ehrlichia muris-like agent were not detected in these endemic ticks. In this study, we show that many nonendemic Ixodes ticks (119/549) are most likely acquired from travel to a different geographic region. We report cases of conventionally recognized nonhuman feeders (I. spinipalpis and I. angustus) parasitizing humans. The highest pathogen prevalence in I. spinipalpis may indicate a larger public health threat than previously thought, and the enzootic life cycle and pathogenicity of B. lanei warrant further study.

Ehrlichia muris is an agent of human ehrlichiosis. To determine its geographic spread in the United States, during 2016-2017, we tested 8,760 ticks from 45 states. A distinct clade of E. muris found in 3 Ixodes cookei ticks from the northeastern United States suggests transmission by these ticks in this region.