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Authors: Natalia I. Brothers, Jonathan M. Zenilman, MD, John N. Aucott, MD

Infrared thermography (IRT) is a non-invasive imaging technology that visualizes heat patterns in the body. IRT measures deviations from baseline body temperature that correspond to areas of increased peripheral perfusion. Previous studies have established its use in medicine to detect areas of inflammation, infection, and tumor growth. The use of IRT in Lyme disease is novel. The present study explored infrared imaging as a tool for capturing the presence of erythema migrans (EM) lesions in patients diagnosed with Lyme disease. Examination of three adult study participants revealed these lesions as round or oval areas of increased heat and prominent venous drainage. IRT images are able to identify single and disseminated EM lesions. This study highlights the potential of IRT to improve EM identification.

Author: Justin Brueggeman

Despite decades of research into Borrelia burgdorferi immunopathogenesis, less is
known about antigen-specific T cell responses to this bacterial pathogen than the role of
antibodies. Studying antigen-specific T cell responses to B. burgdorferi has been challenging due to the relatively few immunological tools available. To address this, we have engineered B.
burgdorferi to express a well-characterized CD4+ T cell epitope from chicken ovalbumin (Bb-
OVA). Using adoptively transferred OVA-specific T cells from OT-II TCR transgenic mice, we
show that these OVA-specific T cells proliferate during infection in an antigen-specific manner
in response to Bb-OVA but not Bb-control. Using a variety of markers, we have characterized
these antigen-specific T cells during various stages of infection, and the sites to which OVAspecific cells migrate. This tool will enable us to better characterize functional aspects of
antigen-specific T cell responses during infection, such as T cell homing and the effector
mechanisms responsible for both protection and immunopathology.

Author: Matthew Butnaru

Tick-borne diseases, including Lyme disease caused by Borrelia burgdorferi, present a growing public health concern. Advancing our understanding of tick biology at the cellular level is essential for uncovering mechanisms of pathogen transmission, immune responses, and signaling. Genetic studies can contribute to this understanding. However, research in whole ticks is limited by the species' long life cycle and reliance on blood meals. Cell-based studies offer a valuable alternative for functional genomic studies.

We have developed a platform for genome-wide pooled CRISPR screening in cultured Ixodes scapularis cells, enabling large-scale genetic studies. Our approach leverages recombination-mediated cassette exchange (RMCE) to efficiently integrate and express sgRNAs delivered as plasmids. To establish this system, we first introduced recombination sites via transposase-mediated integration and confirmed the inserted sites are competent for RMCE. We then further engineered the cel line to stably express Cas9-expressing, laying the foundation for CRISPR studies.

This presentation will highlight key methodological advances and challenges we faced in adapting CRISPR and other gene editing technologies for use in tick cells and conducting high-throughput screens. We will also share findings from proof-of-concept screens and discuss how the platform can be used to study diverse biological topics. Altogether, the platform enables scalable genetic studies directly in cells from I. scapularis, providing a powerful new tool for large-scale functional genomics studies relevant to fundamental tick biology, tick-borne disease, and intervention strategies

Authors: Dezmond Cole, Erin Goley

Rickettsia parkeri is a member and tractable model of the Spotted Fever Group (SFG) rickettsiae, obligate intracellular bacteria responsible for globally emerging tick-borne diseases in humans, with severity ranging from mild to fatal. These pathogens manipulate host cellular machinery to promote their dissemination, notably through actin-based motility (ABM). ABM is a critical virulence mechanism used by SFG rickettsiae to spread intracellularly from cell to cell and disseminate within a host. This process involves direct manipulation of the host cytoskeleton, creating unipolar actin comet tails that propel the bacteria through the host cytosol. A key bacterial effector in rickettsial ABM is Sca2, which localizes to the bacterial cell pole where it directly polymerizes host actin against the bacterial surface. However, the regulation of Sca2 specifically, and ABM more generally, remains unclear. We hypothesized that the regulation of actin-based motility is achieved through its integration with bacterial morphogenesis. To test this, we assessed the efficiency of actin tail formation following pharmacological and genetic perturbations to bacterial cell shape. Our findings show that disruption to the elongasome causes cell rounding and complete inhibition of actin tail formation. Round bacteria recruit peripheral actin clouds, but 0% form tails. These data suggest a critical link between bacterial morphogenesis and actin-based motility. Future work will focus on understanding the molecular basis of this relationship and the conservation of morphogenesis-regulated actin-based motility in pathogenic Burkholderia species. This work suggests that bacterial morphogenesis plays a vital role in regulating intracellular virulence, unveiling a new layer of fundamental bacterial biology.

Authors: Jessica Crooker, Dakota Paine, Xinru Wang,  Saravanan Thangamani

Powassan Virus Lineage II, also called Deer Tick Virus (DTV), is an encephalitic orthoflavivirus transmitted by Ixodes scapularis ticks. In patients who get the neuroinvasive disease, DTV has a mortality rate of 15% and leads to neurologic sequelae in 50% of survivors. These sequelae can include recurrent headaches, muscle wasting, and paralysis. Most of our understanding of DTV comes from research done using the prototypical laboratory strain that was isolated in the late 1990s from Wisconsin. Currently circulating strains provide an opportunity to study viral evolution and to ensure studies investigating DTV pathogenesis and disease mechanisms reflect what is occurring in clinical cases. To gain a better understanding of epidemiologically relevant DTV in the Northeastern US, 103 human-encountered ticks that tested positive for DTV were collected through the Upstate Tick Testing Program from 2019-2024. Virus-positive ticks were homogenized, and the resulting tick-homogenates were isolated via intra-cranial inoculation and subsequent culture in BHK and Vero cells to mimic the isolation of the prototypical strain. As of 2025, for 26 isolates genome sequencing was performed and genetic variations between circulating strains and the prototypical laboratory strain were analyzed in the context of geographic, temporal, and coinfection data. For 15 key isolates, viral kinetics were investigated in vitro using mammalian and tick-cell lines. To understand changes in early infection course in the skin between isolates, an ex vivo human skin model was infected with the isolates. In vivo alterations in I. scapularis acquisition and transmission of virus were studied to better understand alterations in vector dynamics. Finally, using an in vivo mouse model, we investigated the clinical course of disease, viral replication, and viral tropism in a subset of the isolates to better understand alterations in disease pathogenesis. Our preliminary analysis indicates these isolates are phenotypically and genotypically distinct from the laboratory strain. In addition, we have 

Author: Alexander Girgis 

Post-treatment Lyme Disease (PTLD) is a poorly understood complication of Borrelia burgdorferi infection with significant patient morbidity. Characterized by fatigue, generalized myalgias, and cognitive impairment, PTLD symptomatology closely resembles long COVID and other post-acute infection syndromes. While prior studies suggest immune dysregulation as a factor in PTLD pathogenesis, the mechanisms underlying its heterologous presentation and severity remain unclear. To associate symptom burden with discrete immune phenotypes, we applied factor analysis to self-reported symptom data from 272 PTLD patients to generate patient subgroups.  We then immunophenotyped peripheral blood cells of these individuals and 28 healthy controls through 18-parameter flow cytometry and cytokine profiling to associate PTLD status and the newly defined subgroups with specific immune states. Our PTLD cohort had fewer circulating CXCR5+ CD4+ Naïve T cells relative to healthy controls (5.2% vs. 8.3%, Padj < 0.001). These cells were positively associated with musculoskeletal pain in PTLD subjects, but not healthy controls. This and additional immunophenotypic alterations, including an increased prevalence of CXCR3+ CCR4- CCR6- CD8 T cells (43.1% vs. 25.7%, Padj < 0.01), permitted the creation of an elastic net classifier which identified PTLD with moderate efficacy (AUC 0.83). Measurement of cytokines did not reveal associations with PTLD and did not improve the performance of the model. While we were mostly unable to identify immune features which distinguished patient subgroups, factor analysis revealed multiple associations between immune cell frequency and the severity of specific symptoms.  Collectively, our findings add to growing evidence of immune dysfunction as a prominent feature of PTLD.

Author: Anna Hickman

Antiphospholipid antibodies have been linked to autoimmune and inflammatory disorders such as Lupus and Multiple Sclerosis. We previously identified antibodies against both borrelial and host lipids acute Lyme Disease Infection. Using clinical samples from acute and Post-Treatment Lyme Disease (PTLD), we conducted a serosurvey of these antibodies using an anti-lipid ELISA. Several antibodies were found to be elevated in the early stages of disease, while some rose in patients with PTLD. Titers of antiphosphatidylserine in patients with PTLD are elevated compared to controls and lookalike conditions, whereas other markers are transiently elevated in the early stages of disease and then decline. Since anti-lipid producing B Cells are typically short lived, these antibodies are expected to have a shorter half-life than existing diagnostic antibodies. Therefore we are investigating anti-lipid antibodies as biomarkers of PTLD, to provide proof of cure, and diagnosis of reinfection. 

Authors: Isaiah Hoffland, Sanjana Kannan, Irina Shatz, David Sullivan

Treatment of severe Babesia disease in immunocompromised individuals with approved combination therapy has a high recrudescence rate and presents the need for alternate pharmacological approaches. Here we established a pharmacokinetic and pharmacodynamic Babesia microti immunocompromised mouse model for both high and low parasitemia. The stringent SCID mouse model places reliance on the drugs for curative reduction with antibody absence. We have defined minimal single dose tafenoquine cure at 30 mg/kg in a high (20%-40%) starting parasitemia (~ 4 billion parasites) model and 15 mg/kg in the low (<1%) parasitemia model. Atovaquone (300 mg/kg daily) cures zero mice after 10-14 days in high parasitemia model and 75% in low parasitemia model.  Proguanil (20 mg/kg) addition to atovaquone is curative in the low parasitemia model. Interestingly we observe slight antagonism of artesunate with tafenoquine curative killing. Tafenoquine eliminates atovaquone-resistant B. microti. The macrolides, azithromycin  (120 mg/kg) and cethromycin (120 mg/kg) show delay in reduction in high parasitemia model. Combination studies with subcurative doses of tafenoquine are ongoing. Work in progress involves construction of two firefly luciferase transfection plasmids for B. microti genomic integration to enable an alternate means for parasitemia measurements.

Authors: Nerina Jusufovic, Keira Schinaman, Andrew C. Krusenstjerna, Jessamyn P. Morris, and Brian Stevenson

The Lyme disease spirochete, Borrelia burgdorferi, must integrate environmental cues to properly regulate gene expression and maintain survival during the enzootic life cycle. B. burgdorferi has a two-component signaling system which produces the signaling molecule c-di-GMP. Upon binding of this molecule by PlzA, the only universally encoded c-di-GMP binding protein in B. burgdorferi, expression of c-di-GMP responsive genes is modulated. PlzA and c-di-GMP are required for B. burgdorferi survival in the tick vector and maintaining the enzootic life cycle. Despite the importance of PlzA, the mechanism of this regulator was previously unknown. Our group recently identified that PlzA is a c-di-GMP dependent nucleic acid binding protein. Molecular docking of PlzA with nucleic acids identified several, potential key residues for binding. Mutagenesis of residues in these regions impacted PlzA-DNA binding affinity. Further, we have identified promiscuous cyclic di-nucleotide binding by PlzA, the first instance of a PilZ domain protein binding a molecule other than c-di-GMP. PlzA showed intermediate affinity for 3’3’ cGAMP, the bacterial derivative to mammalian cGAMP, indicating that PilZ domain proteins could recognize molecules beyond 3’5’ c-di-GMP and potentially cross-talk with other bacterial signaling systems. B. burgdorferi plzA-mutant strains are currently being produced to determine the consequences of aberrant PlzA-nucleic acid binding function on borrelial physiology. The presented work characterizes PlzA nucleic acid binding properties ultimately to better define the PlzA regulon. Our studies will further inform mechanisms by which the Lyme disease pathogen regulates gene expression for infection.

Authors: Oleksandra Kepple DVM, PhD, Sreeja Puthumana M.S, Amanda R. Walsh, DVM,  Mohamed Sallam, PhD

Intensifying climate variability, deforestation, and wildfire events are significantly reshaping the distribution and ecological dynamics of tick-borne pathogens (TBPs) in the rainforest regions of Colombia.

Wildfire frequency has increased in localized regions in Colombia, contributing to habitat stress. Additionally, severe climatic stress and heatwave conditions are accelerating environmental changes on a large scale. These pressures are shifting tick habitats and accelerating their developmental cycles. At the same time, wildlife displacement is increasing interactions between ticks, wildlife, livestock, and human populations, heightening the risk of zoonotic spillover.

Authors: Andrew Krusenstjerna, Erika Smith, Mathilde Gonin, Dezmond Cole,  Trung Nguyen, Erin Goley

Bacteria of the order Rickettsiales are obligate intracellular organisms. Members of this branch of ⍺-proteobacteria colonize a wide range of arthropods, contributing mutualistic or parasitic roles. Vector-mediated transmission of these bacteria to vertebrate hosts can result in various pathologies. The Spotted Fever Group (SFG) within the genus Rickettsia contains species that pose significant threats to human health. R. parkeri, the causative agent of a mild tick-borne spotted fever, is an emerging model for the study of SFG rickettsiae. Due to the lack of genetic tools, much of the biology of Rickettsia has yet to be elucidated. We have recently developed a streamlined Himar1 mariner transposon that leverages spectinomycin resistance and a bright green fluorescent protein (AausFP1) to produce a saturated transposon library for R. parkeri. Thus far, we have mapped 79 mutants in protein-coding genes using this approach, with ~85% being in genes not hit in past attempts. With this library, we seek to understand rickettsial morphogenesis and signal transduction. Currently, we are investigating the role of the bacterial signaling molecule cyclic-di-GMP (cdG). Disruption of the histidine kinase gene pleC that regulates the synthesis of cdG results in a small plaque phenotype.  We have found that this plaque size defect is not due to reduced actin-based motility, which promotes cell-to-cell spread and is a critical virulence determinant of SFG rickettsiae. We plan to implement mass spectrometry and c-di-GMP riboswitch reporters to further investigate this signaling module's role in rickettsial pathogenesis.   

Author: Olivia Looker, Grace Sarver

In an effort to assess the prevalence of the pathogens that cause Lyme disease and Anaplasmosis in the western MD/PA/WV region, a total of 523 black-legged deer ticks (Ixodes scapularis) were donated from private citizens. The ticks were identified and their genomic DNA extracted.  Polymerase chain reaction (PCR) was used to amplify the ospA gene, a marker exclusively present in Borrelia burgdorferi, the causative agent for Lyme disease. Additionally, the GE 16S rRNA gene was amplified and used to facilitate the identification of ticks carrying  Anaplasma phagocytophilum. PCR results were visualized by gel electrophoresis.  Results of tick testing were analyzed and then sent to the public in the form of an anonymous survey. The survey aimed to communicate findings regarding the prevalence of tick-borne diseases, raise awareness, gather feedback concerning personal preventative measures, and explore the implications of the escalating tick population on public health and personal experiences. Here, we share the tick surveillance data that was collected and the public survey results.

Authors: Barbara Birkaya, Ahana Byne, Fatema Hashemi, Rocio Cornero, Paul Russo, Hope McIntyre, Lance Liotta, Alessandra Luchini

The cause of symptom persistence months to years after patients experience acute Lyme disease is one of the most salient unanswered questions in the field of Lyme disease research.  We hypothesized that Borrelia-derived extracellular vesicles (BEVs) serve as vehicles for maintaining antigenic pressure in the human host. We isolated and characterized BEVs from Borrelia burgdorferi, afzelii, and garinii cultures. Approximately 10,000 BEVs were isolated per spirochete over a 10-day period, with sizes ranging from 130 to 170 nm and a distinctive double-membrane structure, which is characteristic of spirochetal architecture. BEV proteomics and lipidomics analyses revealed 58 proteins related to DNA and RNA maintenance, chemotaxis, ion transport, energy metabolism, chaperones, membrane lipoproteins, and pro-inflammatory lipids, suggesting a role in immune activation. In the immunoassays, BEVs were positive for Borrelia peptidoglycan, p66, and FlaB. DNA analysis confirmed the presence of genetic material, including genes that are potentially involved in immunomodulation. We developed novel monoclonal antibodies targeting conserved epitopes of Borrelia FlaB and p66 proteins and validated their specificity against Borrelia without cross-reactivity to human and microbiome proteins. These antibodies detected BEVs in urinary extracellular vesicles (EVs) of patients with acute Lyme and post-treatment Lyme disease syndrome (PTLDS), thus supporting the systemic circulation of BEVs in the human host. In human HMC3 microglial cells, exposure to BEVs triggered a significant increase in gene expression, particularly of immune-related genes, such as interferon-alpha, aconitate decarboxylase 1, and toll-like receptor 2. Immunofluorescence studies confirmed the upregulation of microglial activation markers and NRF2 nuclear translocation. Furthermore, BEVs impaired the phagocytic efficiency of HMC3 cells, an effect augmented by co-treatment with IL-33. These findings support the hypothesis that BEVs act as persistent antigenic reservoirs and potent inducers of neuroinflammation, potentially explaining the chronic symptoms observed in patients with PTLDS. This study opens new avenues for the development of diagnostic tools and therapeutic strategies targeting vesicle-mediated pathology in Lyme disease.

Authors: Roy Faiman, Vasiliki Ikonomidou, Thomas Jenkins, Sameerah Talafha, Hannah Markle, Autumn Goodwin, Georgia Constantine, Pooja Bala Nehru, Margaret Glancey, Roland Duhaime, Thomas Mather, Tristan Ford

Tick-borne diseases pose significant public health challenges globally. In the United States Lyme disease is the most prevalent vector-borne illness. Other prominent tick-borne diseases in the US include Rocky-Mountain Spotted Fever, Ehrlichiosis, Anaplasmosis, and Babesiosis. Accurate tick identification is essential for effective surveillance to understand risk profiles and disease prevention and mitigation efforts. Vectech has continued development on a smart imaging system for instant tick identification named IDX, which is cross-functional for other arthropod vectors as well. Vectech’s IDX system is designed for professional public health markets to support vector surveillance activities. In its current version, it achieves a macro f1-score of ~93% across a dataset of 31 prominent domestic and international tick species. The precision is predominantly challenged by intra-genus errors, particularly within the 8 Rhipicephalus species, which contribute to 29% of the total error, and the 7 Amblyomma species, accounting for 29% of the error. Notably, 74% of IDX's total error for tick species arises from intra-genus discrepancies, primarily due to the closely related morphologies of species within these genera. In collaboration with partners across the US, Vectech is exploring differentiating Dermacentor variabilis and D. similis in IDX; preliminary results show a 96% accuracy in distinguishing between the two cryptic species. Additionally, Vectech is developing a mobile application to support the general public in identifying ticks found on their person to support personal risk assessment. A large-scale tick image dataset (n≈36,000) sourced from the University of Rhode Island’s TickSpotters citizen science initiative supported development of a preliminary object detection algorithm achieving 88% recall and 99.9% precision, with small tick size in the photo as the primary determinant of error. Current species classification efforts are ongoing, with expected limitations: (1) underrepresentation of species besides Dermacentor variabilis, Ixodes scapularis, and Amblyomma americanum; and (2) morphological variation due to sex, developmental stages and engorgement status. By the end of this development phase, Vectech anticipates the rollout of products that will expand the capacity of tick surveillance and public health responses, significantly contributing to the mitigation of tick-borne diseases in the United States.

Zachary Osterwind, Maureen Miller, Valeria De Giorgi

The oligosaccharide galactose-alpha-1,3-galactose (alpha-gal) is universally produced by mammals, excluding certain primates and humans. Saliva of the Lone Star tick (Amblyomma americanum) contains alpha-gal that can trigger the development of an IgE-mediated food allergy to mammalian meat and derived products known as alpha-gal syndrome (AGS). The blood type B antigen is structurally similar to alpha-gal potentially allowing cross-reactivity between alpha-gal IgE and B red blood cells. It is speculated that alpha-gal sensitized type A and O recipients of type B blood product transfusions (platelets, plasma, cryoprecipitate) may experience allergic transfusion reactions (ATRs) to type B red blood cells. The objective of this study was to evaluate the quality of evidence on the role of AGS in ATRs and organize the information into a comprehensive toolkit. A narrative review of peer-reviewed articles and gray literature on the topic was performed in PubMed and Google without publication language or date restrictions using “alpha-gal syndrome” and “blood transfusion reaction” or “allergy.” The preliminary results were used for a citation search. Overall, the search yielded 216 results, with 45 results reviewed. Two medical chart reviews and one commentary letter specifically discussing AGS and ATRs were found. This literature search yielded few results regarding the hypothesized mechanism of allergic reaction, though alpha-gal sensitization is known to be increasing in prevalence in the United States. This toolkit and other clinical studies investigating the alpha-gal sensitization phenomenon may assist blood bankers managing transfusion reactions in regions such as Washington, D.C. with Lone Star tick presence.

Authors: Lise E. Nigrovic, Meagan M. Ladell, Laura L. Chapman, Fran Balamuth, Desiree N. Neville, Anupam B. Kharbanda, Stephanie M. Hadley, and Amy D. Thompson 

Background: As Lyme disease serology can take several days, alternate diagnoses need to be excluded while awaiting results. Our goal was to describe the impact of the initial evaluation for children with suspected Lyme disease. 

Methods: We performed a prospective cohort study of children 1 to 21 years of age who presented to one of eight Pedi Lyme Net emergency departments between 2015 and 2024 for suspected Lyme disease. We defined Lyme disease with either a clinician diagnosed erythema migrans (EM) lesion measuring at least 5 cm in diameter or positive two-tier Lyme disease serology within 30 days of enrollment. We defined Lyme disease stage as follows: early-localized (single EM lesion), early-disseminated (multiple EM lesions, facial palsy, meningitis, carditis) or late (arthritis). 

Results: Of 5,533 children enrolled, 1,372 (24.8%) had confirmed Lyme disease. The median age was 8 years (interquartile range 5-13 years) and 3,132 (56.6%) were male. Of those with symptoms of early-disseminated Lyme disease, 332 (13.5%) had a lumbar puncture performed and, of those with symptoms of late Lyme disease, 223 (8.3%) had an invasive joint procedure (arthrocentesis and/or joint wash-out). Overall, 1,367 (24.4%) were hospitalized (median duration of hospital stay was 2 days, interquartile range 1-4 days) and 472 (8.5%) received parenteral antibiotics. 

Conclusions:  Evaluated for suspected pediatric Lyme disease has substantial impact on many children. An accurate rapid Lyme disease diagnostic would reduce impact and allow prompt initiation of appropriate treatment.

Authors: Trung Nguyen, Erin Goley
Bacteria can exploit cell polarity to compartmentalize and spatiotemporally coordinate biochemical processes. In many species, this is driven by the polar organizing protein PopZ, which forms a biomolecular condensate at the cell pole and recruits factors that direct processes like chromosome segregation and morphogenesis. The Spotted Fever Group (SFG) of the Rickettsia genus encompasses tick-borne, obligate intracellular human pathogens causing potentially fatal infections. Rickettsia parkeri (Rp), which elicits a mild disease, is a tractable SFG model. Yet, there is a knowledge gap in Rp cell biology fundamentals. Though lacking morphological polarity, Rp’s encodes PopZ and 3 of PopZ’s 12 known client proteins in related species. We hypothesize RpPopZ drives polarity via a unique client repertoire to support growth and/or host interactions. We first validated RpPopZ’s direct interaction with the 3 predicted client proteins. Next, we used quantitative imaging analyses to show that RpPopZ and two of its direct interactors: the centromere anchor ParB, and the predicted PopZ regulator ZitP display distinct cell cycle-regulated localization patterns, suggesting unique functions of RpPopZ in polarity establishment during Rp’s cell cycle. 

To comprehensively determine RpPopZ’s interactome, we are conducting miniTurbo-based proximity labeling. Excitingly, our preliminary data showed miniTurbo-PopZ-specific enrichment of new potential polar proteins that include components of the type IV secretion system and secreted effectors involved in host interactions, as well as cell cycle-related proteins. We will continue to build tools to study how RpPopZ and related polarized processes are integrated into the cell cycle. This work will advance studies of PopZ-associated pathogenic processes and may facilitate therapeutic developments against rickettsial infections and other pathogens that deploy PopZ to support growth and environmental interactions. 

Keywords: cell polarity, intracellular pathogens, host-pathogen interaction, cell cycle regulation, PopZ biomolecular condensate

Authors: Jennifer Platt, DrPH; Genevieve Weseman, MS; Melissa Olivadoti, PhD, Tina Merritt, MD

Alpha-gal Syndrome cases continue to pose a growing threat to public health, with many patients reporting symptoms and reactions long after initial tick-bite. This research presents six years of self-reported data from over 4,000 individuals diagnosed with AGS across the United States, capturing trends in symptom progression, diagnostic challenges, and quality-of-life impacts. Findings reveal consistent patterns of symptoms across all body systems—especially neurological, musculoskeletal, and cognitive issues—that are often under recognized in clinical settings. Patient narratives highlight gaps in timely diagnosis and treatment, underscoring the need for improved medical education and public awareness. These insights offer a vital patient-centered perspective to inform future research and healthcare responses.

Authors: Rebekah Taylor, Grace Sarver, Cierra Wilson, Olivia Looker

Since 2017, residents of Western Maryland and surrounding counties in West Virginia and Pennsylvania have been mailing ticks that they have found to the Biology Department at Frostburg State University (FSU). These ticks are identified by students at FSU, who then extract DNA and test for pathogens, namely B. burgdorferi and A. phagocytophilum, by PCR. The data from the testing is uploaded onto an open-access mapping platform called Fieldscope, which allows users to see where the ticks are and what pathogens they carry, as well as engage in sensemaking by applying various filters and maps to the data. Here, we present the results of our study thus far, reveal trends in tick populations in the Western Maryland region, and share stories of community engagement and student learning.

Author: Alannah Taylor

Lyme Disease is the most common tickborne disease in the United States with over 63,000 cases reported in 2022. However, the actual number of cases of Borrelia burgdorferi (Bb) is estimated to be ten-fold higher. Post-Treatment Lyme Disease Syndrome (PTLDS) is a condition where a patient experiences chronic symptoms despite being treated with antibiotics. There is controversy around whether PTLDS is caused by a persistent bacterial infection or post-infection auto-immune reactivity. Some evidence suggests the failure of symptoms to resolve with more aggressive antibiotic therapy could be due to antibiotic tolerance of persistent bacteria. This persistence phenotype is induced and maintained by the bacterial stringent response pathway. We hypothesize that mRNA transcripts encoding stringent response proteins can be isolated from bacteria-derived microvesicles from the serum of mice infected with Bb. B. burgdorferi extracellular vesicle (BbEV) isolation from bacterial cultures and mouse plasma was conducted using size- exclusion chromatography or ultracentrifugation. It was followed by a density gradient to separate the BbEV from mouse-derived EVs. The BbEVs were confirmed by performing a RNA extraction, RT-PCR, and analyzing fractions on the Zetaview particle analyzer. We have been able to confirm the ability to extract extracellular vesicles from bacterial cultures and are finalizing analysis of Bb-infected mouse plasma. In conclusion, while the controversy of the mechanism of PTLDS is still debated, this study is laying the groundwork for utilizing BbEVs in plasma for detection of genes representative of bacterial infection and potentially of pathways promoting persistence that could lead to PTLDS.

Authors: Emma Tilley, Sohan Chacko, Aleah Eschman, Siu-Ping Turk, Ashley Thomas, Nyah Collier, Sheryce Lassiter, Adriana Marques

The blacklegged tick, Ixodes scapularis, is a vector for seven human pathogens within the United States, notably Lyme disease, anaplasmosis, ehrlichiosis, Powassan virus disease, babesiosis and Borrelia miyamotoi disease. Annually, the reported case counts for these diseases continue to rise as the geographic distribution of I. scapularis broadens. As tick exposure incidence rates in humans continue to rise, research exploring acquired tick resistance and tick-associated skin immunity can add to the arsenal of disease prevention. Immune responses to multiple tick bites or multiple tick exposures have been shown to induce tick resistance in animal models and consequently protection against tickborne pathogen transmission. Similar findings are seen in humans, but little is known about the nature of the protective immune response. Our study aims to develop a model for acquired tick resistance in humans, characterize the acquisition of tick-associated skin immunity, and monitor the innate and adaptive immune response of larval I. scapularis tick bite. Volunteers underwent three separate clean, laboratory-bred, larval I. scapularis tick exposure procedures occurring 2-8 weeks apart. At each time point, two skin biopsies at the site of tick bites are performed. One biopsy is used for histopathology and spatial molecular imaging, while the other biopsy is used for either bulk or single cell transcription studies. For each biopsy site, the area of erythema and its blood perfusion as measured by laser speckle contrast imaging, pruritus as measured by a verbal rating scale, and histopathology are being performed. The correlations between the different measurements are being explored and will be used to inform our in-depth investigation into the inflammatory and immune reactions seen in human skin over repeated tick-bite exposures.

Author: Linus Wang

Lyme disease, the most widespread tick-borne disease in North America, is caused by the bacterium Borrelia burgdorferi (Bb). Approximately 10-15% of infections result in neuroborreliosis, common symptoms of which include headaches, facial palsy, and long-term cognitive impairment. Though Bb dissemination is an important step in disease progression, the biological and microenvironmental factors that govern this process remain poorly understood. Previous studies of Bb dissemination focus on assessing Bb transmigration at static time points rather than analyzing the inherently complex dynamic process of extravasation. Furthermore, current in vitro models lack crucial physiological factors such as flow, demonstrating a need for more robust models to study Bb dissemination and understand its dynamics and mechanisms. Here, we use a 3D tissue-engineered microvessel model and perfuse fluorescently-labeled Bb to model vascular dissemination in non-tissue-specific (iEC) and brain-specific (iBMEC) microvessels while acquiring time-lapse images in real time using confocal microscopy. In iECs, extravasation involves two steps: adhesion to the endothelium and transmigration into the extracellular matrix, which can be modulated through glycocalyx degradation or inflammation. In contrast, Bb extravasation in iBMECs is an extremely rare event regardless of glycocalyx degradation or inflammation. In addition, circulating Bb do not induce endothelial activation in iECs or iBMECs, but induce barrier dysfunction in iECs. These findings provide a further understanding of Bb vascular dissemination and demonstrate the use of a novel model to study infectious disease. 

Authors: Bryan T. Murphy, Jacob J. Wiepen, Danielle E. Graham, Selene K. Swanson, Maithri M. Kashipathy, Anne Cooper, Kevin P. Battaile, David K. Johnson, Laurence Florens, Jon S. Blevins, Scott Lovell, and Wolfram R. Zückert

In diderm bacteria, the Lol pathway mediates the periplasmic transport of lipoproteins from the inner membrane (IM) to the outer membrane (OM) and therefore plays an essential role in bacterial envelope homeostasis. After extrusion of modified lipoproteins from the IM by the LolCDE complex, LolA carries lipoproteins through the periplasm and transfers them to the OM lipoprotein insertase LolB, itself a lipoprotein with a LolA-like fold. Yet, LolB homologs appear restricted to β- and γ-proteobacteria and are missing from spirochetes like the Lyme disease pathogen Borrelia burgdorferi, suggesting a different hand-off mechanism at the OM. Here, we solved the crystal structure of the B. burgdorferi LolA homolog BB0346 (LolA_Bb) at 1.9 Å resolution. We identified two major structural deviations in comparative analyses to other solved LolA structures, particularly a unique LolB-like protruding loop domain. LolA_Bb failed to complement an Escherichia coli lolA knockout, even after codon optimization, signal I peptide adaptation, and a C-terminal chimerization which had allowed for complementation with an a-proteobacterial LolA. Analysis of both B. burgdorferi lolA conditional knockout and CRISPRi-mediated knockdown strains indicated that LolA_Bb was essential for growth. Intriguingly, protein localization assays indicated that depletion of LolA_Bb led to an emerging mislocalization of both IM and periplasmic OM lipoproteins, but not surface lipoproteins. Together, these findings further support the presence of two separate primary secretion pathways for periplasmic and surface OM lipoproteins in B. burgdorferi and suggest that the distinct structural features of LolA_Bb allow it to function in a unique LolB-deficient lipoprotein sorting system.