Andrew J. Gaetano Elizabeth S. Danka St. Norbert College, Division of Natural Sciences, De Pere, WI Keywords: Borrelia burgdorferi, Ixodes scapularis, Lyme Disease, Spirochete, Vector-borne infection © 2024 Gaetano, Danka. Fine Focus, 10(1), 9-37. doi: 10.33043/FF.10.1.9-37. Shared with CC-BY-NC-ND 4.0 License. Manuscript received: 28 Feb 2023; accepted 13 July 2023 Abstract Since its recent discovery in the late 1970s, Lyme Disease (LD) has been a growing public health concern, especially in the United States where it accounts for the majority of vector-borne infections each year. The causative agent, Borrelia burgdorferi, is transmitted to humans through the bite of an infected Ixodes tick. This pathogen uses many unique mechanisms to both shield itself from the host immune response and cause disease. Clinically, LD presents in successive phases, with each increasing in severity as the bacterial cells migrate to new tissues and organ systems. On the epidemiological and ecological fronts, limitations in reporting, ecological changes, and a lack of public support hinder accurate surveillance and enhance the spread of the disease. The goal of this literature review is to increase public knowledge of B. burgdorferi, its vector, and the disease it causes, along with suggesting preventative measures to protect individuals who reside in high-risk areas. A collective and coordinated public health effort represents our greatest chance of restraining the LD-causing pathogen. Borrelia burgdorferi: The Deer Tick’s Dark Secret https://creativecommons.org/licenses/by-nc-nd/4.0/ Fine Focus | Volume 1010 Discovery of the Causative Agent of Lyme Disease In 1976, the Connecticut State Department of Health reported an outbreak of an unusual form of arthritis near Lyme, Connecticut (37). The affected individuals experienced recurrent bouts of pain and swelling in large synovial joints (such as the knee) without prior injury. Other clinical presentations included flu-like symptoms and unusual cutaneous lesions called erythema migrans (EM), which had first been described by the German physician Alfred Buchwald in 1883 (75). Although a causative agent had not yet been discovered, this unique combination of signs and symptoms was termed Lyme Disease (LD) (37). Most patients with the disease lived in heavily wooded areas away from the centers of towns, and the onset of their symptoms was often in the summer and early fall. The spatial and temporal distribution of cases hinted that the disease was likely transmitted through an insect vector, but the State Department of Health did not have any additional information. It would take six more years to identify the LD-causing pathogen. In 1982, Wilhelm “Willy” Burgdorfer isolated a spirochete from Ixodes scapularis, the black- legged deer tick (7). This microorganism was soon shown to be responsible for the unique disease in Lyme, Connecticut. When ticks harboring this pathogen fed on New Zealand White rabbits, long-lasting EM-like lesions developed. Indirect immunofluorescence also confirmed that the rabbits produced antibodies specific to these spirochetes. A causal relation- ship between the newly discovered spirochete and LD in humans was established when the serum of clinically diagnosed patients revealed antibodies specific to the pathogen, which was subsequently named Borrelia burgdor- feri. Despite its recent characterization, the earliest confirmed case of LD occurred in the 5,300-year-old Similaun Iceman (“ÖTZI”) found preserved frozen in the Italian Alps (31). Arthritis was observed upon clinical examina- tion, and DNA sequencing of samples from the Iceman confirmed the presence of B. burgdor- feri. While a great deal of work has focused on characterizing the pathogen, many challenges exist on the clinical and epidemiological fronts. Diagnosis and treatment of LD is complicated by B. burgdorferi’s wide array of virulence strategies that allow it to infect multiple organ systems, lie dormant for long periods of time, and resist and suppress the host immune response (20, 47, 48). Additionally, the clinical manifestations can vary widely, which further heightens the challenge for clinicians to make a timely and accurate diagnosis. While a vaccine represents the most effective preventative measure, there are not any currently available on the market. Due to limitations in disease surveillance, the reported number of LD cases in the United States is thought to be significantly lower than the Centers for Disease Control and Preven- tion’s (CDC) annual estimate (34). Despite this, LD represents more than 80% of vector- borne illnesses making it the most common vector-borne disease in the country (3). Making matters worse, the home range of B. burgdor- feri’s vector and its reservoirs are expanding as the ecological landscape continues to change Gaetano & Danka | Borrelia burgdorferi: The Deer Tick’s Dark Secret 11 (73). Approximately 90 million Americans live in states deemed “high risk” by the CDC, and even this high value is likely an underestimate of the total number of individuals at risk (13). Disease prevention depends on increasing public knowledge and awareness of B. burgdor- feri and its disease-causing ability, Ixodes genus ticks and their life cycle, LD and its clinical manifestations, and of available precautionary measures for individuals in high-risk areas. Introduction to B. burgdorferi B. burgdorferi is a Gram-negative bacterium with an inner and outer membrane. Unlike most gram-negative organisms, B. burgdorferi lacks lipopolysaccharides (LPS) in the outer membrane and instead displays other immuno- genic glycolipids (70). The spirochete bores through host tissues using internal periplasmic flagella that confer swimming motility and flat-wave morphology (32). While this flat-wave structure dominates, other pleomorphic forms exist under certain environmental conditions (see “Pleomorphic forms” and Figure 6) (44). The B. burgdorferi genome consists of a single 910,725 base pair linear chromosome with 853 open reading frames whose products are involved in the basic processes of DNA replication, transcription, translation, solute transport, and energy metabolism (23). Due to a lack of biosynthetic genes, the spirochete is an obligate parasite and depends on an arthropod or mammalian host for survival. Aside from hemolysins and drug efflux pumps, B. burgdor- feri lacks common virulence factors and instead relies on dynamic gene regulation to evade and suppress the host immune response (3). As an obligate parasite, B. burgdorferi can be difficult to maintain in common laboratory cultures that do not closely mimic the host environment (1). To overcome this challenge, optimized Barbour-Stoenner-Kelly (BSK) media that contains 6% rabbit serum and a collagen matrix is used to support the growth of B. burgdorferi (62). In vitro cultivation consists of a two-step process whereby a rapidly growing starter culture is used to initiate the growth of a long-term culture with a high bacterial yield. While in vivo studies most accurately represent the conditions that B. burgdorferi naturally encounters, the ability to work with isolated, parasitic bacteria in the laboratory increases the feasibility of research. Lyme Disease The unique virulence and immune evasion strategies of B. burgdorferi manifest clinically in humans as a complex, multi-stage disease. In this section of the review, the life cycle of the arthropod vector, transmission to the mamma- lian host, and clinical manifestations of LD will be described. 1. Vector life cycle Vectors are organisms, often insects, that harbor and transmit pathogens to other hosts. Within the Ixodes genus, black-legged deer ticks (Ixodes scapularis) are the main vectors that transmit B. burgdorferi to reservoirs such as mice and hosts such as humans. These reservoirs vary in their degree of competency to transmit B. burgdorferi back into an uninfected arthropod vector. Ixodes ticks have a complex, two-year, four-stage life cycle, which consists of egg, larva, Fine Focus | Volume 1012 nymph, and adult forms (Figure 1) (19). In the spring, adult female ticks at the end of their life cycle lay eggs that hatch into six-legged larvae within about 60 days. The females lay the eggs on grasses where the larvae will be exposed to mammals like deer and mice after hatching. To progress to the next stage of the life cycle, the larvae require a first blood meal which often comes from the reservoir-competent, white-footed mouse, but may also come from other small animals such as chipmunks, shrews, squirrels, and birds. After larvae feed in the late summer to early fall, they molt into eight-legged nymphs that remain inactive during the winter months. In the spring, the nymphs take a second blood meal which allows them to develop into adults in the summer. As adults, a third feeding, often from the reser- voir-incompetent white-tailed deer (Odocoileus virginianus), is required to reproduce. After feeding, adult male and female ticks copulate on the deer, and females lay their eggs the following spring to complete the life cycle. 2. Route of transmission For humans to develop LD, a tick harboring B. burgdorferi must bite the human and take a blood meal (7). The tick’s first feeding as a larva is unable to cause infection because the bacteria cannot be passed down in eggs and the arthro- pod can only acquire the spirochete through feeding (19). Therefore, larvae must take a blood meal from an infected, reservoir-com- petent organism to obtain the pathogen before transmission to humans is possible. If this first blood meal contains B. burgdorferi, the pathogen will colonize the tick midgut and the bacterial cells lose their motility. A secondary feeding during the nymph stage is required Figure 1 The Ixodes genus tick life cycle. Note. A red blood cell indicates that a blood meal is required to progress to the following stage. The blue, yellow, red, and orange bars represent winter, spring, summer, and fall, respectively. Timeline is based on reference 19. Gaetano & Danka | Borrelia burgdorferi: The Deer Tick’s Dark Secret 13 for B. burgdorferi to replicate, restore motility, and migrate to the tick’s salivary glands where it becomes primed for transmission. Thus, nymphs represent the earliest stage of the tick life cycle where B. burgdorferi transmission resulting in LD in humans is possible. To locate a host, ticks use a maneuver called questing where they climb to the top of grasses and other small plants, extend their front legs, and latch on to a passing animal (Figure 2B) (18). After finding a host, the tick migrates to a suitable location to take a blood meal. For humans, one study determined the distribution of tick attachment to be 9% head-neck, 5% arm, 24% stomach/groin, 7% back, 18% chest/shoul- der, 25% leg/foot, and 12% hip (Figure 2A) (25). After a tick begins feeding, transmission of B. burgdorferi is not immediate. There is a positive correlation between the duration of vector attachment and the probability of B. burgdorferi colonization and disease (57). In a murine study exploring this relationship, infection was established in 7% of mice after 36 hours, 25% after 42 hours, and 75% after 48 hours. This Figure 2 Tick questing behavior leads to attachment to hosts. Note. Distribution of tick attachment sites on humans (A) and representation of the tick questing behavior that is used to seize a suitable host for a blood meal (B) (18, 25). Fine Focus | Volume 1014 time-dependent transmission demonstrates the importance of rapid tick removal after attach- ment as a preventative measure against LD. While there is no evidence that human-to-hu- man transmission of B. burgdorferi is possible, there are many published cases of gestational LD with negative outcomes such as miscarriage, death following birth, and congenital abnor- malities (72). However, a systematic review of these published cases concluded that most reports contained blinding issues, had missing or limited information on the mother’s clinical symptoms, or used diagnostic methods that are no longer considered reliable. Therefore, additional research using reliable methods is necessary to determine the effects of gestational LD and the consequences it may have on women in their childbearing years. 3. Disease progression LD progresses in three distinct phases termed early localized, early disseminated, and late disseminated infection (48). The basic signs, symptoms, and commonly affected tissues corresponding to each phase are presented in Figure 3, and each phase is described in depth below. The first clinical manifestation of early localized B. burgdorferi infection is usually a slowly expanding cutaneous rash called erythema migrans (EM) (48). EM presents 7-14 days after exposure at the site of tick attachment in 70-90% of cases. The rash begins as a small red papule that takes on a bullseye appearance as it expands to an average diameter of 15 centi- meters. EM is usually asymptomatic, but other flu-like symptoms such as headache, fatigue, malaise, and fever may occur. Early localized LD usually lasts for a few days to a month. Early disseminated infection usually occurs three weeks to several months after the onset of primary EM, and it typically lasts 3-10 weeks (48). During this phase, B. burgdorferi Figure 3 LD signs, symptoms, and commonly affected tissues. Note. Lyme disease progresses through three distinct stages: early localized (A), early disseminated (B), and late disseminated infection (C) (48). Each stage is characterized by different signs and symptoms that reflect the spread of the pathogen from the initial site of infection. Gaetano & Danka | Borrelia burgdorferi: The Deer Tick’s Dark Secret 15 disseminates to the central nervous system (CNS), the cardiovascular system, and other cutaneous regions which leads to secondary EM away from the initial site. CNS involvement, termed Lyme neuroborreliosis, occurs in about 15% of untreated cases (22). Colonization of the nervous system can occur through penetration of the blood-brain barrier, migration from the peripheral nervous system, or movement through the cerebrospinal fluid. Common signs of neurologic involvement include bilateral cranial nerve palsies of the face, meningitis, and encephalitis. Cognitive impairment, psychiatric disturbances, and even seizures have been observed in patients with early disseminated LD (41). Cardiac involvement, termed Lyme carditis, typically occurs a few weeks to several months after EM onset in about 4-10% of untreated cases (21). Signs of Lyme carditis include prolonged PR interval, atrioventricular (AV) block, myocarditis, intra- ventricular conduction disturbances, bundle branch block, and congestive heart failure (50). Patients presenting with Lyme carditis may require electrocardiogram (ECG) monitoring and temporary or permanent pacemakers for AV block (74). General flu-like symptoms also accompany the early disseminated phase of infection (48). Late disseminated infection, also called chronic LD, occurs months to years after the onset of primary EM and can last many years if untreated (48). Late-stage disease is marked by chronic, intermittent arthritis which occurs in about 80% of untreated individuals, along with continued neurologic, cardiac, and cutaneous manifestations (76, 30). Lyme-associated arthritis results from acute swelling and erythe- ma in the joints, most commonly the knee (5). Excessive inflammation, infection-induced autoimmunity, and failure to down-regulate the inflammatory response are factors that contrib- ute to chronic LD. Virulence of B. burgdorferi in Hosts Bacterial pathogens utilize several methods to spread through and damage host tissues. B. burgdorferi is no exception, and in this section of the review a few of the many disease-caus- ing characteristics of this spirochete will be discussed. 1. Plasmid-derived virulence In addition to the single, linear chromosome, the B. burgdorferi genome contains at least 17 linear and circular plasmids (lp and cp, respectively) (23). While some of these extrach- romosomal DNA molecules have demonstrated critical roles in disease progression, others remain uncharacterized. A cell’s plasmid profile is its unique collection of plasmids. Long-term in vitro cultivation of B. burgdorferi results in concurrent changes to both plasmid profile and murine infectivity (64). Over time, the total number of plasmids within each cell decreases, which coincides with a drop in virulence. The degree to which the virulence of B. burgdorferi depends on its plasmid profile was expanded upon in a study that examined the infective phenotypes of a collection of clonal mutants, each with a different combination of plasmids (60). High-infectivity was observed in the presence of both linear plasmid 25 (lp25) and 28-1 (lp28-1), intermediate-infectivity was observed in the presence of lp25 and the absence of lp28-1, and Fine Focus | Volume 1016 low-infectivity was observed in the absence of lp25 independent of lp28-1 (Table 1). While some B. burgdorferi plasmids have been linked to highly infective phenotypes, others take on different roles (60). For example, cp26 is required to cause disease, but its presence does not affect the degree of infectivity. Other plasmids such as cp9 and lp21 are not associ- ated with infectivity at all. Further character- ization of B. burgdorferi plasmids linked to infectivity will lead to a greater understanding of the organism’s requirements for infection and disease. 2. Temperature-dependent gene regulation B. burgdorferi relies on both an arthropod vector and a mammalian host for survival. Upon transmission from vector to host, the spirochete experiences a temperature change from 23˚C to 37˚C. This change activates genetic regulatory mechanisms that allow B. burgdorferi to adapt to its surrounding environ- ment and successfully establish a mammalian infection (65). Outer surface proteins (Osp) are immunogenic lipoproteins found on the cell surface of B. burgdorferi. OspC, a known antiphagocytic factor, is required to establish a mammalian infection (8). In unfed ticks at 23˚C, B. burgdor- feri predominantly expresses ospA (65). After the tick takes a 37˚C mammalian blood meal, the spirochete’s osp expression profile changes to ospC. This was confirmed by examining B. burgdorferi-infected murine serum which contained antibodies specific to OspC. One explanation for this temperature-dependent gene regulation relies on the topology of the ospC-containing cp26 plasmid. At 23˚C, the plasmid is in a supercoiled state which blocks the transcriptional machinery from accessing the ospC locus. In vitro work demonstrated that upon exposure to warm (37˚C) mammalian serum the supercoiling of cp26 is reversed permitting expression of ospC. Another explanation for the temperature-de- pendent regulation of ospC involves small regulatory RNAs (sRNA) (40). RpoS is an alternative sigma factor involved in the initia- Table 1 B. burgdorferi infectivity as it relates to linear and circular plasmid profile. Infectivity-associated Not infectivity-associated Always present Infective phenotype*, # lp25 lp28-1 cp9 lp21 cp26 High + + +/- +/- + Intermediate + - +/- +/- + Low - +/- +/- +/- + Note. * + plasmid must be present for the given infective phenotype, - plasmid must be absent for the given phenotype, +/- plasmid does not affect infective phenotype #Data from reference 60 Gaetano & Danka | Borrelia burgdorferi: The Deer Tick’s Dark Secret 17 tion of ospC transcription. Expression of rpoS is regulated by DsrAAB, a temperature-sensitive sRNA. At 23˚C, the rpoS transcript contains a secondary stem-loop structure that occludes the ribosome binding site (RBS) preventing translation of the sigma factor. Also at 23˚C, DsrAAB exhibits secondary structure which prevents interactions between the sRNA and the rpoS transcript that are necessary for high levels of gene expression (Figure 4A). At 37˚C, DsrAAB loses its secondary structure which allows it to post-transcriptionally regulate the rpoS transcript. This results in loss of the rpoS stem-loop structure, RBS availability, and active translation of RpoS sigma factors (Figure 4B). The high level of RpoS expression at 37˚C greatly enhances the expression of ospC, which helps facilitate transmission of B. burgdorferi from the arthropod vector to the mammalian host. Since the discovery of this molecular thermom- eter, over 1000 other B. burgdorferi-sRNAs have been identified, many of which demon- strate temperature-sensitivity (59). These molecules likely contribute to genetic regulatory mechanisms that promote B. burgdorferi trans- mission by allowing the pathogen to adapt to changing environmental conditions. 3. Motility B. burgdorferi possesses periplasmic bundles Figure 4 Temperature-dependent expression of ospC. Note. A temperature shift accompanies the transmission of B. burgdorferi from the arthropod vector to the mammalian host (40). At 23˚C in the unfed tick, secondary structure of the sRNA DsrAAB prevents translation of the rpoS transcript, resulting in low levels of ospC expression (A). At 37˚C in the mammalian host, DsrAAB secondary structure is reversed, allowing for the production of RpoS and OspC (B). Fine Focus | Volume 1018 of flagella that originate from basal bodies at its terminal ends (32). These structures confer flat-wave morphology and corkscrew swimming motility, which allow the spirochete to move both forward and backward as it bores through host tissues. This unique ability is integral to pathogenicity because it allows B. burgdorferi to disseminate throughout the human body, which results in the wide range of clinical manifesta- tions associated with LD. The flaB and fliG genes are required for flagel- lar functioning (68). The flaB gene encodes the major flagellar filament protein FlaB. flaB mutants are nonmotile, have a straight, bacillus morphology rather than the classic flat-wave morphology, and exhibit decreased viability in both the mammalian host and arthropod vector. The fliG gene encodes the C-ring at the base of the flagellar basal body, which is important for rotational torque generation. Inactivation of fliG results in reduced motility and infectivity despite proper assembly of the flagellar filament (38, 39). These results indicate that periplasmic flagella of B. burgdorferi play an important role in transmission and infectivity. 4. Chemotaxis Some microorganisms use chemotaxis to migrate toward a chemoattractant or away from a chemorepellent in the environment. B. burgdorferi utilizes chemotaxis to colonize arthropod vectors and to infect mammalian hosts. Figure 5 Salp12 salivary protein serves as a chemoattractant for B. burgdorferi. Note. Attachment of the I. scapularis tick to a host allows for the release of Salp12 salivary protein into the host (46). B. burgdorferi swims up the Salp12 concentration gradient to encounter the tick. This process promotes colonization of the tick midgut, which will allow B. burgdorferi to be transmitted to the next mammal that the tick feeds on. Gaetano & Danka | Borrelia burgdorferi: The Deer Tick’s Dark Secret 19 The Salp12 salivary protein of the Ixodes scapularis tick serves as a chemoattractant for the bacterial cells that helps them colonize the arthropod vector (46). When uninfected ticks take a blood meal from an infected reservoir such as the white-footed mouse (Peromyscus leucopus), Salp12 diffuses into the host, attract- ing resident B. burgdorferi cells (Figure 5). This allows the bacteria to enter and colonize the arthropod midgut. Knockdown of salp12 results in a significant reduction of B. burgdorferi colonization of the tick, which demonstrates the importance of chemotaxis for transmission of the pathogen to its vector. To colonize the mammalian host, a chemotactic response that involves the B. burgdorferi cheA2 gene is required (69). This gene encodes the histidine kinase of a two-component regulatory system that controls the directional rotation of B. burgdorferi flagella. Mutations in cheA2 result in unidirectional movement and failure to be attracted into the mammalian host upon tick attachment. Interestingly, while cheA2 mutants are unable to establish an infection in mammals, they retain the ability to colonize ticks. Together, these studies show the key role that chemotaxis plays during transmission of B. burgdorferi to the human host. Innate Immune Evasion The innate immune system is a nonspecific, noninducible line of defense that protects fungi, animals, and plants against a broad range of pathogens. B. burgdorferi uses many methods to evade this first line of defense. 1. Complement inactivation The complement cascade is a tightly regulated pathway of sequentially activated proteins used to identify and eliminate pathogens through opsonization, phagocytosis, and formation of the membrane attack complex (MAC). B. burgdorferi expresses several Osps that disrupt this pathway including the surface lipoprotein BBK32, which binds and inactivates the C1 protease complex (2). This is the initiating component of the complement cascade, and its inactivation prevents all downstream steps. BBK32 mutants exhibit decreased virulence, which demonstrates the importance of comple- ment inactivation for successful infection. Other Osps involved in complement disruption such as OspA, OspC, and CspA function by convert- ing the blood protein plasminogen to plasmin, which is a known inhibitor of the cascade (24, 27, 55). 2. Antimicrobial peptide resistance Antimicrobial proteins and peptides (AMPs) are produced by the host immune system to defend against pathogenic bacteria. Lactoferrin is an AMP that inhibits microbial growth by scaveng- ing free iron, which is a cofactor required by most bacteria (9). B. burgdorferi avoids the effects of lactoferrin by using a manganese cofactor for biological redox reactions instead of iron (2). Cathelicidin is another AMP produced by many mammalian cells. While this molecule usually functions by interacting with cell surface components to disrupt microbial membrane integrity, it exhibits limited binding to the B. burgdorferi outer membrane (63). Additionally, the B. burgdorferi BBA57 surface protein has demonstrated the ability to downregulate the expression of some AMPs such as bactericidal/ permeability-increasing protein (BPI), further promoting its virulence in hosts (6). Fine Focus | Volume 1020 3. Phagocyte interference The host immune response relies on phagocytic macrophages and dendritic cells to engulf and destroy foreign matter. While phagocytes effectively clear B. burgdorferi when exposed to purified bacterial cells in vitro, the pathogen can successfully evade these effects in vivo (16). This is likely due to the upregulation of the anti-inflammatory cytokine IL-10 in phagocytes upon B. burgdorferi engulfment. Normally, IL-10 functions to dampen the host immune response after pathogen clearance to prevent endogenous tissue damage. B. burgdorferi-in- duced premature overproduction of IL-10 inhibits the production of proinflammatory immune factors that are critical to the host’s defense. Macrophages deficient in the ability to produce IL-10 generate greater levels of proin- flammatory cytokines during B. burgdorferi infection, which promote phagocytic events that aid in removal of the pathogen (16). These findings demonstrate the important role that reprogramming the host immune response plays during B. burgdorferi infection. 4. Pleomorphic forms B. burgdorferi shows pleomorphism, which is the ability to alter cellular morphology. This is often used by organisms to survive in extreme environments. In addition to the dominant flat-wave morphology, B. burgdorferi has been observed in other forms including blebs, round bodies (RB), and biofilm-like (BFL) aggregates (Figure 6) (44). At 37˚C, nearly all B. burgdorferi cells are found in their dominant flat-wave or spirochetal form. Environmental stress signals such as extreme pH, high temperatures, and high levels of reactive oxygen species (ROS) result in a conversion of flat-wave cells to other forms such as RBs, which have reduced metabolic requirements (47). Subsequent removal of B. burgdorferi from these unfavorable conditions causes a reversion back to the dominant Figure 6 B. burgdorferi cells can be found in four distinct morphologies. Note. Schematic representation of B. burgdorferi pleomorphic forms based on images obtained from differential interference contrast (DIC) microscopy (44). Flat-wave spirochetes (A), blebs (B), round bodies (RB; C), and biofilm-like aggregates (BFL; D) are not drawn to scale. Gaetano & Danka | Borrelia burgdorferi: The Deer Tick’s Dark Secret 21 flat-wave morphology. Additionally, low levels of BFL aggregates are thought to exist in all environmental conditions where, like biofilms, they promote attachment to host tissues and resist phagocytosis (44). These findings support the idea that B. burgdorferi enhances its surviv- al by altering its morphology as environmental conditions change. The human body contains many microenvi- ronments where B. burgdorferi may exist in different pleomorphic forms. To more success- fully diagnose and treat LD, it will be important to continue exploring the physiological niches where each morphology dominates. This will guide the pharmacological development of novel treatments that more precisely and accurately target each distinct morphology of B. burgdorferi. Adaptive Immune Evasion Unlike the nonspecific innate immune system, the adaptive immune system defends against specific pathogens using antibodies and immune cells produced in response to a past exposure. B. burgdorferi disrupts the normal functioning of the adaptive immune system in many ways, three of which will be described in this review. 1. Germinal center disruption During an infection, antigen-presenting cells (APC) display segments of immunogenic proteins from a phagocytosed pathogen on the major histocompatibility complex class II (MHC II) molecules on their cell surface. Next, these APCs present the antigens to B- and T-lymphocytes in secondary lymphoid tissues such as the spleen and lymph nodes. In these tissues, germinal centers form, and it is within these structures that antibody-produc- ing plasma cells and B-lymphocytes develop to confer long-term immunity. During a B. burgdorferi infection, the host immune system generates structurally defective, short-lived germinal centers that are unable to generate high quantities of antibody-producing immune cells (20). This leaves the host immunosup- pressed and allows B. burgdorferi to cause further infection. 2. Antibody class switching Several classes of immunoglobulins (Igs) exist, each with a unique function in the host immune response. During an infection, the host’s ability to shift production from one Ig class to another is useful in targeting pathogenic microorganisms located in multiple tissue types. Upon B. burgdorferi infection, pentameric IgM molecules are produced in high quantities, while monomeric IgG production is suppressed (28). IgG is the major circulating Ig found in the blood, and downregulating its production results in a less effective immune response. This is yet another way that B. burgdorferi manip- ulates the host immune response to further propagate an infection. 3. Antigenic variation B. burgdorferi modifies its immunogenic cell surface proteins in a process called antigenic variation, which promotes evasion from the host’s adaptive immune response (15, 79). For example, the immunoreactive VlsE surface lipoprotein encoded on lp28-1 undergoes frequent modification (Figure 7). Upstream of the vlsE locus are 15 silent vls cassettes that randomly recombine into the expressed region Fine Focus | Volume 1022 of the gene during B. burgdorferi infection. This results in a mosaic VlsE with an estimated 1040 possible variants. Continuous modification of the structure of this surface lipoprotein prevents previously generated immune factors from functioning properly. Incidence and Reporting of Lyme Disease Understanding the epidemiology of vector- borne diseases such as LD is critical for disease prevention. The incidence rate and geographic distribution of infections provides health officials with pertinent information that can be used to organize public health efforts in high-risk areas. This section of the review will focus on LD surveillance, limitations in report- ing, and ecological challenges that exacerbate the spread of disease. 1. CDC case definition The CDC has published clinical guidelines that define a LD diagnosis (12). The 2022 case definition requires specific clinical and labora- tory criteria to be met. Clinically, a patient must present at least one early or late-stage manifes- tation including EM, arthritis in one or more joints, nervous system abnormalities (lympho- cytic meningitis, facial palsy, or unexplainable encephalomyelitis), or cardiovascular involve- ment (atrioventricular conduction defects). The laboratory criteria include at least one of the following: isolation of B. burgdorferi in culture, detection of B. burgdorferi by polymerase chain reaction (PCR), detection of B. burgdorferi antigens by immunohistochemical assay, or a positive two-tier serology test. 2. Surveillance data Since its discovery, LD has been recognized throughout the world, particularly in Europe and Asia, and outside of the Northeast or New England region of the United States (67). Although surveillance in other countries is difficult, there are an estimated 85,000 cases annually in Europe, with the majority occurring in Germany, Austria, Slovenia, and Sweden. Figure 7 vls cassette recombination leads to highly varied VlsE proteins. Note. Antigenic variation of the B. burgdorferi immunogenic VlsE surface lipoprotein encoded on lp28-1 is accomplished through recombination events between the upstream, silent vls cassettes and the downstream expressed region (15). This allows the pathogen to continuously modify the structure of the expressed antigen, which provides a mechanism of immune evasion. https://ndc.services.cdc.gov/case-definitions/lyme-disease-2022/ https://ndc.services.cdc.gov/case-definitions/lyme-disease-2022/ https://ndc.services.cdc.gov/case-definitions/lyme-disease-2022/ Gaetano & Danka | Borrelia burgdorferi: The Deer Tick’s Dark Secret 23 In the US, LD is the most common vector- borne disease affecting an estimated 476,000 people annually from 2010-2018 (33, 34, 52). Most cases present in just a few states, with the highest incidences being reported in the Northeast, Mid-Atlantic, and upper Midwest regions (Figure 8) (67). According to the CDC, most cases occur in the summer months from June to August, which coincides with nymphal scavenging (13). In the Northeast, there have been drastic spikes in disease occurrence, particularly in Maine where the incidence quadrupled between the years 2005 and 2015 (67). Northern expansion of LD is occurring, and this trend is expected to persist as the suitable habitat for ticks continues to expand (54). In the Midwest, cases are on the rise with the majority concentrated in Wisconsin, Minne- Figure 8 3-year (2018-2020) average LD incidence by state. Note. The CDC considers a state “high risk” if it has greater than 10 confirmed cases of LD per 100,000 persons for three reporting years. In this review, states with 1-10 cases per 100,000 persons are consid- ered to have moderate risk. This figure is based on data reported in reference 13. Fine Focus | Volume 1024 sota, and northern Illinois (67). According to the CDC, Wisconsin had the fourth highest incidence of LD in 2019 behind Pennsyl- vania, New York, and New Jersey (13). Past models predicted further spread of Ixodes ticks throughout the Midwest into northern Michigan, the Ohio River Valley, and northwest Minnesota, which has indeed been observed in recent years (26). In the Southeast, LD incidence is relatively low despite vectors being well established in areas such as coastal Florida, South Carolina, North Carolina, and Georgia (67). In this region, Ixodes affinis and I. minor ticks harbor B. burgdorferi, but these species lack the quest- ing ability of I. scapularis and rarely feed on humans (4). Reported north to south gene flow of I. scapularis raises the possibility of altered southeastern tick behavior to that of the quest- ing northern ticks, which could lead to a greater incidence of LD in this region in the future (78). 3. Limitations in reporting LD has been a nationally reportable disease since 1991 (10). This means that physicians are required to report cases to state and local health departments who relay this informa- tion to the CDC. Due to the disease’s recent characterization, the CDC points out limita- tions in surveillance that prevent the accurate estimation of LD incidence. These include both under-reporting in high incidence areas and over-reporting in low incidence areas due to clinical misclassification, inconsistencies in the funding and practices of health departments from one state to the next, and the collection of LD data based on area of residence rather than the location of exposure. The latter leads to the misinterpretation of surveillance data for tourists who account for a significant proportion of cases. Furthermore, the LD case definition has undergone five modifications since deemed nationally reportable in 1991, which makes it more difficult for clinicians to stay current with reporting guidelines. Due to these limitations, the CDC estimates that the actual number of annual LD cases is about 10 times greater than what is reported. The recent COVID-19 pandemic has also greatly affected the reporting of many diseases including LD (36). While surveys indicated that Americans spent more time outdoors in 2020 than in 2019 putting them at greater risk for LD, the CDC reported about half the number of confirmed cases (13). One group of researchers successfully predicted this discrep- ancy before the CDC published their 2020 incidence data (43). Their study looked at the online traffic of the CDC’s tick removal website as an indirect quantifier of tick encounters (14). In 2020, the most recent year with published LD data, there were 25% more online visits than in 2019 suggesting that more individuals found themselves at risk for developing LD. Conversely, emergency department visits for tick bites and the frequency of LD diagnostic testing were significantly reduced in 2020. This was likely due to health officials and clinicians being preoccupied during the initial spread of SARS-CoV-2 in the spring and early summer of 2020, which coincided with the peak seasons for tick bites. On top of this, many patients delayed or avoided seeking out healthcare for more minor affiliations, due to fear of contracting COVID-19 or contributing to overburdened healthcare systems (36). This Gaetano & Danka | Borrelia burgdorferi: The Deer Tick’s Dark Secret 25 sudden change in LD reporting was not consis- tent with long-term, epidemiological trends, which suggests that the COVID-19 pandemic significantly affected reporting. An organized public health effort is needed to address this discrepancy in LD reporting and restore a high standard of surveillance. 4. Climate and ecological challenges After the colonial period in North America, deforestation, agricultural expansion, and urbanization drastically altered the landscape, which primed the area for LD spread (73). One way this has occurred is through shifts in predator community dynamics (35). Coyotes have replaced populations of both large and small predators such as wolves, bears, and foxes. The reduction in the number of foxes, which are more efficient predators of small mammals than coyotes, has decreased the amount of predation faced by B. burgdorferi’s main reservoir, the white-footed mouse. This has allowed the white-footed mouse to expand its home range, which has broadened the potential area of B. burgdorferi transmission to humans. Addition- ally, the reduction of large predator populations along with vast agricultural expansion has allowed deer populations to flourish, increasing the reproductive range of LD vectors. The gradual increase in the temperature of the Earth’s atmosphere also plays a key role in host/reservoir expansion. Models used to study these effects suggest dramatic expansions of Ixodes tick populations, which will broaden the range of B. burgdorferi (56). Over the next four decades, the geographic distribution of the white-footed mouse is predicted to expand northward by about 300 kilometers (61). If this manifests, it will have significant effects on the distribution of LD. Disease Treatment and Prevention Complete eradication of B. burgdorferi is implausible given its many hosts, reservoirs, and vectors. Rather, proper treatment of affected individuals and public health efforts to increase awareness of preventative measures in high-risk areas represent our best strategy to minimize the incidence of LD. 1. Treatment Early localized infection is often treated with oral antibiotics such as doxycycline, amoxicillin, cefuroxime, or azithromycin (76). Pregnant women and children under the age of eight should avoid the use of doxycycline due to its adverse effects on bone development (29). Early disseminated infection is treated based on the affected tissues. Patients with Lyme carditis or severe Lyme neuroborreliosis often receive intravenously administered ceftriaxone or cefotaxime, followed by one of the oral antibiotic regimens used to treat early localized infections (77). When only mild nervous system involvement such as isolated facial nerve palsy presents, oral antibiotic treatment usually suffices (76). Treatment for late disseminated, chronic LD also depends on the presented signs and symptoms. If arthritis occurs without neuro- logic involvement, oral antibiotics are usually administered. If cardiac or neurologic involve- ment persists from the early disseminated phase of infection, intravenous antibiotic treatment may be necessary (76). Fine Focus | Volume 1026 Prophylactic antibiotics are often prescribed in cases of suspected B. burgdorferi exposure after a tick bite. In a randomized clinical trial with patients that had removed an Ixodes genus tick within 72 hours, a single dose of doxycycline was 87% effective at preventing EM (49). This demonstrates the importance of immediate medical attention after a possible B. burgdorferi exposure to prevent long-term LD. 2. Vector-focused approach While the elimination of Ixodes genus ticks is highly unlikely due to their vast, expanding range, local measures can be taken to greatly reduce the chances of human-tick interactions. One such method involves the use of carbaryl, an insecticide that is also lethal to arachnids, which has proven extremely effective at elimi- nating tick populations (66). Unfortunately, this is a broad-spectrum insecticide that also kills moths, beetles, cockroaches, ants, and mosqui- toes, which could have negative ecological effects (51). This downside of the use of carbaryl should be weighed against the positive impact of treating outdoor public gathering spaces and private landscapes to limit B. burgdorferi exposures (Figure 9A). Permethrin is another insecticide that can be used against ticks, which poses little to no ecological threat since it is applied to clothing rather than broadly to the environment. Perme- thrin-based treatment of clothes and shoes has proven effective at preventing tick bites, killing ticks upon attachment, and preventing the transmission of pathogens such as B. Figure 9 LD preventative measures. Note. The common methods for preventing LD include targeting reservoirs and vectors as well as best practices for humans who may have encountered ticks. These measures include the treatment of landscapes with insecticides (A), frequent self-examination and prompt removal of attached ticks (B), immediate medical intervention after exposure (C), and vaccine development (D) (17, 49, 57, 66). Gaetano & Danka | Borrelia burgdorferi: The Deer Tick’s Dark Secret 27 burgdorferi (45). In a controlled study of the effectiveness of permethrin-treated outdoor gear, individuals wearing treated clothing received 3.36 times less tick bites than those wearing untreated clothing. Additionally, of the ticks attached to subjects, 97.6% were alive upon removal from individuals wearing untreated clothing, while only 22.6% of ticks were alive upon removal from those wearing permethrin-treated clothing. Outdoor gear can be purchased pre-treated with permethrin, or the insecticide can be applied at home carefully following the CDC’s recommendations which include wearing protective gloves, reading the directions before application, allowing clothing to dry before use, and avoiding direct exposure to skin (11). Other behavioral preventative measures should be taken to minimize the chances of acquiring LD. Frequent self-examination for ticks during and after outdoor activities in high-risk areas is critical (Figure 9B). As previously mentioned, there is a positive, nonlinear relationship between the duration of tick attachment and LD outcomes (57). Therefore, prompt removal of ticks and immediate prophylactic antibiotic treatment significantly decreases the chances of B. burgdorferi transmission (Figure 9C). Additionally, tucking pant legs into socks and covering bare skin in the outdoors are good strategies to prevent tick bites. Insect repellents that contain N, N-diethyl-meta-toluamide, commonly known as DEET, may also be helpful, but the effectiveness and duration of efficacy are inconsistent compared to other insecticides like permethrin (45). 3. Reservoir-focused approach Small rodent reservoirs such as the white-foot- ed mouse play a critical role in transmission of B. burgdorferi to its arthropod vector. While culling reservoir populations has been proposed, this would likely have negative ecological consequences. Instead, researchers have turned to the treatment of reservoirs to reduce populations of ticks and therefore the LD-causing spirochete (42). This has been attempted through the dispersal of perme- thrin-treated cotton in tick-infected areas, which is used by mice as nesting material. While this strategy significantly reduced B. burgdorferi transmission, additional research and development is needed to optimize the efficacy of this reservoir-focused treatment. 4. Vaccination efforts While vector- or reservoir-focused approaches may be beneficial, immunization represents the most effective method of disease prevention (Figure 9D). In 1998, the United States Food and Drug Administration (FDA) approved a recombinant B. burgdorferi OspA vaccine called LYMErix (58). This vaccine functions by inducing the production of antibodies that target and eliminate B. burgdorferi as it enters the body during a tick bite. Less than four years after becoming available to the public, LYMErix was removed from the market due to low sales, anti-vaccine backlash, safety concerns, and class-action lawsuits. Although the FDA investigated the safety concerns and concluded there was a lack of evidence for the claims, the vaccine has not been available to the public since early 2002 (53). Fine Focus | Volume 1028 Currently, companies such as Valneva are devel- oping new LD vaccines designed to specifically protect individuals against North American and European strains of B. burgdorferi (17). These VLA15 protein subunit vaccines use the immunoreactive C-terminus of the OspA protein to induce protective immunity. They have proven effective in murine models and are currently undergoing human clinical trials. Wide-spread public acceptance of these vaccines in development represents our greatest potential defense against LD. Additionally, researchers have begun to explore wildlife vaccination as an approach to LD prevention (71). OspA-based oral bait vaccines for reservoir populations of white-footed mice have demonstrated the ability to reduce the risk of LD in preliminary studies. These vaccines elicit an immune response that protects these animals from infection and reduces transmis- sion of the pathogen to its arthropod vector. Decreasing the number of B. burgdorferi-har- boring vectors in the environment will reduce the chances of human infection. Once the efficacy of wildlife vaccination is optimized, this may represent a successful long-term strategy to disease prevention. Conclusion B. burgdorferi, the causative agent of LD, is a highly complex parasite that uses an arthropod vector for transmission to mammalian hosts. The high rate of infection achieved by this spirochete is mainly derived from its unique ability to both evade and disrupt various aspects of the host immune response as it disseminates throughout the body. Clinically, LD has a variable presentation which makes it difficult Glossary of Abbreviations AMP: antimicrobial protein/peptide BFL: biofilm-like (aggregates) BSK: Barbour-Stoenner-Kelly media CDC: Centers for Disease Control and Prevention cp: circular plasmid EM: erythema migrans FDA: United States Food and Drug Administration Ig: immunoglobulin lp: linear plasmid LD: Lyme Disease LYMErix: OspA Lyme Disease vaccine Osp: outer surface protein RB: round bodies sRNA: small regulatory RNA for physicians to diagnose and treat their patients. In the United States, LD is a major public health concern, especially in the upper Midwest and Northeast regions. Surveillance limita- tions, a lack of public awareness, and failed vaccination efforts in the past represent some of the major challenges to disease prevention. Enhancing the general public’s understanding of the risks associated with outdoor activities and providing individuals in high-risk areas with everyday preventative measures can decrease the incidence of LD in the short-term. In the long-term, widespread acceptance of novel vaccines is likely our most promising solution. While LD presents several clinical and epidemi- ological challenges, a collective and coordinated public health effort represents our greatest chance of controlling the deer tick’s dark secret. Gaetano & Danka | Borrelia burgdorferi: The Deer Tick’s Dark Secret 29 References 1. Ahmed N. 2014. Cultivation of parasites. Tropical Parasitology. 4(2): 80-89. doi:10.4103/2229- 5070.138534. Url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4166808/ 2. Anderson C, and Brissette C. 2021. The Brilliance of Borrelia: Mechanisms of Host Immune Evasion by Lyme Disease-Causing Spirochetes. Pathogens. 10(3): 281. doi:10.3390/patho- gens10030281. Url: https://www.mdpi.com/2076-0817/10/3/281 3. Ante V, Farris L, Saputra E, Hall A, O’Bier N, Chavez A, Marconi R, Lybecker M, Hyde J. 2021. The Borrelia burgdorferi Adenylate Cyclase, CyaB, Is Important for Virulence Factor Produc- tion and Mammalian Infection. Frontiers in Microbiology. 25(12): 676192. doi:https:// doi.org/10.3389/fmicb.2021.676192. Url: https://www.frontiersin.org/articles/10.3389/ fmicb.2021.676192/full 4. Arsnoe I, Hickling G, Ginsberg H, McElreath R, Tsao J. 2015. Different Populations of Black- legged Tick Nymphs Exhibit Differences in Questing Behavior That Have Implications for Human Lyme Disease Risk. PLOS ONE. 10(5): e0127450. doi:https://doi.org/10.1371/ journal.pone.0127450. Url: https://journals.plos.org/plosone/article?id=10.1371/journal. pone.0127450 5. Arvikar S, Steer A. 2015. Diagnosis and Treatment of Lyme Arthritis. Infectious Disease Clinics of North America. 29(2): 269-280. doi:10.1016/j.idc.2015.02.004. Url: https://www.ncbi.nlm. nih.gov/pmc/articles/PMC4443866/ 6. Bernard Q, Smith A, Yang X, Koci J, Foor S, Cramer S, Zhuang X, Dwyer J, Lin Y, Mongodin E, Marques A, Leong J, Anguita J, Pal U. 2018. Plasticity in early immune evasion strat- egies of a bacterial pathogen. PNAS. 115(16): E3788-E3797. doi:https://doi.org/10.1073/ pnas.1718595115. Url: https://www.pnas.org/doi/full/10.1073/pnas.1718595115 7. Burgdorfer W, Barbour A, Hayes S, Benach J, Grunwaldt E, Davis J. 1982. Lyme Disease-A Tick-Borne Spirochetosis? Science. 216: 1317-1319. doi:10.1126/science.7043737. Url: https://escholarship.org/content/qt9vj3t37b/qt9vj3t37b_noSplash_13d2b56576d2ac- 55d60a1dd32ddc3074.pdf 8. Carrasco S, Troxell B, Yang Y, Brandt S, Li H, Sandusky G, Condon K, Serezani C, Yang X. 2015. Outer Surface Protein OspC Is an Antiphagocytic Factor That Protects Borrelia burgdorferi from Phagocytosis by Macrophages. Infection and Immunity. 83(12): 4848-4860. doi:https:// doi.org/10.1128/IAI.01215-15. Url: https://journals.asm.org/doi/10.1128/IAI.01215-15 9. Cavestro G, Ingegnoli A, Aragona G, Lori V, Mantovani N, Altavilla N, Dal Bo N, Pilotto A, Bertele A, Franze A, Di Mario F, Borghi L. 2002. Acta Biomedica. 73(5-6): 71-3. Url: https:// pubmed.ncbi.nlm.nih.gov/12643075/#:~:text=Lactoferrin%20is%20an%20iron%20 binding,ions%20available%20for%20microorganism%27s%20metabolism https://doi.org/10.4103%2F2229-5070.138534 https://doi.org/10.4103%2F2229-5070.138534 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4166808/ https://dx.doi.org/10.3390%2Fpathogens10030281 https://dx.doi.org/10.3390%2Fpathogens10030281 https://www.mdpi.com/2076-0817/10/3/281 https://doi.org/10.3389/fmicb.2021.676192 https://doi.org/10.3389/fmicb.2021.676192 https://www.frontiersin.org/articles/10.3389/fmicb.2021.676192/full https://www.frontiersin.org/articles/10.3389/fmicb.2021.676192/full https://doi.org/10.1371/journal.pone.0127450 https://doi.org/10.1371/journal.pone.0127450 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127450 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127450 https://dx.doi.org/10.1016%2Fj.idc.2015.02.004 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4443866/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4443866/ https://doi.org/10.1073/pnas.1718595115 https://doi.org/10.1073/pnas.1718595115 https://www.pnas.org/doi/full/10.1073/pnas.1718595115 https://doi.org/10.1126/science.7043737 https://escholarship.org/content/qt9vj3t37b/qt9vj3t37b_noSplash_13d2b56576d2ac55d60a1dd32ddc3074.pdf https://escholarship.org/content/qt9vj3t37b/qt9vj3t37b_noSplash_13d2b56576d2ac55d60a1dd32ddc3074.pdf https://doi.org/10.1128/IAI.01215-15 https://doi.org/10.1128/IAI.01215-15 https://journals.asm.org/doi/10.1128/IAI.01215-15 Fine Focus | Volume 1030 10. Centers for Disease Control and Prevention (CDC). Last reviewed November 15, 2022. Lyme Disease: Surveillance explained and available data. Accessed March 18, 2022. Url: https:// www.cdc.gov/lyme/stats/survfaq.html#:~:text=Limitations%20of%20surveillance%20data&- text=Not%20every%20case%20of%20Lyme,occur%20in%20low%20incidence%20areas. 11. Centers for Disease Control and Prevention (CDC). Last reviewed February 5, 2020. CDC-TV: What You Need to Know about Permethrin-transcript. Accessed January 7, 2023. Url: https:// www.cdc.gov/cdctv/injuryviolenceandsafety/permethrin-transcript.html 12. Centers for Disease Control and Prevention (CDC): Division of Health Informatics and Surveil- lance. Last reviewed August 30, 2021. Lyme Disease (Borrelia burgdorferi) 2022 Case Definition. Accessed March 17, 2022. Url: https://ndc.services.cdc.gov/case-definitions/ lyme-disease-2022/ 13. Centers for Disease Control and Prevention (CDC), National Center for Emerging and Zoonotic Infectious Disease (NCEZID), Division of Vector-Borne Diseases (DVBD). Last reviewed August 29, 2022. Lyme Disease: Data and surveillance. Accessed March 18, 2022. Url: https://www.cdc.gov/lyme/datasurveillance/index.html?CDC_AA_ refVal=https%3A%2F%2Fwww.cdc.gov%2Flyme%2Fstats%2Findex.html 14. Centers for Disease Control and Prevention (CDC), National Center for Emerging and Zoonotic Infectious Disease (NCEZID), Division of Vector-Borne Diseases (DVBD). Last reviewed May 13, 2022. Ticks: Removing a tick. Accessed January 10, 2023. Url: https://www.cdc.gov/ticks/ removing_a_tick.html 15. Chaconas G, Castellanos M, Verbey T. 2020. Changing of the guard: How the Lyme disease spirochete subverts the host immune response. Journal of Biological Chemistry. 295(2): 301-313. doi:https://doi.org/10.1074/jbc.REV119.008583. Url: https://www.jbc.org/article/ S0021-9258(17)48327-X/fulltext 16. Chung Y, Zhang N, Wooten R. 2013. Borrelia burgdorferi Elicited-IL-10 Suppresses the Production of Inflammatory Mediators, Phagocytosis, and Expression of Co-Stimulatory Receptors by Murine Macrophages and/or Dendritic Cells. PLOS ONE. 8(12): e84980. doi:https://doi.org/10.1371/journal.pone.0084980. Url: https://journals.plos.org/plosone/ article?id=10.1371/journal.pone.0084980 17. Comstedt P, Schuler W, Meinke A, Lundberg U. 2017. The novel Lyme borreliosis vaccine VLA15 shows broad protection against Borrelia species expressing six different OspA serotypes. PLOS ONE. doi:https://doi.org/10.1371/journal.pone.0184357. Url: https://journals.plos.org/ plosone/article/file?id=10.1371/journal.pone.0184357&type=printable https://www.cdc.gov/cdctv/injuryviolenceandsafety/permethrin-transcript.html https://www.cdc.gov/cdctv/injuryviolenceandsafety/permethrin-transcript.html https://ndc.services.cdc.gov/case-definitions/lyme-disease-2022/ https://ndc.services.cdc.gov/case-definitions/lyme-disease-2022/ https://www.cdc.gov/lyme/datasurveillance/index.html?CDC_AA_refVal=https%3A%2F%2Fwww.cdc.gov%2Flyme%2Fstats%2Findex.html https://www.cdc.gov/lyme/datasurveillance/index.html?CDC_AA_refVal=https%3A%2F%2Fwww.cdc.gov%2Flyme%2Fstats%2Findex.html https://www.cdc.gov/ticks/removing_a_tick.html https://www.cdc.gov/ticks/removing_a_tick.html https://doi.org/10.1074/jbc.REV119.008583 https://www.jbc.org/article/S0021-9258(17)48327-X/fulltext https://www.jbc.org/article/S0021-9258(17)48327-X/fulltext https://doi.org/10.1371/journal.pone.0084980 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0084980 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0084980 https://doi.org/10.1371/journal.pone.0184357 https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0184357&type=printable https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0184357&type=printable Gaetano & Danka | Borrelia burgdorferi: The Deer Tick’s Dark Secret 31 18. Cook M. 2014. Lyme borreliosis: a review of data on transmission time after tick attachment. International Journal of General Medicine. 8: 1-8. doi:10.2147/IJGM.S73791. Url: https:// www.dovepress.com/lyme-borreliosis-a-review-of-data-on-transmission-time-after-tick-atta- peer-reviewed-fulltext-article-IJGM 19. Diuk-Wasser M, Vannier E, Krause P. 2016. Coinfection by Ixodes Tick-Borne Pathogens: Ecological, Epidemiological, and Clinical Consequences. Trends in Parasitology. 32(1): 30-42. doi:https://doi.org/10.1016/j.pt.2015.09.008. Url: https://www.cell.com/trends/parasitology/ fulltext/S1471-4922(15)00210-X?_returnURL=https%3A%2F%2Flinkinghub.elsevier. com%2Fretrieve%2Fpii%2FS147149221500210X%3Fshowall%3Dtrue 20. Elsner R, Hastey C, Olsen K, Baumgarth N. 2015. Suppression of Long-Lived Humoral Immunity Following Borrelia burgdorferi Infection. PLOS PATHOGENS. 11(7): e1004976. doi:https:// doi.org/10.1371/journal.ppat.1004976. Url: https://journals.plos.org/plospathogens/ article?id=10.1371/journal.ppat.1004976 21. Fish A, Pride Y, Pinto D. 2008 Lyme Carditis. Infectious Disease Clinics of North America. 22:275-288. Url: https://www.cdc.gov/lyme/resources/fish2008-508.pdf 22. Ford L, Tufts D. 2021. Lyme Neuroborreliosis: Mechanisms of B. burgdorferi Infection of the Nervous System. Brain Sciences. 11(6): 789. doi:10.3390/brainsci11060789. Url: https://www. mdpi.com/2076-3425/11/6/789 23. Fraser C, Casjens S, Huang W, Sutton G, Clayton R, Lathigra R, White O, Ketchum K, Dodson R, Hickey E, Gwinn M, Dougherty B, Tomb J, Fleischmann R, Richardson D, Peterson J, Kerlavage A, Quachenbush J, Salzburg S, Hanson M, Vugt R, Palmer N, Adams M, Gocayne J, Weidman J, Utterback T, Watthey L, McDonald L, Artiach P, Bowman C, Garland S, Fujii C, Cotton M, Horst K, Roberts K, Hatch B, Smith H, Venter J. 1997. Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature. 390: 580-586. doi:https://doi. org/10.1038/37551. Url: https://www.nature.com/articles/37551 24. Fuchs H, Wallich R, Simon M, Kramer M. 1994. The outer surface protein A of the spirochete Borrelia burgdorferi is a plasmin(ogen) receptor. Proc. Natl. Acad. Sci. USA. 91:12594–12598. doi:10.1073/pnas.91.26.12594. Url: https://www.pnas.org/doi/epdf/10.1073/pnas.91.26.12594 25. Gunduz A, Turkmen S, Turedi S, Nuhoglu I, Topbas M. 2008. Tick Attachment Sites. Wilderness and Environmental Medicine. 19(1):4-6. doi:https://doi.org/10.1580/06-WEME-BR-067.1. Url: https://www.wemjournal.org/article/S1080-6032(08)70142-X/fulltext 26. Hahn M, Jarnevich C, Monaghan A, Eisen R. 2016. Modeling the Geographic Distribution of Ixodes scapularis and Ixodes pacificus in the Contiguous United States. Journal of Medical Entomology. 53(5): 1176-1191. doi:https://doi.org/10.1093/jme/tjw076. Url: https://academic. oup.com/jme/article/53/5/1176/1751790 https://dx.doi.org/10.2147%2FIJGM.S73791 https://www.dovepress.com/lyme-borreliosis-a-review-of-data-on-transmission-time-after-tick-atta-peer-reviewed-fulltext-article-IJGM https://www.dovepress.com/lyme-borreliosis-a-review-of-data-on-transmission-time-after-tick-atta-peer-reviewed-fulltext-article-IJGM https://www.dovepress.com/lyme-borreliosis-a-review-of-data-on-transmission-time-after-tick-atta-peer-reviewed-fulltext-article-IJGM https://doi.org/10.1016/j.pt.2015.09.008 https://www.cell.com/trends/parasitology/fulltext/S1471-4922(15)00210-X?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS147149221500210X%3Fshowall%3Dtrue https://www.cell.com/trends/parasitology/fulltext/S1471-4922(15)00210-X?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS147149221500210X%3Fshowall%3Dtrue https://www.cell.com/trends/parasitology/fulltext/S1471-4922(15)00210-X?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS147149221500210X%3Fshowall%3Dtrue https://doi.org/10.1371/journal.ppat.1004976 https://doi.org/10.1371/journal.ppat.1004976 https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1004976 https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1004976 https://www.cdc.gov/lyme/resources/fish2008-508.pdf https://dx.doi.org/10.3390%2Fbrainsci11060789 https://www.mdpi.com/2076-3425/11/6/789 https://www.mdpi.com/2076-3425/11/6/789 https://doi.org/10.1038/37551 https://doi.org/10.1038/37551 https://www.nature.com/articles/37551 https://www.pnas.org/doi/epdf/10.1073/pnas.91.26.12594 https://doi.org/10.1580/06-WEME-BR-067.1 https://www.wemjournal.org/article/S1080-6032(08)70142-X/fulltext https://doi.org/10.1093/jme/tjw076 https://academic.oup.com/jme/article/53/5/1176/1751790 https://academic.oup.com/jme/article/53/5/1176/1751790 Fine Focus | Volume 1032 27. Hallstrom R, Haupt K, Kraiczy P, Hortschansky P, Wallich C, Zipfel P. 2010. Complement Regulator—Acquiring Surface Protein 1 of Borrelia burgdorferi Binds to Human Bone Morphogenetic Protein 2, Several Extracellular Matrix Proteins, and Plasminogen. The Journal of Infectious Disease. 202(3):490-498. doi:https://doi.org/10.1086/653825. Url: https://academic.oup.com/jid/article/202/3/490/831946 28. Hastey C, Elsner R, Barthold S, Baumgarth N. 2012. Delays and Diversions Mark the Devel- opment of B Cell Responses to Borrelia burgdorferi Infection. The Journal of Immunology. 188:5612-5622. doi:https://doi.org/10.4049/jimmunol.1103735. Url: https://journals.aai.org/ jimmunol/article/188/11/5612/86779/Delays-and-Diversions-Mark-the-Development-of-B 29. Holmes N, Charles P. 2009. Safety and Efficacy Review of Doxycycline. SAGE Journals- Clinical medicine Insights: Therapeutics. doi:https://doi.org/10.4137/CMT.S2035. Url: https:// journals.sagepub.com/doi/10.4137/CMT.S2035 30. Hu L, Steere A, Hall K. 2023. Patient education: Lyme disease symptoms and diagnosis (Beyond the Basics). UpToDate. Url: https://www.uptodate.com/contents/lyme-disease-symp- toms-and-diagnosis-beyond-the-basics/print 31. Kean W, Tocchio S, Kean M, Rainsford K. 2012. The musculoskeletal abnormalities of the Similaun Iceman (“ÖTZI”): clues to chronic pain and possible treatments. Inflammo- pharmacology. 21: 11-20. doi:10.1007/s10787-012-0153-5. Url: https://link.springer.com/ article/10.1007/s10787-012-0153-5 32. Kudryashev M, Cyrklaff M, Baumeister W, Simon M, Wallich R, Frischknecht F. 2009. Compar- ative cryo-electron tomography of pathogenic Lyme disease spirochetes. Molecular Microbi- ology. 71(6): 1415-1434. doi:https://doi.org/10.1111/j.1365-2958.2009.06613.x. Url: https:// onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2009.06613.x 33. Kugeler K, Jordan R, Schulze T, Griffith K, Mead P. 2015. Will Culling White-Tailed Deer Prevent Lyme Disease? Zoonoses and Public Health. 63(5): 337-345. doi:https://doi.org/10.1111/ zph.12245. Url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912954/ 34. Kugeler K, Schwartz A, Delorey M, Mead P, Hinckley A. 2021. Estimating the Frequency of Lyme Disease Diagnoses, United States, 2010-2018. Emerging Infectious Diseases. 27(2): 616-619. doi:10.3201/eid2702.202731. Url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7853543/ 35. Levi T, Kilpatrick A, Mangel M, Wilmers C. 2012. Deer, predators, and the emergence of Lyme disease. PNAS. 109(27). doi:https://doi.org/10.1073/pnas.1204536109. Url: https://www. pnas.org/doi/full/10.1073/pnas.1204536109 36. Lindquist S, Goldoft M. 2021. Impact of a Pandemic on Disease Reporting. epiTRENDS. 26(1). Url: https://doh.wa.gov/sites/default/files/legacy/Documents/5100//420-002-epi- trends2021-01.pdf https://doi.org/10.1086/653825 https://academic.oup.com/jid/article/202/3/490/831946 https://doi.org/10.4049/jimmunol.1103735 https://journals.aai.org/jimmunol/article/188/11/5612/86779/Delays-and-Diversions-Mark-the-Development-of-B https://journals.aai.org/jimmunol/article/188/11/5612/86779/Delays-and-Diversions-Mark-the-Development-of-B https://doi.org/10.4137%2FCMT.S2035 https://journals.sagepub.com/doi/10.4137/CMT.S2035 https://journals.sagepub.com/doi/10.4137/CMT.S2035 https://www.uptodate.com/contents/lyme-disease-symptoms-and-diagnosis-beyond-the-basics/print https://www.uptodate.com/contents/lyme-disease-symptoms-and-diagnosis-beyond-the-basics/print https://dx.doi.org/10.1007%2Fs10787-012-0153-5 https://link.springer.com/article/10.1007/s10787-012-0153-5 https://link.springer.com/article/10.1007/s10787-012-0153-5 https://doi.org/10.1111/j.1365-2958.2009.06613.x https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2009.06613.x https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2009.06613.x https://doi.org/10.1111/zph.12245 https://doi.org/10.1111/zph.12245 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912954/ https://dx.doi.org/10.3201%2Feid2702.202731 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7853543/ https://doi.org/10.1073/pnas.1204536109 https://www.pnas.org/doi/full/10.1073/pnas.1204536109 https://www.pnas.org/doi/full/10.1073/pnas.1204536109 https://doh.wa.gov/sites/default/files/legacy/Documents/5100//420-002-epitrends2021-01.pdf https://doh.wa.gov/sites/default/files/legacy/Documents/5100//420-002-epitrends2021-01.pdf Gaetano & Danka | Borrelia burgdorferi: The Deer Tick’s Dark Secret 33 37. Lloyd D. 1976. Circular Letter #12-32 To: Directors of Health (Lyme Disease PSA). State of Connecticut: State Department of Health. Hartford, Connecticut, 06115. Url: https://portal. ct.gov/-/media/Departments-and-Agencies/DPH/dph/infectious_diseases/lyme/1976circu- larletterpdf.pdf 38. Lloyd S, Tang H, Wang X, Hillings S, Blair D. 1995. Torque Generation in the Flagellar motion of Escherichia coli: Evidence of a Direct Role for FliG but Not for FliM or FliN. Journal of Bacteriology. 178(1): 223-231. doi:https://doi.org/10.1128/jb.178.1.223-231.1996. Url: https:// journals.asm.org/doi/epdf/10.1128/jb.178.1.223-231.1996 39. Lowder B, Duyvesteyn M, Blair D. 2005. FliG Subunit Arrangement in the Flagellar Rotor Probes by Targeted Cross-Linking. Journal of Bacteriology. 187(16): 5640-5647. doi:10.1128/ JB.187.16.5640-5647.2005. Url: https://journals.asm.org/doi/10.1128/JB.187.16.5640- 5647.2005 40. Lybecker M, Samuels D. 2007. Temperature-induced regulation of RpoS by a small RNA in Borrelia burgdorferi. Molecular Microbiology. 64(4) 1075-1089. doi:https://doi.org/10.1111/ j.1365-2958.2007.05716.x. Url: https://onlinelibrary.wiley.com/doi/10.1111/j.1365- 2958.2007.05716.x 41. Markeljevic J, Sarac H, Rados M. 2011. Tremor, seizures and psychosis as presenting symptoms in a patient with chronic lyme neuroborreliosis (LNB). Collegium Antropologicum. Suppl 1:313- 8. Url: https://pubmed.ncbi.nlm.nih.gov/21648354/ 42. Mather T, Ribeiro J, Spielman A. 1987. Lyme disease and babesiosis: acaricide focused on poten- tially infected ticks. The American Journal of Tropical Medicine and Hygiene. 36(3): 609-614. doi:10.4269/ajtmh.1987.36.609. Url: https://pubmed.ncbi.nlm.nih.gov/3555140/ 43. McCormick D, Kugeler K, Marx G, Jayanthi P, Dietz S, Mead, P, Hinckley A. 2021. Effects of COVID-19 Pandemic on Reported Lyme Disease, United States, 2020. Emerging Infectious Diseases. 27(10): 2715-2717. Doi: 10.3201/eid2710.210903. Url: https://www.ncbi.nlm. nih.gov/pmc/articles/PMC8462321/#:~:text=Despite%20ongoing%20exposure%2C%20 Lyme%20disease,changes%20in%20healthcare%2Dseeking%20behavior. 44. Merilainen L, Herranen A, Schwarzbach A, Gilbert L. 2015. Morphological and biochemical features of Borrelia burgdorferi pleomorphic forms. Microbiology. 161(Pt 3): 516–527. doi:10.1099/mic.0.000027. Url: https://www.microbiologyresearch.org/content/journal/ micro/10.1099/mic.0.000027 45. Miller N, Rainone E, Dyer M, Gonzales M, Mather T. 2011. Tick Bite Protection With permethrin-Treated Summer-Weight Clothing. Journal of Medical Entomology. 48(2): 327-333. Doi: https://doi.org/10.1603/ME10158. Url: https://academic.oup.com/jme/ article/48/2/327/893233?login=false https://portal.ct.gov/-/media/Departments-and-Agencies/DPH/dph/infectious_diseases/lyme/1976circularletterpdf.pdf https://portal.ct.gov/-/media/Departments-and-Agencies/DPH/dph/infectious_diseases/lyme/1976circularletterpdf.pdf https://portal.ct.gov/-/media/Departments-and-Agencies/DPH/dph/infectious_diseases/lyme/1976circularletterpdf.pdf https://doi.org/10.1128/jb.178.1.223-231.1996 https://journals.asm.org/doi/epdf/10.1128/jb.178.1.223-231.1996 https://journals.asm.org/doi/epdf/10.1128/jb.178.1.223-231.1996 https://dx.doi.org/10.1128%2FJB.187.16.5640-5647.2005 https://dx.doi.org/10.1128%2FJB.187.16.5640-5647.2005 https://journals.asm.org/doi/10.1128/JB.187.16.5640-5647.2005 https://journals.asm.org/doi/10.1128/JB.187.16.5640-5647.2005 https://doi.org/10.1111/j.1365-2958.2007.05716.x https://doi.org/10.1111/j.1365-2958.2007.05716.x https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2007.05716.x https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2007.05716.x https://pubmed.ncbi.nlm.nih.gov/21648354/ https://doi.org/10.4269/ajtmh.1987.36.609 https://pubmed.ncbi.nlm.nih.gov/3555140/ https://doi.org/10.3201%2Feid2710.210903 https://dx.doi.org/10.1099%2Fmic.0.000027 https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000027 https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000027 https://doi.org/10.1603/ME10158 https://academic.oup.com/jme/article/48/2/327/893233?login=false https://academic.oup.com/jme/article/48/2/327/893233?login=false Fine Focus | Volume 1034 46. Murfin K, Kleinbard R, Aydin M, Salazar S, Fikrig E. 2019. Borrelia burgdorferi chemotaxis toward tick protein Salp12 contributes to acquisition. Ticks and Tick-borne Diseases. 10(5):1124-1134. doi:https://doi.org/10.1016/j.ttbdis.2019.06.002. Url: https://www.science- direct.com/science/article/abs/pii/S1877959X19300950?via%3Dihub 47. Murgia R, Cinco M. 2004. Induction of cystic forms by different stress conditions in Borrelia burgdorferi. Journal of Pathology, Microbiology and Immunology. 112(1):57- 62. doi:10.1111/j.1600-0463.2004.apm1120110.x. Url: https://pubmed.ncbi.nlm.nih. gov/14961976/ 48. Murray T, Shapiro E. 2012. Lyme Disease. Clinics in Laboratory Medicine. 30(1):311- 328. doi:10.1016/j.cll.2010.01.003. Url: https://www.ncbi.nlm.nih.gov/pmc/articles/ PMC3652387/ 49. Nadelman R, Nowakowski J, Fish D, Falco R, Freeman K, McKenna D, Welch P, Marcus R, Aguero-Rosenfeld M, Dennis D, Wormser G, Tick Bite Study Group. 2001. Prophylaxis with Single-Dose Doxycycline for the Prevention of lyme Disease after an Ixodes scapularis Tick Bite. New England Journal of Medicine. 345: 79-84. doi:10.1056/NEJM200107123450201. Url: https://pubmed.ncbi.nlm.nih.gov/11450675/ 50. Nagi K, Joshi R, Thakur R. 1996. Cardiac Manifestations of Lyme Disease: a review. Canadian Journal of Cardiology. 12(5): 503-506. PMID: 8640597. Url: https://europepmc.org/article/ med/8640597 51. National Pesticide Information Center (NPIC). Last reviewed 2003. Carbaryl: General Fact Sheet. Accessed January 9, 2023. Url: http://npic.orst.edu/factsheets/carbgen.pdf 52. Nelson, Christina A. et al. September 2015. Incidence of Clinician-Diagnosed Lyme Disease, United States, 2005-2010. Emerging Infectious Diseases. 21(9): 1625-1631. doi:10.3201/ eid2109.150417 53. Nigrovic L, Thompson K. 2007. The Lyme vaccine: a cautionary tale. Epidemiology & Infection. 135(1): 1-8. Doi: 10.1017/S0950268806007096. Url: https://www.ncbi.nlm.nih.gov/pmc/ articles/PMC2870557/ 54. Ogden N, Barker I, Francis C, Heagy A, Lindsay L, Hobson K. 2015. How far north are migrant birds transporting the tick Ixodes scapularis in Canada? Insights from stable hydrogen isotope analyses of feathers. Ticks and Tick-borne Diseases. 6(6): 715-720. doi:https://doi. org/10.1016/j.ttbdis.2015.06.004. Url: https://www.sciencedirect.com/science/article/abs/pii/ S1877959X15001119?via%3Dihub 55. Onder O, Humphrey P, McOmber B, Korobova F, Francella N, Greenbaum D, Brisson D. 2012. OspC Is a Potent Plasminogen Receptor on the Surface of Borrelia burgdorferi. Journal of Biological Chemistry (Cell Biology). 287(20):16860-16868. doi:https://doi.org/10.1074/jbc. M111.290775. Url: https://www.jbc.org/article/S0021-9258(20)60819-5/fulltext https://doi.org/10.1016/j.ttbdis.2019.06.002 https://www.sciencedirect.com/science/article/abs/pii/S1877959X19300950?via%3Dihub https://www.sciencedirect.com/science/article/abs/pii/S1877959X19300950?via%3Dihub https://pubmed.ncbi.nlm.nih.gov/14961976/ https://pubmed.ncbi.nlm.nih.gov/14961976/ https://dx.doi.org/10.1016%2Fj.cll.2010.01.003 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3652387/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3652387/ https://pubmed.ncbi.nlm.nih.gov/11450675/ https://europepmc.org/article/med/8640597 https://europepmc.org/article/med/8640597 http://npic.orst.edu/factsheets/carbgen.pdf https://dx.doi.org/10.3201%2Feid2109.150417 https://dx.doi.org/10.3201%2Feid2109.150417 https://doi.org/10.1017%2FS0950268806007096 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2870557/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2870557/ https://doi.org/10.1016/j.ttbdis.2015.06.004 https://doi.org/10.1016/j.ttbdis.2015.06.004 https://www.sciencedirect.com/science/article/abs/pii/S1877959X15001119?via%3Dihub https://www.sciencedirect.com/science/article/abs/pii/S1877959X15001119?via%3Dihub https://doi.org/10.1074/jbc.M111.290775 https://doi.org/10.1074/jbc.M111.290775 https://www.jbc.org/article/S0021-9258(20)60819-5/fulltext Gaetano & Danka | Borrelia burgdorferi: The Deer Tick’s Dark Secret 35 56. Ostfeld R, Brunner J. 2015. Climate change and Ixodes tick-borne disease of humans. The Royal Society Publishing; Philosophical Transactions B. 370:20140051. doi:https:// doi.org/10.1098/rstb.2014.0051. Url: https://royalsocietypublishing.org/doi/10.1098/ rstb.2014.0051 57. Piesman J. 1993. Dynamics of Borrelia burgdorferi transmission by nymphal Ixodes dammini ticks. Journal of Infectious Disease. 167(5):1082-5. doi:10.1093/infdis/167.5.1082. Url: https://pubmed.ncbi.nlm.nih.gov/8486940/ 58. Poland G. 2001. Prevention of Lyme Disease: A Review of the Evidence. Mayo Clinic Proceedings. 76(7): 713-724. doi:https://doi.org/10.4065/76.7.713. Url: https://www.mayoclinicproceed- ings.org/article/S0025-6196(11)65002-7/fulltext 59. Popitsch N, Bilusic I, Rescheneder P, Schroeder R, Lybecker M. 2017. Temperature-dependent sRNA transcriptome of the Lyme disease spirochete. BMC Genomics. 18: 28. Doi: 10.1186/ s12864-016-3398-3. Url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5216591/ 60. Purser J, Norris S. 2000. Correlation between plasmid content and infectivity in Borrelia burgdorferi. PNAS. 97(25): 13865-13870. doi:https://doi.org/10.1073/pnas.97.25.13865. Url: https://www.pnas.org/doi/full/10.1073/pnas.97.25.13865 61. Roy-Dufresne E, Logan T, Simon J, Chmura G, Millien V. 2013. Poleward Expansion of the White-Footed Mouse under Climate Change: Implications for Spread of Lyme Disease. PLOS ONE. 8(11): e80724. doi:https://doi.org/10.1371/journal.pone.0080724. Url: https:// journals.plos.org/plosone/article?id=10.1371/journal.pone.0080724 62. Sapi E, Pabbati N, Datar A, Davies E, Rattelle A, Kuo B. 2013. Improved Culture Conditions for the Growth and Detection of Borrelia from Human Serum. International Journal of Medical Sciences. 10(4): 362-376. doi: 10.7150/ijms.5698. Url: https://www.ncbi.nlm.nih.gov/pmc/ articles/PMC3590594/pdf/ijmsv10p0362.pdf 63. Sarkar A, Tilly K, Stewart P, Bestor A, Battisti J, Rosa P. 2009. Borrelia burgdorferi Resistance to a Major Skin Antimicrobial Peptide Is Independent of Outer Surface Lipoprotein Content. Antimicrobial Agents and Chemotherapy. 53(10): 4490-4494. doi:10.1128/AAC.00558-09. Url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2764146/ 64. Schwan T, Burgdorfer W, Garon C. 1988. Changes in infectivity and plasmid profile of the Lyme disease spirochete, Borrelia burgdorferi, as a result of in vitro cultivation. Infection and Immunology. 56(8): 1831–1836. doi:10.1128/iai.56.8.1831-1836.1988. Url: https://journals. asm.org/doi/epdf/10.1128/iai.56.8.1831-1836.1988 65. Schwan T, Piesman J, Golde W, Dolan M, Rosa P. 1995. Induction of an outer surface protein on Borrelia burgdorferi during tick feeding. PNAS. 92: 2909-2913. doi:10.1073/pnas.92.7.2909. Url: https://www.pnas.org/doi/epdf/10.1073/pnas.92.7.2909 https://doi.org/10.1098/rstb.2014.0051 https://doi.org/10.1098/rstb.2014.0051 https://royalsocietypublishing.org/doi/10.1098/rstb.2014.0051 https://royalsocietypublishing.org/doi/10.1098/rstb.2014.0051 https://pubmed.ncbi.nlm.nih.gov/8486940/ https://doi.org/10.4065/76.7.713 https://www.mayoclinicproceedings.org/article/S0025-6196(11)65002-7/fulltext https://www.mayoclinicproceedings.org/article/S0025-6196(11)65002-7/fulltext https://doi.org/10.1186%2Fs12864-016-3398-3 https://doi.org/10.1186%2Fs12864-016-3398-3 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5216591/ https://doi.org/10.1073/pnas.97.25.13865 https://www.pnas.org/doi/full/10.1073/pnas.97.25.13865 https://doi.org/10.1371/journal.pone.0080724 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080724 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080724 https://doi.org/10.7150%2Fijms.5698 https://doi.org/10.1128%2FAAC.00558-09 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2764146/ https://dx.doi.org/10.1128%2Fiai.56.8.1831-1836.1988 https://journals.asm.org/doi/epdf/10.1128/iai.56.8.1831-1836.1988 https://journals.asm.org/doi/epdf/10.1128/iai.56.8.1831-1836.1988 https://dx.doi.org/10.1073%2Fpnas.92.7.2909 https://www.pnas.org/doi/epdf/10.1073/pnas.92.7.2909 Fine Focus | Volume 1036 66. Stafford K. 1991. Effectiveness of Carbaryl Applications for the Control of Ixodes dammini Nymphs in an Endemic Residential Area. Journal of Medical Entomology. 28(1): 32-36. doi:https://doi.org/10.1093/jmedent/28.1.32. Url: https://academic.oup.com/jme/article-ab- stract/28/1/32/2220853?redirectedFrom=fulltext 67. Stone B, Tourand Y, Brissette C. 2017. Brave New Worlds: The Expanding Universe of Lyme Disease. Vector Borne and Zoonotic Diseases. 17(9): 619-629. doi:10.1089/vbz.2017.2127. Url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576071/ 68. Sultan S, Manne A, Stewart P, Bestor A, Rosa P, Charon N, Motaleb M. 2013. Motility Is Crucial for the Infectious Life Cycle of Borrelia burgdorferi. Infection and Immunology. 81(6): 2012-2021. doi:https://doi.org/10.1128/IAI.01228-12. Url: https://journals.asm.org/ doi/10.1128/IAI.01228-12 69. Sze C, Zhang K, Kariu T, Pal U, Li C. 2012. Borrelia burgdorferi Needs Chemotaxis To Establish Infection in Mammals and To Accomplish Its Enzootic Cycle. American Society for Microbi- ology. 80(7): 2485-2492. doi:https://doi.org/10.1128/IAI.00145-12. Url: https://journals.asm. org/doi/10.1128/IAI.00145-12 70. Takayama K, Rothenberg R, Barbour A. 1987. Absence of lipopolysaccharide in the Lyme disease spirochete, Borrelia burgdorferi. Infection and Immunity. 55(9): 2311-2313. doi:https:// doi.org/10.1128/iai.55.9.2311-2313.1987. Url: https://journals.asm.org/doi/epdf/10.1128/ iai.55.9.2311-2313.1987 71. Voordouw M, Tupper H, Onder O, Devevey G, Graves C, Kemps B, Brisson D. 2013. Reductions in Human Lyme Disease Risk Due to the Effects of Oral Vaccination on Tick-to-Mouse and Mouse-To-Tick Transmission. Vector Borne and Zoonotic Diseases. 13(4): 203-214. Doi: 10.1089/vbz.2012.1003. Url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3610442/ 72. Waddell L, Greig J, Lindsay L, Hinckley A, Ogden N. 2018. A systematic review on the impact of gestational Lyme disease in humans on fetus and newborn. PLOS ONE. 13(11) e0207067. doi:10.1371/journal.pone.0207067. Url: https://journals.plos.org/plosone/article?id=10.1371/ journal.pone.0207067 73. Walter K, Carpi G, Caccone A, Diuk-Wasser M. 2017. Genomic insights into the ancient spread of Lyme disease across North America. Nature: ecology and evolution. 1569-1576 doi:10.1038/ s41559-017-0282-8. Url: https://www.nature.com/articles/s41559-017-0282-8 74. Wang C, Chacko S, Abdollah H, Baranchuk A. 2018. Treating Lyme carditis high-degree AV block using a temporary-permanent pacemaker. Annals of Noninvasive Electrocardiology. 224(3): e12599. doi:10.1111/anec.12599. Url: https://onlinelibrary.wiley.com/doi/epdf/10.1111/ anec.12599 https://doi.org/10.1093/jmedent/28.1.32 https://academic.oup.com/jme/article-abstract/28/1/32/2220853?redirectedFrom=fulltext https://academic.oup.com/jme/article-abstract/28/1/32/2220853?redirectedFrom=fulltext https://dx.doi.org/10.1089%2Fvbz.2017.2127 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576071/ https://doi.org/10.1128/IAI.01228-12 https://journals.asm.org/doi/10.1128/IAI.01228-12 https://journals.asm.org/doi/10.1128/IAI.01228-12 https://doi.org/10.1128/IAI.00145-12 https://journals.asm.org/doi/10.1128/IAI.00145-12 https://journals.asm.org/doi/10.1128/IAI.00145-12 https://doi.org/10.1128/iai.55.9.2311-2313.1987 https://doi.org/10.1128/iai.55.9.2311-2313.1987 https://journals.asm.org/doi/epdf/10.1128/iai.55.9.2311-2313.1987 https://journals.asm.org/doi/epdf/10.1128/iai.55.9.2311-2313.1987 https://dx.doi.org/10.1089%2Fvbz.2012.1003 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3610442/ https://dx.doi.org/10.1371%2Fjournal.pone.0207067 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0207067 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0207067 http://dx.doi.org/10.1038/s41559-017-0282-8 http://dx.doi.org/10.1038/s41559-017-0282-8 https://www.nature.com/articles/s41559-017-0282-8 https://dx.doi.org/10.1111%2Fanec.12599 https://onlinelibrary.wiley.com/doi/epdf/10.1111/anec.12599 https://onlinelibrary.wiley.com/doi/epdf/10.1111/anec.12599 Gaetano & Danka | Borrelia burgdorferi: The Deer Tick’s Dark Secret 37 75. Weber K, Burgdorfer W, Schierz G. 1993. Aspects of Lyme Borreliosis: The Historical Road to the Discovery of Borrelia burgdorferi. Germany: Springer Berlin, Heidelberg. Url: https://link. springer.com/chapter/10.1007/978-3-642-77614-4_2 76. Wright W, Riedel D, Talwani R, Gillian B. 2012. Diagnosis and Management of Lyme Disease. American Family Physician. 85(11): 1086-1093. doi:https://www.aafp.org/afp/2012/0601/ afp20120601p1086.pdf. Url: https://pubmed.ncbi.nlm.nih.gov/22962880/ 77. Yeung C, Baranchuk A. 2019. Diagnosis and Treatment of Lyme Carditis: JACC Review Topic of the Week. Journal of the American College of Cardiology. 74(21): 2709-2711. doi:https:// doi.org/10.1016/j.jacc.2018.11.035. Url: https://www.sciencedirect.com/science/article/pii/ S0735109718394427?via%3Dihub 78. Zee J, Piesman J, Hojgaard A, Black W. 2015. Nuclear Markers Reveal Predominantly North to South Gene Flow in Ixodes scapularis, the Tick Vector of the Lyme Disease Spirochete. PLOS ONE. 10(11): e0139630. doi:https://doi.org/10.1371/journal.pone.0139630. Url: https:// journals.plos.org/plosone/article?id=10.1371/journal.pone.0139630 79. Zhang J, Norris S. 1998. Genetic Variation of the Borrelia burgdorferi Gene vlsE Involves Cassette-Specific, Segmental Gene Conversion. Infection and Immunology. 66(8): 3698–3704. doi:10.1128/IAI.66.8.3698-3704.1998. Url: https://journals.asm.org/ doi/10.1128/IAI.66.8.3698-3704.1998 https://link.springer.com/chapter/10.1007/978-3-642-77614-4_2 https://link.springer.com/chapter/10.1007/978-3-642-77614-4_2 https://www.aafp.org/afp/2012/0601/afp20120601p1086.pdf https://www.aafp.org/afp/2012/0601/afp20120601p1086.pdf https://pubmed.ncbi.nlm.nih.gov/22962880/ https://doi.org/10.1016/j.jacc.2018.11.035 https://doi.org/10.1016/j.jacc.2018.11.035 https://www.sciencedirect.com/science/article/pii/S0735109718394427?via%3Dihub https://www.sciencedirect.com/science/article/pii/S0735109718394427?via%3Dihub https://doi.org/10.1371/journal.pone.0139630 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139630 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139630 https://doi.org/10.1128/iai.66.8.3698-3704.1998 https://journals.asm.org/doi/10.1128/IAI.66.8.3698-3704.1998 https://journals.asm.org/doi/10.1128/IAI.66.8.3698-3704.1998