Borrelia miyamotoi BipA-like protein, BipM, is a candidate serodiagnostic antigen distinguishing between Lyme disease and relapsing fever Borrelia infections.

A Borrelia miyamotoi gene with partial homology to bipA of relapsing fever spirochetes Borrelia hermsii and Borrelia turicatae was identified by a GenBank basic alignment search analysis. We hypothesized that this gene product may be an immunogenic antigen as described for other relapsing fever Borrelia (RFB) and could serve as a serological marker for B. miyamotoi infections. The B. miyamotoi gene was a truncated version about half the size of the B. hermsii and B. turicatae bipA with a coding sequence of 894 base pairs. The gene product had a calculated molecular size of 32.7 kDa (including the signal peptide). Amino acid alignments with B. hermsii and B. turicatae BipA proteins and with other B. miyamotoi isolates showed conservation at the carboxyl end. We cloned the B. miyamotoi bipA-like gene (herein named bipM) and generated recombinant protein for serological characterization and for antiserum production. Protease protection analysis demonstrated that BipM was surface exposed. Serologic analyses using anti-B. miyamotoi serum samples from tick bite-infected and needle inoculated mice showed 94 % positivity against BipM. The 4 BipM negative serum samples were blotted against another B. miyamotoi antigen, BmaA, and two of them were seropositive resulting in 97 % positivity with both antigens. Serum samples from B. burgdorferi sensu stricto (s.s.)-infected mice were non-reactive against rBipM by immunoblot. Serum samples from Lyme disease patients were also serologically negative against BipM except for 1 sample which may have indicated a possible co-infection. A recently published study demonstrated that B. miyamotoi BipM was non-reactive against serum samples from B. hermsii, Borrelia parkeri, and B. turicatae infected animals. These results show that BipM has potential for a B. miyamotoi-infection specific and sensitive serodiagnostic to differentiate between Lyme disease and various RFB infections.

Structural Elucidation of a Protective B Cell Epitope on Outer Surface Protein C (OspC) of the Lyme Disease Spirochete, Borreliella burgdorferi.

Outer surface protein C (OspC) plays a pivotal role in mediating tick-to-host transmission and infectivity of the Lyme disease spirochete, Borreliella burgdorferi. OspC is a helical-rich homodimer that interacts with tick salivary proteins, as well as components of the mammalian immune system. Several decades ago, it was shown that the OspC-specific monoclonal antibody, B5, was able to passively protect mice from experimental tick-transmitted infection by B. burgdorferi strain B31. However, B5's epitope has never been elucidated, despite widespread interest in OspC as a possible Lyme disease vaccine antigen. Here, we report the crystal structure of B5 antigen-binding fragments (Fabs) in complex with recombinant OspC type A (OspCA). Each OspC monomer within the homodimer was bound by a single B5 Fab in a side-on orientation, with contact points along OspC's α-helix 1 and α-helix 6, as well as interactions with the loop between α-helices 5 and 6. In addition, B5's complementarity-determining region (CDR) H3 bridged the OspC-OspC' homodimer interface, revealing the quaternary nature of the protective epitope. To provide insight into the molecular basis of B5 serotype specificity, we solved the crystal structures of recombinant OspC types B and K and compared them to OspCA. This study represents the first structure of a protective B cell epitope on OspC and will aid in the rational design of OspC-based vaccines and therapeutics for Lyme disease. IMPORTANCE The spirochete Borreliella burgdorferi is a causative agent of Lyme disease, the most common tickborne disease in the United States. The spirochete is transmitted to humans during the course of a tick taking a bloodmeal. After B. burgdorferi is deposited into the skin of a human host, it replicates locally and spreads systemically, often resulting in clinical manifestations involving the central nervous system, joints, and/or heart. Antibodies directed against B. burgdorferi's outer surface protein C (OspC) are known to block tick-to-host transmission, as well as dissemination of the spirochete within a mammalian host. In this report, we reveal the first atomic structure of one such antibody in complex with OspC. Our results have implications for the design of a Lyme disease vaccine capable of interfering with multiple stages in B. burgdorferi infection.

Analysis of variable major protein antigenic variation in the relapsing fever spirochete, Borrelia miyamotoi, in response to polyclonal antibody selection pressure.

Borrelia miyamotoi is a tick-transmitted spirochete that is genetically grouped with relapsing fever Borrelia and possesses multiple archived pseudogenes that encode variable major proteins (Vmps). Vmps are divided into two groups based on molecular size; variable large proteins (Vlps) and variable small proteins (Vsps). Relapsing fever Borrelia undergo Vmp gene conversion at a single expression locus to generate new serotypes by antigenic switching which is the basis for immune evasion that causes relapsing fever in patients. This study focused on B. miyamotoi vmp expression when spirochetes were subjected to antibody killing selection pressure. We incubated a low passage parent strain with mouse anti-B. miyamotoi polyclonal antiserum which killed the majority population, however, antibody-resistant reisolates were recovered. PCR analysis of the gene expression locus in the reisolates showed vsp1 was replaced by Vlp-encoded genes. Gel electrophoresis protein profiles and immunoblots of the reisolates revealed additional Vlps indicating that new serotype populations were selected by antibody pressure. Sequencing of amplicons from the expression locus of the reisolates confirmed the presence of a predominant majority serotype population with minority variants. These findings confirm previous work demonstrating gene conversion in B. miyamotoi and that multiple serotype populations expressing different vmps arise when subjected to antibody selection. The findings also provide evidence for spontaneous serotype variation emerging from culture growth in the absence of antibody pressure. Validation and determination of the type, number, and frequency of serotype variants that arise during animal infections await further investigations.

Evaluation of Immunocompetent Mouse Models for Borrelia miyamotoi Infection.

Borrelia miyamotoi is a relapsing fever spirochete that is harbored by Ixodes spp. ticks and is virtually uncharacterized, compared to other relapsing fever Borrelia vectored by Ornithodoros spp. ticks. There is not an immunocompetent mouse model for studying B. miyamotoi infection in vivo or for transmission in the vector-host cycle. Our goal was to evaluate B. miyamotoi infections in multiple mouse breeds/strains as a prelude to the ascertainment of the best experimental infection model. Two B. miyamotoi strains, namely, LB-2001 and CT13-2396, as well as three mouse models, namely, CD-1, C3H/HeJ, and BALB/c, were evaluated. We were unable to observe B. miyamotoi LB-2001 spirochetes in the blood via darkfield microscopy or to detect DNA via real-time PCR post needle inoculation in the CD-1 and C3H/HeJ mice. However, LB-2001 DNA was detected via real-time PCR in the blood of the BALB/c mice after needle inoculation, although spirochetes were not observed via microscopy. CD-1, C3H/HeJ, and BALB/c mice generated an antibody response to B. miyamotoi LB-2001 following needle inoculation, but established infections were not detected, and the I. scapularis larvae failed to acquire spirochetes from the exposed CD-1 mice. In contrast, B. miyamotoi CT13-2396 was visualized in the blood of the CD-1 and C3H/HeJ mice via darkfield microscopy and detected by real-time PCR post needle inoculation. Both mouse strains seroconverted. However, no established infection was detected in the mouse organs, and the I. scapularis larvae failed to acquire Borrelia after feeding on CT13-2396 exposed CD-1 or C3H/HeJ mice. These findings underscore the challenges in establishing an experimental B. miyamotoi infection model in immunocompetent laboratory mice. IMPORTANCE Borrelia miyamotoi is a causative agent of hard tick relapsing fever, was first identified in the early 1990s, and was characterized as a human pathogen in 2011. Unlike other relapsing fever Borrelia species, B. miyamotoi spread by means of Ixodes ticks. The relatively recent recognition of this human pathogen means that B. miyamotoi is virtually uncharacterized, compared to other Borrelia species. Currently there is no standard mouse-tick model with which to study the interactions of the pathogen within its vector and hosts. We evaluated two B. miyamotoi isolates and three immunocompetent mouse models to identify an appropriate model with which to study tick-host-pathogen interactions. With the increased prevalence of human exposure to Ixodes ticks, having an appropriate model with which to study B. miyamotoi will be critical for the future development of diagnostics and intervention strategies.

Modification of the multiplex plasmid PCR assay for Borrelia miyamotoi strain LB-2001 based on the complete genome sequence reflecting genomic rearrangements differing from strain CT13-2396.

The genome of Borrelia spp. consists of an approximate 1 megabase chromosome and multiple linear and circular plasmids. We previously described a multiplex PCR assay to detect plasmids in the North American Borrelia miyamotoi strains LB-2001 and CT13-2396. The primer pair sets specific for each plasmid were derived from the genome sequence for B. miyamotoi strain CT13-2396, because the LB-2001 complete sequence had not been generated. The recent completion of the LB-2001 genome sequence revealed a distinct number of plasmids (n = 12) that differed from CT13-2396 (n = 14). Notable was a 97-kilobase plasmid in LB-2001, not present in CT13-2396, that appeared to be a rearrangement of the circular plasmids of strain CT13-2396. Strain LB-2001 contained two plasmids, cp30-2 and cp24, that were not annotated for strain CT13-2396. Therefore, we re-evaluated the original CT13-2396-derived multiplex PCR primer pairs and determined their location in the LB-2001 plasmids. We modified the original multiplex plasmid PCR assay for strain LB-2001 to include cp30-2 and cp24. We also determined which LB-2001 plasmids corresponded to the amplicons generated from the original CT13-2396 primer sets. These observations provide a more precise plasmid profile based on the multiplex PCR assay and reflect the complexity of gene rearrangements that occur in B. miyamotoi strains isolated from the same geographic region.

A transwell assay method to evaluate Borrelia burgdorferi sensu stricto migratory chemoattraction toward tick saliva proteins.

We developed a transwell assay to quantify migration of the Lyme disease agent, Borrelia burgdorferi sensu stricto (s.s.), toward Ixodes scapularis salivary gland proteins. The assay was designed to assess B. burgdorferi s.s. migration upward against gravity through a transwell polycarbonate membrane overlaid with 6% gelatin. Borreliae that channeled into the upper transwell chamber in response to test proteins were enumerated by flow cytometry. The transwell assay measured chemoattractant activity for B. burgdorferi s.s. from salivary gland extract (SGE) harvested from nymphal ticks during bloodmeal engorgement on mice 42 h post-attachment and saliva collected from adult ticks. Additionally, SGE protein fractions separated by size exclusion chromatography demonstrated various levels of chemoattractant activity in the transwell assay. Sialostatin L, and Salp-like proteins 9 and 11 were identified by mass spectrometry in SGE fractions that exhibited elevated activity. Recombinant forms of these proteins were tested in the transwell assay and showed positive chemoattractant properties compared to controls and another tick protein, S15A. These results were reproducible providing evidence that the transwell assay is a useful method for continuing investigations to find tick saliva components instrumental in driving B. burgdorferi s.s. chemotaxis.

Borrelia miyamotoi strain LB-2001 retains plasmids and infectious phenotype throughout continuous culture passages as evaluated by multiplex PCR.

Borrelia miyamotoi is a tick-borne spirochete of the relapsing fever borrelia group and an emerging pathogen of public health significance. The genomes of relapsing fever borreliae and Lyme disease borreliae consist of multiple linear and circular plasmids in addition to the chromosome. Previous work with B. burgdorferi sensu lato found diminished infectivity upon continuous in vitro culture passage that was attributable to plasmid loss. The effect of long-term culture passage on B. miyamotoi is not known. We generated a series of plasmid-specific primer sets and developed a multiplex PCR assay to detect the 14 known plasmids of B. miyamotoi North American strains LB-2001 and CT13-2396. We assessed the plasmid content of B. miyamotoi LB-2001 over 64 culture passages spanning 15 months and determined that strain LB-2001 retained all plasmids upon prolonged in vitro cultivation and remained infectious in mice. We also found that strain LB-2001 lacks plasmid lp20-1 which is present in strain CT13-2396. These results suggest that B. miyamotoi remains genetically stable when cultured and passaged in vitro.

Characterization of a Borrelia miyamotoi membrane antigen (BmaA) for serodiagnosis of Borrelia miyamotoi disease.

Borrelia miyamotoi is a tick-borne pathogen that causes Borrelia miyamotoi disease (BMD), an emerging infectious disease of increasing public health significance. B. miyamotoi is transmitted by the same tick vector (Ixodes spp.) as B. burgdorferi sensu lato (s.l.), the causative agent of Lyme disease, therefore laboratory assays to differentiate BMD from Lyme disease are needed to avoid misdiagnoses and for disease confirmation. We previously performed a global immunoproteomic analysis of the murine host antibody response against B. miyamotoi infection to discover antigens that could serologically distinguish the two infections. An initial assessment identified a putative lipoprotein antigen, here termed BmaA, as a promising candidate to augment current research-based serological assays. In this study, we show that BmaA is an outer surface-associated protein by its susceptibility to protease digestion. Synthesis of BmaA in culture was independent of temperature at either 23 °C or 34 °C. The BmaA gene is present in two identical loci harbored on separate plasmids in North American strains LB-2001 and CT13-2396. bmaA-like sequences are present in other B. miyamotoi strains and relapsing fever borrelia as multicopy genes and as paralogous or orthologous gene families. IgM and IgG antibodies in pooled serum from BMD patients reacted with native BmaA fractionated by 2-dimensional gel electrophoresis and identified by mass spectrometry. IgG against recombinant BmaA was detected in 4 of 5 BMD patient serum samples as compared with 1 of 23 serum samples collected from patients with various stages of Lyme disease. Human anti-B. turicatae serum did not seroreact with recombinant BmaA suggesting a role as a species-specific diagnostic antigen. These results demonstrated that BmaA elicits a human host antibody response during B. miyamotoi infection but not in a tested group of B. burgdorferi-infected Lyme disease patients, thereby providing a potentially useful addition for developing BMD serodiagnostic tests.

Evaluation of Patient IgM and IgG Reactivity Against Multiple Antigens for Improvement of Serodiagnostic Testing for Early Lyme Disease.

Serologic testing is the standard for laboratory diagnosis and confirmation of Lyme disease. Serodiagnostic assays to detect antibodies against Borrelia burgdorferi, the agent of Lyme borreliosis, are used for detection of infection. However, serologic testing within the first month of infection is less sensitive as patients' antibody responses continue to develop. Previously, we screened several B. burgdorferi in vivo expressed antigens for candidates that elicit early antibody responses in patients with Stage 1 and 2 Lyme disease. We evaluated patient IgM seroreactivity against 6 antigens and found an increase in sensitivity without compromising specificity when compared to current IgM second-tier immunoblot scoring. In this study, we continued the evaluation using a multi-antigen panel to measure IgM plus IgG seroreactivity in these early Lyme disease patients' serum samples. Using two statistical methods for calculating positivity cutoff values, sensitivity was 70 and 84-87%, for early acute and early convalescent Lyme disease patients, respectively. Specificity was 98-100% for healthy non-endemic control patients, and 96-100% for healthy endemic controls depending on the statistical analysis. We conclude that improved serologic testing for early Lyme disease may be achieved by the addition of multiple borrelial antigens that elicit IgM and IgG antibodies early in infection.

Immunoproteomic analysis of Borrelia miyamotoi for the identification of serodiagnostic antigens.

The tick-borne spirochete, Borrelia miyamotoi, is an emerging pathogen of public health significance. Current B. miyamotoi serodiagnostic testing depends on reactivity against GlpQ which is not highly sensitive on acute phase serum samples. Additionally, anti-B. miyamotoi antibodies can cross-react with C6 antigen testing for B. burgdorferi, the causative agent of Lyme disease, underscoring the need for improved serological assays that produce accurate diagnostic results. We performed an immunoproteomics analysis of B. miyamotoi proteins to identify novel serodiagnostic antigens. Sera from mice infected with B. miyamotoi by subcutaneous inoculation or tick bite were collected for immunoblotting against B. miyamotoi membrane-associated proteins separated by 2-dimensional electrophoresis (2DE). In total, 88 proteins in 40 2DE immunoreactive spots were identified via mass spectrometry. Multiple variable large proteins (Vlps) and a putative lipoprotein were among those identified and analyzed. Reactivity of anti-B. miyamotoi sera against recombinant Vlps and the putative lipoprotein confirmed their immunogenicity. Mouse anti-B. burgdorferi serum was cross-reactive to all recombinant Vlps, but not against the putative lipoprotein by IgG. Furthermore, antibodies against the recombinant putative lipoprotein were present in serum from a B. miyamotoi-infected human patient, but not a Lyme disease patient. Results presented here provide a comprehensive profile of B. miyamotoi antigens that induce the host immune response and identify a putative lipoprotein as a potentially specific antigen for B. miyamotoi serodetection.

Evaluation of in vivo expressed Borrelia burgdorferi antigens for improved IgM serodiagnosis of early Lyme disease.

Improved serologic tests are needed for accurate diagnosis and proper treatment of early stage Lyme disease. We evaluated the 3 antigens currently used for 2-tiered IgM immunoblot testing (FlaB, OspC, and BmpA) in combination with 3 additional antigens (BBA65, BBA70, and BBA73) and measured the sensitivity and specificity against a serum repository of positive and negative controls. Using 3 statistical methods for positivity cutoff determinations and scoring criteria, we found increased sensitivities for early Lyme disease when 2 of 6 antigens were positive as compared with the 2 of 3 antigen IgM criteria currently used for second-tier immunoblot scoring. Specificities for negative controls were comparable or superior to using 2 of 3 antigens. These results indicate that IgM sensitivity and specificity of serological testing for Lyme disease in the early stages of illness can be improved by employing antigens that target the initial host antibody responses.

Human neuroglial cells internalize Borrelia burgdorferi by coiling phagocytosis mediated by Daam1.

Borrelia burgdorferi, the agent of Lyme borreliosis, can elude hosts' innate and adaptive immunity as part of the course of infection. The ability of B. burgdorferi to invade or be internalized by host cells in vitro has been proposed as a mechanism for the pathogen to evade immune responses or antimicrobials. We have previously shown that B. burgdorferi can be internalized by human neuroglial cells. In this study we demonstrate that these cells take up B. burgdorferi via coiling phagocytosis mediated by the formin, Daam1, a process similarly described for human macrophages. Following coincubation with glial cells, B. burgdorferi was enwrapped by Daam1-enriched coiling pseudopods. Coiling of B. burgdorferi was significantly reduced when neuroglial cells were pretreated with anti-Daam1 antibody indicating the requirement for Daam1 for borrelial phagocytosis. Confocal microscopy showed Daam1 colocalizing to the B. burgdorferi surface suggesting interaction with borrelial membrane protein(s). Using the yeast 2-hybrid system for identifying protein-protein binding, we found that the B. burgdorferi surface lipoprotein, BBA66, bound the FH2 subunit domain of Daam1. Recombinant proteins were used to validate binding by ELISA, pull-down, and co-immunoprecipitation. Evidence for native Daam1 and BBA66 interaction was suggested by colocalization of the proteins in the course of borrelial capture by the Daam1-enriched pseudopodia. Additionally, we found a striking reduction in coiling for a BBA66-deficient mutant strain compared to BBA66-expressing strains. These results show that coiling phagocytosis is a mechanism for borrelial internalization by neuroglial cells mediated by Daam1.

Immunization of mice with Borrelia burgdorferi lp54 gene encoded recombinant proteins does not provide protection against tick transmitted infectious challenge.

The Borrelia burgdorferi outer surface membrane proteins BBA65, BBA66, BBA69, BBA70, and BBA73 were tested for their ability to confer protection against B. burgdorferi infection challenge. Mice were immunized with recombinant forms of the proteins singly or in combinations. Following initial protein inoculation and booster injections, seroconversion was confirmed prior to B. burgdorferi challenge by tick bite. Despite mice having high antibody titers for each antigen, no significant protections against the challenge infections were observed. These results demonstrate that these recombinant proteins were not protective and reflects the challenges confronted to identify effective novel vaccine candidates for Lyme disease.

Evaluation of Selected Borrelia burgdorferi lp54 Plasmid-Encoded Gene Products Expressed during Mammalian Infection as Antigens To Improve Serodiagnostic Testing for Early Lyme Disease.

Laboratory testing for the diagnosis of Lyme disease is performed primarily by serologic assays and is accurate for detection beyond the acute stage of the infection. Serodiagnostic assays to detect the early stages of infection, however, are limited in their sensitivity, and improvement is warranted. We analyzed a series of Borrelia burgdorferi proteins known to be induced within feeding ticks and/or during mammalian infection for their utility as serodiagnostic markers against a comprehensive panel of Lyme disease patient serum samples. The antigens were assayed for IgM and IgG reactivity in line immunoblots and separately by enzyme-linked immunosorbent assay (ELISA), with a focus on reactivity against early Lyme disease with erythema migrans (EM), early disseminated Lyme neuroborreliosis, and early Lyme carditis patient serum samples. By IgM immunoblotting, we found that recombinant proteins BBA65, BBA70, and BBA73 reacted with early Lyme EM samples at levels comparable to those of the OspC antigen used in the current IgM blotting criteria. Additionally, these proteins reacted with serum samples from patients with early neuroborreliosis and early carditis, suggesting value in detecting early stages of this disease progression. We also found serological reactivity against recombinant proteins BBA69 and BBA73 with early-Lyme-disease samples using IgG immunoblotting and ELISA. Significantly, some samples that had been scored negative by the Centers for Disease Control and Prevention-recommended 2-tiered testing algorithm demonstrated positive reactivity to one or more of the antigens by IgM/IgG immunoblot and ELISA. These results suggest that incorporating additional in vivo-expressed antigens into the current IgM/IgG immunoblotting tier in a recombinant protein platform assay may improve the performance of early-Lyme-disease serologic testing.

pncA and bptA are not sufficient to complement Ixodes scapularis colonization and persistence by Borrelia burgdorferi in a linear plasmid lp25-deficient background.

The complex segmented genome of Borrelia burgdorferi is comprised of a linear chromosome along with numerous linear and circular plasmids essential for tick and/or mammalian infectivity. The pathogenic necessity for specific borrelial plasmids has been identified; most notably, infections of the tick vector and mammalian host both require linear plasmid 25 (lp25). Genes carried on lp25, specifically bptA and pncA, are postulated to play a role for B. burgdorferi to infect and persist in Ixodes ticks. In this study, we complemented an lp25-deficient borrelial strain with pncA alone or pncA accompanied by bptA to evaluate the ability of the complemented strains to restore larval colonization and persistence through transstadial transmission relative to that of wild-type B. burgdorferi. The acquisition of the complemented strains by tick larvae from infected mice and/or the survival of these strains was significantly decreased when assayed by cultivation and quantitative PCR (qPCR). Only 10% of the pncA-complemented strain organisms were found by culture to survive 17 days following larval feeding, while 45% of the pncA- and bptA-complemented strain organisms survived, with similar results by PCR. However, neither of the complemented B. burgdorferi strains was capable of persisting through the molt to the nymphal stage as analyzed by culture. qPCR analyses of unfed nymphs detected B. burgdorferi genomes in several nymphs at low copy numbers, likely indicating the presence of DNA from dead or dying cells. Overall, the data indicate that pncA and bptA cannot independently support infection, suggesting that lp25 carries additional gene(s) or regulatory elements critical for B. burgdorferi survival and pathogenesis in the Ixodes vector.

Evaluation of the Borrelia burgdorferi BBA64 protein as a protective immunogen in mice.

The Borrelia burgdorferi bba64 gene product is a surface-localized lipoprotein synthesized within mammalian and tick hosts and is involved in vector transmission of disease. These properties suggest that BBA64 may be a vaccine candidate against Lyme borreliosis. In this study, protective immunity against B. burgdorferi challenge was assessed in mice immunized with the BBA64 protein. Mice developed a high-titer antibody response following immunization with soluble recombinant BBA64 but were not protected when challenged by needle inoculation of culture-grown spirochetes. Likewise, mice passively immunized with an anti-BBA64 monoclonal antibody were not protected against needle-inoculated organisms. BBA64-immunized mice were subjected to B. burgdorferi challenge by the natural route of a tick bite, but these trials did not demonstrate significant protective immunity in either outbred or inbred strains of mice. Lipidated recombinant BBA64 produced in Escherichia coli was assessed for possible improved elicitation of a protective immune response. Although inoculation with this antigen produced a high-titer antibody response, the lipidated BBA64 also was unsuccessful in protecting mice from B. burgdorferi challenge by tick bites. Anti-BBA64 antibodies raised in rats eradicated the organisms, as evidenced by in vitro borreliacidal assays, thus demonstrating the potential for BBA64 to be effective as a protective immunogen. However, passive immunization with the same monospecific rat anti-BBA64 polyclonal serum failed to provide protection against tick bite-administered challenge. These results reveal the challenges faced in not only identifying B. burgdorferi proteins with potential protective capability but also in producing recombinant antigens conducive to preventive therapies against Lyme borreliosis.

Borrelia burgdorferi bba66 gene inactivation results in attenuated mouse infection by tick transmission.

The impact of the Borrelia burgdorferi surface-localized immunogenic lipoprotein BBA66 on vector and host infection was evaluated by inactivating the encoding gene, bba66, and characterizing the mutant phenotype throughout the natural mouse-tick-mouse cycle. The BBA66-deficient mutant isolate, Bb(ΔA66), remained infectious in mice by needle inoculation of cultured organisms, but differences in spirochete burden and pathology in the tibiotarsal joint were observed relative to the parental wild-type (WT) strain. Ixodes scapularis larvae successfully acquired Bb(ΔA66) following feeding on infected mice, and the organisms persisted in these ticks through the molt to nymphs. A series of tick transmission experiments (n = 7) demonstrated that the ability of Bb(ΔA66)-infected nymphs to infect laboratory mice was significantly impaired compared to that of mice fed upon by WT-infected ticks. trans-complementation of Bb(ΔA66) with an intact copy of bba66 restored the WT infectious phenotype in mice via tick transmission. These results suggest a role for BBA66 in facilitating B. burgdorferi dissemination and transmission from the tick vector to the mammalian host as part of the disease process for Lyme borreliosis.

Borrelia burgdorferi visualized in Ixodes scapularis tick excrement by immunofluorescence.

The enzootic cycle of Borrelia burgdorferi, the etiologic agent of Lyme disease, involves Ixodes spp. ticks and vertebrates. Resident tick Borrelia, harbored inside the midgut, are eventually expelled with the tick's saliva into the vertebrate host when a tick consumes a blood meal. During this 4- to 5-day feeding period I. scapularis will defecate onto the host's skin. Previously we detected borrelial DNA in tick feces throughout engorgement. In this study we report the microscopic examination for B. burgdorferi in nymphal excrement. Using immunofluorescence assays, we observed Borrelia in all mouse skin and capsule fecal swabs tested, although we could not culture the spirochetes. These results update our previous analysis by revealing that spirochetes can also be visualized in tick excrement. Furthermore, the results emphasize that borrelial contamination by defecation is a possibility, and that caution should be exercised by researchers investigating pathogen/host/vector interactions. The biological significance of the presence of non-culturable Borrelia in tick feces during engorgement is unclear.

Saliva, salivary gland, and hemolymph collection from Ixodes scapularis ticks.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) (1-8). To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick (3,9-13). For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle (14,15). Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva (9,16-19). As a tick feeds the host typically responds with a strong hemostatic and innate immune response (11,13,20-22). Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding (3,11,20,21,23). The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens (3,20,24-27). To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Analysis of Borrelia burgdorferi Surface Proteins as Determinants in Establishing Host Cell Interactions.

Borrelia burgdorferi infection causes Lyme borreliosis in humans, a condition which can involve a systemic spread of the organism to colonize various tissues and organs. If the infection is left untreated by antimicrobials, it can lead to manifestations including, arthritis, carditis, and/or neurological problems. Identification and characterization of B. burgdorferi outer membrane proteins that facilitate cellular attachment and invasion to establish infection continue to be investigated. In this study, we sought to further define putative cell binding properties of surface-exposed B. burgdorferi proteins by observing whether cellular adherence could be blocked by antibodies. B. burgdorferi mixed separately with monoclonal antibodies (mAbs) against outer surface protein (Osp) A, OspC, decorin-binding protein (Dbp) A, BBA64, and RevA antigens were incubated with human umbilical vein endothelial cells (HUVEC) and human neuroglial cells (H4). B. burgdorferi treated with anti-OspA, -DbpA, and -BBA64 mAbs showed a significant decrease in cellular association compared to controls, whereas B. burgdorferi treated with anti-OspC and anti-RevA showed no reduction in cellular attachment. Additionally, temporal transcriptional analyses revealed upregulated expression of bba64, ospA, and dbpA during coincubation with cells. Together, the data provide evidence that OspA, DbpA, and BBA64 function in host cell adherence and infection mechanisms.