Kevin S. Brandt

Comparison of disseminated and nondisseminated strains of Borrelia burgdorferi sensu stricto in mice naturally infected by tick bite.

ABSTRACT Clinical isolates of Borrelia burgdorferi sensu stricto have been categorized into disseminated and nondisseminated groups based on distinct ribosomal spacer restriction fragment length polymorphism genotypes (RSTs). In order to determine whether transmission by tick bite would alter the dissemination dynamics and disease produced by distinct genotypes, disseminated isolates (RST1), nondisseminated isolates (RST3), and a standard laboratory strain (B-31) were established in a murine cycle utilizing infections transmitted by ticks. B-31 spirochetes circulated in the blood of inbred C3H/HeJ mice longer than in the blood of outbred mice. The majority of C3H mice exposed to RST1-infected ticks contained cultivable spirochetes in their blood for up to 17 days; in contrast, mice exposed to RST3 isolates demonstrated a precipitous decline in infection after day 7 postexposure. A quantitative PCR (q-PCR) assay demonstrated that the densities of spirochetes in blood were similar for the RST1 and RST3 isolates, except during the 2nd week postexposure, when the RST1 isolates displayed a markedly higher density in blood. Spirochete load in the heart and bladder of infected mice was measured by q-PCR at 8 weeks postexposure; the numbers of spirochetes in these tissues were similar for mice infected with either disseminated or nondisseminated strains. Similarly, histopathology samples of heart, bladder, and joint tissue obtained at 8 weeks postexposure did not reveal greater pathology in mice infected with the disseminated isolates. We conclude that although the spirochetemia induced by tick-transmitted disseminated isolates was more intense and of longer duration than that induced by nondisseminated isolates, the resultant pathologies produced by these strains were ultimately similar.

Borrelia burgdorferi bba66 Gene Inactivation Results in Attenuated Mouse Infection by Tick Transmission

ABSTRACT 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, BbΔA66, 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 BbΔA66 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 BbΔA66-infected nymphs to infect laboratory mice was significantly impaired compared to that of mice fed upon by WT-infected ticks. trans-complementation of BbΔA66 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.

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

ABSTRACT 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.

Evaluation of the Borrelia burgdorferi BBA64 Protein as a Protective Immunogen in Mice

ABSTRACT 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.

pncA and bptA Are Not Sufficient To Complement Ixodes scapularis Colonization and Persistence by Borrelia burgdorferi in a Linear Plasmid lp25-Deficient Background

ABSTRACT 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.

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 ticks 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 hosts 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.

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.

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.