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Lyme Disease in Virginia

  1. J Infect Dis. 2010 Apr 1;201(7):1084-95.

    BBA52 facilitates Borrelia burgdorferi transmission from feeding ticks to murine hosts.

    Kumar M, Yang X, Coleman AS, Pal U.

    Department of Veterinary Medicine, University of Maryland, and Virginia-Maryland Regional College of Veterinary Medicine, College Park, Maryland 20742, USA.

    Borrelia burgdorferi, the pathogen of Lyme borreliosis, persists in nature through a tick-rodent transmission cycle. A selective assessment of the microbial transcriptome, limited to gene-encoding putative membrane proteins, reveals that bba52 transcription in vivo is strictly confined to the vector-specific portion of the microbial life cycle, with the highest levels of expression noted in feeding ticks and with swift down-regulation noted in mice. bba52 deletion did not affect murine disease as assessed by the genesis of arthritis and carditis or long-term persistence of pathogens in mice or ticks. However, bba52 deficiency did impair microbial transitions between hosts and vector, defects that could be fully rescued when bba52 expression was genetically restored to the original genomic locus. These studies establish that BBA52 facilitates vector-host transitions by the pathogen and therefore is a potential antigenic target for interference with transmission of B. burgdorferi from ticks to mammalian hosts.

    PMCID: PMC2832101 [Available on 2011/4/1] PMID: 20170377 [PubMed - indexed for MEDLINE]

  2. FEMS Immunol Med Microbiol. 2009 Nov 23. [Epub ahead of print]

    Identification and molecular characterization of a cyclic-di-GMP effector protein, PlzA (BB0733): additional evidence for the existence of a functional cyclic-di-GMP regulatory network in the Lyme disease spirochete, Borrelia burgdorferi.

    Freedman JC, Rogers EA, Kostick JL, Zhang H, Iyer R, Schwartz I, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, Richmond, VA, USA.

    Abstract The Borrelia burgdorferi Rrp1 protein is a diguanylate cyclase that controls a regulon consisting of approximately 10% of the total genome. Because Rrp1 lacks a DNA-binding domain, its regulatory capability is most likely mediated through the production of bis-(3'-5')-cyclic dimeric GMP (c-di-GMP). C-di-GMP binds to and activates the regulatory activity of proteins that harbor a PilZ domain. The occurrence of a PilZ domain within a protein is not in and of itself sufficient to convey c-di-GMP binding, as other structural aspects of the protein are important in the interaction. In this study, we have assessed the expression and c-di-GMP binding ability of the sole PilZ domain-containing protein of B. burgdorferi B31, PlzA. PlzA was determined to be upregulated by tick feeding and to be expressed during mammalian infection. The gene is highly conserved and present in all Borrelia species. Analyses of recombinant PlzA demonstrated its ability to bind c-di-GMP and site-directed mutagenesis revealed that this interaction is highly specific and dependent on Arg residues contained within the PilZ domain. In summary, this study is the first to identify a c-di-GMP effector molecule in a spirochete and provides additional evidence for the existence of a complete c-di-GMP regulatory network in the Lyme disease spirochete, B. burgdorferi.

    PMID: 20030712 [PubMed - as supplied by publisher]

  3. Infect Immun. 2009 Oct;77(10):4396-405. Epub 2009 Jul 20.

    Comparative analysis of the properties and ligand binding characteristics of CspZ, a factor H binding protein, derived from Borrelia burgdorferi isolates of human origin.

    Rogers EA, Abdunnur SV, McDowell JV, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, Richmond, VA 23298-0678, USA.

    Borrelia burgdorferi CspZ (BBH06/BbCRASP-2) binds the complement regulatory protein factor H (FH) and additional unidentified serum proteins. The goals of this study were to assess the ligand binding capability of CspZ orthologs derived from an extensive panel of human Lyme disease isolates and to further define the molecular basis of the interaction between FH and CspZ. While most B. burgdorferi CspZ orthologs analyzed bound FH, specific, naturally occurring polymorphisms, most of which clustered in a specific loop domain of CspZ, prevented FH binding in some orthologs. Sequence analyses also revealed the existence of CspZ phyletic groups that correlate with FH binding and with the relationships inferred from ribosomal spacer types (RSTs). CspZ type 1 (RST1) and type 3 (RST3) strains bind FH, while CspZ type 2 (RST2) strains do not. Antibody responses to CspZ were also assessed. Anti-CspZ antibodies were detected in mice by week 2 of infection, indicating that there was expression during early-stage infection. Analyses of sera collected from infected mice suggested that CspZ production continued over the course of long-term infection as the antibody titer increased over time. While antibody to CspZ was detected in several human Lyme disease serum samples, the response was not universal, and the titers were generally low. Vaccination studies with mice demonstrated that while CspZ is immunogenic, it does not elicit an antibody that is protective or that inhibits dissemination. The data presented here provide significant new insight into the interaction between CspZ and FH and suggest that there is a correlation between CspZ production and dissemination. However, in spite of its possible contributory role in pathogenesis, the immunological analyses indicated that CspZ is likely to have limited potential as a diagnostic marker and vaccine candidate for Lyme disease.

    PMCID: PMC2747962 PMID: 19620346 [PubMed - indexed for MEDLINE]

  4. Mol Microbiol. 2009 Mar;71(6):1551-73. Epub 2009 Jan 23.

    Rrp1, a cyclic-di-GMP-producing response regulator, is an important regulator of Borrelia burgdorferi core cellular functions.

    Rogers EA, Terekhova D, Zhang HM, Hovis KM, Schwartz I, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia Commonwealth University, Richmond, VA, USA.

    Two-component systems (TCS) are universal among bacteria and play critical roles in gene regulation. Our understanding of the contributions of TCS in the biology of the Borrelia is just now beginning to develop. Borrelia burgdorferi, a causative agent of Lyme disease, harbours a TCS comprised of open reading frames (ORFs) BB0419 and BB0420. BB0419 encodes a response regulator designated Rrp1, and BB0420 encodes a hybrid histidine kinase-response regulator designated Hpk1. Rrp1, which contains a conserved GGDEF domain, undergoes phosphorylation and produces the secondary messenger, cyclic diguanylate (c-di-GMP), a critical signaling molecule in numerous organisms. However, the regulatory role of the Rrp1-Hpk1 TCS and c-di-GMP signaling in Borrelia biology are unexplored. In this study, the distribution, conservation, expression and potential global regulatory capability of Rrp1 were assessed. rrp1 was found to be universal and highly conserved among isolates, co-transcribed with hpk1, constitutively expressed during in vitro cultivation, and significantly upregulated upon tick feeding. Allelic exchange replacement and microarray analyses revealed that the Rrp1 regulon consists of a large number of genes encoded by the core Borrelia genome (linear chromosome, linear plasmid 54 and circular plasmid 26) that encode for proteins involved in central metabolic processes and virulence mechanisms including immune evasion.

    PMCID: PMC2843504 PMID: 19210621 [PubMed - indexed for MEDLINE]

  5. PLoS One. 2008;3(12):e4101. Epub 2008 Dec 31.

    Spent culture medium from virulent Borrelia burgdorferi increases permeability of individually perfused microvessels of rat mesentery.

    Zhou X, Miller MR, Motaleb M, Charon NW, He P.

    Department of Physiology and Pharmacology, West Virginia University, Morgantown, WV, USA.

    BACKGROUND: Lyme disease is a common vector-borne disease caused by the spirochete Borrelia burgdorferi (Bb), which manifests as systemic and targeted tissue inflammation. Both in vitro and in vivo studies have shown that Bb-induced inflammation is primarily host-mediated, via cytokine or chemokine production that promotes leukocyte adhesion/migration. Whether Bb produces mediators that can directly alter the vascular permeability in vivo has not been investigated. The objective of the present study was to investigate if Bb produces a mediator(s) that can directly activate endothelial cells resulting in increases in permeability in intact microvessels in the absence of blood cells. METHODOLOGY/PRINCIPAL FINDINGS: The effects of cell-free, spent culture medium from virulent (B31-A3) and avirulent (B31-A) B. burgdorferi on microvessel permeability and endothelial calcium concentration, [Ca(2+)](i), were examined in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). Endothelial [Ca(2+)](i), a necessary signal initiating hyperpermeability, was measured in Fura-2 loaded microvessels. B31-A3 spent medium caused a rapid and transient increase in Lp and endothelial [Ca(2+)](i). Within 2-5 min, the mean peak Lp increased to 5.6+/-0.9 times the control, and endothelial [Ca(2+)](i) increased from 113+/-11 nM to a mean peak value of 324+/-35 nM. In contrast, neither endothelial [Ca(2+)](i) nor Lp was altered by B31-A spent medium. CONCLUSIONS/SIGNIFICANCE: A mediator(s) produced by virulent Bb under culture conditions directly activates endothelial cells, resulting in increases in microvessel permeability. Most importantly, the production of this mediator is associated with Bb virulence and is likely produced by one or more of the 8 plasmid(s) missing from strain B31-A.

    PMCID: PMC2605548 PMID: 19116656 [PubMed - indexed for MEDLINE]

  6. J Bacteriol. 2009 Jan;191(2):600-7. Epub 2008 Nov 14.

    The flat-ribbon configuration of the periplasmic flagella of Borrelia burgdorferi and its relationship to motility and morphology.

    Charon NW, Goldstein SF, Marko M, Hsieh C, Gebhardt LL, Motaleb MA, Wolgemuth CW, Limberger RJ, Rowe N.

    Department of Microbiology, Immunology and Cell Biology, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia 26506-9177, USA. ncharon@hsc.wvu.edu

    Electron cryotomography was used to analyze the structure of the Lyme disease spirochete, Borrelia burgdorferi. This methodology offers a new means for studying the native architecture of bacteria by eliminating the chemical fixing, dehydration, and staining steps of conventional electron microscopy. Using electron cryotomography, we noted that membrane blebs formed at the ends of the cells. These blebs may be precursors to vesicles that are released from cells grown in vivo and in vitro. We found that the periplasmic space of B. burgdorferi was quite narrow (16.0 nm) compared to those of Escherichia coli and Pseudomonas aeruginosa. However, in the vicinity of the periplasmic flagella, this space was considerably wider (42.3 nm). In contrast to previous results, the periplasmic flagella did not form a bundle but rather formed a tight-fitting ribbon that wraps around the protoplasmic cell cylinder in a right-handed sense. We show how the ribbon configuration of the assembled periplasmic flagella is more advantageous than a bundle for both swimming and forming the flat-wave morphology. Previous results indicate that B. burgdorferi motility is dependent on the rotation of the periplasmic flagella in generating backward-moving waves along the length of the cell. This swimming requires that the rotation of the flagella exerts force on the cell cylinder. Accordingly, a ribbon is more beneficial than a bundle, as this configuration allows each periplasmic flagellum to have direct contact with the cell cylinder in order to exert that force, and it minimizes interference between the rotating filaments.

    PMCID: PMC2620816 PMID: 19011030 [PubMed - indexed for MEDLINE]

  7. Clin Pediatr (Phila). 2008 Oct;47(8):833-5. Epub 2008 Jun 2.

    Lyme arthritis presenting as transient synovitis of the hip.

    Saulsbury FT.

    Department of Pediatrics, Division of Immunology and Rheumatology, University of Virginia Health System, Charlottesville, Virginia 22908, USA. fts@virginia.edu

    Transient synovitis of the hip is a common cause of hip pain in children. The etiology of transient synovitis of the hip is unknown. Lyme arthritis is characterized by brief, often recurrent episodes of oligoarthritis. Lyme arthritis most often affects a single knee, but hip involvement is uncommon. This report describes 2 children with Lyme arthritis who presented with features of transient synovitis of the hip. Lyme arthritis should be considered in the differential diagnosis of transient synovitis of the hip in children.

    PMID: 18519920 [PubMed - indexed for MEDLINE]

  8. J Bacteriol. 2008 Mar;190(6):1912-21. Epub 2008 Jan 11.

    Borrelia burgdorferi uniquely regulates its motility genes and has an intricate flagellar hook-basal body structure.

    Sal MS, Li C, Motalab MA, Shibata S, Aizawa S, Charon NW.

    Department of Microbiology, Immunology, and Cell Biology, West Virginia University, Robert C. Byrd Health Sciences Center, Morgantown, WV 26506-9177, USA.

    Borrelia burgdorferi is a flat-wave, motile spirochete that causes Lyme disease. Motility is provided by periplasmic flagella (PFs) located between the cell cylinder and an outer membrane sheath. The structure of these PFs, which are composed of a basal body, a hook, and a filament, is similar to the structure of flagella of other bacteria. To determine if hook formation influences flagellin gene transcription in B. burgdorferi, we inactivated the hook structural gene flgE by targeted mutagenesis. In many bacteria, completion of the hook structure serves as a checkpoint for transcriptional control of flagellum synthesis and other chemotaxis and motility genes. Specifically, the hook allows secretion of the anti-sigma factor FlgM and concomitant late gene transcription promoted by sigma28. However, the control of B. burgdorferi PF synthesis differs from the control of flagellum synthesis in other bacteria; the gene encoding sigma28 is not present in the genome of B. burgdorferi, nor are any sigma28 promoter recognition sequences associated with the motility genes. We found that B. burgdorferi flgE mutants lacked PFs, were rod shaped, and were nonmotile, which substantiates previous evidence that PFs are involved in both cell morphology and motility. Although most motility and chemotaxis gene products accumulated at wild-type levels in the absence of FlgE, mutant cells had markedly decreased levels of the flagellar filament proteins FlaA and FlaB. Further analyses showed that the reduction in the levels of flagellin proteins in the spirochetes lacking FlgE was mediated at the posttranscriptional level. Taken together, our results indicate that in B. burgdorferi, the completion of the hook does not serve as a checkpoint for transcriptional regulation of flagellum synthesis. In addition, we also present evidence that the hook protein in B. burgdorferi forms a high-molecular-weight complex and that formation of this complex occurs in the periplasmic space.

    PMCID: PMC2258876 PMID: 18192386 [PubMed - indexed for MEDLINE]

  9. Hum Vaccin. 2007 Nov-Dec;3(6):281-9. Epub 2007 Jul 2.

    An octavalent lyme disease vaccine induces antibodies that recognize all incorporated OspC type-specific sequences.

    Earnhart CG, Marconi RT.

    Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia, USA.

    Lyme disease is the most common vector-borne disease in North America and Europe and, if untreated, has significant arthritic, cardiac, dermatological and neurological sequelae. There is no currently available human Lyme disease vaccine. Outer surface protein C, because of its antigenicity, protective ability, and expression characteristics has emerged as a promising second generation vaccine candidate; however, significant sequence heterogeneity has impeded its development. Analyses of OspC sequences have revealed the existence of stable phylogenetic clusters or types, and that the type-defining sequence variation occurs within defined domains of the protein. Recent data indicating that immunodominant, and potentially protective OspC epitopes are located in these hypervariable regions has allowed development of a tetravalent, epitope-based, chimeric vaccine. In this report, we have extended that previously described tetravalent construct to include four additional OspC types. We demonstrate that the construct is highly immunogenic, and elicits type-specific antibodies that recognize each of the eight incorporated OspC type-specific epitopes. Antibody raised to the octavalent construct readily binds to the surface of strains expressing each component OspC type, indicating that the incorporated epitopes are presented on the surface of intact cells. In addition, the construct elicits antibody isotypes associated with complement-dependent bactericidal activity. These results represent an important step forward in the design of a broadly protective polyvalent OspC-based Lyme disease vaccine.

    PMID: 17921702 [PubMed - indexed for MEDLINE]

  10. Infect Immun. 2007 Nov;75(11):5272-81. Epub 2007 Sep 10.

    Delineation of species-specific binding properties of the CspZ protein (BBH06) of Lyme disease spirochetes: evidence for new contributions to the pathogenesis of Borrelia spp.

    Rogers EA, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, Richmond, VA 23298-0678, USA.

    Borrelia burgdorferi CspZ (TIGR open reading frame designation, BBH06) is part of a functionally related group of proteins that bind one or more members of the factor H (FH) protein family. In this report we assess the conservation, distribution, properties, and ligand binding abilities of CspZ from the three main Borrelia species associated with Lyme disease infections in humans. CspZ (also referred to as BbCRASP-2 in the literature) was found to be highly conserved at the intraspecies level but divergent at the interspecies level. All CspZ orthologs that originated from B. burgdorferi isolates bound FH from a diverse group of mammals. In contrast, CspZ derived from B. garinii and B. afzelii did not. Regardless of the Borrelia species of origin, all CspZ proteins tested bound to unknown approximately 60-kDa serum proteins produced by different mammals. To further define the molecular basis for the differential binding of CspZ orthologs to host proteins, DNA sequence, truncation, and site-directed mutagenesis analyses were performed. DNA sequence analyses revealed that B. garinii and B. afzelii CspZ orthologs possess a 64-amino-acid N-terminal domain that is absent from B. burgdorferi CspZ. However, binding analyses of recombinant proteins revealed that this domain does not in and of itself influence ligand binding properties. Truncation and mutagenesis analyses further revealed that the key determinants required for ligand binding are discontinuous and that the presentation of the ligand binding pocket is dependent on alpha helices with high coiled-coil formation probability. The data presented here provide insight into the molecular basis of CspZ-ligand interactions and suggest that CspZ orthologs from diverse Borrelia species can contribute to the host-pathogen interaction through their interaction with serum proteins.

    PMCID: PMC2168308 PMID: 17846117 [PubMed - indexed for MEDLINE]

  11. Methods Enzymol. 2007;422:438-47.

    Phosphorylation assays of chemotaxis two-component system proteins in Borrelia burgdorferi.

    Motaleb MA, Miller MR, Li C, Charon NW.

    Department of Microbiology, West Virginia University, Morgantown, West Virginia, USA.

    Borrelia burgdorferi has a complex chemotaxis signal transduction system with multiple chemotaxis gene homologs similar to those found in Escherichia coli and Bacillus subtilis. The B. burgdorferi genome sequence encodes two cheA, three cheY, three cheW, two cheB, two cheR, but no cheZ genes. Instead of cheZ, B. burgdorferi contains a different CheY-P phosphatase, referred to as cheX. The multiple B. burgdorferi histidine kinases (CheA1 and CheA2) and response regulators (CheY1, CheY2, and CheY3) possess all the domains and functional residues found in E. coli CheA and CheY, respectively. Understanding protein phosphorylation is critical to unraveling many biological processes, including chemotaxis signal transduction, motility, growth control, metabolism, and disease processes. E. coli, Salmonella enterica serovar Typhimurium, and B. subtilis chemotaxis systems have been studied extensively, providing models to understand chemotaxis signaling in the Lyme disease spirochete B. burgdorferi. Both genetic approaches and biochemical analyses are essential in understanding its complex two-component chemotaxis systems. Specifically, gene inactivation studies assess the importance of specific genes in chemotaxis and motility under certain conditions. Furthermore, biochemical approaches help determine the following in vitro reactions: (1) the extent that the histidine kinases, CheA1 and CheA2, are autophosphorylated using ATP; (2) the transfer of phosphate from CheA1-P and CheA2-P to each CheY species; and (3) the dephosphorylation of each CheY-P species by CheX. We hypothesize that characterizing protein phosphorylation in the B. burgdorferi two-component chemotaxis system will facilitate understanding of how the periplasmic flagellar bundles located near each end of B. burgdorferi cells are coordinately regulated for chemotaxis. During chemotaxis, these bacteria run, pause (stop/flex), and reverse (run again). This chapter describes protocols for assessing B. burgdorferi CheA autophosphorylation, transfer of phosphate from CheA-P to CheY, and CheY-P dephosphorylation.

    PMID: 17628153 [PubMed - indexed for MEDLINE]

  12. Methods Enzymol. 2007;422:421-37.

    Isolation and characterization of chemotaxis mutants of the Lyme disease Spirochete Borrelia burgdorferi using allelic exchange mutagenesis, flow cytometry, and cell tracking.

    Motaleb MA, Miller MR, Bakker RG, Li C, Charon NW.

    Department of Microbiology, West Virginia University, Morgantown, West Virginia, USA.

    Constructing mutants by targeted gene inactivation is more difficult in the Lyme disease organism, Borrelia burgdorferi, than in many other species of bacteria. The B. burgdorferi genome is fragmented, with a large linear genome and 21 linear and circular plasmids. Some of these small linear and circular plasmids are often lost during laboratory propagation, and the loss of specific plasmids can have a significant impact on virulence. In addition to the unusual structure of the B. burgdorferi genome, the presence of an active restriction-modification system impedes genetic transformation. Furthermore, B. burgdorferi is relatively slow growing, with a 7- to 12-h generation time, requiring weeks to obtain single colonies. The beginning part of this chapter details the procedure in targeting specific B. burgdorferi genes by allelic exchange mutagenesis. Our laboratory is especially interested in constructing and analyzing B. burgdorferi chemotaxis and motility mutants. Characterization of these mutants with respect to chemotaxis and swimming behavior is more difficult than for many other bacterial species. We have developed swarm plate and modified capillary tube assays for assessing chemotaxis. In the modified capillary tube chemotaxis assay, flow cytometry is used to rapidly enumerate cells that accumulate in the capillary tubes containing attractants. To assess the swimming behavior and velocity of B. burgdorferi wild-type and mutant cells, we use a commercially available cell tracker referred to as "Volocity." The latter part of this chapter presents protocols for performing swarm plate and modified capillary tube assays, as well as cell motion analysis. It should be possible to adapt these procedures to study other spirochete species, as well as other species of bacteria, especially those that have long generation times.

    PMID: 17628152 [PubMed - indexed for MEDLINE]

  13. Clin Vaccine Immunol. 2007 May;14(5):628-34. Epub 2007 Mar 14.

    OspC phylogenetic analyses support the feasibility of a broadly protective polyvalent chimeric Lyme disease vaccine.

    Earnhart CG, Marconi RT.

    Department of Microbiology and Immunology, Center for the Study of Biological Complexity, Medical College of Virginia at Virginia Commonwealth University, Richmond, VA 23298-0678, USA.

    Using available Borrelia outer surface protein C (OspC) sequences, a phylogenetic analysis was undertaken to delineate the number of antigenic domains required for inclusion in a broadly protective, chimeric, OspC-based Lyme disease vaccine. The data indicate that approximately 34 would be required and that an OspC-based vaccinogen is feasible.

    PMCID: PMC1865620 PMID: 17360854 [PubMed - indexed for MEDLINE]

  14. Vaccine. 2007 Apr 30;25(17):3419-27. Epub 2007 Jan 17.

    Construction and analysis of variants of a polyvalent Lyme disease vaccine: approaches for improving the immune response to chimeric vaccinogens.

    Earnhart CG, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, Richmond, VA 23298-0678, USA.

    There is currently no Lyme disease vaccine commercially available for use in humans. Outer surface protein C (OspC) of the Borrelia has been widely investigated as a potential vaccinogen. At least 38 OspC types have been defined. While the antibody response to OspC is protective, the range of protection is narrow due to the localization of protective epitopes within OspC type-specific domains. To develop a broadly protective vaccine, we previously constructed a tetravalent chimeric vaccinogen containing epitopes from OspC types A, B, K, and D. While this construct elicited bactericidal antibody against strains bearing each of the four OspC types, its solubility was low, and decreasing IgG titer to epitopes near the C-terminus of the construct was observed. In this report, construct solubility and immunogenicity were increased by dialysis against an Arg/Glu buffer. We also demonstrate the immunogenicity of the construct in alum. To further optimize epitope-specific immune responses, several constructs were generated with differing epitope organization or with putative C-terminal protective motifs. Analyses of murine antibody titers and isotype profiles induced by these constructs revealed that while the C-terminal tags did not enhance antibody titer, specific epitope reorganization and reiteration did. These analyses provide important information that can be exploited in the development of chimeric vaccinogens in general.

    PMCID: PMC2696934 PMID: 17239505 [PubMed - indexed for MEDLINE]

  15. Appl Environ Microbiol. 2007 Feb;73(4):1180-8. Epub 2006 Dec 15.

    Identification of specific chemoattractants and genetic complementation of a Borrelia burgdorferi chemotaxis mutant: flow cytometry-based capillary tube chemotaxis assay.

    Bakker RG, Li C, Miller MR, Cunningham C, Charon NW.

    Department of Microbiology, Immunology and Cell Biology, West Virginia University, Morgantown, WV 26506-9177, USA.

    Measuring the chemotactic response of Borrelia burgdorferi, the bacterial species that causes Lyme disease, is relatively more difficult than measuring that of other bacteria. Because these spirochetes have long generation times, enumerating cells that swim up a capillary tube containing an attractant by using colony counts is impractical. Furthermore, direct counts with a Petroff-Hausser chamber is problematic, as this method has a low throughput and necessitates a high cell density; the latter can lead to misinterpretation of results when assaying for specific attractants. Only rabbit serum and tick saliva have been reported to be chemoattractants for B. burgdorferi. These complex biological mixtures are limited in their utility for studying chemotaxis on a molecular level. Here we present a modified capillary tube chemotaxis assay for B. burgdorferi that enumerates cells by flow cytometry. Initial studies identified N-acetylglucosamine as a chemoattractant. The assay was then optimized with respect to cell concentration, incubation time, motility buffer composition, and growth phase. Besides N-acetylglucosamine, glucosamine, glucosamine dimers (chitosan), glutamate, and glucose also elicited significant chemoattractant responses, although the response obtained with glucose was weak and variable. Serine and glycine were nonchemotactic. To further validate and to exploit the use of this assay, a previously described nonchemotactic cheA2 mutant was shown to be nonchemotactic by this assay; it also regained the wild-type phenotype when complemented in trans. This is the first report that identifies specific chemical attractants for B. burgdorferi and the use of flow cytometry for spirochete enumeration. The method should also be useful for assaying chemotaxis for other slow-growing prokaryotic species and in specific environments in nature.

    PMCID: PMC1828676 PMID: 17172459 [PubMed - indexed for MEDLINE]

  16. Clin Vaccine Immunol. 2006 Oct;13(10):1162-5.

    Analysis of antibody response in humans to the type A OspC loop 5 domain and assessment of the potential utility of the loop 5 epitope in Lyme disease vaccine development.

    Buckles EL, Earnhart CG, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, 1112 E. Clay St., McGuire Hall, Richmond, VA 23298-0678, USA.

    The OspC protein of Borrelia burgdorferi is an immunodominant antigen. Here we demonstrate that the loop 5 domain of type A OspC is surface exposed, elicits bactericidal antibody in mice, and is antigenic in humans. The data suggest that loop 5 may be suitable for inclusion in a polyvalent, chimeric OspC vaccinogen.

    PMCID: PMC1595320 PMID: 17028218 [PubMed - indexed for MEDLINE]

  17. Vaccine. 2007 Jan 5;25(3):466-80. Epub 2006 Aug 8.

    Development of an OspC-based tetravalent, recombinant, chimeric vaccinogen that elicits bactericidal antibody against diverse Lyme disease spirochete strains.

    Earnhart CG, Buckles EL, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, Richmond, VA 23298-0678, USA.

    Lyme disease is the most common arthropod-borne disease in North America and Europe. At present, there is no commercially available vaccine for use in humans. Outer surface protein C (OspC) has antigenic and expression characteristics that make it an attractive vaccine candidate; however, sequence heterogeneity has impeded its use as a vaccinogen. Sequence analyses have identified 21 well defined OspC phyletic groups or "types" (designated A-U). In this report we have mapped the linear epitopes presented by OspC types B, K, and D during human and murine infection and exploited these epitopes (along with the previously identified type A OspC linear epitopes) in the development of a recombinant, tetravalent, chimeric vaccinogen. The construct was found to be highly immunogenic in mice and the induced antibodies surface labeled in vitro cultivated spirochetes. Importantly, vaccination induced complement-dependent bactericidal antibodies against strains expressing each of the OspC types that were incorporated into the construct. These results suggest that an effective and broadly protective polyvalent OspC-based Lyme disease vaccine can be produced as a recombinant, chimeric protein.

    PMID: 16996663 [PubMed - indexed for MEDLINE]

  18. Infect Immun. 2006 May;74(5):3030-4.

    Evidence that the BBA68 protein (BbCRASP-1) of the Lyme disease spirochetes does not contribute to factor H-mediated immune evasion in humans and other animals.

    McDowell JV, Hovis KM, Zhang H, Tran E, Lankford J, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, Richmond, VA 23298-0678, USA.

    BBA68 (BbCRASP-1) of the Lyme disease spirochetes binds human factor H (FH) and FH-like protein 1 (FHL-1). Here we assess transcription of the BBA68 gene and production of BBA68 in infected mice and humans using real-time reverse transcriptase PCR and immunoblotting. The species specificity of FH binding to BBA68 was also tested. The data suggest that BBA68 does not play an important role in immune evasion in animals.

    PMCID: PMC1459725 PMID: 16622245 [PubMed - indexed for MEDLINE]

  19. Infect Immun. 2006 Mar;74(3):1967-72.

    Selective binding of Borrelia burgdorferi OspE paralogs to factor H and serum proteins from diverse animals: possible expansion of the role of OspE in Lyme disease pathogenesis.

    Hovis KM, Tran E, Sundy CM, Buckles E, McDowell JV, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, 1112 E. Clay St., Richmond, VA 23298-0678, USA.

    The binding of Borrelia burgdorferi OspE, OspF, and family 163 (Elp) proteins to factor H/factor H-like protein 1 (FHL-1) and other serum proteins from different animals was assessed. OspE paralogs bound factor H and unidentified serum proteins from a subset of animals, while OspF and Elp proteins did not. These data advance our understanding of factor H binding, the host range of the Lyme spirochetes, and the expanding role of OspE in pathogenesis.

    PMCID: PMC1418677 PMID: 16495576 [PubMed - indexed for MEDLINE]

  20. Vector Borne Zoonotic Dis. 2005 Winter;5(4):383-9.

    Distribution of borreliae among ticks collected from eastern states.

    Taft SC, Miller MK, Wright SM.

    Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, Ohio, USA.

    Lyme disease is the most commonly reported vector-borne disease in the United States and is transmitted by Borrelia burgdorferi-infected Ixodes species. The disease is typically characterized by an erythema migrans (EM) rash at the site of tick feeding. EM rashes have also been associated with feeding by Amblyomma americanum ticks despite evidence suggesting that they are incompetent vectors for Lyme disease. In 1996, a Borrelia organism only recently cultivated in the laboratory was described in A. americanum ticks and designated B. lonestari. This Borrelia is believed to be the etiologic agent of a novel Lyme-like disease, southern tick associated rash illness (STARI). This study examined ticks collected from eight eastern states to evaluate the epidemiology of B. lonestari, B. burgdorferi, and their tick hosts. Three hundred individual or small pool samples were evaluated from tick genera that included Amblyomma, Ixodes, and Dermacentor. DNA was extracted following tick homogenization and the polymerase chain reaction (PCR) was performed using primers derived from the flagellin gene that amplify sequences from both B. burgdorferi and B. lonestari. Species specific digoxigenin labeled probes were designed and used to differentiate between B. burgdorferi and B. lonestari. Borrelia DNA was detected in approximately 10% of the A. americanum and I. scapularis tick samples, but none was present in any of the Dermacentor samples tested. Positive samples were detected in ticks collected from Kentucky, Maryland, Massachusetts, New Jersey, New York, and Virginia. This is the first known report of B. lonestari from Massachusetts and New York and the first detection in I. scapularis. This suggests that B. lonestari and its putative association with STARI may be more widespread than previously thought.

    PMID: 16417434 [PubMed - indexed for MEDLINE]

  21. Infect Immun. 2005 Dec;73(12):7869-77.

    Demonstration of OspC type diversity in invasive human lyme disease isolates and identification of previously uncharacterized epitopes that define the specificity of the OspC murine antibody response.

    Earnhart CG, Buckles EL, Dumler JS, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, Richmond, VA 23298-0678, USA.

    Outer surface protein C (OspC) of the Lyme disease spirochetes is an important virulence factor that has potential utility for vaccine development. Of the 21 OspC types that have been identified, it has been postulated that types A, B, I, and K are specifically associated with invasive infections. Through an analysis of isolates collected from patients in Maryland we found that OspC types C, D, and N are also associated with invasive infections. This observation suggests that there is greater diversity in the group of OspC types associated with invasive infection than has been previously suggested. Detailed knowledge of the antigenic structure of OspC is essential for vaccine development. To determine if the antibody response to OspC is type specific, recombinant proteins of several different OspC types were immunoblotted and screened with sera from mice infected with isolates having known OspC types. These analyses revealed a high degree of specificity in the antibody response and suggested that the immunodominant epitopes of OspC reside in the variable domains of the protein. To localize these epitopes, OspC fragments were generated and screened with serum collected from infected mice. These analyses led to identification of previously uncharacterized epitopes that define the type specificity of the OspC antibody response. These analyses provide important insight into the antigenic structure of OspC and also provide a basis for understanding the variable nature of the antibody response to this important virulence factor of the Lyme disease spirochetes.

    PMCID: PMC1307023 PMID: 16299277 [PubMed - indexed for MEDLINE]

  22. J Bacteriol. 2005 Dec;187(23):7963-9.

    CheX is a phosphorylated CheY phosphatase essential for Borrelia burgdorferi chemotaxis.

    Motaleb MA, Miller MR, Li C, Bakker RG, Goldstein SF, Silversmith RE, Bourret RB, Charon NW.

    Department of Microbiology, Immunology, and Cell Biology, Health Sciences Center, West Virginia University, Morgantown, 26506-9177, USA.

    Motility and chemotaxis are believed to be important in the pathogenesis of Lyme disease caused by the spirochete Borrelia burgdorferi. Controlling the phosphorylation state of CheY, a response regulator protein, is essential for regulating bacterial chemotaxis and motility. Rapid dephosphorylation of phosphorylated CheY (CheY-P) is crucial for cells to respond to environmental changes. CheY-P dephosphorylation is accomplished by one or more phosphatases in different species, including CheZ, CheC, CheX, FliY, and/or FliY/N. Only a cheX phosphatase homolog has been identified in the B. burgdorferi genome. However, a role for cheX in chemotaxis has not been established in any bacterial species. Inactivating B. burgdorferi cheX by inserting a flgB-kan cassette resulted in cells (cheX mutant cells) with a distinct motility phenotype. While wild-type cells ran, paused (stopped or flexed), and reversed, the cheX mutant cells continuously flexed and were not able to run or reverse. Furthermore, swarm plate and capillary tube chemotaxis assays demonstrated that cheX mutant cells were deficient in chemotaxis. Wild-type chemotaxis and motility were restored when cheX mutant cells were complemented with a shuttle vector expressing CheX. Furthermore, CheX dephosphorylated CheY3-P in vitro and eluted as a homodimer in gel filtration chromatography. These findings demonstrated that B. burgdorferi CheX is a CheY-P phosphatase that is essential for chemotaxis and motility, which is consistent with CheX being the only CheY-P phosphatase in the B. burgdorferi chemotaxis signal transduction pathway.

    PMCID: PMC1291287 PMID: 16291669 [PubMed - indexed for MEDLINE]

  23. W V Med J. 2005 May-Jun;101(3):126-30.

    Tick exposure and Lyme disease at a summer camp in Maryland.

    Sarwari AR, Strickland T, Pena C, Burkot TR.

    Dept. of Medicine, West Virginia University School of Medicine, Morgantown, USA.

    After investigating an outbreak of Lyme Disease among counselors at a summer camp in Kent County, Maryland in 1994, we wanted to determine the incidence of Lyme Disease (LD) at the camp the following summer and identify risk factors for tick exposure. Any ticks that were detected on campers' skin or clothing were collected by the camp nurse and we studied them for infection with Borrelia burgdorferi. In addition, we sent detailed questionnaires home with the 1,623 campers. A total of 537 campers returned the questionnaire and 200 had found ticks on their skin or clothing while at camp. Risks were analyzed using logistic regression models. Participation in the ropes-course, night-trip and camp-out events significantly increased the risk of tick exposure (OR 1.6 to 2.3). Six cases of LD were identified among campers, which was an estimated incidence of 3.3 per 1,000 campers/10-14 day camp session, and two counselors had LD. Of the 238 ticks collected mainly in June-July 1995, 19% were identified as Ixodes scapularis larvae and nymphs; 11% of the latter were infected with B. burgdorferi. The risk for LD in campers and staff was much higher than that of the general population despite the use of tick-exposure precautions. Focused interventions need to be put in place in summer camps to prevent transmission of LD.

    PMID: 16161531 [PubMed - indexed for MEDLINE]

  24. Vector Borne Zoonotic Dis. 2005 Summer;5(2):101-9.

    The dog as a sentinel for human infection: prevalence of Borrelia burgdorferi C6 antibodies in dogs from southeastern and mid-Atlantic States.

    Duncan AW, Correa MT, Levine JF, Breitschwerdt EB.

    Department of Clinical Sciences, North Carolina State University College of Veterinary Medicine (NCSU-CVM), Raleigh, North Carolina 27606, USA.

    Lyme disease is the most frequently reported human vector-associated disease in the United States. Infection occurs after the bite of an Ixodid tick that is infected with Borrelia burgdorferi. Dogs have often been reported to serve as effective sentinel animals to assess the risk of human B. burgdorferi infection. Based on published data of human Lyme disease case numbers and our clinical impressions, we hypothesized that canine exposure to B. burgdorferi would be lower in North Carolina when compared to the exposure in Virginia, Maryland, and Pennsylvania. To address this hypothesis, we evaluated B. burgdorferi exposure status utilizing a specific and sensitive C6 peptide-based enzyme-linked immunosorbent assay. Our convenience sample included 1,666 canine serum samples submitted to the Vector-Borne Disease Diagnostic Laboratory from North Carolina (n = 987), Virginia (n = 472), Maryland (n = 167), and Pennsylvania (n = 40). Comparisons among states were made using the Chisquare test or the Fisher's exact test; p-values were adjusted for multiple comparisons using the Bonferroni correction. A Chi-square test for trend was used to determine if there was an increase in the frequency of seroreactors associated with the geographical origin of the samples. The proportion of seroreactive dogs in North Carolina was markedly lower (p < 0.008) than that observed in dogs from Virginia, Maryland, and Pennsylvania. These results support the hypothesis that B. burgdorferi transmission seems to occur infrequently in North Carolina dogs as compared to dogs residing in other southeastern and mid-Atlantic states. Furthermore, they support the utility of dogs as a sentinel to characterize the risk of B. burgdorferi transmission to humans in a defined geographical location.

    PMID: 16011425 [PubMed - indexed for MEDLINE]

  25. Clin Pediatr (Phila). 2005 Jun;44(5):419-21.

    Lyme arthritis in 20 children residing in a non-endemic area.

    Saulsbury FT.

    Division of Immunology and Rheumatology, Department of Pediatrics, University of Virginia Health System, Charlottesville, Virginia 22908, USA.

    In non-endemic areas of the country, Lyme disease may not be considered in children who present with arthritis. This report details the clinical features of Lyme arthritis in 20 children residing in central Virginia. All patients presented with transient, often recurrent oligoarthritis of large joints, particularly the knee. Most patients were referred with a presumptive diagnosis of juvenile rheumatoid arthritis (JRA). This report reiterates the clinical presentation of Lyme arthritis in children and reminds physicians to consider the diagnosis of Lyme arthritis in children who present with acute arthritis even if they reside in a non-endemic area of the country. In addition, it differentiates the clinical presentation of Lyme arthritis from JRA.

    PMID: 15965548 [PubMed - indexed for MEDLINE]

  26. J Bacteriol. 2005 Feb;187(4):1317-23.

    Putative coiled-coil structural elements of the BBA68 protein of Lyme disease spirochetes are required for formation of its factor H binding site.

    McDowell JV, Harlin ME, Rogers EA, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia Commonwealth University, Richmond, VA 23298, USA.

    Factor H and factor H like-protein 1 (FHL-1) are complement regulatory proteins that serve as cofactors for the factor I-mediated cleavage of C3b. Some Lyme disease and relapsing fever spirochete species bind factor H to their surface to facilitate immune evasion. The Lyme disease spirochetes produce several factor H binding proteins (FHBPs) that form two distinct classes. Class I FHBPs (OspE orthologs and paralogs) bind only factor H, while class II FHBPs (BBA68) bind both factor H and FHL-1. BBA68 belongs to a large paralogous protein family, and of these paralogs, BBA69 is the member most closely related to BBA68. To determine if BBA69 can also bind factor H, recombinant protein was generated and tested for factor H binding. BBA69 did not exhibit factor H binding ability, suggesting that among family 54 paralogs, factor H binding is unique to BBA68. To identify the determinants of BBA68 that are involved in factor H binding, truncation and site-directed mutational analyses were performed. These analyses revealed that the factor H binding site is discontinuous and provide strong evidence that coiled-coil structural elements are involved in the formation of the binding site.

    PMCID: PMC545637 PMID: 15687195 [PubMed - indexed for MEDLINE]

  27. Vector Borne Zoonotic Dis. 2004 Fall;4(3):221-9.

    The dog as a sentinel for human infection: prevalence of Borrelia burgdorferi C6 antibodies in dogs from southeastern and mid-Atlantic states.

    Duncan AW, Correa MT, Levine JF, Breitschwerdt EB.

    Department of Clinical Sciences, North Carolina State University, College of Veterinary Medicine, Raleigh, North Carolina 27606, USA.

    Lyme disease is the most frequently reported human vector-associated disease in the United States. Infection occurs after the bite of an Ixodid tick that is infected with Borrelia burgdorferi. Dogs have often been reported to serve as effective sentinel animals to assess the risk of human B. burgdorferi infection. Based on published data of human Lyme disease case numbers and our clinical impressions, we hypothesized that canine exposure to B. burgdorferi would be lower in North Carolina when compared to the exposure in Virginia, Maryland, and Pennsylvania. To address this hypothesis, we evaluated B. burgdorferi exposure status utilizing a specific and sensitive C6 peptide-based enzyme-linked immunosorbent assay. Our convenience sample included 1,666 canine serum samples submitted to the Vector Borne Disease Diagnostic Laboratory from North Carolina (n = 987), Virginia (n = 472), Maryland (n = 167), and Pennsylvania (n = 40). Comparisons among states were made using the Chi-square test or the Fisher's exact test; p-values were adjusted for multiple comparisons using the Bonferroni correction. A Chi-square test for trend was used to determine if there was an increase in the frequency of seroreactors associated with the geographical origin of the samples. The proportion of seroreactive dogs in North Carolina was markedly lower (p < 0.008) than that observed in dogs from Virginia, Maryland, and Pennsylvania. These results support the hypothesis that B. burgdorferi transmission seems to occur infrequently in North Carolina dogs as compared to dogs residing in other southeastern and mid-Atlantic states. Furthermore, they support the utility of dogs as a sentinel to characterize the risk of B. burgdorferi transmission to humans in a defined geographical location.

    PMID: 15631067 [PubMed - indexed for MEDLINE]

  28. J Bacteriol. 2005 Jan;187(1):175-84.

    bdrF2 of Lyme disease spirochetes is coexpressed with a series of cytoplasmic proteins and is produced specifically during early infection.

    Zhang H, Raji A, Theisen M, Hansen PR, Marconi RT.

    Department of Microbiology and Immunology, Center for the Study of Biological Complexity, Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia 23298-0678, USA.

    The Bdr proteins are polymorphic inner membrane proteins produced by most Borrelia species. In Borrelia burgdorferi B31MI, the18 bdr genes form three subfamilies, bdrD, bdrE, and bdrF. The production of at least one of the Bdr paralogs, BdrF2, is up-regulated in host-adapted spirochetes, suggesting a role for the protein in the mammalian environment. Here, we demonstrate using reverse transcriptase (RT) PCR that BBG29, BBG30, BBG31, and BBG32, which reside upstream of bdrF2, are cotranscribed with bdrF2 as a five-gene operon. While the functions of most of these proteins are unknown, BBG32 encodes a putative DNA helicase. Real-time RT-PCR analyses demonstrated higher levels of bdrF2 transcript relative to other genes of the operon, suggesting that bdrF2 may also be transcribed independently from an internal promoter. Internal promoters were detected using the 5' rapid amplification of cDNA ends system. The putative promoter associated with bdrF2 was found to be highly similar in sequence to the multiple promoters associated with the ospC gene. Real-time RT-PCR analyses, performed to assess the expression of these genes in infected mice, revealed that genes of the bdrF2 locus are expressed only during early infection, suggesting a role in the establishment of infection. To further characterize the proteins encoded by the bdrF2 locus, which have unknown functions, the cellular localizations of these proteins were determined by Triton X-114 extraction and phase partitioning. BBG29 and BBG31 were found to be cytoplasmic. To determine if these proteins elicit an antibody (Ab) response during infection, immunoblot analyses were performed. Abs to these proteins were not detected. Based on the analyses presented here, we offer the hypothesis that BdrF2 and other proteins encoded by the operon form an inner-membrane-associated protein complex that may interact with DNA and which carries out its functional role during transmission or the early stages of infection.

    PMCID: PMC538826 PMID: 15601701 [PubMed - indexed for MEDLINE]

  29. J Immunol. 2004 Dec 15;173(12):7471-80.

    Demonstration of the involvement of outer surface protein E coiled coil structural domains and higher order structural elements in the binding of infection-induced antibody and the complement-regulatory protein, factor H.

    McDowell JV, Wolfgang J, Senty L, Sundy CM, Noto MJ, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, Richmond, VA 23298, USA.

    Factor H (fH) is an important regulator of the alternative complement cascade. Several human pathogens have been shown to bind fH to their surface, a process that facilitates immune evasion or cell to cell interaction. Among the pathogens that bind fH are some Borrelia species associated with Lyme disease and relapsing fever. The fH-binding proteins of the Lyme spirochetes form two classes (I and II). In Borrelia burgdorferi B31MI, class I includes the outer surface protein E (OspE) paralogs, L39, N38, and P38, whereas the class II group includes A68 and additional proteins that have not yet been identified. To identify the OspE determinants involved in fH and OspE-targeting infection-induced Ab (iAb) binding, deletion, random, and site-directed mutagenesis of L39 were performed. Mutations in several different regions of L39 abolished fH and or iAb binding, indicating that separable domains and residues of OspE are required for ligand binding. Some of the mutants that lost the ability to bind fH, iAb, or both had only a single amino acid change. Site-directed mutagenesis of three putative coiled coil motifs of OspE revealed that these higher order structures are required for fH binding but not for iAb binding. The data presented within demonstrate that the binding of fH and iAb to the OspE protein is mediated by higher order structures and protein conformation. These studies advance our understanding of fH binding as a virulence mechanism and facilitate ongoing efforts to use fH-binding proteins in the development of microbial vaccines.

    PMID: 15585873 [PubMed - indexed for MEDLINE]

  30. J Bacteriol. 2004 Jun;186(12):3703-11.

    The decrease in FlaA observed in a flaB mutant of Borrelia burgdorferi occurs posttranscriptionally.

    Motaleb MA, Sal MS, Charon NW.

    Department of Microbiology, Immunology, and Cell Biology, West Virginia University, Box 9177, Robert C. Byrd Health Sciences Center, Morgantown, WV 26506-9177, USA. ncharon@hsc.wvu.edu

    The Lyme disease bacterium Borrelia burgdorferi is a motile spirochete with a flat-wave morphology. The periplasmic flagella, which are situated between the outer membrane sheath and cell cylinder, are essential for both the cell's wavy shape and motility. Here we focus on the structure and regulation of its periplasmic flagella. Previous studies have suggested that the periplasmic flagella consist of a polymer of the major filament protein FlaB and a minor protein, FlaA. We used immunoprecipitation methodology to present further evidence that FlaA is indeed a flagellar protein. In addition, in contrast to FlaA of the spirochete Brachyspira hyodysenteriae, B. burgdorferi FlaA did not impact the overall helical shape of the periplasmic flagella. We have previously shown that B. burgdorferi lacks the sigma factor-dependent cascade control of motility gene transcription found in other bacteria. To begin to understand motility gene regulation in B. burgdorferi, we examined the effects of an insertion mutation in flaB on the amounts of proteins encoded by motility genes. Of several motility gene-encoded proteins examined, only the amount of FlaA was decreased in the flaB mutant; it was 13% compared to the wild-type amount. Real-time reverse transcriptase PCR analysis indicated that this inhibition was not the result of a decrease in flaA mRNA. In addition, protein stability analysis suggested that FlaA was turned over in the flaB mutant. Our results indicate that the lack of FlaB negatively influences the amount of FlaA found in the cell and that this effect is at the level of either translational control or protein turnover.

    PMCID: PMC419964 PMID: 15175283 [PubMed - indexed for MEDLINE]

  31. Mil Med. 2003 Dec;168(12):1011-4.

    Lyme disease reporting for Navy and Marine Corps (1997-2000).

    McGinnis J, Bohnker BK, Malakooti M, Mann M, Sack DM.

    Preventive Medicine Directorate, Navy Environmental Health Center, 620 John Paul Jones Circle, Portsmouth, VA 23708, USA.

    Reported cases of Lyme disease for Navy and Marine Corps personnel during 1997-2000 are presented from data collected in the Naval Disease Reporting System and the Defense Medical Epidemiological Database. Naval Disease Reporting System identified 210 case subjects; 60% were men, 49% were family members, and 37% were active duty, and most originated in the second quarter of the calendar year. States reporting the greatest number of reports were Connecticut (44%), North Carolina (16%), Rhode Island (10%), and Virginia (10%), which was generally consistent with national figures and the concentration of military populations. Incidence rates from Defense Medical Epidemiological Database for Lyme disease were generally higher for active duty personnel than reported civilian rates. Areas for improvement for Naval Disease Reporting System are identified and include additional emphasis on complete reporting on patient history and on Lyme disease antibody testing results. These findings suggest that Lyme disease is an important disease in military medicine, particularly in the eastern United States.

    PMID: 14719627 [PubMed - indexed for MEDLINE]

    32.
  32. Pharmacy and immunization services: pharmacists' participation and impact.

    Kamal KM, Madhavan SS, Maine LL.

    Department of Pharmaceutical Systems and Policy, School of Pharmacy, West Virginia University, Morgantown 26506-9510, USA.

    OBJECTIVES: To conduct a follow-up to the National Pharmacist Immunization Survey of 1998 to determine changes in pharmacist involvement in immunizations and obstacles to pharmacy-based immunization services and to assess the descriptive information about pharmacy-based immunization services provided. DESIGN: Cross-sectional mail survey. SETTING: United States. PARTICIPANTS: A randomly selected national sample of 6,000 pharmacists. INTERVENTIONS: None. MAIN OUTCOME MEASURES: An updated version of the 1998 study questionnaire was used to collect data about pharmacists' current involvement in adult or childhood immunizations, perceived obstacles to such involvement, and characteristics of pharmacist-administered immunization services. RESULTS: Four mailings in fall 2001 yielded a response rate of 21.2% (1,266 completed, usable surveys out of 5,958 deliverable surveys). Immunization activities that reportedly increased during this period, compared with results from the 1998 survey, include counseling about adult immunizations (increase from 11.9% to 14.7%), nurse-administered childhood immunizations (6.3% to 7.8%), nurse-administered adult immunizations (16.2% to 30.2%), pharmacist-administered childhood immunizations (0.9% to 1.3%), pharmacist-administered adult immunizations (2.2% to 6.8%), and immunization promotion (18.9% to 27.3%). Only counseling for childhood immunizations appears to have decreased slightly, from 13.4% to 8.9%. Willingness to provide all of the above immunization services also increased during the 1998-2001 period. In addition to flu shots and pneumococcal vaccines, pharmacists were administering vaccines for hepatitis A and B, Lyme disease, tetanus, and chicken pox, but flu shots accounted for the majority of immunizations being administered. CONCLUSION: Pharmacist involvement in childhood and adult immunizations has increased significantly in the last few years. Pharmacists perceived obstacles to their involvement in immunizations as less problematic.

    PMID: 12952311 [PubMed - indexed for MEDLINE]

  33. J Clin Microbiol. 2003 Aug;41(8):3905-10.

    Analysis of the ability of spirochete species associated with relapsing fever, avian borreliosis, and epizootic bovine abortion to bind factor H and cleave c3b.

    McDowell JV, Tran E, Hamilton D, Wolfgang J, Miller K, Marconi RT.

    Department of Microbiology and Immunology, Center for the Study of Biological Complexity, Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia 23298, USA.

    Some Borrelia species associated with Lyme disease bind the complement-regulatory protein factor H (fH), a process that may aid in immune evasion. In this report we demonstrate that some Borrelia species associated with relapsing fever bind fH, but not those associated with avian borreliosis and epizootic bovine abortion. Cell-bound fH was also found to mediate cleavage of exogenously supplied human C3b, demonstrating the biological relevance of fH binding and its possible importance in the pathogenesis of the relapsing-fever spirochetes.

    PMCID: PMC179811 PMID: 12904415 [PubMed - indexed for MEDLINE]

  34. Infect Immun. 2003 Jun;71(6):3597-602.

    Comprehensive analysis of the factor h binding capabilities of borrelia species associated with lyme disease: delineation of two distinct classes of factor h binding proteins.

    McDowell JV, Wolfgang J, Tran E, Metts MS, Hamilton D, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, Richmond 23298-0678, USA.

    Some Lyme disease spirochete isolates can bind complement regulatory protein factor H (fH), a process that may allow evasion of complement-mediated killing. Here we demonstrate significant differences in the fH binding capabilities of species of the Borrelia burgdorferi sensu lato complex. The percentages of B. burgdorferi, B. afzelii, and B. garinii bacteria that bound fH in either enzyme-linked immunosorbent assays or affinity ligand binding immunoblot assays were 100, 83, and 29%, respectively. The fH binding protein profiles were examined and found to exhibit variability among isolates and to form two distinct classes. Differences in fH binding ability may contribute to the differences in pathogenesis and clinical course observed upon infection with different species of the B. burgdorferi sensu lato complex.

    PMCID: PMC155754 PMID: 12761145 [PubMed - indexed for MEDLINE]

  35. Infect Immun. 2003 Jun;71(6):3587-96.

    Analysis of the OspE determinants involved in binding of factor H and OspE-targeting antibodies elicited during Borrelia burgdorferi infection in mice.

    Metts MS, McDowell JV, Theisen M, Hansen PR, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, Richmond 23298-0678, USA.

    Immune evasion by Lyme spirochetes is a multifactorial process involving numerous mechanisms. The OspE protein family undergoes antigenic variation during infection and binds factor H (fH) and possibly FHL-1/reconectin. In Borrelia burgdorferi B31MI, the OspE family consists of three paralogs: BBL39 (ErpA), BBP38, and BBN38 (ErpP). BBL39 and BBP38 are identical and therefore are referred to here as BBL39. The goals of this study were to assess the specificity of the antibody (Ab) response to the OspE paralogs and to identify the domains or determinants of OspE that are required for the binding of fH and OspE-targeting Abs that develop during infection. Here we demonstrate that at least some of the anti-OspE Abs produced during infection are paralog specific and that Ab binding requires conformational determinants whose formation requires both the N- and C-terminal domains of OspE. The binding of fH to OspE was also found to be dependent on conformational determinants. It is also demonstrated here that all of the OspE paralogs expressed by B. burgdorferi B31MI are capable of binding fH. The binding of fH to members of the OspF protein family was also assessed. In contrast to an earlier report, no binding of BBO39 or BBR42 to human fH was detected. Lastly, a series of competitive binding enzyme-linked immunosorbent assay analyses, designed to determine if fH and infection serum Abs bind to the same sites on OspE, revealed that these ligands interact with different regions of OspE.

    PMCID: PMC155734 PMID: 12761144 [PubMed - indexed for MEDLINE]

  36. Infect Immun. 2002 Dec;70(12):7033-41.

    Environmental regulation and differential production of members of the Bdr protein family of Borrelia burgdorferi.

    Roberts DM, Caimano M, McDowell J, Theisen M, Holm A, Orff E, Nelson D, Wikel S, Radolf J, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, Richmond 23298-0678, USA.

    Borrelia burgdorferi B31MI carries 18 plasmid-carried genes that form the bdr gene family. The bdr genes of B. burgdorferi encode proteins that form three distinct subfamilies, the BdrD, BdrE, and BdrF subfamilies. bdr orthologs have been demonstrated to be carried by all Borrelia species analyzed, and their widespread distribution suggests that they play an important genus-wide functional role. The biological rationale for maintaining 18 bdr alleles has not been defined. It is our hypothesis that specific paralogs function in different environments and are differentially expressed in response to environmental conditions. As a first step in testing this hypothesis, the production patterns of the Bdr proteins in spirochetes grown under a variety of conditions were assessed through immunoblot analyses. The influence of temperature, serum deprivation, tick feeding, and the mammalian environment on Bdr production was evaluated. These analyses revealed that the synthesis of some Bdr paralogs is environmentally regulated. The production of BdrF(2,) BdrF(1), BdrE(4), and BdrE(5) were upregulated in host-adapted bacteria, while the production levels of other Bdr paralogs were influenced by temperature and serum starvation. These observations suggest that different Bdr paralogs function in different biological environments and provide insight into the biological basis for maintaining multiple members of this gene family.

    PMCID: PMC132981 PMID: 12438383 [PubMed - indexed for MEDLINE]

  37. Adolesc Med. 2002 Oct;13(3):663-81.

    Neuropsychological sequelae of adolescent infectious diseases.

    Obrecht RE, Patrick PD.

    Department of Psychiatric Medicine, University of Virginia Health System, Charlottesville, Virginia 22908-0203, USA.

    This article discusses the neuropsychological sequelae of adolescent infectious diseases. Primary care physicians are encouraged to extend their clinical activities beyond the primary medical care aspects of the infectious disease process to encompass a comprehensive, multidisciplinary, continuum of health care approach. Patient, disease, and socioecologic parameters are the foundation of this approach. This article is designed to help primary care physicians appreciate the complexity of neuropsychological infectious disease issues in the adolescent. Human immunodeficiency virus 1 (HIV-1) is emphasized because the legion of related sequelae demands a comprehensive health care approach and serves as a model for discussing other principal infectious diseases such as encephalitis (particularly Lyme disease) and bacterial meningitis.

    PMID: 12270806 [PubMed - indexed for MEDLINE]

  38. Infect Immun. 2002 Aug;70(8):4196-203.

    Evidence that the variable regions of the central domain of VlsE are antigenic during infection with lyme disease spirochetes.

    McDowell JV, Sung SY, Hu LT, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, Richmond 23298-0678, USA.

    It has been postulated that the vls system of the Lyme disease spirochetes contributes to immune evasion through antigenic variation. While it is clear that vlsE undergoes sequence change within its variable regions at a high frequency during the early stages of infection, a definitive role in immune evasion has not been demonstrated. In this report we assessed the murine and human humoral immune response to recombinant (r)-VlsE variants that originally arose during infection in mice. Immunoblot analyses of r-VlsE variants were conducted by using serum samples collected from mice infected with Borrelia burgdorferi clones that carried different vlsE variants. All of the r-VlsE variants were recognized by infection sera regardless of the identity of the infecting clone or isolate. In addition, all variants were immunoreactive with a panel of human Lyme disease patient serum samples. It is evident from these analyses that the infection-induced VlsE variants share common epitopes that reside within conserved segments of these proteins. However, preabsorption experiments revealed that the variable regions of the central domain of VlsE, which undergo rapid mutation during infection, also influence the antigenic properties of the protein. A subset of the antibodies elicited against vlsE variants that differ in the sequences of their variable regions were found to be variant specific. Hence, in spite of a robust antibody response to conserved segments of VlsE, infection-induced sequence changes within the variable regions alter the antigenicity of VlsE. These results provide the first direct evidence of antigenic variation in the VlsE protein.

    PMCID: PMC128138 PMID: 12117928 [PubMed - indexed for MEDLINE]

  39. Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6169-74.

    Asymmetrical flagellar rotation in Borrelia burgdorferi nonchemotactic mutants.

    Li C, Bakker RG, Motaleb MA, Sartakova ML, Cabello FC, Charon NW.

    Department of Microbiology, Immunology, and Cell Biology, Health Sciences Center, Box 9177, West Virginia University, Morgantown, WV 26506-9177, USA.

    The Lyme disease spirochete Borrelia burgdorferi has bundles of periplasmic flagella subpolarly located at each cell end. These bundles rotate in opposite directions during translational motility. When not translating, they rotate in the same direction, and the cells flex. Here, we present evidence that asymmetrical rotation of the bundles during translation does not depend upon the chemotaxis signal transduction system. The histidine kinase CheA is known to be an essential component in the signaling pathway for bacterial chemotaxis. Mutants of cheA in flagellated bacteria continually rotate their flagella in one direction. B. burgdorferi has two copies of cheA designated cheA1 and cheA2. Both genes were found to be expressed in growing cells. We reasoned that if chemotaxis were essential for asymmetrical rotation of the flagellar bundles, and if the flagellar motors at both cell ends were identical, inactivation of the two cheA genes should result in cells that constantly flex. To test this hypothesis, the signaling pathway was completely blocked by constructing the double mutant cheA1kan cheA2ermC. This double mutant was deficient in chemotaxis. Rather than flexing, it failed to reverse, and it continually translated only in one direction. Video microscopy of mutant cells indicated that both bundles actively rotated. The results indicate that asymmetrical rotation of the flagellar bundles of spirochetes does not depend upon the chemotaxis system but rather upon differences between the two flagellar bundles. We propose that certain factors within the spirochete localize at the flagellar motors at one end of the cell to effect this asymmetry.

    PMCID: PMC122921 PMID: 11983908 [PubMed - indexed for MEDLINE]

  40. Psychiatr Clin North Am. 2002 Mar;25(1):1-16.

    Psychiatric presentations of non-HIV infectious diseases. Neurocysticercosis, Lyme disease, and pediatric autoimmune neuropsychiatric disorder associated with streptococcal infection.

    Schneider RK, Robinson MJ, Levenson JL.

    Departments of Psychiatry and Internal Medicine, Division of Consultation-Liaison Psychiatry, Medical College of Virginia, Campus of Virginia Commonwealth University, Richmond, Virginia, USA. rkschnei@hsc.vcu.edu

    Infectious diseases can cause an array of symptoms, including psychiatric symptoms. Psychiatrists serving the medically ill need to be aware not only of classic infectious diseases (e.g., neurosyphilis and HIV), but also of less commonly discussed infectious diseases (e.g., NCC, PANDAS, and Lyme disease). These examples represent an internationally endemic disease (e.g., NCC), a probable immunogenetic disease (e.g., PANDAS), and a frequently overdiagnosed and overtreated disease (Lyme disease).

    PMID: 11912935 [PubMed - indexed for MEDLINE]

  41. J Bacteriol. 2001 Oct;183(20):5855-61.

    Evidence for the contribution of point mutations to vlsE variation and for apparent constraints on the net accumulation of sequence changes in vlsE during infection with Lyme disease spirochetes.

    Sung SY, McDowell JV, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia 23298-0678, USA.

    In the Lyme disease spirochetes, both the ospE and vlsE gene families have been demonstrated to undergo sequence variation during infection. To further investigate the mechanisms associated with the generation of vls variation, single-nucleotide polymorphism and subsequent DNA sequence analyses were performed on the vlsE gene and its paralog, BBJ51, a related gene with a frameshift mutation. These analyses focused on a series of postinfection clonal populations obtained from mice infected with Borrelia burgdorferi B31MIpc or its clonal derivative, B31MIc53. vlsE, but not BBJ51, was found to undergo sequence changes during infection. Consistent with that reported previously (J.-R. Zhang et al., Cell 89:275-285, 1997) many of the sequence changes appear to have arisen through gene conversion events and to be localized to the variable regions of vlsE. However, analysis of the vlsE nucleotide sequences revealed that some sequence changes were the result of point mutations, as these changes did not have potential contributing sources in the vls cassettes. To determine if sequence changes accumulate in vlsE over long-term infection, the vlsE genes of clonal populations recovered after 7 months of infection in mice were analyzed. While new sequence changes developed, a significant number of these changes resulted in the restoration of the vlsE sequence of the original infecting clone. In addition, we noted that some positions within the variable regions (VR) are stable even though the cassettes possess residues that could contribute to sequence variation through gene conversion. These analyses suggest that the total number of amino acid sequence changes that can be maintained by VlsE levels off during infection. In summary, in this report we demonstrate that the development of point mutations serves as a second mechanism by which vlsE sequence variation can be generated and that the capacity for vlsE variation, while still significant, is less than previously postulated.

    PMCID: PMC99662 PMID: 11566983 [PubMed - indexed for MEDLINE]

  42. Infect Immun. 2001 Aug;69(8):4831-8.

    Demonstration of the genetic stability and temporal expression of select members of the lyme disease spirochete OspF protein family during infection in mice.

    McDowell JV, Sung SY, Price G, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia 23298-0678, USA.

    Infection with Lyme disease spirochetes can be chronic. This suggests that the spirochetes are capable of immune evasion. In a previous study we demonstrated that the ospE gene family, which is one of three gene families whose members are flanked at their 5' end by the highly conserved upstream homology box (UHB) element, undergoes mutation and rearrangement during infection. This results in the generation of antigenically distinct variants that may contribute to immune evasion. In this study we have assessed the genetic stability of the UHB-flanked ospF gene family during infection in mice. Using postinfection clonal populations of Borrelia burgdorferi B31MI, PCR amplicons were generated for three members of the ospF gene family after a 3-month infection time frame. The amplicons were analyzed by single-nucleotide polymorphism pattern analysis and DNA sequencing. Members of the ospF gene family were found to be stable during infection, as no mutations or rearrangements were detected. An analysis of the humoral immune response to these proteins during infection revealed that the immune response to each is specific and that there is a delayed humoral immune response to some OspF protein family members. These analyses suggest that there is a temporal component to the expression of these genes during infection. In addition to a possible contribution to immune evasion, members of the OspF protein family may play specific roles at different stages of infection.

    PMCID: PMC98571 PMID: 11447157 [PubMed - indexed for MEDLINE]

  43. Infect Immun. 2001 Jun;69(6):3670-7.

    Analysis of mechanisms associated with loss of infectivity of clonal populations of Borrelia burgdorferi B31MI.

    McDowell JV, Sung SY, Labandeira-Rey M, Skare JT, Marconi RT.

    Department of Microbiology and Immunology, School of Medicine, Medical College of Virginia at Virginia Commonwealth University, Richmond 23298-0678, USA.

    Numerous studies have provided suggestive evidence that the loss of plasmids correlates with the loss of infectivity of the Lyme disease spirochetes. In this study we have further investigated this correlation. Clonal populations were obtained from the skin of a mouse infected for 3 months with a clonal population of Borrelia burgdorferi B31MI. The complete plasmid compositions of these populations were determined using a combination of PCR and Southern hybridization. The infectivities of clones differing in plasmid composition were tested using the C3H-HeJ murine model for Lyme disease. While several clones were found to be noninfectious, a correlation between the loss of a specific plasmid and loss of infectivity in the clones analyzed in this report was not observed. While it is clear from recent studies that the loss of some specific plasmids results in attenuated virulence, this study demonstrates that additional mechanisms also contribute to the loss of infectivity.

    PMCID: PMC98365 PMID: 11349029 [PubMed - indexed for MEDLINE]

  44. Mil Med. 2001 Feb;166(2):191-3.

    A Naval Academy midshipman with ehrlichiosis after summer field exercises in Quantico, Virginia.

    Rooney TB, McGue TE, Delahanty KC.

    Naval Ambulatory Care Center, Internal Medicine, Newport, RI 02841, USA.

    A case of human ehrlichiosis (caused by infection with Ehrlichia chaffeensis) is presented. The patient was a female Naval Academy midshipman with a 26-day history of daily field training with the U.S. Marines near Quantico, Virginia. She presented with a several-day history of myalgias, fever, and frontal headache. During her clinical course, she developed fever to 104 degrees F, dry cough, dyspnea on exertion, arthralgias, and nephrotic syndrome. She did not develop a rash. Laboratory studies were significant for thrombocytopenia, equivocal Lyme enzyme immunosorbent assay with a negative confirmatory western immunoblot, equivocal Rocky Mountain spotted fever acute serology without a convalescent increase in immunoglobulin G, and immunoglobulin G/immunoglobulin M serology positive for human monocytic ehrlichiosis. She manifested known sequelae for this emerging disease, including dyspnea, pedal edema, increased transminases, and nephrotic syndrome.

    PMID: 11272720 [PubMed - indexed for MEDLINE]

  45. Proc Natl Acad Sci U S A. 2000 Sep 26;97(20):10899-904.

    Borrelia burgdorferi periplasmic flagella have both skeletal and motility functions.

    Motaleb MA, Corum L, Bono JL, Elias AF, Rosa P, Samuels DS, Charon NW.

    Department of Microbiology and Immunology, Health Sciences Center, West Virginia University, Box 9177, Morgantown, WV 26506, USA.

    Bacterial shape usually is dictated by the peptidoglycan layer of the cell wall. In this paper, we show that the morphology of the Lyme disease spirochete Borrelia burgdorferi is the result of a complex interaction between the cell cylinder and the internal periplasmic flagella. B. burgdorferi has a bundle of 7-11 helically shaped periplasmic flagella attached at each end of the cell cylinder and has a flat-wave cell morphology. Backward moving, propagating waves enable these bacteria to swim in both low viscosity media and highly viscous gel-like media. Using targeted mutagenesis, we inactivated the gene encoding the major periplasmic flagellar filament protein FlaB. The resulting flaB mutants not only were nonmotile, but were rod-shaped. Western blot analysis indicated that FlaB was no longer synthesized, and electron microscopy revealed that the mutants were completely deficient in periplasmic flagella. Wild-type cells poisoned with the protonophore carbonyl cyanide-m-chlorophenylhydrazone retained their flat-wave morphology, indicating that the periplasmic flagella do not need to be energized for the cell to maintain this shape. Our results indicate that the periplasmic flagella of B. burgdorferi have a skeletal function. These organelles dynamically interact with the rod-shaped cell cylinder to enable the cell to swim, and to confer in part its flat-wave morphology.

    PMCID: PMC27121 PMID: 10995478 [PubMed - indexed for MEDLINE]

  46. J Bacteriol. 2000 Aug;182(15):4222-6.

    Analysis of the cellular localization of Bdr paralogs in Borrelia burgdorferi, a causative agent of lyme disease: evidence for functional diversity.

    Roberts DM, Theisen M, Marconi RT.

    Department of Microbiology and Immunology, School of Medicine, Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia 23298-0678, USA.

    The bdr (Borrelia direct repeat) gene family of the genus Borrelia encodes a polymorphic group of proteins that carry a central repeat motif region containing putative phosphorylation sites and a hydrophobic carboxyl-terminal domain. It has been postulated that the Bdr proteins may anchor to the inner membrane via the C-terminal domain. In this study, we used cellular fractionation methodologies, salt and detergent treatments, and immunoblot analyses to assess the association of the Bdr proteins with the cellular infrastructure in both Borrelia burgdorferi (a Lyme disease spirochete) and B. turicatae (a relapsing fever spirochete). Triton X-114 extraction and partitioning experiments demonstrated that most Bdr paralogs are associated with the inner membrane-peptidoglycan complex. Analyses of cells treated with the highly chaotropic bile salt detergent deoxycholic acid demonstrated that some Bdr paralogs may also interact with the peptidoglycan, as evidenced by their tight association with the insoluble cellular matrix. In addition, immunoprecipitation (IP) experiments revealed an enhanced IP of all Bdr paralogs when the cell lysates were boiled prior to addition of the precipitating antibody. Furthermore, some Bdr paralogs were accessible to antibody in the IP experiments only in the boiled cell lysates. These observations suggest that different Bdr paralogs may carry out different structural-functional roles. Demonstration of the inner membrane localization of the Bdr proteins and of the differences in nature of the interaction of individual Bdr paralogs with the cell infrastructure is an important step toward defining the functional role of this unique protein family in the genus Borrelia.

    PMCID: PMC101917 PMID: 10894730 [PubMed - indexed for MEDLINE]

  47. J Fam Pract. 2000 May;49(5):397, 465.

    What is the long-term prognosis for patients with Lyme disease?

    Greenawald MH, Vaughan S.

    Carilion Family Practice Residency Program, Roanoke, Virginia, USA. mgreenawald@carilion.com

    PMID: 10836768 [PubMed - indexed for MEDLINE]

  48. Emerg Infect Dis. 2000 Mar-Apr;6(2):110-22.

    The bdr gene families of the Lyme disease and relapsing fever spirochetes: potential influence on biology, pathogenesis, and evolution.

    Roberts DM, Carlyon JA, Theisen M, Marconi RT.

    Medical College of Virginia at Virginia Commonwealth University, School of Medicine, Richmond, VA 23298-0678, USA.

    Erratum in: Emerg Infect Dis 2000 Jul-Aug;6(4):441.

    Species of the genus Borrelia cause human and animal infections, including Lyme disease, relapsing fever, and epizootic bovine abortion. The borrelial genome is unique among bacterial genomes in that it is composed of a linear chromosome and a series of linear and circular plasmids. The plasmids exhibit significant genetic redundancy and carry 175 paralogous gene families, most of unknown function. Homologous alleles on different plasmids could influence the organization and evolution of the Borrelia genome by serving as foci for interplasmid homologous recombination. The plasmid-carried Borrelia direct repeat (bdr) gene family encodes polymorphic, acidic proteins with putative phosphorylation sites and transmembrane domains. These proteins may play regulatory roles in Borrelia. We describe recent progress in the characterization of the Borrelia bdr genes and discuss the possible influence of this gene family on the biology, pathogenesis, and evolution of the Borrelia genome.

    PMCID: PMC2640845 PMID: 10756144 [PubMed - indexed for MEDLINE]

  49. Infect Immun. 2000 Mar;68(3):1319-27.

    Mutation and recombination in the upstream homology box-flanked ospE-related genes of the Lyme disease spirochetes result in the development of new antigenic variants during infection.

    Sung SY, McDowell JV, Carlyon JA, Marconi RT.

    Department of Microbiology, Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia 23298-0678, USA.

    The ospE gene family of the Lyme disease spirochetes encodes a polymorphic group of immunogenic lipoproteins. The ospE genes are one of several gene families that are flanked by a highly conserved upstream sequence called the upstream homology box, or UHB, element. Earlier analyses in our lab demonstrated that ospE-related genes are characterized by defined hypervariable domains (domains 1 and 2) that are predicted to be hydrophilic, surface exposed, and antigenic. The flanking of hypervariable domain 1 by DNA repeats may indicate that recombination contributes to ospE diversity and thus ultimately to antigenic variation. Using an isogeneic clone of Borrelia burgdorferi B31G (designated B31Gc1), we demonstrate that the ospE-related genes undergo mutation and rearrangement during infection in mice. The mutations that develop during infection resulted in the generation of OspE proteins with altered antigenic characteristics. The data support the hypothesized role of OspE-related proteins in immune system evasion.

    PMCID: PMC97285 PMID: 10678944 [PubMed - indexed for MEDLINE]

  50. Microb Pathog. 2000 Feb;28(2):89-105.

    Evolutionary and molecular analyses of the Borrelia bdr super gene family: delineation of distinct sub-families and demonstration of the genus wide conservation of putative functional domains, structural properties and repeat motifs.

    Carlyon JA, Roberts DM, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, Richmond, VA 23298-0678, USA.

    B. turicatae, a causative agent of relapsing fever, carries a polymorphic gene family that is homologous to the bdr gene family of the Lyme disease spirochetes (previously referred to as the rep+ or ORF-E gene family). Here we demonstrate that bdr related genes are widely distributed among pathogenic Borrelia species and exist as large, polymorphic, plasmid carried, gene families. Twenty distinct bdr alleles were identified in isolates of the relapsing fever spirochete, B. hermsii, and were localized to linear plasmids. Cloning and sequence analyses demonstrate that the putative Bdr functional domains (i.e. the phosphorylation motifs and the transmembrane C-terminal domain) are conserved across the genus while other regions of these proteins exhibit variability. An assessment of the evolutionary relationships among all known Bdr protein sequences obtained from five pathogenic Borrelia species revealed that there are distinct Bdr sub-families. The recognition of distinct phyletic clusters serves as the basis of a revised and simplified nomenclature for the bdr proteins that can be applied genus wide. At the biological level the delineation of multiple bdr sub-families within isogeneic populations raises the possibility that there may be functional partitioning among alleles. In summary, the distribution and conservation of the Bdr proteins suggests that they are important in the biology/pathogenesis of the Borrelia at the genus wide level. Copyright 2000 Academic Press.

    PMID: 10644495 [PubMed - indexed for MEDLINE]

  51. J Clin Microbiol. 1999 Dec;37(12):3965-70.

    Genetic analysis of Borrelia garinii OspA serotype 4 strains associated with neuroborreliosis: evidence for extensive genetic homogeneity.

    Marconi RT, Hohenberger S, Jauris-Heipke S, Schulte-Spechtel U, LaVoie CP, Rössler D, Wilske B.

    Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia 23298-0678, USA.

    Infection with Borrelia garinii outer surface protein (Osp) A serotype 4 strains has been correlated with the development of neuroborreliosis in Lyme borreliosis patients in Europe. OspA serotype 4 isolates have been recovered primarily from human cerebrospinal fluid, suggesting a tropism for this environment. Previous studies with monoclonal antibodies directed against OspA and OspC demonstrated that OspA serotype 4 strains are antigenically closely related. In view of the pronounced antigenic and genetic variability that has been noted in the Osps of other Borrelia isolates, we sought to determine if OspA serotype 4 strains represent a recently emerged clonal lineage of B. garinii. Toward this goal, a representative group of OspA serotype 4 strains was analyzed for traits that typically exhibit hypervariability among isolates that cause Lyme borreliosis. The following criteria were assessed: (i) ospC sequences, (ii) plasmid composition, (iii) genomic restriction fragment length polymorphism (RFLP) patterns, and (iv) the RFLP patterns of the upstream homology box (UHB) element which flanks members of the UHB gene family at their 5' end. Collectively, these analyses demonstrate genetic homogeneity, suggesting that OspA serotype 4 strains are a recently emerged clonal lineage with an apparent tropism for the central nervous system.

    PMCID: PMC85856 PMID: 10565915 [PubMed - indexed for MEDLINE]

  52. J Med Entomol. 1999 Sep;36(5):578-87.

    Ticks and antibodies to Borrelia burgdorferi from mammals at Cape Hatteras, NC and Assateague Island, MD and VA.

    Oliver JH Jr, Magnarelli LA, Hutcheson HJ, Anderson JF.

    Institute of Arthropodology & Parasitology, Georgia Southern University, Statesboro 30460, USA.

    Results of a survey for ixodid ticks and/or serum antibodies to Borrelia burgdorferi from 14 species of small to large mammals from eastern coastal areas of the United States are presented. Most samples were obtained from July 1987 through June 1989 (excluding December-March) at 3 locales: Assateague Is. National Seashore, Worcester Co., MD., and Accomack Co., VA. (approximately 38 degrees 05' N 75 degrees 10' W), and Cape Hatteras National Seashore, Dare Co., NC (approximately 35 degrees 30' N 76 degrees 35' W). Hosts sampled included opossums (Didelphis virginiana), least shrews (Cryptotis parva), gray foxes (Urocyon cinereoargenteus), red foxes (Vulpes vulpes), raccoons (Procyon lotor), feral cats (Felis sylvestris), feral horses (Equus caballus), sika deer (Cervus nippon), rice rats (Oryzomys palustris), white-footed mice (Peromyscus leucopus), meadow voles (Microtus pennsylvanicus), house mice (Mus musculus), norway rats (Rattus norvegicus) and jumping mice (Zapus hudsonius). An indirect fluorescent antibody test was used for testing sera from opossums, raccoons, and feral cats; enzyme-linked immunosorbent assays were used for sera from foxes, horses, deer, and house and white-footed mice. Antibodies to B. burgdorferi were found in all species tested from each locale. Seasonal data reinforce the contention that P. leucopus is a suitable sentinel species for B. burgdorferi. Ticks on hosts included Ixodes scapularis Say, I. texanus Banks, Dermacentor variabilis (Say), D. albipictus (Packard), and Amblyomma americanum (L.). Males comprised approximately 0-22 and 60-81% of Ixodes sp. and Amblyomma-Dermacentor adults collected from hosts, respectively. All stages of A. americanum, adult D. variabilis, and larval I. scapularis were collected from vegetation. The highest seropositivity rate (67%) was recorded for 45 P. leucopus at Assateague during July, approximately 1 mo. after peak nymphal I. scapularis intensity. Borrelia burgdorferi was isolated from 6 nymphal and 12 female I. scapularis collected from P. leucopus and C. nippon, respectively, on Assateague.

    PMID: 10534951 [PubMed - indexed for MEDLINE]

  53. J Med Entomol. 1998 Sep;35(5):629-38.

    Reported distribution of Ixodes scapularis and Ixodes pacificus (Acari: Ixodidae) in the United States.

    Dennis DT, Nekomoto TS, Victor JC, Paul WS, Piesman J.

    Division of Vector-Borne Infection Diseases, Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, Fort Collins, CO 80522, USA.

    Lyme disease, caused by infection with Borrelia burgdorferi, is the most frequently reported arthropod-borne disease in the United States. To develop a national map of the distribution of the vectors of B. burgdorferi to humans (Ixodes scapularis Say and Ixodes pacificus Cooley & Kohls ticks), we sent questionnaires to acarologists, health officials, and Lyme disease researchers; surveyed the 1966-1996 MEDLINE data base; and reviewed 1907-1995 National Tick Collection data. Tick collection methods cited included flagging and dragging, deer surveys, small- and medium-sized mammal surveys, CO2 baiting, and receipt of tick submissions. A total of 1,058 unique, county-specific I. scapularis and I. pacificus records was obtained. Tick populations were classified as "reported" (< 6 ticks and 1 life stage identified) or "established" (> or = 6 ticks or > 1 life stage identified). Established populations of I. scapularis were identified in 396 counties in 32 states in the eastern and central United States, whereas established populations of I. pacificus were found in 90 counties in 5 western states. Counties with established populations were most concentrated in the northeastern, upper northcentral, and west-coastal states but were also clustered in southeastern and Gulf-coastal states. A less concentrated distribution was found in the south-central states. Reports were notably missing from all but a few counties in Ohio, West Virginia, western Virginia and North Carolina, Kentucky, and Tennessee. They were absent in the Great Plains and Rocky Mountain regions and from large areas of western states east of the Cascade and Sierra Nevada cordilleras. These data are useful for identifying areas of Lyme disease risk, for targeting Lyme disease prevention strategies, and for monitoring trends in spatial distribution of Lyme disease vector ticks.

    PMID: 9775584 [PubMed - indexed for MEDLINE]

  54. Infect Immun. 1998 Oct;66(10):4656-68.

    Genetic divergence and evolutionary instability in ospE-related members of the upstream homology box gene family in Borrelia burgdorferi sensu lato complex isolates.

    Sung SY, Lavoie CP, Carlyon JA, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia of Virginia Commonwealth University, Richmond, Virginia 23298-0678, USA.

    A series of related genes that are flanked at their 5' ends by a conserved upstream sequence element called the upstream homology box (UHB) have been identified in Borrelia burgdorferi. These genes have been referred to as the UHB or erp gene family. We previously demonstrated that among a limited number of B. burgdorferi isolates, the UHB gene family is variable in composition and organization. Prior to this report the UHB gene family in other species of the B. burgdorferi sensu lato complex had not been studied, and if this family is important in the pathogenesis or biology of the Lyme disease spirochetes, then a wide distribution among species and isolates of the B. burgdorferi sensu lato complex would be expected. To assess this, we screened for the UHB element by Southern hybridization and determined its restriction fragment length polymorphism (RFLP) patterns. The UHB element was found to be carried by all B. burgdorferi sensu lato complex species tested (B. burgdorferi, B. garinii, B. afzelii, B. japonica, B. valaisiana sp. nov., and B. andersonii), but the RFLP patterns varied widely at both the inter- and intraspecies levels. Variation in both the number and size of the hybridizing restriction fragments was evident. PCR analyses also revealed the presence of polymorphic, ospE-related alleles in many isolates. Sequence analyses identified the molecular basis of the polymorphisms as being primarily insertions and deletions. Sequence variation and the insertions and deletions were found to be clustered in two distinct domains (variable domains 1 and 2). In many isolates variable domain 1 is flanked by direct repeat elements, some as long as 38 bp. Computer analyses of the deduced amino acid sequences encoded within variable domain 1 predict them to be hydrophilic, surface exposed, and antigenic. The analyses conducted here suggest that the UHB gene family, as evidenced by the variable UHB RFLP patterns, is not evolutionarily stable and that the polymorphic ospE alleles are derived from a common ancestral gene which has been modified through mutation or recombination events. The characterization of ospE-related genes of the UHB gene family among B. burgdorferi sensu lato species will prove important in attempts to construct a model for UHB gene family organization and in deciphering the role of the UHB gene family in the biology and pathogenesis of the Lyme disease spirochetes.

    PMCID: PMC108573 PMID: 9746562 [PubMed - indexed for MEDLINE]

  55. J Bacteriol. 1998 Sep;180(18):4974-81.

    Cloning and molecular characterization of a multicopy, linear plasmid-carried, repeat motif-containing gene from Borrelia turicatae, a causative agent of relapsing fever.

    Carlyon JA, Marconi RT.

    Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia 23298-0678, USA.

    Borrelia turicatae is one of several spirochete species that can cause relapsing fever. Here, we describe the identification and characterization of a gene from B. turicatae and other relapsing-fever spirochetes that exhibits homology with the rep+ and ORF-E gene families of the Lyme disease spirochetes. This gene, which we have designated repA, encodes a putative protein of 30.2 kDa with an isoelectric point of 4.69. The central region of RepA harbors a series of amino acid repeat motifs which exhibit homology with casein kinase 2 phosphorylation sites. Through Southern hybridization analyses, we demonstrate that repA (or a closely related sequence) is multicopy in the relapsing-fever spirochetes and is carried on variably sized linear plasmids in both Borrelia parkeri and B. turicatae. Transcriptional analyses demonstrate that repA is expressed, albeit at low levels, during in vitro cultivation of B. turicatae. Transcriptional start site analysis revealed that repA is preceded by a consensus ribosomal binding site and an appropriately spaced promoter element. The sequence conservation, unique features, and multicopy status of repA and its homologs suggest that RepA may play an important genus-wide role in the biology of the Borrelia.

    PMCID: PMC107528 PMID: 9733706 [PubMed - indexed for MEDLINE]

  56. J Med Entomol. 1998 Jan;35(1):54-8.

    Occurrence of Ixodes scapularis (Acari: Ixodidae) on a selected segment of the Appalachian Trail.

    Oliver J, Howard JJ.

    New York State Department of Health, SUNY College of Environmental Science and Forestry, Syracuse 13210, USA.

    A 918-km section of the Appalachian National Scenic Trail from the West Virginia-Maryland border to the Massachusetts-Vermont border was surveyed for the presence of Ixodes scapularis Say. The trail and its edges were drag-sampled during 4 hikes between May and October 1991. Trips were designed to survey areas of the Appalachian Trail when I. scapularis might be questing and to revisit states endemic for Lyme disease during differing times. After sampling for ticks, meteorological and ecological characteristics were measured at each site. In total, 1,776 km of the Appalachian Trail were hiked during 88 d and resulted in sampling 489 sites. All life stages of Ixodes scapularis (n = 46) were collected from 21 sites within a 331-km range of the Appalachian Trail between Salisbury, CT, to Delaware Water Gap, PA. This segment of Appalachian Trial is easily accessible to a large urban population and should be posted with tick warning signs to alert the public to the presence of I. scapularis.

    PMID: 9542345 [PubMed - indexed for MEDLINE]

  57. J Bacteriol. 1998 May;180(9):2418-25.

    Structure and expression of the FlaA periplasmic flagellar protein of Borrelia burgdorferi.

    Ge Y, Li C, Corum L, Slaughter CA, Charon NW.

    Department of Microbiology and Immunology, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown 26506-9177, USA.

    The spirochete which causes Lyme disease, Borrelia burgdorferi, has many features common to other spirochete species. Outermost is a membrane sheath, and within this sheath are the cell cylinder and periplasmic flagella (PFs). The PFs are subterminally attached to the cell cylinder and overlap in the center of the cell. Most descriptions of the B. burgdorferi flagellar filaments indicate that these organelles consist of only one flagellin protein (FlaB). In contrast, the PFs from other spirochete species are comprised of an outer layer of FlaA and a core of FlaB. We recently found that a flaA homolog was expressed in B. burgdorferi and that it mapped in a fla/che operon. These results led us to analyze the PFs and FlaA of B. burgdorferi in detail. Using Triton X-100 to remove the outer membrane and isolate the PFs, we found that the 38.0-kDa FlaA protein purified with the PFs in association with the 41.0-kDa FlaB protein. On the other hand, purifying the PFs by using Sarkosyl resulted in no FlaA in the isolated PFs. Sarkosyl has been used by others to purify B. burgdorferi PFs, and our results explain in part their failure to find FlaA. Unlike other spirochetes, B. burgdorferi FlaA was expressed at a lower level than FlaB. In characterizing FlaA, we found that it was posttranslationally modified by glycosylation, and thus it resembles its counterpart from Serpulina hyodysenteriae. We also tested if FlaA was synthesized in a spontaneously occurring PF mutant of B. burgdorferi (HB19Fla-). Although this mutant still synthesized flaA message in amounts similar to the wild-type amounts, it failed to synthesize FlaA protein. These results suggest that, in agreement with data found for FlaB and other spirochete flagellar proteins, FlaA is likely to be regulated on the translational level. Western blot analysis using Treponema pallidum anti-FlaA serum indicated that FlaA was antigenically well conserved in several spirochete species. Taken together, the results indicate that both FlaA and FlaB comprise the PFs of B. burgdorferi and that they are regulated differently from flagellin proteins of other bacteria.

    PMCID: PMC107184 PMID: 9573194 [PubMed - indexed for MEDLINE]

  58. FEMS Microbiol Lett. 1997 Aug 15;153(2):425-31.

    Molecular characterization of a flagellar/chemotaxis operon in the spirochete Borrelia burgdorferi.

    Ge Y, Charon NW.

    Department of Microbiology and Immunology, West Virginia University, Robert C. Byrd Health Sciences Center, Morgantown 26506-9177, USA.

    A chemotaxis gene cluster from Borrelia burgdorferi, the spirochete that causes Lyme disease, was cloned, sequenced, and analyzed. This cluster contained three chemotaxis gene homologs (cheA, cheW and cheY) and an open reading frame we identified as cheX. Although the major functional domains for B. burgdorferi CheW and CheY were well conserved, the size of cheW was significantly different from the homolog of other bacteria. Phylogenetic analysis of CheY indicated that B. burgdorferi constitutes a distinct branch with Treponema pallidum and is closely associated with Archea and Gram-positive bacteria. RT-PCR analysis indicated that the chemotaxis genes and the upstream flagellar gene flaA constitute an operon. Western blot analysis using antibody to Escherichia coli CheA resulted in two reactive proteins in the cell lysates of B. burgdorferi that is consistent with two cheA homologs being present in this organism. The results taken together suggest both similarities and differences in the chemotaxis apparatus of B. burgdorferi compared to those of other bacteria.

    PMID: 9271872 [PubMed - indexed for MEDLINE]

  59. Infect Immun. 1997 Jul;65(7):2992-5.

    FlaA, a putative flagellar outer sheath protein, is not an immunodominant antigen associated with Lyme disease.

    Ge Y, Charon NW.

    Department of Microbiology and Immunology, West Virginia University, Morgantown 26506-9177, USA.

    FlaA was recently found to be associated with flagellar filaments of Borrelia burgdorferi. We tested whether antibodies to this protein are a good indicator of infection, as antibodies to FlaA proteins in other spirochetal infections show an increase in titer. Although overproduction of intact FlaA was highly toxic to Escherichia coli, truncated proteins which lacked the N-terminal signal sequence could be successfully overexpressed. Immunoblotting with sera from mammalian hosts infected with B. burgdorferi indicated that FlaA is not an immunodominant antigen in Lyme disease. However, sera from two patients reacted with both recombinant and native FlaA protein, suggesting that B. burgdorferi FlaA was antigenic and expressed in vivo.

    PMCID: PMC175421 PMID: 9199479 [PubMed - indexed for MEDLINE]

  60. Gene. 1997 Apr 21;189(2):195-201.

    Identification of a large motility operon in Borrelia burgdorferi by semi-random PCR chromosome walking.

    Ge Y, Charon NW.

    Robert C. Byrd Health Sciences Center, Department of Microbiology and Immunology, West Virginia University, Morgantown 26506-9177, USA.

    Motility has been implicated in the invasive process of Borrelia burgdorferi (Bb), the etiologic agent of Lyme disease. To identify Bb motility related genes, we used a method termed 'semi-random PCR chromosome walking' (SRPCW) to walk through a large motility gene cluster. The major advantage of this approach over other PCR walking methods is that it employs a secondary PCR amplification of cloned fragments which can be readily sequenced and analyzed. Starting with a primer specific to flgE, we identified and sequenced 14 open reading frames (ORFs) spanning 11 kb downstream of the flgE gene. The genes identified include flbD, motA, motB, fliL, fliM, fliN, fliZ, fliP, fliQ, fliR, flhB, flhA, flhF and flbE. Twelve of the deduced proteins shared extensive homology with flagellar proteins from other bacteria. The gene products and order of genes within this cluster are most similar to those of Treponema pallidum (Tp) and Bacillus subtilis (Bs). One of the unique genes identified, flbD, demonstrated homology to an ORF from the same operon of Tp. Another ORF, flbE, showed similarity to genes from both Tp and Bs. RT-PCR and primer extension analysis revealed that this gene cluster is transcribed as a single unit indicating that it is part of a large motility operon spanning more than 21 kb. Antisera to Escherichia coli and Salmonella typhimurium FliN, FliM, FlhB and FlhA reacted with proteins of the predicted molecular weights in cell lysates of Bb. The results suggest that the flagellar system is highly conserved in evolution and thus underscore the importance of motility in bacterial survival and pathogenesis.

    PMID: 9168127 [PubMed - indexed for MEDLINE]

  61. J Bacteriol. 1997 Apr;179(7):2289-99.

    Molecular characterization of a large Borrelia burgdorferi motility operon which is initiated by a consensus sigma70 promoter.

    Ge Y, Old IG, Saint Girons I, Charon NW.

    Department of Microbiology and Immunology, West Virginia University, Robert C. Byrd Health Sciences Center, Morgantown 26506-9177, USA.

    A large motility operon, referred to as the flgB operon, was identified, characterized, and mapped at 310 to 320 kb on the linear chromosome of the spirochete Borrelia burgdorferi. This is the first report that a sigma70-like promoter rather than a sigma28-like promoter is involved in the transcription of a major motility operon in bacteria. From these results in conjunction with results from a previous study (Y. Ge and N. W. Charon, Gene, in press), we have identified 26 genes in this operon that are relevant to motility and flagellar synthesis. With few exceptions, the gene order and deduced gene products were most similar to those of other spirochetes and Bacillus subtilis. Primer extension analysis indicated that transcription initiated from a conserved sigma70-like promoter immediately upstream of flgB; this promoter mapped within the heat-shock-induced protease gene hslU. Reverse transcriptase PCR analysis indicated that a single transcript of 21 kb initiated at this promoter and extended through flgE and (with our previous results) onto the putative motility gene flbE. The flgB promoter element had strong activity in both Escherichia coli and Salmonella typhimurium. As expected, a mutant of S. typhimurium with an inactivated flagellum-specific sigma28 factor did not affect the function of this promoter. Western blot analysis indicated that B. burgdorferi recombinant FliG and FliI were antigenically similar to those of E. coli and other spirochetes. Although complementation of E. coli or S. typhimurium fliG or fliI mutants with the B. burgdorferi genes was unsuccessful, B. burgdorferi recombinant FliI completely inhibited flagellar synthesis and motility of wild-type E. coli and S. typhimurium. These results show that spirochete motility genes can influence flagellar synthesis in other species of bacteria. Finally, Western blot analysis with sera from infected humans and animals indicated a weak or nondetectable response to recombinant FliG and FliI. These results indicate that these antigens are not favorable candidate reagents to be used in the diagnosis of Lyme disease.

    PMCID: PMC178966 PMID: 9079915 [PubMed - indexed for MEDLINE]

  62. Gene. 1996 Feb 2;168(1):73-5.

    FliH and fliI of Borrelia burgdorferi are similar to flagellar and virulence factor export proteins of other bacteria.

    Ge Y, Old I, Saint Girons I, Yelton DB, Charon NW.

    Department of Microbiology, West Virginia University, Health Sciences Center, Morgantown 26506-9177, USA.

    Two motility genes (fliH and fliI) of the Lyme disease spirochete Borrelia burgdorferi were cloned, physically mapped and sequenced, FliH and FliI showed extensive homology to the proteins involved in the export of flagellar components and to virulence factors found in both animal and plant bacterial pathogens. The results suggest that the flagellar apparatus and associated protein export pathway are well conserved in evolution.

    PMID: 8626068 [PubMed - indexed for MEDLINE]

  63. J Clin Microbiol. 1995 Sep;33(9):2427-34.

    Identification of novel insertion elements, restriction fragment length polymorphism patterns, and discontinuous 23S rRNA in Lyme disease spirochetes: phylogenetic analyses of rRNA genes and their intergenic spacers in Borrelia japonica sp. nov. and genomic group 21038 (Borrelia andersonii sp. nov.) isolates.

    Marconi RT, Liveris D, Schwartz I.

    Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0678, USA.

    Borrelia spp. associated with Lyme disease possess an rRNA gene organization consisting of a single 16S rRNA gene followed by a spacer of several kilobases and a tandem repeat of a 23S (rrl)-5S (rrf) rRNA gene cluster. The restriction fragment length polymorphism (RFLP) patterns for these genes have been widely used to classify Lyme disease spirochete isolates. We analyzed the rRNA gene organization and sequences for two Ixodes ovatus isolates from Japan (IKA2 and HO14) and two group 21038 isolates associated with Ixodes dentatus ticks or rabbits from North America (isolates 21038 and 19857). This analysis revealed unique polymorphisms not previously described in other Lyme disease spirochete isolates. The molecular basis of these polymorphisms was determined by Southern blotting and PCR analyses. Only one continuous copy of the rrl-rrf gene cluster was identified in isolates IKA2, 19857, and 21038. The second rrl-rrf gene cluster is entirely absent from the IKA2 genome. In isolates 19857 and 21038, an intervening sequence is present, resulting in a fragment rrlB gene. The insertion site of this intervening sequence element differed in each isolate. While isolates 19857 and 21038 were found to carry a fragmented rrlB gene, they lacked rrfB. To determine if these rRNA polymorphisms were indicative of an underlying phylogenetic divergence, sequence analysis of the 16S rRNA (rrs) genes was conducted. The phylogenies inferred from rrs sequence analysis suggest that the polymorphisms resulted from recent mutational events. In addition, the phylogenetic analyses also support the proposed species status of Borrelia japonica sp. nov. and indicate that isolates of genomic group 21038 belong to a previously undescribed species for which we propose the nomenclature Borrelia andersonii sp. nov.

    PMCID: PMC228430 PMID: 7494041 [PubMed - indexed for MEDLINE]

  64. Am J Trop Med Hyg. 1995 Aug;53(2):123-33.

    Borrelia burgdorferi in eastern Virginia: comparison between a coastal and inland locality.

    Sonenshine DE, Ratzlaff RE, Troyer J, Demmerle S, Demmerle ER, Austin WE, Tan S, Annis BA, Jenkins S.

    Department of Biological Sciences, Old Dominion University, Norfolk, Virginia, USA.

    In Virginia, Borrelia burgdorferi was more prevalent in a site along the Atlantic Ocean, near Maryland, than in an inland site near Williamsburg and Yorktown. At the coastal site on Assateague Island, B. burgdorferi was isolated from 4.2% of 475 animals sampled, including four species of small mammals. Serologic tests indicated that 25-37% of the small rodents assayed had been exposed to B. burgdorferi. Immunofluorescence antibody assays specific for B. burgdorferi showed spirochete infection in Ixodes scapularis and Dermacentor variabilis but not in other species of ticks also examined from this site. At another coastal site (Parramore Island), no evidence of Peromyscus leucopus was found, no immature specimens of I. scapularis were collected, and no isolations were made from numerous raccoons or small mammals sampled. Borrelia burgdorferi infection was found in one I. cookei nymph, but not in numerous specimens of I. scapularis or other tick species from this locality. At the inland site between Williamsburg and Yorktown, B. burgdorferi was isolated from two small mammal species and antibodies to B. burgdorferi were found in only 7-10% of the small mammals sampled. Ixodes scapularis were less abundant at this locality than at the Assateague Island site. Borrelia burgdorferi spirochetes were found in I. scapularis and a single nymph of Amblyomma americanum, but not in any of numerous specimens of four other species. Infection with B. burgdorferi was found in 20% of unfed adult I. scapularis from vegetation, but in only 0.2% of numerous adults from hunter-killed deer. Infection in immature ticks was much lower than at Assateague Island. Borrelia burgdorferi may be more prevalent along the Atlantic coast than in inland areas. Isolations, seroprevalence, immature I. scapularis densities, and spirochete infection rates in ticks were higher at the Assateague Island site than the Williamsburg/Yorktown site. Consequently, the risk of human exposure to Lyme disease may be higher in some parts of the coastal area than elsewhere in Virginia. Overall, B. burgdorferi is less intense in Virginia than in the northeastern United States.

    PMID: 7677212 [PubMed - indexed for MEDLINE]

  65. Acad Emerg Med. 1995 Aug;2(8):751-6.

    Case conference: complete heart block in a young man.

    Huff JS, Syverud SA, Tucci MA.

    Department of Emergency Medicine, Eastern Virginia Graduate School of Medicine, Norfolk, USA.

    Erratum in: Acad Emerg Med 1996 Sep;3(9):905.

    Comment in: Acad Emerg Med. 1996 Sep;3(9):905. Acad Emerg Med. 1998 Feb;5(2):196.

    A previously healthy 32-year-old man presented to the ED in complete heart block. Ischemic, infectious, and inflammatory conditions were considered in the differential diagnosis. Management options for complete heart block, the etiology of heart block in young adults, and treatment guidelines are reviewed.

    PMID: 7584757 [PubMed - indexed for MEDLINE]

  66. J Am Vet Med Assoc. 1993 Dec 1;203(11):1524-8.

    Current understanding of Borrelia burgdorferi infection, with emphasis on its prevention in dogs.

    Kazmierczak JJ, Sorhage FE.

    National Association of State Public Health Verterinarians, Virginia Department of Health, Office of Epidemiology, Richmond 23218.

    PMID: 8288471 [PubMed - indexed for MEDLINE]

  67. W V Med J. 1992 Mar;88(3):102.

    Lyme disease case studies.

    Paulk DA.

    Valley Health Care, Inc., Mill Creek, W.Va.

    Lyme disease is the commonest tick-borne disease in the United States. Since the Mill Creek Clinic opened in March 1991, we have diagnosed two cases of the total five cases of Lyme disease reported in West Virginia.

    PMID: 1574875 [PubMed - indexed for MEDLINE]

  68. Curr Probl Cardiol. 1991 Jun;16(6):377-442.

    The heart as a target organ of immune injury.

    Hastillo A, Willis HE, Hess ML.

    Division of Cardiology, Medical College of Virginia, Virginia Commonwealth University, Richmond.

    Over the last 10 years, our knowledge of immunologically mediated processes involving the myocardium appears to have made quantum leaps. New and important disease entities such as AIDS have appeared and the cardiologist now becomes an important member of the "AIDS team." Our understanding of "older diseases" such as sarcoidosis, Lyme disease, systemic lupus and other connective tissue syndromes has significantly increased. The concept of high-dose steroid therapy for these processes may, in fact, turn out to be futile and more selective, as less dangerous immunosuppression is being introduced. This concept has significantly advanced in the field of cardiac transplantation where immunosuppression has now been usurped by specific immunotherapy aimed at selective aspects of the immune sequence. New and exciting concepts will emerge from the molecular biology laboratory that will have direct bearing on the management of patients with cardiovascular disorders. This information explosion will force the cardiovascular physician to become more in tune with the world of immunology and molecular biology. Many obvious, significant problems remain, such as accelerated atherosclerosis in the transplant patient and the role of myocarditis in the patient with heart failure. However, it will truly be an exciting decade in which to work and watch the unraveling of these mysteries and hopefully, the study of today's problems will give way to solutions and a clearer understanding of the heart as a target of immune injury.

    PMID: 1914512 [PubMed - indexed for MEDLINE]

  69. J Med Entomol. 1991 Jan;28(1):186-9.

    Parasitization of humans in West Virginia by Ixodes cookei (Acari: Ixodidae), a potential vector of Lyme borreliosis.

    Hall JE, Amrine JW Jr, Gais RD, Kolanko VP, Hagenbuch BE, Gerencser VF, Clark SM.

    Department of Microbiology and Immunology, West Virginia University, Morgantown 26506.

    In 32 collections, two larvae, 33 nymphs, and one adult female Ixodes cookei Packard were collected from humans in West Virginia from August 1987 to May 1990. Most were attached. The ticks were found in 14 counties and were the most abundant Ixodes found biting humans. One nymphal I. cookei was removed from the left axilla of a 39-yr-old woman who lives and works in Monongalia and Marion counties, W. Va. The bite was the center of an expanding erythematous lesion reaching 4 cm in diameter, clearing centrally, and typical of erythema migrans. This association and the near absence of Ixodes dammini Spielman, Clifford, Piesman & Corwin from the state suggests the possibility that I. cookei may be an important vector of Lyme borreliosis in West Virginia. In five separate collections, five nymphal Ixodes dentatus Marx were removed from humans in four counties, implicating this species as a potential minor vector of Lyme borreliosis in West Virginia.

    PMID: 2033612 [PubMed - indexed for MEDLINE]

  70. Am J Med Sci. 1990 Nov;300(5):283-7.

    Epidemiology of Lyme disease in Virginia.

    Heimberger T, Jenkins S, Russell H, Duma R.

    Division of Infectious Diseases, Medical College of Virginia, Virginia Commonwealth University, Richmond.

    Prior to January 1986, only one case of Lyme disease was reported from Virginia. In 1986-87, however, the Virginia Department of Health observed an increase in reports of suspected Lyme disease by physicians, despite the fact that Ixodes dammini is not highly prevalent in the Virginia tick population. Twenty-eight cases of Lyme disease were identified in Virginia, of which eight cases occurred in 1986 and 20 in 1987. Lyme disease appears to be increasing in frequency in Virginia and moving southward along the Eastern Atlantic Seaboard.

    PMID: 2240015 [PubMed - indexed for MEDLINE]

  71. J Rheumatol. 1990 Sep;17(9):1193-4.

    Prevalence of antibody to Borrelia burgdorferi in children with juvenile rheumatoid arthritis.

    Saulsbury FT, Katzmann JA.

    Department of Pediatrics, University of Virginia Health Sciences Center, Charlottesville.

    Lyme arthritis and juvenile rheumatoid arthritis (JRA) share a number of clinical features. Our study was performed in order to determine the prevalence of antibody to Borrelia burgdorferi in 50 children with JRA who reside in a nonendemic area. Three patients were weakly reactive and one patient was reactive when tested using an enzyme immunoassay to detect serum antibody to B. burgdorferi. No patient, however, had definitive serologic evidence of B. burgdorferi infection by Western blot analysis. We conclude that the prevalence of antibody to B. burgdorferi is very low in children with JRA who reside in a nonendemic area.

    PMID: 2290160 [PubMed - indexed for MEDLINE]

  72. J Med Entomol. 1990 Jul;27(4):671-80.

    Computer simulation of Rocky Mountain spotted fever transmission by the American dog tick (Acari: Ixodidae).

    Cooksey LM, Haile DG, Mount GA.

    Insects Affecting Man and Animals Research Laboratory, USDA-ARS, Gainesville, Florida 32604.

    A computer model was developed for simulation of the transmission of Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF), by the American dog tick, Dermacentor variabilis (Say). The model of RMSF was combined with a model for population dynamics of the American dog tick and included simulation of infection and transmission of rickettsiae between ticks and host mammals and transmission of RMSF to humans. The model simulated the effects of biotic and environmental variables such as weather, host density, habitat, transovarial transmission, fecundity of infected ticks, and infectivity level of ticks and mammals. Some parameters in the model were fitted by iterative simulations to produce realistic rates of R. rickettsii infection in adult ticks and small and medium-sized mammal hosts. Parameters also were fitted to yield the historical average number of RMSF cases for Virginia. Comparisons of the simulated and actual number of cases for nine other states indicated a reasonable level of validity for the model. A theoretical tick density threshold of 252 unfed adult ticks/ha for transmission of RMSF was determined from a relationship between rate of transmission to humans and density of ticks. The transmission threshold can be used for additional modeling efforts to study the effects of management technologies on tick densities and RMSF human cases. The model can serve as a framework for modeling other tick-borne diseases such as Lyme disease, babesiosis, and heartwater.

    PMID: 2388242 [PubMed - indexed for MEDLINE]

  73. Mo Med. 1990 Feb;87(2):86-8.

    Lyme carditis. Severe conduction disorder.

    Mukharji J, DiGrazia J, Galetto D, Vetrovec GW.

    Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond.

    Lyme disease in most cases occurs in the states of Connecticut, Wisconsin, Oregon, California, Missouri and parts of the northeastern coast. Showing the exception to the rule, the authors discuss a case in which a patient acquired the disease on the Eastern Shore of Virginia.

    PMID: 2304446 [PubMed - indexed for MEDLINE]

  74. Am Fam Physician. 1988 Jun;37(6):95-104.

    Tick-borne diseases.

    Petri WA Jr.

    University of Virginia School of Medicine, Charlottesville.

    Tick-borne diseases have their peak incidence in the spring and summer. The different infections caused by tick vectors have certain geographic locations and unique clinical presentations. The most common tick-transmitted infection is Lyme disease. Early diagnosis of tick-borne disease is essential so that effective and, in some cases, lifesaving antibiotic therapy can be instituted. Preventive measures are simple.

    PMID: 3289344 [PubMed - indexed for MEDLINE]

  75. J Rheumatol. 1987 Aug;14(4):772-6.

    IgM rheumatoid factor in Lyme disease: correlation with disease activity, total serum IgM, and IgM antibody to Borrelia burgdorferi.

    Kujala GA, Steere AC, Davis JS 4th.

    Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville.

    We tested the sera of 50 patients with Lyme disease for IgM-rheumatoid factor (IgM-RF) using a sensitive ELISA. Levels of IgM-RF greater than 3 SD above the mean of normal subjects were found in 2 of 15 patients with erythema chronicum migrans, 7 of 10 with neurologic abnormalities, and 7 of 25 with Lyme arthritis (p = 0.038). Only 2 of these sera were positive by latex agglutination. In contrast, none of the 23 control patients with osteoarthritis, ankylosing spondylitis, or Reiter's syndrome had positive tests. The levels of IgM-RF correlated with disease activity (p = 0.002), total serum IgM levels (p = 0.002), and specific IgM antibody titers to Borrelia burgdorferi (p = 0.006). IgM-RF reactivity was absorbed with heat aggregated IgG (HAGG), but the titer of specific IgM antibody was insignificantly affected by this procedure. Thus, small amounts of RF are produced at certain times in many patients with Lyme disease, and IgM-RF production appears to be linked to the specific IgM response.

    PMID: 3668982 [PubMed - indexed for MEDLINE]

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