Cardiac involvement in non-human primates infected with the Lyme disease spirochete Borrelia burgdorferi

To investigate cardiac involvement in the non-human primate (NHP) model of Lyme disease, we inoculated 39 adult Macaca mulatta with Borrelia burgdorferi sensu stricto strains N40 (BbN40) by needle (N=22, 14 immunocompetent (IC), seven permanently immunosuppressed (IS), and four transiently immunosuppressed (TISP)) or by tick-bite (N=4, all TISP) or strain 297 (Bb297) by needle (N=2 IS), or with B. garinii strains Pbi (N=4, 2 TISP and 2 IS), 793 (N=2, TISP) or Pli (N=2, TISP). Five uninfected NHPs were used as controls. Infection and inflammation was studied in the hearts and the aorta removed at necropsy 2-32 months after inoculation by (1) H&E and trichrome-staining; (2) immunohistochemistry and digital image analysis; (3) Western blot densitometry; and (4) TaqMan RT-PCR. All NHPs inoculated with BbN40 became infected and showed carditis at necropsy. The predominant cells were T cells, plasma cells, and macrophages. There was increased IgG and IgM in the heart independent of immunosuppression. The B-cell chemokine BLC was significantly increased in IS-NHPs. There was increased deposition of the complement membrane attack complex (MAC) in TISP and IS-NHPs. The spirochetal load was very high in all BbN40-inoculated IS-NHPs but minimal if any in IC or TISP NHPs. Double-immunostaining revealed that many spirochetes in the heart of BbN40-IS NHPs had MAC on their membranes. We conclude that carditis in NHPs infected with B. burgdorferi is frequent and can persist for years but is mild unless they are immunosupressed.     

中国莱姆病伽氏疏螺旋体蛋白免疫印迹法诊断标准的研究

目的研究中国莱姆病伽氏疏螺旋体基因型蛋白免疫印迹法（WB）的阳性诊断标准。方法采用中国莱姆病螺旋体伽氏疏螺旋体基因型参照菌株PD91作抗原，建立WB检测方法。采用Gel—Pro凝胶分析软件进行结果分析。病例组和对照组各组蛋白条带的频数分布比较采用x^2检验或Fisher’s精确检验，WB检测莱姆病阳性诊断标准确定采用ROC曲线。结果共检测127例莱姆病病例和504例对照的血清标本，经统计学分析得出我国莱姆病伽氏疏螺旋体检测中WB阳性诊断标准为：对于IgG P83／100、P58、P39、P30、OspC、P17、P66、OspA中至少有一条蛋白条带显色即可诊断为阳性，此标准敏感度为73．2％，特异度为99．4％；对于IgM P83／100、P58、OspA、P30、OspC、P17、P41中至少有一条蛋白条带显色则可诊断为阳性，此标准敏感度为50．4％，特异度为93．1％。结论建立了中国莱姆病伽氏疏螺旋体基因型WB阳性诊断标准，用于莱姆病的WB诊断具有较好的特异性。     

Rapid typing of Borrelia burgdorferi sensu lato species in specimens from patients with different manifestations of Lyme borreliosis

To further investigate the pathogenic potential of different Borrelia burgdorferi genospecies, specimens from 27 patients with different manifestations of Lyme borreliosis were analyzed by PCR and reverse line blotting (RLB). In samples from Lyme arthritis patients, B. burgdorferi sensu stricto was predominantly identified, while in patients with neuroborreliosis or acrodermatitis, Borrelia garinii and Borrelia afzelii, respectively, were exclusively detected. The results demonstrate that PCR-RLB is a valuable tool for epidemiological and pathogenetic studies of Lyme borreliosis.     

Western blot analysis of sera from Lyme borreliosis patients according to the genomic species of theBorrelia strains used as antigens

Sera of 52 Lyme borreliosis patients classified according to their clinical features were analysed by Western blot using as antigensBorrelia strains belonging to three recently described genomic species. The antibody response was demonstrated to be homologous within each genospecies. Serum reactivity was studied for each of the type strainsBorrelia burgdorferi sensu stricto (strain B31^T),Borrelia garinii (strain 20047^T) and group VS461. Seven of 15 sera (46.6 %) of patients with menin-goradiculitis showed preferential reactivity withBorrelia garinii (strain 20047^T), all of 8 sera (100 %) of patients with acrodermatitis chronica atrophicans showed preferential reactivity with group VS461 (strain VS461) and 8 of 16 sera (50 %) of patients with arthritis showed preferential reactivity withBorrelia burgdorferi sensu stricto (strain B31^T). The presence of a strong response to OspA and OspB proteins ofBorrelia burgdorferi sensu stricto was found only in this last group of patients. These results suggest that there are clinical implications of the recently described modifications in the taxonomy ofBorrelia burgdorferi.     

Absence of lipopolysaccharide in the Lyme disease spirochete, Borrelia burgdorferi

We were unable to demonstrate the presence of the classic enterobacterium-type lipopolysaccharide in the cells of the Lyme disease spirochete, Borrelia burgdorferi B31. This finding was primarily based on chemical analysis and the absence of free lipid A upon mild acid hydrolysis of the appropriate cell extracts. These results do not preclude the possible existence of an unusual lipopolysaccharide-like compound(s) in B. burgdorferi.     

New method for detection of Borrelia burgdorferi antigen complexed to antibody in seronegative Lyme disease

Serologic tests for Lyme disease are problematic. Because of cross-reactive antigens Borrelia burgdorferi (Bb) shares with other organisms, Lyme disease can be overdiagnosed. However, in addition to specificity problems, serologic tests for early Lyme disease can be falsely negative due to lack of sensitivity of ELISAs and Western blots. Most routine antibody tests are designed to detect free antibodies, and in early, active disease, circulating antibodies may not be free in serum but sequestered in complexes with the antigens which originally triggered their production. This difficulty may be overcome by first isolating immune complexes (IC) from the serum and using this fraction for testing. Free Borrelia-specific antibodies can then be liberated from the immune complexes which may enhance test sensitivity in patients with active disease. We developed a technique that captures the antibody component of IC on immunobeads, and subsequently releases the antigen component of IC. Immunoblotting with monoclonal antibody detected at least one antigen to be OspA, thus definitively demonstrating a Borrelia-specific antigen in circulating IC in early Lyme disease. This test is also useful in demonstrating Bb antigen in otherwise seronegative Lyme disease patients.     

Involvement of birds in the epidemiology of the Lyme disease agent Borrelia burgdorferi.

Borrelia burgdorferi, the causative agent of Lyme disease, was isolated from the liver of a passerine bird, Catharus fuscescens (veery), and from larval Ixodes dammini (tick) feeding on Pheucticus ludovicianus (rose-breasted grosbeak) and Geothlypis trichas (common yellowthroat). In indirect immunofluorescence antibody tests, isolates reacted with polyclonal and monoclonal (H5332) antibodies. Studies on the DNA composition of the veery liver isolate and the strain cultured from an I. dammini larva indicated that both were B. burgdorferi and not Borrelia anserina or Borrelia hermsii. The veery liver isolate infected hamsters and a chick. In contrast, B. anserina infected chicks but not hamsters. B. burgdorferi is unique among Borrelia spp. in being infectious to both mammals and birds. We suggest that the cosmopolitan distribution of B. burgdorferi may be caused by long-distance dispersal of infected birds that serve as hosts for ticks.     

Changes in temporal and spatial patterns of outer surface lipoprotein expression generate population heterogeneity and antigenic diversity in the Lyme disease spirochete,Borrelia burgdorferi

Borrelia burgdorferi differentially expresses many of the OspE/F/Elp paralogs during tick feeding. These findings, combined with the recent report that stable B. burgdorferi infection of mammals occurs only after 53 h of tick attachment, prompted us to further analyze the expression of the OspE/F/Elp paralogs during this critical period of transmission. Indirect immunofluorescence analysis revealed that OspE, p21, ElpB1, ElpB2, and OspF/BbK2.11 are expressed in the salivary glands of ticks allowed to feed on mice for 53 to 58 h. Interestingly, many of the spirochetes in the salivary glands that expressed abundant amounts of these antigens were negative for OspA and OspC. Although prior reports have indicated that OspE/F/Elp orthologs are surface exposed, none of the individual lipoproteins or combinations of the lipoproteins protected mice from challenge infections. To examine why these apparently surface-exposed lipoproteins were not protective, we analyzed their genetic stability during infection and their cellular locations after cultivation in vitro and within dialysis membrane chambers, mimicking a mammalian host-adapted state. Combined restriction fragment length polymorphism and nucleotide sequence analyses revealed that the genes encoding these lipoproteins are stable for at least 8 months postinfection. Interestingly, cellular localization experiments revealed that while all of these proteins can be surface localized, there were significant populations of spirochetes that expressed these lipoproteins only in the periplasm. Furthermore, host-specific signals were found to alter the expression patterns and final cellular location of these lipoproteins. The combined data revealed a remarkable heterogeneity in populations of B. burgdorferi during tick transmission and mammalian infection. The diversity is generated not only by temporal changes in antigen expression but also by modulation of the surface lipoproteins during infection. The ability to regulate the temporal and spatial expression patterns of lipoproteins throughout infection likely contributes to persistent infection of mammals by B. burgdorferi.     

Spinal cord involvement in the nonhuman primate model of Lyme disease

Lyme borreliosis is a multisystemic disease caused by infection with various genospecies of the spirochete Borrelia burgdorferi. The organs most often affected are the skin, joints, the heart, and the central and peripheral nervous systems. Multiple neurological complications can occur, including aseptic meningitis, encephalopathy, facial nerve palsy, radiculitis, myelitis, and peripheral neuropathy. To investigate spinal cord involvement in the nonhuman primate (NHP) model of Lyme borreliosis, we inoculated 25 adult Macaca mulatta with B. burgdorferi sensu strictu strains N40 by needle (N=9) or by tick (N=4) or 297 by needle (N=2), or with B. burgdorferi genospecies garinii strains Pbi (N=4), 793 (N=2), or Pli (N=4) by needle. Immunosuppression either transiently (TISP) or permanently (IS) was used to facilitate establishment of infection. Tissues and fluids were collected at necropsy 7-24 weeks later. Hematoxylin and eosin staining was used to study inflammation, and immunohistochemistry and digital image analysis to measure inflammation and localize spirochetes. The spirochetal load and C1q expression were measured by TaqMan RT-PCR. The results showed meningoradiculitis developed in only one of the 25 NHP's examined, TISP NHP 321 inoculated with B. garinii strain Pbi. Inflammation was localized to nerve roots, dorsal root ganglia, and leptomeninges but rarely to the spinal cord parenchyma itself. T cells and plasma cells were the predominant inflammatory cells. Significantly increased amounts of IgG, IgM, and C1q were found in inflamed spinal cord. Taqman RT-PCR found spirochetes in the spinal cord only in IS-NHP's, mostly in nerve roots and ganglia rather than in the cord parenchyma. C1q mRNA expression was significantly increased in inflamed spinal cord. This is the first comprehensive study of spinal cord involvement in Lyme borreliosis.     

Identification and functional characterization of complement regulator-acquiring surface protein 1 of the Lyme disease spirochetes Borrelia afzelii and Borrelia garinii

Complement regulator-acquiring surface protein 1 (CRASP-1) is the dominant factor-H-like protein 1 (FHL-1)- and factor-H-binding protein of Borrelia burgdorferi and is suggested to contribute to persistence of the pathogen. The prototype CRASP-1 of B. burgdorferi sensu stricto (CRASP-1Bb) has been formerly characterized. As shown recently, serum-resistant Borrelia afzelii strains express a unique FHL-1 and factor H-binding protein, designated CRASP-1Ba. Here, we describe for the first time the isolation and functional characterization of the gene encoding the full-length CRASP-1Ba of 28 kDa, which, upon processing, is predicted to be 26.4 kDa. CPASP-1Ba of B. afzelii spirochetes is associated with a genetic locus encoding the orthologous gbb54 gene family that maps to the linear plasmid of approximately 54 kb. Ligand affinity blotting techniques demonstrate that both native and recombinant CRASP-1Ba molecules strongly bind to FHL-1 and much more weakly to factor H. The FHL-1 and factor-H-binding site in CRASP-1Ba is shown to be localized to a 12-amino-acid residue domain at the C terminus of the protein. For comparison, the corresponding cspA-like gene(s) of a serum-sensitive Borrelia garinii strain has also been cloned and characterized. Most notably, two CRASP-1-related B. garinii proteins were identified; however, both molecules bind only weakly to FHL-1 and not at all to factor H. The present identification of the binding site of CRASP-1Ba represents an important step forward in our understanding of the pathogenesis of Lyme disease and may be helpful to design therapeutic regimens to interfere with complement evasion strategies of human pathogenic Borrelia strains.     

Measurement of antibodies to the Borrelia burgdorferi flagellum improves serodiagnosis in Lyme disease.

The isolation of Borrelia burgdorferi flagella and an enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin G (IgG) and IgM to the B. burgdorferi flagellum are described. The diagnostic performance of the flagellum ELISA for serodiagnosis of Lyme disease was compared with the performance of a traditional whole cell B. burgdorferi sonic extract ELISA. We examined sera and cerebrospinal fluid (CSF) from 56 patients with lymphocytic meningoradiculitis (Bannwarth's syndrome), the most frequent secondary-stage manifestation of Lyme disease in Europe. Two hundred healthy individuals and patients with aseptic meningitis, encephalitis, Guillain-Barré syndrome, and syphilis served as controls. The flagellum ELISA was significantly more sensitive than the sonic extract ELISA. The diagnostic sensitivities were increased from 41.1 to 76.8% (P less than 0.01) for IgG and from 35.7 to 67.9% (P less than 0.05) for IgM detection in serum. The increase in sensitivity was most pronounced in patients with a short duration of disease (less than 20 days after onset). The diagnostic specificity increased for IgG detection but was almost unaltered for IgM. The flagellum ELISA did not improve the diagnostic sensitivity of measuring antibodies to borreliae in CSF, most likely owing to the low level of unspecific antibodies in CSF compared with serum. The cross-reactivity of sera and CSF from patients with syphilis decreased significantly. The flagellum antigen of B. burgdorferi shows no strain variation, is easy to purify in sufficient quantity, and is therefore a suitable reference antigen for routine serodiagnosis of Lyme disease.     

Temperature-related differential expression of antigens in the Lyme disease spirochete, Borrelia burgdorferi.

Previous studies have demonstrated that Borrelia burgdorferi in the midguts of infected ticks shows increased expression of the antigenic outer surface protein OspC after the ticks have ingested a blood meal. This differential expression is at least partly due to a change in temperature, as an increase in OspC levels is also observed when cultures are shifted from 23 to 35 degrees C. Immunoblotting of bacterial lysates with sera from infected mice indicated that the levels of several additional antigens were also increased in bacterial cultures shifted to 35 degrees C; we have identified one antigen as OspE. We have also observed differential expression of OspF, which has been proposed to be coexpressed in an operon with the gene encoding OspE.     

Disulfide-mediated oligomer formation in Borrelia burgdorferi outer surface protein C, a critical virulence factor and potential Lyme disease vaccine candidate

Borrelia burgdorferi OspC is an outer membrane lipoprotein required for the establishment of infection in mammals. Due to its universal distribution among B. burgdorferi sensu lato strains and high antigenicity, it is being explored for the development of a next-generation Lyme disease vaccine. An understanding of the surface presentation of OspC will facilitate efforts to maximize its potential as a vaccine candidate. OspC forms homodimers at the cell surface, and it has been hypothesized that it may also form oligomeric arrays. Here, we employ site-directed mutagenesis to test the hypothesis that interdimeric disulfide bonds at cysteine 130 (C130) mediate oligomerization. B. burgdorferi B31 ospC was replaced with a C130A substitution mutant to yield strain B31::ospC(C130A). Recombinant protein was also generated. Disulfide-bond-dependent oligomer formation was demonstrated and determined to be dependent on C130. Oligomerization was not required for in vivo function, as B31::ospC(C130A) retained infectivity and disseminated normally. The total IgG response and the induced isotype pattern were similar between mice infected with untransformed B31 and those infected with the B31::ospC(C130A) strain. These data indicate that the immune response to OspC is not significantly altered by formation of OspC oligomers, a finding that has significant implications in Lyme disease vaccine design.     

Detection of Borrelia burgdorferi in patients with Lyme disease by the polymerase chain reaction.

Borrelia burgdorferi, the causative agent of Lyme disease, was detected in patients' serum by DNA amplification using the polymerase chain reaction (PCR). B burgdorferi was pelleted from serum samples by centrifugation (10,000 x g for 10 minutes) and lysed by treatment with ammonium hydroxide (100 degrees C for 15 minutes). Two pairs of "nested" PCR primers complementary to the gene encoding a major outer surface protein (OSP A) of B burgdorferi were used in DNA amplification under standard PCR conditions (Perkin-Elmer Cetus). Two out of five patients with erythema migrans, the characteristic primary skin lesion associated with early Lyme disease, were positive by the PCR. This method could form the basis of a useful routine laboratory test in those cases of early Lyme disease where conventional serological testing commonly yields equivocal or false negative results.     

Molecular detection of persistent Borrelia burgdorferi in the urine of patients with active Lyme disease.

Current diagnostic tests for Lyme disease (LD) are dependent upon the host serologic response and are insensitive early in infection and, possibly, following antibiotic therapy. We cloned a library of Borrelia burgdorferi 297 DNA and studied one clone, Ly-1, for its potential in diagnostic and pathogenic studies. Using pulsed-field electrophoresis, we demonstrated that Ly-1 is of chromosomal origin and estimated that the B. burgdorferi chromosome is approximately 1,100 kb in size. The 3.7-kb Ly-1 clone hybridizes with geographically diverse strains of B. burgdorferi. No cross hybridization occurs with DNA from human cells, Escherichia coli, Staphylococcus aureus, Clostridium difficile, or the closely related B. hermsii. We used a dot blot assay to detect 100 pg of B. burgdorferi DNA. We partially determined the nucleotide sequence of Ly-1 and used it to select and synthesize oligonucleotides for use in the polymerase chain reaction (PCR). Two different primer pairs were found to amplify DNA from nine geographically diverse isolates. We could detect 10 fg (less than 10 molecules) of B. burgdorferi or less than five spirochetes added to human urine. Finally, we were able to use the PCR to detect B. burgdorferi DNA in the urine of four of eight patients with suspected active LD (three with arthritis and one with neurologic manifestations), all of whom responded to antibiotic treatment. In contrast, those patients who were PCR negative either had inactive disease or had been appropriately treated and did not respond to additional antibiotics, and all four control urine specimens were PCR negative. We conclude that B. burgdorferi DNA can be sensitively detected by the PCR with the primers and methods we describe and that the urinary tract is a site of persistent infection in some cases of human LD, an observation of potential diagnostic and pathogenic importance.     

Reassessment of a midwestern Lyme disease focus for Borrelia burgdorferi and the human granulocytic ehrlichiosis agent

Previous studies from the late 1980s defined the risk of human Lyme disease by determining the prevalence of Borrelia burgdorferi infection in Ixodes scapularis ticks and Peromyscus sp. mice captured from areas around La Crosse, Wis. High percentages of B. burgdorferi-infected I. scapularis ticks and P. leucopus mice were common in areas located north of Interstate 90 but were not detected in areas south of this major east-west thoroughfare. In this study, we reevaluated the extent of B. burgdorferi infection. High percentages of mice captured from sites north of the interstate were still infected with B. burgdorferi. In addition, B. burgdorferi was recovered from 12 (67%) of 18 mice captured from a site well south of the highway. However, none of 104 mice or 713 I. scapularis ticks captured from the study sites were infected with Ehrlichia spp. The results confirmed the continued high risk for humans to contract infection with B. burgdorferi and the significant southward expansion of the area in which Lyme disease is endemic. In contrast, the risk of acquiring human granulocytic ehrlichiosis remains minimal despite the abundance of appropriate vector ticks and reservoir rodents.     

Ticks and biting insects infected with the etiologic agent of Lyme disease, Borrelia burgdorferi.

Members of 18 species of ticks, mosquitoes, horse flies, and deer flies were collected in southeastern Connecticut and tested by indirect fluorescent-antibody staining methods for Borrelia burgdorferi, the etiologic agent of Lyme disease. An infection rate of 36.2% (116 tested), recorded for immature Ixodes dammini, exceeded positivity values for all other arthropod species. Prevalence of infection for hematophagous insects ranged from 2.9% of 105 Hybomitra lasiophthalma to 14.3% of seven Hybomitra epistates. Infected I. dammini larvae and nymphs coexisted with infected Dermacentor variabilis (American dog tick) immatures on white-footed mice (Peromyscus leucopus), but unlike I. dammini, none of the 55 adult American dog ticks collected from vegetation harbored B. burgdorferi. Groups of 113 field-collected mosquitoes of Aedes canadensis and 43 Aedes stimulans were placed in cages with uninfected Syrian hamsters. Of these, 11 females of both species contained B. burgdorferi and had fed fully or partially from the hamsters. No spirochetes were isolated from the hamsters, but antibodies were produced in one test animal.     

Variable serum immunoglobulin responses against different Borrelia burgdorferi sensu lato species in a population at risk for and patients with Lyme disease.

Borrelia burgdorferi sensu lato species display considerable antigenic polymorphism. In order to evaluate the importance of this antigenic heterogeneity in the serodiagnosis of Lyme disease, the serum immunoglobulin G response in 148 healthy individuals from an area in northern Sweden where Lyme disease is endemic and in 40 American patients with Lyme disease was assessed. In a seroprevalence study, the control group included 173 individuals from a region of northern Sweden where Lyme disease is not endemic. The two enzyme immunoassays used were based on outer membrane-associated proteins of either B. burgdorferi sensu stricto or Borrelia garinii. The Swedish populations were also screened for antiflagellum seroreactivity. The individuals from the area of endemicity were significantly more seropositive for the subcellular protein fraction of the local B. garinii isolate NBS16 than the control group (11.5 versus 2.9%; P = 0.005) but were not significantly more positive for the other antigens used. In contrast, American patients with Lyme disease were significantly more reactive against the North American B. burgdorferi sensu stricto strain B31 than against B. garinii NBS16 (57.5 versus 15.0%; P = 0.0001). Immunoblot analysis suggests that the borrelial outer surface protein C is involved in triggering the production of species-specific antibody during localized Lyme disease. We conclude that a species-specific immune response develops during infection with Lyme disease Borrelia spp. Thus, the reliability of a serological investigation of Lyme disease increases when one measures antibody titers against the outer membrane proteins of Lyme disease Borrelia spp. occurring in a particular geographic region.