The enolase of Borrelia burgdorferi is a plasminogen receptor released in outer membrane vesicles.

The agent of Lyme disease, Borrelia burgdorferi, has a number of outer membrane proteins that are differentially regulated during its life cycle. In addition to their physiological functions in the organism, these proteins also likely serve different functions in invasiveness and immune evasion. In borreliae, as well as in other bacteria, a number of membrane proteins have been implicated in binding plasminogen. The activation and transformation of plasminogen into its proteolytically active form, plasmin, enhances the ability of the bacteria to disseminate in the host. Outer membrane vesicles of B. burgdorferi contain enolase, a glycolytic-cycle enzyme that catalyzes 2-phosphoglycerate to form phosphoenolpyruvate, which is also a known plasminogen receptor in Gram-positive bacteria. The enolase was cloned, expressed, purified, and used to generate rabbit antienolase serum. The enolase binds plasminogen in a lysine-dependent manner but not through ionic interactions. Although it is present in the outer membrane, microscopy and proteinase K treatment showed that enolase does not appear to be exposed on the surface. However, enolase in the outer membrane vesicles is accessible to proteolytic degradation by proteinase K. Samples from experimentally and tick-infected mice and rabbits as well as from Lyme disease patients exhibit recognition of enolase in serologic assays. Thus, this immunogenic plasminogen receptor released in outer membrane vesicles could be responsible for external proteolysis in the pericellular environment and have roles in nutrition and in enhancing dissemination.

Phylogenetic analysis of a virulent Borrelia species isolated from patients with relapsing fever.

Multilocus sequence analysis (MLSA) was used to clarify the taxonomic status of a virulent Borrelia organism previously isolated from patients with relapsing fever and from ticks in Spain that is designated the Spanish relapsing fever (SRF) Borrelia. This species has been used extensively in experimental infection models because of its continued virulence. Seven genes were amplified to analyze the phylogenetic relationships among several Spanish isolates of SRF Borrelia and other relapsing fever Borrelia species. The genes targeted in this study included rrs and flaB, which have commonly been used in phylogenetic studies; the rrf-rrl intergenic spacer (IGS), which is highly discriminatory; and four additional genes, p66, groEL, glpQ, and recC, which are located on the chromosome and which have therefore evolved in a clonal way. The species included in this study were Borrelia duttonii, B. recurrentis, B. crocidurae, and B. hispanica as Old World Borrelia species and B. turicatae and B. hermsii as New World Borrelia species. The results obtained by MLSA of the SRF Borrelia on the basis of 1% of the genomic sequence data analyzed confirmed that the SRF Borrelia isolates are B. hispanica. However, the prototype isolates of B. hispanica used in this study have an uncertain history and display unique phenotypic characteristics that are not shared with the SRF Borrelia. Therefore, we propose to use strain SP1, isolated from a relapsing fever patient in 1994 in southern Spain, as the type strain for B. hispanica.

The important and diverse roles of antibodies in the host response to Borrelia infections.

Antibodies are of critical importance in the host response to tick-borne Borrelia species that cause relapsing fever and Lyme disease. Recent studies on the role of various B cell subsets in the host response to Borrelia, complement-independent, bactericidal antibodies, and diagnostics led to this review that focuses on the array of functions that antibodies to Borrelia can perform.

Lyme disease-a tick-borne spirochetosis?

A treponema-like spirochete was detected in and isolated from adult Ixodes dammini, the incriminated tick vector of Lyme disease. Causally related to the spirochetes may be long-lasting cutaneous lesions that appeared on New Zealand White rabbits 10 to 12 weeks after infected ticks fed on them. Samples of serum from patients with Lyme disease were shown by indirect immunofluorescence to contain antibodies to this agent. It is suggested that the newly discovered spirochete is involved in the etiology of Lyme disease.

Natural Distribution of the Ixodes dammini spirochete.

Spirochetes believed to be the cause of Lyme disease were isolated from white-footed mice and white-tailed deer, the preferred natural hosts of Ixodes dammini, the tick vector. Evidence suggests that deer act as a reservoir of the disease and provide an overwintering mechanism for both spirochetes and adult ticks. Some tick larvae may acquire the spirochete by transovarial passage and the nymphal stage may transmit the disease to humans.

Identification and characterization of an endoflagellar antigen of Borrelia burgdorferi.

The 41-kD antigen of Borrelia burgdorferi is an immunodominant protein that is recognized early by antibodies in sera from Lyme disease patients and known to be associated with the endoflagella. We identified the 41-kD endoflagellar antigen to be a single polypeptide with an apparent isoelectric point (pI) of 6.5 by two-dimensional (2-D) electrophoresis. This polypeptide, which we designated P41F alpha, was heavily labeled by 125I in 2-D autoradiographs of B. burgdorferi whole-cell lysates and was recognized by a murine monoclonal antibody (MCB1) and human antisera in 2-D immunoblots. NH2-terminal sequence analysis showed 80% homology between P41F alpha and the 33-kD endoflagellar protein of Treponema pallidum. Results of indirect immunofluorescence assays (IFA), Triton X-114 phase partitioning, and agglutination studies suggested a possible surface exposure of the polypeptide. Silver stained 2-D gels also revealed the presence of another 41-kD species, with an apparent pI of 6.6 (designated P41 beta), which was not radioiodinated in 2-D autoradiographs, and was not recognized by MCB1 or human antisera. NH2-terminal sequence analysis of P41 beta revealed no homology with P41F alpha, leading to the conclusion that they are not related.

Interaction between Borrelia burgdorferi and endothelium in vitro.

During the pathogenesis of Lyme disease, Borrelia burgdorferi spreads hematogenously from the site of a tick bite to several tissues throughout the body. The specific mechanism of spirochete emigration is presently unknown. Using cultured human umbilical vein endothelial cells, we found that Borrelia burgdorferi bound to the endothelial cells and to the subendothelial matrix. Low passage isolates adhered 22-30-fold greater than a strain maintained in culture continuously. Spirochete binding to subendothelial matrix was inhibited 48-63% by pretreatment of the matrix with anti-fibronectin antiserum. Spirochete migration across endothelial monolayers cultured on amniotic membrane was increased when the monolayers were damaged by chemical or physical means. Electron microscopic examination of spirochete-endothelial interactions demonstrated the presence of spirochetes in the intercellular junctions between endothelial cells as well as beneath the monolayers. Scanning electron microscopy identified a mechanism of transendothelial migration whereby spirochetes pass between cells into the amniotic membrane at areas where subendothelium is exposed.

The plasminogen activation system enhances brain and heart invasion in murine relapsing fever borreliosis.

The role of the plasminogen activation system (PAS) was investigated during the course of infection of a relapsing fever Borrelia species in plasminogen-deficient (plg -/-) and control (plg +/+ and plg +/-) mice. Subcutaneous inoculation of 10(4) spirochetes resulted in a peak spirochetemia five days after infection with 20-23 x 10(6) organisms per milliliter of whole blood in all mice, indicating that the PAS had no effect on the development of this phase of the infection. Anemia, thrombocytopenia, hepatitis, carditis, and splenomegaly were noted in all mice during and immediately after peak spirochetemia. Fibrin deposition in organs was noted in plg -/- mice but not in controls during these stages. Significantly greater spirochetal DNA burdens were consistently observed in the hearts and brains of control mice 28-30 days after infection, as determined by PCR amplification of this organism's flagellin gene (flaB), followed by quantitative densitometry. Furthermore, the decreased spirochetal load in brains of plg -/- mice was associated with a significant decrease in the degree of inflammation of the leptomeninges in these mice. These findings indicate a role for the PAS in heart and brain invasion by relapsing fever Borrelia, resulting in organ injury.

Glycolytic enzyme operon of Borrelia burgdorferi: characterization and evolutionary implications.

The genes encoding three enzymes of the glycolytic pathway have been identified and sequenced completely in Borrelia burgdorferi sensu stricto and partially in B. hermsii. They are clustered on the chromosome into an operon with a single putative promoter and are arranged downstream of this promoter in the following order: gapdh (glyceraldehyde-3-phosphate dehydrogenase), pgk (phosphoglycerate kinase), and tpi (triosephosphate isomerase). gapdh and pgk are separated by 19 bp of intergenic sequence and pgk and tpi are separated by only 1 bp. Each of the three genes contains a putative RBS 6-7 bp upstream of each respective translational (ATG) start codon. The deduced protein encoded by gapdh consists of 335 amino acids (aa) with a predicted MW of 36,400, that of pgk is 393 aa (MW of 42,156) and that of tpi is 290 aa (MW of 27,683). The aa sequences of each of the three enzymes share 58.4% (GAPDH), 52.8% (PGK) and 46.1% (TPI) identity with respective enzymes from other prokaryotic organisms. Phylogenetic analyses based on these universal and conserved proteins support the hypothesis that spirochetes are an ancient and distinct eubacterial phylum.

The effect of age on the infection and immunoresponsiveness of mice to Babesia microti.

5he effect of age on the immunological responses to Babesia microti infection in the mouse was investigated. Aged mice experienced reduced and delayed peak parasitemias compared to younger animals; however, the old mice failed to clear the parasites from the blood and experienced fluctuating parasitemias until death. Babesiosis produced suppression of responses to nonspecific B and T cell mitogens concomitant with rising autoantibody plaque forming cells reactive with untreated and bromelain modified mouse erythrocytes. Similar observations of increased susceptibility to babesiosis with age and immunosuppression have been made in human babesiosis. Thus, the murine model for this hemoprotozoan infection is faithful to the human immunological responses.

Borrelia burgdorferi and other related spirochetes bind to galactocerebroside.

Spirochetes are agents of neurologic disease that may utilize specific neural cell surface molecules for adhesion. Borrelia burgdorferi, the etiologic agent of Lyme disease, bound to galactocerebroside (GalCer) in numbers that were two- to threefold greater than to ceramide and glucocerebroside, and four- to fivefold greater than to sphingosine, psychosine, sulfatide, cholesterol, and three membrane phospholipids. The adherence was greater to GalCer and ceramide with a higher content of alpha-hydroxyl fatty acids. Treponema phagedenis Reiter and Borrelia hermsii also bound to GalCer. The binding of B burgdorferi to GalCer was inhibited in a concentration-dependent manner by rabbit polyclonal and murine monoclonal antibodies to this glycosphingolipid component of myelin. The monoclonal antibody to GalCer also inhibited adhesion of the organisms to Schwann cells. Neither free D or L monosaccharides nor the lectin peanut agglutinin inhibited binding. Since B burgdorferi and other spirochetes cause neurologic disease, these results suggest a role for GalCer as a binding site in both the central and peripheral nervous systems.

Detection of Borrelia burgdorferi-specific antigen in antibody-negative cerebrospinal fluid in neurologic Lyme disease.

OBJECTIVE: To determine the potential of detection in CSF of specific Borrelia burgdorferi antigen, OspA, as a marker of infection in neurologic Lyme disease and compare this with the detection of antibody. DESIGN: CSF from 83 neurologic patients in an area highly endemic for Lyme disease was examined prospectively for (1) OspA by antigen capture ELISA and Western blot employing monoclonal antibodies, and for (2) B burgdorferi antibodies by ELISA. RESULTS: Of the 35 of 83 (42%) patients who were positive for OspA antigen in their CSF, 15 (43%) were antigen positive despite being antibody-negative in CSF. Seven of these 15 (47%) had otherwise normal routine CSF analyses. Six of these 15 (40%) patients met strict CDC surveillance criteria for Lyme disease; four (27%) patients had seroconversion coincident with new neurologic problems; and three (20%) with characteristic syndromes for Lyme disease were seronegative, but had complexed antibody to B burgdorferi. The final two patients (13%) were seropositive and had unexplained neurologic problems not characteristic of Lyme disease. CONCLUSIONS: B burgdorferi antigen can be detected in CSF that is otherwise normal by conventional methodology, and can be present without positive CSF antibody. Since CSF antigen implies intrathecal seeding of the infection, the diagnosis of neurologic infection by B burgdorferi should not be excluded solely on the basis of normal routine CSF or negative CSF antibody analyses.

Babesiosis in splenectomized adults. Review of 22 reported cases.

Since 1957, there have been 22 reported cases of human babesiosis in splenectomized persons, representing about one third of all clinical human babesiosis. Splenectomy had been performed one month to 36 years (mean 8.7 years, median 6.0 years) earlier for a variety of reasons. Four of the seven European cases were from Babesia divergens whereas 12 of the 15 United States cases were from B. microti. Most of the 22 patients had moderate to severe clinical disease including hemolytic anemia, yet all but six recovered. Three patients had transfusion-acquired babesiosis. Treatments employed included the use of chloroquine, quinine, pyrimethamine, pentamidine, clindamycin, dialysis, and exchange transfusion. Splenectomized and/or otherwise immunocompromised hosts should be advised to avoid visiting endemic areas for babesiosis such as Nantucket Island or Martha's Vineyard in Massachusetts and Shelter Island and other parts of Long Island, New York. Babesiosis must be considered as one of the not uncommon organisms responsible for the postsplenectomy sepsis syndrome and one for which there is no current prophylaxis.

Seroprevalence and seroconversion for tick-borne diseases in a high-risk population in the northeast United States.

PURPOSE: To determine the prevalence of serologic reactivity, the 1-year incidence of seroconversion, and the frequency of multiple infections, and their associations with symptoms in a group of volunteers at high risk for tick-borne infections in New York state. METHODS: We performed a seroepidemiologic study of Lyme borreliosis, 2 of the ehrlichioses, Rocky Mountain spotted fever, and babesiosis among 671 participants who lived or worked in a high-risk area (mainly in eastern Long Island, New York) for tick-borne diseases. Sera were collected in the winters of 1994 and 1995. Signs and symptoms of tick-borne disease were monitored monthly by mail and telephone. Lyme borreliosis serologies were done by enzyme-linked immunosorbent assay and Western blot. Rocky Mountain spotted fever serologies were initially screened using Dip-S-Ticks, followed by specific indirect immunofluorescence. Ehrlichiosis serologies were determined by epifluorescent microscopy, as were antibodies to Babesia microti. RESULTS: Of the 671 participants, 88 (13%) had antibodies to > or = 1 tick-borne organisms, including 34 (5% of the total) with antibodies to Borrelia burgdorferi. Twenty-seven participants had evidence of exposure to B. burgdorferi at baseline. Seven participants (1%) seroconverted during the course of the study, 5 of whom were symptomatic for Lyme borreliosis. Antibodies to spotted fever group rickettsiae were seen in 28 participants (4%), 22 of whom were positive at baseline and 6 of whom seroconverted during the observation period. None of the seropositive patients had any symptoms or signs of infection. Twenty-four participants (3%) had serologic evidence of exposure to Ehrlichia (all but one to Ehrlichia equi); 5 (0.7%) seroconverted during the observation period, including 3 subjects who were asymptomatic. Antibodies to B. microti were seen in 7 participants (1%), including one asymptomatic seroconversion during the year of observation. There was evidence of possible dual infection in 5 patients. CONCLUSION: In a high-risk population, there was evidence of exposure to 5 tick-borne pathogens; however, many infections were asymptomatic, and coinfections were rare.

Lyme disease in childhood: clinical and epidemiologic features of ninety cases.

In 1982 and 1983 practicing pediatricians in a Lyme disease-endemic county, reported 90 cases of Lyme disease among children 19 years of age and younger (median age, 9 years). Three-fourths of the children had initial symptom onset in the summer months, with peak incidence in July. Infection occurred twice as often in boys than in girls, and tick bites were recalled by less than half (49%) of the children or parents. Erythema chronicum migrans was present in two-thirds (67%) of the cases with median onset 7 days after a definite tick bite. Arthritis or arthralgia occurred in 59% and neurologic symptoms, especially seventh nerve palsy, occurred in 14%. Asymmetric involvement of a few large joints, especially the knee, was most commonly reported for those with joint involvement. antibiotics were prescribed for 79% of the children, three-fourths of whom were treated with oral penicillin. Initial diagnosis of Lyme disease is usually made on clinical grounds alone because serologic tests are often negative. Serologic tests for antibody to Borrelia burgdorferi were more often positive in cases with neurological or joint involvement, in addition to erythema chronicums migrans (80%), than in cases presenting with erythema chronicums migrans only.

The pathogenesis of Lyme disease.

Lyme disease affects several major organ systems and leads to chronic illness. The pathogenesis of this disease appears to be centered around the long-term persistence of the organisms in tissues. In Lyme disease, isolations of B. burgdorferi are rare. It is thought that few organisms actually invade the host and that host mediators amplify the inflammatory response. Immune and nonimmune phagocytosis leading to bacterial killing occurs in Lyme disease. This organism shows preference for cell surfaces and tissues which may explain the paucity of isolations but also displays characteristic nonspecificity in its adherence to eukaryotic cells. This lack of specificity may explain its capacity to reside and injure vastly different tissues. Autoimmune mechanisms may coincide with spirochetal persistence in the pathogenesis of chronic Lyme disease.

Occupational risk of Lyme disease in endemic areas of New York State.

Although Lyme disease (LD) is the most common tick-borne disease in the United States, little is known about the frequency of and risk factors for infection with Borrelia burgdorferi in occupational groups. In 1986, we recruited primarily outdoor workers from six employee groups in southeastern New York where LD is endemic. Of 414 participants who completed questionnaires and had blood samples tested for antibodies against B. burgdorferi by ELISA and Western immunoblot, 27 (6.5%) were seropositive, but only 14 of the 27 reported previous symptoms of LD. Persons who spent more than 30 hours per week outdoors during leisure were 2.5 times more likely to be seropositive than those who did not (p = .02). Those with a history of outdoor employment were twice as likely to be seropositive as those without such a history, although this finding was not statistically significant (p = .70). However, the seroprevalence rate for the employees was 5.9 times higher than the rate for a comparison group of anonymous blood donors from the same region of New York (p less than .001). These results suggest that there was a relatively high rate of seropositivity for the employee groups and that infection was frequently asymptomatic and associated with outdoor exposure.

Biological activity of Borrelia burgdorferi antigens.

OSP-A (approximately 31 kDa) and flagellins (approximately 41 kDa) are prominent antigens of Borrelia burgdorferi. Both OSP-A and flagellins are immunogenic in patients and in experimentally infected mice and hamsters, but the kinetics of antibody formation to each vary considerably between the species. The role of eluted OSP-A and flagellins in the cellular immune response, chemotaxigenesis, and cytoadherence was measured. Eluted OSP-A and flagellins stimulated the proliferation of normal and infected mouse splenocytes but only the peripheral mononuclear cells of patients. Both OSP-A and flagellins induced human neutrophil chemotaxis, but at significantly reduced levels as compared to other known chemotactic peptides. Live B. burgdorferi adhere to HEp-2 cells in culture. OSP-A and the flagellins are involved in adherence; monoclonal antibodies to determinants in these proteins partially inhibited adherence. Cytoadherence was also partially inhibited by treatment of the cells with tunicamycin and sialidase.

Babesiosis in Long Island. Host-parasite relationships of rodent- and human-derived Babesia microti isolates in hamsters.

The effects of splenectomy and size of inoculum on response of hamsters to three isolates of Babesia microti (two rodent- and one human-derived) from Long Island were studied. Splenectomy of hamsters did not enhance susceptibility to the rodent isolates of B. microti at a dosage of 5 X 10(7) parasites. Larger parasite inocula produced shorter prepatent periods and slightly shorter duration of infection in intact hamsters. Inoculum size was not contributory to mortality of hamsters or to pathogenesis. Hamsters showed profound anemia with depressed hematocrit and hemoglobin values and erythrocyte counts. Moderate leucocytosis was seen just prior to peak parasitemia, with immature polymorphonuclear cells predominating. Infections in hamsters lasted for 14--17 weeks. As determined by the parameters studied, the three isolates appear to be identical.

Immunological relationships of Long Island isolates of Babesia microti.

Studies to detect strain differences among two rodent-derived and one human-derived Babesia microti isolates from Long Island were undertaken, using various methods. Superinfection experiments using the homologous and heterologous isolates showed cross-protection. All hamsters were resistant to superinfection challenges of increasing dosages of both the homologous and heterologous isolates. Attempts to infect other laboratory animals with the Long Island isolates of B. microti were successful in intact and splenectomized Sprague-Dawley rats and questionable in Swiss mice. Nylar and CFW mice as well as CFW and Wistar intact and splenectomized rats were refractory to B. microti isolates from Long Island. Indirect fluorescence tests using convalescent sera from six Long Island cases of babesiosis showed no titer differences with tests using the three Long Island antigens as well as the Gray strain antigens. The rise of hamster IgG anti-B. microti antibody was followed by indirect immunofluorescence done at different parasitemia levels. The IgG antibody in hamsters was detected early in the course of infection, rose rapidly concurrent with increasing parasitemia, and became stable at high titers for the duration of the infection. IgG antibody titers were unaffected by homologous superinfection challenges.