Structural analysis of the variable major proteins of Borrelia hermsii.

Borrelia hermsii undergoes spontaneous antigenic variation in vivo and in vitro. Serotype specificity is associated with expression of one of a family of molecular weight-variable proteins, the pI proteins. We studied the structure of the pI proteins as well as the molecular weight-invariable pII proteins of three serotypes of B. hermsii HS1: C, 7, and 21. The techniques used were one-dimensional (1-D) mapping of Staphylococcus aureus V8 protease-generated peptides and two-dimensional (2-D) mapping of alpha-chymotrypsin-generated peptides. The pI and pII proteins were isolated by excision of polypeptides from stained polyacrylamide gel electropherograms. The 1-D peptide patterns were visualized by fluorography of intrinsically [14C]leucine-labeled proteins or by silver stain. Before 2-D mapping, polypeptides in excised gel fragments were labeled with 125I in the presence of chloramine-T. We also compared the 2-D peptide maps of pI proteins, pI7 and pI21, after their surface-exposed portions were radioiodinated using 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril (Iodogen). The I-D and 2-D peptide maps demonstrated the following: (a) pI proteins of the three serotypes have few V8 protease- or chymotrypsin-generated peptides in common, and (b) pI proteins of each serotype appear to be identical. The findings suggest that pI protein variability derives from extensive differences in the amino acid sequences of these proteins.

Antigenic variability of Borrelia burgdorferi.

Borrelia burgdorferi strains (six isolates from North America and 28 isolates from Europe) were analyzed by physicochemical and immunological methods. By SDS-PAGE, all Borrelia burgdorferi strains tested had two major proteins with constant molecular weights of 60 and 41 kDa and one, two, or three variable low molecular weight proteins (OspA = 30-32 kDa, OspB = 34-36 kDa, pC = 21-22 kDa). All combinations--except OspB alone or OspB/pC--were observed. Borrelia burgdorferi strains were different from relapsing fever borreliae by strong reactivity with OspA- and/or pC-specific polyclonal antibodies, whereas relapsing fever borreliae were only weakly reactive. Among 25 Borrelia burgdorferi isolates, seven different serotypes of Borrelia burgdorferi were defined according to their reactivity in the Western blot with three monoclonal OspA-specific antibodies (H5332, H3TS, and LA5), four OspA- or OspB-specific polyclonal antibodies, and 12 polyclonal antibodies against whole borreliae. Antigenic differences between European CSF and skin isolates were observed, all skin isolates (n = 11) belonging to serotype 2 in contrast to only two out of seven CSF isolates. CSF isolates were antigenically heterogenous (serotypes 1, 2, 3, 4, and 5). Serotypes 6 and 7 were represented by two tick isolates, and the other European tick isolates are not yet fully characterized. Antigenic differences between European and North American strains may play a role in differences in the clinical picture of Lyme borreliosis.

Attenuation of Borrelia anserina by serial passage in liquid medium.

Borrelia anserina (Sakharoff) was successfully grown in a liquid medium (Barbour-Stoenner-Kelly) for 39 passages. By the 12th serial passage in medium, infectivity of B anserina for chicks was lost. Electron microscopy did not reveal structural differences between non-infective and infective cultured organisms. Changes in the protein profiles were found by electrophoresis as the organisms were passed in culture.

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.

Lack of endotoxin in Borrelia hispanica and Treponema pallidum.

Borrelia hispanica from infected guinea pigs and Treponema pallidum from testicular syphilomas of rabbits were assayed for the presence of endotoxin with the Limulus lysate test. A suspension of Borrelia, containing 1.3 X 10(8) spirochetes/ml, was nonreactive both when it was tested as intact organisms, and when tested after disruption of the spirochetes by sonication. Eight different suspensions of treponemes, ranging from 0.6 X 10(9) to 3 X 10(9) treponemes/ml, were negative at a 1:10 dilution and were no more active than control suspensions of normal rabbit testes. Therefore, it was concluded that T. pallidum, as well as the Borrelia, possessed no endotoxin.

Changes in infectivity and plasmid profile of the Lyme disease spirochete, Borrelia burgdorferi, as a result of in vitro cultivation.

In vitro cultivation of Borrelia burgdorferi, the etiologic agent of Lyme spirochetosis, allows for the isolation and growth of this bacterium from infected tissues. However, continuous cultivation in modified Kelly medium causes a reduction in the number of detectable plasmids and the loss of infectivity in the white-footed mouse, Peromyscus leucopus. In an unpassaged culture of B. burgdorferi, nine plasmids were present, including seven linear plasmids ranging in size from 49 to 16 kilobases (kb) and two circular plasmids of 27 and 7.6 kb. The 7.6-kb circular and 22-kb linear plasmids were no longer detectable in spirochetes noninfective in white-footed mice, suggesting that a gene(s) encoding for factors responsible for infection may be present on one or more of these extrachromosomal elements. Furthermore, changes in spirochetal proteins and lipopolysaccharide-like material were observed also during early cultivation and may be related to loss of infectivity.

Enzyme-linked immunosorbent assays for Lyme disease: reactivity of subunits of Borrelia burgdorferi.

We prepared fractions of Borrelia burgdorferi, the etiologic agent of Lyme disease, from cultured spirochetes and used them as antigen in an enzyme-linked immunosorbent assay (ELISA) for IgG antibody. Polystyrene plates coated with an extract containing major proteins with apparent molecular masses of 34, 39, 59, and 68 kilodaltons had comparable sensitivity but greater specificity than plates coated with whole cells. Of the 33 serum specimens from individuals with Lyme disease that reacted with whole cells of B. burgdorferi in the class-specific ELISA, 30 (91%) remained positive when this extract was used. Cross-reactivity was minimal with antibody to treponemes. Use of subunit antigens may improve serological diagnosis of Lyme disease.

Heterogeneity in immunoblot patterns obtained by using four strains of Borrelia burgdorferi and sera from naturally exposed dogs.

This study evaluated the differences in immunoblot patterns when serum specimens from seropositive dogs were reacted against four strains of Borrelia burgdorferi. Intense bands were consistently detected for the 83-, 66-, 61- to 60-, 41-, and 31-kilodalton regions for all four strains. Most variations were observed in the regions of 45 to 34 and 26 to 15 kilodaltons. Adsorption studies suggested that one reason for the observed variability was a difference in proteins among the organisms, rather than a difference in migration of proteins. Therefore, knowledge and consistency of the test antigen are essential when evaluating and comparing canine immunoblot patterns to B. burgdorferi, but for diagnostic purposes all of the serum samples would have been considered positive regardless of the strain used.

Isolation of the outer envelope from Borrelia burgdorferi.

Borrelia burgdorferi consists of an inner protoplasmic cylinder, containing the genome and cytoplasmic elements, surrounded by a number of axial filaments, all completely encased within a multiple-layered outer envelope structure (OE). In this study, a sodium dodecyl sulfate-mediated technique was used to isolate the OE from Borrelia burgdorferi in an attempt to better understand this structure in terms of its antigenic reactivity to Lyme disease patient sera. Electron microscopic evidence suggested that the OE product was relatively free of other spirochete cellular components. SDS-PAGE analysis indicated that the electrophoretic pattern of the OE was consistent with that of the remaining protoplasmic cylinder (PC) and whole spirochete controls. Antigenic determinants in the OE were recognized by sera from Lyme disease patients in Western blots. Chemical analysis of the OE revealed a composition of 45.90% protein, 50.75% lipid and 3.33% carbohydrate. The OE comprised 16.5% by lyophilized dry weight of the whole spirochete. Antigenic determinants located within or associated with this structure are likely to play a significant role in the development of immunity in the infected host.

Heterogeneity of major proteins in Lyme disease borreliae: a molecular analysis of North American and European isolates.

We examined 46 isolates of Borrelia burgdorferi, the etiologic agent of Lyme disease and related disorders, with polyacrylamide gel electrophoresis and monoclonal antibodies. Our attention was on the OspA proteins, which are major proteins of the spirochete. There were at least four discernible phenotypes of the OspA protein. While 25 North American isolates were, with one exception, homogeneous in the type of OspA protein that they produced, 21 European isolates were heterogeneous in the types of OspA proteins represented. Only three European strains resembled North American strains in their OspA phenotype. Application of a deoxyribonucleic acid probe for an ospA gene demonstrated that the arrangement of ospA-associated sequences in the DNA differed between isolates.

Chemical and biologic characterization of a lipopolysaccharide extracted from the Lyme disease spirochete (Borrelia burgdorferi).

A lipopolysaccharide (LPS) was isolated from the Lyme disease spirochete by a modification of the hot phenol-water method. The material was composed of 45% carbohydrate, 8% protein, 44% lipid A, and 1% 3-deoxy-D-mannooctulosonic acid and accounted for approximately 1.5% of the cellular dry weight. The isolated LPS possessed several biologic activities characteristic of endotoxins. The LPS was pyrogenic for rabbits, mitogenic for human mononuclear cells and murine splenocytes, capable of clotting limulus lysate, and cytotoxic for murine macrophages. LPS extracted from Borrelia burgdorferi by the petroleum-ether:chloroform:liquid-phenol procedure was also characterized. The results show that the Lyme disease spirochete contains a hitherto unknown LPS that is biologically active in vitro, and the expression of such activities in vivo may play an important role in the pathogenesis of Lyme disease. Some of the clinical manifestations of other spirochetal disease may be explained by similar endotoxins in those organisms. To our knowledge this is the first report of an LPS extracted from a spirochete that is known to be a human pathogen.

Endotoxicity of newly recognized gram negative organism: a collective review.

A comprehensive picture of the in part personal investigations concerning an Endotoxin-like activity associated with some recently recognized Gram negative significant pathogens has been drawn. Suspensions of heat-killed organisms (or endotoxic lipopolysaccharide when available) have been tested using as markers of endotoxicity the Limulus assay and the skin Shwartzman reaction in rabbit. Some general considerations are finally set forth on the possible role of the surface structures in mediating (with other virulence factors) the damage in the host.

Prevalence of Borrelia burgdorferi in white-footed mice and Ixodes dammini at Fort McCoy, Wis.

Borrelia burgdorferi, the causative agent of Lyme disease, was isolated from 15 of 17 white-footed mice (Peromyscus leucopus) and 54 of 82 subadult Ixodes dammini from Fort McCoy, Wis. Of the 47 isolates tested, all reacted in indirect fluorescent-antibody tests with monoclonal antibodies directed against a surface protein of B. burgdorferi (approximate molecular weight, 31,000) and flagellins that are common to all Borrelia species. Indirect fluorescent-antibody reactions were variable when an antibody that binds to a surface protein with an approximate molecular weight of 34,000 was tested. The major proteins of isolates from ticks and mice had approximate molecular weights of 31,000, 34,000, and 41,000. Antibodies to B. burgdorferi were present (titer, greater than or equal to 1:64) in 16 of 97 white-tailed deer (Odocoileus virginianus). The mean number of subadult I. dammini on mice captured in June 1986 was 6.5, and the mean number of adult I. dammini on deer killed in November 1986 was 5.9. The presence of ticks and the high prevalence of I. dammini and mice infected with B. burgdorferi establish that Fort McCoy is an area in which the Lyme disease agent is highly endemic, even though there have been relatively few documented cases in humans. The low number of reported cases in humans may be a result of National Guard and reserve unit personnel returning home to civilian life and having symptoms expressed subsequently, or it could be due to misdiagnosis or nonreporting.