Alan G. Barbour

The Spirochetal Etiology of Lyme Disease

We recovered a newly recognized spirochete from the blood, skin lesions (erythema chronicum migrans [ECM]), or cerebrospinal fluid of 3 of 56 patients with Lyme disease and from 21 of 110 nymphal or adult lxodes dammini ticks in Connecticut. These isolates and the original one from l. dammini appeared to have the same morphologic and immunologic features. In patients, specific IgM antibody titers usually reached a peak between the third and sixth week after the onset of disease; specific IgG antibody titers rose slowly and were generally highest months later when arthritis was present. Among 40 patients who had early disease only (ECM alone), 90 per cent had an elevated IgM titer (greater than or equal to 1:128) between the ECM phase and convalescence. Among 95 patients with later manifestations (involvement of the nervous system, heart, or joints), 94 per cent had elevated titers of IgG (greater than or equal to 1:128). In contrast, none of 80 control subjects had elevated IgG titers, and only three control patients with infectious mononucleosis had elevated IgM titers. We conclude that the I. dammini spirochete is the causative agent of Lyme disease.

Spirochetes Isolated from the Blood of Two Patients with Lyme Disease

We isolated spirochetes from the blood of 2 of 36 patients in Long Island and Westchester County, New York, who had signs and symptoms suggestive of Lyme disease. The spirochetes were morphologically similar and serologically identical to organisms recently found to infect lxodes dammini ticks, which are endemic to the area and have been epidemiologically implicated as vectors of Lyme disease. In both patients, there was a rise in specific antispirochetal antibodies in paired specimens of serum. We conclude that the l. dammini spirochete has an etiologic role in Lyme disease.

Antigenic variation in Lyme disease borreliae by promiscuous recombination of VMP-like sequence cassettes.

Abstract We have identified and characterized an elaborate genetic system in the Lyme disease spirochete Borrelia burgdorferi that promotes extensive antigenic variation of a surface-exposed lipoprotein, VlsE. A 28 kb linear plasmid of B. burgdorferi B31 (lp28–1) was found to contain a vmp -like sequence ( vls ) locus that closely resembles the variable major protein ( vmp ) system for antigenic variation of relapsing fever organisms. Portions of several of the 15 nonexpressed (silent) vls cassette sequences located upstream of vlsE recombined into the central vlsE cassette region during infection of C3H/HeN mice, resulting in antigenic variation of the expressed lipoprotein. This combinatorial variation could potentially produce millions of antigenic variants in the mammalian host.

Molecular analysis of linear plasmid-encoded major surface proteins, OspA and OspB, of the lyme disease spirochaete Borrelia burgdorferi

The ospA and ospB genes encode the major outer membrane proteins of the Lyme disease spirochaete Borrelia burgdorferi. The deduced translation products from the ospA and ospB genes were: (OspA) 273 amino acids long with a molecular weight of 29334, and (OspB) 296 amino acids long with a molecular weight of 31739. The two Osp proteins showed a great degree of sequence similarity indicating a recent evolutionary event. Molecular analysis and sequence comparison of OspA and OspB with other proteins revealed a sequence similarity to the signal peptides of prokaryotic lipoproteins. These are the first sequences from Borrelia and provide interesting data on the evolutionary relationship between spirochaetes and other species as well as providing potential for spirochaete diagnostics and vaccines.

Profiling of temperature-induced changes in Borrelia burgdorferi gene expression by using whole genome arrays.

ABSTRACT Borrelia burgdorferi is the etiologic agent of Lyme disease, the most prevalent arthropod-borne disease in the United States. The genome of the type strain, B31, consists of a 910,725-bp linear chromosome and 21 linear and circular plasmids comprising 610,694 bp. During its life cycle, the spirochete exists in distinctly different environments, cycling between a tick vector and a mammalian host. Temperature is one environmental factor known to affect B. burgdorferi gene expression. To identify temperature-responsive genes, genome arrays containing 1,662 putative B. burgdorferi open reading frames (ORFs) were prepared on nylon membranes and employed to assess gene expression in B. burgdorferi B31 grown at 23 and 35°C. Differences in expression of more than 3.5 orders of magnitude could be readily discerned and quantitated. At least minimal expression from 91% of the arrayed ORFs could be detected. A total of 215 ORFs were differentially expressed at the two temperatures; 133 were expressed at significantly greater levels at 35°C, and 82 were more significantly expressed at 23°C. Of these 215 ORFs, 134 are characterized as genes of unknown function. One hundred thirty-six (63%) of the differentially expressed genes are plasmid encoded. Of particular interest is plasmid lp54 which contains 76 annotated putative genes; 31 of these exhibit temperature-regulated expression. These findings underscore the important role plasmid-encoded genes may play in adjustment of B. burgdorferi to growth under diverse environmental conditions.

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.

An ecological approach to preventing human infection: Vaccinating wild mouse reservoirs intervenes in the Lyme disease cycle

Many pathogens, such as the agents of West Nile encephalitis and plague, are maintained in nature by animal reservoirs and transmitted to humans by arthropod vectors. Efforts to reduce disease incidence usually rely on vector control or immunization of humans. Lyme disease, for which no human vaccine is currently available, is a commonly reported vector-borne disease in North America and Europe. In a recently developed, ecological approach to disease prevention, we intervened in the natural cycle of the Lyme disease agent (Borrelia burgdorferi) by immunizing wild white-footed mice (Peromyscus leucopus), a reservoir host species, with either a recombinant antigen of the pathogen, outer surface protein A, or a negative control antigen in a repeated field experiment with paired experimental and control grids stratified by site. Outer surface protein A vaccination significantly reduced the prevalence of B. burgdorferi in nymphal blacklegged ticks (Ixodes scapularis) collected at the sites the following year in both experiments. The magnitude of the vaccines effect at a given site correlated with the tick infection prevalence found on the control grid, which in turn correlated with mouse density. These data, as well as differences in the population structures of B. burgdorferi in sympatric ticks and mice, indicated that nonmouse hosts contributed more to infecting ticks than previously expected. Thus, where nonmouse hosts play a large role in infection dynamics, vaccination should be directed at additional species.

Niche Partitioning of Borrelia burgdorferi and Borrelia miyamotoi in the Same Tick Vector and Mammalian Reservoir Species

The Lyme borreliosis agent Borrelia burgdorferi and the relapsing fever group species Borrelia miyamotoi co-occur in the United States. We used species-specific, quantitative polymerase chain reaction to study both species in the blood and skin of Peromyscus leucopus mice and host-seeking Ixodes scapularis nymphs at a Connecticut site. Bacteremias with B. burgdorferi or B. miyamotoi were most prevalent during periods of greatest activity for nymphs or larvae, respectively. Whereas B. burgdorferi was 30-fold more frequent than B. miyamotoi in skin biopsies and mice had higher densities of B. burgdorferi densities in the skin than in the blood, B. miyamotoi densities were higher in blood than skin. In a survey of host-seeking nymphs in 11 northern states, infection prevalences for B. burgdorferi and B. miyamotoi averaged approximately 0.20 and approximately 0.02, respectively. Co-infections of P. leucopus or I. scapularis with both B. burgdorferi and B. miyamotoi were neither more nor less common than random expectations.

Migratory Passerine Birds as Reservoirs of Lyme Borreliosis in Europe

Birds host vector ticks and Borrelia species and vary in effectiveness as reservoirs.

Human Borrelia miyamotoi infection in the United States.

A tickborne pathogen, Borrelia miyamotoi, has recently been shown to cause disease in humans. A serologic survey showed that infection with this organism is not uncommon in the Northeast.