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.

/pdf/lyme-disease-a-tick-borne-spirochetosis-etxhj723da.pdf

Lyme disease-a tick-borne spirochetosis?

The successful isolation and cultivation of Lyme disease spirochetes traces its lineage to early attempts at cultivating relapsing fever borreliae. Observations on the growth of Lyme disease spirochetes under different in vitro conditions may yield important clues to both the metabolic characteristics of these newly discovered organisms and the pathogenesis of Lyme disease.

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Isolation and cultivation of Lyme disease spirochetes.

We have determined that Borrelia burgdorferi strain B31 MI carries 21 extrachromosomal DNA elements, the largest number known for any bacterium. Among these are 12 linear and nine circular plasmids, whose sequences total 610 694 bp. We report here the nucleotide sequence of three linear and seven circular plasmids (comprising 290 546 bp) in this infectious isolate. This completes the genome sequencing project for this organism; its genome size is 1 521 419 bp (plus about 2000 bp of undetermined telomeric sequences). Analysis of the sequence implies that there has been extensive and sometimes rather recent DNA rearrangement among a number of the linear plasmids. Many of these events appear to have been mediated by recombinational processes that formed duplications. These many regions of similarity are reflected in the fact that most plasmid genes are members of one of the genome's 161 paralogous gene families; 107 of these gene families, which vary in size from two to 41 members, contain at least one plasmid gene. These rearrangements appear to have contributed to a surprisingly large number of apparently non-functional pseudogenes, a very unusual feature for a prokaryotic genome. The presence of these damaged genes suggests that some of the plasmids may be in a period of rapid evolution. The sequence predicts 535 plasmid genes ≥300 bp in length that may be intact and 167 apparently mutationally damaged and/or unexpressed genes (pseudogenes). The large majority, over 90%, of genes on these plasmids have no convincing similarity to genes outside Borrelia, suggesting that they perform specialized functions.

https://onlinelibrary.wiley.com/doi/epdf/10.1046/j.1365-2958.2000.01698.x

A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi.

A large amount of knowledge has been acquired since the original descriptions of Lyme borreliosis (LB) and of its causative agent, Borrelia burgdorferi sensu stricto. The complexity of the organism and the variations in the clinical manifestations of LB caused by the different B. burgdorferi sensu lato species were not then anticipated. Considerable improvement has been achieved in detection of B. burgdorferi sensu lato by culture, particularly of blood specimens during early stages of disease. Culturing plasma and increasing the volume of material cultured have accomplished this. Further improvements might be obtained if molecular methods are used for detection of growth in culture and if culture methods are automated. Unfortunately, culture is insensitive in extracutaneous manifestations of LB. PCR and culture have high sensitivity on skin samples of patients with EM whose diagnosis is based mostly on clinical recognition of the lesion. PCR on material obtained from extracutaneous sites is in general of low sensitivity, with the exception of synovial fluid. PCR on synovial fluid has shown a sensitivity of up to >90% (when using four different primer sets) in patients with untreated or partially treated Lyme arthritis, making it a helpful confirmatory test in these patients. Currently, the best use of PCR is for confirmation of the clinical diagnosis of suspected Lyme arthritis in patients who are IgG immunoblot positive. PCR should not be used as the sole laboratory modality to support a clinical diagnosis of extracutaneous LB. PCR positivity in seronegative patients suspected of having late manifestations of LB most likely represents a false-positive result. Because of difficulties in direct methods of detection, laboratory tests currently in use are mainly those detecting antibodies to B. burgdorferi sensu lato. Tests used to detect antibodies to B. burgdorferi sensu lato have evolved from the initial formats as more knowledge on the immunodominant antigens has been collected. The recommendation for two-tier testing was an attempt to standardize testing and improve specificity in the United States. First-tier assays using whole-cell sonicates of B. burgdorferi sensu lato need to be standardized in terms of antigen composition and detection threshold of specific immunoglobulin classes. The search for improved serologic tests has stimulated the development of recombinant protein antigens and the synthesis of specific peptides from immunodominant antigens. The use of these materials alone or in combination as the source of antigen in a single-tier immunoassay may someday replace the currently recommended two-tier testing strategy. Evaluation of these assays is currently being done, and there is evidence that certain of these antigens may be broadly cross-reactive with the B. burgdorferi sensu lato species causing LB in Europe.

Diagnosis of Lyme Borreliosis

A review of reports on the genetic and phenotypic characteristics of strains of the spirochete which causes Lyme disease revealed that these organisms are representative of a new species of Borrelia. We propose the name Borrelia burgdorferi for this species. The type strain of B. burgdorferi is strain B31 (= ATCC 35210). In two separate studies the guanine-plus-cytosine content of the deoxyribonucleic acid of the type strain was determined to be 29.0 to 30.5 mol% (thermal denaturation method).

/pdf/borrelia-burgdorferi-sp-nov-etiologic-agent-of-lyme-disease-5c46o0jraj.pdf

Borrelia burgdorferi sp. nov.: Etiologic Agent of 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.

Spirochetes isolated from the blood of two patients with Lyme disease.

The outer surface protein, OspC, is highly variable in Borrelia burgdorferi sensu stricto, the agent of Lyme disease. We have shown that even within a single population OspC is highly variable. The variation of ospA and ospC in the 40 infected deer ticks collected from a single site on Shelter Island, New York, was determined using PCR-SSCP. There is very strong apparent linkage disequilibrium between ospA and ospC alleles, even though they are located on separate plasmids. Thirteen discernible SSCP mobility classes for ospC were identified and the DNA sequence for each was determined. These sequences, combined with 40 GenBank sequences, allow us to define 19 major ospC groups. Sequences within a major ospC group are, on average, <1% different from each other, while sequences between major ospC groups are, on average, approximately 20% different. The tick sample contains 11 major ospC groups, GenBank contains 16 groups, with 8 groups found in both samples. Thus, the ospC variation within a local population is almost as great as the variation of a similar-sized sample of the entire species. The Ewens-Watterson-Slatkin test of allele frequency showed significant deviation from the neutral expectation, indicating balancing selection for these major ospC groups. The variation represented by major ospC groups needs to be considered if the OspC protein is to be used as a serodiagnostic antigen or a vaccine.

https://www.genetics.org/content/genetics/151/1/15.full.pdf

Genetic Diversity of ospC in a Local Population of Borrelia burgdorferi sensu stricto

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.

https://science.sciencemag.org/content/sci/220/4594/321.full.pdf

Natural Distribution of the Ixodes dammini spirochete

Amblyomma americanum is a likely secondary vector of Lyme disease in New Jersey. Ticks of this species were removed from the site of the characteristic skin lesion known as erythema chronicum migrans on two patients with the disease, and the Lyme disease spirochete was isolated from nymphs and adults of this species. That A. americanum is a potential vector is supported by its similarities to Ixodes dammini, the known tick vector, in seasonal distribution and host utilization. The extensive range of A. americanum may have great implications for potential Lyme disease transmission outside known endemic areas.

https://science.sciencemag.org/content/sci/224/4649/601.full.pdf

Edward M. Bosler

Papers

Spirochetes isolated from the blood of two patients with Lyme disease.

Genetic Diversity of ospC in a Local Population of Borrelia burgdorferi sensu stricto

Natural Distribution of the Ixodes dammini spirochete

Amblyomma americanum: a potential vector of Lyme disease in New Jersey

Spirochete-associated arthritis (Lyme disease) in a dog.