Our cell culture and molecular, immunological, and electron microscopy data all indicate that we have made the first isolation of B. lonestari in culture; we have designated this strain LS-1. We were able to cultivate this fastidious organism using a tick cell line and a source of organisms from naturally infected lone star ticks. The spirochetes were found to possess molecular characteristics consistent with the organism tentatively named B. lonestari and morphological and IFA staining properties distinct from those of B. burgdorferi.
The flagellin B gene sequence of our B. lonestari
isolate (LS-1) is identical to B. lonestari flaB
sequences previously reported from lone star ticks and white-tailed deer in that it lacks a 3-base insert at positions 330 to 332 (4
). Although the LS-1 isolate differs in this respect from the single flaB
sequence of B. lonestari
reported from a human clinical case (23
), the significance of this 3-base insert is not clear. Nucleotide differences have been observed at four base positions in the 16S rRNA gene among the reported sequences of B. lonestari
(= B. barbouri
). Our LS-1 isolate 16S rRNA gene sequence matched one of the two published sequences for B. lonestari
at three of these base positions and both B. lonestari
sequences at the fourth position (base 522) (10
). The published sequence for B. barbouri
contained a single base difference at position 522, but because this was a partial sequence (355 bp) that did not include bases 698, 1025, and 1201, nucleotide similarity could not be evaluated at those positions (41
). While it is important to note them, these differences are minor, and both the 16S rRNA and flagellin B gene sequences of the LS1 spirochetes support the identification of the organisms we have cultured as B. lonestari.
We also report here the first ultrastructural description of B. lonestari.
The LS-1 organisms had typical spirochete morphology and characteristics consistent with Borrelia
). Their lengths, although variable, also fell within the range of Borrelia
spp. The ends of LS-1 organisms, like those of Borrelia anserina
and Borrelia recurrentis
, were tapered, but the tips were relatively blunt in comparison to those of B. burgdorferi;
unlike B. anserina
, strain LS-1 was not regularly waved (15
). Cytoplasmic protrusions or blebs on the surfaces of LS-1 organisms were consistent with protrusions that have been described for other spirochetes (7
TEM showed multiple sections of spirochetes inside vacuole-like, membrane-bound organelles within the ISE6 cells. Transverse organism sections that shared the same periplasmic flagellar bundle were evidently the same organism; other apparently unassociated sections may have been the same organism or another organism within the same organelle. The presence of multiple membrane-bound organelles within the same ISE6 cell, as well as single spirochetes that appeared to be free within the cytoplasm, suggests that B. lonestari may replicate within membrane-bound organelles of cells and then emerge from the organelles and cells. Spirochetes seen in close association with ISE6 cells on SEM, often indenting the host cell membrane, may represent the attachment and subsequent invasion of ISE6 cells, suggesting a possible dependence on the host tick cell. The interactions between these spirochetes and their growth in culture are among many significant life history characteristics that await investigation.
The findings in this study establish the critical foundation necessary for further investigations of B. lonestari that were previously hampered by the lack of a live isolate. Although culture isolation of B. lonestari from human cases of STARI is necessary to demonstrate a causative link to the illness, our results are an important step toward a greater understanding of STARI and the variable presentation of this Lyme disease-like illness. Cultured organisms are critical for the development of accurate human diagnostic assays; this will allow investigation of the clinical manifestations of infection in humans, as well as further our understanding of the epidemiology and natural history of B. lonestari, including host and vector competence, natural maintenance cycles, and geographical distribution. In addition, an in vitro source of organisms is now available for controlled experiments using animal hosts as models for human infection, as well as tick transmission studies. The knowledge gained from studies using cultured B. lonestari will be fundamental in differentiating between B. lonestari and B. burgdorferi infections and ultimately in revealing the true occurrence of Lyme disease and STARI in the southeastern and south-central United States.