First culture isolation of Borrelia lonestari, putative agent of southern tick-associated rash illness

Abstract

Southern tick-associated rash illness (STARI) is a Lyme disease-like infection described in patients in the southeastern and south-central United States, where classic Lyme disease is relatively rare. STARI develops following the bite of a lone star tick (Amblyomma americanum) and is thought to be caused by infection with an "uncultivable" spirochete tentatively named Borrelia lonestari. In this study, wild lone star ticks collected from an area where B. lonestari is endemic were cocultured in an established embryonic tick cell line (ISE6). The cultures were examined by dark-field microscopy for evidence of infection, and spirochete identity and morphology were evaluated by flagellin B and 16S rRNA gene sequence, by reaction to Borrelia-wide and B. burgdorferi-specific monoclonal antibodies, and by electron microscopy. Live spirochetes were first visualized in primary culture of A. americanum ticks by dark-field microscopy 14 days after the cell culture was inoculated. The sequences of the flagellin B and 16S rRNA genes of cultured spirochetes were consistent with previously reported sequences of B. lonestari. The cultured spirochetes reacted with a Borrelia-wide flagellin antibody, but did not react with an OspA antibody specific to B. burgdorferi, by indirect fluorescent antibody testing. Electron microscopy demonstrated organisms that were free and associated with ISE6 cells, with characteristic Borrelia sp. morphology. This study describes the first successful isolation of B. lonestari in culture, providing a much needed source of organisms for the development of diagnostic assays and forming a basis for future studies investigating the role of the organism as a human disease agent.

FIG. 1.

Bootstrap consensus (1,000 times) of phylogenetic tree of Borrelia sp. 16S rDNA gene sequences generated using maximum-parsimony analysis with close-neighbor interchange. The number at each node indicates the percentage of times that node was supported by bootstrap analysis.

FIG. 2.

Immunofluorescence antibody and Giemsa staining of cultured spirochetes. (A) Immunofluorescence antibody staining of spirochetes isolated from lone star ticks using Borrelia-wide anti-flagellin antibody (H9724). (B) ISE6 tick cell cultures of spirochetes were Giemsa stained; spirochetes are present free and attached to ISE6 cells. Magnification, ×1,000.

FIG. 3.

Transmission electron micrographs of cultured spirochetes. (A) Spirochetes are present free and associated with ISE6 cells (arrows). Transverse sections of intracellular spirochetes showing periplasmic flagella (PPF) are evident within membrane-bound vacuoles. (B) Bundles of flagella can be seen running along oblique and transverse sections of spirochetes (arrowheads). (C) Spirochete and ISE6 cell in close association, with loss of resolution at the site of contact (arrow). The trilaminar membrane (TLM), consisting of an outer sheath, cell wall, and inner cytoplasmic membrane, is also seen more directly. Bars, 500 (A), 200 (B), and 100 (C) nm.

FIG. 4.

Scanning electron micrographs of cultured spirochetes. (A) Single spirochete with characteristic tapered ends, relatively flat wavelength, and a cytoplasmic bleb. (B) The arrow points to a spirochete with a counterclockwise helix (left handed; moving away from the observer); also evident are spirochetes entangled with each other and closely associated with the ISE6 cell. (C) Greater magnification of a cytoplasmic bleb (arrow).

FIG. 5.

Transmission electron micrographs of negatively stained spirochetes.(A)Spirochete with a prominent outer membrane extending over one end. (A and B) Flagella can be seen running along the axis of the spirochete.(B)Flagellar bundles are evident on either side of the curvature of the spirochete (bars). (C) Greater magnification of the spirochete from panel B reveals the trilaminar membrane (TLM) with evidence of multiple flagella running periplasmically and more clearly demonstrates a single flagellar bundle (bars). Bars,200 (A), 100 (B), and 50 (C)nm.