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.

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Amblyomma americanum (lone star tick) is known or suspected to vector several organisms that are implicated as human pathogens, including Ehrlichia chaffeensis, E. ewingii, and Borrelia lonestari. These three agents have also been detected in white-tailed deer (Odocoileus virginianus). Because northeastern Georgia has a high abundance of both lone star ticks and white-tailed deer, and one of these organisms, E. chaffeensis, is already known to be endemic in the area, we assayed individual adult A. americanum, collected during the spring of 2001, 2002, and 2003, for these three organisms. A total of 400 ticks were dissected and tissues assayed by polymerase chain reaction (PCR) using Ehrlichia species-specific and Borrelia genus-wide primers. Of ticks tested, 2.0% (8/398) had evidence of E. chaffeensis, 4.8% (19/398) had evidence of E. ewingii, and 1.0% (4/398) had evidence of B. lonestari. Borrelia sp. spirochetes were also visualized by an indirect fluorescent antibody test, using an anti-flagellin monoclonal antibody (H9724), in a total of 10.7% (32/300) of ticks tested in 2003. These results reconfirm the presence of E. chaffeensis and establish evidence of E. ewingii and B. lonestari in questing adult A. americanum ticks from northeastern Georgia. Detection of at least two of the three organisms in ticks collected each year suggests that people in northeastern Georgia are at risk of infection with these organisms.

The infection dynamics of the ticktransmitted organism Ehrlichia chaffeensis were investigated in white-tailed deer (Odocoileus virginianus) using different routes of inoculation. Six deer were each inoculated with 5.4ϫ10 6 DH82 cells infected with E. chaffeensis (Arkansas strain) by three different routes: intravenous (nϭ2), subcutaneous (nϭ2), and intradermal (nϭ2). Two control deer were inoculated with uninfected cells. Infections were monitored for 54 days and were continued in one deer from each E. chaffeensis inoculated group for an additional 31 days. All deer inoculated with E. chaffeensis seroconverted (Ն1: 64) and became 16S rDNA polymerase chain reaction and/or cell culture positive by post-inoculation day 15. There was no apparent difference in susceptibility to infection between deer inoculated by different routes for the first 50 days based on detection of E. chaffeensis infection by PCR assay of blood or culture isolation. These results demonstrate infection of deer by intradermal and subcutaneous routes for the first time.