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Journal of Clinical Microbiology, March 2004, p. 1163-1169, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1163-1169.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

First Culture Isolation of Borrelia lonestari, Putative Agent of Southern Tick-Associated Rash Illness

Andrea S. Varela,1 M. Page Luttrell,2 Elizabeth W. Howerth,3 Victor A. Moore,1 William R. Davidson,2,4 David E. Stallknecht,1,2 and Susan E. Little1*

Department of Medical Microbiology and Parasitology,1 Southeastern Cooperative Wildlife Disease Study,2 Department of Pathology, College of Veterinary Medicine,3 Warnell School of Forest Resources, University of Georgia, Athens, Georgia 306024

Received 25 August 2003/ Returned for modification 5 October 2003/ Accepted 5 November 2003

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.

* Corresponding author. Mailing address: Department of Medical Microbiology and Parasitology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602. Phone: (706) 542-8447. Fax: (706) 542-0059. E-mail: slittle{at}vet.uga.edu.

Journal of Clinical Microbiology, March 2004, p. 1163-1169, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1163-1169.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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