JCM Accepts, published online ahead of print on 21 May 2008

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J. Clin. Microbiol. doi:10.1128/JCM.02197-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

In situ detection of Anaplasma spp. by DNA target-primed rolling circle amplification of a padlock probe and intracellular colocalization with immunofluorescently labeled host cell von Willebrand factor

Heather L. Wamsley^* and Anthony F. Barbet

University of Florida, College of Veterinary Medicine, Department of Infectious Diseases and Pathology, Gainesville, FL 32610

^* To whom correspondence should be addressed. Email: wamsleyh{at}vetmed.ufl.edu.

Endothelial cell culture and preliminary immunofluorescent staining of Anaplasma-infected tissues suggest that endothelial cells may be an in vivo nidus of mammalian infection. To investigate endothelial cells and other potential cryptic sites of Anaplasma spp. infection in mammalian tissues, a sensitive and specific, isothermal, in situ technique to detect localized Anaplasma gene sequences using rolling circle amplification of circularizable, linear, oligonucleotide probes (padlock probes) was developed. Cytospin preparations of uninfected or Anaplasma-infected cell cultures were examined using this technique. Via fluorescence microscopy, the technique described here, and a combination of differential interference contrast microscopy or von Willebrand factor immunofluorescence, A. phagocytophilum and A. marginale were successfully localized in situ within intact cultured mammalian cells. This work represents the first application of this in situ method for detection of a microorganism and forms the foundation for future applications of this technique to detect, localize, and analyze Anaplasma nucleotide sequences in the tissues of infected mammalian and arthropod hosts and in cell cultures.