This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wamsley, H. L.
Right arrow Articles by Barbet, A. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wamsley, H. L.
Right arrow Articles by Barbet, A. F.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, July 2008, p. 2314-2319, Vol. 46, No. 7
0095-1137/08/$08.00+0     doi:10.1128/JCM.02197-07
Copyright © 2008, American Society for Microbiology. 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 {triangledown}

Heather L. Wamsley* and Anthony F. Barbet

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

Received 13 November 2007/ Returned for modification 26 December 2007/ Accepted 13 May 2008

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 potentially cryptic sites of Anaplasma sp. infection in mammalian tissues, a sensitive and specific isothermal in situ technique to detect localized Anaplasma gene sequences by 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 and von Willebrand factor immunofluorescence, Anaplasma phagocytophilum and Anaplasma marginale were successfully localized in situ within intact cultured mammalian cells. This work represents the first application of this in situ method for the 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.

* Corresponding author. Mailing address: University of Florida, College of Veterinary Medicine, 2015 SW 16th AV, Box 100103C, Gainesville, FL 32610. Phone: (352) 392-2235, ext. 5848. Fax: (352) 392-2938. E-mail: wamsleyh{at}vetmed.ufl.edu

{triangledown} Published ahead of print on 21 May 2008.

Journal of Clinical Microbiology, July 2008, p. 2314-2319, Vol. 46, No. 7
0095-1137/08/$08.00+0     doi:10.1128/JCM.02197-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»