Mammalian host cell reservoirs during Anaplasma infection

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 sites of Anaplasma infection in mammalian tissues, a sensitive and specific, in situ rolling-circle amplification technique to detect localized Anaplasma gene sequences was developed. Via the technique described here and von Willebrand factor immunofluorescence, A. phagocytophilum and A. marginale were successfully localized in situ within cultured mammalian cells. This is the first application of this in situ method for detection of a microorganism and forms the foundation for applications of this technique to detect, localize, and analyze Anaplasma nucleotide sequences in the tissues of infected hosts and in cell cultures.;Three Anaplasma-infection trials using immunocompetent dogs and cattle were performed to investigate different aspects of endothelial cells as they relate to Anaplasma life cycles and to further describe clinical aspects of Anaplasma pathogenesis. Four Beagles were inoculated with A. phagocytophilum from different sources, allowed to develop chronic infection, and treated with doxycycline. Regardless of isolate or duration of doxycycline treatment, A. phagocytophilum DNA remained detectable for several months in blood and tissues, though 8 organisms were not identified microscopically. This is the first infection of dogs using cultured endothelial cells as the source of inoculum, the first demonstration of molecular evidence of chronic, persistent infection in blood and tissues of subclinical dogs despite doxycycline treatment, and the first investigation of endothelial cells as a potential in vivo source of A. phagocytophilum during chronic canine infection using in situ rolling-circle amplification. Two steers were inoculated with A. marginale by tick-feeding transmission and were euthanized at different points within the parasitemic cycle. The tissue distribution of A. marginale during peak and trough parasitemia was described using real-time PCR, though organisms were not identified in tissues microscopically. This is the first survey of A. marginale tissue distribution after tick-transmission and the first investigation in immunocompetent cattle of endothelial cells as a potential in vivo source of A. marginale in tick-bite sites and distant tissues using three techniques (immunofluorescence, electron microscopy, in situ rolling-circle amplification).