In vivo Endothelial Cell Infection by Anaplasma marginale
Type of DegreeThesis
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Anaplasmosis is an arthropod-borne hemoparasitic disease of cattle and other ruminants. The causative agent is the gram negative bacterium, Anaplasma marginale. Infection of bovine erythrocytes by A. marginale has been well established in vivo, as well as in vitro. Recently, A. marginale has been propagated in vitro in bovine and primate vascular endothelial cell cultures. This finding provides evidence that infected endothelial cells may initiate MHC-Class-I restricted CTL responses in infected cattle. To determine the extent to which endothelial cells are susceptible to A. marginale infection in vivo, a dual staining technique was applied to tissues from a splenectomized calf experimentally inoculated with 109 organisms from the St. Maries strain of A. marginale. Sections of kidney, lung, and hemal lymph node were collected, embedded in freezing compound, frozen in isopentane/liquid nitrogen, and cryosectioned at 5 microns. Sections were co-labeled with monoclonal antibody ANAF16C1, recognizing A. marginale major surface protein 5 (MSP5) conjugated to fluorescein isothiocyanate (FITC) or Alexa Fluor 488 and a polyclonal rabbit antibody against human von Willebrand Factor (an endothelial cell marker) conjugated to tetramethylrhodamine isothiocyanate (TRITC) or Alexa Fluor 568. Nuclei were stained with 284nM 4’, 6-diamidino-2-phenylindole, dihydrochloride (DAPI). Sections were evaluated by conventional wide field fluorescence microscopy using a Nikon Eclipse E800 and confocal fluorescence microscopy using a BioRad MRC 1024 Scanning Laser Confocal Microscope. As expected, non-endothelial cells within vascular lumens were the major reservoir for A. marginale. In addition, A. marginale fluorescence co-localized to capillary endothelial cells of the kidney, lung, and hemal lymph node. These results suggest that endothelial cells may serve as a cellular reservoir for A. marginale in vivo, and have implications for both pathogenesis and immune mechanisms.