Abstract
A simple colorimetric assay, on the basis of bioreduction of the substrate XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt) by dehydrogenases of actively metabolizing mammalian cells, was assessed for measurement of in vitro killing of canine macrophage-like cells by Ehrlichia canis infection. Formation of a highly colored, water soluble formazan which resulted from bioreduction by the test cells was significantly enhanced by phenazine methosulfate, an electron coupling agent. Normal cells markedly reduced XTT in the presence of phenazine methosulfate, yielding a post-/pre-bioreduction absorbance ratio of >4.0 under the assay conditions tested. E. canis-infected cells, however, markedly lost the capability for bioreduction, producing a post-/pre-bioreduction absorbance ratio as low as 2.0. Colorimetric assay results correlated with E. canis infections as tested by an indirect fluorescent antibody assay ( R = 0.95). This colorimetric assay involved a single step and results could be obtained in 1 h. Thus, the microtiter colorimetric assay was able to quantitate killing of the cells by E. canis infection with the advantages of reliability and simplicity. It will also prove valuable in facilitating the development of other in vitro assays in ehrlichiosis research.
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