Western blot analysis of sera reactive to human monocytic ehrlichiosis andhuman granulocytic ehrlichiosis agents

Laboratory diagnosis of human ehrlichioses is routinely made by an indirect immunofluorescence assay (IFA) using cultured ehrlichia-infected whole cel ls as antigen. Concern has been raised that incorrect diagnoses of human mo nocytic ehrlichiosis (HME) or human granulocytic ehrlichiosis (HGE) may be made on the basis of serologic cross-reactivity between Ehrlichia chaffeens is and the agent of HGE. The present study examined whether two recombinant major outer membrane proteins, rP30 and rP44, that were previously shown t o be sensitive and specific serodiagnostic antigens for HME and HGE, respec tively, could be used to discriminate IFA dually reacting sera. Thirteen du ally IFA-reactive sera, three sera that were IFA positive only with E. chaf feensis, and three sera that were IFA positive only with the HGE agent were examined by Western immunoblot analysis using purified whole organisms and recombinant proteins as antigens. All 16 E. chaffeensis IFA-positive sera reacted with rP30. However, none of these sera reacted with rP44, regardles s of IFA reactivity with the HGE agent. The three HGE-agent-only IFA-positi ve sera reacted only with rP44, not with rP30. Western immunoblotting using purified E. chaffeensis and the HGE agent as antigens suggested that heat shock and other proteins, but not major outer membrane proteins, cross-reac t between the two organisms. Therefore, Western immunoblot analysis using r P44 and rP30 may be useful in discriminating dually HME and HGE IFA-reactiv e sera.