Identification and characterization of survival mechanisms of the human granulocytic ehrlichiosis agent and identification and characterization of an Ehrlichia risticii isolated from aquatic insects and horse isolates

Ehrlichiae are obligate intracellular bacteria causing mammalian disease and replicating in membrane-bound cytoplasmic inclusions (morulae). The HGE agent replicates in granulocytes. These studies investigated mechanisms utilized by the HGE agent to survive in the human neutrophils by characterizing inclusions by double immunofluorescence labeling with markers specific to host cell cytoplasmic compartments. We found that E. chaffeensis resided in early endosomes while HGE agent inclusions did not resemble endosomes, lysosomes, or Golgi-derived vesicles.; The ability of the HGE agent to inhibit the production of superoxide anion (O₂−), a toxic antibacterial metabolite, was examined. Using ferricytochrome c reduction and LDCL assays, we found the HGE agent reversibly inhibited extracellular and intracellular production of O₂− in human neutrophils in response to receptor-dependent and -independent stimuli in a dose-dependent manner. Results showed that both membrane and total cellular levels of p22 phox, a membrane bound enzyme component, was decreased in the presence of the HGE agent suggesting a novel mechanism to rapidly inhibit as well as maintain NADPH oxidase inhibition.; This research also examined the diagnosis of Potomac horse fever (PHF) caused by Ehrlichia risticii. We examined sensitivities of nested PCR, culture, and the IFA test. Results revealed that nested PCR is as sensitive as culture isolation for determining infection and that vaccine failure is common.; Finally, we examined the ability of aquatic insects from an endemic area to transmit E. risticii to susceptible horses via oral ingestion. Clinical signs compatible with PHF were seen in two experimentally infected horses. E. risticii was isolated in culture from blood samples prior to sequencing of 16S rRNA and 51-kDa genes obtained from isolates, blood samples, and aquatic insects. Sequences identifying an identical strain of E. risticii throughout the study provided evidence that oral transmission of disease via ingestion of aquatic insects is an important route of natural infection.