We describe the cultivation of the agent of human granulocytic ehrlichiosis in cell culture. The ability to isolate this organism should lead to a better understanding of the biology, treatment, and epidemiology of this emerging infection.
Background Ehrlichiosis is a clinically important, emerging zoonosis. Only Ehrlichia chaffeensis and E. ewingii have been thought to cause ehrlichiosis in humans in the United States. Patients with suspected ehrlichiosis routinely undergo testing to ensure proper diagnosis and to ascertain the cause. Methods We used molecular methods, culturing, and serologic testing to diagnose and ascertain the cause of cases of ehrlichiosis. Results On testing, four cases of ehrlichiosis in Minnesota or Wisconsin were found not to be from E. chaffeensis or E. ewingii and instead to be caused by a newly discovered ehrlichia species. All patients had fever, malaise, headache, and lymphopenia; three had thrombocytopenia; and two had elevated liver-enzyme levels. All recovered after receiving doxycycline treatment. At least 17 of 697 Ixodes scapularis ticks collected in Minnesota or Wisconsin were positive for the same ehrlichia species on polymerase-chain-reaction testing. Genetic analyses revealed that this new ehrlichia species is closely related to E. muris. Conclusions We report a new ehrlichia species in Minnesota and Wisconsin and provide supportive clinical, epidemiologic, culture, DNA-sequence, and vector data. Physicians need to be aware of this newly discovered close relative of E. muris to ensure appropriate testing, treatment, and regional surveillance. (Funded by the National Institutes of Health and the Centers for Disease Control and Prevention.)
Human granulocytic ehrlichiosis (HGE) is a febrile tick-borne illness caused by a recently discovered intracellular bacterium remarkable for its tropism for professionally phagocytic neutrophils. Monoclonal antibodies against the P-selectin binding domain of the leukocyte P-selectin glycoprotein ligand, PSGL-1, prevented HGE cell binding and infection, as did enzymatic digestion of PSGL-1. Furthermore, simultaneous neoexpression in nonsusceptible cells of complementary DNAs for both PSGL-1 and its modifying alpha-(1,3) fucosyltransferase, Fuc-TVII, allowed binding and infection by HGE. Thus, the HGE bacterium specifically bound to fucosylated leukocyte PSGL-1. Selectin mimicry is likely central to the organism's unique ability to target and infect neutrophils.
Human granulocytic ehrlichiosis (HGE) is a newly recognized tickborne infection of humans (1-3). HGE is a febrile illness characterized by reductions in blood leukocyte and platelet counts and the presence of colonies of infecting organisms (morulae) proliferating within cytoplasmic vacuoles of circulating neutrophils. No other organism is known to replicate within human granulocytes. Our laboratory recently isolated and propagated the causative agent of this disease using the human promyelocytic leukemia cell line HL60 (4). In addition, we found that the agent can grow in primary bone marrow granulocytic and monocytic precursors, blood neutrophils, and nonactivated monocytes (5).The clinical and in vitro tropism of this obligate intracellular parasite for myelomonocytic cells suggested that specific cell-surface molecules are required for adhesion, cell signaling, and entry into a permissive intracellular compartment. Of interest, sialyl Lewis x ([NeuAcα(2− 3)GalΒ1−4(Fucα1-3)GlcNAc, or CD15s]), which serves as a ligand to initially tether leukocytes to endothelial cell Eselectin (6, 7), is richly expressed on both HL60 cells and on those bone marrow and blood cells permissive for the HGE agent (8-11), where it modifies a variety of lipid and protein molecules. We hypothesized that cell-surface sialylated Lewis x might be important for infection by HGE. Methods HGE infection, cultivation, and antibody blocking studies.A cell-free bacterial inoculum of HGE was prepared from fully infected HL60 cells, propagated as described (4). The infected cells, at a density of 10 6 per ml, were passed through a 25-gauge needle three times, and cellular debris were removed by centrifugation at 700 g for 5 min. The supernatant was centrifuged at 1,236 g for 5 min, and the resultant bacterial pellet was resuspended in 10 µl supernatant per ml of the original culture. Counting in a Petroff-Hausser chamber revealed ∼10 7 organisms per 10 µl. Infections were carried out by coincubating 10 µl of this cell-free HGE preparation with 10 6 cells for a multiplicity of infection of ∼10 organisms per cell used in all experiments. For studies to determine whether bacterial binding requires calcium, both HL60 cells and the bacterial inoculum were separately incubated in HBSS with or without calcium for 20-70 min before coincubation, as described later here. For blocking studies, 10 µg (or the quantity specified) of the monoclonal antibody (MAB) tested (or control diluent or murine IgM alone) was added to 1 ml of 10 6 HL60 cells (or bone marrow progenitors, peripheral blood monocytes, or granulocytes, prepared as described; ref. 5) in medium and incubated at room temperature for 1 h. Blocking studies were performed six times with similar results. As noted, antibody blocking studies were also performed at 4°C or 37°C. Cells were centrifuged at 200 g for 5 min, 900 µl of supernatant saved, the cells resuspended in the 100 µl of medium remaining, and 10 µl of the HGE bacteria added and then coincubated for 15 min at room temperature. The cells w...
Background: Anaplasma phagocytophilum (Ap) is an obligate intracellular bacterium and the agent of human granulocytic anaplasmosis, an emerging tick-borne disease. Ap alternately infects ticks and mammals and a variety of cell types within each. Understanding the biology behind such versatile cellular parasitism may be derived through the use of tiling microarrays to establish high resolution, genome-wide transcription profiles of the organism as it infects cell lines representative of its life cycle (tick; ISE6) and pathogenesis (human; HL-60 and HMEC-1).
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