ISOLATION OF THE EQUINE GRANULOCYTIC EHRLICHIOSIS AGENT, EHRLICHIA-EQUI, IN TICK CELL-CULTURE

The equine granulocytic ehrlichiosis agent, Ehrlichia equi, is closely related or identical to the human granulocytic ehrlichiosis (HGE) age nt, Both are suspected of being transmitted by ticks, We have successf ully isolated E. equi in a cell line, IDE8, derived from a putative ve ctor, the tick Ixodes scapularis, Peripheral blood leukocytes from an experimentally infected horse were inoculated onto IDE8 monolayers. Cu ltures were incubated in a candle jar at 34 degrees C in tick cell cul ture medium with NaHCO3 and an organic buffer [3-(N-morpholino)-propan esulfonic acid] (MOPS). Within 2 weeks, infected cells were detected i n Giemsa-stained culture samples, and the organisms subsequently sprea d to uninfected cells in the cultures, E. equi was passaged serially b y transferring a portion of an infected culture to new cell layers eve ry 2 to 3 weeks. The identity of the organisms was confirmed by PCR us ing oligonucleotide primers specific for E. equi and the HGE agent and by immunocytology, Homologous equine antibodies and human anti-HGE co nvalescent serum recognized E. equi grown in tick cell culture, Electr on microscopy revealed electron-lucent and -dense ehrlichia-like forms developing within host cell endosomes. E. equi passaged twice in tick cell culture retained infectivity and pathogenicity for the equine ho st, as demonstrated by intravenous inoculation of a suspension of infe cted tick cells and subsequent reisolation from peripheral blood, in f ulfillment of Koch's postulates, The horse developed severe clinical s igns, i.e., fever, inappetence. thrombocytopenia, icterus, and limb ed ema, typical of granulocytic equine ehrlichiosis, within 1 week.