Isolation of the equine granulocytic ehrlichiosis agent, Ehrlichia equi, in tick cell culture

Abstract

The equine granulocytic ehrlichiosis agent, Ehrlichia equi, is closely related or identical to the human granulocytic ehrlichiosis (HGE) agent. Both are suspected of being transmitted by ticks. We have successfully isolated E. equi in a cell line, IDE8, derived from a putative vector, the tick Ixodes scapularis. Peripheral blood leukocytes from an experimentally infected horse were inoculated onto IDE8 monolayers. Cultures were incubated in a candle jar at 34؇C in tick cell culture medium with NaHCO 3 and an organic buffer [3-(Nmorpholino)-propanesulfonic acid] (MOPS). Within 2 weeks, infected cells were detected in Giemsa-stained culture samples, and the organisms subsequently spread to uninfected cells in the cultures. E. equi was passaged serially by transferring a portion of an infected culture to new cell layers every 2 to 3 weeks. The identity of the organisms was confirmed by PCR using oligonucleotide primers specific for E. equi and the HGE agent and by immunocytology. Homologous equine antibodies and human anti-HGE convalescent serum recognized E. equi grown in tick cell culture. Electron microscopy revealed electron-lucent and-dense ehrlichialike forms developing within host cell endosomes. E. equi passaged twice in tick cell culture retained infectivity and pathogenicity for the equine host, as demonstrated by intravenous inoculation of a suspension of infected tick cells and subsequent reisolation from peripheral blood, in fulfillment of Koch's postulates. The horse developed severe clinical signs, i.e., fever, inappetence, thrombocytopenia, icterus, and limb edema, typical of granulocytic equine ehrlichiosis, within 1 week.

A 16S rRNA-based PCR assay for detection and identification of granulocytic Ehrlichia species in dogs, horses, and cattle

“…Rather few laboratory diagnostic methods are available. Cultivation of granulocytic ehrlichiae has recently been demonstrated in cultures of different cell lines (10,19), but it is still not a routine method for the clinical laboratory. In the acute febrile phase, granulocytic inclusions can usually be demonstrated in circulating neutrophils; however, this method is impaired by subjective errors, leading to both false-positive and false-negative results.…”

Unique macrophage and tick cell-specific protein expression from the p28/p30-outer membrane protein multigene locus in Ehrlichia chaffeensis and Ehrlichia canis

“…The Ixodes scapularis cell line, ISE6, is one of the two tick cell lines that are currently used for the assessment of the impact of tick cell environment on the growth of Ehrlichia and Anaplasma species pathogens (Munderloh et al, 1996;Bell-Sakyi et al, 2000;Jauron et al, 2001;Blouin et al, 2002;Singu et al, 2005). As I. scapularis is a non-vector tick for both E. chaffeensis and E. canis (Anderson et al (1993), the validity of the cell line to serve as a good model system is a concern.…”

Cell cycle effects and induction of apoptosis caused by infection of HL-60 cells with human granulocytic ehrlichiosis pathogen measured by flow and laser scanning cytometry

“…As is evident, ehrlichiae manifested their presence predominantly in the cytoplasm. Aggregates of the organism were seen within the infected cells often forming characteristic morulae-like structures (21,22). In some cells the pathogen was located peripherally, apparently in association with the plasma membrane.…”