Development of a polyclonal competitive enzyme-linked immunosorbent assay for detection of antibodies to Ehrlichia ruminantium

A polyclonal competitive enzyme-linked immunosorbent assay (PC-ELISA) is described for detection of antibodies to Ehrlichia (Cowdria) ruminantium by using a soluble extract of endothelial cell culture-derived E. ruminantium as the antigen and biotin-labeled polyclonal goat immunoglobulins as the competitor. For goats, the diagnostic sensitivity and specificity were both 100% with a cutoff of 80% inhibition (80 PI), with detection of antibodies for 550 days postinfection. For cattle, diagnostic sensitivity and specificity were 86 and 100%, respectively, with a cutoff of 50 PI and 79 and 100% with a cutoff of 70 PI. Cross-reactions with high-titer experimental or field antisera to other Ehrlichia and Anaplasma species were observed at up to 68 PI in cattle and up to 85 PI in sheep, and therefore to exclude these cross-reactions, cutoffs of 70 PI for bovine serology and 85 PI for small-ruminant serology were selected. Application of the PC-ELISA to bovine field sera from South Africa gave a higher proportion of positive results than application of the murine macrophage immunofluorescent antibody test or indirect ELISA, suggesting a better sensitivity for detection of recovered cattle, and results with bovine field sera from Malawi were consistent with the observed endemic state of heartwater and the level of tick control practiced at the sample sites. Reproducibility was high, with average standard deviations intraplate of 1.2 PI and interplate of 0.6 PI. The test format is simple, and the test is economical to perform and has a level of sensitivity for detection of low-titer positive bovine sera that may prove to be of value in epidemiological studies on heartwater

Growth of Cowdria ruminantium, the causative agent of heartwater, in a tick cell line

The tick-borne rickettsia Cowdria ruminantium has been propagated continuously for over 500 days in the Ixodes scapularis tick cell line IDE8 by using the Gardel isolate from bovine endothelial cells as an inoculum. Infection of the tick cells was confirmed by PCR, karyotyping, electron microscopy, and reinfection of bovine cells