The aim of this work was to evaluate the minimal protective dose of the inactivated vaccine against heartwater. In order to conduct a reliable vaccination trial, an in vitro quantification method of Ehrlichia ruminantium (ER) challenge doses was developed. In experimental conditions, homologous Gardel challenges with 2.1 x 104 to 9.4 x 104 live ER elementary bodies were reproducible and mimicked a natural challenge. Similar results were obtained when animals were challenged with 3 x 104 live elementary bodies from five different ER strains. A 28-fold reduction of the vaccine dose did not decrease protection when compared to the conventional dose of inactivated vaccine. Two injections of 35 [mu]g of ER antigen induced good protection against heartwater. Moreover, we found that the amount of IFN[gamma] secreted in blood from vaccinated animals after in vitro stimulation with ER antigen was not a reliable predictor of survival and could not be used to test the potency of the inactivated vaccine. © 2006 Elsevier Ltd. All rights reserved.