The reactivity of IgG in animal sera to the VAR2CSA protein expressed on IE surface was measured by flow-cytometry (FCM) using a protocol modified from [39] . Briefly, the parasite culture was enriched for late trophozoite and schizont stage parasites in a strong magnetic field (MACS, Miltenyi). Aliquots of 2610 5 IE in a total volume of 100 ml were labelled by ethidium bromide and sequentially exposed to (1) 15 ml animal serum and (2) 1:100 dilutions of FITC labelled secondary antibodies specific for IgG from the individual species: Mouse (FL2000, Vector); rat (62-9511, Invitrogen) and rabbit (FL1000, Vector). As a negative control, IE were incubated both with sera from non-immunized animals and without animal serum and then exposed to secondary antibodies specific against IgG from different animals spec. Data from 5000 infected cells (ethidium bromide positive) was collected on a FC500 flowcytometer (Beckman Coulter). The median FITC fluorescence intensity was determined using Winlist Software (Verity Software House).