Field trial of a competitive ELISA based on Babesia bovis MSA-2c for the serological diagnosis of bovine babesiosis in Argentina

DOMINGUEZ M, ECHAIDE I, DE ECHAIDE ST, WILKOWSKY S, ZABAL O, SCHNITTGER L, FLORIN-CHRISTENSEN M

Congreso; TTP7: Seventh Ticks and Tick-borne pathogens International Conference; 2011

Infections by Babesia bovis, one of the etiological agents of bovine babesiosis, cause important losses and limit cattle production in the Argentine North West and North East as well as in several tropical and subtropical areas of the world. In regions of Argentina endemic for the B. bovis tick vector, Rhipicephalus (Boophilus) microplus, calf sera are annually monitored to detect areas of enzootic instability and take, if necessary, strategic measures such as vaccination. The Merozoite Surface Antigen 2-c (MSA-2c) is an immunodominant protein, located on the surface of B. bovis merozoites with exposed B-cell epitopes that are conserved among geographic isolates. Thus, this antigen can be useful for the development of serological diagnostic tools. In previous work, monoclonal antibodies against a recombinant form of MSA-2c were generated, one of which (MAb H9P2C2) proved useful to discriminate between B. bovis positive and negative sera in a pilot competitive ELISA (cELISA). In this work, the conditions for this assay were re-evaluated with respect to antigen and MAb concentration, the choice of blocking reagent, and serum dilutions. Those conditions that gave maximal differences between positive and negative reference sera were selected. Once the test was optimized, a cut-off value was determined by ROC analysis using B. bovis-positive and negative sera diagnosed as such using indirect immunofluorescence assay (IFA), which has been validated by OIE as a gold standard test for bovine babesiosis. Positive samples (n=104) were either from endemic regions or experimentally infected animals, and negative sera (n=253), came either from tick-free regions, experimentally infected with B. bigemina or naturally infected with Anaplasma marginale. Considering a cut-off value of 29.5%, expressed as percentage of inhibition, the sensitivity and specificity of the method resulted in 96.2% and 98%, respectively. Comparison between IFA and MSA-2c-cELISA was carried out by calculation of the Kappa index, using additional sera of bovines from endemic regions (n=211), that had or had not suffered natural infection; and from tick-free regions (n=92). The Kappa index obtained was of 0.8325 (very good agreement). These results indicate that the developed cELISA is an efficient method for the detection of parasite-specific antibodies and provides a useful tool for the control of bovine babesiosis. Financed by INTA (AESA 203961, CONICET, ANPCyT (PICT 2002-00054), the European Commission (INCO 003691) and Asoc. Coop. EEA Rafaela 426100.