Epidemiological Studies of Equine Piroplasmosis in two regions of Argentina.

ASENZO G; WILKOWSKY S; BARRANDEGUY, M; MESPLET M; BENITEZ D; FLORIN-CHRISTENSEN, M.

Buenos Aires, Argentina

Congreso; VI International Conference of Tick and Tick-borne Diseases; 2008

Theileria equi, a tick–borne protozoon, is one of the causative agents of equine piroplasmosis, a disease that affects horses, mules, donkeys and zebras, giving rise to fever, reduced appetite, malaise and general weakening. Importantly, equine piroplasmosis-free status is mandatory for the international trade of horses. Although Argentina is a major exporter of horses, there is very scarce epidemiological information about equine piroplasmosis in this country, information that would be critical for the implementation of control strategies. To this aim, we have developed an indirect ELISA to detect anti-Theileria equi antibodies in horse sera, based on the conserved surface antigen EMA-1. A recombinant form of a 229 amino acid fragment of EMA-1 was produced in E. coli using the pBAD/thioTOPO expression system, purified by affinity chromatography in Ni-agarose and used for the development of this iELISA. The conditions that gave maximal differences between positive and negative reference sera were as follows: 50 mg of recombinant protein was used per well, blocking was carried out with 0.5% BSA/0.1% Tween-20 in PBS; sera were used in a 1:100 dilution, and peroxidase-labeled anti-equine IgG (1:1000) was used as secondary antibody, followed by ABTS, as substrate, and absorbancy measurements at 405 nm. Blocking and incubation with sera were carried out at 37ºC with shaking. For the calculation of the cut-off the results obtained with this iELISA on 63 negative and 117 positive sera were compared in a ROC curve with those obtained with a commercial cELISA recommended by the OIE. A concordance test was applied and the К value obtained was of 0.79 (significance: acceptable). Once this iELISA was set up, it was applied to analyze horse serum samples from two Argentine regions: the town of Mision Laishi in the province of Formosa (n=159), a tick-endemic region of the NE of the country, and the province of Entre Rios (n=84), situated North of the province of Buenos Aires. The positivity percentages obtained in these two regions were of 88.05 % and 17.86 %, respectively. The preliminary prevalence obtained in Entre Rios is surprisingly high, considering that this province is considered to be tick-free. The iELISA developed in this work can constitute a useful tool for further epidemiological studies of equine piroplasmosis.