Evaluation of an Antigen Enzyme Immunoassays Test with Monoclonal Antibody for Diagnosis of Tryapanosoma Evansi in Horses in Argentina

MONZON,C.M

PARIS

Simposio; Annual Meeting of the OIE ad hoc Group on Non Tsetse Transmitted Animal Trypanosomes; 2004

Oficina Internacional de Epizootias (OIE)

“Evaluation of an Antigen-Detection Enzyme Immunoassays Test with Monoclonal Antibody for Diagnosis of Trypanosoma Evansi in Horses in Argentina” Carlos Manuel Monzón Cátedra de Parasitología-Facultad de Ciencias de la Salud-Universidad Nacional de Formosa (U.Na.F.) Concejo Nacional de Investigaciones Científicas y Técnicas (CONICET) E-mail: cedivef@satlink.com A double antibody sandwich immunosorbent assay (ELISA) technique has been developed to diagnose Trypanosoma evansi infection in horses (Surra), based on circulating antigen detection. The assay uses as capture reagent the immunoglobulins fractions obtained from a goat immunised with T.evansi, while the detection system was prepared from of a monoclonal antibody (Mab) directed against internal T.evansi antigen and secondarily a peroxidase conjugate anti-mouse IgM (m-chain specific), antibody developed in goat. Previous results indicated that the Mab used in the test did not react with Trypanosoma cruzi, Babesia equi and B.caballi the main horse’s parasites protozoan in the sub-tropical area of Argentina. In an experimentally T.evansi infected horse circulating antigens were first detected by Ag-ELISA 16 days after infection, thereafter antigens levels showed a progressive increase, the test being also positive even when parasites could not be detected. Diminazene aceturate failed to detain the disease and ELISA-Ag remained positive; contrarily, antigens were cleared from serum 30 days after a successful Suramin drug treatment (Figure 1). The test was evaluated with sera samples of 156 horses from four groups infected with T.evansi; of which 86 horses were positive using standard parasite detection methods (SPDM).  The Ag- ELISA sensitivity rate varied between 70-91% for the different groups (Table 1).  As negative controls, 269 horses from the free area of this parasite were used which were all negative for T.evansi antibodies. ELISA–Ag results were expressed in terms of percent positivity (PP) comparative to a reference control serum (CP+++ 100%). The difference between a positive and a negative test was based in the histogram of the frequency distribution of the results, using sera from infected and non-infected horses (Figure 2). A 15PP gave a sensitivity of 81% for confidence interval (CI), of 95% between 71% to 88% and specificity of 98% for a CI between 95.5% and 99.3%. There was a positive association between infected animals and positive ELISA-Ag test (Kappa = 0.83 IC 95%: 0.76-0.90; (P=0.000). The inter-assay coefficient of variation (CV) expressed as PP, for the CP++, CP+ and CN was 5.4%, 10.6% and 43%, while de intra-assay CV for each of the above sera was 1.6 %, 0.97 % and 0.36 respectively. Positive and negative predictive values were determined for a 50% prevalence rate at 0.01%. In the four horse groups with T.evansi Ag-ELISA was positive in 70 of 86 (81%) parasite-positive horses by SPDM. Interestingly also was positive in 18 of 70  (25 %) parasite-negative horses, while a combination of SPDM and ELISA-Ag tests detected as infected 104 animals, indicating that these methods constitute a good combination to increase test sensitivity. Results obtained represent the first evaluation of this ELISA-Ag and the preliminary information generated so far indicate its usefulness for diagnosing T.evansi in horses in the subtropical area of Argentina.