Preliminary results of a competition ELISA to detect Anaplasma marginale antibodies

SARLI, MACARENA; VALENTINI, BEATRIZ S.; NOVOA, MARÍA B.; THOMPSON, CAROLINA S.; TORIONI-DE-ECHAIDE, SUSANA M.; PRIMO, MARÍA E.

Mar del Plata

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2019

Sociedades de Biociencias

Bovine anaplasmosis is caused by the intraerythrocytic rickettsia Anaplasma marginale. It is an arthropod-borne hemolytic disease characterized by high lethality in adults. The disease is expanding and it is a threat to cattle industry. Live vaccine based on A. centrale, a less pathogenic species, is used to prevent acute anaplasmosis. Competition ELISA (cELISA) is used to assess rodeo epidemiological status through the detection of anti-MSP5 antibodies. A. marginale MSP5 (MSP5m) and A. centrale MSP5 (MSP5c) have 91% of identity and cELISA does not differentiate A. marginale-infected from A. centrale-vaccinated cattle. In this work, a cELISA (cELISAm) was developed to detected specific A. marginale antibodies. cELISAm is based in the antigen A. marginale MSP5 and the Am6 monoclonal antibody. Am6 recognized an A. marginale specific epitope, absent in A. centrale. ELISA plates were coated with MSP5m (1μg/well) overnight at 4ºC. After blocked, the plates were incubated with serum samples diluted 1:2 in PBST/10% fat-free dried milk containing 35 μg/ml MSP5c. Then, Am6 1/2500 and anti-mouse IgG peroxidase conjugate 1/3000 in PBS were added consecutively. The reaction was revealed with ABTS/H2O2. Results were expressed as inhibition percentage (%I). A total of 744 serum samples, previously classified by cELISA and nPCR, were analyzed by cELISAm: 497 negative, 185 A. marginale positive and 62 A. centrale positive serum samples. Sensitivity and specificity of the cELISAm were 97% and 95%, respectively with a cutoff >18 %I. The 65% of A. centrale-vaccinated cattle were not detected by cELISAm. Preliminary results showed that the cELISAm developed, allows us to detect with high sensitivity A. marginale-infected cattle decreasing the cross reaction for A. centrale. The identification of the specific epitope recognized for Am6 and the use of the polypeptide of this epitope in a cELISA with Am6, could enhance the performance of this specific cELISAm in the future.