Blood sera were tested for the presence of anti-T. gondii IgG antibodies, using an in-house ELISA in flat-bottom 96-well microplates as previously described (Bahrami et al. 2019).

The microplates (Greiner, Germany) were coated with 50 µL of tachyzoite antigen of T. gondii RH strain (Razi institute, Iran), diluted in carbonate buffer (Sigma-Aldrich), pH 9.6 (1:50), and incubated overnight at 4 °C. The excess antigen was removed by washing the microplates with phosphate buffered saline (PBS) (Sigma-Aldrich). Blocking was then performed with blocking buffer (5% skimmed milk) for 60 min at 37 °C. Having washed the microplates, we added 100 µL of sera (1:100 in PBS with Tween 20 or PBST) and the microplates were then incubated for 120 min at 37 °C. The wells were washed again as above, then 50 µL of alkaline phosphatase-labeled anti-goat/sheep conjugate (Sigma-Aldrich) was added (1:500 in PBST) and incubated for 60 min at 37 °C. Once the micrplates were washed, 50 µL of substrate solution (10 mg/mL 4-nitrophenyl phosphate in 10 mL diethanolamine buffer, pH 9.6) was added and the microplates were incubated for 30 min at room temperature. The reaction was stopped with 50 µL of hydrochloric acid (20%). Negative control was obtained from a new-born sheep. After gaining the approval of the local ethics committee for the study protocol, the sheep were infected by a two-step intramuscular and subdermal injection of live tachyzoites to obtain a positive control sample. The sensitivity and specificity of this in-house ELISA test were 90.1% and 85.9%, respectively. The absorbance was read at 450 nm after 30 min using an automatic microplate reader (ELX800-Biotek, Biotek). The cut-off of optical densities (OD) was calculated using the method of Hillyer et al. (1992) including the mean OD of negative control sera plus two standard deviations.

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