Serological analysis
A modified agglutination test (MAT) was used to detect immunoglobulin G (IgG) specific for T. gondii as previously described by Desmonts and Remington (1980), and Dubey and Desmonts (1987). Formalin-fixed tachyzoites were used as antigen (2x108 tachyzoites/mL from KeraFAST Inc., Boston, MA, USA. Catalog No. EH2002). Sera were serially diluted in buffered saline solution (1X PBS) from 1/4 to 1/8192 in 96-well round bottom microplates and 25 ul of antigen mixture (2.5 ml alkaline buffer, 35 ul 2-mercaptoethanol, 50 ul Evans blue dye (2 mg/ml in H2O) and 150 ul T. gondii MAT antigen) were added to each well. The microplates were covered with sealing tape and incubated at 37°C for 24 hours and subsequently for 4 hours at 4°C. A positive reaction was characterized by a complete “mesh” of agglutination and a clear bottom, while sedimentation of antigen suspension in the form of a blue pellet at the bottom of the well was considered to be a negative reaction. Sera from mice and/or humans with a titer of 1/200 or greater were included as positive controls and negative controls were also included with each group of processed sera. All antibody titers that were 1/16 or higher were reported as positive. Titer values from captive versus free ranging primates were analyzed using a Mann-Whitney two tailed test to know if there are significant differences at a 0.05 alpha value.