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.