Fluorescent bead-based serological detection of Toxoplasma gondii infection in chickens

Background: Especially free-ranging chickens are frequently exposed to Toxoplasma gondii. They are sensitive indicators for environmental contamination with T. gondii oocysts. The detection of infected birds relies primarily on serological assays. Methods: Here, we established a bead-based multiplex assay (BBMA) using the Luminex technology for the specific and sensitive detection of T. gondii infections in chickens. Recombinant biotinylated T. gondii surface antigen 1 (TgSAG1bio) bound to streptavidin-conjugated magnetic Luminex beads served as antigen. Specific serum antibodies were detected by a fluorophore-coupled secondary antibody. Beads of differing color codes were conjugated with anti-chicken IgY or chicken serum albumin and served for each individual sample as an internal positive or negative control, respectively. The assay was validated with sera from experimentally and naturally infected chickens. The results were compared to those from reference methods, including other serological tests and bioassay in mice. Results: In experimentally infected chickens, all chickens were positive by magnetic-capture PCR (100%, n=45/45) and the vast majority (98.5%, n=65/66) of inoculated birds tested seropositive in the BBMA. Most, but not all inoculated and TgSAG1bio-BBMA-positive chickens were also positive in two previously established TgSAG1-ELISAs, an immunofluorescence assay (IFAT) and in a modified agglutination test (MAT). All non-inoculated control animals (n=28) tested negative. In naturally exposed chickens, the TgSAG1bio-BBMA showed a high sensitivity (98.5%; 95% Confidence Interval: 90.7-99.9%) and specificity (100%; 85.0-100%) relative to a reference standard established using results obtained with ELISA, IFAT and MAT. Almost all naturally exposed chickens that were positive in the mouse bioassay or by PCR tested positive in the TgSAG1bio-BBMA (93.5%, 77.1-98.9%), while all mouse bioassay- or PCR-negative chickens remained negative (100%, 85.0-100%). Conclusions: The TgSAG1bio-BBMA represents a suitable method for the detection of T. gondii infections in chickens with high sensitivity and specificity, which is comparable or even superior to other tests. Since assays based on this methodology allow for the simultaneous analysis of a single biological sample with respect to multiple analytes, the described assay may represent one component in future multiplex assays for broad serological monitoring of poultry and other farm animals, including pigs or small ruminants, for various pathogens.