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

Abstract

Background: Free-ranging chickens are often infected with Toxoplasma gondii and seroconvert upon infection. This indicates environmental contamination with T. gondii. Methods: Here, we established a bead-based multiplex assay (BBMA) using the Luminex technology for the detection of T. gondii infections in chickens. Recombinant biotinylated T. gondii surface antigen 1 (TgSAG1 bio) bound to streptavidin-conjugated magnetic Luminex beads served as antigen. 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 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, PCRs and bioassay in mice. Results: In experimentally infected chickens, the vast majority (98.5%, n = 65/66) of birds tested seropositive in the BBMA. This included all chickens positive by magnetic-capture PCR (100%, n = 45/45). Most, but not all inoculated and TgSAG1 bio-BBMA-positive chickens were also positive in two previously established TgSAG1-ELISAs (TgSAG1-ELISA SL , n = 61/65; or TgSAG1-ELISA SH , n = 60/65), or positive in an immunofluorescence assay (IFAT, n = 64/65) and in a modified agglutination test (MAT, n = 61/65). All non-inoculated control animals (n = 28/28, 100%) tested negative. In naturally exposed chickens, the TgSAG1 bio-BBMA showed a high sensitivity (98.5%; 95% confidence interval, CI: 90.7-99.9%) and specificity (100%; 95% CI: 85.0-100%) relative to a reference standard established using ELISA, IFAT and MAT. Almost all naturally exposed chickens that were positive in bioassay or by PCR tested positive in the TgSAG1 bio-BBMA (93.5%; 95% CI: 77.1-98.9%), while all bioassay-or PCR-negative chickens remained negative (100%; 95% CI: 85.0-100%). Conclusions: The TgSAG1 bio-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 a component in future multiplex assays for broad serological monitoring of poultry and other farm animals for various pathogens.

Prevalence of Toxoplasma gondii in retail fresh meat products from free-range chickens in Spain

“…Despite meat being considered an important source of human exposure to T. gondii , chickens are not regarded as a high-risk vector, because typically their meat is well cooked ( 16 ). Nonetheless, specific recipes (such as chicken carpaccio, barbecued chicken or chicken sashimi) or products (sausages) call for procedures in which meat may not be heat treated sufficiently to inactivate the parasite ( 9 ).…”

Development of an Immunochromatographic Test Based on Rhoptry Protein 14 for Serological Detection of Toxoplasma gondii Infection in Swine

“…Whereas several new laboratory diagnostic methods have been established for the serological detection of T. gondii in animals, they are not yet used clinically at a large scale. A novel luciferase-linked antibody capture assay (LACA) [ 34 ] and a fluorescent bead-based multiplex assay [ 35 ] were recently established for the individually serological diagnosis of T. gondii in pigs and chickens. In addition, a miniaturized protein microarray assay was recently developed for analysis of antibodies in meat juice and blood serum with high sample throughput for the slaughter of pigs [ 36 ].…”

Toxoplasma gondii in Foods: Prevalence, Control, and Safety

“…Duong et al (2020) developed a Luciferase-linked antibody capture assay (LACA) to detect T. gondii in serum chicken, and they obtained high sensitivity (90.5%) and specificity (95.4%) [ 85 ]. The best results were obtained by Fabian et al (2020) that recently developed a new serological method that was named bead-based multiple assay (BBMA) using the Luminex technology with high sensibility (98.5%) and specificity (100%) relative to a reference of ELISA, IFAT, and MAT [ 53 ].…”