Development and Field Validation of a Double-Antigen Sandwich Colloidal Gold Immunochromatographic Strip for Detection of Toxoplasma gondii Antibodies in Multiple Host Species.

Toxoplasmosis, a globally prevalent zoonosis caused by Toxoplasma gondii (T. gondii), poses major threats to both human and animal health, leading to reproductive losses in livestock and severe disease in immunocompromised individuals. Although enzyme-linked immunosorbent assay (ELISA) and PCR are widely used for diagnosis and surveillance, they may be limited by turnaround time, laboratory instrumentation, and, in the case of serological assays, the need for species-specific reagents. To enable rapid, equipment-minimal detection applicable to multiple host species, we developed a point-of-care (POC) double-antigen sandwich colloidal gold immunochromatographic assay (GICA) based on recombinant surface antigen 2 (rSAG2) of T. gondii. In this assay, rSAG2 served both as the capture antigen immobilized on the test line and as the colloidal gold-conjugated detection probe. Key parameters, including conjugation pH, antigen loading, and buffer composition, were systematically optimized. The resulting strip showed a detection limit of a 1:40 serum dilution, and no cross-reactivity was observed with sera positive for 23 common pathogens from different host species. Good repeatability and storage stability for 4 months at 4°C were also observed. For field evaluation, 409 clinical serum samples from six animal groups (100 chickens, 68 dogs, 30 cats, 81 pigs, 80 yaks, and 50 sheep) were tested, and seropositivity rates ranged from 2.9% to 31.3% across the sampled groups. In a subset of chicken (n = 46) and dog (n = 44) sera tested in parallel with the corresponding commercial ELISA kits, the rSAG2-GICA showed good preliminary agreement, with overall agreement rates of 91.3% and 97.7%, respectively. Collectively, this rSAG2-based GICA shows potential as a rapid and practical tool for on-site serological screening of T. gondii antibodies across multiple host species and may support epidemiological surveillance in diverse animal populations.