Development and evaluation of recombinant dense granule 14 for serological diagnosis of Toxoplasma gondii infection in domestic cats

Toxoplasma gondii is a widespread zoonotic protozoan parasite, with domestic cats as definitive hosts. To improve serodiagnosis surveillance and control, this study aimed to develop and evaluate an indirect enzyme-linked immunosorbent assay (iELISA) using recombinant Tg GRA14 protein for the serological detection of T. gondii in domestic cats. The Tg GRA14 gene (nucleotides 114–858 encoding amino acids 38–286) was synthesized and cloned into the pET-28a vector containing an N-terminal FLAG tag. The recombinant plasmid was transformed into E. coli BL21, and protein expression was purified via anti-FLAG affinity chromatography. Protein integrity, antigenicity and identity was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), blotting, while mass spectrometry verified protein identity. The purified Tg GRA14 protein was used as a coating antigen in iELISA, which was applied to 149 feline serum samples and compared with results from the indirect fluorescent antibody test (IFAT). The recombinant Tg GRA14 protein (approximately 29 kDa) was successfully expressed and purified, with confirmed antigenicity and 99.6–100% identity by mass spectrometry. The iELISA detected 38.3% seroprevalence in feline samples, compared to 32.9% by IFAT, and showed high diagnostic performance with 95.9% sensitivity, 90.0% specificity, and a kappa value of 0.824. These results indicate that the iELISA based on recombinant Tg GRA14 is a highly sensitive and specific diagnostic tool for detecting T. gondii infection in domestic cats, which has strong potential for application in epidemiological surveillance and serological screening programs.