Characterization and evaluation of a recombinant multiepitope peptide antigen MAG in the serological diagnosis of Toxoplasma gondii infection in pigs

Background: Toxoplasmosis caused by Toxoplasma gondii is a serious disease threatening human and animal health. People can be infected with T. gondii by ingesting raw pork contaminated with cysts or oocysts. Serological test is a sensitive and specific method usually used for large-scale diagnosis of T. gondii infection in humans and animals (such as pigs). Commercial pig Toxoplasma antibody ELISA diagnostic kits are expensive, which limits their use; moreover, the wide antigen composition used in these diagnostic kits is still unclear and difficult to standardize. The multiepitope peptide antigen is a novel diagnostic marker, and it has potential to be developed into more accurate and inexpensive diagnostic kits.

Methods: The synthetic multiepitope antigen (MAG) cDNA encoding a protein with epitopes from five T. gondii-dominant antigens (SAG1, GRA1, ROP2, GRA4, and MIC3) was designed, synthesized, and expressed in Escherichia coli BL21 (DE3) strain. The recombinant protein was detected through western blot with pig anti-T. gondii-positive and -negative serum, and then IgG enzyme-linked immunosorbent assay (ELISA) named MAG-ELISA was designed. The MAG-ELISA was evaluated in terms of specificity, sensitivity, and stability. The MAG-ELISA was also compared with a commercial PrioCHECK^® Toxoplasma Ab porcine ELISA (PrioCHECK ELISA). Finally, the trend of pig anti-T. gondii IgG levels after artificial infection with RH tachyzoites was evaluated using MAG-ELISA and two other ELISA methods (rMIC3-ELISA and PrioCHECK ELISA).

Results: MAG antigen could be specifically recognized by pig anti-T. gondii-positive but not -negative serum. MAG-ELISA showed high diagnostic performance in terms of specificity (88.6%) and sensitivity (79.1%). MAG-ELISA could be used for detecting anti-T. gondii IgG in the early stage of T. gondii infection in pigs (at least 7 days after artificial infection).

Conclusions: Our results suggest that MAG antigen can be applied to specifically recognize anti-T. gondii IgG in pig, and MAG-ELISA has the potential for large-scale screening tests of T. gondii infection in pig farms and intensive industries.