Abstract
BackgroundToxoplasmosis 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.MethodsThe 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).ResultsMAG 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).ConclusionsOur 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.Graphical abstract
Highlights
Toxoplasmosis caused by Toxoplasma gondii is a serious disease threatening human and animal health
The multiepitope antigen (MAG) cDNA used in the indirect enzyme-linked immunosorbent assay (ELISA) was synthesized (TSINGKE Biological Technology), and the synthetic cDNA fragment was cloned into the pGEX-KG expression vector and expressed in Escherichia coli BL21(DE3) strain
To improve the performance of MAG-ELISA, we attempted to optimize the procedures related to MAG antigen, pig serum, buffer, secondary antibody, and ELISA substrate, and others
Summary
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). Toxoplasma gondii is an apicomplexan intracellular protozoan parasite, and it can infect all warm-blooded vertebrates, including humans and domestic animals [1]. This parasite threatens human and animal health especially for pregnant and in immunocompromised individuals [2, 3]. Humans can be infected with T. gondii by ingesting food and raw pork contaminated with cysts or oocysts [4, 5]. The development of simple, inexpensive, and sensitive diagnostic tests for T. gondii detection in pigs is crucial to reduce the risk of toxoplasmosis in humans and pigs
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