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

Abstract

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. Graphical abstract

Mining the Proteome of Toxoplasma Parasites Seeking Vaccine and Diagnostic Candidates

“…It seems that, according to the heterogeneity of host immune responses against Toxoplasma infection and the possible challenges for selecting appropriate diagnostic markers, the combination of immunogens (synthetic multiepitope antigen) may be useful for the design of diagnostic tests in human toxoplasmosis [94,164]. In addition, recent advances in our knowledge of parasite genetics and gene manipulation, key antigenic epitopes, strain variation, delivery systems and induction of immune responses are considered participating insights for the development of new vaccines which may be more efficient against toxoplasmosis [165].…”

“…Furthermore, the integrated recombinant multi-epitope antigens of T. gondii (SAG1, ROP1, and GRA7) might be useful to develop clinical diagnostic kits for acute and chronic toxoplasmosis [93]. In addition, as a synthetic multiepitope antigen, the recombinant forms of several proteins, including SAG1, ROP2, GRA1, GRA4 and MIC3, have been also considered useful to design a potential ELISA test with specificity and sensitivity of 88.6% and 79.1%, respectively [94].…”

Exposure to Toxoplasma gondii in Asian Elephants (Elephas maximus indicus) in Thailand

“…The kappa value between LAT and TgGRA8-iELISA revealed a moderate agreement (κ = 0.42), with low sensitivity and specificity. Recently, multi-epitope or chimeric antigens have been used as an alternative approach to address the need for standardizing and increasing the sensitivity and specificity of serodiagnostic tests for animal toxoplasmosis [25,26,38]. Therefore, the application of a single recombinant protein or a mixture of recombinant proteins in an ELISA test to diagnose T. gondii infection in elephants needs to be developed and evaluated.…”

Recombinant multiepitope proteins expressed in Escherichia coli cells and their potential for immunodiagnosis

“…infantum -positive serum samples/50 healthy dog serum samples/30 serum samples from non- L . infantum diseases ELISA Sensitivity: 94% Specificity: 86% Direct agglutination PQ10 was recognized by 92% of asymptomatic and 96% of symptomatic infected dogs [ 80 ]/Iran Human toxoplasmosis/ Toxoplasma gondii pQE30 E. coli BL21(DE3) cells Indirect ELISA 95 toxoplasmosis-positive serum samples/25 serum samples from healthy individuals/6 serum samples from non-toxoplasmosis diseases Sensitivity: 72.6% Specificity: 90.3% [ 81 ]/Iran Pig toxoplasmosis/ Toxoplasma gondii MAG E. coli BL21(DE3) cells ELISA 82 toxoplasmosis-positive serum samples/127 healthy pig serum samples Sensitivity: 79.1% Specificity: 88.6% [ 82 ]/China Canine visceral leishmaniasis/ Leishmania infantum P1P2P3 E. coli BL21(DE3) cells ELISA 50 L . infantum -positive serum samples/50 healthy dog serum samples/14 serum samples from non- L .…”