Toxoplasma gondii is an apicomplexan intracellular protozoan parasite, and it can infect any warm-blooded vertebrates including humans and domestic animals . Toxoplasmosis caused by T. gondii infection threatens human and animal health especially for pregnant and in immunocompromised individuals [2, 3]. Human could be infected with T. gondii by ingesting food and raw pork meat contaminated with cysts or oocysts [4, 5]. Pork is the main meat source in many countries, such as China. Many epidemiological investigations show that pig farms and intensive industries possess high prevalence and parasite load through PCR detection and serological test, but the detection of T. gondii in pigs is usually not taken seriously in many pig farms and intensive industries for expensive diagnosis cost and high error rate [6, 7, 8]. Therefore, 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.
The diagnosis approach of toxoplasmosis has been constantly evolving, including traditional techniques (etiology, immunology, and imaging diagnosis) and many emerging molecular techniques. The etiological diagnosis of toxoplasmosis is relatively time-consuming since it involves the isolation of numerous disease materials and requires considerable skills to get reliable results. Thus, it is impossible to apply etiological diagnosis for large-scale clinical test in pig farms and intensive industries. Imaging diagnosis is mainly applied to cerebral and ocular toxoplasmosis through large medical equipment including computed tomography (CT), magnetic resonance imaging (MRI), nuclear imaging and ultrasonography (US), but imaging diagnostic results might not be reliable, and it requires expert interpretation . Molecular techniques are widely applied to the epidemiological survey and clinical diagnosis of toxoplasmosis for their accuracy and sensitivity . Molecular technique used for toxoplasmosis diagnosis is a high-sensitivity nucleic acid detection method of parasites in biological samples, and it overcomes the limitations of the serological tests, in addition, molecular technique mainly includes PCR, nested PCR, Real-time PCR, loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA) assay [11, 12, 13]. However, parasite nucleic acid detection involving DNA extraction tends to be expensive, and it is only accessible to the laboratory. Immunological detection is common method to determine the immune status of host by examining the change patterns of several different specific antibodies (IgA, IgM, IgG and IgE) after T. gondii infection [1, 14]. The common immunological method of toxoplasmosis diagnosis includes enzyme-linked immunosorbent assays (ELISA), modified agglutination test (MAT), and others [15, 16, 17].
ELISA is a serological detection that can be easily performed on a large scale, and many commercial kits are available to detect specific immunoglobulins (Igs) after T. gondii infection. The solid phase antigen used for ELISA includes crude tachyzoite antigen, Escherichia coli recombinant antigen, and chimeric peptide antigen. Although toxoplasma lysate antigen (TLA) possesses high levels of sensitivity and specificity in ELISA, there are the problems with TLA such as false positive results, standardization difficulty, unclear antigen composition, and complex and expensive TLA preparation [18, 19]. It is impossible to detect all serologically positive individuals by using one or several Escherichia coli recombinant antigens, because the expression patterns of genes from different T. gondii strains are diverse during different infection stages . Synthetic multiepitope antigen also known as chimeric antigen is a new generation of recombinant product for ELISA, and it contains multiple immunoreactive epitopes from several dominant antigens of T. gondii. Multiepitope antigens have been widely used for toxoplasmosis diagnosis, for example, synthetic multiepitope antigens are applied to detect anti-T. gondii IgG and IgM, and AMA1-SAG2-GRA1-ROP1 chimeric antigens are used for detecting specific antibodies of human and mouse in the early and chronic T. gondii infection . The chimeric antigen technology has been developed for the serological diagnosis of the Trypanosoma cruzi infection caused by another protozoan parasite, the cutaneous anthrax caused by Bacillus anthracis, the human T-lymphotropic virus type I (HTLV-1) infection,and others [22, 23, 24, 25]. However, few studies have been conducted to evaluate chimeric antigens for serodiagnosis of T. gondii in pigs and to design an ELISA kit using synthetic antigens for the large-scale diagnosis of toxoplasmosis in pig farms and intensive industries.
Many T. gondii proteins are mainly secreted outside through three specific organelles (rhoptry, dense granule, and microneme), some of which could well activate host immune system. T. gondii surface antigen 1 (SAG1), as a highly immunogenic protein, is mainly distributed on the tachyzoite surface by glycosyl-phosphatidylinositol anchoring [26, 27]. Dense granule protein 1 and 4 (GRA1 and GRA4) secreted by T. gondii have good antigenicity [28, 29, 30, 31]. Rhoptry protein 2 (ROP2) belonging to ROP2-protein family is expressed in three stages (tachyzoites, bradyzoites and sporozoites) of T. gondii life cycle, and this protein induces a strong antibody response in mice and humans [32, 33]. Microneme protein 3 (MIC3), as an adhesion molecule, expressed in T. gondii could be recognized by anti-T. gondii positive serum. Mice immunized with recombinant pseudorabies viruses expressing MIC3 can produce high level of anti-T. gondii IgG to provide effective protection against T. gondii challenge in BALB/c mouse model . Though these antigens (SAG1, GRA1, ROP2, GRA4 and MIC3) have been well documented to stimulate host immunity, little work has been done to determine whether the chimeric antigen with their T cell and (or) B cell epitopes is a good diagnostic marker for toxoplasmosis in pig farms and intensive industries.
To develop an efficient and low-cost ELISA kit for toxoplasmosis diagnosis in pigs, our study synthesized a multiepitope antigen (MAG) gene from 5 T. gondii dominant antigen genes (SAG1, GRA1, ROP2, GRA4 and MIC3) and evaluated the chimeric protein expressed by this MAG. A multiepitope protein encoded by MAG was designed, expressed, and purified from Escherichia coli BL21 (DE3) strain. The reactivity of MAG antigen was determined through western blot, and MAG protein was strongly recognized by pig anti-T. gondii positive serum, but not by negative serum. Horseradish peroxidase (HRP)-conjugated recombinant protein A/G was applied as secondary antibody in our ELISA since this recombinant protein has strong IgG binding ability, and it is less pH-dependent than Protein A or Protein G alone, thus performing better at pH 5–8 [35, 36]. The optimized MAG-ELISA was applied to detect 209 pig serum samples. The overall coincidence rate between MAG-ELISA and a commercial PrioCHECK ELISA was 78.47%. Furthermore, MAG-ELISA could diagnose positive IgG at 7 days post pig artificial peritoneal infection with RH tachyzoites with diagnosis results obtained earlier than those obtained with the commercial ELISA kit. Therefore, our MAG-ELISA has the potential for large-scale diagnosis of T. gondii infection in pig farms and intensive industries.