Characterization and Evaluation of a Recombinant Multiepitope Peptide Antigen MAG in the Serological Diagnosis of Toxoplasma Gondii Infection in Pigs

doi:10.21203/rs.3.rs-582733/v1

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Research Article

Characterization and Evaluation of a Recombinant Multiepitope Peptide Antigen MAG in the Serological Diagnosis of Toxoplasma Gondii Infection in Pigs

https://doi.org/10.21203/rs.3.rs-582733/v1

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published 17 Aug, 2021

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Background

Toxoplasmosis caused by toxoplasma gondii (T. gondii) is a serious disease threatening human and animal health. People could be infected with T. gondii by ingesting raw pig meat 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). Since commercial pig toxoplasma antibody ELISA diagnostic kits are too expensive, it is difficult to use them widely, moreover, the native antigen composition used in these diagnostic kits is still unclear and difficult to standardize. The multiepitope peptide antigen is novel diagnostic marker, and it has the potential to be developed into more accurate and inexpensive diagnostic kits.

Methods

The synthetic multiepitope antigen (MAG) gene encoding a protein with epitopes from 5 T. gondii dominant antigen (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, 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 level after artificially infection with RH tachyzoites was evaluated through 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 possessed a high diagnostic performance in terms of specificity and sensitivity. The overall coincidence rate between MAG-ELISA and a commercial PrioCHECK Toxoplasma antibody ELISA was 78.47%. 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 could be applied to specifically recognize anti-T. gondii IgG in pig, and MAG-ELISA has the potential for large-scale diagnosis of T. gondii infection in pig farms and intensive industries.

Parasitology

Toxoplasma gondii

pig

synthetic multiepitope antigen

indirect ELISA

serological detection

Toxoplasma gondii is an apicomplexan intracellular protozoan parasite, and it can infect any warm-blooded vertebrates including humans and domestic animals [1]. 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 [9]. Molecular techniques are widely applied to the epidemiological survey and clinical diagnosis of toxoplasmosis for their accuracy and sensitivity [10]. 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 [20]. 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 [21]. 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 [34]. 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.

Ethics statement

Our study was authorized by the animal ethical committee from Huazhong agricultural university in China with the number HZAUSW-2012-001. Pig serum samples were provided by the animal parasite laboratory of Huazhong agricultural university. Pigs for T. gondii artificial infection were provided by the pig farm of Huazhong agricultural university. Finally, all pigs were sacrificed through euthanasia after T. gondii infection experiment.

MAG recombinant chimeric antigen

The MAG gene used in the indirect ELISA was synthesized (TSINGKE Biological Technology), then the synthetic gene was cloned into the pGEX-KG vector, and expressed in Escherichia coli BL21(DE3) strain. MAG recombinant protein fused with glutathione S-transferase (GST) was induced with IPTG (isopropyl β-D-1-thiogalactopyranoside), and then purified by glutathione-based affinity chromatography (GE Healthcare Life Sciences, USA). Finally, the purified MAG antigens were quantified by enhanced BCA protein assay kit (Beyotime Biotechnology, China).

Western immunoblot analysis

The purified MAG protein was separated by SDS-PAGE to identify the protein molecular weight through protein molecular weight marker (ThermoFisher scientific), and analyzed by western blot. The Immobilon-PSQ PVDF membrane (0.2 µm pore size, Millipore, USA) onto which purified protein was transferred was incubated with pig anti-T. gondii positive or negative serum, and then detected with HRP-conjugated rabbit anti-swine IgG (H + L) (Frdbio bioscience & technology, China).

Pig artificial infection with tachyzoite

“Chang xin” binary miscellaneous commodity pigs (any gender, weight of 15-20kg) bred by the pig farm of Huazhong agricultural university were applied to perform RH (type I strain) tachyzoite artificial infection by intraperitoneal injection. Four pigs were randomly assigned into two groups with two pigs in treatment group and control group. Two pigs in treatment group were intraperitoneally injected with 5 million tachyzoites for T. gondii infection. The 2 pigs in control group were intraperitoneally injected with equal volume of sterile 0.9% NaCl. Serum samples were all collected from treatment group and control group at 0, 2, 4, 7, 14, 21, 28, 35, 42, and 49 day. The serum sample at 3 day before T. gondii infection was also collected for subsequent detection.

Optimization of ELISA procedure

ELISA was performed as described preciously [20]. The 96-well flat bottom microtiter plates (BIOFIL, China) were coated overnight at 4℃ with 100 ul per well of coating buffer (25mM carbonate buffer, PH9.6) containing MAG purified antigen. The 96 well plate was washed 3 times with wash buffer (PBS with 0.05% Tween 20). Blocking buffer (BSA in PBS) was added, then the plate was incubated for a period of time at 37℃. After three times rewash as described above, 100ul of pig serum diluted with serum dilution buffer was added to each well, and the plates were incubated for a period of time at 37℃. After being washed as described above, 100ul of HRP conjugated recombinant protein A/G diluted in blocking buffer was added to each well, and the plates were incubated for a period of time at 37℃. After being washed as described above, color was developed for a period of time at 37℃ in dark room after the addition of 100ul per well of the substrate solution containing 3, 3’, 5, 5’- tetramethylbenzidine (TMB, Sigma-Aldrich, USA) and H₂O₂. Finally, the OD630 value was measured with microplate reader Bio-Tek ELx-800 (BioTek Instruments, USA). All the tests were performed in duplicate wells.

To obtain the optimal dilution ratio of MAG-coated antigen and pig serum, the cross-titration was performed at serial dilution ratios of MAG antigen (1:100, 1:200, 1:400, 1:800, 1:1600, and 1:3200), T. gondii positive serum, and negative pig serum samples (1:20, 1:40, 1:80, 1:160, 1:320 and 1:640). The OD630 was measured and the ratio of P/N (positive/negative serum) was calculated at different dilution ratios. The dilution ratios of MAG antigen and pig serum with the maximum value of P/N was designated to be their optimal dilution ratio.

The optimal blocking buffer concentration (0.5%, 1.0%, 1.5%, and 2.0% BSA) and the optimal blocking time (30, 40, 50, and 60 mins) were determined as described above. The optimal serum dilution buffer (0.1% BSA, 0.5% BSA, and PBS containing 0.05% Tween 20) was determined by the maximum value of P/N. The optimal coating time of pig serum (30, 40, 50, and 60 mins) was determined by the maximum value of P/N under the above optimal conditions. The optimal dilution ratio (1:3000, 1:4000, 1:5000, 1:6000, 1:7000 and 1:8000) and the optimal reaction time (30, 40, 50 and 60 mins) of HRP-conjugated recombinant protein A/G were also determined through cross-titration. The optimal reaction time of TMB substrate solution (5, 7.5, 10 and 12.5 mins) was determined by the maximum value of P/N. The cut-off value of MAG-ELISA was determined referring to the OD630 mean value of 25 T. gondii negative pig sera under the optimal conditions (Cut-off = mean + 3SD).

Evaluation of MAG-ELISA

The specificity of MAG-ELISA was assessed by the diagnosis of 5 pig common virus sera (swine fever virus (SFV), porcine reproductive and respiratory disease virus (PRRSV), pseudorabies virus (PrV), porcine circovirus (PCV), and foot-and-mouth disease virus (FMDV)), and the control group including pig anti-T. gondii positive and negative sera. The sensitivity of MAG-ELISA was determined by measuring OD630 at serial dilution concentrations of pig anti-T. gondii positive sera. The coefficient of variation (CV) of repeated tests within batch and between batches was calculated and confirmed respectively through 6 samples (one anti-T. gondii positive and five negative pig sera). The stability was tested by the destruction experiments in which MAG-coated plates were placed at 37℃ for 12, 24, 36, 48, 60, and 72h, respectively.

Coincidence test

The 209 pig serum samples were tested by MAG-ELISA and a commercial PrioCHECK® Toxoplasma Ab porcine ELISA (Prionics, Switzerland), respectively. The coincidence of MAG-ELISA versus PrioCHECK ELISA was assessed according to two ELISA results.

Procedure of rMIC3-ELISA

The rMIC3-ELISA was performed in a previously reported ELISA method for detecting pig toxoplasmosis [37]. Recombinant entire T. gondii microneme protein 3 (rMIC3) fused with GST tag was expressed and purified as coated antigen. The 96-well plates were coated with rMIC3 at 3.40ug/ml, and pig serum samples were diluted at 1:160. The cut-off value of rMIC3-ELISA was defined as 0.40. The OD630 value was measured as described above.

Characterization of recombinant multiepitope antigen and Optimization of MAG-ELISA procedure

Our study selected 6 epitopes from 5 reported T. gondii dominant antigen genes (SAG1, GRA1, ROP2, GRA4, and MIC3) and designed a new chimeric recombinant multiepitope antigen (MAG) gene by concatenating peptides containing T cell and (or) B cell epitopes (Table 1, Fig. 1A). MAG was synthesized and cloned into pGEX-KG vector for prokaryotic expression. The MAG protein with GST tag (43.3KD) was successfully expressed in Escherichia coli BL21 (DE3) and purified through glutathione-based affinity chromatography. Western blot results showed the MAG protein was recognized specifically by pig anti-T. gondii positive serum but not by negative serum (Fig. 1B).

Table 1

Sources of chimeric antigen MAG
Gene	Epitope	Strain	Sequence	Position
SAG1	S1	GT1	TCPDKKSTA	59–67
SAG1	S2	GT1	ILPKLTENPWQ	246–256
GRA1	G1	GT1	DTMKSMQRDED	104–114
ROP2	R2	GT1	PGDVVIEELFNRIPETSV	197–214
GRA4	G4	GT1	SGLTGVKDSSS	235–245
MIC3	M3	GT1	KRTGCHAFRE…SCKCDNGYSG	233–310

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. The cross-titration showed that the optimal dilution ratio of MAG-coated antigen was 1:100 (0.91 ug/well) and that of pig serum was 1:160 (Fig. 2A). The maximum P/N was obtained when MAG-coated plate was incubated with 1.0% BSA for 30 mins under the optimal condition described above (Fig. 2B). We also found 0.5% BSA was optimal serum dilution buffer, compared to 0.1% BSA and phosphate solution containing 0.05% Tween 20 (Fig. 2C). The optimal pig serum incubation time was determined to be 40 mins (Fig. 2D). HRP-conjugated recombinant protein A/G exhibited optimal performance when the plates were incubated at 1:8000 for 60 mins (Fig. 2E). The optimal color development was obtained after TMB substrate was incubated for 10 mins (Fig. 2F). The cut-off value of MAG-ELISA was set as 0.25 after testing 25 T. gondii negative pig sera under the optimal conditions described above (Fig. 2G). The subsequent MAG-ELISA was performed according to the above-mentioned optimal procedures.

Evaluation of MAG-ELISA

To evaluate the feasibility of our MAG-ELISA, we also examined its specificity, sensitivity, stability, and others. The OD630 values of the 5 common pig viruses (SFV, PRRSV, PrV, PCV and FMDV) were significantly lower than the cut-off value of MAG-ELISA (Fig. 3A), indicating our ELISA did not exhibit obvious cross reaction with these 5 common pig viruses, which confirmed the good specificity of MAG-ELISA. By measuring OD630 at serial dilutions of T. gondii positive pig serum, we found that pig anti-T. gondii positive serum was still determined as positive even at the dilution ratio of 1:320, indicating the sensitivity of MAG-ELISA was adequate (Fig. 3B). The CV of repeated tests within batches and between batches was all below 10%, suggesting the good stability of our MAG-ELISA (Fig. 3C). After placing MAG-coated plates at 37℃ for 72h, the OD630 only dropped by less than 20%. (Fig. 3D). We further compared MAG-ELISA with PrioCHECK ELISA by testing 209 pig serum samples simultaneously, and found their coincidence rate was 66.67% for T. gondii positive results, 91.84% for T. gondii negative results, and the total coincidence rate was 78.47% (Table 2).

Table 2

Coincidence between MAG-ELISA and PrioCHECK ELISA for detecting *T. gondii* infection
		PrioCHECK ELISA
		Positive	Negative	Total
MAG-ELISA	Positive	74	8	82
MAG-ELISA	Negative	37	90	127
	Total	111	98	209

MAG-ELISA detection of Pig artificial infection with type I tachyzoite

MAG-ELISA and 2 other ELISA methods (rMIC3-ELISA and PrioCHECK ELISA) were applied to detect pig anti-T. gondii IgG level after artificial infection with RH tachyzoites. We collected pig serum samples from infection group and control group at day 0, day 2, day 4, day 7, day 14, day 21, day 28, day 35, day 42 and day 49 after infection and at day 3 before infection, respectively. MAG-ELISA detected anti-T. gondii positive IgG in the early stage of pig infection (at least 7 days), which was earlier than the results obtained by rMIC3-ELISA and PrioCHECK ELISA (Fig. 4). Two weeks after artificial infection, our MAG-ELISA indicated that the overall level of anti-T. gondii IgG was gradually decreased, and rMIC3-ELISA and PrioCHECK ELISA showed that the level of anti-T. gondii IgG reached their maximum value at day 35 post infection. Therefore, MAG-ELISA had the potential to be applied for early detection of T. gondii infection in pig farms and intensive industries.

Humans can be infected by toxoplasma gondii through digesting raw or uncooked meats (such as pork). Infection with toxoplasma gondii can lead to reproductive disorders in sows (such as miscarriage, stillbirths, and weak fetuses). Poor meat quality would threaten human health and cause serious economic losses to the livestock industry. T. gondii could survive in many pig tissues such as heart, lungs, and brain for a long time in the form of cyst. Pork meat is considered to be one of the main sources of human toxoplasmosis [4, 38]. The high prevalence of T. gondii is found in pig farms, especially organic farms and intensive pig industries [39, 40]. Therefore, it is necessary to develop a highly sensitive, specific, and inexpensive kit for the large-scale diagnosis of pig toxoplasmosis in farms and intensive industries.

ELISA is a common method for the large-scale detection of infectious disease mainly caused by pathogenic bacteria, viruses, and parasites. The specificity and sensitivity of ELISA depend mainly on the coated antigen. All the common coated antigens such as TLA and recombinant antigen have their own limitations to some extent. For example, the composition of TLA is poorly understood, and TLA preparation procedure is complex and infectious to operator. Although recombinant protein antigen of T. gondii is very useful for serodiagnosis of toxoplasmosis, one recombinant antigen could detect only one type of anti-T. gondii IgG, and the diagnosis result may be affected by the expression level of antigen gene[41]. Thus, the application of these antigens in ELISA may cause high error rate in large-scale detection. The chimeric antigen encoded by synthetic multiepitope antigen gene is a new generation of recombinant products, and it has competitive advantages since it contains more immunoreactive epitopes from several dominant antigens of T. gondii than conventional recombinant antigen. Chimeric antigen with the epitope from no more than 3 T. gondii genes used in ELISA has been reported to perform well in toxoplasmosis diagnosis [19]. To further improve the performance of ELISA, our MAG-ELISA used a chimeric antigen containing 6 epitopes of T and B cells from 5 T. gondii dominant antigens (SAG1, GRA1, ROP2, GRA4, and MIC3). SAG1 is only highly expressed in acute infection, but GRA1, ROP2, ROP4, and MIC3 are expressed in acute and chronic T. gondii infection, endowing MAG antigen with the potential to detect pig anti-T. gondii IgG in two different infection periods. Our results showed that MAG-ELISA possessed high specificity, sensitivity, and repeatability. The test of 209 pig serum samples indicated that the total coincidence rate of MAG-ELISA and PrioCHECK ELISA was only 78.47% with a coincidence of 66.67% for T. gondii positive results and a coincidence of 91.84% for T. gondii negative results (Table 2). The difference in the seroprevalence of T. gondii infection between MAG-ELISA and PrioCHECK ELISA was significant in pigs. The low total IgG level in MAG-ELISA might be attributed to the inconsistence in the levels of anti-T. gondii individual IgGs against these 6 epitopes at different infection stages. Furthermore, false positive results of the PrioCHECK ELISA might be caused in the case that some sample test results were close to cut-off value due to the unclear component of TLA antigen.

We further compared the obtained levels of anti-T. gondii IgGs after artificial infection with RH tachyzoites through 3 different ELISA (MAG-ELISA, rMIC3-ELISA, and PrioCHECK ELISA). Our result showed that MAG-ELISA could detect T. gondii infection at day 7 after infection, which was earlier than the other 2 ELISA methods. However, the level of anti-T. gondii IgG obtained through MAG-ELISA was inconsistent with those through rMIC3-ELISA and PrioCHECK (Fig. 4). The reasons for the above-mentioned inconsistence might be as follows. First, pig anti-T. gondii IgGs against the epitope of SAG1, GRA1, ROP2, GRA4, and MIC3 respectively possessed inconsistent levels after RH artificial infection. Second, the IgG trend in MAG-ELISA could represent the overall level of several anti-T. gondii IgGs. Thus, our MAG-ELISA should be more reliable than the ELISA using only one recombinant antigen. However, the MAG-ELISA could detect anti-T. gondii IgG only within 1–7 weeks after artificial infection with detection time span shorter than that of other 2 ELISA methods, which might be due to the low expression of the antigen genes corresponding to some epitopes. Previous studied also indicated that antibody responses to GRA1 and MIC3 were very weak, or even absent six weeks post infection [42].

MAG could specifically recognize pig anti-T. gondii IgG, thus MAG-ELISA has a potential for diagnosing toxoplasma infection in pigs. The MAG-ELISA would be conducive to improving the accuracy and reducing cost of toxoplasmosis diagnosis in pig farms and intensive industries. MAG-ELISA can monitor the change trend of anti-T. gondii IgG after pig artificial infection, which increases our knowledge about the T. gondii infection in pigs. Further work is also needed to further optimize the performance of the MAG-ELISA and verify its diagnosis effect in detecting T. gondii acute and chronic infection in pigs and other animals (such as pets).

We reported here MAG, as a synthetic multiepitope antigen, could recognize pig anti-T. gondii positive but not negative serum. MAG-ELISA possessed a high diagnostic performance in terms of specificity and sensitivity. The overall coincidence rate between MAG-ELISA and a commercial PrioCHECK ELISA was good. MAG-ELISA has the potential for large-scale diagnosis of T. gondii infection in pig farms and intensive industries.

T. gondii: toxoplasma gondii; MAG: synthetic multiepitope antigen; ELISA: enzyme-linked immunosorbent assay; PrioCHECK ELISA: PrioCHECK® Toxoplasma Ab porcine ELISA; TLA: toxoplasma lysate antigen; SAG1: T. gondii surface antigen 1; GRA1: dense granule protein 1; GRA4: dense granule protein 4; ROP2: rhoptry protein 2; MIC3: microneme protein 3; HRP: horseradish peroxidase; GST: glutathione S-transferase; TMB: 3, 3’, 5, 5’- tetramethylbenzidine; P/N: positive/negative serum; SFV: swine fever virus; PRRSV: porcine reproductive and respiratory disease virus; PrV: pseudorabies virus; PCV: porcine circovirus; FMDV: foot-and-mouth disease virus; CV: coefficient of variation; rMIC3: recombinant entire T. gondii microneme protein 3.

Acknowledgements

We thank the Laboratory Animal Center of Huazhong Agricultural University for its services. Great gratitude goes to linguistics professor Ping Liu from Huazhong Agriculture University, Wuhan, China for her work at English editing and language polishing.

Authors’ contributions

YQZ conceived this project, designed the experiment and reviewed the manuscript. YLS, YJZ and KP performed the experiment, analyzed and interpreted the data and drafted the manuscript. BS, RF and MH participated in designing the experiment and reviewing the manuscript. All authors read and approved the final manuscript.

Funding

Our work was supported by the National Key Research and Development Program of China (Grant No.2017YFD0501304) and the National Natural Science Foundation of China (Grant No. 31272553 to YQZ).

Availability of data and materials

All relevant data are within the manuscript and its Supporting Information files.

Ethics approval and consent to participate

Our study was authorized by the animal ethical committee from Huazhong agricultural university in China with the number HZAUSW-2012-001.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Robert-Gangneux F, Dardé ML. Epidemiology of and diagnostic strategies for toxoplasmosis. Clin Microbiol Rev. 2012;25 2:264-96; doi: 10.1128/cmr.05013-11.
Pappas G, Roussos N, Falagas ME. Toxoplasmosis snapshots: global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis. Int J Parasitol. 2009;39 12:1385-94; doi: 10.1016/j.ijpara.2009.04.003.
Torgerson PR, Mastroiacovo P. The global burden of congenital toxoplasmosis: a systematic review. Bull World Health Organ. 2013;91 7:501-8; doi: 10.2471/blt.12.111732.
Gisbert Algaba I, Verhaegen B, Jennes M, Rahman M, Coucke W, Cox E, et al. Pork as a source of transmission of Toxoplasma gondii to humans: a parasite burden study in pig tissues after infection with different strains of Toxoplasma gondii as a function of time and different parasite stages. Int J Parasitol. 2018;48 7:555-60; doi: 10.1016/j.ijpara.2017.12.009.
Condoleo R, Rinaldi L, Sette S, Mezher Z. Risk Assessment of Human Toxoplasmosis Associated with the Consumption of Pork Meat in Italy. Risk Anal. 2018;38 6:1202-22; doi: 10.1111/risa.12934.
Santoro M, Viscardi M, Sgroi G, DʼAlessio N, Veneziano V, Pellicano R, et al. Real-time PCR detection of Toxoplasma gondii in tissue samples of wild boars (Sus scrofa) from southern Italy reveals high prevalence and parasite load. Parasit Vectors. 2019;12 1:335; doi: 10.1186/s13071-019-3586-5.
Foroutan M, Fakhri Y, Riahi SM, Ebrahimpour S, Namroodi S, Taghipour A, et al. The global seroprevalence of Toxoplasma gondii in pigs: A systematic review and meta-analysis. Vet Parasitol. 2019;269:42-52; doi: 10.1016/j.vetpar.2019.04.012.
Tao Q, Wang Z, Feng H, Fang R, Nie H, Hu M, et al. Seroprevalence and risk factors for Toxoplasma gondii infection on pig farms in central China. J Parasitol. 2011;97 2:262-4; doi: 10.1645/ge-2646.1.
Rostami A, Karanis P, Fallahi S. Advances in serological, imaging techniques and molecular diagnosis of Toxoplasma gondii infection. Infection. 2018;46 3:303-15; doi: 10.1007/s15010-017-1111-3.
Switaj K, Master A, Skrzypczak M, Zaborowski P. Recent trends in molecular diagnostics for Toxoplasma gondii infections. Clin Microbiol Infect. 2005;11 3:170-6; doi: 10.1111/j.1469-0691.2004.01073.x.
Gutierrez J, O'Donovan J, Williams E, Proctor A, Brady C, Marques PX, et al. Detection and quantification of Toxoplasma gondii in ovine maternal and foetal tissues from experimentally infected pregnant ewes using real-time PCR. Vet Parasitol. 2010;172 1-2:8-15; doi: 10.1016/j.vetpar.2010.04.035.
Fallahi S, Seyyed Tabaei SJ, Pournia Y, Zebardast N, Kazemi B. Comparison of loop-mediated isothermal amplification (LAMP) and nested-PCR assay targeting the RE and B1 gene for detection of Toxoplasma gondii in blood samples of children with leukaemia. Diagn Microbiol Infect Dis. 2014;79 3:347-54; doi: 10.1016/j.diagmicrobio.2014.02.014.
Wu YD, Xu MJ, Wang QQ, Zhou CX, Wang M, Zhu XQ, et al. Recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip for detection of Toxoplasma gondii in the environment. Vet Parasitol. 2017;243:199-203; doi: 10.1016/j.vetpar.2017.06.026.
Zhang K, Lin G, Han Y, Li J. Serological diagnosis of toxoplasmosis and standardization. Clin Chim Acta. 2016;461:83-9; doi: 10.1016/j.cca.2016.07.018.
Turunen H, Vuorio KA, Leinikki PO. Determination of IgG, IgM and IgA antibody responses in human toxoplasmosis by enzyme-linked immunosorbent assay (ELISA). Scand J Infect Dis. 1983;15 3:307-11; doi: 10.3109/inf.1983.15.issue-3.12.
Li S, Maine G, Suzuki Y, Araujo FG, Galvan G, Remington JS, et al. Serodiagnosis of recently acquired Toxoplasma gondii infection with a recombinant antigen. J Clin Microbiol. 2000;38 1:179-84.
Desmonts G, Remington JS. Direct agglutination test for diagnosis of Toxoplasma infection: method for increasing sensitivity and specificity. J Clin Microbiol. 1980;11 6:562-8.
Pishkari S, Shojaee S, Keshavarz H, Salimi M, Mohebali M. Evaluation of Toxoplasma gondii soluble, whole and excretory/secretary antigens for diagnosis of toxoplasmosis by ELISA test. J Parasit Dis. 2017;41 1:289-91; doi: 10.1007/s12639-016-0794-1.
Ferra B, Holec-Gąsior L, Kur J. Serodiagnosis of Toxoplasma gondii infection in farm animals (horses, swine, and sheep) by enzyme-linked immunosorbent assay using chimeric antigens. Parasitol Int. 2015;64 5:288-94; doi: 10.1016/j.parint.2015.03.004.
Dai J, Jiang M, Wang Y, Qu L, Gong R, Si J. Evaluation of a recombinant multiepitope peptide for serodiagnosis of Toxoplasma gondii infection. Clin Vaccine Immunol. 2012;19 3:338-42; doi: 10.1128/cvi.05553-11.
Ferra BT, Holec-Gąsior L, Gatkowska J, Dziadek B, Dzitko K, Grąźlewska W, et al. The first study on the usefulness of recombinant tetravalent chimeric proteins containing fragments of SAG2, GRA1, ROP1 and AMA1 antigens in the detection of specific anti-Toxoplasma gondii antibodies in mouse and human sera. PLoS One. 2019;14 6:e0217866; doi: 10.1371/journal.pone.0217866.
Leony LM, Freitas NEM, Del-Rei RP, Carneiro CM, Reis AB, Jansen AM, et al. Performance of recombinant chimeric proteins in the serological diagnosis of Trypanosoma cruzi infection in dogs. PLoS Negl Trop Dis. 2019;13 6:e0007545; doi: 10.1371/journal.pntd.0007545.
Varshney A, Puranik N, Kumar M, Pal V, Padmaja J, Goel AK. An ELISA using a recombinant chimera of protective antigen and lethal factor for serodiagnosis of cutaneous anthrax in India. Biologicals. 2019;57:55-60; doi: 10.1016/j.biologicals.2019.01.001.
Mosadeghi P, Heydari-Zarnagh H. Development and Evaluation of a Novel ELISA for Detection of Antibodies against HTLV-I Using Chimeric Peptides. Iran J Allergy Asthma Immunol. 2018;17 2:144-50.
Cardinal MV, Sartor PA, Gaspe MS, Enriquez GF, Colaianni I, Gürtler RE. High levels of human infection with Trypanosoma cruzi associated with the domestic density of infected vectors and hosts in a rural area of northeastern Argentina. Parasit Vectors. 2018;11 1:492; doi: 10.1186/s13071-018-3069-0.
Tomavo S, Schwarz RT, Dubremetz JF. Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens. Molecular and Cellular Biology. 1989;9 10:4576-80; doi: 10.1128/mcb.9.10.4576.
Peptide-Based Monoclonal Antibody Production Against SAG1 (P30) Protein of Toxoplasma gondii. Monoclonal Antibodies in Immunodiagnosis and Immunotherapy. 2020;39 2:51-6; doi: 10.1089/mab.2019.0041.
Cesbron-Delauw MF, Guy B, Torpier G, Pierce RJ, Lenzen G, Cesbron JY, et al. Molecular characterization of a 23-kilodalton major antigen secreted by Toxoplasma gondii. Proc Natl Acad Sci U S A. 1989;86 19:7537-41; doi: 10.1073/pnas.86.19.7537.
Beghetto E, Pucci A, Minenkova O, Spadoni A, Bruno L, Buffolano W, et al. Identification of a human immunodominant B-cell epitope within the GRA1 antigen of Toxoplasma gondii by phage display of cDNA libraries. Int J Parasitol. 2001;31 14:1659-68; doi: 10.1016/s0020-7519(01)00288-0.
Wang Y, Wang G, Ou J, Yin H, Zhang D. Analyzing and identifying novel B cell epitopes within Toxoplasma gondii GRA4. Parasit Vectors. 2014;7:474; doi: 10.1186/s13071-014-0474-x.
Altcheh J, Diaz NS, Pepe CM, Martin V, Nigro M, Freilij H, et al. Kinetic analysis of the humoral immune response against 3 Toxoplasma gondii-recombinant proteins in infants with suspected congenital toxoplasmosis. Diagn Microbiol Infect Dis. 2006;56 2:161-5; doi: 10.1016/j.diagmicrobio.2006.03.012.
Chardès T, Bourguin I, Mevelec MN, Dubremetz JF, Bout D. Antibody responses to Toxoplasma gondii in sera, intestinal secretions, and milk from orally infected mice and characterization of target antigens. Infect Immun. 1990;58 5:1240-6; doi: 10.1128/iai.58.5.1240-1246.1990.
Saavedra R, Becerril MA, Dubeaux C, Lippens R, De Vos MJ, Hérion P, et al. Epitopes recognized by human T lymphocytes in the ROP2 protein antigen of Toxoplasma gondii. Infect Immun. 1996;64 9:3858-62; doi: 10.1128/iai.64.9.3858-3862.1996.
Nie H, Fang R, Xiong BQ, Wang LX, Hu M, Zhou YQ, et al. Immunogenicity and protective efficacy of two recombinant pseudorabies viruses expressing Toxoplasma gondii SAG1 and MIC3 proteins. Vet Parasitol. 2011;181 2-4:215-21; doi: 10.1016/j.vetpar.2011.04.039.
Eliasson M, Olsson A, Palmcrantz E, Wiberg K, Inganäs M, Guss B, et al. Chimeric IgG-binding receptors engineered from staphylococcal protein A and streptococcal protein G. J Biol Chem. 1988;263 9:4323-7.
Sikkema. An Fc-Binding Protein. Amer Biotech Lab. 1989;7:42.
Jiang T, Yao B, Zhao J. Detection of the antibody against Toxoplasma gondii using the indirect ELISA with the recombinant MIC3. Journal of Anhui Agricultural Sciences. 2009;37 34:17286-7.
Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol. 2000;30 12-13:1217-58; doi: 10.1016/s0020-7519(00)00124-7.
Pipia AP, Varcasia A, Dessì G, Panzalis R, Gai C, Nonnis F, et al. Seroepidemiological and biomolecular survey on Toxoplasma gondii infection on organic pig farms. Parasitol Res. 2018;117 5:1637-41; doi: 10.1007/s00436-018-5823-9.
Gazzonis AL, Marangi M, Villa L, Ragona ME, Olivieri E, Zanzani SA, et al. Toxoplasma gondii infection and biosecurity levels in fattening pigs and sows: serological and molecular epidemiology in the intensive pig industry (Lombardy, Northern Italy). Parasitol Res. 2018;117 2:539-46; doi: 10.1007/s00436-017-5736-z.
Holec-Gasior L. Toxoplasma gondii recombinant antigens as tools for serodiagnosis of human toxoplasmosis: current status of studies. Clin Vaccine Immunol. 2013;20 9:1343-51; doi: 10.1128/cvi.00117-13.
Verhelst D, De Craeye S, Dorny P, Melkebeek V, Goddeeris B, Cox E, et al. IFN-γ expression and infectivity of Toxoplasma infected tissues are associated with an antibody response against GRA7 in experimentally infected pigs. Vet Parasitol. 2011;179 1-3:14-21; doi: 10.1016/j.vetpar.2011.02.015.

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Characterization and Evaluation of a Recombinant Multiepitope Peptide Antigen MAG in the Serological Diagnosis of Toxoplasma Gondii Infection in Pigs

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Journal Publication

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Ethics statement

MAG recombinant chimeric antigen

Western immunoblot analysis

Pig artificial infection with tachyzoite

Optimization of ELISA procedure

Evaluation of MAG-ELISA

Coincidence test

Procedure of rMIC3-ELISA

Results

Characterization of recombinant multiepitope antigen and Optimization of MAG-ELISA procedure

Evaluation of MAG-ELISA

MAG-ELISA detection of Pig artificial infection with type I tachyzoite

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

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Journal Publication

Version 1