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Logo of parasitvectBioMed CentralBiomed Central Web Sitesearchsubmit a manuscriptregisterthis articleParasites & VectorsJournal Front Page
 
Parasit Vectors. 2016; 9: 374.
Published online 2016 June 29. doi:  10.1186/s13071-016-1643-x
PMCID: PMC4928332

Twenty-six circulating antigens and two novel diagnostic candidate molecules identified in the serum of canines with experimental acute toxoplasmosis

Abstract

Background

The protozoan Toxoplasma gondii is a pathogen that causes severe opportunistic disease in a wide range of hosts. Efficient methods to diagnose acute T. gondii infection are essential for the administration of appropriate treatments and to reduce economic losses. In animals with acute infections, circulating antigens (CAgs) were detected as early as two days post-infection; these CAgs were reliable diagnostic indicators of acute infection. However, only a limited number of CAgs have been identified to date. The objective of this study was to identify a broader spectrum of CAgs and to explore novel diagnostic candidates in serum.

Methods

A canine model of acute toxoplasmiosis was established. For this purpose, six dogs were infected by intraperitoneal inoculation of tachyzoites. The CAgs spectrum in the serum was identified with the immunoprecipitation-shotgun approach. Two CAgs with low homology to other species, coronin protein (TgCOR) and ELMO protein (TgELMO), were heterologously expressed in Escherichia coli. Polyclonal antibodies against these two proteins were prepared, and the presence of these proteins in the serum was verified by Western blotting. The two CAgs were detected and evaluated by indirect ELISA methods.

Results

The CAgs levels peaked between two and five days after inoculation, and twenty-six CAgs were identified. Western blotting showed the presence of the two proteins in the serum during acute infection. Based on ELISA tests, the two CAgs were detected during acute infection.

Conclusions

We identified twenty-six CAgs in the serum of canines with experimental acute toxoplasmosis and discovered two novel diagnostic candidates. We also provide new insights into the diagnosis of acute toxoplasmosis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13071-016-1643-x) contains supplementary material, which is available to authorized users.

Keywords: Toxoplasma gondii, Acute infection, Proteomics, Circulating antigens, Diagnostic candidates

Background

Toxoplasma gondii can cause serious opportunistic infections in a wide range of hosts [1]. This species has tremendous disease-causing potential because it can invade any nucleated cell, resulting in lysis of the host cells [1]. The symptoms of T. gondii acute infection in humans range from mild flu-like symptoms in most people to more severe complications in immunocompromised individuals or following trans-placental transmission to a foetus [2, 3]. During the first or second trimester of pregnancy, infection with T. gondii could potentially lead to the parasite passing through the placenta to the foetus, resulting in mental retardation, retinochoroiditis, blindness, and even death [4, 5]. The available therapies of acute toxoplasmosis are not fully effective. Thus, it is crucial to diagnose T. gondii infection and make efforts to reduce toxoplasmosis transmission [6]. If an acute infection cannot be diagnosed in time, the optimal period for treatment will also be missed [7].

Laboratory tests are the main diagnostic method for acute T. gondii infection. Indirect (serological methods) and direct (PCR, in situ hybridization, isolation and histology) methods have been used to diagnose acute T. gondii infection [8]. In general, serological diagnosis methods are based on the detection of specific antibodies (IgG, IgM and IgA) [9, 10]. Antibodies are commonly produced in the late stage of acute infection or during chronic infection. Detection based on molecular biology methods is limited because specific equipment is required that is not universally available in clinical laboratories. Isolation and identification of the parasite requires extensive experience and is time-consuming. The lack of efficient diagnostic methods for acute T. gondii infection can lead to therapy failure, and there is an urgent need to identify new diagnostic candidates.

The T. gondii invasion process is rapid and dynamic and relies on the secretion of numerous proteins from micronemes, rhoptries and dense granules [11]. Excretory/secretory antigens (ESA) are a group of antigens from T. gondii that represent the majority of circulating antigens (CAgs) in the sera of hosts during acute toxoplasmosis and the reactivation of infection [1214]. Recent evidence strongly suggests that CAgs can serve as serological markers to diagnose acute infection [15, 16]. To date, only a limited number of CAgs have been identified. Moreover, the identification of CAgs is meaningful for the diagnosis of acute T. gondii infections. The aim of this study was to identify the spectrum of T. gondii CAgs using the immunoprecipitation-LC-MS/MS technique. We also evaluated the use of antibodies to identify novel candidate molecules for the detection of acute toxoplasmosis by ELISA tests.

Methods

T. gondii, cell culture and animal preparations

The T. gondii RH strain is an international standard virulent strain that is widely used for Toxoplasma analysis. A virulent strain is also advantageous to successfully establish an acute T. gondii infection model. The T. gondii strain RH and Vero cells were stored in our laboratory. Vero cells were propagated at 37 °C in a 5 % CO2 atmosphere in DMEM (Dulbecco modified Eagle medium) supplemented with 10 % FBS, 2 mmol/l glutamine, 100 kU/l streptomycin, and 400 kU/l penicillin. T. gondii were grown and maintained in Vero cells.

Female BALB/c mice (~25 g body weight) were purchased from the Shanghai Laboratory Animal Centre at the Chinese Academy of Science (Shanghai, China). Six clean female beagles (3 months old; ~4 kg body weight) without pathogens that obviously interfere with scientific experiments were purchased from the Shanghai Xingang Laboratory Animal Farm (Shanghai, China). All of the animals used in the experiments were raised in a sterilized room and fed sterilized food and water at the Animal Laboratory Centre at the Shanghai Veterinary Research Institute. The study was approved by the Animal Care and Use Committee of the Shanghai Veterinary Research Institute. The animals were handled in strict accordance with the animal protection law of the People’s Republic of China (released on 09/18/2009) and the National Standards for Laboratory Animals in China (executed on 05/1/2002).

Excretory/secretory antigens and antisera preparation

Excretory/secretory antigens were prepared according to previously described methods [14]. Briefly, 3 × 109 purified tachyzoites were suspended in 15 ml DMEM and incubated at 37 °C for 2 h. Tachyzoites were removed by centrifugation at 1,000 g for 15 min (at 4 °C). The supernatant was supplemented with a protease inhibitor cocktail (Merck, Darmstadt, Germany, Cat No. 535140) and concentrated to 500 μl using a 3 kDa centrifugal filter (Merck, Cat No. UFC900308). The ESA protein concentration was determined using a DC protein assay reagent package (Bio-Rad, Hercules, USA, Cat No. 500-0120). Two groups of two female BALB/c mice were immunized. Two mice were immunised with EAS and two mice were immunised with PBS as a control. The 206 adjuvant (Seppic, Paris, France) was used according to the manufacturer’s instructions. In total, 100 μg ESA was injected into each mouse at 0, 2, 4, 6 and 8 weeks. Equal doses of PBS were used to immunize the control group mice. Blood sera were sampled from the tails of mice at 10 days after each immunization. To detect the ESA antisera, immunoblotting and ELISA assays were performed using standard methods.

Development of acute infection models

Three dogs that were intraperitoneally inoculated with 1 × 109 purified tachyzoites were treated as the test group. Three dogs that were intraperitoneally inoculated with the same volume of sterile PBS were treated as a control group. A 3 ml volume of blood was collected twice per day between days 1 and 3 and once per day between days 4 and 18. Blood was then collected once every 3 days until two months had elapsed. Each blood sample was divided into 2 tubes (one tube for serum separation and one tube for sodium citrate anticoagulation). The sera were used to detect CAgs or to purify CAgs by immunoprecipitation. Centrifuged sera (4,000 rpm for 10 min) from blood not treated with an anticoagulant were supplemented with a protease inhibitor cocktail (Merck, Cat No. 535140) and stored at -80 °C until use. The anticoagulant blood was used to extract DNA for T. gondii detection.

Evaluation of an acute infection model by ELISA and nested PCR

To test for CAgs, an ELISA assay was performed using a commercial Circulating Antigen Detection ELISA (CA-ELISA) kit (Combined Company, Shenzhen, China). The results are presented as the mean value and standard error (SE) of the sample absorbance from 3 dog groups.

To detect T. gondii in the blood, a genomic DNA extraction kit (SBS Gentech, Shanghai, China) was used to extract DNA from whole blood samples. Nested-PCR was conducted as described in our earlier study [17]. The protozoa rDNA sequence contains 5'-IGS-18S rDNA-ITS1-5.8S rDNA-ITS2-28S rDNA-IGS-3'. Based on the sequence of the ITS1-5.8S rDNA-ITS2 gene (GenBank Accession No. X75453) of T. gondii, two pairs of nested PCR primers were designed. The forward primer 5'-ACC TTT GAA TCC CAA GCA-3' and the reverse primer 5'-TAA ATC GGA CAA ACG CCC-3' were used to amplify an 855 bp fragment representing the outer amplicon. The forward primer 5'-TTT GCA TTC AAG AAG CGT G-3' and the reverse primer 5'-AAG GTG CCA TTT GCG TTC-3' were used to amplify a 432 bp fragment representing the inner amplicon. The primers were used at 0.4 μM each in the 25 μl reaction system. Each reaction used 1 μl of the extracted DNA, 9.5 μl double-distilled water, 12.5 μl of the 2 × PCR mix (Dongsheng Biotech, Guangzhou, China), which consisted of 100 mM KCl, 20 mM Tris-HCl, 3 mM MgCl2, 400 μM dNTPs and 0.1U/μl Taq DNA polymerase. After an initial denaturation at 94 °C for 5 min, amplification consisted of 30 cycles of denaturation at 94 °C for 50 s, annealing at 55 °C for 40 s and extension at 72 °C for 45 s. Finally, the DNA fragments were extended at 72 °C for 10 min.

Immunoprecipitation

Serum (10 μl) was sampled on days 2 and 3 post-challenge with T. gondii. Sera from each of the 3 beagles were combined (60 μl). The mixed serum was diluted with 540 μl sterile PBS (pH 7.4). Protein A agarose beads (100 μl, 25 %, Beyotime, Nantong, China, Cat No. P2006) were added, and the sample was stirred at 4 °C for 2 h to eliminate serum antibodies. The mixture was centrifuged at 4 °C at 2,500 rpm for 5 min. After that, the supernatant was collected. Protein G agarose beads (100 μl, 25 %, Beyotime, Cat No. P2009) and 5 μl (1 μg) normal mouse IgG was added to eliminate non-specific binding and then the sample was agitated at 4 °C for 2 h. After centrifugation, the supernatant was collected as described above. Then, 5 μl ESA antiserum was added to the supernatant and the mixture was stirred at 4 °C overnight. Protein G agarose beads (100 μl, 25 %, Beyotime) were added and then stirred at 4 °C for 3 h. The mixture was centrifuged at 2,500 rpm for 5 min to collect the beads. The beads were washed 3 times with PBS and then boiled with 60 μl sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer for 5 min. The sample was centrifuged at 2,500 rpm for 5 min, and the supernatant was collected for LC-MS/MS detection and 8 % polyacrylamide gel electrophoresis analysis.

Filter-aided proteome preparation

The supernatant was supplemented with DTT (to a final concentration of 100 mM) and boiled for 5 min. After cooling, 200 μl UA buffer (8 M Urea, 150 mM Tris-HCl, pH 8.0) was added. The sample was then mixed. The liquid was transferred to a 10 kDa Millipore Amicon Ultra-4 centrifugal filter device and centrifuged at 14,000 rpm for 15 min. Another 200 μl UA buffer were added, and the sample was centrifuged as described above. The filtrate was discarded, and 100 μl iodoacetamide (50 mM iodoacetamide in UA) was added. The tube was shaken at 600 rpm for 1 min and then incubated at room temperature in the dark for 30 min. After being centrifuged at 14,000 rpm for 10 min, the solution was dialyzed against 100 μl UA buffer twice and then dialyzed against 100 μl dissolution buffer (25 mM NH4HCO3) 3 times. The mixture treated with 40 μl buffered trypsin (2 μg trypsin in 40 μl dissolution buffer) and then incubated at 37 °C for 18 h. The incubated solution was centrifuged at 14,000 rpm for 10 min, and the supernatant was used for LC-MS/MS analysis [18].

Liquid chromatography (LC) - electrospray ionization (ESI) tandem MS (MS/MS) analysis using a Q Exactive mass spectrometer

Experiments were performed using a Q Exactive mass spectrometer coupled to an Easy nLC (Thermo Fisher Scientific, Waltham, USA). The peptide mixture was loaded onto a C18-reverse phase column (15 cm long, 75 μg inner diameter) packed in-house with RP-C18 5 μm resin in buffer A (0.1 % formic acid in HPLC-grade water). Peptides were separated with a linear gradient of buffer B (0.1 % Formic acid in 84 % acetonitrile) at a flow rate of 250 nl/min over 60 min. MS data were acquired using a data-dependent top10 method by dynamically choosing the most abundant precursor ions from the survey scan (300–1800 m/z) for HCD (Higher-energy collisional dissociation) fragmentation. Determination of the target value was based on predictive Automatic Gain Control (pAGC). The dynamic exclusion duration was 20 s. The survey scans were acquired at a resolution of 70,000 at 200 m/z. The resolution for the HCD spectra was set to 17,500 at 200 m/z. The normalized collision energy was 27 eV. The underfill ratio, which specifies the minimum percentage of the target value likely to be reached at the maximum fill time, was defined as 0.1 %. The instrument was run with the peptide recognition mode enabled.

ESI mass spectrometry data analysis

The MS/MS spectra were used to search the UniProt database (26,517 sequences, downloaded on June 13th, 2014) with the MASCOT engine (Matrix Science, version 2.2). To identify proteins, the following options were used: Peptide mass tolerance = 20 ppm; MS/MS tolerance = 0.1 Da; Enzyme = Trypsin; Missed cleavage = 2; Fixed modification: carbamidomethyl (C); Variable modification: Oxidation (M); mascot score  20; and FDR < 0.01 at the peptide and protein levels.

Bioinformatics analysis

To better understand the biological functions of the identified proteins, GO annotation was performed based on BLAST results using the ToxoDB (http://toxodb.org/toxo/), QuickGO (http://www.ebi.ac.uk/QuickGO) and DAVID v. 6.7 (http://david.abcc.ncifcrf.gov) databases [1921].

Protein expression and antibody production

To further verify the CAgs components present in the serum, coronin protein (TgCOR) and ELMO/CED 12 family protein (TgELMO), which both have low homology to other species were selected for further evaluation of their diagnostic value. The secondary structures of TgCOR (GenBank Accession No. EPT25607.1) and TgELMO (GenBank Accession No. XP_002367438.1) were analysed using Protean DNAStar software. Hydrophilicity, surface accessibility and antigenicity prediction schemes were used. TgCOR and TgELMO were expressed using an E. coli (BL 21) expression system and the pET-28a-c(+) vector. The recombinant plasmids were sent to Invitrogen Company for full-length sequencing. The protein-coding regions, primers, restriction enzymes and cut sites, expression data and the theoretical molecular weights of the proteins are shown in Table 1. The transformed cells with expression vectors were grown in LB medium containing 50 μg/ml ampicillin at 37 °C with vigorous shaking until the optical density (600 nm) of the medium reached 0.6. The expression was induced by 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and the culture was incubated at 20 °C for 18 h. Collected cells were lysed by sonication. Recombinant proteins isolated from inclusion bodies were refolded by a refolding kit (Novagen, Darmstadt, Germany, CAT No. 70123-3) and purified using Ni-NTA His-Bind resin (Novagen). Recombinant proteins were dialysed and endotoxin was removed using the Triton-X114 method [22]. Briefly, Triton X-114 was added to the protein solution (final concentration of Triton X 114 was 2 %, W/V). After being mixed by vortex, the solution was incubated on ice for 15 min and then at 50 °C for 5 min. The supernatant was collected after centrifugation (12,000 g, 1 min, 30 °C. Protein concentrations were measured using a DC protein assay reagent kit (Bio-Rad, USA). BALB/c mice were immunized with each protein at a dose of 100 μg per mouse. The immunization procedures and the detection of antibody titres (Table 1) were performed as described above. Blood samples were collected 10 days after the second booster injection.

Table 1
Information of expressed protein

Verification of circulating antigens in the sera in an acute infection model

To verify the presence of the identified CAgs in the sera of animals with acute T. gondii infections, sera from the test group were analysed by Western blotting at a dilution of 1:10. Mixed serum from the control group was used as a negative control. Antiserum for TgCOR and TgELMO was treated as the primary antibody at a dilution of 1:2,000 and 1:4,000, respectively. HRP-conjugated goat anti-mouse IgG (Jackson, USA) was used as a secondary antibody at a dilution of 1:4,000. The detection of signals on the membranes was performed using an Enhanced Chemiluminescence (ECL) Kit (Thermo Fisher Scientific, Waltham, USA).

Analysis of the diagnostic value of TgCOR and TgELMO proteins

To evaluate the use of ELISA methods for clinical diagnosis, sera collected from the 6 dogs in the initial 16 days post-infection were assessed for CAgs. Microwell plates with 96 wells were coated (100 μl/well) with serum samples in carbonate buffer at a dilution of 1:50. TgCOR and TgELMO antiserum was used as the primary antibody at a dilution of 1:1,000 and 1:2,000, respectively. HRP-conjugated goat anti-mouse IgG (Jackson, USA) was used as a secondary antibody at a dilution of 1:4,000. The results are presented as the absorbance value for each sample.

Results

An acute model of T. gondii infection in canines using inoculation with tachyzoites was established successfully

The T. gondii strain RH is lethal when used to inoculate mice; the death rate is also high in rabbit models of acute RH strain infection. Small rabbits are also unsuitable for the continuous collection of large amounts of blood. To ensure the collection of adequate amounts of sera, an acute canine infection model was established in this trial. Our early experiments showed that dogs (~7 kg body weight) did not display symptoms of acute infection if inoculated with less than 1 × 108 tachyzoites. In this study, the dogs showed fever symptoms 24 h post-T. gondii infection (Fig. 1a). Toxoplasma gondii and CAgs could be detected in the blood after infection. The levels of CAgs peaked between 2 and 5 days after inoculation. The curve of CAgs levels was similar to that of body temperature. CAgs were gradually cleared away concomitant with the disappearance of T. gondii (Fig. 1b, c). These results demonstrate the successful development of an acute T. gondii infection model.

Fig. 1
Analyses of body temperature, CAgs and nested-PCR. a Body temperature between 1 to 16 days post-infection. b Sera from days 0 to 16 were tested for CAgs levels using a commercial Circulating Antigen Detection ELISA (CA-ELISA) Kit. Values represent ...

Circulating antigens enriched and purified by immunoprecipitation were identified by LC-MS/MS

ESA antibodies could be used to identify numerous ESA components with good immunogenicity (Fig. 2). The molecular weights of the vast majority of identified CAgs were greater than 15 kDa (Fig. 3a). In total, 220 protein groups were identified by LC-MS/MS (Additional file 1). Among these proteins, the unique peptide counts for 26 proteins were equal to or greater than 2. The proteins identified by immunoprecipitation-shotgun analysis were classified into the following major categories: micronemal proteins (MIC1, MIC3 and MIC4), dense granule proteins (GRA1 and GRA5), surface antigens (SAG1 and SAG2), novel CAg proteins (coronin, ELMO and ribosomal-ubiquitin protein L40) and others (Table 2).

Fig. 2
ESA antibodies were tested by ELISA and immunoblotting analyses. a Levels of ESA antibodies after each immunization. Two groups of 2 female BALB/c mice were immunized five times at 2-week intervals. PBS was used to immunize 2 mice in the control group. ...
Fig. 3
SDS-PAGE analysis and GO annotation of CAgs enriched and purified by immunoprecipitation. a Circulating antigens enriched and purified by immunoprecipitation were analysed by SDS-PAGE (8 %). Lane 1: marker; Lane 2: immunoprecipitation supernatant. ...
Table 2
CAg proteins identified by LC-MS/MS after IP enrichment and purification with ESA antibodies

Bioinformatics Analysis

To better understand the functions of CAgs in biological processes during acute T. gondii infection, GO analysis was performed on 26 high-confidence proteins. Twenty proteins were successfully annotated based on biological process or cellular component and classified accordingly (Table 2 and Fig. 3b). Among the annotated proteins, 25 % were involved in protein metabolism, 20 % were involved in binding, 15 % were involved in carbohydrate metabolism, 10 % were involved in oxidation-reduction processes, 10 % localized to extracellular regions, and 5 % were involved in responses to stress, nucleoside metabolism processes and phagocytosis. The other 5 % were membrane components.

The two CAgs were detected during acute infection by T. gondii

Considering the results of the BLAST analyses of 8 novel CAgs, we chose 2 (coronin and ELMO) with low homology to other species to further assess their diagnostic value. Secondary structure prediction revealed that TgCOR and TgELMO had excellent hydrophilicity and antigenicity in the regions H211–A621 and G1613–L1916, respectively. Peptides containing good hydrophilic domains and antigen epitopes were successfully expressed in an E. coli system (Fig. 4a, b). Mixed sera from the test group and control group were analysed by Western blotting. Antiserum for TgCOR or TgELMO was treated as the primary antibody, respectively. The results showed that the 2 novel CAgs identified in this study were present in the sera from canines with acute infections (Fig. 4c). The two circulating antigens reached the highest level between day 2 and day 5. However, TgCOR and TgELMO were undetectable at 12 and 14 days after inoculation, respectively (Figs. 5a, b). TgELMO in the blood was detectable longer than TgCOR, which might be due to the slower clearance rate and lower ELISA detection background of TgELMO.

Fig. 4
Expression and Western blotting of TgCOR and TgELMO. a, b Recombinant TgCOR and TgELMO expressed in E. coli were purified with Ni-NTA His-Bind resin. c Western blotting of TgCOR and TgELMO. Mixed serum samples from the test group on day 3 were analysed ...
Fig. 5
Detection of TgCOR and TgELMO in serum with the ELISA method using antisera. The average OD value for negative samples was multiplied by 2.1 to obtain the cut-off value. The horizontal lines represent cut-off values. The two circulating antigens in the ...

Discussion

Diagnosis of acute T. gondii infection

Molecular biological methods (PCR, real-time PCR and Loop-mediated isothermal amplification) can be used to diagnose acute T. gondii infection [23, 24]. However, these methods are limited because specific equipment is required. Serological methods have been widely used because they are easy to perform. To date, diagnosis of T. gondii infection is mainly based on IgG detection in the blood. However, IgG is only detectable at thirteen days after infection [25]. Also, IgG persists in the blood for a long period and might not represent an acute infection [25]. IgM ELISA and complement fixation tests are the most sensitive methods currently [9], but newborns might not produce IgM antibodies, and IgM production might be delayed because maternal IgG can be transmitted to the foetus through the placenta. Because acute infection might be caused by a recurrent infection, IgM is also rarely produced in immunocompromised patients [10, 26].

As primary toxoplasmosis usually manifests as a flu-like illness in immunocompetent individuals, these cases are often not brought to the attention of the medical personnel and symptoms soon pass without chemotherapy. However, when T. gondii infection spreads in humans and animals in an epidemic manner or occurs in immunosuppressed individuals, rapid diagnosis of acute toxoplasmosis becomes particularly important. In cases where acutely infected individuals do seek medical attention, the drugs available for treating influenza do not effectively kill T. gondii tachyzoites. Inefficient diagnosis of acute T. gondii infection will lead to the spread and treatment delays of toxoplasmosis. We also propose to detect the CAgs of T. gondii if the patients experience flu-like symptoms before or during pregnancy. If CAgs tests are positive, emergency strategies such as drug therapies should be taken.

The presence of circulating antigens in blood represents an acute infection

Serological diagnosis of acute T. gondii infection is usually based on the detection of specific antibodies (IgM). However, the presence of IgM antibodies is not always indicative of active toxoplasmosis. IgM can be produced for 6 months after acute infection and remain in the body fluid for up to 18 months or even a few years [2729]. The presence of IgM during the chronic phase of T. gondii infection might lead to unnecessary diagnostic risks and treatment side effects. These risk factors might even lead to the termination of pregnancy [27, 30].

Previous studies have developed acute T. gondii infection models in mice or rabbits by intraperitoneal inoculation of tachyzoites. CAgs can be successfully detected with serological methods in animal models [28, 31] and CAgs have also been detected in the blood of rabbits and pigs infected by oral inoculation of oocysts [25]. In the sera of women and children with acute infection, CAgs are also detectable [32]. The previous studies demonstrate that the route of infection does not affect CAgs production. In this study, acute T. gondii infection of canines was successfully modelled by intraperitoneal inoculation of tachyzoites. CAgs, markers of acute infection, were also detected in this model.

Acute T. gondii infection can be diagnosed by direct detection of CAgs [33]. The diagnosis of acute T. gondii infection by CAgs detection is more sensitive than testing for the whole parasite [34]. Although CAgs are partly neutralized by blood antibodies, immunogenic CAgs remain in the blood and can be detected using high levels of specific antibodies [28]. Previous ELISA results showed that the amount of CAgs in women and children with acute infection are significantly higher than that of chronic patients and healthy individuals, which show no differences [32]. CAgs do not persist in the blood for a long period of time [28].

Selection of multiple CAgs for diagnosis can improve the reliability of diagnostic results

Some CAgs have been used to diagnose acute T. gondii infection [31]. SAG1, SAG2, MIC1, MIC3 and GRA1 are proteins identified in this study that have also been studied for use in diagnostic assays [35]. The screening of multiple diagnostic markers is helpful to improve the sensitivity and specificity of diagnosis. The dynamic change of diagnostic candidate molecules in the blood reflects the course of an acute infection. First, the amount of antigen used for serological diagnosis must achieve the minimum detection limit. Multiple molecules represent a large range of CAgs and are more stable than single CAg molecule in the blood. Identification of multiple CAgs and candidate diagnostic molecules will improve the sensitivity of detection for acute infection. Secondly, identification of the protein profile for CAgs will improve the screening range of highly specific antigens and minimize the effect of nonspecific binding on diagnosis. This study identified 26 CAgs components and analysed the preliminary application of two proteins for the detection of CAgs to provide a theoretical basis for the development of sensitive, specific and convenient immunological diagnostic techniques. Furthermore, this study showed that TgCOR and TgELMO are detectable with serological methods during active toxoplasmosis. The two antigens change in a manner consistent with body temperature and T. gondii levels in blood and disappear within five days after the disappearance of T. gondii in the blood.

However, whether antibodies against two recombinant proteins can be widely used to diagnosis acute infection and whether they can be used to effectively distinguish acute and chronic infections in clinical applications requires a large clinical trial for verification. Diagnosis of Toxoplasma by detecting specific CAg also has the potential to reduce the subsequent molecular diagnostic procedures during acute infection. In general, rapid and accurate diagnosis of acute infection will lead to appropriate therapeutic interventions.

Conclusions

In summary, we identified 26 circulating antigens in the sera of canines infected with T. gondii. Among these CAgs molecules, 2 novel candidates were identified as diagnostic antigens for Toxoplasma. Based on the described CAgs, diagnostic methods were developed. Further analysis of these methods will be performed in the future and represents improvement in the detection efficiency of Toxoplasma.

Abbreviations

CAg, circulating antigen; DDT, DL-dithiothreitol; DMEM, Dulbecco modified Eagle medium; ECL, enhanced chemiluminescence; ESA, excretory/secretory antigens; GO, gene ontology; GRA, dense granule antigen; HCD, Higher-energy collisional dissociation; IP, immunoprecipitation; LC, liquid chromatography; MIC, micronemal protein; MS/MS, tandem mass spectrometry; NC, nitrocellulose; SAG, surface antigen; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis.

Acknowledgments

We gratefully thank Yunbing Duan, associate Prof. Tao Li and associate Prof. Houshuang Zhang from Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science for technical assistance. We also acknowledge the Shanghai Applied Protein Technology Co., Ltd. for the technology support.

Funding

This work was supported by National Special Research Program for Non-profit Trades (Agriculture) (Grant No. 201303045), and Basic Foundation for Scientific Research of State-level Public Welfare Institutes of China (Grant No. 2013JB10).

Authors’ contributions

XJX, WQ and HKH participated in the experiment design and wrote the manuscript. XJX and JW analysed the data. XJX, CYJ, ZHJ, XY and QYB carried out the experiments. XJX and LYC carried out bioinformatics analysis. All authors read and approved the final version of the manuscript.

Authors’ details

1College of Veterinary Medicine, Nanjing Agricultural University, No. 1, Weigang, Xuanwu District, Nanjing 210095, P. R. China. 2Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, No. 518, Ziyue Road, Minhang District, Shanghai 200241, P. R. China. 3College of Food Sciences, Shanghai University, No. 99, Shangda Road, Baoshan District, Shanghai 200444, P. R. China.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

This study was approved by the Animal Care and Use Committee of the Shanghai Veterinary Research Institute. The animals were handled in strict accordance with the animal protection law of the People’s Republic of China (released on 09/18/2009) and the National Standards for Laboratory Animals in China (executed on 05/1/2002).

Additional file

Additional file 1: Table S1.(27K, xlsx)

List of CAg proteins identified by LC-MS/MS after IP enrichment and purification with ESA antibodies. (XLSX 27 kb)

Contributor Information

Junxin Xue, moc.621@nixnujeux.

Wei Jiang, moc.361@99wjiewgnaij.

Yongjun Chen, nc.ca.irvhs@nuJgnoYnehC.

Yingchun Liu, nc.ca.irvhs@nuhcgniyuil.

Huajing Zhang, moc.361@321oahiniz.ut.

Yan Xiao, moc.361@21608891iznay.

Yuanbiao Qiao, moc.621@99oaibnauy.

Kehe Huang, nc.ude.uajn@gnauhhk.

Quan Wang, nc.ca.irvhs@nauqgnaw.

References

1. Hunter CA, Sibley LD. Modulation of innate immunity by Toxoplasma gondii virulence effectors. Nat Rev Microbiol. 2012;10:766–778. doi: 10.1038/nrmicro2858. [PMC free article] [PubMed] [Cross Ref]
2. Crone KG, Muraski MB, Skeel JD, Love-Gregory L, Ladenson JH, Gronowski AM. Between a rock and a hard place: Disclosing medical errors. Clin Chem. 2006;52:1809–1814. doi: 10.1373/clinchem.2006.072678. [PubMed] [Cross Ref]
3. Chiesa C, Panero A, Osborn JF, Simonetti AF, Pacifico L. Diagnosis of neonatal sepsis: A clinical and laboratory challenge. Clin Chem. 2004;50:279–287. doi: 10.1373/clinchem.2003.025171. [PubMed] [Cross Ref]
4. Kravetz JD, Federman GD. Toxoplasmosis in pregnancy. Am J Med. 2005;118:212–216. doi: 10.1016/j.amjmed.2004.08.023. [PubMed] [Cross Ref]
5. Montoya JG, Remington JS. Management of Toxoplasma gondii infection during pregnancy. Clin Infect Dis. 2008;47:554–566. doi: 10.1086/590149. [PubMed] [Cross Ref]
6. Mahmoudvand H, Dezaki E, Soleimani S, Baneshi M, Kheirandish F, Ezatpour B, et al. Seroprevalence and risk factors of Toxoplasma gondii infection among healthy blood donors in south-east of Iran. Parasite Immunol. 2015;37:362–367. doi: 10.1111/pim.12198. [PubMed] [Cross Ref]
7. Gras L, Wallon M, Pollak A, Cortina-Borja M, Evengard B, Hayde M, et al. Association between prenatal treatment and clinical manifestations of congenital toxoplasmosis in infancy: a cobort study in 13 European centres. Acta Paeduatr. 2005;94:1721–1731. doi: 10.1080/08035250500251999. [PubMed] [Cross Ref]
8. Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet. 2004;363:1965–1976. doi: 10.1016/S0140-6736(04)16412-X. [PubMed] [Cross Ref]
9. Kodym P, Machala L, Rohacova H, Sirocka B, Maly M. Evaluation of a commercial IgE ELISA in comparison with IgA and IgM ELISAs, IgG avidity assay and complement fixation for the diagnosis of acute toxoplasmosis. Clin Microbiol Infect. 2007;13:40–47. doi: 10.1111/j.1469-0691.2006.01564.x. [PubMed] [Cross Ref]
10. Gutiérrez J, Rodriguez M, Maroto C. A study of IgM antibodies in diagnosis of acute infection by Toxoplasma gondii in Spain. Ser Immunother Infect Dis. 1996;8:85–88. doi: 10.1016/S0888-0786(96)80004-7. [Cross Ref]
11. Carruthers VB. Host cell invasion by the opportunistic pathogen Toxoplasma gondii. Acta Trop. 2002;81:111–122. doi: 10.1016/S0001-706X(01)00201-7. [PubMed] [Cross Ref]
12. Meira CS, Vidal JE, Costa-Silva TA, Frazatti-Gallina N, Pereira-Chioccola VL. Immunodiagnosis in cerebrospinal fluid of cerebral toxoplasmosis and HIV-infected patients using Toxoplasma gondii excreted/secreted antigens. Diagn Microbiol Infect Dis. 2011;71:279–285. doi: 10.1016/j.diagmicrobio.2011.07.008. [PubMed] [Cross Ref]
13. Mattos CC, Meira CS, Ferreira AI, Frederico FB, Hiramoto RM, Jr GC, et al. Contribution of laboratory methods in diagnosing clinically suspected ocular toxoplasmosis in Brazilian patients. Diagn Microbiol Infect Dis. 2011;70:362–366. doi: 10.1016/j.diagmicrobio.2011.02.002. [PubMed] [Cross Ref]
14. Zhou XW, Kafsack BFC, Cole RN, Beckett P, Shen RF, Carruthers VB. The opportunistic pathogen Toxoplasma gondii deploys a diverse legion of invasion and survival proteins. J Biol Chem. 2005;280:34233–34244. doi: 10.1074/jbc.M504160200. [PMC free article] [PubMed] [Cross Ref]
15. Liang L, Doskaya M, Juarez S, Caner A, Jasinskas A, Tan XL, et al. Identification of potential serodiagnostic and subunit vaccine antigens by antibody profiling of toxoplasmosis cases in Turkey. Mol Cell Proteomics. 2011;10(7):M110.006916. doi:10.1074/mcp.M110.006916. Epub 2011 Apr 21. [PMC free article] [PubMed]
16. Wang YH, Li XR, Wang GX, Yin H, Cai XP, Fu BQ, et al. Development of an immunochromatographic strip for the rapid detection of Toxoplasma gondii circulating antigens. Parasitol Int. 2011;60:105–107. doi: 10.1016/j.parint.2010.11.002. [PubMed] [Cross Ref]
17. Wang Q, Jiang W, Chen YJ, Shi JL, Li XT. Prevalence of Toxoplasma gondii antibodies, circulating antigens and DNA in stray cats in Shanghai. Parasite Vector. 2012;5:190. doi: 10.1186/1756-3305-5-190. [PMC free article] [PubMed] [Cross Ref]
18. Wisniewski JR, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis. Nat Methods. 2009;6:359–362. doi: 10.1038/nmeth.1322. [PubMed] [Cross Ref]
19. Gajria B, Bahl A, Brestelli J, Dommer J, Fischer S, Gao X, et al. ToxoDB: an integrated Toxoplasma gondii database resource. Nucleic Acids Res. 2008;36:D553–D556. doi: 10.1093/nar/gkm981. [PMC free article] [PubMed] [Cross Ref]
20. Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4:44–57. doi: 10.1038/nprot.2008.211. [PubMed] [Cross Ref]
21. Dimmer EC, Huntley RP, Alam-Faruque Y, Sawford T, O’Donovan C, Martin MJ, et al. The UniProt-GO annotation database in 2011. Nucleic Acids Res. 2012;40:D565–D570. doi: 10.1093/nar/gkr1048. [PMC free article] [PubMed] [Cross Ref]
22. Aida Y, Pabst MJ. Removal of endotoxin from protein solutions by phase separation using Triton X-114. J Immunol Methods. 1990;132:191–195. doi: 10.1016/0022-1759(90)90029-U. [PubMed] [Cross Ref]
23. Iqbal J, Khalid N. Detection of acute Toxoplasma gondii infection in early pregnancy by IgG avidity and PCR analysis. J Med Microbiol. 2007;56:1495–1499. doi: 10.1099/jmm.0.47260-0. [PubMed] [Cross Ref]
24. Lin Z, Zhang Y, Zhang H, Zhou Y, Cao J, Zhou J. Comparison of loop-mediated isothermal amplification (LAMP) and real-time PCR method targeting a 529-bp repeat element for diagnosis of toxoplasmosis. Vet Parasitol. 2012;185:296–300. doi: 10.1016/j.vetpar.2011.10.016. [PubMed] [Cross Ref]
25. Hassl A, Auer H, Hermentin K, Picher O, Aspöck H. Experimental studies on circulating antigen of Toxoplasma gondii in intermediate hosts: criteria for detection and structural properties. Zbl Bakt Hyg. 1987;263:625–634. [PubMed]
26. Logar J, Novaak-antolic Z, Zore A, Cerer V, Likar M. Incidence of congenital toxoplasmosis in the Republic of Slovenia. Scand J Infect. 1992;24:105–108. doi: 10.3109/00365549209048408. [PubMed] [Cross Ref]
27. Liesenfeld O, Press C, Montoya JG, Gill R, Isaac-Renton JL, Hedman K, et al. False-positive results in immunoglobulin M (IgM) Toxoplasma antibody tests and importance of confirmatory testing: the Platelia Toxo IgM Test. J Clin Microbiol. 1997;35:174–178. [PMC free article] [PubMed]
28. Van Knapen F, Panggabean SO. Detection of circulating antigen during acute infections with Toxoplasma gondii by enzyme-linked immunosorbent assay. J Clin Microbiol. 1977;6:545–547. [PMC free article] [PubMed]
29. Thulliez P, Remington JS, Santoro F, Ovlaque G, Sharma S, Desmonts G. A new agglutination reaction for the diagnosis of the developmental stage of acquired toxoplasmosis. Pathol Biol. 1986;34:173–177. [PubMed]
30. Montoya JG, Liesenfeld O, Kinney S, Press C, Remington JS. VIDAS test for avidity of Toxoplasma-specific immunoglobulin G for confirmatory testing of pregnant women. J Clin Microbiol. 2002;40:2504–2508. doi: 10.1128/JCM.40.7.2504-2508.2002. [PMC free article] [PubMed] [Cross Ref]
31. Asai T, Kim TJ, Kobayashi M, Kojima S. Detection of nucleoside triphosphate hydrolase as a circulating antigen in sera of mice infected with Toxoplasma gondii. Infect Immun. 1987;55:1332–1335. [PMC free article] [PubMed]
32. Hassan MM, Mansour SA, Atta M, Shalaby MM, Seksaka MA, Awad A. The importance of detecting circulating Toxoplasma antigens in human cases. J Egypt Soc Parasitol. 1997;27:27–34. [PubMed]
33. Raizman RE, Neva FA. Detection of circulating antigen in acute experimental infections with Toxoplasma gondii. J Infect Dis. 1975;132:44–48. doi: 10.1093/infdis/132.1.44. [PubMed] [Cross Ref]
34. Petray P, Bonardello N, Clark R, Agranatti M, Corral R, Grinstein S. Evaluation of an ELISA technique for detection of antigens and circulating immune complexes of Trypanosoma cruzi by a field study in an endemic zone of Argentina. Rev Inst Med Trop Sao Paulo. 1992;34:141–147. doi: 10.1590/S0036-46651992000200010. [PubMed] [Cross Ref]
35. Weiss LM, Kim K. Toxoplasma gondii: the model apicomplexan: perspectives and methods. In: Petersen E, Liesenfeld O, editors. Clinical disease and diagnostics. London: Elsevier; 2007. pp. 81–100.
36. Brecht S, Carruthers VB, Ferguson DJP, Giddings OK, Wang G, Jakle U, et al. The Toxoplasma micronemal protein MIC4 is an adhesin composed of six conserved apple domains. J Biol Chem. 2001;276:4119–4127. doi: 10.1074/jbc.M008294200. [PubMed] [Cross Ref]
37. Marchant J, Cowper B, Liu Y, Lai L, Pinzan C, Marq JB, et al. Galactose recognition by the apicomplexan parasite Toxoplasma gondii. J Biol Chem. 2012;287:16720–16733. doi: 10.1074/jbc.M111.325928. [PMC free article] [PubMed] [Cross Ref]
38. Cerede O, Dubremetz JF, Soete M, Deslee D, Vial H, Bout D, et al. Synergistic role of micronemal proteins in Toxoplasma gondii virulence. J Exp Med. 2005;201:453–463. doi: 10.1084/jem.20041672. [PMC free article] [PubMed] [Cross Ref]
39. Lin J, Lin X, Yang GH, Wang Y, Peng BW, Lin JY. Toxoplasma gondii: Expression of GRA1 gene in endoplasmic reticulum promotes both growth and adherence and modulates intracellular calcium release in macrophages. Exp Parasitol. 2010;125:165–171. doi: 10.1016/j.exppara.2010.01.010. [PubMed] [Cross Ref]
40. Lecordier L, Mercier C, Sibley LD, Cesbron-Delauw MF. Transmembrane insertion of the Toxoplasma gondii GRA5 protein occurs after soluble secretion into the host cell. Mol Biol Cell. 1999;10:1277–1287. doi: 10.1091/mbc.10.4.1277. [PMC free article] [PubMed] [Cross Ref]
41. He XL, Grigg ME, Boothroyd JC, Garcia KC. Structure of the immunodominant surface antigen from the Toxoplasma gondii SRS superfamily. Nat Struct Biol. 2002;9:606–611. [PubMed]
42. Wang HL, Li YQ, Yin LT, Meng XL, Guo M, Zhang JH, et al. Toxoplasma gondii protein disulfide isomerase (TgPDI) is a novel vaccine candidate against Toxoplasmosis. PLoS One. 2013;8:e70884. doi: 10.1371/journal.pone.0070884. [PMC free article] [PubMed] [Cross Ref]
43. Liwak U, Ananvoranich S. Toxoplasma gondii: Over-expression of lactate dehydrogenase enhances differentiation under alkaline conditions. Exp Parasitol. 2009;122:155–161. doi: 10.1016/j.exppara.2009.01.016. [PubMed] [Cross Ref]
44. Boucher JI, Jacobowitz JR, Beckett BC, Classen S, Theobald DL. An atomic-resolution view of neofunctionalization in the evolution of apicomplexan lactate dehydrogenases. Elife. 2014;3:e02304. [PMC free article] [PubMed]
45. Skillman KM, Ma CI, Fremont DH, Diraviyam K, Cooper JA, Sept D, et al. The unusual dynamics of parasite actin result from isodesmic polymerization. Nat Commun. 2013;4:2285. doi: 10.1038/ncomms3285. [PMC free article] [PubMed] [Cross Ref]
46. Mun HS, Aosai F, Norose K, Chen M, Hata H, Tagawa Y, et al. Toxoplasma gondii Hsp70 as a danger signal in Toxoplasma gondii-infected mice. Cell Stress Chaperon. 2000;5:328–335. doi: 10.1379/1466-1268(2000)005<0328:TGHAAD>2.0.CO;2. [PMC free article] [PubMed] [Cross Ref]
47. Holmes M, Liwak U, Pricop I, Wang X, Tomavo S, Ananvoranich S. Silencing of tachyzoite enolase 2 alters nuclear targeting of bradyzoite enolase 1 in Toxoplasma gondii. Microbes Infect. 2010;12:19–27. doi: 10.1016/j.micinf.2009.09.010. [PubMed] [Cross Ref]
48. Assossou O, Besson F, Rouault JP, Persat F, Ferrandiz J, Mayencon M, et al. Characterization of an excreted/secreted antigen form of 14-3-3 protein in Toxoplasma gondii tachyzoites. Fems Microbiol Lett. 2004;234:19–25. doi: 10.1111/j.1574-6968.2004.tb09508.x. [PubMed] [Cross Ref]
49. Jewett TJ, Sibley LD. Aldolase forms a bridge between cell surface adhesins and the actin cytoskeleton in apicomplexan parasites. Mol Cell. 2003;11:885–894. doi: 10.1016/S1097-2765(03)00113-8. [PubMed] [Cross Ref]
50. Harris MT, Mitchell WG, Morris JC. Targeting protozoan parasite metabolism: glycolytic enzymes in the therapeutic crosshairs. Curr Med Chem. 2014;21:1668–1678. doi: 10.2174/09298673113206660286. [PubMed] [Cross Ref]
51. Donaldson TM, Cassera MB, Ho MC, Zhan C, Merino EF, Evans GB, et al. Inhibition and structure of Toxoplasma gondii purine nucleoside phosphorylase. Eukaryot Cell. 2014;13:572–579. doi: 10.1128/EC.00308-13. [PMC free article] [PubMed] [Cross Ref]
52. Grininger M. Perspectives on the evolution, assembly and conformational dynamics of fatty acid synthase type I (FAS I) systems. Curr Opin Struct Biol. 2014;25:49–56. doi: 10.1016/j.sbi.2013.12.004. [PubMed] [Cross Ref]

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