Toxoplasma gondii: Detection of MIC10 antigen in sera of experimentally infected mice

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Experimental Parasitology 118 (2008) 362–371 www.elsevier.com/locate/yexpr

Toxoplasma gondii: Detection of MIC10 antigen in sera of experimentally infected mice George Dautu a, Akio Ueno a, Aracelis Miranda b, Sophie Mwanyumba c, Biscah Munyaka d, Gabriella Carmen e, Tatsuya Kariya a, Yoshitaka Omata f, Atsushi Saito f, Xuenan Xuan a, Makoto Igarashi a,* a

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, 080-8555 Japan b Gorgas Memorial Institute for Health Research, Panama c National Public Health Laboratory Services, Kenya d Kenya Medical Research Institute (KEMRI), P.O. Box 54840-00200 Nairobi, Kenya e Servicio Nacional de salud y Calidad Animal, 595-(21)-584-496 San Lorenzo, Paraguay f Department of Basic Veterinary Science, School of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, 080-8555 Japan Received 5 June 2007; received in revised form 20 September 2007; accepted 24 September 2007 Available online 1 October 2007

Abstract We developed a sandwich ELISA for the detection of circulating Toxoplasma gondii MIC10 antigens. In T. gondii culture supernatant, MIC10 was detected in a growth dependent manner. Mice were infected with a lethal dose of either a virulent RH strain, an avirulent Beverley strain or a sub-lethal dose of a PLK strain of T. gondii. MIC10 appeared 2 days after infection and increased gradually in the sera of RH-infected mice. A detectable but significantly lower amount of MIC10 was observed in the sera of mice infected intraperitoneally with Beverley tachyzoites. In contrast, the MIC10 antigen in mice sera following oral infection with Beverley cysts was below detectable levels during the course of the experiment. In sera of PLK-infected mice, MIC10 was predominantly observed between late acute and early chronic phase. Our data show that the kinetics of circulating MIC10 differs depending on the strain and route of infection.  2007 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Toxoplasma gondii; Circulating antigen; Sandwich ELISA; MIC10, Microneme protein-10

1. Introduction Toxoplasma gondii is an obligate intracellular protozoan parasite belonging to the phylum Apicomplexa. T. gondii is one of the major opportunistic pathogens which can cause fatal toxoplasmic encephalitis in immunocompromised patients such as AIDS patients (Luft and Remington, 1992; Dzierszinski et al., 1999). The seroprevalence of IgG antibody to T. gondii in humans differs depending on countries or areas and can range from less than 10% to more than 60% (Naito et al., 2007). In Japan, for example, this seroprevalence was previously reported to be at *

Corresponding author. Fax: +81 155 49 5643. E-mail address: [email protected] (M. Igarashi).

0014-4894/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2007.09.010

9.3% in healthy individuals (Yamaoka and Konishi, 1993) and 1.07% of AIDS patients were reported to develop toxoplasmic encephalitis (Kano et al., 2004). Toxoplasmosis is caused by three major strains of T. gondii which are categorized as type I (includes strains like RH which are highly virulent), type II (includes avirulent strains like PLK and Beverley) and type III (includes avirulent strains like VEG and CTG). The type I strain or type I-like strains are associated with severe or atypical ocular toxoplasmosis in infected immunocompetent adults and are also overrepresented in congenital infection (Grigg and Boothroyd, 2001). On the other hand, type II strains account for most toxoplasmosis cases in immunocompromised patients (Grigg and Boothroyd, 2001; Howe et al., 1997). It is also worthwhile to mention that type III strains

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are also found in patients with ocular toxoplasmosis (Fux et al., 2003). In domestic animals, especially sheep and goats, the parasite can cause abortions and stillbirth resulting in significant losses to the livestock industry (Buxton, 1998). Furthermore, the tissue cysts of T. gondii in meat of infected livestock are an important source of infection in humans (Buffolano et al., 1996). The diagnosis of recently acquired toxoplasmosis has been done traditionally by detecting specific immunoglobulin (Ig) M antibodies or by demonstrating a significant increase in specific IgG antibodies, or both. However, the high prevalence of specific IgG antibody to T. gondii among normal individuals in most populations (Remington and Desmonts, 1990) and the sustained persistence, in some people, of specific IgM antibodies has complicated the interpretation of serological tests when toxoplasmosis is suspected (van Loon et al., 1983; Brooks et al., 1987; Del Bono et al., 1989; Bobic et al., 1991). For this reason, there is an increasing demand for newer, more accurate diagnostic markers to distinguish recent from past T. gondii infections. Recently, several reports have shown how to detect acute toxoplasmosis by the IgG avidity test and the existence of specific IgA or E antibodies (Suzuki et al., 2001; Kodym et al., 2007). Several reports have emphasized the value of detecting circulating antigen to T. gondii for diagnosis of the early phase of toxoplasmosis (van Knapen and Panggabean, 1977; Brooks et al., 1985; Asai et al., 1987; Acebes et al., 1994; Hafid et al., 1995; Villavedra et al., 2001). However, few reports have appeared in the literature regarding the diagnostic potential of T. gondii circulating antigens (Attallah et al., 2006). The origin of circulating antigens could be as a result of parasite degradation, active secretion by tachyzoites, bradyzoites in the cysts, shedding of membrane components or a combination of all previous mechanisms (Cesbron-Delauw and Capron, 1993). Microneme proteins (MICs) are actively discharged from parasites upon the apical attachment to a host cell and play a role in host cell attachment and penetration (Tomley and Soldati, 2001). MIC10, one of the members of MICs, is a small 18 kDa protein lacking a putative adhesive and transmembrane domain (Hoff et al., 2001), and was previously shown to be released by T. gondii parasites (Zhou et al., 2005). The present study was designed to assess the usefulness of sandwich ELISA as a diagnostic method for detection of MIC10 antigen by exploring the time course of changes in the serum level of experimentally infected mice.

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Nos. N59901–N99999, W00001–W00143, W05897–W06460, W35481–W35691, W63000–W66462, W96583–W96820, W99405–W99830, AA009304–AA009405, AA011884–AA012644, AA037889–AA037942, U72327–U72329, T62239–T62475, AW702178–AW703613, BG657138–BG661027, BI921045– BI921090, BI946571–BI946588, CA955183–CA955618, CA961801–CA962422, CB299937–CB303434, CB333476– CB333771, CB367803–CB368333, CB368902–CB369135, CB370032–CB370338, CB370611–CB371114, CB372018– CB372283, CB373342–CB373482, CD216790–CD217544, CD240550–CD240708, CF120340–CF121080, CF245762– CF249018, CF266495–CF269258, CF340749–CF341985, CF370793–CF371098, CF453443–CF453444, CK730349– CK738218, CN121380–CN123810, CN194697–CN200102, CN613745–CN621555, CN658034–CN660031, CN779737– CN781903, CO052971–CO058125, CO390367–CO391146, CO511155–CO511687, CO720933–CO722176, CO742628– CO745536, CO905955–CO906326, CV122487–CV122733, CV512448–CV514344, CV548806–CV553866, CV582089– CV582762, CV652796–CV655371, CV700481–CV704168, and CV793522–CV793584, were included in the analysis. Blast search analysis was performed using the T. gondii gene encoding microneme proteins (Accession Nos.: MIC1, Z71786, introns were removed; MIC2, U62660, introns were removed; MIC3, AJ132530; MIC4, AF143487; MIC5, Y09782, introns were removed; MIC6, AF110270; MIC7, AF357911; MIC8, AF353165; MIC9, AF353166; MIC10, AF293654; MIC11, AF539702; AMA1, AF010264; M2AP, AF364813, introns were removed) as queries. The number of clones identical to corresponding microneme genes were determined and expressed as percentages from the calculation of the total number of ESTs deposited in the dbEST/Genbank. 2.2. Animals Six to eight weeks old female BALB/c mice (H-2d) and a 3-month-old male white Japanese rabbit used in our experiments were purchased from CLEA, Japan. The BALB/c mice were used both for anti-MIC10 antibody production and infection studies whereas the white Japanese rabbit was used for anti-MIC10 antibody production. The experiments were conducted in accordance with the standards relating to the care and management of experimental animals promulgated by Obihiro University of Agriculture and Veterinary Medicine (Hokkaido, Japan). 2.3. Parasites

2. Materials and methods 2.1. Expression sequence tagged (EST) database analysis ESTs of nonsubtracted/unnormalized libraries from T. gondii tachyzoites were used to analyze the expression profile of genes encoding microneme proteins. A total of 79,267 ESTs, deposited into the dbEST/Genbank under Accession

Toxoplasma gondii RH (virulent), Beverley (avirulent), PLK (avirulent) strains and Neospora c (N. caninum) NC1 strain were used in this study. Tachyzoites of all strains were maintained in our laboratory through serial passage in Vero cells grown in modified Eagle’s medium (Sigma–Aldrich, UK) supplemented with 5% fetal calf serum. Cysts of T. gondii Beverley strain used for challeng-

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ing the mice were obtained from the brains of orally infected ICR mice (CLEA, Japan) and maintained by monthly passage. 2.4. Toxoplasma and neospora lysate antigen Toxoplasma gondii and Neospora caninum lysates were prepared from T. gondii RH strain and N. caninum NC1 strain tachyzoites as follows. Intracellular tachyzoites were passed through a 27-G needle 5 times, filtered through a Millex SV 5-lm membrane (Millipore, USA) to remove host cell debris, washed with phosphate buffered saline (PBS) and adjusted to 1 · 108 tachyzoites/ ml of PBS. The parasites were disrupted by three freeze–thaw cycles and the crude extract was clarified by centrifugation at 2500g for 15 min. The resulting soluble fraction was filtered through a Millex GV 0.22-lm filter unit (Millipore, USA). The protein concentration in the supernatant was measured using the Coomassie protein assay reagent kit using bovine serum albumin as a standard (Pierce, USA) and kept at 80 C until use.

2.6. Generation of polyclonal anti-MIC10 antibodies The MIC10 antibodies were produced in white Japanese rabbit and BALB/c mice. Rabbit immunization was done as follows. The rabbit was immunized subcutaneously at three different inoculation sites with 500 lg of recombinant GST-MIC10 protein emulsified in an equal volume of Freund’s complete adjuvant (Sigma–Aldrich, UK). Two weeks later, the rabbit was immunized with the same dose of antigen emulsified with incomplete Freund’s adjuvant (Sigma– Aldrich, UK). On day 28, the rabbit was immunized with one more doses of antigen with incomplete adjuvant. The rabbit was sacrificed 7 days after the last immunization and blood for sera preparation was colleted. The serum collected was tested using Western blot analysis. BALB/c mice immunization was done in the same way with the only difference being the amount of antigen given i.e., 50 lg in the case of mice, and the route of administration (intraperitoneal for mice). The sera were tested in the same way as described above. The rabbit anti-MIC10 antibody was affinity purified by a Protein G column as per the manufacturer’s instructions (HiTrap Protein G HP, GE Healthcare Bio-Sciences, USA).

2.5. Generation of recombinant MIC10 protein 2.7. SDS–PAGE and Western blot analysis Toxoplasma gondii MIC10 cDNA containing an entire coding region (Hoff et al., 2001) was amplified by polymerase chain reaction (PCR) from Beverley strain total RNA as a template using primers 5 0 -TTGGATCCAT GGCGCTTTCTTCTTTG-3 0 and 5 0 -TTCTCGAGCTAC ATTGATTTCCTGCGTC-3 0 . The PCR was performed as per the manufacturer’s instructions (one step RNA PCR kit, Takara, Japan). The reaction was done for 1 cycle at: 50 C for 30 min, 94 C for 2 min then for 30 cycles at: 94 C for 30 s, 55 C for 30 s, 72 C for 1 min, and finally for 1 cycle at: 72 C for 10 min. After the reaction, the sample was kept at 4 C. The amplified product was purified by phenol extraction followed by ethanol precipitation. After dissolving in TE buffer (10 mM Tris, pH 7.5, 1 mM EDTA), the cDNA was double digested with BamHI and XhoI rescriction enzymes (Toyobo, Japan). The digested product was electrophoresed on a 1% agarose gel, purified by a gel extraction kit (Qiagen, USA) as per the manufacturer’s instructions and subcloned into the BamHI and XhoI sites of pGEX6P-2 (GE Healthcare Bio-Sciences). The resulting plasmid was transformed in Escherichia coli strain BL21(DE3)pLysS cells (Novagen, USA) and the recombinant protein was expressed as GST fused protein herein referred to as recombinant GST-MIC10. The fusion protein was produced by IPTG induction (1 mM, WAKO, Japan) and affinity purified by glutathione Sepharose (GE Healthcare Bio-Sciences, USA). The eluted fraction was dialyzed against PBS and the amount of recombinant protein was evaluated using both SDS– polyacrylamide gel electrophoresis (PAGE) and a Coomassie protein assay reagent kit (Pierce, USA).

The recombinant GST-MIC10 and T. gondii tachyzoites (RH strain) were dissolved in SDS–PAGE sample buffer (62.5 mM Tris, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.02% bromophenol blue), heated at 95 C for 5 min and separated on a 12% Polyacrylamide gel as previously described (Laemmli, 1970). For recombinant protein analysis, visualization of separated protein was done by staining the gel with Coomassie brilliant blue R250 (MP Biomedicals, France). The LMW calibration kit (GE Healthcare Bio-Sciences, USA) was used as a molecular mass standard. In order to assess if the produced polyclonal antibodies in mice and rabbit could identify the native MIC10 antigen in T. gondii lysate, Western blot analysis was done as follows. After the SDS–PAGE, the separated proteins were transferred to Nylon membrane (Immobilon, Millipore, USA). Non-specific binding sites were blocked by incubating the transferred membrane in PBS containing 1% skimmed milk (milk-PBS) for 1 h at room temperature. The membrane was washed three times with PBS containing 0.5% Tween 20 (PBS-T) and probed with anti-MIC10 polyclonal antibodies raised in either mice or rabbit at a dilution ratio of 1:100 in milk-PBS at room temperature for 1 h. After washing the membrane with PBS-T three times, bound antibodies were detected using anti-mouse (from sheep) or rabbit (from donkey) IgG conjugated with horseradish peroxidase (GE Healthcare Bio-Sciences, USA) diluted 1:1000 in milk-PBS. After 1-h incubation at room temperature, the membrane was washed three times with PBS-T. Peroxidase activity was revealed by using a substrate mixture of 0.25 mg/ml diaminobenzidine tetrahydrochloride (Sigma–Aldrich, UK),

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100 mM Tris, pH 7.5 and 0.05% H2O2 or chemo luminescence (ECL Western Blotting Detection Reagents, GE Healthcare Bio-Sciences, USA). A molecular mass standard (SeeBlue plus2 pre-stained standard, Invitrogen, USA) was used. 2.8. Enzyme linked immunosorbent assay (ELISA) Levels of specific IgG antibodies in mouse and rabbit serum samples were determined as follows. In brief, 96 wells of flat-bottom micro-titer plates (Maxisorp, Nunc, Denmark) were coated overnight at 4 C with recombinant GST-MIC10 at 5 lg/ml in 50 mM sodium carbonate buffer (pH 9.6). The plates were washed six times with PBS-T. Non-specific binding sites were blocked with milk-PBS for 1 h at room temperature. The plates were then washed once with PBS-T and subsequently 50 ll of mice or rabbit sera of indicated concentration in milk-PBS was added and incubated at 37 C for 1 h. After incubation, the plates were washed six times with PBS-T. The bound antibodies were detected by incubating at 37 C for 1 h with sheep anti-mouse IgG or donkey anti-rabbit IgG-conjugated with horseradish peroxidase (GE Healthcare Bio-Sciences, USA) diluted 1:2000 in milk-PBS. Finally the plates were washed six times with PBS-T and the bound peroxidase enzyme activity was revealed by adding 100 ll/well of ABTS substrate i.e., 3 mg 2,2 0 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Sigma– Aldrich, UK), in ABTS buffer (0.1 M citric acid monohydrate, 0.2 M disodium hydrogen phosphate), pH 4.0 and 1 ll H2O2 per 10 ml of buffer. The absorbance at 415 nm in each well was measured using a MTP-500 microplate reader (Corona Electrical, Japan). 2.9. Detection of MIC10 antigen After several optimization trials, the following sandwich ELISA was performed for detecting MIC10 antigen. The purified rabbit anti-MIC10 antibody was diluted in absorption buffer at a concentration of 2 lg/ml. The diluted antibodies were added (100 ll/well) into the wells of the 96-well flat-bottom microplates (Maxisorb, Nunc, Denmark). The plates were covered and incubated at 4 C overnight for absorption of the antibodies. The plates were washed five times with PBS-T and non-specific binding sites were blocked with milk-PBS. After 1-h incubation at room temperature, the blocking solution was discarded and 50 ll of samples of appropriate dilutions in milk-PBS was added to each well. Following incubation for 1 h at room temperature, the plates were washed five times with PBS-T. The polyclonal anti-MIC10 antibody raised in mice immunized with recombinant GST-MIC10 (diluted 1:500 in milk-PBS) was added to each well and then incubated at room temperature for 1 h. After 1 h incubation, the plates were washed five times with PBS-T. Horseradish peroxidase conjugated anti-mouse IgG (diluted 1:1000 in milk-PBS, GE Healthcare Bio-Sciences, USA) was incubated at room

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temperature for 1 h. After washing with PBS-T five times, 100 ll of ABTS substrate as described above was added to each well. After incubation at room temperature for 30 min, the absorbance at 415 nm was read using a microplate reader (MTP-500, Corona Electric, Japan). The amount of MIC10 antigen was expressed as optical density (OD) values or was estimated from a standard curve created by recombinant GST-MIC10 protein. 2.10. In vitro growth assay of T. gondii For cell culture, modified Eagle’s medium (Sigma– Aldrich, UK) supplemented with 5% fetal calf serum was used. An anti-toxoplasma drug, pyrimethamine, was purchased from Sigma–Aldrich (UK). Human primary fibroblast monolayer was cultured in a 96-well plate at 37 C in a 5% CO2 incubator. Freshly prepared T. gondii parasites of RH strain tachyzoites were inoculated into three different culture plates containing monolayers of human primary fibroblast cells with different numbers of parasites (10,000, 1000, 100) being added to each plate. The cultures were incubated for 2 h and then replaced with culture medium with or without pyrimethamine. Infected cells were incubated at 37 C in a 5% CO2 incubator and culture supernatants were collected on day 1, 2, 3 and 4 for the determination of MIC10 antigen. 2.11. Murine experimental toxoplasmosis Four groups of BALB/c mice were arranged according to the T. gondii strain (16 mice for RH tachyzoites, 10 mice for PLK tachyzoites, 9 mice for Beverley tachyzoites and 9 mice for Beverley cysts) used for infection. For tachyzoite infection, 1000 tachyzoites of RH strain and 2000 each of Beverley and PLK strains were used, whereas for cyst infection, 20 cysts of avilurent Beverley strain were used. Tachyzoite infection was done intraperitoneally whereas cyst infection was done orally. Sera obtained from non-infected mice served as negative controls. Serum samples were kept at 80 C until use. 2.12. Statistical analysis Levels of significance were determined by the MannWhitney test. Statistical analysis was carried out using Prism 3 software (Graph Pad, USA). 3. Results 3.1. MIC10 was most frequently found in the EST database We analyzed the expression sequence tagged (EST) database in order to determine the expression profile of T. gondii genes encoding microneme proteins. As shown in Fig. 1, the MIC10 gene was most frequently found in the tachyzoite cDNA library among genes encoding microneme proteins. This was consistent with the result shown

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Frequency (%)

1.0 0.8 0.6 0.4 0.2

MI C1 MI C2 MI C3 MI C4 MI C5 MI C6 MI C7 MI C8 MI C9 MI C1 0 MI C1 1 AM A1 M2 AP

0

Fig. 1. Frequency of cDNAs encoding microneme proteins found in the EST database. BLAST searches were performed using indicated cDNAs as query sequences and the number of clones was expressed as percentages from the calculation of the total number of ESTs deposited in the dbEST/ Genbank.

by others (Zhou et al., 2005). A total of 79,267 ESTs, 822 clones (1.04%) were identical to the MIC10 sequence. 3.2. Development of sandwich ELISA system for the detection of MIC10 antigen The recombinant GST-MIC10 protein produced in E. coli had a molecular mass of 49 kDa as shown in Fig. 2a. Since the GST protein is 26 kDa, this result is in agreement with the predicted molecular mass of 23 kDa of immature MIC10 protein. The specificities and sensitivities of produced antibodies were examined by Western blot and ELISA analysis. Both antibodies were able to detect 18 kDa MIC10 protein in T. gondii lysate (Fig. 2b). The circulating MIC10 antigen at 18 kDa and possible degradation products at lower bands in sera of RH-

a

infected mice were observed as shown in Fig. 2c. The titer of anti-MIC10 activity in sera produced in mice and rabbit was compared with that of PLK-infected chronic mice sera (Table 1). The difference of specific activity between MIC10 immunized mice sera and chronic mice sera was about 1:20,000 and chronic mouse sera contained less than 10 ng/ml of specific antibody when compared with rabbit antibody. In order to establish the sensitivity and linearity of the assay, we set up a standard curve using different concentrations of recombinant GST-MIC10 fusion protein. Optical density (OD) readings were plotted against the concentration of the protein preparations (Fig. 3a). The co-efficient of variation (CV) for intra- and inter-assay determined were less than 10% and 12%, respectively (data not shown). A lower detection limit of the assay was determined as 0.2 ng/ml of MIC10 protein (Fig. 3a and data not shown). To examine the specificity of the assay, different concentrations of T. gondii and closely related N. caninum tachyzoite lysates were subjected to the assay as described Table 1 Titer of anti-MIC10 antibody by ELISA analysis Serum sample

Dilution/ concentration

OD415 (mean ± SD)

Normal mouse (n = 1) Normal rabbit (n = 1) PLK chronic (90 dpi) (n = 8)a MIC10 immunized mice (n = 3)

·50 ·50 ·50

<0.01 <0.01 0.15 ± 0.14

·10,000 ·100,000 ·1,000,000 20 ng/ml 2 ng/ml 0.2 ng/ml

2.74 ± 0.05 0.97 ± 0.23 0.13 ± 0.04 2.91 2.16 0.6

MIC10 immunized rabbit, Purified (n = 1)

a PLK chronic mice sera used in this experiment were obtained from samples in Section 3.5 of this study.

b

Mol. Wt (kDa)

1

2

c

Mol. Wt (kDa) 98

97 66

1

2

3

1

2

3

Mol. Wt (kDa) 64

64 50 50 36 36 22

45 30

22 16 16

Fig. 2. SDS–PAGE and Western blot analysis of recombinant and endogenous MIC10 protein. (a) SDS–PAGE analysis of recombinant GST-MIC10 protein. (Lane 1) Molecular weight marker. (Lane 2) Recombinant GST-MIC10 protein as indicated by arrowhead. (b) Western blot analysis of MIC10 protein (arrowhead) in T. gondii lysate using MIC10 immunized mouse (lane 2) and rabbit (lane 3) serum. Molecular weight marker is shown in lane 1. (c) Detection of circulating MIC10 antigen in RH infected (lane 1) or uninfected (lane 2) mouse serum by Western blot using MIC10 immunized rabbit serum. Molecular weight marker is shown in lane 3. Arrowhead indicates 18 kDa MIC10 protein. Arrows indicate possible degradation products of MIC10 protein.

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above. As shown in Fig. 3b, the MIC10 antigen was detected in T. gondii lysates in a dose-dependent manner. On the other hand, no reaction was observed in N. caninum lysate at any concentration. In order to evaluate the effect of anti-MIC10 antibody in serum samples, the recombinant GST-MIC10 protein (5 ng/ml; black column, 1 ng/ ml; grey column, 0.2 ng/ml; white column, Fig. 3c) was added in milk-PBS containing indicated concentrations of produced mouse or rabbit anti-MIC10 antibodies and subjected to the assay. As shown in Fig. 3c, the concentration of more than or equal to 1:1600 dilution of GST-MIC10 immunized mouse serum and less than 6 ng/ml of rabbit anti-MIC10 antibody did not affect the signal significantly. 3.3. Application for in vitro growth assay The assay system was used to examine the growth kinetics of T. gondii tachyzoites in vitro. MIC10 antigen was

a

OD reading at 415 nm

detected 2 days (10,000 parasites), 3 days (1000 parasites) or 4 days (100 parasites) after infection and reached a plateau in 4 days (10,000 parasites) (Fig. 4a). Next, we tested the assay system to see the growth inhibition by the antitoxoplama drug, pyrimethamine in vitro. As shown in Fig. 4b, MIC10 was below the detectable level in the presence of pyrimethamine at more than 0.4 lM and the IC50 value was estimated at about 0.15 lM. 3.4. Detection of MIC10 antigen in sera of mice infected with a lethal dose of T. gondii In order to evaluate the diagnostic potential of the assay system in the diagnosis of acute toxoplasmosis, we infected mice with a lethal dose of T. gondii. Sera were collected as described in Section 2. The amounts of MIC10 antigen in serum samples were examined using the assay system. In the sera of RH-infected mice, the MIC10 antigen was

b 1.2

GST-TgMIC10

1.2

T. gondii

1.0

1.0

0.8

0.8

0.6

0.6

0.4

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0.2

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0

0 0

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4

6

8

10

N. caninum

0

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Addition of mouse serum (dilution)

x100

0.6

x200

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x400

0.8

x800

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x1,600

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x3,200

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OD reading at 415 nm

6

8

10

(Concentration, μg/ml)

(Concentration, ng/ml)

c

367

0

3

6

12

25

50

100

Addition of rabbit antibody (ng/ml)

Fig. 3. Evaluation of sensitivity and specificity of MIC10 sandwich ELISA. (a) Recombinant GST-TgMIC10 protein was diluted as indicated and applied to the assay. (b) Each T. gondii and N. caninum lysate was diluted as indicated and applied to the assay. (c) Recombinant GST-MIC10 (5 ng/ml; black column, 1 ng/ml; grey column, 0.2 ng/ml; white column) was mixed with an indicated dilution/concentration of either MIC10 immunized mouse (left panel) or rabbit (right panel) antibody and applied to the assay. Results represent mean values ± SD from triplicate (a) or duplicate (b and c) samples. Where no error bars are shown, the error was less than the symbol size.

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a

b

16

3

14

TgMIC10 (ng/ml)

TgMIC10 (ng/ml)

12 10 8 6 4

2

1

2 0

0 0

1

2

3

0

4

Days after infection

0.1

0.2

0.3

0.4

0.5

0.6

Pyrimethamine concentration (μM)

Fig. 4. In vitro growth assay of T. gondii. (a) Either 10,000 (circle), 1000 (square) or 100 (triangle) T. gondii RH strain parasites were used to infect each well of a confluent human fibroblast culture in a 96-well plate. Culture supernatants were collected at indicated days and applied to the assay. (b) One thousand T. gondii RH strain parasites were inoculated into each well of a confluent human fibroblast culture in a 96-well plate with varying concentrations of pyrimethamine. Three days after inoculation, culture supernatants were collected and the amount of MIC10 antigen was determined as described in Section 2. Results represent mean values ± SD from triplicate (a) or duplicate (b) samples.

cumbed to the infection by day 10 in this group (data not shown).

detected on day 2 and reached a plateau on day 5. The maximum concentration of MIC10 in serum was about 150 ng/ml (Fig. 5a). In the sera of avirulent Beverley strain tachyzoite-infected mice, the MIC10 antigens were detectable in all samples 5 days post infection and increased thereafter, but the level was much lower (10 ng/ml, maximum) than that observed in sera of RH-infected mice (Fig. 5b). Out of nine mice infected, three succumbed to the infection on day 7 and the rest (6 mice) on day 8. However, no obvious correlation was observed between MIC10 antigen levels and the survival days (data not shown). In the sera of avirulent Beverley strain cyst-infected mice, the MIC10 antigen was below detectable levels during the course of the experiment (Fig. 5b) with the exception of one mouse which showed a low level of the antigen (6 ng/ml) on day 9. Six out of nine mice eventually suc-

a

3.5. Kinetics of MIC10 antigen in sera of mice infected with a sub-lethal dose of T. gondii To examine the kinetics of the amount of MIC10 antigen in sera during the acute and chronic phases, we infected mice with a sub-lethal dose of T. gondii. Sera samples were collected and subjected to the assay system with a view of detecting MIC10 (Fig. 6). The MIC10 antigen was detected in sera of all infected mice on day 10–15 and thereafter increased gradually. At the end of the experiment on day 90, MIC10 antigen was still detectable in 3 out of 8 infected surviving mice. Initially 10 mice were infected with a sublethal dose of T. gondii. During the course of infection,

b

180

12

160 10

TgMIC10 (ng/ml)

TgMIC10 (ng/ml)

140 120 100 80 60 40

8 6 4 2

20 0

0 0

1

2

3

4

5

Days after infection

6

7

8

0

2

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Days after infection

Fig. 5. Detection of MIC10 antigen in mouse sera of lethal toxoplasmosis. (a) MIC10 concentration in sera of mice after intraperitoneal inoculation with a lethal dose of virulent RH strain (1000 tachyzoites). Two mice were sacrificed each day and serum samples were collected. The amount of MIC10 in sera was determined as described in Section 2. Results are expressed as means ± SD from each of two samples. (b) MIC10 concentration in sera of mice after inoculation with a lethal dose of either avirulent Beverley strain tachyzoites (2000 parasites intraperitoneally, white circle) or cysts (20 cysts per orally). Results are expressed as mean ± SD (from each of nine mice).

G. Dautu et al. / Experimental Parasitology 118 (2008) 362–371 40 36

TgMIC10 (ng/ml)

32 28 24 20 16 12 8 4 0 0

20

40

60

80

100

Days after infection

Fig. 6. Kinetics of the MIC10 antigen in mouse sera of sub-lethal toxoplasmosis. MIC10 antigen concentration in sera of mice after intraperitoneal inoculation with a sub-lethal dose of 2000 tachyzoites of avirulent PLK strain. Results are expressed as the mean (±SD, not shown) from duplicate sample determination.

one mouse died on day 21, the other on day 35. However, the amounts of MIC10 antigen in sera of these mice were not significantly different from those in survived mice (data not shown). 4. Discussion The diagnostic usefulness of detection of Toxoplasma antigen in the blood of infected subjects was previously investigated by several groups (van Knapen and Panggabean, 1977; Brooks et al., 1985; Asai et al., 1987; Acebes et al., 1994; Hafid et al., 1995; Villavedra et al., 2001; Attallah et al., 2006). In this work, we have developed an assay system that can detect MIC10 antigen and we further evaluated its diagnostic potential both in vitro and in vivo. First, we chose the target antigen among various microneme proteins through analysis of the EST database. By analyzing the EST database, MIC10 was found to be the most abundant in the T. gondii tachyzoite cDNA library. This finding was in agreement with results previously reported by others (Zhou et al., 2005). MIC10 belongs to the microneme protein family and is secreted from micro-organelles, the micronemes, of T. gondii tachyzoites. Mature MIC10, whose function is unknown, is an 18 kDa protein shown to be expressed preferentially in tachyzoites rather than bradyzoites (Hoff et al., 2001) and was previously shown to be released from parasites (Zhou et al., 2005). Unlike other microneme proteins, MIC10 lacks the putative adhesive and transmembrane domain that is capable of binding to host cell receptor(s) and hence this raises the possibility that MIC10 could diffuse from the infection site in the tissue and become accessible as a circulating antigen (Hoff et al., 2001). Therefore, we produced a pair of polyclonal antibodies against MIC10 in mouse and rabbit and employed them in sandwich ELISA for MIC10 detection. The sensitivity of the developed sandwich ELISA was

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determined by using recombinant MIC10 protein as an antigen. From the results, our assay system could detect as little as 0.2 ng/ml of the recombinant GST-MIC10 protein (Fig. 3a). The specificity of the assay system was further determined by using various concentrations of T. gondii and closely related N. caninum lysates as antigens. From the results, the developed assay system was highly specific for T. gondii but not for N. caninum lysate because no reaction was seen with N. caninum lysate at any concentration (Fig. 3b). Most T. gondii-infected subjects have antibodies, and if present in sera, could mask the detection of circulating antigens. Therefore, we were interested in assessing the inhibitory effect on MIC10 antigen detection on the presence of anti-MIC10 antibody in the samples. To examine this possibility, the recombinant GST-MIC10 protein was diluted in either the mouse or rabbit antiMIC10 antibodies and finally the developed assay was used to detect MIC10 protein. When the recombinant MIC10 protein was added to mouse anti-MIC10 sera (at a dilution more than 1:1,600) or purified rabbit anti-MIC10-antibody (less than 6 ng/ml), no obvious sequestration of signals was observed (Fig. 3c). The specific activity of anti-MIC10 antibody in chronically infected mouse sera was below the level observed in recombinant MIC10-immunized mice and rabbit sera as described above (Table 1). Therefore, inhibition of signals by antibody produced in infected mice was most likely negligible. Our results (Fig. 3a, b and c) show that the developed MIC10 sandwich ELISA is sensitive and highly specific for T. gondii MIC10 antigen. We were also interested in evaluating the application of the assay on the in vitro culture samples. This was important because we wanted to determine if there was a relationship between T. gondii growth and the release of MIC10 antigen in culture supernatants. Sandwich ELISA was used to detect the amount of MIC10 in culture supernatants and our data show a good correlation between the amounts of MIC10 detected in culture supernatants and the growth of T. gondii tachyzoites (Fig. 4a). The addition of varying concentrations (0.1–0.6 lM) of anti-toxoplasma drug, pyrimethamine affected the growth of the T. gondii tachyzoites and subsequently the release of the MIC10 antigen (Fig. 4b). A concentration more than 0.4 lM of pyrimethamine completely inhibited the release of MIC10 antigen (Fig. 4b). The IC50 was about 0.15 lM where values between 0.15 and 1.6 lM have been reported previously (Pfefferkorn and Pfefferkorn, 1977; Fichera et al., 1995; McFadden et al., 1997; Gubbels et al., 2003). These sets of data show that the release of MIC10 antigen is dependent on the growth and multiplication of the T. gondii tachyzoites in vitro. We evaluated the amount of MIC10 antigen in sera of mice infected with three strains of T. gondii namely RH (type I, virulent), Beverley (type II, avirulent) and PLK (type II, avirulent). The amount of MIC10 antigen in the sera of mice challenged with virulent RH strain tachyzoite was much higher than that in avirulent Beverley strain tachyzoite-infected mice (P < 0.05, Fig. 5a and b).

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Previous reports show that the important feature of infections by type I (such as RH) strains in mice is that they rapidly disseminate and reach high tissue burdens even from very low initial inoculums (Gavrilescu and Denkers, 2001; Mordue et al., 2001; Barragan and Sibley, 2002). We therefore attribute the difference of the amount of sera MIC10 antigen between RH and Beverley strains to the rapid dissemination and ability by the RH strain to reach high tissue burden faster than the Beverley strain. In addition, intraperitoneal and oral infection of mice with the avirulent Beverley strain showed different kinetics of serum MIC10 antigen levels. In the intraperitoneally infected mice with tachyzoites of the Beverley strain, a detectable amount of MIC10 antigen was observed in the sera of all mice by day 6 (Fig. 5b, white circle). On the other hand, only one out of nine mice orally infected with cysts of the Beverley strain showed a detectable amount of MIC10 antigen in sera on day 9 (Fig. 5b, black solid circle). The amount of MIC10 antigen in the sera of mice challenged with tachyzoites of the Beverley strain was significantly higher than in the mice challenged with Beverley cysts (P < 0.05). We attribute this difference to the route of infection and the stage of the parasite used for infection (tachyzoites and encysted bradyzoites). We assume that tachyzoites (the invasive stage of T. gondii) rapidly disseminated and reached a high tissue burden with the subsequent release of a detectable amount of MIC10 antigen. On the other hand, we assume that in the oral route, the liberation of bradyzoites from the cysts in the gastrointestinal tract and their subsequent dissemination and multiplication was slow. Another possibility could be that the parasites by oral infection preferentially may disseminate through the lymphatic route rather than the hematogenous route. This could have led to low or below detectable levels of free circulating MIC10 antigen. Because oral infection with cysts closely represents a natural course of infection, higher sensitivity of the assay may be required for detection of the signals. Production and use of monoclonal or polyclonal antibody with higher sensitivity could solve this problem. In Fig. 6, we show the kinetics of the MIC10 antigen in sera of mice infected with a sub-lethal dose of the PLK strain. All mice showed the MIC10 antigens in sera 10–15 days after infection. The kinetics of MIC10 antigen varied between mice and the level was sustained in 3 out of 8 mice until the end of the experiment on day 90. The correlation between MIC10 antigen levels and intensity of toxoplasma infection is a matter of question. Between virulent and avirulent strain, higher intensity of infection caused by the RH strain showed a significantly higher amount of circulating MIC10 antigen. On the other hand, in the group of PLK-infected mice, the amount of MIC10 antigen in sera was not significantly different between mice that survived and mice that succumbed to infection. Therefore, there was no correlation between mortality and the level of MIC10 antigen in this experimental setting.

In the future, further evaluation of the assay will be carried out by determining the amount of MIC10 antigen in the sera of patients showing toxoplasmosis. In addition, the amount of MIC10 antigen in cerebral or amniotic fluids of such patients will be investigated. Acknowledgments This work was supported in part by a Grant-in-Aid from the Research and Development program for New Bio-industry Initiatives. References Acebes, M.V., Diez, B., Garcia-Rodriguez, J.A., Viens, P., Cisterna, R., 1994. Detection of circulating antigens in the diagnosis of acute toxoplasmosis. American Journal of Tropical Medicine and Hygiene 51, 506–511. Asai, T., Kim, T., Kobayashi, M., Kojima, S., 1987. Detection of nucleoside triphosphate hydrolase as a circulating antigen in sera of mice infected with Toxoplasma gondii. Infection and Immunity 55, 1332–1335. Attallah, A.M., Ismail, H., Ibrahim, A.S., Al-Zawawy, L.A., El-Ebiary, M.T., El-Waseef, A.M., 2006. Immunochemical identification and detection of a 36-kDa Toxoplasma gondii circulating antigen in sera of infected women for laboratory diagnosis of toxoplasmosis. Journal of Immunoassay and Immunochemistry 27, 45–60. Barragan, A., Sibley, L.D., 2002. Transepithelial migration of Toxoplasma gondii is linked to parasite motility and virulence. Journal of Experimental Medicine 195, 1625–1633. Bobic, B., Sibalic, D., Djurkovic-Djakovic, O., 1991. High levels of IgM antibodies specific for Toxoplasma gondii in pregnancy 12 years after primary toxoplasma infection. Case report. Gynecologic and Obstetric Investigation 31, 182–184. Brooks, R.G., Sharma, S.D., Remington, J.S., 1985. Detection of Toxoplasma gondii antigens by a dot-immunobinding technique. Journal of Clinical Microbiology 21, 113–116. Brooks, R.G., McCabe, R.E., Remington, J.S., 1987. Role of serology in the diagnosis of toxoplasmic lymphadenopathy. Reviews of Infectious Diseases 9, 1055–1062. Buffolano, W., Gilbert, R.E., Holland, F.J., Fratta, D., Palumbo, F., Ades, A.E., 1996. Risk factors for recent toxoplasma infection in pregnant women in Naples. Epidemiology and Infection 116, 347–351. Buxton, D., 1998. Protozoan infections (Toxoplasma gondii, Neospora caninum and Sarcocystis spp) in sheep and goats: recent advances. Veterinary Research 29, 289–310. Cesbron-Delauw, M.F., Capron, A., 1993. Excreted/secreted antigens of Toxoplasma gondii: their origin and role in the host-parasite interaction. Research in Immunology 144, 41–44. Del Bono, V., Canessa, A., Bruzzi, P., Fiorelli, M.A., Terragna, A., 1989. Significance of specific immunoglobulin M in the chronological diagnosis of 38 cases of toxoplasmic lymphadenopathy. European Journal of Clinical Microbiology 27, 2133–2135. Dzierszinski, F., Popescu, O., Toursel, C., Slomianny, C., Yahiaoui, B., Tomavo, S., 1999. The protozoan parasite Toxoplasma gondii expresses two functional plant-like glycolytic enzymes. Implications for evolutionary origin of apicomplexans. The Journal of Biological Chemistry 274, 24888–24895. Fichera, M.E., Bhopale, M.K., Roos, D.S., 1995. In vitro assays elucidate peculiar kinetics of clindamycin action against Toxoplasma gondii. Antimicrobial Agents and Chemotherapy 39, 1530–1537. Fux, B., Rodrigues, C.V., Portela, R.W., Silva, N.M., Su, C., Sibley, D., Vitor, R.W.A., Gazzinelli, R.T., 2003. Role of cytokines and major histocompatibility complex restriction in mouse resistance to infection

G. Dautu et al. / Experimental Parasitology 118 (2008) 362–371 with a natural recombinant strain (type I-III) of Toxoplasma gondii. Infection and Immunity 71, 6392–6401. Gavrilescu, L.C., Denkers, E.Y., 2001. IFN-gamma overproduction and high level apoptosis are associated with high but not low virulence Toxoplasma gondii infection. Journal of Immunology 167, 902–909. Grigg, M.E., Boothroyd, J.C., 2001. Rapid identification of virulent type I strains of the protozoan pathogen Toxoplasma gondii by PCRrestriction fragment length polymorphism analysis at the B1 gene. Journal of Clinical Microbiology 39, 398–400. Gubbels, M-J., Li, C., Striepen, B., 2003. High-throughput growth assay for Toxoplasma gondii using yellow fluorescent protein. Antimicrobial Agents and Chemotherapy 47, 309–316. Hafid, J., Tran Manh Sung, R., Raberin, H., Akono, Z.Y., Pozzetto, B., Jana, M., 1995. Detection of circulating antigens of Toxoplasma gondii in human infection. American Journal of Tropical Medicine and Hygiene 52, 336–339. Hoff, E.F., Cook, S.H., Sherman, G.D., Harper, J.M., Ferguson, D.J.P., Dubremetz, J-F., Carruthers, V.B., 2001. Toxoplasma gondii: cloning and characterization of a novel 18-kDa secretory antigen, TgMIC10. Experimental Parasitology 97, 77–88. Howe, D.K., Honore, S., Derouin, F., Sibley, L.D., 1997. Determination of genotypes of Toxoplasma gondii strains isolated from patients with toxoplasmosis. Journal of Clinical Microbiology 35, 1411–1414. Kano, S., Genka, I., Yoshida, K., Oka, S., Ito, T., Sato, I., Katakura, M., Mamiya, H., Watanabe, S., Kamihira, A., Shirasaka, T., Yamamoto, M., Miyamura, T., 2004. Parasitic disease complicated with HIV/ AIDS patients in Japan. Clinical Parasitology 15, 95–98, in Japanese. Kodym, P., Machala, L., Rohacova, H., Sirocka, B., Maly, M., 2007. 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. Clinical Microbiology and Infection 13, 40–47. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 277, 680–685. Luft, B.J., Remington, J.S., 1992. Toxoplasmic encephalitis in AIDS. Clinical Infectious Diseases 15, 211–222. McFadden, D.C., Seeber, F., Boothroyd, J.C., 1997. Use of Toxoplasma gondii expressing b-galactosidase for colorimetric assessment of drug activity in vitro. Antimicrobial Agents and Chemotherapy 41, 1849–1853. Mordue, D.G., Monroy, F., La Regina, M., Dinarello, C.A., Sibley, L.D., 2001. Acute toxoplasmosis leads to lethal overproduction of Th1 cytokines. Journal of Immunology 167, 4574–4584.

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Naito, T., Inui, A., Kudo, N., Matsumoto, N., Fukuda, H., Isonuma, H., Sekigawa, I., Dambara, T., Hayashida, Y., 2007. Seroprevalence of IgG anti-Toxoplasma antibodies in asymptomatic patients infected with human immunodeficiency virus in Japan. Internal Medicine 46, 1149–1150. Pfefferkorn, E.R., Pfefferkorn, E.C., 1977. Specific labeling of intracellular Toxoplasma gondii with uracil. Journal of Protozoology 24, 449–453. Remington, J.S., Desmonts, G., 1990. Toxoplamosis. In: Remington, J.S., Klein, J.O. (Eds.), Infectious Diseases of the Fetus and Newborn Infant. WB Saunders Company, Philadelphia, pp. 89–195. Suzuki, L.A., Rocha, R.J., Rossi, C.L., 2001. Evaluation of serological markers for the immunodiagnosis of acute acquired toxoplasmosis. Journal of Medical Microbiology 50, 62–70. Tomley, F.M., Soldati, D.S., 2001. Mix and match modules: structure and function of microneme proteins in apicomplexan parasites. Trends in Parasitology 17, 81–88. van Knapen, F., Panggabean, S.O., 1977. Detection of circulating antigen during acute infections with Toxoplasma gondii by enzymelinked immunosorbent assay. Journal of Clinical Microbiology 6, 545–547. van Loon, A.M., van der Logt, J.T.M., Heessen, F.W.A., van der Veen, J., 1983. Enzyme-linked immunosorbent assay that uses labeled antigen for detection of immunoglobulin M and A antibodies in toxoplasmosis: comparison with indirect immunofluorescence and double-sandwich enzyme-linked immunosorbent assay. Journal of Clinical Microbiology 17, 997–1004. Villavedra, M., Rampoldi, C., Carol, H., Baz, A., Battistoni, J.J., Nieto, A., 2001. Identification of circulating antigens, including an immunoglobulin binding protein, from Toxoplasma gondii tissue cyst and tachyzoites in murine toxoplasmosis. International Journal for Parasitology 31, 21–28. Yamaoka, M., Konishi, E., 1993. Prevalence of antibody to Toxoplasma gondii among inhabitants under different geographical and climatic conditions in Hyogo Prefecture, Japan. Japanese Journal of Medical Science and Biology 46, 121–129. Zhou, X.W., Kafsack, B.F.C., Cole, R.N.C., Beckett, P., Shen, R.F., Carruthers, V.B., 2005. The opportunistic pathogen Toxoplasma gondii deploys a diverse legion of invasion and survival proteins. The Journal of Biological Chemistry 280, 34233–34244.