Introduction

Toxoplasma gondii is an obligate intracellular parasite that is capable of infecting a variety of mammals and birds and causing toxoplasmosis. T. gondii is an important food-borne parasite, and the main route of transmission from animals to humans is through the consumption of infected meat (Nielsen et al. 1999). In some countries, pork is the most common meat consumed, and some ethnic groups consume raw pork; thus, pigs are considered to be the primary source of human infection with T. gondii in the world (Bezerra et al. 2012; Dubey et al. 1994; Paştiu et al. 2013). In addition, due to gross lesions in infected animals leading to their being condemned at slaughter or due to the expense associated with treatment for or the weight loss associated with clinical toxoplasmosis, toxoplasmosis is a source of significant economic loss for swine farmers. Therefore, the development of an effective vaccine for controlling this infection is an important goal because of the worldwide public health and economic repercussions of T. gondii infections.

As an apicomplexan parasite, T. gondii contains three morphologically distinct secretory or ganelles (rhoptries, micronemes, and dense granules). Rhoptries and micronemes discharge their proteins during cell invasion, and dense granule exocytosis occurs after invasion and continues during the intracellular residence of the organism (Carruthers 2002; Carruthers et al. 1997). This phenomenon allows parasites to discharge prodigious amounts of proteins into the vacuolar space enclosed by the parasitophorous vacuolar membrane, which surrounded the parasites inside the cells (Assossou et al. 2004). These proteins are named excreted–secreted antigens (ESAs), which have attracted growing interest because of their possible roles in nutrient uptake during multiplication and in host immunity (Darcy et al. 1988; Prigione et al. 2000). During T. gondii infection, ESAs might be one of the first targets of the immune system. Several authors have stressed the primary role played by tachyzoite ESAs in stimulating the host immune system. ESAs are highly immunogenic (Carruthers 2002; Decoster et al. 1988; Khosroshahi et al. 2012; Prigione et al. 2000; Qu et al. 2013; Tao et al. 2013) and can induce either antibody-dependent or cell-mediated immune responses (Darcy et al. 1988; Zenner et al. 1999). The level of protection provided by either whole proteins (Daryani et al. 2003; Duquesne et al. 1990) or individual (native or recombinant) proteins (Godard et al. 1994; Mevelec et al. 1998; Ram et al. 2013; Scorza et al. 2003) has been assayed in experimental toxoplasmosis models. It has been suggested that ESAs are good candidates for the development of immunization strategies.

However, although there have been promising results using ESAs in immunization strategies in murine models, no published reports have examined whether the immunization of pigs with ESAs confers resistance against T. gondii infection. In this study, we used the pig as a large animal model to further investigate the extent of the immune response generated by ESAs of the Gansu Jingtai strain (GJS) of T. gondii in combination with Freund's adjuvant.

Materials and methods

T. gondii strain

T. gondii type I GJS was isolated from a pig with acute toxoplasmosis in our laboratory (Chen et al. 1982). GJS tachyzoites were obtained from the ascites of Kunming mice infected by intraperitoneal injection and were used for challenge as well as antigen preparation.

Antigen preparation

To prepare a toxoplasma lysate (TLA), GJS tachyzoites were obtained from the peritoneal fluid of infected Kunming mice as described previously (Vercammen et al. 2000). This material was passed twice through a 26-gauge needle. The parasites were washed, resuspended in phosphate-buffered saline (PBS), and sonicated (1-min pulse, 1 min of cooling, 150 W) with an ultrasonic disintegrator. The protein concentration of TLA was determined using the Bio-Rad DC protein assay.

ESAs were recovered from the supernatant of cell cultures infected with tachyzoites as previously described (Costa-Silva et al. 2008) with minor modifications. Briefly, Vero cells were washed five times with RPMI-1640 medium without FBS, supplemented with fresh medium, and then infected with 107 GJS tachyzoites for each milliliter of culture. After 48 h, the culture supernatants were harvested and filtered with a 0.22-μm pore size filter. The supernatants, which contained the ESAs, were concentrated in a Speed Vac SPD111V for 5 h. The concentration of ESAs was estimated by determining the difference between the protein concentrations of ESAs and the control preparations, which were processed in the same manner as the culture flasks prepared for recovery of the ESAs but without infection with tachyzoites. The concentrated ESAs and the control preparations were used for immunizations.

Experimental animals

Forty healthy pigs (7–9 weeks old), which were Toxoplasma seronegative as determined by enzyme-linked immunosorbent assay (ELISA), were purchased from a pig farm in Lanzhou, China. The pigs in this study were raised on a conventional farm or in experimental isolation units. The experimental protocol was approved by the ethical Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

Immunization of pigs

Forty pigs were divided into two groups (20 pigs per group): an immunization group (numbered I1–I20) and a control group (numbered C1–C20). The pigs in the immunization group were injected subcutaneously in the neck twice with 2 mg of ESAs emulsified in Freund's adjuvant (Sigma) with a month interval between the injections. The pigs in the control group received a suspension of culture supernatants of uninfected Vero cells and Freund's adjuvant. The first immunization was performed in Freund's complete adjuvant, and the booster immunization was administered in Freund's incomplete adjuvant. At day 15 before the boost and at days 15, 60, 90, and 150 after the boost, blood samples were collected from the pigs (I16–I20 and C16–C20).

Challenge infection of pigs

Fifteen pigs from each group (I1–I15 and C1–C15) were challenged intraperitoneally with 107 tachyzoites 30 days after the boost. Clinical signs and body temperatures of pigs I1–I5 and C1–C5 were recorded before and after the challenge. Then the pigs (I1–I5 and C1–C5) were euthanatized at day 60 post-infection (PI), and the muscles (tongue, skeletal muscle, diaphragm, and heart) were collected to investigate T. gondii tissue cysts by mouse bioassay.

Pigs I6–I15 and C6–C15 were euthanatized at days 2, 3, 4, 5, 6, 7, 10, 14, 21, and 35 PI. A complete post mortem examination was performed, and the presence of visible lesions was recorded.

Sodium dodecyl sulfate–polyacrylamide gels and Western blot analysis

The ESAs (20 μg per line) were separated by 12 % polyacrylamide gels. The proteins were transferred to nitrocellulose membranes, cut into 3- to 4-mm-wide strips, blocked for 1 h with 5 % skim milk–PBS, and incubated with pig serum (at 1:200 dilution) at room temperature. After 1 h, the strips were washed with PBS and incubated for 1 h at room temperature with horseradish peroxidase-conjugated rabbit anti-pig IgG (at 1:500 dilution). After washing, the strips were developed with an ECL Western blot substrate (Pierce Biotechnology) by following the manufacturer's directions.

Enzyme-linked immunosorbent assay

Total antigen-specific antibodies were measured by ELISA. Briefly, 96-well microtiter plates (flat bottom, low binding, Corning) were coated with TLA at 10 μg/ml in 50 mM sodium carbonate buffer (pH 9.6). Pig sera were diluted 1:200 in PBS and used as the primary antibody. The rabbit anti-pig peroxidase-conjugated IgG (Sigma) diluted 1:40,000 in PBS was used as the secondary antibody, with o-phenylenediamine and hydrogen peroxide as substrates. Optical density was read at 490 nm (OD490 nm) using an ELISA plate reader. On day 15 after the boost, IgG1 and IgG2a antibody determinations were also performed as described for total antibodies, except that peroxidase-conjugated IgG1 and IgG2a (Shanghai Yueyan Biological Technology Co., Ltd), diluted 1:2000, were used as the secondary antibodies.

Evaluation of the cellular immune response

On day 15 after the boost, peripheral blood monomorphonuclear cells (PBMCs) were separated using a lymphocyte separation medium (Beijing Solarbio Science & Technology Co., Ltd.). The PBMC suspension was incubated with 10 μg/ml TLA according to the previously described protocol (Wang et al. 2011). Cells cultivated in the medium alone served as the negative controls. OD490 nm was measured after 4 days using the standard MTS method (Promega, USA). Lymphocyte proliferative responses were quantified using the stimulation index, which was calculated as the ratio of the OD490 nm of stimulated cells to the OD490 nm of the unstimulated cells.

Additionally, the IFN-γ and IL-4 levels in the serum samples on day 15 after the boost were also measured using commercial ELISA kits (Jingmei, Biotech Co. Ltd., Shenzhen, China) according to the manufacturer's instructions. Cytokine concentrations were determined by reference to standard curves that were calculated using known amounts of pig recombinant IFN-γ and IL-4.

Bioassay of pig tissues for T. gondii

The muscle samples (12.5 g each of tongue, skeletal muscle, diaphragm, and heart) were bioassayed for the presence of T. gondii tissue cysts as described previously (Dubey 1998). Briefly, each sample was homogenized in 250 ml of saline solution (0.14 M NaCl). Then 250 ml of pepsin solution was added and incubated at 37 °C for 1 h. The homogenate was filtered through two layers of gauze and centrifuged at 1,180×g for 10 min. The sediment was resuspended in 20 ml PBS (pH 7.2), and 15 ml of 1.2 % sodium bicarbonate (pH 8.3) was added and centrifuged as above. The sediment was resuspended in 5 ml of antibiotic saline solution (1,000 U penicillin and 100 ml of streptomycin/ml of saline solution) and inoculated with a volume of 1 ml subcutaneously into five mice (25–30 g).

Impression smears of lung tissue from the mice that died were stained with Giemsa and examined microscopically. Blood samples were collected from the mice that survived 60 days after inoculation, and the brain of each mouse was examined microscopically for T. gondii tissue cysts. The serum from each mouse was diluted at 1:16 and 1:64 and examined for T. gondii antibodies using IFA.

Statistical analysis

All statistical analyses were performed using SPSS 11.5 software. The results are expressed as the mean ± standard deviation (SD). The mean of each variable, including the antibody OD values, rectal temperatures, and cytokine expression levels, were compared between the immunization and the control groups using the Mann–Whitney U test. The significant differences in the mouse bioassay were evaluated by the chi-square test. P values less than 0.05 were considered to be statistically significant.

Results

Humoral immune responses in pigs immunized with ESAs

At different time points after the immunization, serum was harvested and tested by ELISA using TLA (Fig. 1). The levels of anti-T. gondii antibodies started rising at day 15 before the boost and were significantly higher than those in the control group (P = 0.009). Furthermore, the antibody responses of the immunized pigs continued to increase for 15 days after the boost, and the levels of anti-T. gondii antibodies remained high throughout the study. In contrast, the OD values of anti-T. gondii antibodies for the control group remained low during the course of the experiment (P < 0.01). IgG subclasses are depicted in Fig. 2. IgG1 and IgG2a values were significantly higher in the immunized group compare to those in the control group (P < 0.01). In Western blot analysis of ESAs after SDS-PAGE, sera from the immunized pig (strip 2) especially reacted with ESAs of high molecular weight (about 34–116 kDa; Fig. 3).

Fig. 1
figure 1

The OD values as measured by ELISA using TLA of T. gondii antibodies (mean ± SD, n = 5 pigs per group) in serum taken from pigs at days 15, 45, 90, 120, and 180 after the first immunization. At day 15 after the first immunization, the OD values reached 0.480 ± 0.036 for immunized pigs and 0.225 ± 0.015 for control pigs, P = 0.009. The Mann–Whitney U test was used to compare the immunization and control groups. Immunization group pigs received ESAs plus adjuvant immunizations. Control group pigs only received the control immunization (Vero cell culture supernatant plus adjuvant)

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Fig. 2
figure 2

IgG subclass in sera from the immunized group on day 15 after the boost (OD values = mean ± SD, n = 5 pigs per group). The Mann–Whitney U test was used to compare the immunization and control groups (P < 0.01)

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Fig. 3
figure 3

SDS-PAGE (a) and Western blot (b) analysis of ESAs. Lane M protein molecular weight marker, Control Vero cell culture supernatant, Strip 1 pooled serum from immunized pig with Vero cell culture supernatant plus adjuvant, Strip 2 pooled serum from immunized pig with ESAs, Strip 3 pooled serum from normal pig

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Cellular immune responses in pigs immunized with ESAs

A proliferation assay was performed on PBMCs collected 15 days after the boost with ESAs. As shown in Table 1, the immunized group of pigs produced vigorous lymphocyte responses compared with the negative control group (P = 0.008). Meanwhile, the immunized pigs produced significantly higher levels of IFN-γ and IL-4 than the control pigs (P = 0.008 and 0.008, respectively).

Table 1 Cytokine production and lymphocyte proliferation in pigs immunized with ESAs
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Effective protection against T. gondii infection in pigs immunized with ESAs

The immunized pigs that were challenged with infection 30 days after the boost showed no clinical signals of disease, except for brief low-grade fever (≤40.5 °C) at 6 days PI. In contrast, all the control pigs developed clinical signs of toxoplasmosis after challenge. The first clinical signs were observed at 3 days PI and persisted until day 9 PI, and they included ocular secretions followed by coughing, anorexia, prostration, and high fever. These signs were most pronounced at day 6 after challenge, when the pigs showed severe prostration and high body temperatures (mean = 41.1 ± 0.5 °C), as shown in Fig. 4. At days 3, 4, 5, 6, 7, and 8 PI, the rectal temperatures of the immunized pigs were significantly lower than those of the control pigs (P = 0.012, 0.016, 0.008, 0.047, 0.021, and 0.036, respectively).

Fig. 4
figure 4

The rectal temperatures of pigs following intraperitoneal challenge with 107 tachyzoites at 30 days after the boost. The results are expressed as the mean ± SD (n = 5 pigs per group). The rectal temperatures were significantly different between the immunized and control groups at days 3, 4, 5, 6, 7, and 8 PI (P = 0.012, 0.016, 0.008, 0.047, 0.021, and 0.036, respectively). The Mann–Whitney U test was used to compare the immunized and control groups. Immunization group pigs had received ESAs plus adjuvant immunizations. Control group pigs had only received the control immunization (Vero cell culture supernatant plus adjuvant)

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The visible and distinct lesions characteristic of acute T. gondii infection (enlarged mesenteric lymph nodes, an enlarged spleen with focal necrosis, and enlarged lungs with miliary or focal necrosis and off-white lesions) were found in three control pigs (euthanatized at days 4–6 PI) (Fig. 5). The remainder of the control pigs had less severe lesions (moderately enlarged mesenteric lymph nodes or an enlarged spleen with focal necrosis, and enlarged lungs with miliary or focal necrosis and off-white lesions). No lesions were found in the brain, kidney, heart, or intestines. None of the immunized pigs developed lesions over the course of the experiment.

Fig. 5
figure 5

Observed lesions. Mesenteric lymph nodes (a), spleen (b), and lung (c) from immunized pig; mesenteric lymph nodes (d), spleen (e), and lung (f) from control pig

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To evaluate the effect of vaccination on T. gondii cyst development, cysts in the muscles were detected by mouse bioassay. The results are summarized in Table 2. Tissue cysts were not detected in 4/5 (immunization group) and 0/5 (control group) of pigs. The mouse bioassay revealed that T. gondii tissue cysts in the muscles of the immunized pigs were fewer than those in the control pigs (P < 0.01).

Table 2 Results of T. gondii tissue cysts in muscles of pigs as determined by mouse bioassay
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Discussion

Worldwide, food safety has become a central issue in food production and marketing. Consumers demand not only high quality foods but also safe foods. T. gondii is one of the most important zoonotic parasites that can be transmitted by the consumption of infected meat (Paştiu et al. 2013). Because of the risk of toxoplasmosis, it is essential to develop a better vaccine to immunize pigs against T. gondii infection to improve human health and reduce economic loss (Liu et al. 2009). To date, little information is available concerning noninfectious vaccines against toxoplasmosis in pigs (Garcia et al. 2005; Jongert et al. 2008). Thus, our work focuses on the immunogenicity of the ESA vaccine and its ability to protect pigs against T. gondii infection.

The mechanism of the humoral response in the effective prevention of toxoplasmosis has been documented (Butcher and Denkers 2002; Konishi and Nakao 1992; Vercammen et al. 1999; Mineo et al. 1993; Fuhrman et al. 1989). B cells are required for the resistance of host cells to T. gondii (Couper et al. 2005; Denkers and Gazzinelli 1998; Johnson and Sayles 2002; Sayles t al. 2000). Previous experiments have shown that anti-ESA antibodies reacted with a crude preparation of tachyzoite antigens and bound to the surface of the parasite (Costa-Silva et al. 2008). After the boost with ESAs, high levels of anti-T. gondii antibodies were detected by ELISA in the immunized pigs. Immunoblot analysis indicated that these antibodies are mainly directed against the antigens with molecular masses between 34 and 116 kDa. Recently, 37-, 49-, and 65-kDa proteins have been identified as 14-3-3 protein, microneme protein 1 (MIC1), and MIC4, respectively, by our lab (Ye 2013). MICs are released on the parasite surface at the time of invasion and act as major cellular adhesins, participating in parasite reorientation and entry of the parasite into the host cell. It is becoming increasingly apparent that many MICs are found in stable adhesive complexes, which are formed in the endoplasmic reticulum, and normally comprise an escorter protein and one or more soluble effector proteins (Saouros et al. 2005). The first such complex discovered in T. gondii was MIC4–MIC1–MIC6, in which MIC6 (34 kDa) fulfils the role of the escorter protein, whereas MIC1 and MIC4 function as adhesions (Reiss et al. 2001). Therefore, it is reasonable to speculate that the 34-kDa proteins may be MIC6. Despite significant progresses in studying these immunoreactive proteins, only a limited number of proteins have been identified. Thus, there is a need for more rigorous studies on the antigenic components of T. gondii ESAs.

Different cytokines, such as IFN-γ and IL-4, have been shown to play an important role in the resistance to early and late stages of infection with T. gondii parasites (Fux et al. 2003; Liu et al. 2009; Wu et al. 2012). IFN-γ may inhibit the replication of T. gondii within macrophages and somatic cells (Denkers and Gazzinelli 1998; Hakim et al. 1991; Reichmann et al. 1997) and can confer protection against lethal T. gondii challenge (Gazzinelli et al. 1991; Suzuki et al. 1990; Suzuki and Joh 1994; Suzuki et al. 1988). Solano Aguilar et al. (2001) also reported that IFN-γ production participates in the development of the response to an acute T. gondii infection in swine. It is likely that IFN-γ is required for pigs to survive infection. In the present study, we found that the immunization of pigs with ESAs in Freund's adjuvant induced Th1 cell-mediated immunity and IFN-γ secretion. Additionally, a significant increase in IL-4 production was observed in the serum of the immunized pigs. The role of Th2 cytokines in toxoplasmosis is not fully understood. However, some studies have suggested that IL-4-deficient mice infected with T. gondii have an increased mortality compared to control animals (Roberts et al. 1996; Suzuki et al. 1996). In addition, IL-4 has been shown to modulate the replication of tachyzoites in murine macrophages (Swierczynski et al. 2000).

In this study, we investigated the outcome of acute T. gondii infection in pigs immunized with ESAs. The GJS used to challenge, which was originally isolated from a pig with clinical signs of toxoplasmosis, is one of the most pathogenic strains of the parasite in mice. Pigs intraperitoneally inoculated with 107 GJS tachyzoites develop the clinical signs of toxoplasmosis (coughing, anorexia, prostration, and high fever). In the present study, the pigs immunized with ESAs remained clinically normal, except for a brief low-grade fever, following intraperitoneal challenge with 107 tachyzoites, while the control pigs showed clinical signs of toxoplasmosis.

An important consideration when developing a vaccine for pigs is whether the vaccine reduces or eliminates tissue cyst formation in immunized pigs, consequently potentially reducing human exposure by this route. In the present study, pigs immunized with ESAs had a significantly lower number of tissue cysts compared to controls. It is therefore possible that the immune responses observed in these animals could protect against tissue cyst formation.

In conclusion, our results revealed that the immunization with ESAs supplemented with a strong adjuvant to induce a Th1 response (Freund's adjuvant) elicits a strong and specific immune response and an effective, highly significant protection against T. gondii infection. Moreover, the response elicited by the immunization with ESAs was capable of reducing the levels of cysts in the muscle. The production of tissue cyst-free meat would be advantageous because it would reduce the risk of human exposure to T. gondii. Our findings suggest that immunization with ESAs is a promising strategy for combating toxoplasmosis in pigs.