Identification and Application of Epitopes in EtMIC1 of Eimeria tenella Recognized by the Monoclonal Antibodies 1-A1 and 1-H2

Eimeria tenella microneme-1 protein (EtMIC1) has been proposed to be a transmembrane protein, but this characteristic has not yet been confirmed experimentally. Furthermore, despite EtMIC1 being an important candidate antigen, its key epitope has not been reported.

ABSTRACT

Eimeria tenella microneme-1 protein (EtMIC1) has been proposed to be a transmembrane protein, but this characteristic has not yet been confirmed experimentally. Furthermore, despite EtMIC1 being an important candidate antigen, its key epitope has not been reported. Here, two linear B-cell epitopes of EtMIC1, ⁹¹LITFATRSK⁹⁹ and ⁶⁹⁸ESLISAGE⁷⁰⁵, were identified by Western blotting using specific monoclonal antibodies (MAbs) and were named epitope I (located in the I-domain) and epitope CTR (located in the CTR domain), respectively. Sequence comparative analyses of these epitopes among Eimeria species that infect chickens showed that epitope I differs greatly across species, whereas epitope CTR is relatively conserved. Point mutation assay results indicate that all the amino acid residues of the epitopes recognized by MAb 1-A1 or 1-H2 are key amino acids involved in recognition. Comparative analyses of indirect immunofluorescence assay (IFA) results for MAbs 1-A1 and 1-H2 under both nonpermeabilization and permeabilization conditions indicate that epitope I is located on the outer side of the sporozoite surface membrane whereas epitope CTR is located on the inner side, together providing experimental evidence that EtMIC1 is a transmembrane protein. IFA also labeled the EtMIC1 protein on the parasitophorous vacuole membrane and on the surface of schizonts, which suggests that the EtMIC1 protein may play an important role in parasitophorous vacuole formation and E. tenella development. Immunoprotective efficacy experiments revealed that epitope I has good immunogenicity, as evidenced by its induction of high serum antibody levels, blood lymphocyte proliferation, and CD4⁺ blood lymphocyte percentage.

INTRODUCTION

Microneme proteins of apicomplexan parasites secreted by microneme organelles are usually associated with parasite motility and with recognition and binding to and invasion of host cells (1–3). To date, nine Eimeria tenella microneme proteins (E. tenella microneme-1 protein [EtMIC1] to EtMIC7 and AMA1 and AMA2) have been identified. As the first identified microneme protein in E. tenella (4), EtMIC1 has been studied by several research groups. Their work indicated that EtMIC1 could be secreted into the culture medium during cell invasion in vitro (5) and that it interacts with EtMIC2 (6). Liu et al. confirmed that EtMIC1 proteins are expressed in all developmental stages of E. tenella (7). A bioinformatics analysis showed that EtMIC1 is homologous to Toxoplasma gondii MIC2 (TgMIC2), which is comprised of a single von Willebrand factor A (vWA)–integrin-like A/I-domain and five thrombospondin (TSP) type 1 repeats (4). Identification by bioinformatics of a possible membrane-spanning region positioned before the C-terminal cytoplasmic tail suggests that EtMIC1 may be a transmembrane protein, but this has not yet been confirmed experimentally. The localizations of EtMIC1 in sporozoites, merozoites, and schizonts of E. tenella have not been reported yet due to a lack of appropriate molecular tools.

In the present study, we identified two EtMIC1 epitopes by using monoclonal antibodies (MAbs) developed in our previous study that specifically recognize EtMIC1 (MAbs 1-A1 and 1-H2) (7). We labeled the epitopes in the different developmental stages of E. tenella using these specific MAbs. Our results provide experimental evidence that EtMIC1 is a transmembrane protein and that it might be involved in the development of the E. tenella parasitophorous vacuole (PV).

RESULTS

Identification of epitopes recognized by MAbs 1-A1 and 1-H2.

We first aimed to identify the epitopes of EtMIC1 that are recognized by MAbs 1-A1 and 1-H2. These MAbs were each separated from ascites fluid with a high titer and specificity. To map the epitope position of MAb 1-A1 precisely, 11 overlapping fragments of the EtMIC1 gene were designed (Fig. 1A) and expressed successively in Escherichia coli BL21(DE3). Western blotting results show that MAb 1-A1 reacted with EtMIC1-1-1 and with fragments A-2-1, A-3-2, and A-4-2 (Fig. 1B), suggesting that the epitope recognized by MAb 1-A1 is located at amino acids 93 to 101. To identify the smallest sequence corresponding to the epitope to determine the precise position of the epitope, eight fragments were designed by removal of the amino acids from both ends of the sequence, one amino acid at a time (Fig. 1A). Western blotting results show that MAb 1-A1 reacted with fragments A-5-5, A-6-1, and A-6-2 but not with A-5-1, A-5-2, A-5-3, A-5-4, or A-6-3. Thus, based on the Western blotting results, the exact position of the epitope recognized by MAb 1-A1 was deduced to be ⁹¹LITFATRSK⁹⁹ in the EtMIC1 protein and named epitope I. Using the same approach, the exact position of the epitope recognized by MAb 1-H2 was deduced to be ⁶⁹⁸ESLISAGE⁷⁰⁵ in the EtMIC1 protein CTR domain and named epitope CTR (Fig. 1C).

FIG 1.

Identification of epitopes recognized by MAbs 1-A1 and 1-H2. (A) Summary of the design of overlapping EtMIC1 fragments. The accurate positions of the epitope recognized by MAb 1-A1 (red) and 1-H2 (green) were deduced to be ⁹¹LITFATRSK⁹⁹ and ⁶⁹⁸ESLISAGE⁷⁰⁵, respectively. (B) Western blot analysis using MAb 1-A1 as the primary antibody for various overlapping EtMIC1 fusion proteins expressed in E. coli BL21(DE3). (C) Western blot analysis using MAb 1-H2 as the primary antibody for various overlapping EtMIC1 fusion proteins expressed in E. coli BL21(DE3).

All amino acids of the ⁹¹LITFATRSK⁹⁹ and ⁶⁹⁸ESLISAGE⁷⁰⁵ epitopes are key residues.

To define the key residues contributing to the reactivity of the epitope to MAb 1-A1 or 1-H2, eight or seven mutagenesis epitope peptides with a glutathione S-transferase (GST) tag, respectively, were designed and expressed successively in E. coli BL21. The levels of reactivity of these mutagenesis epitope peptides to MAbs 1-A1 and 1-H2 were then examined. Western blotting performed with MAb 1-A1 or 1-H2 resulted in a strong signal for wild-type epitope I (Fig. 2A) or CTR (Fig. 2B), respectively, but no signal was observed for the mutagenesis epitope peptides. Because alanine substitution at any of the identified epitope amino acids in epitope I or CTR completely disrupted their interaction with MAb 1-A1 or 1-H2, all amino acids in these epitopes are likely crucial residues.

FIG 2.

Analysis of the epitope central sequence and alignment of the epitope peptide in different Eimeria species. (A) Western blot analysis using MAb 1-A1 as the primary antibody for alanine substitution mutants to determine the crucial residues of epitope I. M, protein marker; M-I-1-8, the mutagenesis epitope I peptides; A-6-1, positive control. (B) Western blot analysis using MAb 1-H2 as the primary antibody for alanine substitution mutants to determine the crucial residues of epitope CTR. M, protein marker; M-CTR-1-7, the mutagenesis epitope CTR peptides; H-5-1, positive control. (C) Sequence alignments of the epitope peptides in different Eimeria species that infect chickens. (D) Western blot analysis of the reactivity between the seven chicken Eimeria species epitope I peptide and MAb 1-A1. (E) Western blot analysis of the reactivity between the seven chicken Eimeria species epitope CTR peptide and MAb 1-H2.

The two MAbs recognized the epitope peptides of E. tenella but not those of six other chicken Eimeria species.

The amino acid sequences of epitopes in different chicken Eimeria species were analyzed. As shown in Fig. 2C, the I epitopes in E. tenella are similar to those in Eimeria necatrix or Eimeria mitis and Eimeria praecox, with only one amino acid difference, but the various versions of this epitope differed greatly across the seven Eimeria species. The amino acid sequence of epitope CTR is conserved in E. tenella, E. necatrix, E. mitis, and Eimeria acervulina and has only one amino acid substitution compared with the CTR epitopes in Eimeria brunetti, E. praecox, and Eimeria maxima. These results suggest that epitope I differs among different chicken Eimeria species, whereas epitope CTR is conserved in seven Eimeria species. To detect the reactivity of the seven chicken Eimeria epitopes to MAb 1-A1 or 1-H2, seven epitope peptides with a GST tag were expressed successively. The levels of reactivity of these epitope peptides to MAbs 1-A1 and 1-H2 were detected by Western blotting. The results showed that the two MAbs reacted with the E. tenella epitope peptides but not with other six chicken Eimeria species (Fig. 2D and E).

EtMIC1 is a transmembrane protein.

The results of a bioinformatics analysis showed that epitope I is located in the I-domain (a domain outside the sporozoite cell membrane) and that epitope CTR is situated in the CTR domain (a domain inside the cytoplasm). To confirm that EtMIC1 is a membrane protein, two MAbs were applied to detect the localization of EtMIC1 in sporozoites. When sporozoites were incubated with MAb 1-A1 under nonpermeabilizing conditions, EtMIC1 exhibited a distribution pattern throughout the membranes of sporozoites (in phosphate-buffered saline [PBS]) (Fig. 3A, panel a; apical). However, epitope CTR could not be labeled by staining with its specific MAb (1-H2) (Fig. 3A, panel b). When incubated with MAb 1-A1 or 1-H2 under permeabilizing conditions, the sporozoites were labeled on the apical end of the parasites (Fig. 3A, panels c and d). These results suggest that epitope CTR is cytoplasmic and that EtMIC1 is a transmembrane protein.

FIG 3.

EtMIC1 localization in parasites as visualized by indirect immunofluorescence analysis. (A) Sporozoites (in PBS) were immunostained with MAbs 1-A1 (a) and 1-H2 (b) under nonpermeabilizing conditions and with 1-A1 (c) and 1-H2 (d) under permeabilizing conditions. (B) Analysis of the EtMIC1 localization details in parasites immunostained with MAb 1-A1 (a to h), visualized with FITC (green), and counterstained with DAPI (blue). (a) Unpermeabilized sporozoites (SZ; incubated in DMEM). (b) Unpermeabilized second-generation merozoites (MZ). (c) Unpermeabilized immature second-generation schizonts (iSC). (d) Unpermeabilized mature second-generation schizonts (mSC). (e) Permeabilized SZ. (f) Permeabilized MZ. (g) Permeabilized iSC. (h) Permeabilized mSC. (C) Analysis of the EtMIC1 localization details in parasites immunostained with MAb 1-H2. (a) Unpermeabilized SZ (incubated in DMEM). (b) Permeabilized SZ (incubated in DMEM).

The localizations of EtMIC1 in sporozoites, second-generation merozoites, immature second-generation schizonts, and mature second-generation schizonts of E. tenella were also examined by staining with MAb 1-A1 (Fig. 3B). When sporozoites were incubated in culture medium, EtMIC1 protein expression was increased and the proteins were labeled by MAb 1-A1 on the surface of sporozoites (Fig. 3B, panel a). EtMIC1 also exhibited a distribution pattern throughout the membrane of second-generation merozoites (Fig. 3B, panel b). Because the EtMIC1 proteins are located in the microneme, the fluorescence intensity increased at the apical end of sporozoites (Fig. 3B, panel e) and second-generation merozoites (Fig. 3B, panel f) under permeabilizing conditions. The immature second-generation schizonts and mature second-generation schizonts were labeled with MAb 1-A1. The results showed that EtMIC1 could be labeled on the surface of immature second-generation schizonts (Fig. 3B, panels c and g) and mature second-generation schizonts (Fig. 3B, panel d). It was also labeled on the membrane of merozoites in mature second-generation schizonts under permeabilizing conditions (Fig. 3B, panel h). Additionally, the sporozoites could be successfully immunostained by MAb 1-H2 during incubation in culture medium under nonpermeabilizing conditions (Fig. 3C, panel a) or permeabilizing conditions (Fig. 3C, panel b), which suggested that the full-length EtMIC1 protein could be secreted to the outer side of the sporozoites.

EtMIC1 is involved in the formation of the PV.

MAb 1-A1 was used to label EtMIC1 during an infection of DF1 cells performed with E. tenella sporozoites. At 0.5 h postinfection, the sporozoites penetrated the host cell, and the EtMIC1 protein exhibited a homogenous distribution pattern throughout the sporozoite membranes (Fig. 4A). EtMIC1 was immunostained by MAb 1-A1 on the PV membrane at 4 h (Fig. 4C and E) and 36 h (Fig. 4D) postchallenge. No immunofluorescence was observed on the sporozoites that were labeled without primary antibodies (Fig. 4B).

FIG 4.

Immunofluorescence analysis of EtMIC1 localization in E. tenella-infected DF-1 cells. Details of parasites immunostained with MAb 1-A1 are indicated. Intracellular sporozoites were subjected to immunofluorescence labeling using MAb 1-A1 as the primary antibody at 0.5 h postchallenge (A), 4 h postchallenge (C and E), or 36 h postchallenge (D). (B) Intracellular sporozoites were subjected to immunofluorescence labeling without primary antibody as a control. hpi, hour postinfection.

Protective efficacy of recombinant EtMIC1 protein.

The efficacy of immunization was evaluated on the basis of body weight gain, lesion score, and oocyst shedding. No chickens in any groups died following an E. tenella challenge. With regard to body weight gain, cecal lesion score, and oocyst shedding results (Table 1), there were no significant differences among the epitope I, I-domain, and EtMIC1 vaccine groups, but all of these groups showed better results than the members of the nonimmunized challenged (PBS-II) group. The anticoccidial index (ACI) value was calculated for each group, and the results are summarized in Table 1. The ACI calculated for epitope I group (ACI: 162.01) is similar to that calculated for the I-domain immunization group (ACI: 164.85) but lower than that calculated for the EtMIC1 immunization group (ACI: 168.69). Together, these results show that the epitope I peptide is capable of moderate activity against challenge with 10,000 E. tenella oocysts and suggest that epitope I may be the key antigenic epitope of the I-domain.

TABLE 1.

Protective effect of the epitope peptide, I-domain, and EtMIC1 against oocysts of E. tenella infection in chicken^a

Group	Avg body wt gain (g)	Oocyst shedding (×106/g)	Lesion score	ACI
I-domain	117.9 ± 2.97c	2.910 ± 0.44a	1.93 ± 0.26a	164.85
Epitope I	115.0 ± 3.35bc	3.277 ± 0.10a	1.88 ± 0.23a	162.01
Epitope CTR	102.6 ± 3.69a	5.12 ± 0.21b	2.63 ± 0.18b	134.76
MIC1	120.3 ± 4.73c	2.76 ± 0.28a	1.63 ± 0.18a	168.69
GST	98.16 ± 4.54a	7.91 ± 0.17c	2.75 ± 0.16b	110.04
PBS-II	97.65 ± 3.19a	8.17 ± 0.11c	2.88 ± 0.23b	108.34
PBS-I	126.6 ± 3.53c	0	0	200

^aThe PBS-I group was immunized with PBS without E. tenella challenge; the PBS-II group was immunized with PBS and challenged with E. tenella. Values with different letters in same column are significantly different (P < 0.05). Levels of activity are ranked as follows: excellent activity, ACI > 180; moderate activity, 179 > ACI > 160; limited activity, 159 > ACI > 120; nonactivity, ACI < 120.

To evaluate the humoral immune and cell-mediated immune responses stimulated by the recombinant epitope peptides, the serum antibody levels and blood lymphocyte (BL) proliferation ability and subpopulations were measured. At 0 days postchallenge, the serum antibody levels in the epitope I, I-domain, and EtMIC1 groups were significantly (P < 0.01) higher than those in the other groups, and the serum antibody levels continued to increase significantly at 5 days and 10 days postchallenge (P < 0.01) (Fig. 5A). These results indicate that epitope I peptides could induce a relatively high level of serum antibody that might partially explain their protective effect against the symptoms of E. tenella infection, whereas epitope CTR may be a nonprotective epitope. The results of the proliferation ability and blood lymphocyte subpopulation assays show that the epitope I, I-domain, and EtMIC1 groups had proliferation responses to concanavalin A (ConA) that were significantly higher than those seen with the other groups (Fig. 5B) and that the CD4⁺ blood lymphocyte percentages were higher in these groups (Fig. 5C). The percentage of CD8⁺ lymphocytes was lower in the epitope I and I-domain groups but was significantly higher in the EtMIC1 group than in the other groups at 0 days postchallenge (Fig. 5D).

FIG 5.

Effects of recombinant protein vaccines on the serum antibody level and blood lymphocyte proliferation and subpopulations. (A) The level of serum antibody IgG was detected by ELISA at 0 days, 5 days, and 10 days postchallenge (d P.I.). Horseradish peroxidase-conjugated mouse anti-chicken IgG was used as the secondary antibody, and the OD₄₅₀ value was determined. (B) Blood lymphocyte responses to ConA in vitro on day 0 postchallenge. PBLs were seeded into 96-well microtiter plates with 100 μl of culture medium containing 10 μl of ConA (0.5 mg/ml) and were then incubated in 5% CO₂ at 37°C for 60 h. A Cell Counting Kit-8 assay kit was used for the detection of lymphocyte proliferation, and the OD₄₅₀ was measured. (C) The percentage of CD4⁺ cells in the blood. (D) The percentage of CD8⁺ cells in the blood. PBLs were incubated with mouse monoclonal antibodies against chicken CD4⁺ and CD8⁺ cells at 4°C for 1 h and were then analyzed using flow cytometry. Each bar represents means ± standard deviations (SD) (n = 3), and each bird was analyzed in triplicate. *, P < 0.05 (representing the difference revealed by comparisons to the PBS-I group).

DISCUSSION

An epitope is the smallest unit recognized by the immune system. It usually consists of 5 to 22 amino acids, and it plays an important role in protein function. The amino acid residues of epitopes are usually conserved, but they can be variable depending on the function of the protein and the internal environment. When exposed to diversified and multivariant living environments, some epitope amino acids may change to adapt to variable environments, thus resulting in a corresponding change in the function or antigenicity of proteins, such as escape from antibodies or a low level of cross-reaction by antibodies directed against the homologous epitope in different serotypes or species (8, 9). For example, previous work found that mutations in the A¹⁷⁵, P¹⁷⁶, and S¹⁷⁸ residues of epitope ¹⁷³LPAPTS¹⁷⁸ (10) and in the I⁷⁷ of epitope ⁷⁵GEIILT⁸⁰ (11) in the VP1 protein, which is the most external and immunodominant capsid protein, of duck hepatitis A virus (DHAV) greatly decreased cross-reactions among different serotypes. Su et al. speculated that mutations in the neutralizing epitopes of the porcine epidemic diarrhea virus (PEDV) S protein may represent the evolution of viral escape from antibodies (12). Additionally, Tkaczyk et al. reported that the MEDI4893 epitope of alpha toxin (AT) is highly conserved due in part to its role in AT pore formation (13). In the present study, epitope I (⁹¹LITFATRSK⁹⁹) of EtMIC1 was found to vary greatly among species of Eimeria that infect chickens. This may because epitope I is located in the extracellular domain of EtMIC1, which faces and interacts directly with the various living environments of different host cells. The other epitope investigated in this work, epitope CTR (⁶⁹⁸ESLISAGE⁷⁰⁵), is located within the cytoplasmic tail, which interacts with parasite aldolase and activates the actinomyosin system (14, 15), and is highly conserved across Eimeria species.

Thrombospondin-related anonymous protein (TRAP), which was first discovered in Plasmodium falciparum in 1988 (16), is considered to be a type I integral membrane protein (17). Homologous proteins, such as TgMIC2, EtMIC1, NcMIC2, and BbTRAP2, were later identified in other apicomplexan parasites (4, 18–20). Plasmodium TRAP protein is stored within the Plasmodium microneme and is released onto the cell surface following contact with a host cell, after which it is translocated to the posterior of the sporozoite during penetration (17). Here, we found that the TRAP homologous protein EtMIC1 in E. tenella could be substained on the apical membrane of sporozoites (incubated in PBS) when the parasites were immunostained, under nonpermeabilizing conditions, by MAb 1-A1 but not by MAb 1-H2. The peripheral contours of sporozoites (incubated in culture medium) and merozoites were clearly labeled following the staining of EtMIC1 epitope I with its specific MAb. These results suggest that epitope I is located on the outer side of the sporozoite surface membrane, whereas epitope CTR is located on the inner side of the cytoplasm, thus providing evidence that EtMIC1 is a transmembrane protein.

As surface proteins, the TRAP family proteins are considered to be parasite cell surface ligands that are essential for gliding motility and for adhesion to and invasion of host cells (21–23). The Toxoplasma homologue TgMIC2 was reported to bind to heparin specifically and to play an important role in Toxoplasma invasion of host cells (17, 24). During host cell attachment, TgMIC2 is initially secreted at the apical end of parasites and is progressively capped behind the moving junction. At the end of host cell invasion, the TgMIC2 proteins are no longer detectable on either the parasite membrane or the host cell surface (25). Those findings suggest that the TgMIC2 protein is mainly related to the adhesion and invasion of T. gondii. In the present study, EtMIC1 was found to be secreted at the apical end of sporozoites at 0 h postinfection, after which it was observed throughout the whole sporozoite membrane following incubation in culture medium. This finding is consistent with those pointing to the subcellular localization of TgMIC2. Interestingly, EtMIC1 was easily detected on the sporozoite surface after penetrating the host cell. Our data suggest that EtMIC1 not only participates in the process of invasion, similarly to TgMIC2 in T. gondii, but also might play an important role in parasite intracellular survival.

The PV is an interface between the parasite and its host cell; it plays key roles in parasite multiplication, nutrient acquisition, waste product disposal, and host immune response evasion (26, 27). After parasites push themselves forward into host cells, the PV forms through the invagination of the host plasma membrane, and sporozoites replicate in the PV during the intracellular cycle (28–30). To render the PV incapable of destruction by the endosome/lysosome and evade host immune responses, host transmembrane proteins are eliminated, and numerous parasite proteins are secreted into the PV membrane (31, 32). It has been reported that dense granule proteins (GRA proteins) play a major role in T. gondii PV development (33). Several GRA proteins associated with the PV, such as GRA3, GRA5, GRA14, GRA17, GRA23, and GRA33, have been identified (34–39). In Plasmodium species, a limited number of PV membrane proteins, including exported protein 1 (EXP1), early transcribed membrane protein (ETRAMP), Plasmodium translocon for exported proteins (PTEX), and serine/threonine protein phosphatase UIS, have been identified (40, 41). Shi et al. (42) studied the dynamic development of PV of E. tenella; however, there are no reports about the composition of E. tenella PV. Here, we found that EtMIC1 was secreted into the PV membrane at 4 h postchallenge and was then exhibited on the circle of second-generation schizonts. These results suggest that the EtMIC1 protein might play an important role in E. tenella PV formation and might be associated with sporozoite intracellular development.

Epitopes are the modules within antigens to which an antibody specifically binds. They are short amino acid sequences in a protein that can sometimes induce a more direct and potent immune response against pathogen infection (43, 44) than the whole cognate protein (45). Immunization with an epitope peptide or passive immunization with MAbs that specifically recognize the chicken coccidium protein was found to be capable of inducing partial immunity against chicken coccidiosis (46–49). These findings demonstrate the important role of epitopes in immunity against infection by Eimeria species. In the present study, the protective efficacy of two epitope peptides against E. tenella infections was evaluated. The results indicate that epitope I displayed higher immunogenicity and protective efficacy, as demonstrated by higher body weight gains and significantly lower fecal oocyst shedding levels and lesion scores along with higher serum IgG and lymphocyte proliferation levels and a higher percentage of CD4⁺ lymphocytes. Previous work has shown that the A/I domain is likely optimally positioned to interact with cell receptors (50). Epitope I (located in the I-domain) could induce protection against an E. tenella challenge by inducing a high level of serum IgG antibodies. These data suggest that epitope I is the key amino acid residue of the EtMIC1 I-domain and may be associated with sporozoite invasion.

In conclusion, we identified two epitopes of EtMIC1 and examined the key amino acid residues of those epitopes. Epitope I, located within a domain outside the sporozoite cell membrane, varies among chicken Eimeria species. In contrast, epitope CTR is situated within a domain inside the cytoplasm and is conserved among chicken-infecting Eimeria species. EtMIC1 may play a role in PV formation and in E. tenella development. Notably, because epitope I is the key EtMIC1 epitope, it has protective efficacy similar to that of the I-domain.

MATERIALS AND METHODS

Ethics statement.

The study protocol and all associated animal studies were approved by the Animal Care and Use Committee of Shandong Agricultural University, Tai’an, China (SCUC permission no. SDAUA-2015-015).

Strains, parasites, and experimental animals.

PGEX-6p-1 (TaKaRa) vectors were used to obtain a recombinant GST-tagged protein. The PET30a-EtMIC1 plasmid (stored in our laboratory) was used to express His-tagged EtMIC1 protein and used as the template for PCR. Escherichia coli Top10 strains (Invitrogen, Carlsbad, CA, USA) were used as the host for recombinant plasmids in the cloning experiment, and E. coli BL1(DE3) strains (Invitrogen) were used for the protein expression assay. MAbs (1-A1 and 1-H2) that specifically recognize EtMIC1 protein were prepared in our laboratory (7). Yeast cells displaying EtMIC1 (stored in our laboratory) were used to test the specificity of MAbs separated from fluid by yeast surface display assay.

Wild-type E. tenella strain SD-01 was stored in our laboratory (7). Propagation and purification of sporulated oocysts were performed as previously described (51). Sporozoites were prepared from sporulated oocysts and were purified by chromatography over columns packed with nylon wool and DE-52 cellulose. Second-generation merozoites and schizonts were collected from ceca at 120 h postchallenge.

One-day-old male Hy-Line variety brown layer chickens (Dongyue Poultry, Tai’an, China) were reared in isolated cabinets under coccidian parasite-free conditions and provided with coccidiostat-free feed and water. BALB/c female mice aged 4 to 5 weeks were purchased from Vital River Laboratories (Beijing, China).

Preparation of ascites fluid in BALB/c mice.

To obtain high-titer MAbs, ascites fluid was prepared from BALB/c mice. Ten BALB/c mice were injected intraperitoneally with 0.5 ml of liquid paraffin. At 7 to 10 days later, approximately 10⁶ positive hybridoma cells were injected into sensitized mouse peritoneal cavities. Ascites fluid containing abundant MAbs was then obtained and stored at −80°C for later use. The titer and specificity of MAbs were detected by enzyme-linked immunosorbent assay (ELISA), Western blotting, and yeast surface display assays following methods that were described previously (52).

Identification of accurate linear epitope positions.

To map the linear epitope recognized by MAb 1-A1, GST-tagged EtMIC1 fragments were designed and expressed in E. coli BL21. First, three overlapping fragments of EtMIC1 were amplified with specific primers (see Table S1 in the supplemental material) to construct the recombinant plasmids PGEX-EtMIC1-1-1, PGEX-EtMIC1-1-2, and PGEX-EtMIC1-1-3. After transforming these recombinant plasmids into E. coli BL21, the accurate position of the epitope recognized by MAb 1-A1 or 1-H2 was analyzed by Western blotting, utilizing MAb 1-A1 or 1-H2, respectively, as the primary antibody.

On the basis of the results of the Western blot detection assay described above, three fragments (A-2-1, A-2-2, and A-2-3) from the positive-testing region were designed for use in determining the epitope position (Table S1). These recombinant proteins were detected by Western blotting using MAbs as described above. Subsequently, two fragments (A-3-1 and A-3-2) were designed to determine the position of the epitopes (Table S1). Additionally, three fragments (A-4-1, A-4-2, and A-4-3) from the positive-testing region were designed to determine the position of the epitopes (Table S1). Finally, eight fragments (A-5-1, A-5-2, A-5-3, A-5-4, A-5-5, A-6-1, A-6-2, and A-6-3) were designed by deleting amino acids from both sides one by one until the accurate position of the epitopes was determined (Table S1). The linear epitope recognized by MAb 1-H2 was determined via the same method.

Identification of the central sequence of the epitope for MAb 1-A1 or 1-H2.

To identify the key amino acid residues for antigen binding to MAb 1-A1 or 1-H2, the epitope amino acids were substituted for alanine one by one. Sequences with BamHI and XhoI restriction sites were designed (Table S2). The 20-μl reaction volume contained 18 μl of PCR-grade water and 2 μl of primer mix (10 μM concentration per primer). After incubation of the products for 30 min at 60°C, the products were cloned into plasmid PGEX-6p-1 to construct recombinant plasmids PGEX-M-I-1, PGEX-M-I-2, PGEX-M-I-3, PGEX-M-I-4, PGEX-M-I-5, PGEX-M-I-6, PGEX-M-I-7, and PGEX-M-I-8 or PGEX-M-CTR-1, PGEX-M-CTR-2, PGEX-M-CTR-3, PGEX-M-CTR-4, PGEX-M-CTR-5, PGEX-M-CTR-6, and PGEX-M-CTR-7. The central sequence of the epitope was obtained by Western blotting as described above.

The conservation of the two epitopes was determined via the alignment of homogenous MIC1 proteins from different Eimeria species that infect chickens. The epitope peptides of seven chicken Eimeria species, fused with the GST tag, were expressed and analyzed by SDS-PAGE (data not shown). To identify the reactivity between the epitope peptides of the seven chicken Eimeria species and the MAbs, the GST tag recombinant plasmids were constructed as described above. The primers used are summarized in Table S3. The reactivity between recombinant peptide and MAbs was detected by Western blotting as mentioned above.

Localization of EtMIC1 by immunofluorescence.

Sporozoites were incubated for 40 min at 41°C in either PBS or complete Dulbecco’s modified Eagle medium (DMEM). Fresh sporozoites, second-generation merozoites, or second-generation schizonts were fixed with 4% paraformaldehyde on glass slides for 30 min. The samples were divided into two groups. One group was permeabilized with 1% Triton X-100–PBS for 10 min, whereas the other group was left unpermeabilized. After being blocked with 2.5% bovine serum albumin, the samples were washed three times with PBS and then incubated with MAb 1-A1 or 1-H2. After three washes with PBS, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG antibody (Sigma, USA) (1:200 dilution) was added to the samples with 10 μg/ml of DAPI (4′,6-diamidino-2-phenylindole) (Solarbio) and incubated for 1 h. After being washed with PBS, the cells were examined by fluorescence microscopy (Eclipse microscope; Nikon, Japan) or confocal microscopy (Leica; Germany). Samples labeled without primary antibodies were used as nonspecific binding controls.

DF-1 cells maintained on glass slides in a 12-well plate were infected by purified sporozoites. At 6 h postchallenge, the cells were washed with PBS to remove sporozoites that had not penetrated the host cells. The samples were fixed, permeabilized, and stained exactly as described above, using MAb 1-A1 as the primary antibody.

Active immunization with recombinant epitope peptide.

To determine the immunogenicity of the two epitopes identified in this study, the epitopes, epitope peptides, I-domain, and EtMIC1 GST-tagged recombinant protein were used as antigens to immunize chickens. The epitopes were administered in a series of six doses to increase the antigenicity. The I-domain fragment and full-length EtMIC1 with the signal peptide removed were amplified from the PET30a-EtMIC1 plasmid using specific primers and were then cloned into the PGEX-6p-1 plasmid. The positive-testing plasmids were transformed into E. coli BL21(DE3), and the GST-tagged recombinant protein was obtained by induction with isopropyl-β-d-thiogalactopyranoside (IPTG). After being purified using a ProteinIso GST resin kit (TransGen Biotech), the identity of the GST-tagged recombinant protein was confirmed by SDS-PAGE analysis. The protein concentrations were confirmed using a Bio-Rad protein assay kit (Bio-Rad, USA).

A total of 210 chickens were randomly divided into seven groups (30 chickens/group). The immune program was conducted as described previously (53). Briefly, at 7 days of age, the chickens were immunized subcutaneously with purified recombinant GST-tagged protein in Freund’s complete adjuvant (FCA; Sigma). PBS-I and PBS-II groups were immunized with PBS–FCA. At 7 days postimmunization, the chickens were administered a booster immunization of the purified recombinant protein in Freund’s incomplete adjuvant (FIA; Sigma). PBS-I and PBS-II groups were immunized with PBS–FIA. At 7 days after the secondary immunization, all the chickens, except for those in the PBS-I group, were administered 10,000 E. tenella oocysts by oral gavage.

Evaluation of protective efficacy.

The protective efficacy was evaluated based on the survival rate, body weight gain, oocyst count per gram of feces, cecal lesion score, and ACI. The survival rate was calculated by dividing the number of surviving chickens by the number of initial chickens in each group. The body weight gain of the chickens in each group was determined by comparing the weights measured at 10 days postchallenge and 0 days postchallenge. Oocyst output was estimated as described previously (54). The cecal lesion scores of the chickens (n = 8) were recorded at 6 days postchallenge as described previously by Chen et al. (53). The ACI was calculated for each group as described previously (53). The immunoprotective effect seen with each group was evaluated by the following standards: ACI of >180 indicated excellent activity, ACI of 160 to 179 indicated moderate activity, ACI of 120 to 159 indicated limited activity, and ACI of <120 indicated no activity.

Serum antibody levels.

At 0 days and 5 days postchallenge, the serum antibodies of three chickens per group were detected by ELISA as described previously (53). Briefly, the IgG level was estimated using ELISA plates coated with recombinant proteins (500 ng/well), along with horseradish peroxidase-conjugated (HRP) rabbit anti-chicken IgG (Sigma) as the secondary antibody. All the samples were analyzed in triplicate, and the optical density at 450 nm (OD₄₅₀) was determined using an automated microplate reader (Biotek, USA).

Blood lymphocyte proliferation assay and subpopulation assessment.

Peripheral blood lymphocytes (PBLs) were isolated from three chickens per group at 7 days after the secondary immunization using lymphocyte separation medium (Solarbio, USA). The isolated PBLs were then seeded into 96-well microtiter plates at 2 × 10⁵ cells per well in triplicate, with 100 μl of 1640 medium containing 5 μg of ConA (Sigma). After incubation in 5% CO₂ at 37°C for 60 h, 10 μl of Cell Counting Kit-8 assay kit (Slarbio, China) solution was added to each well. Following incubation for 2 h at 37°C, the OD₄₅₀ was measured using an automated microplate reader (Biotek).

To measure the percentages of CD4⁺ and CD8⁺ cells among the CD3⁺ cells, the PBLs were incubated with phycoerythrin (PE)-conjugated mouse anti-chicken CD3 antibody (Southern Biotech, USA) and FITC-conjugated mouse anti-chicken CD4 antibody (Southern Biotech) or FITC-conjugated mouse anti-chicken CD8α antibody (Southern Biotech) at 4°C for 1 h. After being washed with PBS, the PBLs were subjected to fluorescence-activated cell sorter (FACS) analysis (FACSC 2000; BD).

Statistical analyses.

The data are presented as means ± standard deviations. Statistical analyses were performed using the GraphPad Prism statistical software package for Windows, version 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). P values of <0.05 were considered to indicate statistically significant differences.