Identification of common immunodominant antigens of Eimeria tenella, Eimeria acervulina and Eimeria maxima by immunoproteomic analysis

Clinical chicken coccidiosis is mostly caused by simultaneous infection of several Eimeria species, and host immunity against Eimeria is species-specific. It is urgent to identify common immunodominant antigen of Eimeria for developing multivalent anticoccidial vaccines. In this study, sporozoite proteins of Eimeria tenella, Eimeria acervulina and Eimeria maxima were analyzed by two-dimensional electrophoresis (2DE). Western bot analysis was performed on the yielded 2DE gel using antisera of E. tenella E. acervulina and E. maxima respectively. Next, the detected immunodominant spots were identified by comparing the data from MALDI-TOF-MS/MS with available databases. Finally, Eimeria common antigens were identified by comparing amino acid sequence between the three Eimeria species. The results showed that analysis by 2DE of sporozoite proteins detected 629, 626 and 632 protein spots from E. tenella, E. acervulina and E. maxima respectively. Western bot analysis revealed 50 (E. tenella), 64 (E. acervulina) and 57 (E. maxima) immunodominant spots from the sporozoite 2DE gels of the three Eimeria species. The immunodominant spots were identified as 33, 27 and 25 immunodominant antigens of E. tenella, E. acervulina and E. maxima respectively. Fifty-four immunodominant proteins were identified as 18 ortholog proteins among the three Eimeria species. Finally, 5 of the 18 ortholog proteins were identified as common immunodominant antigens including elongation factor 2 (EF-2), 14-3-3 protein, ubiquitin-conjugating enzyme domain-containing protein (UCE) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In conclusion, our results not only provide Eimeria sporozoite immunodominant antigen map and additional immunodominant antigens, but also common immunodominant antigens for developing multivalent anticoccidial vaccines.


INTRODUCTION
Avian coccidiosis, a major parasitic disease of chickens worldwide, was caused by intestinal infection of Eimeria spp. [1]. It causes reduction in weight gain and poor feed-conversion, and death of the chickens, leading to an estimated annual economic loss of more than US$3 billion to the global poultry [2,3]. The species of E. tenella, E. acervulina and E. maxima are the most important in terms of global disease burden and economic impact [2,4].
Present control strategy against this disease relies on anticoccidial drugs and live vaccines containing virulent or attenuated strains of Eimeria [5]. However, chemical residues, emergence of drug-resistant parasites and the high cost associated with the development of new drugs results in serious problems. Moreover, the live vaccines have inherent production limitations, risk of vaccinal pathogenicity as well as the potential reversion to a pathogenic form, and cost issues [2,6,7]. Thus, new vaccines containing either defined immunodominant antigens or based on recombinant DNA technology have

Research Paper
been or are being developed [2,8,9]. Clinical coccidiosis is mainly caused by co-infection with multiple species of Eimeria [10,11], hence, a practical novel anticoccidial vaccine should contain the common antigens among Eimeria or antigens from multiple Eimeria species. Therefore, exploring immunodominant antigens, especially common antigens of Eimeria, is essential for developing novel vaccine against the simultaneous infection clinically.
Here, we described immunoproteomic analysis of Eimeria tenella, Eimeria acervulina and Eimeria maxima. A batch of immunodominant antigens was identified, with 33, 27 and 25 found in E. tenella, E. acervulina and E. maxima, respectively. Eighteen ortholog proteins and 5 common immunodominant antigens across the three Eimeria species were identified. Our results provide additional immunodominant antigens and common antigens for the development of multivalent vaccines against Eimeria.

Sporozoite 2DE gel profile of E. tenella, E. acervulina and E. maxima
The separation by 2-DE of 400 μg solubilized sporozoite proteins detected 629, 626 and 632 spots of E. tenella, E. acervulina and E. maxima, respectively. Most spots were located between 13 and 140 kDa ( Figure 1). Analysis with ImageMaster 2D Platinum (Version 5.0, GE Amersham) revealed 22 spots shared among all these species.

Detection of immunodominant spots by Western blot
Sporozoite 2DE gels of E. tenella, E. acervulina and E. maxima were analyzed by western blot using the corresponding antisera of these Eimeria species separately. Western blot profiles of the 2DE gel were shown in Figure 2. Immunodominant spots were observed on the western blot profiles of the three Eimeria species. Comparison with ImageMaster 2D Platinum revealed that 50 (E. tenella), 64 (E. acervulina) and 57 (E. maxima) immunodominant spots had high similarity between the 2DE gel profile and western blot profile. When the same western blot was probed with sera from negative control chickens, no proteins were detected ( Figure 3).

Immunodominant proteins analysis and identification using NCBI and Uniport database
All the immunodominant spots (171) detected by western blot were analyzed by MALDI-TOF-MS/MS. The obtained peptide mass fingerprint dates were submitted to MASCOT Sequence Query server (http://www. matrixscience.com) for identification against nonredundant NCBI database (http://www.ncbi.nlm.nih.gov/BLAST) and the uniprot database (http://www.uniprot.org/). Identification required a MASCOT confidence interval of 95%. As shown in Table 1, 112 spots were identified in the databases as corresponding to 85 Eimeria proteins, including 33 of E. tenella, 27 of E. acervulina and 25 of E. maxima. Fifty-four out of the 85 immunodominant proteins were 18 kinds of ortholog proteins among the three Eimeria species. Table 2 showed amino acids similarity of the 18 ortholog proteins between the three Eimeria species. All the ortholog proteins shared sequence similarity of more than 63% between the three Eimeria species except peroxiredoxin. Five of the ortholog proteins even shared sequence similarity of more than 93% between the three Eimeria species, namely, elongation factor 2 (EF-2), 14-3-3 protein, ubiquitin-conjugating enzyme domaincontaining protein (UCE), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transhydrogenase. Therefore, the five proteins were identified as common immunodominant antigens among the three Eimeria species. Since there were no matched proteins in the database, 59 spots were not identified successfully.

DISCUSSION
The immunity elicited by infections with Eimeria is species specific and an effective recombinant vaccine should include common protective antigens among several Eimeria species [12][13][14]. Some researchers have reported several Eimeria common antigens. Talebi reported a conserved immunodominant protein band (45 kDa) among sporulated oocysts of five Eimeria species (E. acervulina, E. maxima, E. necatrix, E. praecox and E. tenella) recognized by chicken anti-E. maxima serum. Sasai and colleagues reported a common antigen present on the apical complex of all chicken Eimeria sporozoites [15]. Constantinoiui and colleagues reported a highly conserved apical antigen among Eimeria species [13]. However, the reported common antigens are not specific. In the present study, we identified at least 5 specific Eimeria common immunodominant antigens by immunoproteomic analysis. Our research provided additional candidate common antigens for developing multivalent vaccines against simultaneous infection by several Eimeria species.
Since antibodies could confer protective immunity against Eimeria [17][18][19][20], in addition, the induced antibodies are relatively long lasting and easy for collection [13,17,21]. Therefore, in some previous reports, Eimeria antisera were used to identify immunodominant Eimeria antigens. For example, Réfega et al. obtained a total of 119 cDNA clones by immunoscreening E. tenella libraries using intestinal antibodies [22]. Laurent et al. screened a 19-kilodalton antigen present in several Eimeria species using sera raised to E. acervulina or E. tenella [23]. Some immunodominant antigens were screened from cDNA libraries by corresponding Eimeria antisera,   E. maxima. Soluble proteins (400 μg) from sporozoite of the three species were resolved by IEF over a broad, non-linear pH 3, 10 range followed by molecular mass on a 12.5% w/v acrylamide gel under denaturing conditions. Protein spots are visualized using silver stain. such as TA4 [24], LPMC-61 [25], rhomboid proteins ETRH01 of E. tenella [26] and so on, and these identified immunodominant antigen were further demonstrated to be able to confer protection against Eimeria challenge [27,28]. Therefore, we also used the Eimeria antisera to screen the immunodominant antigens of Eimeria species and obtained 85 immunodominant proteins.
Some of the identified antigens have been demonstrated to be able to confer protection against Eimeria challenge, such as lactate dehydrogenase [32], microneme 2 [33], microneme 7 [34] and so on. Therefore, the immunodominant antigens identified in this study have the potential for conferring protection against Eimeria challenge.
In this study, most of the identified immunodominant antigens shared orthologous relationships across the three Eimeria species. Nearly all the ortholog proteins shared amid acid sequence similarity of more than 63%, furthermore, five proteins shared sequence similarity of more than 93% and were identified as common immunodominant proteins among the three Eimeria species. In addition, we compared the available amid acid sequence of the five identified common immunodominant proteins among 7 chicken Eimeria species, and found that nearly all the sequence similarity among the 7 Eimeria species were more than 90% except E. mitis UCE, indicating the 5 protein were highly conserved among 7 chicken Eimeria. Our further studies demonstrated the identified common antigen 14-3-3 protein and GAPDH could confer effective protection against challenge by several Eimeria species (unpublished data). Taken together, our data demonstrated that immunoproteomics screening could be an efficient approach for identifying common immunodominant proteins of Eimeria species. Biological functions of the five common antigens have been generally described in some protozoa. Eukaryotic elongation factor 2 plays crucial role in the elongation stage of mRNA translation in eukaryotes, by mediating the translocation of the ribosome relative to the mRNA after addition of each amino acid to the nascent chain [35,36]. The 14-3-3 proteins are a family of conserved regulatory molecules expressed in all eukaryotic cells. And play important roles in extensively regulatory processes, such as mitogenic signal transduction, apoptotic cell death, cell cycle control, and protein localization [37,38]. E. tenella 14-3-3 protein could interact with the telomerase RNA-binding domain of telomerase reverse transcriptase [39]. UCE is a member of the family of ubiquitin-conjugating (E2) enzymes characterized by the presence of a highly conserved ubiquitin-conjugating (UBC) domain. E2 enzymes are well-conserved in eukaryotes and involved in Ub/UBL-modification pathways, and play central roles in processes like regulating protein degradation, function, and localization, thereby controlling the biology of the eukaryotic cell [40]. GAPDH is a key glycolytic enzyme in the process of metabolism of coccidian, as several pathogenic protozoa entirely depend on glycolysis as the source of ATP in the host [41,42]. Transhydrogenase catalyses transhydrogenation between analogues of NAD(H) and NADP(H). A transhydrogenase was found to be located in the Eimeria refractile body and might function in relation with the ATP hydrolysis and respiration in the process of oocysts sporulation [43]. However, their specific biological functions in Eimeria need further studies.
In previous studies, the similarities of conserved or common antigens ranged from 70% to 99% [44][45][46]. In theory, the higher similarity a protein among several species is, the more conserved the protein is. Thus, we used a high threshold of 93% to define the common antigens. Certainly, we can use a lower threshold less than 93%. If so, more antigens would be identified as common antigens.
It has been reported that some of the five common immunodominant antigens were protective in protozoa and other parasites. Toxoplasma gondii 14-3-3 protein was proved to be a potential vaccine candidate against toxoplasmosis [47]. Leishmania elongation factor 2 was identified as T cell-stimulating antigen and might constitute potential vaccine candidates for leishmaniasis [48]. Elongation factor 1-Alpha was reported as protective antigen in Toxoplasma gondii and Cryptosporidium parvum [49,50]. GAPDH was proved to confer protection against Haemonchus contortus and Schistosoma mansoni [51,52]. Protozoal GAPDHs were suggested as a potential antiparasitic targets in Plasmodium falciparum [41,42], Leishmania mexicana [53], Trypanosoma brucei and Trypanosoma cruzi [54,55]. Chen et al. [56] reported that immunization with recombinant UCE induced protection against Taenia pisiformis. Our subsequent studies demonstrated that 14-3-3 protein and GAPDH could confer protection against coinfection of E. tenella, E. acervulina and E. maxima (unpublished data). Taken together, the five common immunodominant antigens could be selected as vaccine candidates against Eimeria.
We provided reference maps of sporozoite immunodominant proteins for E. tenella, E. acervulina and E. maxima. In some previous studies, sporozoites protein 2DE profiles of Eimeria have been reported [29,[57][58][59][60]. However, sporozoites immunodominant protein 2DE profiles were seldom reported. de Venevelles et al. analyzed the sporozoite 2DE map of E. tenella and detected approximately 50 immunodominant protein spots. However, they only identified a few of the immunodominant spots by mass spectrometry [29]. In this study, 50

Ethics statement
Animal experiments were conducted following the guidelines of the Animal Ethics Committee, Nanjing Agricultural University, China. All animal experiments were evaluated and approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University (approval number: 2012CB120750).

Chickens and parasites
New-hatched Hy-Line layer chickens (commercial breed W-36) were reared in sterilized wire cages under coccidian-free conditions and provided daily with coccidiostat-free feed and water until the end of experiment.
Oocysts of E. tenella, E. acervulina and E. maxima were propagated, harvested and sporulated using a previously described protocol [61], and then stored in 2% (w/v) potassium dichromate solution at 4°C no longer than 2 weeks. Purity of the parasites was determined by PCR based on the internal transcribed spacer-1 (ITS-1) as previously described [62,63]. Sporozoites of the parasites were harvested from sporulated oocysts by in vitro excystation and purified over nylon wool and DE-52 cellulose columns [61].

Antisera preparation
Three antisera were prepared by inoculation of twoweek-old chickens with pure coccidian. Chickens were orally inoculated 6 times at 3-day intervals with 1 × 10 4 sporulated oocysts of E. tenella, E. acervulina or E. maxima per chicken. Negative control birds were maintained under the same conditions and inoculated with distilled water. One week post the last inoculation, blood was collected from wing vein of the chickens. Subsequently, the sera were collected and determined by ELISA. A seventh even eighth dose would be given unless titers of the sera were beyond 1: 64. Sera were stored at −20°C for Western blot analysis. Meanwhile, serum was collected from uninfected chickens as negative control [21,64].
For 2DE, 400μg of sporozoite proteins were loaded onto analytical and preparative gels. The Ettan IPGphor Isoelectric Focusing System (GE Amersham) and pH 3-10 immobilized pH gradient (IPG) strips (13 cm, nonlinear; GE Healthcare) were used for isoelectric focusing (IEF). The IPG strips were rehydrated for 12 h in 250 μl of rehydration buffer containing the protein samples. IEF was performed in four steps: 30V for 12 h, 500 V for 1 h, 1000 V for 1 h, and 8000 V for 8 h. The gel strips were equilibrated for 15 min in equilibration buffer (50 mM Tris-HCl (pH 8.8), 6 M urea, 2% SDS, 30% glycerol, and 1% DTT). This step was repeated using the same buffer with 4% iodoacetamide in place of 1% DTT. The strips were then subjected to the seconddimensional electrophoresis after transfer onto 12.5% SDS-polyacrylamide gels. Electrophoresis was performed using the Hofer SE 600 system (GE Amersham). The 2DE was performed twice for each sample simultaneously, and one of obtained gels was used for immunoblot analysis, while the other one prepared for silver staining.

Western blot and image analysis
Proteins in the 2DE gel were transferred electrophoretically onto a 0.45 μm pore size polyvinylidene fluoride (PVDF) membrane (GE Healthcare, USA) for 2 h at 100V using a TE62 Tank Transfer Unit system (GE Healthcare, USA). Membranes were then blocked in 5% skim milk in PBS containing 0.05% Tween 20 (PBST) for 1h at room temperature with gently swinging. And then was incubated with anti-E. tenella, anti-E. acervulina and anti-E. maxima sera (1:100) for 2 h at 37°C. The uninfected chicken serum was used to test another membrane as a negative control. After frequent washing with PBST, the membrane were incubated with secondary antibody of Goat anti-chicken IgG (1:2000, PTG Inc., USA) for 2 h at 37°C. Finally, 3, 3′-diaminobenzidine (DAB, Sigma) was added to visualize the immunodominant protein spots, according to the manufacturer's instructions.
Blots were scanned using TyphoonTMFLA 9500 (GE Amersham, USA). Through ImageMaster 2D Platinum (Version 5.0, GE Amersham, USA), the spots on the membranes were matched to their orthologs in 2DE gels stained using a modified silver staining methods compatible with subsequent mass spectrometric analysis [66].

Two-dimensional gel excision, Tryptic Digestion, and Desalting
All the the immunodominant spots on the PVDF membranes were excised from 2D gels from the preparative gels. Subsequently, the protein spots were destained for 20 min in 30 mM potassium ferricyanide/100mM sodium thiosulfate (1:1 v/v) and washed with Milli-Q waters. The spots were incubated in 0.2 M NH 4 HCO 3 for 20 min and then lyophilized. Each spot was digested overnight in 12.5 ng/µl trypsin in 25 mM NH 4 HCO 3 . The peptides were extracted three times with 60% acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA). The extracts were pooled and dried completely by a vacuum centrifuge.

MS analysis of protein spot and database searches
MS analysis of protein spot was performed by APT (Applied ProteinTechnology co. ltd, Shanghai, China). MS and MS/MS data for protein identification were obtained by using a MALDI-TOF-TOF instrument (5800 proteomics analyzer; Applied Biosystems). Instrument parameters were set using the 4000 Series Explorer software (Applied Biosystems). The MS spectra were recorded in reflector mode in a mass range from 800 to 4000 with a focus mass of 2000. MS was used a CalMix5 standard to calibrate the instrument (ABI 4700 Calibration Mixture). For one main MS spectrum 25 subspectra with 125 shots per subspectrum were accumulated using a random search pattern. For MS calibration, autolysis peaks of trypsin ([M+H]+842.5100 and 2,211.1046) were used as internal calibrates, and up to 10 of the most intense ion signals were selected as precursors for MS/ MS acquisition, excluding the trypsin autolysis peaks and the matrix ion signals. In MS/MS positive ion mode, for one main MS spectrum 50 subspectra with 50 shots per subspectrum were accumulated using a random search pattern. Collision energy was 2 kV, collision gas was air, and default calibration was set by using the Glu1-Fibrino-peptide B ([M+H] + 1,570.6696) spotted onto Cal 7 positions of the MALDI target. Combined peptide mass fingerprinting PMF and MS/MS queries were performed by using the MASCOT search engine 2.2 (Matrix Science, Ltd.) embedded into GPS-Explorer Software 3.6 (Applied Biosystems) on the database of uniprot Eimeria or NCBI with the following parameter settings: 100 ppm mass accuracy, trypsin cleavage one missed cleavage allowed, carbamidomethylation set as fixed modification, oxidation of methionine was allowed as variable modification, MS/MS fragment tolerance was set to 0.4 Da. a GPS Explorer protein confidence index ≥ 95% were used for further manual validation.

Authors' contributions
SXK designed the study and critically revised the manuscript. LXR, YRF and XLX helped in the study design and analyzed the data. LLR performed the laboratory tests. HXM contributed to the preparation and purity identification of Eimeria species and wrote the draft. LJH, LWY and JYH contributed to the separation of the Eimeria sporozoites antigen. TD, TL and YXC contributed to the preparation of the antisera and western blot analysis. All authors read and approved the final version of the manuscript.