Molecular characterization and protective efficacy of a new conserved hypothetical protein of Eimeria tenella

Eimeria tenella is an obligate intracellular parasite that actively invades cecal epithelial cells of chickens. This parasite encodes a genome of more than 8000 genes. However, more than 70% of the gene models for this species are currently annotated as hypothetical proteins. In this study, a conserved hypothetical protein gene of E. tenella, designated EtCHP18905, was cloned and identified, and its immune protective effects were evaluated. The open reading frame of EtCHP18905 was 1053bp and encoded a protein of 350 amino acids with a molecular weight of 38.7kDa. The recombinant EtCHP18905 protein (rEtCHP18905) was expressed in E. coli. Using western blot, the recombinant protein was successfully recognized by anti GST-Tag monoclonal antibody and anti-sporozoites protein rabbit serum. Real-time quantitative PCR analysis revealed that the EtCHP18905 mRNA levels were higher in sporozoites than in unsporulated oocysts, sporulated oocysts and second-generation merozoites. Western blot analysis showed that EtCHP18905 protein expression levels were lower in sporozoites than in other stages. Immunofluorescence analysis indicated that the EtCHP18905 protein was located on the surface of sporozoites and second-generation merozoites. Inhibition experiments showed that the ability of sporozoites to invade host cells was significantly decreased after treatment with the anti-rEtCHP18905 polyclonal antibody. Vaccination with rEtCHP18905 protein was able to significantly decrease mean lesion scores and oocyst outputs as compared to non-vaccinated controls. The results suggest that the rEtCHP18905 protein can induce partial immune protection against infection with E. tenella and could be an effective candidate for the development of new vaccines.


Introduction
Avian coccidiosis in the poultry industry is a common disease caused by obligate apicomplexan parasites of the genus Eimeria. At present, coccidiosis has historically been controlled by anticoccidial drugs and live vaccines [34]. However, longterm prophylactic drug usage has promoted drug-resistance. As pressure to reduce drug use in poultry production intensifies industry-wide, novel vaccination strategies are needed. Hence, new strategies such as DNA vaccines and subunit vaccines have received widespread attention. Therefore, it is urgent to find novel immunoprotective antigens.
Eimeria tenella is one of the 7 recognized species of Eimeria that infect chickens. More than 8000 genes of E. tenella have been identified throughout the genome [30]. Significant progress has been achieved over the past few several years identifying E. tenella genes involved in development, differentiation, virulence, and susceptibility to therapy. However, the identification of most genes in the genome remains unknown [2,30]. The genome of the Houghton strain of E. tenella has been sequenced [30]. The data show that more than 70% of gene models are currently annotated as hypothetical proteins in E. tenella [2]. These conserved proteins may be important for invasion, development or the E. tenella life cycle. In 2016, Zhai et al. characterized the conserved protein EtCHP559 and studied the function and immunogenicity of EtCHP559 [47]. However, there are many conserved proteins that have neither been studied nor tested for their function.
In the present study, a new conserved hypothetical protein of E. tenella, EtCHP18905 (NCBI reference sequence accession number: XP_013231819), was cloned and recombinant protein GST-EtCHP18905 (rEtCHP18905) was expressed in an Escherichia coli BL21 (DE3) expression system. Polyclonal anti-rEtCHP18905 antibodies were generated and used to localize EtCHP18905 in parasites by immunofluorescence and to assess inhibitory effects in an in vitro assay. The results of the present study indicate that EtCHP18905 may participate in parasite invasion, growth and development.

Ethics considerations
All experiments involving animals were approved by the Institutional Animal Care and Use Committee of Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences (approval no. SHVRI-SZ-20180106-3), and were conducted in strict compliance following the recommendations outlined in the Guide for the Care and Use of Laboratory Animals.

Animals, parasites and cells
One-day-old Chinese Pudong yellow broilers were obtained from Shanghai Fuji Biological Technology Co., Ltd and reared in steel cages with a wire floor. Animals were provided with water and feed ad libitum. The birds were placed in a coccidiafree environment. BALB/c mice were purchased from Shanghai Lingchang Biological Technology Co., Ltd. New Zealand rabbits were obtained from Shanghai SLAC Laboratory Animal Co. Ltd.
Eimeria tenella (CAAS21111601) was obtained from the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The parasites were propagated by inoculating 2-week-old chickens, as previously described [40]. Unsporulated (UO) and sporulated oocysts (SO) were obtained and purified using standard procedures [35]. Sporozoites (Spz) were purified in vitro from cleaned SO [28]. Second-generation merozoites (Mrz) were collected from the cecal mucosa scraped from the cecum and the cecal contents of chickens at 115 h post inoculation (p.i.) and then purified with Percoll [35].
The chicken fibroblast cell line DF-1 (ATCC CRL-12203) was used for in vitro inhibition and immunofluorescence assays [19].

Cloning and sequence analysis of EtCHP18905
Total RNA was extracted from Spz using TRIzol reagent (TaKaRa, Tokyo, Japan) according to the manufacturer's protocol. Total RNA was extracted from 2.0 Â 10 7 Spz, counted with a hemocytometer using TRIzol reagent (TaKaRa), according to the manufacturer's protocol. RNA samples were resuspended in diethylpyrocarbonate (DEPC) treated water. Complementary DNA (cDNA) was generated from the total RNA with oligo dT primer and SuperScript™ III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA).

Expression and purification of the recombinant EtCHP18905 protein
A 1053-bp fragment of EtCHP18905 was amplified from the plasmid pGEM-T-EtCHP18905 with the primers: forward primer, 5 0 -GGAATTCATGGACCGAGACCGTCGCTC -3 0 ; reverse primer, 5 0 -GCGTCGACGC TGTGGGGGCTCG-GGTCG -3 0 , and ligated into the pGEX-4T-1 vector at the EcoRI and SalI cloning sites (underlined). The recombinant pGEX-4T-EtCHP18905 plasmid was transformed into E. coli BL21 (DE3) cells (Tiangen, Beijing, China). rEtCHP18905 expression in E. coli was induced by addition of 0.8 mM Isopropyl-b-D-1-thiogalactopyranoside (IPTG; Sigma, St Louis, MO, USA) to the bacteria culture after the OD 600 of the culture reached 0.6 at 37°C. The bacteria were collected by centrifugation at 8000 Âg for 10 min at 4°C. The bacteria were lysed by sonication and then the bacteria lysates were analyzed by 12% (w/v) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The rEtCHP18905 protein was purified by cut SDS polyacrylamide gel [3]. The concentration of the sample was determined using a BCA protein assay kit (Beyotime, Haimen, China). The protein was stored at À20°C for later analysis.

Generation of anti-rEtCHP18905 and anti-sporozoite polyclonal serum
The Spz proteins were prepared using sonication as described by Jiang et al. [19]. To generate polyclonal antibodies, either 50 lg or 200 lg of the purified rEtCHP18905 protein or the Spz protein was mixed with Freund's complete adjuvant (Sigma) in a 1:1 mixture and injected into six-week-old BALB/c mice or two-month-old New Zealand white rabbits, respectively. After two weeks, mice and rabbits were immunized with the same dose of antigen emulsified with Freund's incomplete adjuvant (Sigma). And then, the rabbits and mice were re-boosted four times at intervals of 1 week. Finally, the polyclonal antibody serum was collected and stored at À80°C until use. Pre-immune serum was collected from the rabbits' ear vein before immunization for further use as the negative control.
EtCHP18905 transcript levels in different developmental stages of E. tenella Transcription profiles of EtCHP18905 at different developmental stages of E. tenella (UO, SO, Spz, and Mrz) were determined using real-time quantitative PCR (qPCR). Total RNAs were isolated by TRIzol reagent (Invitrogen) from UO, SO, Spz, and Mrz. RNA preparations were treated with RNase-free DNase I (Invitrogen) to remove DNA contamination. In brief, 1.0 Â 10 7 purified UO or SO were oscillated and broken in 500 lL of TRIzol with equal volume of 710-1180 lm glass beads (Sigma) for 10 min (4000 rpm). A total of 2.0 Â 10 7 Spz or Mrz were lysed in 500 lL of TRIzol. Total RNAs were precipitated with isopropanol and washed with 75% ethanol and then resuspended in DEPC treated water. cDNA was synthesized with SuperScript II reverse transcriptase (Invitrogen) and random primers (Invitrogen). qPCR was performed with SYBR1 Green I dye (Takara) on a StepOne™ Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). qPCR primers for EtCHP18905 were: 5 0 -TCCC-CTCAAGCCCCTCATACAGT -3 0 (forward) and 5 0 -CCA-GCACTAAGTCCACTGAACGC -3 0 (reverse). A housekeeping gene of E. tenella, 18S ribosomal RNA, was used as the reference gene and was amplified using the primers 5 0 -TGTAGTGGAGTCTTGGTGATTC -3 0 (forward) and 5 0 -CCTGCTGCCTTCCTTAGATG -3 0 (reverse). The reactions for each sample were performed in triplicate, and the experiment was repeated three times. The transcription levels were quantified with the 2 ÀDDCt method [27].

EtCHP18905 protein expression in four development stages of E. tenella
Total proteins were prepared from four life cycle stages of E. tenella using a commercially available cell-lysis buffer for western blot and immunoprecipitation (Beyotime). Protein concentrations were determined with a BCA protein assay kit (Beyotime). The protein lysate from each sample was separated by SDS-PAGE and transferred to PVDF membrane (Merck Millipore). Membranes were blocked for 2 h with 5% (w/v) skimmed milk powder in PBS, followed by incubation with mouse polyclonal anti-rEtCHP18905 (1:100) and mouse monoclonal anti-a-tubulin (1:5000) at 37°C for 2 h, respectively. Secondary antibodies, HRP-conjugated Affinipure Goat anti-Mouse IgG (H + L) (1:5000) (Proteintech, Rosemont, IL, USA) were incubated at room temperature for 45 min, and bands were detected using ChemiDoc (Bio-Rad, Hercules, CA, USA). a-tubulin (Sigma) was used as an internal reference for protein extracts at each stage. For comparative quantitative protein expression profile analysis, the resulting images were analyzed by Image J (Rawak Software Inc., Stuttgart, Germany) software.

Localization of EtCHP18905 by indirect immunofluorescence
The location of EtCHP18905 in Spz, Mrz and parasites invaded DF-1 cells were assessed by indirect immunofluorescence assay (IFA) with anti-rEtCHP18905, as previously described [19]. Six-well plates (Corning Inc., Corning NY, USA) precoated with coverslips were seeded with DF-1 cells (2 Â 10 5 cells per well). These cells were sequentially cultured in complete medium (CM, DMEM with 10% fetal bovine serum and 100 units/mL penicillin/streptomycin) at 37°C and 5% CO 2 for 24 h. Freshly cleaned Spz (6 Â 10 5 parasites per well) were added to invade and develop in the DF-1 cells. The coverslips were collected and washed at 2, 48 and 72 h p.i., respectively. Subsequently, all the coverslips were fixed in 4% paraformaldehyde for 20 min, permeabilized with 1% Triton X-100 in PBS for 15 min, and then blocked with 2% bovine serum albumin in PBS overnight at 4°C. After washing four times, rabbit anti-rEtCHP18905 polyclonal antibody (1:100 dilution) with PBS was added for incubation at 37°C for 2 h. Then the goat anti-rabbit IgG fluorescein isothiocyanate (FITC)-conjugated antibody (1:500 dilution) (Sigma) was added, and the samples were incubated at 37°C for 1 h. Nuclei of parasites and cells were stained with 15 lg/mL 4, 6-diamidino-2-phenylindole (DAPI) (Beyotime) for 30 min at room temperature. At the end of each step, all the coverslips were washed three times in PBS. The coverslips were placed on glass slides using 60 lL of Fluoromount Aqueous Mounting Medium (Sigma) and observed by a laser scanning confocal microscope (Zeiss LSM800 microscope, Carl Zeiss, Germany). Spz were incubated in PBS or CM for 2 h at 41°C, and air dried on coverslips before fixation. Mrz were also incubated in PBS for observation. Spz or Mrz were evenly smeared onto glass coverslips to localize the protein in Spz and Mrz. After air drying, the coverslips were prepared for immunofluorescence using the method described above.

Invasion inhibition assays
Invasion inhibition assays were performed to investigate whether anti-rEtCHP18905 affects Spz invasion of DF-1 cells [19]. Rabbit IgG against rEtCHP18905 and GST protein were purified using protein A + G agarose (Beyotime) and the concentration of IgG was determined by a BCA Protein Assay Kit (Beyotime). DF-1 cells (3 Â 10 5 cells per well) were cultured in flat-bottomed 24-well plates (Corning) in CM at 37°C and 5% CO 2 for 12 h. Freshly cleaned E. tenella Spz were labeled for 15 min using carboxyfluorescein diacetate succinimidyl ester (CFDA SE) (Invitrogen), according to the manufacturer's protocol. Then, the labeled Spz were incubated with purified rabbit anti-rEtCHP18905 IgG at different concentrations (100, 200, or 300 lg/mL) for 2 h at 37°C and added to infect DF-1 cells at 41°C, 5% CO 2 for 12 h. Naïve rabbit serum IgG (Sigma, USA) and rabbit anti-GST IgG were used as the negative control and GST control. Labeled Spz incubated with no antibody were the positive control. The cells were washed, trypsinized, harvested, and analyzed on a flow cytometer (model Cytomics FC500; Beckman Coulter, Brea, CA, USA). All assays were performed in triplicate. The inhibition rate was calculated based on the invasion rate and the positive control [17].

Immunization experimental design
Broilers at 7 days of age were randomly divided into four groups and each group included 12 birds. Broilers were inoculated with a subcutaneous injection of 50 lg or 100 lg of purified rEtCHP18905 protein emulsified in Montanide ISA 71 adjuvant (Seppic, Puteaux, France) in a 3:7 mixture [18]. The challenged and unchallenged control birds were immunized with PBS emulsified in Montanide ISA 71 adjuvant. A booster immunization was given one week later with the same amount of components as the first immunization. Subsequently, 7 days after the last immunization, 1 Â 10 4 SO of E. tenella were given to all the birds except for the unchallenged control birds. Unchallenged control chickens were given PBS orally.

Evaluation of immune protection
The efficacy of immunization was evaluated by the average body weight gain, mean lesion scores, fecal oocyst output, and percentage reduction of oocyst excretion. Body weight was measured on days 0 and 8 post challenge. Fecal samples were collected daily from days 6 to 8 post challenge. Oocysts per gram of fecal sample were counted using a McMaster chamber [9]. The percentage reduction of oocyst excretion was calculated by the formula: (number of oocysts from the challenged-unvaccinated group À number of oocysts from the challenged-vaccinated group)/number of oocysts from the challenged-unvaccinated group Â 100% [31]. The ceca of each group were collected separately. Intestinal lesions were scored according to the method of Johnson and Reid [20].

Preparation of the serum
The blood of broilers in each group was collected on day 8 post challenge. For the serum IgG, cytokines, sCD4 and sCD8 determination, the sera were separated from isolated blood samples. In brief, the blood samples were incubated at 37°C for 1 h, and centrifuged at 1000 Âg for 5 min at 4°C to separate the serum.

Determination of serum antibody levels
The serum IgG against rEtCHP18905 levels were detected by ELISA at day 8 post challenge, as described previously [24]. Briefly, 96-well microtiter plates (Corning) were coated with purified rEtCHP18905 (10 lg/well) and incubated overnight at 4°C. After three washes with PBS-T, the plate was blocked with PBS containing 1% BSA for 2 h at 37°C, and then the plate was washed with PBS-T. The plates were incubated with the serum samples diluted 1:25 in PBS (50 lL/well) for 2 h at 37°C. After washing five times with PBS-T, the secondary antibodies of HRP-donkey-anti-chicken IgG antibody (50 lL/well) (Sigma) (1:5,000 dilution) was added and incubated for 2 h at 37°C. The plates were washed five times with PBS-T and developed with 3,3 0 ,5,5 0 -tetramethylbenzidine. Optical densities at 450 nm (OD450) were determined on a microplate spectrophotometer. All assays were performed in triplicate.

Statistical analysis
SPSS version 22 (SPSS, Chicago, IL, USA) was used to analyze body weight gain, mean lesion scores, and fecal oocyst output and oocyst reduction ratio. GraphPad Prism version 6.0 (GraphPad, La Jolla, CA, USA) was used to analyze real-time quantitative PCR (qPCR), invasion inhibition, and antibody and cytokine levels. Differences among groups were analyzed by one-way analysis of variance (ANOVA) and Duncan's multiple range test (p < 0.05 was considered significantly different). The lesion scores were compared by the nonparametric Kruskal-Wallis test.

Results
Characterization of the EtCHP18905 sequence By analysis of the sequence, the open reading frame (ORF) was 1053 bp and found to encode a protein of 350 amino acids with a predicted molecular mass of 38.7 kDa. Based on BLASTp analysis, the sequence obtained shared 100%, 84.68%, 52.48%,    (Fig. 1), respectively. Analysis of the amino acid sequence showed that a transmembrane domain (amino acid sequence 73-92) was found in the deduced protein, but no signal peptide was detected. Structural module and conservative structure predictions indicated that the protein contains seven casein kinase II phosphorylation sites, four protein kinase C sites, three N-glycosylation sites, three N-myristoylation sites, two cAMP and cGMP-dependent protein kinases phosphorylation sites and a tyrosine kinase phosphorylation site (Fig. 2).

Expression and purification of recombinant EtCHP18905
SDS-PAGE analysis showed that the recombinant protein was expressed in E. coli BL21 cells successfully and found mainly in the precipitate. After purification by cut SDS polyacrylamide gel, the expected protein band of 64.7 kDa (including with GST tag) (Fig. 3A) was observed in the SDS-acrylamide gel. Western blot analysis indicated that the recombinant protein was recognized by the anti-GST monoclonal antibody (Fig. 3B) and rabbit sera against Spz of E. tenella (Fig. 3C, lane 2). Normal rabbit serum failed to detect any protein of the expected molecular weight of rEtCHP18905 (Fig. 3C, lane 4).
Transcription of EtCHP18905 at different developmental stages of E. tenella qPCR results showed that the levels of EtCHP18905 mRNA in UO, SO and Mrz were similar (p > 0.05), which were significantly lower than those in the Spz (p < 0.05) (Fig. 4).

EtCHP18905 protein expression level in E. tenella
Western blot analysis results indicated that EtCHP18905 expression levels were higher in the UO and SO than Mrz and Spz stages, and EtCHP18905 expression levels were lowest in the Spz stage (Fig. 5).

Localization of EtCHP18905 during in vitro infection
Indirect immunofluorescence results showed that in Spz incubated with PBS, EtCHP18905 was distributed on the surface of Spz (Fig. 6A). EtCHP18905 uniformly distributed throughout the cytoplasm in whole Spz after incubation in CM (Fig. 6B). After Spz were added to DF-1 cells for 2 h and 48 h, EtCHP18905 was concentrated on the surface of Spz (Figs. 6C and 6D). After infection for 72 h, EtCHP18905 was evenly distributed in most areas of the parasite (Fig. 6E). Moreover, EtCHP18905 was primarily located on the surface of Mrz (Fig. 6F).

In vitro invasion inhibition assay
In vitro invasion inhibition assay results showed that the inhibition rate was 28% at an antibody concentration of 300 lg/mL (Fig. 7). Compared with naïve rabbit IgG and GST control groups, the inhibition effect after pretreatment with anti-rEtCHP18905 IgG was significant (p < 0.01). In contrast, naïve rabbit sera IgG and GST control groups did not have a significant effect on invasion by E. tenella Spz.

Protective efficacy of vaccination on E. tenella challenge
Body weight gain, cecal lesion scores, oocyst output and the percentage reduction of oocyst excretion are summarized in Table 1. The results showed that non-immunized challenged control groups exhibited significantly reduced weight gain compared with all immunized groups and the unchallenged control group chickens (p < 0.05) after challenge. The cecal lesion  scores of chickens immunized with rEtCHP18905 were significantly lower than those of the challenged control. The oocyst output was reduced after immunization with rEtCHP18905. Chickens immunized with rEtCHP18905 presented significantly higher percentage reduction of oocyst excretion compared with the challenged controls (p < 0.05).

IgG titers and cytokine, sCD4 and sCD8 concentrations in sera of immunized chickens
The results in Figure 8A show that serum from chickens immunized with rEtCHP18905 had significantly higher levels of IgG antibody (p < 0.001) compared with the challenged control group. sCD4 levels in chickens from the two immunized groups were not significantly higher (p > 0.05) than the challenged control group, but were significantly lower than the unchallenged group (Fig. 8B). No significant differences (p > 0.05) of sCD8 and IL-10 were observed between the immunized and unimmunized-challenged group (Figs. 8C and 8E). The IFN-c levels in the 100 lg rEtCHP18905-immunized group were higher but not significantly than challenged control (Fig. 8D). IL-17 100-lg-immunized group and TGF-b1 in chickens from the two immunized groups all showed significantly higher levels (p < 0.05) compared with the unchallenged group (Figs. 8F and 8G).

Discussion
In the present report, a new gene of conserved hypothetical protein from E. tenella was cloned and characterized. The 1053 bp ORF was shown to encode a 350 amino acid polypeptide of~38.7 kDa. Sequence analysis showed that the protein had no signal peptide, and amino acids 73-92 formed a transmembrane region. Given this observation, it is speculated that the protein is anchored to the membrane. Bioinformatics analysis predicted that the protein contains seven tyrosine kinase phosphorylation sites, four protein kinase C sites, three N-glycosylation sites, three N-myristoylation sites, two cAMP and cGMP-dependent proteins kinase phosphorylation sites, and one tyrosine kinase phosphorylation site. These sites and structures indicate that the function of this protein may be regulated by post-translational modifications. The BLASTp results showed that the deduced amino acid sequence of EtCHP18905 was 100% homologous to the E. tenella conserved hypothetical protein (NCBI reference sequence accession number: XP_013231819, GeneID: ETH_00018905) and 84% homologous to the E. necatrix conserved hypothetical protein (NCBI reference sequence accession number: XP_013438465). These results indicate that CHP in E. tenella and CHP in E. necatrix have high homology. Eimeria tenella and E. necatrix are the most pathogenic species among the species represented in the genus Eimeria, which cause severe tissue damage to the host intestine [33]. In addition, the EtCHP18905 protein is a putative interacting protein of the EtCDPK3 that we screened by a yeast two-hybrid system. In this series of experiments, although we were not able to show an interaction between EtCDPK3 and EtCHP using GST pull-down and Co-IP methods (data not shown), we speculated that this protein may be involved in invasion of host cells and development of E. tenella.
The mRNA and protein levels of EtCHP18905 were examined in four different developmental stages. According to qPCR, the EtCHP18905 gene was most prominent in Spz of E. tenella, and only weakly detected in UO and Mrz. These results showed that the EtCHP18905 gene was transcribed predominantly at a distinct phase of the E. tenella life cycle. However, western blot showed that protein levels were weakest for EtCHP18905 in SO. A previous study revealed that the ratios between protein and mRNA are mainly determined by translation and protein degradation in a cell [8]. However, the two processes of translation and protein degradation are highly regulated at the overall and gene-specific level [8]. Their study revealed that 15-70% of the variation is explained by posttranscriptional and posttranslation regulation and by measurement errors [5]. Hence, this may explain why the mRNA level of EtCHP18905 is inconsistent with the protein level. However, the specific reasons for this difference need to be investigated further.
The localization of the EtCHP18905 protein in different developmental stages of parasite development was also investigated using an antibody raised against the rEtCHP18905. Indirect immunofluorescence showed that EtCHP18905 was located on the membrane of E. tenella Spz and Mrz stages, which was consistent with the transmembrane protein structure data indicating that the protein was anchored to the membrane. Moreover, immunofluorescence showed that staining was stronger in the Mrz stage. Western blot analysis showed that the expression levels of EtCHP18905 in the Mrz stage was higher than in the Spz stage. However, this result was inconsistent with the results at the transcription level. This may be due to posttranslational modifications [29]. Results from previous studies have suggested that mRNA abundance is a poor indicator of the levels of the corresponding protein [1,10,13]. Furthermore, the expression of EtCHP18905 increased after the Spz invaded DF-1 cells for 2 h. Thus, this protein might function in Spz invasion or schizonts evolution. The results of the in vitro experiments confirm this. In vitro invasion inhibition assays using Figure 7. Inhibition of sporozoite invasion in vitro by anti-rEtCHP18905. Anti-rEtCHP18905, rabbit anti-rEtCHP18905 IgG; NA, naïve rabbit sera IgG; GST control, rabbit anti-GST IgG. The symbol "*" represents p < 0.05, "**" represents p < 0.01, and "***" represents p < 0.001 for comparison of treatment with anti-rEtCHP18905 and naïve rabbit sera IgG and anti-GST IgG at the same concentration. The error bars indicate the standard deviation. All assays were performed in triplicate.
polyclonal antibody against rEtCHP18905 showed partial blockage of the invasion of Spz into cells. Inhibition of sporozoites was modest at 28%, at antibody concentrations of 300 lg/mL. Likewise, the rate of invasion inhibition increased with the increase of anti-rEtCHP18905 IgG concentration. Previous studies have shown that polyclonal antibodies can significantly inhibit Spz invading DF-1 cells [7,11,19,48]. In 2016, Zhai et al. found that rabbit antiserum against rEtCHP559 can block invasion of host cells by Spz [47]. The above results suggest that EtCHP18905 is related to invasion. This new antigen might be useful for identification of novel vaccine targets, thus improving the knowledge of immunogenic proteins in E. tenella.
In previous research, many DNA and recombinant protein vaccines have been reported to induce immuno-protection to live parasite challenge [18,43,44]. In the present study, following infection challenge, the body weight gain of nonimmunized chickens was reduced significantly compared with immunized chickens. Moreover, chickens immunized with rEtCHP18905 had significantly lower lesion scores and fecal oocyst output compared to non-immunized birds. Previous studies of Eimeria spp. proteins have shown that a similar effect can be produced after immunization with recombinant protein or recombinant plasmid [25,26]. The data presented here showed that immunization with rEtCHP18905 could produce partial protection against live E. tenella infection. However, the difference of oocysts output between challenged control groups and the two immunized groups may be under reality. This was probably due to widespread tissue damage and severe hemorrhage that prevents a large number of merozoites from reinfecting intestinal epithelial cells, resulting in a decrease in average oocyst production [12]. It, therefore, appears likely that the differences in lesion score and oocyst output can be caused by merozoite loss [12]. Furthermore, the decrease in oocyst output is not as high as in previous studies [16,47]. Thus, the effect of rEtCHP18905 in reducing oocyst shedding should be researched further.
Humoral immunity in the immune response against coccidiosis is usually considered to play a minor role [45]. However, as early as 2008, Constantinoiu et al. pointed out that humoral immunity may also contribute to protective immune responses [4]. Their study revealed that chicken infected with an attenuated strain of E. tenella mount an antibody response to all lifecycle stages. High levels of antibodies against Spz and Mrz were detected in infected chickens inoculated with SO of E. tenella [4]. Moreover, in 1994, Smith also found that antibodies could inhibit parasite development and provide passive immune protection [36]. They found that there was an excellent correlation between antibody titer and protection. Oral infection of hens with E. maxima oocysts caused production of antibodies which were passed into the egg yolk and subsequently to hatchlings. The total number of oocysts excreted in the feces of chicks from eggs has decreased after infection with E. maxima oocysts [36]. Huang et al. [16] reported that birds immunized with the E. maxima rMIC7 protein and pVAX1-MIC7 exhibited higher IgG concentrations than the PBS and pVAX1 controls. In the present study, the IgG concentrations of the rEtCHP18905-immunized chickens were significantly higher than the negative controls. These findings confirmed that in rEtCHP18905-immunized chickens, certain humoral immune responses were induced.
Cell-mediated immunity plays a major role against coccidiosis [39]. The concentration of soluble sCD4 and sCD8 in serum is consistent with the number of CD4+ and CD8+ lymphocytes that produce them [41,46]. Previous studies reported that levels of sCD4 and sCD8 were higher in experimental groups immunized with rEmSAG and rEmMIC7 compared with the control [16,26]. However, the results presented here showed that serum sCD4 and sCD8 were not significantly different in immunized chickens compared to non-immunized controls. This suggests that rEtCHP18905 could not stimulate the recruitment of T-cell subpopulations. IFN-c is reported to be related to protective immune responses to avian coccidiosis [23]. A previous study found that IFN-c concentrations were higher in EmMIC7-vaccinated birds [16]. However, rEtCHP18905 increased the serum concentrations of IFN-c but not significantly compared to the control group. Nevertheless, the relative importance of each cytokine type in inducing immune challenge could not be inferred from these data.
One of the Th2-type cytokines, IL-10 and IL-17 are also involved in immune response to coccidial challenge. IL-10 has been shown to be crucial for control of Eimeria infections [32]. Wu et al. observed that serum IL-10 levels increased on day 5 after infection with E. tenella [42]. However, in this study, there was no significant level of IL-10 detected in groups immunized with rEtCHP18905. Previous reports have shown that the immunization of animals with DNA vaccines produced higher levels of IL-17 [14,37]. In previous vaccination trials, IL-17 concentrations in the vaccinated groups were significantly higher than those of the unvaccinated groups [16,26]. In the current study, the concentrations of IL-17 in the immunized groups were also significantly higher than the non-immunized groups. The previous study, and our data, together confirm that IL-17 might be associated with protective immunity to coccidiosis. However, the specific functions of IL-10 and IL-17 in the immunity to coccidiosis requires further investigation.
The Treg-type cytokines TGF-b, produced by Treg cells, have been shown to regulate immunosuppression mechanisms  [21]. Two earlier reports showed that TGF-b was significantly higher in chickens immunized with recombinant EbAMA1 [38,49]. The same result was demonstrated by Liu et al. [26]. Likewise, in the present study, the levels of the TGF-b1 in the rEtCHP18905-immunized groups were significantly higher than the non-immunized group. These findings together confirm that TGF-b1 may function in coccidiosis-induced immune pathways. In summary, Th2-type cytokines can down-regulate the expression levels of Th1-type cytokines and regulate the immune response [15]. In the present study, IL-17 and TGF-b1 levels increased following immunization. In contrast, sCD4, sCD8 and IL-10 did not increase significantly. Moreover, higher IgG concentrations were detected in the EtCHP18905 vaccinated chickens. Thus, EtCHP18905 might be a supplementary candidate, alongside other proteins that stimulate cellular immunity for the development of new vaccines to combat E. tenella infection in chickens.

Conclusions
In this study, EtCHP18905 was amplified, expressed and characterized. Its location on Spz and Mrz was determined. Anti-rEtCHP18905 antibodies could reduce the rate of Spz invasion. The results of animal immune protection assays indicated that vaccination with rEtCHP18905 was capable of eliciting both humoral immunity and cell-mediated immunity, providing moderate protective immunity against E. tenella. However, the exact roles of EtCHP18905 in coccidial infections require further investigation.