The prokaryotic expression vector pET-32a was purchased from Novagen (Darmstadt, Germany), and the eukaryotic expression vector pVAX1 (Figure 1C) was purchased from Invitrogen (Carlsbad, CA, United States). E. tenella, E. acervuline, and E. maxima were isolated from Jiangsu Province of China (JS). And their purity were determined with ITS1-PCR as described previously (Jenkins et al., 2006; Haug et al., 2007). Oocysts of E. tenella, E. acervuline, and E. maxima were propagated, harvested and sporulated using a previously described protocol 7 days prior to the challenge infection (Tomley, 1997). New-hatched Hy-Line layer chickens (commercial breed W-36) were raised in a sterilized room under coccidia-free conditions until the end of the experiment. Food and water without anti-coccidial drugs were available. Thirty-day-old rats (SD) were obtained from the Comparative Medicine Centre, Yangzhou University, Yangzhou, China. 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).

Sporulated oocysts of E. acervulina and E. maxima were broken to release sporocysts by whirl mix with micro glass balls (Tomley, 1997). Next, total RNA was extracted from the released sporocysts of E. acervulina and E. maxima using E.Z.N.A.^® Total RNA Kit Maxi Kit (OMEGA, Norcross, GA, United States), respectively. Figure 1A showed schematic of the cloning strategy. The cDNAs were synthesized by reverse transcription (RT) reaction with the specific primers for E. acervulina GAPDH (EaGAPDH) and E. maxima GAPDH (EmGAPDH) (see Table 1). PCR products were cloned into pMD-19T vector (TaKaRa, Dalian, China) to construct pMD-19T-EaGAPDH and pMD-19T-EmGAPDH. The recombinant plasmids were identified by endonuclease digestion and sequencing. The nucleotide sequences were analyzed online with a basic alignment search tool (BLAST)¹.

The EaGAPDH and EmGAPDH genes were cloned into the prokaryotic expression vector pET-32a, and transformed into E. coli BL21 (DE3), respectively. The recombinant proteins were expressed and purified with His Trap^TM FF (GE Healthcare, United States) following introduction of the kit. The purities of protein were determined by SDS-PAGE and protein concentrations were estimated using Pierce^TM BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, United States). The proteins were diluted in PBS buffer with a concentration of 400 μg/ml. Stocks proteins were prepared and stored at –80°C until further use.

Rat anti-EaGAPDH and anti-EmGAPDH sera were prepared as following. Rats were vaccinated subcutaneously in the back with 200 μg (400 μg/ml) recombinant proteins of EaGAPDH and EmGAPDH emulsified in 0.5 ml of Freund’s Adjuvant Complete (Sigma–Aldrich, Merck KGaA, Darmstadt, Germany) at 30 days of age separately. Two weeks later, two doses of proteins (200 μg, 400 μg/ml) emulsified in 0.5 ml of Freund’s Adjuvant Incomplete (Sigma–Aldrich) were given at an interval of 1 week. A forth even fifth dose would be given unless the titers of sera were beyond 1: 64. The antisera were collected and then stored at –20°C for further Western blot analysis.

Chicken Antisera were obtained after two oral infections of chickens with sporulated oocysts of E. acervulina and E. maxima at 10-days intervals and bled 10 days after the last infection (Song et al., 2017). Serum was collected from the uninfected chicken as empty control. Recombinant proteins of EaGAPDH and EmGAPDH were analyzed by Western blot assay with anti-E. acervulina and anti-E. maxima chicken sera as first antibody, respectively. Briefly, recombinant proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, United States). The membrane was incubated with anti-E. acervulina and anti-E. maxima chicken sera (1: 100) and horseradish peroxidase (HRP)-conjugated Goat anti-chicken IgG (1:2000, Sigma–Aldrich) as secondary antibody. The bound antibody was detected using 3, 30-diaminobenzidine (DAB) (Song et al., 2015b).

Recombinant plasmids pVAX-EaGAPDH and pVAX-EmGAPDH were generated with the eukaryotic expression vector pVAX1. The cloning scheme was shown in Figure 1B. Briefly, fragments of EaGAPDH and EmGAPDH were excised from the plasmids of pMD19-EaGAPDH and pMD19-EmGAPDH by BamHI and XhoI (TaKaRa) digestion and ligated into pVAX1 at the same enzyme sites to construct pVAX-EaGAPDH and pVAX-EmGAPDH, respectively. The resulting plasmids were confirmed by endonuclease cleavage and sequence analysis.

Chickens were intramuscularly injected with the recombinant plasmids pVAX-EaGAPDH and pVAX-EmGAPDH in thighs at 14 days of age separately. Seven days later, about 0.5 g of the injected muscle was cut for transcription and expression detection of the GAPDH genes. Meanwhile the same site of muscles from non-injected chickens was selected as control.

Total mRNA from the injected muscle was extracted and digested by DNase I (TaKaRa) to remove the residual plasmid. With the specific primers for GAPDH genes (Table 1), RT-PCR were performed with the product RNA as template and electrophoresis in 1% agarose gel was performed to examine the transcription of EaGAPDH and EmGAPDH.

The injected muscle was treated with RIPA solution [0.1 mol/L phenylmethylsulfonyl fluoride (PMSF), 50 mmol/L Tris–HCl, 150 mmol/L NaCl, 1% Nonnidet P-40, 0.1% SDS] for 3 h. After centrifugation at 13,000 rpm, the supernatant was collected for use. Then Western blots were performed to detect the expressed proteins with anti-EaGAPDH and anti-EmGAPDH rat sera as primary antibody.

The proportions of spleen T lymphocyte subpopulations of CD4+ and CD8+ from the vaccinated chickens were analyzed using flow cytometry technique. One week after the primary and booster vaccination, spleens were collected from five chickens of each group to prepare the spleen lymphocytes as described by Sasai et al. (2000). The spleens were cut into scraps and gently pushed through a mesh screen and cells were washed with calcium and magnesium-free Hank’s balanced salt solution (HBSS). Lymphocytes were collected using a separation solution (TBD, Tianjin, China) according to the manufacturer’s protocol. Lymphocytes suspensions (1 × 10⁶ cells/ml) were incubated with anti-chicken CD3-FITC, anti-chicken CD3-FITC + anti-chicken CD8-PE, anti-chicken CD3-FITC + anti-chicken CD4-PE (SouthernBiotech, Birmingham, AL, United States) in the dark for 30 min at 4°C separately. Subsequently, the cells were washed twice with PBS by centrifugation (500 ×g, 10 min, 4°C) and re-suspended with cold PBS and analyzed with a BD FACSCalibur flow cytometer (BD, Franklin Lakes, NJ, United States). A lymphocyte specific gating was set according to forward and side scatter profiles. The proportions of CD4+ and CD8+ T lymphocytes were determined as described by (Song et al., 2010).

The transcription levels of IFN-γ, IL-2, IL-4 TNFSF15, IL-17, and TGF-β4 were determined by qPCR. Total RNA was extracted from the isolated lymphocytes of spleen from chickens (five per group) using E.Z.N.A.^® Total RNA Kit Maxi Kit (OMEGA, Norcross, GA, United States). Lymphocytes cDNA was synthetized using HiScript II Q RT SuperMix (Vazyme, Nanjing, China). Chicken GAPDH gene was used as an internal control. The primers for qPCR were presented in Table 2 (Song et al., 2010). Amplifications were conducted in 20 μl reaction mixture containing 1 μl of cDNA, 10 μl of 2 × ChamQ^TM SYBR^® qPCR Master Mix (Vazyme, Nanjing, China), and 0.5 μM primers. Reaction was performed in an ABI PRISM 7300 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, United States), with an initial denaturation at 95°C for 30 s, followed by 40 cycles at 95°C for 10 s, 60°C for 30 s and followed by a melting curve program at 95°C 15 s, 60°C 15 s, 95°C 15 s. Each reaction was analyzed in triplicate and GAPDH was used as endogenous control. A negative control, in which template cDNA was replaced by PCR-grade water was included with all qPCR assays. In this study, the 2^-ΔΔCT method was used to estimate the relative quantification of cytokine gene mRNA compared with the internal control gene of GAPDH (n-fold change to the water control group) (Livak and Schmittgen, 2001). Validation experiment was performed by running a dilution series of the cDNA to evaluate the amplification efficiencies of the target genes and reference genes (Song et al., 2010). The corresponding real-time efficiency (E) of one cycle in the exponential phase was calculated by the following formula:

As the efficiency of the cytokine primers were not 100% (Song et al., 2010), Pfaffl correction was done for these RT-PCR analyses.

After 1 week of primary and booster vaccination, whole blood from five chickens per group was collected from wing vein of the chickens and the sera were collected for determining the GAPDH-specific IgG antibody response by indirect ELISA (Song et al., 2017). Briefly, flat-bottomed 96-well plates (MarxiSorp, Nunc, Denmark) were coated overnight at 4°C with 100 μl solution per well of recombinant EaGAPDH or EmGAPDH (50 mg/ml) in 0.05 M carbonate buffer, pH 9.6. The plates were washed with 0.01 M PBS containing 0.05% Tween-20 (PBS-T) and blocked with 5% skim milk powder (SMP) in PBS-T for 2 h at 37°C. The plates were incubated for 2 h at 37°C with 100 μl of the serum samples diluted 1:50 in PBS-T with 1% SMP in duplicate. After three washes, the plates were incubated for 1 h at room temperature with 100 μl/well of HRP conjugated donkey anti-chicken IgG anti-body (Sigma–Aldrich) diluted 1:1000 in 2% SMP in PBS-T. Color development was carried out with 3, -3′, 5, 5′-tetramethylbenzidine (TMB) (Sigma-Aldrich), and the optical density at 450 nm (OD450) was determined with a microplate spectrophotometer. All serum samples were investigated by ELISA at the same time under the same conditions and were included on one plate.

At 14 days of age, chickens were weighted and randomly divided into 17 groups of 30 chicks each (see Table 3). The schematic of vaccination was shown in Figure 2B. Experimental groups were intramuscularly vaccinated with 100 μg (0.5 μg/μl) of plasmids pVAX-EaGAPDH and pVAX-EmGAPDH in thigh, respectively. The vector control group was injected with 100 μg (0.5 μg/μl) of pVAX1, the challenged and unchallenged control groups were injected with sterile PBS buffer with the same administration route as the experimental groups. A booster vaccination was given 1 week later with the same amount of components as the primary vaccination. At 28 days of age, chickens were weighed individually and orally challenged with E. tenella (5 × 10⁴/bird), E. acervulina (10 × 10⁴ /bird), E. maxima (10 × 10⁴/bird), and mixed Eimeria species (5 × 10⁴ E. tenella/bird, 10 × 10⁴ E. acervulina/bird, 10 × 10⁴ E. maxima/bird) separately (Holdsworth et al., 2004), except the unchallenged control groups. Unchallenged control chickens were given PBS orally (see Table 3). Five to seven days, post challenge the chickens were weighed individually. Twenty chickens of each group were slaughtered for lesion score (Johnson and Reid, 1970). Additionally, the enteric contents of the chickens were collected separately and oocysts per gram of content (OPG) were counted using a McMaster chamber. The rest ten chickens were used to count the oocysts of the fecal samples during 5–10 days post-challenge. Efficacy of immunization was evaluated on the basis of survival rate, lesion score, body weight gain, oocyst decrease ratio and the ACI as described by Song et al. (2015a). The ACI is rated “good” when 180 or more, “moderate” when 160–179, and “poor” when below 160 (McManus et al., 1968).

One-way analysis of variance (ANOVA) with Duncan’s multiple range tests were used for the determination of statistical significance by using the SPSS statistical package (SPSS for Windows 16, SPSS Inc., Chicago, IL, USA). Differences among groups were tested and p < 0.05 was considered to indicate a significant difference.

As shown in Figure 3, the open reading frames (ORFs) of EaGAPDH and EmGAPDH are composed 1020 nucleotides with predicted molecular weights of 37.4 kDa. BLAST analysis revealed that EaGAPDH and EmGAPDH shared similarities of 99% in nucleotides and amino acid sequences with the genes in NCBI (Gene ID: EaGAPDH 25337292; EmGAPDH 25268815), respectively. GAPDH shared similarities of more than 86% in amino acid among five species of chicken coccidia (Figure 4). The recombinant proteins of EaGAPDH and EmGAPDH were expressed as His₆ tag fusion proteins. SDS–PAGE revealed prominent protein bands about 55.4 kDa (Figure 5A, lanes 1 and 3) which were consistent with molecular weight sum of fusion protein of pET-32a vector (18 kDa) and recombinant proteins of EaGAPDH or EmGAPDH (37.4 kDa). The purification of the recombinant proteins showed single band with molecular weight of 55.4 kDa by SDS–PAGE (Figure 5A, lanes 2 and 4). Western blot assay revealed specific band of 55.4 kDa (Figure 5B, lanes 1 and 3), indicating EaGAPDH and EmGAPDH recombinant proteins were recognized by with anti-E. acervulina and anti-E. maxima chicken sera, respectively.

Plasmids pVAX-EaGAPDH and pVAX-EmGAPDH were generated using the eukaryotic expression vector pVAX1. The endonuclease cleavage and sequence analysis indicated that plasmids of pVAX-EaGAPDH and pVAX-EmGAPDH were constructed successfully.

Transcriptions of GAPDH gene in the injected muscles were detected using RT-PCR assay. With the specific primers of EaGAPDH and EmGAPDH, bands of approximately 1020 bp were detected from the muscle injected with pVAX-EaGAPDH and pVAX-EmGAPDH, respectively (Figure 6A, lanes 1 and 4). No specific DNA bands were detected in pVAX1 injected and non-injected control samples (Figure 6A, lanes 2 and 5; Figure 6A, lanes 3 and 6). This result indicated that GAPDH was transcribed in the injected muscles of chickens.

Expression the GAPDH genes in the injected muscle was detected by Western blot. As shown in Figure 6B, ant-EaGAPDH and ant-EmGAPDH sera reacted with protein band of approximately 37.4 kDa from the pVAX-EaGAPDH and pVAX-EmGAPDH injected muscles, respectively (Figure 6B, lanes 1 and 3). No specific band was detected in non-injected control samples (Figure 6B, lanes 2 and 4). This result indicated the eukaryotic recombinant plasmid pVAX-EaGAPDH and pVAX-EmGAPDH were expressed in the injected muscles of chickens.

To determine the possible effects induced by GAPDH on T lymphocytes subpopulation, the proportions of CD4⁺/CD3⁺ and CD8⁺/CD3⁺ T cells in spleens of the vaccinated chickens were evaluated by flow cytometry (Figure 7). One week after the primary vaccination, administration with pVAX-EaGAPDH and pVAX-EmGAPDH significantly increased the proportion of CD4⁺/CD3⁺ and CD8⁺/CD3⁺ T cells compared to the pVAX1 and PBS control (p < 0.05). No significant difference was observed on the proportion of CD4⁺/CD3⁺ and CD8⁺/CD3⁺ T lymphocytes subpopulation between pVAX1 and PBS control group (p > 0.05). No significant difference was observed on the proportion of CD8⁺/CD3⁺ T lymphocyte subtype between pVAX-EaGAPDH and pVAX-EmGAPDH group (p > 0.05). However, pVAX-EmGAPDH significantly increased the proportion of CD4⁺/CD3⁺ T cells compared with pVAX-EaGAPDH group (p < 0.05). The similar result was observed one week after the booster vaccination, except that no significant difference was observed on the proportion of CD4⁺/CD3⁺ T cells between pVAX-EaGAPDH and pVAX-EmGAPDH group (p > 0.05).

To determine the possible effects induced by GAPDH on cytokine production, the transcription levels of IFN-γ, IL-2, IL-4 TNFSF15, IL-17, and TGF-β4 were determined by qPCR (Figure 8). One week after the primary vaccination, chickens immunized with recombinant plasmids pVAX-EaGAPDH and pVAX-EmGAPDH showed a significant switch in their cytokine profiles, with a significant increase in IFN-γ, IL-2, IL-4 TNFSF15, IL-17, and TGF-β4 compared with the pVAX1 and PBS control (p < 0.05). No significant difference was observed between pVAX1 and PBS control group (p > 0.05). The similar result was observed one week after the booster vaccination.

To determine the possible effects induced by GAPDH on antibody response, levels of GAPDH-specific antibodies were determined by ELISA. One week after the primary vaccination, sera from chickens immunized with recombinant plasmids pVAX-EaGAPDH or pVAX-EmGAPDH showed significantly high level of IgG antibody (p < 0.05) compared to that of pVAX1 and PBS controls, whereas IgG antibody of chicken immunized with pVAX1 and PBS was not significantly induced (p > 0.05) (Figure 9). The similar result was observed one week after the booster vaccination (Figure 10).

Protective efficacies of pVAX-EaGAPDH and pVAX-EmGAPDH were evaluated by challenge with E. tenella, E. acervulina, E. maxima, or mixed oocysts of the three Eimeria species, respectively. As shown in Table 3, body weight gains were significantly reduced in challenged and pVAX1 control group compared with the unchallenged control group (p < 0.05). Chickens vaccinated with pVAX-EaGAPDH and pVAX-EmGAPDH displayed significantly increased weight gains compared to chickens in challenged and pVAX1 control groups (p < 0.05). The oocyst outputs of pVAX-EaGAPDH and pVAX-EmGAPDH vaccinate chickens were significantly lower than those of challenged and pVAX1 control group (p < 0.05). Significant alleviations in enteric lesions were observed in pVAX-EaGAPDH and pVAX-EmGAPDH immunized chickens compared to those of challenged and pVAX1 control group (p < 0.05). Group of chickens immunized with recombinant plasmids pVAX-EaGAPDH and pVAX-EmGAPDH resulted in ACI more than 160 showing moderate protections against E. tenella, E. acervulina, E. maxima, and mixed infection of the three Eimeria species.