14-day-old specific-pathogen-free (SPF) White Leghorns were purchased from Beijing Boehringer Ingelheim Vital Biotechnology Co., Ltd. (Beijing, China) and housed in coccidia-free isolators under controlled conditions. All chicks were reared under SPF conditions with ad libitum access to food and tap water. The basal grower diet was based on Crude protein (≥19.5%), Crude fiber (≤5.5%), Crude ash (≤8.0%), Calcium (0.80~1.20%), Total phosphorus (≥0.30%), Sodium chloride (0.30~0.80), and Methionine(0.45~0.90%). Two-month-old New Zealand White rabbits were obtained from Beijing Baierkangnate Experimental Rabbit Breeding Biotechnology Development Co., Ltd. (Beijing, China). All rabbits were screened for Eimeria infection by three consecutive days of fecal examination and subsequently maintained under coccidia-free conditions. Throughout the experiment, animals were monitored daily by veterinarians for general health status, including feed and water intake, behavior, posture, and any signs of clinical symptoms such as diarrhea, weight loss, lethargy, or signs of distress. Animals that reached predetermined humane endpoints were humanely euthanized to minimize suffering. The Shanxi virulent strain of E. tenella (SX-01) was propagated and maintained via cecal passage in SPF chickens at the Veterinary Pathology Laboratory of the College of Veterinary Medicine, Shanxi Agricultural University.

The collection of merozoites was performed according to previously described protocols (14, 15). Briefly, ten 20-day-old SPF chickens were orally inoculated with 1 × 10⁴ oocysts each. At 120 hours post-infection, the chickens were euthanized via CO₂ asphyxiation. Caecal tissues were collected, finely minced, and incubated with hyaluronidase (Solarbio, Shanghai, China) at 37°C for 60 min. The resulting mixture was filtered and centrifuged at 3000 rpm for 5 min to collect the precipitate. The pellet was incubated with red blood cell lysis buffer (Beyotime, Shanghai, China) at 4°C for 10 min, and merozoites were subsequently purified by density gradient centrifugation.

Total RNA was extracted from E. tenella merozoites using TRIzol reagent ((Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Subsequently, cDNA was synthesized using the PrimeScript™ RT reagent kit with gDNA Eraser (TaKaRa, Shiga, Japan), according to the manufacturer’s instructions for genomic DNA removal and reverse transcription. The gDNA removal step was conducted at 42°C for 2 min, followed by reverse transcription at 37°C for 15 min and 85°C for 5 s. The resulting cDNA was stored at –80°C until further use (16).

Specific primers were designed to amplify the E. tenella MIF (EtMIF) gene sequence based on GenBank ID DQ323515.1, and were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. (Suzhou, China). The primer sequences were as follows: EtMIF forward primer, 5’-GCTCGAGTTAACCAAACACGCGGGAACC-3’; and reverse primer, 5’-GGCTAGCATGCCACTGTGCCAGATCG-3’. PCR products were purified and cloned into the pMD18-T vector (Takara, Shiga, Japan) according to the manufacturer’s instructions for the gel extraction kit. A single colony was selected and cultured for plasmid extraction and sequencing. The colonies with correct insertions were chosen for subsequent experiments. The EtMIF gene fragment was amplified by RT-PCR from RNA isolated from merozoites of E. tenella (Shanxi virulent strain). The pMD18-T-EtMIF plasmid and the pET-28a(+) vector (Sangon Biotech, Shanghai, China) were digested with NheI and XhoI restriction enzymes (Takara, Shiga, Japan) and purified. The EtMIF gene fragment was then ligated into the pET-28a(+) vector, and the recombinant plasmid was confirmed by DNA sequencing. The plasmid with the correct sequence was designated pET-28a(+)-EtMIF.

The recombinant pET-28a(+)-EtMIF plasmids were transformed into Escherichia coli BL21 (DE3) competent cells (TransGen, Beijing, China). Single colonies were selected and cultured in LB medium (Solarbio, Beijing, China) supplemented with 50 μg/mL kanamycin at 37°C and 170 rpm until reaching the mid-logarithmic phase (approximately 1 h). The culture was then inoculated into 500 mL of fresh LB medium (Solarbio, Beijing, China) supplemented with 50 μg/mL kanamycin and incubated at 37°C with shaking at 200 rpm. When the OD600 of the culture reached approximately 0.5, isopropyl β-D-1-thiogalactopyranoside (IPTG; Solarbio, Beijing, China) was added to a final concentration of 1 mM to induce protein expression.

Following IPTG induction, the bacterial cells were harvested by centrifugation and lysed by sonication on ice. The lysate was centrifuged to separate the soluble and insoluble fractions. The rEtMIF protein was purified using Ni-NTA affinity chromatography (Sangon, Shanghai, China), The Ni-NTA column was pre-equilibrated with 5 mL of deionized water and 5 mL of binding buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 8.0), after which the supernatant was slowly loaded onto the column. After washing with 3 mL of binding buffer, the column was sequentially eluted with 3 mL volumes of elution buffer containing increasing concentrations of imidazole (50, 100, 150, 200, and 500 mM). The collected elution fractions were dialyzed stepwise against PBS to remove imidazole, with the buffer replaced every 4 h over a 12-hour period. To further concentrate the sample, the dialyzed protein was centrifuged in 3 kDa ultrafiltration tubes at 6000 rpm for 40 min at 4 °C and reduced to a final volume of approximately 200 μL. Endotoxin in the purified rEtMIF protein was removed using a Protein Endotoxin Removal Kit (Beyotime, Shanghai, China), and residual endotoxin levels were measured using a Chromogenic LAL Endotoxin Assay Kit (Beyotime, Shanghai, China). Its molecular weight was assessed by 12% SDS-PAGE. Additionally, its concentration was quantified using a BCA Protein Assay Kit (Beyotime, Shanghai, China). The purified rEtMIF protein was aliquoted and stored at −80°C for subsequent experiments (17). Rigorous measures were implemented to minimize the risk of contamination during protein preparation, including ultraviolet irradiation, high-temperature treatment, and the use of sterile, nuclease-free, and endotoxin-free consumables.

During the experiment, 1×10⁴ sporulated E. tenella oocysts were orally administered to 20-day-old SPF chickens via crop instillation. Each chick was subsequently challenged with 1×10⁴ sporulated oocysts every 7 days for a total of four times. Blood samples were collected via cardiac puncture, incubated at 37°C for 1 h and then at 4°C for 12 h. Samples were centrifuged at 3000 rpm for 10 min, and serum was separated to prepare anti-E. tenella positive serum. Serum was aliquoted and stored at −80°C until use. Serum from uninfected rabbits was collected as a negative control.

Three two-month-old New Zealand White rabbits were selected and underwent four immunizations. For the first immunization, the positive group received the rEtMIF protein was emulsified with complete Freund’s adjuvant (CFA, Sigma, St Louis MO, USA) at a 1:1 volume ratio using a homogenizer (FLUKO, Shanghai, China) on ice, whereas the negative group was immunized with PBS emulsified with CFA. Subsequent immunizations for both groups employed incomplete Freund’s adjuvant (IFA) instead of CFA. All emulsifications were performed at a 1:1 volume ratio. Subcutaneous immunizations were performed bilaterally along the spine at a dose of 200 µg per rabbit, with a total of four immunizations. Before each immunization, 30 µL of blood was collected from the ear vein, serum was isolated, and antibody titers were determined. The interval between immunizations was 14 days. Seven days after the final immunization, blood was collected via cardiac puncture, incubated at 37°C for 1 h followed by overnight incubation at 4°C, and centrifuged at 3000 rpm for 10 min to collect the serum. The collected serum was heat-inactivated at 56°C for 30 min and stored at −80°C until use. Serum from uninfected rabbits was collected simultaneously as a negative control.

Indirect immunofluorescence assay was performed according to the method described by Wang et al. (18). The collected E. tenella merozoites were smeared onto glass slides and fixed with 4% paraformaldehyde (Sangon Biotech, Shanghai, China) at room temperature for 10 min. After fixation, the samples were permeabilized with 1% Triton X-100 (Beyotime, Shanghai, China) at room temperature for 10 min. Slides were washed three times with PBST (Solarbio, Beijing, China), with 5-min incubations between washes. Samples were blocked with 5% bovine serum albumin (BSA; Beyotime, Shanghai, China) at 37°C for 2 h. The polyclonal antibody against rEtMIF and rabbit negative serum (both diluted 1:800 in PBST) were applied and incubated overnight at 4°C. After washing the slides three times with PBST, the samples were incubated with FITC-labeled goat anti-rabbit IgG secondary antibody (1:5000 dilution in PBST; Abmart, Shanghai, China) at 37°C in the dark for 40 min. Subsequently, the slides were stained with Hoechst 33342 (Beyotime, Shanghai, China) at room temperature for 10 min, washed three times with PBST with 5-min incubations between washes, mounted with an anti-fade mounting medium, and observed under an inverted fluorescence microscope.

One hundred 1-day-old SPF chickens were selected. Chickens with similar body weights were randomly allocated into five groups (n = 20 per group), with each group further divided into two cages. As shown in Figure 1 , at 14 and 21 days of age, chickens were subcutaneously immunized in the neck with rEtMIF protein emulsified with Montanide™ ISA 71 VG (ISA 71 VG, Seppic, Paris, France) at doses of 50, 100, or 150 µg. The challenged and unchallenged control groups were simultaneously injected with PBS emulsified with ISA 71 VG. All emulsifications were prepared by mixing the adjuvant and rEtMIF protein (in PBS) at a 70:30 weight ratio, and the resulting emulsion was repeatedly passed through a 0.9 G20 injection needle 20 times to ensure uniformity. Seven days later, chickens in the immunized and challenged control groups were orally inoculated with 1×10⁴ sporulated E. tenella oocysts, whereas chickens in the unchallenged control group received PBS orally ( Table 1 ). Body weights were recorded on days 14, 21, 28 (before challenge), and 35 (7 days post-challenge). On day 35, fecal samples were collected for oocysts counting, ceca were dissected for lesion scoring, and cardiac blood samples were collected.

In vivo immunoprotective parameters of the rEtMIF protein included clinical, pathological, and parasitological parameters. Clinical parameters included weight gain, survival rate, and mortality rate, which were directly recorded and calculated. Pathological evaluation was based on cecal lesion scores ranging from 0 (none) to 4 (severe), assessed independently by three observers according to the criteria described by Johnso (19). Parasitological parameters included oocyst output and anticoccidial index (ACI) (20). Oocyst output was quantified as the number of oocysts per gram (OPG) of cecal content using the McMaster counting technique (21, 22), and the oocyst index was subsequently calculated. The oocyst index was then calculated based on the following OPG ranges: an OPG ≤ 0.1 was scored as 0; 0.1–1.0 as 1; 2.0–5.0 as 10; 6.0–10.0 as 20; and OPG ≥ 11.0 was scored as 40 (23). The ACI served as a comprehensive parameter for evaluating anticoccidial efficacy and was calculated using the following formula: ACI = (relative weight gain rate + survival rate) − (mean lesion score×10−oocyst index). An ACI ≥ 180 indicated good protection, 160 ≤ ACI < 180 indicated moderate protection, 120 ≤ ACI < 160 indicated limited protection, and ACI < 120 indicated no protection.

At 14, 21, and 28 days of age, three chickens were randomly selected. Blood was collected via cardiac puncture, and serum samples were obtained by centrifugation and stored at −80°C until use. The levels of antigen-specific IgY were evaluated by enzyme-linked immunosorbent assay (ELISA). Briefly, polystyrene 96-well flat-bottom microtiter plates were coated with 100 µL/well of rEtMIF (1 µg/mL) in coating buffer at 37°C for 1 h, followed by overnight incubation at 4°C. After four washes with PBS containing 0.05% Tween 20 (PBST, pH 7.4), the plates were blocked with 1% bovine serum albumin (BSA; Beyotime, Shanghai, China) at 37°C for 1 h. After four additional washes with PBST, sera diluted in 0.1% BSA-PBST were added and incubated at 37°C for 1.5 h. Plates were then washed again, followed by incubation with 100 µL of HRP-conjugated rabbit anti-chicken IgY (1:10000 dilution in PBST; Bioss, Beijing, China) at 37°C for 1 h. After washing, 50 µL/well of TMB substrate (Bioss, Beijing, China) was added and incubated for 20 min. The enzymatic reaction was terminated by adding 50 µL/well of stop solution, and the optical density (OD450) was measured using a microplate reader. All serum samples were tested in triplicate. Serum levels of IL-8, TNF-α, IL-1, and IFN-γ were measured using chicken cytokine ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer’s instructions, and absorbance was read at 450 nm using a microplate reader. Data were obtained from three independent experiments.

All data were analyzed using SPSS version 22.0 (IBM Corp., Armonk, NY, USA) and expressed as mean ± standard deviation (SD). Statistical significance was evaluated using one-way ANOVA followed by Tukey’s post hoc test to assess group differences. Graphs were generated using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). Each experiment was repeated at least three times. Differences were considered statistically significant at P < 0.05.

The CDS region of the EtMIF gene was amplified by PCR using cDNA as the template, and a specific band of 348 bp was obtained ( Figure 2A ). The PCR product was then cloned into the pMD-18T vector, and positive colonies were selected. Following plasmid extraction and double digestion, bands corresponding to the vector (2692 bp) and the target gene (348 bp) were observed ( Figure 2B ). The target fragment was excised from the pMD-18T vector and subcloned into the pET-28a(+) vector. Positive colonies were screened by colony PCR, followed by double digestion identification and sequencing analysis ( Figures 2C, D ). The prokaryotic expression vector pET-28a(+)-EtMIF was successfully constructed.

After IPTG induction, the expression of rEtMIF protein was analyzed by SDS-PAGE. Coomassie Brilliant Blue staining revealed that rEtMIF was highly expressed after IPTG induction at 37°C for 4 h ( Figure 3A ). After purification, the rEtMIF protein was predominantly present in the soluble fraction, and a target band of approximately 12 kDa was detected ( Figure 3B ). The optimal imidazole elution concentration was determined to be 500 mmol/L ( Figure 3C ). Western blot analysis showed that the rEtMIF protein was specifically recognized by monoclonal antibodies against the 6×His tag, with a target band appearing at approximately 12 kDa, confirming the successful recombinant expression of the rEtMIF protein in vitro ( Figure 3D ).

Western blot analysis showed that the recombinant rEtMIF protein was recognized by E. tenella-positive chicken serum ( Figure 4A ) and by rabbit anti-rEtMIF polyclonal antibodies ( Figure 4B ). A specific band of approximately 12 kDa was observed, while no reactivity was detected with negative control sera from chickens and rabbits. These results indicate that EtMIF is a native antigen of E. tenella and a promising candidate for vaccine development. Indirect immunofluorescence assay (IFA) revealed that EtMIF was localized both on the surface and within the cytoplasm of E. tenella merozoites ( Figure 4C ).

In the challenged control and immunized groups, bloody stools were observed, along with lethargy, reduced appetite, and ruffled feathers. Compared to the challenged control group, the immunized groups exhibited alleviated clinical symptoms. No clinical symptoms were observed in the unchallenged (blank) group. No deaths occurred in any of the experimental groups.

There were no significant differences in average body weight among the groups before the challenge, indicating that immunization with rEtMIF had no adverse effect on chicken growth. On day 7 post-infection, the 50 µg rEtMIF immunized group showed significantly higher weight gain compared to the challenged control group (P < 0.01), while the 100 µg group also showed significantly higher weight gain (P < 0.05). No significant difference was observed between the 150 µg rEtMIF group and the challenged control group ( Table 2 ).

Similarly, the cecal lesion score of the 50 µg rEtMIF group was significantly lower than that of the challenged control group (P < 0.01), and the 100 µg rEtMIF group also showed a significant reduction in lesion scores (P < 0.05). No significant difference in lesion scores was observed in the 150 µg rEtMIF group ( Table 2 , Figures 5A, B ).

Compared to the challenged control group, chickens immunized with rEtMIF protein exhibited a significant reduction in oocyst output (P < 0.05) ( Figure 5C ). Notably, the anticoccidial index (ACI) of the 50 µg rEtMIF immunized group exceeded 160, whereas the ACI values of the 100 µg and 150 µg groups remained below 160. These findings indicate that the 50 µg dose provided moderate protective efficacy and represented the optimal immunization dose among the three tested doses against E. tenella infection.

Seven days after the first immunization, the levels of IgY antibodies in the serum of the rEtMIF immunized groups at different doses were significantly higher than those in the challenged control group (P < 0.05), with the 150 µg rEtMIF group exhibiting the highest antibody levels. Seven days after the booster immunization, the IgY antibody levels in the rEtMIF immunized groups remained significantly higher than those of the challenged control group, and antibody levels after the booster immunization were significantly higher than after the primary immunization (P < 0.05, Figure 6 ). These results demonstrate that rEtMIF is a native antigen of E. tenella capable of eliciting a strong humoral immune response to combat E. tenella infection.

Serum levels of IL-1, IL-8, TNF-α, and IFN-γ were measured using ELISA kits. No significant differences were observed among groups before immunization. Prior to the booster immunization, IL-1 levels were significantly higher in the 100 µg and 150 µg rEtMIF immunized groups compared to the challenged control group (P < 0.05), while IFN-γ and TNF-α levels were significantly higher in the 50 µg and 100 µg rEtMIF groups (P < 0.05). Additionally, IL-8 levels were significantly higher in all rEtMIF immunized groups compared to the challenged control group (P < 0.01). Before challenge, the levels of IL-1, IL-8, and IFN-γ in each rEtMIF immunized group were significantly higher than those in the challenged control group (P < 0.01), and TNF-α levels were also significantly higher (P < 0.05) ( Figure 7 ).