Inoculum and drug
Oocysts of E. tenella (Luoyang strain) were passaged before inoculation, and the obtained oocysts was sporulated in 2.5% K2Cr2O7 solution at 29 °C for 48 h. Diclazuril ( > 99%; Shanghai Veterinary Research Institute, CAAS, China) was supplied at a dose of 1 mg/kg in broiler feed.
Experimental chickens and treatment
One-day-old male Chinese Yellow broiler chickens were obtained from Gonghua Commercial Hatchery, Luoyang, China. The chickens were kept in wire-floored batteries for 14 days under coccidiosis-free conditions. On the 14th day, healthy 90 chickens were randomly divided into two groups of 45 with three biological replicates of 15 in each group: (1) Chickens that were challenged with E. tenella sporulated oocysts and received commercial diet without drugs were included to the infected/control group. (2) Chickens that were challenged with E. tenella sporulated oocysts and treated with 1 mg/kg diclazuril in feed from 96 h to 120 h were included to the infected/diclazuril group. Inoculation was performed with a dose of 8 × 104 sporulated oocysts/chicken on day 14. The experimental scheme conformed strictly to the guidelines of the Institutional Animal Care and Use Committee (No. 201) of Henan University of Science and Technology (Luoyang, Henan, China).
Preparation of the second-generation merozoites
At 120 h postinoculation, chickens were killed through CO2 asphyxiation, and the pooled caecal tissues from 10 randomly selected chickens in each replicate were used for the preparation of the second-generation merozoites in accordance with a previously described method [19-21]. Briefly, caecal tissues were cut into pieces, then incubated with enzyme digestion solution (0.12 mol/L NaCl, 3.0 mmol/L K2HPO4·3H2O, 9 mmol/L CaCl2, 1 mg/mL hyaluronidase, 1 mg/mL BSA and 0.02 mol/L Tris; pH = 7.4) at 37 °C for 60 min. After the filtration and centrifugation of the digestion mixture, the collected sediment was incubated with red blood cell lysis buffer (Beyotime, Shanghai) at 4 °C for 10 min. Lysates were removed through centrifugation, and the merozoites were collected through density gradient centrifugation. The collected merozoite samples were subjected to total RNA preparation, Western blot and immunofluorescence assay successively. Three samples in each group were used for each experiment.
Total RNA preparation and cDNA synthesis
The total RNA of merozoites was extracted with TRIzol® Reagent (Invitrogen, USA) according to the manufacturer’s procedure, and cDNA was synthesised with EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing).
Amplification of the EtCRK2 gene
According to the sequence (GenBank: AY508221.1), specific primers P1: 5′-AAGGGACTTACGGAGTGGTTTA-3′ and P2: 5′-TGAATTTACGTGAATATGTTGG-3′ were designed to amplify the EtCRK2 gene and an ORF from a cDNA template. Amplified fragments were separated through 1% agarose gel electrophoresis and isolated using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 according to the manufacturer’s instructions. The purified amplified fragments were ligated into the pMD-19T vector and then transformed into Escherichia coli strain DH5α. The recombinant clone of pMD-19-EtCRK2 was identified through PCR and sequenced by Shanghai Sangon Biotech Co., Ltd. The positive plasmid was named pMD-19-EtCRK2.
Prokaryotic expression and purification of EtCRK2
Using pMD-19-EtCRK2 as a template, the ORF of EtCRK2 was amplified using primers containing EcoR I (P2-F: 5′-CGGGAATTCATGGAGCGCTACAAGA-3′) or Hind III (P2-R: 5′-TCTTGTAGCGCTCCATGAATTCCCG-3′) restriction sites. Amplification products and pET-28a (+) were digested by EcoR I and Hind III prior to ligation. The recombinant plasmid pET-28a-EtCRK2 was transformed into E. coli Rosetta (DE3) competent cells. Expression was induced at 37 °C with 0.5 mM IPTG, recombinant EtCRK2 (rEtCRK2) protein was purified through Ni-NTA His Bind Resin affinity chromatography (Novagen, Germany) and then analysed using SDS-PAGE.
Polyclonal antibody preparation
For the production of polyclonal antibodies, purified rEtCRK2 protein was used as an antigen in subsequent immune procedures. rEtCRK2 protein emulsified with the same volume of Freund’s complete adjuvant (Sigma-Aldrich) was injected into New Zealand rabbits at a dose of 500 µg/rabbit. After 2 weeks, purified rEtCRK2 protein emulsified with Freund’s incomplete adjuvant (Sigma-Aldrich) was injected to the rabbits at a dose of 500 µg/rabbit for secondary immunisation. After a 2 week hiatus, the third and fourth rounds of immunisation were performed separately. Ten days after the last immunisation, serum samples were collected for the determination of specificity antibody titer. The specificity of the antibody was determined with Western blot. Briefly, 25 ng of recombinant protein was used for SDS-PAGE, and preimmunisation rabbit serum and antiserum were used as the primary antibodies. HRP conjugated goat antirabbit IgG (Biolab, Beijing) was subsequently used. HRP activity was revealed by enhanced chemiluminescence system using BeyoECL Plus substrate (P0018S, Beyotime Biotechnology, China). The antibody titer was analysed with indirect enzyme linked immunosorbent assay (ELISA) [38]. Briefly, 500 ng/well rEtCRK2 protein was used to coat 96-well plates at 4 °C overnight, and the antiserum diluted with PBS at 1:2K, 1:4K, 1:8K, 1:16K, 1:32K, 1:64K, 1:128K, 1:256K, 1:512K, 1:1024K and 1:2048K were incubated with the antigen at 37 °C for 2 h. After washing with TBST, horse radish peroxidase (HRP)-conjugated goat antirabbit IgG were added to all microwells at 37 °C for 2 h. After washing, the substrate solution tetramethylbenzidine was added, and the microplate was read at 450 nm in a microplate reader (Multiskan FC, Thermo Scientific, USA). Preimmunisation rabbit serum and PBS were used as the control.
EtCRK2 mRNA expression analysis
The mRNA expression level of EtCRK2 was quantified through real-time PCR with a CFX96 touch real-time PCR system (Bio-Rad, America) and TB Green® Premix Ex Taq™ GC (Perfect Real Time; Takara, Beijing). Each reaction was performed in triplicate, and the entire experiment was carried out in triplicate. E. tenella 18S rRNA was used as the control. The primer sequences are shown in Table 1. Relative mRNA expression was determined using the ΔΔ Ct method.
Western blot analysis
Purified merozoites were treated with RIPA lysis buffer (Beyotime, Shanghai) for Western blot analysis and determined using a BCA protein assay kit (Cwbio, Beijing) for concentration assessment. Pyrolysis products were dissolved in SDS-PAGE sample buffer (Beyotime, Shanghai), heated at 96 °C for 5 min, separated on 12% SDS-PAGE and electrotransferred to a polyvinylidene difluoride membrane (Membrane Solutions, USA). The membrane was detected with rabbit antiserum against EtCRK2 as the primary antibody or anti-β-tubulin monoclonal antibody (1:1000 dilution, K200059 M, Solarbio, China), followed by HRP conjugated goat antirabbit IgG ((Biolab, Beijing). HRP activity was revealed by enhanced chemiluminescence system using BeyoECL Plus substrate (P0018S, Beyotime Biotechnology, China) and Image J Software. The independent experiments were performed in triplicate.
Immunofluorescence test
In accordance with our previously described method with minor modifications [22], the merozoites were prepared into smears and fixed with 4% paraformaldehyde. The merozoites were washed three times with PBS, then permeabilised with 1% Triton X-100 (Sangon-Biotech, Shanghai) and blocked with 2% BSA-PBS at 4 °C overnight. Rabbit antiserum against EtCRK2 (1:2000 dilution) was used as the primary antibody at 37 °C for 1 h, and FITC-conjugated goat antirabbit IgG (Servicebio, Wuhan) with 1:100 dilution was used as the second antibody at 37 °C in the dark for 1 h. Finally, the merozoites were stained with 4′,6′-diamidino-2-phenylindole (Boster, China) at room temperature for 30 min, and 50 μL of anti-fade mounting medium (Sangon Biothch, Shanghai) was used to close the coverslip. Images were visualised with a confocal laser scanning microscope (LSM 800, ZEISS) at a lex/em of 492 nm/520 nm and 358 nm/461 nm.
Statistical analysis
Data were expressed as mean ± standard deviation. Student’s t test was used in statistical analyses. Values of P < 0.05 and P < 0.01 were considered significant.