Inoculum and Drug
Ocysts of E. tenella (Luoyang strain) were passaged before inoculation. Diclazuril (>99%) (Shanghai Veterinary Research Institute, CAAS, China) was supplied at a dose of 1 mg/kg in broiler feed.
Chickens and Treatment
1-day-old male Chinese Yellow broiler chickens were obtained from Gonghua Commercial Hatchery, Luoyang, China. The chickens were kept on wire-floored batteries for 14 days under coccidiosis-free conditions. On the 14th days, healthy 90 chickens with similar weight were randomly divided into two groups of 45 with 3 biological replicates of 15 in each group: (1) Chickens that were challenged with E. tenella sporulated oocysts and that received commercial diet without drugs were designated as 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 designated as the infected/diclazuril group. Inoculation was performed with a dose of 8×104 sporulated oocysts/chicken on days 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 post inoculation, chickens killed by CO2 asphyxiation, cecal tissues from randomly selected 10 chickens pooled in each replicate were used for preparation of the second-generation merozoites in accordance with our previously described method [19-21]. The collected merozoites samples were subjected for RNA preparation, Western blot and immunofluorescence assay, respectively. Thus, there were three samples in each group for each experiment.
Total RNA Preparation and cDNA Synthesis
In accordance with the manufacturer’s procedure, the total RNA of merozoites was extracted with TRIzol® Reagent (Invitrogen, USA), and cDNA was synthesized by using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing).
Amplification of EtCRK2 Gene
Based the sequence form GenBank: AY508221.1, specific primers P1: 5'-AAGGGACTTACGGAGTGGTTTA-3' and P2: 5'-TGAATTTACGTGAATATGTTGG-3' were used to amplify EtCRK2 gene including an open reading frame (ORF) from a cDNA template. Amplified fragments were separated through 1% agarose gel electrophoresis and isolated using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (Takara, Beijing) following the manufacturer’s instructions. The purified amplifying fragments were ligated into the pMD-19T vector (Takara, Beijing), and then transformed into Escherichia coli strain DH5α (Takara, Beijing). The recombinant clone of pMD-19-EtCRK2 was identified through polymerase chain reaction (PCR) and sequenced by Sangon Biotech (Shanghai) Co., Ltd. The positive plasmid was named pMD-19-EtCRK2.
Prokaryotic Expression and Purification of EtCRK2
Using positive pMD-19-EtCRK2 as 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 that were 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 analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Polyclonal Antibody Preparation
To produce polyclonal antibody, purified rEtCRK2 protein was used as antigen in the following 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 mg/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 mg/rabbit for secondary immunization. Then, at the interval of a 2-week hiatus, the third and fourth immunizations were performed separately. Ten days after the last immunization, serum samples were collected and determined by indirect enzyme linked immunosorbent assay (ELISA) . Briefly, 500 ng/well recombinant protein was used to coat the 96-well plates at 4 °C overnight, the antiserum were diluted with PBS at 1∶2K, 1∶4K, 1∶8K, 1∶16K, 1∶32K, 1∶64K, 1∶128K, 1∶256K, 1∶512K, 1∶1024K, 1∶2048K, and then incubated with antigens at 37 °C for 2 hours. After washing by TBST, Horse Radish Peroxidase (HRP)-conjugated goat anti-rabbit IgG were added to all microwells at 37 °C for 2 hours. After washing, the substrate solution tetra-methyl ben-zidine (TMB) was added and the microplate was read at 450 nm in a microplate reader (Multiskan FC, Thermo Scientific, USA). Preimmunization rabbit serum and PBS were used as the control. The specificity of antibody was determined by Western blot, briefly, 25 ng recombinant protein was used for loading for SDS-PAGE, preimmunization rabbit serum and antiserum was used as the primary antibodies followed by HRP conjugated goat anti-rabbit IgG (Biolab, Beijing). HRP activity was revealed by enhanced chemiluminescence system using BeyoECL Plus substrate (P0018S, Beyotime Biotechnology, China)
EtCRK2 mRNA Expression Analysis
The mRNA expression level of EtCRK2 was quantified through real-time PCR using the 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 by using the ΔΔ Ct method.
Western Blot Assay
Purified merozoites were treated with RIPA lysis buffer (Beyotime, Shanghai) for Western blot analysis and determined by 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 then electrotransferred to a polyvinylidene difluoride membrane (Membrane Solutions, USA). The membrane was detected with rabbit antiserum against EtCRK2 as the primary antibodies or anti-β-tubulin monoclonal antibody (1:1000 dilution, K200059 M, Solarbio, China), followed by HRP conjugated goat anti-rabbit 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.
In accordance with our previously described method with minor modifications , the merozoites were prepared into smears and fixed with 4% paraformaldehyde. After washing three times with PBS, the merozoites were permeabilized with 1% Triton X-100 (Sangon-Biotech, Shanghai) and blocked with 2% BSA-PBS. Rabbit antiserum against EtCRK2 (1∶2000 dilution) was used as the primary antibody, and FITC-conjugated goat anti-rabbit IgG (Servicebio, Wuhan) with 1∶100 dilution was used as the second antibody. Finally, the merozoites were stained with 4’,6’-diamidino-2-phenylindole (DAPI) (Boster, China). Images were visualized with a confocal laser scanning microscope (LSM 800, ZEISS) at an lex/em of 492 nm/520 nm and 358 nm/461 nm, respectively.
Data were expressed as mean ± standard deviation. Student’s t test was used for statistical analyses. Values of P < 0.05 and P < 0.01 were considered significant.