This study was reviewed and approved by the Care and Use of Laboratory Animals Department of the Xinjiang Academy of Agricultural and Reclamation Sciences (Shihezi, China) (Approval No. 2019-012, April 9, 2019). All animals were handled in strict accordance with the animal protection laws of the People’s Republic of China (a draft of the animal protection law was released on September 18, 2009) and the National Standards for Laboratory Animals in China (executed on January 5, 2002).
Female BALB/c mice aged 6–8 weeks were purchased from the Laboratory Animal Center of Xin Jiang Medical University (Xingjiang, China). Nine 8-month-old beagle dogs of mixed breed were obtained from the Institute of Musk Deer Breeding.
Hydatid cysts were collected from the livers of naturally infected sheep at an abattoir in Urumqi, Xinjiang Province, China. The fertility of the cysts was confirmed by observing the protoscoleces (PSCs) within the cysts under light microscopy. Once identified, the PSCs were separated and treated as previously described . Briefly, 2000 PSCs were cultured in 1 ml of Roswell Park Memorial Institute 1640 medium with 10% bovine serum albumin (Hyclone, Logan, UT, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Echinococcus granulosus adult worms were obtained from an 8-month-old dog 28 days after being artificially infected with PSCs.
The cDNA sequence of EgHCDH was downloaded from the National Centre for Biotechnology Information (NCBI), and the physicochemical parameters were analyzed using ProtParam of ExPASY (https://web.expasy.org/protparam/). The open reading frames (ORFs) of EgHCDH were analyzed using ORF Finder (https://www.ncbi.nlm.nih.gov/orfnder/). Signal peptides and transmembrane areas were predicted using the SignalP server available online (http://www.cbs.dtu.dk/services/SignalP-3.0/) and the TMHMM-2.0 software package (http://www.cbs.dtu.dk/services/TMHMM-2.0/). Tertiary (three-dimensional) structures were modeled using SWISS-MODEL (http://swissmodel.expasy.org/).
EgHCDH-similar sequences were aligned, and phylogenetic trees were constructed using MEGA software (version 5.05) with the neighbor-joining method .
Expression and purification of EgHCDH
Total RNA was extracted using an RNA prep Pure Tissue Kit (Nanjing Vazyme Biotech, Nanjing, China). First-strand cDNA was synthesized using a reverse transcription system kit (Nanjing Vazyme Biotech). The full coding sequence of the non-membrane region of EgHCDH was amplified using the primers 5′- CGG GAT CCA TGT CAG CCG GTG CTG G-3′ (BamHI) and 5′-GAC GTC GAC TCA CTG TTT TTC CTT GAC AAT GCG C-3′ (SalI). Amplification reactions were performed using the following cycling conditions: pre-denaturation at 95 °C, 5 min; then denaturation at 95 °C/30 s, 62 °C/30 s, 72 °C/1 min; with a final extension at 72 °C, 5 min. Through sequencing, digestion and ligation, EgHCDH was ligated into the pET32a (+) plasmid (Novagen, Darmstadt, Germany) and transformed into Escherichia coli BL21 (DE3) cells (Tiangen, Beijing, China). Protein expression was induced with 1 mM isopropyl-1-thio-β-d-galactopyranoside at 37 °C for 6 h. The rEgHCDH protein was purified using Ni2+ affinity chromatography (Bio-Rad, Hercules, CA, USA), with the the purity of the final product determined by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The concentrations of the purified protein were determined using a NanoDrop 2000c spectrophotometer (Bio-Rad).
Preparation of polyclonal antibodies
Each mouse was subcutaneously immunized with 50 µg rEgHCDH emulsified in Freund’s complete adjuvant (Sigma-Aldrich), followed by three repeated inoculations with 50 µg rEgHCDH emulsified in Freund’s incomplete adjuvant (Sigma-Aldrich) every 2 weeks. Control mice were immunized with phosphate-buffered saline (PBS). Sera were collected at post-immunization days 0, 14, 28, 42 and 56.
Total proteins extracted from PSCs and rEgHCDH protein were separated by 10% SDS-PAGE and then transferred onto PVDF membranes. The membranes were blocked in 5% (w/v) skim milk at 37 °C for 2 h and then incubated with E. granulosus-infected dog sera or anti-rEgHCDH mouse sera (1:100 v/v dilutions) overnight at 4 °C. The membranes were then washed and incubated with horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG or rabbit anti-dog IgG (1:5000 v/v dilution) for 2 h. Signals were visualized using an ECL kit (Pierce ECL Western Blotting Substrate; Thermo Scientific, Waltham, MA, USA) and a molecular imaging system (Bio-Rad).
Reverse-transcription quantitative PCR
Total RNA was extracted from PSCs and strobilated worms, and cDNA was synthesized as described above. Reverse-transcription quantitative PCR (RT-qPCR) was performed on a LightCycler 96 real-time fluorescent quantitative PCR system (Bio-Rad). The SYBR Green I dye was used, and the primers for EgHCDH were 5′-TAG AGA TGT GGG AGC GTT GC-3′ and 5′-TCC GTA ACC GCA CTT TTT GC-3′. Expression of the actin gene was used as an internal control for normalization. Primers specific to E. granulosus actin were 5′-CGC ATC GGT CGT CTT GTG TT-3′ and 5′-CGG TAA TCC TGT GGC TGT CAA T-3′. The data were analyzed using the 2−ΔΔCT method .
To determine the tissue location of EgHCDH, fresh PSCs and adult worms were first fixed in 4% paraformaldehyde phosphate buffer overnight, followed by permeabilization with 1% Triton X-100 for 30 min and then soaking in 0.01% Triton X-100 for 1 h at 4 °C. After three washes with 0.01× PBS, the sections were blocked in 5% (w/v) skim milk at 37 °C for 2 h, then incubated with anti-rEgHCDH mouse IgG or native mouse IgG (1:100 v/v dilutions in PBS + Tween-20 [PBST]) overnight at 4 °C, washed again and finally incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (H + L) (1:1000 v/v dilution in PBST) at room temperature for 2 h in the dark. After four washes with PBST, the sections were examined under a fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany).
Vaccination and parasite challenge
Nine beagle dogs were randomly divided into three groups. Group 1 was vaccinated with rEgHCDH mixed with Quil A adjuvant (Sigma-Aldrich); Group 2 was vaccinated with Quil A adjuvant only (Sigma-Aldrich); and Group 3 was vaccinated with PBS as a control group. One dose of vaccine comprised 200 μg of soluble rEgHCDH and 100 μg of Quil A in 825 μl of PBS. The mixture was stirred overnight at 4 °C prior to being used as vaccine. One week after the last immunization, all three groups were orally challenged with 100,000 PSCs. For safety reasons, all dogs were euthanized and necropsied at 28 days post-infection and E. granulosus worms were collected and counted .
Indirect enzyme-linked immunosorbent assay
The enzyme-linked immunosorbent assay (ELISA) conditions were optimized by checkerboard titration of the rEgHCDH antigen and sera. The purified rEgHCDH protein (5 μg/ml) was diluted in 0.1 M carbonate buffer (pH 9.6). The ELISA plates were coated with the diluted antigen solution overnight at 4 °C, then washed with PBST and incubated with 5% skim milk for 2 h at 37 °C. The wells were then washed thoroughly and incubated with 100 μl of serum samples (1:80) in PBST at 37 °C for 1.5 h. After washing, the HRP-labeled rabbit anti-dog IgG (1:3000; Solarbio, Beijing, China) was added to the plates and incubated at 37 °C for 1.5 h, following which the wells were washed again and incubated with the substrate 3, 3′, 5, 5′-tetramethylbenzidine (Tiangen, Beijing, China) at 37 °C for 15 min. Finally, the reaction was stopped with 100 μl of 1 M H2SO4 and the optical density at 450 nm was determined.
Detection of cytokines
The immune stimulation effect of rEgHCDH protein in dogs was evaluated using quantitative ELISA on day 28 post-challenge. Dog cytokine ELISA Quantitation kits (Jianglaibio, Shanghai, China) were used to quantify interleukin (IL)-1, IL-4, IL-5, IL-6, and interferone gamma (IFN-γ). An ELISA strip in its aluminum foil bag was left at to equilibrate at room temperature for 60 min; then the strip was removed from the bag and placed in the the standard sample well. Each well received 50 μl of dog serum at a different concentration. Next, 100 μl of HRP-labeled antibody was added to each well. The reaction wells were sealed with a sealing film and incubated at 37 °C for 60 min. The liquid was then discarded, and the plate was patted dry using absorbent paper. Each well was filled with 350 μl of PBST and left to stand for 1 min. The detergent was removed, the plate was patted dry using absorbent paper and the detergent washing step was repeated five times. Each well was then incubated with 50 μl of substrate A and 50 μl of substrate B at 37 °C for 15 min, following which 50 μl of termination solution was added to each well, and the optical density value of each well was measured at 450 nm within 15 min.
All statistical analyses were performed using SPSS version 22.0 software (SPSS IBM Corp., Armonk, NY, USA). All data analyses and graphs were performed using GraphPad Prism 6.0 software package (GraphPad Software Inc., San Diego, CA, USA). Differences between groups were considered significant if the P value was ≤ 0.05. All experiments were repeated a minimum of three separate times.