C. sinensis metacercariae were collected from naturally infected Pseudorasbora parva in the endemic area of Qiqihar, Heilongjiang province, China. Muscular tissue was digested with artificial digestive juice (1% pepsin-hydrochloric acid, Aladdin, China). White Japanese rabbits were purchased from Yisi Experimental Animal Technology Corporation (Changchun City, Jilin Province, China). Fecal examination was conducted before selection to exclude any prior infection with C. sinensis. This study was approved by the Animal Health, Animal Care, and Use Committee of the Heilongjiang Bayi Agricultural University. Twenty rabbits (8-9-month-old) negative for C. sinensis were selected and randomly divided into two groups: control group and C. sinensis-infected group (n=10 each). Rabbits in the experimental group were infected orally with 500 viable metacercariae, while rabbits in the control group were mock-inoculated with 0.85% w/v NaCl solution without metacercariae. After 18 days of infection, the feces of rabbits were collected for fecal examination. Blood samples from each animal were collected aseptically into tubes without anticoagulant, and at 7, 14, 35, and 77 dpi, sera were separated by centrifugation and preserved at -80°C for further use. Positive sera for F. hepatica and S. japonicum were both obtained from artificially infected rabbits. The rabbits were orally infected with 40 viable F. hepatica metacercariae, and the sera were obtained at 90 dpi, stored at the College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University. After 42 dpi, positive sera of S. japonicum were acquired from rabbits, which infected with 1000 ± 10 S. japonicum cercariae, the sera were provided by the Laboratory Animal Center of Shanghai Veterinary Research Institute, Chinese Academy of Agriculture Sciences.

CSESPs were prepared according to standard procedures (Pak et al., 2017). Briefly, C. sinensis adults were separated from the bile ducts of infected rabbits, washed three times with PBS (stored at 37°C), and preincubated in Locke’s medium (NaCl 8.9 g, KCl 0.42 g, NaHCO₃ 0.2 g, and CaCl₂ 0.24 g (wt/vol) at 37°C and 5% CO₂ for 1 h. Thereafter, the parasites were transferred to fresh medium and incubated for 48 h; the medium was changed at 6-h intervals. Finally, the collected cultures were centrifuged at 10 000 g for 30 min at 4°C, to obtain the supernatant, filtered (0.22 μm), and stored at -80°C.

The protein A/G plus-agarose immunoprecipitation kit (Santa Cruz Biotechnology, USA) was used for Co-IP according to the manufacturer’s instructions. Briefly, 200 μL of serum positive and negative (7, 14, 35, and 77 dpi) for C. sinensis was added into tubes; rabbit sera positive for S. japonicum and F. hepatica were handled in the same manner. Protein A/G plus-agarose beads (30 μL) were added to each tube and incubated overnight at 4°C. The beads were pelleted by centrifugation at 1,000 g for 30 s at 4°C. Five hundred micrograms C. sinensis ESPs (500 µg) was precleared by incubation with protein A/G plus-agarose beads, and incubated for 1.5 h at 4°C. The pellet, collected by centrifugation at 1,000 × g for 5 min at 4°C, was washed three times with PBS buffer, and resuspended in 50 μL SDS loading buffer. The samples were then boiled at 100°C for 10 min. Ten microliters of the sample was analyzed by SDS-PAGE, and the remaining 40 μL was stored for mass spectrometry identification.

The stained protein bands were cut into 1 mm³ pieces and washed twice with 200 µL of mass spectrometry (MS) water for 10 min. The gels were detained with 50% acetonitrile (ACN) in 50 mM ammonium bicarbonate (ABC), dehydrated by washing with 100% ACN until the gel turned white, and then washed twice with 200 µL of MS water for 10 min/wash. ACN was added to induce dehydration, which occurred until the colloidal particles turned white. Thereafter, they were vacuum dried for 10 min. Proteins on the gels were treated with 200 µL of 10 mM DTT for 1 h at 37°C and subsequently alkylated with 200 µL of 55 mM iodoacetamide (IAM) for 30 min in the dark. The gels were then washed with digested buffer and treated with ACN, as described above. The suspension was washed with the following solutions: MS water (once), ACN (once), MS water (once), and ACN (once) for 10 min/wash, each experiment group was performed three times.

Peptides were separated by liquid chromatography using an Acclaim Pep Map 100 column and an EASY-Spray column on an EASY-NLC 1,000 system. The flow rate was set to 0.600 μL/min, and gradient elution was performed for 88 min. The gradient was generated using mobile phase A, which comprised of 100% ddH₂O containing 0.1% formic acid, and mobile phase B, which consisted of 100% ACN containing 0.1% formic acid. Label-free mass spectrometry was performed using a QE HF-X mass spectrometer. The scan events were composed of one single full MS scan and a 3 s MS/MS scan dependent on the previous scan data. The spray voltages were set at 3.8 kV, and the heated capillary temperature was 320°C. The parameters of the MS/MS scan were as follows: resolution, 15,000; auto gain control target, under 2 × 10⁴; maximum isolation time, 30 ms; and normalized collision energy, 27%, each experiment group was performed three times.

Specific proteins were analyzed using Proteome Discoverer 2.4.1.15. Trypsin was employed as the enzyme, which cleaved after all lysine and arginine residues, with up to two missed cleavages allowed. Carbamidomethylating of cysteine was specified as fixed modification, and protein N-terminal acetylation, oxidation of methionine, and pyro-glutamate formation from glutamine were considered as variable modifications for all groups. The raw file of the mass spectrum was identified and analyzed using the commercial software, Max Quant (Thermo Fisher Scientific, Waltham, MA, USA). The precursor ion mass tolerance was set to 15 ppm and the fragment ion mass tolerance was set to 0.02 Da. All data were searched as a single batch with PSM and protein FDR set to 1% using a target decoy approach. The search parameters were as follows: species, C. sinensis; dynamic modification, oxidation; mass tolerance of the precursor ion, ± 15 ppm; fragment ion mass tolerance, ± 0.5 Da; and protein false discovery rate (FDR), 0.01. The maximum number of missed cleavages was two. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the proteins of C. sinensis was performed to select the most significant pathway and to analyze the relationship between the abundance of specific proteins of different pathways and the different periods of infection.

Cloning of cDNAs encoding myoferlin from C. sinensis was achieved using the Uniprot database (protein ID H2KUF2). Further, the bioinformatics, including domains, antigen epitopes, and associated interacting proteins, was analyzed. Ultimately, to achieve a better expression of the protein, it was truncated into two parts: Myof1 (aa 160-aa 660) and Myof2 (aa 600-aa 870). The two coding regions of myoferlin were amplified by polymerase chain reaction (PCR) using the following primers: 5’-GGA TCC ACC ATA AAG GAT GTC CGT CA-3’ and 5’-CTC GAG CAG ACA ATG ACT CGT AGC TCA T-3’ (Cs-Myof1), or 5’-GGA TCC CTA CCA CTA GTA AAA GAG CAC G-3’ and 5’-CTC GAG CAC AAC CAA AAG AAC GAT GTC TC-3’ (Cs-Myof2), the underline represents restriction enzyme cutting sites (BamHI, GGA TCC and XhoI, CTC GAG). The resulting DNA was digested, purified, and ligated into the BamHI and XhoI cloning sites of the pET32a plasmid designed for expression. Recombinant plasmids (pET32a-CsMyof1 and pET32a-CsMyof2) were transformed into E. coli BL21 (DE3) cells, and positive clones were selected. Following induction with 1 mM IPTG, bacterial cells were harvested and lysed by sonication in PBS buffer. The lysate was centrifuged at 12,000 g for 20 min at 4°C, and the supernatant was collected. The fusion protein was purified by gel cutting, frozen, and melted five times using liquid nitrogen repeatedly. After centrifugation at 12,000 g for 20 min at 4°C, the supernatant was collected for further use.

The purified recombinant CsMyof1 and CsMyof2 were subjected to SDS-PAGE (12% gel) and transferred to a Hybond-C pure NC membranes (Immobilon-P, 0.45 μm; Millipore). The membranes were subsequently blocked with blocking buffer (5% w/v skim milk in PBS-T buffer) overnight at 4°C. The sera of rabbits infected with C. sinensis, S. japonicum, F. hepatica, and the negative sera were used as primary antibodies at a dilution of 1:200; the sera were incubated with membranes for 2 h at 37°C. Thereafter, the membranes were washed three times with PBS-T buffer for 30 min. Further incubation was performed using goat anti-rabbit IgG antibody conjugated with HRP at a dilution of 1:5,000 in blocking buffer for 1 h. After washing five times with PBS-T buffer, the membranes were treated with a diaminobenzidine substrate solution for 10 min.

The metacercariae of C. sinensis were collected from Pseudorasbora parva ( Figure 1A ). Fecal examination and morphological examination of eggs were carried out ( Figure 1B ). The first identification was performed at 19 dpi, and all infections were confirmed at 25 dpi. After the infected rabbits were sacrificed, the worms were detected in their hepatobiliary tract ( Figure 1C ); however, no worms were observed in control rabbits. C. sinensis was collected and made into films ( Figure 1D ), which complies with its typical characteristics, such as the branched testicles and S-shaped excretory sac. The model of rabbits infected with C. sinensis was successfully established, and sera at different periods of infection (7, 14, 35, and 77 dpi) were collected. Adult C. sinensis worms were collected and cultured in vitro for 48 h to prepare the ESPs. The majority of proteins among the ESPs ranged in molecular weight from 10 to 170 ku ( Figure S1A ). A total of 334 proteins were obtained, among which 254 annotated proteins were obtained from the C. sinensis protein library by BLAST homology comparison and Uniport identification ( Figure S1B ).

An immuno-proteomic approach was used to identify the proteins secreted by C. sinensis, specifically using infection sera to pull down the C. sinensis ESPs that might be involved in host-parasite interactions. The results of the Co-IP assay revealed that the antibodies from serum at the periods of 7, 14, 35, and 77 dpi, could recognize and pull down the specific proteins from C. sinensis ESPs, with most proteins ranging in molecular weight from 15 to 170 ku ( Figure 2 ). There were 32, 18, 39, and 35 proteins in the first round of differential screening between positive and negative serum samples ( Figure 3A ). The second round of screening was carried out by comparing and analyzing different types of positive sera, including F. hepatica, S. japonicum, and C. sinensis, based on the first screening ( Figure 3B ). According to the LC-MS/MS analysis, the obtained data was compared with the C. sinensis protein library to screen the differential proteins in each period. There were 13, 9, 16, and 15 types of proteins specific to the periods of 7, 14, 35, and 77 dpi with C. sinensis, respectively ( Tables 1 – 4 ). Five proteins could only be detected in the early stage (7 dpi), which may explain their induction of an early response in the host, and three proteins were found to be specifically co-purified in all four periods of C. sinensis ( Table 5 ), including dynein light chain-1, dynein light chain-2, and myoferlin, which were characterized by label-free quantification and functional analysis. These three proteins are involved in body movement and energy metabolism, and were found to display high expression at different stages, thereby providing energy for C. sinensis. Myoferlin, an important protein related to tumors, is a member of the ferlin family and is a type II transmembrane protein with a single transmembrane domain at the C terminus.

Twenty-seven proteins were detected in C. sinensis, but not in F. hepatica and S. japonicum, where they participate in host-parasite interactions. KEGG analysis revealed that thease proteins were mainly involved in oxidative phosphorylation, membrane transport, signal transduction, and metabolism. The abundance of proteins was different in each period of infection; cystoskeleton, oxidative phosphorylation, and mRNA surveillance pathway-related proteins were highly expressed at the early infection stage (7 dpi), and may be involved in early immune evasion ( Figure 4A ). In the middle stage of parasite infection (14 dpi), membrane trafficking related proteins were evidently increased, and may play important roles in providing energy for parasites ( Figure 4B ). NOD-like receptor signaling pathway, glutathione metabolism, and lysosome-related proteins were highly expressed at the late infection stage (35 dpi and 77 dpi); lysosomes secreted by C. sinensis can effectively inactivate the proteins released by hosts to protect themselves ( Figures 4C, D ).

Myoferlin has two same domains: protein kinase C conserved region 2 (aa415-aa514, aa655-aa783). Regions with significant homology to the C2 domain have been identified in many proteins. The C2 domain is thought to be involved in calcium-dependent phospholipid binding and membrane-targeting processes, such as subcellular localization. Myoferlin also has two low-complexity domains (domain I, aa254-aa265 and II aa388-aa399) and a transmembrane region (aa887-aa909) ( Figure S2A ). The B cell epitopes of Myoferlin were predicted via IEDB (http://tools.iedb.org/), which resulted in ten linear epitopes ( Table S1 ) and five discontinuous epitopes ( Table S2 ). The predicted scores were all greater than 0.5, which indicated that the protein has a potential for binding antigens. The protein network interactions of myoferlin were also predicted (https://string-db.org/), which resulted in a total of 11 associated proteins ( Figure S2B ). Among these relative proteins, ATPase (ASAN1) is required for the post-translational delivery of tail-anchored proteins to the endoplasmic reticulum. ATPase also recognizes and selectively binds to the transmembrane domain of TA proteins in the cytosol. Vesicle-associated membrane protein 2 (VAMP2), a member of the SNARE family, is the first synaptobrevin studied in synaptic vesicles, and is regarded as a molecule that plays a key role in the process of cell growth, neurotransmission, hormone secretion, and insulin-dependent glucose uptake.

To achieve a better expression, myoferlin was truncated into two parts (Myof1 and Myof2) according to the antigenic epitopes provided by bioinformatics. The Myof1 and Myof2 (1,500 bp and 810 bp) genes were amplified using specific oligonucleotide primers ( Figure 5A ) and sub-cloned into the E. coli pET32a expression vector. After sub-cloning, the size of the inserted DNA was confirmed by restriction digestion with BamHI and XhoI ( Figure 5B ). Subsequently, the cloned Myof1 and Myof2 were successfully expressed in E. coli BL21 (DE3) cells. The recombinant proteins were purified by gel cutting and analyzed by SDS-PAGE ( Figure 6A ). The molecular weights of the recombinant proteins were determined to be approximately 60 ku (Myof1) and 31 ku (Myof2). The sera from rabbits infected with C. sinensis could be probed at different levels, and the sera positive for S. japonica, F. hepatica, and from naive rabbit could hardly be probed ( Figure 6B ), which indicates that myoferlin may be a potential antigen for the detection of clonorchiasis.