This study was conducted in accordance with the National Guidelines for Experimental Animal Welfare (Minister of Science and Technology, China, 2006). The animal experimental protocols were approved by the Life Science Ethics Committee, Zhengzhou University (No. SCXK 2015–0005).

Characterization of TsDNase II and their capacity to induce protective immunity was carried out based on the scheme (Figure 1).

The T. spiralis isolate (ISS534) was maintained in mice in our laboratory, which was originally obtained from an infected domestic pig in Henan Province, China. Female BALB/c mice, which were 6 weeks old, were purchased from the Experimental Animal Center of Henan Province. Intestinal epithelial cells (IECs) were prepared from normal fetal mouse intestines, and they were susceptible to the invasion by T. spiralis larvae (Ren et al., 2011; Long et al., 2015).

The mice experimentally infected with 300 T. spiralis ML were killed at 42 days post-infection (dpi). The carcasses were minced and digested at 43°C for 4 h with 0.33% pepsin (1:31000; Sigma) and 1% HCl. After filtration and sedimentation, the ML were counted by stereomicroscopy (Li et al., 2010). The IIL was collected from small intestines at 6 hours post-infection (hpi) (Liu R.D. et al., 2015). Sixty mice were orally infected with 5000 ML. Thirty mice were sacrificed at 3 dpi, and the rest were sacrificed at 6 dpi. The small intestine was cut along its length, washed by phosphate buffered saline (PBS), cut into pieces, and incubated in PBS at 37°C for 1.5 h on a 300 μm sieve. The AWs were released from the intestinal debris and collected by filtration with a 200 μm sieve and were subjected to differential sedimentation for 30 min. After washing using PBS supplemented with 100 U penicillin/ml and 100 μg streptomycin/ml, the worms were centrifuged at 600 ×g for 10 min, collected, and counted by microscopy (Sun et al., 2015). The NBL were collected from 6 days old female AWs, which were cultivated for 24 h in RPMI-1640 medium with 10% fetal bovine serum (FBS) (Long et al., 2015; Wu et al., 2016).

Crude extracts of worms of various stages (ML, IIL, 3 and 6 days old AWs, NBL) and 3 days old AW ES proteins were prepared (Wang et al., 2011; Li et al., 2015). Briefly, different worms were suspended in deionized water. The worms underwent five cycles of freezing-thawing. The worms were homogenized on ice in a glass tissue grinder, and then the worm fragments were further homogenized with ultra-sonication (200 W, 3 s, 99 times, 0°C). The supernatant was centrifuged at 15,000 g for 20 min at 4°C and was collected.

The complete cDNA sequence of TsDNase II-1 (GenBank: AY963695.1) and TsDNase II-7 (GenBank: AY963701.1) were analyzed by the open reading frame (ORF) finder program in the NCBI website. The conserved domains of the two proteins were predicted using the Conserved Domain Database of NCBI (Long et al., 2014). Amino acid sequences of TsDNase II-1 and TsDNase II-7 were submitted to an online tool¹ for predicting their molecular weights (MWs) and isoelectric points (pI). The signal peptide was predicted using the SignalP 4.1 Server (Petersen et al., 2011). The amino acid sequences were aligned with DNase II of other Trichinella species and other organisms using Clustal X, and phylogeny was estimated using the neighbor-joining (NJ) method by MEGA 6.0 (Liu L.N. et al., 2013; Zhang et al., 2015).

Total RNA of T. spiralis worms of different stages (ML, IIL, 3 and 6 days old AWs, and NBL) was extracted by Trizol (Invitrogen) and then transcribed into first-strand cDNA with the PrimeScript RT Reagent Kit (Takara) (Ren et al., 2013). The transcription levels of TsDNase II-1 and TsDNase II-7 genes were quantified by qPCR (Liu R.D. et al., 2013). Specific primers for qPCR of TsDNase II-1 (5′-TCTGCAACAGTACCCGCTTTC-3′; 5′-GGCGCAGTCAAGTTCTTATGCTA-3′) and TsDNase II-7 (5′-GAGAAAATCATGCGCTGGTC-3′; 5′-ATTACATGCACAGTAAA CAGTAG-3′) were designed by Primer Premier 5. The transcription levels of TsDNase II-1 and TsDNase II-7 genes were standardized by the transcription level of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5′-AGATGCTCCTATGTTGGTTA TGGG-3′; 5′-GTCTTTTGGGTTGCCGTTGTAG-3′) and then determined by the comparative Ct (2^-ΔΔCt) method (Schmittgen and Livak, 2008; Sun et al., 2018a). Each experiment was repeated three times, and triplicates of each sample were performed.

Total RNA was extracted from 3 days old AWs using Trizol (Invitrogen). The complete cDNA sequences of TsDNase II-1 and TsDNase II-7 were amplified by PCR with the specific primers carrying BamHI and HindIII restriction enzyme sites (in bold) (TsDNase II-1: 5′-CAGGATCCGATTACCAATGCAAAGAACA-3′ and 5′-CCAAGCTTTTAGGTGCATCCA TCCAAGT-3′; TsDNase II-7: 5′-CGGGATCCGACTTCCAATGTTTCCAAGA-3′ and 5′-CCCAAGCTTTTAGGTGCATGCATCCAAGT-3′). The PCR products were cloned into the vector pMD19-T (Takara, China) and then subcloned into the vector pQE-80L (Novagen, United States) with an N-terminal His₆ tag. The recombinant plasmids were transformed into the E. coli DH5α strain (Sangon Biotech, China) for expression. The Ni-NTA-Sefinose resin (Sangon Biotech, China) was used to purify rTsDNase II-1 and rTsDNase II-7. After purification, rTsDNase II-1 and rTsDNase II-7 were identified by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Liu et al., 2017).

Twenty-two mice were injected subcutaneously with 20 μg rTsDNase II-1 or rTsDNase II-7 emulsified with complete Freund’s adjuvant. Two boost immunizations were carried out at an interval of 10 days using the same amount of rTsDNase II-1/7 emulsified with incomplete Freund’s adjuvant (Cui et al., 2013c; Song et al., 2018a). The immunized mice were bled at 10 days after the final immunization, and the serum samples were isolated. Pre-immune serum and T. spiralis infection serum were collected from mice before infection and at day 42 post-infection and were used as controls for the in vitro invasion test and in vitro antibody-dependent cell-mediated cytotoxicity (ADCC) experiment.

Samples consisted of crude extracts of ML, IIL, 3 and 6 days old AW, NBL, and AW ES proteins, rTsDNase II-1, and rTsDNase II-7; these proteins were analyzed by SDS-PAGE with 10% polyacrylamide gels (Wang L. et al., 2013). The gel was subsequently transferred onto nitrocellulose membranes (Millipore, United States) (Wang et al., 2012). The membrane was blocked at 37°C for 1 h with 5% skim milk in tris buffered saline with Tween 20 (TBST) (pH 7.4) and then incubated overnight at 4°C with 1:100 dilutions of anti-rTsDNase II-1 serum or anti-rTsDNaseII-7 serum. Anti-mouse IgG-horse radish peroxidase (HRP) conjugate (Southern Biotechnology, United States) was diluted to 1:10,000 and incubated with the membranes at 37°C for 1 h. The substrate was 3, 3′-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich) (Wang B. et al., 2013; Yang et al., 2015).

Anti-rTsDNase II-1 serum and anti-rTsDNase II-7 serum were utilized to detect the expression and localization of natural TsDNase II-1 and TsDNase II-7 in the T. spiralis worms of various stages (Zhang et al., 2013; Sun et al., 2018b). The worms were fixed using 4% paraformaldehyde, then embedded in paraffin after dehydration, and microtomed into 3 μm-thickness sections. The indirect immunofluorescence test (IIFT) with intact worms and their sections was performed (Liu et al., 2018). The worms and sections were blocked with 5% goat serum for 1 h and then incubated with 1:10 dilution of different sera (anti-rTsDNase II-1 or anti-rTsDNase II-7 serum, T. spiralis infection serum, or pre-immune serum). Subsequently, they were reacted for 1 h at 37°C with anti-mouse IgG-FITC conjugate (1:100; Santa Cruz Biotechnology, United States) and finally observed under fluorescent microscopy (Olympus, Tokyo, Japan) (Cui et al., 2015b).

To observe the suppression effects of anti-rTsDNase II-1 and anti-rTsDNase II-7 sera on the invasion of intestinal epithelial cells (IECs) by T. spiralis, the IIL were utilized for the invasion test (Yang et al., 2015). The IECs were grown to confluence on 6-well culture plates. One hundred and fifty IIL were firstly mixed with 2 ml DMEM semisolid medium with 1.75% agarose, and then the mixture containing larvae was added onto the IEC monolayer (Xu et al., 2018). Before use, the medium was supplemented with 1:50 to 1:800 dilutions of anti-rTsDNase II-1 or anti-rTsDNase II-7 sera and 1:50 dilutions of pre-immune serum or T. spiralis-infected mouse serum (ManWarren et al., 1997). After being incubated for 2 h at 37°C, the invaded worms within the monolayer were examined. The invaded and migrated larvae within the monolayer were counted as invaded larvae, whereas the larvae suspended in media were numbered as non-invaded larvae (Cui et al., 2015a; Zhang et al., 2016). Four independent tests for anti-rTsDNase II-1 or anti-rTsDNase II-7 sera, infection serum, and pre-immune serum were performed, and three repeats were served to evaluate larva invasion rate for each kind of serum (Long et al., 2015).

The cytotoxicity of anti-rTsDNase II-1 and anti-rTsDNase II-7 antibodies on the NBL were evaluated as reported before (Moskwa, 1999; Liu L.N. et al., 2015). One hundred and fifty NBL were cocultured for 72 h at 37°C with 1 × 10⁵ mouse peritoneal exudate cells (PECs) in a 96-well plate in the presence of various sera (1:5 to 1:1,000 dilutions of anti-rTsDNase II-1 serum, anti-rTsDNase II-7 serum, pre-immune serum, or T. spiralis-infected serum). The larva viability was assessed according to its mobility and morphology under light microscopy. The live NBL were mobile and wriggling, and the dead NBL were straight and immobile or disintegrated (Jiang et al., 2012). The cytotoxicity was calculated as the percentage of dead NBL to total number of NBL in each experiment (Liu et al., 2018).

One hundred mice were divided into five groups of 20 animals each. Vaccination groups of mice were inoculated subcutaneously with 20 μg proteins (rTsDNase II-1, rTsDNase II-7, or 1:1 mixed of these two proteins). Equal volume of Freund’s adjuvant was used to emulsify the rTsDNase II protein, and the mice were boosted twice at an interval of 10 days (Gu et al., 2017). Two control groups received only adjuvant or PBS. Tail blood was collected on 0, 10, 20, and 30 days after the first vaccination (Liu et al., 2017).

Indirect enzyme-linked immunosorbent assay (ELISA) with rTsDNase II-1, rTsDNase II-7, their mixtures, or AW ES proteins was used to detect serum specific total IgG and its subclass (IgG1 and IgG2a) in vaccinated mice (Long et al., 2014; Pompa-Mera et al., 2014). Plate was coated with 1 μg/ml of rTsDNase II or 2.5 μg/ml AW ES proteins at 4°C overnight and then blocked with 5% nonfat milk in PBST. After washing, the plate was incubated with immune serum (1:100) at 37°C for 1 h. After washing again, HRP-conjugated anti-mouse IgG, IgG1, or IgG2a (1:5,000; Sigma-Aldrich) was added and incubated for another 1 h. The compound o-phenylenediamine dihydrochloride (OPD; Sigma-Aldrich) was used as the substrate for HRP. The optical density (OD) at 490 nm was determined by an ELISA reader (Tecan, Schweiz, AG, Switzerland).

To evaluate immune protection, each mouse was orally administrated with 300 ML at 10 days following the third immunization. Intestinal AWs were collected from 10 mice at 5 dpi (Yang et al., 2010), and the ML at 42 dpi were recovered by artificial digestion of the carcasses of the other 10 mice (Liu et al., 2015a). Immune protection was evaluated according to the number of AWs or ML recovered from the vaccinated group compared with those from the PBS group (Liu et al., 2015b; Xu et al., 2017).

The statistical analyses of the data were performed using SPSS for Windows, version 17.0. Pearson correlation analysis was used for analyzing the relationship between serum dilutions and the inhibition of IIL invasion or ADCC. The analyses of intragroup or intergroup differences of transcription levels in the different stages of T. spiralis, cytotoxicity, specific antibody responses, and immune protection were performed using one-way ANOVA or Student’s t-test. For parasite invasion of IECs assay, the analyses of intergroup differences were performed by chi-square test. All data were expressed as the mean ± standard deviation (SD), and the differences were considered significant at P < 0.05.

The full-length of the TsDNase II-1 cDNA sequence was 1221 bp, and the predicted ORF (1–1044 bp) encoded 347 amino acids. The predicted MW of TsDNase II-1 is 38.06 kDa with a pI of 8.85. The complete TsDNase II-7 cDNA sequence was 1161 bp long, and the predicted ORF (4–1050 bp) encoded 348 amino acids. The predicted MW of TsDNase II-7 is 38.22 kDa, and its pI is 7.56. In addition, TsDNase II-1 and TsDNase II-7 have signal peptides, and both the cleavage sites are positioned between 18 and 19 amino acids. The putative active sites and the predicted catalytic sites of TsDNase II-1 and TsDNase II-7 are shown in Figure 2. The phylogenetic analysis of TsDNase II-1 and TsDNase II-7 with the DNase II of other organisms shows that TsDNase II-1 has the closest evolutionary status with T. nelsoni and Trichinella genotype T9, while TsDNase II-7 is genetically related to Trichinella murrelli and Trichinella britovi (Figure 3).

A qPCR analysis was performed to quantify the transcription of the TsDNase II-1 and TsDNase II-7 genes in the T. spiralis worms of various stages (ML, IIL, 3 days old AW, 6 days old AW, and NBL), and the results showed that the transcription of TsDNase II-1 and TsDNase II-7 was detected at all the developmental stages of T. spiralis. The TsDNase II-1 transcription level in 3 days old AWs was statistically significantly higher than those of the worms in the other stages (ML, IIL, 6 days old AW, and NBL) (P < 0.05) (Figure 4A). The TsDNase II-7 transcription level in 3 days old AWs was obviously higher than those of the ML and IIL stages but lower than those of the 6 days old AW and NBL stages (P < 0.05) (Figure 4B).

The cloning and sequencing results revealed that the cDNA ORF of TsDNase II-1 and TsDNase II-7 without signal peptides were about 987 bp (57–1044 bp) and 990 bp long (61–1050 bp), and the predicted MW were 36.01 kDa and 36.17 kDa, respectively. The bacteria harboring pQE-80L/TsDNase II-1 or pQE-80/TsDNase II-7 were induced for 6 h at 37°C with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). On SDS-PAGE analysis, it was observed that the rTsDNase II-1 and rTsDNase II-7 proteins have the consistent MW with the predicted combined size (Figure 5).

Western blot analysis revealed that rTsDNase II-1 and rTsDNase II-7 were recognized by anti-rTsDNase II-1 and anti-rTsDNase II-7 sera, respectively. Both natural TsDNase II-1 and TsDNase II-7 proteins in the crude extracts of the different stages of T. spiralis other than ML and 3 days old AW ES proteins were identified using the anti-rTsDNase II-1 serum or the anti-rTsDNase II-7 serum (Figure 6), indicating that TsDNase II-1 and TsDNase II-7 were expressed in the different stages of T. spiralis but not in the ML stage.

Results of IIFT with intact worms using anti-rTsDNase II-1 and anti-rTsDNase II-7 sera revealed that green fluorescence staining was homogenously distributed along the external surface of the 3 and 6 days old AWs and NBL, whereas no staining was detected on the cuticles of the ML and IIL stages (Figure 7). Moreover, immunostaining was observed at early, midterm, and late embryo stages (Supplementary Figure 1). After worm sections were incubated using anti-rTsDNase II-1 or anti-rTsDNase II-7 sera, immunostaining was located at the epicuticles and embryos of the 3 days old AWs and stichosome of IIL but not on the ML tissue sections.

After incubating with the IEC monolayer for 2 h, the invaded larvae and non-invaded larvae were observed and numbered (Figures 8A,B). When 1:50 dilutions of different sera were used, the larval invasion rate in the monolayer supplemented with anti-rTsDNase II-1 serum, anti-rTsDNase II-7 serum, infection serum, pre-immune serum, and PBS were 51.97, 58.82%, 37.50%, 83.02%, and 87.42%, respectively (Supplementary Table 1). The inhibition rate of pre-immune serum, anti-rTsDNase II-1 serum, anti-rTsDNase II-7 serum, and infection serum were 4.01%, 40.13%, 32.65%, and 56.69%, respectively. The inhibition of immune serum and T. spiralis infection serum were distinctly greater than those of pre-immune serum (χ12 = 105.012, χ22 = 70.118, P < 0.0001). The inhibition effects of immune sera had a correlation with dilutions of anti-rTsDNase II-1 serum (r = -0.881, P < 0.0001) and anti-rTsDNase II-7 serum (r = -0.891, P < 0.0001) and exhibited a decreasing trend with a serum dilution increase (F₁ = 20.749, F₂ = 27.655, P < 0.0001). Nevertheless, we failed to observe the evident inhibition of pre-immune serum on larva invasion (Figure 8C).

The ADCC assay revealed that after incubation at 37°C for 72 h, anti-rTsDNase II-1 and anti-rTsDNase II-7 sera mediated the adhering of the PECs to the NBL and the subsequent killing of the NBL (Figure 9). When 1:100 dilutions of anti-rTsDNase II-1 and anti-rTsDNase II-7 sera were used, the ADCC resulted in the remarkable death of NBL (66.84% and 63.39% cytotoxicity), in comparison with the NBL treated by pre-immune serum (15.30%, t₁ = 26.420, t₂ = 22.599, P < 0.0001). The cytotoxicity had a correlation with the dilutions of anti-rTsDNase II-1 (r = -0.927, P < 0.0001) and anti-rTsDNase II-7 sera (r = -0.771, P < 0.0001). The cytotoxicity had a decreasing trend following the increase of serum dilutions (F₁ = 69.569, F₂ = 68.231, P < 0.0001).

Specific total IgG and its subclass (IgG1 and IgG2a) antibodies in serum from mice vaccinated with rTsDNase II-1, rTsDNase II-7, or their mixtures were measured by ELISA at different time points after first vaccination. Total IgG and its subclass IgG1 and IgG2a of anti-rTsDNase II-1 and anti-rTsDNase II-7 antibodies and the antibodies against the mixture of these two proteins increased obviously after the second vaccination and reached the highest level following the third vaccination (Figure 10), but no obvious detectable specific antibodies were identified in mice inoculated with only adjuvant or PBS. Mice vaccinated with rTsDNase II-1 exhibited higher levels of IgG1 than IgG2a at 20 and 30 days after vaccination (t_20d = 13.318, t_30d = 56.608, P < 0.0001), which was similar to the levels of IgG1 and IgG2a in mice vaccinated with rTsDNase II-7 (t_20d = 17,507, t_30d = 22.466, P < 0.0001) or rTsDNase II-1 + rTsDNase II-7 (t_20d = 7.821, t_30d = 24.942, P < 0.0001), indicating that the predominant IgG subclass triggered with the two rTsDNase II proteins was IgG1; however, the IgG2a response was also elicited after the second vaccination, and the concurrent Th1/Th2 immune response was elicited by immunization with rTsDNase II.

When specific antibodies of vaccinated mice were assayed by ELISA using AW ES proteins, the total IgG level was not significantly different between mice vaccinated with rTsDNase II-1 and rTsDNase II-1 + rTsDNase II-7 (P < 0.01), but total IgG level of the above-mentioned two groups of vaccinated mice was higher than those of mice vaccinated with rTsDNase II-7 (P > 0.05). The IgG1 level was not statistically different among the three groups of vaccinated mice (F = 1.889, P > 0.05); the IgG2a level also was not significantly different between the two groups of mice vaccinated with rTsDNase II-1 or rTsDNase II-7 (t = 0.564, P > 0.05), but the IgG2a level of mice vaccinated with rTsDNase II-1 + rTsDNase II-7 was higher than those of mice vaccinated with rTsDNase II-1 (t = 2.171, P < 0.05) and rTsDNase II-7 (t = 2.510, P < 0.05).

The protection was investigated in mice immunized with rTsDNase II-1, rTsDNase II-7, or the mixture of these two proteins after the larval challenge infection. The results demonstrated that vaccination of mice with rTsDNase II-1, rTsDNase II-7, or the mixture of these two proteins produced a 40.36%, 34.86%, and 49.54% reduction in AWs at 5 dpi (F = 4.028, P < 0.05), in comparison with the PBS group. The AW burden of the three groups of immunized mice was significantly lower than those of the mice that received only adjuvant (t₁ = 3.593, t₂ = 2.922, t₃ = 5.513, P < 0.01). The reduction of muscle larva burden in the three groups of vaccinated mice at 42 dpi was 50.43%, 42.33%, and 57.79%, separately (F = 1.408, P > 0.05), compared with the PBS group (Table 1). The muscle larva burden of the three groups of immunized mice was obviously lower than those of the adjuvant group (t₁ = 6.812, t₂ = 5.363, t₃ = 6.815, P < 0.001).