Molecular characterization of a Trichinella spiralis elastase-1 and its potential as a diagnostic antigen for trichinellosis

Background Trichinella spiralis muscle larval (ML) excretion/secretion (ES) antigen is the most widely used diagnostic antigen of trichinellosis, but preparation of ES antigen requires collecting worms from infected animals, and detection of specific IgG against ML ES antigen may result in a false negative at the early stage of infection. The aim of the study was to characterize T. spiralis elastase-1 (TsEla) and to evaluate its potential as diagnostic antigen for trichinellosis. Methods The complete cDNA sequences of the TsEla gene were cloned and expressed, and recombinant (rTsEla) was purified. TsEla transcription and expression in different T. spiralis life-cycle stages was investigated by qPCR and western blotting, and its location in the nematodes was evaluated using an immunofluorescence assay (IFA). The antigenicity of rTsEla was investigated by western blotting analysis and ELISA. Anti-Trichinella IgG, IgM and IgE of experimentally infected mice and specific IgG antibodies of trichinellosis patients were assayed by rTsEla-ELISA and ES-ELISA. Results The results of the qPCR and western blotting showed that TsEla was expressed in various T. spiralis life stages. Natural TsEla was detected in the soluble proteins and ES proteins of different life stages. IFA revealed that TsEla was identified in the whole nematodes of various stages, especially in the cuticle, stichosome and genital primordium of the parasite. Serum anti-Trichinella IgM, IgG and IgE in infected mice was first detected by rTsEla-ELISA at 6, 10 and 12 days post-infection (dpi), and reached 100% at 8, 14 and 14 dpi, respectively. When rTsEla-ELISA and ES-ELISA were used to detect anti-Trichinella IgG in sera of trichinellosis patients, the sensitivity was 97.37% (37/38) and 89.74% (34/38) (P > 0.05), and the specificity was 99.10% (220/222) and 98.20% (218/222), respectively (P > 0.05). The rTsEla cross-reacted with only one serum sample out of 20 samples from paragonimiasis patients and 7 samples from clonorchiasis patients. Conclusions rTsEla is valuable to early diagnosis of trichinellosis and could be an alternative diagnostic antigen to the ML ES antigens.


Background
Trichinellosis is a globally distributed food-borne parasitic disease. Humans can become infected by ingesting raw or semi-cooked meat containing Trichinella muscle larvae (ML) [1]. Human Trichinella infection has been reported in 55 countries worldwide and trichinellosis is regarded to be a neglected zoonotic parasitosis. During 1986-2009, 65,818 cases and 42 deaths caused by trichinellosis were recorded in 41 countries [2,3]. The main etiological agent of this disease is the common species T. spiralis. From 2004-2009, 15 trichinellosis outbreaks were documented in the Chinese mainland which consisted of 1387 cases and 4 deaths, with the dominating infection source being pork [4][5][6]. Trichinellosis has become an important food-borne zoonosis with direct risk for food safety and public health [7,8].
After the infected animal meat is eaten, infectious T. spiralis muscle larvae are released from their capsules under the digestion of gastric fluid, they develop in the intestine into infectious larvae, which intrude intestinal epithelium and, after 4 molts, mature into the adult stage (AW) [9,10]. The main symptom of the intestinal phase of trichinellosis is a non-specific gastroenteritis, with abdominal pain, diarrhea, nausea and/or vomiting, which may last up to 1 week. In 2-3 weeks after infection, each fertilized female deposits about 1500 newborn larvae (NBL), which migrate, penetrate and encapsulate in the host's skeletal muscle (acute or muscle stage) [11]. The acute stage results in an inflammatory reaction in response to the larval penetration of the muscles, and its obvious manifestations are fever, eyelid/facial oedema, myalgia, and eosinophilia [12,13].
Because of the non-specific manifestations, clinical diagnosis of trichinellosis is difficult [12]. Muscle biopsy or detection of specific IgG against Trichinella is the most commonly used definitive diagnostic method for human Trichinella infection, but examination of muscle biopsies for detection of the parasite is insufficiently sensitive to find Trichinella larvae in the early and mild stages of infection [14]. The International Commission on Trichinellosis (ICT) recommended an ELISA with ML excretion/secretion (ES) antigen to detect anti-Trichinella IgG [15], but the primary weakness of an muscle larval (ML) ES antigen is the potential of false-negatives due to specific IgG in the early stage of T. spiralis infection, which usually occurs in a 2-3 week window, because the major ML antigenic epitopes are stage-specific and can therefore only be probed by a ML stage-specific antibody [16]. The intestinal infective larvae (IIL) ES proteins are first exposed to the host's enteral local mucosal and systematic immune system which triggers the generation of Trichinella-specific antibodies [17,18]. The IIL ES proteins may contain early sero-diagnostic markers for T. spiralis infection [19]. Hence, the novel diagnostic antigens need to be exploited from the IIL ES proteins.
By immunoproteomics, several serine proteases have been identified in T. spiralis IIL and ML ES or surface proteins recognized using early infection serum [20][21][22][23][24]. Furthermore, T. spiralis serine proteases were highly expressed in IIL surface proteins relative to that of ML [25]. In this study, a novel T. spiralis elatase-1 gene (TsEla, GenBank: EFV56917.1) belonging to the serine protease family was obtained from the T. spiralis draft genome [26], cloned and expressed. Characteristics of the TsEla were investigated and the prospective serodiagnostic value of recombinant TsEla (rTsEla) for trichinellosis was evaluated using serum samples from infected animals and patients.

Worm collection and protein preparation
At 42 days post-infection (dpi), T. spiralis-infected mouse muscles were digested using 0.33% pepsin and 1% HCl to recover the ML [29,30]. The IIL were collected from infected mouse intestine at 6 h post-infection (hpi) [25]. AW were acquired from duodenum and jejunum at 3 and 6 dpi, respectively [31]. Adult females were cultivated in RPMI-1640 medium containing 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 24 h, and then NBL were recovered [32]. The somatic crude or ES protein of NBL, ML, IIL and AW were prepared as previously described [33,34].

Bioinformatics analysis of TsEla
The complete TsEla cDNA sequences were retrieved from GenBank (EFV56917.1). The characteristics of the TsEla gene were analyzed using bioanalysis software [39]. The tertiary structure and functional site of TsEla protein was predicted using PyMOL and CN3D software [40]. TsEla amino acid sequences were compared with elastases from other organisms by means of Clustal X [41]. The GenBank accession number of elastases from other organisms were as follows:  3. The maximum parsimony method was used to construct a phylogenetic tree of the elastase sequences using MEGA7.0 [42].

Preparation of polyclonal antibodies against rTsEla
Fifteen mice were immunized subcutaneously with 20 μg rTsEla mixed 1:1 (v/v) with complete Freund's adjuvant. Three booster immunizations were administered using the same dose of rTsEla mixed with incomplete Freund's adjuvant at a two-week interval [46]. Two weeks after the final boost, 100 μl of tail blood was taken from immunized mice, and anti-rTsEla serum was isolated [47].

Immunofluorescence assay (IFA)
To locate TsEla in worm tissues, IFA was performed with T. spiralis whole worms and their cross-sections [54,55]. The integrated parasite was fixed in cold acetone for 20 min and 2 μm worm cross-sections were cut using a microtome. The whole worms and cross-sections were blocked with 1% bovine serum albumin (BSA) and probed by anti-rTsEla serum diluted at 1:10 at 37 °C for 2 h. After washing with PBS, they were incubated at 37 °C for 1 h with cy3/FITC anti-mouse IgG conjugate (1:100; Santa Cruz Biotech, Dallas, Texas, USA), and examined using fluorescence microscopy (Olympus, Tokyo, Japan) [56].

Application of rTsEla-ELISA to assay Trichinella-specific antibodies
Anti-Trichinella IgG, IgM and IgE of experimentally infected mice and specific IgG antibodies of trichinellosis patients were assayed by ELISA with rTsEla (rTsEla-ELISA) and ML ES antigens (ES-ELISA) [57,58]. Optimal dilutions of antigens and sera were first determined using a checkerboard titration. The optimal antigen coating concentration was 2.0 μg/ml rTsEla for detecting specific IgG and IgM, 3.5 μg/ml rTsEla for detecting IgE, and 2.5 μg/ml ES antigens for IgG, IgM and IgE. Microtiter plates were coated with the above mentioned antigens, and blocked using 5% skimmed milk at 37 °C for 2 h. After washes with PBST, the plates were probed with mouse sera (1:100 for IgG and 1:50 for IgM and IgE), and human sera diluted at 1:200 for detecting three antibodies, subsequently incubated at 37 °C for 1 h with HRPlabelled anti-human/mouse IgG antibody (1:10,000; Southern Biotech). Following washes, the plates were developed using o-phenylenediamine dihydrochloride (OPD; Sigma-Aldrich) with 30% H 2 O 2 for 30 min, and the reaction was stopped using 2M H 2 SO 4 . The absorbance (optical density, OD) at 492 nm was assayed with a microplate reader (TECAN, Schweiz, AG, Switzerland), and all serum samples were tested in duplicate. The ratio < 2.1 of tested serum/negative serum OD values was considered as negative and the ratio ≥ 2.1 as positive [59,60]. For detecting mouse serum IgG, IgM and IgE, the cut-off values for rTsEla-ELISA were 0.25, 0.24 and 0.21, respectively; and those for ES-ELISA were 0.29, 0.27 and 0.23, respectively. The cut-off values of rTsEla-ELISA and ES-ELISA for detecting human IgG were 0.34 and 0.61, respectively.

Statistical analysis
SPSS 21.0 software was used to statistically analyze the data. Data are shown as the arithmetic mean ± standard deviation (SD). TsEla transcription and expression in different T. spiralis life stages were compared using one-way ANOVA. The difference between groups was determined by Chi-square test. A statistical difference was considered significant at P < 0.05.

Bioinformatics analysis of TsEla
Complete TsEla coding sequence consisted of 1350 bp encoding 449 amino acids (aa), with a molecular weight (MW) of 47.3 kDa and isoelectric point (pI) of 8.88. A signal peptide at 19 aa was detected by Signal P 4.1 Server. TMHMM prediction revealed that TsEla has no transmembrane domain, but has a functional domain named as Tryp_SPc located at 38-314 aa. The TsEla homology comparison of the TsEla gene with those of other species or genotypes of Trichinella is shown in Fig. 1. The TsEla amino acid sequence had an identity of 87.2, 81.6, 78.1, 77.9, 77.6, 75.4 and 72.0% with elastase of the 7 species with encapsulated larvae (T. nelsoni, T. nativa, T. britovi, T9, T6, T. murrelli and T8), and an identity of 70.6 and 67.3% with species with non-encapsulated larvae (T. pseudospiralis and T. zimbabwensis, respectively).
Phylogenetic analysis of TsEla with elastase-1 from other nematodes is shown in Fig. 2a. The phylogenetic tree shows the monophyletic group of above-mentioned 10 Trichinella species/gene types, except for the recently described T. patagoniensis from Argentina. According to the phylogenetic analysis of elastase, T. spiralis has a closer evolutionary relationship with the species of the genus with encapsulated larvae. The structure prediction revealed that TsEla has 5 α-helices and 37 β-strands, a catalytic site Ile, and three serine protease-specific active sites (His, Asp and Ser) (Fig. 2b, c). To assess the murine humoral response to rTsEla, anti-rTsEla IgG antibody in immunized mice was measured by ELISA. The results indicated anti-rTsEla IgG was induced by immunization with rTsEla. The specific IgG titer was up to 1:10 5 after the fourth immunization, demonstrating that the rTsEla has good immunogenicity. Western blotting revealed that three bands of native TsEla with 40.9-59.8 kDa in soluble protein of different T. spiralis life stages (NBL, ML, IIL, 3d and 6d AW) were detected by anti-rTsEla serum (Fig. 4b), 2-9 bands of native TsEla with 27.8-62.6 kDa in ML, IIL and 3d AW ES proteins was also probed using anti-rTsEla serum (Fig. 4c). Eight bands of native TsEla with 25.9-52.8 kDa in ML soluble proteins, three bands of native TsEla with 44.3-52.8 kDa in ML ES proteins, and rTsEla were detected by anti-rTsEla serum, but not by non-infected serum (Fig. 4d), indicating that TsEla was expressed at diverse T. spiralis stages, and it is a secretory protein of this nematode.

qPCR analysis of TsEla gene transcription in different life stages of T. spiralis
Transcription of the TsEla gene in different life stages of T. spiralis was detected by qPCR, the results revealed that the TsEla gene was transcribed at all T. spiralis stages (NBL, ML, IIL, 3 d and 6 d AW) (Fig. 5). The transcription level in the ML stage was obviously higher than those of the other stages (F (4, 10) = 287.184, P < 0.001), and the TsEla level in the IIL stage was higher than those in the NBL and AW stage (P < 0.0001), but lower than that in the ML stage (P < 0.0001).

Expression and localization of TsEla in different T. spiralis stages by IFA
IFA was used to detect the expression and localization of TsEla in T. spiralis stages. IFA with entire worms detected positive fluorescence staining in the epicuticle of ML, early IIL (3, 6 and 12 h IIL) and NBL by anti-rTsEla serum, but no immunostaining was found in late IIL (15 and 24 h IIL) and 3 d AW (Fig. 6), suggesting that native TsEla was expressed in the epicuticle of ML, early IIL and NBL, but not in the epicuticle of late IIL and AW stages. When worm cross-sections were used in IFA, fluorescence staining was distributed within the whole body in ML, 6 h IIL and 3 d AW, especially in the cuticle, stichosome and genital primordium of the parasites (Fig. 7). No fluorescence staining in ML was observed by incubation with mouse pre-infected serum. Fig. 1 Sequence alignment of Trichinella spiralis elastase gene (XP_003377838.1) with other Trichinella species or genotypes. Clustal X and BOXSHADE were used to analyze the sequences, obvious differences were observed in different species/genotypes. Black shading represents the residues identical to TsEla, and grey shading shows the conservative substitutions

Assay of Trichinella-specific IgG in sera of mice infected with Trichinella and other parasites
Trichinella-specific IgG in serum samples from mice infected with T. spiralis and other parasites was tested using rTsEla-ELISA and ES-ELISA; the result is shown in Table 1. The detection rate of specific IgG using two antigens was 100% of mice infected with 300 T. spiralis ML at 35 dpi. Both antigens did not cross-react with sera from mice infected with T. gondii, Capillaria hepatica, A. cantonensis, Clonorchis sinensis and S. japonicum. However, ML ES antigen had a cross-reactivity with one serum (4.8%) from sparganum-infected mice. Specific IgG in serum from mice infected with 300 larvae of other Trichinella species were also measured using two antigens ( Table 2). The IgG detection rate with two antigens had also no significant difference in mice infected with the other four Trichinella species ( χ 2 (1) = 0.317, P = 0.573).

Discussion
Detection of circulating antigen or DNA from live Trichinella worms is an ideal early diagnostic technique for detecting Trichinella infection. However, the content of Trichinella circulating antigen and DNA in serum samples is usually low and difficult to detect [16,61]. In addition, the easy degrading and short duration of Trichinella DNA leads to its non-application as a diagnostic marker for Trichinella infection [62,63]. Hence, novel diagnostic antigens for early Trichinella infection need to be explored.
Elastases are the trypsin-like proteins belonging to the family of serine proteases, they can degrade host's various macromolecular substances such as keratin, laminin, type IV collagen, fibronectin and elastin [64]. The elastases are involved in larva invasion, molting and digestion, and may exert a major action for parasite invasion [65,66]. Elastases are likely significant target antigens for eliciting an immune response at an early stage of infection, and may be used as diagnostic markers for early parasitic diseases. Nevertheless, studies on elastases were mainly focused on Schistosoma spp. [67,68]; we found no reports of studies on T. spiralis elastase in the literature.
In the present study, the complete TsEla sequence was cloned and expressed in E. coli. Bioinformatics analysis showed that the TsEla had a signal peptide and one functional domain of three serine protease-specific active sites. Sequence analysis showed that TsEla has 87.2% and 81.6% identity with the elastase of T. nelsoni and T. nativa, respectively. The phylogenetic tree indicated TsEla had a close evolutionary relationship with elastase of T. nelsoni, and demonstrated a monophyletic group of 10 Trichinella species/gene types, except for T. patagoniensis which was recently found in a cougar from Argentina [69]. Following purification, the rTsEla was highly immunogenic and used to produce anti-rTsEla serum. Immunization of mice with rTsEla triggered the obvious antibody responses to rTsEla. qPCR revealed that TsEla transcription was detected in various T. spiralis stages (ML, IIL, AW and NBL), indicating that TsEla was transcribed at every life-cycle stage of this nematode, and the TsEla transcription level at ML and IIL stages was remarkably greater than that of other stages as shown in Fig. 5. Western blotting showed that several native TsEla protein bands in ML somatic and ES proteins were identified by anti-rTsEla serum, it is likely because TsEla might have various isoforms, or this protein was   possibly processed by post-translational modifications and processing, or because TsEla is one member of the Trichinella serine protease superfamily and possesses the same antigenic epitopes [13,36,70].
Western blotting results also revealed that native TsEla in ML, IIL and 3 d AW ES proteins were recognized by anti-rTsEla serum. The results of IFA with intact worms showed that native TsEla was mainly expressed in the epicuticle of ML, early IIL (3-12 h IIL) and NBL, but not in late IIL (15-24 h IIL) and AW. The native TsEla was distributed in the whole worms of ML, 6 h IIL and 3 d AW, especially in the cuticle, stichosome and genital primordium of the parasite when worm cross-sections were used. Concerning the TsEla location, some differences in 3 d adults occurred between Figs. 6 and 7c, it is likely because when whole worms were used in the IFA, their surface cuticle was intact, and the inner antigens were not exposed, and not recognized by anti-rTsEla serum. Whereas when the cross-sections of 3 d adult worms were prepared, the worm surface cuticle (epicuticle) might be destroyed by the hot paraffin in the process of worm embedding, so the inner antigens were exposed and therefore recognized and immuno-stained. These results suggest that TsEla is a secretory protein which is likely derived from surface/ES protein of this nematode.  Previous studies have demonstrated that other T. spiralis serine proteases (TspSP-1.2, TsSP and Ts31) were also expressed in the cuticle and ES products of the parasitic nematode [13,36,48], suggesting that TsEla is an indispensable protein of parasite-host interaction, and might have an important action in larva invasion and eliciting an early immune response. However, the rTsEla enzyme activity and functions need to be further investigated.
In order to evaluate the rTsEla potential for serodiagnosis of trichinellosis, an rTsEla-ELISA was established, and the results were comparatively analyzed with those of a conventional ML ES-ELISA. Our results revealed that anti-Trichinella IgM, IgG and IgE in infected mice were first detected by rTsEla-ELISA at 6, 10, 12 dpi, and reached 100% at 8, 14 and 14 dpi, respectively. When the ES-ELISA was used, anti-Trichinella IgM, IgG and IgE in infected mice were first detected at 6, 10, 12 dpi, and reached 100% at 8, 18 and 14 dpi, respectively, indicating that anti-Trichinella IgM levels rapidly elevated during the early phase of Trichinella infection, but declined quickly from the beginning of 26 dpi when the larvae were encapsulated in the skeletal muscles [11]. The results suggest that detection of anti-Trichinella IgM is valuable only for early trichinellosis [16,71]. Because of the short half-life in serum samples, IgE had no advantage to conventional IgG detection [12,72]. The amount and avidity of IgG antibodies was found to be most useful for serodiagnosis of trichinellosis [73], the seroconversion time following primary Trichinella infection was dependent on the infectious dose and muscle larva burden, anti-Trichinella IgG was usually detected approximately 2-3 weeks post-infection in infected animals, and may remain for many years [74].
Interestingly, Trichinella-specific IgG in 100% of mice infected with 200 T. spiralis larvae were detected using rTsEla-ELISA as early as 14 dpi, but the ES-ELISA could not detect all of the infected mice prior to 18 dpi. Our results suggest that TsEla as a surface/ES protein of ML and early IIL was exposed with host's immune system at an early stage of the infection, triggering an early specific IgG response which continued to the muscle stage [17,25].
In order to ascertain the sensitivity and specificity of rTsEla for detecting anti-Trichinella IgG, rTsEla-ELISA was used to test serum samples from patients with trichinellosis and other parasitoses, and the results were compared with ES-ELISA. The results showed that the sensitivity of rTsEla-ELISA and ES-ELISA was 97.37% and 89.47%, respectively (P > 0.05). When the serum samples from patients at the early stage of trichinellosis at 19 dpi were examined, the sensitivity of rTsEl-ELISA (92.31%) was higher than 76.92% of ES-ELISA, but there was no statistically significant difference (P > 0.05); this result is likely because only a few of the early serum samples were assayed. Specificity of rTsEla and ES antigens was 99.10% and 98.20%, respectively (P > 0.05), when the two antigens were used to test Trichinella-specific IgG in sera from patients with other helminthiases and healthy individuals. rTsEla cross-reacted with only one out of 20 paragonimiasis patients and seven clonorchiasis patients. Because of the long-time storage and repeated freezing/ thawing of these serum samples, anti-Trichinella IgM in our patients' sera were not detected (data not shown). Sensitivity and specificity of rTsEla are comparative to those of recombinant Ts31 and TsSP proteins [13,36]. Moreover, preparation of ML ES antigens requires collecting the infectious larvae from experimentally infected animals, which is inconvenient due to labor, time and costs [32]. Recombinant antigens can be produced in vitro in large quantities and may be used as a diagnostic antigen in a standardized ELISA to diagnose trichinellosis [58]. Therefore, the rTsEla is worthy of early serodiagnosis of trichinellosis, and could be useful as a potential alternative diagnostic antigen to the ML ES antigens.

Conclusions
Our study indicates that TsEla was transcribed and expressed in all life-cycle stages of T. spiralis, it was likely derived from the nematodeʼs surface and ES proteins, and located in the whole worm body. The rTsEla had a good immunogenicity. The sensitivity and specificity of rTsEla for assaying Trichinella-specific IgG and IgM are comparable to the commonly used muscle larval ES antigens. The rTsEla is a worthy candidate for early serodiagnosis of trichinellosis and could be a potential alternative antigen to the ML ES antigens. However, more serum samples from patients at an early stage of Trichinella infection are needed to further ascertain its sensitivity and specificity.