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Trichinellosis is an important food-borne parasitic zoonosis throughout the world. At present, the mechanisms of Trichinella spiralis infection remain unclear. Acquiring detailed information on the host-Trichinella interaction would be beneficial for the development of new strategies for trichinellosis control. Circulating miRNAs are stably detectable in the blood of humans and animals infected with parasites. Circulating miRNAs might regulate the expression of target genes in pathological responses during infection and might be novel potential biomarkers of parasitic diseases. In the present study, a total of ten differentially expressed circulating mouse miRNAs with |log 2 (fold change)| ≥ 1.0 and FDR < 0.01 were found during T. spiralis infection, of which five were upregulated and five were downregulated. GO and KEGG analyses showed that the target genes of the ten miRNAs were enriched in many signalling pathways, especially focal adhesion, MAPK pathway, and so on. The results of qRT-PCR showed that among the five upregulated miRNAs, mmu-miR-467a-3p and mmu-miR-467d-3p expression in mouse serum reached a peak at 30 days post-infection (dpi). The expression of mmu-miR-376b-3p and mmu-miR-664-3p increased significantly at 18 dpi and then decreased at 30 dpi. The expression of mmu-miR-292a-5p gradually decreased from 12 to 30 dpi. Among the 5 downregulated miRNAs, mmu-miR-199a-5p expression was significantly downregulated at 30 dpi, while the expression levels of the other four miRNAs (mmu-miR-455-5p, mmu-miR-125b-5p, mmu-miR-125a-5p, and mmu-miR-615-3p) were significantly lower compared with the control, showing a steady downregulation at different phases of infection. These findings will help to further understand the host-Trichinella interaction and provide promising serum biomarkers for trichinellosis.

The drug albendazole (ABZ) has a positive effect against Trichinella spiralis infection and has been used for the treatment and prevention of trichinellosis in humans and animals. However, the molecular mechanism ofthe effects of ABZ on T. spiralis remains unknown. Albendazole sulfoxide (ABZSO) is the main intermediary metabolic product of ABZ, and it is often used as a substitute for ABZ in metabolism and bioavailability research. Herein, isobaric tagging reagents for relative and absolute quantification (iTRAQ)-based LC-MS/MS analysis was used to identify the effect of ABZSO on the proteome of T. spiralis muscle larvae in vitro. 3795 proteins were quantified from 22974 unique peptides. Comparative proteomics analysis displayed that 417 proteins were remarkably differentially expressed in ABZSO-treated larvae, of which 213 proteins were up-regulated and 204 proteins were down-regulated. Quantitative real-time PCR of ten randomly-selected genes verified the proteomic data. Gene ontology annotation and KEGG pathway analysis showed that most of the differentially expressed proteins were involved in cell apoptosis, signal pathway, amino acid metabolism, protein synthesis/assembly/degradation and other biological processes. This study firstly provided the comprehensive proteomics data of T. spiralis in response to ABZSO, and would help us to deeply understand the molecular mechanism of ABZSO effects on T. spiralis., (Copyright © 2017. Published by Elsevier B.V.)

As we all know, invasion of host intestinal epithelium is very important for T. spiralis to complete successfully their life cycle. However, the mechanisms that the intestinal infective larvae (IIL) invade and migrate in the intestinal epithailial cells (IECs) remain unclear until now. The related researches have been hindered since a readily operable in vitro normal model. In our earlier study, an in vitro normal IEC invasion model was established for the first time, and the abilities of the normal IECs to initiate mucosal inflammatory responses to invasion by the IIL in vitro were evaluated in this study. When the IIL were overlaid on the normal mouse IEC monolayers, they quickly within seconds invaded the monolayers and move within the IECs, leaving trails of damaged cells. Then the larvae were found to have started their molting at 12 h, and the complete cuticle was found at 24 h. The percentage of the first molt in the larvae was about 62.3%, and the percentage of the 2nd-4th molt was about 38.2% at 36 h. Real-time PCR showed that the mRNA levels of interleukin-1β (IL-1β), IL-8, epithelial neutrophil-activating peptide 78 (ENA-78), inducible nitric oxide synthase (iNOS), and monocyte chemotactic protein 2 (MCP-2) were elevated in the IECs after 7 h of infection after invasion by the IIL, and their levels were enhanced with the increase of larvae number. No changes in tumor necrosis factor-α (TNF-α) mRNA were observed after the IIL invasion. Secretion increases of IL-1β and IL-8 from the IEC monolayers invaded by T. spiralis were also detected by ELISA. Secretion increases of proinflammatory cytokines and inflammatory mediators in normal IECs can launch the acute inflammatory in response to the IIL invasion. This study would be helpful in further investigating the relationship between the host and T. spiralis, and the immune escape mechanisms of the niche established by T. spiralis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of this study was to investigate the functions of Trichinella spiralis Nudix hydrolase (TsNd) during the larval invasion of intestinal epithelial cells (IECs), development and survival in host by RNAi. The TsNd-specific double-stranded RNA (dsRNA) was designed to silence the expression of TsNd in T. spiralis larvae. DsRNA were delivered to the larvae by soaking incubation or electroporation. Silencing effect of TsNd transcription and expression was determined by real-time PCR and Western blotting, respectively. The infectivity of larvae treated with dsRNA was investigated by the in vitro larval invasion of IECs and experimental infection in mice. After being soaked with 40 ng/μl of dsRNA-TsNd, the transcription and expression level of TsNd gene was inhibited 65.8% and 56.4%, respectively. After being electroporated with 40 ng/μl of dsRNA-TsNd, the transcription and expression level of TsNd gene was inhibited 74.2% and 58.2%, respectively. Silencing TsNd expression by both soaking and electroporation inhibited significantly the larval invasion of IECs in a dose-dependent manner (r1 = -0.96798, r2 = -0.98707). Compared with the mice inoculated with untreated larvae, mice inoculated with larvae soaked with TsNd dsRNA displayed a 49.9% reduction in adult worms and 39.9% reduction in muscle larvae, while mice inoculated with larvae electroporated with TsNd dsRNA displayed a 83.4% reduction in adult worms and 69.5% reduction in muscle larvae, indicating that electroporation has a higher efficiency than soaking in inhibiting the larval development and survival in mice. Our results showed that silencing TsNd expression in T. spiralis inhibited significantly the larval invasion and survival in host., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Trichinella spiralis Nudix hydrolase (TsNd) was identified by screening a T7 phage display cDNA library from T. spiralis intestinal infective larvae (IIL), and vaccination of mice with recombinant TsNd protein (rTsNd) or TsNd DNA vaccine produced a partial protective immunity. The aim of this study was to identify the characteristics and biological functions of TsNd in the process of invasion and development of T. spiralis larvae. Transcription and expression of TsNd gene at all developmental stages of T. spiralis were observed by qPCR and immunofluorescent test (IFT). The rTsNd had the Nd enzymatic activity to dGTP, NAD, NADP and CoA. Its kinetic properties on the preferred substrate dGTP were calculated, and the Vmax, Km, and kcat/Km values at pH 8.0 were 3.19 μM min(-1) μg(-1), 370 μM, and 144 s(-1) M(-1), respectively, in reaction matrix containing 5 mM Zn(2+) and 2 mM DTT. The rTsNd was active from 25 °C to 50 °C, with optimal activity at 37 °C. rTsNd was able to bind specifically to mouse intestinal epithelial cells (IECs) and promoted the larval invasion of IECs, whereas anti-rTsNd antibodies inhibited the larval invasion of IECs in a dose-dependent manner. Anti-rTsNd antibodies could kill T. spiralis infective larvae by an ADCC-mediated mechanism. Our results showed that the rTsNd protein was able to interact with host IECs, had the Nudix hydrolasing activity and the enzymatic activity appeared to be essential indispensable for the T. spiralis larval invasion, development and survival in host., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Previous studies showed that Trichinella spiralis Nudix hydrolase (TsNd) bound to intestinal epithelial cells (IECs), and vaccination of mice with rTsNd or TsNd DNA produced a partial protective immunity against T. spiralis infection. In this study, three TsNd specific small interfering RNA (siRNA) were designed to silence the expression of TsNd in T. spiralis larvae. SiRNAs were delivered to the larvae by electroporation. Silencing effect of TsNd transcription and expression was determined by real-time PCR and Western blotting, respectively. The infectivity of the larvae treated with siRNA was investigated by the in vitro larval invasion of IECs and experimental infection in mice. The results showed that siRNAs were efficiently delivered into T. spiralis larvae through electroporation. Real-time PCR and Western blotting showed that transcription and expression level of TsNd gene was inhibited 73.3 and 76.7 %, respectively, after being electroporated with 2 μM of siRNA-275 for 1 day. Silencing TsNd expression inhibited significantly the larval invasion of IECs (P < 0.01) and was in a dose-dependent manner (r = -0.97941). The mice with infected larvae treated with TsNd siRNA displayed a 63.6 % reduction in intestinal adult worms and 68.8 % reduction in muscle larval burden compared with mice infected with control siRNA-treated larvae. Our results showed that silencing TsNd expression in T. spiralis significantly reduced the larval infectivity and survival in host.

We have previously reported that Trichinella spiralis Nudix hydrolase (TsNd) bound to intestinal epithelial cells (IECs), and the vaccination of mice with recombinant TsNd protein (rTsNd) produced a partial protective immunity against challenge infection in mice. In this study, the full-length cDNA sequence of TsNd gene was cloned into the eukaryotic expression plasmid pcDNA3.1, and the recombinant TsNd DNA was transformed into attenuated Salmonella typhimurium strain ⊿cyaSL1344. Oral immunization of mice with TsNd/S. typhimurium elicited a significant local mucosal IgA response and a systemic Th1/Th2 immune response. Cytokine profiling also showed a significant increase in the Th1 (IFN-γ, IL-2) and Th2 (IL-4, 10) responses in splenocytes of immunized mice upon stimulation with the rTsNd. The oral immunization of mice with TsNd/S. typhimurium displayed a statistically significant 73.32% reduction in adult worm burden and a 49.5% reduction in muscle larvae after challenge with T. spiralis muscle larvae, compared with PBS control group. Our results demonstrated that TsNd DNA delivered by attenuated live S. typhimurium elicited a local IgA response and a mixed Th1/Th2 immune response, and produced a partial protection against T. spiralis infection in mice., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Previous study showed that Trichinella spiralis proteasome subunit beta type-7 (Tspst) gene is an up-regulated gene in intestinal infective larvae (IIL) compared to muscle larvae (ML), which was screened by using suppression subtractive hybridization (SSH) and confirmed by real-time PCR. Tspst may be related to the larval invasion of intestinal epithelial cells (IECs). The aim of this study was to identify Tspst and to investigate its immune protection against intestinal T. spiralis infection., Methods: The Tspst gene encoding a 29 kDa protein from T. spiralis infective larvae was cloned, and recombinant Tspst protein (rTspst) was produced in an Escherichia coli expression system. The rTspst was used to immunize BALB/c mice. Anti-rTspst antibodies were used to determine the immunolocolization of Tspst in the parasite. Transcription and expression of Tspst at T. spiralis different developmental stages were observed by RT-PCR and immunofluorescence test (IFT). The in vitro or in vivo immune protection of anti-rTspst serum or rTspst against intestinal T. spiralis infection in BALB/c mice was evaluated., Results: Anti-rTspst serum recognized the native Tspst protein with 29 kDa in ML crude antigens. Transcription and expression of gene was observed at all T. spiralis different developmental stages (IIL, adult worms, newborn larvae, and ML). An immunolocalization analysis identified Tspst in the cuticle and internal organs of the parasite. An in vitro invasion assay showed that, when anti-rTspst serum, serum of mice infected with T. spiralis or normal mouse serum were added to the medium, the invasion rate of the infective larvae in an IEC monolayer was 25.2%, 11.4%, and 79%, respectively (P < 0.05), indicating that anti-rTspst serum partially prevented the larval invasion of IECs. After a challenge infection with T. spiralis muscle larvae, mice immunized with rTspst conferred a 45.7% reduction in adult worm burden in intestines., Conclusions: In the present study, Tspst was first identified and characterized. Tspst is an invasion-related protein of T. spiralis IIL and could be considered as a potential vaccine candidate antigen against intestinal T. spiralis infection that merits further study.

The activation of Trichinella spiralis muscle larvae (ML) by exposure to intestinal contents or bile and the intestinal epithelial cells (IECs) themselves are two pivotal requirements for the in vitro larval invasion of IECs. However, it is yet unknown which genes are involved in the process of larval invasion. The purpose of the present study was to analyze the differentially expressed genes of T. spiralis larvae activated by bile and cultured with IECs by using real-time polymerase chain reaction. Ten T. spiralis genes encoded the proteins produced by the larvae after co-culture with IECs were selected. Compared with untreated ML, four genes were up-regulated in both bile-activated and cell-cultured larvae, including calcium-dependent secretion activator (Csa; 2.55- and 16.04-fold, respectively), multi cystatin-like domain protein precursor (Mcd; 4.36 and 52), serine protease (Sp; 2.03 and 20.02), and intermediate filament protein ifa-1 (Ifa 1; 2 and 3.31). The expression of two genes, enolase (Eno; 1.51) and ribosomal protein S6 kinase beta-1 (Rsk; 1.49), was up-regulated only in cell-cultured larvae, not in bile-activated larvae. The expression of secreted 5'-nucleotidase (5 N; 1.42) and putative serine protease (Psp; 1.41) was up-regulated in bile-activated larvae, but was not changed or down-regulated after cultured with IECs. ATP synthase F1, beta subunit (ATPase; 0.58 and 0.51) and serine protease precursor (Spp; 0.42 and 0.65) were down-regulated in both bile-activated and cell-cultured larvae. This study provide some differentially expressed genes among the untreated (normal), bile-activated and cell-cultured larvae of T. spiralis. The up-regulated genes might be related with the larval invasion of IECs, but their exact biological functions need to be further investigated. This study will be helpful to further elucidate the molecular mechanism of the invasion of IECs by T. spiralis larvae and to better understand the interaction between parasite and host enterocytes.

Although it has been known for many years that T. spiralis muscle larvae (ML) can not invade intestinal epithelial cells unless they are exposed to the intestinal milieu and activated into intestinal infective larvae (IIL), which genes in IIL are involved in the process of invasion is still unknown. In this study, suppression subtractive hybridization (SSH) was performed to identify differentially expressed genes between IIL and ML. SSH library was constructed using cDNA generated from IIL as the 'tester'. About 110 positive clones were randomly selected from the library and sequenced, of which 33 T. spiralis genes were identified. Thirty encoded proteins were annotated according to Gene Ontology Annotation in terms of molecular function, biological process, and cellular localization. Out of 30 annotated proteins, 16 proteins (53.3%) had binding activity and 12 proteins (40.0%) had catalytic activity. The results of real-time PCR showed that the expression of nine genes (Ts7, Ndr family protein; Ts8, serine/threonine-protein kinase polo; Ts11, proteasome subunit beta type-7; Ts17, nudix hydrolase; Ts19, ovochymase-1; Ts22, fibronectin type III domain protein; Ts23, muscle cell intermediate filament protein OV71; Ts26, neutral and basic amino acid transport protein rBAT and Ts33, FACT complex subunit SPT16) from 33 T. spiralis genes in IIL were up-regulated compared with that of ML. The present study provide a group of the potential invasion-related candidate genes and will be helpful for further studies of mechanisms by which T. spiralis infective larvae recognize and invade the intestinal epithelial cells.

Serine proteases are found in the excretory-secretory (ES) products from Trichinella spiralis muscle larvae, have collagenolytic and elastolytic activities, and may be related to the larval invasion of intestinal epithelial cells. In this study, the serine protease gene (TspSP-1.2, GenBank accession No. EU302800) encoding a 35.5 kDa protein from T. spiralis was cloned, and recombinant TspSP-1.2 protein was produced in an Escherichia coli expression system. An anti-TspSP-1.2 serum recognized the native protein migrating at 35.5 kDa by the Western blotting of the crude or ES antigens from muscle larvae at 42 days post infection. An immunolocalization analysis identified TspSP-1.2 in the cuticle and internal organs of the parasite. Transcription and expression of the TspSP-1.2 gene was observed at all developmental stages of T. spiralis (adult worms, newborn larvae, pre-encapsulated larvae and muscle larvae). An in vitro invasion assay showed that, when anti-TspSP-1.2 serum, serum of infected mice and normal mouse serum were added to the medium, the invasion rate of the infective larvae in an HCT-8 cell monolayer was 33.0%, 89.4%, and 96.2%, respectively (P<0.05), indicating that the anti-TspSP-1.2 serum partially prevented the larval invasion of intestinal epithelial cells. After a challenge infection with T. spiralis infective larvae, mice immunized with the recombinant TspSP-1.2 protein displayed a 34.92% reduction in adult worm burden and 52.24% reduction in muscle larval burden. The results showed that the recombinant TspSP-1.2 protein induced a partial protective immunity in mice and could be considered as a potential vaccine candidate against T. spiralis infection., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Although it has been known for many years that Trichinella spiralis initiates infection by penetrating the columnar epithelium of the small intestine, the mechanisms by which T. spiralis infective larvae recognize and invade the intestinal epithelial cells (IECs) are unknown. It is speculated that the molecular interactions between the parasite and host enterocytes may mediate the recognition and invasion of IECs by T. spiralis. However, no Trichinella proteins that interact with the enterocytes have been identified previously. The aim of this study was to identify Trichinella proteins that bind to IECs by screening a T7 phage display cDNA library constructed using messenger RNA from T. spiralis intestinal infective larvae. Following five rounds of biopanning, sequencing, and bioinformatics analysis, ten T. spiralis proteins (Tsp1-Tsp10) with significant binding to normal mouse IECs were identified. The results of the protein classification showed that six proteins (Tsp1, calcium-transporting ATPase 2 protein; Tsp4, ovochymase-1; Tsp6, T-complex protein 1 subunit eta; Tsp7, glycosyl hydrolase family 47; Tsp8, DNA replication licensing factor MCM3; and Tsp10, nudix hydrolase) of these T. spiralis proteins were annotated with putative molecular functions. Out of the six proteins, five have catalytic activity, four have binding activity, and one has transporter activity. Anti-Tsp10 antibodies prevented the in vitro partial invasion of IECs by infective larvae and the mice immunized with the recombinant phage T7-Tsp10 showed a 62.8 % reduction in adult worms following challenge with T. spiralis muscle larvae. Although their biological functions are not yet fully known, these proteins might be related to the larval invasion of host enterocytes. Future experiments will be necessary to ascertain whether these proteins play important roles in the recognition and invasion of host enterocytes. The construction and biopanning of Trichinella phage display libraries provide a novel approach for searching for candidate genes that are related to invasion and for identifying protein interactions between parasite and host.

Trichinellosis is a public health problem and is considered an emerging/re-emerging disease in various countries. The etiological agent of trichinellosis is the nematode Trichinella, which infects humans, domestic animals and wildlife. A veterinary vaccine could be an option to control the disease in domestic animals. Although several vaccine candidates have shown promising results, a vaccine against trichinellosis remains unavailable to date. Phage particles are especially ideal vaccine delivery vehicles because they do not interfere with the immune response against the displayed peptide antigens, and, if anything, are more likely to efficiently direct antigen expression to professional antigen-presenting cells. In this study, Tsp10 polypeptide, which was encoded by a cDNA fragment of Trichinella spiralis intestinal infective larvae and was found to bind to normal mouse intestinal cells, was displayed on the surface of T7 phage. Anti-Tsp10 antibodies were able to recognize the native Tsp10 protein mainly localized to the stichosome of T. spiralis. Mice immunized with the recombinant phage T7-Tsp10 showed a 62.8% reduction in adult worms and a 78.6% reduction in muscle larvae following challenge with T. spiralis muscle larvae. Our results demonstrate that the vaccination with Tsp10 polypeptide displayed by T7 phage elicits the Th2-predominant immune responses and produces a significant protection against T. spiralis infection in mice. These findings suggest that phage display is a simple, efficient, and promising tool to express candidate vaccine antigens for immunization against T. spiralis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In this study, a sandwich enzyme-linked immunosorbent assay (ELISA) based on IgY (egg yolk immunoglobulin) and monoclonal antibody (mAb) against excretory-secretory (ES) antigens of Trichinella spiralis muscle larvae was developed for detection of circulating antigens (CAg) in serum from mice infected with T. spiralis. The IgY-mAb sandwich ELISA involved the use of chicken antibody IgY as a capture antibody and mouse mAb 35B9 as a detecting antibody. This method was able to detect as little as 1 ng/mL of ES antigens added to normal mouse serum. A group of 15 mice was orally inoculated with 500 T. spiralis muscle larvae per animal and the serum samples were daily taken during 1-49 days post-infection (dpi). The level of CAg was detectable as early as 3 dpi in the sera from infected mice, increased gradually, and reached two peaks with detection rate of 40% at 13 dpi and 100% at 24 dpi, respectively. The anti-Trichinella antibodies was first detected in 33.3% of the infected mice at 3 week post-infection (wpi), and reached a peak positive rate of 100% at 5 wpi. Moreover, the infected mice were treated with abendazole at 5 weeks post-infection, and the serum levels of CAg in treated group began to increase rapidly at 2 days post-treatment (dpt) and reached a peak with detection rate of 100% (10/10) at 8 dpt, and then decreased gradually. By 42 dpt, the CAg levels decreased to the undetected level, but the anti- Trichinella antibodies were still detected in 100% of the infected mice. The novel assay appears to be sensitive for detection of antigens of T. spiralis and should be valuable to the early diagnosis and evaluation of the efficacy of chemotherapy in trichinellosis.

The first human case with trichinellosis was reported in 1964 in Tibet, China. However, up to the present, the etiological agent of trichinellosis has been unclear. The aim of this study was to identify a Tibet Trichinella isolate at a species level by PCR-based methods. Multiplex PCR revealed amplicon of the expected size (173 bp) for Trichinella spiralis in assays containing larval DNA from Tibet Trichinella isolate from a naturally infected pig. The Tibet Trichinella isolate was also identified by PCR amplification of the 5S ribosomal DNA intergenic spacer region (5S ISR) and mitochondrial large-subunit ribosomal RNA (mt-lsrDNA) gene sequences. The results showed that 2 DNA fragments (749 bp and 445 bp) of the Tibet Trichinella isolate were identical to that of the reference isolates of T. spiralis. The Tibet Trichinella isolate might be classifiable to T. spiralis. This is the first report on T. spiralis in southwestern China.

It has been known for many years that Trichinella spiralis initiates infection by penetrating the columnar epithelium of the small intestine; however, the mechanisms used by the parasite in the establishment of its intramulticellular niche in the intestine are unknown. Although the previous observations indicated that invasion also occurs in vitro when the infective larvae are inoculated onto cultures of intestinal epithelial cells (e.g., human colonic carcinoma cell line Caco-2, HCT-8), a normal readily manipulated in vitro model has not been established because of difficulties in the culture of primary intestinal epithelial cells (IECs). In this study, we described a normal intestinal epithelial model in which T. spiralis infective larvae were shown to invade the monolayers of normal mouse IECs in vitro. The IECs derived from intestinal crypts of fetal mouse small intestine had the ability to proliferate continuously and express specific cytokeratins as well as intestinal functional cell markers. Furthermore, they were susceptible to invasion by T. spiralis. When inoculated onto the IEC monolayer, infective larvae penetrated cells and migrated through them, leaving trails of damaged cells heavily loaded with T. spiralis larval excretory-secretory (ES) antigens which were recognized by rabbit immune sera on immunofluorescence test. The normal intestinal epithelial model of invasion mimicking the natural environment in vivo will help us to further investigate the process as well as the mechanisms by which T. spiralis establishes its intestinal niche.