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Trichinellosis is a major foodborne parasitosis caused by Trichinella spiralis. In the present study, a serine protease gene from an adult T. spiralis (Ts-Adsp) cDNA library was cloned, expressed in Escherichia coli and purified by Ni-affinity chromatography. Previous studies of our laboratory have found that mice vaccinated with recombinant Ts-Adsp protein (rTs-Adsp) exhibited partial protection against T. spiralis infection. In this study, the protective effect of rTs-Adsp against T. spiralis infection in pigs was further explored. The cell-mediated and humoral immune responses induced by rTs-Adsp were measured, including the dynamic trends of specific antibody levels (IgG, IgG1, IgG2a and IgM), as well as the levels of cytokines (IFN-γ, IL-2, IL-4, and IL-10) in the serum. Moreover, the changes in T lymphocytes, B lymphocytes, and neutrophils were measured to evaluate cellular immune responses in pigs vaccinated with rTs-Adsp. The results indicated that a Th1-Th2 mixed immune response with Th1 predominant was induced by rTs-Adsp after vaccination. Flow cytometric analysis showed that the proportions of CD4+ T cells, B cells, and neutrophils in the immunized groups were significantly increased. Furthermore, pigs vaccinated with rTs-Adsp exhibited a 50.9% reduction in the muscle larvae burden, compare with pigs from the PBS group five weeks after challenged. Our results suggested that rTs-Adsp elicited partial protection and it could be a potential target molecule for preventing and controlling Trichinella transmission from pigs to human. Author summary: Trichinellosis is a global foodborne parasitic disease caused by consuming raw or poorly cooked meat. The porcine products are the most common source. Therefore, it will have a great significance for public health security and human health to prevent and control the trichinellosis. We previously found that mice vaccinated with recombinant Adsp protein (rTs-Adsp) exhibited partial protection against T. spiralis infection. In this study, the protective effect of rTs-Adsp against challenge infections with T. spiralis in pigs was further explored. We found that rTs-Adsp elicited partial protection and it could be an important target molecule for preventing and controlling T. spiralis transmission from pigs to human. [ABSTRACT FROM AUTHOR]

The excretory-secretory products (ESP) released by muscle stage of Trichinella spiralis have been suggested to be involved in nurse cell formation. However, the molecular mechanisms by which ESP modulate nurse cell formation remain unclear. In the present study, the ability of ESP of muscle larvae of T. spiralis (ML-ESP) to influence the proliferation and differentiation of murine myoblasts and the mechanisms were evaluated in vitro using C2C12 myoblast cell line, which were incubated for various times under grow or differentiation culture medium containing various concentrations of ML-ESP. The results indicated that ML-ESP promoted myoblast proliferation in a dose-dependent manner and increased the expression of the cell-cycle regulator cyclin D1 as well as that of proliferating cell nuclear antigen (PCNA). Conversely, ML-ESP inhibited the differentiation of these cells, which was evidenced by a reduction in the levels of MHC and MRFs expression (MyoD and myogenin) as well as that of p21. In addition, ML-ESP also inhibited the phosphorylation of p38 MAPK in differentiating C2C12 myoblast. Taken together, these results imply that certain critical mediators contained in ML-ESP inhibit myogenesis through enhancing skeletal myoblasts proliferation and down-regulating the expression of MRFs as well as involving p38 MAPK signalling pathway, which provides insight into the mechanisms utilised by T. spiralis to interfere normal wound repair in infected muscle cells and affect nurse cell formation. [ABSTRACT FROM AUTHOR]

• Characterized infectivity of muscle larvae during the early stages of infection. • Th2-predominant immune response was induced in Trichinella spiralis infection. • The body weight of infected pigs compared with the control group was increased. • Anti- Trichinella spiralis IgM antibody was detected earlier than IgG antibody. The objective of the present study was to investigate the infectivity and antibody response of four Trichinella species (Trichinella spiralis , Trichinella britovi , Trichinella pseudospiralis and Trichinella murrelli) in experimentally infected pigs. A total of 120 Large White pigs (30 animals per group) were inoculated with 10,000 muscle larvae (ML) of T. spiralis , T. britovi , T. pseudospiralis , and T. murrelli. The pigs were sacrificed at 12–21 days post-infection (dpi) to examine the viability and infectivity of ML. A total of 54 Large White pigs (6 animals per group) were inoculated with 25, 50, 100, 200, 400, 600, 800, 1000 and 10,000 T. spiralis ML. The pigs were sacrificed, and the average numbers of larvae per gram (lpg) from six different muscle tissues were calculated at 120 dpi. The results showed that the larvae first be detectable for T. spiralis , T. britovi , and T. pseudospiralis at 16 dpi, 17 dpi, and 16 dpi, respectively. Viable larvae and average lpg were significantly increased with time from 17 to 21 dpi. The T. spiralis ML burden was dependent of the inoculation dose with an average lpg of 0.003, 0.005, 0.007, 0.17, 0.82, 2.89, 4.90, 28.30 and 226.18, respectively. The IgG antibody response was dose-dependent to generate and increased throughout the experimental period. And the IgG1 isotype was significantly higher than IgG2a, which meant that T. spiralis infection induced the Th2 immune response. The time of detecting IgM antibodies was significantly earlier than IgG antibody detection. These results provide important information in the primary characterization of pigs infected with Trichinella. [ABSTRACT FROM AUTHOR]

Trichinella spiralis (T. spiralis) is a widely distributed pathogenic microorganism that causes trichinellosis, a disease that has the potential of causing severe harm to their host. Numerous studies have demonstrated that autophagy can be triggered by microbial infection, such as bacteria, viruses, protozoa, and parasitic helminths. However, it's still unknown whether autophagy can facilitate host resistance to T. spiralis infection. The present study examined the role of autophagy in striated muscle cell transformation following infection with T. spiralis in BALB/c mice. Transmission electron microscopy (TEM) was used to detect the production of the host diaphragm autophagosome after T. spiralis infection, and changes in the protein and transcriptional levels of autophagic marker proteins were also detected. The significance of autophagy in T. spiralis infection, namely inhibition of T. spiralis growth, was preliminarily evaluated by conducting in vivo experiments using autophagy inhibitors. Besides, we studied the effect of excretory-secretory products (ES) of T. spiralis on autophagy of C2C12 myoblasts. The changes in protein and gene expression levels in autophagy-related pathways in vitro and in vivo were measured as further evidence. The results showed that T. spiralis infection induced autophagy in the host muscle cells. Meanwhile, ES inhibited autophagy of myoblasts in vitro, but this did not affect the cell viability. The upregulation and downregulation of autophagy-related factors in skeletal muscle cells may indicate an adaptive mechanism providing a comfortable niche for the parasite. Author summary: Autophagy, a intracellular degradation system, is a kind of unique phenomenon in eukaryotic cells. The commonly referred autophagy is the process of forming autophagosomes by wrapping the cytoplasmic components with double-membrane structure, and then fusing with lysosomes to degrade the internal substances of the cell. Autophagy can be induced by various pathogens including parasites. When the body is infected with intracellular parasites, the host cell can remove the parasites by autophagy. However, parasites have also evolved defence mechanisms that use autophagy in host cells to promote growth. These can be seen in some intracellular parasitic infections such as Toxoplasma gondii and Plasmodium. Although the role of autophagy in other parasitic infections has been revealed, it remains unclear whether autophagy is involved in the invasion process by Trichinella. We investigated the role of Trichinella infection on host muscle cells autophagy and the effect of autophagosome formation on the survival of T. spiralis. Understanding the role of autophagy in the interaction between parasitic infection and host cell is of great significance for the prevention and treatment of Trichinella infection and the development of anti-parasite drugs. [ABSTRACT FROM AUTHOR]

Background: Trichinellosis is a serious food-borne parasitic zoonosis, thus finding high quality antigens is the key to serodiagnosis of trichinosis. This article reports the characterization and sensitivity of four recombinant proteins expressed by four genes (Wn10, Zh68, T668, and Wm5) from different developmental stages of Trichinella spiralis for the diagnosis of trichinellosis in mice. Methods: This study was conducted in Jilin University and National Institute of Parasitic Diseases of Chinese Center for Disease Control and Prevention in 2017-2018. The structures and functions of the proteins encoded by four genes were predicted by bioinformatics analysis. The four genes were cloned and expressed, and the recombinant proteins were purified. Anti-Trichinella IgM and IgG antibodies in the sera of mice infected with T. spiralis from 1-45 d post-infection (dpi) were evaluated by ELISA. Results: The optimal antigen epitopes of four proteins (P1, P2, P3, and P4) encoded by the four genes from T- and B-cells were predicted, and four purified recombinant proteins (r-P1, r-P2, r-P3, and r-P4) were successfully produced. For IgM, the antibody levels detected by the four recombinant antigens were approximately equal to the cut-off value. Anti-Trichinella IgG antibodies were first detected by r-P1 at 8 dpi, followed by r-P2, r-P3, and r-P4 at 10 dpi, 14 dpi, and 16 dpi, respectively, and the antibody levels remained high until 45 dpi. Conclusion: The recombinant antigens r-P1, r-P2, r-P3, and r-P4 could be antigens that react with antibodies, they showed high sensitivity in the detection of anti-Trichinella IgG antibodies in mice. Among these proteins, r-P1 may be a candidate antigen for the detection of anti-Trichinella IgG antibodies in the early infection phase and exhibited the best sensitivity among the antigens. [ABSTRACT FROM AUTHOR]

Background: Several studies have reported the roles of Trichinella spiralis extracellular vesicles in immune regulation and pathogen diagnosis. Currently, the T. spiralis muscle larvae excretory/secretory product (Ts-ML-ES) is the antigen recommended by the International Commission on Trichinellosis (ICT) for serological diagnosis of trichinellosis. However, it can only be used to detect middle and late stages of infections, and cross-reactions with other parasite detections occur. Therefore, there is a need to identify antigens for specific detection of early stage trichinellosis. Methods: Extracellular vesicles of T. spiralis muscle larvae (Ts-ML-EVs) were isolated by ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, flow cytometry and western blot. Ts-ML-EVs protein profiles were analyzed by LC-MS/MS proteomics for identification of potential antigens (Ts-TTPA). Ts-TTPA were cloned into pMAL-c5X vector and expressed as recombinant proteins for evaluation of potential as detected antigens by western blot and ELISA. Results: Isolated Ts-ML-EVs were round or elliptic (with diameters between 110.1 and 307.6 nm), showing a bilayer membrane structure. The specific surface markers on the Ts-ML-EVs were CD81, CD63, enolase and the 14-3-3 protein. A total of 53 proteins were identified by LC-MS/MS, including a variety of molecules that have been reported as potential detection and vaccine candidates. The cDNA of Ts-TTPA selected in this study has a total length of 1152 bp, encoding 384 amino acids with a molecular weight of 44.19 kDa. It contains a trypsin domain and can be recognized by anti-His antibody. It reacted with swine sera infected with 10,000 T. spiralis at 15, 25, 35 and 60 days post-infection (dpi). At 10 μg/ml, this antigen could detect T. spiralis antibodies from the swine sera at 13 dpi. There were no cross-reactions with the swine sera infected with other parasites including Clonorchis sinensis, Toxoplasma gondii, Taenia suis, Ascaris suis and Trichuris suis. Conclusions: This study identifies potential early stage detection antigens and more thoroughly characterizes a serine protease domain-containing protein. Extracellular vesicle proteins may be explored as effective antigens for the early stage detection of trichinellosis. [ABSTRACT FROM AUTHOR]

Background: Trichinella nematodes are globally distributed food-borne pathogens, in which Trichinella spiralis is the most common species in China. Microsatellites are a powerful tool in population genetics and phylogeographic analysis. However, only a few microsatellite markers were reported in T. spiralis. Thus, there is a need to develop and validate genome-wide microsatellite markers for T. spiralis. Methods: Microsatellites were selected from shotgun genomic sequences using MIcroSAtellite identification tool (MISA). The identified markers were validated in 12 isolates of T. spiralis in China. Results: A total of 93,140 microsatellites were identified by MISA from 9267 contigs in T. spiralis genome sequences, in which 16 polymorphic loci were selected for validation by PCR with single larvae from 12 isolates of T. spiralis in China. There were 7–19 alleles per locus (average 11.25 alleles per locus). The observed heterozygosity (HO) and expected heterozygosity (HE) ranged from 0.325 to 0.750 and 0.737 to 0.918, respectively. The polymorphism information content (PIC) ranged from 0.719 to 0.978 (average 0.826). Among the 16 loci, markers for 10 loci could be amplified from all 12 international standard strains of Trichinella spp. Conclusions: Sixteen highly polymorphic markers were selected and validated for T. spiralis. Primary phylogenetic analysis showed that these markers might serve as a useful tool for genetic studies of Trichinella parasites. [ABSTRACT FROM AUTHOR]

The gut microbiota plays an important role in parasite-host interactions and the induction of immune defense responses. Trichinella spiralis is an important zoonotic parasite that can directly or indirectly interact with the host in the gut. Changes in the gut microbiota following infection with T. spiralis and the role of the gut microbiota in host immune defense against T. spiralis infection were investigated in our study. 16S rRNA sequencing analysis revealed that infection with T. spiralis can reduce the diversity of the gut microbiota and alter the structure of the gut microbiota during early infection, which was restored when the worm left the gut. Antibiotic treatment (ABX) and fecal bacterial transplantation (FMT) were used to investigate the role of the gut microbiota in the host expulsion response during infection with T. spiralis. We found that ABX mice had a higher burden of parasites, and the burden of parasites decreased after fecal bacterial transplantation. The results of flow cytometry and qPCR revealed that the disturbance of the gut microbiota affects the proportion of CD4+ T cells and the production of IL-4, which weakens Th2 responses and makes expulsion difficult. In addition, as the inflammatory response decreased with the changes of the microbiota, the Th1 response also decreased. The metabolomic results were in good agreement with these findings, as the levels of inflammatory metabolites such as ceramides were reduced in the ABX group. In general, T. spiralis infection can cause changes in the gut microbiota, and the presence or absence of microbes may also weaken intestinal inflammation and the expulsion of T. spiralis by affecting the immune response of the host. Author summary: Due to the complexity of intestinal environment and function, we are interested in the relationship between Trichinella spiralis and the gut microbiota. In our study, we reported the effects of T. spiralis infection on the gut microbiota of the host and gut microbiota intervention in T. spiralis expulsion by influencing the immune status of the host. On the one hand, we demonstrated that the infection of T. spiralis in the intestinal stage could lead to a decrease in the diversity of the gut microbiota and changes in community structure, and these results are conducive to T. spiralis invasion. On the other hand, the gut microbiota plays an important role in T. spiralis infection. The balance of gut microbiota is largely involved in the invasion and colonization of T. spiralis and the state of host immunity. Our findings further elucidated the relationship among host, parasite and gut microbiota, and provided new insights into the prevention and control of T. spiralis. In addition, the study also provides a new basis for the important role of the gut microbiota in pathogen infection and host homeostasis. [ABSTRACT FROM AUTHOR]

Abstract Background Therapeutic potential of helminth have been shown to have a protective effect on immune-mediated diseases such as Crohn's disease (CD), which is associated with increased production of T helper cell type 1. However, helminth therapy is unacceptable to patients due to side-effects and the fear of parasites. As helminths regulate the cellular immune responses through innate cells such as dendritic cells (DCs), cellular immunotherapy has been considered a therapeutic option to treat CD. Methods Bone marrow-dendritic cells were generated, enriched and treated with Trichinella spiralis muscle larval excretory/secretory products (Ts -MLES). DCs maturation was measured by flow cytometry and cytokine production of DCs were measured by ELISA. Colitis was generated by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) solution. For adoptive transfer, Ts -MLES treated-DCs injected intravenously 24 h prior to TNBS challenge. Disease activity index (DAI) including weight loss, diarrhea, and bloody stool were measured. Colon segments were stained with hematoxylin and eosin (H.E.) and periodic acid schiff (PAS) staining for histological damage scoring. The relative mRNA expression of cytokines in colon was analyzed by RT-PCR. Cytokine production in colon was measured by ELISA. Splenocytes were separated and cytokine profiles including Th1 (IFN-γ), Th2 (IL-4, IL-13), and Treg subsets (IL-10, TGF-β) were analyzed by flow cytometry. Results Ts -MLES regulated the maturation and cytokine production of DCs. Ts -MLES -DC ameliorated the severity of the TNBS-induced colitis. In the colon and the spleen, Ts -MLES-DC decreased IFN-γ (Th1) significantly and increased Th2 (IL-4, IL-13)- and Treg (IL-10, TGF-β)- related cytokines. Conclusions Ts -MLES-DC ameliorated the severity of the TNBS-induced colitis through decreasing IFN-γ. Ts -MLES-DC skewed the Th1-mediated response toward the Th2 type and regulatory T cell response. Highlights • T. spiralis MLES (Ts -MLES) regulated DCs phenotype. • Ts -MLES-DC ameliorated the severity of the TNBS-induced colitis • Ts -MLES-DC inhibited Th1 immune response while enhancing the Th2 and Treg immune response in TNBS-induced colitis in vivo. [ABSTRACT FROM AUTHOR]

Abstract Serine proteases have been identified as important molecules that are involved in many parasitic infections, and these molecules have also been suggested to play important roles in Trichinella spiralis infections. In the present study, the antigenic serine protease gene Ts-ADSp-7 , which was screened from a cDNA library of Trichinella spiralis Adults at 3 days post-infection (p.i.), was cloned and expressed in Escherichia coli. The encoded protein, Ts-ADSp-7, revealed a potential trypsin-like serine protease domain but lacked substrate banding site at position 227 and protease activity. Transcription could be detected in the Adult and muscle larval stage but not in the newborn larval stage, where no fluorescent signal was detected. Western blot analysis revealed that the 3 days p.i. Adults and muscle larvae could secrete Ts-ADSp-7. Interestingly, strong fluorescent signal of Ts-ADSp-7 could be detected in the nucleoli of the enlarged muscle cell nuclei from 12 to 16 days p.i. and in the β-stichosomes of the muscle larvae from 16 to 35 days p.i.. The coagulation assay indicated that Ts-ADSp-7 could inhibit intrinsic coagulation pathway. Regarding the putatively important function of the serine protease in the helminth infection to hosts, a total of 81 serine proteases were found in the parasite and mainly comprised eight subfamilies. These subfamilies exhibited high similarity to transmembrane serine protease, coagulation factor XI, lipocalin, guanylin, ceropin, kallikrein, and plasminogen. Moreover, stage specificity was detected in several subfamilies. In summary, the putatively inactive serine protease-like protein Ts-ADSp-7 could inhibit blood coagulation, and the protein is located in the enlarged nuclei of nurse cells during capsule formation. Furthermore, members of the serine protease family in the parasite might be important molecules in the parasite-host interaction. Graphical abstract Image Highlights • The antigenic serine protease gene Ts-AdSp-7 of Trichinella spiralis adult is transcripted in adult and muscle larval stage. • Ts-AdSp-7 was detected in the enlarged nuclei of myocytes in the capsule. • Ts-AdSp-7 was the main molecule in ES products of Trichinella spiralis adults that inhibit blood coagulation. • 81 serine proteases of Trichinella spiralis exhibited diversity of structures, stage-specificity, and potential functions. [ABSTRACT FROM AUTHOR]

Trichinella spiralis (T. spiralis) derived extracellular vesicles (EVs) have been proposed to play a key role in regulating the host immune responses. In this study, we provided the first investigation of EVs proteomics released by T. spiralis muscle larvae (ML). T. spiralis ML EVs (Ts-ML-EVs) were successfully isolated and characterized by transmission electron microscopy (TEM) and western blotting. Using liquid chromatograph mass spectrometer (LC-MS/MS) analysis, we identified 753 proteins in the Ts-ML-EVs proteome and annotated by gene ontology (GO). These proteins were enriched in different categories by GO, kyoto encyclopedia of genes and genomes (KEGG) and domain analysis. GO enrichment analysis indicated association of protein deglutathionylation, lysosomal lumen and serine-type endopeptidase inhibitor activity with proteins which may be helpful during parasite-host interaction. Moreover, KEGG enrichment analysis revealed involvement of Ts-ML-EVs proteins in other glycan degradation, complement and coagulation cascades, proteasome and various metabolism pathways. In addition, BALB/c mice were immunized by subcutaneous injection of purified Ts-ML-EVs. Ts-ML-EVs group demonstrated a 23.4% reduction in adult worms and a 43.7% reduction in ML after parasite challenge. Cellular and humoral immune responses induced by Ts-ML-EVs were detected, including the levels of specific antibodies (IgG, IgM, IgE, IgG1 and IgG2a) as well as cytokines (IL-12, IFN-γ, IL-4 and IL-10) in serum. The results showed that Ts-ML-EVs could induce a Th1/Th2 mixed immune response with Th2 predominant. This study revealed a potential role of Ts-ML-EVs in T. spiralis biology, particularly in the interaction with host. This work provided a critical step to against T. spiralis infection based on Ts-ML-EVs. Author summary: Extracellular vesicles (EVs) play an important role in cell-cell communication. They can transport functional molecules to target tissues and cells. During parasite infection, EVs provide suitable diagnostic makers and vaccine antigens for as well as inducing host immune responses. In Trichinella spiralis (T. spiralis), we previous proved that Ts-ML-EVs exerted immunomodulatory effect that inhibited experimental colitis in mice. Based on these results, we tested the protein composition of Ts-ML-EVs and the immune protective effect of Ts-ML-EVs. Our study showed that Ts-ML-EVs contained a number of immune-related proteins and it could be potential vaccine antigen for preventing T. spiralis infection. [ABSTRACT FROM AUTHOR]

Background: Deoxyribonuclease II (DNase II) is a well-known acidic endonuclease that catalyses the degradation of DNA into oligonucleotides. Only one or a few genes encoding DNase II have been observed in the genomes of many species. 125 DNase II-like protein family genes were predicted in the Trichinella spiralis (T. spiralis) genome; however, none have been confirmed. DNase II is a monomeric nuclease that contains two copies of a variant HKD motif in the N- and C-termini. Of these 125 genes, only plancitoxin-1 (1095 bp, GenBank accession no. XM_003370715.1) contains the HKD motif in its C-terminus domain. Methodology/Principal Findings: In this study, we cloned and characterised the plancitoxin-1 gene. However, the sequences of plancitoxin-1 cloned from T. spiralis were shorter than the predicted sequences in GenBank. Intriguingly, there were two HKD motifs in the N- and C-termini in the cloned sequences. Therefore, the gene with shorter sequences was named after plancitoxin-1-like (Ts-Pt, 885 bp) and has been deposited in GenBank under accession number KF984291. The recombinant protein (rTs-Pt) was expressed in a prokaryotic expression system and purified by nickel affinity chromatography. Western blot analysis showed that rTs-Pt was recognised by serum from T. spiralis-infected mice; the anti-rTs-Pt serum recognised crude antigens but not ES antigens. The Ts-Pt gene was examined at all T. spiralis developmental stages by real-time quantitative PCR. Immunolocalisation analysis showed that Ts-Pt was distributed throughout newborn larvae (NBL), the tegument of adults (Ad) and muscle larvae (ML). As demonstrated by DNase zymography, the expressed proteins displayed cation-independent DNase activity. rTs-Pt had a narrow optimum pH range in slightly acidic conditions (pH 4 and pH 5), and its optimum temperature was 25°C, 30°C, and 37°C. Conclusions: This study indicated that Ts-Pt was classified as a somatic protein in different T. spiralis developmental stages, and demonstrated for the first time that an expressed DNase II protein from T. spiralis had nuclease activity. Author Summary: Deoxyribonuclease II (DNase II) is classified into a unique family of nucleases and mediates the degradation of DNA associated with apoptosis. Although DNase II activity was first observed in 1947, and has been studied biochemically and enzymatically since the 1960s, only recently has genetic information on the enzyme been reported. Compared with enzymes from other species, including C. elegans, the DNase II-like protein family of the parasitic nematode T. spiralis has expanded remarkably, with an estimated 125 genes found in the draft genome of T. spiralis. However, none of these proteins have been confirmed by biochemical studies. This study describes Ts-Pt, a DNase II protein that is expressed in different T. spiralis developmental stages. The recombinant protein purified via a prokaryotic expression system displayed in vitro nuclease activity, as determined by DNase zymography. The exact function and mechanisms of Ts-Pt should be further explored in vivo. [ABSTRACT FROM AUTHOR]

Background: Deoxyribonuclease II (DNase II) is a well-known acidic endonuclease that catalyses the degradation of DNA into oligonucleotides. Only one or a few genes encoding DNase II have been observed in the genomes of many species. 125 DNase II-like protein family genes were predicted in the Trichinella spiralis (T. spiralis) genome; however, none have been confirmed. DNase II is a monomeric nuclease that contains two copies of a variant HKD motif in the N- and C-termini. Of these 125 genes, only plancitoxin-1 (1095 bp, GenBank accession no. XM_003370715.1) contains the HKD motif in its C-terminus domain. Methodology/Principal Findings: In this study, we cloned and characterised the plancitoxin-1 gene. However, the sequences of plancitoxin-1 cloned from T. spiralis were shorter than the predicted sequences in GenBank. Intriguingly, there were two HKD motifs in the N- and C-termini in the cloned sequences. Therefore, the gene with shorter sequences was named after plancitoxin-1-like (Ts-Pt, 885 bp) and has been deposited in GenBank under accession number KF984291. The recombinant protein (rTs-Pt) was expressed in a prokaryotic expression system and purified by nickel affinity chromatography. Western blot analysis showed that rTs-Pt was recognised by serum from T. spiralis-infected mice; the anti-rTs-Pt serum recognised crude antigens but not ES antigens. The Ts-Pt gene was examined at all T. spiralis developmental stages by real-time quantitative PCR. Immunolocalisation analysis showed that Ts-Pt was distributed throughout newborn larvae (NBL), the tegument of adults (Ad) and muscle larvae (ML). As demonstrated by DNase zymography, the expressed proteins displayed cation-independent DNase activity. rTs-Pt had a narrow optimum pH range in slightly acidic conditions (pH 4 and pH 5), and its optimum temperature was 25°C, 30°C, and 37°C. Conclusions: This study indicated that Ts-Pt was classified as a somatic protein in different T. spiralis developmental stages, and demonstrated for the first time that an expressed DNase II protein from T. spiralis had nuclease activity. [ABSTRACT FROM AUTHOR]

Background: Trichinella spiralis is a zoonotic food-borne parasite. A disease caused by infection with T. spiralis is called trichinellosis in humans. It is important to investigate the epidemic situation and the surveillance of herds and then prevent infection in humans. Therefore, this study is to develop a rapid and sensitive diagnostic method for on-site test in domestic and wild animals. Methods: Upconverting phosphor nanoparticles (UCNPs), an excellent optical label, were conjugated with the excretory-secretory (ES) antigens from T. spiralis muscle larvae (ML) or goat anti-rabbit IgG, and a lateral flow (LF) assay based on these probes (UCNPs-ES/goat anti-rabbit IgG) was developed for the rapid and sensitive detection of anti-T. spiralis IgG antibodies in pig serum. The assay is named the UPT-LF-ES assay. In addition, the probes were characterized, and the assay was optimized. A cut-off threshold of the assay was also identified by using 169 known negative pig samples. Performance of the assay to T. spiralis with different infective numbers, cross-reactivity with other parasitic infections, the single-blinded experiment, and coincidence were evaluated with the assay. Results: The UPT-LF-ES assay was successfully constructed and optimized based on the probes of UCNPs-ES/goat anti-rabbit IgG. In the pigs infected with 100, 1000, and 10,000 ML, positive results were first presented at 35 days post-infection (dpi), 30 dpi, and 25 dpi, respectively. The assay had no cross-reaction with other parasitic infections. A single-blinded experiment indicated that the sensitivity and specificity of the UPT-LF-ES assay were 100% and 100%, respectively, the area under the receiver operating characteristic (ROC) curve was 1.000. In addition, the value detected by the UPT-LF-ES assay was significantly different between positive and negative samples. Moreover, compared with the "gold standard" magnetic stirrer method, the coincidence rate of the UPT-LF-ES assay was 87.27%, and the kappa (K) coefficient was 0.7454, showing a substantial agreement. Conclusions: The UPT-LF-ES assay is a useful point-of-care test (POCT) with T. spiralis in the detection of pig, which contributes to preventing human trichinellosis. [ABSTRACT FROM AUTHOR]

Background: Parasites of the genus Trichinella are the pathogenic agents of trichinellosis, which is a widespread and severe foodborne parasitic disease. Trichinella spiralis resides primarily in mammalian skeletal muscle cells. After invading the cells of the host organism, T. spiralis must elude or invalidate the host's innate and adaptive immune responses to survive. It is necessary to characterize the pathogenesis of trichinellosis to help to prevent the occurrence and further progression of this disease. The aims of this study were to elucidate the mechanisms of nurse cell formation, pathogenesis and immune evasion of T. spiralis, to provide valuable information for further research investigating the basic cell biology of Trichinella-infected muscle cells and the interaction between T. spiralis and its host. Methods: We performed transcriptome profiling by RNA sequencing to identify global changes at 1, 3, 7, 10 and 15 days post-infection (dpi) in gene expression in the diaphragm after the parasite entered and persisted within the murine myocytes; the mice were infected by intravenous injection of newborn larvae. Gene expression analysis was based on the alignment results. Differentially expressed genes (DEGs) were identified based on their expression levels in various samples, and functional annotation and enrichment analysis were performed. Results: The most extensive and dynamic gene expression responses in host diaphragms were observed during early infection (1 dpi). The number of DEGs and genes annotated in the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology databases decreased significantly in the infected mice compared to the uninfected mice at 3 and 7 dpi, suddenly increased sharply at 10 dpi, and then decreased to a lower level at 15 dpi, similar to that observed at 3 and 7 dpi. The massive initial reaction of the murine muscle cells to Trichinella infection steadied in the later stages of infection, with little additional changes detected for the remaining duration of the studied process. Although there were hundreds of DEGs at each time point, only 11 genes were consistently up- or downregulated at all 5 time points. Conclusions: The gene expression patterns identified in this study can be employed to characterize the coordinated response of T. spiralis-infected myocytes in a time-resolved manner. This comprehensive dataset presents a distinct and sensitive picture of the interaction between host and parasite during intracellular infection, which can help to elucidate how pathogens evade host defenses and coordinate the biological functions of host cells to survive in the mammalian environment. [ABSTRACT FROM AUTHOR]

Human trichinellosis is acquired by eating raw or undercooked meats carrying muscle larvae of Trichinella spp. Toll-like receptors (TLRs) are essential components of the innate immune system. However, little is known about the potential application of TLR agonists for immunotherapy against Trichinella spiralis (T. spiralis) infection. Here, we evaluated the effects of four TLR agonists (i.e., TLR3, TLR4, TLR8, and TLR9 agonists) on T. spiralis infection in mice. The reduction rate of worm burden showed that TLR3 agonist poly(I:C) significantly reduced T. spiralis infection rather than TLR4, TLR8, and TLR9 agonists (p < 0.05). Moreover, TLR3 showed a continuous high-level of expression during 6–35 days post infection (dpi). The levels of interferon-gamma (IFN-γ), interleukin (IL)-2, and IL-6 increased significantly in mice serum compared with control group after treatment with TLR3 agonist at 0, 3, 6, 9, 12, 15, 18, 21, 28, and 35 dpi (p < 0.05). A significant decreasing trend was also detected in levels of IL-10 and IL-4 after treatment with TLR3 agonist compared with control group at 0, 3, 6, 9, 12, 15, 18, 21, 28, and 35 dpi (p < 0.05). Overall, this study suggested that TLR3-targeted therapies might be effective on worm burden reduction by regulation of the cytokine levels in the mice infected with T. spiralis. [ABSTRACT FROM AUTHOR]

The excretory-secretory product (ESP) of Trichinella spiralis (T. spiralis) has been reported to inhibit the growth of various tumor cells, but the mechanism is not yet clear. To explore the effect and mechanism of ESP on liver cancer cells, tumor models were established with H22 cells and then infected with T. spiralis. The results showed that the growth of tumors in mice infected with T. spiralis was significantly inhibited. ESP from adult worms or muscle larvae were then incubated with H22 cells in vitro , and it was found that the ESP could inhibit cell proliferation and promote apoptosis. Subsequently, apoptosis-related proteins in stimulated H22 cells were evaluated, and ESP was found to induce cell apoptosis through the mitochondrial pathway. Additionally, Th-related cytokines were investigated in vivo , and the results showed that the levels of Th1 cytokines were significantly increased in the early stage of T. spiralis infection, while Th2 cytokines increased later than Th1 cytokines, implying that Th1 cytokines with antitumor effects may play a role in inhibiting tumor growth at early stage. In short, ESP can directly induce tumor cell apoptosis and indirectly inhibit tumor cell growth through the host immune system, which may be the antitumor mechanism of T. spiralis infection. [ABSTRACT FROM AUTHOR]

• Nod-like receptor pyrin domain containing 3 (NLRP3) expression is elevated in CD4+ T cells at 7 and 14 dpi. • NLRP3 participates in the control of the Th2 immune response during Trichinella spiralis infection. • NLRP3 influences the Th2 cell-mediated host defenses against Trichinella spiralis. The inflammasome is a key line of immune defense against invading infectious pathogens. However, knowledge of the role of nod-like receptor pyrin domain containing 3 (NLRP3) in Trichinella spiralis infection which characteristically induces T-helper 2 (Th2) immune responses is sparse. In this study, we investigated the role of NLRP3 in the protection against T. spiralis infection through the Th2 immune response. We show that NLRP3 expression in CD4+ T cells was significantly increased at 7 days post-infection of T. spiralis. Compared to wild-type (WT) CD4+ T cells, the expression of IL-4 mRNA was reduced in NLRP3−/− CD4+ T cells, however, the expression of IFN-γ mRNA was comparable between the two groups. Consistently, ELISA and flow cytometry analysis showed that NLRP3−/− CD4+ T cells secreted lower levels of IL-4 than CD4+ T cells from WT mice, whilst the levels of IFN-γ secreted by NLRP3−/− CD4+ T cells were of similar levels to those secreted by WT CD4+ T cells. In addition, we observed a significant reduction of IL-4 and IL-13 by ELISA in NLRP3 −/− mice at 1, 2 and 4 weeks post-infection. Furthermore, we found that adult worm survival was substantially prolonged and muscle larvae burden was significantly increased in NLRP3 −/− mice. We further show that NLRP3 promotes the host defense against T. spiralis through its participation in the differentiation of Th2 cells. These findings provide novel insights into parasite expulsion and highlight the importance of NLRP3 in the host defense against T. spiralis. [ABSTRACT FROM AUTHOR]

• Trichinella spiralis extracellular vesicles (Ts -EVs) intervened with the inflammatory responses of DSS-induced colitis in mice; • Ts -EVs inhibited M1 macrophage polarization in DSS-induced colitis; • Ts -EVs targeted both the MAPK and NF-κB pathways to regulate host immunity in DSS-induced colitis. Extracellular vesicles (EVs) are membranous containers released by cells that are powerful agents of intercellular communication. EVs have been described for various parasites and are associated with tissue inflammation. Several studies have demonstrated that parasite EVs can have either pro- or anti-inflammatory impacts, depending on the type of parasite. To evaluate the immunomodulatory properties of EVs produced by Trichinella spiralis (T. spiralis), we established a mouse model with dextran sulphate sodium (DSS)-induced colitis. The muscle larvae of T. spiralis were cultured in vitro and the released EVs were isolated by ultracentrifugation. T. spiralis EVs (Ts -EVs) were characterized according to morphology, size and constituent surface proteins (CD63, Enolase and Hsp70). Mice were treated with water containing 3% DSS after last intraperitoneal injection of Ts -EVs. Disease activity index (DAI), macroscopic and histopathological scores of Ts -EVs group was lower than DSS group. And Ts -EVs prevented the increase in the expression of TNF-α, IFN-γ, IL-17A and IL-1β observed in the colon of DSS-treated mice. In contrast, upregulation of IL-4, IL-10, TGF-β and IL-13 expression was detected in Ts -EVs+DSS group. In addition, Ts -EVs increased the infiltration of alternatively activated (M2) macrophages into the colon. The expression of CD206 (M2 marker) in the mesenteric lymph nodes (MLN) of mice with colitis increased in Ts -EVs+DSS group. Furthermore, Ts -EVs interfered with both the NF-κB and MAPK signalling pathways. Taken together, our findings demonstrate that Ts -EVs can affect the development of inflammation in DSS-induced colitis by inhibiting M1 macrophage polarization, due to their immunomodulatory ability. [ABSTRACT FROM AUTHOR]

• The recombinant serine protease from newborn larval stage (NBL-SP) of trichinella spirails was expressed, purified and the endotoxin in NBL-SP was removed;. • NBL-SP alleviated the severity of TNBS-induced colitis in mice;. • NBL-SP skewed the Th1 and Th17 response induced by TNBS towards the Th2 and treg response. Inflammatory bowel disease (IBD) is a complex immune-mediated disease of gastrointestinal tract that is mainly driven by Th1/Th17 immune response. "Helminth therapy" has emerged, and helminth-derived immunoregulatory molecules are being used as safe and new therapeutic antigens for IBD. Recombinant serine protease (SP) from newborn Trichinella spiralis (T. spiralis) larvae (NBL) was expressed and purified. BALB/c mice were immunized with NBL-SP at 100 µg three times at an interval of 5 days. Experimental colitis was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) administration. The disease activity index (DAI) and macroscopic and microscopic scores of the colon were assessed to identify the effect of NBL-SP on experimental colitis. Cytokine production in the serum was analysed by meso scale discovery (MSD). Cytokine production in the colon was detected by ELISA. CD4+ T cell differentiation was measured by flow cytometry. NBL-SP alleviated TNBS-induced colitis in mice. The DAI, macroscopic and microscopic scores and colon length all showed a positive intervention effect of NBL-SP on experimental colitis. NBL-SP can weaken the increase in IFN-γ, TNF-α and IL-17 production as well as CD4+ IFN-γ+ T cell and CD4+IL-17+ T cell populations induced by colitis. Furthermore, the levels of Th2-related cytokines (IL-4, IL-5) and regulatory cytokines (IL-10, TGF-β) were elevated meanwhile the ratio of regulatory T cells (Tregs) and CD4+ IL-4 + T cells were increased by NBL-SP. NBL-SP of T. spiralis had a potential protective effect against IBD. NBL-SP skewed the Th1 and Th17-mediated response towards the Th2 and Treg response. [ABSTRACT FROM AUTHOR]

• rTs-ADSp-7 alleviated the severity of TNBS-induced colitis. • rTs-ADSp-7 increased the population of Th2 and Treg cells in TNBS-induced colitis mice. • rTs-ADSp-7 protein has the potential to treat colitis or other Th1 immune-mediated diseases. Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), is a chronic autoimmune disease. At present, worms and their products has been shown to have protective effects on immune-mediated diseases. Therefore, we aimed to investigate the effect of the recombination Trichinella spiralis (T. spiralis, Ts) adult serine protease-like protein r Ts -ADSp-7 on a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced CD mouse model. Colitis was induced by intrarectal administration of a TNBS solution. The disease activity index (DAI), which included weight loss, diarrhoea, and bloody stool, was measured. Colon segments were stained with haematoxylin and eosin (H.E.) for histopathological score. Cytokine release in the serum was analysed by meso scale discovery (MSD). Cytokine release in the colon was detected by ELISA. Splenocytes were separated, and the cytokine profiles of Th1 (IFN-γ), Th2 (IL-4), Th17 (IL-17A) and Treg cells were analysed by flow cytometry. Our result showed that rTs-ADSp-7 reduced the clinical disease activity of TNBS-induced colitis in mice. In addition, we found that r Ts -ADSp-7 reduced the production of Th1- and Th17-related cytokines while upregulating the expression of Th2- and Treg-related cytokines in TNBS-induced colitis mice. r Ts -ADSp-7 also increased the population of Th2 and Treg cells in TNBS-induced colitis mice. r Ts -ADSp-7 alleviated the severity of TNBS-induced colitis while balancing the CD4+ T cell immune response. r Ts -ADSp-7 has therapeutic potential for colitis treatment and can be used as a helminth-derived protein therapy for CD or other Th1 immunity-mediated diseases. [ABSTRACT FROM AUTHOR]

• SERS combined with PCA-LDA has great potential in trichinellosis detection. • PCA-LDA analyses achieved a high diagnostic accuracy. • The ROC curve illustrated the performance of PCA-LDA analyses and the value is 0.977. Based on blood serum surface-enhanced Raman spectroscopy (SERS) analysis, this paper proposed a simple and unlabeled non-invasive serum detection for T. spiralis infection. Serum samples were collected and analyzed from 40 rats at 0 days post infection (dpi) (normal rats), 19 uninfected rats, and 16 rats infected with T. spiralis at 28 dpi, using SERS measurements. Multivariate statistical techniques, such as linear discriminant analysis (LDA) and principal components analysis (PCA), were used to analyze and identify the obtained blood serum SERS spectra. The diagnosis algorithms, based on PCA-LDA, achieved a diagnostic sensitivity of 87.5%, a specificity of 94.7%, and an accuracy of 91.4% for separating the samples infected with T. spiralis from the control samples. This exploratory study demonstrated that colloidal Ag NPs-based SERS serum analysis technique combined with PCA-LDA has a great potential in improving the detection of T. spiralis infection and onsite screening. [ABSTRACT FROM AUTHOR]