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Previous studies have shown that recombinant Trichinella spiralis galectin (rTsgal) is characterized by a carbohydrate recognition domain sequence motif binding to beta-galactoside, and that rTsgal promotes larval invasion of intestinal epithelial cells. Galactomannan is an immunostimulatory polysaccharide composed of a mannan backbone with galactose residues. The aim of this study was to investigate whether galactomannan inhibits larval intrusion of intestinal epithelial cells and enhances antibody-dependent cellular cytotoxicity (ADCC), killing newborn larvae by polarizing macrophages to the M1 phenotype. The results showed that galactomannan specially binds to rTsgal, and abrogated rTsgal facilitation of larval invasion of intestinal epithelial cells. The results of qPCR, Western blotting, and flow cytometry showed that galactomannan and rTsgal activated macrophage M1 polarization, as demonstrated by high expression of iNOS (M1 marker) and M1 related genes (IL-1β, IL-6, and TNF-α), and increased CD86+ macrophages. Galactomannan and rTsgal also increased NO production. The killing ability of macrophage-mediated ADCC on larvae was also significantly enhanced in galactomannan- and rTsgal-treated macrophages. The results demonstrated that Tsgal may be considered a potential vaccine target molecule against T. spiralis invasion, and galactomannan may be a novel adjuvant therapeutic agent and potential vaccine adjuvant against T. spiralis infection.

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 V max , K m , and k cat /K m 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. [ABSTRACT FROM AUTHOR]

We have previously reported that Trichinella spiralis glutathione-S-transferase (TsGST) gene is an up-regulated gene in intestinal infective larvae (IIL) compared to muscle larvae (ML). In this study, the TsGST gene was cloned, and recombinant TsGST (rTsGST) was produced. Anti-rTsGST serum recognized the native TsGST by Western blotting in crude antigens of ML, adult worm (AW) and newborn larvae (NBL) of T. spiralis , but not in ML excretory–secretory (ES) antigens. Expression of TsGST was observed in all different developmental stages (IIL, AW, NBL and ML). An immunolocalization analysis identified TsGST in the cuticle, stichosome and genital primordium of the parasite. The rTsGST had GST enzymatic activity. After a challenge infection with T. spiralis larvae, mice immunized with rTsGST displayed a 35.71% reduction in adult worms and a 38.55% reduction in muscle larvae. The vaccination of mice with rTsGST induced the Th1/Th2-mixed type of immune response with Th2 predominant (high levels of IgG1) and partial protective immunity against T. spiralis infection. [ABSTRACT FROM AUTHOR]

Highlights: [•] A serine protease gene (TspSP-1.2) of T. spiralis was cloned and characterized. [•] The serine protease was located in cuticle and internal organs of the parasite. [•] Transcripts for the protease were observed at all developmental stages of T. spiralis. [•] Anti-TspSP-1.2 sera partially prevented the larval invasion of intestinal cells. [•] Recombinant TspSP-1.2 protein induced partial protective immunity in mice. [ABSTRACT FROM AUTHOR]

Abstract: Although it has been known for many years that Trichinella spiralis initiates infection by invading intestinal epithelium, the mechanisms by which the parasite invades the intestinal epithelium are unknown. The purpose of this study was to screen the invasion-related proteins among the increased proteins of intestinal epithelial cells after culture with T. spiralis and to study their molecular functions. The proteins of HCT-8 cells which cultured with T. spiralis infective larvae were analyzed by SDS-PAGE and Western blot. Results showed that compared with proteins of normal HCT-8 cells, four additional protein bands (115, 61, 35 and 24kDa) of HCT-8 cells cultured with the infective larvae were recognized by sera of the mice infected with T. spiralis, which may be the invasion-related proteins released by the infective larvae. Three bands (61, 35 and 24kDa) were studied employing shotgun LC–MS/MS. Total 64 proteins of T. spiralis were identified from T. spiralis protein database by using SEQUEST searches, of which 43 (67.2%) proteins were distributed in a range of 10–70kDa, and 26 proteins (40.6%) were in the range of pI 5–6. Fifty-four proteins were annotated according to Gene Ontology Annotation in terms of molecular function, biological process, and cellular localization. Out of 54 annotated proteins, 43 proteins (79.6%) had binding activity and 23 proteins (42.6%) had catalytic activity (e.g. hydrolase, transferase, etc.), which might be related to the invasion of intestinal epithelial cells by T. spiralis. The protein profile provides a valuable basis for further studies of the invasion-related proteins of T. spiralis. [Copyright &y& Elsevier]

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 (r 1 = −0.96798, r 2 = −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. [ABSTRACT FROM AUTHOR]

The aim of this study was to identify the biological characteristics and functions of a putative Trichinella spiralis glutathione S-transferase (TspGST). The results of real-time PCR and immunofluorescent test (IFT) showed that the TspGST gene was expressed at all of T. spiralis different developmental stages (muscle larvae, intestinal infective larvae, adult worms and newborn larvae). When anti-rTspGST serum, mouse infection serum, and pre-immune serum were added to the medium, the inhibition rate of the larvae penetrated into the intestinal epithelial cells (IECs) was 25.72%, 49.55%, and 4.51%, respectively ( P  < 0.01). The inhibition of anti-rTspGST serum on larval invasion of IECs was dose-dependent ( P  < 0.05). Anti-rTspGST antibodies killed T. spiralis newborn larvae by an ADCC-mediated mechanism. Our results showed that the TspGST seemed to be an indispensable protein for T. spiralis invasion, growth and survival in host. [ABSTRACT FROM AUTHOR]

• The mice were intraperitoneally injected with 200 mM mannose for 4 weeks. • Mannose reduced enteral adult and muscle worm burden after T. spiralis infection. • Mannose also relieved inflammation of intestine and muscle of infected mice. • The protection of mannose might be not related to host's immune responses. • Mannose could be considered as a novel adjuvant against early Trichinella infection. Trichinellosis is a major zoonotic parasitosis which is a vital risk to meat food safety. It is requisite to exploit new strategy to interdict food animal Trichinella infection and to obliterate Trichinella from food animals to ensure meat safety. Mannose is an oligosaccharide that specifically binds to the carbohydrate-recognition domain of C-type lectin; it has many physiological functions including reliving inflammation and regulating immune reaction. The purpose of this study was to investigate the suppressive role of mannose on T. spiralis larval invasion and infection, its effect on intestinal and muscle inflammation, and immune responses after challenge. The results showed that compared to the saline-treated infected mice, the mannose-treated infected mice had less intestinal adult and muscle worm burdens, mild inflammation of intestine and muscle of infected mice. The levels of specific anti- Trichinella IgG (IgG1/IgG2a), IgA and sIgA in mannose-treated infected mice were obviously inferior to saline-treated infected mice (P < 0.01). Furthermore, the levels of two cytokines (IFN-γ and IL-4) in mannose-treated infected mice were also significantly lower than the saline-treated infected mice (P < 0.01). The protective effect of the mannose against Trichinella infection might be not related to specific antibody and cellular immune responses. The above results demonstrated that mannose could be considered as a novel adjuvant therapeutic agent for anti- Trichinella drugs to block larval invasion at early stage of Trichinella infection. [ABSTRACT FROM AUTHOR]

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 . [ABSTRACT FROM AUTHOR]

• A novel Trichinella spiralis cathepsin L (TsCL) was cloned and expressed. • TsCL is expressed in various stage and mainly located in cuticle, stichosome, midgut and embryos. • rTsCL has the capacity to specially bind with intestinal epithelial cells (IECs). • rTsCL promoted the larval invasion of IECs. • RNAi impaired larval invasive ability, development and female fecundity. Cathepsin L is one member of cysteine protease superfamily and widely distributed in parasitic organisms, it plays the important roles in worm invasion, migration, nutrient intake, molting and immune evasion. The objective of this study was to investigate the biological characteristics of a novel cathepsin L from Trichinella spiralis (TsCL) and its role in larval invasion, development and reproduction. TsCL has a functional domain of C1 peptidase, which belongs to cathepsin L family. The complete TsCL sequence was cloned and expressed in Escherichia coli BL21. The rTsCL has good immunogenicity. RT-PCR and Western blotting analysis showed that TsCL was transcribed and expressed at different T. spiralis phases (e.g., muscle larvae, intestinal infectious larvae, adult worms and newborn larvae). Immunofluorescence test revealed that TsCL was principally localized in the cuticle, stichosome, midgut and female intrauterine embryos of the nematode. rTsCL has the capacity to specially bind with intestinal epithelial cells (IECs) and the binding sites was located in the cytoplasm. rTsCL promoted larval penetration into IEC, while anti-rTsCL antibodies inhibited the invasion. The silencing of TsCL gene by specific dsRNA significantly reduced the TsCL expression and enzyme activity, and also reduced larval invasive ability, development and female reproduction. The results showed that TsCL is an obligatory protease in T. spiralis lifecycle. TsCL participates in worm invasion, development and reproduction, and may be regarded as a potential candidate vaccine/drug target against T. spiralis infection. [ABSTRACT FROM AUTHOR]

• Silencing of Trichinella spiralis glutathione S-transferase (TsGST) suppresses its expression and activity. • Specific siRNA inhibits larval invasion of enterocytes and isolated intestine. • Knockdown of TsGST gene impairs the worm development and reproduction. • TsGST is a new candidate molecular target for anti- Trichinella vaccines. The purpose of this study was to determine the biological function of a Trichinella spiralis glutathione S-transferase (TsGST) in larval invasion and development by RNA interference (RNAi). The TsGST-specific siRNA 366 was transfected into T. spiralis muscle larvae (ML) via electroporation. At 1 day following transfection, the larval TsGST mRNA and protein expressions were reduced by 40.09 and 65.22 % (P < 0.05), respectively. The enzymatic activity of natural TsGST in siRNA-transfected ML was also suppressed by 45% compared with PBS group (P < 0.05). Silencing of the TsGST significantly inhibited the ability of larvae to invade intestinal epithelium cells (IECs) and isolated intestine. After challenge with siRNA-366-treated ML, the infected mice exhibited a 62.82% reduction of intestinal adult worms, and 65.03 % reduction of muscle larvae compared to the PBS group. Besides, the length of adults, newborn larvae and muscle larvae was significantly shorter than that of control siRNA and PBS group; the female fecundity of siRNA 366 group was lower than those of control siRNA and PBS group (P < 0.05). The results revealed that the specific RNAi significantly reduced the expression and enzymatic activity of TsGST, inhibited the larval invasive and developmental capacity, and impaired the female fecundity. The results further confirmed that TsGST plays a crucial role in the T. spiralis life cycle and it might be a potential molecular target for anti- Trichinella vaccines. [ABSTRACT FROM AUTHOR]

• TsCX gene was transcribed and expressed at diverse Trichinella spiralis stages. • There was a specific binding between TsCX and intestinal epithelial cells (IEC). • TsCX binding to IEC was located in IEC cytoplasm. • rTsCX promoted T. spiralis larva invasion of IEC. • TsCX is a potential target of vaccines against T. spiralis enteral stages. The aim of this study was to ascertain the characteristics of a Trichinella spiralis cathepsin X (TsCX) and its role on larval invasion of intestinal epithelial cells (IECs). The full-length of TsCX cDNA sequence was cloned and expressed in Escherichia coli BL21. The results of RT-PCR, IFA and Western blot revealed that TsCX was expressed at T. spiralis muscle larvae (ML), intestinal infective larvae, adult worm and newborn larvae, and it was located in whole worm section. The results of Far western and confocal microscopy demonstrated that there was a specific binding of rTsCX and IEC, and the binding site was located within the IEC cytoplasm. rTsCX promoted T. spiralis larval invasion of mouse IECs while anti-rTsCX antibody inhibited larval invasion into the IECs. Silencing TsCX by specific siRNA reduced the TsCX expression and larval invasive capacity. These results indicated that TsCX specifically binds to IECs and promotes larval invasion of intestinal epithelia, and it might be a potential target of vaccines against enteral stages of T. spiralis. [ABSTRACT FROM AUTHOR]