Serology survey of Ascaris suum and Trichinella spiralis in rural pigs in Southwestern Mexico.

BACKGROUND: Parasitic diseases of pigs are a public and veterinary health problem. Helminths influence pork production, whereas backyard pigs can transmit these parasites. OBJECTIVES: This work aimed to investigate the prevalence of antibodies against Ascaris suum and Trichinella spiralis in backyard pigs from Jamiltepec, Region de la Costa, Oaxaca, in Southwestern Mexico. METHODS: Six hundred sixty-four serum samples were obtained from backyard pigs from 23 rural villages distributed in 5 municipalities; samples were taken in a non-probabilistic manner with the owner's consent. The presence of serum antibodies against a total extract of A. suum adult worm was determined by ELISA. In contrast, antibodies to the excretion-secretion products of the T. spiralis muscle larva were determined by Western blot. RESULTS: The global seroprevalence for A. suum was 5.12% and 2.41% for T. spiralis; however, antibodies were only found in 8 villages and distributed in 3 municipalities. The highest frequency of positivity for Ascaris was found in the municipality of Santa Catarina Mechoacán (13.01%), whereas, in Santa María Huazalotitlán, the highest frequency of positivity for Trichinella was found (5.75%). In San Andrés, frequencies were 7.23% and 4.82%, respectively. No statistical differences were observed between populations. CONCLUSIONS: Our data suggest that helminth transmission is restricted by locality. However, further studies must be conducted to understand the factors limiting this transmission to promote pork meat production in parasite-free zones.

Crystal structure of Trichinella spiralis calreticulin and the structural basis of its complement evasion mechanism involving C1q.

Helminths produce calreticulin (CRT) to immunomodulate the host immune system as a survival strategy. However, the structure of helminth-derived CRT and the structural basis of the immune evasion process remains unclarified. Previous study found that the tissue-dwelling helminth Trichinella spiralis produces calreticulin (TsCRT), which binds C1q to inhibit activation of the complement classical pathway. Here, we used x-ray crystallography to resolve the structure of truncated TsCRT (TsCRTΔ), the first structure of helminth-derived CRT. TsCRTΔ was observed to share the same binding region on C1q with IgG based on the structure and molecular docking, which explains the inhibitory effect of TsCRT on C1q-IgG-initiated classical complement activation. Based on the key residues in TsCRTΔ involved in the binding activity to C1q, a 24 amino acid peptide called PTsCRT was constructed that displayed strong C1q-binding activity and inhibited C1q-IgG-initiated classical complement activation. This study is the first to elucidate the structural basis of the role of TsCRT in immune evasion, providing an approach to develop helminth-derived bifunctional peptides as vaccine target to prevent parasite infections or as a therapeutic agent to treat complement-related autoimmune diseases.

The effects of anti-lung cancer in nude mice by a fully human single-chain antibody against associated antigen Ts7TMR between A549 cells and Trichinella spiralis.

Lung cancer is a dangerous disease that is lacking in an ideal therapy. Here, we evaluated the anti-lung cancer effect in nude mice of a fully human single-chain antibody (scFv) against the associated antigen 7 transmembrane receptor (Ts7TMR), which is also called G protein-coupled receptor, between A549 cells and Trichinella spiralis (T. spiralis). Our data showed that anti-Ts7TMR scFv could inhibit lung cancer growth in a dose-dependent manner, with a tumour inhibition rate of 59.1%. HE staining did not reveal any obvious tissue damage. Mechanistically, immunohistochemical staining revealed that the scFv down-regulated the expression of PCNA and VEGF in tumour tissues. Overall, this study found that anti-Ts7TMR scFv could inhibit A549 lung cancer growth by suppressing cell proliferation and angiogenesis, which may provide a new strategy for treating lung cancer.

The prophylactic and therapeutic impact of Trichinella spiralis larvae excretory secretory antigens- loaded Ca-BTC metal organic frameworks on induced murine colitis.

Background: Inflammatory bowel disease is an autoimmune disease that affects the gut. T. spiralis larvae (E/S Ags) loaded on calcium-benzene-1,3,5-tricarboxylate metal-organic frameworks (Ca-BTC MOFs) were tested to determine whether they might prevent or cure acetic acid-induced murine colitis. Methods: T. spiralis larvae E/S Ags/Ca-BTC MOFs were used in prophylactic and therapeutic groups to either precede or follow the development of murine colitis. On the seventh day after colitis, mice were slaughtered. The effect of our target antigens on the progress of the colitis was evaluated using a variety of measures, including survival rate, disease activity index, colon weight/bodyweight, colon weight/length) ratios, and ratings for macroscopic and microscopic colon damage. The levels of inflammatory cytokines (interferon-γ and interleukin-4), oxidative stress marker malondialdehyde, and glutathione peroxidase in serum samples were evaluated. Foxp3 T-reg expression was carried out in colonic and splenic tissues. Results: T. spiralis larvae E/S Ags/Ca-BTC MOFs were the most effective in alleviating severe inflammation in murine colitis. The survival rate, disease activity index score, colon weight/length and colon weight/bodyweight ratios, and gross and microscopic colon damage scores have all considerably improved. A large decrease in proinflammatory cytokine (interferon-γ) and oxidative stress marker (malondialdehyde) expression and a significant increase in interleukin-4 and glutathione peroxidase expression were obtained. The expression of Foxp3+ Treg cells was elevated in colonic and splenic tissues. Conclusion: T. spiralis larvae E/S Ags/Ca-BTC MOFs had the highest anti-inflammatory, antioxidant, and cytoprotective capabilities against murine colitis and might be used to develop new preventative and treatment strategies.

Helminth alleviates COVID-19-related cytokine storm in an IL-9-dependent way.

Hyperactivation of pro-inflammatory type 1 cytokines (e.g., tumor necrosis factor alpha [TNF-α] and interferon gamma [IFN-γ]) mirrors the inflammation of coronavirus disease 2019. Helminths could alleviate excessive immune responses. Here, helminth Trichinella spiralis (Ts) infection was shown to protect against TNF-α- and IFN-γ-induced shock. Mechanistically, Ts-induced protection was interleukin-9 (IL-9) dependent but not IL-4Rα. Recombinant IL-9 treatment not only improved the survival of wild-type mice with TNF-α- and IFN-γ-induced shock but also that of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected K18-human angiotensin-converting enzyme 2 (hACE2) mice, emphasizing the significance of IL-9 in alleviating cytokine storm syndromes during SARS-CoV-2 infection. Interestingly, Ts excretory/secretory (TsES)-induced protection was also observed in SARS-CoV-2 infection, indicating that identifying anti-inflammatory molecules from TsES could be a novel way to mitigate adverse pathological inflammation during pathogen infection.IMPORTANCESevere coronavirus disease 2019 (COVID-19) is linked to cytokine storm triggered by type 1 pro-inflammatory immune responses. TNF-α and IFN-γ shock mirrors cytokine storm syndromes, including COVID-19. Helminths (e.g., Trichinella spiralis, Ts) can potently activate anti-inflammatory type 2 immune response. Here, we found that helminth Ts-induced protection against TNF-α and IFN-γ shock was IL-9 dependent. Treatment with recombinant IL-9 could protect against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in K18-hACE2 mice. Helminth Ts excretory/secretory (TsES) products also ameliorated SARS-CoV-2 infection-related cytokine storm. In conclusion, our study emphasizes the significance of IL-9 in protecting from cytokine storm syndromes associated with SARS-CoV-2 infection. Anti-inflammatory molecules from TsES could be a new source to mitigate adverse pathological inflammation associated with infections, including COVID-19.

Trichinella spiralis Larval Extract as a Biological Anti-Tumor Therapy in a Murine Model of Ehrlich Solid Carcinoma.

Trichinella spiralis (T. spiralis) is an immunomodulating parasite that can adversely affect tumor growth and extend host lifespan. The aim of this study was to elucidate the mechanisms by which T. spiralis larval antigens achieve this effect using Ehrlich solid carcinoma (ESC) murine model. Assessment was done by histopathological and immunohistochemical analysis of caspase-3, TNF-α, Ki-67 and CD31. Additionally, Bcl2 and Bcl2-associated protein X (Bax) relative gene expression was assessed by molecular analysis for studying the effect of T. spiralis crude larval extract (CLE) antigen on tumor necrosis, apoptosis, cell proliferation and angiogenesis. We found that both T. spiralis infection and CLE caused a decrease in the areas of necrosis in ESC. Moreover, they led to increased apoptosis through activation of caspase-3, up-regulation of pro-apoptotic gene, Bax and down-regulation of anti-apoptotic gene, Bcl2. Also, T. spiralis infection and CLE diminished ESC proliferation, as evidenced by decreasing Ki-67. T. spiralis infection and CLE were able to suppress the development of ESC by inhibiting tumor proliferation, inducing apoptosis and decreasing tumor necrosis, with subsequent decrease in tumor metastasis. T. spiralis CLE antigen may be considered as a promising complementary immunotherapeutic agent in the treatment of cancer.

Eosinophilic Pleural Effusion Secondary to Trichinella spiralis Infection in a Patient with Systemic Sclerosis: A Case Report.

BACKGROUND Scleroderma is a chronic autoimmune disease characterized by angiopathy, autoimmunity, and fibrosis. One form of scleroderma, systemic sclerosis, is characterized by diffuse skin lesions and visceral involvement. Eosinophilic pleural effusion is a rare complication attributed to a large array of diseases. We present a case of a man with underlying systemic sclerosis who developed eosinophilic pleural effusion as a complication of associated Trichinella spiralis infection. CASE REPORT A 49-year-old man presented for bilateral inflammatory radio-ulnar-carpal joint pain, paresthesia of the hands and forearms and a 2-week history of right posterior aching thoracic pain and night sweats. The physical examination revealed sclerodermatous skin involvement of the hands, forearms, and forehead, sclerodactyly, Raynaud's phenomenon, and telangiectasias, together with muffled cardiac sounds and right basal abolishment of the vesicular breath sounds. Imagistic evaluation showed the presence of pleuro-pericardial fluid. A thoracocentesis highlighted the presence of an exudative eosinophilic pleural effusion. Laboratory findings showed leukocytosis, with elevated neutrophil and eosinophil counts. The patient was tested for a parasitic infection, but initially the results were negative. He started anti-inflammatory treatment, but no reduction of the pleural fluid was observed. Subsequent evaluation revealed specific anti-trichinella IgG antibodies. Albendazole and corticosteroid therapy were initiated, which resulted in remission of the symptoms. CONCLUSIONS This report highlights the possibility of developing rare or even not-until-now seen complications when 2 etiologically different diseases are associated. The physician should carefully assess the situation to find and resolve the underlying causes.

Helminth-induced impairment of humoral immunity differently contribute to their anti-arthritic effects in mice: Comparison of Schistosoma mansoni and Trichinella spiralis.

AIMS: We have previously reported reduction of anti-type II collagen (IIC) IgG levels in collagen-induced arthritis (CIA) by Schistosoma mansoni (Sm) and Trichinella spiralis (Ts). To clarify the contribution of the impairment of humoral immunity to their anti-arthritic activities, we herein investigated the relationship between anti-IIC IgG levels and arthritic swelling in Sm- or Ts-infected mice. METHODS AND RESULTS: Male DBA/1J mice were infected with Sm cercariae or Ts muscle larvae prior to the IIC immunization. In the Sm-infected mice, paw swelling and anti-IIC IgG levels were continuously lower than those of non-infected control group. In contrast, arthritic swelling in the Ts-infected mice only decreased in the early phase of CIA progression, despite the continued impairment of anti-IIC IgG production throughout the experimental period. Correlation coefficients between residual paw swelling and anti-IIC IgG titers were similar or higher in the Sm group than in the control group, but were similar or lower in the Ts group than in the control group. CONCLUSION: The down-modulations of anti-IIC IgG levels by the two parasitic infections and the correlation analyses suggest that the anti-arthritic activity of Sm was primarily attributed to the modulation of IgG-independent arthritogenic mechanisms and secondarily to the impairment of anti-IIC IgG production. In contrast, Ts could alleviate CIA mainly via the impairment of antibody production.

Superb bio-effectiveness of Cobalt (II) phthalocyanine and Ag NPs adorned Sm-doped ZnO nanorods/cuttlefish bone to annihilate Trichinella spiralis muscle larvae and adult worms: In-vitro evaluation.

Herein, innovative biocides are designed for the treatment of Trichinella spiralis muscle larvae (ML) and adult worms. Samarium-doped ZnO nanorods (Sm-doped ZnO) are stabilized onto the laminar structure of cuttlefish bone (CB) matrix and adorned by either Ag NPs or cobalt phthalocyanine (CoPc) species. Physicochemical characteristics of such nanocomposites are scrutinised. Adorning of Sm-doped ZnO/CB with Ag NPs shortens rod-like shaped Sm-doped ZnO nanoparticles and accrues them, developing large-sized detached patches over CB moiety. Meanwhile, adorning of Sm-doped ZnO/CB by CoPc species degenerates CB lamellae forming semi-rounded platelets and encourages invading of Sm-doped ZnO nanorods deeply inside gallery spacings of CB. Both nanocomposites possess advanced parasiticidal activity, displaying quite intoxication for ML and adult worms (≥88% mortality) within an incubation period of <48 h at concentrations around 200 µg/ml. CoPc@Sm-doped ZnO/CB nanocomposite exhibits faster killing efficiency of adult worms than that of Ag@Sm-doped ZnO/CB at a concentration of ∼75 µg/ml showing entire destruction of parasite after 24 h incubation with the former nanocomposite and just 60% worm mortality after 36 h exposure to the later one. Morphological studies of the treated ML and adult worms show that CoPc@Sm-doped ZnO/CB exhibits a destructive impact on the parasite body, creating featureless and sloughed fragments enriched with intensive vacuoles. Hybridization of cuttlefish bone lamellae by CoPc species is considered a springboard for fabrication of futuristic aggressive drugs against various food- and water-borne parasites.

Light microscopical and parasitological analyses revealed the beneficial effects of silver nanoparticles and various myrrh extracts against Trichinella spiralis infection in mice.

Trichinella spiralis infection is a food-borne zoonotic disease caused by nematodes that dwell in the tissues, presenting a significant public health concern. This study aimed to evaluate the effectiveness of different treatments including silver nanoparticles (AgNPs), myrrh biosynthesized AgNPs "AgNPs synthesized using plant-based green technologies", myrrh extract, and myrrh essential oil, as alternative treatments against T. spiralis infection. Parasitological, histopathological, and cytotoxicity assessments were conducted to investigate the effects of various concentrations of these treatments in reducing the populations of adult worms and larvae during both the intestinal and muscular phases of T. spiralis-infected mice. The results showed that the highest antihelminthic efficacy against the intestinal phase of T. spiralis was achieved by myrrh extract (86.66%), followed closely by AgNPs (84.96%) and myrrh AgNPs (82.51%) at higher concentrations (800 mg/kg for myrrh extract, 40 µg/mL for AgNPs, and 40 µg/mL for myrrh AgNPs). While the group treated with myrrh essential oil showed the lowest percentage of adult reduction (78.14%). However, all treatments demonstrated comparable effects in reducing the larvae population in the muscle phase. Histopathological examination of the tissues revealed compelling evidence of the effectiveness of AgNPs, particularly when prepared with myrrh. Additionally, a comprehensive assessment of the cytotoxicity of AgNPs indicated low toxicity levels. This study supports that AgNPs synthesized using plant-based green technologies hold therapeutic potential for the treatment of T. spiralis infection. These findings present a promising avenue for the development of novel antiparasitic drugs that are both effective and safe. RESEARCH HIGHLIGHTS: Myrrh extract has the highest antihelminthic efficacy against the intestinal phase of T. spiralis. Histopathological examination of the tissues revealed compelling evidence of the effectiveness of AgNPs, particularly when prepared with myrrh. During intestinal phase of T. spiralis, varying levels of nanoparticle precipitation were detected in the liver, brain, lung, and intestine. During the muscular phase, the highest amount of AgNPs precipitation was detected in the liver, followed by the brain, and lung.

Efficacy of silver nanoparticles against Trichinella spiralis in mice and the role of multivitamin in alleviating its toxicity.

Trichinellosis is a worldwide zoonotic disease. The majority of currently available anti-trichinellosis medications exhibit inadequate efficacy. The efficacy of a natively prepared new formulation of silver nanoparticles (Ag-NPs) was evaluated in the treatment of Trichinella spiralis (T. spiralis) infection in mice alone and combined with multivitamin-mineral (MM). After investigating the product's biological and pharmacological characteristics, its therapeutic dose was estimated to be Ag-NPs at 21.5 mg/kg B.W. This dose was orally inoculated to experimentally infected mice at 3-5 days post-inoculation (dpi) against the mature worms, at 8-10 dpi against the newborn larvae, and at 33-35th dpi against the encapsulated larvae. Each treatment's efficacy was assessed by scarifying control and treated mice 3 days post-treatment. The drug alone or in supplement form has a high trichinocidal effect exceeding that of the reference drug. Early treatment (3-5 dpi) by Ag-NPs or Ag-NPs + MM and albendazole revealed high efficacy against the intestinal stage, reaching 93.3%, 94.7%, and 90.6% for the three treatments, respectively. The materials causing a significant (P-value < 0.001) decrease in the mean encapsulated larvae reached 86.61%, 89.07%, and 88.84%/gm of muscles using the three treatments, respectively. Moreover, all larvae extracted from Ag-NPs-treated groups failed to induce infection post-inoculation in new mice. Additionally, combining the material with MM proved to overcome the reversible adverse effects of silver material on the estimated redox parameters and liver and kidney biomarkers, denoting its ability to alleviate Ag-NP toxicity. In conclusion, the high trichinocidal effect of Ag-NPs against the adult and encapsulated larvae during a short inoculation period introduced Ag-NPs as an alternative to other nematicidal drugs.

Nanocurcumin: A Promising Therapeutic Candidate for Experimental Trichinellosis.

In our pursuit of an alternative drug against Trichinella spiralis, we assessed the effectiveness of nanocurcumin in alleviating pathogenesis, parasitological factors, MMP-9 levels, and its expression in the enteral and parenteral phases of infection. The nanocurcumin particles, with a spherical shape and a size of 100 ± 20 nm, were used in the study. Eighty mice were divided into four groups: the control group, the untreated infected group, the nanocurcumin-treated group, and the albendazole-treated group. The nanocurcumin-treated group exhibited a statistically significant increase in the percentage of lymphocytes, along with a reduction in neutrophils, monocytes, and eosinophils compared to the untreated, infected group. Both the nanocurcumin (87.2 and 97.3%) and the albendazole-treated groups (99.8 and 98.2%) showed a significant reduction in the mean number of intestinal worms and encysted larvae, respectively. The treated groups exhibited normal intestinal villi, suppression of the inflammatory process, and fewer instances of degenerated larvae in the diaphragm and muscle compared to the untreated, infected group. Immunohistochemistry and ELISA analyses revealed a significant downregulation of MMP-9 levels in the intestines and muscles of the treated groups. Our data demonstrate that nanocurcumin contains highly versatile molecules capable of modulating biological activity against inflammation and its pathway markers.

Galactomannan inhibits Trichinella spiralis invasion of intestinal epithelium cells and enhances antibody-dependent cellular cytotoxicity related killing of larvae by driving macrophage polarization.

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-1ß, 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.

Title: Le galactomannane inhibe l'invasion par Trichinella spiralis des cellules de l'épithélium intestinal et améliore la cytotoxicité cellulaire dépendante des anticorps tuant les larves en activant la polarisation des macrophages. Abstract: Des études antérieures ont montré que la galectine recombinante de Trichinella spiralis (rTsgal) est caractérisée par un motif de séquence de domaines de reconnaissance des glucides se liant au bêta-galactoside, et que la rTsgal favorise l'invasion larvaire des cellules épithéliales intestinales. Le galactomannane est un polysaccharide immunostimulateur composé d'un squelette mannane avec des résidus galactose. Le but de cette étude était de déterminer si le galactomannane inhibe l'intrusion larvaire des cellules épithéliales intestinales et améliore la cytotoxicité cellulaire dépendante des anticorps (CCDA) tuant les larves nouvelles-nées en polarisant les macrophages au phénotype M1. Les résultats ont montré que le galactomannane se liait spécialement au rTsgal et supprimait la facilitation du rTsgal sur l'invasion larvaire des cellules épithéliales intestinales. Les résultats de la qPCR, du Western blot et de la cytométrie en flux ont montré que le galactomannane et le rTsgal activaient la polarisation des macrophages M1, comme le démontre la forte expression de l'iNOS (marqueur de M1) et des gènes liés à M1 (IL-1ß, IL-6 et TNF-α), et l'augmentation des macrophages CD86+. Le galactomannane et le rTsgal ont également augmenté la production de NO. La capacité de destruction de la CCDA médiée par les macrophages sur les larves était également significativement améliorée dans les macrophages traités au galactomannane et au rTsgal. Les résultats ont démontré que Tsgal pourrait être considéré comme une molécule cible potentielle d'un vaccin contre l'invasion par T. spiralis, et que le galactomannane pourrait être un nouvel agent thérapeutique adjuvant et un adjuvant vaccinal potentiel contre l'infection à T. spiralis.

Serine protease inhibitor from the muscle larval Trichinella spiralis ameliorates non-alcoholic fatty liver disease in mice via anti-inflammatory properties and gut-liver crosstalk.

Trichinella spiralis is recognized for its ability to regulate host immune responses. The serine protease inhibitor of T. spiralis (Ts-SPI) participates in T. spiralis-mediated immunoregulatory effects. Studies have shown that helminth therapy exhibits therapeutic effects on metabolic diseases. In addition, we previously found that T. spiralis-derived crude antigens could alleviate diet-induced obesity. Thus, Ts-SPI was hypothesized to alleviate non-alcoholic fatty liver disease (NAFLD). Herein, recombinant Ts-SPI (rTs-SPI) was prepared from the muscle larvae T. spiralis. The relative molecular mass of rTs-SPI was approximately 35,000 Da, and western blot analysis indicated good immunoreactivity. rTs-SPI ameliorated hepatic steatosis, inflammation, and pyroptosis in NAFLD mice, which validated the hypothesis. rTs-SPI also reduced macrophage infiltration, significantly expanded Foxp3+ Treg population, and inactivated TLR4/NF-κB/NLRP3 signaling in the liver. Furthermore, rTs-SPI treatment significantly shifted the gut microbiome structure, with a remarkable increase in beneficial bacteria and reduction in harmful bacteria to improve gut barrier integrity. Finally, Abx-treated mice and FMT confirmed that gut-liver crosstalk contributed to NAFLD improvement after rTs-SPI treatment. Taken together, Taken together, these findings suggest that rTs-SPI exerts therapeutic effects in NAFLD via anti-inflammatory activity and gut-liver crosstalk.

The neuroprotective potential of curcumin on T. Spiralis infected mice.

BACKGROUND: Trichinella spiralis can affect the brain by inducing inflammatory and vascular changes. Drug management with the antiparasitic drug albendazole can be enhanced by natural compounds such as curcumin. The potential benefit of curcumin as an adjuvant to albendazole in the management of cerebral affection during experimental T. spiralis infection was evaluated. Animals received either curcumin 150 mg/Kg, albendazole 50 mg/Kg or a combination of both drugs. Animal groups receiving treatment were compared with infected and non-infected control groups. Blood levels of reduced glutathione (GSH) and dopamine were measured, and brain tissue expression of cyclooxygenase-2 enzyme (COX-2) and CD34 was assessed by immunohistochemistry. RESULTS: T. spiralis infection resulted in a state of oxidative stress, which was improved by albendazole and curcumin. Also, both drugs restored the peripheral dopamine level, which was decreased in infected non-treated mice. Curcumin was also found to be efficient in improving brain pathology and reducing local COX-2 and CD 34 expression. CONCLUSIONS: Inflammatory and pathological changes during neurotrichinosis can be improved by the addition of curcumin to conventional anti-parasitic drugs.

*Inactivation of encysted muscle larvae of Trichinella spiralis in pigs using Mebendazole.

We evaluated the effect of 4 anthelmintic treatments on the viability of Trichinella spiralis encysted muscle larvae (ML) 55 days post infection (PI) in experimentally infected pigs. Muscle larvae were isolated from pig muscle by artificial digestion after oral treatment of pigs with Levamisole (8 mg/kg, daily for 5 days) and Mebendazole (50 mg/kg, daily for 5 days); Doramectin (0.3 mg/kg, single IM injection), and Moxidectin (0.5 mg/kg, single pour on). Isolated larvae from treated pigs were orally inoculated into mice to assess viability of ML from each treatment. Only Mebendazole treatment of pigs significantly reduced ML viability in mice. The effect of timing of the effective Mebendazole treatment on ML from a longer term infection was then examined in a second experiment. Analysis revealed that Mebendazole treatment of pigs with 250 mg/kg over 3 days (83 mg/kg/day) or 5 days (50 mg/kg/day) reduced numbers of ML recovered from pig tissues compared to untreated, infected controls, and rendered ML non-infective to mice; Mebendazole treatment of pigs with 250 mg/kg in a single dose was not effective in reducing ML numbers recovered from pigs or in impacting ML infectivity to mice. An examination of the lowest effective dose of Mebendazole on encysted ML was determined in a third experiment. Mebendazole of pigs with 5, 50, or 100 mg/kg over 3 days demonstrated that 5 or 50 mg/kg over 3 days insufficient to reduce infectivity in recovered ML, while 100 mg/kg (and 83 g from experiment 2) over 3 days significantly reduces infectivity of ML. This procedure provides a means to evaluate the efficacy of various anthelmintic treatments on the viability of Trichinella spiralis ML in pig tissues, and identified Mebendazole, at 83-100 mg/kg administered over a 3-5 day period as an anthelmintic which renders encysted Trichinella spiralis ML from pig tissues non-infective. As risk from Trichinella significantly impacts acceptance of pork from pasture-raised pigs, these data provide a method, especially for producers of these high-risk pigs, to eliminate the potential of Trichinella transmission from infected pork.

A novel trypsin of Trichinella spiralis mediates larval invasion of gut epithelium via binding to PAR2 and activating ERK1/2 pathway.

BACKGROUND: Proteases secreted by Trichinella spiralis intestinal infective larvae (IIL) play an important role in larval invasion and pathogenesis. However, the mechanism through which proteases mediate larval invasion of intestinal epithelial cells (IECs) remains unclear. A novel T. spiralis trypsin (TsTryp) was identified in IIL excretory/secretory (ES) proteins. It was an early and highly expressed protease at IIL stage, and had the potential as an early diagnostic antigen. The aim of this study was to investigate the biological characteristics of this novel TsTryp, its role in larval invasion of gut epithelium, and the mechanisms involved. METHODOLOGY/PRINCIPAL FINDING: TsTryp with C-terminal domain was cloned and expressed in Escherichia coli BL21 (DE3), and the rTsTryp had the enzymatic activity of natural trypsin, but it could not directly degrade gut tight junctions (TJs) proteins. qPCR and western blotting showed that TsTryp was highly expressed at the invasive IIL stage. Immunofluorescence assay (IFA), ELISA and Far Western blotting revealed that rTsTryp specifically bound to IECs, and confocal microscopy showed that the binding of rTsTryp with IECs was mainly localized in the cytomembrane. Co-immunoprecipitation (Co-IP) confirmed that rTsTryp bound to protease activated receptors 2 (PAR2) in Caco-2 cells. rTsTryp binding to PAR2 resulted in decreased expression levels of ZO-1 and occludin and increased paracellular permeability in Caco-2 monolayers by activating the extracellular regulated protein kinases 1/2 (ERK1/2) pathway. rTsTryp decreased TJs expression and increased epithelial permeability, which could be abrogated by the PAR2 antagonist AZ3451 and ERK1/2 inhibitor PD98059. rTsTryp facilitated larval invasion of IECs, and anti-rTsTryp antibodies inhibited invasion. Both inhibitors impeded larval invasion and alleviated intestinal inflammation in vitro and in vivo. CONCLUSIONS: TsTryp binding to PAR2 activated the ERK1/2 pathway, decreased the expression of gut TJs proteins, disrupted epithelial integrity and barrier function, and consequently mediated larval invasion of the gut mucosa. Therefore, rTsTryp could be regarded as a potential vaccine target for blocking T. spiralis invasion and infection.

A novel Trichinella spiralis serine proteinase disrupted gut epithelial barrier and mediated larval invasion through binding to RACK1 and activating MAPK/ERK1/2 pathway.

BACKGROUND: Gut epithelium is the first natural barrier against Trichinella spiralis larval invasion, but the mechanism by which larval penetration of gut epithelium is not completely elucidated. Previous studies showed that proteases secreted by T. spiralis intestinal infective larvae (IIL) degraded tight junctions (TJs) proteins of gut epithelium and mediated larval invasion. A new T. spiralis serine proteinase (TsSPc) was identified in the IIL surface proteins and ES proteins, rTsSPc bound to the intestinal epithelial cell (IECs) and promoted larval invasion of IECs. The aim of this study was to characterize the interacted proteins of TsSPc and IECs, and to investigate the molecular mechanisms of TsSPc mediating larval invasion of gut mucosa. METHODOLOGY/PRINCIPAL FINDING: IIFT results showed natural TsSPc was detected in infected murine intestine at 6, 12 hours post infection (hpi) and 3 dpi. The results of GST pull-down, mass spectrometry (MS) and Co-IP indicated that rTsSPc bound and interacted specifically with receptor for activated protein C kinase 1 (RACK1) in Caco-2 cells. rTsSPc did not directly hydrolyze the TJs proteins. qPCR and Western blot showed that rTsSPc up-regulated RACK1 expression, activated MAPK/ERK1/2 pathway, reduced the expression levels of gut TJs (occludin and claudin-1) and adherent protein E-cad, increased the paracellular permeability and damaged the integrity of intestinal epithelial barrier. Moreover, the RACK1 inhibitor HO and ERK1/2 pathway inhibitor PD98059 abolished the rTsSPc activating ERK1/2 pathway, they also inhibited and abrogated the rTsSPc down-regulating expression of occludin, claudin-1 and E-cad in Caco-2 monolayer and infected murine intestine, impeded larval invasion and improved intestinal epithelial integrity and barrier function, reduced intestinal worm burdens and alleviated intestinal inflammation. CONCLUSIONS: rTsSPc bound to RACK1 receptor in gut epithelium, activated MAPK/ERK1/2 pathway, decreased the expression of gut epithelial TJs proteins and disrupted the epithelial integrity, consequently mediated T. spiralis larval invasion of gut epithelium. The results are valuable to understand T. spiralis invasion mechanism, and TsSPc might be regarded as a vaccine target against T. spiralis invasion and infection.

Pumpkin seeds (Cucurbita pepo subsp. ovifera) decoction promotes Trichinella spiralis expulsion during intestinal phase via "Weep and Sweep" mechanism.

Trichinosis is a zoonotic disease of communal health concern as it instigated human outbreaks in several countries. Besides, the development of resistance, traditional therapy has numerous antagonistic effects. Thereby, finding efficient natural alternatives is required. In comparison to albendazole, this study evaluated the impact of pumpkin decoction on Trichinella spiralis in experimentally infected mice. The anthelmintic action of pumpkin decoction (500 mg/kg) was determined using T. spiralis infected mice in enteric phase for 5 days. Pumpkin decoction anthelmintic activity fortified by mixing with honey (1:1). Pumpkin decoction and Pumpkin decoction-honey mixture were evaluated by comprising with reference drug, albendazole (50 mg/kg). The T. spiralis adult count was significantly lower in all treated groups, with the pumpkin decoction-honey mixture showing the largest reduction (83.2%) when compared to the infected group (P ≤ 0.001). The intestinal histological changes and the level of COX-2 expression in the intestinal tissue were both significantly reduced in the same group. The pumpkin decoction improved the immune response, as evidenced by a significant decrease in nitric oxide (NO) and tumor necrosis factor (TNF-α) and a significant increase in the expression of the transforming growth factor (TGF-1ß) and interleukin-17 (IL-17). The pumpkin decoction's anthelmintic action was facilitated by the TGF-1ß and IL-17-driven Weep and Sweep mechanism. Both administration of pumpkin decoction beside honey showed the best treatment group that resulted in high infection reduction besides amelioration of biochemical markers and restoration of histological to normal state. In conclusion, pumpkin decoction is highly effective against T. spiralis which could be a promising alternative herbal drug and the pumpkin decoction effect was higher in the case of combination with honey.