Anti-osteosarcoma effect of antiserum against cross antigen TPD52 between osteosarcoma and Trichinella spiralis

Background Trichinella spiralis (T. spiralis) is a parasite occurring worldwide that has been proven to have antitumour ability. However, studies on the antitumour effects of cross antigens between the tumour and T. spiralis or antibodies against cross antigens between tumours and T. spiralis are rare. Methods To study the role of cross antigens between osteosarcoma and T. spiralis, we first screened the cDNA expression library of T. spiralis muscle larvae to obtain the cross antigen gene tumour protein D52 (TPD52), and prepared fusion protein TPD52 and its antiserum. The anti-osteosarcoma effect of the anti-TPD52 antiserum was studied using cell proliferation and cytotoxicity assays as well as in vivo animal models; preliminary data on the mechanism were obtained using western blot and immunohistochemistry analyses. Results Our results indicated that TPD52 was mainly localized in the cytoplasm of MG-63 cells. Anti-TPD52 antiserum inhibited the proliferation of MG-63 cells and the growth of osteosarcoma in a dose-dependent manner. The tumour inhibition rate in the 100 μg treatment group was 61.95%. Enzyme-linked immunosorbent assay showed that injection of anti-TPD52 antiserum increased the serum levels of IFN-γ, TNF-α, and IL-12 in nude mice. Haematoxylin and eosin staining showed that anti-TPD52 antiserum did not cause significant pathological damage. Apoptosis of osteosarcoma cells was induced by anti-TPD52 antiserum in vivo and in vitro. Conclusions Anti-TPD52 antiserum exerts an anti-osteosarcoma effect by inducing apoptosis without causing histopathological damage. Graphical abstract

Characterization of a Novel Glutamine Synthetase From Trichinella spiralis and Its Participation in Larval Acid Resistance, Molting, and Development

Trichinella spiralis is a major foodborne parasite worldwide. After the encapsulated muscle larvae (ML) in meat are ingested, the ML are liberated in the stomach of the host and activated into intestinal infectious larvae (IIL), which develop into adult worm after molting four times. A novel glutamine synthetase (TsGS) was identified from T. spiralis IIL at 10 h post-infection, but its biological role in T. spiralis life cycle is not clear. The aim of this study was to investigate the biological characteristics of TsGS and its functions in larval acid resistance, molting, and development. TsGS has a glutamine synthetase (GS) catalytic domain. Complete TsGS sequence was cloned and expressed in Escherichia coli BL21. rTsGS has good immunogenicity. qPCR and Western blotting showed that TsGS was highly expressed at IIL stage, and immunofluorescence revealed that TsGS was principally localized at the cuticle and intrauterine embryos of this nematode. rTsGS has enzymatic activity of natural GS to hydrolyze the substrate (Glu, ATP, and NH4+). Silencing of TsGS gene significantly reduced the IIL survival at pH 2.5, decreased the IIL burden, and impeded larval molting and development. The results demonstrated that TsGS participates in T. spiralis larval acid resistance, molting and development, and it might be a candidate vaccine target against Trichinella molting and development.

Characterization of a novel cysteine protease in Trichinella spiralis and its role in larval intrusion, development and fecundity

The aim of this study was to investigate the biological properties of a novel gut-specific cysteine protease in Trichinella spiralis (TsGSCP) and its role in larval intrusion, development and fecundity. TsGSCP has a functional C1 peptidase domain; C1 peptidase belongs to cathepsin B family. The TsGSCP gene cloned and expressed in Escherichia coli BL21 showed intensive immunogenicity. qPCR and Western blotting revealed that TsGSCP mRNA and protein were expressed at various T. spiralis stages, but their expression levels in intestinal infectious larvae (IIL) were clearly higher than those in muscle larvae (ML), adult worms (AWs) and new-born larvae (NBL). Indirect immunofluorescence (IIF) analysis showed that TsGSCP was primarily located at the outer cuticle and the intrauterine embryos of this parasite. rTsGSCP showed the ability to specifically bind with IECs, and the binding site is within the IEC cytoplasm. rTsGSCP accelerated larval intrusion into host intestinal epithelial cells (IECs), whereas anti-rTsGSCP antibodies suppressed larval intrusion; the acceleration and suppression was induced by rTsGSCP and anti-rTsGSCP antibodies, respectively, in a dose-dependent manner. When ML were transfected with TsGSCP-specific dsRNA, TsGSCP expression and enzymatic activity were reduced by 46.82 and 37.39%, respectively, and the capacity of the larvae to intrude into IECs was also obviously impeded. Intestinal AW burden and adult female length and fecundity were significantly decreased in the group of mice infected with dsRNA-transfected ML compared to the control dsRNA and PBS groups. The results showed that TsGSCP plays a principal role in gut intrusion, worm development and fecundity in the T. spiralis lifecycle and might be a candidate target for vaccine development against Trichinella intrusion and infection.

Development of a rapid and sensitive immunochromatographic strip based on EuNPs-ES fluorescent probe for the detection of early Trichinella spiralis-specific IgG antibody in pigs

Trichinellosis, which is caused by nematodes of the genus Trichinella, is one of the most important zoonotic parasite diseases in the world. A rapid and sensitive immunochromatographic strip (ICS) based on Eu (III) nanoparticles (EuNPs) was developed for the detection of Trichinella spiralis (T. spiralis) infection in pigs. T. spiralis muscle larvae excretory secretory or preadult worm excretory secretory (ML-ES or PAW-ES) antigens were conjugated with EuNPs probes to capture T. spiralis-specific antibodies in pig sera, after which the complex bound to mouse anti-pig IgG deposited on the test line (T-line), producing a fluorescent signal. In the pigs infected with 100, 1000 and 10 000 ML, seroconversion was first detectable for the EuNPs-ML-ES ICS at 30, 25 and 21 days post-infection (dpi) and for the EuNPs-PAW-ES ICS at 25, 21 and 17 dpi. These results show that EuNPs-PAW-ES ICS detects anti-Trichinella IgG in pigs 4–5 days earlier that test using ML-ES antigens. Our ICS have no cross reaction with other parasite infection sera. Furthermore, the detection process could be completed in 10 min. This study indicated that our ICS can be used for the detection of the circulating antibodies in early T. spiralis infection and provide a novel method for on-site detection of T. spiralis infection in pigs.

Low Prevalence of Cysticercosis and Trichinella Infection in Pigs in Rural Cambodia

Cysticercosis and Trichinella spp. infection are parasitic zoonoses prevalent among pigs in Southeast Asia, where pork is the most important source of meat. In rural Cambodia, many pigs are raised extensively in family backyards, and information regarding the prevalence in rural small-scale pig production is very limited. This study was conducted in four provinces in north-eastern Cambodia to determine the seroprevalence of porcine cysticercosis and Trichinella spp. infection in rural villages, and to identify possible risk factors. Only households with less than 10 pigs above three months old were eligible. In total, 139 households participated, and 242 blood samples were collected. Farmers were interviewed about food and hygiene habits, disease knowledge and practices. The serum samples were analysed by ELISA to determine antigens to Taenia spp. cysticerci or antibodies to Trichinella spp. muscle larvae. Positivity among the pigs was 11.2% (95% CI 7.5–15.8) for Taenia spp. cysts and 2.5% (95% CI 0.9–5.4) for Trichinella spp. Cysticerci were more common in the province Preah Vihear (p < 0.001) than in the other provinces. Risk factors associated with porcine cysticercosis were management systems for the pigs and access to human faeces (p < 0.001). Trichinella spp. infection in pigs was more common in the province Ratanakiri (p = 0.001). The main risk factor associated with Trichinella spp. transmission was feeding pigs with food waste (p = 0.048). More men had heard about cysticercosis than women (p = 0.002), and men also consumed undercooked pork meat to a greater extent (p = 0.004). Although the present study is relatively small, several risk factors could be identified for porcine infection with Taenia spp. and Trichinella spp., which can be used to guide future interventions to improve both porcine and human health in these provinces.

Therapeutic efficacy of Albendazole and Mefloquine alone or in combination against early and late stages of Trichinella spiralis infection in mice

Summary This study aimed to determine the effectiveness of mefl oquine alone or combined with albendazole in reduced doses against T. spiralis infection. One hundred and twenty albino mice were orally infected with 200 T. spiralis larvae/mouse. Drugs were administered during the enteral phase on days 1 to 3 and on the chronic phase on days 35 to 37 post-infection, and mice were sacrificed, respectively, at days 7 or 48 post-infection to count mature intestinal worms or encysted muscle larvae. The effect of the treatment on the histology of the target organs of each phase, intestine and diaphragm, was also evaluated. A signifi cant decrease in intestinal worms was found in all treated groups relative to the untreated control group at a peak of 93.7% in the combination albendazole-mefl oquine group. Results in all treated groups demonstrated a signifi cant decrease in muscle larvae relative to untreated control groups, achieving 86.2 % in the combined albendazole-mefl oquine group. There was a marked improvement in the intestinal and muscular architecture in all treated groups compared to the non-treated control group. Notably, the albendazole-mefl oquine group showed an almost complete recovery. The combined albendazole-mefl oquine low dose regimen had the highest effect on reducing parasite burden and restoring normal histological architecture.

RNAi-mediated silencing of Trichinella spiralis glutaminase results in reduced muscle larval infectivity

Trichinella spiralis is an important foodborne parasitic nematode distributed worldwide that infects humans and animals. Glutaminase (GLS) is an important gene in the glutamine-dependent acid resistance (AR) system; however, its role in T. spiralis muscle larvae (ML) remains unclear. The present study aimed to characterize T. spiralis GLS (TsGLS) and assess its function in T. spiralis ML AR both in vitro and in vivo using RNA interference. The results indicated that native TsGLS (72 kDa) was recognized by anti-rTsGLS serum at the muscle larvae stage; moreover, an immunofluorescence assay confirmed that TsGLS was located in the epidermis of ML. After silencing the TsGLS gene, the relative expression of TsGLS mRNA and the survival rate of T. spiralis ML were reduced by 60.11% and 16.55%, respectively, compared to those in the PBS and control groups. In vivo AR assays revealed that the worm numbers at 7 and 35 days post-infection (dpi) decreased by 61.64% and 66.71%, respectively, compared to those in the PBS group. The relative expression of TsGLS mRNA in F1 generation T. spiralis ML was reduced by 42.52%, compared to that in the PBS group. To the best of our knowledge, this is the first study to report the presence of the glutamine-dependent AR system in T. spiralis. Our results indicate that TsGLS plays a crucial role in the T. spiralis AR system; thus, it could be used as a potential candidate target molecule for producing vaccines against T. spiralis infection.

Primary characterization of the immune responses in Tibetan pigs infected with Chinese Tibet isolate of Trichinella spiralis

Background Trichinellosis, caused by Trichinella spiralis, is a serious foodborne parasitic zoonosis. Tibetan pig is an infrequent, endemic plateau pig species, mainly distributed in Tibet Plateau, China. Because of the free-range system, Tibetan pigs are at risk of infection with Trichinella. The present study aimed to primarily profile the characteristics of T. spiralis infection in Tibetan pigs, including IgG levels, larvae burdens, and cytokines. Results The immune responses to Chinese Tibet T. spiralis isolate infection in Tibetan pigs with different doses were investigated in a tracking duration of 49 days. The muscle larvae per gram (lpg) were evaluated at 105 days post-infection (dpi). The results showed that the mean larval number of T. spiralis in Tibetan pigs increased with infective dose, with average lpg values of 3.5, 50.4 and 115.6 for Tibetan pigs infected with 200, 2,000, and 20,000 muscle larvae (ML) of T. spiralis. The anti-Trichinella IgG increased with inoculum dose and dpi, and peaked at 49 dpi. The kinetics of cytokines in the sera was detected by microarray, including interferon-γ (IFN-γ), interleukin (IL)-1β, IL-8, IL-12, IL-4, IL-6, IL-10, Granulocyte-macrophage Colony Stimulating Factor (GM-CSF), tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β1. The Th1/Th2 mixed cytokines were detectable in all samples. Interleukin-12 demonstrated the highest concentration compared to other cytokines and peaked at 42 dpi. Almost all cytokines were maintained at a high level at 42 dpi. Additionally, we also report a Trichinella seropositive rate of 43.9 % (18 out of 41) from field samples of Tibetan pigs. Conclusions The present study showed an increased Th1/Th2 mixed cytokines in Tibetan pigs elicited by T. spiralis. The high seroprevalence of Trichinella infection in field samples of Tibetan pigs further raises serious concern for the prevention and control of trichinellosis in this host for public health safety.

Influence of chemically and biosynthesized silver nanoparticles on in vitro viability and infectivity of Trichinella spiralis muscle larvae

Background Trichinellosis is a serious worldwide parasitic zoonosis. The available therapy for the treatment of Trichinella spiralis is not satisfactory. This work aimed at evaluating of the in vitro effect of silver Therefore, the recovery of effective treatment is required.nanoparticles (AgNPs) on muscle larvae of Trichinella. Methodology / principal finding The present study investigated the larvicidal properties of chemical and myrrh AgNPs on muscle larvae (ML) of T. spiralis. The used AgNPs were chemically prepared using NaBH4 as reducing agent and biosynthesized using methanolic myrrh extract. Characterization of synthesized AgNPs was monitored via UV-Vis spectrophotometry, Fourier transform infrared spectroscopy and transmission electron microscopy (TEM) studies. The ML incubated with AgNPs at concentrations ranged from 1μg/ml to 20μg/ml. Conclusions /Significance Chemical and biosynthesized AgNPs revealed marked larvicidal effect against ML of Trichinella. Additionally, this in vitro study showed degenerative changes affecting the cuticle of AgNPs treated ML. The effectiveness of AgNPs on the infectivity of Trichinella ML was also assessed. The results showed complete inhibition of the infectivity of ML exposed to sublethal doses of chemical and myrrh prepared AgNPs when used to infect animal models. This is the first report where myrrh synthesized AgNPs have been tested for their anthelminthic activity against Trichinella in an in vitro model.