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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.

The excretory-secretory products (ESPs) released by the muscle-larvae (ML) stage of Trichinella spiralis have been suggested to be involved in nurse cell formation. However, the molecular mechanisms by which ML-ESPs modulate nurse cell formation remain unclear. Macrophages exert either beneficial or deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for skeletal muscle repair, notably, via their actions on myogenic precursor cells. However, these interactions during T. spiralis infection have not been characterized. In the present study, the ability of conditioned medium (CM) from J774A.1 macrophages treated with ML-ESPs to influence the differentiation of murine myoblasts, and the mechanisms of this influence, were investigated in vitro. The results showed that the expression of Myogenic Regulatory Factors (MRFs) MyoD and myogenin, myosin heavy chain (MyHC), and the p21 cyclin-dependent kinase inhibitor were reduced in CM treated cells compared to their expression in the control group. These findings indicated that CM inhibited myoblast differentiation. Conversely, CM promoted myoblast proliferation and increased cyclin D1 levels. Taken together, results of our study suggested that CM can indirectly influence myoblast differentiation and proliferation, which provides a new method for the elucidation of the complex mechanisms involved in cell-parasite and cell-cell interactions during T. spiralis infection.

A clone, designated L20h-Ts3, was selected by immunoscreening of cDNA libraries of Trichinella spiralis worms collected 14h, 20h and 48h post-infection (p.i.) from mice intestines. L20h-Ts3 encodes the full-length of a conserved hypothetical protein of 13.1kDa involving putative interaction with the immune system. PCR analysis showed that L20h-Ts3 mRNA is constitutively expressed throughout T. spiralis life cycle and not restricted to intestinal stages. The L20h-Ts3 fusion protein was obtained in an Escherichia coli expression system and purified by Ni-affinity chromatography before inoculation into mice in order to produce polyclonal antibodies. Then, immunohistochemical study and Western blot analysis revealed its presence within the stichosome of T. spiralis and in excretory/secretory products strengthening a putative fundamental role for the parasite's survival such as host tissue invasion or modification of the host muscular cell phenotype. L20h-Ts3 fusion protein was recognized in Western blot as soon as 15–20 days p.i. by sera from pigs experimentally infected with 20,000 muscle larvae (ML) of T. spiralis. Thus, an indirect L20h-Ts3 ELISA was designed and evaluated using sera from experimentally infected pigs by comparison with the only ELISA currently available for trichinellosis purposes. A gain of precocity from 7 up to 14 days and detection up to 25 weeks p.i. was possible with the L20h-Ts3 ELISA offering a large window for trichinellosis detection. The L20h-Ts3 ELISA was less effective in the case of low infections in pigs. Nevertheless, these results show that the L20h-Ts3 ELISA has a real interest due to its precocity and stability of detection in time. The association of the L20h-Ts3 fusion protein with other antigenic proteins identified previously could appreciably improve the serological test and facilitate its standardization.

Parasite-induced and parasite-regulated larval capsule formation and host immunosuppression are two major characteristics that are unique in Trichinella spp. infections, but the molecule(s) and mechanism(s) that mediate these processes remain largely unknown. Trichinella pseudospiralis and Trichinella spiralis, are obviously different with respect to these two characteristics. A comparative study of these two species, in particular their antigen expression profiles at different developmental stages (the main molecules involved in the cross-talk or interaction between each parasite and its host), may help us better understand the parasite molecules and mechanisms involved. Here, we constructed cDNA libraries from T. pseudospiralis adults (Ad), newborn larvae (NBL) and muscle larvae (ML) mRNA and screened them with pig anti-T. pseudospiralis serum collected 26, 32 and 60 days post-infection (p.i.). The most abundant antigens were found to vary among life-cycle stages. Pyroglutamy peptidase 1-like and 6-phosphogluconolactonase-like genes predominated in the Ad stage and a serine protease (SS2-1-like gene) predominated in NBL similar to that observed in T. spiralis. Muscle larvae expressed proteasome activator complex subunit 3-like and 21 kDa excretory/secretory protein-like genes. This study indicated that parasites of two species may utilise different molecules and mechanisms for larvae capsule formation and host immunosuppression during their infections. Proteins of antigenic genes identified in this study may be also good candidates for diagnosis, treatment or vaccination for T. pseudospiralis infection, and also for the differential diagnosis of two species' infections.

The goal of this work was to identify novel, early antigens present in Trichinella spiralis. To this end, a cDNA library generated from 3-day old adult worms (Ad3) was immunologically screened using serum from a pig infected with 20,000 muscle larvae. The serum was obtained from multiple, time course bleeds coinciding with early worm development. Seventeen positive clones were isolated using serum obtained at 20 days post infection (dpi). All clones corresponded to one gene that exhibited high sequence identity with the T. spiralis ATP-dependent RNA helicase DDX19B which is involved in parasite growth and development. In addition, nine additional positive clones representing 5 unique genes were identified when the library was screened with 30 dpi serum; four of these five genes displayed high similarity with members of a putative T. spiralis serine protease family known to be involved in host invasion and host-parasite interactions. The remaining gene aligned with the T. spiralis hypothetical ORF 11.30. The identification of these antigens provides potential candidates for the early diagnosis of trichinellosis and for the development of a vaccine against this parasite.

Mast cell (MC) hyperplasia and activation are prominent features in Trichinella spiralis infection. Indeed a temporal correlation has been shown between the kinetics of intestinal mastocytosis, release of inflammatory mediators from MC, and adult worm loss, which constitutes a major component of the defense against T. spiralis infection. It is well known that during the intestinal phase of trichinellosis, muscle larvae (ML) and adult worms (AD) enter into contact with the host; however, interaction with MC may also occur during migration of newborn larvae (NBL). Therefore, it is plausible that antigens from these developmental stages could activate MC. We have previously demonstrated by in vitro assays that T. spiralis muscle larval (TSL-1) antigens activate MC through an Ig-independent mechanism leading to the release of histamine, MC protease 5, IL-4 and TNF alpha. In this work we evaluated whether total antigens from AD or NBL could activate unsensitized MC and we compared this activation with the activation seen when MC are stimulated with TSL-1 antigens. MC activation was also tested with affinity chromatography purified antigens from NBL using the monoclonal antibody CE-4 that recognizes NBL surface components. The results obtained in this study showed that AD total extracts and TSL-1 antigens induced the release of histamine but not beta-hexosaminidase from unsensitized MC, suggesting a selective secretion of MC mediators. In contrast, NBL total extracts or purified NBL antigens did not induce the release of either histamine or beta-hexosaminidase from MC. Interestingly, AD and ML are the stages that interact with the host during the intestinal phase of infection. The mechanisms involved in TSL-1 and AD activation of unsensitized MC may function together with other mechanisms of MC activation in host protection against T. spiralis.

Previous studies in southeastern Europe reported a high incidence of human trichinellosis and a high prevalence of Trichinella infection in animals in countries like Bulgaria, Croatia, Romania and Serbia. The aim of this study was, using routine Trichinella test data in pig and game animals, to investigate the extent of Trichinella infection in slaughtered animals in Romania, over the period of 1997–2004, and to identify possible differences in prevalence among the various regions of Romania. Trichinella infection data were obtained from trichinelloscopic examinations of domestic (backyard and industrial reared pigs) and game species (wild boar and bears). A marked difference between Transylvania and other counties of Romania, observed for human trichinellosis, was taken into account when analyzing Trichinella epidemiological data. A cumulative prevalence of 8 cases/10 4 animals tested for pigs, 9 cases/10 3 animals tested for wild boars, and 13.1 cases/10 2 animals tested for bears was obtained for the 8 years period. Analysis of animal prevalence data demonstrated a geographical heterogeneity: whereas Trichinella prevalence in pigs is much lower in Transylvania than in the other counties, Trichinella prevalence in game animals is similar for the different regions. This observation suggests that, in Romania, rather than the levels of the parasite circulating in domestic and game animals, it was changes in the social and political structure of Romania in the 1990s, combined with inadequate meat inspection practices that were the main contributors to these geographic variations.

Trichinella spiralis can cause immunosuppression during the intestinal phase of early infection. However, changes in the peripheral blood during T. spiralis early infection remain unclear. Here, select immune cells in mice infected with 500 muscle larvae (ML) of T. spiralis during the intestinal phase of infection were studied. First, the recovery rates of the intestinal worms and female fecundity were determined, and the results showed that the intestinal worms were completely eliminated at 17 days post-infection (dpi) and that large numbers of new-born larvae (NBL) were generated from 5 to 9dpi. Using flow cytometry, it was shown that the number of CD4+ T cells and CD8+ T cells increased over the entire intestinal phase, except on 7dpi when CD4+ T cells decreased significantly compared to the control groups. Although both CD4+ and CD8+ T cells increased, CD8+ T cells increased more than CD4+ T cells, leading to a lower CD4+/CD8+ ratio compared to the control group. Subsequently, a depression of the proliferative response of T cells to concanavalin A (Con A) was noticed at 7 and 11dpi. Although the proliferative response of B cells to LPS was enhanced, the number of B cells from mouse peripheral blood stimulated by T. spiralis antigens showed no differences with the control group prior to 11dpi. The expression of CD14 on monocyte-macrophages decreased during the same period, which meant that the antigen-presenting response was reduced in the immune system of the infected mice. Moreover, the alternatively activated macrophages were induced in T. spiralis early infection. These data provide a better understanding of the development of the intestinal immune response in mice infected with T. spiralis.

The immune protective response developed by swine against Trichinella spiralis is not yet fully understood, particularly at the mucosal level. This study aimed to characterise intestinal immunity to T. spiralis by comparison with the systemic response in specifically pathogen-free pigs. For this purpose, the kinetics of cytokine and antibody production were assessed in the intestinal mucosa and serum of swine infected with T. spiralis for up to 60 days post-infection (dpi). An ex vivo model of jejunum mucosa culture was used to collect the supernatant as a source of antibodies (Abs). Mucosal antibodies were observed by Western blot from 15 dpi, while serum antibodies were expressed from 20 dpi. Both sources of antibodies initially recognized a 110 kDa protein, followed by the identification of 35, 43/46 and 55/59 kDa proteins. IgG1 and IgA antibodies were strongly expressed within the mucosa. The expression levels of Type 1 (IFN-gamma, IL-12), Type 2 (IL-4, IL-6), pro-inflammatory (TNF-alpha) and regulatory (IL-10, TGF-beta) cytokines were assessed by RT-PCR in the intestinal mucosa and spleen. Both IL-10 and IFN-gamma mRNA levels were increased in mucosa, whereas IL-6 and IL-12 mRNA were expressed in spleen. Taken together, these results demonstrated a mixed Type 1/Type 2 profile, the Type 2 profile being dominant in the mucosa.

International audience; We developed a PCR-based assay using a single primer pair to amplify the 5S ribosomal DNA intergenic spacer region to identify Trichinella isolates. In our method, amplified products are directly sequenced on both strands and compared to GenBank sequences. Using this method, we were able to identify Trichinella spiralis, T britovi and T nativa. This method permits rapid species identification of Trichinella isolates; however, further evaluation is required before recommending this approach for routine use.

Trichinella spiralis infection confers effective resistance to tumor cell expansion. In this study, a T7 phage cDNA display library was constructed to express genes encoded by T. spiralis. Organic phase multi-cell screening was used to sort through candidate proteins in a transfected human chronic myeloid leukemia cell line (K562) and a human hepatoma cell line (H7402) using the display library. The protein encoded by the A200711 gene was identified and analyzed using protein analysis software. To test the antitumor effects of A200711, variations in cell proliferation and apoptosis were monitored after recombinant pEGFP-N1-A200711 was transfected into H7402 cells. The results show that the expressed target gene successfully induced apoptosis in H7402 cells as measured by Hoechst-PI staining, MTT assay (p

We assessed a serological method for detecting Trichinella infection in horses, specifically, an ELISA using three antigens to detect anti- Trichinella IgG (i.e. a synthetic tyvelose glycan–BSA (stg-BSA) antigen, an excretory/secretory (ES) antigen, and a crude worm extract (CWE) antigen). Serum samples were collected from 2502 horses (433 live horses from Romania and 2069 horses slaughtered in Italy and originating from Italy, Poland, Romania, and Serbia). Serum samples were also taken from horses experimentally infected with different doses of T. spiralis and T. murrelli larvae, as controls. The cut-off value of ELISA was determined on serum samples from 330 horses from Trichinella -free regions of Italy, which were also examined by artificial digestion of preferential-muscle samples. In the experimentally infected horses, the stg-BSA and ES antigens were less sensitive than the CWE antigen. Trichinella spiralis showed a higher immunogenicity than T. murrelli , and the IgG immunoresponse was dose-dependent. The kinetics of anti- Trichinella IgG were similar among all experimentally infected horses. No circulating antibodies were detected 4–5 months after experimental infection, although these horses still harbored infective larvae. Depending on the antigen used, for 4–7 of the 330 horses from Trichinella -free areas, the optical density (OD) of the serum sample was higher than the cut-off value, yet these samples were negative when subjected to Western Blot. Similar results were obtained for the 1739 horses slaughtered in Italy (originating from Italy, Poland, Romania, and Serbia) and the 433 live Romanian horses. Of the 4 horses with muscle larvae, only one was positive by ELISA and Western Blot. Because the anti- Trichinella IgG remain circulating for only a short period of time, whereas the larvae remain infective for longer periods, serology cannot be used for either diagnosing Trichinella infection in horses or estimating the prevalence of infedction. Artificial digestion of at least 5 g of preferential-muscle tissue continues to be the method of choice at the slaughterhouse for preventing equine-borne trichinellosis in humans.

After the initial report in 1976 of a trichinellosis epidemic caused by the consumption of infected horsemeat, 12 other outbreaks have been described in Europe. Since the first serious human outbreak several experiments have confirmed the susceptibility of horses to Trichinella species and the rapid disappearance of specific antibodies in this host that prevents the use of serological methods for routine screening. A review of the distribution of parasite burdens in muscles of naturally or experimentally infected horses indicates that the tongue is the most likely sample to contain detectable numbers of Trichinella larvae in low level infections. Requirements for testing of horsemeat are specified in legislation of the European Union, and other recommendations are published elsewhere. The EEC directives have evolved into very specific requirements which specify the testing of at least 5 g of tongue, masseter or diaphragm per horse using a pooled digestion assay. More recently, France has revised the requirement for sample size to 10 g for horsemeat originating from countries with high prevalence of Trichinella . To address the continuing outbreaks of human trichinellosis due to infected horsemeat, the development and implementation of a quality assurance system for testing is being considered.

Freeze-tolerance of encapsulated Trichinella muscle larvae (ML) is mainly determined by Trichinella species, but is also influenced by host species, the age of the infection and the storage time and temperature of the infected meat. Moreover, the freeze-tolerance of the encapsulated species appears to be correlated to the development of thick capsule walls which increases with age. An extended infection period and the muscle composition in some hosts (e.g. herbivores) may provide freeze-avoiding matrices due to high carbohydrate contents. The present experiment compares freeze-tolerance of Trichinella spiralis and Trichinella britovi ML in wild boar meat 24 weeks post inoculation (wpi). Three groups of four wild boars were infected with 200, 2000 or 20,000 ML of T. britovi (ISS 1575), respectively. Additionally, three wild boars were inoculated with 20,000 ML of T. spiralis (ISS 004) and two animals served as negative controls. All wild boars were sacrificed 24 wpi. Muscle samples of 70 g were stored at -21°C for 19, 30 and 56 h, and for 1-8 weeks. Larvae were recovered by artificial digestion. Their mobilities were recorded using Saisam(®) image analysis software and their infectivities were evaluated using mouse bioassays. Samples frozen for 19, 30 and 56 h allowed recovery of mobile ML, but samples frozen for 1 or 2 weeks did not. Correspondingly, only T. spiralis and T. britovi larvae isolated from wild boar meat frozen for 19, 30 and 56 h established in mice. This study showed that freezing at -21°C for 1 week inactivated T. spiralis and T. britovi ML encapsulated in wild boar meat for 24 weeks.

Infection of mice with Trichinella spiralis redirects the mucosal immune system from a Th1 to a protective Th2 response with a reduction in the severity of trinitrobenzesulfonic acid-induced colonic damage. T. spiralis infection induced IL-10 production in a dose-dependent manner in oxazolone (OXZ)-induced colitis. This phenomenon may be responsible for the lack of efficacy of T. spiralis in the treatment of OXZ-induced colitis. These results indicate that if the source of increased IL-10 production is identified and addressed, T. spiralis may alter the Th2 response.

Monitoring of Toxoplasma infection in animals destined for human consumption is a great challenge for human toxoplasmosis prevention. This study aimed to compare results obtained from a naturally infected population of sheep using different tests and targeting an original matrix: meat samples and muscle fluids collected at the slaughterhouse. A commercial ELISA test was performed on diaphragm fluids from 419 ovine carcasses collected at the slaughterhouse. A MAT (modified agglutination test) was performed on heart fluids obtained from the same animals. In addition, all hearts were bioassayed in mice. Serological test agreement, the relative sensitivity of ELISA MAT and mouse bioassay as well as a correlation between titres and parasite isolation probability were statistically evaluated. The overall agreement (kappa coefficient=0.64) of ELISA on diaphragm fluids and MAT on heart fluids is substantial and subsequently both tests can be used for epidemiological studies. Relative sensitivity was higher for MAT performed on cardiac fluids (90%) than ELISA on diaphragm fluid (61%). For both serological tests, relative sensitivity is lower in lambs younger than 12 months. Relative sensitivity of mouse inoculation was 42%. A significant correlation was obtained between increasing MAT titres and probability to isolate live parasite from the heart. When the fluid titre was higher than 1:16, parasites were isolated in 65% of cases. When it was lower, isolation failed in 95% of the cases. According to our results, cardiac fluids appear to be a relevant matrix for toxoplasmosis survey in meat.

Herbivorous animals are usually, by virtue of their diet, outside the major transmission cycles of Trichinella spp. However, since 1975, the year of the first report of human trichinellosis caused by the consumption of infected horse meat, the domestic horse has appeared as a novel vector of Trichinella spp. infection to humans, with 15 outbreaks documented in France and Italy. Romania, one of the main countries exporting horses into the European Union (EU), experienced a dramatic increase of Trichinella spp. infection in both domestic pigs and humans in the 1990s. Some Trichinella spiralis-infected horses were exported to the EU during this period. The aim of this study was to evaluate the prevalence of Trichinella spp. infections in horses from Romania using both direct and indirect tests. Of 3000 serum samples tested in 2001, none were positive by ELISA using three different Trichinella antigens (crude; excretory/secretory, ES; stg-BSA antigens). Of 2992 serum samples tested in 2002, 17 (0.56%) showed optical density values higher than the cut-off in an ELISA using ES antigens and one was confirmed by western blot (WB). Four of the 17 ELISA positive horses, including the horse with a confirmed serology by WB, were subjected for intensive meat examination at slaughter, but no Trichinella spp. larvae were detected. Further, no Trichinella spp. larvae were detected by trichinelloscopy and artificial digestion of 25,838 horses slaughtered in Alexandria and Timisoara between 2001 and 2004. The false positive results obtained by serology confirm the previous work on the unreliability of serology for detection of Trichinella spp. infection in horses. Furthermore, the lack of detection of Trichinella spp. infected horses by artificial digestion, suggests a very low prevalence of infection in horses in Romania.