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Inflammatory bowel disease and allergic asthma, as typical immune-mediated inflammatory diseases (IMIDs), are associated with immune imbalance caused by complex interactions among environmental, genetic and bacterial factors. The changing immune imbalance of IMIDs not only causes serious pathological damages but also increases the difficulty of treatment. Helminths or helminth-derived molecules have been increasingly employed to treat IMIDs due to their immunoregulatory ability. Since helminth infection is not an appropriate treatment direction due to the complex immunoregulation and safety concerns, one of the new therapies is to harness the immunoregulation induced by the identified helminth-derived molecules using immune indexes as a guide. This review discusses the pathogenesis of inflammatory bowel disease and allergic asthma, and summarizes the therapeutic effect of helminths and the immunoregulatory mechanisms induced by helminth-derived molecules proposing therapeutic regimens.

Background Major prevention trials for Alzheimer’s disease (AD) are now focusing on multidomain lifestyle interventions. However, the exact combination of behavioral factors related to AD pathology remains unclear. In 2 cohorts of cognitively unimpaired individuals at risk of AD, we examined which combinations of personality traits, neuropsychiatric symptoms, and cognitive lifestyle (years of education or lifetime cognitive activity) related to the pathological hallmarks of AD, amyloid-β, and tau deposits. Methods A total of 115 older adults with a parental or multiple-sibling family history of sporadic AD (PREVENT-AD [PRe-symptomatic EValuation of Experimental or Novel Treatments for AD] cohort) underwent amyloid and tau positron emission tomography and answered several questionnaires related to behavioral attributes. Separately, we studied 117 mutation carriers from the DIAN (Dominant Inherited Alzheimer Network) study group cohort with amyloid positron emission tomography and behavioral data. Using partial least squares analysis, we identified latent variables relating amyloid or tau pathology with combinations of personality traits, neuropsychiatric symptoms, and cognitive lifestyle. Results In PREVENT-AD, lower neuroticism, neuropsychiatric burden, and higher education were associated with less amyloid deposition (p = .014). Lower neuroticism and neuropsychiatric features, along with higher measures of openness and extraversion, were related to less tau deposition (p = .006). In DIAN, lower neuropsychiatric burden and higher education were also associated with less amyloid (p = .005). The combination of these factors accounted for up to 14% of AD pathology. Conclusions In the preclinical phase of both sporadic and autosomal dominant AD, multiple behavioral features were associated with AD pathology. These results may suggest potential pathways by which multidomain interventions might help delay AD onset or progression.

Trichinella spiralis is recognized for its ability to regulate host immune responses via excretory/secretory (ES) products. Serine protease inhibitors (serpins) play an important role in ES product-mediated immunoregulatory effects during T. spiralis infection. In this study, the immunoregulatory properties of a serpin derived from T. spiralis (Ts-serpin) were explored in BALB/c mice. The results showed that naturally occurring Ts-serpin was detected in the stichosomes of muscle larvae and adult worms. Moreover, enhancing (by injection of a soluble-expressed recombinant Ts-serpin [rTs-serpin]) or blocking (by passive immunization with anti–rTs-serpin serum) the effects of Ts-serpin changed the levels of cytokines related to inflammation induced by T. spiralis infection in the serum, mesenteric lymph nodes, and peritoneal cavity, which then led to a change in the adult worm burden in early T. spiralis infection. Moreover, the phenotypic changes in peritoneal macrophages were found to be related to Ts-serpin–mediated immunoregulation. Furthermore, a STAT6 activation mechanism independent of IL-4Rα has been found to regulate protein-mediated alternative activation of bone marrow–derived macrophages and mimic the immunoregulatory role of Ts-serpin in T. spiralis infection. Finally, the anti-inflammatory properties of rTs-serpin and bone marrow–derived macrophage alternative activation by rTs-serpin were demonstrated using a trinitrobenzene sulfonic acid–induced inflammatory bowel disease model. In summary, a protein-triggered anti-inflammatory mechanism was found to favor the survival of T. spiralis in the early stage of infection and help to elucidate the immunoregulatory effects of T. spiralis on the host immune response.

In September 2017, a severe trichinellosis outbreak occurred in Cambodia after persons consumed raw wild pig meat; 33 persons were infected and 8 died. We collected and analyzed the medical records for 25 patients. Clinical signs and symptoms included myalgia, facial or peripheral edema, asthenia, and fever. We observed increased levels of creatine phosphokinase and aspartate aminotransferase-, as well as eosinophilia. Histopathologic examination of muscle biopsy specimens showed nonencapsulated Trichinella larvae. A Trichinella excretory/secretory antigen ELISA identified Trichinella IgM and IgG. Biopsy samples were digested and larvae were isolated and counted. PCR for the 5S rDNA intergenic spacer region and a multiplex PCR, followed by sequencing identified the parasite as Trichinella papuae. This species was identified in Papua New Guinea during 1999 and in several outbreaks in humans in Thailand. Thus, we identified T. papuae nematodes in humans in Cambodia.

Parasites have developed many strategies to ensure their development, multiplication, and dissemination, including the use of reservoir hosts that are often nondomesticated species. Despite drastic reductions in their populations, wild birds remain widespread worldwide and could constitute some of these reservoirs. We focused on the identification of wild bird species harboring parasite stages in their muscles. Breast muscles of 327 birds of 27 different species were collected at three different sites in France. After artificial digestion, isolated nematode larvae were identified by PCR sequencing or restriction fragment length polymorphism (PCR-RFLP). Toxocara cati was identified mainly in birds of prey. The presence of anti-Toxoplasma antibodies was investigated by modified agglutination test on muscle fluids. Anti-Toxoplasma antibodies were detected in 65 out of 166 samples from various bird species. Avifauna, particularly birds of prey, could help on the surveillance of parasite circulation and play a role as sentinel species.

We describe a small family outbreak of trichinellosis caused by the consumption of raw ham from a wild boar (Sus scrofa) hunted in the northern Alps of France in February 2022. Out of the six people, aged 3–69 years, who consumed the meat, three were confirmed cases, and three were suspected cases. Eosinophilia detected in four people was the hallmark that drove the diagnosis. Three patients presented with myalgia, two with intense and prolonged chest pain, and one with elevated troponin. One patient presented with dermographism during treatment. Anti-Trichinella IgG were detected in three symptomatic individuals after about ten weeks. One patient had negative serology and no symptoms, but was on long-term corticosteroid therapy. Trichinella britovi larvae (8.3 larvae/g) were detected in the wild boar meat remnants. Trichinellosis is rare in France, but this family outbreak is reminiscent of the circulation of this pathogen in wild animals, highlighting the need to inform hunters about the risk of infection linked to the consumption of raw meat of game animals, and about the need for veterinary inspection of game meat. The consumption of raw meat outside controlled circuits is a practice not devoid of risks, which justifies raising the awareness of hunters, doctors, and medical biologists.

International audience; Background During the last decade, the scientific community has begun to investigate the composition and role of gut microbiota in normal health and disease. These studies have provided crucial information on the relationship between gut microflora composition and intestinal parasitic infection, and have demonstrated that many enteric pathogen infections are associated with altered gut microflora composition. In this study, we investigated the effects of Cryptosporidium parvum infection (zoonotic protozoan affecting a large range of vertebrates) on both qualitative and quantitative composition of gut microbiota in a CD-1 neonatal mouse model. Methods 5-day-old neonate mice were experimentally infected with 105Cryptosporidium parvum Iowa oocysts by oesophageal gavage. The intestinal microbiota of both infected (Cp+) and uninfected (Cp−) mice groups was examined by high-throughput sequencing of the bacterial 16S rDNA gene V3–V4 hypervariable region. Results The most consistent change in the microbiota composition of Cp+ mice was the increased proportion of bacterial communities belonging to the Phylum Bacteroidetes. In contrast, the microbiota of Cp− mice was associated with increased proportions of several Firmicutes and Actinobacteria phyla members. Conclusion For the first time, our study provides evidence of an association between cryptosporidial infection and gut dysbiosis, thus contributing valuable knowledge to the as-yet little-explored field of Cryptosporidium–microbiota interactions in a neonatal mouse model.

Parasites are complex pathogens, particularly in terms of their antigens, which can vary with life cycle stage and location in the host. Three main biological stages in Trichinella genus were described but it is an oversimplification of the complex Trichinella cycle. Trichinella antigens and immunodominant epitopes have been studied using various strategies requiring immunological reagents or the selection of specific gene targets to be expressed in several systems. Here, we review the Trichinella antigens used for vaccine development and their temporal and spatial variation with the parasite life cycle and in the parasite body. The first antigen studies were devoted to defining antigen profiles reacting with serum samples from various hosts. Trichinella antigens have been classified according to their immunoreactivity with different monoclonal antibodies obtained with various immunization protocols. Based on our knowledge of the Trichinella genome, specific targets have been evaluated. The first fully analyzed immunodominant epitope was s-tyvelose (3,6-dideoxy- d -arabinohexose), a carbohydrate initially found only in the Trichinella genus, but several other linear peptide epitopes have also been described. We discuss their use in vaccine developments taking into account the specific challenge of a foodborne zoonotic parasite. Recently, new data obtained from the use of recombinant antigens offer new challenges for vaccine development, but most studies focus only on rodent models and are far from the scale-up development stage. Adjuvants are of utmost importance in vaccine development and must be well designed to the target host. RNA vaccines may be more appropriate in the future. Ultimately, nematodes with an aborted cycle may be the ideal vaccine candidate in target animals with a local protective immune response and strong immunity, as in natural infection.

International audience; To ensure that meat from livestock and game is safe for human consumption, European legislation lays down rules for mandatory testing. Helminth larvae are a category of zoonotic foodborne pathogens that can contaminate meat. Among helminths, the only zoonotic nematode regulated in Europe regarding meat inspection isTrichinellaspp.. It is precisely during Trichinella testing that other potentially zoonotic larvae can be found. Due to current lack of tools, their identification is often very complicated. Nematode larvae other thanTrichinella, recovered from artificial digestions of pig and wild boar muscles from France and Germany, were subjected to a newly developed two-step identification scheme, which includes both morphological examination and molecular assays. The first step is a general orientation towards a broad taxonomic group; the second step consists of targeted identification based on the results of first step. Different parasites were identified, some of which were not zoonotic such asMetastrongylusspp. andAngiostrongylus vasorum, but others are known to be zoonotic such asToxocara cati,Ascaris suum, andUncinaria stenocephala. The strategy is efficient for the identification of nematode larvae recovered from muscles but could also be applied for larvae from other sources.

Ethnopharmacological Relevance: An ethnobotanical survey was conducted in three regions of the country (two rural and one urban), using a questionnaire focussing on anti-diarrheal therapeutic habits. Methodology: Information about the plants has been recorded (local name, organs or part(s) of the plant used, therapeutic indications, harvesting methods, methods of administration, side effects, etc.). Collection of the plants was done in collaboration with traditional practitioners and identification of the specimens was conducted at the National Floristic Center (Ivory coast). During the ethnobotanical survey, twelve traditional healers and nine breeders were interviewed which all declared to treat diarrhoea with plants. During this survey, twenty-seven species belonging to eighteen different families of plants were reported for anti-diarrhoeal use in humans or animals. They were all harvested on the field and a herbarium of each species was prepared in duplicate, one stored at the National Floristic Centre of the Felix Houphouët-Boigny University and the other at the Pasteur Institute of Ivory coast. Results: Decoction was the most common mode of preparation used by practionioners and the oral route remained also the main way of administration of plants by healers. These plants are also used in other countries for the same or other purpose. Conclusion: This study shows that traditional medicinal plants play an important role in the treatment of diarrhoea in Ivory coast. It provides basis for future studies to assess, biological and chemical potential of these plants.

Background: More than a hundred species of mammals, birds, and reptiles are infected by nematodes of the Trichinella genus worldwide. Although, Trichinella spp. are widely distributed in neighboring countries including Georgia, Azerbaijan, Turkey and Iran, no study was conducted in Armenia since 1980’s. Methods: In 2017-2018, five muscle samples belonging to Armenian lynx, otter, wild boar, fox and wolf were tested for Trichinella spp. and recovered larvae were identified by multiplex PCR technique. Results: Twenty-six larvae/gram and one larva/gram were found in lynx and fox samples respectively. They were identified as T. britovi. Conclusion: So far only two species were identified in Armenia, T. spiralis and T. pseudospiralis, and this is the first time that T. britovi is reported in Armenia.

Summary The wild boar (Sus scrofa) has a wide geographical distribution and can be an important source of Trichinella spp. infection in humans in Romania. The objective of this study was to identify the presence of Trichinella spp. in the wild boar population in Bihor County, Romania. Eighty four plasma and diaphragm samples, collected from wild boars, were included in this study. Artificial digestion, ELISA and Western blot were performed on these specimens. All diaphragm samples were negative for Trichinella larvae in artificial digestion, while in ELISA, 54 (64.2 %) plasma samples were positive and 6 (7.1 %) plasma samples were doubtful. Western blot was performed on 26 plasma samples from which only 6 (23.0 %) gave a positive result. Serological evidences indicate the presence of Trichinella spp. in wild boars from western Romania. Therefore, human consumers might be at risk to ingest Trichinella larvae, even in low numbers.

Trichinellosis is a rare parasitic zoonosis in the European Union. Meat from backyard pigs was the common source for a trichinellosis outbreak caused by Trichinella spiralis, which occurred in France and Serbia in the beginning of 2017. An epidemiological study was conducted in France and Serbia to determine the extent of the outbreak, to identify its source and to implement control measures. Three cases were exposed in Serbia and brought back to France pork delicatessen which they shared with relatives and friends. Around 47 individuals were exposed to the parasitised meat in France and Serbia and 20 cases of trichinellosis were reported (nine in France and 11 in Serbia). Nine of them were female. The diagnosis was delayed, in part because the parasitosis was not known by most physicians, which led to complications in the French cases such as facial paralysis and pulmonary embolism. Health alerts and survey networks are indispensable at a European level to control the disease.

International audience; Cryptosporidiosis is a zoonotic disease caused by species in the genus Cryptosporidium. In young ruminants, Cryptosporidium parvum causes economically significant disease with mild to severe clinical signs and occasional death. The typical clinical course in animals aged 1-3 weeks old is acute diarrhoea. Currently there are no available treatments that are fully effective against cryptosporidiosis in either humans or animals. Therefore there is a critical need for the development of new therapeutic agents. We adapted two in vitro culture systems (HCT-8 and Caco-2 cell lines) for C. parvum infection to investigate the "anticryptosporidial" activity of two chitosans; Chitosan NAG and Chitosan Mix. Chitosan-a naturally-occurring polysaccharide compound-has been found to be active against a variety of diseases, possessing both antimicrobial and anticancer properties. We investigated both chitosan's toxicity and effects on C. parvum in the two in vitro models. To evaluate chitosan's effects on oocyst shedding in vivo, CD-1 neonate mice were orally inoculated with C. parvum oocysts (Iowa strain), treated with chitosan, and compared to infected non-treated animals. Paromomycin, a classical drug used in veterinary medicine, was used as a reference compound. Immunofluorescence techniques were used to analyse the parasites. Our results showed significant reductions in Cryptosporidium oocyst viability (> 95%) after oocyst pre-incubation with either paromomycin (P < 0.001), Chitosan Mix or Chitosan NAG (P < 0.001), for 24 hat 37 degrees C. Additionally, paromomycin, Chitosan Mix, and Chitosan NAG significantly inhibited C. parvum multiplication in HCT-8 and Caco-2 cell lines (P < 0.005). These effects were dose-dependent. In in vivo studies, treatment with both chitosans (Chitosan NAG, Chitosan Mix) or paromomycin sulfate significantly reduced parasite shedding in infected treated newborn mice (-56%, -34.5% and -58%, respectively). In conclusion, these findings provide the first in vitro and in vivo evidence of the anticryptosporidial activities of this natural polysaccharide.

Background The zoonotic nematode Trichinella britovi was discovered in two neighboring Mediterranean islands of Corsica and Sardinia, almost simultaneously at the beginning of the 21st century. An epidemiological link between the two parasite populations was generally assumed. In 2015, an outbreak of trichinellosis in Nice, the South of France, was reportedly caused by the consumption of raw pork delicatessen imported from Corsica. The aims of the present study were to investigate, by multilocus genotype (MLG) analyses, the hypothesis of the common origin of the Corsican and Sardinian T. britovi foci and to trace “from fork to farm” the origin of the pork product, which caused a trichinellosis outbreak in mainland France in 2015. Methods Sixty-three T. britovi isolates were collected from animals and pork products of Sardinia and Corsica islands and from mainland of Italy, France and Spain. We analyzed genetic variability at four polymorphic microsatellite loci by two independent algorithms, the Bayesian and multivariate analyses, to evaluate the genetic relationships of 1367 single larvae. Results Trichinella britovi isolates of the two islands showed different genetic structures and the Bayesian analysis revealed a different membership of the two insular populations. Furthermore, two geographically separate genetic groups were identified among Corsican isolates. Lastly, the origin of the pork delicatessen marketed in Nice was linked to a breeder-butcher in Corsica. Conclusions The low level of genetic admixture of the insular T. britovi isolates suggests that this pathogen colonized the two islands by separate events. On the other hand in Corsica, although the isolates share the same genetic structure, geographically separate isolates showed different membership. We suggest the MLG analysis as a suitable method in supporting epidemiological investigations to trace “from fork to farm” insular populations of T. britovi. Electronic supplementary material The online version of this article (10.1186/s13071-018-2939-9) contains supplementary material, which is available to authorized users.

Trichinella spiralis typically infects domestic swine, wild boar and occasionally horses, has a cosmopolitan distribution, and consequently is most frequently associated with food-borne outbreaks of trichinellosis in humans. Trichinella murrelli is typically found in wild carnivores in temperate areas of North America, where it has been responsible for outbreaks of human trichinellosis due to consumption of infected wild game. There has previously been only indirect evidence of natural infection with T. murrelli in a horse originating from Connecticut and implicated in an outbreak of trichinellosis in France in 1985. We describe a T. murrelli infection detected during routine testing of a horse from the USA imported to Canada for slaughter and export to the European Union (EU). Approximately 5 or more larvae per gram were recovered from digested tongue and diaphragm samples and identified as T. murrelli by PCR. This case provides the first direct evidence of naturally acquired T. murrelli infection in a horse, and further supports the potential food safety risk posed by this parasite species. It is the first instance in Canada of the detection of a Trichinella-infected horse via routine post-mortem testing. Trichinella spiralis-infected horses have been similarly detected by regulatory testing in France, and further details of two such previously reported cases are also provided here. The cases described herein underscore the importance of continued vigilance in quality assured food safety testing of horse meat to mitigate the risk of human trichinellosis.

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.

International audience; Toxoplasmosis is a zoonotic disease caused by the parasite Toxoplasma gondii. Up to a third of the global human population is estimated to carry a T. gondii infection, which can result in severe complications in immunocompromised individuals and pregnant women. Humans and animals can become infected by ingesting either tissue cysts containing T. gondii bradyzoites, from raw or undercooked meat, or sporulated oocysts from environmental sources. T. gondii oocysts are released in the faeces of cats and other felids, which are the parasite's definitive hosts, leading to environmental contamination. Therefore, vaccination of the feline host against T. gondii is an interesting strategy to interrupt the parasitic life cycle and subsequently limit contamination of intermediate hosts. With this goal in mind, we tested in cats, an attenuated live strain of T. gondii deleted for the Mic1 and Mic3 genes (Mic1-3KO) that was previously shown to be an efficient vaccine candidate in mouse and sheep models. Subcutaneous or oral vaccination routes induced a high specific antibody titer in the cat sera, indicating that the Mic1-3KO strain is immunogenic for cats. To assess protection induced by the vaccine candidate strain, we followed oocysts shedding by vaccinated cats, after oral challenge with a T. gondii wild-type strain. Surprisingly, a high antibody titer did not prevent cats from shedding oocysts from the challenge strain, regardless of the vaccination route. Our results show that the Mic1-3KO vaccine candidate is immunogenic in the feline host, is well tolerated and safe, but does not confer protection against oocysts shedding after natural infection with wild type T. gondii. This result highlights the particular relationship between T. gondii and its unique definitive host, which indicates the need for further investigations to improve vaccination strategies to limit environmental and livestock contaminations.

15 páginas, 3 figuras y 2 tablas., Environmental and anthropogenic changes are expected to promote emergence and spread of pathogens worldwide. Since 2013, human urogenital schistosomiasis is established in Corsica island (France). Schistosomiasis is a parasitic disease affecting both humans and animals. The parasite involved in the Corsican outbreak is a hybrid form between Schistosoma haematobium, a human parasite, and Schistosoma bovis, a livestock parasite. S. bovis has been detected in Corsican livestock few decades ago raising the questions whether hybridization occurred in Corsica and if animals could behave as a reservoir for the recently established parasite lineage. The latter hypothesis has huge epidemiological outcomes since the emergence of a zoonotic lineage of schistosomes would be considerably harder to control and eradicate the disease locally and definitively needs to be verified. In this study we combined a sero-epidemiological survey on ruminants and a rodent trapping campaign to check whether schistosomes could shift on vertebrate hosts other than humans. A total of 3,519 domesticated animals (1,147 cattle; 671 goats and 1,701 sheep) from 160 farms established in 14 municipalities were sampled. From these 3,519 screened animals, 17 were found to be serologically positive but were ultimately considered as false positive after complementary analyses. Additionally, our 7-day extensive rodent trapping (i.e. 1,949 traps placed) resulted in the capture of a total of 34 rats (Rattus rattus) and 4 mice (Mus musculus). Despite the low number of rodents captured, molecular diagnostic tests showed that two of them have been found to be infected by schistosomes. Given the low abundance of rodents and the low parasitic prevalence and intensity among rodents, it is unlikely that neither rats nor ruminants play a significant role in the maintenance of schistosomiasis outbreak in Corsica. Finally, the most likely hypothesis is that local people initially infected in 2013 re-contaminated the river during subsequent summers, however we cannot definitively rule out the possibility of an animal species acting as reservoir host., JB received a grant from the ANSES (Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail) (https://www.anses.fr/fr). This study is set within the framework of the "Laboratoire d’Excellence (LABEX)" TULIP (ANR-10-LABX-41).

Trichinellosis is a serious foodborne zoonotic disease. It is an important threat to public health in both developing and developed countries. Human infections are strongly associated with consuming undercooked meat containing infective Trichinella larvae. The development of serological tools has enabled seroepidemiological studies and contributed to our knowledge on the importance of this parasite. Serological tests can also help the diagnosis of parasite infections in humans and the surveillance of animals. Generally speaking, serological techniques include detection methods for specific antibodies and for circulating parasite antigens in the serum or tissue fluids. Here, we present a comprehensive review of various methods used in the detection of antibodies against Trichinella and circulating parasite antigens in animals and humans., Highlights • Trichinellosis is considered as a public health problem in several countries around the world. • The development of immunodiagnostic techniques has promoted the conduct of seroepidemiological studies. • This review provides insight into the evolution of these techniques.

Trichinella spiralis typically infects domestic swine, wild boar and occasionally horses, has a cosmopolitan distribution, and consequently is most frequently associated with food-borne outbreaks of trichinellosis in humans. Trichinella murrelli is typically found in wild carnivores in temperate areas of North America, where it has been responsible for outbreaks of human trichinellosis due to consumption of infected wild game. There has previously been only indirect evidence of natural infection with T. murrelli in a horse originating from Connecticut and implicated in an outbreak of trichinellosis in France in 1985. We describe a T. murrelli infection detected during routine testing of a horse from the USA imported to Canada for slaughter and export to the European Union (EU). Approximately 5 or more larvae per gram were recovered from digested tongue and diaphragm samples and identified as T. murrelli by PCR. This case provides the first direct evidence of naturally acquired T. murrelli infection in a horse, and further supports the potential food safety risk posed by this parasite species. It is the first instance in Canada of the detection of a Trichinella-infected horse via routine post-mortem testing. Trichinella spiralis-infected horses have been similarly detected by regulatory testing in France, and further details of two such previously reported cases are also provided here. The cases described herein underscore the importance of continued vigilance in quality assured food safety testing of horse meat to mitigate the risk of human trichinellosis., Highlights • First direct evidence of natural infection with Trichinella murrelli in a horse • First detection in Canada of a Trichinella-infected horse via regulated testing • Trichinella murrelli should be considered a potential food safety risk in horsemeat. • Horse tongue and diaphragm are effective for detecting T. murrelli and T. spiralis. • Screening of horse carcasses by digestion assay mitigates risk of trichinellosis.

An immunodominant serine protease ofTrichinella spiralisnamed NBL1 showed encouraging potential in early diagnosis of trichinellosis in pigs and elicited protective immune responses during infection of animals. To further define serological reagents for diagnostic use, the specific epitopes on NBL protein recognized by the antibody responses of different susceptible hosts need to be defined. The present study described comprehensive mapping of immunodominant linear epitopes in the antigenic region (NBL-C, the C-terminal part of the protein) using various serum samples obtained from three kinds of hosts – pig, wild boar and mice. We identified six peptides which were commonly recognized by sera from pigs experimentally infected withTrichinellaand pigs immunized with rNBL1-C; five and four peptides were recognized by sera from wild boars and mice infected withTrichinella,respectively. Three peptides (NBL1-6, -7 and -9) were commonly recognized by antisera in all three hosts, which share the sequence PSSGSRPTYP. We also found that one peptide (NBL1-12) was only recognized by antibodies from pigs immunized with rNBL1-C. The identification of specific epitopes targeted by the host antibody response is important both for understanding the natural response to infection and for the development of subunit vaccines and diagnostic tools for trichinellosis.

Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we described the serine proteases that have been identified in parasitic helminths, including nematodes (Trichinella spiralis, T. pseudospiralis, Trichuris muris, Anisakis simplex, Ascaris suum, Onchocerca volvulus, O. lienalis, Brugia malayi, Ancylostoma caninum, and Steinernema carpocapsae), cestodes (Spirometra mansoni, Echinococcus granulosus, and Schistocephalus solidus), and trematodes (Fasciola hepatica, F. gigantica, and Schistosoma mansoni). Moreover, the possible biological functions of these serine proteases in the endogenous biological phenomena of these parasites and in the host-parasite interaction were also discussed.

Giardia duodenalis (syn. G. lamblia, G. intestinalis) is the protozoan parasite responsible for giardiasis, the most common and widely spread intestinal parasitic disease worldwide, affecting both humans and animals. After cysts ingestion (through either contaminated food or water), Giardia excysts in the upper intestinal tract to release replicating trophozoites that are responsible for the production of symptoms. In the gut, Giardia cohabits with the host's microbiota, and several studies have revealed the importance of this gut ecosystem and/or some probiotic bacteria in providing protection against G. duodenalis infection through mechanisms that remain incompletely understood. Recent findings suggest that Bile-Salt-Hydrolase (BSH)-like activities from the probiotic strain of Lactobacillus johnsonii La1 may contribute to the anti-giardial activity displayed by this strain. Here, we cloned and expressed each of the three bsh genes present in the L. johnsonii La1 genome to study their enzymatic and biological properties. While BSH47 and BSH56 were expressed as recombinant active enzymes, no significant enzymatic activity was detected with BSH12. In vitro assays allowed determining the substrate specificities of both BSH47 and BSH56, which were different. Modeling of these BSHs indicated a strong conservation of their 3-D structures despite low conservation of their primary structures. Both recombinant enzymes were able to mediate anti-giardial biological activity against Giardia trophozoites in vitro. Moreover, BSH47 exerted significant anti-giardial effects when tested in a murine model of giardiasis. These results shed new light on the mechanism, whereby active BSH derived from the probiotic strain Lactobacillus johnsonii La1 may yield anti-giardial effects in vitro and in vivo. These findings pave the way toward novel approaches for the treatment of this widely spread but neglected infectious disease, both in human and in veterinary medicine.

Lysine acetylation is a dynamic and highly conserved post-translational modification that plays a critical role in regulating diverse cellular processes. Trichinella spiralis is a foodborne parasite with a considerable socio-economic impact. However, to date, little is known regarding the role of lysine acetylation in this parasitic nematode. In this study, we utilized a proteomic approach involving anti-acetyl lysine-based enrichment and highly sensitive mass spectrometry to identify the global acetylated proteome and investigate lysine acetylation in T. spiralis. In total, 3872 lysine modification sites were identified in 1592 proteins that are involved in a wide variety of biological processes. Consistent with the results of previous studies, a large number of the acetylated proteins appear to be involved in metabolic and biosynthetic processes. Interestingly, according to the functional enrichment analysis, 29 acetylated proteins were associated with phagocytosis, suggesting an important role of lysine acetylation in this process. Among the identified proteins, 15 putative acetylation motifs were detected. The presence of serine downstream of the lysine acetylation site was commonly observed in the regions surrounding the sites. Moreover, protein interaction network analysis revealed that various interactions are regulated by protein acetylation. These data represent the first report of the acetylome of T. spiralis and provide an important resource for further explorations of the role of lysine acetylation in this foodborne pathogen.

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.

International audience; To date, no information is available about the presence of Cryptosporidium spp. in French sheep, nor their potential role as zoonotic reservoirs. A total of 23 fecal samples were collected from diarrheic lambs (< 11 days old) from seven randomly selected farms. Cryptosporidium-oocysts were detected microscopically with Direct Immunofluorescence Assays (DFA) in 23/23 (100%) of fecal samples. PCR-RFLP of the 18S rRNA gene was used to determine species in all samples, and only Cryptosporidium parvum was identified. Isolates were subtyped by sequencing the 60 kDa glycoprotein (gp60) gene. Two zoonotic subtypes within the IIa subtype family were identified, including IIaA15G2R1 (22/23) and IIaA16G3R1 (1/23). This study reports for the first time the identification and genotyping of zoonotic C. parvum subtypes from lambs in France. Sheep could thus play an important role as potential reservoirs for this zoonotic protist.

Cryptosporidium is an obligate intracellular protist parasite infecting a wide range of vertebrate hosts and causes significant intestinal disease in both animals and humans, as some species are zoonotic. Cattle and especially calves have been identified as one of the most common reservoirs of this protist. However, little is known about the genetics of Cryptosporidium in calves in some regions of France. The aim of this study was to detect and isolate Cryptosporidium spp. in faecal samples from naturally infected pre-weaned calves (≤45 days-old) in France. A total of 35 diarrhoeic pre-weaned calf faecal samples were collected from 26 dairy cattle farms in six departments (French administrative provinces). Cryptosporidium presence was established by microscopically screening samples for oocystes with an immunofluorescent (DFA) staining method. DFA-positive samples were then analysed by PCR-RFLP and 18S rRNA gene sequencing to determine species. Cryptosporidium parvum-positive samples were subtyped via nested PCR analysis of a partial fragment of the 60 kDa glycoprotein (gp60) gene product. Data were then integrated into phylogenetic tree analysis. DFA revealed the presence of Cryptosporidium oocysts in 31 out of 35 (88%) samples. Combined with 18S rRNA gene analysis results, C. parvum was detected in 30 samples. Subtyping analysis in 27/30 samples (90%) of the C. parvum isolates revealed two zoonotic subtype families, IIa (24/27) and IId (3/27). Four subtypes were recognised within the subtype family IIa, including the hypertransmissible IIaA15G2R1 subtype that is the most frequently reported worldwide (21/27), IIaA17G3R1 (1/27), IIaA17G1R1 (1/27), and IIaA19G1R1 (1/27). Two subtypes were recognised within the IId subtype family including IIdA22G1 (2/27) and IIdA27G1 (1/27). These findings illustrate the high occurrence of Cryptosporidium in calves in dairy herds and increase the diversity of molecularly characterised C. parvum isolates with the first description of IIaA17G3R1, IIaA19G1R1, and IId subtypes in France. The presence of zoonotic C. parvum subtype families (IIa, IId) in this study suggests that pre-weaned calves are likely to be a significant reservoir of zoonotic C. parvum, and highlights the importance of animal to human cryptosporidiosis transmission risk. Further molecular studies in calves and small ruminants from other French regions are required to better understand the epidemiology of cryptosporidiosis in France., Highlights • Faecal samples from pre-weaned diarrheal calves were analysed • Cryptosporidium spp. was detected in 30 samples out of 35. • C. parvum was the only species identified • Two zoonotic subtype families were identified: IIa and IId • The hyper-transmissible IIaA15G2R1 was the dominant C. parvum subtype

The presence of the mesocercarial stage of Alaria alata (Goeze, 1792) in wild boar meat represents a potential risk for human, but little is known about the circulation of mesocercaria in wild boar populations. Routine Trichinella inspection, mandatorily performed in wild boar in France, also allowed detecting mesocercaria. We analyzed the results of this detection in the carcasses of 27,582 wild boars hunted in 2007-2011, in 502 hunting areas of the Rhine valley. Prevalence was globally low (0.6%), but 12% of the hunting areas were affected. These were clustered in lowlands of the Rhine valley, and prevalence strongly decreased with increasing elevation. In the lowlands, prevalence doubled between 2007 and 2011. This time trend and the geographic aggregation of positive wild boars suggest risk management measures based on targeted surveillance, control and prevention.

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.

International audience; Toxoplasma gondii (Nicolle et Manceaux, 1908) is an obligate intracellular, parasitic protozoan within the phylum Apicomplexa that causes toxoplasmosis in mammalian hosts (including humans) and birds. Since meat of wild boar, Sus scrofa (Linnaeus), has been demonstrated to be a potential source of human infection, a careful evaluation of the prevalence of infection with T. gondii in hunted animals is needed to protect public health. In the Var area in southeastern France, we performed a spatio-temporal survey in order to investigate the prevalence of IgG antibodies in wild boars shot by hunters in the Canjuers military camp during two subsequent hunting seasons. Of 841 wild boars screened, antibodies (IgG) to T. gondii (modified agglutination test, cut-off 1 : 6) were found in 141 (16.8%) muscle extract samples. A significant association (p < 0.001) was found between positivity and age, but not gender, and hunting districts. The results obtained indicate that consumption of raw or undercooked meat from wild boars carries an important risk of infection with T. gondii. Wild boars may be considered as a bioindicator of parasite circulation in this ecosystem.

SUMMARYThe aim of this study was to assess the seroprevalence of the Toxoplasma gondii parasite in pork produced in France, and to determine infection risk factors. An innovative survey was designed based on annual numbers of slaughtered pigs from intensive and outdoor farms in France. A total of 1549 samples of cardiac fluids were collected from pig hearts to determine seroprevalence using a Modified Agglutination Test. Of those, 160 hearts were bio-assayed in mice to isolate live parasites. The overall seroprevalence among fattening pigs was 2·9%. The adjusted seroprevalence in pigs from intensive farms was 3·0%; the highest in sows (13·4%); 2·9% in fattening pigs and 2·6% in piglets. Adjusted seroprevalence in fattening animals from outdoor farms was 6·3%. Strains were isolated from 41 animals and all were genotyped by Restriction Fragment Length Polymorphism as type II. Risk-factor analysis showed that the risk of infection was more than three times higher for outdoor pigs, and that sows’ risk was almost five times higher than that of fattening animals. This study provides further evidence of extensive pork infection with T. gondii regardless of breeding systems, indicating that farm conditions are still insufficient to guarantee ‘Toxoplasma-free pork’.

Alaria alata (Goeze, 1792), a trematode that parasitizes canids, usually needs two intermediate hosts to complete its life cycle: an aquatic freshwater snail and an amphibian. Although many studies have been undertaken on the wild boar's role as paratenic host, owing to the potential threat to human health, few have sought to identify the snails that act as first intermediate hosts in natural conditions. Adopting a molecular approach, with specific markers for a portion of the second internal transcribed spacer (ITS-2), we detected haplotypes of A. alata furcocercariae in two snail species (Planorbis planorbis and Anisus vortex), identified by molecular analysis (ribosomal 18S, mitochondrial 16S and COI). This study provides the first description of snails naturally emitting A. alata furcocercaria in Western Europe.

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

The trematode Alaria alata is a cosmopolite parasite found in red foxes (Vulpes vulpes), the main definitive host in Europe. In contrast only few data are reported in wild boars (Sus scrofa), a paratenic host. The aim of this paper is to describe the importance and distribution of Alaria alata mesocercariae in wild boars, information is given by findings of these larvae during Trichinella mandatory meat inspection on wild boars’ carcasses aimed for human consumption. More than a hundred cases of mesocercariae positive animals are found every year in the East of France. First investigations on the parasite’s resistance to deep-freezing in meat are presented in this work.

Each year, more than 167 million pigs in the European Union (EU) are tested for Trichinella spp. under the current meat hygiene regulations. This imposes large economic costs on countries, yet the vast majority of these pigs test negative and the public health risk in many countries is therefore considered very low. This work reviewed the current Trichinella status across the EU as well as the national level of monitoring and reporting. It also reviewed which animal species were affected by Trichinella and in which species it should be surveyed. This information was used to design a cost-effective surveillance programme that enables a standardised monitoring approach within the EU. The proposed surveillance programme relies on identifying sub-populations of animals with a distinct risk. Low-risk pigs are finisher pigs that originate from so-called controlled housing. All other pigs are considered high-risk pigs. Controlled housing is identified by the application of a specific list of management and husbandry practices. We suggest that member states (MS) be categorised into three classes based on the confidence that Trichinella can be considered absent, in the specified sub-population of pigs above a specified design prevalence which we set to 1 per million pigs. A simple and transparent method is proposed to estimate this confidence, based on the sensitivity of the surveillance system, taking into account the sensitivity of testing and the design prevalence. The probability of detecting a positive case, if present, must be high (>95 or >99%) to ensure that there is a low or negligible risk of transmission to humans through the food chain. In MS where the probability of a positive pig is demonstrated to be negligible, testing of fattening pigs from a sub-population consisting of pigs from controlled housing can be considered unnecessary. Furthermore, reduced testing of finishers from the sub-population consisting of pigs from non-controlled housing might even be considered, if conducted in conjunction with a proportionate sampling scheme and a risk-based wildlife surveillance programme where applicable. The proposed surveillance programme specifies the required number of samples to be taken and found negative, in a MS. A MS with no data or positive findings will initially be allocated to class 1, in which all pigs should be tested. When a MS is able to demonstrate a 95% or 99% confidence that Trichinella is absent, the MS will be allocated to class 2 or 3, in which the testing requirement is lower than in class 1.