Trichinella infection in Serbia from 2011 to 2020: a success story in the field of One Health.

In Serbia, modern pork production systems with implemented control measures, including the detection of Trichinella larvae in meat (ISO18743), have eliminated farmed pork from pigs slaughtered at abattoirs as a source of trichinellosis. Epidemiological data from 2011 to 2020 indicate that the number of human cases and the number of infected domestic pigs has decreased significantly. Over the years, pork was the most frequent source of human infection. Cases generally occurred in small family outbreaks, and the infection was linked to consumption of raw or undercooked pork from backyard pigs. In most of the outbreaks, T. spiralis was the aetiological agent of infection, but in 2016, a large outbreak was caused by consumption of uninspected wild boar meat containing T. britovi larvae. To achieve safe pork, it is important that consumers of pork from animals raised in backyard smallholdings and of wild game meat are properly educated about the risks associated with consumption of untested meat. Laboratories conducting Trichinella testing should have a functional quality assurance system to ensure competency of analysts and that accurate and repeatable results are achieved. Regular participation in proficiency testing is needed.

Trichinellosis in Serbia has become a rare event - one outbreak with pulmonary complications.

An outbreak of trichinellosis occurred in Stari Banovci, a settlement in the municipality of Stara Pazova, Srem, Republic of Serbia, in March-April 2019. A total of 28 persons were exposed and trichinellosis was confirmed in 24 of them. This outbreak involved members of eight families, their relatives and friends. The infection, due to Trichinella spiralis (Owen, 1835), was associated with consumption of pork sausages procured in a local butcher's shop. The trace-back study revealed that the meat originated from swine that was raised on a small farm belonging to the owner of the shop, who did not have permission from the Veterinary Directorate for slaughtering animals and who put on the market sausages prepared from uninspected meat. Trichinellosis was accompanied by typical clinical symptoms. However, the unusual occurrence of high percentage of pulmonary complications was noticed. The description of this outbreak indicates that medical practitioners should initiate treatment immediately in cases of high suspicion of trichinellosis, even if the serology is negative, to prevent the complications of the disease. In spite of significant achievements in the control of Trichinella infection among domestic pigs and humans in the last 10 years, it is obvious that such cases of breeding animals under inappropriate conditions, slaughtering them without approval and placing uninspected pork on the market represent a source of sporadic outbreaks in Serbia.

Harnessing immunomodulatory mechanisms of Trichinella spiralis to design novel nanomedical approaches for restoring self-tolerance in autoimmunity.

The rapid increase in the prevalence of autoimmune diseases in recent decades, especially in developed countries, coincided with improved living conditions and healthcare. Part of this increase could be ascribed to the lack of exposure to infectious agents like helminths that co-evolved with us and display potent immune regulatory actions. In this review we discussed many investigations, including our own, showing that Trichinella spiralis via its excretory-secretory products attenuate Th1/Th17 immunopathological response in autoimmunity and potentiate the protective Th2 and or regulatory T cell response, acting as an effective induction of tolerogenic dendritic cells (DCs), and probably mimicking the autoantigen in some diseases. A recent discovery of T. spiralis extracellular vesicles (TsEVs) suggested that inducing a complex regulation of the immune response requires simultaneous delivery of different signals in nano-sized packages. Indeed, different artificial nanomedical approaches discussed here suggested that co-delivery of multiple signals via nanoparticles is the most promising strategy for the treatment of autoimmune diseases. Although a long way is ahead of us before we could completely replicate natural nano-delivery systems which are both safe and potent in restoring self-tolerance, a clear path is being opened from a careful examination of parasite-host interactions.

DC-SIGN signalling induced by Trichinella spiralis products contributes to the tolerogenic signatures of human dendritic cells.

Tolerogenic dendritic cells (tolDCs) are central players in the maintenance of immune tolerance and thereby have been identified as the most favourable candidates for cell therapy of autoimmune diseases. We have recently shown that excretory-secretory products (ES L1) released by Trichinella spiralis larvae induce stable human tolDCs in vitro via Toll-like receptor 2 (TLR2) and TLR4. However, engagement of these receptors did not fully explain the tolerogenic profile of DCs. Here, we observed for the first time that dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) interacts with highly glycosylated ES L1 and contributes to the generation of ES L1-induced tolDCs. Blocking DC-SIGN interfered with the ES L1-induced higher expression of CD40 and CCR7 and the production of IL-10 and TGF-ß by DCs. The cooperation of TLR2, TLR4 and DC-SIGN receptors is of importance for the capacity of DCs to prime T cell response toward Th2 and to induce expansion of CD4+CD25+Foxp3+ T cells, as well as for the production of IL-10 and TGF-ß by these cells. Overall, these results indicate that induction of tolDCs by ES L1 involves engagement of multiple pattern recognition receptors namely, TLR2, TLR4 and DC-SIGN.

A common source for a trichinellosis outbreak reported in France and Serbia in 2017.

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.

Antibodies in sera from multiple sclerosis patients recognize Trichinella spiralis muscle larvae excretory-secretory antigens.

Helminths, as complex pathogens, possess a large number of different epitopes, some of which may be similar to the epitopes of the host. Besides being the cause for the activation of self-reactive immune cells, molecular mimicry may also be the cause for the expansion of regulatory T cells, crucial for the host tolerance of self-antigens. Amelioration of experimental autoimmune encephalomyelitis (EAE), animal model of multiple sclerosis (MS), caused by Trichinella spiralis infection or application of its muscle larvae excretory-secretory products (ES L1), was achieved through activation of Th2 and regulatory responses. The present study aimed to reveal whether the cause of observed immunomodulation could be the existence of shared epitopes between ES L1 antigens and auto-antigens. Serum samples from 92 MS patients were tested in Western blot for the reactivity toward components of ES L1. Immunoglobulins from the sera of MS patients recognized several ES L1 components, but 45, 49 and 58 kDa proteins dominated others by the frequency of interaction. According to the logistic regression analysis, these interactions were statistically significantly associated with MS, regardless of the disease phenotype or severity. Selected molecules might share homology with self-antigens and as such are worthy of further investigation in terms of potential immunomodulatory capacity and involvement in the parasite's provoked amelioration of EAE.

Trichinella spiralis muscle larvae release extracellular vesicles with immunomodulatory properties.

AIMS: Extracellular vesicles (EVs) represent a newly discovered but universal communication tool between cells or organisms. However, few data exist on nematode EVs and none for Trichinella spiralis. Here, we aimed to investigate whether T spiralis muscle larvae produce EVs, whether they carry immunomodulatory proteins and whether they have a role in immunomodulation as a component of excretory-secretory muscle larvae products (ES L1). METHODS AND RESULTS: EVs were enriched from conditioned medium of T spiralis muscle larvae. Transmission electron microscopy images showed T spiralis EVs to be 30-80 nm in size, and Western blot confirmed the presence of two out of three glycoproteins with the immunodominant epitope characteristic for muscle larvae of the genus Trichinella. Using a peripheral blood mononuclear cell (PBMC) stimulation assay, it was shown that these EVs elevated production of IL10 and IL6. CONCLUSION: T spiralis muscle larvae produce EVs. Those EVs carry immunomodulatory proteins and have the capacity independently to induce regulatory responses in the same way as the T spiralis excretory-secretory muscle larvae products from which they were isolated.

Functionalization-dependent effects of cellulose nanofibrils on tolerogenic mechanisms of human dendritic cells.

BACKGROUND: Cellulose nanofibrils (CNF) are attractive nanomaterials for various biomedical applications due to their excellent biocompatibility and biomimetic properties. However, their immunoregulatory properties are insufficiently investigated, especially in relation to their functionalization, which could cause problems during their clinical application. METHODS: Using a model of human dendritic cells (DC), which have a central role in the regulation of immune response, we investigated how differentially functionalized CNF, ie, native (n) CNF, 2,2,6,6-tetramethylpiperidine 1-oxyl radical-oxidized (c) CNF, and 3-aminopropylphosphoric acid-functionalized (APAc) CNF, affect DC properties, their viability, morphology, differentiation and maturation potential, and the capacity to regulate T cell-mediated immune response. RESULTS: Nontoxic doses of APAcCNF displayed the strongest inhibitory effects on DC differentiation, maturation, and T helper (Th) 1 and Th17 polarization capacity, followed by cCNF and nCNF, respectively. These results correlated with a specific pattern of regulatory cytokines production by APAcCNF-DC and their increased capacity to induce suppressive CD8+CD25+IL-10+ regulatory T cells in immunoglobulin-like transcript (ILT)-3- and ILT-4- dependent manner. In contrast, nCNF-DC induced predominantly suppressive CD4+CD25hiFoxP3hi regulatory T cells in indolamine 2,3-dioxygenase-1-dependent manner. Different tolerogenic properties of CNF correlated with their size and APA functionalization, as well as with different expression of CD209 and actin bundles at the place of contact with CNF. CONCLUSION: The capacity to induce different types of DC-mediated tolerogenic immune responses by functionalized CNF opens new perspectives for their application as well-tolerated nanomaterials in tissue engineering and novel platforms for the therapy of inflammatory T cell-mediated pathologies.

Trichinella spiralis Excretory-Secretory Products Induce Tolerogenic Properties in Human Dendritic Cells via Toll-Like Receptors 2 and 4.

Trichinella spiralis, as well as its muscle larvae excretory-secretory products (ES L1), given either alone or via dendritic cells (DCs), induce a tolerogenic immune microenvironment in inbred rodents and successfully ameliorate experimental autoimmune encephalomyelitis. ES L1 directs the immunological balance away from T helper (Th)1, toward Th2 and regulatory responses by modulating DCs phenotype. The ultimate goal of our work is to find out if it is possible to translate knowledge obtained in animal model to humans and to generate human tolerogenic DCs suitable for therapy of autoimmune diseases through stimulation with ES L1. Here, the impact of ES L1 on the activation of human monocyte-derived DCs is explored for the first time. Under the influence of ES L1, DCs acquired tolerogenic (semi-matured) phenotype, characterized by low expression of HLA-DR, CD83, and CD86 as well as moderate expression of CD40, along with the unchanged production of interleukin (IL)-12 and elevated production of IL-10 and transforming growth factor (TGF)-ß, compared to controls. The interaction with DCs involved toll-like receptors (TLR) 2 and 4, and this interaction was mainly responsible for the phenotypic and functional properties of ES L1-treated DCs. Importantly, ES L1 potentiated Th2 polarizing capacity of DCs, and impaired their allo-stimulatory and Th1/Th17 polarizing properties. Moreover, ES L1-treated DCs promoted the expansion of IL-10- and TGF-ß- producing CD4+CD25hiFoxp3hi T cells in indolamine 2, 3 dioxygenase (IDO)-1-dependent manner and increased the suppressive potential of the primed T cell population. ES L1-treated DCs retained the tolerogenic properties, even after the challenge with different pro-inflammatory stimuli, including those acting via TLR3 and, especially TLR4. These results suggest that the induction of tolerogenic properties of DCs through stimulation with ES L1 could represent an innovative approach for the preparation of tolerogenic DC for treatment of inflammatory and autoimmune disorders.

Cell-based Tolerogenic Therapy, Experience from Animal Models of Multiple Sclerosis, Type 1 Diabetes and Rheumatoid Arthritis.

Cell-based tolerogenic therapy is a promising approach for the treatment of autoimmune diseases and transplant rejection. Regulatory T cells and tolerogenic dendritic cells have been particularly explored in the treatment of various autoimmune disorders in experimental models of disease. Although some of these cells have already been tested in a limited number of clinical trials, there is still a need for preclinical research on tolerogenic cells in animal models of autoimmunity. This review will focus on the relevance of data obtained from studies in experimental animal models for the use of tolerogenic cell-based therapy in humans. Also, perspectives for further improvement of tolerogenic cell preparation towards enhanced suppressive activity and stability of the cells will be discussed.

Minimum Information about T Regulatory Cells: A Step toward Reproducibility and Standardization.

Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications.

Minimum information about tolerogenic antigen-presenting cells (MITAP): a first step towards reproducibility and standardisation of cellular therapies.

Cellular therapies with tolerogenic antigen-presenting cells (tolAPC) show great promise for the treatment of autoimmune diseases and for the prevention of destructive immune responses after transplantation. The methodologies for generating tolAPC vary greatly between different laboratories, making it difficult to compare data from different studies; thus constituting a major hurdle for the development of standardised tolAPC therapeutic products. Here we describe an initiative by members of the tolAPC field to generate a minimum information model for tolAPC (MITAP), providing a reporting framework that will make differences and similarities between tolAPC products transparent. In this way, MITAP constitutes a first but important step towards the production of standardised and reproducible tolAPC for clinical application.

The first report of Trichinella pseudospiralis presence in domestic swine and T. britovi in wild boar in Bosnia and Herzegovina.

The Balkans is endemic for nematodes of the genus Trichinella in both domestic and wild animals. The high prevalence of these zoonotic pathogens in animals linked with the food habits to consume raw meat and meat derived products resulted in a very high prevalence of trichinellosis in humans living in this European region. In spite of numerous epidemiological investigations carried out in this region, very few information is available on the Trichinella species circulating in Bosnia and Herzegovina. Trichinella spp. larvae were isolated from a domestic pig reared in a backyard and from a hunted wild boar whose meat had been the source of trichinellosis in one case. Both Trichinella pseudospiralis and T. spiralis have been identified in the domestic pig, whereas, T. britovi was detected in the wild boar. While, T. spiralis is the Trichinella species most frequently detected in domestic pigs, T. pseudospiralis has been previously documented in domestic pigs only three times in Russia, Slovakia and Croatia. The detection of T. britovi in the wild boar confirms that this nematode is the most frequent species circulating among wildlife of Europe.

Necrosis and apoptosis in Trichinella spiralis-mediated tumour reduction.

It is known that infection with different pathogens, including helminths, can alter the progression of malignant or other diseases. We studied the effect of chronic Trichinella spiralis infection or muscle larvae excretory-secretory (ES L1) antigens on the malignant tumour growth in the mouse melanoma model system in vivo and in vitro. Our results confirmed that chronic infection with T. spiralis possesses the capacity to slow down the progression of tumour growth, resulting in an impressive reduction in tumour size. We found that the phenomenon could, at least partially, be related to a lower level of tumour necrosis compared to necrosis present in control animals with progressive malignancy course. An increased apoptotic potential among the low percentage of cells within the total tumour cell number in vivo was also observed. ES L1 antigen, as a parasitic product that is released during the chronic phase of infection, reduced the survival and slightly, but significantly increased the apoptosis level of melanoma cells in vitro. Our results imply that powerful Trichinella anti-malignance capacity does not rely only on necrosis and apoptosis but other mechanisms through which infection or parasite products manipulate the tumor establishment and expansion should be considered.

Secretory Products of Trichinella spiralis Muscle Larvae and Immunomodulation: Implication for Autoimmune Diseases, Allergies, and Malignancies.

Trichinella spiralis has the unique ability to make itself "at home" by creating and hiding in a new type of cell in the host body that is the nurse cell. From this immunologically privileged place, the parasite orchestrates a long-lasting molecular cross talk with the host through muscle larvae excretory-secretory products (ES L1). Those products can successfully modulate parasite-specific immune responses as well as responses to unrelated antigens (either self or nonself in origin), providing an anti-inflammatory milieu and maintaining homeostasis. It is clear, based on the findings from animal model studies, that T. spiralis and its products induce an immunomodulatory network (which encompasses Th2- and Treg-type responses) that may allow the host to deal with various hyperimmune-associated disorders as well as tumor growth, although the latter still remains unclear. This review focuses on studies of the molecules released by T. spiralis, their interaction with pattern recognition receptors on antigen presenting cells, and subsequently provoked responses. This paper also addresses the immunomodulatory properties of ES L1 molecules and how the induced immunomodulation influences the course of different experimental inflammatory and malignant diseases.

Immunomodulatory effects of Trichinella spiralis-derived excretory-secretory antigens.

Helminth-derived products, either released into the circulation during the course of the infection or isolated after in vitro cultivation of the parasite and applied by the injection, are able to suppress the host immune response to autoantigens and allergens, but mechanisms could differ. Prophylactic application of Trichinella spiralis excretory-secretory muscle larvae (ES L1) products ameliorates experimental autoimmune encephalomyelitis (EAE) with the same success as infection did. However, a shift to the Th2-type response in the periphery and in the central nervous system, accompanied by activation of regulatory mechanisms, had a striking, new feature of increased proportion of unconventional CD4(+)CD25(-)Foxp3(+) regulatory cells both in the periphery and in the central nervous system of animals treated with ES L1 before the induction of EAE.

Glycans expressed on Trichinella spiralis excretory-secretory antigens are important for anti-inflamatory immune response polarization.

Trichinella spiralis muscle larvae excretory-secretory antigens (ES L1) are most likely responsible for the induction of immune response during infection by this parasitic. The antigens bear carbohydrate structures that may contribute to immune system activation resulting in a Th2/anti-inflammatory immune response. We show that T. spiralis glycans affect the expression and the production of IL-4 and IL-10 in vivo. Alteration of carbohydrate structures on ES L1 altered dendritic cell (DC) maturation. Periodate treatment of ES L1 led to the reduction in both ERK and p38 phosphorylation which may be the cause of reduced IL-10 and IL-12p70 production. In vitro priming of naïve T cells with DCs stimulated with native and periodate-treated ES L1 emphasized the importance of intact glycans for IL-10 production. We conclude that T. spiralis glycans affect the anti-inflammatory environment and can interfere with the development of inflammatory diseases.

Helminths: Immunoregulation and Inflammatory Diseases-Which Side Are Trichinella spp. and Toxocara spp. on?

Macropathogens, such as multicellular helminths, are considered masters of immunoregulation due to their ability to escape host defense and establish chronic infections. Molecular crosstalk between the host and the parasite starts immediately after their encounter, which influences the course and development of both the innate and adaptive arms of the immune response. Helminths can modulate dendritic cells (DCs) function and induce immunosuppression which is mediated by a regulatory network that includes regulatory T (Treg) cells, regulatory B (Breg) cells, and alternatively activated macrophages (AAMs). In this way, helminths suppress and control both parasite-specific and unrelated immunopathology in the host such as Th1-mediated autoimmune and Th2-mediated allergic diseases. However, certain helminths favour the development or exacerbation of allergic responses. In this paper, the cell types that play an essential role in helminth-induced immunoregulation, the consequences for inflammatory diseases, and the contrasting effects of Toxocara and Trichinella infection on allergic manifestations are discussed.

Trichinella spiralis shares epitopes with human autoantigens.

Like other helminths, Trichinella spiralis has evolved strategies to allow it to survive in the host organism, including the expression of epitopes similar to those present in either expressed or hidden host antigens. To identify T. spiralis-derived antigens that are evolutionarily conserved in the parasite and its host and that could be responsible for its evasion of the host immune response, we examined the reactivity of six different types of autoantibodies to T. spiralis larvae from muscle. T. spiralis antigens that share epitopes with human autoantigens were identified by assessing the cross-reactivity of autoantibody-containing serum samples with T. spiralis antigens in the absence of specific anti-parasite antibodies. Of the 55 autoantibody-containing human serum samples that we analysed by immunohistological screening, 24 (43.6%) recognised T. spiralis muscle larvae structures such as the subcuticular region, the genital primordium or the midgut. Using Western blots, we demonstrated that the same sera reacted with 24 protein components of T. spiralis muscle larvae excretory-secretory L1 antigens. We found that the human autoantibodies predominantly bound antigens belonging to the TSL1 group; more specifically, the autoantibody-containing sera reacted most frequently with the 53-kDa component. Thus, this protein is a good candidate for further studies of the mechanisms of T. spiralis-mediated immunomodulation.

Trichinella spiralis shares epitopes with human autoantigens

Like other helminths, Trichinella spiralis has evolved strategies to allow it to survive in the host organism, including the expression of epitopes similar to those present in either expressed or hidden host antigens. To identify T. spiralis-derived antigens that are evolutionarily conserved in the parasite and its host and that could be responsible for its evasion of the host immune response, we examined the reactivity of six different types of autoantibodies to T. spiralis larvae from muscle. T. spiralis antigens that share epitopes with human autoantigens were identified by assessing the cross-reactivity of autoantibody-containing serum samples with T. spiralis antigens in the absence of specific anti-parasite antibodies. Of the 55 autoantibody-containing human serum samples that we analysed by immunohistological screening, 24 (43.6%) recognised T. spiralis muscle larvae structures such as the subcuticular region, the genital primordium or the midgut. Using Western blots, we demonstrated that the same sera reacted with 24 protein components of T. spiralis muscle larvae excretory-secretory L1 antigens. We found that the human autoantibodies predominantly bound antigens belonging to the TSL1 group; more specifically, the autoantibody-containing sera reacted most frequently with the 53-kDa component. Thus, this protein is a good candidate for further studies of the mechanisms of T. spiralis-mediated immunomodulation.