Trichinella britovi outbreak: Epidemiological, clinical, and biological features.

OBJECTIVES: We aimed to describe the first cases of human trichinellosis due to Trichinella britovi in Serbia. A large trichinellosis outbreak due to the consumption of wild boar meat products took place during the 2015-2016 winter. PATIENTS AND METHODS: In January 2016, the Department of Infectious Diseases in Uzice examined 111 individuals with clinical and biological signs of trichinellosis, of whom 19 were hospitalized. Trichinella species identification was performed by multiplex PCR. Serodiagnosis was performed using immunofluorescence antibody assay, indirect ELISA, and Western Blot as confirmatory tests. RESULTS: The main symptoms included myalgia (83%), weakness (82%), joint pain (80%), fever (77%), facial edema (74%), and diarrhea (23%). Eosinophil levels>500/µl were observed in 98% of patients. Elevated CPK levels were detected in 71% of patients and elevated LDH levels in 75%. Three patients had cardiac complications. Treatment included mebendazole, nonsteroidal anti-inflammatory drugs, and corticosteroids. Anti-Trichinella antibodies were observed in 89.7% of patients two months after disease onset, including all hospital-treated patients. Among them, serum positivity detected one year later was 100%. CONCLUSIONS: This outbreak highlighted communication failures, from hunters to consumers. Awareness should be raised on the relation between trichinellosis and game meat. Trichinella species detection is important for adequate outbreak recording and could contribute to better understanding the clinical and serological signs of T. britovi infection.

Application of dendritic cells stimulated with Trichinella spiralis excretory-secretory antigens alleviates experimental autoimmune encephalomyelitis.

The parasitic nematode, Trichinella spiralis (T. spiralis), exerts an immunomodulatory effect on the host immune response through excretory-secretory products (ES L1) released from encysted muscle larvae. Our model of combined T. spiralis infection and experimental autoimmune encephalomyelitis (EAE) in Dark Agouti (DA) rats demonstrated a significant reduction in EAE severity in infected animals. Recently, we have created an immune status characteristic for the live infection by in vivo application of dendritic cells (DCs) stimulated with ES L1 products of T. spiralis muscle larvae. Moreover, these cells were able to ameliorate EAE when applied 7 days before EAE induction. ES L1-stimulated DCs increased production of IL-4, IL-10 and TGF-ß, and decreased production of IFN-γ and IL-17, both at the systemic level and in target organs. A significant increase in the proportion of CD4+CD25+Foxp3+ T cells was found among spleen cells, and CNS infiltrates from DA rats treated with ES L1-stimulated DCs before EAE induction, compared to controls injected with unstimulated DCs. Regulatory T cells, together with elevated levels of IL-10 and TGF-ß, are most likely involved in restraining the production of Th1 and Th17 cytokines responsible for autoimmunity and thus are responsible for the beneficial effect of ES L1-educated DCs on the course of EAE. Our results show that ES L1 antigen-stimulated DCs are able not only to provoke, but also to sustain anti-inflammatory and regulatory responses regardless of EAE induction, with subsequent amelioration of EAE, or even protection from the disease.

Trichinella spiralis antigens prime mixed Th1/Th2 response but do not induce de novo generation of Foxp3+ T cells in vitro.

Many parasitic helminth infections induce Th2-type immune responses and engage the regulatory network. In this study, we specifically investigated the influence of antigens derived from different life stages of the helminth Trichinella spiralis on the polarization of naive CD4(+) T cells by dendritic cells. Results obtained from C57BL/6 mice showed that T. spiralis derived antigens have the capacity to induce bone marrow-derived dendritic cells to acquire an incompletely mature phenotype that promotes a significant proliferation of naive CD4(+) T cells and a mixed Th1/Th2 cytokine profile with the predominance of Th2 cytokines. Increased production of IL-4, IL-9, IL-10 and IL-13 accompanied increased IFN-γ. Furthermore, dendritic cells pulsed with T. spiralis antigens did not induce an increase in the population of Foxp3(+) T regulatory cells. Although other helminth antigens have demonstrated the capacity to induce de novo generation of Foxp3(+) T regulatory cells, here our in vitro studies provide no evidence that T. spiralis antigens have this capacity.

Mechanisms of modulation of experimental autoimmune encephalomyelitis by chronic Trichinella spiralis infection in Dark Agouti rats.

Trichinella spiralis is a helminth that provokes Th2 and anti-inflammatory type responses in an infected host. Our previous studies using Dark Agouti (DA) rats indicated that T. spiralis infection reduced experimental autoimmune encephalomyelitis (EAE) severity in rats. The aim of this study was to analyse the mechanisms underlying EAE suppression driven by T. spiralis infection. Reduced clinical and histological manifestations of the disease were accompanied by increased IL-4 and IL-10 production and decreased IFN-gamma and IL-17 production in draining lymph node cells. This indicates that T. spiralis infection successfully maintains a Th2 cytokine bias regardless of EAE induction. High IL-10 signifies parasite-induced anti-inflammatory and/or regulatory cell responses. Transfer of splenic T cell-enriched population of cells from T. spiralis-infected rats into EAE immunized rats caused amelioration of EAE and in some cases protection from disease development. This population of cells contained higher proportion of CD4(+) CD25(+) Foxp3(+) regulatory cells and produced high level of IL-10 when compared with uninfected rats.

Application of GIS in epizootiological surveillance of swine trichinellosis in one endemic district in Serbia.

Application of new tools for epizootiological investigations in veterinary medicine, such as Geographical Information Systems (GIS), offers a new approach and possibilities for the eradication or control of infectious diseases. GIS is particularly useful for research conducted in small areas strongly impacted by man. Trichinellosis is a world-wide zoonosis, which is endemic in some European countries, Balkan district and Serbia in particular. There are very few data on GIS application in the field of trichinellosis. We here present the application of GIS for mapping Trichinella spp. occurrence and spatial and temporal patterns of Trichinello infection in one endemic district in Serbia. Settlements with trichinellosis were marked and particular points of interest were designated. Data on prevalence of Trichinella infection in domestic swine accompanied by location of foci indicated the existence of disease geographical stationarity. This first report on GIS application in Serbia will facilitate trichinellosis surveillance and monitoring of Trichinella spp. circulation among domestic pigs, and populations of synanthropic and sylvatic animals.

Strain difference in susceptibility to Trichinella spiralis infection between dark agouti and albino oxford rats.

The influence of host genetics on the susceptibility to primary Trichinella spiralis infection has been extensively studied in a mouse model, but has not been clarified for rats. Analyses of interstrain and intrastrain genetic variation in response to infectious agents could be beneficial not only for elucidating the genetic basis of host resistance/susceptibility, but for revealing immune response mechanisms as well. The aim of this study was to analyse interstrain differences in worm burdens and cytokine production between Albino Oxford (AO) and Dark Agouti (DA) rats in muscle phase of T. spiralis infection. Clear strain-dependent variation was observed in the number of T. spiralis larvae per gram (lpg) of muscle tissue where values for DA rats (626.7+/-171lpg) vastly exceeded those found in AO rats (49.8+/-25.9lpg, p<0.001). Differences between the strains were also noticed in key cytokine levels. In infected AO rats, the cytokine production remained in favor of Th1 type response, while infected DA rats showed a shift towards a Th2 type response. The level of regulatory IL-10 was significantly increased only in T. spiralis infected DA rats. Our results provide evidence that DA rats express higher susceptibility to T. spiralis infection in comparison to AO rats with respect to muscle larvae burden. The infection in DA rats was accompanied by the production of anti-inflammatory cytokines, while the response of AO rats was characterized by a proinflammatory type of immune response.

The lateral flow card test: an alternative method for the detection of Trichinella infection in swine.

A novel lateral flow card (TS-Card pork) test was developed for the serological detection of Trichinella infected pigs. Based on extensive studies performed in Romania during 1999-2000 this test proved to be highly specific sensitive, rapid (3-12 minutes) and easy to use (no need for laboratory facilities). It can be used both for the detection of Trichinella infection in carcasses and for epizooliological studies using a variety of samples including whole or dried blood, serum, or tissue fluids. The TS-Card pork test, used as a screening test, can be the foundation of an on-farm or field based inspection system to significantly improve food safety in countries with a high prevalence of Trichinella in pigs or other food animal species. The results presented are also promising for application of the test in an on-line laboratory based inspection system since the speed of the test allows sufficient time to rail out suspected hog carcasses during the slaughter process.

Immunodiagnosis of Trichinella infection in the horse.

From 1998 to 2000, 5,267 horse sera were collected from several Trichinella regions in Romania. Sera were initially screened in laboratories in Romania, Serbia and Italy with an ELISA and a Western blot (Wb) using an excretory/secretory (ES) antigen and several conjugates (protein A, protein G, and sheep or goat anti-horse). Differences in serology results were obtained among the different conjugates and also between ELISA and Wb. Depending on the test used, specific antibodies were found at a prevalence rate of 3-6% of horses. Serum samples classified as positive were tested again by ELISA using a synthetic tyvelose glycan-BSA antigen, in Italy. All serum samples tested using this antigen were negative; in contrast, serum samples from experimentally infected horses were positive with the glycan antigen. The negative results obtained with the glycan antigen are consistent with the low prevalence of horse trichinellosis reported in the literature. Based on these results, further studies are needed to validate immunodiagnostic tests to detect Trichinella infection in horses.

Present status of trichinellosis in Yugoslavia: Serbia.

Trichinellosis was recognized almost one century ago as a health and animal husbandry problem in Serbia. In the last 10 years, trichinellosis has been expanding from three endemic regions (Srem, Macva and Negotinska Krajina) to neighbouring regions. The infection rate detected by veterinary inspection in 1999 year was 0.17% in slaughtered swines. Simultaneously, the number of infected humans increased three-five times in comparison with the period 1980-1990. For instance, 555 individuals were registered in 1999 as infected after the consummation of non-inspected pork from domestic swine or wild boar. Prevalence of trichinellosis in wild animals was examined more than a 20 years ago. The trichinellosis in horses has not been detected in the country, but infected horses imported from Serbia were detected in France and Italy.