Two trichinellosis outbreaks in Serbia - challenging diagnosis due to a potential co-infection with Toxocara spp.

In Serbia, in most cases, small family outbreaks of trichinellosis occur due to the consumption of untested infected meat from domestic pigs that are raised and slaughtered in the backyards of small individual farms. The aim of this study is to present data regarding 24 patients involved in two outbreaks that occurred in two neighbouring districts in Serbia during a closely related period of time in August 2014. The source of infection in the first outbreak was undercooked pork, while raw pork sausages caused the second outbreak. Meat samples and sausages were Trichinella spp. positive by artificial digestion. With the aim to obtain a second opinion and validate the serological findings discovered at the Public Health Institute Nis, all samples were sent to the National Reference Laboratory for Trichinellosis, INEP. Serodiagnosis showed that 21 persons were positive (87.5%) and three (12.5%) were negative for anti-Trichinella antibodies, while 15 patients fulfilled the trichinellosis case definition. Western blot analysis (using an epitope unique for the muscle larvae stage of the Trichinella genus) confirmed the diagnosis of trichinellosis in five patients. Six patients also had specific antibodies against Toxocara canis (T. canis). Due to the fact that in endemic foci in Serbia there is the presence of Trichinella spiralis and T. canis and that these two infections could be asymptomatic, we consider that trichinellosis cases were irrefutably proven. The dilemma about the existence of co-infection with T. canis remained open due to the lack of clinical findings.

Efficacy of an aerosol-resistant pepsin powder used in artificial digestion for the detection of Trichinella larvae in meat.

Trichinellosis is an important worldwide foodborne zoonosis. The gold standard test to detect Trichinella spp. larvae in muscle samples of animals intended for human consumption is the artificial digestion method. Handling and dispensing of conventional pepsin powder present significant safety risks for analysts. The use of pepsin powder that is resistant to aerosolization should alleviate these safety concerns. The aim of this study was to compare the efficacy of an aerosol-resistant pepsin powder to conventional pepsin powder in the artificial digestion method. Proficiency samples of pork diaphragm containing specific numbers of viable Trichinella spiralis larvae were tested in two laboratories. The results revealed that aerosol-resistant pepsin was simple, effective and convenient to use, and showed good solubility and larval recovery that met the requirements of the European Union regulation EU 2015/1375. Overall, the efficacy of the aerosol-resistant pepsin was comparable to the conventional pepsin and safer for analysts.

Long lasting immunity in trichinellosis - insight from a small study group.

Trichinellosis in humans is most often caused by the parasite Trichinella spiralis. The clinical course of the disease is diverse and the symptoms can vary from mild to severe. Symptoms usually disappear within a few months, but encysted larvae in the muscles can cause myalgia and weakness that last for years. However, the existence of chronic trichinellosis as a disease is still debatable. This study presents the results obtained at the National Reference Laboratory for Trichinellosis - INEP, Serbia. The study was conducted to assess the immunoserological, biochemical and symptomatic disease parameters of twelve patients who acquired trichinellosis 13 and 18 years ago, respectively. They were involved in two T. spiralis outbreaks in Serbia, at the village of Kumane and the city of Belgrade (nine and three patients, respectively). Results indicated the presence of specific anti-Trichinella antibodies in 83% of the total number of patients. However, while the humoral immune response to Trichinella lasted for more than a decade reaching almost two decades after the acute infection phase (7/9 and 3/3 respectively, in two outbreaks), persistence of chronic muscular pain, as the most prolonged symptom of trichinellosis, could be found in the majority of patients from the Kumane outbreak (7/9). As a consequence, these patients suffered from limitations in daily living activities for the same period of time. The results presented in this paper are our contribution to the view that trichinellosis as a chronic disease with symptoms exists and may be related to the severity of the disease in the acute phase.

A competitive enzyme-linked immunosorbent assay for rapid detection of antibodies against Trichinella spiralis and T. britovi - one test for humans and swine.

Infection with parasites from the Trichinella genus occurs in many vertebrates but disease only occurs in humans (trichinellosis). Humans are infected after the consumption of raw or undercooked meat from infected wild or domestic animals (usually swine or horses). Using the monoclonal antibody (mAb) 7C2C5, specific for an epitope unique to the muscle larvae of the genus Trichinella, we have developed a competitive enzyme-linked immunosorbent assay (c-ELISA) that enables the rapid detection of Trichinella-specific antibodies in sera originating from two different host species (human, swine) infected with either Trichinella spiralis or Trichinella britovi. This novel c-ELISA exhibited 100% specificity and sensitivity, as confirmed by a Western blot test. The assay is easy to use (one incubation step), and the time required for the procedure (45 min) is shorter than in any other ELISA format. This test could be useful for both the detection and surveillance of Trichinella infections.

Immunomodulatory potential of particular Trichinella spiralis muscle larvae excretory-secretory components.

Excretory-secretory antigens of Trichinella spiralis muscle larvae can induce the semi-matured status of rat dendritic cells. This may at least partly be the consequence of transient activation of extracellular signal-regulated kinases 1/2 (ERK1/2). Here we investigated the potential of several components of excretory-secretory antigens (native fraction containing 45, 49 and 53kDa proteins and recombinant Tsp53, representing one of the constituents of this fraction) to demonstrate previously observed effects of excretory-secretory antigens on dendritic cells in vitro, characterised by establishment of a particular phenotype (very low MHC II expression, moderate CD86 expression and significant ICAM-1 expression) and functional properties (low production of pro-inflammatory cytokine IL-12p70, and high production of IL-10 and TGF-ß). Dendritic cells activated by these components were able to provoke proliferation of naïve T cells and their polarisation towards Th2 and anti-inflammatory responses. The investigated antigens had almost the same capacity to induce IL-4 and IL-10 production from T cells as excretory-secretory antigens, but failed to induce significant TGF-ß synthesis. It could be concluded that the investigated excretory-secretory antigens components can largely reproduce the immunomodulatory effects of the complete excretory-secretory antigens and therefore may be considered as molecules important for creation of the anti-inflammatory milieu achieved by the parasite.

Trichinella infection in Serbia in the first decade of the twenty-first century.

In Serbia, infection with Trichinella spp. has been recognized as a human health and animal husbandry problem for almost a century. The rate of swine infection gradually decreased from 0.14% to 0.02% between 2001 and 2010. For the past 5 years, Trichinella infections among swine were detected at levels higher than 0.05% in 3 districts of Serbia while prevalence persisted at lower levels for the rest of the country. During this 10-year period, there were 2257 cases of human trichinellosis, including 3 deaths; however, a significant decrease in the number of cases was reported during the last 5 years (fewer than 200 cases per year). The fact that prevalence data presented here are similar to prevalence data from 1990 indicates that this period of 10 years was needed to overcome the re-emergence of Trichinella infection in swine and humans that occurred during the last decade of the previous century.

Trichinella infections in different host species of an endemic district of Serbia.

Trichinella infections are endemic in the Balkan region of Europe. Though trichinellosis and agents thereof are serious problems for human health and animal husbandry, only a limited number of Trichinella isolates from Serbia have been identified at the species level so far. The aim of the present study was the surveillance and monitoring of Trichinella in domestic pigs and wild animals from the endemic district of Branicevo. Investigations performed during the 2009-2010 period revealed Trichinella infections in 344 out of 282,960 (0.12%) domestic pigs. Among wildlife, Trichinella infections were detected in 11 out of 94 (11.7%) wild boars (Sus scrofa), 7 out of 57 (12.3%) red foxes (Vulpes vulpes), 7 out of 13 (53.8%) golden jackals (Canis aureus), and in all three examined wolves (Canis lupus). Trichinella spiralis and Trichinella britovi were the only two species identified. T. britovi was identified in 31% of isolates from wildlife of the Branicevo district and T. spiralis was found in 53% of wild animals; mixed infections were observed in 16% of the animals examined. Findings form the basis of an information campaign for veterinary services, pig owners and the hunter's associations about the risk of the transmission of these zoonotic agents. The application of control programs as established at the Veterinary Specialist Institute of Pozarevac resulted in a decline in Trichinella infections among domestic pigs and the absence of human trichinellosis in the last three years in the Branicevo district.

The impact of Trichinella spiralis excretory-secretory products on dendritic cells.

Parasitic nematode Trichinella spiralis exert immunomodulatory effect on the host immune response through excretory-secretory products (ES L1) released from the encysted muscle larvae. Rat bone-marrow derived dendritic cells (DCs) stimulated with ES L1 antigens acquire semi-matured status and induce Th2 and regulatory responses in vitro and in vivo. Priming naïve T cells in vitro with ES L1 pulsed DCs caused strong Th2 polarization, accompanied by elevated production of regulatory cytokines IL-10 and TGF-ß and no increase in the proportion of CD4+CD25+Foxp3+ among the effector T cell population. In vivo T cell priming resulted in mixed Th1/Th2 cytokine response, with the dominance of the Th2 type and elevated levels of regulatory cytokines. Significant increase in the proportion of CD4+CD25+Foxp3+ cells was found among recipient's spleen cells. We have achieved to create immune status characteristic for the live infection by in vivo application of DCs educated with ES L1 antigens.

Trichinella prevalence in swine in an endemic district in Serbia: epidemiology and control.

Endemic trichinellosis is re-emerging in Serbia and it is a serious problem both from the perspective of human health and animal husbandry. The widespread appearance of human trichinellosis is attributed to a high prevalence of Trichinella infection in domestic animals, especially swine. Epidemiological data presented in this paper were collected during a 12-year period (1995-2006) at small private swine farms in the region of Branicevo, Serbia, where a high Trichinella prevalence in slaughter pigs (0.57%) has been detected. To further monitor Trichinella prevalence in swine, a serological survey, using ELISA, was performed in 2006. Of 916 swine tested by ELISA, Trichinella specific antibodies were detected in 15 (1.64%), while suspect results were obtained in 10 (1.09%). Positive or suspect animals originated from all parishes except one (Pozarevac). Our results point to the need for systematic monitoring in pigs to achieve a better control of trichinellosis in Serbia.

Trichinella spiralis: nurse cell formation with emphasis on analogy to muscle cell repair.

Trichinella infection results in formation of a capsule in infected muscles. The capsule is a residence of the parasite which is composed of the nurse cell and fibrous wall. The process of nurse cell formation is complex and includes infected muscle cell response (de-differentiation, cell cycle re-entry and arrest) and satellite cell responses (activation, proliferation and differentiation). Some events that occur during the nurse cell formation are analogous to those occurring during muscle cell regeneration/repair. This article reviews capsule formation with emphasis on this analogy.

Characterization of rat bone marrow dendritic cells initially primed by Trichinella spiralis antigens.

Pathogen-derived products have the capacity to induce maturation of bone marrow-derived dendritic cells (BMDCs)into populations of effectors cells that polarize Th cells toward Th1 or Th2 phenotype via different mechanisms. Since those mechanisms are not entirely clear for helminths, and almost completely unknown for Trichinella spiralis(TS), we started an investigation of the effects of TS antigens (four different antigens isolated from all three life-cycle stages of parasite)on maturation of BMDCs and their potential to present TS antigens. The expression of MHC class II, costimulatory molecules CD86, CD54, IL-10 and IL-12p70 cytokine production were measured after 2 days of BMDCs cultivation with TS antigens. While parasitic antigens did not significantly alter the expression of MHC II, most of them, except crude muscle larvae antigens, up-regulated the expression of costimulatory molecules. BMDCs, primed with all TS antigens, released increased amounts of IL-10 and decreased amounts of IL-12p70. BMDCs, primed with TS antigens, induced significant proliferation of syngeneic TS sensitized lymph nodes cells and also stimulated the production of IL-4 by T cells purified from of TS infected DA rats. The results indicate that TS stimulated BMDCs leads to the polarization of the immune response towards regulatory and Th2 type.

Trichinella spiralis: modulation of experimental autoimmune encephalomyelitis in DA rats.

Helminth infection has a potent systemic immunomodulatory effect on the host immune response, which also affects the development of autoimmune diseases. We investigated the dose-dependent influence of Trichinella spiralis infection on experimental autoimmune encephalomyelitis (EAE). Our model of concomitant T. spiralis infection and EAE demonstrates that established infection of Dark Agouti (DA) rats with the parasite causes amelioration of the clinical course of induced EAE in a dose-dependent way. Infection with T. spiralis L1 stage muscle larvae (TSL1) reduced the severity of the autoimmune disease as judged by lower maximal clinical score, cumulative index, duration of illness and degree of mononuclear cell infiltration in T. spiralis infected animals compared to control, EAE-induced group. This study provides a valuable model of worm infection to investigate helminth-induced regulatory mechanisms for optimal benefit to the host.

Anti-Trichinella antibodies detected in chronically infected horses by IFA and Western blot, but not by ELISA.

In the Balkan countries, where trichinellosis is a re-emerging zoonosis, it is of great importance to determine Trichinella infection prevalence among the major hosts, including horses. One method for monitoring prevalence is serological surveillance; however, the validity of serological methods in horses is not well understood. The dynamics of anti-Trichinella IgG production and circulating excretory/secretory (ES) antigens were investigated in three horses experimentally-infected with Trichinella spiralis. Horses were slaughtered at 32 week post infection (p.i.). Low worm burdens were found in all three animals. Anti-Trichinella IgG was detected up to 32 weeks p.i. by an indirect immunofluorescence assay (IFA) and by Western blot (Wb), but not by ELISA. The ELISA test detected antibodies for only a short period of time (up to 18 weeks p.i. using ES antigen or up to 20 weeks p.i. using tyvelose-BSA antigen). The presence of circulating muscle larvae ES antigen in sera of infected horses was observed by dot blot from the 4th week p.i. up to the 32nd week p.i.

[Carcinoembryonic antigen in chronic otitis media with cholesteatoma]. / Karcinoembrionski antigen kod hronicnog otitisa medije s holesteatomom.

INTRODUCTION: Carcinoembryonic antigen (CEA) is a cell surface glycoprotein and member of immunoglobuline superfamily. Recent studies pointed out that this glycoprotein also belonged to the family of intercellular adhesive molecules (ICAMs). The presence and increased expression of CEA is noticed in chronic inflammation of the skin, gall bladder and extrahepatic biliary tract. According to our knowledge, this glycoprotein has not been studied previously in chronic inflammations of the middle ear mucosa. The purpose of this study was to investigate the presence and distribution of carcinoembryonic antigen (CEA) in chronic otitis media with cholesteatoma. MATERIALS AND METHODS: The immunohistochemical analysis with high affinity monoclonal antibodies against human carcinoembryonic antigen (MEDIX Bochemica AB, Finland) was performed in 71 mucosal samples with chronic inflammation and in 8 samples of the clinically healthy middle ear mucosa. The mucosal specimens were taken intraoperatively from different regions of the middle ear cleft during surgery of chronic otitis media with cholesteatoma. The control samples were obtained during surgery of traumatic perforation of the ear drum. For control staining irrelevant monoclonal antibodies were used (Anti-h-PRL clone code 5601: OY Medix Biochemica AB, Finland). The complex antibody-CEA antigen was visualized by marked Ig mice antibody (HRPO Inep, Zemun) and diaminobenzidine (DAB) as chromogene. After being prepared through this specific immunohistochemical analysis, all mucosal specimens were analysed under the light microscope. RESULTS: Carcinoembryonic antigen (CEA) was not detected in non-inflamed, clinically healthy mucosal specimens of the middle ear mucosa. The 71 samptes taken from mastoid, attic, promontory and protympanon in chronic otitis media with cholesteatoma showed positive staining for CEA expression in 56 (79%) biopsy specimens, while in 15 (21%) CEA has not been identified. CEA was present in squamous epithelium of cholesteatoma, then in fibrous stroma and in endothelial cells of blood vessels of the lamina propria in the middle ear mucosa. DISCUSSION: Chronic otitis media with cholesteatoma is an invasive disease with aggressive destruction of mucosal and bony structures of the middle ear. The pathophysiological mechanism of that kind of chronic inflammation has not been clarified. Studies which analysed excessive expression of CEA in chronic inflammation of the skin pointed out that CEA may induce hyperproliferation of keratinocytes. The findings in our study confirmed the high level of expression of CEA in squamous epithelium of the middle ear cholesteatoma. The endothelial cells of blood vessels and stroma of lamina propria, as well as stroma of the granulation tissue were stained positively with labelled CEA antibodies. The results of this study showed that CEA was present in chronic inflammation of the middle ear mucosa and in the squamous epithelium of cholesteatoma. The precise function of the increased expression of CEA in chronic otitis media with cholesteatoma will be the subject of our further studies.

[Susceptibility and the immune response to Trichinella spiralis infection]. / Prijemsivost i imunski odgovor na infekciju Trichinella spiralis-om.

The aim of this study was to assess a possible association between parameters of immune response and susceptibility/resistance to T. spiralis infection. The immune response to T. spiralis was monitored in two inbred strains of mice (BALB/c and C57Bl/6), as well as in the most important natural host--swine, by analysing the changes in main lymphocyte populations, the appearance of specific antibodies and worm burden recovery. In the murine model, the lower level of muscle larvae worm burden recovery in mice expressed greater resistance (BALB/c) was associated with manifested clinical signs, earlier development of humoral antibody response and a persistently higher level of CD4/CD8 T cell ratio, compared to T. spiralis-susceptible mice (C57Bl/6). In swine, high susceptibility to infection was also associated with lack of clinical symptoms, a delayed development of an antibody response and persistently low CD4/CD8 T cell ratio.