Trichinella proficiency testing in Southeastern European countries.

In some Southeastern European (SEE) countries, like Serbia, Romania, and Bulgaria, trichinellosis is one of the most important foodborne zoonotic diseases. In those countries, EU regulation and local authorities require the staff of laboratories performing official controls on meat to be properly trained and to check their competence by participating regularly in proficiency testing (PT). PTs are organized by National Reference Laboratories for Trichinella of each country and involve all official laboratories testing meat. In Romania and Bulgaria, the organization of PT for the detection of Trichinella larvae in meat by Magnetic Stirrer Method (MSM) started in 2012. In Croatia and Serbia PT was first organized in 2015 and 2017, respectively. This study presents data on the performance obtained by official laboratories of SEE countries that organize PT at national level and compares the performance obtained by laboratories belonging to different countries. Results suggest that the constant participation in PT leads to an increase in the performance of participating laboratories by positively affecting the staff accuracy in sample testing by MSM. Since the percentage of recovered larvae was in some cases suboptimal (<80%) and occasionally very poor (<40%), there is room for improvement. The regular participation in PT by laboratories involved in official controls on meat intended for human consumption is fundamental to guarantee consumer safety.

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.

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.

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.

Dendritic cells acquire tolerogenic properties at the site of sterile granulomatous inflammation.

Subcutaneous implantation of polyvinyl sponges represents a suitable model for studying the mechanisms of acute and chronic inflammation, granulomatous foreign-body reaction, as well as wound healing. Using such a model in rats, we studied the phenotypic and functional characteristics of dendritic cells (DC). DC were purified from the sponge exudate using a combination of separation gradients, adherence to plastics, and immunomagnetic sorting. We have shown that the number of DC progressively increased in the sponges, reaching maximal values at day 10 after implantation, followed by their decrease thereafter. Inflammatory DC expressed MHC class II molecules and myeloid markers CD11b, CD11c, and CD68. A subset of DC expressed CD4, R-MC46, DEC-205, R-MC17, and CCR1. Compared to DC isolated in the early phase of inflammation (day 6 DC), DC in the late stage of inflammation (day 14 DC) had a lower capability to stimulate the proliferation of allogeneic lymphocytes and CD4(+) T cells. This finding correlated with the downregulation of CD80, CD86, and CD54 expression and the increased proportion of plasmacytoid MHC class II(+) His 24(+) His 48(+) DC. The suppression of allogeneic lymphocyte proliferation was abrogated by the treatment of DC with lipopolysaccharide. In addition, day 14 DC exerted tolerogenic capability in co-culture with allogenic CD4(+) T cells. These results correlated with the increased levels of IL-10 and TGF-beta in culture supernatants and the sponge exudate.