Trichinella britovi recombinant proteins produced in Pichia pastoris expression system for specific IgG antibody detection in the sera of mice and pigs infected with Trichinella spp.

Trichinellosis, a disease caused by infection with Trichinella spp, poses an economic problem in the animal sector and a recurrent health problem for humans. Discovering the new diagnostic tests may be achieved by identification and production of species- and stage-specific recombinant proteins of Trichinella genus which are recognized by the host antibodies after infection. In this study the T. britovi proteins identified earlier in excretory-secretory (ES) products: CTRL, ES21 and HSP20, were cloned and produced using a eukaryotic Pichia pastoris system. Their immunodiagnostic properties were verified by measuring the abundance of specific IgG antibodies in sera from mice and pigs experimentally infected with T. britovi or T. spiralis. The rTbCTRL and the rTbES21 proteins were more effectively produced and stable than rTbHSP20. The most sensitive protein for serodiagnostic purposes occurred to be CTRL; anti-rTbCTRL IgG level increased at 41 days post infection (dpi) in pigs infected with T. britovi and 45 dpi for those infected with T. spiralis. The rTbES21 protein was the most specific for the T. britovi species, as no antibody titers were observed in pigs infected with T. spiralis. Following the multiple-antigen strategy, the combination of rTbCTRL + rTbES21 was applied in ELISA, but no significant difference in IgG level was detected in the tested conditions.

Inactivation of Trichinella muscle larvae at different time-temperature heating profiles simulating home-cooking.

BACKGROUND: Trichinellosis is caused by consumption of raw or undercooked meat containing infective Trichinella muscle larvae (ML). Only few studies on heat-inactivation of Trichinella ML are available in literature and more validated data concerning heat inactivation is needed to improve the risk estimation. OBJECTIVE AND METHODS: The aim of the present study was to evaluate the two in vitro methods "staining" and "morphological examination" as proxies for Trichinella ML heat inactivation in comparison with the mouse bioassay method to get more insight in the relationship between heat, heating time and inactivation of Trichinella ML. The second aim was to evaluate whether these methods could replace the bioassay in the light of ongoing animal use reduction in lifescience research. Tubes containing quantified live Trichinella ML were exposed to heat profiles ranging from 40 to 80 °C. Subsequently, inactivation was evaluated using both methylene blue staining and morphological examination, which was validated by bioassay. Results were used to model Trichinella inactivation. RESULTS: Trichinella muscle larvae exposed to 60 °C or higher for 12-12.5 min were not infective to mice. We found that morphological examination was more consistent with the bioassay than methylene blue staining. Modelled inactivation fitted experimental data consistently. Moreover, this study shows that larval Trichinella morphology may be used in situations where bioassays are not possible or prohibited. CONCLUSIONS: The relationship between heat and inactivation of larvae obtained from this study could be used in Trichinella QMRA models to improve quantification of the risk of Trichinella infection.

Clinical and epidemiological descriptions from trichinellosis outbreaks in Bulgaria.

Bulgaria is one of European countries where trichinellosis continues to be regularly diagnosed and registered. The clinical and epidemiological features of 72 cases of trichinellosis associated with five outbreaks caused by Trichinella spiralis and Trichinella britovi between 2009 and 2011, are described. At hospital admission, patients were often initially treated with antibiotics, without any improvement. A range of signs and symptoms were recorded, including: myalgia, elevated temperature, arthralgia, difficulty with movement, facial oedema, conjunctival hyperaemia, ocular haemorrhages, diarrhoea, skin rash, headache, and fatigue. Due to the variable clinical course of the disease, the diagnostic process for trichinellosis is often complex and difficult. This means the diagnosis may be established late for an appropriate treatment, potentially leading to a severe course of the disease with complications. Laboratory abnormalities were expressed by marked eosinophilia (97.2%), leucocytosis (70.8%), elevated serum creatine phosphokinase levels (82%), and antibody-positive results by ELISA and indirect hemagglutination. Patients were treated with albendazole (Zentel) 10 mg/kg for 7-10 days. In two outbreaks, the aetiological agent was T. spiralis, in one outbreak T. britovi, and an unknown Trichinella species in the fourth outbreak. The sources of infection were domestic pigs, probably fed with scraps and offal of wild game. In one outbreak, T. spiralis was also detected in brown rats trapped close to where the pig had been raised in the backyard. These epidemiological factors are relevant in considering implementation of targeted control programmes.

Human trichinellosis caused by Trichinella britovi in Greece, and literature review.

Trichinellosis is a cosmopolitan zoonotic parasitic disease caused by the nematodes of the genus Trichinella, through the consumption of raw or semi-raw infected meat from swine, horses and wild animals. This disease has been sporadically reported in Greece since 1946. The aim of the present study was to describe a trichinellosis case in a patient hospitalized in northern Greece, in 2017. A 47-year-old male was admitted to hospital with intense generalized myalgia, periorbital swelling, fever, exhaustion and anorexia. Biochemical and haematological profile showed eosinophilia and elevated creatine phosphokinase (CPK). Anti-Trichinella spp. IgG and IgM antibodies were detected by serology and Trichinella spp. larvae were found in two muscle biopsies by compressorium and histological examination. A larva collected from the muscle biopsy was identified as Trichinella britovi by polymerase chain reaction (PCR). Albendazole (400 mg twice per day × 10 days) was administered and the clinical condition of the patient promptly improved. This is the first identification of T. britovi in a patient in Greece.

TRICHINELLA INFECTIONS IN RED FOXES (VULPES VULPES) AND GOLDEN JACKALS (CANIS AUREUS) IN SIX DISTRICTS OF SERBIA.

Wild animals, including red foxes (Vulpes vulpes) and golden jackals (Canis aureus), are the most important reservoirs of Trichinella spp. Although the red fox is considered one of the main reservoirs of Trichinella spp. in Europe, only a few animals have been examined in Serbia. The present study assessed Trichinella spp. infection in red foxes and golden jackals from the six districts in Serbia. Thirty-seven carcasses of red foxes and 13 carcasses of golden jackals shot during the official hunting season were examined. Larvae of Trichinella spp. were detected in 13 (35%) of 37 red foxes and in 8 (61%) of 13 golden jackals. Trichinella spiralis and Trichinella britovi were the only two species identified after a multiplex polymerase chain reaction analysis. Trichinella britovi infection was detected in 85% of red foxes and in 38% of golden jackals, and T. spiralis was detected in 15% of red foxes and in 63% of golden jackals. The findings emphasize the need for an active surveillance program for Trichinella spp. infection in wildlife in Serbia and the whole of the Balkans, with special attention on the red fox because it is widespread and occurs in high densities.

A Trichinella britovi outbreak in the Northern Alps of France: investigation by a local survey network.

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.

Title: Un foyer de Trichinella britovi dans les Alpes du Nord françaises : investigation par un réseau local de prospection. Abstract: Nous décrivons une épidémie familiale de trichinellose causée par la consommation de jambon cru d'un sanglier (Sus scrofa) chassé dans le nord des Alpes françaises en février 2022. Sur les six personnes âgées de 3 à 69 ans qui ont consommé la viande, trois étaient des cas confirmés, et trois étaient des cas suspects. L'éosinophilie détectée chez quatre personnes a permis d'évoquer le diagnostic. Trois patients présentaient des myalgies, et deux des douleurs thoraciques intenses et prolongées dont un avec une troponine élevée. Un patient a présenté un dermographisme pendant le traitement. Des IgG anti-Trichinella ont été détectées chez trois individus symptomatiques après environ dix semaines. Un des patients avait une sérologie négative et aucun symptôme mais était sous corticothérapie au long cours. Des larves de Trichinella britovi (8,3 larves/g) ont été détectées dans les restes du jambon de sanglier incriminé. La trichinellose est rare en France, mais cette épidémie familiale rappelle la circulation de cet agent pathogène chez les animaux sauvages, qui nécessite d'informer les chasseurs sur les risques d'infections liés à la consommation de viande crue de gibier, et de préconiser un contrôle vétérinaire des viandes de gibier. La consommation de viande crue en dehors des circuits contrôlés est une pratique non dénuée de risques, qui justifie une sensibilisation des chasseurs, médecins et biologistes médicaux.

First Identification and Molecular Characterization of Trichinella britovi (Nematoda: Trichinellidae) from the Pine Marten (Martes martes Linnaeus, 1758) in Romania.

Trichinella spp. are etiological zoonotic agents spread throughout the world and affect mammals, birds, and reptiles; they evolve via two cycles: domestic and sylvatic. Martes martes is a carnivorous nocturnal mammal from the family Mustelidae. In Romania, this host is widespread in all forests of the country. Martes martes has an extremely voracious appetite, feeding on fruit and also on a variety of small animals, including rodents such as mice and rats. The aim of this study was the identification and molecular characterization of Trichinella larvae isolated from the muscle tissue of Martes martes collected in different counties of Romania. The muscle samples were examined via artificial digestion, and the larvae were identified at the species level via multiplex PCR. The presence of larvae belonging to Trichinella britovi, a species frequently identified in wild carnivores in temperate zones, was observed. Although T. britovi has been already reported in several host species in Romania, this is the first time this species has been observed in a Martes martes specimen. This finding contributes to our knowledge about the host species involved in the maintenance of the Trichinella sylvatic cycle in Romania, and it confirms that this parasite is consistently present in the wild fauna of this country.

Trichinella spp. detection in hunted wild boar (Sus scrofa) diaphragm biopsies in Central Italy.

Trichinellosis is a globally diffused foodborne parasitic disease caused by nematodes of the Trichinella complex. During evolution, guided by ecological interactions, natural selection, and biochemical "intelligence", these parasites developed admirable strategies to infect the host's organism. One of the most fascinating is represented by the nurse cell formation in muscular tissue (e.g., diaphragm, skeletal muscle, extrinsic ocular muscles, etc.). This strategy allowed the parasite to adapt and conquer the wider host species spectrum, including ungulates and humans. Consumption of undercooked meat from infected wild ungulates constitutes the most important source of infection for the human species. In this study, we show the prevalence of Trichinella spp. in hunted wild boars (Sus scrofa) in Central Italy. During the hunting season 2021/2022 in the province of Rieti, 554 wild boar diaphragm biopsies were collected for Trichinella spp. screening, in accordance with Regulation EU 1375/2015. An artificial digestion method was used for the detection of Trichinella spp. larval forms. The results revealed a positivity of 0.18% (1/554), and molecular biology identification demonstrated the presence of Trichinella britovi in the positive sample. This species is the most diffused in wild ungulate populations in Central Italy and the most frequently isolated in human patients with trichinellosis from this area, showing a close epidemiological relation between Homo sapiens and Sus scrofa for Trichinella spp. diffusion in an ecosystem. Epidemiological surveillance, in receptive animal species destined for human consumption and at any One Health level, represents the main "winning" strategy in the control of this worldwide, widespread foodborne parasitic disease.

Trichinella britovi in wild boar meat from Italy, 2015-2021: A citizen science approach to surveillance.

As a result of the increase of game meat intended for human consumption through Europe, a plethora of food-borne diseases, including trichinellosis, may occur in consumers, posing a relevant public health threat. Thus, this study aims to a citizen science approach to monitor the occurrence of Trichinella spp. in wild boar meat intended for human consumption, evaluating the risk of infection for consumers. Following the European Regulation 2015/1375 (laying down specific rules on official controls for Trichinella in meat), from 2015 to 2021, hunters (n = 478) were involved to collect diaphragm pillar samples of wild boars from mainland southern Italy, which were tested for Trichinella spp. L1 larvae via HCl-pepsin digestion and Multiplex PCR. Overall, 139,160 animals were collected (average of 19,880 per year), being 14 (i.e., 0.01%) tested positive to Trichinella britovi by the combined biochemical and molecular approach. An average larval burden of 28.4 L1 per gram of meat was found (minimum 3.2 - maximum 132.6). A statistically significant difference was found in the prevalence according to hunting seasons (p < 0.01, with higher values in 2016 and 2021) and regions of the study area (p < 0.01). No statistically significant decrease in the prevalence of T. britovi throughout the study period was found (p = 0.51), except in Apulia region (p < 0.01). These findings revealed a stable prevalence of T. britovi in wild boar meat intended for human consumption, suggesting a risk of infection for consumers, especially hunters and local markets users. Citizen science surveillance models could be promoted to improve trichinellosis control and prevention in a One Health perspective.

The illegal rearing and slaughtering of pigs in the wild on the Mediterranean island of Sardinia favor an increase in the biomass of Trichinella britovi in wild boars (Sus scrofa) but do not affect the serological prevalence of infection.

BACKGROUND: Worms of the nematode genus Trichinella are zoonotic pathogens with a worldwide distribution. The first report of Trichinella on the Mediterranean island of Sardinia was for Trichinella britovi, one of the four species of this genus circulating in Europe, which was identified in 2005 following an outbreak of trichinellosis in humans due to the consumption of pork from pigs reared in the wild. Since then, T. britovi larvae have been repeatedly isolated from free-ranging pigs, foxes (Vulpes vulpes) and wild boars (Sus scrofa) sampled in the central-eastern region of the island (Orgosolo municipality), but have never been isolated from samples from other areas of the island. The aim of this study was to investigate the parasitological and serological prevalence of T. britovi infection in wild boars in Sardinia over space [eight wild boar hunting management units (HMUs)] and time (seven wild boar hunting seasons). METHODS: Muscle and serum samples of boars killed in the 2014-2015 to 2020-2121 hunting seasons were collected from eight HMUs of central and south-western Sardinia. Trichinella sp. larvae were detected by artificial digestion of predilection muscles. A total of 4111 serum samples of wild boar were collected from the investigated HMUs and tested by enzyme-linked immunosorbent assay as a screening test and by western blot as a confirmatory test using excretory/secretory antigens. RESULTS: Trichinella britovi muscle larvae were detected in six (0.03%) of the 17,786 wild boars tested. All of the Trichinella sp.-positive wild boars had been hunted in Orgosolo municipality (central-eastern area of the island), except for one, hunted in a neighboring municipality. An overall serological prevalence of 3.8% (95% confidence interval, 3.3-4.5) was detected by western blot. No statistical differences were detected between the HMUs where T. britovi larvae were detected in wild boars, foxes, and free-ranging pigs and those where wild boars, foxes and free-ranging pigs tested negative. CONCLUSIONS: The serological prevalence did not vary between the wild boar populations in which the larval load was detectable by artificial digestion (Orgosolo municipality) and those in which the larval load was below the detection limit. Furthermore, the serological prevalence of anti-Trichinella immunoglobulin G in the wild boar populations remained constant during the study period, which covered seven wild boar hunting seasons. As the transmission events (i.e., the serological prevalence) are stable, the high biomass of the parasite in Orgosolo municipality can only have arisen as a consequence of factors independent of its natural cycle, i.e., the presence of a high number of free-ranging pigs, and the concomitant presence of African swine fever, due to illegal pig slaughtering in the field. This epidemiological situation suggests that the natural cycle of T. britovi may be influenced by inappropriate pig husbandry and slaughtering practices.

Wolf Is Back: A Novel Sensitive Sentinel Rejoins the Trichinella Cycle in the Western Alps.

Trichinella is a foodborne parasite whose wildlife reservoirs are represented by carnivores and omnivores with predatory and scavenger behavior. The aim of the present study was to investigate the occurrence of Trichinella infection in grey wolves (Canis lupus) that recolonized the Western Alps from the end of the past century, and discuss the epidemiological role played by this apex predator in the early phases of its return. During the period 2017-2022, diaphragm samples were obtained from 130 individuals collected in the frame of a wolf mortality survey. Trichinella larvae were found in 15 wolves (11.53%) with a parasite intensity of 11.74 larvae per gram. Trichinella britovi was the only species identified. This is the first prevalence survey of Trichinella in wolves recolonizing the Alps. Results suggest that, in this particular biotope, the wolf has rejoined the Trichinella cycle and has the potential to play an increasingly important role as maintenance host. Arguments in favor and against this perspective are discussed and knowledge gaps highlighted. The calculated Trichinella larval biomass in the estimated wolf population roaming in Northwest Italy will serve as baseline value to explore possible shifts in the relative importance of wolves as Trichinella reservoir within the regional carnivore community. Finally, wolves re-colonizing the Alps already appear as sensitive sentinels to monitor the risk of Trichinella zoonotic transmission by infected wild boar meat.

Antiparasitic Action of Lactobacillus casei ATCC 393 and Lactobacillus paracasei CNCM Strains in CD-1 Mice Experimentally Infected with Trichinella britovi.

Nematodes of the genus Trichinella are among the most widespread parasites of domestic and wild omnivores and predatory animals. The present study aimed to evaluate the antiparasitic effect of Lactobacillus casei ATCC 393 (original) and L. paracasei CNCM in CD-1 mice experimentally infected with Trichinella britovi. Four groups of 20 mice (10 females and 10 males/group) were used, with two control (C) groups and two experimental (E) groups, in which each animal received a daily oral dose of 100 µL of 105 CFU/mL probiotics in Ringer's solution. On day 7, all mice (except the negative control group) were infected orally with Trichinella (100 larvae/animal) as well as the two probiotics. On day 9 post-infection (p.i.), 10 mice/group were euthanized, and the presence of adult parasites in the intestinal content and wall was tested. On day 32 p.i., 10 mice/group were euthanized, then trichinoscopy and artificial digestion were performed to assess the muscle infection with T. britovi. On day 9 p.i., the experimental group pretreated with L. casei ATCC 393 (6.3 ± 3.03) showed a significantly lower number of adult parasites in the intestinal wall compared with the positive control group (24.6 ± 4.78). Additionally, a significantly lower adult parasite count in the intestinal wall was registered in female mice pretreated with L. paracasei CNCM (7.4 ± 4.71) compared to female mice from the positive control (29.0 ± 5.17). No statistically relevant results were obtained concerning the male mice or the data obtained at 32 days p.i., irrespective of mice gender.

Immunoproteomic Analysis of Trichinella britovi Proteins Recognized by IgG Antibodies from Meat Juice of Carnivores Naturally Infected with T. britovi.

Infection with Trichinella nematodes elicits non-specific and specific immune responses; these depend on the dose of infection, the nematode, and the host species. Few studies have examined the presence of specific antibodies against Trichinella spp. in the meat juice of wild animals. The aims of the study were to determine the prevalence of antibodies against Trichinella spp. in meat juice and to identify the specific proteins reacting with the meat juice from free-living carnivores naturally infected with the parasite. Meat juice samples were taken from foxes, badgers, raccoon dogs, and martens and tested with indirect ELISA. Antibodies against Trichinella spp. were detected in 10% of foxes and 46% of raccoon dogs. The ELISA results were confirmed by immunoblot, which revealed different protein patterns in meat juice from red foxes, raccoon dogs, and badgers. The most frequently observed bands were sent for further analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the detection of Trichinella britovi immunogenic proteins. The results confirm the presence of proteins such as serine protease and heat shock proteins associated with Trichinella infection. These findings provide that meat juice is a useful matrix for proteomic analysis.

Insight into Trichinella britovi Infection in Pigs: Effect of Various Infectious Doses on Larvae Density and Spatial Larvae Distribution in Carcasses and Comparison of the Detection of Anti-T. britovi IgG of Three Different Commercial ELISA Tests and Immunoblot Assay.

There is limited information available on the Trichinella britovi (T. britovi) muscle larvae (ML) distribution in pig muscle and the humoral immune response of pigs infected with moderate doses of this parasite; therefore, this study investigated the infectivity of a Polish strain of T. britovi for pigs, the antibody response of this host to various doses of T. britovi, and the efficiency of three different commercial ELISA kits and an immunoblot assay at detecting anti-T. britovi IgG. No significant differences in terms of the infection level of particular muscles or of whole carcasses between pigs infected with 3000 and those infected with 5000 ML of T. britovi were observed. The highest intensity of T. britovi infection was reported in the diaphragm pillars. The larvae of T. britovi showed homogeneous distribution with respect to the muscle side. Statistically, specific IgG antibodies against excretory-secretory (ES) antigens of Trichinella ML were first detected by all ELISA protocols on day 36 post infection; however, individual pig results showed some differences between the three tests applied. A significant increase in the level of anti-T. britovi IgG was observed between days 36 and 41 post infection, and from day 45 until day 62 after T. britovi infection, production of these antibodies reached its plateau phase. No positive correlation was found between the anti-T. britovi IgG level and the larvae density in 15 different muscles. Sera of T. britovi-infected pigs showed reactivity with T. britovi ML ES antigens of 62, 55, and 52 kDa. The results provide novel information on spatial larvae distribution in muscles and the humoral immune response of pigs exposed to two different doses of a Polish strain of T. britovi, extend knowledge on serological diagnostic tools which may be introduced in veterinary practice for the detection of T. britovi infections in pig production, and offer practical solutions for meat hygiene inspectors in the field at sampling sites when examining pig carcasses for Trichinella.

First report of Trichinella pseudospiralis in a wolf (Canis lupus italicus).

Within the genus Trichinella, Trichinella pseudospiralis is the only recognized non-encapsulated species known to infect mammals and birds. In October 2020, larvae recovered from muscle tissues of a wolf (Canis lupus italicus) originating from Molise Region, Central Italy, were molecularly confirmed as those of Trichinella britovi and T. pseudospiralis. This is the first detection of T. pseudospiralis from a wolf. In Italy, this zoonotic nematode was detected in a red fox (Vulpes vulpes), three birds (Strix aluco, Athene noctua, Milvus milvus) and five wild boars (Sus scrofa), and was also identified as the etiological agent of a human outbreak of trichinellosis in 2015. Since T. pseudospiralis is rarely reported from carnivore mammals in comparison to the encapsulated species frequently detected in these hosts, this finding opens the question of the role of carnivores as reservoirs for this parasite.

Reprint of: The European badger, Meles Meles, as a new host for Trichinella britovi in Romania.

The European badger, Meles meles (Carnivora, Mustelidae) is a widespread opportunistic omnivorous mammal. Its food spectrum comprises a wide variety of plants and small animals, occasionally including rodents, such as mice or rats. Considering that rodents are known to play a key role in the life cycle of Trichinella spp., the aims of this study were to investigate the occurrence of these parasites in badgers from Romania and to identify the species. Overall, 61 badgers originating from 14 counties were examined by trichinoscopy and artificial digestion. For species determination, the positive muscle samples, and the larvae recovered from the artificial digestion were used for DNA isolation, and further processed by multiplex PCR. A single badger, originating from Sibiu County, Central Romania, was found positive for Trichinella spp. Five cysts were identified using trichinoscopy: four in the diaphragm and one in the foreleg muscles. Artificial digestion revealed an infection rate of 70 larvae/100 g of muscle. The PCR indicated the occurrence of T. britovi, which is the most commonly detected species in wild carnivores in temperate areas. Although T. britovi has previously been reported in Romania, this represents the first report of its occurrence in the European badger in the country. However, the low prevalence indicates a minor reservoir role of this species.

The first analysis of Trichinella spiralis and Trichinella britovi adult worm excretory-secretory proteins by two-dimensional electrophoresis coupled with LC-MS/MS.

The two most common Trichinella species present in European countries are Trichinella spiralis and Trichinella britovi. The consumption of raw or undercooked meat with invasive larvae results in trichinellosis. Currently, the most commonly used sources for detecting specific anti-Trichinella antibodies is ELISA with the muscle larvae (ML) excretory-secretory (E-S) proteins. However, these serological methods cannot be efficiently applied at early stage of infection. The aim of the current study was to identify the common and species-specific E-S proteins of T. spiralis and T. britovi adult worms which could have potential for accurate diagnosis of Trichinella infection at an early stage of invasion. Different sets of immunoreactive proteins were identified in T. spiralis and T. britovi proteomes by a combination of two-dimensional electrophoresis (2-DE), immunoblot and LC-MS/MS analysis. Polyubiquitin-B, a possible enoyl-CoA hydratase/isomerase YngF or polyubiquitin-like protein was found to be common for both species; gene ontology analysis confirmed its involvement in proteolysis, oxidation-reduction and translation processes, as well as in molecular transport. These molecules, being secreted or excreted at an early stage of parasite development, may play a critical role in the processes occurring during the initial steps of the host invasion and hence be suitable for diagnostic test development.

Exploiting the potential of 2D DIGE and 2DE immunoblotting for comparative analysis of crude extract of Trichinella britovi and Trichinella spiralis muscle larvae proteomes.

The Trichinella genus poses an interesting puzzle for researchers, having diverged very early in the evolution of the nematodes. The Trichinella spiralis proteome is a cosmopolitan and well-studied model of Trichinella; however, Trichinella britovi also circulates in the sylvatic environment and both species infect humans, resulting in the development of trichinellosis. Few experiments have examined the proteins belonging to the T. britovi proteome. The aim of the present study was to compare the protein expression profiles of crude extracts of T. spiralis and T. britovi muscle larvae using a highly-sensitive two-dimensional differential in-gel electrophoresis (2D DIGE) technique coupled with 2DE immunoblotting. Selected immunoreactive protein spots were then identified by liquid chromatography coupled with mass spectrometry analysis (LC-MS/MS), and their function in Trichinella and the host-parasite interaction was determined by gene ontology analysis. Spots common to both T. spiralis and T. britovi, spots with different expressions between the two and spots specific to each species were labelled with different cyanine dyes. In total, 196 protein spots were found in both proteomes; of these 165 were common, 23 expressed exclusively in T. spiralis and 8 in T. britovi. A comparative analysis of volume ratio values with Melanie software showed that among the common spots, nine demonstrated higher expression in T. spiralis, and 17 in T. britovi. LC-MS/MS analysis of 11 selected spots identified 41 proteins with potential antigenic characteristics: 26 were specific for T. spiralis, six for T. britovi, and eight were found in both proteomes. Gene Ontology analysis showed that the identified T. spiralis proteins possess hydrolytic endopeptidase, endonuclease and transferase activities. Similarly, most of the T. britovi proteins possess catalytic activities, such as lyase, hydrolase, isomerase and peptidase activity. The applied 2D DIGE technique visualized Trichinella spp. protein spots with different molecular weights or isoelectric point values, as well as those with different expression levels. The identified immunoreactive proteins participate in multiple processes associated with host muscle cell invasion and larval adaptation to the host environment. Their reactivity with the host immune system makes them possible candidates for the development of a novel trichinellosis diagnostic test or vaccine against helminthiasis caused by T. spiralis or T. britovi.

The First Report of Trichinella britovi in Armenia.

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.

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.