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Background Domesticated pigs are the main source of Trichinella sp. infections for humans, particularly when reared in backyards or free-ranging. In temperate areas of southern Europe, most pigs are farmed under controlled housing conditions, but sows and sometimes fattening pigs have access to outdoors to improve animal welfare. The aim of the present study was to investigate whether outdoor access of breeding pigs farmed under controlled housing conditions can represent a risk for Trichinella sp. transmission when the farm is located in an agricultural area interspersed with wooded areas and badlands, where Trichinella spp. could be present in wildlife. Methods Serum samples were collected from 63 breeding sows and one boar before and after their access to an open fenced area for 2 months and from 84 pigs that never had outdoor access. Samples were screened for anti-Trichinella antibodies by ELISA, and positive sera were confirmed using Western blot (Wb) excretory/secretory antigens. To detect Trichinella sp. larvae, muscle tissues from serologically positive and negative pigs were tested by artificial digestion. Results Thirteen (20.6%) sows and one boar tested positive with both ELISA and Wb. No larvae were detected in muscle samples of serologically positive and serologically negative pigs. Positive serum samples were then tested by Wb using crude worm extract as antigens. The Wb banding pattern displayed was that characteristic of encapsulated species (Trichinella spiralis or Trichinella britovi). Conclusions The detection of anti-Trichinella antibodies without larvae in the pig muscles, supported by epidemiological data, suggests that pigs may have been exposed to T. britovi. This study stresses the importance of instigating monitoring systems at farm level to prevent Trichinella sp. transmission and to investigate, through a landscape parasitological study, the suitability of a site before the planting of a high containment level pig farm in which the sows can have outside access to improve their welfare during pregnancy. Graphical abstract

Background Domesticated and wild swine play an important role as reservoir hosts of Trichinella spp. and a source of infection for humans. Little is known about the survival of Trichinella larvae in muscles and the duration of anti-Trichinella antibodies in pigs with long-lasting infections. Methods Sixty pigs were divided into three groups of 20 animals and infected with 10,000 larvae of Trichinella spiralis, Trichinella britovi or Trichinella pseudospiralis. Four pigs from each group were sacrificed at 2, 6, 12, 18 and 24 months post-infection (p.i.) and the number of larvae per gram (LPG) of muscles was calculated. Serum samples were tested by ELISA and western blot using excretory/secretory (ES) and crude antigens. Results Trichinella spiralis showed the highest infectivity and immunogenicity in pigs and larvae survived in pig muscles for up to 2 years p.i. In these pigs, the IgG level significantly increased at 30 days p.i. and reached a peak at about 60 days p.i., remaining stable until the end of the experiment. In T. britovi-infected pigs, LPG was about 70 times lower than for T. spiralis at 2 months p.i. and only very few infecting larvae were detected at 6 months p.i., whereas no larvae were detected at 12, 18 and 24 months p.i. At 6 months p.i., degenerated/calcified larvae and cysts were detected in the muscles by trichinoscopy and histology. The IgG pattern showed by T. britovi-infected pigs was similar to that of T. spiralis-infected pigs, although seroconversion occurred some days later. The larval burden of T. pseudospiralis was slightly greater than for T. britovi at 2 months p.i., but no larvae were detected at 6 and 12 months p.i. In T. pseudospiralis-infected pigs, seroconversion occurred slowly, as in T. britovi-infected pigs. The IgG level showed a significant drop at 6 months p.i. and declining to the cut-off value at 12 months p.i. Conclusions The longer survival of T. spiralis in pigs in comparison with the other two species highlights its exceptional dissemination potential. These results provide an explanation of the controversial data collected by parasitological and serological tools in the course of epidemiological investigations.

We undertook the first study systematically evaluating the risk of Anisakis-sensitization in Croatian fish-processing workers and potential genetic susceptibility to anisakiasis. Anti-Anisakis IgE seroprevalence and risk factors for 600 employees of Croatian fish processing facilities and 466 blood donor controls, were assessed by indirect ELISA targeted with: recombinant Ani s 1 and Ani s 7 allergens, an Anisakis crude extract, the commercial ImmunoCAP kit, and questionnaires. Genetic susceptibility to anisakiasis was evaluated by genotypisation of human leukocytes alleles (HLA). Anti-Anisakis seropositive and a fraction of negative subjects were also assessed by ELISA and Western Blot (WB) for IgG seroprevalence to Trichinella spp. Overall, the observed anti-Anisakis seroprevalence inferred by indirect ELISA was significantly higher in fish processing workers (1.8%, 95% CI 0.9–3.3%) compared to the controls (0%, 0–0.8%). Seven out of 11 Ani s 1 and Ani s 7-positives and none of selected 65 negative sera, tested positive on whole-Anisakis extract (ImmunoCAP), whereas Anisakis crude extract ELISA detected 3.9% (2.4–6.0%) seropositives in fish processing workers, three (14%) of which showed IgE reactivity to milk proteins. The highest risk associated with Anisakis-sensitization among workers was fishing in the free time, rather than any of attributes related to the occupational exposure. Although no association was observed between anti-Anisakis seropositivity and wearing gloves or protective goggles, the majority of workers (92%) wore protective gloves, minimizing the risk for Anisakis sensitization via skin contact. Six HLA alleles within DRB1 gene were significantly associated with seropositivity under dominant, allelic or recessive models. All sera confirmed negative for anti-Trichinella spp. IgG. The study exhaustively covered almost all marine fish processing workers in Croatia, reflecting real-time Anisakis sensitization status within the industry, already under the influence of wide array of allergens., Author summary Anisakiasis is a human disease caused by ingestion of live Anisakis spp. larvae by raw seafood. Fish processing and aquaculture workers (approximately 60 million people world-wide) are at occupational risk of becoming sensitized to Anisakis. This is the first study systematically evaluating the risk of Anisakis-sensitization in Croatian fish-processing workers and potential genetic susceptibility to anisakiasis in the analysed population. Observed seroprevalence was significantly higher in fish processing workers (1.8%) compared to the controls (0%). Surprisingly, the highest risk associated with Anisakis-sensitization among workers was fishing in their free time, rather than any of attributes related to the occupational exposure. While no association was observed between anti-Anisakis seropositivity and wearing gloves or protective goggles, the majority of workers (92%) wore protective gloves, minimizing the risk for Anisakis sensitization via skin contact. Additionally, seroprevalence to another important food-borne helminth, Trichinella spp. assessed in all Anisakis-positive subjects showed to be negative. Almost all marine fish processing workers in Croatia were part of this research, reflecting real-time Anisakis-sensitization status within the industry. This sets a baseline for the future tracking of sensitization in the sector already under the influence of wide array of allergens.

Trichinellosis is a zoonotic disease due to the ingestion of raw or undercooked meat from animals infected with the larvae of nematodes belonging to the genus Trichinella. In January–February 2015, an outbreak of trichinellosis occurred in Genoa, Northern Italy. The epidemiological link was traced back to a dinner served at an agritourism farm on 31 December 2014, where a majority of the 52 guests had consumed the ‘beef’ steak tartare. The source of infection was not traced; however, it was noted that the amount of beef purchased officially for providing at the dinner did not correspond with that served, suggesting that meat of a different origin had been added to the beef to prepare the steak tartare. Clinical and laboratory data of 30 individuals out of the 52 (57.7%), of which four were hospitalized, were consistent with that of the case definition of trichinellosis. Western blot patterns of the sera from patients with confirmed trichinellosis were similar to the diagnostic pattern identified for the reference sera of Trichinella pseudospiralis but different from those of the control sera tested for patients infected with Trichinella spiralis and Trichinella britovi. Identification of T. pseudospiralis as the aetiological agent responsible for the outbreak of trichinellosis using an indirect tool represents an advancement in the epidemiological investigation of this zoonotic disease.

• Zoonotic nematodes of the genus Trichinella are known to occur in countries bordering the Mediterranean Sea. For example, Trichinella spiralis was reported in pigs and humans at the turn of the Second World War in Sicily and Trichinella britovi in pigs, red foxes and dogs from Corsica and Sardinia since 2004. Following the discovery of T. britovi in Corsica and Sardinia, a question arose as to whether this species was also present in Sicily which is located only 3.14 km from continental Italy. To address this query, we investigated the presence of anti-Trichinella antibodies in the serum of Nebrodi black pigs, a breed that is bred in the wild in the Nebrodi Regional Park, a protected area of the island rich in flora and fauna. Blood samples were collected from 112 Nebrodi black pigs from five farms. Sera were tested by ELISA and ELISA positive sera were confirmed by Western blot (Wb) using excretory/secretory antigens. Eighteen (16.1%) serum samples belonging to 17 fattening pigs and 1 boar tested positive by Wb. Positive sera tested by Wb using crude worm extract antigens (CWE), displayed a banding pattern similar to the CWE-Wb pattern of T. spiralis and T. britovi reference pig sera but different to that of T. pseudospiralis reference pig sera. No larvae were detected in muscles of serologically positive pigs by artificial digestion. The presence of anti-Trichinella antibodies in the absence of larvae in the muscles, suggests that the pigs were infected with T. britovi and not T. spiralis whose larvae survive in the muscles for at least two years. These results suggest that T. britovi is circulating in Nebrodi Regional Park in Sicily. ELISA testing may constitute a suitable tool for large-scale screening of Trichinella spp. infection in free-ranging pigs, when ELISA-positive sera are confirmed by Wb. Free-ranging animals can act as sentinels for the presence of zoonotic nematodes of the genus Trichinella in wildlife.

Background Cryptosporidium parvum is a major cause of diarrhea in children and ruminants at the earliest stages of life. Maternal antibodies represent the main shield of neonate mammals for most of the infections. Two recombinant antigens (SA35 and SA40), portions of two C. parvum proteins, were tested for their ability to induce immune responses in adult mice and for protection on neonate BALB/c mice born from females immunised by mucosal delivery of both peptides. Methods Adult BALB/c mice were intraperitoneally immunised with SA35 and SA40, separately or mixed, and their immune response was characterised. Furthermore, BALB/c pregnant mice were immunised by mucosal delivery with an SA35/40 mix, before and during pregnancy. Soon after birth, their offspring were infected with two doses (1 × 105 and 5 × 103) of C. parvum oocysts and the parasitic burden was determined at 5 and 9 days post-infection. Results Intraperitoneal immunisation with SA35 and SA40 induced specific IgG and IgG1 in serum, specific IgA in the intestinal mucosa, increase of CD3+/CD4+ and CD30+ cells in splenocytes, which produced IFN-γ. Neonates born from immunised mice and infected with 1 × 105 oocysts showed a significant reduction of oocysts and intestinal forms (23 and 42%, respectively). A reduction of all parasitic forms (96%; P

Background Trichinellosis is a meat-borne zoonotic disease caused by parasites of the genus Trichinella. To date, 12 taxa have been described. The identification of Trichinella species is crucial in order to identify the possible source of infection, the geographical origin of the parasite and to assess risk of infection for domestic pigs and humans. Specific identification of the etiological agent is not always feasible using direct methods since the source of infection can be untraceable. The aim of this study was to develop a diagnostic tool to infer the causative Trichinella species using western blot patterns of sera derived from infected animal and human hosts. Methods Sera from mice experimentally infected with Trichinella spiralis, Trichinella britovi, Trichinella pseudospiralis and Trichinella papuae were tested by western blot using homologous and heterologous crude worm extracts (CWE) and a highly sensitive detection system based on chemiluminescence. In addition, sera from pigs experimentally infected with T. spiralis, T. britovi and T. pseudospiralis and from patients with confirmed T. spiralis, T. britovi and T. pseudospiralis infections, were also included. Results Sera from mice infected with one Trichinella species reacted with CWE proteins from all four investigated species. Likewise, sera derived from pigs and humans infected with one Trichinella species reacted with CWE proteins from all the three investigated species. Using T. spiralis CWE, sera from T. pseudospiralis-infected hosts yielded a characteristic pattern of reactivity using Wb, which differed to that produced by T. spiralis/T. britovi- or T. papuae-infected host sera. Conclusions The present study suggests that western blot using T. spiralis CWE may be a useful tool to distinguish Trichinella infections caused by T. pseudospiralis from those caused by T. spiralis or T. britovi. This method may support epidemiological investigations, particularly when the source of infection is not traceable. Electronic supplementary material The online version of this article (10.1186/s13071-018-3244-3) contains supplementary material, which is available to authorized users.

To determine the reproducibility and robustness of an ELISA to detect anti-Trichinella IgG in pig sera which was previously validated at the Community Reference Laboratory for Parasites (CRLP), a ring trial was organized involving European and extra-European reference laboratories for Trichinella. The sensitivity and specificity of the assay determined by the CRLP validation resulted to be 100% and 98.29%, respectively. The assay was reproducible, moreover, based on the receiver-operator characteristic (ROC) curve, the sensitivity and specificity of the assay reached 97.5% and 96.9%, respectively. The analysis of the differences in optical density (OD) between duplicates indicated a high repeatability of the ELISA with about 95% of the differences between −0.16 and 0.17 absorbance units. The accuracy of the test was determined by calculating the area under the ROC curve (AUC). Overall, the ELISA index (IE) showed a very high accuracy (AUC = 0.9965) and it performed significantly better than the mean of the duplicated ODs (AUC = 0.9387). Of the 21 participating laboratories, nine performed the test without any modification of the original protocol, and 14 with some modifications. Of the laboratories that followed the protocol exactly, three produced false-negatives; whereas of the laboratories that modified the protocol, five produced false-negatives (differences between these two groups of laboratories were not significant, p = 0.18). When comparing these two groups of laboratories, the AUCs were very similar (0.9988 and 0.9955, respectively). Finally, a normal mixed multiple model effect was used to evaluate if the IE obtained was only related to the serum or to other parameters such as the laboratory, dilution of the serum tested and application of the proposed protocol. The variability found in the test results was mainly due to the serum samples. The assay proposed is robust and reproducible and can be used for monitoring the lack of Trichinella infection in domestic pigs.

Background Nematode parasites of the genus Trichinella are important foodborne pathogens transmitted by ingestion of striated muscles harbouring infective larvae. Wild carnivorous and omnivorous animals are the most important reservoirs of these parasites. Hunting activities play an important role in Trichinella spp. epidemiology. The aim of the present work was to assess if serological detection of anti-Trichinella IgG in hunting dogs can be a tool to indirectly monitor Trichinella spp. infections in wildlife. Methods An ELISA and a Western blot (Wb) were developed and validated. To validate the assays, serum samples were collected from 598 dogs considered to be Trichinella-free, 15 naturally infected dogs, and six experimentally infected foxes. Sera were tested by ELISA with Trichinella spiralis excretory/secretory antigens. The diagnostic sensitivity and specificity of ELISA were 100 % (95 % CI: 83.89–100 %) and 95.65 % (95 % CI: 93.69–97.14 %), respectively. Sera from Trichinella-infected dogs/foxes tested by Wb showed a three-band pattern ranging from 48 to 72 kDa. Since the prevalence of Toxocara canis is very high in dogs, the specificity of the ELISA and Wb was further assessed by testing sera for anti-T. canis IgG using T. canis excretory/secretory antigens. No cross-reactivity was observed. To evaluate the test’s reliability in the field, serum samples were collected from wild boar hunting dogs from Central Italy where Trichinella britovi was circulating among wildlife. Results Out of 384 hunting dog sera, 189 (49.2 %) tested positive by ELISA and of these, 56 (29.6 %) tested positive by Wb, showing an overall prevalence of 14.6 % (56/384) in the wild boar hunting dog population of the investigated area. The serological prevalence in hunting dogs was significantly (P

Serology to monitor Trichinella spp. infection in pigs reared in controlled system has been claimed as a possible diagnostic tool. However, no international biological standards or reference materials exist to validate in house tests or commercial kits, and to improve the inter-laboratory comparability for the serological detection of anti-Trichinella IgG in pigs. In this work, potential reference sera have been prepared from four experimentally infected pigs. Sera were tested, aliquot, lyophilized, and maintained at +4°C. Since one of the prerequisites for the development of any reference material is to plan and execute stability studies, isochronous studies for short and long term stability testing were carried out to evaluate the possible degradation effects of transportation and storage. The stability of the lyophilized serum samples at +4°C, was arbitrarily assumed. For the short term stability study, two units were stored at -20°C, +4°C, +20°C, and +50°C for 0, 1, 2, and 4 weeks, and then tested in duplicate. For the long term stability study, the same number of units and replicates per unit were stored at -80°C, -20°C, and +4°C for 0, 6, 12, 18 and 24 months. In both studies, unit samples were selected randomly and tested on the same day under repeatability conditions. The linear regression versus time for each serum at each studied temperature was analyzed and then slopes were tested for significance. Further, uncertainty of the short and long term stability was calculated for a shelf life period of one week and three years, respectively. For all sera but one, and for all the studied temperatures but +50°C, the data from the short term stability study indicate the absence of a significant trend that would hint at degradation. The slopes of the regression lines did not significantly vary from zero. Even if the uncertainty of the short term stability was variable among serum samples, the rate of degradation was considered acceptable. For the long term stability, slopes of the regression lines of two serum samples significantly varied from zero, indicating a trend of possible degradation during storage. The percentage of degradation deducted from the uncertainty of the long term study varied; however, two serum samples showed the lower rate of degradation at all the assayed temperatures. The most suitable temperatures for dispatching serum samples are -20°C, +4°C and +20°C; whereas, -20°C and -80°C are suitable temperatures for serum storage.

The ITRC is the official reference laboratory of both the International Commission on Trichinellosis (since 1988) and the International Office of Epizootics (since 1992). The ITRC was created as a repository for Trichinella strains and as a source of materials and information for international research in 1988. To date, about 900 isolates of human and animal origin from throughout the world have been examined and identified by new procedures developed at the ITRC or in collaboration with other institutions. Using material from this collection, the ITRC has provided a complete revision of the systematics of the genus Trichinella. The ITRC database can be consulted by accessing the web-site: www.simi.iss.it/trichinella/index.htm.

For 60 years, the islands of the Mediterranean basin were considered to be Trichinella-free. In April 2005, an outbreak of human trichinellosis due to the consumption of infected pork involved 11 persons in the villages of Orgosolo and Lanusei (Nuoro province) on the island of Sardinia (Italy). We conducted an investigation to identify free-range and backyard pigs and other humans with Trichinella infection in the area of the 2005 outbreak. We also tested wild animals from various parts of Sardinia. In December 2005, eight persons were found to have been infected, and in May 2007 there was a single case of infection. The sources of all infections were domestic pigs. Artificial digestion of muscle samples from 681 pigs (325 free-range and 356 backyard pigs) revealed Trichinella sp. larvae in four sows (1.2%). All larvae, including those from the consumed pork products, were identified as Trichinella britovi. All infected pigs originated from the Orgosolo municipality. None of the 6188 wild boars (Sus scrofa) or 13 red foxes (Vulpes vulpes) examined were positive for Trichinella sp., suggesting that this parasite is restricted to free-range pigs. The origin of infected animals on Sardinia remains to be determined, although it could be related to the presence of T. britovi-infected animals on the island of Corsica (France).

Trichinellosis is a zoonotic disease caused by the consumption of raw or semiraw meat from different animals harboring Trichinella larvae in their muscles. Since there are no pathognomonic signs, diagnosis can be difficult; for this reason, serology is important. The objective of this study was to validate an enzyme-linked immunosorbent assay (ELISA) using excretory/secretory antigens to detect anti- Trichinella immunoglobulin G antibodies in human sera. A total of 3,505 human serum samples were tested. A receiver-operator characteristic (ROC) curve analysis was performed. The accuracy of the test was determined by calculating the area under the curve, which was equal to 0.999, indicating high accuracy. The coefficient of variation calculated for data from four serum samples in eight working sessions was no higher than 5% for the positive sera or 14% for the negative sera. Moreover, the analysis of the differences in optical density between duplicates indicated a high repeatability for the ELISA. At the ROC optimized cutoff, the sensitivity and specificity of the test were, respectively, 99.2% and 90.6% (specificity of 95.6% when excluding the samples from multiparasitized persons from Tanzania). The validated ELISA showed good performance in terms of sensitivity, repeatability, and reproducibility, whereas the specificity was limited. These results suggest that this test is suitable for detecting anti- Trichinella antibodies in human sera for diagnostic purposes, whereas its use in epidemiological surveys could be questionable.

Domestic and sylvatic trichinellosis have frequently been documented in European regions of Russia, with the highest prevalence reported in wolves (Canis lupus). From 1998 to 2000, 75 carcasses of wolves shot by hunters were tested for Trichinella larvae, and 73 (97.3 %) of them were found to be positive. This very high prevalence of infection, the highest ever detected in a natural population of carnivores, could be explained by the human impact on the natural ecosystem. In fact, the diet of wolves living in the region under study mainly consists of carcasses of dogs and wolves, which are left in the forest or used as bait by hunters.

Since opisthorchiasis does not show pathognomonic signs or symptoms, physicians can have serious problems to make a differential diagnosis of this infection in non endemic areas, in particular when there is a simultaneous occurrence with other seasonal infections. Moreover, symptomatic infections due to O. felineus can last a few weeks and then the signs and symptoms disappear, but the worms survive in the bile ducts for years causing hepatobiliary diseases including hepatomegaly, cholangitis, fibrosis of the periportal system, cholecystitis, and gallstones. Consequently, an early diagnosis prevents chronicity and loss of working days. The detection of specific antibodies has been considered as a complementary tool to the fecal examination to establish the definitive diagnosis of this infection and for the follow up. Therefore the aim of this work was the development and validation of an enzyme-linked immunosorbent assay (ELISA) using excretory/secretory antigens (ESA) from O. felineus adult worms to detect anti-Opisthorchis IgG in human sera. A total of 370 human sera were tested: 144 sera from persons with a confirmed diagnosis of opisthorchiasis, 110 sera from healthy Italian people, and 116 sera from people with other parasitic or non-parasitic infections. Results were analyzed by receiver-operator characteristic (ROC) curve analysis. The accuracy of the test, calculated by the area under curve (AUC), yielded a 0.999 value, indicating the high performance of the test. The sensitivity was 100% (95% CI: 97.40% to 100%) and no false-negative sera were detected; the specificity was 99.09% (95% CI: 95.02% to 99.83%). The validated ELISA shows a good performance in terms of sensitivity, repeatability and reproducibility, and it is suitable to detect anti-Opisthorchis IgG in human sera for diagnostic purposes and for the follow up to assess the efficacy of drug treatment.