Search

Human trichinellosis is a disease caused by nematode worms of the genus Trichinella. In Italy, as well as in most other European countries, notification of Trichinella infections in humans is mandatory; however, no information is available on the number of cases occurring annually. The aim of the present study was to retrospectively evaluate the burden of trichinellosis in Italy from 2005 to 2016. Hospital discharge records (HDRs) showing the code for trichinellosis (124) were registered and screened. Results were then compared with yearly reports issued by the Italian National Reference Laboratory for Trichinella (NRLT), with reports from the European Centre for Disease Prevention and Control (ECDC), and with literature data. A total of 102 HDRs revealed that the 124 code was erroneously reported in 72 (70.6%) records. Out of the 30 (29.4%) records with a correct diagnosis of trichinellosis, nine cases were reported by HDRs only, 21 cases were documented by both HDRs and the NRLT, whereas the NRLT documented 100 additional cases. In the studied period, the average yearly incidence was 0.01 cases per 100,000 inhabitants. This study highlights the limitations of using HDRs to obtain a clear picture of the prevalence and incidence of trichinellosis in Italy. These findings demonstrate the need to intensify the surveillance system for trichinellosis through the development of an Italian registry. This would allow the identification of patients with severe infections and pauci-symptomatic patients, and would avoid the need for clinical analyses and unnecessary treatments, reducing the resulting economic burden on the Italian National Health Service.

A preliminary survey of Trichinella spp. infection was conducted in Colombian swine herds between 2014 and 2016. A total of 1,773 pigs reared on farms under controlled housing conditions and processed in 34 slaughterhouses were tested either by the artificial digestion of pooled muscle samples (n = 1,173) or by serology (n = 600). In addition, 550 rats trapped on 29 swine farm premises were also tested by artificial digestion. No positive pig samples were detected. Similarly, no Trichinella spp. muscle larvae were detected in rats. These results are in agreement with the lack of historical Trichinella infection reports in domestic and wild animals and humans in Colombia. However, a more extensive epidemiological investigation and a continuous surveillance program are needed to continue declaring swine herds in Colombia free of Trichinella infection.

Purpose: Opisthorchis felineus is a trematode causing a foodborne infection transmitted by raw freshwater fish belonging to Cyprinidae family. Human outbreaks in Italy dated back to 2003-2011 and involved lakes of Central Italy. The aim of this study is to report epidemiological and clinical characteristics of the human opisthorchiasis outbreak occurred in Central Italy in 2022 comparing it with previous events., Methods: We report cases diagnosed from June to December 2022 in Perugia hospital thanks to serological and molecular tests and direct examination of feces., Results: Sixty-seven individuals were traced back by epidemiological investigation. Forty-seven received a diagnosis of opisthorchiasis, of which 45 were confirmed cases and two were considered as probable cases. These 47 individuals attended a Trasimeno lakeshore restaurant in May 2022. All but 20 presented symptoms, mostly fever. Sixteen (15 confirmed and 1 probable) cases required hospitalization. Feces examination revealed Opisthorchis spp. eggs in 35/45 (78%) confirmed cases. Thirty individuals underwent to serology and molecular stool test: 5 (16.7%) results positive to the former, 1 (3.3%) to the latter while 4 (13.3%) to both. Laboratory tests, available in 28 patients, showed eosinophilia in 82.1%, increase of alanine aminotransferase, gamma-glutamyl transferase and alkaline phosphatase in 64.3%, 75% and 67.9%, respectively. Because of pharmacy shortage of praziquantel, 22 patients were treated with albendazole, of which 13 failed clearing the parasite., Conclusion: Opisthorchiasis still represents a challenging diagnosis, in particular for asymptomatic patients. Albendazole may lead to treatment failure. Control measures in known endemic areas should be implemented., Trial Registration: number 27,498/23/ON, approved by Ethical Committee of Umbrian Region in 09.13.2023., (© 2024. The Author(s).)

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., (© 2021. The Author(s).)

Human trichinellosis can be diagnosed by a combination of medical history, clinical presentation, and laboratory findings, and through detection of anti-Trichinella IgG in the patient's sera. ELISA using excretory-secretory (E/S) antigens of Trichinella spiralis larvae is currently the most used assay to detect Trichinella spp. antibodies. Bead-based assay can detect antibodies to multiple antigens concurrently; the ability to detect antibody to T. spiralis using a bead assay could be useful for diagnosis and surveillance. We developed and evaluated a bead assay to detect and quantify total IgG or IgG4 Trichinella spp. antibodies in human serum using T. spiralis E/S antigens. The sensitivity and specificity of the assay were determined using serum from 110 subjects with a confirmed diagnosis of trichinellosis, 140 subjects with confirmed infections with other tissue-dwelling parasites, 98 human serum samples from residents of the United States with no known history of parasitic infection, and nine human serum samples from residents of Egypt with negative microscopy for intestinal parasites. Sensitivity and specificity were 93.6% and 94.3% for total IgG and 89.2% and 99.2% for IgG4, respectively. Twelve percent of sera from patients with confirmed schistosomiasis reacted with the IgG Trichinella bead assay, as did 11% of sera from patients with neurocysticercosis. The Trichinella spp. bead assay to detect IgG total antibody responses has a similar performance as the Trichinella E/S ELISA. The Trichinella spp. bead assay shows promise as a method to detect trichinellosis with a possibility to be used in multiplex applications.

In 2012, WHO/FAO ranked 24 foodborne parasites (FBP) using multicriteria decision analysis (MCDA) to provide risk assessors with a basis for prioritising control of highly ranked FBP on the global level. One conclusion was that ranking may differ substantially per region. In Europe, the same methodology was used to rank FBP of relevance for Europe. Of the 24 FBP, the top-five prioritised FBP were identified for Europe as Echinococcus multilocularis , Toxoplasma gondii , Trichinella spiralis , E. granulosus , and Cryptosporidium spp., all of which are zoonotic. The objective of the present study was to provide an overview of surveillance and reporting systems in Europe for these top five prioritised FBP in the human and animal populations, to identify gaps, and give recommendations for improvement. Information on the surveillance systems was collected from 35 European countries and analysed according to the five different regions. For most FBP, human surveillance is passive in most countries and regions in Europe and notification differs between countries and regions. Adequate surveillance programmes for these FBP are lacking, except for T. spiralis , which is notifiable in 34 countries with active surveillance in susceptible animals under EU directive. Although human and animal surveillance data are available for the five prioritised FBP, we identified a lack of consistency in surveillance and reporting requirements between national experts and European bodies. Recommendations for improved surveillance systems are discussed., Competing Interests: Authors declare that they have no conflict of interest., (© 2021 The Authors. Published by Elsevier Ltd on behalf of World Federation of Parasitologists.)

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., (© 2020 The Authors. Zoonoses and Public Health published by Wiley-VCH GmbH.)

Herbivores can be accidental hosts for the zoonotic nematode parasites Trichinella spp., which are endemic at high prevalence in wildlife in northeastern Europe. Using direct and indirect detection methods for Trichinella spp., we investigated samples from 463 wild moose (Alces alces) harvested by hunters in Estonia in 2015. A total of 460 moose were tested directly by artificial digestion of diaphragm muscle, 463 moose were tested indirectly by enzyme-linked immunosorbent assay (ELISA), and 34 moose also by western blot. Positive-control reference sera were from other host species. Nematode larvae were found in six muscle samples; five of which were pooled samples. None of the larvae were identified as Trichinella spp., based on their morphology and molecular analyses. Twelve moose (2.6%) were positive by ELISA, but none were positive by the western blot test. Trichinella spp. infection was not detected, but ELISA results may suggest Trichinella spp. exposure in a small proportion of moose in Estonia., (© Wildlife Disease Association 2021.)

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.

Objectives: To report on relevant national surveillance systems of (N)CC and taeniasis (the infection with the adult tapeworm) in the European Union/European Economic Area and to assess the magnitude of (N)CC occurrence by retrieving information on cases for the period 2000-2016., Methods: (N)CC cases were retrieved via national reporting systems, a systematic literature search, contact with clinicians and a search for relevant 'International Statistical Classification of Diseases and Related Health Problems' (ICD)-based data., Results: Mandatory notification systems for (N)CC were found in Hungary, Iceland and Poland. Ten cases were reported in Poland and none in Hungary and Iceland. Through the systematic literature review and information given by clinicians, 263 individual and 721 aggregated (N)CC cases from 19 European countries were identified. ICD-based data were obtained from five countries. From 2000 to 2016, a total of 3489 cases (N)CC cases were coded: 832 in Italy, eight in Latvia, 357 in Portugal, 2116 in Spain and 176 in Sweden., Conclusion: Despite being classified as a possible eradicable disease, (N)CC is still diagnosed across Europe, yet its true extent and impact remain unclear., (© 2020 The Authors Tropical Medicine & International Health Published by John Wiley & Sons Ltd.)

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 × 10 5 and 5 × 10 3 ) 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 × 10 5 oocysts showed a significant reduction of oocysts and intestinal forms (23 and 42%, respectively). A reduction of all parasitic forms (96%; P < 0.05) was observed when neonates were infected with 5 × 10 3 oocysts., Conclusions: SA35 and SA40 peptides induce specific humoral and cell-mediated immune responses to C. parvum in adult mice. Moreover, mucosal administration of the SA35/40 mix in pregnant mice reduces C. parvum burden in their litters.

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.

Serological methods are widely used for detection of infections in animals and humans. The recommendations provided here take into account the best current methods for the serological detection of Trichinella infection. They are based on current scientific information including unpublished data from laboratories with relevant expertise in this field. These recommendations represent the official position of the International Commission on Trichinellosis (ICT) regarding acceptable methods for the use and interpretation of serology testing for Trichinella infection in animals and humans. The ICT does not recommend use of serological methods for testing individual carcasses of animals at slaughter for assuring food safety. For detection of human infections, for epidemiological studies in animals and humans, and for monitoring Trichinella infection in swine, the ICT recommends ELISA using excretory/secretory (ES) antigens. These antigens are obtained from the in-vitro maintenance of Trichinella spiralis muscle larvae and are recognized by sera from hosts infected by all Trichinella species and genotypes identified thus far. In most situations, positive results obtained by ELISA should be confirmed by western blot. Serological assays should be properly standardized and validated for their intended purpose. The components of the test that are critical for maintaining suitable performance should be identified and appropriately checked. Users of commercial tests should verify that the test has been adequately evaluated by an independent body. Serology is useful for detecting Trichinella in animals and humans but its limitations need to be taken into account when interpreting the results., (© 2019 The Authors.)

Background: Toxoplasmosis is one of the most frequent parasitic infections in animals causing reproductive disorders and thus notable economic losses in productivity. Among food animals, pigs along with sheep and goats possess the highest incidence of Toxoplasma gondii cysts in meat, and play a role as a source of human infection., Methods: The commercial ELISA kit (PrioCHECK® Toxoplasma Ab SR, Prionics Schlieren-Zurich, Switzerland) for the detection of specific antibodies against T. gondii in swine serum was compared with a commercial IFAT (indirect fluorescent antibody test) (Toxo-Spot IF, bioMérieux, France), used as the reference test., Results: The kappa value obtained comparing the results performed on sera by ELISA with the by IFAT was 1. By a receiver operating characteristics curve analysis, the commercial ELISA had a relative sensitivity of 100%, and a relative specificity of 100% respect to IFAT., Conclusions: The commercial ELISA showed a very good agreement with the commercial IFAT in the detection of serum antibodies to Toxoplasma in pigs. Our study confirmed the usefulness of the commercial ELISA kit for the detection of antibodies against T. gondii in pigs, representing a valuable tool to improve the diagnostic activity for T. gondii in swine populations at the farm level or at the slaughterhouse, contributing to the control of this widespread infection.

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.

Human cysticercosis (CC) is a parasitic zoonosis caused by the larval stage (cyst) of the Taenia solium. Cysts can establish in the human central nervous system (neurocysticercosis, NCC) and other organs and tissues; they also develop in pigs, the natural intermediate host. Human taeniosis may be caused by T. solium, Taenia saginata and Taenia asiatica tapeworms; these infections are usually asymptomatic, but show a significant relevance as they perpetuate the parasites' life cycle, and, in the case of T. solium, they are the origin of (N)CC. In European Union (EU) member states and associated countries, the occurrence of autochthonous T. solium cases is debated, and imported cases have significantly increased lately; the status of T. asiatica has been never reported, whereas T. saginata is prevalent and causes an economic impact due to condemned carcasses. Based on their effects on the EU society, the specific diagnosis of these pathologies is relevant for their prevention and control. The aims of this study were to know the diagnostic tests used in European laboratories for human taeniosis/cysticercosis by means of a questionnaire, to determine potential gaps in their detection, and to obtain preliminary data on the number of diagnosed taeniosis/CC cases.

Background: Anisakis simplex is the only fishery-product associated parasite causing clinical allergic responses in humans so far. However, other anisakids, due to the presence of shared or own allergens, could also lead to allergic reactions after sensitization. The aim of this study was to determine if Pseudoterranova decipiens belonging to the family Anisakidae has allergenic activity and is able to induce sensitization after oral administration in a murine (BALB/c mice) model., Results: The ingestion of A. pegreffii proteins by BALB/c mice, which had been previously sensitized by intraperitoneal inoculation with the corresponding live L3 larvae, triggers signs of allergy within 60 min, whereas P. decipiens did to a lesser extent. Beside symptoms, allergic reactions were furtherly supported by the presence of histamine in sera of sensitized mice. Specific IgG1 and IgE responses were detected in sera of all sensitized mice from week four. Specific IgG2a response was detected in sera from mice sensitized to P. decipiens. After polyclonal or specific activation with anti-CD3/anti-CD28 or antigens, respectively, splenocytes from mice infected i.p. with A. pegreffii or P. decipiens larvae showed significantly higher production of IL-10 than naïve mice. After stimulation with specific antigens, significantly higher IL-5 and IL-13 amounts were produced by specific antigen stimulated splenocytes than by the naïve cells; only P. decipiens proteins induced IFN-ɣ., Conclusions: The overall results suggest that infection with P. decipiens can sensitize mice to react to subsequent oral challenge with anisakid proteins, as described for A. simplex (sensu stricto) and A. pegreffii infections. The results show that anisakid proteins induce a dominant Th2 response, although P. decipiens could also induce a mixed type 1/type 2 pattern.

Trichinella spp. are relevant zoonotic pathogens in Estonia. The aim of this nationwide cross-sectional study was to estimate the seroprevalence of Trichinella spp. in domestic pigs (Sus scrofa domestica) and hunted wild boars (Sus scrofa). Serum samples from 374 pigs, originating from 14 farms, and meat juice samples from 470 wild boars were tested for immunoglobulin G antibodies against Trichinella excretory/secretory antigens using a commercial enzyme-linked immunosorbent assay (ELISA). Antibodies against Trichinella were not detected in the domestic pigs, indicating effective parasite control strategies in the farms. By contrast, 42.1% of the wild boars tested positive, indicating substantial infection pressure in the sylvatic cycle. Further analysis of a subset of the wild boar samples, using another ELISA and Western blot, yielded a confirmed seroprevalence estimate of 17.4%. A substantial proportion of wild boars in Estonia had evidence of exposure to Trichinella spp. and may have carried infective larvae. Undercooked Estonian wild boar meat is a potential source of Trichinella spp. infections to humans and other hosts.

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 < 0.001) associated with the hunting district's altitude. This is in agreement with previous investigations, which had shown that the prevalence of T. britovi in wildlife was higher in mountainous areas than in lowland areas of Italy., Conclusion: The results suggest that the circulation of Trichinella spp. among wildlife can be monitored by testing sera from hunting dogs, which could act as sentinel animals of Trichinella spp. circulation in wildlife.

The wild boar is an important source of trichinellosis for people in European countries as a large number of hunted animals escape veterinary control. In November 2012, uncooked sausages made with meat from wild boar were consumed by 38 persons in a village of the Lucca province (Tuscany region, Italy). Of them, 34 were serologically positive, 32 developed clinical signs and symptoms of trichinellosis, and two were asymptomatic. Trichinella britovi larvae were detected in vacuum-packed sausages made with the same batch of sausages consumed raw which had been prepared with meat from wild boar hunted in the Lucca province. As no case of trichinellosis had been reported in this region during the last 20 years, the regional public health authority considered the risk for this zoonosis to be negligible and put in place a surveillance programme on Trichinella spp. in indicator animals (mainly foxes and including wild boar for private consumption), by testing only a percentage of heads. The experience from this outbreak shows that the definition of a region with a negligible risk for Trichinella infection is not applicable to wild boar and stresses the need to test all Trichinella-susceptible wild animals intended for human consumption and to implement risk communication to consumers and hunters., (© 2014 Blackwell Verlag GmbH.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Trichinella spp. infections in wild boar (Sus scrofa), one of the main sources of human trichinellosis, continue to represent a public health problem. The detection of Trichinella spp. larvae in muscles of wild boar by digestion can prevent the occurrence of clinical trichinellosis in humans. However, the analytical sensitivity of digestion in the detection process is dependent on the quantity of tested muscle. Consequently, large quantities of muscle have to be digested to warrant surveillance programs, or more sensitive tests need to be employed. The use of indirect detection methods, such as the ELISA to detect Trichinella spp. infections in wild boar has limitations due to its low specificity. The aim of the study was to implement serological detection of anti-Trichinella spp. antibodies in meat juices from hunted wild boar for the surveillance of Trichinella spp. infections., Methods: Two tests were used, ELISA for the initial screening test, and a specific and sensitive Western blot (Wb) as a confirmatory test. The circulation of anti-Trichinella IgG was determined in hunted wild boar muscle juice samples in 9 provinces of 5 Italian regions., Results: From 1,462 muscle fluid samples, 315 (21.5%, 95% C.I. 19.51-23.73) were tested positive by ELISA. The 315 ELISA-positive muscle fluid samples were further tested by Wb and 32 (10.1%, 95% C.I. 7.29-13.99) of these were positive with a final seroprevalence of 2.2% (95% C.I 1.55-3.07; 32/1,462). Trichinella britovi larvae were detected by artificial digestion in muscle tissues of one (0.07%, 95%C.I. 0.01-0.39) out of the 1,462 hunted wild boars. No Trichinella spp. larvae were detected in Wb-negative wild boar. From 2006 to 2012, a prevalence of 0.017% was detected by muscle digestion in wild boar hunted in the whole Italian territory., Conclusions: The combined use of both serological methods had a sensitivity 31.4 times higher than that of the digestion (32/1,462 versus 1/1,462), suggesting their potential use for the surveillance of the Trichinella spp. infection in wild boar populations.

Trichinella surveillance in wildlife relies on muscle digestion of large samples which are logistically difficult to store and transport in remote and tropical regions as well as labour-intensive to process. Serological methods such as enzyme-linked immunosorbent assays (ELISAs) offer rapid, cost-effective alternatives for surveillance but should be paired with additional tests because of the high false-positive rates encountered in wildlife. We investigated the utility of ELISAs coupled with Western blot (WB) in providing evidence of Trichinella exposure or infection in wild boar. Serum samples were collected from 673 wild boar from a high- and low-risk region for Trichinella introduction within mainland Australia, which is considered Trichinella-free. Sera were examined using both an 'in-house' and a commercially available indirect-ELISA that used excretory-secretory (E/S) antigens. Cut-off values for positive results were determined using sera from the low-risk population. All wild boar from the high-risk region (352) and 139/321 (43.3%) of the wild boar from the low-risk region were tested by artificial digestion. Testing by Western blot using E/S antigens, and a Trichinella-specific real-time PCR was also carried out on all ELISA-positive samples. The two ELISAs correctly classified all positive controls as well as one naturally infected wild boar from Gabba Island in the Torres Strait. In both the high- and low-risk populations, the ELISA results showed substantial agreement (k-value=0.66) that increased to very good (k-value=0.82) when WB-positive only samples were compared. The results of testing sera collected from the Australian mainland showed the Trichinella seroprevalence was 3.5% (95% C.I. 0.0-8.0) and 2.3% (95% C.I. 0.0-5.6) using the in-house and commercial ELISA coupled with WB respectively. These estimates were significantly higher (P<0.05) than the artificial digestion estimate of 0.0% (95% C.I. 0.0-1.1). Real-time PCR testing of muscle from seropositive animals did not detect Trichinella DNA in any mainland animals, but did reveal the presence of a second larvae-positive wild boar on Gabba Island, supporting its utility as an alternative, highly sensitive method in muscle examination. The serology results suggest Australian wildlife may have been exposed to Trichinella parasites. However, because of the possibility of non-specific reactions with other parasitic infections, more work using well-defined cohorts of positive and negative samples is required. Even if the specificity of the ELISAs is proven to be low, their ability to correctly classify the small number of true positive sera in this study indicates utility in screening wild boar populations for reactive sera which can be followed up with additional testing., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Crocodiles are known reservoir hosts of Trichinella papuae and Trichinella zimbabwensis, two zoonotic parasites that also infect mammals. Since commercial crocodile farming represents a key source of income in several countries, it is important to monitor this nematode infection in both farmed crocodiles and in breeding stocks which are frequently introduced from the wild. For this purpose, an indirect ELISA was developed to detect the anti-Trichinella immune response in crocodile sera. New Zealand rabbits were immunized with pooled sera from non-infected farmed crocodiles in the presence of Freund's complete adjuvant. The anti-crocodile serum was then conjugated with horseradish peroxidase. Serum samples from four Nile crocodiles (Crocodylus niloticus) experimentally infected with T. zimbabwensis and from four uninfected crocodiles were used to set up the ELISA. The larval burden per gram of muscle tissue was determined by muscle biopsy. The test was performed on serum samples from an additional 15 experimentally infected crocodiles as well as eight wild Nile crocodiles. Among the 19 experimentally infected crocodiles, seroconversion was observed in 11 animals. The highest antibody response was observed six weeks post infection (p.i.), but in most of these animals, antibodies were not detectable after six weeks p.i. even though live larvae were present in the muscles up to six months p.i., (Copyright © 2013 Elsevier B.V. All rights reserved.)

An antigen detection kit (Trichin-L), based on latex agglutination and developed by the Bio-Rad company was validated at five European laboratories. The validation parameters included specificity, sensitivity, robustness and reproducibility. Specificity was evaluated by testing parasite antigens from five non-Trichinella parasites in addition to the Trichinella genus. To evaluate sensitivity, 10 pork samples spiked with 1, 3, 6 or 15 Trichinella larvae were tested in each laboratory. To evaluate the robustness of the test, the solubilized antigens were maintained at room temperature and tested at different times. Reproducibility was assessed in each laboratory using 40, 100g minced pork samples, each spiked with Trichinella spiralis. The use of larval homogenates obtained from the Trichin-L kit as a template for parasite identification at the species level by a multiplex PCR, was also evaluated. The results showed a high specificity and sensitivity where solubilized antigens maintained their stability and reactivity for up to three days. Reproducibility was high, as similar results were obtained in the five laboratories. The larval homogenates obtained using the Trichin-L kit were successfully used in multiplex PCRs to identify Trichinella species., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

Trichinellosis is a zoonotic disease caused by parasites of the genus Trichinella, which have a cosmopolitan distribution. For diagnostic purposes, a confirmatory test for ELISA-positive human and pig sera such as Western blotting is required, due to the high number of ELISA false positive sera. The objective of this study was to identify the Trichinella-specific antigens most frequently recognized by sera from Trichinella-infected humans and pigs, so as to define a distinctive pattern of Trichinella infection in sera from infected hosts using Western blots which allow false positive sera to be distinguished from true positive sera. Using excretory/secretory antigens, 450 human sera were tested by Western blotting: 150 from persons with a confirmed diagnosis of trichinellosis and 300 from persons who did not have trichinellosis but who tested positive by ELISA (i.e., false positives). We also tested 210 pig sera: (i) 30 from pigs experimentally infected with Trichinella spiralis; (ii) 90 from naturally T. spiralis-infected pigs; and (iii) 90 from pigs not infected with Trichinella, as shown after artificial digestion of the diaphragm pillars, yet which tested positive by ELISA (i.e., false positives). All true positive sera (i.e., sera from persons with confirmed trichinellosis as well as sera from naturally and experimentally infected pigs), reacted with a three-band pattern ranging in size from 48-72kDa. A distinctive pattern for recognizing Trichinella spp. infections in humans and pigs by Western blots is defined; it shows a sensitivity of 100% and it allows sera from Trichinella-infected humans and pigs to be distinguished from sera from persons and pigs that were not infected with Trichinella spp. (100% specificity)., (Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Trichinellosis is a foodborne disease caused by the consumption of raw meat and raw meat-derived products from swine, horse and some game animals infected with nematode worms of the genus Trichinella. Between June 2006 and February 2011, 16 million domestic pigs and 0.22 million wild boars (Sus scrofa) were tested for Trichinella sp. in Hungary. Trichinella infection was not found in any pigs slaughtered for public consumption. Nevertheless, Trichinella spiralis was detected in four backyard pigs when trace back was done following a family outbreak. Trichinella infection was demonstrated in 17 wild boars (0.0077%). Larvae from wild boars were identified as Trichinella britovi (64.7%), T. spiralis (29.4%) and Trichinella pseudospiralis (5.9%). Although the prevalence of Trichinella sp. infection in wild boars and domestic pigs is very low, the spatial analysis reveals that the level of risk differs by region in Hungary. Most of the T. britovi infected wild boars (63.6%) were shot in the north-eastern mountain area of Hungary; whereas domestic pigs and wild boars infected with T. spiralis were detected only in the southern counties bordering Croatia and Romania. In the north-western and central counties, the prevalence of Trichinella infection seems to be negligible., (Copyright © 2011 Elsevier B.V. All rights reserved.)

There is much evidence to indicate the ability of Indinavir (IND) to reduce Cryptosporidium parvum infection in both in vitro and in vivo models. However, there are limitations to the administration of IND as such, due to its renal toxicity and the high rate of metabolism and degradation. We aimed to encapsulate IND in biodegradable poly (D,L-lactide-co-glycolide) nanoparticles (Np) and to engineer their surface by conjugation with an anti-Cryptosporidium IgG polyclonal antibody (Ab). Tetramethylrhodamine-labelled Np were loaded with IND and modified by conjugation with an Ab. The IND-loaded modified Np (Ab-TMR-IND-Np) did not show any change, as demonstrated by chemical analysis studies. Simultaneous addition of 50μM Ab-TMR-IND-Np and excysted oocysts to the cell culture resulted in complete inhibition of the infection. In C. parvum-infected cells, the extent to which the infection decreased depended on the duration of treatment with the Ab-TMR-IND-Np. The antibody-engineered Np loaded with IND were able to target C. parvum in infected cells and therefore might represent a novel therapeutic strategy against Cryptosporidium sp. infection. Moreover, the use of Np as an IND delivery device, allows the development of a more appropriate dose formulation thereby reducing the IND side effects.

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 (I(E)) 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 I(E) 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.

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.

A case of gongylonemiasis in a 31-year-old woman of Granada, Spain, is reported. The worm, clearly belonging to Gongylonema genus, could not be identified definitively as Gongylonema pulchrum, the species usually described in humans.