A T-cell diagnostic test for cystic echinococcosis based on Antigen B peptides.

Cystic echinococcosis (CE) immunodiagnosis is still imperfect. We recently set-up a whole-blood test based on the interleukin (IL)-4 response to the native Antigen B (AgB) of Echinococcus granulosus. However, AgB is encoded by a multigene family coding for five putative subunits. Therefore, the aims of this study were to analyse the IL-4 response to peptides spanning the immunodominant regions of the five AgB subunits and to evaluate the accuracy of this assay for CE diagnosis. Peptides corresponding to each subunit were combined into five pools. A pool containing all peptides was also used (total pool). IL-4 evaluated by enzyme-linked immunosorbent assay was significantly higher in patients with CE compared to those without (NO-CE subjects) when whole-blood was stimulated with AgB1 and with the total pool. Moreover, IL-4 levels in response to the total pool were significantly increased in patients with active cysts. Receiver Operator Curve analysis identified a cut-off point of 0.59 pg/mL predicting active cysts diagnosis with 71% sensitivity and 82% specificity in serology-positive CE patients. These data, if confirmed in a larger cohort, offer the opportunity to develop new diagnostic tools for CE based on a standardized source of AgB as the peptides.

T-cell clones in human trichinellosis: Evidence for a mixed Th1/Th2 response.

In humans, studies on the cellular immune response against Trichinella are scarce. Aim of this study was to characterize the cytokine profile of T cells specific for Trichinella britovi in trichinellosis patients. Peripheral blood mononuclear cells (PBMC) were obtained from five patients involved in a trichinellosis outbreak caused by T. britovi, which occurred in 2013 in Tuscany (Italy). All the patients resulted positive for Trichinella-specific IgG, IgE and presented eosinophilia. T cells were investigated for their proliferation to excretory/secretory antigens from Trichinella spiralis muscle larvae (TsES) and for their cytokine profile. A total of 284 CD4+ and 42 CD8+ T-cell clones were obtained from the TsES-specific T-cell lines from PBMC. All T-cell clones proliferated in response to mitogen. Of the 284 CD4+ T-cell clones generated from TsES-specific T-cell lines, 135 (47%) proliferated significantly to TsES; 26% CD8+ T-cell clones showed proliferation to TsES. In the series of the 135 TsES-specific CD4+ clones, 51% expressed a Th2 profile, 30% a Th0 and 19% Th1. In the series of the 11 TsES-specific CD8+ T-cell clones, 18% were Tc2, 45% Tc0 and 36% Tc1. In human trichinellosis, the cellular immune response is, during the chronic phase, mixed Th1/Th2.

Present status of laboratory diagnosis of human taeniosis/cysticercosis in Europe.

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.

The occurrence of Trichinella species in the cougar Puma concolor couguar from the state of Colorado and other regions of North and South America.

Trichinella species are zoonotic nematodes that infect wild carnivores and omnivores throughout the world. We examined the prevalence and species of Trichinella infections in cougars (Puma concolor couguar) from Colorado, United States. Tongues from cougars were examined by pepsin-HCl artificial digestion to detect Trichinella spp. larvae. The species or genotype of individual worms was identified by multiplex polymerase chain reaction (PCR). Trichinella spp. larvae were detected in 17 of 39 cougars (43.6% (28.7-59.5%)). Five of the cougars (12.8%) were infected with T. murrelli, 3 (7.7%) were infected with T. pseudospiralis, and 1 (2.6%) had Trichinella genotype T6. Trichinella spp. larvae from eight cougars were not identified at the species level, due to degraded DNA. The high prevalence of Trichinella spp. in cougars from Colorado and reports of the parasite in other populations of Puma spp. suggest that this large predator is a key mammalian reservoir.

The Italian registry of cystic echinococcosis (RIEC): the first prospective registry with a European future.

Cystic echinococcosis (CE), a worldwide zoonosis, is highly endemic in southern and eastern Europe. Its actual prevalence is unknown due to the lack of efficient reporting systems designed to take into account the particular features of the disease. Neglect of CE makes diagnosis and clinical management difficult outside referral centres, with inconsistencies in clinical practice and often unnecessary procedures carried out that have associated risks and costs. The Italian registry of CE (RIEC) is a prospective multicentre registry of CE patients seen from January 2012 in Italian health centres; data are voluntarily submitted to the registry. Its aims are to show the prevalence of CE in Italy, bring the importance of this infection to the attention of health authorities, encourage public health policies towards its control, and stimulate biological, epidemiological and clinical research on CE. From January 2012 to February 2014, a total 346 patients were enrolled in 11 centres, outnumbering national reports of many CE-endemic European countries. We discuss preliminary data and challenges of the RIEC, template for the European registry of CE, which has been implemented within the Seventh Framework Programme project HERACLES (Human cystic Echinococcosis ReseArch in CentraL and Eastern Societies) since September 2014.

Molecular differentiation of Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis by pyrosequencing.

Nematodes of the genus Trichinella which infect wildlife and domestic animals show a cosmopolitan distribution. These zoonotic parasites are the aetiological agents of a severe human disease, trichinellosis. Twelve taxa are recognized in the Trichinella genus, but they cannot be identified by morphology since they are sibling species/genotypes. For epidemiological studies, it is extremely important to identify each taxon since they have different distribution areas and host ranges. In the present study, polymerase chain reaction (PCR) amplification of the mitochondrial large subunit ribosomal RNA (lsu-RNA) gene coupled with a pyrosequencing technique was developed to distinguish among four Trichinella species: Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis. A PCR method was used to amplify the lsu-RNA of Trichinella sp. larvae in mouse muscles and single larvae collected from infected muscles by digestion. The results show that the four Trichinella species can be distinguished by using 26 nucleotides in the target region and the method is sensitive enough to identify individual larvae. The pyrosequencing provides a simple, rapid and high-throughput tool for the differentiation of Trichinella species.

Matrix metalloproteinase (MMP)-2 and MMP-9 as inflammation markers of Trichinella spiralis and Trichinella pseudospiralis infections in mice.

Trichinella spiralis and Trichinella pseudospiralis exhibit differences in the host-parasite relationship such as the inflammatory response in parasitized muscles. Several studies indicate that matrix metalloproteinases (MMPs) represent a marker of inflammation since they regulate inflammation and immunity. The aim of this study was to evaluate the serum levels of gelatinases (MMP-9 and MMP-2) in mice experimentally infected with T. spiralis or T. pseudospiralis, to elucidate the involvement of these molecules during the inflammatory response to these parasites. Gelatin zymography on SDS polyacrilamide gels was used to assess the serum levels and in situ zymography on muscle histological sections to show the gelatinase-positive cells. In T. spiralis infected mice, the total MMP-9 serum level increased 6 days post-infection whereas, the total MMP-2 serum level increased onward. A similar trend was observed in T. pseudospiralis infected mice but the MMP-9 level was lower than that detected in T. spiralis infected mice. Significant differences were also observed in MMP-2 levels between the two experimental groups. The number of gelatinase positive cells was higher in T. spiralis than in T. pseudospiralis infected muscles. We conclude that MMP-9 and MMP-2 are markers of the inflammatory response for both T. spiralis and T. pseudospiralis infections.

Trichinella britovi in the jackal Canis aureus from south-west Iran.

Trichinellosis is an important helminthic food-borne zoonosis, which is caused by nematodes of the genus Trichinella. Although, Trichinella spp. has been detected frequently in Iranian wildlife, this parasitic infection is not considered a major public health problem. This is largely because Islamic codes forbid consumption of pork meat in this country. However, knowledge about this zoonotic pathogen is important because human trichinellosis has been documented in countries where most of the population is Muslim. The aims of the present work were to investigate whether Trichinella spp. was still circulating in wildlife of the Khuzestan Province (south-west Iran) about 30 years after the first investigation, to identify the aetiological agent at the species level by molecular analyses, and to review the literature on Trichinella spp. in animals of Iran. During the winter 2009-2010, muscle samples from 32 road-killed animals (14 dogs and 18 jackals, Canis aureus) were collected. Muscle samples were digested and Trichinella sp. larvae were isolated from two jackals. The Trichinella sp. larvae have been identified as Trichinella britovi by molecular analyses. These results confirm that T. britovi is the prevalent species circulating in wild animals of Iran.

Integrating animal health surveillance and food safety: the example of Anisakis.

Nematodes of the genera Anisakis and Pseudoterranova (family Anisakidae) are zoonotic parasites for which marine mammals (e.g., whales, dolphins, porpoises, seals, sea lions, walruses) act as final hosts, and crustaceans, cephalopods and fish as intermediate and/or paratenic hosts. In humans, the ingestion of Anisakidae larvae can result in infection with live larvae, an allergic reaction to Anisakidae allergens (even when dead larvae are ingested), or both. Worldwide, more than 2000 infections are diagnosed in humans every year, yet most of the infections and allergic reactions are undiagnosed. A very high prevalence of anisakid larvae has been found in many commercially important species of fish, cephalopods and crustaceans. Preventive measures for anisakiosis focus on post-harvest handling.

A large outbreak of Opisthorchis felineus in Italy suggests that opisthorchiasis develops as a febrile eosinophilic syndrome with cholestasis rather than a hepatitis-like syndrome.

We describe the greatest Italian human acute opisthorchiasis outbreak acquired from eating raw tenches. Out of 52 people with suspected opisthorchiasis, 45 resulted in being infected. The most frequent symptoms and laboratory findings were fever, abdominal pain and eosinophilia. Seven tri-phasic computed tomography (CT) scans were done, showing multiple hypodense nodules with hyper-enhancement in the arterial phase. All patients took one day of praziquantel 25 mg/kg TID without failures. Reported symptoms suggested a febrile eosinophilic syndrome with cholestasis rather than a hepatitis-like syndrome. It seems common to find hepatic imaging alterations during acute opisthorchiasis: CT scan could be the most suitable imaging examination. Even if stool test remains the diagnostic gold standard, we found earlier positivity with the serum antibody test. Without previous freezing, the consumption of raw freshwater fish should be avoided.

The loading of labelled antibody-engineered nanoparticles with Indinavir increases its in vitro efficacy against Cryptosporidium parvum.

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.

Investigation on a focus of human trichinellosis revealed by an atypical clinical case: after wild-boar (Sus scrofa) pork consumption in northern Italy.

Trichinellosis is one of the most serious foodborne parasitic zoonoses in Europe. Wild carnivorous and omnivorous hosts are the main reservoirs of Trichinella spp. nematodes in nature. In the winter of 2008-2009, an atypical clinical case of trichinellosis occurred for the consumption of pork from a wild boar (Sus scrofa) hunted in southwestern Alps in Italy. The symptomatic individual showed delayed development of oedemas in the lower limbs and eosinophilia, which appeared three months after infection. Muscle samples harboured 3.8 larvae/g, which were identified as Trichinella britovi. During the epidemiological investigation, anti-Trichinella IgG were detected in five hunters.

Trichinella nativa in Iceland: an example of Trichinella dispersion in a frigid zone.

In most Arctic and subarctic regions, Trichinella nativa is a common zoonotic pathogen circulating among wild carnivores. The polar bear (Ursus maritimus) is one of the most important reservoirs for T. nativa in frigid zones. In Iceland, Trichinella infection has never been detected in the local wildlife, despite the presence of one of the host species, the arctic fox (Alopex lagopus). In 2008, one of two polar bears that had swum to Iceland's coast was found to have been infected with Trichinella sp. (8.5 larvae/g in the tongue, 6.8 larvae/g in the masseter and 4.4 larvae/g in the diaphragm); the larvae were identified as T. nativa. This is the second report of Trichinella infection in polar bears that reached the Icelandic coast. In the present work, we describe this case of infection and discuss the epidemiological features that have allowed T. nativa to spread in Arctic regions.

Trichinella zimbabwensis in a naturally infected mammal.

Trichinella zimbabwensis has been detected in wild and farmed Nile crocodiles (Crocodylus niloticus) and in wild monitor lizards (Varanus niloticus) of several African countries, but it has never been detected in mammals in nature, in spite of its infectivity to rodents, pigs, foxes and monkeys under laboratory conditions. The aim of this work was to describe the first detection of T. zimbabwensis in a naturally infected lion (Panthera leo) of the Kruger National Park (KNP) of South Africa. The sequence of the expansion segment V, a highly variable non-coding sequence of the large subunit ribosomal RNA of the genus Trichinella, of larvae from the lion was identical to that of larvae of T. zimbabwensis collected from a Nile crocodile originating from the same locality as the lion, suggesting a possible transmission of this parasite between mammals and reptiles. The KNP proves to be a very interesting area for parasites of the genus Trichinella since three taxa (Trichinella nelsoni, Trichinella T8 and T. zimbabwensis) circulate among the wildlife of this protected area.

Incorrect sequencing and taxon misidentification: an example in the Trichinella genus.

Molecular analyses such as polymerase chain reaction (PCR) and sequencing are very useful for taxon identification, especially when morphological characters useful for identifying taxa are lacking. However, the use of molecular tools can be the source of taxon misidentification if they are not correctly applied and the results are not critically evaluated and compared with the literature and GenBank data. We describe a case of misidentification of a taxon of the genus Trichinella due to sequencing mistakes, lack of reference material and selection of a single molecular marker. A Trichinella sp. isolate from an Iranian wild boar (Sus scrofa) was identified as belonging to the Nearctic species Trichinella murrelli, through the molecular analysis of the 5S rRNA intergenic spacer region. A successive molecular identification of the same isolate was performed by the International Trichinella Reference Centre in Rome, Italy, using the 5S rRNA intergenic spacer region, the LSU rDNA expansion segment five, and the internal transcribed spacers 1 and 2. According to these analyses, the Iranian isolate belonged to Trichinella britovi, a Palaearctic species already described in Iran.

Human trichinellosis in Hungary from 1965 to 2009.

Human trichinellosis was first documented in Hungary in 1891 and then there were an increased number of reports up to 1964 when the most severe outbreak occurred. After that, no information was available on the international literature on human trichinellosis which occurred from 1965 up to the present years. The aim of this study was to collect all the data available in Hungarian official data-sources on human trichinellosis which occurred from 1965 up to 2009 in Hungary. Furthermore, a comparative analysis was performed on the different serological tests used along the 45 years of investigation. In the period in question, 573 infections were documented in Hungary. Of them, 57 occurred in the years 1965-69, 130 in 1970-79, 302 in 1980-89, 27 in 1990-99, and 57 in 200009. The most common sources of infection were pork from backyard pigs and hunted wild boars. Sporadic cases and small family outbreaks marked the last ten years. The comparison of serological tests shows that the ELISA is a good test for the first screening, but ELISA-positive serum samples should be confirmed by western blot except for clinically clear-cut cases.

Trichinella britovi and Trichinella spiralis mixed infection in a horse from Poland.

Trichinella infections in horses continue to represent a health problem and, despite the rarity of infection, it is necessary to continue to control properly horse meat. In 2008, a 10-year-old horse imported from Poland to Italy for consumption found to have been positive at the digestion test. Both Trichinella britovi and Trichinella spiralis larvae in a proportion of 4:1 were detected in the horse muscles. This is the first report of a mixed Trichinella species infection in a horse. The epidemiological investigation revealed that the infected horse originated from a small farm about 120km from Warsaw and the horse owner had bought the horse at a horse market. The findings suggest that the horse was fed more than once with infected meat.

Validation of a Western Blot for the detection of anti-Trichinella spp. antibodies in domestic pigs.

Trichinellosis is a zoonotic disease in humans caused by Trichinella spp. According to international regulations and guidelines, serological surveillance can be used to demonstrate the absence of Trichinella spp. in a defined domestic pig population. Most enzyme-linked immunosorbent assay (ELISA) tests presently available do not yield 100% specificity, and therefore, a complementary test is needed to confirm the diagnosis of any initial ELISA seropositivity. The goal of the present study was to evaluate the sensitivity and specificity of a Western Blot assay based on somatic Trichinella spiralis muscle stage (L1) antigen using Bayesian modeling techniques. A total of 295 meat juice and serum samples from pigs negative for Trichinella larvae by artificial digestion, including 74 potentially cross-reactive sera of pigs with other nematode infections, and 93 meat juice samples from pigs infected with Trichinella larvae were included in the study. The diagnostic sensitivity and specificity of the Western Blot were ranged from 95.8% to 96.0% and from 99.5% to 99.6%, respectively. A sensitivity analysis showed that the model outcomes were hardly influenced by changes in the prior distributions, providing a high confidence in the outcomes of the models. This validation study demonstrated that the Western Blot is a suitable method to confirm samples that reacted positively in an initial ELISA.

The relationship between the presence of antibodies and direct detection of Toxoplasma gondii in slaughtered calves and cattle in four European countries.

In cattle, antibodies to Toxoplasma gondii infection are frequently detected, but evidence for the presence of T. gondii tissue cysts in cattle is limited. To study the concordance between the presence of anti-T. gondii IgG and viable tissue cysts of T. gondii in cattle, serum, liver and diaphragm samples of 167 veal calves and 235 adult cattle were collected in Italy, the Netherlands, Romania and the United Kingdom. Serum samples were tested for anti-T. gondii IgG by the modified agglutination test and p30 immunoblot. Samples from liver were analyzed by mouse bioassay and PCR after trypsin digestion. In addition, all diaphragms of cattle that had tested T. gondii-positive (either in bioassay, by PCR on trypsin-digested liver or serologically by MAT) and a selection of diaphragms from cattle that had tested negative were analyzed by magnetic capture quantitative PCR (MC-PCR). Overall, 13 animals were considered positive by a direct detection method: seven out of 151 (4.6%) by MC-PCR and six out of 385 (1.6%) by bioassay, indicating the presence of viable parasites. As cattle that tested positive in the bioassay tested negative by MC-PCR and vice-versa, these results demonstrate a lack of concordance between the presence of viable parasites in liver and the detection of T. gondii DNA in diaphragm. In addition, the probability to detect T. gondii parasites or DNA in seropositive and seronegative cattle was comparable, demonstrating that serological testing by MAT or p30 immunoblot does not provide information about the presence of T. gondii parasites or DNA in cattle and therefore is not a reliable indicator of the risk for consumers.

Spatial distribution of Trichinella britovi, T. pseudospiralis and T. spiralis in red foxes (Vulpes vulpes) in Hungary.

The red fox (Vulpes vulpes) is considered one of the main reservoir of Trichinella spp. in Europe. As limited information on Trichinella infection in wildlife of Hungary is available, 2116 red foxes, representing more than 3% of the estimated fox population of the country, were screened to detect Trichinella larvae by a digestion method. Trichinella larvae from the 35 positive foxes were identified by a multiplex PCR as Trichinella britovi (30 isolates, 85.7%), Trichinella spiralis (4 isolates, 11.4%), and Trichinella pseudospiralis (1 isolate, 2.9%). The true mean intensity of T. britovi, T. spiralis and T. pseudospiralis larvae in lower forelimb muscles was 23.6, 3.5 and 13.5larvae/g, respectively. T. spiralis was detected only in the southern and eastern regions. The non-encapsulated T. pseudospiralis was recorded for the first time in Hungary. Although the overall true prevalence of Trichinella infection in foxes was only 1.8% (95% confidence interval, CI=1.5-2.1%), the spatial analysis reveals different risk regions. In the north-eastern counties bordering Slovakia and Ukraine (21% of the Hungarian territory), the true prevalence of Trichinella infection is significantly higher than that observed in other regions (6.0%, CI=4.8-7.1%). In the southern counties bordering Croatia, Serbia and Romania (41% of the Hungarian territory), the true prevalence of Trichinella infection is moderate (1.4%, CI=1.0-1.8%). In the north-western and central counties (38% of Hungarian territory), the prevalence of Trichinella infection is significantly lower (0.2%, CI=0.1-0.4%) than that of the other regions. Based on the statistical analysis and the evaluation of epidemiological data, none of the counties can be considered free of Trichinella infection. In the past decade, Trichinella infection has been detected only in few backyard pigs, and only few wild boar-related autochthonous infections in humans were described. Nevertheless, these results highlight the need of the maintenance of a strict monitoring and control programmes on Trichinella infection in farmed and hunted animals of Hungary.