Edoardo Pozio’s research while affiliated with Istituto Superiore di Sanità and other places

A total of 120 muscle tissues from three horses naturally infected with Trichinella spiralis were examined. The head was the most infected site. In particular, the muscles harbouring the highest number of larvae were: musculus buccinator (12, 411 and 1183 larvae g ⁻¹ ), the tongue (11, 615 and 1749 larvae g ⁻¹ ), m. levator labii maxillaris (17, 582 and 1676 larvae g ⁻¹ ), and the masseter (4.9, 289 and 821 larvae g ⁻¹ ). Compared with the diaphragm, the number of larvae per gram was from 3.5 to 6.8 times higher in the tongue, from 3.5 to 6.5 higher in m. levator labii maxillaris , and from 2.5 to 4.6 higher in m. buccinator . Of the examined muscles, the diaphragm had from the 6th to the 15th highest level of infection (3.1, 166 and 256 larvae g ⁻¹ ). Published data from experimentally infected horses confirm these results, suggesting that efforts to detect predilection sites should focus on the head muscles.

Clonorchiasis and opisthorchiasis are helminthic diseases caused by the liver flukes Clonorchis sinensis, Opisthorchis felineus and Opisthorchis viverrini, respectively. Humans acquire these trematode infections by consuming raw or partially cooked freshwater fish infected with the larval stage metacercariae. More than 45 million people most of whom in Asia, have been estimated to be infected. These infections are prevalent in developing countries and are closely linked to poverty, pollution, and population growth, as well as to cultural food habits and tradition. However, people living in industrialised countries are not exempted to acquire these pathogens due to an increasing consumption of freshwater raw fish. Besides being the etiological agents of helminthic diseases, C. sinensis and O. viverrini have been classified as class I carcinogens, since they are the causative agents of cholangiocarcinoma in chronically infected people.KeywordsClonorchiasisopisthorchiasis Clonorchis sinensis Opisthorchis felineus Opisthorchis viverrini zoonosischolangiocarcinoma

The main reservoir hosts of nematodes of the genus Trichinella are wild carnivores, although most human infections are caused by the consumption of pork. This group of zoonotic parasites completes the entire natural life cycle within the host organism. However, there is an important phase of the cycle that has only been highlighted in recent years and which concerns the permanence of the infecting larvae in the striated muscles of the host carcasses waiting to be ingested by a new host. To survive in this unique biological niche, Trichinella spp. larvae have developed an anaerobic metabolism for their survival in rotting carcasses and, for some species, a resistance to freezing for months or years in cold regions. Climate changes with increasingly temperatures and reduction of environmental humidity lower the survival time of larvae in host carcasses. In addition, environmental changes affect the biology and ecology of the main host species, reducing their number and age composition due to natural habitat fragmentation caused by increasing human settlements, extensive monocultures, increasing number food animals, and reduction of trophic chains and biodiversity. All of these factors lead to a reduction in biological and environmental complexity that is the key to the natural host-parasite balance. In conclusion, Trichinella nematodes can be considered as an indicator of a health natural ecosystem.

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.

Trichinella spiralis has historically been deemed “the pig parasite” owing to its initial classification within a monospecific genus. However, in recent years, the genus has expanded to include 10 distinct species and at least 3 different genotypes whose taxonomic status remains unstipulated. In contrast to T. spiralis, however, most of these sylvatic species and genotypes do not infect pigs well. Inasmuch as morphological characters cannot be used to define species within this genus, earlier classifications were based upon host and geographical ranges, biological characters, and the presence or absence of a collagen capsule that surrounds the muscle stage larvae. Later, isoenzymes, DNA gel fragmentation patterns and DNA probes were used to help in identification and classification. Today, amidst the “-omics” revolution, new molecular and biochemical-based methodologies have improved detection, differentiation and characterization at all levels including worm populations. These efforts have discernably expanded immunological, epidemiological, and genetic studies resulting in better hypotheses on the evolution of the genus, and on global events, transmission cycles, host associations, and biogeographical histories that contributed to its cosmopolitan distribution. Reviews of this sort are best begun with a background on the genus; however, efforts will divert to the most recent knowledge available on the taxonomy, phylogeny, epidemiology and biochemistry that define this genus in the 21st century.

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

The scientific basis that led to the development of a multispecies concept within the Trichinella genus originated in the 1950s, when scientists began reporting an increasing number of host-specific peculiarities among different geographic isolates. This led to speculation that important geographic variability existed within Trichinella spiralis, the only species in the genus at that time. Comparative infection results sparked great interest among investigators and led to similar studies using various geographic isolates of the parasite. In 1972, the Russian scientists V.A. Britov, S.N. Boev and B.L. Garkavi, described three new species: Trichinella nativa, Trichinella nelsoni and Trichinella pseudospiralis. This shattered the concept that the genus Trichinella was monospecific and widened the host range to include birds. The description of these new species generated an intense debate over their taxonomic validity because there were no clear morphological differences among them and because the concept of sibling species had not yet been accepted by parasitologists. The resolution of the taxonomic issues was facilitated by the adoption of new biochemical and molecular techniques for systematics research. In 1992, the first study comparing 152 isolates from various host species and geographical regions identified eight distinct taxa, coded T1 through T8; four of these represented the previously proposed species and included one new species, Trichinella britovi (T3). During the past 27 years, an increasing number of investigations in different geographical regions and hosts coupled with the availability of new and highly sensitive molecular techniques have allowed the description of four new species; Trichinella murrelli (T5), Trichinella papuae (T10), Trichinella zimbabwensis (T11) and Trichinella patagoniensis (T12), and two new genotypes Trichinella T9 and T13. Thus, the taxonomic status of Trichinella T6, T8, T9 and T13 remain unresolved. These new technologies have also advanced a more complete phylogenetic, zoogeographical and epidemiological knowledge base for future work.

Foodborne parasites, most of which are zoonotic, represent an important human health hazard. These pathogens which include both protozoa (e.g., Cryptosporidium spp., Cyclospora cayetanensis, Toxoplasma gondii) and helminths (e.g., liver and intestinal flukes, Fasciola spp., Paragonimus spp., Echinococcus spp., Taenia spp., Angiostrongylus spp., Anisakis spp., Ascaris spp., Capillaria spp., Toxocara spp., Trichinella spp., Trichostrongylus spp.), have accompanied the human species since its origin and their spread has often increased due to their behavior. Since both domesticated and wild animals play an important role as reservoirs of these pathogens the increase/decrease of their biomasses, migration, and passive introduction by humans can change their epidemiological patterns. It follows that globalization and climate change will have a tremendous impact on these pathogens modifying their epidemiological patterns and ecosystems due to the changes of biotic and abiotic parameters. The consequences of these changes on foodborne parasites cannot be foreseen as a whole due to their complexity, but it is important that biologists, epidemiologists, physicians and veterinarians evaluate/address the problem within a one health approach. This opinion, based on the author's experience of over 40 years in the parasitology field, takes into consideration the direct and indirect effects on the transmission of foodborne parasites to humans.

This study identified helminth species of wild boar ( Sus scrofa ) originating from northeastern and northwestern regions of Tunisia using 297 lungs, 297 livers, 264 intestinal tracts, 120 samples of muscle tissue (tongue, masseter, diaphragm, inter-costal) and 232 faecal samples derived from a total of 591 animals. Host gender was registered for the lung and liver wild boar group, which included 163 males and 134 females. All animals, excluding those used to retrieve muscular samples, were classified into three age classes, <2 ( n = 212), 2–3 ( n = 208) and ⩾4 years old ( n = 141). Helminth fauna of the examined wild boar included 14 parasite species: one trematode (adult, Brachylaemus suis ), three cestodes (metacestodes of Echinococcus granulosus , Taenia hydatigena cysticercus, adult, Hymenolepis diminuta ), nine nematodes (adults of Metastrongylus apri , Metastrongylus pudendotectus , Ascarops strongylina , Globocephalus urosubulatus , Physocephalus sexalatus , Gnathostoma hispidum , Gongylonema pulchrum and eggs of Strongyloides ransoni and Capillaria spp.) and one acanthocephalan (adult, Macracanthorhynchus hirudinaceus ). Trichinella larvae were not recovered from any of the 30 wild boar examined. Results showed a 73.5% global prevalence of infection with visceral helminths, 67.3% of which were lung and hepatic infections and 80.3% of helminths were recovered from the gastrointestinal tract. The most prevalent parasite was M. hirudinaceus (61.7%) while the highest intensity of infection was observed for Metastrongylus spp. The most prevalent cestode was E. granulosus (18.9%). This is the first detailed study on helminth infections of wild boar from a North African country.

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.