E Pozio

Istituto Superiore di Sanità, Roma, Latium, Italy

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Publications (410)976.94 Total impact

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    ABSTRACT: Trichinella spp. are zoonotic parasites transmitted to humans by the consumption of raw or insufficiently cooked meat of different animal species. The most common source of infection for humans is meat from pigs and wild boar (Sus scrofa). The aim of the present work was to evaluate the incidence of Trichinella spp. infections in wild boar hunted in Latvia over a 38 year interval (1976 to 2013). A total 120,609 wild boars were individually tested for Trichinella spp. by trichinoscopy and, in case of negativity, by artificial digestion of 25 g muscles, in the 1976-2005 period, and by artificial digestion of 25-50 g muscles in the 2006-2013 period. Trichinella spp. larvae were identified at the species level by multiplex PCR. In the study period, the overall prevalence of infected wild boar was 2.5%. Trichinella britovi was the predominant (90%) species. The incidence of Trichinella spp. infection in wild boar exhibited two different trends. From 1976 to 1987, the incidence of infected/hunted wild boar increased from 0.23% to 2.56%, then it decreased to 0.19 in 1994. Thereafter, the incidence fluctuated between 0.05% and 0.37%. A statistically significant (P < 0.05) correlation (r = 0.54; p = 0.0199) was found between the trend of Trichinella spp. incidence in hunted wild boar and the number of snow cover days from 1976 to 1993. From 1997 to 2013, the estimated wild boar population of Latvia increased by 4.9 times and the hunting bag by 9.7 times, with a stable incidence of Trichinella spp. in the population. It follows that the biomass of Trichinella spp. larvae and of T. britovi, in particular, increased. The incidence trends of Trichinella spp. in wild boar could be related to the role played by the snow in reducing the thermal shock and muscle putrefaction which increases the survival of the larvae in muscle tissues of carrion in the 1976-1993 period; and, in the 1997-2013 period, to the increased biomass of Trichinella spp. due to the increased carnivore populations, which are the main reservoirs of these parasites.
    Parasites & Vectors 12/2015; 8(1):753. DOI:10.1186/s13071-015-0753-1 · 3.43 Impact Factor
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    ABSTRACT: Several species of protozoa cause acute or chronic gastroenteritis in humans, worldwide. The burden of disease is particularly high among children living in developing areas of the world, where transmission is favored by lower hygienic standards and scarce availability of safe water. However, asymptomatic infection and polyparasitism are also commonly observed in poor settings. Here, we investigated the prevalence of intestinal protozoa in two small fishing villages, Porto Said (PS) and Santa Maria da Serra (SM), situated along the river Tietê in the State of São Paolo, Brazil. The villages lack basic public infrastructure and services, such as roads, public water supply, electricity and public health services. Multiple fecal samples were collected from 88 individuals in PS and from 38 individuals in SM, who were asymptomatic at the time of sampling and had no recent history of diarrheal disease. To gain insights into potential transmission routes, 49 dog fecal samples (38 from PS and 11 from SM) and 28 river water samples were also collected. All samples were tested by microscopy and PCR was used to genotype Giardia duodenalis, Blastocystis sp., Dientamoeba fragilis and Cryptosporidium spp. By molecular methods, the most common human parasite was Blastocystis sp. (prevalence, 45% in PS and 71% in SM), followed by D. fragilis (13.6% in PS, and 18.4% in SM) and G. duodenalis (18.2% in PS and 7.9% in SM); Cryptosporidium spp. were not detected. Sequence analysis revealed large genetic variation among Blastocystis samples, with subtypes (STs) 1 and 3 being predominant, and with the notable absence of ST4. Among G. duodenalis samples, assemblages A and B were detected in humans, whereas assemblages A, C and D were found in dogs. Finally, all D. fragilis samples from humans were genotype 1. A single dog was found infected with Cryptosporidium canis. River water samples were negative for the investigated parasites. This study showed a high carriage of intestinal parasites in asymptomatic individuals from two poor Brazilian villages, and highlighted a large genetic variability of Blastocystis spp. and G. duodenalis.
    Parasites & Vectors 12/2015; 8(1). DOI:10.1186/s13071-015-0714-8 · 3.43 Impact Factor
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    Frontiers in Microbiology 11/2015; 6(e108329-1305). DOI:10.3389/fmicb.2015.01305 · 3.99 Impact Factor
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    ABSTRACT: Trichinellosis is a zoonotic disease caused by Trichinella muscle larvae (ML) through ingestion of raw or undercooked meat. To date, 12 taxa are recognized in this genus, of which four are circulating in Europe (Trichinella spiralis, Trichinella nativa, Trichinella britovi and Trichinella pseudospiralis). T. spiralis and T. britovi circulate in European wildlife and occur simultaneously in the same host species. The possibility of hybrid formation between T. britovi and T. spiralis has hardly been addressed and so far, results of experimental hybridisation attempts between T. britovi and T. spiralis are inconclusive. The aim of the present study was to analyse molecular polymorphisms of single T. spiralis and T. britovi (ML) from natural infections based on nuclear 5S rDNA intergenic spacer region (5S rDNA-ISR) and mitochondrial cytochrome c oxidase 1 (CO1) gene sequences. Six haplotypes of the 5S rDNA intergenic spacer region (5S rDNA-ISR) and 14 of the cytochrome c oxidase 1 (CO1) gene were demonstrated in 89 individual T. britovi ML from Latvia and Poland. In contrast, only two haplotypes were observed at both 5S rDNA-ISR and CO1 of 57 individual T. spiralis ML from Polish wild boar and red foxes. Moreover, this study demonstrates hybridisation in eight individual ML between T. britovi and T. spiralis under natural conditions in four Polish wild boar and two red foxes, revealed by combining 5S rDNA-ISR and CO1 sequence information of individual Trichinella ML. To our knowledge, this is the first report of interspecies hybridisation between T. spiralis and T. britovi under field conditions.
    Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 10/2015; 36. DOI:10.1016/j.meegid.2015.10.005 · 3.02 Impact Factor
  • S. Zoltán · G. Marucci · T. Zoltán · E. Pozio · S. Tamás ·
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    ABSTRACT: To investigate the spatial distribution of the Trichinella spp. and the factors influencing their circulation in Hungary, 4086 red foxes (Vulpes vulpes) and 0.32 million wild boars (Sus scrofa) were tested for Trichinella spp. infection in Hungary from 2006 to 2014. Trichinella spp. larvae from 86 (2.1%) foxes and 58 (0.02%) wild boars were identified by multiplex PCR as Trichinella britovi, Trichinella spiralis or Trichinella pseudospiralis. T. britovi was the dominant species in both foxes and wild boars (87.5% and 67.3%) followed by T. spiralis (11.2% and 31.0%) and T. pseudospiralis (1.3% and 1.7%). There was no correlation between environmental parameters in the home range of foxes and wild boars and the T. spiralis larval counts, but there was a positive correlation between the boundary zone of Hungary and T. spiralis infection. These results indicate that the distribution of T. spiralis in the Hungarian wildlife is determined by the transborder transmission of the parasite from the surrounding endemic countries. Based on the statistical analysis, non-agricultural areas and the mean annual temperature were the major determinants of the spatial distribution of T. britovi in Hungary. The positive relationship with non-agricultural areas can be explained by the generalist feeding behaviour including scavenging of foxes in these areas. The negative relationship with the mean annual temperature can be attributed to the slower decomposition of wildlife carcasses favouring a longer survival of T. britovi larvae in the host carrion and to the increase of scavenging of foxes.
    Magyar Allatorvosok Lapja 08/2015; 137(8):495-506. · 0.19 Impact Factor
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    ABSTRACT: The zoonotic nematode Trichinella britovi has been documented in animals and/or humans of the Mediterranean islands of Corsica and Sardinia since 2004. From 2005 to 2007 in the Sardinia island, several surveys had shown that T. britovi was circulating among backyard and free-ranging pigs reared in the Orgosolo municipality but all attempts had failed to detect this parasite in wild susceptible animals. The aim of the present work was to investigate the circulation of T. britovi in pigs and wildlife of the Orgosolo municipality, and of surrounding municipalities and provinces in the 2010-2014 slaughtering/hunting seasons. The results show that the T. britovi circulation was still restricted to the Orgosolo municipality with a prevalence of 2.6% in free-ranging pigs and 0.2% in backyard pigs but, for the first time, this parasite was detected also in 0.4% of wild boar, and 27.6% of red foxes. No infection was detected in backyard pigs, wild boar, and red foxes of the other municipalities and provinces. Since 1978, African swine fever is endemic in Sardinia and foci of this virus are still active in the investigated areas favoring cannibalism and, consequently, the T. britovi transmission, due to the high mortality rate caused by this virus. This is the first documented report on the transmission of T. britovi between the domestic and the sylvatic cycle. The health authority of the island must provide a service to dispose animal carcasses and offal, stamping out illegal free-ranging pigs, and train hunters and pig owners to manage waste and by-products according to the EU regulations. Copyright © 2015 Elsevier B.V. All rights reserved.
    Veterinary Parasitology 07/2015; 212(3). DOI:10.1016/j.vetpar.2015.07.020 · 2.46 Impact Factor
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    ABSTRACT: Nematode worms of the genus Trichinella are zoonotic parasites with a worldwide distribution. The majority of the biomass of these nematodes circulates among wildlife, but when humans fail in the proper management of domestic animals and wildlife, Trichinella infections are transmitted from the sylvatic to the domestic environment. Such failures occur in Romania, where a high prevalence of Trichinella spiralis has been detected in domestic pigs. The aim of the present study was to provide data about the prevalence of Trichinella spp. infections in red foxes (Vulpes vulpes) that are hunted in Romanian counties, in which the prevalences of Trichinella spp. infection in backyard and free-ranging pigs range from 0.17 to 2.5%, to determine the role played by this carnivore species in the transmission of the parasite to domestic cycle. A total of 121 animals from 45 hunting grounds of three counties were screened to detect Trichinella spp. larvae by the digestion method. Infections were detected in 26 (21.5%) foxes from 18 (40%) hunting grounds of the three counties (13/67 in Arad, 1/3 in Hunedoara, and 12/51 in Timiş). The mean larval density was 10.5 larvae per gram. Of the 25 successfully tested samples, the Trichinella larvae from 24 isolates were identified as T. britovi (96%), and the larvae from one isolate were identified as T. spiralis (4%). No mixed infections were recorded. The present results revealed that the red fox should be considered an important T. britovi reservoir in the sylvatic cycle; in contrast, the detection of only a single T. spiralis-positive isolate suggests that red foxes play a minor role in the epidemiology of the domestic cycle in the investigated area of western Romania. Copyright © 2015. Published by Elsevier B.V.
    Veterinary Parasitology 07/2015; 212(3). DOI:10.1016/j.vetpar.2015.06.032 · 2.46 Impact Factor
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    Accreditation and Quality Assurance 06/2015; DOI:10.1007/s00769-015-1142-3 · 0.97 Impact Factor
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    ABSTRACT: The flagellated protozoan Giardia duodenalis is a worldwide parasite causing giardiasis, an acute and chronic diarrheal disease. Metabolism in G. duodenalis has a limited complexity thus making metabolic enzymes ideal targets for drug development. However, only few metabolic pathways (i.e, carbohydrates) have been described so far. Recently, the parasite homolog of the mitochondrial-like glycerol-3-phosphate dehydrogenase (gG3PD) has been identified among the interactors of the g14-3-3 protein. G3PD is involved in glycolysis, electron transport, glycerophospholipids metabolism and hyperosmotic stress response, and is emerging as promising target in tumor treatment. In this work, we demonstrate that gG3PD is a functional flavoenzyme able to convert glycerol-3-phosphate into dihydroxyacetone phosphate and that its activity and the intracellular glycerol level increase during encystation. Taking advantage of co-immunoprecipitation assays and deletion mutants, we provide evidence that gG3PD and g14-3-3 interact at the trophozoite stage, the intracellular localization of gG3PD is stage dependent and it partially co-localizes with mitosomes during cyst development. Finally, we demonstrate that the gG3PD activity is affected by the antitumoral compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX), that results more effective in vitro at killing G. duodenalis trophozoites than the reference drug metronidazole. Overall, our results highlight the involvement of gG3PD in processes crucial for the parasite survival thus proposing this enzyme as target for novel anti-giardial interventions.
    Frontiers in Microbiology 06/2015; 06. DOI:10.3389/fmicb.2015.00544 · 3.99 Impact Factor
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    ABSTRACT: Giardiosis is a common gastrointestinal infection caused by the flagellate Giardia duodenalis, and affects both humans and animals, worldwide. Animals are infected with both zoonotic and host-specific G. duodenalis assemblages, and their role in the transmission of the infection to humans has been a subject of intense research and debate. Conventional PCR assays are appropriate to determine G. duodenalis assemblages, but lack sensitivity for the detection of mixed infections. Previous surveys demonstrated the occurrence of mixed infections with G. duodenalis assemblage A and B in humans, and with assemblages A and E in cattle, but are likely to be underestimated. In this study, we designed a set of assemblage-specific primers by exploiting sequence variability in homologous genes from assemblages A, B and E. Primers were designed to amplify fragments of different size that generated different melting curves from each assemblage in real-time PCR (rt-PCR) experiments. The assay has been tested on a large panel of human and farm animal isolates, and shown to possess high specificity (no cross reactions observed) and sensitivity (detection limit close to 20 copies). Therefore, this assay can be useful to detect zoonotic and host-specific G. duodenalis assemblages in fecal samples from farm animals, particularly when a large number of samples is to be tested. Copyright © 2015. Published by Elsevier B.V.
    Veterinary Parasitology 04/2015; 211(1-2). DOI:10.1016/j.vetpar.2015.04.017 · 2.46 Impact Factor
  • M J Zamora · M. Alvarez · J. Olmedo · M C Blanco · E. Pozio ·
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    ABSTRACT: Nematode worms of the genus Trichinella are zoonotic parasites circulating in most continents, including Europe. In Spain, Trichinella spiralis and Trichinella britovi are highly prevalent in wildlife but seldom in domestic pigs. In Portugal, only T. britovi was documented in wild carnivores. In the period 2006-2013 in Spain, 384 (0.0001%) pigs and 1399 (0.20%) wild boars (Sus scrofa) were positive for Trichinella spp. larvae, which were identified as T. spiralis or T. britovi. In 2014, Trichinella pseudospiralis larvae were isolated from a wild boar hunted in the Gerona province, Cataluña region, North-East of Spain, near the border to France. This is the first report of T. pseudospiralis in the Iberian peninsula, which suggests a broad distribution area of this zoonotic nematode in Europe. Since larvae of this Trichinella species do not encapsulate in the host muscles, they can be detected only by artificial digestion of muscle samples. T. pseudospiralis is the only Trichinella species infecting both mammals and birds. Birds can spread this pathogen over great distances including islands triggering new foci of infections in areas previously considered at low risk for this pathogen. Copyright © 2015 Elsevier B.V. All rights reserved.
    Veterinary Parasitology 04/2015; 210(3-4). DOI:10.1016/j.vetpar.2015.04.004 · 2.46 Impact Factor
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    ABSTRACT: Trichinellosis is a zoonotic parasitic disease with a worldwide distribution. The aim of this work was to describe the epidemiological and clinical data of five outbreaks of trichinellosis, which affected ethnic minorities living in remote mountainous areas of northwestern Vietnam from 1970 to 2012. Trichinellosis was diagnosed in 126 patients, of which 11 (8.7%) were hospitalized and 8 (6.3%) died. All infected people had consumed raw pork from backyard and roaming pigs or wild boar at wedding, funeral, or New Year parties. The short incubation period (average of 9.5 days), the severity of the symptoms, which were characterized by diarrhea, abdominal pain, fever, myalgia, edema, weight loss, itch, and lisping, and the high mortality suggest that patients had ingested a high number of larvae. The larval burden in pigs examined in one of the outbreaks ranged from 70 to 879 larvae/g. These larvae and those collected from a muscle biopsy taken from a patient from the 2012 outbreak were identified as Trichinella spiralis. Data presented in this work show that the northern regions of Vietnam are endemic areas for Trichinella infections in domestic pigs and humans. © The American Society of Tropical Medicine and Hygiene.
    The American journal of tropical medicine and hygiene 04/2015; 92(6). DOI:10.4269/ajtmh.14-0570 · 2.70 Impact Factor
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    ABSTRACT: Taenia solium taeniasis/cysticercosis is a neglected zoonotic disease complex occurring primarily in developing countries. Though claimed eradicated from the European Union (EU), an increasing number of human neurocysticercosis cases is being detected. Risk factors such as human migration and movement of pigs/pork, as well as the increasing trend in pig rearing with outside access are discussed in this review. The entry of a tapeworm carrier into the EU seems a lot more plausible than the import of infected pork. The establishment of local transmission in the EU is presently very unlikely. However, considering the potential changes in risk factors, such as the increasing trend in pig farming with outdoor access, the increasing human migration from endemic areas into the EU, this situation might change, warranting the establishment of an early warning system, which should include disease notification of taeniasis/cysticercosis both in human and animal hosts. As currently human-to-human transmission is the highest risk, prevention strategies should focus on the early detection and treatment of tapeworm carriers, and should be designed in a concerted way, across the EU and across the different sectors. Copyright © 2015 Elsevier B.V. All rights reserved.
    Veterinary Parasitology 03/2015; DOI:10.1016/j.vetpar.2015.03.006 · 2.46 Impact Factor
  • Edoardo Pozio ·
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    ABSTRACT: Nematodes of the genus Trichinella are widely distributed throughout the world in omnivorous and carnivorous animals (mammals, birds, and reptiles) and in incidental hosts. To prevent the transmission of these zoonotic parasites to humans, meat samples from Trichinella spp. susceptible animals are tested at the slaughterhouse or in game processing plants. The aim of the present review was to collect documented cases on Trichinella infected animals, meat, or meat derived products which reached the international trade or were illegally introduced from one to another country in personal baggage. In the course of the last 60 years in the international literature, there have been 43 reports of importation of Trichinella spp. infected animals or meat, most of which (60%, 26/43) related to live horses or their meat. Meat or meat derived products from pigs, wild boar and bears, account only for 18.6% (8/43), 4.7% (3/43), and 14.3% (6/43), respectively. However, only live horses or their meat intended for human consumption, meat from a single wild boar, and live polar bears caught in the wild for zoos, were imported through the international market; whereas, meat from pigs, wild boars and bears were illegally introduced in a country in personal baggage. Trichinella infected animals or meat which were officially or illegally introduced in a country were the source of 3443 Trichinella infections in humans in a 40-year period (1975-2014). Most of these infections (96.8%) have been linked to horsemeat consumption, whereas meat from pigs, wild boars and bears accounted only for 2.2%, 0.7% and 0.3% of cases, respectively. This review shows the Trichinella spp. risk in the international animal and meat trade has been linked mainly to horses and only one time to wild boar, if they carcasses are not adequately tested, whereas pigs and other wild animals or their derived products infected with Trichinella spp. are unlikely to reach the international market by the official animal and meat trade. Copyright © 2015 Elsevier B.V. All rights reserved.
    Veterinary Parasitology 02/2015; DOI:10.1016/j.vetpar.2015.02.017 · 2.46 Impact Factor
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    ABSTRACT: Background Parasites of the genus Trichinella are zoonotic nematodes common in carnivores throughout the world. We determined the prevalence and species of Trichinella infections in Florida panthers (Puma concolor coryi).Methods Tongues from Florida panthers were collected at necropsy and examined by pepsin-HCl artificial digestion for infection with Trichinella spp. DNA was extracted from larvae and multiplex PCR using Trichinella species-specific primers was used to genotype the worms.Results Trichinella spp. larvae were detected in 24 of 112 (21.4%; 14.6%¿30.3%) panthers. Sixteen of the panthers (14.3%) were infected with T. pseudospiralis, 1 (0.9%) was infected with T. spiralis, and 2 (1.8%) had mixed infections of T. pseudospiralis and T. spiralis. Trichinella spp. larvae from 5 panthers were not identified at the species level due to degraded DNA.Conclusions This is the highest prevalence of T. pseudospiralis detected in North America up to now and suggests the Florida panther is a key mammalian reservoir of this parasite in southern Florida. Trichinella pseudospiralis can infect both mammals and birds indicting the source of infection for Florida panthers could be broader than believed; however, birds represent a small percentage (0.01%) of the cat¿s diet. Since wild pigs (Sus scrofa) can be parasitized by both T. pseudospiralis and T. spiralis and that these swine can comprise a large portion (~40%) of a panther¿s diet in Florida, we believe that Florida panthers acquired these zoonotic parasites from feeding on wild pigs.
    Parasites & Vectors 02/2015; 8(1):67. DOI:10.1186/s13071-015-0674-z · 3.43 Impact Factor
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    ABSTRACT: 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.
    Veterinary Parasitology 01/2015; 208(3-4). DOI:10.1016/j.vetpar.2015.01.012 · 2.46 Impact Factor
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Publication Stats

9k Citations
976.94 Total Impact Points


  • 1980-2015
    • Istituto Superiore di Sanità
      • Department of Infectious, Parasitic and Immune-mediated Diseases
      Roma, Latium, Italy
  • 2012
    • Université de Montpellier
      Montpelhièr, Languedoc-Roussillon, France
  • 2010
    • National Institute of Health Dr. Ricardo Jorge
      Oporto, Porto, Portugal
  • 2009
    • Bundesinstitut für Risikobewertung
      • Department of Biological Safety
      Berlín, Berlin, Germany
  • 2008
    • Agenzia di Sanità Pubblica della Regione Lazio
      Roma, Latium, Italy
    • University of Alberta
      • Department of Biological Sciences
      Edmonton, Alberta, Canada
  • 2006
    • Danish Veterinary and Food Administration
      Glostrup, Capital Region, Denmark
  • 2004
    • University of Zagreb
      • Department of Parasitology and Parasitic Diseases
      Zagrabia, Grad Zagreb, Croatia
  • 2002
    • University of Helsinki
      • Faculty of Veterinary Medicine
      Helsinki, Uusimaa, Finland
  • 2001-2002
    • Università di Pisa
      Pisa, Tuscany, Italy
    • Texas A&M University - Kingsville
      NQI, Texas, United States
  • 1998
    • Chulalongkorn University
      • Faculty of Medicine
      Siayuthia, Bangkok, Thailand
  • 1997
    • Universidad de Extremadura
      Ara Pacis Augustalis, Extremadura, Spain
  • 1990-1997
    • Università degli Studi di Perugia
      • Department of Experimental Medicine and Biochemical Sciences
      Perugia, Umbria, Italy
  • 1993-1996
    • Sapienza University of Rome
      Roma, Latium, Italy
    • University of Pavia
      Ticinum, Lombardy, Italy
  • 1995
    • Ministry of Health, Italy
      Roma, Latium, Italy
    • University of Naples Federico II
      • Department of Translational Medical Sciences
      Napoli, Campania, Italy