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Hepatitis E virus (HEV) is the causative agent of hepatitis E, an emerging public health infection which has an increasing incidence across Europe. Because of the apparent lack of species barriers, HEV was characterized as a zoonotic agent. Swine are recognized as the main reservoir, but HEV is also found in wild animals such as ungulates, lagomorphs, and bats. Our work aimed at detecting the HEV presence in wild fauna in two hunting areas of Northern Italy (Parma and Sondrio areas) with different environmental and anthropic characteristics to investigate its possible role as reservoir. Liver samples were collected from wild boars, red deer, roe deer and chamois, and viral identification was carried out by One-Step RT Real-time PCR. Positive samples were genotyped, and phylogenetic analysis was performed. The virus was found only in the wild boar population, with different prevalence and subtypes in the two areas (14% HEV3a and 1.2% close to HEV3f in Parma and Sondrio, respectively). Wild ruminants seem otherwise to pose a marginal risk. Given the high pig farm density in the Parma area, and expansion of the wild boar population, continuous monitoring of the strains circulating in wildlife is crucial.

Background SARS CoV2 pandemic has generated interest in research of virus spread sources, apart from human contagion. In particular different studies are performed to understand surfaces involvement; during the epidemic event, different types of studies on surfaces have been performed. However, there is little information regarding the presence of the virus in food; wastewater contamination and poor hygiene practices could be the sources of viral contamination in handled food products. The aim of this work was to analyze different types of food for Sars CoV 2 presence. Methods 390 samples of molluscs, 353 vegetables, 10 meat, 72 water, 169 berries, 16 seafood products and ready-to-eat dishes were analysed (2020-2021). Samples were selected from those provided for routine analysis during the pandemic period. The preparation was done based on ISO 15216-2 indications, followed by extraction with Nuclisense®Minimag® system; real-time PCR was applied to detect the Sars CoV2 virus Open Reading Frame 1ab, with two different master mixes. Results All samples tested were negative for ORF1ab detection. Many hypothesis can be made about negative Results: it could be possible that contamination levels in water, or on surfaces in contact with food, are too low to generate a detection signal; again, handling practices may be sufficient to avoid the transmission of the virus to food. Otherwise, the selected genetic target and the Real-time PCR performed, could have been not so efficient in detecting virus as expected. Conclusions In this preliminary work, food does not seem to be a substrate for Sars CoV2 contamination; other food categories or transformation/production processes could be investigated to confirm the obtained results. Also, different targets and amplification systems would have to be evaluated to verify the rule of foodstuffs as Sars CoV2 vehicles. Key messages Primary production and manual processed food don't seem to be at risk for SARS CoV2 contamination. Food has not been identified as a source of Sars CoV2 spreading.

To observe the prevalence of contamination by hepatitis A virus (HAV) and norovirus (NoV) in different food types, 9242 samples were analyzed over a 6-year period (January 2014–December 2019). Samples were from routine official activities by Competent Authorities (CAs) and Food Business Operators, according to Hazard Analysis and Critical Control Points plans. Analyses were performed in accordance with European and Italian regulations. Food types were obtained from different production areas of Italy, and ranged from mollusks, ready-to-eat (RTE) and packaged vegetables, frozen berries, tap water, fruit and RTE fruit salads, and processed and preserved foods. No risk management plans were set by the authors’ laboratory, because they were still adopted by conferring customers. Analyses were conducted according to ISO/TS 15216-2:2013 (ISO in Part 2: Method for Qualitative Detection. International Organization for Standardization, Geneva, 2013). The data showed that 2.25% (95% CI: 2.0–2.6) of samples were contaminated by at least one virus type, and that the most detected pathogen was NoV GII (89.50% of all positives). Mollusks (filter-feeding animals) were the most contaminated category (92.31% of all positives) not only by NoV or HAV individually, but also by multiple HAV/NoV contaminations consisting of 22.59% of all positives. For NoV, there was a significant correlation between shellfish positivity and season, with the autumn–winter period being the most associated with risk. Conversely, berries, drinking water and RTE vegetables, previously linked to several outbreaks, showed a low rate of contamination. These results from data collection have implications for the improvement of sampling plans for HAV and NoV by Italian CAs, and by food-producing and distribution operators. Moreover, these findings obtained by a standardized qualitative method contribute the collection of data aimed at establishing new microbiological criteria not yet foreseen (but advocated) by current European rules.

Hepatitis E virus (HEV) is a singlestrand RNA virus that causes an acute viral hepatitis in humans. Among its eight recognized genotypes, HEV-3 and HEV-4 are zoonotic, infecting humans, pigs and wild boars. Recently, HEV-3 has been also detected in red deer, which represents another reservoir of HEV. Consumption of raw pork products (mainly liver sausages), undercooked wild boar meat, raw wild boar liver and deer meat has been responsible for foodborne HEV human worldwide. From November 2018 to March 2019, liver samples collected from 97 wild boars hunted in Emilia-Romagna region (Northern Italy) were tested for HEV RNA. The hunting area included two territories for an extension of 33 km2, named A (about 13 km2, natural park, deciduous wood) and B (about 20 km2, cultivated fields in proximity of a river) areas. Distance between the two areas ranged between 8 to 10 km. A total of 73 wild boars were hunted in area A, and 24 in area B. HEV RNA was detected by Realtime RT–PCR in 23/73 liver samples of wild boars living in area A only (31.5% - 95% CI: 22.0-42.8%). The HEV sequences (n=13) clustered within genotype 3. The majority of positives belonged to animals < 12 months (12/25; 48%), followed by subadults (13-24 months) (7/16; 43.8%) and adults (4/32; 12.5%). This difference was found to be statistically significant (p=0.0024). In absence of pig farms, the restriction of HEV-positive animals to a well-defined territory of 13 km2 (Boschi di Carrega Regional Park) could hypothetically be related to the presence of red deer (Cervus elaphus), which lived in area A at the beginning of the hunting season. Further studies are needed to confirm or deny our hypothesis.

Purpose. In 2013/2014, Italy experienced one of the largest community-wide prolonged outbreaks of hepatitis A virus (HAV) throughout the country. The article provides a comprehensive description of the outbreak and the investigation carried out by a multidisciplinary National Task Force, in collaboration with regional and local public health authorities. Control strategies of food-borne HAV infection in both the human and food sectors are also described. Methodology. Enhanced human epidemiological and microbiological surveillance together with microbiological monitoring of HAV in food and trace-back investigation were conducted. Results. A total of 1803 HAV cases were identified from 1 January 2013 to 31 August 2014, in Italy. Sequencing was possible for 368 cases (20.4 %), mostly collected between 1 January 2013 and 28 February 2014, and 246 cases (66.8 %) harboured an HAV outbreak strain. Imported frozen berries contaminated with HAV were identified as the vehicle of the outbreak which also involved many other European countries in 2013 and 2014. Epidemiological evidence obtained through a case–control study was supported by the finding of a 100 % nucleotide similarity of the VP1/2A sequences of HAVs detected in human and food samples. Trace-back investigation revealed an extremely complex supplying network with no possibility for a point source potentially explaining the vast contamination of berries found in Italy. Conclusion. The investigation benefited from an excellent collaboration among different sectors who shared proactively the available information. Our findings highlight the importance of considering frozen berries among the highest risk factors for HAV.

The rising prevalence of celiac disease leads to an increased demand of “gluten-free” products. A survey study on the gluten content and on the microbiological quality of “gluten-free” flour, and processing flour products, was carried out from 2010 to 2015 in Northern Italy. Overall 12,419 samples were analyzed, and 94.7% contained a gluten concentration less than 5 mg kg −1 (lower limit of detection). Only 0.1% of samples showed a gluten concentration above 80 mg kg −1 (maximum limit of detection). In the remaining 5.2%, the gluten concentration was between 5 and 80 mg kg −1 , underlining how a gluten-free diet completely devoid of gluten is unrealistic. The microbiological quality of these products was investigated. Overall, the majority of samples revealed microbial loads of less than 1 l g CFU g −1 (lower limit of detection). High levels of spoilage bacteria were found in egg-containing products. Total mesophilic bacteria were counted in all analyzed food categories with concentrations up to about 6, 8 and 9 l g CFU g −1 in dry pasta, flours and egg products respectively. Listeria monocytogenes was found only in one sample, whereas Salmonella spp. was never found. Buckwheat flour was the most frequently contaminated product by presumptive Bacillus cereus , with a prevalence of 12.5%. Also, a contamination by Coagulase-Positive Staphylococci was found during this investigation, especially in buckwheat dry pasta and flour and in egg dry pasta, with a prevalence of 54.7%. This study aimed to enhance the knowledge about the “gluten-free” products which are still poorly studied, even if their impact on the food market is increasingly considerable.

Background Norovirus (NoV) is the leading cause of acute gastroenteritis in developed countries. Transmission is through direct contact, unsanitary food handling or ingestion of contaminated water or food. Shellfish bioaccumulate NoV and current post-harvest depuration is not effective for its removal. Materials and Methods A monitoring programme for NoV in bivalve shellfish harvested from Northern Adriatic Sea was initiated in 2016. From January 2016 to March 2019, 418 samples including 257 Manila clams, 73 mussels, 46 striped clams, and 42 oysters, were examined. Mollusc samples were tested for NoV genogroups I (NoV GI) and II (NoV GII) contamination by RealTime RT-PCR according to ISO 15216-2. Results Ninety-three out of 418 tested samples (22.2%) were contaminated by at least one NoV genogroup, the simultaneous presence of the two genogroups was detected in 19/418 of the cases. Positive samples were distributed among the tested species as follow: 29/73 (39.7%) mussels, 51/257 (19.5%) Manila clams, 8/42 (19.0%) oysters, 5/46 (10.9%) striped clam. In 2016 (n = 135), 2017 (n = 122), 2018 (n = 132) and in the first trimester of 2019 (n = 12), prevalence of NoV was 6.7%, 24.6%, 31.8% and 41.4%, respectively. NoV GII was largely predominant being detected, alone or in association with GI, in 98.9% of the contaminated samples. On the other hand, GI prevalence increased from 1.5% (2016) to 12.1% in 2018, maybe reflecting a higher circulation of this genogroup in production environments. The vast majority of positive samples (91.4%) were detected during the cold season (November to March). Conclusions Overall positive samples have increased over the years (from 6.7% in 2016 to 31.8% in 2018). NoV GII was the most frequently detected genogroup, but NoV GI prevalence significantly raised in 2018. The routine application of quantitative RT-PCR (ISO 15216-1) to determine the viral load in bivalve molluscs would expand knowledge on potential for foodborne transmission. Key messages Overall positive samples have increased over the years. The routine application of quantitative PCR to determine the viral load in bivalve molluscs would expand knowledge on potential for foodborne transmission.

Issue/problem EU Regulation regarding fresh poultry meat considers only S. enteritidis or S. typhimurium as Food Safety Criteria. On the contrary, the isolation of other Salmonella spp. in this food matrices is considered compliant. Several bibliographical sources indicate an increase in the isolation trend of S. infantis in the poultry industry and in derived products. Description of the problem Episodes of food poisoning caused by fresh chicken meat contaminated with S. infantis have been recently reported. According to CDC, last year in USA, an outbreak caused by a S. infantis strain, resistant to multiple antibiotics, ended with 129 sick people, 25 hospitalizations and a death. National Salmonellosis Control Plan for Poultry 2019-21 establishes in Italy the abatement conditions for poultry farms in case of positive isolation of S. enteritidis, S. typhimurium and S. infantis. Results In 2018, the National Health System disposed the analysis of batches of fresh chicken meat from Poland, sampled in a cold storage facility in Northern Italy. Food Control Dept. of IZSLER analysed 156 samples for a total of 780 sampling units (s.u.), for Salmonella spp.: 72 samples were positive (46,15%;corresponding to 222 s.u. - 28,46%). 42 strains were serotyped as S. enteritidis, 149 as S. infantis, 26 as S. newport, 4 as S. kentucky and 1 as S. mbandaka. According to EU Regulation, batches positive for S. enteritidis were indicated as not compliant, but batches positive to other Salmonella were considered regular. Lessons EU legislation allows the circulation of chicken meat contaminated with S. infantis. Chicken meat should be consumed after appropriate well cooking eliminating this hazard, but it does not protect the consumer from the risk of cross-contamination of tools and surfaces at the household level. It’s therefore fundamental the application of correct processing practices in order to prevent the spreading of Salmonella in home kitchens and limit dangerous episodes of infection. Key messages S. infantis trend of isolation in the poultry industry is increasing. application of GMPs in home cooking is fundamental to limit food outbreaks of infection.

Mammalian orthoreoviruses (MRVs) are emerging infectious agents that may affect wild animals. MRVs are usually associated with asymptomatic or mild respiratory and enteric infections. However, severe clinical manifestations have been occasionally reported in human and animal hosts. An insight into their circulation is essential to minimize the risk of diffusion to farmed animals and possibly to humans. The aim of this study was to investigate the presence of likely zoonotic MRVs in wild ungulates. Liver samples were collected from wild boar, red deer, roe deer, and chamois. Samples originated from two areas (Sondrio and Parma provinces) in Northern Italy with different environmental characteristics. MRV detection was carried out by PCR, confirmation by sequencing and typing for MRV type 3, which has been frequently associated with disease in pigs, were carried out for positive samples. MRV prevalence was as high as 45.3% in wild boars and 40.6% in red deer in the Sondrio area, with lower prevalence in the Parma area (15.4% in wild boars). Our findings shed light on MRV occurrence and distribution in some wild species and posed the issue of their possible role as reservoir.

The classical microbiological method for detection of Listeria monocytogenes requires around 7 days for final confirmation, and due to perishable nature of RTE food products, there is a clear need for an alternative methodology for detection of this pathogen. This study presents an international (at European level) ISO 16140-based validation trial of a non-proprietary real-time PCR-based methodology that can generate final results in the following day of the analysis. This methodology is based on an ISO compatible enrichment coupled to a bacterial DNA extraction and a consolidated real-time PCR assay. Twelve laboratories from six European countries participated in this trial, and soft cheese was selected as food model since it can represent a difficult matrix for the bacterial DNA extraction and real-time PCR amplification. The limit of detection observed was down to 10 CFU per 25 of sample, showing excellent concordance and accordance values between samples and laboratories (>75%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (82.75%, 96.70% and 97.62%, respectively) when the results obtained for the real-time PCR-based methods were compared to those of the ISO 11290-1 standard method. An interesting observation was that the L. monocytogenes detection by the real-time PCR method was less affected in the presence of Listeria innocua in the contaminated samples, proving therefore to be more reliable than the reference method. The results of this international trial demonstrate that the evaluated real-time PCR-based method represents an excellent alterative to the ISO standard since it shows a higher performance as well as reduce the extent of the analytical process, and can be easily implemented routinely by the competent authorities and food industry laboratories.

Berries have repeatedly been associated with outbreaks of hepatitis A virus (HAV) infection. The fruits are usually minimally processed in the food industry due to their delicate nature. While washing treatments partially remove enteric viruses, the commonly used chemical additives produce toxic by-products. A valid alternative to preserve the food safety of these products could be the use of essential oils (EOs). EOs exert antimicrobial activity and do not interfere with the nutritional characteristics of food products. We investigated the efficacy of four essential oils, lemon (Citrus limon), sweet orange (Citrus sinensis), grapefruit (Citrus paradisi), and rosemary cineole (Rosmarinus officinalis chemotype 1.8 cineole) in reducing viral loads of HAV in soft fruits. Mixed fruit berries were inoculated with 106.74 TCID50/ml of HAV, and treated with four different EOs (0.5% lemon, 0.1% sweet orange and grapefruit, and 0.05% rosemary) for 1 h at room temperature. Virus infectivity was then assessed by titration assays for its ability to grow on cell cultures. A statistically significant reduction in HAV titer on the fruit surface was observed after treating the berries with EOs of lemon (2.84 log TCID50/ml), grapefruit (2.89 log TCID50/ml), and rosemary cineole (2.94 log TCID50/ml). Rosemary cineole was the most effective EO in reducing viral titer on berries, followed by grapefruit EO. These results improve our knowledge about the antiviral activity of these EOs and highlight their potential use in fresh produce sanitation.

The recent outbreak of Salmonella Agona linked to the consumption of infant formula (powdered formula) has rekindled the attention about the correct procedures for preparation and use of these products. International guidelines have already been published so far, particularly in association with Cronobacter sakazakii in early 2000s. FAO/WHO suggested to reconstitute formula with water at no less than 70°C. We therefore contaminated powdered formula with low levels of Salmonella spp and C sakazakii to evaluate the pathogens inactivation during the formula preparation using water at 70°C. In these conditions we observed a survival of both pathogens, indicating that the suggested recommendations may be not enough to guarantee the safety of this product. Higher temperatures are needed to reduce the biological risk, even if it may be not easily realized in actual domestic conditions. Moreover, the impact on the nutritional value of reconstituted formulas should be evaluated.

Silter cheese is a traditional hard cheese, produced in Valcamonica, Brescia, Italy. A total of 426 lactic strains isolated from Silter were analyzed to determine their probiotic characteristics. 274 out of 426 strains were found to produce bacteriocins against at least one of eight different pathogens (Salmonella enterica, Listeria monocytogenes, Salmonella derby, Salmonella thyphimurium, Salmonella napoli, Staphylococcus aureus, E. coli O157:H7, Salmonella enteritidis). In addition, 211 of 274 bactericin-producer strains adhered to Caco-2 cells and were characterized by RiboPrinter, revealing predominance of Enterococcus faecalis (26%) and Enterococcus durans-faecium (22%). These findings suggest that Silter may qualify as an important source of feasible probiotic strains.

Hepatitis A virus (HAV) was detected in two samples of mixed frozen berries linked to Italian hepatitis A outbreak in April and September 2013. Both viruses were fully sequenced by next-generation sequencing and the genomes clustered with HAV complete genomes of sub-genotype IA with nucleotide identities of 95–97 %.

Fresh vegetables and their ready-to-eat (RTE) salads have become increasingly recognized as potential vehicles for foodborne diseases. The EU Reg. 1441/2007 establishes microbiological criteria for bacterial pathogens for products placed on the market during their shelf-life (i.e. Salmonella spp., Listeria monocytogenes) for pre-cut fruits and vegetables (RTE) whilst it does not address the problem of contamination by enteric viruses. In this study we investigated the contamination by hepatitis A virus (HAV), hepatitis E virus (HEV) and norovirus (NoV) in 911 ready-to-eat vegetable samples taken from products at retail in Apulia and in Lombardia. The vegetable samples were tested using validated real-time PCR (RT-qPCR) assays, ISO standardized virological methods and ISO culturing methods for bacteriological analysis. The total prevalence of HAV and HEV was 1.9% (18/911) and 0.6% (6/911), respectively. None of the samples analysed in this study was positive for NoV, Salmonella spp. or Listeria monocytogenes. The detection of HAV and HEV in RTE salads highlights a risk to consumers and the need to improve production hygiene. Appropriate implementation of hygiene procedures is required at all the steps of the RTE vegetable production chain and this should include monitoring of emerging viral pathogens.

The classical microbiological method for detection of Salmonella spp. requires more than five days for final confirmation, and consequently there is a need for an alternative methodology for detection of this pathogen particularly in those food categories with a short shelf-life. This study presents an international (at European level) ISO 16140-based validation study of a non-proprietary Real-Time PCR-based method that can generate final results the day following sample analysis. It is based on an ISO compatible enrichment coupled to an easy and inexpensive DNA extraction and a consolidated Real-Time PCR assay. Thirteen laboratories from seven European Countries participated to this trial, and pork meat was selected as food model. The limit of detection observed was down to 10 CFU per 25 g of sample, showing excellent concordance and accordance values between samples and laboratories (100%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (100%) when the results obtained for the Real-Time PCR-based methods were compared to those of the ISO 6579:2002 standard method. The results of this international trial demonstrate that the evaluated Real-Time PCR-based method represents an excellent alternative to the ISO standard. In fact, it shows an equal and solid performance as well as it reduces dramatically the extent of the analytical process, and can be easily implemented routinely by the Competent Authorities and Food Industry laboratories.

Noroviruses and rotaviruses from a gastroenteritis outbreak affecting >300 people near Garda Lake (Northern Italy) in 2009 were investigated. Characterization of viruses from 40 patient stool samples and 5 environmental samples identified three distinct rotavirus and five norovirus genotypes; two of the latter were detected in both patient and environmental samples.

Vibrio parahaemolyticus is an autochthonous microorganism of the marine environments, frequently isolated from seafood, including bivalve mollusks, and a human pathogen responsible for gastroenteritis outbreaks and sporadic cases. The mechanism of pathogenicity has yet to be comprehensively determined, but two haemolysins (TDH and TRH) have been recognized as virulence factors. The aims of this work were: a) the evaluation of the …

Hepatitis E virus (HEV) is an important public health concern in many developing countries and it occurs in sporadic forms in industrialized areas. With the discovery of swine HEV in pigs, which is genetically closely related to human HEV, hepatitis E is considered to be a zoonotic disease. To investigate the circulation of HEV within a distinct area of Lombardy region (Northern Italy), 17 pig farms were subjected to monitoring study by collection of fresh stool samples each represented by ground-pooled specimens. In particular, three distinct types of breeding farms were focused, represented by farrow to weaning, farrow to finish and fattening farms, respectively. Epidemiological data confirm that in Europe the seroprevalence in pigs, more than 9 month of age, ranges from 51.4 to 75%, while in 3-9 months fatteners is about 38%. In France and Italy, the positivity among farms is respectively 30 and 97.4% and the seroprevalence in Italy is 50.2%. Since HEV viremia was typically observed in the early period of life in swine, faeces were collected in boxes containing weaning pigs. For the study, 183 stool samples were collected and amplifications were performed with universal primers specific for the ORF2 region of genome. Twentyeight samples resulted positive to HEV RNA and genotyping demonstrated that they were closely related to HEV strains belonging to genotype 3 and circulating in Europe. Comparison with reference strains from GenBank excluded their similarity to genotype 1, 2 or 4 confirming that genotype 3 strains are circulating in Europe. Since it was demonstrated that swine act as a reservoir for HEV, and since many strains into HEV genotype 3 share a strong molecular similarity to human HEV, it was important to detect the presence of HEV in a restricted area with a very high density of pigs.

In animal nutrition, mycotoxins are important feed contaminants. Among them, ochratoxins (OTA) and aflatoxins (AF) have several biological activities, including acute toxicity, teratogenicity, mutagenicity, immunotoxicity and carcinogenicity (Creepy, 2002). Obviously, toxicity of mycotoxins is a risk factor not only for animals but for humans, too (Hussein et al., 2001). Deficiencies of certain nutritional factors e.g. micronutrients may predispose or enhance individuals to the toxic effects of mycotoxins (Atroshi et al., 2002). It is well known that antioxidants like vitamin E, protect cellular membranes from oxidative damage.........

In early May 2013, Germany reported through the Epidemic Intelligence Information Systems (EPIS) and the Early Warning and Response Systems (EWRS) seven Hepatitis A (HAV) cases associated with a travel history in Northern Italy. Following the alert, the Italian Integrated Epidemiological System of Acute Viral Hepatitis (SEIEVA) reported also an increase of HAV cases in the same area in 2013. Cases identified were infected with HAV genotype IA with an identical “outbreak” sequence (KF182323). To monitor HAV cases …

The presence of foodborne pathogens (Salmonella spp., Listeria monocytogenes, Escherichia coli O157:H7, thermotolerant Campylobacter, Yersinia enterocolitica and norovirus) in fresh leafy (FL) and ready-to-eat (RTE) vegetable products, sampled at random on the Italian market, was investigated to evaluate the level of risk to consumers. Nine regional laboratories, representing 18 of the 20 regions of Italy and in which 97.7% of the country's population resides, were involved in this study. All laboratories used the same sampling procedures and analytical methods. The vegetable samples were screened using validated real-time PCR (RT-PCR) methods and standardized reference ISO culturing methods. The results show that 3.7% of 1372 fresh leafy vegetable products and 1.8% of 1160 "fresh-cut" or "ready-to-eat" (RTE) vegetable retailed in supermarkets or farm markets, were contaminated with one or more foodborne pathogens harmful to human health.

Consumption of raw or insufficiently cooked mussels contaminated with hepatitis A virus (HAV) is a major cause of infection to humans. The origin of mussels commonly used for the preparation of marinated seafood salads is often unknown, since different producers worldwide undergo a precooking treatment at the original collection site with methods and parameters not always indicated. These treatments could be insufficient for the inactivation of HAV, which is characterized by a high temperature resistance. Both high hydrostatic pressure (HHP) and marinade treatments have been shown to affect HAV vitality. In this study, two treatments (HHP and marinating) were combined in order to assess a potential synergistic effect on the virus vitality. A kinetic test was conducted by subjecting the experimentally-contaminated mussels (HAV titre: 10(6)/ml TCID50) to marinating, and to different HHP treatment (4,000; 5,000; and 6,000 bar for 1, 5, and 9 min). Virus post-treatment vitality was assessed by its ability to grow on cell cultures and by quantitative real-time RT-PCR to evaluate virus resistance under such conditions. Marinating treatment alone (final pH 4.3, and NaCl 2 %) did not inactivate the virus. On the other hand, the use of HHP treatment alone on non-marinated HAV-contaminated mussels was effective only above 5,000 bar for 5 min. The results of the present study elucidate the synergistic effect of a combination between marination and HHP treatments on the inactivation of the virus.

In the last years, consequently to EC Regulation no. 1924/2006 on nutrition and health claims made on foods, some Italian food businnes operators (FBOs) leaders in the meat sector, invested in research to develop innovative products such as low fat salami, containing up to 30% less fat than the traditional one. For FBOs it is essential to demonstrate for each production process whether the substrate allows the growth of L. monocytogenes and whether L. monocytogenes could reach or exceed the limit of 100 cfu g–1 at the end of the shelf life, as stated by EC Regulation no. 2073/2005. In the present study, the growth potential of L. monocytogenes during the shelf life of low fat salami packed in modified atmosphere was evaluated. The results show that the product is unable to support the growth of pathogen, even if the storage temperature is between 8 and 12°C.

In September 2011, an acute gastroenteritis outbreak affected 33 children in Northern Italy. Patients had drunk river water during an excursion. Identical GI.4 norovirus genomes were detected from one patient's stools and from the river water. Improper discharge of human sewage into the river may have caused this waterborne outbreak.

Norovirus (NoV) are increasingly important as etiological agents of gastrointestinal infections. Consumption of bivalve molluscs and ready-to-eat fishery products is one of the most common ways of acquiring NoV foodborne infections, and the rise of outbreaks of viral gastroenteritis represents an important health problem that is also responsible for economic losses. The aim of this work was to define the prevalence of NoV contamination in preserved fishery products and in shellfish commercialized in Italy, taking into account the results obtained during 9 years of survey (2003-2011) and paying special attention to the regions more involved in national production. A total of 4463 samples were examined (2310 mussels, 1517 clams, 510 oysters, 22 other shellfish species, 104 preserved seafood products) and the average positivity rate for NoV presence was 4.1% and ranged from 0.6% in 2007 to 9.8% in 2003 and from 1.9% in preserved seafood products to 4.7% in mussels. Genetic characterization of circulating strains showed a prevalence of genogroup II genotypes, including GII.b and GII.e polymerase types and different GII.4 variants. This information could contribute to the optimization of risk-based sampling strategies for NoV contamination in seafood, taking into account variability in different species and from year to year.

Aims: To investigate the presence of enteric viruses [hepatitis A (HAV) and norovirus (NoV)] in shellfish harvested from the deltaic area of the Po river in relation to environmental factors. Methods and Results: Fortnightly sampling of shellfish was carried out in two lagoon areas (category B production areas) and one sea area (category A). Environmental parameters in the lagoon and hydrometric level of the tributary river were monitored throughout the sampling period. Samples (n = 120) were analysed for bacterial (E. coli and Salmonella) and viral (HAV and NoV) contamination; samples from category B areas were analysed before and after purification treatment. All the samples were negative for HAV whereas 10 samples (8AE3%), all harvested in the lagoon areas, were positive for NoV. Sequencing identified the strains as genotypes II.4 and II.b. None of the samples was found to be contaminated after depuration. Conclusions: The monitoring showed a low frequency of NoV presence; viral contamination, detected exclusively in shellfish collected from the deltaic area (category B), could be influenced by the flow of the tributary river. Significance and Impact of the Study: The data collected are useful for the design of targeted prevention strategies and for the modulation of control plans after meteorological events.

Incidence and circulation of different strains of hepatitis A and Norovirus in shellfish were studied on 235 samples (Tapes philippinarum, Mytilus galloprovincialis, Ostrea spp. and Chlamys spp.) obtained from different sites, representing the shellfish production areas of the northern Adriatic sea. Shellfish were harvested in the period of one year and, after depuration, were examined for bacterial (Escherichia coli and Salmonella) and viral (HAV and NoV) contamination. Viral contamination was present on average in 22% of samples: specifically, 6% of samples tested positive for HAV, 14% for NoV and 2% for both viruses. None of the samples revealed the presence of Salmonella, and in most of them (93%) the number of E. coli was below the European legislation limit of 230 MPN/100 g. T. philippinarum was the species most often contaminated, as well as being the only species in which the legal limit for E. coli was, in some cases, exceeded. Both HAV and NoV contamination were detected throughout the year; NoV detection was slightly more frequent during winter months, but positive samples were also present in summer. The sequencing of the PCR products showed the circulation of only one HAV genotype (IA) and four different NoV genotypes (Hawaii, Melksham, Lordsdale and GGIIb) with a prevalence of the GGIIb genotype in the second period of the monitoring.

The "Istituti Zooprofilattici" are an important network whose main function is the monitoring of animal health as well as food. As a result of the recent improvements in aquaculture technology interest in the safety of seafood is increasing. Therefore, the purpose of this study was to set up diagnostic methods for the detection of virus contamination, as well as the use of in vitro techniques able to identify the different toxins. The results have allowed the development of molecular biology assays which, together with the isolation in cell cultures, can detect contaminations/infections by the hepatitis A virus and the most common enteroviruses. Moreover, specific selected cell lines have led to the detection of different toxins. These laboratory methods will be used in order to control seafood safety.