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Background In recent decades, the celiac disease showed a gradual increase in prevalence. Therefore, there was a raised demand of gluten-free products. EU Reg. 1169/2011 states that 20 mg.kg-1 (ppm) is the maximum gluten content in food for celiac people, and that “gluten-free” labelling must be put on compliant food packages. This work is a study on different food categories, aiming at verifying the safety of analyzed samples. Methods Totally, 4615 gluten-free-labelled specimens were collected from January 2019 to April 2022 (pasta, 2944; cured meat, 566; flours/bakery, 489; sweets, 125 and other matrices, 491). A commercially available E.L.I.S.A. kit, according to the AOAC 2012.01-2016 method, was used to quantify gluten. Results In 97% of samples (4475) the gluten content was Conclusions The increased prevalence of celiac disease and the consumers’ perception that a gluten-free diet gives benefits, lead to a greater demand of gluten-free products. In this study, 99.4% of samples were compliant with the gluten-free labelling and safe for celiac consumers. The 0.32% had a gluten content between 20 and 80 ppm, still considered “compliant”, according to the EU Reg. 828/2014 that defines as very low gluten containing (thus edible for some celiac groups), those products with a gluten content Key messages • A continuous surveillance of gluten-free-labelled food products is very important to prevent risks for celiac consumers. • The foodstuffs distributed in the two considered regions are mainly safe for celiac patients.

Background Norovirus is an enteric virus with foodborne transmission. Bivalve shellfish are a main source of infections and outbreaks, which in the last years are showing an increasing trend. Therefore, in Italy a voluntary based monitoring plan to control the safety of bivalve shellfish was set up at provincial level. This study describes the occurrence and distribution of Norovirus in different areas of Northern Italy seas. Methods Between the years 2018-2020 622 samples (n = 173 oysters, n = 139 mussels, n = 300 clams, n = 10 other bivalve shellfish) were tested by OneStep RT Real Time PCR for Norovirus GI and GII and quantified according to the ISO 15216:2017. Positive samples were further analyzed to determine genotype by sequencing of the ORF1/ORF2 junction of the Norovirus genome. Results A total of 111 (18%) samples were positive for Norovirus (mussels 22%; oysters 18%; clams 16%, other molluscs 20%), positive samples were found mainly in the winter season. The mean contamination was 103 ge/µL and ranged 100-105 ge/µL. Thirty-two samples were typeable (GI n = 10; GII n = 22) with GI samples belonging to four genotypes and GII samples belonging to five genotypes. GII.4, particularly Sydney 2012 subtype (found in three oysters and three clams samples), was the most prevalent, followed by GII.3. Often more than one genotype was found in samples collected simultaneously in the same sampling site. The virus was found with relatively high occurrence both in samples collected close to the coast (38%) and in those harvested from farms located 3-6 nautical miles offshore (73%). Conclusions The overall prevalence of Norovirus in edible shellfish, particular those to be eaten raw, is moderately high (18%). No difference in contamination was highlighted among the different species or different sampling sites. One of the most prevalent variants found (i.e. GII.4 Sydney 2012) is a leading cause of infections worldwide. Key messages Given the moderately high proportion of positive samples (18%), oysters (which are often eaten raw), regardless of the origin, represent the main risk of infection. The presence of genotypes frequently involved in human infections strengthens the need for ongoing monitoring, which should be extended to all provinces involved in shellfish farming.

Ricotta cheese is a ready-to-eat product with properties (pH >6.0, aw >0.98-0.99) and moisture content (75-80%) that may pose a risk to public health due to postprocess contamination by several bacterial pathogens, including Arcobacters. The objective of the study was to evaluate the behavior of Arcobacter butzleri and Arcobacter cryaerophilus in ricotta cheese during its shelf life assuming postprocessing contamination. Two types of ricotta cheese, artisanal water buffalo (WB) and industrial cow milk ricotta cheese, were experimentally contaminated with A. butzleri and A. cryaerophilus and the count was monitored at 2 different temperatures (6°C and 12°C) during shelf life of 5 d for WB cheese and 22 d for industrial ricotta cheese. In WB ricotta cheese the A. butzleri count remained stable during the 5 d of storage at 6°C, whereas a moderate but significant decrease was observed in A. cryaerophilus count. The counts of both species increased when WB ricotta cheese was stored at 12°C. In industrial ricotta cheese stored at 6°C, a significant reduction was observed both in A. butzleri and A. cryaerophilus counts during the 22-d storage period; at 12°C storage, a count increase was observed for both Arcobacter species up to d 14 of storage after which the log cfu/g count resulted constant until d 22 of storage. The ability of A. butzleri and A. cryaerophilus to survive at 6°C and to grow at 12°C in ricotta cheese has significant food safety implications.

A quantitative risk assessment (RA) was developed to estimate haemolytic-uremic syndrome (HUS) cases in paediatric population associated with the consumption of raw milk sold in vending machines in Italy. The historical national evolution of raw milk consumption phenomenon since 2008, when consumer interest started to grow, and after 7Âyears of marketing adjustment, is outlined. Exposure assessment was based on the official Shiga toxin-producing Escherichia coli O157:H7 (STEC) microbiological records of raw milk samples from vending machines monitored by the regional Veterinary Authorities from 2008 to 2014, microbial growth during storage, consumption frequency of raw milk, serving size, consumption preference and age of consumers. The differential risk considered milk handled under regulation conditions (4°C throughout all phases) and the worst time–temperature field handling conditions detected. In case of boiling milk before consumption, we assumed that the risk of HUS is fixed at zero. The model estimates clearly show that the public health significance of HUS cases due to raw milk STEC contamination depends on the current variability surrounding the risk profile of the food and the consumer behaviour has more impact than milk storage scenario. The estimated HUS cases predicted by our model are roughly in line with the effective STEC O157-associated HUS cases notified in Italy only when the proportion of consumers not boiling milk before consumption is assumed to be 1%. Raw milk consumption remains a source of E.Âcoli O157:H7 for humans, but its overall relevance is likely to have subsided and significant caution should be exerted for temporal, geographical and consumers behaviour analysis. Health education programmes and regulatory actions are required to educate people, primarily children, on other STEC sources.

The safety of raw milk sold in Northern Italy was investigated in relation to hygiene quality parameters and presence of Salmonella spp., Listeria monocytogenes, thermotolerant Campylobacter, and Verocytotoxin producing Escherichia coli O157:H7. The performance of different analytical methods used-official culture method (ISO), modified Bacteriological Analytical Manual cultural method (mBAM), and polymerase chain reaction (PCR)-was evaluated. The presence of Mycobacterium avium subsp. paratuberculosis (Map) was investigated only by PCR. All samples met regulations for alkaline phosphatase and inhibitory substance, while 18% and 44.8% of samples collected from vending machines had, respectively, somatic cell count (SCC) >300,000/mL and total bacterial count (TBC) >50,000 CFU/mL. The correlation between hygienic quality parameters in samples collected from bulk tank and vending machines showed a significant increase of TBC in vending machines meaning that raw milk was mishandled during distribution and sale. All pathogens investigated were detected in raw milk sold at vending machines; a total of five samples (5%) had at least one pathogen, of which two were detected by PCR and three by mBAM. None of the samples was positive by cultural ISO methods. Even if the comparison of analytical methods showed that none performs significantly better than the others, testing a higher volume of milk (25 versus 210 mL) affects significantly the detection rate of pathogens. Three samples (3%) were positive for Map, suggesting that raw milk is a significant source of Map exposure for consumers. The observed TBC increase and the detection of several pathogenic bacteria pose questions on the safety of raw milk; the use of ISO seems inefficient in detecting a low contamination level of pathogens in milk and consequently not appropriate as official method for testing. In order to ensure consumer's safety, a new approach for the raw milk chain is required.

12 Large-White-Landrace piglets were subdivided in four groups of 3 and housed in separate units. The piglets of three groups were inoculated with the 86/27V 6C2 thymidine kinase negative (TK−) mutant of pseudorabies virus (PRV), by different routes. A second inoculation with the same mutant was given to the pigs 21 days later. The animals of a fourth group were left as uninoculated controls. 21 days following the second inoculation with the TK− mutant all pigs were challenge infected with the virulent PRV. On post challenge day (PCD) 30 all pigs were killed and samples for virus detection and histology were taken from several organs. The inoculated TK− mutant of PRV did not induce any ill effects in the pigs except a transient febrile reaction in some animals. Virus was recovered from nasal swabbings from one pig 2 days after the first inoculation of the mutant. After challenge exposure with virulent PRV, the TK− mutant-inoculated pigs were apparently protected, whereas the control pigs all were severely affected and recovered very slowly over 3 weeks. Virus was isolated from the nasal swabbings from the TK− mutant-inoculated pigs on PCDs 2 and 4, whereas the nasal swabbings from the control piglets were all positive for virus from PCD 2 through PCD 10. DNA analysis of the virus recovered showed a pattern identical to that of the virulent PRV. Histologic lesions were found in the respiratory and the central nervous systems, however, the lesions in the TK− mutant-inoculated pigs were much milder compared to those registered for the control pigs. Virus was not isolated from any of the tissue samples that were tested, but viral DNA with sequences typical of PRV genome was detected by PCR in all samples of trigeminal ganglia from either the TK− mutant-inoculated pigs or from the controls.

A quantitative risk assessment was developed to describe the risk of campylobacteriosis and hemolytic uremic syndrome (HUS) linked to consumption of raw milk sold in vending machines in Northern Italy. Exposure assessment considered the microbiological status of dairy farms, expected milk contamination, storage conditions from bulk tank to home storage, microbial growth during storage, destruction experiments, consumption frequency of raw milk, age of consumers, serving size, and consumption preference. The differential risk between milk handled under regulation conditions (4°C throughout all phases) and the worst field handling conditions was considered. The probability of Campylobacter jejuni infection was modeled with a single-hit dose-response beta-Poisson model, whereas for HUS an exponential dose-response model was chosen and two probabilities were used to model the higher susceptibility of children younger than 5 years old. For every 10,000 to 20,000 consumers each year, the models predicted for the best and worst storage conditions, respectively, 2.12 and 1.14 campylobacteriosis cases and 0.02 and 0.09 HUS cases in the 0- to 5-year age group and 0.1 and 0.5 HUS cases in the5-year age group. The expected pediatric HUS cases do not differ considerably from those reported in Italy by the Minister of Health. The model developed may be a useful tool for extending the assessment of the risk of campylobacteriosis and HUS due to raw milk consumption at the national level in Italy. Considering the epidemiological implications of this study, the risk of illness linked to raw milk consumption should not be ignored and could be reduced by the use of simple measures. Boiling milk before consumption and strict control of temperatures by farmers during raw milk distribution have significant effects on campylobacteriosis and HUS and are essential measures for risk management.

The direct sale by farmers of raw milk for human consumption has been allowed in Italy since 2004. The aim of this study was to evaluate the behaviour of selected foodborne pathogens in raw milk sold in vending machines, in field handling conditions, and during shelf-life from production to consumption. Temperature of storage of raw milk in 33 farms authorized to produce and sell raw milk were investigated from farm to vending machine delivery, together with consumer habits in one province of the Emilia-Romagna region of northern Italy. Failure to maintain appropriate low temperatures during shelf-life was recorded and 43% of consumers did not boil milk before consumption. Listeria monocytogenes, Escherichia coli O157:H7, Salmonella Typhimurium and Campylobacter jejuni strains were inoculated into raw milk samples, and the best (4°C as established by law) and worst temperature storage conditions detected (variable temperature) were simulated. Boiling tests were performed for each pathogen considered at high and low levels of contamination. Results showed an increase in L. monocytogenes in milk stored at 4°C and at variable temperatures recorded in shelf-life monitoring, an increase in E. coli O157:H7 and S. Typhimurium at variable temperatures but not at 4°C, and a decrease in C. jejuni in all storage conditions. Boiling milk is effective in making it safe for consumers. This study provides evidence that appropriate handling of raw milk, maintaining low temperatures, together with consumer education concerning boiling raw milk before consumption are key factors in preventing foodborne infections linked to raw milk consumption, and helps assess the risk of foodborne infection linked to raw milk consumption.

Water buffalo mozzarella cheese (WBMC) is a fresh pasta filata cheese produced from whole chilled buffalo milk. Although pasteurization of milk and the use of defined starter cultures are recommended, traditional technology involving the use of unpasteurized milk and natural whey cultures is still employed for WBMC production in Italy. The aim of this study were to assess the behaviour of Salmonella Typhimurium during the production of artisan water buffalo mozzarella cheese and during its shelf life under different temperature conditions. Raw milk was inoculated with S. Typhimurium and the evolution of S. Typhimurium count during production and shelf life was monitored. In artisan WBMC production technology S. Typhimurium multiplied in the curd during ripening, but its growth rate expressed in log CFU/g/h was lower than the growth rate reported by theoretical predictions. Stretching proved to be a process with good repeatability and able to reduce S. Typhimurium contamination by 5.5 Log CFU/g. The intrinsic characteristics of traditional WBMC proved to be unable to obstacolate the growth of S. Typhimurium during storage in the case of thermal abuse. Control of raw milk contamination and a proper refrigeration temperature are key factors in reducing the risk for consumers.

Twenty‐day‐old susceptible chickens were divided into three groups; two were vaccinated with inactivated, water in oil emulsified La Sota strain of Newcastle disease virus (NDV) obtained from a bovine embryo kidney (BS/BEK) cell line and from chicken embryos, respectively. The third unvaccinated group represented the control. At 30‐day intervals subgroups were exposed to the Herts 33 virulent NDV strain. Serological and clinical findings showed no appreciable difference in the immunogenicity of the antigen from either culture systems and no significant differences could be observed in its ability to protect against ND challenge.

Ochratoxin A (OTA), a mycotoxin frequently present in food and feedstuffs, produces a wide range of toxic effects, including cell death via lipid peroxidation. In one human and four animal cell lines we determined the half lethal concentration (LC50) of OTA, its effect on reactive oxygen species (ROS) production, and its ability to induce cytochrome p450 activity. We also examined the protective effect of α-tocopherol and all-trans-retinol in the most sensitive cell lines (i.e. bovine mammary epithelia, for which LC50was 0·8μg/ml (24h), and Madin Darby canine kidney, for which LC50was 4·3μg/ml (48h)). Pre-incubation for 3h with either antioxidant significantly (PP

The lentogenic La Sota strain of Newcastle disease virus (NDV) was adapted to grow in the BS/BEK cell line of bovine embryo kidney origin with a infectious titre similar to that in chicken embryos. No modification in the biological properties was detected after serial passages in the cell line, as indicated by CPE and by size and shape of the plaques. By contrast, the intracerebral pathogenicity index test, determined in 1-day-old chicks, was lower than for La Sota grown in chicken embryos. However, the immunogenicity of the cell culture adapted virus did not show any variation as demonstrated by serological response and by protection following challenge with virulent NDV. Accordingly, it appears that La Sota grown in cell cultures has retained its biological and immunological characteristics and if these results are confirmed by field trials and long term protection tests, the use of the BS/BEK cell line could be an alternative to chicken embryos for the cultivation of NDV.

We have tested amplified fragment length polymorphism (AFLP) technology, in comparison with isoenzyme analysis, for the simultaneous detection of inter- and intraspecific cell line cross-contaminations (CCCs) in the cell line collection held at the Istituto Zooprofilattico della Lombardia e dell'Emilia Romagna. Isoenzyme analysis identified four cases of interspecific CCCs. In a single experiment, AFLP was able to identify the species of origin of all cell lines for which a reference genomic deoxyribonucleic acid was available and to detect five interspecific contaminations. Four CCCs confirmed data on isoenzymes, whereas the fifth CCC was detected in a species for which isoenzyme analysis was noninformative. In addition, AFLP was able to identify the putative source of the contaminations detected. The utility of the technology in the detection of intraspecific cell line contaminations depends on the number of cell lines that have to be distinguished in a specific species and on the availability of highly informative fingerprinting systems. In mice, a single AFLP primer pair produced 16 polymorphisms and distinguished all the 15 strains of mouse cell lines analyzed. In humans, 18 AFLPs identified 83 different profiles in the 159 cell lines analyzed. Amplified fragment length polymorphism can conveniently be applied for cell line fingerprinting in species for which hypervariable markers are not available. In species for which a highly informative multiplex of microsatellite markers is available, AFLP can still provide a useful and cheap tool for simultaneously testing inter- and intraspecific contaminations.