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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.

Mycobacterium avium subsp. paratuberculosis (MAP), the agent of paratuberculosis in ruminants, is suspected to be involved in the aetiology of some human diseases. Notably, the consumption of milk and dairy products is considered to be the main route of human exposure to MAP because of its ability to survive during pasteurization and manufacturing processes. The aim of this study was to investigate, through a microbiological challenge test, the survival of MAP during the manufacturing and ripening period of two Italian hard cheeses, Parmigiano Reggiano and Grana Padano, made from raw bovine milk. The challenge test was performed in two different phases: the creaming phase and the manufacturing phase. The creaming phase, which is the first step of cheese production, was reproduced in the laboratory employing raw cow's milk spiked with a MAP reference strain at a final concentration of 5.58 log10 CFU/mL. After the creaming at 18 °C and 27 °C for 12 h, a decrease of 0.80 log10 and 0.77 log10 was observed in partially skimmed milk, respectively. In the second phase, two batches of raw cow's milk (1000 L each) were inoculated with MAP reference and wild strains, respectively. Then, the entire manufacturing process for Parmigiano Reggiano and Grana Padano, both of Protective Designation of Origin (PDO), was reproduced in an experimental cheese factory, starting from a concentration in milk of 5.19 ± 0.01 and 5.28 ± 0.08 log10 CFU/mL of MAP reference and wild strains, respectively. Heating the curd at 53 °C for 20 min did not affect MAP survival, however a significant decrease (p Taking into account the poor data availability about MAP survival in hard cheeses, this study may improve the knowledge regarding the effect of the cheese manufacturing process on the MAP dynamics, supporting also the safety of traditional raw milk hard cheeses.

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.

Aims: The aim of the study was to evaluate the behaviour of Listeria monocytogenes in the conditioning liquid of packaged water buffalo mozzarella cheese (WBMC). Methods and Results: The conditioning liquid was contaminated with L. monocytogenes, and the contaminated samples were stored at four different storage temperatures: 5 and 10°C for 22 days; 20°C for 9 days; 20°C for 3 days and then at 5°C for 6 days. The results showed that L. monocytogenes concentration decreased when contaminated samples were stored at 5°C. When WBMC was stored at 20°C and at 10°C, L. monocytogenes started to grow after a lag phase of 3 and 10 days, respectively. When samples were stored at variable temperature conditions, L. monocytogenes numbers showed a lag phase of 5 days. Conclusions: Use of a conditioning liquid characterized by acidity and a correct storage temperature is able to counteract pathogen replication during shelf life. A high concentration of lactic acid bacteria was associated with effective control of L. monocytogenes but the role of lactic acid bacteria in WBMC conditioning liquid requires further investigation. Significance and Impact of the Study: According to European regulations, food producers should be able to justify decision-making on the shelf life assigned to their products, taking into account reasonable storage conditions and use by consumers. The results of the trial yielded information for producers of WBMC and similar cheeses for decision-making on product shelf life.

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.

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.

Water buffalo mozzarella cheese (WBMC) is a fresh stretched cheese produced from whole chilled buffalo milk. Although pasteurization of milk and the use of defined starter cultures are recommended, traditional technology involving unpasteurized milk and natural whey cultures is still employed for WBMC production in Italy. The purpose of this study was to assess the behavior of Arcobacter butzleri during WBMC production and storage under different temperature conditions (5, 10, and 20 °C). Raw milk was experimentally inoculated with one reference strain and two isolates of A. butzleri, and the count was monitored during WBMC production and storage. The bacterial count of A. butzleri decreased during curd ripening (from 7.83 log colony-forming units (CFU)/g to 4.14 log CFU/g in about 4 h) and a further decrease (4 log CFU/g) was observed at the end of curd stretching. During storage testing, A. butzleri was never detected by direct plating, whereas it was recovered from 12 of the total 162 WBMC until the end of storage testing by enrichment. The results revealed that A. butzleri is able to survive during WBMC production and storage at different temperature conditions. Consequently, traditional WBMC produced from raw milk could represent a potential source of Arcobacter infection for humans.

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 aim of the study was to evaluate the behaviour of Listeria monocytogenes in the conditioning liquid of packaged water buffalo mozzarella cheese (WBMC).The conditioning liquid was contaminated with L. monocytogenes, and the contaminated samples were stored at four different storage temperatures: 5 and 10 °C for 22 days; 20 °C for 9 days; 20 °C for 3 days and then at 5 °C for 6 days. The results showed that L. monocytogenes concentration decreased when contaminated samples were stored at 5 °C. When WBMC was stored at 20 °C and at 10 °C, L. monocytogenes started to grow after a lag phase of 3 and 10 days, respectively. When samples were stored at variable temperature conditions, L. monocytogenes numbers showed a lag phase of 5 days.Use of a conditioning liquid characterized by acidity and a correct storage temperature is able to counteract pathogen replication during shelf life. A high concentration of lactic acid bacteria was associated with effective control of L. monocytogenes but the role of lactic acid bacteria in WBMC conditioning liquid requires further investigation.According to European regulations, food producers should be able to justify decision-making on the shelf life assigned to their products, taking into account reasonable storage conditions and use by consumers. The results of the trial yielded information for producers of WBMC and similar cheeses for decision-making on product shelf life.

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

Two pig cell lines derived from kidney and trachea tissues and referred to as newborn swine kidney (NSK) and newborn pig trachea (NPTr) were established following serial culture of primary cells. They were characterized by an epithelial-like morphology, high capacity to replicate and stability of the cell monolayer for several days after seeding. Their modal chromosome number was modified in comparison to that of primary swine cells and they both displayed a transforming potential in vitro and displayed oncogenicity in nude mice. Infection with pig endogenous retroviruses was detected. Almost all the swine viruses tested, i.e., pseudorabies virus, pig parvovirus, hog cholera virus, transmissible gastroenteritis virus of swine, encephalomyocarditis virus, swine vesicular disease virus and the enteroviruses, except pig reproductive respiratory syndrome virus, were capable of replicating in the new cell lines with titres similar to the ones detected in the reference culture systems. Furthermore, all the selected influenza virus sub-types isolated from human, swine and avian species replicated with cytopathic effect in NSK and NPTr cells, whereas, of all the equine influenza viruses tested only the Miami and Suffolk sub-types replicated.

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.

Summary Two mutant strains of pseudorabies virus (PRV) were selected from the virulent 86/27V virus treated with chemical drugs. The viruses, named 6A1 and 6C2, respectively, appeared to be unable to express thymidine kinase function, as demonstrated by the autoradiography test. They showed a reduced virulence for some susceptible animal species (chickens, mice, rabbits, calves, lambs and piglets) and virus was isolated sporadically. The mutant viruses appeared to be able to protect animals against infection with the virulent strain of PRV. At gross, as well as at histological examination, no lesions in apparatus, system and tissues were detected in pigs inoculated with 6A1 and 6C2 viruses. By contrast, rabbits treated with 6C2 mutant strain presented lymphomononucleated cuffs, microgliosis, and neuronophagia in some areas of the brain. This focal spreading, together with the absence of neuronal necrosis and intranuclear inclusions, suggest an infection induced by a modified strain of PRV.

A newly established cell strain capable of continuous cultivation derived from newborn pig thyroid (NPTh), is discussed. The karyotype study revealed that the cell line has a heteroploid chromosome pattern, but the altered karyotype did not seem to be related to malignancy. It is suggested that the cells may be useful for investigations connected with the porcine viruses.

A cell line (BS/BEK) which was obtained from bovine embryo kidney tissue, when studied at its 140th passage level it showed the following properties: 1. An epithelial-like morphology, possessing a heteroploid karyotype with a modal chromosome number ranging between 70 and 75 chromosomes. 2. It failed to produce tumors in mice and in hamster. 3. It was shown to be ready susceptible to the replication of several viral agents originated from a variety of animal species. 4. It was not contaminated by mycoplasma or other bacterial spp.

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.

We have developed a fibroblastic-like continuous culture of newborn pig kidney (NPK). The current cell line was serially passaged 160 times and appeared to be well suited for production and assay of a number of viruses affecting pigs, such as pig parvovirus, pseudorabies and transmissible gastroenteritis. The cell line appeared aneuploid, with a modal chromosome number of 36 and induced tumors, classified as fibrosarcoma, in athymic mice.

The study aim was to assess the behaviour of Listeria monocytogenes during the production and shelf life of artisan water buffalo mozzarella cheese (WBMC) under different storage conditions. Raw milk was deliberately contaminated by L. monocytogenes and the evolution of L. monocytogenes count during production and shelf life was monitored. In traditional WBMC production technology L. monocytogenes can multiply in the curd during ripening, but its growth rate expressed in log CFU/g/h is lower than the growth rate reported by theoretical predictions. Stretching proved to be a process with good repeatability and able to reduce L. monocytogenes contamination by about 2 Log CFU/g. The intrinsic characteristics of traditional WBMC proved to be able to obstacolate the growth of L. monocytogenes during storage even in the case of severe thermal abuse.