Global surveillance for norovirus identified in 2012 the emergence of a novel pandemic GII.4 variant, termed Sydney 2012. In Italy, the novel pandemic variant was identified as early as November 2011 but became predominant only in the winter season 2012-2013. Upon sequencing and comparison with strains of global origin, the early Sydney 2012 strains were found to differ from those spreading in 2012-2013 in the capsid (ORF2) putative epitopes B, C and D, segregating into a distinct phylogenetic clade. At least three residues (333, 340 and 393, in epitopes B, C and D, respectively) of the VP1 varied among Sydney 2012 strains of different clades. These findings suggest that the spread of the pandemic variant in Italy during the winter season 2012-2013 was due to the introduction of strains distinct from those circulating at low frequency in the former winter season and that similar strains were also circulating elsewhere worldwide.

Considering its widespread distribution in marine environments, its fast replication times and low infectious doses and the rapid spread of its strains in recent years, intensive and continuous monitoring of potentially pathogenic Vibrio parahaemolyticus is strongly recommended in order to assess the human health risk arising from shellfish consumption. The lack of epidemiological data points to the need to develop specific methods for detectingV. parahaemolyticus. In this note, the authors compare two platingmedia currently available for isolating V. parahaemolyticus in shellfish. Both approaches involve pre-enrichment of V. parahaemolyticus. One uses thiosulphate-citrate-bile salt sucrose (TCBS) as the isolation medium, while the other uses a chromogenic medium (CHROMagar Vibrio). Next, biochemical identification of isolates was performed with API 20E, followed by PCR assay aimed at the toxR gene to confirm the cultural and biochemical identification. Comparison of the two methods highlighted that CHRO-Magar Vibrio is more accurate and specific than TCBS. The analysis of data from 160 shellfish samples showed an accuracy and specificity of just 51% and 71% for TCBS compared with 88% and 95% for CAV

This study provides data on the prevalence of potentially pathogenic Aeromonas spp. in ready-to-eat (RTE) seafood products by evaluating the occurrence of Aeromonas spp. and the presence of virulence-associated genes. Aeromonas spp. was detected in 57 ⁄ 81 (70.3%) RTE seafood samples. Specifically, Aeromonas spp. was highlighted in 19 ⁄ 21 (90.5%) sushi, in 18 ⁄ 21 (85.7%) sea salad, 11 ⁄ 12 (91.7%) surimi and 9 ⁄ 12 (75%) peeled shrimp samples. Aeromonas spp. was not observed in marinated anchovies and octopus salad samples. Then, PCRs aimed at the hlyA, aerA, alt and ast genes, encoding, respectively, haemolysin A, aerolysin, aeromonas labile temperature cytotonic enterotoxin and aeromonas stable temperature cytotonic entero- toxin, demonstrated a widespread distribution of these genes among Aeromonas isolates. The results underline the need to implement an adequate control plan performing an intensive and continuous monitoring to guarantee the human health.

This study provides data on the prevalence of potentially pathogenic Bacillus cereus in foods from catering kitchens by evaluating the occurrence of B. cereus and the presence of virulence-associated genes. B. cereus was detected in 72⁄250 (28.8%) food samples. Specifically, B. cereus was highlighted in 34⁄74 (45.9%) pastries, 16 ⁄ 40 (40%) rice samples, 4 ⁄ 38 (10.5%) potato meals, 6 ⁄ 54 (11.1%) mozzarella samples and 12 ⁄ 44 (27.3%) meat meals. PCRs aimed at the hbl (C, D, A, B), nhe (A, B, C), bceT and cytK genes demonstrated a widespread distribution of the toxin-encoding genes among B. cereus isolates. The results highlight the frequent failure of control measures in catering kitchens and the need for intensive and continuous monitoring in order to assess the human health risk, as proposed by Regulation (EC) no. 1441⁄2007 on microbiological criteria for foodstuffs.

Aims: This study evaluated the application of polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) for the detection of Vibrio parahaemolyticus in shellfish. Methods and Results: The PCRs were selected to amplify a species-specific sequence region. In particular, internal tl biotin-labelled oligonucleotide probe was used to capture the DIG-labelled PCR products. Next, the probe PCR product hybrids, immobilized on a streptavidin-coated microtiter plate, were detected with peroxidase-conjugated anti-digoxigenin antibody (anti-DIG-POD) and the colorimetric peroxidase substrate ABTS [2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] using an ELISA plate reader. Conclusions: The PCR-ELISA system described is a feasible, sensitive method for the direct and specific detection of V. parahaemolyticus in shellfish samples. Compared with gel-based detection methods, PCR-ELISA in this study increased sensitivity by 100-fold for V. parahaemolyticus. Significance and Impact of the Study: The PCR-ELISA described may be used for potential rapid detection in routine shellfish analysis for the seafood industry. The sector requires simultaneous large-scale sample screenings to monitor contamination levels in processing plants and evaluate the performance of the hazard analysis and critical control point (HACCP) system. PCR-ELISA also proved to be economical, with a cost of about 9 Euros per sample, and the quick assay taking 8h to complete starting from DNA extraction. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

The Italian Ministry of Agricultural, Food and Forestry Policies (MiPAAF) Decree dated 31 January 2008, which reports the Italian name for fish species of commercial interest, establishes that baccalà can be obtained exclusively from G. macrocephalus (Pacific cod) and G. morhua (Atlantic cod). This paper describes the COI-based DNA identification system to verify the substitution or misbranding of gadoid fish species and, consequently, its concordance with the labels on salted cod fillets shown as baccalà and on battered cod chunks labelled as bocconcini di baccalà. The analysis of interpretable sequences revealed that 55/65 dried salted cod fillet samples were detected as belonging to the family Gadidae, while 10/65 samples appeared to belong to the Lotidae family, while among battered cod chunks labelled as bocconcini di baccalà, the post-sequencing data analysis shows that the labels were completely wrong, with 28/40 samples from Pollachius virens and 12/40 samples from Brosme brosme. The substitution rate for products labelled on the market as baccalà in this study raises significant issues relating to food safety and consumer protection.

Norovirus (NoV) is one of the major causes of diarrhoeal disease with epidemic, outbreak and sporadic patterns in humans of all ages worldwide. NoVs of genotype GII.4 cause nearly 80-90 010 of all NoV infections in humans. Periodically, some GII.4 strains become predominant, generating major pandemic variants. Retrospective analysis of the GII.4 NoV strains detected in Italy between 2007 and 2013 indicated that the pandemic variant New Orleans 2009 emerged in Italy in the late 2009, became predominant in 2010-2011 and continued to circulate in a sporadic fashion until April 2013. Upon phylogenetic analysis based on the small diagnostic regions A and C, the late New Orleans 2009 NoVs circulating during 2011-2013 appeared to be genetically different from the early New Orleans 2009 strains that circulated in 2010. For a selection of strains, a 3.2 kb genome portion at the 3' end was sequenced. In the partial ORF1 and in the full-length ORF2 and ORF3, the 2011-2013 New Orleans NoVs comprised at least three distinct genetic subclusters. By comparison with sequences retrieved from the databases, these subclusters were also found to circulate globally, suggesting that the local circulation reflected repeated introductions of different strains, rather than local selection of novel viruses. Phylogenetic subclustering did not correlate with changes in residues located in predicted putative capsid epitopes, although several changes affected the P2 domain in epitopes A, C, D and E.

Given the considerable economic loss to beekeepers worldwide and the possible public health implications related to the presence of antibiotics in honey, an American Foulbrood (AFB) monitoring/prevention program for Paenibacillus larvae is regarded as essential. This study investigates the occurrence and distribution of P. larvae genotypes in honey and brood combs from Apulia (Italy). Genotyping of P. larvae isolates using ERIC-PCR generated a total of four different ERIC banding patterns (ERIC-A, ERIC-B, ERIC-C, ERIC-D), including fragments ranging from 200 to 3000 bp. Considering that the genotype has an influence on P. larvae infections and multi-genotype infections of colonies or apiaries may increase the complexity of P. larvae infections by influencing the type and speed of the development of clinical symptoms, the findings of the present study could be helpful for training veterinarians, bee inspector's extension staff, and beekeepers, thus improving the detection of AFB infections in the field.

The potential of high pressure processing to inactivate hepatitis A virus (HAV) within Mediterranean mussels (Mytilus galloprovincialis) and blue mussels (Mytilus edulis) was evaluated. HAV was bioaccumulated within mussels to approximately 6-log10 PFU by exposure of mussels to HAV-contaminated seawater. After shucking, 5 min pressure treatments of 300, 325, 350, 375, and 400 MegaPascals (MPa) were performed at room temperature (18–22°C). For blue mussels, log10 PFU reductions of HAV averaged 2.1 and 3.6 for treatments of 350 and 400 MPa, while for Mediterranean mussels reductions of 1.7 and 2.9 log10 PFU MPa were observed for equivalent treatments. These results demonstrate that high pressure processing is capable of inactivating HAV within mussels.

Human astroviruses (HAstVs) are important enteric pathogens and can be classified genetically and antigenically into eight types. During surveillance of HAstVs in Italy, type-4 HAstVs were detected only sporadically and found to cluster into two distinct genetic groups. Upon sequence analysis of the 3' end of the polymerase gene (ORF1b) and of the full-length ORF2, the 2008 type-4 HAstV strains were characterised as a novel ORF2 genetic lineage, designated as 4c. The 2008 type-4 HAstVs also shared the ORF1b gene with similar HAstV-4c strains detected globally, thus displaying a conserved ORF1b/ORF2 asset. By interrogation of the databases, this novel lineage 4c accounted for 60.8% of the type-4 strains identified worldwide and the vast majority of recent type-4 HAstVs. The 2002 type-4 HAstVs displayed a type-4b ORF2, whereas in the ORF1b they resembled type-1 HAstVs. This inconsistency suggests a possible recombinant origin, with the RNA switch taking place upstream the ORF1b/ORF2 junction region. Also, recombination likely played a role in the diversification of the ORF2 of the three type-4 lineages. Multi-target analysis is required for appropriate characterisation and identification of recombinant HAstVs.

In winter 2015-16, norovirus GII.17 Kawasaki 2014 emerged as a cause of sporadic gastroenteritis in children in Italy. Median patient age was higher for those with GII.17 than GII.4 infection (55 vs. 24 months), suggesting limited cross-protection for older children.

Considering that powdered infant milk formula effectively supports the growth of numerous pathogens, this study investigates the prevalence of potentially pathogenic Bacillus cereus in dried milk products by evaluating the occurrence of B. cereus and the presence of virulenceassociated genes. The approach consisted of enriching, isolating and biochemical identifying isolates, followed by PCR assays aimed at the hbl (C, D, A, B), nhe (A, B, C) and cytK enterotoxin genes coding HBL complex, NHE complex and cytotoxin K, respectively. Among cytKpositive strains, the discrimination of two different forms for cytotoxin K, cytK-1 and cytK-2 was performed. Bacillus cereus was detected in powdered infant milk formula samples. All the strains harbored at least one gene of the cytK, HBL and NHE enterotoxins. Because of an increasing trend in invasive infections by B. cereus in infants and immunocompromised children, our PCR findings highlight the need to implement an adequate control plan in order to guarantee the health of potentially fragile consumers. From a hygiene point of view, intensive and continuous monitoring of potentially pathogenic B. cereus may be crucial for powdered infant milk formula safety and even recommended in order to assess the infant health risk, as proposed by Commission Regulation (EC) no. 1441/2007 on microbiological criteria for foodstuffs. Furthermore, the detection in this study of B. licheniformis, B. subtilis and B. mycoides strains raises significant health issues regarding Bacillus spp. in powdered infant milk formula.

Protothecosis is a potential zoonotic disease associated with bovine mastitis which can be transmitted to humans through contaminated milk. Considering the increasing prevalence of bovine mastitis due to Prototheca species, individual cow milk samples were analyzed using microbiological examination and biomolecular assay. Aspects related to health requirements for milk production, clinical and histological bovine mastitis were also described. The results showed 24/257 (9.3%) culture-positive samples and 42/257 (16.3%) PCR-positive samples. Moreover in 5 cows with somatic cell count over 106/mL presented histological features of mastitis. This study reveals that the presence of Prototheca species in dairy herds was related to the hygienic conditions of the milking equipment, showing an emerging public health issue.

AiV-1 is considered an emerging human enteric pathogens and foodborne transmission has been documented as an important source of exposure for humans, chiefly in relation to non-safe, risky food habits. We surveyed the presence of AiV-1 in retail shellfish, including oysters and mussles, identifying the virus in 3/170 (1.8%) of the analysed samples. The AiV-1 positive samples were of different geographic origin. Upon sequence analysis of a portion of the 3CD junction region, two AiV strains identified from harvesting areas in Northern Italy were characterised as genotype B and displayed 99-100% identity at the nucleotide level to other AiV-1 strains detected in sewages in Central Italy in 2012, suggesting that such strains are stably circulating in Italian ecosystems. Interestingly, a strain identified from mussles harvested in Southern Italy could not be characterised firmly, as inferred in the Bayesian analysis and by sequence comparison, indicating that different AiV strains are also circulating in Italy. Viral contamination in retail shellfish challenges the microbiological guidelines for food control and requires the development and optimization of additional diagnostic and prevention strategies.

Given that changes in consumer food behaviours have led to an increase in the demand for pre-cut ready-to-eat (RTE) vegetables, and that few data are currently available on the occurrence of Arcobacter spp. in such foods, the aim of the present study was to assess the occurrence of Arcobacter spp. that carry virulence-associated genes on pre-cut RTE vegetables, using cultural and molecular methods. Arcobacter was detected using biomolecular iden- tification methods in 44/160 (27.5%) of the samples, of which 40/44 (90.9%) isolates corresponded to A. butzleri and 4/44 (9.1%) to A. cryaerophilus. Studying the incidence of 9 virulence-associated genes revealed the wide- spread distribution of these genes among the Arcobacter isolates tested. The results obtained in our research provided plenty of information on the health risks associated with the direct consumption of raw vegetables, and highlight the need to implement further studies at each level of the produc- tion chain, in order to obtain further information to help protect human health.

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.

Norovirus (NoV) and hepatitis A virus (HAV) are a common cause of gastroenteritis outbreaks associated with consumption of raw shellfish. The majority of NoV infections worldwide are due to geno group II NoVs. The predominant HAV strains belong to sub -genotype IB. A total of 369 bivalve molluscs (294 mussels, 42 clams and 33 oysters) from several retail points and harvesting class -A areas of the Adriatic basin in South Italy, North Italy and Albania (Butrinti Lagoon) were sampled between 2008-2013. All the samples were screened by a hemi-nested RT-PCR specific for NoV geno group II and by a nested RT-PCR for the VP1/2A region of HAV. NoV RNA was detected in 10,5% of samples and ranged from 3% in 2008 to 85% in 2013. HAV RNA was detected in 32,5% of samples and ranged from 90% in 2008 to 3,1% in 2013. The marked decrease in HAV prevalence may be the related to the vaccine-induced immunity, able to interrupt the ecological cycle of HAV. Monitoring the epidemiology of the virus strains circulating in the field is pivotal to develop and assess the efficacy of new control strategies to reduce the risks for public health

Fish authentication is a major concern not only for the prevention of commercial fraud, but also for the assessment of safety risks deriving from the undeclared introduction of potentially dangerous toxic or allergenic substances or environmentally damaging fish where endangered species are involved. Moreover, food authentication might affect the diet of certain groups of consumers, such as followers of religious practices. Considering the authentication of fish products is one of the key issues in food safety, quality and sustainability, the aim of this work was to investigate the prevalence of mislabelling in sole (Solea solea), plaice (Pleuronectes platessa), Atlantic salmon (Salmo salar), and hake (Merluccius merluccius) fillets from markets and supermarkets located in Apulia (Southern Italy) using DNA barcoding. The results of the molecular investigations reveal that 42/98 (42.8%) fillet samples were not correctly labelled. In particular, 12/27 (44.4%) fillets of sole (Solea solea) were identified as belonging to Solea senegalensis. In addition, 13/28 (46.4%) plaice (Pleuronectes platessa) samples were identified as Pangasius hypophtalmus. All Atlantic salmon (Salmo salar) samples were correctly labelled. Post-sequencing data analysis revealed that 17/30 (56.6%) hake fillets (Merluccius merluccius) were not correctly labelled, of which 8/30 samples identified as Merluccius hubbsi, 5/30 samples as Merluccius products and 4/30 as Merluccius capensis. The study reveals a high occurrence of species mislabelling in the prepared fish fillet products, further evidence of the need for increased traceability and assessment of the authenticity of food products.

Considering that several recent cases of human gastroenteritis have been associated with species from the Arcobacter genus, and that few data are currently available about the occurrence of this genus in Italian shellfish, the aim of the present study was to evaluate the occurrence of Arcobacter spp. and the presence of virulence-associated genes. The approach consisted of cultural and biomolecular (multiplex- PCR and 16S-RFLP) methods identifying isolates, followed by PCR assays aimed at the cadF, ciaB, cjl349, irgA, hecA putative virulence genes. Arcobacter spp. was detected in 16/70 (22.8%) shellfish samples. Specifically, Arcobacter spp. was highlighted in 10/42 (23.8%) mussel and in 6/28 (21.4%) clam samples. Subsequently, biomolecular assays revealed Arcobacter butzleri in 12/16 (75%) and Arcobacter cryaerophilus 1B in 4/16 (25%) isolates. PCRs aimed at the five putative virulence genes demonstrated widespread distribution of these genes among Arcobacter isolates and some differences from the results published by other authors. Our research provides more information regarding the health risks associated with the consumption of raw bivalve molluscs and underlines the need to implement an adequate control plan by performing intensive and continuous monitoring in order to guarantee human health

Hepatitis A virus (HAV) was detected in a batch of imported non-packaged frozen redcurrants purchased in a Bari grocery. Sequence and phylogenetic analysis showed the HAV strain clustered tightly with the HAV strain from the 2013 Italian epidemic, providing additional evidence that frozen redcurrants were the main vehicle of the HAV outbreak.

Given the increase in the international trade of packaged frozen fishery products, this study used DNA barcoding to investigate the breaded hake and plaice species, sold in Italian markets. The results of this study generally matched the ingredient list on the food label. Only 6 of the 120 samples were non-compliant. Specifically, breaded merluccius samples match the species shown in the list of ingredients on the label. Of the ‘‘breaded plaice’’ samples, 4/14 contained Lepidopsetta polyxystra and 2/14 Merluccius gayi, thus failing to match the ingredient list on the label. Considering the European legislation indicates that the label must not mislead consumers, but international trade and the use of similar terms for different products makes it complicated when a product from one country is introduced into another in which the niche already exists, clear labeling is strongly recommended in order to ensure that consumers can make conscious choices.

The health and vigour of honeybee colonies are threatened by numerous parasites (such as Varroa destructor and Nosema spp.) and pathogens, including viruses, bacteria, protozoa. Among honeybee pathogens, viruses are one of the major threats to the health and wellbeing of honeybees and cause serious concern for researchers and beekeepers. To tone down the threats posed by these invasive organisms, a better understanding of bee viral infections will be of crucial importance in developing effective and environmentally benign disease control strategies. Here we summarize recent progress in the understanding of the morphology, genome organization, transmission, epidemiology and pathogenesis of eight honeybee viruses: Deformed wing virus (DWV) and Kakugo virus (KV); Sacbrood virus (SBV); Black Queen cell virus (BQCV); Acute bee paralysis virus (ABPV); Kashmir bee virus (KBV); Israeli Acute Paralysis Virus (IAPV); Chronic bee paralysis virus (CBPV). The review has been designed to provide researchers in the field with updated information about honeybee viruses and to serve as a starting point for future research.