Preliminary survey on the occurrence of microplastics in bivalve mollusks marketed in Apulian fish markets.

Microplastics (MPs) are a relevant threat to food safety because they are ingested by humans through various foods. Bivalves are at high risk of microplastic contamination due to their filter-feeding mechanism and pose a risk to consumers as they are ingested whole. In this work, microplastics were detected, quantified, identified, and classified in samples of mussels (Mytilus galloprovincialis) and oysters (Crassostrea gigas) marketed in the Apulia region. The total number of plastic debris was 789 particles in the mussel samples and 270 particles in the oyster samples, with size ranging from 10 to 7350 µm. Fragments with size within the category of 5-500 µm were the predominant findings in both species, with blue as the predominant color in mussels and transparent in oysters; most of the debris was polyamide and nylon polymers in the mussels and chlorinated polypropylene in the oysters. These results show that mussel and oyster samples purchased at fish markets are contaminated with microplastics. The sources may be diverse and further studies are needed to assess the impact of the marketing stage on microplastic contamination in bivalves to better define the human risk assessment associated with microplastic exposure from bivalves consumption.

DNA metabarcoding for identification of species used in fish burgers.

The absence of morphological identification characters, together with the complexity of the fish supply chain make processed seafood vulnerable to cases of species substitution. Therefore, the authentication and the traceability of such products play a strategic role in ensuring quality and safety. The aim of the present study was to detect species used in the production of multi-species fish burgers and to evaluate mislabelling rates, using a DNA metabarcoding approach by sequencing a fragment of the 16S rRNA mitochondrial gene. The study highlighted the presence of 16 marine and 2 mammalian taxa with an overall mislabelling rate of 80%, including cases of species substitution, the undeclared presence of molluscs and of taxa whose use is not permitted by current Italian legislation. The presence of swine DNA as well as the inclusion of undeclared taxa potentially causing allergies raise concerns regarding consumer safety and protection regarding ethical or religious issues. Overall, the study shows that the application of DNA metabarcoding is a promising approach for successfully enforcing traceability systems targeting multi-species processed food and for supporting control activities, as a guarantee of an innovative food safety management system.

Occurrence of hepatitis E virus (HEV) in Calabrian wild boars.

Hepatitis E virus (HEV) is an emerging pathogen in industrialized countries. HEV infections in humans are mainly related to the HEV-3 genotype, predominant in Europe and widespread in wild boars' food products. However, there are little relevant data around HEV prevalence in wild boars, although they are considered the main HEV reservoir and used for typical food products such as liver sausages. Our study aimed to assess HEV occurrence and genetic variability in Calabrian wild boars hunted in the central and ionic area of Catanzaro's province. A total of 86 wild boar liver samples were analyzed showing an overall HEV RNA prevalence of 26.7% (23/86). All positive samples were characterized molecularly as genotype 3 and predicted as HEV-3c subtype despite the shortness of fragment employed for the molecular analysis. This data is in line with previous studies conducted in Europe highlighting the public health concern of these results. Biomolecular methods performed in our study detected only the HEV RNA positivity of analyzed samples without information about the virus viability. Consequently, it is not possible to fully estimate the risk related to the consumption of wild boar's liver sausages or wild boar meat products. Our results highlight the need for further studies in order to investigate the virus viability and to link wild boar's meat consumption with HEV human seroprevalence in Italian regions (Abruzzo, Lazio, Campania and Calabria) where typical wild boar's products are consumed. In this way, the Competent Authorities could perform a complete risk assessment, implement risk management and establish proper measures to ensure the public health and prevent relative human disease.

Occurrence and characterization of Arcobacter spp. from ready-to-eat vegetables produced in Southern Italy.

Given that the number of foodborne illness outbreaks linked to the consumption of ready-to-eat vegetables has been widely documented and considering that data on the occurrence of Arcobacter spp. in such foodstuffs are lacking, the aim of the present study was to evaluate the presence of Arcobacter spp. and the occurrence of virulence factors as well as to genotype Arcobacter spp. in ready-to-eat (RTE) vegetable samples, using cultural and biomolecular assays. Arcobacter spp. was detected in 16/110 (14.5%) samples, with A. butzleri being detected in 15/16 and A. cryaerophilus in 1/16 isolates. PCRs aimed at the nine putative virulence genes demonstrated widespread distribution of such genes among A. butzleri and A. cryaerophilus isolates. In addition, multilocus sequence type (MLST) analysis revealed a low genetic diversity within the arcobacters isolates. The results underline the need to develop an appropriate surveillance system based on biomolecular characterization for an integrated microbiological risk assessment of ready-toeat vegetables, and consequently of composite foods.

Short communication: Detection of undeclared presence of bovine milk in buffalo yogurt.

The authenticity of buffalo (Bubalus bubalis) dairy products is a focal issue, considering the increasing demand for buffalo milk products. Therefore, the aim of this study was to investigate the undeclared presence of bovine (Bos taurus) milk in buffalo yogurt, to understand which risk factors might make the product vulnerable to fraud. Real-time PCR assay showed the undeclared presence of bovine DNA in addition to buffalo DNA in 18 of 72 samples. Given the widespread lack of data on the presence of undeclared milk species in buffalo dairy products, the study provides a significant insight into the incidence of fraud in the buffalo dairy field. The data from this study could help improve the analysis of food safety risks along the buffalo milk supply chain and in the dairy processing industry, perceived as being highly vulnerable to food fraud, and prioritize target areas for food policy making to steer and enforce European food fraud regulations.

Determining the Authenticity of Shark Meat Products by DNA Sequencing.

Given that the global shark meat market is poised to grow in future years, the aim of this study was to use DNA sequencing of the cytochrome c oxidase I (COI) and NADH dehydrogenase subunit 2 (NADH2) mitochondrial genes to examine the market of shark meat products in Italy. This made it possible to analyze patterns of species utilization and commercialization of threatened, endangered and/or prohibited species, focusing on fraudulent activities in the shark food chain in order to propose seafood safety and environmental sustainability solutions. The study shows that the labeling of shark meat products generally lacks comprehensive information, thus making it difficult for consumers to make informed purchasing decisions and fails to comply with European Union (EU) legislation regarding seafood labelling. Molecular investigation reveals a high mislabeling rate (45.4%), highlighting widespread use of cheaper species either in order to replace species that are better known and more popular, or else in order to sell various threatened species. Considering that seafood mislabeling can circumvent the management of sustainable fisheries and facilitate Illegal, Unreported and Unregulated (IUU) fishing, the routine use of genetic analysis should be encouraged among control and enforcement agencies in order to implement effective management measures. This would help to build a species-specific reporting system for all catches, and enhance control measures, in order to prevent illegal activities connected with shark catches and trade around the world.

Evaluation of the animal welfare during religious slaughtering.

Plasma cortisol and its metabolites are physiological indicators for stress assessment and slaughtering method may affect their levels, playing an important role in the correct acidification of meat. The aim of the study was to determine and compare plasma cortisol values in animals slaughtered using traditional procedures, which include stunning (using captive bolt pistol), with those in animals slaughtered using Halal method, which does not involve stunning. The study was carried out on a total of 60 Charolais male beef cattle of eight months of age, bred in free paddock outdoors. The animals were divided into two experimental groups, each consisting of 30 individuals, on the basis of the slaughtering method, i.e. traditional or Halal, to verify the whole production chain and to ensure that the product conformed to Muslim rules. Plasma cortisol levels (detected by Elisa test) were evaluated at two different times of animal productive life: on the farm, one week before slaughter (T0) and during bleeding (T1). The 30 calves slaughtered after stunning showed plasma cortisol values of 4.06±1.94 and 43.72±12.09 nmol/L, respectively on the farm and during exsanguination. Conversely, the average values found in the 30 calves subjected to ritual slaughter were 3.26±1.01 and 88.81±41.02 nmol/L. The study demonstrated that throughout the animal's productive life (from pasture to slaughter) the greatest variation between slaughter with and without stunning was observed during bleeding.

Phenotype and genomic background of Arcobacter butzleri strains and taxogenomic assessment of the species.

In this study the phenotypic and genomic characterization of two Arcobacter butzleri (Ab) strains (Ab 34_O and Ab 39_O) isolated from pre-cut ready-to-eat vegetables were performed. Results provided useful data about their taxonomy and their overall virulence potential with particular reference to the antibiotic and heavy metal susceptibility. These features were moreover compared with those of two Ab strains isolated from shellfish and a genotaxonomic assessment of the Ab species was performed. The two Ab isolated from vegetables were confirmed to belong to the Aliarcobacter butzleri species by 16S rRNA gene sequence analysis, MLST and genomic analyses. The genome-based taxonomic assessment of the Ab species brought to the light the possibility to define different subspecies reflecting the source of isolation, even though further genomes from different sources should be available to support this hypothesis. The strains isolated from vegetables in the same geographic area shared the same distribution of COGs with a prevalence of the cluster "inorganic ion transport and metabolism", consistent with the lithotrophic nature of Arcobacter spp. None of the Ab strains (from shellfish and from vegetables) metabolized carbohydrates but utilized organic acids and amino acids as carbon sources. The metabolic fingerprinting of Ab resulted less discriminatory than the genome-based approach. The Ab strains isolated from vegetables and those isolated from shellfish endowed multiple resistance to several antibiotics and heavy metals.

Genomic Characterization of Arcobacter butzleri Isolated From Shellfish: Novel Insight Into Antibiotic Resistance and Virulence Determinants.

Arcobacter (A.) butzleri is an emerging pathogenic microorganism, whose taxonomy has been recently suggested to be emended to the Aliarcobacter (Al.) butzleri comb. nov. Despite extensive taxonomic analysis, only few fragmented studies have investigated the occurrence and the prevalence of virulence and antibiotic resistance determinants of this species in strains isolated from shellfish. Herein we report for the first time the whole genome sequencing and genomic characterization of two A. butzleri strains isolated from shellfish, with particular reference to the antibiotic, heavy metals and virulence determinants. This study supported the taxonomic assignment of these strains to the Al. butzleri species, and allowed us to identify antibiotic and metal resistance along with virulence determinants, also additional to those previously reported for the only two A. butzleri strains from different environments genomically characterized. Moreover, both strains showed resistance to ß-lactams, vanocomycin, tetracycline and erythromycin and susceptibility to aminoglycosides and ciprofloxacin. Beside enlarging the availability of genomic data to perform comparative studies aimed at correlating phenotypic differences associated with ecological niche and geographic distribution with the genetic diversity of A. butzleri spp., this study reports the endowment of antibiotic and heavy metal resistance and virulence determinants of these shellfish-isolated strains. This leads to hypothesize a relatively high virulence of A. butzleri isolated from shellfish and prompt the need for a wider genomic analysis and for in vitro and in vivo studies of more strains isolated from this and other ecological niches, to unravel the mechanism of pathogenicity of this species, and the potential risk associated to their consumption.

Arcobacter spp. in bovine milk: An emerging pathogen with potential zoonotic risk.

The aim of the present study was to assess the prevalence and genetic characteristics of Arcobacter spp. in bovine bulk tank milk produced in Apulia Region (Italy). Samples collected from 396 dairy farms, after enrichment in a selective broth, were subjected to an Arcobacter genus - specific Real Time PCR. Positive broths, previously filtered, were seeded on Karmali, MCCD and Columbia Blood Agar plates; presumptive Arcobacter spp. colonies were identified using an amplification and sequencing method and then characterized by Multi-Locus Sequence Typing (MLST). Prevalence of Arcobacter spp. in bovine milk samples was 5% (20/396); A. butzleri was the only isolated species, in agreement with previous studies that reported A. butzleri as the most commonly recovered species in milk and dairy products. MLST analysis of the 20 A. butzleri strains identified 81 alleles and 16 STs. Consistent with previous studies, MLST revealed a high level of heterogeneity between the A. butzleri isolates and confirmed the high discriminatory power of this method and its suitability for epidemiological investigations. This study confirmed the importance of raw milk as a possible source of Arcobacter spp. for humans.

DNA-based approach for species identification of goat-milk products.

Considering that mislabeled milk products have been widely reported throughout the world and that the authentication of food components is one of the key issues in food safety and quality, the aim of this study was to use DNA-based methods to investigate the prevalence of mislabeling among goat-milk products and, consequently, how far the ingredients matched the labels. The study reveals a high degree of species mislabeling in milk products (80%), underlining the need to enhance dairy traceability practices, so as to guarantee product authenticity, and provide reliable information to consumers.

Kosher slaughter paradigms: Evaluation of slaughterhouse inspection procedures.

Slaughter by Jewish religious rite is the killing of an animal by cutting the trachea and oesophagus and major blood vessels using a very sharp blade. This operation is subject to strict rules laid down by religious authorities that characterize its sacredness. The aim of the study was to evaluate the specific criteria inherent in the Jewish religious rite, by analysing reject rates during the different phases. In this study, 52.4% of the carcasses failed to quality as Kosher, with 22.9% being rejected due to pulmonary lesions and only 3% for miscuts. The study also revealed legal vacuums in the field of labelling rules.

Occurrence of emerging food-borne pathogenic Arcobacter spp. isolated from pre-cut (ready-to-eat) vegetables.

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 identification 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 widespread 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 production chain, in order to obtain further information to help protect human health.

Occurrence of potentially pathogenic arcobacters in shellfish.

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.

Packaged frozen fishery products: species identification, mislabeling occurrence and legislative implications.

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.

Occurrence of Mislabelling in Prepared Fishery Products in Southern Italy.

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.

Occurrence of mislabeling in meat products using DNA-based assay.

Considering that the authentication of food contents is one of the most important issues for the food quality sector, and given the increasing demand for transparency in the meat industry followed the horsemeat scandal in Europe, this study investigates processed-meat products from Italian markets and supermarkets using the mitochondrial cytochrome b gene qualitative PCR identification system in order to verify any species substitution or mislabeling. The results revealed a high substitution rate among the meat products, highlighting a mislabeling rate of 57 %, and consequently, considerable discordance with the indications on the labels, which raises significant food-safety and consumer-protection concerns.

Detection of Arcobacter spp. in Mytilus galloprovincialis Samples Collected from Apulia Region.

The aim of the study was to evaluate the occurrence of Arcobacter spp. in 20 samples of Mytilus galloprovincialis purchased at fish markets in Apulia region. The detection of Arcobacter spp. was performed, after selective enrichment, on modified charcoal cefoperazone deoxycholate (mCCD) agar supplemented with Cefoperazone, Amphotericin B and Teicoplanin (CAT). In 6 out of the 20 tested samples the presence of Arcobacter spp. was found and confirmed by genus-based polymerase chain reaction. All the isolates were identified as belonging to the species Arcobacter butzleri using 16S rDNA sequencing and BLAST online. The results represent the first report in Italy of A. butzleri detection in marketed Mytilus galloprovincialis. The survey underlines the epidemiological importance of A. butzleri as an emerging pathogen, and highlights that mussels should be considered as a potential cause of foodborne disease outbreak.

Occurrence of Prototheca spp. in cow milk samples.

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.

Synthesis and aggregation properties of a novel enzymatically resistant nucleoamino acid.

In this work, we describe the synthesis, evaluation of some biological properties, such as DNA- and RNA-binding ability and in sero stability, as well as the supramolecular assembly of a novel nucleoamino acid based on L-spinacine. More particularly, a thymine-containing L-spinacine derivative was synthesized in liquid phase by a simple peptide-coupling procedure. Subsequently, nucleic acid and Cu(2+)-binding ability, as well as self-assembly properties of the novel nucleoamino acid, were investigated by spectroscopy (CD and UV) and laser light scattering which furnished interesting information on the assembly of supramolecular networks based on the peptidyl nucleoside analog. Finally, nucleoamino acid enzymatic stability was studied and a half life of about 7 days was found in the presence of fresh human serum.