The EU Interreg Project "ADRINET": Assessment of Well-Known and Emerging Pollutants in Seafood and Their Potential Effects for Food Safety.

Anthropogenic activities lead to the spread of chemicals and biological materials, including plastic waste, toxic metals, and pharmaceuticals, of which the impact on the Mediterranean Sea is of high concern. In this context, the EU Interreg Italy-Albania-Montenegro Project "ADRINET (Adriatic Network for Marine Ecosystem) _244" (2018-2020) arises. It aims to carry out biomonitoring campaigns in the main commercial interest of fish and cephalopod species, such as Sparus aurata, Dicentrarchus labrax, Sepia spp., and Loligo spp. sampled in three different subregions of the Mediterranean Sea. The presence of the main environmental contaminants, such as cadmium, microplastics, and antibiotics was investigated in these seafood samples. Contamination by cadmium and antibiotics in the seafood investigated in our study was negligible. However, a high value of microplastics was detected in the stomach and gut of Sparus aurata and Dicentrarchus labrax. Overall, even though the presence of microplastics needs to be investigated by further studies, the results confirmed that the environmental conditions of the three bays investigated by the ADRINET project partners (Italy, Albania, Montenegro) are positive and not affected by intensive anthropogenic activity.

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.

Determination of antibiotic residues in honey in relation to different potential sources and relevance for food inspection.

Honey contaminations could derive from intensive agriculture and industrial activities, but also from beekeeper treatments. In EU no MRLs for antibiotics in honey are set, only a minimum required performance limit for chloramphenicol of 0.3 µg kg-1 is recommended. Screening tests are available, characterised by their rapidity and simple use. Due to their high rate of false positives and the need to meet zero tolerance levels for antibiotics, their presence in samples was investigated using a liquid chromatography High Resolution Mass Spectrometry (LC-HRMS) multiclass antibiotic residue method, comparing the results with those of previous screening tests. The confirmatory method showed good sensitivity: CCα and CCß ranging from 0.03 to 4.80 ng g-1 and from 0.12 to 5.56 ng g-1, respectively. Ninety-eight honey samples from different geographical areas, analysed by two screening tests, showed a high percentage of false positives. This is fundamental to guarantee honey safety, especially, for organic production.

Pesticides and Environmental Contaminants in Organic Honeys According to Their Different Productive Areas toward Food Safety Protection.

Monitoring contaminant residues in honey helps to avoid risks to human health, as it is a natural product widely consumed in all population groups, including the most vulnerable, such as children and the elderly. This is important for organic honey production that may be negatively influenced by geographical area pollution. Considering the importance of collecting data on the occurrence of various xenobiotics in different geographical areas, this study aimed to investigate the presence of contaminant residues (persistent organic pollutants (POPs) and pesticides, including glyphosate and metabolites) in organic honey samples from different production areas using different analytical methods, in order to confirm their incidence and possible impact on the food safety traits of organic production. Regarding POPs, traces of benzofluoroanthene and chrysene were detected in honey from intensive orchards and arable lands. Traces of all polychlorobiphenyl (PCB) congeners were detected at different percentages in almost all of the samples, regardless of the origin area. Traces of polybromodiphenylethers (PBDE 28, 33, and 47) were found in different percentages of samples from all of the geographical areas examined. Traces of organochlorines (OCs) and organophosphates (OPs) were identified in honey samples belonging to all of the geographical areas. No glyphosate, glufosinate, and aminomethylphosphonic acid (AMPA) residues were detected.

Pilot Study of the Relationship between Deck Level and Journey Duration on Plasma Cortisol, Epinephrine and Norepinephrine Levels in Italian Heavy Pigs.

The aim of this pilot study was to evaluate the relationship between journey duration, deck level and activation patterns of the hypothalamic-pituitary-adrenocortical axis (HPA) and sympathetic adrenal medullary system (SAM) in pigs. A total of 90 pigs were examined. The animals came from three different Italian farms associated with the same slaughterhouse located in Bari (Apulia region-Italy). A group of thirty animals was transported from Pordenone (11 h journey); a second group was transported from Terni (6.5 h journey); a third group was transported from Benevento (3 h journey). The animals were transported in the same vehicle, which complied with the structural characteristics indicated in the Council Regulation (EC) No. 1/2005. The truck was composed of a lorry and a trailer, each one divided into three decks. Only the animals transported in the trailer were tested for the study. Before transportation, blood samples were collected on each farm, at 6:00 a.m., from 30 pigs randomly selected out of 135 pigs ready to be transported. Blood samples were also collected during slaughter to evaluate plasma cortisol, epinephrine and norepinephrine, around 6:00 a.m. A journey duration of 11 h was associated with significantly higher plasma concentrations of stress hormones compared with shorter journeys. This increase was proportional to the journey duration, with the pigs travelling for 6.5 h displaying intermediate concentrations between those noticed after 3 h and 11 h journeys. The interaction between deck and journey distance was not significant on epinephrine, norepinephrine or cortisol levels collected at arrival. There was a significant effect of deck level on norepinephrine levels (p < 0.0001), a tendency to influence epinephrine levels (p = 0.073) but no effect on cortisol levels (p = 0.945). Overall, we observed that an 11 h-long journey seemed to impact negatively on pigs' HPA-SAM activity, likely requiring the animals to spend more time in the lairage facilities to recover.

Evaluation of the Occurrence of False Aneurysms During Halal Slaughtering and Consequences on the Animal's State of Consciousness.

This study evaluated the occurrence of false aneurysms and reflexes in bovines, associated with the point along the neck where the cut was performed. The survey was carried out on a total of 1200 male beef cattle, belonging mainly to the Charolais and Limousin breeds, aged between twelve and twenty-four months. In the slaughterhouse, three operators, identified by convention as Operator C4, Operator C2, and Operator C1, performed the Halal slaughtering. Each operator carried out the slaughter of 400 animals and, on the basis of his own professional training, performed the cutting of the vessels at different points along the neck: Operator C4 between the third and fourth cervical vertebrae; Operator C2 at the level of the second cervical vertebra; and Operator C1 at the first cervical vertebra. The occurrence of false aneurysms was assessed on the basis of the different cutting site used by the three operators. Then, the evaluation of consciousness indicators, that is, rhythmic breathing and eye response, closely related to a slow bleeding process and to a delayed loss of brain function, was carried out 90 s post-bleeding. The group of cattle slaughtered by Operator C4 had a prevalence of false aneurysms of 10.25%. Conversely, the other two groups of cattle slaughtered by Operators C2 and C1 showed false aneurysm formation in 7.25% of each case. Further, 37.5% of the animals (18 for Operator C4, 17 for Operator C2, and 16 for Operator C1) with signs of consciousness 90 s after sticking were consequent to the onset of false aneurysms and, more precisely, they were owing to a late second cut of the vessel carried out by the operator, when false aneurysms occurred. The results of the research showed that the cutting point chosen by the operator is a key element in determining the onset of false aneurysms.

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.

Occurrence of antibiotic residues in Apulian honey: potential risk of environmental pollution by antibiotics.

The presence of antibiotic residues in honey is widely documented and is attributed almost exclusively to improper beekeeping practices, due to the frequent use of drugs for the treatment of beehive diseases. Therefore, the aim of our research was to evaluate the presence of antibiotics in honeycomb using the Anti-Microbial Array II (AM II) and IV (AM IV) method and to assess the relationship between environmental context and antibiotic residues in honey. The results show the presence of antibiotic residues in 26/50 honey from brood nests samples, confirming the impact of environmental contamination on the health quality of this food product. In addition, subsequent analyses conducted on positive samples reveal the instability over time of antimicrobial molecules in honey. These results highlight the need for further studies in order to understand all likely sources of contamination and to implement a comprehensive safety management plan for honey.

Electrochemical Preparation of Synergistic Nanoantimicrobials.

The rapid spreading of resistance among common bacterial pathogens towards the misused antibiotics/disinfectant agents has drawn much attention worldwide to bacterial infections. In light of this, the present work aimed at the realization of core-shell nanoparticles possessing remarkable antimicrobial properties thanks to the synergistic action of the metal core and the disinfectant shell. Copper nanoparticles stabilized by benzalkonium chloride were prepared, characterized, and implemented in poly-vinyl-methyl ketone to obtain nanoantimicrobial composite coatings. Bioactivity tests are reported, proving the excellent disinfectant properties of the proposed nanomaterials, as compared to one of the well-known and strongest silver-based nanoantimicrobials. Applications are also briefly described.

Simultaneous Quantitative Detection of Six Families of Antibiotics in Honey Using A Biochip Multi-Array Technology.

Chemical residues of veterinary drugs such as streptomycin, chloramphenicol, macrolides, sulphonamides, tetracyclines, quinolones and aminoglycosides and other contaminants such as pesticides and heavy metals have been found in honey, leading to concerns for human health. Indeed, there is a growing interest in their presence and persistence in the environment because low levels of antibiotics may favour the proliferation of antibiotic-resistant bacteria. Moreover, antibiotics present in honey may produce residues in foodstuffs, causing adverse effects on humans such as allergic reactions, toxic effects and damage to the central nervous systems. For food and health/safety reasons, antibiotic drugs are not authorized for the treatment of honey bees in the EU, even though these antimicrobial drugs have been approved in many third-party countries. For this reason, contaminated honey products can still be found in European markets. Therefore, there is a need to develop a precise, accurate and sensitive analytical method that may be used to simply and rapidly detect these compounds in honey. The aim of our study was to detect the presence of antibiotics in Apulian honey using the Anti-Microbial array II (AM II) as an innovative screening method to test the health quality of honey and honey products.

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.

Investigation of Industrial Polyurethane Foams Modified with Antimicrobial Copper Nanoparticles.

Antimicrobial copper nanoparticles (CuNPs) were electrosynthetized and applied to the controlled impregnation of industrial polyurethane foams used as padding in the textile production or as filters for air conditioning systems. CuNP-modified materials were investigated and characterized morphologically and spectroscopically, by means of Transmission Electron Microscopy (TEM), and X-ray Photoelectron Spectroscopy (XPS). The release of copper ions in solution was studied by Electro-Thermal Atomic Absorption Spectroscopy (ETAAS). Finally, the antimicrobial activity of freshly prepared, as well as aged samples-stored for two months-was demonstrated towards different target microorganisms.

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.

Genetic heterogeneity and recombination in type-3 human astroviruses.

Human astroviruses (HAstVs) are important enteric pathogens and can be classified genetically and antigenically into eight types. During molecular surveillance for HAstVs in Italy, sequence analysis of the diagnostic region C (about 400 nucleotide in length), located on the capsid (ORF2) gene, identified a novel type-3 strain. Upon sequencing of the full-length ORF2, the type-3 HAstV strain was characterized as a novel ORF2 genetic lineage, designated as 3c. By converse, in the ORF1b the virus was more similar to type-1 HAstVs, rather than to type-3 strains, suggesting a recombination nature, with the crossover site being mapped to the ORF1b/ORF2 junction region. Region C sequences of similar type-3 HAstV identified from European and extra-European countries were retrieved in the databases, suggesting the global distribution of this novel type-3 lineage.