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.

Evaluation of the persistence of SARS-CoV-2 (ATCC® VR-1986HK™) on two different food contact materials: flow pack polyethylene and polystyrene food trays.

Even though SARS-CoV-2's primary transmission pathway is person-to-person, the role played by surfaces and food contact materials in carrying viral RNA should be further explored. For this purpose, the study aimed to investigate the persistence of SARS-CoV-2 using the strain ATCC® VR-1986HK™ on flow pack polyethylene (FPP) and polystyrene food trays (PFT). Samples of FPP and PFT were contaminated with heat-inactivated SARS-CoV-2 and were incubated at a temperature of 24 ± 1 °C and at controlled relative humidity (RH 65%). The experimental design included analyses at the time 0, 3, 6, 12, 24, 36, 48 and after every 24 h until the viral RNA was no longer detectable. The results showed a significant decrease (P < 0.001) in viral copy numbers on PFT within 3 h (24% reduction) and, at 72 h, the viral RNA had fallen below the limit of detection. Regarding the FPP, it was necessary to wait 24 h for a significant decrease (P = 0.015) in the viral load (14% reduction), while the detection threshold was reached at 96 h. These findings showed that the viral RNA persists longer on flow pack polyethylene samples than on polystyrene food trays, thus highlighting the importance of material characteristics in the persistence of SARS-CoV-2.

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.

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.

Classification of bovine carcasses: New biometric remote sensing tools.

Slaughtering plants approved by the European Union have specific processes to guarantee that beef carcasses or halfcarcasses, of no less than eight months of age, are provided with health mark and then classified and identified according to the EU carcass classification grid. This classification is based on three criteria: i) Category, ii) Conformation (SEUROP) and iii) Fat Cover (FC). At the end of the classification process each carcass is given a code, consisting of two letters and a number: this operation is called Identification. The aim of our study was to evaluate how the European beef carcass quality classification is determined according to the experience of the personnel involved, then comparing the results with those yielded by the Android platform application. West Systems, through its West- Zootech division, has developed an Android platform application (SEUROP APP) that allows SEUROP and FC classification with a smart-phone. The photo taken with the smart-phone will yield the necessary angular parameters to determine the conformation class depending on the animal's muscular mass and based on the convexity of some areas on the half-carcass. It also evaluates the ratio between surface of lean tissue and total carcass surface in order to determine the fat cover and complete the classification. The SEUROP APP was able to obtain objective measurements for as much as 84% of the assessments made during the research and development phase.

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.

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.

Consumer attitudes towards animal welfare and their willingness to pay.

The interest of European consumers towards animal welfare can be influenced by several variables, both related to the consumers themselves and to the different countries of the EU. In order to assess animal welfare at farm level, it is essential to develop animal-based measures in accordance with the animals' actual welfare state in terms of their behaviour, health and physiology. The search for valid and reliable indicators is a key objective of several research programs especially for assessing welfare at farm level and the tools may include surveys addressed to farmers. However, there is a need to guarantee financial support for farmers who breed animals in accordance with such welfare conditions, to cover their additional costs. The aim of the study was to investigate the eating habits of Italian consumers regarding meat consumption linked to their knowledge of animal welfare and to their willingness to pay. We investigated consumers' understanding of animal welfare using a questionnaire (based on a list of twenty-three closed-ended questions) designed for collecting data from large numbers of respondents and multivariate statistical analysis. The data in our study showed that the variable with the greatest influence on purchase price was the place of meat purchase. As regards level of education, it appears that people with a high level of education are more concerned about animal welfare and, consequently, are willing to spend a higher price when buying meat. Consumer attention to the animal-welfare issue is on the rise and, in parallel with this growth, there is also a greater willingness to pay, i.e. a surcharge for the products obtained in the respect of animal welfare. This growth is influenced by the awareness and knowledge of the characteristics of animal welfare.

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.

Presence of cadmium residues in muscle, liver and kidney of Bubalus bubalis and histological evidence.

Cadmium (Cd) concentrations were evaluated in the samples of kidney, liver and muscle of sixty-six buffaloes regularly slaughtered. Forty were raised in Campania, in the territory between the province of Naples and Caserta and twenty-six were bred in Apulia, in the province of Bari. Two aliquots were prepared for the renal and hepatic samples: one intended for the chemical analysis and the other one intended for histological investigations. Muscle samples were the subject of purely chemical investigation. In the group of forty animals raised in the Campania region, the limits imposed by EC Reg. 1881/2006 and EC Reg. 488/2014 were exceeded in three renal samples, which showed values of 1.53, 1.22 and 1.1 mg/kg respectively; in three hepatic samples, which presented values of 0.72, 0.64 and 0.61 mg/kg, and in five muscle samples, with values of 0.16, 0.16, 0.09, 0.08 and 0.07, respectively. On the other hand, in the group of animals raised and slaughtered in the province of Bari, none of the twenty-six samples examined exceeded the limits imposed by the European regulations. The histological analysis showed typical, but not pathognomonic lesions in the renal samples from the animals raised in the provinces of Naples and Caserta. The levels of Cd contamination found in the samples examined suggest that it would be correct to exclude from the human consumption, as it happens for the equines, the kidney and the liver, especially from the animals raised in some geographical areas with a high rate of pollution.

Occurrence of Aichi virus in retail shellfish in Italy.

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.

Analysis of Stress Indicators for Evaluation of Animal Welfare and Meat Quality in Traditional and Jewish Slaughtering.

Sixty Charolais male beef cattle of eight months of age were divided into two groups according to the slaughtering method, i.e., traditional or Kosher (religious Jewish rite). The aim of the study was to detect and compare the plasma concentrations of cortisol and catecholamines (dopamine, norepinephrine and epinephrine), by Elisa and HPLC test. These four stress indicators were evaluated during three different stages of each animal productive life: on the farm (step 1), after transportation (step 2) and during bleeding (step 3). The patterns of the parameters measured were similar and, interestingly, revealed significant changes throughout the three steps considered. The greatest variation between the two methods of slaughtering was observed in step 3, where we found a statistically significant difference with all the parameters except epinephrine. In the animals slaughtered by the religious rite, cortisol, dopamine, norepinephrine and epinephrine were 68.70 ± 30.61 nmol/L; 868.43 ± 508.52 ng/L; 3776.20 ± 1918.44 ng/L; and 4352.20 ± 3730.15 ng/L, respectively, versus 45.08 ± 14.15 nmol/L; 513.87 ± 286.32 ng/L; 3425.57 ± 1777.39 ng/L; and 3279.97 ± 1954.53 ng/L, respectively, in the other animals. This suggests that the animals slaughtered by the Kosher rite are subjected to higher stress conditions at the exsanguination phase. The animals slaughtered by the religious Jewish rite showed lower cortisol and catecholamine levels on the farm (step 1) and after transportation to the slaughterhouse (step 2). This was likely because the animals selected at the end of step 1 by the Rabbis for the religious rite are usually the most docile and gentle.

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.

Determination of plasmatic cortisol for evaluation of animal welfare during slaughter.

The plasmatic cortisol levels of 60 eight-month-old calves (Charolais breed) were measured as stress indicators resulting from two types of slaughter: traditional and religious rite. The plasmatic cortisol levels were evaluated during three different stages of their productive life: during growth, after transport and during slaughter. The thirty calves slaughtered after stunning showed plasmatic cortisol values of 4.85±3.2; 36.36±12.2 and 45.08±14.1 nmol/L, during growth, in the slaughterhouse stables and during exsanguination, respectively. Conversely, the average values found in the thirty calves subjected to ritual slaughter were 2.96±1.2; 31.65±25.4 and 68.70±30.6 nmol/L. The results of the study showed that animal welfare should be improved in both forms of slaughter.

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.