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Because the world's wild fish stocks are limited and the market demand is increasing, fish farming has become an alternative food source and a way to reduce costs for consumers. The sale of farmed as wild fish is a fraudulent practice; it is, therefore, important to find new and alternative tools that can help in the fight against fraud to protect consumers and to ensure food traceability. The proteomic profiles of farmed and wild fish differ. With this study we wanted to identify liver protein markers via two-dimensional electrophoresis that would allow us to distinguish wild from farmed gilthead seabream. The liver samples from 32 gilthead seabream, wild and farmed, were stored at −80°C before protein extraction. The samples were subjected to two-dimensional electrophoresis to detect qualitative and quantitative differences. Proteomic analysis showed a protein spot (molecular weight of ∼34 kDa and isoelectric point of ∼6.9) only in the samples from the wild gilthead seabream; liquid chromatography–tandem mass spectrometry identified the spot as ubiquitin. Ubiquitin could be a valid marker to differentiate wild from farmed gilthead seabream; it could be used to ensure continuous monitoring throughout the entire commercial chain and to fight commercial fraud. A proteomic approach to differentiate the method of fish production is proposed. A 2-DE spot, characterized by MS/MS as ubiquitin, differentiated between samples. Liver proteome could be useful to find biological markers. [ABSTRACT FROM AUTHOR]

Abstract Fighting food frauds is a ceaseless challenge because of the constant evolution of fraudulent practices and for the consequences both on consumers' and on globalized trade. In Italy fish is a vulnerable commodity for frauds thanks to the high national production, importation and consumption and it is important to monitor the entire food chain in order to detect and prevent fraudulent actions, such as species substitutions, which is considered the most common fraud in seafood. Aim of this study was to realise a targeted Monitoring Plan to estimate the prevalence of fish species substitutions in Italy. As a first step, Italian fish supply chain, from production to selling, was analysed, in order to identify products and chain points at risk, by reviewing literature and by involving, in two focus groups, food inspectors and representatives of the large scale food distribution system. Then a monitoring plan was designed by sampling three fish species considered at major risk for their economic value and/or large consumption, i.e. tuna, grouper and flat fishes, at different selling points (wholesale markets, retail markets, fish shops and supermarkets). From February to March 2017, 242 samples from fresh, frozen or transformed whole fishes or fillets were sampled in 13 Italian cities (5 in Northern Italy, 2 in Central Italy, 3 in Southern Italy and 3 in the main Islands). Samples were analysed by "FINS" (Forensically Informative Nucleotide Sequencing), using two markers: cytochrome oxidase subunit I gene (COI) as first option and then mitochondrial cytochrome B gene (cytb), if necessary to identify uncertain or unassigned samples. Species substitutions were uncovered in 8.7% of analysed samples, principally related to grouper (prevalence of 14.71%). Supermarkets resulted the selling points with a major number of frauds (prevalence of 12.79%). Substituted species were taxonomically related to those declared on the label and no species harmful for consumers were detected. Results obtained can give to National Authorities a detailed frame of trends in fish substitution frauds in Italy, providing also relevant information to put into effect control measures. Highlights • Targeted Italian Monitoring Plan to estimate the prevalence of fish substitution frauds in Italy. • Results of the first National Monitoring Plans on fish substitutions. • Trends in fish substitution frauds in Italy giving information to put into effect control measures. [ABSTRACT FROM AUTHOR]

Sudan dyes are synthetic azo dyes used by industry in a variety of applications. Classified as carcinogenic, they are not allowed in foodstuffs; however, their presence as adulterants in food products has been regularly reported. Here, we describe an innovative screening method to detect Sudan I, II, III, and IV in tomato sauce, palm oil, and chilli powder. The method entails minimal sample preparation, completely avoiding the liquid chromatography phase, followed by detection and identification through atmospheric pressure chemical ionization time-of-flight mass spectrometry, in positive ionization mode. Analytes were efficiently identified and detected in samples, fortified both with individual analytes and with their mixture, with an error in mass identification less than 5 ppm. Limits of identification of the analytes in the fortified samples were 0.5 to 1 mg/kg, depending on the dye and matrix. The method had a linear range of 0.05 to 5 mg/kg and good linear relationships (R2 > 0.98). Repeatability was satisfactory, with a coefficient of variation lower than 20%. The method was applied to detect the dyes in real adulterated chilli samples, previously found positive by confirmatory high-performance liquid chromatography–mass spectrometry and ELISA, and in commercial products purchased from supermarkets. In all positive samples, analytes were correctly identified with an error in mass identification lower than 5 ppm, while none of the 45 commercial samples analyzed were found to be contaminated. The proposed new assay is sensitive, with a limit of identification, for all the three matrices, complying with the limits defined by the European Union (0.5 to 1 mg/kg) for analytical methods. Compared with conventional methods, the new assay is rapid and inexpensive and characterized by a high throughput; thus, it could be suitable as screening technique to identify Sudan dyes in adulterated food products. [ABSTRACT FROM AUTHOR]

Abstract: The partial PrP gene sequence and the deduced protein of eight cetacean species, seven of which have never been reported so far, have been determined in order to extend knowledge of sequence variability of the PrP genes in different species and to aid in speculation on cetacean susceptibility to prions. Both the nucleotide and the deduced amino acid sequences have been analysed in comparison with some of the known mammalian PrPs. Cetacean PrPs present typical features of eutherian PrPs. The PrP gene from the species of the family Delphinidae gave identical nucleic acid sequences, while differences in the PrP gene were found in Balaenopteridae and Ziphidae. The phylogenetic tree resulting from analysis of the cetacean PrP gene sequences, together with reported sequences of some ungulates, carnivores and primates, showed that the PrP gene phylogenesis mirrors the species phylogenesis. The PrP gene of cetaceans is very close to species where natural forms of TSEs are known. From an analysis of the sequences and the phylogenesis of the PrP gene, susceptibility to or occurrence of prion diseases in cetaceans can not be excluded. [Copyright &y& Elsevier]

Seafood freshness is a quality key parameter due to the short shelf life of this highly perishable foodstuff in relation to safety, nutritional value, availability, and edibility. A variety of physical, microbiological, and chemical methods are used for assessing and predicting seafood quality. Chemical analyses to assess seafood freshness quality are based on the determination of substances like volatile amines that are structurally similar to ammonia and responsible for the fishy odour fish give off several days after catch. Here we report the results of a method to detect and quantify trimethylamine (TMA) by means of direct sampling analysis (DSA) coupled with a high-resolution mass spectrometer with a time of flight detector (TOF). Three different seafood products, Sea bream ( Sparus aurata ), Octopus ( Octopus vulgaris ), and Red mullet ( Mullus barbartus ) purchased fresh from a wholesale fish market (Savona, Ligurian Sea, Italy) were analyzed. The TMA adduct was correctly identified in all samples. Monitoring of TMA levels in the wholesale products from day 0 to day 20 showed an increase in all samples, confirming deterioration of freshness quality. This innovative, simple, and rapid method may prove useful for routine screening analysis and holds promise as a tool to evaluate seafood freshness quality and protect consumers from potential health risks. [ABSTRACT FROM AUTHOR]

Abstract In this study, 111 ready-to-eat anchovy products were collected on the Italian market. The products were molecularly identified through a BLAST analysis of a highly informative cytb fragment amplified by a newly designed primer pair for the genus Engraulis spp. and the mislabelling rate was assessed. In addition, the labels were analysed in the light of the current EU law. Despite only one mislabelling case was observed (mislabelling rate 0.9%), which involved the substitution of the European anchovy (Engraulis encrasicolus) with the low-valuable Peruvian/Chilean anchoveta (Engraulis ringens), the molecular technique developed in this study was proved as suitable tool for detecting species in processed anchovy products. It could be therefore applied to carry out more extensive EU survey aimed at evaluating the mislabelling rate of such products, still poorly covered by a targeted and clear legislation system. Highlights • EU legislation on the labelling of semi-preserved anchovy products presents some gaps. • 111 ready-to-eat anchovy products collected in Italy were molecularly identified. • A primer pair for amplifying 484 bp from cytb of all Engraulis spp. was projected. • Only one mislabelling case was observed (E. encrasicolus replaced with E. ringens). • This technique is a suitable tool to detect species in processed anchovy products. [ABSTRACT FROM AUTHOR]

Authentication of food product ingredients by food control agencies relies on analytical laboratory methods for animal species identification. Such methods must be validated if their results are to be used as acceptable evidence in a court of law. Following the validation guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), we carried out a validation process for the Low Cost and Density (LCD) Array (MEAT 5.0 version) kit for food forensics based on a DNA biochip technology that simultaneously detects 24 animal species. Mixtures of the animal species the kit is designed to detect were prepared at different concentrations and tested on raw/pasteurized, and heated meat and milk matrices. The LCD Array showed high specificity and high sensitivity and it appeared to be robust and repeatable. As such, it provides a useful tool for detecting common adulterants in food items of animal origin. [ABSTRACT FROM AUTHOR]

Small ruminant lentiviruses (SRLV) globally affect welfare and production of sheep and goats and are mainly controlled through elimination of infected animals, independently of the viral kinetics within the single animal. Control programs are based on highly sensitive serological tests, however the existence of low antibody responders leads to the permanent presence of seronegative infected animals in the flock, thus perpetuating the infection. On the other hand, long-term non-progressors show a detectable antibody response not indicative of a shedding animal, suggesting immune contention of infection. In this study, we analyse two goat populations within the same herd, harbouring low or high proviral SRLV loads respectively, both showing a robust antibody response. In vivo findings were confirmed in vitro since fibroblastic cell lines obtained from one high and one low proviral load representative goats, showed respectively a high and a faint production of virus upon infection with reference and field circulating SRLV strains. Differences in virus production were relieved when strain CAEV-Co was used for experimental infection. We analysed LTR promoter activity, proviral load, entry step and production of virus and viral proteins. Intriguingly, proteasomal activity was higher in fibroblasts from low proviral load animals and proteasome inhibition increased viral production in both cell lines, suggesting the implication of active proteasome-dependent restriction factors. Among them, we analysed relative expression and sequences of TRIM5α, APOBEC3 (Z1, Z2, Z3 and Z2-Z3) and BST-2 (Tetherin) and found a global antiviral status in low proviral carriers that may confer protection against viral shedding and disease onset. [ABSTRACT FROM AUTHOR]

The activity budgets of seven captive tigers (Panthera tigris) housed in four zoological gardens (A, B, C, D) were analysed to assess their welfare and to relate it to several variables, including enclosure type, management, and animal history. Behaviours were recorded by instantaneous focal animal sampling at2-min intervals. Data were collected by five observers using an ethogram listing 26 behaviours adapted from the literature. To process the data, the activity budgets of each tiger and the overall activity budget were constructed. On the basis of previous literature, some of the behaviours, listed in the ethogram, were labelled as indicators of diminished welfare and some were labelled as indicators of enhanced welfare. Statistical analysis was carried out to determine in which zoo the tigers were more prone to exhibit indicators of enhanced welfare and which feature(s) had a major effect on their welfare. Over 195 h of data were collected and 5867 observations were recorded. The tigers in zoo A (OR = 4.11, 95% confidence interval [CI] 3.2-5.3) and zoo C (OR = 1.83, 95% CI 1.4-2.4) were more prone to express indicators of enhanced welfare with respect to zoo D as the reference. Among the variables describing animal peculiarity, daily routine management, and enclosure features, the presence of a water pool with clean water was significantly associated with enhanced welfare (OR = 2.04, 95% CI 1.4-3.04). The data suggested that none of the tigers displayed consistent signs of stress and that all experienced a basic welfare status. An essential feature that helped to enhance good animal welfare was a water pool in the enclosure containing clean water. [ABSTRACT FROM AUTHOR]

Abstract: Recently, atypical bovine pestiviruses (BVDV-3) have been identified in batches of contaminated foetal calf serum (FCS) and in naturally infected cattle. During routine screening of FCS by conventional panpestivirus PCR assay, one batch showed traces of pestivirus nucleic acids, and the contaminating virus was typed as BVDV-3-like. Phylogenetic analysis based on three genome regions (5′UTR, Npro and E2) showed that this strain, named IZSPLV_To, clusters in a separate clade with CH_KaHo/cont, a cell culture contaminant detected in Switzerland. This study is the first report of the detection in Italy of a FCS batch contaminated with BVDV-3 and adds more evidence that atypical pestiviruses represent a serious cause for concern in cell culture laboratories, with potential repercussions on BVD control and vaccine biosafety. Our findings suggest that the BE/B2 primers may be able to detect BVDV-3 in a panpestivirus assay, but testing of a larger number of strains is required. [Copyright &y& Elsevier]

A male, six-year-old pudu (Pudu puda) from an Italian zoo was submitted for postmortem examination after sudden death. Necroscopy revealed non-suppurative bronchopneumonia and degeneration of the liver and haemorrhagic lesions of the thymus, pericardium and spleen. Microscopically, multifocal perivascular mononuclear cell infiltrates were observed in the kidneys, lungs, spleen, and the portal triads of the liver. Histological examination of the brain showed meningitis, vasculitis and perivascular cuffs of mononuclear inflammatory cells. A region of the DNA polymerase gene of malignant catarrhal fever viruses was amplified by real-time PCR and nested PCR. PCR products from the tissue samples were sequenced and analysed. The sequences showed 99% similarity with a portion of the caprine herpesvirus 2 DNA polymerase gene. This is the first report of malignant catarrhal fever in a captive pudu. [ABSTRACT FROM AUTHOR]