Implementation of a pilot checklist for control standardization on fishery goods consignments at the Livorno border control post.

To standardize control activities, it is necessary to introduce checklists to support the control of consignments entering the European Union through border control posts (BCPs). This study aimed to develop a pilot checklist for the control of fishery consignments, preliminarily identified as the predominant group of goods entering the Livorno (Italy) BCP. The design of the pilot checklist was preceded by i) a revision of the current European and national legislation on the general and specific objectives of border control activities on fishery products and ii) a comparative analysis of two checklists (one of the Ministry of Health and one of the former Livorno border inspection post) developed on the basis of the repealed legislation. This comparison aimed to define the pilot checklist structure, verification objectives, and selection of assessment scores to be included in defining consignment compliance and acceptability. Once developed, the clarity and ease of use of the first draft of the pilot checklist were verified through its use in a field test during the control of 64 fishery product consignments. 22 regulatory sources (18 European and 4 national) were selected as reference legislation. The pilot checklist was structured as a dynamic "read-do" document based on the workflow of control activities described in the current legislation. The field test was useful in improving the clarity of the verification objectives within the documentary, identity, and physical control sections and in facilitating the use of the checklist and the collection of evidence during the control activity. This study, which focused on fishery products, can provide a practical approach for the development of checklists for all the other categories of goods under the responsibility of BCPs.

Mapping the occurrence of Eustrongylides spp. in fish species caught from six lakes in Central Italy (Tuscany and Latium regions): Implications for local fishery supply chains.

In the present study, in order to expand the knowledge on the distribution of parasites of the genus Eustrongylides in never investigated Italian lacustrine areas of Tuscany and Latium, fish specimens from four Tuscan lakes (Bilancino, Chiusi, Montedoglio and San Cipriano) and two Latium lakes (Bolsena and Bracciano) were collected and analyzed. The parasitological analysis, consisting of a visual inspection followed by a chloro-peptic digestion, was performed on 1650 specimens belonging to 17 species: European perch (Perca fluviatilis), Pumpkinseed (Lepomis gibbosus), Pike-perch (Sander lucioperca), Largemouth black bass (Micropterus salmoides), Pike (Esox lucius), Big-scale sand smelt (Atherina boyeri), Tench (Tinca), European Whitefish (Coregonus lavaretus), Channel catfish (Ictalurus punctatus), Chub (Squalius cephalus), Rudd (Scardinius erythrophthalmus), Common bleak (Alburnus alborella), South European roach (Sarmarutilus rubilio), South European nase (Protochondrostoma genei), Italian riffle dace (Telestes muticellus), Goldfish (Carassius auratus), and Freshwater bream (Abramis brama). Eustrongylides spp. larvae were only found in one specimen of European perch caught in Bracciano Lake and purchased from a local fishmonger (P = 3.3%; MI = 1; MA = 0.03). The only isolated Eustrongylides spp. larva was submitted to molecular analysis of the ITS gene region and identified as E. excisus. This finding adds Bracciano Lake to the list of the several Italian lakes in which nematodes belonging to this genus have been reported. Even if the observed prevalence is currently low, the presence of the parasite in this local freshwater fish supply chain requires FBOs to adopt risk management measures to prevent contaminated products from reaching final consumer. Moreover, future investigations will show if the parasite expands in these areas.

Accidental discovery of a Tetraodontidae (Sphoeroides marmoratus) within a cuttlefish (Sepia officinalis) bought in a fish shop in Italy: risk assessment associated with the presence of Tetrodotoxin.

The discovery of a pufferfish specimen (Tetraodontidae) inside a frozen cuttlefish, purchased by a fishmonger, and caught in the Eastern Central Atlantic (FAO 34) is reported. The consumer, who reported this case to FishLab (Department of Veterinary Sciences, University of Pisa) for investigation, was a student of Veterinary Medicine at the University of Pisa. He recognized the Tetraodontidae because he attended practical lessons on fish morphological identification during the course of food inspection and was aware of the risks to human health linked to the Tetrodotoxin (TTX). In this study, the pufferfish was identified morphologically, using the FAO morphological keys, and molecularly, analyzing two markers, the cytochrome oxidase I (COI) and the cytochrome b genes, by DNA barcoding. The pufferfish was identified morphologically as Sphoeroides spp., and molecularly as Sphoeroides marmoratus using the COI gene (99-100% identity values). Literature reports that S. marmoratus from the Eastern Atlantic contains high concentrations of TTX in the gonads and the digestive tract. However, the possible passage of TTX from fish to other organisms linked to contact or ingestion has never been reported. This represents the first case of a potentially toxic pufferfish entering the market inside another organism. The fact that a student observed this occurrence highlights the key role of citizen science in the management of emerging risks.

An Authentication Survey on Retail Seafood Products Sold on the Bulgarian Market Underlines the Need for Upgrading the Traceability System.

Economically motivated or accidental species substitutions lead to economic and potential health damage to consumers with a loss of confidence in the fishery supply chain. In the present study, a three-year survey on 199 retail seafood products sold on the Bulgarian market was addressed to assess: (1) product authenticity by molecular identification; (2) trade name compliance to the list of official trade names accepted in the territory; (3) adherence of the list in force to the market supply. DNA barcoding on mitochondrial and nuclear genes was applied for the identification of whitefish (WF), crustaceans (C) and mollusks (cephalopods-MC; gastropods-MG; bivalves-MB) except for Mytilus sp. products for which the analysis was conducted with a previously validated RFLP PCR protocol. Identification at the species level was obtained for 94.5% of the products. Failures in species allocation were reconducted due to low resolution and reliability or the absence of reference sequences. The study highlighted an overall mislabeling rate of 11%. WF showed the highest mislabeling rate (14%), followed by MB (12.5%), MC (10%) and C (7.9%). This evidence emphasized the use of DNA-based methods as tools for seafood authentication. The presence of non-compliant trade names and the ineffectiveness of the list to describe the market species varieties attested to the need to improve seafood labeling and traceability at the national level.

Safety and commercial issues in fresh mushrooms and mushroom-based products sold at retail in Tuscany region.

The compliance to European and National safety and labelling requirements relating to the sale of spontaneous and cultivated mushrooms and mushroom-based products in Tuscany was assessed. The evidence was collected by the Mycological Inspectorate of North-West Tuscany Local Health Authority during 90 inspections (from 2016 to 2020) at large-scale distribution stores, wholesalers, and restaurants in 10 cities belonging to 3 provinces, and on the labelling analysis of 98 commercial products collected at retail in 2021. Despite a substantial compliance of the inspected activities and products with the regulatory requirements, critical issues were highlighted: 1) EU legislative gap in the definition of specific measures for the safe sale of spontaneous mushrooms; 2) improper shelf storage temperatures of fresh-cut products; 3) incorrect condition of use on the labels of pre-packaged products; 4) lack of countryof- origin declaration in pre-packaged products. Furthermore, the labelling analysis highlighted that 18.4% and 15.3% of the products presented issues in the validity and correctness of the scientific names respect to national requirements in. A revision of the current EU legislation is needed to guarantee consumers safety, also considering the relevant number of poisoning cases related to false mycetisms (ingestion of edible mushrooms unproperly stored or used). Also, a specific revision and harmonization of the EU labelling of mushrooms would be desirable to protect consumers.

Risks and critical issues related to the discovery on the market of unauthorized live alien species on the Italian territory: Chinese crab (Eriocheir sinensis).

Eriocheir sinensis, Chinese Crab or Chinese Mitten Crab is a catadromous species belonging to the Varunidae family, native to river and estuarine areas of North and South East China and Korea. At European level, E. sinensis is widespread in the main water basins of Central and Northern Europe and, since 2016, it has been included in the list of invasive species important for the European Union and subjected to confinement and eradication measures which include the prohibition of collection, transit and placing on the market of live specimens (Regulation (EC) N° 1143/2014). The Chinese Crab can represent a significant danger for the local ecosystem and for the native biota as well as contributing to the appearance of hydrogeological instability phenomena resulting from the intense excavation and erosion of the riverbanks. The first finding of 5 kg of live specimens of Eriocheir sinensis was recorded in the official control by the UFS (Functional Simple Unit) veterinary public health and food safety of the ASL Toscana Centro at an ethnic catering establishment. The specimens were subjected to seizure, photographed, identified morphologically, and subjected to euthanasia and destruction in accordance with the European requirements for welfare and management of animal by-products. From the sanitary point of view, the dangers associated with the consumption of this crab are mainly biological and chemical therefore, risk communication is fundamental, not only at the level of the competent authorities in the sector, but also for the food business operators.

Urban Consumer Trust and Food Certifications in China.

China has experienced frequent food safety incidents that have undermined consumer trust in the food supply chain. To overcome this problem, China requalified the legislative framework and adopted a comprehensive food certification system over the years. Here, we investigated the influences of food traceability and Chinese certifications (QS/SC-food quality safety market access/production system, hazard-free, green, and organic) on Chinese consumer trust of food safety for different types of products: fish, meat, milk, eggs, and rice. Data were collected through face-to-face surveys conducted in rural and urban Chinese areas. With a sample of 757 questionnaires, we ran a logit model. The results show consumers' uncertainty and skepticism of certifications guaranteeing food safety attributes, especially for animal-based products. We found that price is used as a cue of safety by Chinese consumers. Individuals with higher education seem less influenced by certifications and other cues included in the analysis. The findings demonstrate that Chinese policy makers should implement new strategies to enhance consumer food safety trust, and design policies by considering different categories (e.g., vegetables, meat, fish, etc.) of food.

DNA barcoding for the verification of supplier's compliance in the seafood chain: How the lab can support companies in ensuring traceability.

Food Business Operators (FBOs) rely on laboratory analysis to ensure seafood traceability. DNA barcoding and Forensically Informative Nucleotide Sequencing may represent a support within self-checking programs finalized to suppliers' qualification and products identity certification. The present study aimed at verifying the usefulness of a decisional procedure (decision tree) set up at the FishLab (Department of Veterinary Sciences, University of Pisa, Italy) for seafood species identification by DNA analysis, to cope with FBOs' needs. The decision tree was applied to the analysis of 182 seafood (fish and molluscs) products, conferred to the FishLab by different FBOs between 2014 and 2015 as result of their self-checking activities. The analysis relied on a standard COI gene fragment eventually integrated by the analysis of alternative or supportive molecular targets (cytb and 16S rRNA). It also included a mini-DNA barcoding approach for processed products. Overall, 96.2% of the samples were unambiguously identified at species level using the elective target alone (92.4%) or a multi-target approach (3.8%). The lack of species identification (3.8%) was attributable to the absence of reference sequences or to the low resolution of the molecular targets. Nonetheless, all the molecular results were deemed adequate to evaluate the sample's compliance to the label information. Non-compliances were highlighted in 18.1% of the products. The protocol was proven as an effective supportive tool for the seafood identity verification within the supply chain self-checking activities. In addition, a considerable fraud rate was confirmed and the species most frequently involved in substitution were pointed out.

Seafood Identification in Multispecies Products: Assessment of 16SrRNA, cytb, and COI Universal Primers' Efficiency as a Preliminary Analytical Step for Setting up Metabarcoding Next-Generation Sequencing Techniques.

Few studies applying NGS have been conducted in the food inspection field, particularly on multispecies seafood products. A preliminary study screening the performance and the potential application in NGS analysis of 14 "universal primers" amplifying 16SrRNA, cytb, and COI genes in fish and cephalopods was performed. Species used in surimi preparation were chosen as target. An in silico analysis was conducted to test primers' coverage capacity by assessing mismatches (number and position) with the target sequences. The 9 pairs showing the best coverage capacity were tested in PCR on DNA samples of 53 collected species to assess their amplification performance (amplification rate and amplicon concentration). The results confirm that primers designed for the 16SrRNA gene amplification are the most suitable for NGS analysis also for identification of multispecies seafood products. In particular, the primer pair of Chapela et al. (2002) is the best candidate.

Serum responsiveness to recombinant bovine somatotropin in buffalo: a three-month lactation study using an acid-stripping ELISA for screening.

The misuse of recombinant bovine somatotropin (rbST) to increase milk yield involves buffalo not just cows. Screening methods to identify rbST-treated cattle have already been proposed. However, there have been no studies on prolonged periods with a high number of animals. In this study, we developed a new enzyme-linked immunosorbent assay (ELISA) to measure the serum responsiveness towards rbST, based on an acid-stripping procedure and relatively simple integral calculation dilution curves. We also applied the analysis to 640 serum and 96 milk samples collected from 16 buffalo treated with rbST and 16 controls, over a period of approximately three months. Its suitability as a screening method, in compliance with EU law, was also assessed. A bi-factorial approach was also evaluated, including the measurement of insulin-like growth factor 1 concentration in serum. Results showed that our ELISA could be used on its own for screening purposes, without the need to assess other biomarkers. Copyright © 2016 John Wiley & Sons, Ltd.

Two alternative multiplex PCRs for the identification of the seven species of anglerfish (Lophius spp.) using an end-point or a melting curve analysis real-time protocol.

Anglerfish (Lophius spp.) is consumed worldwide and is an important economic resource though its seven species are often fraudulently interchanged due to their different commercial value, especially when sold in the form of fillets or pieces. Molecular analysis is the only possible mean to verify traceability and counteract fraud. We developed two multiplex PCRs, one end-point and one real-time with melting curve post-amplification analysis, which can even be run with the simplest two-channel thermocyclers. The two methods were tested on seventy-five reference samples. Their specificity was checked in twenty more species of those most commonly available on the market and in other species of the Lophiidae family. Both methods, the choice of which depends on the equipment and budget of the lab, provide a rapid and easy-to-read response, improving both the simplicity and cost-effectiveness of existing methods for identifying Lophius species.

Pentaplex PCR as screening assay for jellyfish species identification in food products.

Salted jellyfish, a traditional food in Asian Countries, is nowadays spreading on the Western markets. In this work, we developed a Pentaplex PCR for the identification of five edible species (Nemopilema nomurai, Rhopilema esculentum, Rhizostoma pulmo, Pelagia noctiluca, and Cotylorhiza tuberculata), which cannot be identified by a mere visual inspection in jellyfish products sold as food. A common degenerated forward primer and five specie-specific reverse primers were designed to amplify COI gene regions of different lengths. Another primer pair targeted the 28SrRNA gene and was intended as common positive reaction control. Considering the high level of degradation in the DNA extracted from acidified and salted products, the maximum length of the amplicons was set at 200 bp. The PCR was developed using 66 reference DNA samples. It gave successful amplifications in 85.4% of 48 ready to eat products (REs) and in 60% of 30 classical salted products (CPs) collected on the market.