New provisions for the labelling of fishery and aquaculture products: Difficulties in the implementation of Regulation (EU) n. 1379/2013

The European Union (EU), within the renewal plan of the Common Fisheries Policy and the Common Market Organization, with the Cape IV of Reg. (EU) n. 1379/2013 have introduced new requirements for the labelling of fisheries and aquaculture products. These, as well as providing consumers with more complete information, integrate the provisions of Reg. (EU) n. 1169/2011 and acts as a tool to prevent frauds and illegal fishing. In this work the new seafood labelling provisions were evaluated, starting from the analysis of the art. 35 of the Chapter IV and comparing it with the previous EU dispositions (Reg. (EC) no. 104/2000 and no. 2065/2001). The exclusion of prepared and processed products and aquatic invertebrates from the application of the mandatory seafood labelling provisions and the role of the mass caterer operators with respect to the labelling requirements were identified as the two major shortcomings that still need to be better addressed by the legislator. Overall, what emerged from this work is that, if on the one hand the European legislation on seafood labelling has achieved important goals, evolving and improving itself, on the other it is still controversial and plagued by the same problems as 15 years ago. Therefore, the authors suggest that the regulation is modified at least extending its scope to all products and to at all stages of the fishery logistic chai

Development of a Simple and Cost-Effective Bead-Milling Method for DNA Extraction from Fish Muscles

In the fish food sector, due to a growing globalization of the market, where intentional and unintentional frauds reach alarming levels, the molecular analysis is increasingly used by both official agencies, to enforce the law on traceability, and private companies, to verify the quality of goods. DNA extraction represents a necessary and critical step for all types of DNA analysis. Among the drawbacks associated with this procedure, there are handling of toxic materials, low DNA yield, and low throughput, due to time-consuming manual procedures. In this work, to overcome some of these problems, we developed an alternative method based on a bead-milling procedure without proteinase K digestion. The new method was then compared with both a salting-out protocol, developed in a previous work, and a commercial kit. Yield, spectrophotometric purity, electrophoretic degradation pattern, and amplificability of the extracted DNA were assessed. In particular, DNA amplificability was evaluated by comparing the band intensity on the gel, after amplification of the 16S rRNA and cytochrome oxidase I genes with a conventional PCR, and the take-off cycles, after amplification of the 16S rRNA gene with a real-time PCR. The results showed that the bead-based method allowed to obtain acceptable amounts of DNA, with good purity and good characteristics of amplificability. Although the salting-out method remains the most effective protocol in terms of pure performances, the bead-milling procedure can be considered a valid alternative, in the light of its lower demand in terms of labor and costs

Evolution of the Anisakis risk management in the European and Italian context

Due to the social and legislative implications, the presence of Anisakis spp. larvae in fishery products has become a concern for both the consumers and the official Control Authorities. The issuance of a large number of provisions, aimed at better managing fish products intended to be consumed raw or almost raw and the associated risks, resulted in a very complicate legal framework. In this work, we analyzed the evolution of the normative through an overview on the local and international legislations, focusing on issues that are of practical interest for Food Business Operators (FBOs) in the fishery chain. In addition, we performed a survey across the Department of Prevention of the Italian Local Health Authorities (LHA) and the main fish markets in Italy to collect the operating procedures and the monitoring plans. Overall, we found many differences, due to the absence of a national reference standard for the management of the Anisakis risk. From this examination, it turns clear that only a participation of all the involved institutions, a strategy of synergistic interventions, as well as a correct training of FBOs, can result in an effective risk management and a proper risk communication, which should overcome states of confusion and unnecessary negative impacts on the economy

DEVELOPMENT OF A PCR-RFLP METOD FOR THE IDENTIFICATION OF SIX SPECIES BELONGING TO THE GENUS LOPHIUS

Nowadays, six of the seven species belonging to the Genus Lophius have an important commercial value in the national and international markets. Usually they are sold beheaded and for this reason they are called tails. This kind of preparation is a limit for the specie-identification by means of the morphological characteristics. The mitochondrial cytochrome b (Cyt b) gene is considered a useful genetic marker to identify fish species. In this work, after obtaining the Cyt b complete sequence of the Lophius species that were missed in the databases, we set up a method based on PCR-RFLP able to identify the six species of Lophius with a commercial denomination in the Italian market

A SURVEY ON THE FRAUDULENT USE OF ANABOLIC SUBSTANCES IN BOVINES SLAUGHTERED IN MOLISE

An investigation has been performed on the fraudulent use of anabolic substances in the Region of Molise. One hundred fourty-four bovines (12-24 months old, 123 males and 21 females) have been included in the survey. Ante-mortem assessment on their behaviour and clinical analysis on some target organs were carried out. After slaughtering, samples of prostate, bulbo-urethral glands, Bartholin's glands, mammary gland, ovaries, thymus and thyroid were collected and processed for an anatomo-histopathological evaluation, as suggested in the guidelines of the Italian National Plan for Residues (PNR) 2009. Overall, the 15% of the subjects analysed have been classified as "suspect", whereas the 44% as "uncertain" and the remaining 59% as "negative". The lesion most frequently found was a serious atrophy of the thymic parenchyma with fat infiltration (15% of males and 14% of females), suggesting a prevalence of an illegal use of cortisonic drugs

DEVELOPMENT OF A PCR-RFLP METOD FOR THE IDENTIFICATION OF SIX SPECIES BELONGING TO THE GENUS LOPHIUS

Nowadays, six of the seven species belonging to the Genus Lophius have an important commercial value in the national and international markets. Usually they are sold beheaded and for this reason they are called tails. This kind of preparation is a limit for the specie-identification by means of the morphological characteristics. The mitochondrial cytochrome b (Cyt b) gene is considered a useful genetic marker to identify fish species. In this work, after obtaining the Cyt b complete sequence of the Lophius species that were missed in the databases, we set up a method based on PCR-RFLP able to identify the six species of Lophius with a commercial denomination in the Italian market

MITOCHONDRIAL CYTOCHROME B SEQUENCING OF LOPHIUS VOMERINUS (Valenciennes, 1837)

The Genus Lophius, belonging to the Lophiidae Family and commercially called anglerfish, has an important commercial value in the national and international markets. The seven species of the Genus are very similar and it is difficult to identify them by means of the traditional taxonomic methods also because fish are often commercialized decapitated. DNA analysis is one of the most useful tools for identifying animal species. In this work, in order to provide some more information on the DNA sequence of the aforesaid species, we use some universal primers to amplify the mitochondrial cytochrome b gene of Lophius vomerinus (Valenciennes, 1837) and obtained the complete sequence by sequencing of PCR products

Bacteriophage Lysin Mediates the Binding of Streptococcus mitis to Human Platelets through Interaction with Fibrinogen

The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. We have previously found that platelet binding by Streptococcus mitis SF100 is mediated by surface components encoded by a lysogenic bacteriophage, SM1. We now demonstrate that SM1-encoded lysin contributes to platelet binding via its direct interaction with fibrinogen. Far Western blotting of platelets revealed that fibrinogen was the major membrane-associated protein bound by lysin. Analysis of lysin binding with purified fibrinogen in vitro confirmed that these proteins could bind directly, and that this interaction was both saturable and inhibitable. Lysin bound both the Aα and Bβ chains of fibrinogen, but not the γ subunit. Binding of lysin to the Bβ chain was further localized to a region within the fibrinogen D fragment. Disruption of the SF100 lysin gene resulted in an 83±3.1% reduction (mean ± SD) in binding to immobilized fibrinogen by this mutant strain (PS1006). Preincubation of this isogenic mutant with purified lysin restored fibrinogen binding to wild type levels. When tested in a co-infection model of endocarditis, loss of lysin expression resulted in a significant reduction in virulence, as measured by achievable bacterial densities (CFU/g) within vegetations, kidneys, and spleens. These results indicate that bacteriophage-encoded lysin is a multifunctional protein, representing a new class of fibrinogen-binding proteins. Lysin appears to be cell wall-associated through its interaction with choline. Once on the bacterial surface, lysin can bind fibrinogen directly, which appears to be an important interaction for the pathogenesis of endocarditis

Sortase A Substrate Specificity in GBS Pilus 2a Cell Wall Anchoring

Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is one of the most common causes of life-threatening bacterial infections in infants. In recent years cell surface pili have been identified in several Gram-positive bacteria, including GBS, as important virulence factors and promising vaccine candidates. In GBS, three structurally distinct types of pili have been discovered (pilus 1, 2a and 2b), whose structural subunits are assembled in high-molecular weight polymers by specific class C sortases. In addition, the highly conserved housekeeping sortase A (SrtA), whose main role is to link surface proteins to bacterial cell wall peptidoglycan by a transpeptidation reaction, is also involved in pili cell wall anchoring in many bacteria. Through in vivo mutagenesis, we demonstrate that the LPXTG sorting signal of the minor ancillary protein (AP2) is essential for pilus 2a anchoring. We successfully produced a highly purified recombinant SrtA (SrtAΔN40) able to specifically hydrolyze the sorting signal of pilus 2a minor ancillary protein (AP2-2a) and catalyze in vitro the transpeptidation reaction between peptidoglycan analogues and the LPXTG motif, using both synthetic fluorescent peptides and recombinant proteins. By contrast, SrtAΔN40 does not catalyze the transpeptidation reaction with substrate-peptides mimicking sorting signals of the other pilus 2a subunits (the backbone protein and the major ancillary protein). Thus, our results add further insight into the proposed model of GBS pilus 2a assembly, in which SrtA is required for pili cell wall covalent attachment, acting exclusively on the minor accessory pilin, representing the terminal subunit located at the base of the pilus

Supramolecular Organization of the Repetitive Backbone Unit of the Streptococcus pneumoniae Pilus

Streptococcus pneumoniae, like many other Gram-positive bacteria, assembles long filamentous pili on their surface through which they adhere to host cells. Pneumococcal pili are formed by a backbone, consisting of the repetition of the major component RrgB, and two accessory proteins (RrgA and RrgC). Here we reconstruct by transmission electron microscopy and single particle image reconstruction method the three dimensional arrangement of two neighbouring RrgB molecules, which represent the minimal repetitive structural domain of the native pilus. The crystal structure of the D2-D4 domains of RrgB was solved at 1.6 Å resolution. Rigid-body fitting of the X-ray coordinates into the electron density map enabled us to define the arrangement of the backbone subunits into the S. pneumoniae native pilus. The quantitative fitting provide evidence that the pneumococcal pilus consists uniquely of RrgB monomers assembled in a head-to-tail organization. The presence of short intra-subunit linker regions connecting neighbouring domains provides the molecular basis for the intrinsic pilus flexibility