Anisakis spp. Larvae in Deboned, in-Oil Fillets Made of Anchovies (Engraulis encrasicolus) and Sardines (Sardina pilchardus) Sold in EU Retailers

Sardina pilchardus and Engraulis encrasicolus are considered the principal target species for commercial fishing in Europe and are widely consumed as semipreserved products. Although they are considered shelf-stable products, if treatment is not correctly applied, their consumption may represent a public health risk in regard to anisakiasis and allergic reactions. Little is known about the prevalence of Anisakis spp. in ripened products. This study aimed to evaluate the presence of Anisakis spp. larvae in deboned, in-oil anchovy and sardine fillets marketed in the EU to assess the influence of processing techniques on the prevalence of larvae. Ninety semipreserved anchovy and sardine products deriving from the Mediterranean Sea or Atlantic Ocean were collected from different EU retailers and examined using chloropeptic digestion to evaluate the presence of larvae and identify them. Thirty nonviable Anisakid larvae—A. pegreffii (30%) and A. simplex (70%)—were found. The frequency of larvae was higher in anchovies (28.8%). The low frequency of parasites found proved that processing technologies can influence the presence of larvae in final products, but it is important that visual inspection is performed only by trained people. The sources of raw materials should be considered in the production flow chart

Levels and congeners distribution of dioxins, furans and dioxin-like PCBs in buffaloes adipose tissues sampled in vivo and milk

The levels of PCDDs, PCDFs and DL-PCBs were analyzed both in milk and adipose tissues sampled "in vivo" from lactating, drying off and heifer buffaloes from a Campania farm which had been impounded by the competent authority owing to the high dioxin levels found in bulk milk. The chemical determination was carried out by HRGC-HRMS using US EPA Method 1613b. The range of WHO-TEQ values for the PCDDs/PCDFs in adipose tissues was 1.79 to 68.64 pg g−1 fat and in milk was 8.33 to 13.95 pg g−1 fat. The contamination profile for dioxins and furans was given by 1,2,3,7,8-PeCDD; 2,3,4,7,8-PeCDF; 1,2,3,6,7,8-HxCDD and 2,3,7,8-TCDD. The levels of DL-PCBs in adipose tissue varied from 1.38 to 20.13 pg g−1 fat while ranged from 8.33 to 13.95 pg g−1 fat in milk. The pattern of DL-PCBs in both matrices was dominated by congeners PCB 126 and PCB 169

Norovirus monitoring in bivalve molluscs harvested and commercialized in southern Italy.

Norovirus (NoV) is the main cause of human nonbacterial gastroenteritis throughout the world. NoVs are classified into five genogroups: GI, GII, GIII, GIV, and GV. NoVs from GI and GII are the most commonly reported NoVs associated with human infections, and raw or undercooked shellfish have been identified as the main potential infection vehicle. European Commission Regulation 2073/2005 defines only bacteriological parameters for use as safety criteria for shellfish because reference methods for detection of viruses are lacking. From July 2007 to April 2010, 163 shellfish samples were collected in southern Italy from harvesting areas, authorized or nonauthorized retailers, and a restaurant after an outbreak of human gastroenteritis. The shellfish were analyzed for the presence of NoVs from GI and GII using the one-step real-time reverse transcription PCR protocol. A total of 94 shellfish samples (57.7%) were positive for the presence of NoV, and GII was the most frequently identified genogroup

The Inhibitory Effect of Plant Extracts on Growth of the Foodborne Pathogen, Listeria monocytogenes

Listeria monocytogenes is a foodborne pathogen responsible for about 1600 illnesses each year in the United States (US) and about 2500 confirmed invasive human cases in European Union (EU) countries. Several technologies and antimicrobials are applied to control the presence of L. monocytogenes in food. Among these, the use of natural antimicrobials is preferred by consumers. This is due to their ability to inhibit the growth of foodborne pathogens but not prompt negative safety concerns. Among natural antimicrobials, plant extracts are used to inactivate L. monocytogenes. However, there is a large amount of these types of extracts, and their active compounds remain unexplored. The aim of this study was to evaluate the antibacterial activity against L. monocytogenes of about 800 plant extracts derived from plants native to dierent countries worldwide. The minimal inhibitory concentrations (MICs) were determined, and scanning electron microscopy (SEM) was used to verify how the plant extracts aected L. monocytogenes at the microscopic level. Results showed that 12 of the plant extracts had inhibitory activity against L. monocytogenes. Future applications of this study could include the use of these plant extracts as new preservatives to reduce the risk of growth of pathogens and contamination in the food industry from L. monocytogenes

Antimicrobial activity evaluation of pure compounds obtained from Pseudoalteromonas haloplanktis against Listeria monocytogenes: Preliminary results

L. monocytogenes is a foodborne pathogen responsible for a serious disease with a high mortality rate, particularly in vulnerable consumers. Recently, the scientific community has shown increasing attention to the search for new natural molecules with antimicrobial activity, aimed at preventing the spread of foodborne diseases. Extremophilic microorganisms, typical of extreme temperature environments, are a valuable source of these molecules. The present work aimed to study the antibacterial activity of four pure compounds derived from a molecule, the pentadecanal, produced by the Antarctic bacterium Pseudoalteromonas haloplanktis, against two different pathotypes of L. monocytogenes. Growth assays were performed in 96-well polystyrene plates with serial dilutions of the tested compounds at different concentrations (0.6, 0.3, 0.15, 0.07 mg/mL). The plates were incubated at 37°C for 24 h, with a spectrophotometric reading at OD 600 nm. Preliminary results of this study showed that pentadecanal inhibits the growth of L. monocytogenes, with a MIC (Minimum Inhibitory Concentration) of 0.6 mg/mL. Acetal, carboxylic acid, and ester did not demonstrate antibacterial activity at the concentrations tested. These findings suggest the possibility of using pentadecanal as a natural antibacterial to improve safety standards along the food supply chain

Comparative mitogenomic analysis of Sparids and evaluation of a new potential DNA barcoding marker for Dentex dentex.

Dentex dentex is one of the most commercially caught Sparidae species in the Mediterranean Sea and Atlantic Ocean. It is very appreciated in European markets and consequently more subjected to species substitution frauds [1]. The currently mitochondrial (mt) DNA sequences used for fish species identification in prepared and processed products are cytochrome b-CYTB, cytochrome c oxidase I-COI, 16S and 12S genes. Recent researches showed that the study of the whole mtDNA allows to identify new, effective and specie-specific barcode markers [2]. In particular, NAD5 gene has high discrimination capacity for Sparidae species. However, the use of all these genes needs amplification and a sequencing process [2,3]. Therefore, a valuable species identification requires many laboratory steps and is time consuming. The aim of this research was to analyze and compare the whole mtDNA sequence of Sparidae species to find a barcoding marker useful to identify the sparid species D. dentex, avoiding the sequencing step. Thirteen Sparidae complete mitogenomes were compared in this study. They were aligned by UGENE software. Hamming Distance algorithm was used to evaluate in percent the genetic dissimilarity among species and genes. Overall mean p-genetic distance analyses were conducted using the Maximum Composite Likelihood model. The nucleotide sequence variability was determined by aligning gene-by-gene sequences of Sparidae species using MEGA 6.0. Primers were designed by eye after multiple alignment of the Sparidae complete mtDNA sequences using BioEdit Sequence Alignment Editor. Primers efficiency for D. dentex identification was tested using PCR reaction. Results of Hamming Distance, nucleotide sequence variability and p-genetic distance analysis showed the potentiality of NAD2 gene as barcode marker for sparids. The PCR reaction confirmed the discrimination capacity of NAD2 gene. In particular, the amplification of the selected NAD2 fragment was possible only for the species D. dentex. In conclusion, NAD2 gene showed high interspecific nucleotide dissimilarity to provide unambiguous results for D. dentex species authentication without sequencing, reducing time, costs and efficiency. In fact, species identification results can be obtained in a few hours of lab work. Therefore, competent national authorities responsible for monitoring and enforcing could improve and make full use of DNA-testing methods in order to deter operators from false labelling of seafood. In agreement with Regulation (EU) 1379/2013, this study contributes to the molecular traceability of fishery products. [1] Katavic et al. Growth performance of pink dentex as compared to four other sparids reared in marine cages in Croatia. Short Communication. Aquaculture International, 8:455–461, 2000. [2] Ceruso et al. Frauds and fish species authentication: study of the complete mitochondrial genome of some Sparidae to provide specific barcode markers, Food Control, accepted for publication, 2019. [3] Armani et al. DNA and Mini-DNA barcoding for the identification of Porgies species (family Sparidae) of commercial interest on the international market. Food Control, 50: 589-596, 2015

Frauds and fish species authentication: Study of the complete mitochondrial genome of some Sparidae to provide specific barcode markers

Sparids have different organoleptic properties that correlate with a wide variety of retail prices in the market. Components of the observed morphology are rarely sufficient for full identification of these fish species, whose authentication requires specialist knowledge. Genetic diversity or variation and their measurements enable molecular methods as one of the most suggested remedies for aliud pro alio frauds. Genetic approaches have the potential for reducing costs and providing correct identification for a large number of market products. Mitochondrial (mt) DNA sequences (16S and 12S ribosome subunits, cytochrome b-CYTB, and cytochrome c oxidase I-COI) have been widely used for fish species identification. Yet, these mtDNA regions perform well for certain species but are less discriminating for others. Here, we report the first study of the whole mtDNA of the perciform fishes of the family Sparidae with the aim to select more efficient barcoding markers for taxonomical discrimination against frauds. For species-level sequence information, we analyzed and compared the whole sequence of thirteen Sparidae mitogenomes, nine publicly available and four recently sequenced ones. In particular, we searched for effective DNA barcode markers for the correct identification of sparid species by looking for interspecific variable regions flanked by conserved sequences for PCR primer design. We found that only four mtDNA genes are devoid of insertions or deletions, which can complicate the process of sequence alignment. Among them, NAD genes show encouraging utility in discriminating closely related sparid species owing to nucleotide sequence variability compared with classical barcodes for species. Discrimination capacity of NAD genes suggests their application as alternative mtDNA tools for the identification of Sparidae fishes. In particular, NAD5 fragments with high interspecific nucleotide sequence divergence were amplified and appear flawless for Sparidae species identification

The Sparidae mitochondrial genomes comparison may provide alternative barcode markers

The molecular markers used for differentiating fish species to detect fraudulent substitutions in prepared and transformed fishery products are mitochondrial (mt) DNA sequence fragments. The most used belong to the genes encoding for ribosomal 16S and 12S subunits, cytochrome b (cytb), and cytochrome c oxidase I (COI). The genetic variability of the complete mt genome of the perciform fishes of the family Sparidae has never been investigated before and remains elusive. Prompted by the aim to identify new specie-specific genetic markers to use against frauds, we analyzed and compared the complete mitogenome of thirteen Sparidae species, including four newly sequenced ones. We searched for mtDNA regions with high interspecific variability (barcodes) flanked by sequences with high sequence conservation (primer). Results showed that the nucleotide sequence variability in NAD group genes (NAD1 39%, NAD2 50%, NAD3 39%, NAD4L 39%, NAD4 43%, NAD5 41%, NAD6 44%) was much higher than in the molecular markers used for species identification, i.e. cyt b (36%), COI (32%) and ribosomal 16S (24%) and 12S (21%). Further, NAD group genes showed a very high discrimination capacity, suggesting their utilization as alternative DNA barcodes for the Sparidae fishes. In particular, the NAD5 gene allows to seek for regions with high nucleotide variability flanked by conserved areas appropriate to design Sparidae-specific primers. This study highlights the importance of complete mtDNA genome comparisons of commercially valuable fish species to identify regions with high (barcode) and low (primer) interspecific nucleotide variation, to be used for species identification of fishery products