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The increasing world market demand for seafood requires an expansion of product categories available to consumers. Inland fish are usually considered having unmarked taste and are less appreciated by consumers; thus, they have low commercial value. Therefore, the marketing of the lake’s fresh and processed fish is limited to the local market and consumers are currently uninformed and mistrustful about these species. In this study, six different fish species were caught in the Fondi lake (Lazio, central Italy): Anguilla anguilla, Tinca tinca, Carassius gibelio, Cyprinus carpio, Micropterus salmoides, Chelon ramada. All the samples were subjected to nutritional and DNA barcoding analysis. Moisture, protein, fat, carbohydrates, ash, and sodium content were measured. As regards the fatty acids profile, the most abundant were MUFAs with the highest value in Anguilla anguilla (45.97%). Oleic acid (C18: 1 n9 cis) was particularly high in Cyprinus carpio (55.46%). The fraction of polyunsaturated fatty acids (PUFA) revealed a higher DHA content (C22: 6 n3) in Anguilla anguilla than the other species (>12 %) while Chelon ramada presented both higher EPA content (C 20: 5 n3) and total fraction of omega 3 PUFAs. Concerning molecular analysis, a 655 bp fragment of cytochrome C oxidase subunit I (COI) gene was successfully used for the identification at the species level using both BOLD and BLAST public databases. The present study gives the basis for improving the knowledge and promoting inland fish’ market and traceability along the supply chain.

Sparid fish species have different commercial values related to their organoleptic features. Mitochondrial (mt) DNA provides a potential tool to distinguish species, but the enrichment of high-quality mtDNA from total genomic DNA is critical to obtain entire mtDNA sequences. Conventional mtDNA isolation is relatively low-cost and proficient. However, high numbers of PCR cycles can lead to artefacts (10-6 mutations/bp). We describe a rapid protocol for mtDNA extraction and enrichment from fish tissues, based on conventional miniprep columns and paramagnetic bead-based purification, without the need to employ PCR amplification. This newly described method generates a substrate for next-generation sequencing (NGS) analysis and is likely to have wider applications for mitochondrial studies in other fish families to help ensure traceability and differentiation of fish with high commercial values.

The growing global diffusion of seafood for human consumption requires always more accurate sanitary and quality controls. Among the globally marketed fish, the species belonging to the family Sparidae are excellent food-fishes of high economic value. The Sparidae family comprises about 38 genera and 159 species, some of which are highly appreciated as seafood. In Italy, the fish species of commercial interest are defined by the law (DM n. 19105 del 22 Settembre 2017). Among them, 41species belong to the Sparidae family. The identification of these species is difficult even when external characters are preserved due to their morphological similarity. Species identification becomes even harder to achieve after industry processing, when distinctive external traits are removed. Nevertheless, despite their similarity, sparid species have different organoleptic quality that corresponds to variable prices in fish markets. Consequently, substitution of commercially important Sparidae species is common. The sparid specie with highest commercial value, Dentex, is often replaced with less expensive species or lower quality alternatives. All this premised, Sparidae species identification requires immediate resolutions. Research on fish mitochondrial DNA (mtDNA, mitogenome) has led to substantial advances in the fields of species authentication and population biology. The main species’ specific DNA sequences used as markers for fish species identification belong to the mitochondrial genes encoding ribosomal 16S and 12S subunits, cytochrome b (Cytb), and cytochrome c oxidase I (COI). However, current research shows that mitochondrial DNA markers perform well for certain species but may be less discriminating for others. Therefore, they cannot be used indiscriminately for the identification of all fish species. This situation has led to the formulation of the proposal to study and analyze the complete mtDNA sequence with the aim to identify mitochondrial markers or multiple marker approaches with higher and more specific discrimination capacity. At present, mtDNA genomes of Sparidae fishes are not conspicuously represented in the scientific literature, which makes difficult to understand if currently used genetic markers are the most effective for sparid species identification. This thesis project had the aim to study and analyze Sparids’ complete mtDNA sequence to identify mitochondrial markers or a multiple marker approach to achieve a higher and more specific discrimination capacity. We focused on the genera Dentex, Pagellus and Pagrus, listed in the Ministerial Decree.

The common dentex (Dentex dentex (Linnaeus, 1758)) is an iconic fish in the Mediterranean diet. Due to its commercial and organoleptic importance, this sparid is highly appreciated in European markets and is often subjected to species substitution frauds. Comparative mitogenomics is a suitable approach for identifying new and effective barcode markers. This study aimed to find a molecular tag useful for unequivocally discriminating the sparid species D. dentex. The comparison of the complete mitochondrial DNA (mtDNA) sequences of 16 sparid species allowed us to highlight the potential of the NAD2 gene for direct identification purposes. Common dentex-specific primers were created and successfully evaluated by end-point and real-rime PCR (Polymerase Chain Reaction) for several fish species, achieving amplification only in the D. dentex. The method proposed in this study appears fast, simple, and inexpensive and requires affordable instrumentation. This approach provides unambiguous results for the common dentex authentication without the sequencing step. The presence/absence assay for D. dentex can be executed in a few hours of lab work. Therefore, national authorities responsible for food safety and traceability could apply and make full use of DNA-testing methods for deterring operators from false seafood declarations.

The sharpsnout seabream Diplodus puntazzo Walbaum, 1792 is a target species of small-scale fishery activities and is cage-cultured for human consumption. Nonetheless, genetic information on this species is limited. We here first sequence its complete mitochondrial genome. The sequence is composed of 16,638 base pairs, accounting for 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 2 non-coding regions (D-loop and L-origin). The overall nucleotide composition is: 27.4% A, 28.9% C, 26.9% T, and 16.8% G. Maximum likelihood analyses placed D. puntazzo close to Acanthopagrus and some Pagellus species.

The white seabream Diplodus sargus (Linnaeus, 1758) is a species of interest for commercial fisheries throughout its range of distribution and it is also reared using aquaculture techniques. Herein, we present the first complete sequence and annotation of the mitochondrial genome of this species. The D. sargus mitogenome is 16,515 base pairs in length and contains 13 protein-coding genes, 2 rRNA, 22 tRNA, and 2 non-coding regions (D-loop and L-origin). The overall nucleotide composition is: 27.3% A, 28.9% C, 26.8% T, and 17.0% G. Maximum likelihood analyses placed D. sargus as a sister species of Diplodus puntazzo. This study provides valuable information for further studying identification methods and evolutionary relationships of Sparidae species.

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 different countries worldwide. The minimal inhibitory concentrations (MICs) were determined, and scanning electron microscopy (SEM) was used to verify how the plant extracts affected 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.

espanolEn este estudio se analizaron los niveles de PCDD, PCDF y DL-PCB en leche y en tejido adiposo de bufalos lactantes muestreado in vivo y en periodo de secado, asi como de vaquillas de una granja de Campania, confiscada por la autoridad competente debido a los altos niveles de dioxina encontrados en la leche a granel. A traves de HRGC-HRMS, utilizando el metodo 1613b de la epa (Agencia para la Proteccion del Medioambiente) de Estados Unidos, se realizo la determinacion quimica. El rango de valores establecido por el WHO-TEQ (equivalente toxico de la Organizacion Mundial de la Salud) para PCDD y PCDF en tejidos adiposos se situa entre 1.79 y 68.64 pg g−1 grasa, ubicandose entre 8.33 y 13.95 pg g−1 grasa en la leche. El perfil de contaminacion para dioxinas y furanos se establecio en 1,2,3,7,8-PeCDD; 2,3,4,7,8-PeCDF; 1,2,3,6,7,8-HxCDD y 2,3,7,8-TCDD. Los niveles de DL-PCB en los tejidos adiposos oscilaron entre 1.38 y 20.13 pg g−1 grasa, mientras que en la leche variaron entre 8.33 y 13.95 pg g−1 grasa. Los congeneres PCB 126 y PCB 169 dominaron el patron de DL-PCB en las dos matrices. EnglishThe 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.

Listeria monocytogenes is a pathogenic bacterium responsible for foodborne illness worldwide. Antimicrobial peptides, or bacteriocins, produced by food-grade lactic acid bacteria can serve as preservatives to prevent Listeria's growth in various foods, including dairy products. This study investigated the anti-listerial activities of bacteriocin-producing lactic acid bacteria, Streptococcus thermophilus B59671, and Lactobacillus plantarum 076. In vitro studies showed that the concentration of pediocin produced by L. plantarum 076 (2560 AU/mL) inhibited the growth of a six-strain cocktail of L. monocytogenes. However, the concentration of thermophilin 110 produced by S. thermophilus B59671 (320 AU/mL) only delayed the growth by ~2 h. Higher concentrations of thermophilin 110 (≥640 AU/mL) suppressed Listeria growth for up to 22 h. Pasteurized skim milk fermented with a co-culture of S. thermophilus B59671 and L. plantarum 076 reduced the number of L. monocytogenes cells by > 4 Log CFU/mL due mainly to the activity of pediocin. The anti-listerial activity was not observed in whey samples collected from pasteurized skim milk fermented with this co-culture but was detected when raw milk was the substrate. Two additional whey preparations, the by-products from commercial bovine and goat raw-milk cheeses, also inhibited Listeria growth and reduced the number of cells following storage at 4 °C for one week. This study showed that a concentrated preparation of thermophilin 110 has potential as an anti-listerial compound. It demonstrated the prospect of using a co-culture of S. thermophilus B59671 and L. plantarum 076 to prevent Listeria contamination in dairy foods. Additionally, results showed that metabolites with antimicrobial activities may be generated during the fermentation of raw milk due to indigenous microflora.

Aims To evaluate the performance of the IQ-Check kits and the USDA Microbiology Laboratory Guidebook (MLG) methods for detection of the top seven Shiga toxin-producing Escherichia coli (STEC) (O157:H7, O26, O45, O103, O111, O121 and O145) in ground beef and both STEC and Salmonella in co-inoculated samples. Methods and Results Ground beef samples inoculated with ~10 CFU of STEC or both STEC and Salmonella Typhimurium were stored at 4°C for 72 h, followed by screening with the IQ-Check and BAX System kit (MLG) methods that employ different enrichment media. STEC and S. Typhimurium were detected after 12 and 18 h and their presence was confirmed by colony isolation. Conclusions Both methods were able to detect STEC in ground beef after 12 h of enrichment in samples inoculated with low levels of the pathogen. STEC and S. Typhimurium can be detected and isolated in co-inoculated ground beef samples. Significance and Impact of the Study The IQ-Check methods are comparable to the MLG methods for detection of STEC and simultaneous detection of STEC and S. Typhimurium in seeded ground beef after a short enrichment time, thus the IQ-Check method can be useful for the food industry for rapid detection of these pathogens.

Il Pagello fragolino (Pagellus erythrinus) è una delle specie di Sparidi più pescate e commercializzate nel Mar Mediterraneo e nell’Oceano Atlantico. Tale specie è molto apprezzata nei mercati Europei ed è spesso soggetta a frodi per sostituzione di specie. Le sostituzioni avvengono soprattutto mediante utilizzo di altri Sparidi con differente pregio commerciale. Le sequenze di DNA mitocondriale (mt) maggiormente utilizzate per l'identificazione di specie ittiche sono il citocromo b-CYTB, la citocromo c ossidasi I-COI, i geni che codificano per le subunità ribosomiali 16S e 12S. Ricerche recenti hanno dimostrato che lo studio dell'intero mtDNA consente di identificare nuovi marcatori molecolari specie-specifici e quindi più efficaci. In particolare, il gene NAD5 ha un'elevata capacità discriminatoria per le specie appartenenti alla famiglia Sparidae. Tuttavia, l'utilizzo di tutti questi markers molecolari richiede diversi steps analitici, come l'amplificazione del frammento target ed il sequenziamento. Quest’ultima fase rappresenta un momento critico dell’identificazione di specie, poiché richiede tempi lunghi. Inoltre, tale analisi deve essere effettuata in laboratori altamente specializzati, poiché la lettura errata di pochi nucleotidi può falsare la corretta identificazione della specie. Scopo di questo studio è stato quello di analizzare e confrontare l'intera sequenza di mtDNA di alcune specie di Sparidi al fine di identificare un marcatore molecolare utile per riconoscere la specie P. erythrinus, evitando la fase di sequenziamento. In questo studio sono stati confrontati i mitogenomi di n°13 Sparidi. L’allineamento degli mtDNA è stato condotto mediante l’utilizzo del software UGENE. L'algoritmo Hamming Distance è stato utilizzato per valutare la percentuale di diseguaglianza genetica tra le specie ed i geni. La p-genetic distance è stata valutata utilizzando il Maximum Composite Likelihood model. La variabilità della sequenza nucleotidica è stata determinata allineando le sequenze gene-by-gene utilizzando MEGA 6.0. I primers sono stati disegnati a mano in seguito ad allineamento multiplo delle sequenze complete di mtDNA mediante il software BioEdit Sequence Alignment Editor. L'efficienza dei primers per l'identificazione di P. erythrinus è stata testata mediante PCR end-point. Sono stati testati n°10 campioni di P. erythrinus con differente provenienza geografica e n°30 campioni di filetti appartenenti ad altre specie ittiche. I risultati della Hamming Distance analysis, della valutazione della p-genetic distance e dell’analisi di variabilità della sequenza nucleotidica hanno consentito di identificare il gene NAD2 come potenziale marcatore molecolare per gli Sparidi. La reazione PCR ha confermato la capacità di discriminazione del gene NAD2. In particolare, l'amplificazione di un frammento selezionato del gene NAD2 è stata possibile solo per la specie P. erythrinus. Tale risultato può consentire di verificare la specie senza necessità di sequenziamento, riducendo tempi e costi delle analisi ed aumentando l’efficienza dei risultati. La valutazione di presenza/assenza della specie P. erythrinus può essere ottenuta in poche ore di lavoro di laboratorio. Ulteriori indagini sono in corso al fine di identificare primers specie-specifici anche per altre specie appartenenti alla famiglia degli Sparidi. In accordo con il Regolamento (UE) 1379/2013, questo studio contribuisce alla tracciabilità molecolare dei prodotti della pesca.

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.

Inclusion of novobiocin as a selective agent for enrichment media and selective agars inhibits the growth of some Shiga toxin-producing Escherichia coli (STEC) strains, particularly non-O157 STEC, which can yield false-negative detection results. Here, we report the draft genomic sequences of seven STEC O111 isolates with different sensitivities to novobiocin.

The common Dentex (Dentex dentex, Linnaeus 1758) has a significant economic importance and is a highly valued table fish in the Mediterranean region. The paucity of genetic information relating to sparids, despite their growing economic value, provides the impetus for exploring the mitogenomics of this fish group. Here, we sequenced D. dentex complete mitochondrial genome. The sequence is comprised of 16,652 bp and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a 2 non-coding regions (D-loop and L-origin). The overall nucleotide composition is: 27.5% of A, 28.7% of C, 26.9% of T, and 16.9% of G.

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.

The complete mitochondrial genome of theAngolan dentex(Dentex angolensis,Poll and Maul, 1953) is16.581bp in length and contains 13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomalRNA genes. The overall base composition ofD. angolesismtDNA is: 27.4% for A, 28% for C, 16.7% forG, 27.9% for T. Phylogenetic analysis indicated thatD. angolensisis most closely related toD. tumifrons.Whole genome sequencing ofD. angolensismitochondrial DNA will contribute to improving knowledgeabout Sparidae evolution.

This study was designed to investigate physicochemical (pH, peroxides) and histological parameters (glycogen reserve, muscle damages), as pre-slaughter stress markers and quality meat evaluators. Ross commercial broilers was processed, either without stunning (NS Lot), or by combining two current levels of 200 mA, 67 V (HV Lot), and 200 mA, 53 V (MV Lot); with two frequencies of 1000 Hz and 800 Hz, using sinusoidal alternating current. The use of high frequency (800 and 1000 Hz) induced the fastest pH decline, already at 3 h postmortem, in Pectoralis major and Quadriceps femoris of MV and HV Lot carcasses (P < 0.05). Moreover HV carcasses showed a lesser production of superoxide free radicals than the NS Lot and MV Lot carcasses (P < 0.05). Changes in the glycogen reserve and the histological muscle damages did not show significant differences in the analysed Lots. The incidence of individual carcass defects, sufficient to cause downgrading or rejection was generally low in each Lot. To assess animal welfare and quality poultry meat the pH monitoring and measurement of superoxide radical production, as additional and feasible parameters, might be markers easier to use in practice at abattoir.

Bivalve molluscs are a well documented source of viral infection. Further data on shellfish viral contamination are needed to implement European Regulations with sanitary measures more effective against viral pathogens. To this aim, 336 samples of bivalve molluscs (185 mussels, 66 clams, 23 oysters and 62 samples from other species) collected in harvesting areas of class A and B of four Italian Regions were analyzed for qualitative and quantitative determination of hepatitis A virus (HAV) and Norovirus (NoV) GI and GII, using real time RT-PCR. The results showed a wide diffusion of viral contamination in the shellfish production areas considered. HAV prevalence was low (0.9%) with contamination levels that varied from 5 to 7 × 10(2)copies/g. On the contrary, NoV showed a high prevalence (51.5%), with a large variability according to the group considered (e.g. 47.8% for Crassostrea in Veneto, 79.7% for Mytilus in Campania, 84.6% for Tapes in Sardinia). NoV contamination affected class A and class B production areas to a different extent, with a statistically significant difference in both contamination prevalence (22.1% vs. 66.3%; p

The aim of this study was to measure the persistence of residues of the pyrethroid insecticide α-cypermethrin (ACYP) in the milk of lactating donkeys following pour-on treatment. Milk was collected from animals (n = 7) before the treatment and at 12, 24, 36, 48, 60, 72 and 84 h post-treatment. The last sampling was taken 7 days post-treatment (168 h). Milk samples were analysed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The analytical method was validated following requirements of Commission Decision 2002/657/EC. All samples showed levels of ACYP below the maximum residue limit (MRL) of 20 μg kg(-1) established for bovine milk (Commission Regulation (EU) No. 37/2010). The results demonstrate that there is minimal partitioning of ACYP into milk in lactating donkeys from pour-on treatment.

The common pandora (Pagellus erythrinus, Linnaeus 1758), one of the most popular sea bream species in the Mediterranean Sea, has high potential for aquaculture development. In this investigation, we analyzed the complete mitochondrial genome of P. erythrinus. The sequence has 16,828 bp in length and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a two non-coding regions (D-loop and L-origin). The overall nucleotide composition is: 27.5% of A, 28.2% of C, 27.5% of T, and 16.8% of G.

To assess the quality of shellfish harvest areas, bivalve mollusk samples from three coastal areas of the Campania region in Southwest Italy were evaluated for viruses over a three-year period (2015&ndash, 2017). Screening of 289 samples from shellfish farms and other locations by qPCR and RT-qPCR identified hepatitis A virus (HAV, 8.9%), norovirus GI (NoVGI, 10.8%) and GII (NoVGII, 39.7%), rotavirus (RV, 9.0%), astrovirus (AsV, 20.8%), sapovirus (SaV, 18.8%), aichivirus-1 (AiV-1, 5.6%), and adenovirus (AdV, 5.6%). Hepatitis E virus (HEV) was never detected. Sequence analysis identified HAV as genotype IA and AdV as type 41. This study demonstrates the presence of different enteric viruses within bivalve mollusks, highlighting the limitations of the current EU classification system for shellfish growing waters.

Listeria monocytogenes is a foodborne pathogen that is difficult to eliminate due to its ability to survive under different stress conditions such as low pH and high salt. To better control this pathogen in food, it is important to understand its survival mechanisms under these stress conditions. LMOf2365_0442, 0443, and 0444 encode for phosphotransferase transport system (PTS) permease (fructose-specific IIABC components) that is responsible for sugar transport. LMOf2365_0445 encodes for glycosyl hydrolase. These genes were induced by high pressure and inhibited under salt treatments; therefore, we hypothesized that genes encoding these PTS proteins may be involved in general stress responses. To study the function of these genes, deletion mutants of the PTS genes (LMOf2365_0442, LMOf2365_0443, and LMOf2365_0444) and the downstream gene LMOf2365_0445 were created in L. monocytogenes strain F2365. These deletion mutants were tested under different stress conditions. The growth of ∆LMOf2365_0445 was increased under nisin (125 μg/mL) treatments compared to the wild-type (P < 0.01). The growth of ∆LMOf2365_0442 in salt (brain-heart infusion medium with 5% NaCl) was significantly increased (P < 0.01), and ∆LMOf2365_0442 showed increased growth under acidic conditions (pH 5.0) compared to the wild-type (P < 0.01). The results from phenotypic arrays demonstrated that some of these mutants showed slightly slower growth under different carbon sources and basic conditions. The results indicate that deletion mutants ∆LMOf2365_0442 and ∆LMOf2365_0445 were more resistant to multiple stress conditions compared to the wild-type, suggesting that they may contribute to the general stress response in L. monocytogenes. An understanding of the growth of these mutants under multiple stress conditions may assist in the development of intervention strategies to control L. monocytogenes in food.

Background/Aims: We studied the association of low birth weight with ultrasound-assessed nonalcoholic fatty liver disease (NAFLD) to test the hypothesis that fetal growth retardation followed by a rapid weight catch-up growth might be an additional factor responsible for liver steatosis via insulin resistance (IR) and/or intra-abdominal fat. Methods: We enrolled 23 children born small for gestational age (SGA) with a rapid catch-up growth within the first 6-12 months, and 24 appropriate for gestational age (AGA) children as controls. All children underwent anthropometric, body composition measurements and evaluation of liver function tests, lipid profile, plasma glucose and insulin levels. Abdominal ultrasonography was performed in order to asses liver steatosis and thickness of subcutaneous and visceral adipose tissue. Results: NAFLD were observed in 8 out of the 23 SGA children (34.8%). IR and visceral fat were significantly increased in children with hepatic steatosis compared to those without. IR index was significantly related to liver steatosis, independently of body mass index standard deviation score and visceral fat. Conclusions: NAFLD should be recognized as an emerging problem in SGA prepubertal children who presented a rapid weight gain in postnatal life, and IR plays the key role. An appropriate diet during pregnancy and in the first year of life might prevent metabolic syndrome and NAFLD in these subjects.

Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx 2e (81%) was the most common Stx variant, followed by stx 1a (14%), stx 2d (3%), and stx 1c (1%). The STEC serogroups that carried stx 2d were O15:H27, O159:H16 and O159:H-. Similar to stx 2a and stx 2c, the stx 2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. The present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections.

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O26 have been associated with sporadic cases and outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. In addition to chromosomal virulence genes, STEC strains usually harbor a large plasmid that carries genes associated with pathogenicity. The complete nucleotide sequence and genetic organization of 6 plasmids carried by STEC O26:H11 strain H30 were determined. The large virulence plasmid (pO26-Vir) was approximately 168 kb in size and contained 196 open reading frames (ORFs). pO26-Vir possesses a mosaic structure and shows similarity to the virulence plasmids in locus of enterocyte effacement (LEE)-negative STEC O113:H21 EH41 (pO113), in E. coli clinical strain C1096 (pSERB1), and in E. coli O157:H7 RIMD 0509952 (pO157). Plasmid pO26-Vir shares several highly conserved regions with pO157 and carries important virulence genes, including toxB, katP, espP, and the hly gene cluster. In addition, pO26-Vir possesses genes encoding for type IV pili (pilL-V). The second largest plasmid, pO26-L (73 kb) contains 101 ORFs. pO26-L carries the tetracycline resistance gene and has regions that show similarity to the E. coli conjugative resistance plasmid NR1. The third largest plasmid, pO26-S4 (5.8 kb), is homologous to the ColE2 colicinogenic plasmid that encodes for colicin E2. The remaining 3 plasmids, pO26-S1 (1.5 kb), pO26-S2 (3.1 kb), and pO26-S3 (4.2 kb), carry very little genetic information except for putative proteins involved in plasmid replication and DNA maintenance. The data presented underscore the diversity among the STEC virulence plasmids and provide insights into the evolution of these plasmids in STEC strains that cause serious human illness.