Fine tuning of a sequencing protocol for species identification of Mytilus spp. specimens

The genus Mytilus comprises eight species of mussels of commercial relevance, which have different geographic origin and distribution and can hybridize in case of coexistence (1). To date, events of habitat contamination with allochthonous species occur with increasing frequency, due mainly to anthropogenic transfer, posing threats to local ecosystems (2). Moreover, such events favour species mislabeling, which is reported as the most frequent issue affecting seafood products (3). To prevent these problems, EU Regulation 1379/2013 promotes the use of DNA-based methods. PCR-RFLP is to date one of the most efficient techniques used in Mytilus spp. species identification (4-5). However, it has some drawbacks linked to limited resolution and subjective interpretation of the results. To improve such aspects, an optimized protocol for the sequencing of short fragments was developed. A total of 50 samples were analyzed. Of them, 12 were DNA samples belonging to various Mytilus spp. that were already identified by PCR-RFLP in a previous study (5). The other 38 were tissue samples, of which 10 belonging to specimens directly collected from production sites in Chile, and 28 belonging to fresh and pre-cooked specimens collected from national market. For these samples, the total DNA was extracted using the DNeasy Mericon Food kit (Qiagen). The target PAP region was amplified from all the 50 samples (DNA and tissue) using the primer pair designed by Satto et al. (4). All PCR products were analyzed through capillary electrophoresis to verify the successful amplification, and then sequenced using Sanger technique. Twenty samples (41%), of which 10 from production site and 10 from market samples, were randomly selected to perform the RFLP analysis to be compared to the sequencing results. Ten out of the 12 species (83.3%) identified by PCR-RFLP in the previous study were confirmed by sequencing. The remaining 2 samples, identified as M. chilensis x M. edulis by PCR-RFLP were instead identified as M. chilensis x M. trossulus by capillary electrophoresis and confirmed by sequencing. For the remaining 38 samples, sequencing identified 20 tissue samples (52.6%) as M. galloprovincialis, 7 as M. chilensis (18.4%), 6 as hybrids M. chilensis x M. galloprovincialis (15.8%), 2 as M. edulis (5.3%), 2 as hybrids M. chilensis x M. trossulus (5.3%) and 1 as hybrid M. galloprovincialis x M. edulis (2.6%). The PCR-RFLP confirmed the sequencing results for 19 of the tested samples and failed the unambiguous identification of one sample which was identified as hybrid by sequencing. Overall, the species identification by PCRRFLP failed in 3 out 32 (9.4%) tested samples (12 DNA and 20 tissue). These findings suggest that the optimized protocol relying on Sanger sequencing has some practical advantages. First, the use of capillary electrophoresis to visualize the PCR products allowed the clear distinction of M. edulis and M. trossulus which, having a difference in amplicon length of only 12 bp (177 bp vs 165 bp), are very hard to discriminate with a standard agarose gel electrophoresis. Moreover, the use of sequencing allowed the unambiguous identification of pure species and hybrids, especially in the case of M. galloprovincialis x M. edulis, in which a double peak at the SNP site is clearly visible. Considering the ever-dropping costs linked to sequence-based technology, we propose this sequencing protocol as a valid, consistent, and reliable alternative to the currently used methods

Newly Designed Primers for the Sequencing of the inlA Gene of Lineage I and II Listeria monocytogenes Isolates

Listeria monocytogenes is a major human foodborne pathogen responsible for listeriosis. The virulence factor Internalin A (inlA) has a key role in the invasion of L. monocytogenes into the human intestinal epithelium, and the presence of premature stop-codons (PMSC) mutations in the inlA gene sequence is correlated with attenuated virulence. The inlA sequencing process is carried out by dividing the gene into three sections which are then reassembled to obtain the full gene. The primers available however were only able to entirely amplify the lineage II isolates. In this study, we present a set of new primers which allow inlA sequencing of isolates belonging to both lineages, since lineage I isolates are the ones most frequently associated to clinical cases. Using newly designed primers, we assessed the presence of inlA PMSCs in food, food processing environments and clinical isolates

Combination therapy with lercanidipine and enalapril reduced central blood pressure augmentation in hypertensive patients with metabolic syndrome

Arterial stiffness and blood pressure (BP) augmentation are independent predictors of cardiovascular events. In a randomized, open, parallel group study we compared the effect on these parameters of combination therapy with an ACE-inhibitor plus calcium channel blocker or thiazide diuretic in 76 hypertensive patients with metabolic syndrome uncontrolled by ACE-inhibitor monotherapy. After 4weeks run-in with enalapril (ENA, 20mg), patients were randomized to a combination therapy with lercanidipine (LER, 10-20mg) or hydrochlorothiazide (HCT, 12.5-25mg) for 24weeks. Aortic stiffness (carotid to femoral pulse wave velocity, PWV), central BP values and augmentation (augmentation index, AIx) were measured by applanation tonometry. The two groups showed similar office and central BP after run-in. Office (ENA/LER: from 149.1±4.9/94.5±1.5 to 131.7±8.1/82.2±5.3; ENA/HCT: from 150.3±4.7/94.7±2.1 to 133.1±7.1/82.8±5.3mmHg) and central BP (ENA/LER 127.4±17.1/85.2±12.1 to 120.5±13.5/80.0±9.5mmHg; ENA/HCT 121.6±13.4/79.3±9.5mmHg) were similarly reduced after 24weeks. PWV was comparable after run-in and not differently reduced by the two treatments (ENA/LER from 8.6±1.5 to 8.1±1.3m/s, p<0.05; ENA/HCT from 8.5±1.2 to 8.2±1.0m/s, p<0.05). Finally, both combinations reduced AIx, but its reduction was significantly greater (p<0.05) in ENA/LER (from 26.8±10.9 to 20.6±9.1%) than in ENA/HCT arm (from 28.2±9.0 to 24.7±8.7%). In conclusion, the combination with LER caused a similar PWV reduction as compared to HCT, but a greater reduction in AIx in hypertensive patients with metabolic syndrome not controlled by ENA alone. These results indicate a positive effect of the combination of ENA/LER on central BP augmentation, suggesting a potential additive role for cardiovascular protection

Isolation and Molecular Evidence of Tunisian Sheep-like Pestivirus (<i>Pestivirus N</i>) in Persistently Infected Sheep in Northern Italy, 2023

Over the last few decades, several pestiviruses have been discovered in ruminants, pigs, and, more recently, in non-ungulate hosts. Consequently, the nomenclature and taxonomy of pestiviruses have been updated. The Tunisian sheep-like pestivirus (TSV, Pestivirus N) is an additional ovine pestivirus genetically closely related to classical swine fever virus (CSFV). In this study, during a survey of pestivirus infections in ovine farms in the Lombardy region of Northern Italy, we identified and isolated a pestivirus strain from a sheep that was found to belong to Pestivirus N species based on its genomic nucleotide identity. The sheep itself and its lamb were found to be persistently infected. We performed molecular characterization and phylogenetic analysis of three viral genomic regions (a fragment of 5′-UTR, partial Npro, and the whole E2 region). In conclusion, these results confirmed circulating TSV in Northern Italy after notification in Sicily, Italy, and France. Correlation with Italian, Tunisian, and French strains showed that detection might have resulted from the trading of live animals between countries, which supports the need for health control measures

Evaluation of the Virulence Potential of <i>Listeria monocytogenes</i> through the Characterization of the Truncated Forms of Internalin A

Listeria monocytogenes is a widespread Gram-positive pathogenic bacterium that causes listeriosis, a rather rare but severe foodborne disease. Pregnant women, infants, the elderly, and immunocompromised individuals are considered particularly at risk. L. monocytogenes can contaminate food and food-processing environments. In particular, ready-to-eat (RTE) products are the most common source associated with listeriosis. L. monocytogenes virulence factors include internalin A (InlA), a surface protein known to facilitate bacterial uptake by human intestinal epithelial cells that express the E-cadherin receptor. Previous studies have demonstrated that the presence of premature stop codon (PMSC) mutations naturally occurring in inlA lead to the production of a truncated protein correlated with attenuate virulence. In this study, 849 L. monocytogenes isolates, collected from food, food-processing plants, and clinical cases in Italy, were typed and analyzed for the presence of PMSCs in the inlA gene using Sanger sequencing or whole-genome sequencing (WGS). PMSC mutations were found in 27% of the isolates, predominantly in those belonging to hypovirulent clones (ST9 and ST121). The presence of inlA PMSC mutations in food and environmental isolates was higher than that in clinical isolates. The results reveal the distribution of the virulence potential of L. monocytogenes circulating in Italy and could help to improve risk assessment approaches