Norovirus occurrence and tissue localization in farmed Mediterranean mussels (Mytilus galloprovincialis) from Sardinia, Italy M. Molotzu, A. Canu, T. Tedde, S. Virgilio, R. Bazzardi, R. Sau, S. Salza, G. Piras, S. Cau, B. Soro, J.G. Pereira, J.C.F. Pantoja, T. Cubeddu, S. Rocca, C. Spanu Food Research International, 2026 Norovirus (NoV) is the leading cause of foodborne gastroenteritis worldwide, with bivalve molluscs representing a major transmission vehicle. This study investigated the occurrence, temporal variability, and tissue localization of NoV in farmed Mytilus galloprovincialis from Sardinia (Italy), integrating long-term surveillance data with experimental histological analyses. A total of 592 mussel samples collected between 2015 and 2025 within official monitoring activities were analyzed by RT-qPCR according to ISO 15216. Overall, NoV RNA was detected in 15.2% of samples. Due to uneven sampling outside the active surveillance period, statistical analyses were restricted to 2015-2018, during which both year and season significantly influenced NoV detection (p < 0.0001), with higher odds observed in 2016 and during spring, and reduced probability in summer. Due to uneven sampling outside the active surveillance period, statistical analyses were restricted to 2015-2018, during which both year and season significantly influenced NoV detection (p < 0.0001), with higher odds observed in 2016 and during spring, and reduced probability in summer. To investigate tissue-specific viral localization, mussels were experimentally exposed under controlled laboratory conditions and analyzed by immunohistochemistry. Norovirus antigens were detected intracellularly within digestive epithelial cells 24 h post-exposure, with additional focal detection in the intestine and gill epithelium, supporting a multistep uptake and retention process associated with filter feeding. Rather than challenging existing detection standards, these findings provide in situ confirmation of tissue-associated viral persistence in M. galloprovincialis and offer mechanistic support for current monitoring approaches. Overall, the integration of epidemiological and histological evidence improves understanding of norovirus persistence in Mediterranean mussel production systems and highlights implications for seasonally informed surveillance and food safety risk management.
DNA metabarcoding for the identification and relative abundance assessment of general and potentially pathogenic bacteria in Sardinian sheep cheese processing environments L. Giagnoni, S. Salza, A. Tondello, G. Zardinoni, Deb. Saptarathi, G. Piras, R. Melillo, A. Cecchinato, P. Stevanato, T. Tedde, A.G. Mudadu, A. Squartini, C. Spanu International Journal of Food Microbiology, 2025 In dairy processing facilities, microbial contamination poses significant challenges to food safety and quality, even under rigorous hygiene protocols as those implemented in sheep milk processing in Sardinia. Specific niches within factories facilitate the persistence of spoilage and pathogenic microorganisms, including Listeria monocytogenes, Salmonella spp., Bacillus cereus, and Pseudomonas spp. Hereby, 16S rRNA-based bacterial metabarcoding was applied to assess bacterial populations in Sardinian cheese processing environments. The scope was to acquire taxonomical information on the total microbiota, including the presence of taxa known to feature also potentially pathogenic strains. Samples were collected from 14 dairy plants distributed throughout Sardinia, analyzing nine areas of the cheese processing line and their workflow pieces, distinguishing food-contact from non-food-contact surfaces, for a total of 253 samples. Five sequencing runs yielded 51,114,952 quality-filtered reads, resulting, upon bioinformatic processing, in 4426 annotated taxonomic lineages. A clear community partition driver within the technical workflow, was identified upon grouping the areas of washing-processing apart from those of salting-aging-shelving. Notably, the genus Halomonas and the family Halomonadaceae dominated and differentiated the microbial composition across samples. Sequences which belong to potential pathogens were instead prevailingly in the opposite sites when compared to salt-adapted biota.
Application of droplet digital PCR for the detection of fish DNA in food products S. Cau, B. Soro, R. Melillo, G. Piras, S. Salza, T. Tedde, F. Pinna, B. Vodret, C. Spanu Food Research International, 2025 Fish is one of the most common causes of food allergy. The global prevalence of fish allergy has increased over the years as a result of the increased fish consumption. In allergic individuals even small amounts of allergen can trigger a life-threatening allergic reaction. Correct food allergen labelling plays a crucial role in consumers protection. Food business operators are required to verify compliance with labelling requirements. In order to do so, they need reliable, specific and sensitive methods for the detection of fish in food products. The present study evaluated the use of a droplet digital PCR assay for the identification of fish allergen in a variety processed foods. The method was developed targeting the ribosomal 18S rRNA gene. The specificity, the limit of detection (LOD) and of quantification (LOQ), dynamic range and selectivity were evaluated. The LOD and LOQ of ddPCR were 0.08 pg/μL and 0.31 pg/μL, respectively. Following optimization and validation of the method, the digital droplet PCR was tested on 37 prepackaged food samples placed on the market. Samples comprised a variety of composite foods, including both animal and plant- origin ingredients. The presence of fish was identified in the ingredient list of some samples, while in others, it was indicated as traces or not listed at all. The presence of fish DNA was detected in 16 out of 18 labeled fish-containing samples (88.9 %), while it was never detected in foods where fish was not declared on the label. The results of this study demonstrate the strong potential of droplet digital PCR (ddPCR) as a highly sensitive and specific method for detecting fish allergens in complex food matrices.
Implementation of digestion and characterization procedures for control standardization on samples of Gilthead Seabream (Sparus aurata) and plastic polymers from the coastal environment of the Asinara Gulf (Sardinia, Italy) Rita Melillo, Roberto Lonis, Giuseppa Lorenzoni, Gabriella Piras, Nicola Cogoni, Simona Cau, Sara Salza, Riccardo Bazzardi, Monica Molotzu, Giuseppe Esposito, Bruna Vodret, Domenico Meloni, Sebastiano Virgilio Applied Food Research, 2025 • A digestion procedure for on animal and plastic samples was implemented. • Plastic samples were characterised by μ-FTIR. • The use of KOH 10 % was the most effective in the digestion of animal tissues. • Trial C (40 °C for 48 h) guaranteed a lower impact on plastics. • Six different polymers were identified by μ-FTIR. The objective of the present study was the implementation of a digestion procedure along with Fourier transform infrared microscopy (μ-FTIR) analysis for control standardization on samples of Gilthead Seabream ( Sparus aurata ) and different types of plastic polymers isolated in the coastal environment of the Asinara Gulf (Sardinia, Italy). The study has been carried out across two steps, the first of which was necessary to evaluate the exact amount of potassium hydroxide (KOH) at 10 % to degrade the organic matrix of gastro-intestinal tract and muscle of Gilthead Seabrams samples as much as possible without causing alterations to the eventual polymers that may be present. Moreover, the possible effects of 10 % KOH at 60 °C for 48 and 72 h and at 40 °C for 48 h were evaluated through the examination of plastic polymers that can be easily ingested by aquatic organisms under the Stereomicroscope and Scanning Electron Microscope (SEM). The second step involved the characterization of these plastic polymers by μ-FTIR.The results showed that the use of 1/10 KOH 10 % proved to be the most effective for the digestion of animal tissues, but at the same time (40 °C for 48 h) able to guarantee a lower impact on plastics and greater time savings. The characterization of plastic polymers by μ-FTIR revealed that the samples isolated from the coastal environment of the Asinara Gulf (Sardinia, Italy) were mostly represented by polyethylene, nylon, polyethylene terephthalate, polystyrene, polypropylene and polyurethane. These plastic polymers can be easily ingested by aquatic organisms and humans are exposed to them via their consumption, but so far, the consequences and potential risks have not yet been quantified. The results of the present study represent a preliminary contribution to the control standardization on samples of Gilthead Seabream ( Sparus aurata ) and different types of plastic polymers isolated in Sardinian coastal environments.
Assessment of Salmonella spp., E. coli, Vibrio parahaemolyticus and algal biotoxins in Pacific Cupped Oysters (Magallana gigas) along with monitoring of phytoplankton and hydrological parameters in water samples from an experimental facility in the Calich Lagoon (Italy) Gabriella Piras, Anna Maria Bazzoni, Giuseppa Lorenzoni, Domenico Meloni, Rita Melillo, Riccardo Bazzardi, Simona Cau, Barbara Soro, Alida Delogu, Sara Salza, Tiziana Tedde, Nicola Fois, Giuseppe Esposito, Sebastiano Virgilio, Alessandro Graziano Mudadu Applied Food Research, 2025 • Hydrological parameters and phytoplankton were investigated. • Biotoxins and pathogenic bacteria in oysters were determined. • The potentially toxic microalga Dinophysis acuminata was found. • No accumulation of biotoxins in oysters was pointed out. • Escherichia coli was always present overlapping the EU limits of 230 MPN/100 g. This study aimed to assess the presence ofof Salmonella spp., Escherichia coli, Vibrio parahaemolyticus and algal biotoxins in Pacific cupped oysters ( Magallana gigas while also monitoring the phytoplankton community (both toxic and non-toxic), as well as key environmental parameters including temperature, dissolved oxygen, chlorophyll a , and pH in the Calich Lagoon (Sardinia, Italy). An experimental pilot farm was set up in the frame of the EU Interreg program “RETRALAGS” and samples were collected from December 2018 to November 2019. Overall, . E. coli was always over the EU limit of 230 MPN/100 g provided for Class A areas Salmonella spp. was never detected and V. parahaemolyticus was detected only in summer samples. Only once the presence of the mixotrophic dinoflagellate Dinophysis acuminata was found, without accumulation of any biotoxins. The results confirmed the suitability of Pacific cupped oysters as bioindicators of the health status of coastal lagoon ecosystems.Microbiological parameter concentrations were strongly influenced by the season of collection and by anthropogenic activities within the lagoon's catchment area, highlighting the dynamic interaction between environmental conditions and human impact.
Environmental monitoring of Listeria monocytogenes contamination in dairy processing facilities combining culturing technique and molecular methods S. Salza, G. Piras, R. Melillo, M. Molotzu, L. Giagnoni, L. Doneddu, A. Tondello, A. Cecchinato, P. Stevanato, A. Squartini, T. Tedde, S. Virgilio, A.G. Mudadu, C. Spanu Lwt, 2024 of the present study was to investigate Listeria contamination in Sardinian sheep cheese-making plants using culture and DNA-based methods. Food and environmental samples were collected from different surfaces of 14 facilities. Samples were collected using pre-moistened sponge swabs. Each site was sampled using two sponges used one for microbiological detection and the other for extraction of total DNA. The DNA was submitted to real time PCR targeting the prs gene for Listeria spp., the gene inlA for Listeria monocytogenes and the gene iap for Listeria innocua . DNA metabarcoding was also conducted on the total DNA. Overall were collected 254 environmental and 36 food samples. The prevalence of Listeria spp. and Listeria monocytogenes was 18.6% and 10.0%, respectively. Samples enrichment coupled with quantitative PCR (qPCR) showed a greater sensitivity than the conventional culture method with an overall prevalence of 12.1% for L. monocytogenes . Both, qPCR and ribosomal DNA-metabarcoding conducted on total DNA extracted from the sponges performed poorly in comparison to the culture-based method detecting the presence of Listeria genus in as little as 7 (2.4%) and 4 (1.4%) samples respectively. The present study supports the use of a culture enrichment coupled with qPCR for routine monitoring of contamination in food premises. • Prevalence of Listeria was assessed in Sardinian cheesemaking plants. • Culture-mediated enrichment and DNA-based methods with no enrichment were used. • Metabarcoding was performed on DNA extracted from environmental samples; • The mean prevalence of L. monocytogenes with enrichment methods was 10.0% • Listeria represented as low as 0.001%-0.028% of the total microbial population
Contaminants of Emerging Concern: Antibiotics Research in Mussels from the Coasts of the Tyrrhenian Sea (Sardinia, Italy) Filomena Dessì, Maria Vittoria Varoni, Elena Baralla, Maria Nieddu, Valeria Pasciu, Gabriella Piras, Giuseppa Lorenzoni, Maria Piera Demontis Animals, 2024 Contaminants of emerging concern (CECs) are compounds found in several environmental compartments whose ubiquitous presence can cause toxicity for the entire ecosystem. Several personal care products, including antibiotics, have entered this group of compounds, constituting a major global threat. It is essential to develop simple and reliable methods by which to quantify these contaminants in several matrices. In this work, mussels were chosen as sentinel organisms to assess environmental pollution and the safety of bivalve mollusk consumption according to the “One Health perspective”. A liquid chromatographic tandem mass spectrometry method (LC-MS/MS) was developed for the quantification of two macrolides, erythromycin (ERY) and azithromycin (AZI), in mussels. This new method was validated according to international guidelines, showing high selectivity, good recoveries (>60% for both of them), sensitivity, and precision. The method was successfully applied for ERY and AZI research in mussels farmed along the Sardinian coasts (Italy), demonstrating itself to be useful for routine analysis by competent authorities. The tested macrolides were not determined in the analyzed sites at concentrations above the limits of detection (LODs). These results demonstrate the food safety of mussels (as concerns the studied antibiotics) and a negligible amount of pollution derived from these drugs in the studied area.
IMPACT OF RAINFALL EVENTS ON COMPLIANCE OF BIVALVE MOLLUSC, HARVESTED IN SARDINIA, ITALY, FOR ESCHERICHIA COLI Veterinarija Ir Zootechnika, 2024
IDENTIFYING AND QUANTIFYING MICROBIAL COMMUNITIES IN SARDINIAN SHEEP CHEESE DAIRIES’ PROCESSING ENVIRONMENTS VIA DNA METABARCODING Veterinarija Ir Zootechnika, 2024
DETERMINATION OF MICROBIOLOGICAL PARAMETERS, ALGAL BIOTOXINS AND TRACE ELEMENTS IN OYSTERS FROM A MEDITERRANEAN LAGOON Veterinarija Ir Zootechnika, 2024
New evidence of pectenotoxins in farmed bivalve molluscs from sardinia (Italy) Alessandro G. Mudadu, Anna Maria Bazzoni, Rita Melillo, Cecilia Teodora Satta, Simona Cau, Virgilio Congiu, Barbara Soro, Riccardo Bazzardi, Giuseppa Lorenzoni, Gabriella Piras, Nadia Bardino, Bruna Vodret, Sebastiano Virgilio Italian Journal of Food Safety, 2021
