Bacterial and viral investigations combined with determination of phytoplankton and algal biotoxins in mussels and water from a Mediterranean coastal lagoon (Sardinia, Italy).

Calich Lagoon is a Mediterranean coastal lagoon located along the northwestern coast of Sardinia (Italy). The connection to marine and fresh water determines the high productivity of this coastal lagoon. Despite its great potential and the presence of natural beds of bivalve mollusks (Mytilus galloprovincialis), the lagoon has not yet been classified for shellfish production. In this study, through a multidisciplinary approach, the presence of several bacterial pathogens (Escherichia coli, Salmonella spp., and Vibrio spp.) and viral pathogens (hepatitis A virus and norovirus genogroups I and II) was evaluated from March 2017 to February 2018. In addition, phytoplankton composition in lagoon waters and associated algal biotoxins (paralytic and diarrhetic shellfish poisoning) in mussels were also monitored. The aim of this study was to provide useful data to improve knowledge about their seasonal presence and to assess the potential risk for public health, as well as to provide input for future conservation and management strategies. In mussels, Salmonella spp. were found in spring, along with E. coli, but Salmonella spp. were not found in autumn or winter, even though E. coli was detected in these seasons. Vibrio parahaemolyticus was found in autumn and winter, but not in spring. Norovirus genogroups I and II were found in winter samples. None of the bacteria were found in summer. Algal biotoxins have never been detected in mussel samples. Among potentially harmful phytoplankton, only Pseudo-nitzschia spp. were present, mainly in summer. The results showed that a possible bacterial and viral contamination, together with the presence of potentially toxic microalgae, is a real problem. Therefore, the development of natural resource management strategies is necessary to ensure the good quality of waters and guarantee the protection of consumers

Identification and quantification of potential microplastics in shellfish harvested in Sardinia (Italy) by using transillumination stereomicroscopy

Plastics are non-biodegradable polymers made up of different groups of petrochemical materials. Several biotic and abiotic factors can change the density of plastic fragmenting it and originating microplastics (MPs). MPs have been defined as small pieces of plastic less than 5 mm in size. Due to their small size, they are an emerging concern in the marine environment since they can be ingested by aquatic organisms, especially filter-feeding organisms, such as bivalve mollusks. Impacts of MPs exposure have been shown at various levels of biological organization, from cellular to tissue to individual and population levels. For example, oxidative stress and inflammation have been observed in copepods and mussels, obstruction and physical damage of the digestive tract were found in fish and swimming behavior alterations, disruption of foraging and feeding behavior and overall reduced fitness and survival were observed in fish and oysters. In addition, MPs can act as a vector for the transfer of chemicals to marine biota. The aim of the present study was the identification and quantification of potential MPs in shellfish harvested in Sardinia (Italy) by using transillumination stereomicroscopy. Bivalves were collected from 4 of the main production areas located along the Sardinian coast and selected according to the principles of the risk assessment. The results of the present study demonstrated the presence of potential MPs in 70% of the analyzed samples: the presence of MPs in bivalve mollusks may pose a threat to food safety, and there is an urgent need to evaluate the potential risks of MPs to human health

Marine phycotoxin levels in shellfish-14 years of data gathered along the Italian coast

Along the Italian coasts, toxins of algal origin in wild and cultivated shellfish have been reported since the 1970s. In this study, we used data gathered by the Veterinary Public Health Institutes (IZS) and the Italian Environmental Health Protection Agencies (ARPA) from 2006 to 2019 to investigate toxicity events along the Italian coasts and relate them to the distribution of potentially toxic species. Among the detected toxins (OA and analogs, YTXs, PTXs, STXs, DAs, AZAs), OA and YTX were those most frequently reported. Levels exceeding regulatory limits in the case of OA (≤2,448 μg equivalent kg-1) were associated with high abundances of Dinophysis spp., and in the case of YTXs (≤22 mg equivalent kg-1) with blooms of Gonyaulax spinifera, Lingulodinium polyedra, and Protoceratium reticulatum. Seasonal blooms of Pseudo-nitzschia spp. occur all along the Italian coast, but DA has only occasionally been detected in shellfish at concentrations always below the regulatory limit (≤18 mg kg-1). Alexandrium spp. were recorded in several areas, although STXs (≤13,782 μg equivalent kg-1) rarely and only in few sites exceeded the regulatory limit in shellfish. Azadinium spp. have been sporadically recorded, and AZAs have been sometimes detected but always in low concentrations (≤7 μg equivalent kg-1). Among the emerging toxins, PLTX-like toxins (≤971 μg kg-1 OVTX-a) have often been detected mainly in wild mussels and sea urchins from rocky shores due to the presence of Ostreopsis cf. ovata. Overall, Italian coastal waters harbour a high number of potentially toxic species, with a few HAB hotspots mainly related to DSP toxins. Nevertheless, rare cases of intoxications have occurred so far, reflecting the whole Mediterranean Sea conditions

Colorimetric Analysis and Determination of Histamine in Samples of Yellowfin Tuna (<i>Thunnus albacares</i>) Marketed in Sardinia (Italy) by a Combination of Rapid Screening Methods and LC-MS/MS

The consumption of fishery products has been steadily increasing in recent decades. Among the quantitatively more important species, the yellowfin tuna (Thunnus albacares), is one of the main at-risk species as regards the possibility to present important levels of histamine and to be associated with the so-called “Scombroid Fish Poisoning”. The main aim of the present study was to evaluate the colorimetric parameters, the occurrence, and the quantification of histamine contamination in yellowfin tuna samples marketed in Sardinia (Italy) by a combination of rapid screening and official control methods. A total of 20 samples of yellowfin tuna loins collected from large retailers, fishmongers and local markets were analyzed for the qualitative and quantitative determination of histamine by the lateral flow test HistaSure™ Fish Rapid Test and LC-MS/MS, respectively. Moreover, all the samples were examined to assess the conformity with the EU rules on labelling and subjected to colorimetric analysis according to the CIE-L*a*b* standard. Visual inspection of yellowfin tuna labels highlighted a 30% of non-compliances. A significant (p L *), redness (a *), and yellowness (b *). The results of histamine occurrence agreed with the food safety criteria (p < 0.00001) was pointed out. Moreover, the two methods showed an agreement rate of 85%. The results of the present study confirmed the utility of lateral flow tests for the fast qualitative determination of histamine in yellowfin tuna. Rapid screening test should be strengthened by comparison with the official method especially in case of uncertain or positive results

Yessotoxin detection in bivalve molluscs: A case study from coastal mussel farms (Sardinia, Italy)

This work reports the first communication relating to the presence of yessotoxins in Mytilus galloprovincialis from coastal mussel farms (Sardinia, western Mediterranean) detected during 2008 and 2013 through a monitoring programme. The paper emphasizes how the changes both in yessotoxin permitted limits and used methods, established by legislation, have influenced the interpretation of the obtained results. Consequently, the samples that resulted negative during 2008 would have been positive until August 2013 and negative from September 2013 up to now, and the samples that were positive in 2013 would have been positive in 2008 and negative nowadays, according to Regulation currently in force. Regular monitoring of biotoxins demonstrated that, although yessotoxins have been rarely present in the past in Sardinia, they may cause toxicity in shellfish. So, it’s important to keep up on legislation’s changing and laboratory methods

Detection of Fish Allergens in Foods Using an In-House Real-Time PCR Targeting the Ribosomal 18S rRNA Gene

Fish is one of the major food allergens which, in sensitised individuals, can cause life-threatening allergic reactions, even when present in small amounts. To protect consumers&rsquo; health, the correct labeling of foods is important. The objective of the present study was to validate an in-house real-time PCR method targeting the ribosomal 18S rRNA gene as universal DNA marker for the detection of fish in foods. The specificity of the primers was assessed on 20 fish species commonly marketed in the Mediterranean basin and other species of molluscs and crustaceans and foods of animal and plant origin. The absolute detection of the method was assessed using DNA extracted from a fish mixture and the SureFood&reg; QUANTARD Allergen 40 reference material. The relative amount was assessed on a fish and b&eacute;chamel sauce blend. Commercial food samples either labelled with or without fish in the ingredient list, were tested for the presence of fish DNA. The primer showed high specificity against the selected fish species. The limit of detection (LOD) and limit of quantification (LOQ) of the in-house method were 0.5 pg/&micro;L and 5 pg/&micro;L, respectively. The relative quantification in fish and b&eacute;chamel blend samples detected a concentration as low as 0.000025%, corresponding to 0.25 mg/kg of fish, indicating the suitability of the method in a food matrix. The presence of fish DNA was always detected in commercial samples in which the presence of fish was listed in the ingredient list. The method was able to detect the presence of fish DNA also in samples in which the presence of fish was indicated as traces or was not declared on the label. The proposed method was demonstrated to be a reliable, specific, and sensitive method for the detection of fish allergens in foods. Therefore, the proposed real-time PCR method could be used as a useful instrument in the verification of compliance with allergen labelling regulations

Correction: Occurrence of harmful algal species and shellfish toxicity in Sardinia (Italy)

Not available

Occurrence of harmful algal species and shellfish toxicity in Sardinia (Italy)

Sardinia (Italy, north-western Mediterranean) is a commercially important producer of edible bivalve molluscs. Since the early 2000s, it was subjected to recurring cases of mussel farm closures due to toxic algal poison. Here, we present the studies on toxin concentrations and the associated potentially toxic phytoplankton distribution and abundances carried out by a regular monitoring programme in Sardinian shellfish areas, from January to May 2015. Diarrheic shellfish poisoning (DSP) toxins were detected in several bivalve molluscs samples, while paralytic shellfish poisoning (PSP) and paralytic shellfish poisoning toxins were present just once, without exceeding the legal limits. Potentially toxic algal species have been constantly present. <em>Pseudo</em>-<em>nitzschia</em> species were present during the entire study often with high abundances, while <em>Dinophysis</em> species reached high densities sporadically. Among PSP phytoplankton, only <em>Alexandrium minutum</em> Halim was found. The data obtained in this study showed an increase in the DSP toxicity in mussels in Sardinia. No clear relation between the occurrence of toxins in shellfish and the presence of potentially toxic algal species was found, although a slight correlation between DSP toxins and <em>Dinophysis</em> species could be supported

Trend of Pathogenic Vibrio parahaemolyticus Occurrences in Bivalve Molluscs Harvested in Sardinian Coastal Environments Between 2011 and 2018

The aim of the present study was to evaluate Vibrio parahaemolyticus occurrences in bivalve molluscs harvested from Sardinian coastal environments between 2013 and 2015. The prevalence of pathogenic V. parahaemolyticus isolates is based on the detection of the two major virulence genes thermostable direct hemolysin (tdh) and thermolabile hemolysin (trh) To assess changes between 2011 and 2018 in the prevalence of V. parahaemolyticus in bivalve molluscs, we compared our results with those of previous investigations. In total, 2,933 samples were collected: 1,079 in 2013, 1,288 in 2014, and 566 in 2015. The mean prevalence of V. parahaemolyticus in shellfish was 3.5% in 2013, 1.7% in 2014, and 3.5% in 2015. The highest percentage of positive samples in 2013 and 2014 was observed in clams (3.5% and 2.7%, respectively), whereas in 2015, it was reported in oysters (15.1%). By comparing the sampling period of 2011–2014 with that of 2015–2018, an increase in the prevalence of V. parahaemolyticus was observed in shellfish (p < 0.05). In parallel, 208 potentially enteropathogenic V. parahaemolyticus strains were identified through the years 2011–2018 and, in particular, 10 trh+ and six tdh+ isolates. Our present study provides information regarding trends of V. parahaemolyticus occurrences in bivalve molluscs harvested from Sardinian coastal environments between 2011 and 2018 suggesting that the prevalence varies depending on the sampling period and shellfish species

A Year of Bio-Monitoring (2021): Presence of Algae of the Genus <i>Alexandrium</i>, <i>Dinophysis</i>, <i>Prorocentrum</i> and Non-Compliance for Paralytic Toxins and Lipophilic Toxins in Bivalve Mollusks Bred in Sardinia (W Mediterranean Sea)

Bivalve mollusk production represents the principal aquaculture activity in Sardinia (40°03′ N, 9°05′ E). In 2021, 859 water samples and 1270 mollusk samples were analyzed. The species Alexandrium minutum caused the accumulation of Paralytic Shellfish Toxins (PST) in three samples of bivalve mollusks. Dinophysis acuminata complex caused the accumulation of lipophilic toxins (LTs) belonging to the okadaic acid group (OAs) in 18 samples of bivalve mollusks. The research of paralytic shellfish toxins (PSTs) in shellfish samples has been carried out with LC-FLD, as mentioned in the AOAC 2005 Official Method 2005.06. The determination of LTs was carried out by LC-MS/MS analysis. DTX2, belonging to the group of OA toxins, was detected for the first time in Sardinia, in mussels sampled in Tortolì. The presence of Dinophysis and Prorocentrum species was correlated with the accumulation of the OA toxin group in bivalve mollusks, showing a certain repeatability at certain times of the year in the areas included in the study. The results of the present study can help to plan and organize more effective bio-monitoring sampling strategies