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.

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' 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® QUANTARD Allergen 40 reference material. The relative amount was assessed on a fish and béchamel 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/µL and 5 pg/µL, respectively. The relative quantification in fish and béchamel 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.

Determination of phytoplankton in water samples, algal biotoxins, microbiological parameters and microplastics in Mediterranean mussels (Mytilus galloprovincialis Lamarck, 1819) from an experimental pilot farm in the Calich Lagoon (Sardinia, Italy).

The aims of this paper were to collect and analyse preliminary data of phytoplankton in the water, biotoxins, Escherichia coli, Salmonella spp., Vibrio spp. and microplastic eventually present in farmed mussels, and to acquire information about the production capability from an experimental pilot farm of the Calich Lagoon. Two sampling sessions were carried out, in February and in May 2019, also monitoring the water condition (pH, temperature, salinity, dissolved oxygen, chlorophyll a). No potentially toxic algae were detected, and moreover no biotoxins (Paralytic Shellfish Poison, Diarrheic Shellfish Poison, Amnesic Shellfish Poison) were found in mussels. E.coli was present with the highest concentration in February (16000 MPN/100g e.p.). Salmonella and Vibrio spp. have not been detected. Almost a microplastic per grams was found, mainly fiber of different colours. Further studies, carried out for several months, will allow to better understand the possible problems related to the production of mussels in a lagoon not yet classified as a shellfish production area.

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.

Presence, Seasonal Distribution, and Biomolecular Characterization of Vibrio parahaemolyticus and Vibrio vulnificus in Shellfish Harvested and Marketed in Sardinia (Italy) between 2017 and 2018.

ABSTRACT: In the present study, we investigated the presence, seasonal distribution, and biomolecular characteristics of Vibrio parahaemolyticus and Vibrio vulnificus in samples of bivalve mollusks (Mytilus galloprovincialis, Crassostrea gigas, and Ruditapes decussatus) harvested and marketed in Sardinia (Italy) between 2017 and 2018. A total of 435 samples were submitted for qualitative determination of Vibrio spp., V. parahaemolyticus, and V. vulnificus. Potentially enteropathogenic isolates were detected with biomolecular methods. The overall prevalence of Vibrio spp. was 7.6%. The highest Vibrio prevalence was found in R. decussatus (8.3%). The prevalences of V. parahaemolyticus and V. vulnificus were 2.7 and 4.8%, respectively. Higher prevalences of V. parahaemolyticus and V. vulnificus were found in R. decussatus (4.2%) and C. gigas (6.2%), respectively. Only two pathogenic V. parahaemolyticus strains were recovered (genotypes: tdh- and trh+; tdh+ and trh-), both from M. galloprovincialis. None of the isolates were tdh+ and trh+. Pathogenic Vibrio infections are often underestimated, and human infections are increasing in Europe. European data on the true distribution of Vibrionaceae are scarce, and the results of the present study highlight the need of constant monitoring to update the distribution of pathogenic vibrios.

Epidemiological survey on the prevalence of Salmonella spp. in the Sardinian pig production chain, using real-time PCR screening method.

The aim of this study was to evaluate the prevalence of Salmonella spp. in the Sardinian pig production chain in order to establish the incidence of monophasic serovariant of Salmonella Typhimurium on isolates with molecular methods (real-time PCR and multiplex PCR). Samples were collected in three EC slaughterhouses, four small slaughterhouses annexed to farmhouses, one meat distribution center, four meat cutting laboratories and four sausage processing plants. A total of 166 samples were collected and analyzed: 46 environmental samples, 48 finishing pigs, 16 piglets, 24 samples of non-processed meat, 28 meat preparations and 4 meat products. All samples were processed with an initial screening using the real-time PCR MicroSEQ® Salmonella spp detection Kit (Applied biosystems, life technologies) and with the TaqMan® Real-time PCR to confirm the kit results. Samples that tested positive for Salmonella spp were confirmed with cultural method using the standard ISO 6579. Positive samples were submitted to phenotypic identification. One colony from each positive sample was serotyped with multiplex PCR method. Salmonella spp was isolated in 7 on 166 samples (4.22 %). Among the positive samples, two came from finishing pigs, two belonged to the category meat preparations, two to meat products, one was an environmental sample. Multiplex PCR confirmed that the collected strains belonged to the species Salmonella Typhimurium (1), Salmonella derby (3) and monophasic serovariant of Salmonella Typhimurium (3).

Toxoplasma gondii and Other Zoonotic Protozoans in Mediterranean Mussel ( Mytilus galloprovincialis) and Blue Mussel ( Mytilus edulis): A Food Safety Concern?

Mediterranean mussels ( Mytilus galloprovincialis) and blue mussels ( Mytilus edulis) are among the most consumed fishery products, but they are frequent vehicles of foodborne infection worldwide. In this study, we investigated the occurrence and seasonality of zoonotic protozoans in mussels farmed or sold at retail outlets in Italy. We collected and tested 1,440 M. galloprovincialis and 180 M. edulis. Pooled samples were molecularly tested for Giardia duodenalis, Cryptosporidium spp., and Toxoplasma gondii and then sequenced. Sixty-two (45.9%; 95% confidence interval, 37.5 to 54.3%) mussel pools tested positive for one or more of the investigated pathogens. Both Mytilus species and samples from all the investigated areas harbored pathogens. Mussels were statistically more contaminated by Cryptosporidium spp., followed by T. gondii and G. duodenalis assemblage A, and M. galloprovincialis was more contaminated than M. edulis ( P < 0.01). Contamination was more likely in mussels at retail outlets ( P < 0.05) than in those from farms and in mussels collected in spring ( P < 0.01) than in other seasons. This is the first report of T. gondii found in M. galloprovincialis in Italy and in M. edulis in Europe. The detection of zoonotic protozoans in a widely consumed food source indicates the need for a more detailed microbiological risk analysis, especially considering that bivalve mollusks are often consumed raw worldwide.

Detection of Pathogenic Yersinia Enterocolitica in Slaughtered Pigs by Cultural Methods and Real-Time Polymerase Chain Reaction.

Healthy pigs carrying pathogenic to human Yersinia enterocolitica strains are the main source of entry into slaughterhouse, where cross-contamination of carcasses can happen. The aim of this work was to determine Y. enterocolitica prevalence in slaughtered pigs, investigating the presence of carriers in relation to carcass contamination. A total of 132 pig samples (tonsils, mesenteric lymph nodes, colon content, carcass surface) were collected from 4 Sardinian slaughterhouses. All the samples were examined by the ISO 10273:2003 method, and the prevalence was also determined by direct plating on CIN Agar. Moreover, to detect the ail positive Y. enterocolitica strains in enrichment broths and isolates a real-time polymerase chain reaction (PCR) was applied. Y. enterocolitica prevalence was 19% with direct plating and 12% with enrichment methods. Carcass surfaces and tonsils prevalence was 5.30% by direct plating, and 5.3% and 2.2%, respectively, by enrichment method. Tonsil samples showed an average contamination level of 3.2×103 CFU/g, while the mean value on carcass was 8.7×102 CFU/g. An overall prevalence of 9.8% of ail positive Y. enterocolitica broths was detected by RT-PCR, that found a higher prevalence in tonsils (7.5%) with respect to cultural methods, confirming the greater sensitivity of this technique when applied for tonsils and faeces samples. The results show a relatively low pathogenic Y. enterocolitica prevalence in pigs slaughtered in Sardinia. Good hygiene measures should be applied at slaughterhouse in order to prevent the entry of carriers and control carcass contamination.

Microbiological Findings in Ready-to-Eat and Precooked Food Distributed in Public Catering Halls in Cagliari Province, Italy.

During this research 159 samples of ready-to-eat and precooked food were examined for the detection of Salmonella spp., Listeria monocytogenes, Escherichia coli, coagulase-positive staphylococci and Enterobacteriaceae, bacterial count. All samples were negative for Salmonella spp. and Listeria monocytogenes; although a low count of coagulase-positive staphylococci (no. 5 samples) and E. coli (no. 3) was found. We proceeded with the identification of the isolates on every sample with high, moderate and low-count of Enterobacteriaceae, to better understand the microbial ecology. The Enterobacteriaceae species most frequently detected were Pantoea spp. (29.62%), Enterobacter cloacae (20.37%) and Serratia liquefaciens (12.96%); other different species of Enterobacteriaceae were detected with a <10% presence. Since there are no legal regulations indicating tolerance or limit values or this kind of microorganisms related to the samples, it would be appropriate, in the presence of high count of Enterobacteriaceae or by type of ready-to-eat and precooked food, to proceed with the identification of every microorganism.