Tetrodotoxin in live bivalve mollusks from Europe: Is it to be considered an emerging concern for food safety?

Tetrodotoxins (TTXs) are a group of potent neurotoxins named after the Tetraodontidae fish family (pufferfish). TTXs have been reported in several animal taxa, both terrestrial and marine. The ingestion of TTX-contaminated flesh can cause serious neurotoxic symptomatology and can eventually lead to death. Traditionally, TTXs have been associated with Asian countries, in particular with pufferfish consumption. However, they have also been reported in bivalve mollusks farmed in the Pacific area and, recently, in European seas. In Europe, different countries have reported TTXs, especially those bordering the Mediterranean Sea. As a consequence, in 2017 the European Food Safety Authority (EFSA) released an opinion with reference to TTX present in marine gastropods and bivalves, proposing a safety limit of 44 µg/kg TTXs in shellfish meat, below which no adverse effects should be observed in humans. Nevertheless, this limit has been exceeded on many occasions in European shellfish and, while for bivalves there have been no registered human intoxications, that is not the case for marine gastropods. However, TTXs have not yet been included in the list of marine biotoxins officially monitored in live bivalve mollusks within the European Union (EU). Thus, the aims of this manuscript are to discuss the increasing occurrence of TTXs in live bivalve mollusks from European sea waters, to acknowledge the still ongoing knowledge gaps that should be covered and to stimulate constructive debate on the eventuality of adopting a shared regulatory context, at least in the EU, for monitoring and managing this potential threat to food safety.

Parallel analysis of miRNAs and mRNAs suggests distinct regulatory networks in Crassostrea gigas infected by Ostreid herpesvirus 1.

BACKGROUND: Since 2008, the aquaculture production of Crassostrea gigas was heavily affected by mass mortalities associated to Ostreid herpesvirus 1 (OsHV-1) microvariants worldwide. Transcriptomic studies revealed the major antiviral pathways of the oyster immune response while other findings suggested that also small non-coding RNAs (sncRNA) such as microRNAs might act as key regulators of the oyster response against OsHV-1. To explore the explicit connection between small non-coding and protein-coding transcripts, we performed paired whole transcriptome analysis of sncRNA and messenger RNA (mRNA) in six oysters selected for different intensities of OsHV-1 infection. RESULTS: The mRNA profiles of the naturally infected oysters were mostly governed by the transcriptional activity of OsHV-1, with several differentially expressed genes mapping to the interferon, toll, apoptosis, and pro-PO pathways. In contrast, miRNA profiles suggested more complex regulatory mechanisms, with 15 differentially expressed miRNAs (DE-miRNA) pointing to a possible modulation of the host response during OsHV-1 infection. We predicted 68 interactions between DE-miRNAs and oyster 3'-UTRs, but only few of them involved antiviral genes. The sncRNA reads assigned to OsHV-1 rather resembled mRNA degradation products, suggesting the absence of genuine viral miRNAs. CONCLUSIONS: We provided data describing the miRNAome during OsHV-1 infection in C. gigas. This information can be used to understand the role of miRNAs in healthy and diseased oysters, to identify new targets for functional studies and, eventually to disentangle cause and effect relationships during viral infections in marine mollusks.

Host-microbiota interactions shed light on mortality events in the striped venus clam Chamelea gallina.

Mass mortalities due to disease outbreaks have recently affected a number of major taxa in marine ecosystems. Climate- and pollution-induced stress may compromise host immune defenses, increasing the risk of opportunistic diseases. Despite growing evidence that mass mortality events affecting marine species worldwide are strongly influenced by the interplay of numerous environmental factors, the reductionist approaches most frequently used to investigate these factors hindered the interpretation of these multifactorial pathologies. In this study, we propose a broader approach based on the combination of RNA-sequencing and 16S microbiota analyses to decipher the factors underlying mass mortality in the striped venus clam, Chamelea gallina, along the Adriatic coast. On one hand, gene expression profiling and functional analyses of microbial communities showed the over-expression of several genes and molecular pathways involved in xenobiotic metabolism, suggesting potential chemical contamination in mortality sites. On the other hand, the down-regulation of several genes involved in immune and stress response, and the over-representation of opportunistic pathogens such as Vibrio and Photobacterium spp. indicates that these microbial species may take advantage of compromised host immune pathways and defense mechanisms that are potentially affected by chemical exposure, resulting in periodic mortality events. We propose the application of our approach to interpret and anticipate the risks inherent in the combined effects of pollutants and microbes on marine animals in today's rapidly changing environment.

Identification of a newly described OsHV-1 µvar from the North Adriatic Sea (Italy).

The surveillance activities for abnormal bivalve mortality events in Italy include the diagnosis of ostreid herpesvirus type 1 (OsHV-1) in symptomatic oysters. OsHV-1-positive oysters (Crassostrea gigas) were used as a source for in vivo virus propagation and a virus-rich sample was selected to perform shotgun sequencing based on Illumina technology. Starting from this unpurified supernatant sample from gills and mantle, we generated 3.5 million reads (2×300 bp) and de novo assembled the whole genome of an Italian OsHV-1 microvariant (OsHV-1-PT). The OsHV-1-PT genome encodes 125 putative ORFs, 7 of which had not previously been predicted in other sequenced Malacoherpesviridae. Overall, OsHV-1-PT displays typical microvariant OsHV-1 genome features, while few polymorphisms (0.08 %) determine its uniqueness. As little is known about the genetic determinants of OsHV-1 virulence, comparing complete OsHV-1 genomes supports a better understanding of the virus pathogenicity and provides new insights into virus-host interactions.

Assessing the health status of farmed mussels (Mytilus galloprovincialis) through histological, microbiological and biomarker analyses.

The Gulf of La Spezia (northern Tyrrhenian Sea, Italy) is a commercially important area both as a shipping port and for mussel farming. Recently, there has been increased concern over environmental disturbances caused by anthropogenic activities such as ship traffic and dredging and the effects they have on the health of farmed mussels. This paper reports the results of microbiological and histological analyses, as well as of measurement of several biomarkers which were performed to assess the health status of mussels (Mytilus galloprovincialis) from four rearing sites in the Gulf of La Spezia. Mussels were collected between October 2015 and September 2016 and histological analyses (including gonadal maturation stage), as well as the presence of pathogenic bacteria (Vibrio splendidus clade, V. aestuarianus and V. harveyi), viruses (Herpes virus and ostreid Herpes virus 1) and protozoa (Marteilia spp., in the summer season only) were carried out on a monthly basis. Conversely, biomarker responses in haemocyte/haemolymph (total haemocyte count, haemocyte diameter and volume, lysozyme and lactate dehydrogenase activities in cell-free haemolymph, and micronuclei frequency) and in gills and digestive gland (cortisol-like steroids and lipid peroxidation levels), were evaluated bimonthly. Microbiological data indicated that mussels contain a reservoir of potentially pathogenic bacteria, viruses and protozoa that in certain environmental conditions may cause a weakening of the immune system of animals leading to mortality episodes. The percentage of parasites detected in the mussels was generally low (9.6% for Steinhausia mytilovum, that is 17 samples out of 177 examined females; 3.4% for Proctoeces maculatus; 0.9% for Mytilicola intestinalis and 2% for ciliated protozoa), while symbiont loads were higher (31% for Eugymnanthea inquilina and Urastoma cyprinae). Interestingly, a previously undescribed haplosporidian was detected in a single mussel sample (0.2%) and was confirmed by in situ hybridization. Cells morphologically similar to Perkinsus sp. trophozoites were observed in 0.7% of the mussels analysed; however, infection with Perkinsus spp. could neither be confirmed by ISH nor by PCR. Different pathological aspects, such as host defence responses and regressive/progressive changes were detected in the gills, digestive glands, gonads and mantle. Only one single case of disseminated neoplasia (0.2%) was observed. As for the biomarker evaluation, the MANOVA analysis revealed the statistically significant effect that the variable "sampling site" had on the biological parameter measured, thus suggesting that the multibiomarker approach was able to differentiate the rearing sites.

Species identification of bivalve molluscs by pyrosequencing.

BACKGROUND: The increase in seafood consumption and the presence of different species of bivalves on the global markets has given rise to several commercial frauds based on species substitution. To prevent and detect wilful or unintentional frauds, reliable and rapid techniques are required to identify seafood species in different products. In the present work, a pyrosequencing-based technology has been used for the molecular identification of bivalve species. RESULTS: Processed and unprocessed samples of 15 species belonging to the bivalve families Pectinidae, Mytilidae, Donacidae, Ostreidae, Pharide and Veneridae were analysed and correctly identified by the developed pyrosequencing-based method according to the homology between query sequences of the 16S ribosomal RNA (16S rRNA) and cytochrome c oxidase I (COI) genes and their correspondent reference libraries. This technique exhibits great potential in automated and high-throughput processing systems, allowing the simultaneous analysis of 96 samples in shorter execution and turnaround times. CONCLUSIONS: The correct identification of all the species shows how useful this technique may prove to differentiate species from different products, providing an alternative, simple, rapid and economical tool to detect seafood substitution frauds. © 2016 Society of Chemical Industry.

Perkinsosis in the clams Ruditapes decussatus and R. philippinarum in the Northeastern Atlantic and Mediterranean Sea: A review.

Perkinsosis is a disease of gastropod and bivalve molluscs caused by protozoan parasites of the Perkinsus genus. These parasites have been responsible for mass mortalities worldwide, with a significant impact in both fisheries and aquaculture, and resulting in severe economic losses. This review focuses mainly on current knowledge of diagnostic methods and on the distribution of Perkinsus spp. in the Northeastern Atlantic and Mediterranean Sea, which infect the grooved carpet shell clam Ruditapes decussatus and the Japanese carpet shell clam Ruditapes philippinarum. The association between perkinsosis and high mortality rates of R. decussatus and R. philippinarum in southern European countries such as Portugal and Italy is discussed as is the role of environmental factors in those mortality outbreaks. The putative introduction of Perkinsus olseni into the Northeastern Atlantic and Mediterranean Sea is also discussed, as are management strategies that could be used to mitigate the impact of perkinsosis in production of R. decussatus and R. philippinarum.

Mortality occurrence and pathogen detection in Crassostrea gigas and Mytilus galloprovincialis close-growing in shallow waters (Goro lagoon, Italy).

The complex interactions occurring between farmed bivalves and their potential pathogens in the circumstances of global climate changes are current matter of study, owing to the recurrent production breakdowns reported in Europe and other regions of the world. In the frame of Project FP7-KBBE-2010-4 BIVALIFE, we investigated the occurrence of mortality and potential pathogens during the Spring-Summer transition in Crassostrea gigas and Mytilus galloprovincialis cohabiting in the shallow waters of one northern Italian lagoon (Sacca di Goro, Adriatic Sea) and regarded as susceptible and resistant species, respectively. In 2011, limited bivalve mortality was detected in the open-field trial performed with 6-12 month old spat whereas subsequent trials with 2-3 month old spat produced almost complete (2012) and considerable (2013) oyster mortality. Macroscopical examination and histology excluded the presence of notifiable pathogens but, in the sampling preceding the massive oyster spat mortality of 2012, a µdeleted variant of OsHV-1 DNA was found in wide-ranging amounts in all analyzed oysters in conjunction with substantial levels of Vibrio splendidus and Vibrio aestuarianus. The large oyster spat mortality with borderline OsHV-1 positivity recorded in 2013 supports the multi-factorial etiology of the syndrome. This is the first report of a OsHV-1 (under a form interpreted as the variant µVar) in the Goro lagoon. Transcriptional host footprints are under investigation to better understand the bivalve response to environmental factors, included viral and bacterial pathogens, in relation to the observed mortalities.

Noroviruses in seafood: a 9-year monitoring in Italy.

Norovirus (NoV) are increasingly important as etiological agents of gastrointestinal infections. Consumption of bivalve molluscs and ready-to-eat fishery products is one of the most common ways of acquiring NoV foodborne infections, and the rise of outbreaks of viral gastroenteritis represents an important health problem that is also responsible for economic losses. The aim of this work was to define the prevalence of NoV contamination in preserved fishery products and in shellfish commercialized in Italy, taking into account the results obtained during 9 years of survey (2003-2011) and paying special attention to the regions more involved in national production. A total of 4463 samples were examined (2310 mussels, 1517 clams, 510 oysters, 22 other shellfish species, 104 preserved seafood products) and the average positivity rate for NoV presence was 4.1% and ranged from 0.6% in 2007 to 9.8% in 2003 and from 1.9% in preserved seafood products to 4.7% in mussels. Genetic characterization of circulating strains showed a prevalence of genogroup II genotypes, including GII.b and GII.e polymerase types and different GII.4 variants. This information could contribute to the optimization of risk-based sampling strategies for NoV contamination in seafood, taking into account variability in different species and from year to year.

Tetrodotoxin in bivalve mollusks: An integrated study towards the comprehension of the influencing factors of a newly native phenomenon.

Tetrodotoxins (TTXs) are potent neurotoxins named after the Tetraodontidae fish family. The ingestion of TTX-contaminated flesh can cause neurotoxic symptoms and can lead to death. In 2017 symptoms the European Food Safety Authority (EFSA) recognized the threat to food safety resulting from TTX exposure via food consumption and, thus, proposed a safety limit of 44 µg/kg of TTX in marine gastropods and bivalves. To date, however, TTXs have not yet been included in the list of biotoxins to be monitored within the European Union, even though, in a few cases, levels of TTX found were higher than the EFSA limit. The origin of TTX production is debated and the roles of both biotic and abiotic factors on TTX-mediated toxic events remain unclear. In order to meet these knowledge requests the present study was aimed to investigate the role of seawater temperature, pH, water conductivity, and oxygen saturation, along with the marine phytoplankton community and the bacterial community of mussels and oysters on the accumulation of TTX and analogues in the bivalves. Abiotic parameters were measured by means of a multi-parametric probe, phytoplankton community was analyzed by optic microscopy while microbial community was described by amplicon metataxonomic sequencing, TTXs concentration in the collected matrices were measured by HILIC-MS/MS. A possible role of seawater pH and temperature, among the investigated abiotic factors, in regulating the occurrence of TTXs was found. Regarding biotic variables, a possible influence of Vibrio, Shewanella and Flavobacteriaceae in the occurrence of TTXs was found. Concurrently, Prorocentrum cordatum cell numbers were correlated to the incidence of TTX in mussels. The results herein collected suggest that environmental variables play a consistent part in the occurrence of TTX in the edible bivalve habitats, and there are also indications of a potential role played by specific bacteria taxa in association with phytoplankton.

Effect of TiO2 Nanoparticle on Bioaccumulation of ndl-PCBs in Mediterranean Mussels (Mitilus galloprovincialis).

The interaction of nanomaterials with pollutants in the marine environment might alter bioavailability, as well as toxicity, of both nanomaterials and pollutants, representing a risk, not only for marine organisms, but also for consumers through the marine food chain.The aim of this study was to evaluate the effect of titanium dioxide nanoparticles (TiO2NPs) in terms of bioaccumulation and toxicity on Mediterranean mussels (Mytilus galloprovincialis) exposed to six-indicator non-dioxin-like polychlorinated biphenyls (ndl-PCBs). Mussels were exposed to ndl-PCBs (20 µg/mL) (groups 3-4) or to a combination of ndl-PCBs (20 µg/mL) and TiO2NPs (100 µg/mL) (groups 5-6) for four consecutive days. TiO2NPs was detected in groups 5-6 (3247 ± 567 and 1620 ± 223 µg/kg respectively), but their presence did not affect ndl-PCBs bioaccumulation in mussels. In fact, in groups 3-4, the concentration of ndl-PCBs (ranging from 3818.4 ± 166.0-10,176 ± 664.3 µg/kg and 2712.7 ± 36.1-9498.0 ± 794.1 µg/kg respectively) was not statistically different from that of groups 5-6 (3048.6 ± 24.0-14,635.9 ± 1029.3 and 5726.0 ± 571.0-9931.2 ± 700.3 µg/kg respectively). Histological analyses showed alterations to the structure of the gill tissue with respect to the control groups, with more severe and diffuse dilatation of the central hemolymphatic vessels of the gill lamellae in groups 5-6 (treated with TiO2NPs and ndl-PCBs concurrently) compared to groups 3-4 (ndl-PCBs only). Finally, in mussels submitted to a seven-day depuration process, most TiO2NPs were eliminated, and NPs had a synergistic effect on ndl-PCBs elimination; as a matter of fact, in groups 5-6, the percentage of concentration was statically inferior to the one observed in groups 3-4. In any case, consumers might be exposed to TiO2NPs and ndl-PCBs (both concurrently and separately) if edible mussels, harvested in a contaminated environment, are consumed without a proper depuration process.

The Occurrence of Freshwater Fish-Borne Zoonotic Helminths in Italy and Neighbouring Countries: A Systematic Review.

In recent years, the consumption of fish products has surged in European countries, being an essential part of a healthy diet. Despite representing a small part of EU production, freshwater fisheries hold considerable significance for lake-dwelling populations and tourists seeking traditional dishes. This increased fish consumption has brought to light potential health risks associated with fish-borne zoonotic helminths (FBZHs), now acknowledged as global food-borne parasites. Fish-borne zoonotic helminths belong to various taxonomic groups, including nematodes (Anisakidae), trematodes (Opisthorchiidae and Heterophyidae), and cestodes (Diphyllobothriidae). More than 50 species of FBZH are known to cause human infections, derived from eating raw or undercooked aquatic foods containing viable parasites. Despite increased attention, FBZHs remain relatively neglected compared to other food-borne pathogens due to factors like chronic disease progression and under-diagnosis. This systematic review concentrates on the prevalence of six freshwater FBZHs (Clinostomum complanatum, Contracaecum rudolphii, Dibothriocephalus latus, Eustrongylides excisus, Opisthorchis felineus, and Pseudamphistomum truncatum) in Italy and neighbouring countries. The study explores the expansion of these parasites, analysing their biological and epidemiological aspects, and the factors that influence their proliferation, such as the increased cormorant population and the lake eutrophication phenomena. In summary, this research highlights the necessity for further research, the development of spatial databases, and the establishment of a unified European policy to effectively manage these multifaceted health concerns. It strongly advocates adopting a One-Health approach to address the growing incidence of parasitic zoonoses within the context of food safety in EU countries.

Investigation of levels of perfluoroalkyl substances in freshwater fishes collected in a contaminated area of Veneto Region, Italy.

The bioaccumulation of 12 perfluoroalkyl substances (PFASs) in 107 freshwater fishes collected during 2017 in waterbodies of a contaminated area in Veneto Region (Italy) was evaluated. The contamination had been previously ascribed to a fluorochemical manufacturing plant that discharged mainly perfluorooctanoic acid (PFOA), among other PFASs, into the surrounding environment. Perfluorooctane sulfonate (PFOS) was the most abundant compound, detected in almost 99% of the fish with an average concentration of 9.23 µg/kg wet weight (w/w). Other detected compounds were perfluoroundecanoic acid (PFUnA) (98%, 0.55 µg/kg w/w), perfluorodecanoic acid (PFDA) (98%, 2.87 µg/kg w/w), perfluorododecanoic acid (PFDoA) (93%, 1.51 µg/kg w/w), and PFOA (79%, 0.33 µg/kg w/w). Bioaccumulation of PFASs was species related, with Italian barbel being the most contaminated, followed by chub, wels catfish, and carp, reflecting animals' habitat use and feeding behavior. A significant negative linear relation between PFAS concentration and fish weight was observed no matter the considered species, with smaller fish having proportionally higher bioaccumulation. PFOS concentrations were strongly correlated with the concentrations of other PFASs, suggesting a similar source of contamination or a contamination from ubiquitous sources. Correlation analysis showed PFOA likely originated from a separated source, unlinked to other PFASs. Although the fishes studied are not usually consumed by local people, with the likely exception of freshwater anglers (and relatives), their consumption has been banned by Veneto Authority since the time this study was conducted. In fact, the study suggests that a medium/high consumption frequency (superior to 1 portion per month) of fish from the investigated area might result in a high exposure to PFASs.

Preliminary multi analytical approach to address geographic traceability at the intraspecific level in Scombridae family.

Globalization of seafood product marketing caused the increase of request of an effective fish traceability that enhances the consumer confidence in food safety. In this study, an integrated multi analytical approach based on two different and independent analytical techniques (carbon and nitrogen stable isotopes and fatty acids analysis) was applied in order to identify different fish species and trace their geographical provenience. The investigation was focused on four species (Thunnus thynnus, Thunnus alalunga, Auxis rochei and Scomber scombrus) belonging to the Scombridae family. The DNA barcoding method confirmed genus and species for S. scombrus and A. rochei, but only genus for T. alalunga and T. thynnus. Carbon and nitrogen stable isotopes results evidenced different fish diets and trophic positions, whereas fatty acids analysis displayed that the unsaturated prevailed (∼60 %) over the saturated compounds with a variation among the species and the geographical area in particular for docosahexaenoic and eicosapentaenoic acids percentage. The principal component analysis applied to stable isotopes and fatty acids evidenced a good discrimination among species and their geographical catching area. This multi-disciplinary analytical approach could represent a promising tool to identify the commercial fish and trace their origin in order to guarantee the health of consumers.

Bioaccumulation and in vivo formation of titanium dioxide nanoparticles in edible mussels.

The aim of this study was to evaluate the bioaccumulation of titanium dioxide nanoparticles (TiO2NPs) in edible mussels bred in polluted artificial seawater. An in vivo study was conducted by exposing mussels to different concentrations of TiO2NPs (0.25 mg/L and 2.5 mg/L) or ionic titanium (1.6 mg/L) for 4 days. Inductively coupled plasma mass spectrometry (ICP-MS) showed titanium presence in all groups proportional to exposure levels (concentration range: 209-1119 µg/kg). Single particle ICP-MS revealed NPs in both TiO2NP treated mussels (concentration range: 231-1778 µg/kg) and in ionic titanium treated mussels (concentration 1574 µg/kg), suggesting potential nanoparticle formation in vivo. These results were confirmed by transmission electron microscopy with energy dispersive X-ray detection. Nonetheless, mussels eliminated more than 70% of the TiO2NPs after 3 days' depuration. These results show the potential for consumer exposure to TiO2NPs when contaminated mussels are consumed without a proper depuration process.

Microbiological and Histological Analysis for the Evaluation of Farmed Mussels (Mytilus galloprovincialis) Health Status, in Coastal Areas of Italy.

Shellfish farming is a relevant economic activity in Italy. The Gulf of La Spezia is one of the major production areas for mussels: the area is characterized by the presence of numerous human activities that could harm the quality of seawater. Additionally, the presence of potentially pathogenic microorganisms may influence the health status of animals, which must be constantly monitored. To have a clear view of the health conditions of the mussels (Mytilus galloprovincialis) farmed in this area, microbiological, parasitological, and histological analyses were performed. The study was conducted from November 2016 to October 2017. Overall, despite the presence of potentially pathogenic microorganisms for mussels, abnormal mortality rates were not reported during the monitoring period and the histological examination revealed no significant lesions. Our study confirms that studying different aspects together is a useful tool for assessing the health conditions of mussels and points out the importance of adverse environmental conditions for the expression of the pathogenicity of microorganisms.

Potential for Genetic Improvement of Resistance to Perkinsus olseni in the Manila Clam, Ruditapes philippinarum, Using DNA Parentage Assignment and Mass Spawning.

The Manila clam Ruditapes philippinarum, a major cultured shellfish species, is threatened by infection with the microparasite Perkinsus olseni, whose prevalence increases with high water temperatures. Under the current trend of climate change, the already severe effects of this parasitic infection might rapidly increase the frequency of mass mortality events. Treating infectious diseases in bivalves is notoriously problematic, therefore selective breeding for resistance represents a key strategy for mitigating the negative impact of pathogens. A crucial step in initiating selective breeding is the estimation of genetic parameters for traits of interest, which relies on the ability to record parentage and accurate phenotypes in a large number of individuals. Here, to estimate the heritability of resistance against P. olseni, a field experiment mirroring conditions in industrial clam production was set up, a genomic tool was developed for parentage assignment, and parasite load was determined through quantitative PCR. A mixed-family cohort of potentially 1,479 clam families was produced in a hatchery by mass spawning of 53 dams and 57 sires. The progenies were seeded in a commercial clam production area in the Venice lagoon, Italy, where high prevalence of P. olseni had previously been reported. Growth and parasite load were monitored every month and, after 1 year, more than 1,000 individuals were collected for DNA samples and phenotype recording. A pooled sequencing approach was carried out using DNA samples from the hatchery broodstock and from a Venice lagoon clam population, providing candidate markers used to develop a 245-SNP panel. Parentage assignment for 246 F1 individuals showed sire and dam representation were high (75 and 85%, respectively), indicating a very limited risk of inbreeding. Moderate heritability (0.23 ± 0.11-0.35 ± 0.13) was estimated for growth traits (shell length, shell weight, total weight), while parasite load showed high heritability, estimated at 0.51 ± 0.20. No significant genetic correlations were found between growth-associated traits and parasite load. Overall, the preliminary results provided by this study show high potential for selecting clams resistant to parasite load. Breeding for resistance may help limit the negative effects of climate change on clam production, as the prevalence of the parasite is predicted to increase under a future scenario of higher temperatures. Finally, the limited genetic correlation between resistance and growth suggests that breeding programs could incorporate dual selection without negative interactions.

Survey, characterization and antimicrobial susceptibility of Clostridium difficile from marine bivalve shellfish of North Adriatic Sea.

Clostridium difficile is a major cause of infectious diarrhea associated to healthcare settings. Community-acquired infections are increasingly reported in the last decade and exposure other than to symptomatic patients rather to contaminated foods or animals is feasible. Occurrence of C. difficile in shellfish raises concern because spores can survive the cooking temperatures given that shellfish is often consumed poorly cooked or raw. Aim of our study was to investigate whether shellfish represents a reservoir of C. difficile human PCR-ribotypes (RTs). 702 shellfish samples of farmed and wild bivalve mollusc species were collected over the 2015-2017 period in North Adriatic Italian Sea to investigate contamination with C. difficile and characterize the isolates in terms of genotypic variability and antimicrobial resistance profile. C. difficile was detected in 16.9% (CI: 14.1%-19.8%) samples: 11.6% mussels and 23.2% clams. Compared to mussels, clams were significantly associated with detection of C. difficile (OR = 2.4, P < 0.01). Overall 113 C. difficile isolates were genotyped and 75 (66.4%) were toxigenic. Fifty-three different RTs were identified. 40.7% C. difficile isolates were among the RTs most commonly involved in human infection in Europe. The profile of antimicrobial susceptibility was determined by E-test; microbiological resistance was frequent against clindamycin (17%), erythromycin (23%), rifampicin (8.8%) and moxifloxacin (10.6%). All isolates were susceptible to metronidazole and one showed MIC > ECOFF for vancomycin. C. difficile strains showed high variety in RTs, most of them already detected in other animals or known as highly virulent and epidemic in humans. These results prompt towards investigating on specific risk mitigation measures against C. difficile and are preliminary for any source attribution and risk assessment study.

Assessment of human enteric viruses in shellfish from the northern Adriatic sea.

Incidence and circulation of different strains of hepatitis A and Norovirus in shellfish were studied on 235 samples (Tapes philippinarum, Mytilus galloprovincialis, Ostrea spp. and Chlamys spp.) obtained from different sites, representing the shellfish production areas of the northern Adriatic sea. Shellfish were harvested in the period of one year and, after depuration, were examined for bacterial (Escherichia coli and Salmonella) and viral (HAV and NoV) contamination. Viral contamination was present on average in 22% of samples: specifically, 6% of samples tested positive for HAV, 14% for NoV and 2% for both viruses. None of the samples revealed the presence of Salmonella, and in most of them (93%) the number of E. coli was below the European legislation limit of 230 MPN/100 g. T. philippinarum was the species most often contaminated, as well as being the only species in which the legal limit for E. coli was, in some cases, exceeded. Both HAV and NoV contamination were detected throughout the year; NoV detection was slightly more frequent during winter months, but positive samples were also present in summer. The sequencing of the PCR products showed the circulation of only one HAV genotype (IA) and four different NoV genotypes (Hawaii, Melksham, Lordsdale and GGIIb) with a prevalence of the GGIIb genotype in the second period of the monitoring.

First report of a fish kill episode caused by pyrethroids in Italian freshwater.

INTRODUCTION: Fish kills are events of strong emotional impact on the population because of the frequent suspicion that they can be the result of serious pollution accidents. As a matter of fact, they are often due to natural occurrences, such as low levels of dissolved oxygen in the water, but in many cases the causes remain unknown. Fish are particularly sensitive to pesticides and pyrethroids are reported to be the most ecotoxicologically active in the aquatic environment. Nevertheless, the reported cases of massive wild fish mortalities due to these toxicants are very few. This paper describes a fish kill episode occurred in the Padua Province (Veneto Region - North Eastern Italy) which involved several fish species and for which it was possible to identify the cause in the presence of pyrethroids in the water. CASE PRESENTATION: When a whitish liquid coming from the rainwater drain of an industrial area was seen to be spilling into a drainage channel, a fish massive mortality was noticed and investigated. The collected water samples showed the presence of relevant concentrations of cypermethrin, permethrin, deltamethrin and tetramethrin. Analyses on the fish tissues revealed the presence of cypermethrin and permethrin at a concentration range of 476-2834µg/kg and 346-2826µg/kg on a lipid basis, respectively. DISCUSSION: According to the results of the performed analyses, we can reasonably state that the described episode had been caused by the exposure of biota to high concentrations of pyrethroids. The present case report significantly contributes to the limited literature available on pesticides-related fish kills. Moreover, it highlights the importance of sharing protocols for fish kill management at a national level, as this would help to better define the roles of the different institutions involved and to improve the investigation and the reporting of these events.