Detection of SARS-CoV-2 RNA in Bivalve Mollusks by Droplet Digital RT-PCR (dd RT-PCR)

Bivalve shellfish are readily contaminated by human pathogens present in waters impacted by municipal sewage, and the detection of SARS-CoV-2 in feces of infected patients and in wastewater has drawn attention to the possible presence of the virus in bivalves. The aim of this study was to collect data on SARS-CoV-2 prevalence in bivalve mollusks from harvesting areas of Campania region. A total of 179 samples were collected between September 2019 and April 2021 and were tested using droplet digital RT-PCR (dd RT-PCR) and real-time RT-PCR. Combining results obtained with different assays, SARS-CoV-2 presence was detected in 27/179 (15.1%) of samples. A median viral concentration of 1.1 × 102 and 1.4 × 102 g.c./g was obtained using either Orf1b nsp14 or RdRp/gene E, respectively. Positive results were unevenly distributed among harvesting areas and over time, positive samples being more frequent after January 2021. Partial sequencing of the spike region was achieved for five samples, one of which displaying mutations characteristic of the Alpha variant (lineage B.1.1.7). This study confirms that bivalve mollusks may bioaccumulate SARS-CoV-2 to detectable levels and that they may represent a valuable approach to track SARS-CoV-2 in water bodies and to monitor outbreak trends and viral diversity.

Life cycle assessment of aquaculture bivalve shellfish production — a critical review of methodological trends

Purpose The increase of shellfish production has raised environmental concerns, i.e., enrichment and redistribution of nutrients and energy consumption. Efforts assessing the environmental burdens arising from the expansion of shellfish production have been made using the life cycle assessment (LCA) methodology. Although LCA has been extensively applied and reviewed in aquaculture systems, shellfish production remains scarcely studied. The objective of this review is to identify methodological trends, highlight gaps and limitations, and provide guidelines for future studies. Methods A systematic literature review was applied to scientific studies published up to 2021. A total of 13 documents were shorted by abstract and full text-screening. Literature meeting the inclusion criteria were further analyzed in six different aspects of a LCA (functional unit, system boundaries, data and data quality, allocation, impact assessment methods, interpretation methods). Discussion and guidelines are provided for each reviewed aspect. Results and discussions Shellfish LCAs differ considerably from other aquaculture studies mainly because shellfish avoids the allocation of impacts derived from the production of fishmeal. Co-products are present when the shellfish is processed, e.g., in canned products. Furthermore, shellfish studies do not take into account the positive credits from the removal of nutrients from the ecosystems and from the valorization of the shellfish waste (shell and organic remains). Limited information was found for countries outside Europe and species different from mussels. Despite the variability on goals and scopes of the studies, methodological trends were found. The local impacts of the shellfish with the farming area and the impacts on biodiversity have not been included into the studies. Conclusions and recommendations Effort should be made in providing the data associated with the fore-background system within the studies in order to improve transparency and to allow the reproduction of the results. Information regarding the natural condition of the cultivation area should be provided as the shellfish production depends mainly on non-anthropogenic conditions. Application of biodiversity assessment methodologies should be encouraged, despite their limitations.

Predicting Harmful Algal Blooms and Impacts on Shellfish Mariculture using Novel Data-Driven Approaches

Harmful algal blooms (HABs) intoxicate and asphyxiate marine life, causing devastating environmental and socio-economic impacts costing at least $8bn/yr globally. Accumulation of phycotoxins from HAB phytoplankton in filter-feeding shellfish can poison human consumers, prompting site harvesting closures if concentrations in shellfish exceed safe levels. To better quantify both long- and short-term HAB risks, we developed novel data-driven approaches to predict phycotoxin concentrations in bivalve shellfish associated with HAB forming Dinophysis species. Our spatiotemporal statistical modelling framework assesses long-term HAB risks for different shellfish species in both data-rich and data-poor locations. This can revolutionise mariculture management by more confidently informing optimal siting of new shellfish operations and safe harvesting periods for businesses. Meanwhile, our machine learning framework forecasts phycotoxin concentrations further into the future than previously possible. Across 6 coastal, estuarine and loch sites, we achieve 87% overall accuracy in predicting future harvesting shutdowns 0-8 weeks ahead.

Bacteriophages with Potential to Inactivate Aeromonas hydrophila in Cockles: In Vitro and In Vivo Preliminary Studies

The recurrent emergence of infection outbreaks associated with shellfish consumption is of extreme importance for public health. The present study investigated the potential application of phages AH-1, AH-4, and AH-5 to inactivate Aeromonas hydrophila, a causative agent of infections in humans associated with bivalve shellfish consumption. The inactivation of A. hydrophila was assessed in vitro, using a liquid culture medium, and in vivo, using artificially contaminated cockles with A. hydrophila ATCC 7966. In the in vitro experiments, all phages were effective against A. hydrophila, but phage AH-1 (with a maximum reduction of 7.7 log colonies forming units CFU/mL) was more effective than phages AH-4 and AH-5 (with reductions of 4.9 and 4.5 log CFU/mL, respectively). The cocktails AH-1/AH-4, AH-1/AH-5, AH-4/AH-5, and AH-1/AH-4/AH-5 were slightly more effective than the single phage suspensions. The phages presented a low emergence rate of phage-resistant mutants. When artificially contaminated cockles were treated in static seawater with phage AH-1, around 44% of the added A. hydrophila (1.0 log CFU/g) was inactivated. The results of this study suggest that phage therapy can be an effective alternative to control human pathogenic bacteria during depuration.

Predominance of human bocavirus genotypes 1 and 2 in oysters in Thailand

Human bocavirus (HBoV) has been recognized as one of important pathogens of respiratory infection and acute gastroenteritis in young children worldwide. HBoV is most likely transmitted by the respiratory route and fecal-oral transmission. Recently, HBoV has been detected in several types of environmental water and bivalve shellfish. However, study of the existence of HBoV in oyster is still undocumented in Thailand. In this study, 144 oyster samples collected from different markets in Chiang Mai, Thailand during 2017 to 2018 were investigated for the presence of HBoV by nested-PCR and sequencing. HBoV was detected in 11 out of 144 samples (7.6%). Nine HBoV-positive samples (81.8%) were identified as genotype 1 (HBoV1) and two (18.2%) as HBoV2. A monthly investigation of HBoV in oyster samples from July 2017 to June 2018 showed that HBoV was sporadically detected in some particular months spanning the rainy and colder season with its peak in January. This study demonstrates the presence and genotype diversity of HBoV in oyster samples in Thailand. The finding contributes to evaluating the risk of foodborne transmission of HBoV and monitoring the outbreak of HBoV in Thailand and other countries. Importance Human bocavirus is recognized as an important cause of respiratory infection and of acute gastroenteritis in children worldwide. Human bocavirus is widely detected in many clinical specimens as well as in several environmental samples. Most of the studies describe the incidence of bocavirus infection in human while only few data are available for the occurrence of human bocavirus in food materials, particularly, in bivalve shellfish. Our findings provide the evidence for the existence and prevalence of human bocavirus in oysters suggesting that further monitoring the potential risk of food and waterborne transmission of this virus to human should be undertaken.

Baseline Health Risk Assessment of Trace Metals in Bivalve Shellfish From Commercial Growing Areas in the Estuaries of Ashtamudi and Vembanad (Kerala, India)

Trace metal concentrations were monitored in the yellow clam (Paphia malabarica), green mussel (Perna viridis) and edible oyster (Crassostrea madrasensis) from growing areas in the Ashtamudi and Vembanad estuaries, Kerala. Ranges of mean annual concentrations (mg/kg) were Ni (0.46–0.65); Co (2.87–3.49); Fe (80.0–119.4); Mn (3.88–9.38); Zn (40.8–76.2); Pb (1.28–2.00); Cu (1.59–4.38). In Ashtamudi, clams had higher mean concentrations of Ni, Co, Fe, Mn and Pb than oysters. Mean concentrations of Ni, Pb (in all species), Zn (in clams and mussels) and Cu (in mussels) did not exceed maximum permissible limits mandated by the Food Safety and Standards Authority of India. Mean Mn concentrations exceeded the World Health Organization guideline in the three species while mean Fe concentrations in clams and oysters did not exceed the guideline. Target hazard quotients were generally ≤ 1, except for a few Pb results in clams and mussels. Although results suggest no health risk to consumers for the reference doses, daily intakes and elements considered, regular monitoring of trace metals is recommended to maintain consumer protection given increasing anthropogenic and climatic pressures on the shellfish growing areas.