Bivalve Molluscan Shellfish Research Articles

Droplet Digital PCR for Precise Quantification of Human Norovirus in Shellfish Associated with Gastroenteritis Illness

Norovirus is the predominant cause of viral gastroenteritis globally with foodborne outbreaks commonly reported. Filter-feeding bivalve molluscan shellfish can become contaminated with norovirus when grown in waters impacted by inadequately treated effluent wastewater, overflows, or other human fecal sources. Contaminated shellfish pose a significant risk to consumers, because combined with a low norovirus infectious dose, oysters and mussels are often eaten raw or lightly cooked resulting in no or minimal virus inactivation, respectively. In addition, shellfish contamination has significant economic impacts on the seafood industry. To improve risk assessments, reverse transcription (RT)-digital droplet PCR (ddPCR) was used to determine the precise norovirus concentrations in 20 shellfish samples, all positive for norovirus genogroup I and/or II (GI or GII) by RT-qPCR, and associated with reported norovirus illness in New Zealand. Using RT-ddPCR, total norovirus GI and/or GII concentrations in shellfish ranged between 44 and 4,630 genome copies (GC)/g digestive tissue. Importantly, 40% (8/20) of shellfish samples contained a total norovirus concentration less than 200 GC/g digestive tissue. In parallel, RNase treatment was applied, prior to viral extraction to remove free RNA, which subsequently led to average reductions in norovirus GC/g concentration of 37.1% and 19.4% for GI and GII, respectively. These RT-ddPCR data provide valuable evidence for risk assessment of contaminated shellfish and evaluation of safety guidelines and highlight issues associated with setting a safe threshold of norovirus in shellfish.

PMAxx-RT-qPCR to Determine Human Norovirus Inactivation Following High-Pressure Processing of Oysters.

Norovirus is the leading cause of viral gastroenteritis globally. While person-to-person transmission is most commonly reported route of infection, human norovirus is frequently associated with foodborne transmission, including through consumption of contaminated bivalve molluscan shellfish. Reverse transcription (RT)-qPCR is most commonly used method for detecting human norovirus detection in foods, but does not inform on its infectivity, posing challenges for assessing intervention strategies aimed at risk elimination. In this study, RT-qPCR was used in conjunction with a derivative of the photoreactive DNA binding dye propidium monoazide (PMAxx™) (PMAxx-RT-qPCR) to evaluate the viral capsid integrity ofnorovirus genogroup I and II (GI and GII) in shellfish following high pressure processing (HPP). Norovirus GI.3 and GII.4 bioaccumulated oysters were subjected to HPP at pressures of 300 and 450MPa at 15°C, and 300, 450 and 600MPa at 20°C. Samples were analysed using both RT-qPCR and PMAxx-RT-qPCR. For each sample, norovirus concentration (genome copies/g digestive tissue) determined by RT-qPCR was divided by the PMAxx-RT-qPCR concentration, giving the relative non-intact (RNI) ratio. The RNI ratio values relate to the amount of non-intact (non-infectious) viruses compared to fully intact (possible infectious) viruses. Our findings revealed an increasing RNI ratio value, indicating decreasing virus integrity, with increasing pressure and decreasing pressure. At 300MPa, for norovirus GI, the median [95% confidence interval, CI] RNI ratio values were 2.6 [1.9, 3.0] at 15°C compared to 1.1 [0.9, 1.8] at 20°C. At 450MPa, the RNI ratio values were 5.5 [2.9, 7.0] at 15°C compared to 1.3 [1.0, 1.6] at 20°C. At 600MPa, the RNI ratio value was 5.1 [2.9, 13.4] at 20°C. For norovirus GII, RT-qPCR and PMAxx-RT-qPCR detections were significantly reduced at 450 and 600MPa at both 15°C and 20°C, with the median [95% CI] RNI ratio value at 300MPa being 1.1 [0.8, 1.6]. Following HPP treatment, the use of PMAxx-RT-qPCR enables the selective detection of intact and potential infectious norovirus, enhancing our understanding of the inactivation profiles and supporting the development of more effective risk assessment strategies.

Persistence of Infectious Human Norovirus in Estuarine Water

Norovirus is the predominant cause of viral acute gastroenteritis globally. While person-to-person is the most reported transmission route, norovirus is also associated with waterborne and foodborne illness, including from the consumption of contaminated bivalve molluscan shellfish. The main cause of shellfish contamination is via the bioaccumulation of norovirus from growing waters impacted by human wastewater. However, data on the persistence of infectious norovirus in the environment are limited due to a lack of a human norovirus culture method in the past. In this study, we applied the recently established method of norovirus replication in human intestinal enteroids to determine the persistence of norovirus in artificial estuarine water at 25 ppt for up to 21 days at 4 °C and 16 °C in the dark. Infectious norovirus was detected for up to 21 days. The relative infectivity declined from 100 to 3% at day 21, with decay rate constants of 0.07 day−1 at 4 °C and 0.17 day−1 at 16 °C. There was no decrease in norovirus titres as measured by reverse transcription-droplet digital PCR (RT-ddPCR), confirming the lack of the relationship between norovirus infectivity and direct detection by PCR. The results confirm that norovirus can remain infectious for at least 3 weeks in an estuarine water environment, presenting associated health risks.

Microbial Spoilage Organisms in Seafood Products: Pathogens and Quality Control

Consuming contaminated seafood can lead to various bacterial illnesses, either from contamination at the source or during processing and distribution. These illnesses can result from bacterial infection or the ingestion of toxins formed in the food before consumption. However, the distinction between the two causes is not always straightforward. For instance, toxins from Clostridium botulinum and Staphylococcus aureus are preformed in food during bacterial growth, while the toxin from Clostridium perfringens is usually formed when the bacteria sporulate in the intestinal tract. Moreover, the toxin from Vibrio cholerae O1 (and O139) is generated as the bacteria multiply in the intestinal tract. Scombrotoxin, a common illness linked to seafood consumption, is caused by the production of histamine and potentially related substances by certain types of Gram-negative bacteria on histidine-rich fish tissue. This topic is examined in greater detail in another chapter of this book, and therefore will not be extensively discussed here. This paper will explore the various types of bacteria that can lead to illness in humans after consuming seafood, the reported incidence of such infections in specific countries with available data. It will also examine both conventional and molecular methods for detecting, identifying, and categorizing these bacteria. There is growing interest in vibrios as the cause of bacterial illnesses linked to the consumption of bivalve molluscan shellfish and other seafood varieties, with a specific focus on this bacterial group.

Impedance method application for number detection Escherichia coli in molluscs testing by official laboratory

Bivalve molluscan shellfish (BMS) have an important role in the transmission of some pathogens responsible for food-borne disease because they are filter-feeding animals capable of greatly concentrating certain pathogenic agents (bacteria, viruses, or parasites) present in the water column, like Escherichia coli. The reference method for E. coli testing in bivalves is the most probable number (MPN) method therefore, as this method has some disadvantages, alternative techniques of equivalent accuracy could be used without the drawbacks of the MPN method such as the direct impedance technique. This method is based on the principle that bacteria produce positively or negatively charged end products causing an impedance variation of the medium that can be used to measure their growth. The present study evaluated the efficiency of the direct impedance measuring technique through a series of laboratory tests performed on 6 kg of clams belonging to two different batches. The preliminary obtained data, compared with the MPN reference method and dd-PCR to validate the alternative method, show that the direct impedance technique has the potential to be used in place of TBX culture for confirming E. coli in MPN assays.

Monitoring and Genotyping of Norovirus in Bivalve Molluscan Shellfish from Northern Italian Seas (2018-2020).

Norovirus (NoV) is an enteric virus with foodborne transmission. Bivalve shellfish are a main source of infections and outbreaks. In Italy a voluntary based monitoring plan to check the safety of bivalve shellfish was set up at provincial level. This study describes the occurrence and distribution of NoV in the Northern Adriatic Sea and in the Ligurian Sea. From October 2018 to September 2020, 807 bivalve shellfish samples (n = 205 oysters, n = 182 mussels, n = 348 clams, n = 72 other bivalve shellfish) were tested by One-Step Retrotranscription Real-time polymerase chain reaction for NoV GI and GII and quantified according to the ISO 15216-2:2013 and ISO 15216-1:2017. Positive samples were further analyzed to determine genotype by sequencing of the ORF1/ORF2 junction of the viral genome. A total of 126 samples were positive for NoV, mussels, and oysters had the highest probability of being positive and positive samples were found mainly in the colder season. Of these samples, 46% were NoV GII, 13% NoV GI, and 40% carried both genogroups. Thirty-seven samples were typeable (GI n = 12 and GII n = 25) with GI samples belonging to four genotypes and GII samples belonging to five genotypes. GII.3 genotype was the most prevalent, followed by GII.4, particularly Sydney 2012 subtype, a leading cause of infections worldwide, was found in three oysters' and three clams' samples. The phylogenetic analysis revealed a high heterogeneity among the species that are scattered in several clusters. Considering the low infectious dose the overall presence of NoV in edible shellfish, particular those to be eaten raw or undercooked, is moderately high. The presence of genotypes frequently involved in human infections strengthens the need for ongoing monitoring, which should be extended at national level.

Toward better monitoring of human noroviruses and F-specific RNA bacteriophages in aquatic environments using bivalve mollusks and passive samplers: A case study.

Monitoring pathogenic enteric viruses in continental and marine water bodies is essential to control the viral contamination of human populations. Human Noroviruses (NoV) are the main enteric viruses present in surface waters and foodstuff. In a context of global change, it is currently a challenge to improve the management of viral pollutions in aquatic environments and thereby limit the contamination of vulnerable water bodies or foodstuffs. The aim of this study is to evaluate the potential of specific accumulation systems for improving the detection of NoV in water bodies, compared to direct water analyses. Passive samplers (Zetapor filters) and three species of bivalve molluscan shellfish (BMS) (Dreissena polymorpha, Mytilus edulis and Crassostreas gigas) were used as accumulation systems to determine their performance in monitoring continental and marine waters for viruses. F-specific RNA bacteriophages (FRNAPH) were also analyzed since they are described as indicators of NoV hazard in many studies. During a one-year study in a specific area frequently affected by fecal pollution, twelve campaigns of exposure of passive samplers and BMS in continental and coastal waters were conducted. Using suitable methods, NoV (genome) and FRNAPH (infectious and genome) were detected in these accumulation systems and in water at the same time points to determine the frequency of detection but also to gain a better understanding of viral pollution in this area. The reliability of FRNAPH as a NoV indicator was also investigated. Our results clearly showed that BMS were significantly better than passive samplers and direct water analyses for monitoring NoV and FRNAPH contamination in water bodies. A dilution of viral pollution between the continental and the coastal area was observed and can be explained by the distance from the source of the pollution. Viral pollution is clearly greater during the winter period, and stakeholders should take this into consideration in their attempts to limit the contamination of food and water. A significant correlation was once again shown between NoV and FRNAPH genomes in BMS, confirming the reliability of FRNAPH as a NoV indicator. Moreover, a strong correlation was observed between NoV genomes and infectious FRNAPH, suggesting recent viral pollution since infectious particles had not been inactivated at sufficient levels in the environment. More generally, this study shows the value of using BMS as an active method for improving knowledge on the behavior of viral contamination in water bodies, the ranking of the contamination sources, and the vulnerability of downstream water bodies.

A Comprehensive Review for the Surveillance of Human Pathogenic Microorganisms in Shellfish.

Bivalve molluscan shellfish have been consumed for centuries. Being filter feeders, they may bioaccumulate some microorganisms present in coastal water, either naturally or through the discharge of human or animal sewage. Despite regulations set up to avoid microbiological contamination in shellfish, human outbreaks still occur. After providing an overview showing their implication in disease, this review aims to highlight the diversity of the bacteria or enteric viruses detected in shellfish species, including emerging pathogens. After a critical discussion of the available methods and their limitations, we address the interest of technological developments using genomics to anticipate the emergence of pathogens. In the coming years, further research needs to be performed and methods need to be developed in order to design the future of surveillance and to help risk assessment studies, with the ultimate objective of protecting consumers and enhancing the microbial safety of bivalve molluscan shellfish as a healthy food.

Dynamics of fecal coliform bacteria along Canada's coast

The vast coastline provides Canada with a flourishing seafood industry including bivalve shellfish production. To sustain a healthy bivalve molluscan shellfish production, the Canadian Shellfish Sanitation Program was established to monitor the health of shellfish harvesting habitats, and fecal coliform bacteria data have been collected at nearly 15,000 marine sample sites across six coastal provinces in Canada since 1979. We applied Functional Principal Component Analysis and subsequent correlation analyses to find annual variation patterns of bacteria levels at sites in each province. The overall magnitude and the seasonality of fecal contamination were modelled by functional principal component one and two, respectively. The amplitude was related to human and warm-blooded animal activities; the seasonality was strongly correlated with river discharge driven by precipitation and snow melt in British Columbia, but such correlation in provinces along the Atlantic coast could not be properly evaluated due to lack of data during winter.

Prevalence and Antimicrobial Resistance of Bacterial Foodborne Pathogens Isolated from Raw Bivalve Molluscs Subjected to Consumption in Poland during a Ten-Year Period.

The aim of this study was to evaluate the microbiological contamination of raw bivalve molluscan shellfish (BMS) available on the Polish market and determinate the antimicrobial resistance of the obtained isolates. A total of 1000 mollusc samples were tested for the presence of Salmonella spp., L. monocytogenes, V. parahaemolyticus, and S. aureus using the ISO standard methods. Additionally, the bacterial isolates' susceptibility to antimicrobials was determined using the minimum inhibitory concentration (MIC) method. The obtained results showed that Salmonella spp. was detected in 31 (3.1%) samples, and 51.6% of the bacterial isolates were classified as Salmonella Typhimurium. A total of 74.2% of the Salmonella isolates were sensitive to all antimicrobial agents, whereas three isolates were multiresistant. L. monocytogenes was isolated from 18 (1.8%) BMS, and the isolates belonged to serogroups IIa, IIb, and IVb. Most of them were resistant to ceftriaxone (77.8%) and oxacillin (55.6%). V. parahaemolyticus was present in 24.2% BMS. These isolates were mainly resistant to ampicillin (77.3%) and streptomycin (64.0%). Moreover, 15.2% of the bivalve molluscs were contaminated with S. aureus. Most isolates belonging to this species were resistant to penicillin (84.9%). A total of 60 (6.0%) bivalve molluscs were contaminated with more than one pathogen simultaneously. In addition, the tested bacteria were more likely to be identified during the warmer period (53.9%) compared to the samples analyzed in colder months (35.7%). The obtained results indicate that raw bivalve molluscs from the Polish market are frequently contaminated with bacterial foodborne pathogens, which may be resistant to antimicrobials.

Application of Next Generation Sequencing on Norovirus‐contaminated oyster samples

Bivalve molluscan shellfish (BMS) contamination with gastroenteritis viruses such as norovirus (NoV) is recognized as a significant public health risk worldwide. These foodborne epidemics are characterized by very low viral concentrations in the implicated foods, and by diverse viruses or multiple NoV strains originating from human sewage, resulting in different strains (co-)infecting the consumers. Next-generation sequencing (NGS) offers promising means to describe the diversity of strains present in BMS or to retrace transmission chains in outbreak settings, but their sensitivity and reproducibility remained to be assessed for this application. In this work, we evaluated the ability of three promising NGS methods to sequence diverse NoV in environmental or food BMS and human stool samples. Using laboratory-prepared samples of known NoV composition, we evaluated the sensitivity, reproducibility, repeatability and selectivity of metabarcoding, capture-based metagenomics and long amplicon sequencing, considering representative NoV strains from genogroup I and II, and the impact of the BMS matrix. The metabarcoding, with separate amplification of polymerase and capsid gene segments followed by Illumina sequencing, was the most sensitive method. It was applied to a selection of 212 BMS samples collected through the European Commission’s NoV baseline survey (BLS), demonstrating a high diversity of NoV sequences found in the BMS which reflect the diversity of NoV strains circulating in the European human population. Besides, a capture-based metagenomics with enrichment of vertebrate viruses was applied on 20 of these BLS samples as well as 20 BMS linked to outbreaks and 10 related human stool samples. In BMS, it yielded NoV sequences compatible with the genomes identified in stool samples, but they were too short to allow definitive confirmation of the infection source. The present report describes NGS methods, including the bioinformatic pipelines, applicable to molecular epidemiology of NoV in BMS, their current limitations and expected outcomes.

SARS-CoV-2 circulation in Croatian wastewaters and the absence of SARS-CoV-2 in bivalve molluscan shellfish

The circulation of SARS-CoV-2 in the environment has been confirmed numerous times, whilst research on the bioaccumulation in bivalve molluscan shellfish (BMS) has been rather scarce. The present study aimed to fulfil the knowledge gap on SARS-CoV-2 circulation in wastewaters and surface waters in this region and to extend the current knowledge on potential presence of SARS-CoV-2 contamination in BMS. The study included 13 archive wastewater and surface water samples from the start of epidemic and 17 influents and effluents from nine wastewater treatment plants (WWTP) of different capacity and treatment stage, sampled during the second epidemic wave. From that period are the most of 77 collected BMS samples, represented by mussels, oysters and warty venus clams harvested along the Dalmatian coast. All samples were processed according to EN ISO 15216-1 2017 using Mengovirus as a whole process control. SARS-CoV-2 detection was performed by real-time and conventional RT-PCR assays targeting E, N and nsp14 protein genes complemented with nsp14 partial sequencing. Rotavirus A (RVA) real-time RT-PCR assay was implemented as an additional evaluation criterion of virus concentration techniques. The results revealed the circulation of SARS-CoV-2 in nine influents and two secondary treatment effluents from eight WWTPs, while all samples from the start of epidemic (wastewaters, surface waters) were negative which was influenced by sampling strategy. All tertiary effluents and BMS were SARS-CoV-2 negative. The results of RVA amplification were beneficial in evaluating virus concentration techniques and provided insights into RVA dynamics within the environment and community. In conclusion, the results of the present study confirm SARS-CoV-2 circulation in Croatian wastewaters during the second epidemic wave while extending the knowledge on wastewater treatment potential in SARS-CoV-2 removal. Our findings represent a significant contribution to the current state of knowledge that considers BMS of a very low food safety risk regarding SARS-CoV-2.

Detection of Rotavirus Strains in Freshwater Clams in Japan.

Bivalve molluscan shellfish like clams and oysters, etc., are capable to bioaccumulate surrounding contaminants from waters into their digestive systems and posing serious threats of food poisoning. Detection of rotaviruses (RVs) in shellfish is of particular importance because RVs are prone to genome reassortment resulting in the emergence of new RV variants that may compromise vaccine safety. Herein, we have detected the wild-type RVs and Rotarix/RotaTeq vaccine strains in freshwater clams collected on the riverside, Kawasaki city, from July 2019 to January 2020 and correlated the detected genotypes with that of gastroenteritis cases of nearby clinics to understand the transmission of RVs in the environment. The wild-type RVs were detected in 62 (64.6%) out of 96 freshwater clams in every study month: July, September, November, and January that are considered as off-season for RV infections. The most frequent genotypes were G2 (42.9%), G8 (28.6%), G3 (14.3%), G1 (7.1%), and G10 (7.1%), which remained comparable with genotypic distribution found in the clinical samples over the last few years indicating that these RVs may accumulate in clams since a long time. However, G10 genotype was detected in clam but not in clinical samples suggesting the presence of asymptomatic infection or RVs could be carried out from a long distance. Importantly, vaccine strains, RotaTeq (1%) but not Rotarix (0%), were also detected in a clam. Attention must be paid to monitoring the potential transmission of wild-type and vaccine RV strains in the environment to prevent the emergence of new variants generated from genome reassortment with vaccine strains.

Risk Assessment of Norovirus Illness from Consumption of Raw Oysters in the United States and in Canada.

Human norovirus (NoV) is the leading cause of foodborne illness in the United States and Canada. Bivalve molluscan shellfish is one commodity commonly identified as being a vector of NoV. Bivalve molluscan shellfish are grown in waters that may be affected by contamination events, tend to bioaccumulate viruses, and are frequently eaten raw. In an effort to better assess the elements that contribute to potential risk of NoV infection and illness from consumption of bivalve molluscan shellfish, the U.S. Department of Health and Human Services/Food and Drug Administration (FDA), Health Canada (HC), the Canadian Food Inspection Agency (CFIA), and Environment and Climate Change Canada (ECCC) collaborated to conduct a quantitative risk assessment for NoV in bivalve molluscan shellfish, notably oysters. This study describes the model and scenarios developed and results obtained to assess the risk of NoV infection and illness from consumption of raw oysters harvested from a quasi‐steady‐state situation. Among the many factors that influence the risk of NoV illness for raw oyster consumers, the concentrations of NoV in the influent (raw, untreated) and effluent (treated) of wastewater treatment plants (WWTP) were identified to be the most important. Thus, mitigation and control strategies that limit the influence from human waste (WWTP outfalls) in oyster growing areas have a major influence on the risk of illness from consumption of those oysters.

Influence of fucosidase-producing bifidobacteria on the HBGA antigenicity of oyster digestive tissue and the associated norovirus binding

Bivalve molluscan shellfish such as oysters are filter feeders and are able to accumulate human noroviruses (NoVs) largely due to the presence of human histo-blood group antigens (HBGAs)-like carbohydrates in their intestine. Since the fucose contents play a key role in the binding of NoVs to HBGAs, this study intended to investigate the influence of fucosidase-producing bifidobacteria on the HBGA antigenicity of oyster digestive tissue and the associated NoV binding. On the contrary to the expected, after a treatment of the oyster digestive tissue extracts with Bifidobacterium bifidum strain JCM 1254, the binding of human NoV GII.4 virus like particles (VLPs) to the oyster digestive tissue extracts enhanced significantly (OD450 from 1.15 ± 0.05 to 1.51 ± 0.02, P < 0.001) in an in vitro direct binding assay. The accumulation of human NoV GII·P16-GII.4 also enhanced significantly in the intestine of B. bifidum JCM 1254 treated oysters from 4.27 ± 0.25 log genomic copies/g oyster digestive tissue to 5.25 ± 0.29 log genomic copies/g oyster digestive tissue (P < 0.005) as observed in an in vivo test. Correspondingly, the type A antigenicity of the oyster digestive tissue extracts enhanced (OD450 from 0.77 ± 0.04 to 1.06 ± 0.05, P < 0.01) after the treatment with B. bifidum JCM 1254. These results could be explained by the substrate specificity of the B. bifidum JCM 1254 associated fucosidases. This study identified an indirect interaction possibly happening between the bacterial microbiota with human NoVs during their transmission in the food systems. We also supplied a potential strategy to mitigate the NoV contamination from shellfish, suppose bacterial strains with specified fucosidase production could be obtained in the future.

Risk Assessment of Pectenotoxins in New Zealand Bivalve Molluscan Shellfish, 2009-2019.

Pectenotoxins (PTXs) are produced by Dinophysis spp., along with okadaic acid, dinophysistoxin 1, and dinophysistoxin 2. The okadaic acid group toxins cause diarrhetic shellfish poisoning (DSP), so are therefore regulated. New Zealand currently includes pectenotoxins within the DSP regulations. To determine the impact of this decision, shellfish biotoxin data collected between 2009 and 2019 were examined. They showed that 85 samples exceeded the DSP regulatory limit (0.45%) and that excluding pectenotoxins would have reduced this by 10% to 76 samples. The incidence (1.3%) and maximum concentrations of pectenotoxins (0.079 mg/kg) were also found to be low, well below the current European Food Safety Authority (EFSA) safe limit of 0.12 mg/kg. Inclusion within the DSP regulations is scientifically flawed, as pectenotoxins and okadaic acid have a different mechanism of action, meaning that their toxicities are not additive, which is the fundamental principle of grouping toxins. Furthermore, evaluation of the available toxicity data suggests that pectenotoxins have very low oral toxicity, with recent studies showing no oral toxicity in mice dosed with the PTX analogue PTX2 at 5000 µg/kg. No known human illnesses have been reported due to exposure to pectenotoxins in shellfish, a fact which combined with the toxicity data indicates that they pose negligible risk to humans. Regulatory policies should be commensurate with the level of risk, thus deregulation of PTXs ought to be considered, a stance already adopted by some countries.

A qPCR-MPN method for rapid quantification of Escherichia coli in bivalve molluscan shellfish

Faecal contamination of bivalve molluscan shellfish (BMS) can lead to infections from enteric pathogens if consumed. Across Europe, the faecal indicator bacteria Escherichia coli, is used to determine contamination of BMS harvesting areas. The reference most probable number (MPN) method for E. coli in BMS takes around 48 h from sample receipt to result. In this study, an alternative method was developed in which the final, E. coli confirmation step in the MPN method (usually carried out on chromogenic TBX agar) was replaced by presence/absence real-time PCR (qPCR). This qPCR-MPN method was directly compared with the reference TBX-MPN method using 194 BMS samples consisting of mussels (Mytilus spp.), Pacific oysters (Crassostrea gigas) and common cockles (Cerastoderma edule). The qPCR-MPN method correlated positively with the TBX-MPN method (Kendall's tau coefficient = 0.812). However, the strength of this correlation varied between BMS species, with mussels having the poorest correlation (0.677) followed by Pacific oysters (0.795) and common cockles (0.890). There were some samples for which the difference between the two methods was higher than might be expected by statistical probability alone. Variations in the way in which the two confirmation methods work may account for much of this variation. This method may serve as an ad hoc, rapid assessment method that is complementary to the official reference method and could be easily implemented in many official control laboratories.

Survey of Tetrodotoxin in New Zealand Bivalve Molluscan Shellfish over a 16-Month Period.

Tetrodotoxin (TTX) is a heat-stable neurotoxin typically associated with pufferfish intoxications. It has also been detected in shellfish from Japan, the United Kingdom, Greece, China, Italy, the Netherlands and New Zealand. A recent European Food Safety Authority (EFSA) scientific opinion concluded that a level of <0.044 mg TTX/kg in marine bivalves and gastropods, based on a 400 g portion size, does not result in adverse effects in humans. There have been no reports of human illness attributed to the consumption of New Zealand shellfish containing TTX. To obtain a greater understanding of its presence, a survey of non-commercial New Zealand shellfish was performed between December 2016 and March 2018. During this period, 766 samples were analysed from 8 different species. TTX levels were found to be low and similar to those observed in shellfish from other countries, except for pipi (Paphies australis), a clam species endemic to New Zealand. All pipi analysed as part of the survey were found to contain detectable levels of TTX, and pipi from a sampling site in Hokianga Harbour contained consistently elevated levels. In contrast, no TTX was observed in cockles from this same sampling site. No recreationally harvested shellfish species, including mussels, oysters, clams and tuatua, contained TTX levels above the recommended EFSA safe guidance level. The levels observed in shellfish were considerably lower than those reported in other marine organisms known to contain TTX and cause human intoxication (e.g., pufferfish). Despite significant effort, the source of TTX in shellfish, and indeed all animals, remains unresolved making it a difficult issue to understand and manage.

Assessing conservation conflict: Does intertidal oyster aquaculture inhibit foraging behavior of migratory shorebirds?

AbstractConservation is increasingly in conflict with human activities due to global human population growth, particularly in areas that support threatened species. Conflicts often impede effective implementation of needed conservation measures and also have implications for social inequality, resource use and economic development. Bivalve molluscan shellfish aquaculture is commonly considered one of the least impactful forms of protein production worldwide but, in some locations, may interfere with essential activities of threatened species such as the stopover ecology of migrating shorebirds. Here we assess the impact of oyster aquaculture as practiced in Delaware Bay (New Jersey, USA) on the presence and foraging behavior of migratory shorebird species of conservation concern. We conducted counts and behavioral observations of shorebirds across a 4.8‐km stretch of the Delaware Bay and tested the effect of regulated aquaculture structures and activities on shorebird presence relative to various environmental factors. We also evaluated differences in mean peck rates for each species within and away from aquaculture areas, and we examined multiple factors influencing foraging rates for each species. For all species, we found that oyster tending reduced the probability of shorebird presence by 1–7%, whereas the untended aquaculture structures had no detectable impact. Foraging rates were mostly influenced by environmental conditions, particularly the presence of competitors (gulls or other shorebirds), and the foraging substrate. None of the focal species substantially altered their time budget or foraging rates in the presence of tended or untended oyster aquaculture. This evidence suggests that intertidal oyster aquaculture and migrating shorebirds can co‐utilize the resource rich intertidal areas on which they occur.

Characterizing the effects of thermal treatment on human norovirus GII.4 viability using propidium monoazide combined with RT-qPCR and quality assessments in mussels

This study investigated the effects of thermal treatment (70, 80, and 90 °C for 5, 10, and 20 min) on the titers of human norovirus (HuNoV) GII.4, a dominant genotype in fresh mussels using propidium monoazide (PMA) pretreatment and real-time reverse transcription quantitative-polymerase chain reaction (RT-qPCR) and the effects of thermal treatment on mussel quality (Hunter colors and sensory properties). Reductions of >1-log10 for HuNoV in PMA-treated mussels required 70 °C for 20 min, 80 °C for 10 min and 90 °C for 5 min. As assessed by PMA/RT-qPCR, the log10 reduction values of HuNoV in PMA-treated samples was significantly reduced (46% or 0.27 log10; P < 0.05) when heated to 90 °C for 20 min compared with non-PMA treated samples under identical conditions. Hunter color ‘L’-, and ‘a’- and ‘b’- values significantly (P < 0.05) decreased and increased, respectively with a stepwise increase of temperature (70–90 °C) and duration of heat treatment (5–20 min). The results of the untrained 7-point hedonic scale of sensory evaluation revealed that mussels heated to 90 °C for 10 min were the best quality sample among all thermally treated mussels. The results suggest that the PMA/RT-qPCR method could be very effective in distinguishing HuNoV viability following high heat treatment for an extended duration (eg, 90 °C for 20 min). Based on these results, the PMA/RT-qPCR method could be very effective in assessing HuNoV viability following high-heat treatment for an extended duration (eg, 90 °C for 20 min). Furthermore, our findings suggest that inactivation of HuNoV GII.4 in the bivalve molluscan shellfish may be accomplished by treatment at 70 °C for 20 min, 80 °C for 10 min, or 90 °C for 5 min. Among all thermal treatments, 90 °C for 10 min was considered the optimal cooking temperature and duration for mussels.