Catherine McLeod

Processing Strategies to Inactivate Enteric Viruses in Shellfish

Noroviruses, hepatitis A and E viruses, sapovirus, astrovirus, rotavirus, Aichi virus, enteric adenoviruses, poliovirus, and other enteroviruses enter shellfish through contaminated seawater or by contamination during handling and processing, resulting in outbreaks ranging from isolated to epidemic. Processing and disinfection methods include shellfish depuration and relaying, cooking and heat pasteurization, freezing, irradiation, and high pressure processing. All the methods can improve shellfish safety; however, from a commercial standpoint, none of the methods can guarantee total virus inactivation without impacting the organoleptic qualities of the shellfish. Noroviruses cause the majority of foodborne viral illnesses, yet there is conflicting information on their susceptibility to inactivation by processing. The inability to propagate and quantitatively enumerate some viral pathogens in vitro or in animal models has led to the use of norovirus surrogates, such as feline calicivirus and murine norovirus. During processing, these surrogates may not mimic the inactivation of the viruses they represent and are, therefore, of limited value. Likewise, reverse transcription-PCR has limited usefulness in monitoring processing effectiveness due to its inability to identify infectious from inactivated viruses. This article (a) describes mechanisms of virus uptake and persistence in shellfish, (b) reviews the state-of-the-art in food processing strategies for the inactivation of enteric viruses in shellfish, (c) suggests the use of combined processing procedures to enhance shellfish safety, (d) highlights limitations in research data derived from virus surrogate studies and molecular assay procedures, and (e) recommends enhanced funding for human volunteer studies and the development of assays to detect viable viruses.

Depuration and Relaying: A Review on Potential Removal of Norovirus from Oysters

Pollution of coastal waters can result in contamination of bivalve shellfish with human enteric viruses, including norovirus (NoV), and oysters are commonly implicated in outbreaks. Depuration is a postharvest treatment involving placement of shellfish in tanks of clean seawater to reduce contaminant levels; this review focuses on the efficacy of depuration in reducing NoV in oysters. There have been many NoV outbreaks from depurated oysters containing around 103 genome copies/g oyster tissue, far exceeding the median infectious dose (ID50). Half of the published NoV reduction experiments showed no decrease in NoV during depuration, and in the remaining studies it took between 9 and 45.5 d for a 1-log reduction-significantly longer than commercial depuration time frames. Surrogate viruses are more rapidly depurated than NoV; the mean number of days to reduce NoV by 1 log is 19, and 7.5 d for surrogates. Thus, surrogates do not appear to be suitable for assessing virological safety of depurated oysters; data on reduction of NoV infectivity during depuration would assist evaluations on surrogate viruses and the impact of methods used. The longer persistence of NoV highlights its special relationship with oysters, which involves the binding of NoV to histo-blood group-like ligands in various tissues. Given the persistence of NoV and on-going outbreaks, depuration as currently performed appears ineffective in guaranteeing virologically safe oysters. Conversely, relaying oysters for 4 wk is more successful, with low NoV concentrations and no illnesses associated with products. The ineffectiveness of depuration emphasizes the need for coastal water quality to be improved to ensure oysters are safe to eat.

Paralytic shellfish toxins, including deoxydecarbamoyl-STX, in wild-caught Tasmanian abalone (Haliotis rubra)

For the first time wild-caught Tasmanian abalone, Haliotis rubra, have been reported to contain paralytic shellfish toxins (PSTs). This observation followed blooms of the toxic dinoflagellate Gymnodinium catenatum. No illnesses were reported, but harvesting restrictions were enforced in commercial areas. Abalone were assayed using HPLC-FLD methodology based on AOAC official method 2005.06. An uncommon congener, deoxydecarbamoyl-STX (doSTX), was observed in addition to regulated PSTs as unassigned chromatographic peaks. A quantitative reference material was prepared from contaminated Tasmanian abalone viscera and ampouled at 54.2 μmol/L. The LD50 of doSTX via intraperitoneal injection was 1069 nmol/kg (95% confidence limits 983-1100 nmol/kg), indicating it is nearly 40 times less toxic than STX. A toxicity equivalence factor of 0.042 was generated using the mouse bioassay. Levels of PSTs varied among individuals from the same site, although the toxin profile remained relatively consistent. In the foot tissue, STX, decarbamoyl-STX and doSTX were identified. On a molar basis doSTX was the dominant congener in both foot and viscera samples. The viscera toxin profile was more complex, with other less toxic PST congeners observed and was similar to mussels from the same site. This finding implicates localised dinoflagellate blooms as the PST source in Tasmanian abalone.

Australian seafood compositional profiles: A pilot study. Vitamin D and mercury content

Given the scarcity of comprehensive nutritional data for Australias >400 commercially produced seafood species a pilot study was undertaken to collect and analyse 22 species of wild and aquaculture seafood in order to develop a model for future comprehensive surveys. The species analysed were: Atlantic salmon, Australian sardine, prawn (six species), barramundi, abalone (three species), blue sprat, burrowing blackfish, gummy shark, oyster (four species), ocean trout and yellowtail kingfish. The analyses undertaken in this pilot study were: moisture, protein, total fat, cholesterol, fatty acids, vitamin C, vitamins A and D, and 21 mineral elements (including total mercury and methyl mercury). The data reported here are for vitamin D and mercury only. Comprehensive data have already been published elsewhere. Issues identified that should be addressed prior to undertaking a more extensive and representative study of the remaining major edible commercial Australian seafood species include: choice of samples and nutrients for analysis, facilities for sample handling and storage, data management and scrutiny, and laboratory quality control.

Digital PCR for Quantifying Norovirus in Oysters Implicated in Outbreaks, France

Using samples from oysters clearly implicated in human disease, we quantified norovirus levels by using digital PCR. Concentrations varied from 43 to 1,170 RNA copies/oyster. The analysis of frozen samples from the production area showed the presence of norovirus 2 weeks before consumption.

Uptake, distribution and depuration of paralytic shellfish toxins from Alexandrium minutum in Australian greenlip abalone, Haliotis laevigata

Farmed greenlip abalone Haliotis laevigata were fed commercial seaweed-based food pellets or feed pellets supplemented with 8 × 10⁵ Alexandrium minutum dinoflagellate cells g⁻¹ (containing 12 ± 3.0 μg STX-equivalent 100 g⁻¹, which was mainly GTX-1,4) every second day for 50 days. Exposure of abalone to PST supplemented feed for 50 days did not affect behaviour or survival but saw accumulation of up to 1.6 μg STX-equivalent 100 g⁻¹ in the abalone foot tissue (muscle, mouth without oesophagus and epipodial fringe), which is ∼50 times lower than the maximum permissible limit (80 μg 100 g⁻¹ tissue) for PSTs in molluscan shellfish. The PST levels in the foot were reduced to 0.48 μg STX-equivalent 100 g⁻¹ after scrubbing and removal of the pigment surrounding the epithelium of the epipodial fringe (confirmed by both HPLC and LC-MS/MS). Thus, scrubbing the epipodial fringe, a common procedure during commercial abalone canning, reduced PST levels by ∼70%. Only trace levels of PSTs were detected in the viscera (stomach, gut, heart, gonad, gills and mantle) of the abalone. A toxin reduction of approximately 73% was observed in STX-contaminated abalone held in clean water and fed uncontaminated food over 50 days. The low level of PST uptake when abalone were exposed to high numbers of A. minutum cells over a prolonged period may indicate a low risk of PSP poisoning to humans from the consumption of H. laevigata that has been exposed to a bloom of potentially toxic A. minutum in Australia. Further research is required to establish if non-dietary accumulation can result in significant levels of PSTs in abalone.

A national survey of marine biotoxins in wild-caught abalone in Australia.

The first national survey of Australian wild-caught abalone was conducted between September 2012 and December 2013. The aim of the survey was to determine the presence of paralytic shellfish toxins (PSTs), amnesic shellfish toxins (ASTs), and diarrhetic shellfish toxins (DSTs) in wild-caught abalone at levels above the current Codex marine biotoxin limits during the 2013 fishing season. Abalone (n = 190) were collected from 68 abalone-fishing blocks for which the combined annual harvest accounts for 80 % of Australian production. Concurrent seawater samples were collected and enumerated for potentially toxic phytoplankton. The foot and viscera tissues of each abalone sample were analyzed separately for PSTs, ASTs, and DSTs. No samples (abalone foot or viscera) contained toxins at levels exceeding the marine biotoxin limits stipulated by Codex. The resulting prevalence estimate suggests that less than 1.6 % of the commercially caught wild abalone population in Australia were contaminated with marine biotoxins at levels above the regulatory limit during the survey period. ASTs were detected at very low (trace) levels in the foot and viscera tissue of four and three abalone samples, respectively. To our knowledge, this represents the first reported detection of domoic acid in Australian abalone. PSTs also were detected at very low levels in 17 samples of abalone foot tissue and 6 samples of abalone viscera. The association between the low levels of ASTs and PSTs detected in abalone and the presence of potential toxin-producing phytoplankton in seawater samples was weak. DSTs were not detected in any abalone despite the detection of very low levels of DST-producing phytoplankton in a small number (9 of 77) of seawater samples. The results of this survey should be useful for public health risk assessments and provide additional evidence that the prevalence of marine biotoxins in Australian wild-caught abalone is very low.

Experimental uptake and depuration of paralytic shellfish toxins in Southern Rock Lobster, Jasus edwardsii

ABSTRACT In October 2012, paralytic shellfish toxins (PST) were detected in the hepatopancreas of Southern Rock Lobsters (Jasus edwardsii) collected from the east coast of Tasmania, Australia. This resulted in the first commercial closure in Australia for this species. Questions were raised on how the toxins were transferred to the lobsters, how long the toxins would persist, whether PST‐contaminated hepatopancreas posed a risk to human health, and what management strategies could be applied. The aim of this study was to investigate whether PST‐contaminated mussels are a potential vector enabling toxin accumulation in J. edwardsii and to collect information on toxin uptake, distribution and depuration rates and toxin profiles under controlled experimental settings. Lobsters were fed mussels naturally contaminated with PST for a period of 28 days in an experimental setting; following this, lobsters were allocated to either fed or starved treatment groups. PST were not detected in the tail tissue of lobsters at any stage of the experiment. Lobster hepatopancreas contained mean levels of 2.4 mg STX.2HCl eq/kg after 28 days of uptake, although substantial variability in total toxicity was observed. The PST profile of the hepatopancreas was similar to that of the contaminated mussels used as feed. Significant differences were noted in the PST depuration rates between fed and starved treatment groups. The daily depuration rate for total PST was estimated to be 0.019 and 0.013 mg STX.2HCl eq/kg for the fed and starved treatment groups respectively using a constant‐rate decay model. After 42 days of depuration, total PST (STX equivalents) levels in the hepatopancreas of all lobsters were below 0.8 mg STX.2HCl eq/kg, which represents the regulatory level applied to bivalves. This result indicates that long‐term holding to depurate PST may potentially be used as a risk management tool. HighlightsUptake and depuration of paralytic shellfish toxins by Southern Rocklobster in an artificial system is described.Toxins accumulated in hepatopancreas but not in tail meat.Total toxicity varied substantially between lobsters.Depuration followed a constant decay curve and was slower in starved lobsters.

Theoretical dietary modelling of Australian seafood species to meet long-chain omega 3 fatty acid dietary recommendations.

Background Several agencies recommend seafood to be consumed 2–3 times per week. In Australia, there is a lack of nutrient composition data for seafood species and it is not known whether including different seafood species in a diet would provide sufficient long-chain omega 3 fatty acids (LC n–3 PUFA) to meet various national recommendations. Objective To utilise recent nutrient composition data for major Australian seafood groups (n=24) with the addition of two tuna options (total n=26) to: (1) determine whether including these species into a diet based on the Australian Guide to Healthy Eating (AGHE) will achieve LC n–3 PUFA recommendations [Adequate Intake (AI: 160 mg/d men, 90 mg/d women)], Suggested Dietary Target (SDT), 500 mg/d Heart Foundation (HF) recommendation and (2) determine the weekly number of servings of seafood to meet recommendations using either lower fat (n=23, <10% total fat) or higher fat (n=3, ≥10% total fat) seafood. Design Two simulation models incorporated all 26 species of seafood or only lower fat seafood into a diet based on the AGHE. Two further models identified the number of servings of lower or higher fat seafood required to meet recommendations. Results Including 2 and 3 servings/week of any seafood would enable 89% of women and 66% of men to meet the AI. Including only lower fat seafood would enable 83% of women and 47% of men to meet the AI. Half a serving/week of higher fat seafood would enable 100% of men and women to meet the AI. Conclusions Including the recommended 2–3 servings of seafood/week requires at least some higher fat seafood to be consumed in order for most men and women to meet the AI. Further messages and nutrition resources are needed which provide options on how to increase intake of LC n–3 PUFA, specifically through consumption of the higher fat seafood.

Paralytic shellfish toxins in Australian Southern Rock Lobster (Jasus edwardsii): Acute human exposure from consumption of hepatopancreas

ABSTRACT Paralytic shellfish toxins (PST) were identified in the hepatopancreas of Southern Rock Lobster (Jasus edwardsii) during Alexandrium tamarense blooms in Tasmania, Australia. Human health risk from PST in lobsters was unknown – this study assesses exposure to PST from hepatopancreas consumption. Lobster hepatopancreas samples collected during blooms (n = 181) were mostly positive for PST (>88%), the highest concentration was 4032 μg STX-2HCl eq/kg. Consumer exposure to PST was estimated using a 2-D Monte Carlo model. Mean PST intake (pi) from hepatopancreas consumption (raw and cooked) was below the lowest-observed-adverse-effect-level (LOAEL) for PST (<2.0 μg/kg bw), however the 97.5th percentile pi for raw meals (2.64 μg/kg bw) exceeded the LOAEL. A total of 4.1% of raw hepatopancreas meals were estimated to exceed the LOAEL. Lobster hepatopancreas consumption during A. tamarense blooms may be concerning for a small proportion of consumers, particularly those that eat large meals at the bloom peak. However, when the model was re-run with PST concentration capped at the bivalve regulatory limit (800 μg STX-2HCl eq/kg) pi decreased, with the 97.5th percentile values below the LOAEL. Thus, issuing public health warnings and harvesting restrictions for lobsters when levels exceed 800 μg STX-2HCl eq/kg would reduce the probability of illness occurring.