Project number: 2017-203
Project Status:
Completed
Budget expenditure: $241,125.00
Principal Investigator: Penelope A. Ajani
Organisation: University of Technology Sydney (UTS)
Project start/end date: 31 Dec 2018 - 30 Nov 2020
Contact:
FRDC

Need

DSTs are a significant, yet largely unquantified issue for Australian shellfish. A significant human poisoning event resulting in 56 hospitalisations from DST contamination of pipis (Plebidonax deltoides) in Ballina NSW in 1997. Since then, DSTs have been a major food safety challenge for the NSW pipi industry, with up to 40% of pipis in an end-product market survey between 2015 - 2017 (n=271) returning positive results for DTX, and two market place samples above the regulatory limit (NSW Food Authority 2018). DSTs have also been detected in shellfish from Tasmania with a review of data in 2015 (n=1710) finding that 0.4% of oysters (Saccostrea glomerata) and 0.6% of mussels (Mytilus planulatus) exceeded the regulatory limit (TSQAP). Since then the incidence of DSTs has increased in Tasmania, with DSTs responsible for a recall of mussels from Spring Bay Seafoods in 2016. Although low risk to date, positive DST detections occur in shellfish from South Australia (Madigan et al. 2006) and Western Australia.

In response to the significant shellfish contamination events with Paralytic Shellfish Toxins (PSTs) from 2012-17, which cost the Tasmanian economy ~$23 million, aquaculture industries are adopting efficient, fast and cost-effective management tools for PSTs and the phytoplankton producing them. This response will mitigate the cost of harvest closures and lead to strong improvements in shellfish safety. This project will build on that successful work by:
• Generating knowledge about commercially available DST test kits and rapid molecular techniques for toxin and species detection;
• Conducting an initial assessment of DST profiles present in Australian shellfish and assess laboratory capabilities to detect these toxins.
• Comparing the efficacy of DST toxin detecting kits across oysters, mussels and pipis.
• Developing a DST qPCR assay for species detection for onsite farm use.
• Providing cost versus benefit analysis of improved testing of DSTs in Tasmanian shellfish.

Objectives

1. Our team boasts extensive experience in the development, management and coordination of large scientific research projects. We use highly rigorous experimental designs and novel molecular techniques to examine phycotoxins and their causative species in relation to the Australian shellfish industry. Our project objectives focus mainly on Tasmania, but also include results that are relevant to the wide Australian shellfish industry and are as follows:OBJECTIVE 1. GENERATE KNOWLEDGE ABOUT COMMERCIAL DST TEST KITS AND RAPID MOLECULAR TECHNIQUES (SUCH AS qPCR) FOR DST TOXIN AND SPECIES DETECTION• Conduct a desktop literature review to determine the most up-to-date information on available commercial test kits, the toxin analogs that they target and their capabilities. • Conduct literature review to determine the most up-to-date information on rapid molecular techniques for the detection of DST producing dinoflagellates belonging to the genus Dinophysis.
2. OBJECTIVE 2. IDENTIFY DST PROFILES PRESENT IN AUSTRALIAN SHELLFISH AND ASSESS LABORATORY CAPABILITIES TO DETECT THESE TOXINS• Define and synthesise information on the precise DST toxin analogue/profiles in Australian shellfish (oysters, mussels and pipis) in relation to the Dinophysis species that are currently present in Australian coastal waters (Dinophysis acuminata, D. caudata, D. fortii, D. tripos, D. acuta, D. ovum, D. truncata, Prorocentrum spp.)• Conduct an initial assessment of analytical laboratories in the detection of DSTs in shellfish (oysters, mussels and pipis). A full spike and recovery quality control assessment program to assess the analytical techniques and to determine whether the type of sample matrix affects toxin detection will be undertaken. Shellfish matrices (oysters, mussels and pipis) will be spiked with known quantities of toxin standards in varying quantities and sent to 4 analytical laboratories (Cawthron Institute, Sydney Institute of Marine Science, Analytical Services Tasmania, and Symbio Laboratories) for LC-MS/MS toxin separation and confirmation.• Assess and conduct statistical analysis on results.• Work with NSW Food Authority and other state regulators/farmers to obtain DSTs positive shellfish from Australia-wide shellfish from farmers (when a toxic episode occurs) and analyse toxin profiles from event sampling. • Report findings to laboratory managers, relevant authorities and all other industry partners/collaborators.
3. OBJECTIVE 3. COMPARE THE EFFICACY OF COMMERCIALLY AVAILABLE TOXIN DETECTING KITS USING RELEVANT SAMPLE MATRICES • Conduct a replicated, quality-controlled laboratory-based study to compare the efficacy of a range of commercially available rapid test kits with DSTs standards spiked in oyster, mussel and pipi samples. Toxin kits to be tested will include Zeulab/Abraxis, Beacon, BioScientific, Biosens/Abraxis, Europroxima, Neogen and Scotia, and any new commercially available kits found during the literature review.
4. OBJECTIVE 4. DEVELOP A QUANTITATIVE PCR ASSAY FOR DINOPHYSIS SPECIES DETECTION FOR POTENTIAL ONSITE FARM USEWith the advent of genomic sequencing and high throughput genetic methods such as qPCR, it is now possible to obtain high-resolution enumeration of harmful algae from environmental samples. Costs for this are now lower, and results can be obtained within hours (or less), or even obtained on site. • Conduct a lab-based study to develop and implement a state of the art, rapid qPCR assay (including QA/QC) for Dinophysis species causing DSTs in Australian shellfish. This assay, similar to that currently used on site by Spring Bay Seafoods for the detection of Alexandrium (Paralytic Shellfish Toxins), will calculate the potential cell concentrations in environmental samples thereby providing real time (early) detection and monitoring of Dinophysis blooms.• Implement and field-test the qPCR assay for on-site use at Spring Bay Seafoods, Tasmania.
5. OBJECTIVE 5. PROVIDE COST VERSUS BENEFIT ANALYSIS OF IMPROVED TESTING OF DSTS IN TASMANIAN SHELLFISH• Conduct an economic impact analysis to estimate the reduction/annual savings in monitoring costs for regulatory authorities in Tasmania by using the most appropriate diagnostic testing for DSTs.• Calculate the reduction in commercial loss and economic impact from potentially harmful DST blooms in Tasmania following the introduction of the most appropriate diagnostic testing.
6. OBJECTIVE 6. CONDUCT A WORKSHOP TO TRAIN INTERESTED SHELLFISH INDUSTRY MEMBERS (TAS) IN THE USE OF RAPID TEST METHODS FOR DINOPHYSIS DETECTION AND DST TEST KITS IN ENVIRONMENTAL SAMPLESAMENDED:• Conduct a workshop to train shellfish industry members (Tas) in the use of the rapid method of qPCR for Dinophysis detection in environmental samples, and seek their advice and feedback on how to best move forward.ORIGINAL:• The project team will implement a training workshop to keep growers and industry abreast of qPCR methods, other research developments in DSTs, and seek their advice and feedback on how to best move forward.

Final report

ISBN: 978-0-646-84668-2
Authors: Penelope A. Ajani Hazel Farrell Gustaaf Hallegraeff Stuart Helleren Phil Lamb Alison Turnbull Anthony Zammit and Shauna A. Murray
Final Report • 2022-04-24
2017-203-DLD.pdf

Summary

This study first examined DSTs in spiked and naturally contaminated shellfish - Sydney Rock Oysters (Saccostrea glomerata), Pacific Oysters (Magallana gigas/Crassostrea gigas), Blue Mussels (Mytilus galloprovincialis) and  Pipis (Plebidonax deltoides/Donax deltoides), using LC-MS/MS  (Liquid Chromatography—tandem Mass  Spectrometry) and  LCMS  (Liquid Chromatography—Mass Spectrometry) in 4  laboratories, and  5 rapid test kits. The rapid test kits included three quantitative ELISA (Enzyme-Linked Immunosorbent Assay) kits  by  BeaconTM,  Eurofins/AbraxisTM and  EuroProximaTM;  a  quantitative PP2A (Protein Phosphatase Inhibition Assay) kit by Eurofins/AbraxisTM, and a qualitative LFA (Lateral Flow Assay) kit by NeogenTM. We  found all toxins in all species could be recovered by all laboratories using LC-MS/MS  and  LC-MS, however, DST recovery at low and mid-level concentrations (<0.1 mg/kg) was variable (0-150%), while recovery at high-level concentrations (>0.86 mg/kg) was higher (60-262%). While no clear differences were observed between shellfish, all kits delivered an unacceptably high (25-100%) level of falsely compliant results for spiked samples (ie. sample spiked above the regulatory limit but resulted in a concentration below  the  regulatory  limit). The Neogen and the PP2A kits performed  satisfactorily for naturally contaminated pipis (0%,  5%  falsely compliant, respectively). Correlations between spiked DSTs and quantitative methods was highest for LC-MS (r2= 0.92) and the PP2A kit (r2= 0.78). Overall, our results do not support the use of any DST rapid test kit as a stand-alone quality assurance measure at this time. We then developed a rapid and  quantitative polymerase chain reaction (qPCR) assay to detect species belonging to the genus Dinophysis in environmental samples. This assay had no cross-reactivity to other closely related species, and an assay efficiency of 91.5% for D. acuminata, 91.3% for D. fortii, 92.4% for D. caudata, and 97.9% for gene fragment based serial dilutions. This novel assay was then evaluated for its potential to detect Dinophysis in environmental samples. The assay was successful in the early detection of a bloom of D. acuminata in the Manning River on 9/2/2019 (~7,441  cell L-1), compared to microscopy counts of 5,300  cellL-1 on 17/2/2019.
 
A cost-benefit analysis of rapid detection of Diarrhetic Shellfish Toxins (DSTs) was subsequently undertaken. This was a case study of the Pacific Oysters (Magallana gigas/Crassostrea gigas) industry in Tasmania with the following aims:
to estimate  the  reduction/annual  savings in  monitoring costs  for  regulatory  authorities by implementing rapid diagnostic testing for DSTs; and
- to calculate the reduction in commercial loss and economic impact from potentially harmful DST blooms in Tasmania following the introduction of the rapid diagnostic testing.
The analysis considered three hypothetical scenarios for implementation of DST rapid testing: 1. Implement Neogen DST rapid kit testing on-farm in Tasmania, replacing confirmatory DST testing in low risk areas in 3 out of 4 weeks; 2: Implement Neogen DST rapid kit testing in laboratory in Tasmania, replacing confirmatory DST testing in low risk areas in 3 out of 4 weeks; 3. Implement qPCR testing on-farm in Tasmania, replacing confirmatory DST testing in low risk areas in 3 out of 4 weeks. Results revealed that, while the qPCR or Neogen technology both offer cost advantages when compared with the business as usual practice (BAU), it was not possible to calculate the exact extent of this without further work to  validate (including number of samples, Quality Assurance and  National Association of Testing Authorities accreditation) the two alternative testing technologies. All scenarios considered, however, represented a net cost saving over 10 years when compared with the current practice of weekly LC MS  tests for DSTs conducted by a  laboratory service provider (BAU). The highest savings occurred under Scenario 2, in which the Neogen technology is centralised in the laboratory and spread across all 24 Tasmanian growing areas. A full validation study covering each of the major testing methods examined (Neogen rapid test, qPCR test) is recommended as an important area of further research. Furthermore, the scenarios considered in this analysis were for domestic supply only, in compliance with potential use under the ASQAP programme. Further considerations would be needed for use in any export programme.

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