FRDC Toxigenic vibrio baselines and optimum storage, transport and shelf-life conditions to inform cold supply chains in the north Australian Tropical Rock Oyster industry

Internationally, Tropical Rock Oysters have a poor safety reputation with vibrios at the top of the list, so it is important that northern Australia¶s emerging TRO industry demonstrates a clear commitment to QA including routine vibrio testing. While a pro-active not reactive approach to vibrio food safety is essential for product assurance and branding, effort needs to be proportional to risk. In northern Australia we know seawater contains up to 42 Vibrio spp. including several known toxigenic species in addition to the human pathogens Vibrio parahaemolyticus (Vp) and V. vulnificus (Vv). We know Vp and Vv concentrations in seawater are higher in the wet season compared to the dry and more shellfish are Vp and Vv positive in the wet season. So if vibrio diversity and abundance in TRO is seasonal (as shown elsewhere), it is likely that Vibrio spp. infections in humans will also follow a seasonal trend. Vibrios are a known issue for TRO aquaculture and we need to fill knowledge gaps for northern Australia including 1) identifying vibrio baselines in TRO and develop tests for toxigenic species, 2) assess post-harvest management to identify the best storage and transport temperatures, and 3) assess TRO shelf life at realistic storage temperatures. We need this information to prepare a vibrio risk profile for TRO in northern Australia to assist initial risk management activities, such as identifying future actions required, and the options for food safety management programmes. This information will provide the developing TRO industry with the knowledge needed to ensure an exemplary reputation, thus giving access to premium markets. While the work we propose here will be used as a framework for the tropical north, it is worth noting that with warming sea temperatures, toxigenic vibrios are expanding their geographic range. This means our work could help future-proof the south because tests and risk profiles we develop for tropical vibrio toxigenic species will be needed if these species expand their geographic range south.

Methods
Objective 1 Measure vibrio baseline in Tropical Rock Oysters and develop tests to vibrio species that are toxigenic to oysters and humans We will measure the vibrio microbiome baseline in TRO in the NT, Qld and WA to identify toxigenic species and develop tests for these known toxigenic species. As part of FRDC project number 2018-005 (PI Matthew Osborne) TRO collections are underway for eight sites in the NT. We propose to use these same samples for this vibrio project if funded. In anticipation and at our own cost we have processed aliquots of these samples from the three sites already sampled in 2019 . Total sample effort for NT will be 8 sites x 4 locations per site x 3 replicates per location x 3 TRO pooled per replicate = 288 TROs/3 = 96 samples for vibrio baseline analysis (each sample is a pool of 3 TRO). Queensland (Bowen) = 1 site as per above = 36 TROs/3 = 12 samples. Western Australia (Shark Bay) = 1 site as per above = 36 TROs/3 = 12 samples. We will analyse these samples using a new high throughput vibrio community diversity method that represents a breakthrough in identifying Vibrio spp. including toxigenic Vibrio spp. in addition to Vibrio parahaemolyticus (Vp) and V. vulnificus (Vv). This new approach targets the Hsp60 gene for high resolution of vibrio spp. in seawater and oysters (King et al 2020 in press) has shown that NT seawater has 42 vibrio species including several known toxigenic species including but not limited to Vp and Vv (Padovan et al in prep).
Tests will be developed to toxigenic vibrios detected in the baseline assessment. Vp and Vv are likely targets because we have detected these previously in oysters in non-harvest areas and they occur in seawater especially in the wet season. We have already developed qPCR tests for total Vp, Vp toxin genes trh and tdh

Objectives

1 Measure vibrio baseline in Tropical Rock Oysters and develop tests to vibrio species that are toxigenic to oysters and humans. 2 Identify optimum storage and transport temperatures to inform best practice post-harvest management. 3 Assess TRO shelf life at realistic storage temperatures to maximise product quality.
4 Use information from objectives 1-3 to produce a risk profile for vibrio in north Australian TRO to assist risk management activities and food safety management programs.

Objective 1 Measure vibrio baseline in Tropical Rock Oysters and develop tests to vibrio species that are toxigenic to oysters and humans We will measure the vibrio microbiome baseline in TRO in the NT, Qld and WA to identify toxigenic species and develop tests for these known toxigenic species. As part of FRDC project number 2018-005 (PI Matthew Osborne) TRO collections are underway for eight sites in the NT. We propose to use these same samples for this vibrio project if funded. In anticipation and at our own cost we have processed aliquots of these samples from the three sites already sampled in 2019 . Total sample effort for NT will be 8 sites x 4 locations per site x 3 replicates per location x 3 TRO pooled per replicate = 288 TROs/3 = 96 samples for vibrio baseline analysis (each sample is a pool of 3 TRO). Queensland (Bowen) = 1 site as per above = 36 TROs/3 = 12 samples. Western Australia (Shark Bay) = 1 site as per above = 36 TROs/3 = 12 samples. We will analyse these samples using a new high throughput vibrio community diversity method that represents a breakthrough in identifying Vibrio spp. including toxigenic Vibrio spp. in addition to Vibrio parahaemolyticus (Vp) and V. vulnificus (Vv). This new approach targets the Hsp60 gene for high resolution of vibrio spp. in seawater and oysters (King et al 2020 in press) has shown that NT seawater has 42 vibrio species including several known toxigenic species including but not limited to Vp and Vv (Padovan et al in prep).
Tests will be developed to toxigenic vibrios detected in the baseline assessment. Vp and Vv are likely targets because we have detected these previously in oysters in non-harvest areas and they occur in seawater especially in the wet season. We have already developed qPCR tests for total Vp, Vp toxin genes trh and tdh and total Vv. If toxigenic species such as (harveyi, fortis, alginolyticus, other) are detected in TRO (we have recently detected several toxigenic Vibrio species in pearl oysters) then semi-quantitative MPN-(q)PCR tests for these toxic species will be developed for seawater and TRO.
Co-Investigators at Charles Darwin University (CDU) (PI Gibb and CI Padovan) and University Technology Sydney (CI Seymour)) with skills in molecular ecology will work in collaboration with Co-Investigators at the NT Department of Primary Industry and Resources (CIs Osborne and Nowland) with skills in TRO husbandry and field logistics to complete objective 1.
Objective 2 Identify optimum storage and transport temperatures to inform best practice post-harvest management We will measure growth or inactivation of Vibrio parahaemolyticus in TROs over a range of relevant temperatures and time. We will also assess oyster condition at time of sampling e.g. alive/dead, meat weight, flesh texture and colour, liquor colour. Live Tropical Rock Oysters (TRO) will be injected with a cocktail of Vp isolates derived from local TROs and stored at different temperatures for up to 21 days. At timed intervals, oysters will be sampled and Vp levels measured along with virulence genes. Data will be analysed to determine optimum storage and transport time and temperatures. Resources permitting, results will be validated using TROs with natural Vp populations. Vp enumeration tests have been optimized in the PIs laboratory and are used routinely.
Co-Investigators at CDU (lead by CI Padovan) with skills in Vp enumeration will work in collaboration with CoInvestigators at the NT Department of Primary Industry and Resources with skills in TRO husbandry and field logistics (CIs Osborne and Nowland) to complete objective 2.
Objective 3 Assess TRO shelf life at realistic storage temperatures to maximise product quality. Live TROs of marketable size will be sourced (site TBD), transported to Adelaide and stored under four temperatures. Temperatures will be chosen after consideration of objective 2 results. To understand natural variability within a batch of TRO, multiple oysters will be assessed. Assessments will be repeated to compare the wet and dry seasons from a single location. Results will be summarised and included in a report on the expected shelf-life of TRO when exposed to various temperatures. For each of the four storage temperatures, TROs will be assessed at six times. The frequency of assessment will be skewed towards the anticipated end of shelf-life. Each time point will involve the assessment of product mortality/product gaping (12 oysters), inter-valvular liquor (12 oysters), organoleptic attributes (six panellists each evaluating two oysters for aroma and visual assessment of flesh and liquor; 12 oysters) and standard plate counts (SPCs; 18 oysters). SPC is used as a microbiological indicator for product quality and the results can help highlight any product handling issues and storage issues. SPCs will be measured in triplicate using a composite sample consisting of six pooled oysters. The composite sampling helps to reduce the variability between results and will also be undertaken in accordance with Australian Standard 5013.11.3 2012 that stipulates that a representative sample shall consist of at least 6 individuals and shall be about 75-100g.
Determination of the quality based shelf-life of TRO requires experienced personnel (lead by CI Pahl) and the sensory assessment will be undertaken in a dedicated sensory facility at SARDI that can control the local environment and reduce interactions between the panellists.
Objective 4 Use information from objectives 1-3 to produce a risk profile for vibrio in north Australian TRO to assist risk management activities and food safety management programs We will produce a risk profile for vibrio in northern Australian Tropical Rock Oysters. Risk profiles are an important tool for risk managers and industry. They provide a summary of all information pertinent to food safety associated with the specific hazard/food combination. The purpose of a risk profile is to assist initial risk management activities, such as identifying future actions required (if any), and the options for food safety management programmes. They also inform the level of resourcing required to control the hazard/food pairing.
The risk profile will address the questions: 1. What is the risk of vibriosis from Tropical Rock Oysters? 2. What control measures are available?