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abstract book

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10 TH INTERNATIONAL CONFERENCE ON MOLLUSCAN SHELLFISH SAFETY
ORGANIZING COMMITTEE
Viviana Cachicas
Carlos Aranda
Enrico Buenaventura
Anthony Zammit Claudia Rozas
Dinorah Medina
ORGANIZING SUBCOMMITTEE
SCIENTIFIC COORDINATOR
Alejandra Goya
Veronica Jurquiza
Carmen Lopez
Orialis Villarroel
Rita Orozco
Jorge Mardonez
M Angelica Larrain
Jaminton Ramirez
Monica Jara
Cristina Martinez
Gonzalo Diaz
Andrea Rivera
Puerto Varas / Chile
15 - 20 March 2015
INTERNATIONAL ADVISORY COMMITTEE IAC
Ron Lee
Iddya Karunasagar
Gary Richards
William Burkhardt
Jesus Romalde
Francesca Leoni
Marcela Costagliola
Michelle Price-Haywart
Jessica Jones
Stephen Jones
Miguel O Ryan
Philipp Hess
Soizick Le Guyader
Elisabetta Suffredini
Dr. Andrew Turner
Ana Gago Martinez
Rachell Hartnell
Angelo De Paola
Irma Rivera
Jaime Martinez Urtaza
Veronica Madrid
Elsa Quiñones
Heriberto Fernandez
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4
BIOTOXIN AND PHYTOPLANKTON
1. Oral Presentation
SOMETHING IS HAPPENING IN FJORDS AND CHANNELS OF SOUTHERN CHILE (41°- 55°): The case of Alexandrium catenella and
Paralytic Shellfish Poison for the past 40 years. Guzmán, L1., 1Departamento de Medio Ambiente, División de Investigación en Acuicultura, Instituto de Fomento Pesquero.
The first record of Alexandrium catenella and Paralytic Shellfish Poison (PSP) was in 1972, then blooms occurred in 1981 and
1989, but restricted to the Magellan region. Since 1991, recurrent A. catenella blooms and PSP outbreaks are annually occurring,
expanding gradually its distribution to the North. Records of this expansion dating from 1992 (45°S) in the Aysén region, 1998 in
the Southeastern sector of Chiloé Island (43°S) and recently was detected in the Northern area of the inner sea of Chiloé (41°48’ºS)
during the Spring of 2009. A. catenella motile stage presence, preceded at least two years, PSP detection in shellfish in its geographic
expansion. Today its distribution encompasses from 41°48’ to 54°55’S. The PSP outbreaks are associated to 35 fatal cases and
about 5 hundreds of intoxicated people, most in the Magellan region (23), situation that has changed since year 2000, since no fatal
cases have occurred and undesirable intoxications have decreased to a few cases. Besides the clear expansion of the species in
its geographic distribution, different studies realize interannual variability in the relative abundance and density of A. catenella, and
toxicity in shellfish; also clear differences between macro-regions and within these regions in the temporal and spatial distribution
of A. catenella and PSP levels. Furthermore, the highest records of PSP toxicity show a clear latitudinally increase, being the highest,
recurrently detected in specific localities of the Magellan region. In addition, there are clear differences, between and within geographic
macro-regions, in the periods with higher probabilities when A. catenella presents density increases and PSP outbreaks. A. catenella, as
an exception may be the numerical dominant species of phytoplankton assemblages, as occurred in the exceptional 2009 bloom, but
usually the species represents a low proportion of total phytoplankton (<1%). The climatic oceanographic hypothesis would explain
this phenomenon in Chilean fjords and channels. Puerto Varas / Chile
15 - 20 March 2015
5
Mussel long-line culture and the risks of toxic Dinophysis in the water column.
Roughan, B1., 1Verification Services Ministry for Primary Industries.
The New Zealand BMS aquaculture industry is heavily reliant on GreenShell™ mussels (Perna canaliculus). Approximately 90,000
tonnes mussels are exported annually worth approximately NZD$200 million per annum. The majority of the production is in the
sheltered waters of the Marlborough Sounds. The mussels grow on a series of dropper ropes hanging down to 15 metres from a
sturdy ‘backbone’ rope that is held up by a row of buoys. Where BMS flesh samples are not tested for marine biotoxins on a weekly basis, the New Zealand programme requires at least weekly
samples of water to be analysed for phytoplankton counts. Seawater samples are taken using an integrated tube method with flesh
taken at the same time should they need to be analysed at the laboratory. Based on the risk profile, many areas routinely test the
flesh weekly for the at-risk toxins. New Zealand has conservative phytoplankton trigger levels which require flesh testing for the
relevant toxin should they be exceeded.
D. acuminata and D. acuta blooms occur annually in some areas of the Marlborough Sounds. Experience from the routine marine biotoxin
monitoring programme has identified significant difference in flesh toxin levels depending on the depth of flesh sampling from the
dropper rope. During 2010 and 2014 bloom events DSP toxins levels varied from nil detect to over the regulatory level of 0.16mg
Okadaic acid equivalents/kg. This paper will highlight the importance of knowing the growing area hydrodynamics, phytoplankton risk profile and adapting sampling
during risk periods to ensure consumers are protected. 6
Operational Forecasting of Harmful Algal Blooms and Toxicity in Ireland
Silke, Joe1., Cusack, Caroline1.,Glenn, Nolan2.,1Marine Environment and Food Safety Services Irish Marine Institute .2Ocean Science and
Information Services Irish Marine Institute.
The shellfish industry depend on having a robust biotoxin programme and appropriate regulations to ensure produce is safe to place
on the market. In Ireland, results from the national biotoxin monitoring programme are distributed direct to the aquaculture industry,
regulatory agencies and public within 24-48 hours of laboratory sample receipt. To ensure that fish farmers can harvest and market
their product in a sustainable manner, they require advance knowledge of impending HAB events. An ideal solution would be an alert
system that would enable farms to improve harvesting practices (eg. get bivalve products onto market prior to intoxication event)
and ensure that protective measures are implemented on time when a HAB is imminent. Such improved decision on risk and crisis
management will encourage industry growth. The EC FP7 funded project “ASIMUTH” developed a short term HAB forecasting (2-3 day)
system for Scotland, Ireland, France, Spain and Portugal. In this presentation we discuss the implementation of this system in Ireland.
The system uses a “HAB Decision Support System” designed to assimilate information (satellite images, model forecasts, ground
data) and via expert interpretation of results reports back to end users. The 2012 and 2013 Irish field campaign will be examined.
This includes collaboration with the Irish coast guard to detect high biomass blooms in Irish shelf waters and model their movements.
Regular reports to predict HABs were prepared and published to inform the industry. The feedback from this exercise demonstrated
the value to the industry and provided extra assurance regarding food safety of bivalves placed on the market.
15 - 20 March 2015
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New insights on the ecophysiology of Alexandrium catenella from Chilean fjords
Mardones, Jorge1., Dorantes-Aranda, Juan Jose1.,Müller, Marius2.,Bolch, Chris1.,Varela, Daniel3.,Hallegraeff, Gustaaf1.,1Institute for
Marine and Antarctic Studies (IMAS) University of Tasmania.2Instituto Oceanográfico University of São Paulo.3i-mar Universidad De
Los Lagos. (Sponsored by BECAS CHILE Program Of The National Commission For Scientific And Technological Research (CONICYT))
Harmful Algal Blooms (HABs) are common natural phenomena that occur along the Chilean coast. In the southern fjords, the
dinoflagellate Alexandrium catenella is the most studied species because of its production of saxitoxin and analogues responsible
for Paralytic Shellfish Poisoning (PSP). Despite the deleterious effects on the local aquaculture, little is known about its ecology in
this complex ecosystem. In vitro experiments were conducted to determine the role of cyst in bloom dynamics, the physiological
response against pCO2/pH variations and the potency of toxic compounds with cytolytic properties in A. catenella. Cyst experiments
using pairwise crosses showed highly variable reproductive compatibility among 10 Chilean Group 1 A. catenella strains; synchronized
encystment with cyst production starting 26 days and terminating 45 days after inoculation; a dormancy period between 80 and 120
days and excystment rate stimulated by low temperature (10-12°C), high salinity (30 psu), N and/or P depletion, and low irradiance
(20 μ photons m-2 s-1). The broad natural pCO2/pH fluctuations in the southern fjords play an important role in the growth response
of A. catenella. Optimum growth and dissolved inorganic carbon (DIC) uptake occurred around pH =8.0, but was negatively affected by
lower and higher pH. Chain-formation (and hence swimming speed) was enhanced under low pCO2/high pH conditions while cell size
(ESD) was negatively correlated with increasing pH. Finally, our results on the ichthyotoxic potency of A. catenella assessed by a fish
gill cell line assay, showed that pure PST fractions (C1&C2, STX, GTX 1&4) had an insignificant role in lytic activity compared to the
synergistic interaction between reactive oxygen species (superoxide anion) and polyunsaturated fatty acids such as DHA. 8
The Red Tide Project as a health monitoring system for the shellfish exposed to Harmful Algal Blooms (HABs)
Castillo, M1.,1ExecutiveDirection of SpecialPrograms Ministry of Health, COFEPRIS, Mexico.
In 2001, theFederalCommission for Protectionagainst Sanitary Risks(COFEPRIS) was created by a governmental decree, as a
decentralized technical, administrativeand operational autonomousbodywithin the Ministryof Health. It is responsible forregulation,
controlandpromotion of health.In 2004, the COFEPRIS created The Red Tide Project. This project its main purpose is the sanitary
control of shellfish exposed to Harmful Algal Blooms (HABs) in the Mexican coasts, through a program of ongoing monitoring which
detectsphytoplankton in seawater, complementary to the sampling of biotoxins in shellfish, and this functions as an early warning
system for detecting HABs. Its objective is to avoid the human consumption of shellfish exposed to such events. For this purpose, a set
of Guidelines have been established to support the Sanitary Authorities of States in Mexico with coastlines to perform a permanent
application of monitoring programs and proceed with actions in the event where HABs are detected. As a result of the application of
the phytoplankton sampling Plan, from 2005 to 2013 98 HABs were identified, 55 of them were potentially toxic for humans, 12
wereichthyotoxic,and 31 werenon-toxic.
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9
Occurrence of phycotoxins in the coast of Santa Catarina, the largest aquaculture mollusks producer in Brazil.
Schramm, Mathias1., Proença, Luis Antonio1.,Alves, Thiago1.,1LAQUA/MPA-Itajaí/SC Instituto Federal de Santa Catarina - Campus
Itajaí.
Analysis of marine biotoxins focusing mollusks safety started in Brazil in mid 90ies with the implemantation of HPLC-DAD and HPLC-FLD
analytical methods and mice bioassay in Santa Catarina. These methods were set in order to provide support for the new growing mussel
and oyster aquaculture in the region. In this first period were detected toxins from groups of okadaic acid, saxitoxin and domoic acid, in
different sources. From 2009, a second period of analysis started with LC-MS/MS methods which enabled the detection of several lipophilic
toxins either in algae or in mollusks extracts. In this paper, we list all the toxins found in the coast of Santa Catarina until now, from both
in phytoplankton and/or mollusks, including: OA, DTX1, DTX2, AZA-1, AZA-2, AZA-7/37, YTX, 45-hyd-YTX, PTX-2, DA, neo-STX, dc-STX,
GTX1,2,3,4,5, dc-GTX-2, dc-GTX-3, SPX-1, OVA-A. These findings were considered to set the toxins scope to be monitored in the Brazilian
legislation regarding mussel sanitation control. At this moment, based on these findings, the groups of toxins officially monitored are: a)
PSP - saxitoxin and congeners; b) DSP - okadaic acid and dinophysistoxins; c) DSP - yessotoxins; d) AZP – azaspiracids; and e) ASP - domoic
acid and congeners, mainly associated to Gymnodinium catenatum, Dinophysis spp., Azadinium spp. and Pseudonitzschia spp., respectively.
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7 .Oral Presentation
Review of three years of monitoring of palytoxin-group toxins in different marine organisms in Villefranche bay on the French
Mediterranean coast
Biré, R1., Amzil, Z2.,Lemée, R3,4.,Trotereau, S1.,Hess, P2.,Delpont, C1.,Brissard, C2.,Krys, S1.,1Department of Chemical contaminants in
food, Pesticides and marine biotoxins unit ANSES, Maisons-Alfort laboratory for food safety.2Phycotoxins Laboratory, Atlantic Centre
IFREMER.3UPMC Univ Paris 06, UMR 7093, LOV, Observatoire Océanologique Villefranche/mer Sorbonne Universités.4LOV, Groupe
Biodiversité et Biogéochimie, Observatoire océanologique Villefranche/mer CNRS, UMR 7093.
Palytoxin (PLTX) and analogues are potent non-protein marine compounds produced by corals of the genus Palythoa and by
dinoflagellates of the genus Ostreopsis. Since the beginning of the 2000s, Ostreopsis blooms have been reported in the Mediterranean
Sea with increasing frequency. In September 2006, divers and swimmers around the Frioul Island, offshore of Marseilles, reported
various symptoms related to the presence of Ostreopsis in the water. Since then, a monitoring system has been set up by the
competent authority along the French Mediterranean coast; when Ostreopsis abundances exceed 4,000 cells/L in the water column,
shellfish are analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) for the presence of PLTX-group toxins.
Currently no regulations exist for these toxins in the European Union or elsewhere, however the European Food Safety Authority
(EFSA) recommends a threshold of 30 µg/kg applicable to shellfish. Other marine organisms were not considered due to the lack of
occurrence data. In 2009, 2010 and 2012, various marine organisms including fishes, echinoderms, gastropods, crustaceans and
cephalopods were sampled in Villefranche bay, on the French Mediterranean coast and analysed by the hemolytic assay and LC-MS/
MS for the presence of PLTX-group toxins. The toxin levels in sea urchins (Paracentrotus lividus), fishes (Sarpa salpa, Mugil cephalus),
crustaceans (Maja squinado) and gastropods (Hexaplex trunculus) were above the EFSA threshold of 30 µg/kg, either once or repeatedly
during the sampling periods. The omnivorous and herbivorous organisms were most contaminated, showing the influence of the diet
in the contamination process. Toxins accumulated almost exclusively in digestive tube for all species, with the exception of octopus
(Octopus vulgaris) which exhibited detectable toxin amounts also in remaining tissues. The toxin profile determined by LC-MS/MS
identified ovatoxin-a as the major compound (90 to 100%).
15 - 20 March 2015
11
Communicating with stakeholders: new tools to meet challenges
Mellano, Grace1., Edwards, Robyn2.,1Aquaculture Management Directorate Fisheries and Oceans Canada.2Domestic Food Safety
Systems Canadian Food Inspection Agency.
The Canadian Shellfish Sanitation Program (CSSP) is a federal food safety program developed to minimize health risks from the
consumption of bivalve molluscan shellfish. The CSSP is administered by the Canadian Food Inspection Agency, Fisheries and Oceans
Canada, and Environment Canada. The three partners collaborate to jointly deliver the program, manage risk and communicate with
stakeholders. Over the years, we have recognized various challenges in providing the public with precise and timely information of
whether areas are open or closed to harvesting. Specifically, interviews with stakeholders such as federal and provincial government
authorities not directly involved in CSSP delivery, industry and the general public, determined that they are looking for a web-based
solution that allows them to quickly and easily access locations and real-time information about where shellfish are safe to harvest.
As a result, the CSSP partners are developing a geospatial web mapping application using ArcGIS and Geocortex Essentials. Led by
Fisheries and Oceans Canada, the application will target distinct end-users: commercial harvesters, the general public, and the CSSP
partners. The application is being designed to comply with Government of Canada accessibility guidelines and will be hosted on DFO’s
network. Official launch is expected in in 2015. This tool will provide real-time information about harvest area openings and closures
and detailed information about each area, such as biotoxin closures. The layered structure of the application will allow for different
degrees of data accessibility depending on the constituent – from the general public to the regulators. Ultimately, this mapping tool
will improve commercial and recreational harvester understanding about where they can safely harvest. Industry will be provided with
enhanced ability to make informed business decisions about harvesting and purchasing product, thus providing both food safety and
economic benefits. 12
THE TRANSITION FROM MOUSE BIOASSAY TO CHEMICAL METHODS FOR THE DETERMINATION OF SAXITOXIN GROUP TOXINS: THE
EURLMB EXPERIENCE
Ben-Gigirey, B.1., Rossignoli, A.1.,Gago-Martinez, A.1,2.,1EURLMB European Union Reference Laboratory for Marine Biotoxins-Spanish
Consumers Affairs, Food Safety and Nutrition Agency. Ministry of Health, Social Affairs and Equality.2Department of Analytical and
Food Chemistry, Chemistry Faculty. , Universidad de Vigo.
The drawbacks of the MBA for marine biotoxins control have been the main reason for the search of analytical alternatives for these
compounds. Three AOAC Official Methods, including two Liquid Chromatographic methods with Fluorescence Detection (LC-FLD) and
a Receptor Binding Assay, have been accepted as alternative to the MBA for the control of STX toxins group (STXs).
The two methods based on a chromatographic separation prior to the FLD of the STXs derivatives differ in the STXs precolumn or
postcolumn derivatisation. Significant efforts have been devoted to the extension and implementation of both methods among
scientists. In particular, the EU Reference Laboratory for Marine Biotoxins (EURLMB) has made considerable efforts on these
implementations, not only through research but also through the organisation and/or participation in different collaborative studies.
The suitability of the AOAC OMA 2005.06 for Official Control prior to its inclusion in the E.U. legislation was evaluated by the EURLMB
in 2006. Since then many attempts have been made among the EURLMB Working Group (WG) experts to identify specific method
drawbacks and to find solutions to overcome them. The WG tasks included, among others, a study for the quantification of GTX6
after hydrolysis, an interlaboratory study for the extension of the method validation for dcGTX2,3, and the evaluation of the method
recovery following an harmonised procedure.
The EURLMB has been also working on the setting up of AOAC 2011.02 OMA and participated in its international AOAC validation, as well
as in the certification study of the first tissue reference material containing STXs. Present efforts are focused on the implementation
of the chromatographic resolution for certain STXs and GTXs.
The EURLMB experience on the coordination of Proficiency Testing Schemes, trainings and validations allowed concluding the
importance of such efforts to a reliable and efficient application of both methods for STXs analysis.
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13
Single laboratory validation of a UPLC-HILIC-MS/MS method for quantitation of paralytic shellfish toxins in twelve commercially
produced bivalve shellfish species
Turner, Andrew1.,Harwood, Tim2., Selwood, Andrew2.,McNabb, Paul2.,Boundy, Michael2.,1Food Safety Centre for Environment, Fisheries
and Aquaculture Science (UK).2Analytical Services Cawthron Institute (NZ). (Sponsored by Queen Elizabeth II Technicians Study Award)
Routine regulatory monitoring of paralytic shellfish toxins (PST) in bivalve shellfish can prove difficult using instrumental techniques.
There is a need for alternative methods that provide faster turnaround times and improved detection limits. A sensitive quantitative
hydrophilic interaction liquid chromatography (HILIC) UPLC-MS/MS method has been developed as a simpler alternative to the precolumn oxidation AOAC 2005.06 and post-column oxidation AOAC 2011.02 liquid chromatography with fluorescence detection
methods, as well as the biological assay AOAC 959.08. This new method employs a novel desalting clean-up procedure with inexpensive
carbon solid phase extraction cartridges. HILIC HPLC-MS/MS has been used previously for PST research purposes, but substantial
sample matrix issues and high detection limits have to date prevented it from being a viable alternative for routine shellfish monitoring
purposes. A single-laboratory validation study was conducted using the new method for the analysis of PST in 12 commercially produced bivalve
shellfish species. PST assessed included saxitoxin (STX), neosaxitoxin (NEO), decarbamoylsaxitoxin (dcSTX), decarbamoylneosaxitoxin
(dcNEO), deoxydecarbamoylsaxitoxin (doSTX), gonyautoxins 1-6 (GTX1-6), decarbamoylgonyautoxin 1-4 (dcGTX1-4), N-sulfocarbamoyl
gonyautoxin 2&3 (C1&2) and N-sulfocarbamoyl gonyautoxin 1&4 (C3&4). Twelve commercially produced bivalve species from both
New Zealand and the United Kingdom were assessed, including mussels, oysters, scallops and clams. Validation studies demonstrated
acceptable method performance characteristics for specificity, linearity, recovery, repeatability and within-laboratory reproducibility.
Limits of detection and quantitation were significantly improved in comparison to current fluorescence-based detection methods and
the method was shown to be robust. The method performed well in comparison with AOAC 2005.06, with evidence obtained from
both comparative analysis of 1,141 PST contaminated samples and successful participation in proficiency testing schemes. 14
Screening of a wide variety of fycotoxins in different matrices using liquid chromatography high resolution mass spectrometry
Gerssen, A1., Klijnstra, Mirjam1.,1Natural Toxins and Pesticides RIKILT - Institute of Food Safety - Wageningen UR.
Phycotoxins such as azaspiracids, microcystins, saxitoxins and ciguatoxins are produced by algae which are naturally occurring in marine
and fresh water. Different types of phycotoxins can end up in matrices like shellfish, water or food supplements. Analytical methods are
available to analyse regulated phycotoxins, however these methods are only suitable for a small specific group of toxins and/or a specific
matrix (mainly shellfish). Therefore, a screening method is developed for all kinds of phycotoxins in different matrices such as shellfish,
fresh and sea water and food supplements. To analyse lipophilic phycotoxins, water samples are cleaned and toxins are concentrated
with use of solid phase extraction (SPE). Due to the chemical properties of hydrophilic phycotoxins, these toxins are not retained on
a C18 SPE cartridge. Therefor the hydrophilic phycotoxins another type of clean-up and concentration step is required. For shellfish
(tissue) samples and food supplements (solids) a different approach is needed. To extract the phycotoxins out of tissue (shellfish)
and food supplements a two-step extraction is used. First methanol is added (lipophilic phycotoxins) followed by extraction using an
ultrasonic disruptor. Second, a mixture of acetonitrile, water, ammonium formate and formic acid is used (hydrophilic phycotoxins)
and vortex mixing. Both extracts are combined. Subsequently, food supplement extracts are cleaned with use of C18 SPE for lipophilic
phycotoxins. Shellfish extracts are analysed with LC-hrMS directly after extraction. For separation, liquid chromatography methods are
developed for lipophilic and hydrophilic phycotoxins. Lipophilic phycotoxins are separated by reversed phased chromatography (C18) as
opposed to the hydrophilic phycotoxins, which are separated with use of a HILIC LC-column. For both methods a gradient with a runtime
of 20 minutes is used with the same mobile phase composition, water, acetonitrile, ammonium formate and formic acid. Subsequently,
the samples are measured with use of a full scan high resolution mass spectrometer (Orbitrap) at a resolution of 50.000 and in
combination with HCD fragmentation. For the developed extraction procedures acceptable recoveries for the known phycotoxins are
obtained between 70% and 150%. Compared to current available methods toxins are well retained and produce acceptable peak shapes
on the LC-column. By using an in house created library and developed software (MetAlign) we were able to screen various sample types
for over 300 different phycotoxins. During the presentation the development and the application of the method will be presented.
15 - 20 March 2015
15
Volunteer-based harmful algae monitoring networks with rapid toxin detection as an added safety layer and managing option for
aquaculture
Cassis, D1., 1Centro de Investigación e Innovación para el Cambio Climático Universidad Santo Tomás / Jellett Rapid Testing.
Shellfish aquaculture operations are affected by extraction closures, harvest delays and loss of consumer confidence produced by
toxic harmful algal blooms, causing upwards of US80 million in the US. These blooms depend on environmental variables and follow
relatively predictable seasonal trends. Climate change seems to be affecting these cycles as unseasonal conditions may lead to the
appearance of species, blooms and toxins not observed before. In 2011 DSP was detected for the first time in British Columbia, and
despite the ongoing toxin monitoring program, 62 people resulted intoxicated. As a result of the negative effects on public health the
BC shellfish industry with support from government agencies (BCCDC, CFIA) requested the creation of the capabilities to confront these
threat through the formation of a volunteer-based harmful algae monitoring program to complement the biweekly toxin monitoring
program. It was based on in situ phytoplankton monitoring supported by on-line taxonomical guides, networking capabilities, and an
event response protocol. This protocol included the use of rapid qualitative tests to confirm the presence of toxins in phytoplankton
and shellfish as a precautionary measure. Soon after its deployment in 2013, this monitoring network scored its first success by
preventing PSP-contaminated shellfish from being harvested in the Strait of Georgia after an Alexandrium bloom was observed soon
after samples were obtained for the regulatory toxin monitoring program. Toxicity in phytoplankton and shellfish samples was detected
through rapid tests and confirmed as above the legal limit several days later though quantitative analysis. These efforts resulted in
the grower´s increased awareness of the safety of their products, an early warning system to prevent harvest of toxic products, and
a proactive and more detailed layer to current toxin monitoring programs that can help manage their harvests. The design and initial
results of this network are hereby presented.
16
Mode of action-based bioassays for marine biotoxins causing gastrointestinal disturbances
Bodero, M1., Bovee, Toine 2.,Hoogenboom, Ron 1.,Hendriksen , Peter1.,1Toxicology-RIKILT Wageningen .2Toxicology and Bioassays
-RIKILT Wageningen .
The presence of marine biotoxins in shellfish is a risk for consumers and requires constant monitoring of production areas and products.
In several countries (including Chile), the method of detection is the Mouse Bioassay (MBA), where mice are injected with an extract
of the sample, and the occurrence of death or other signs and symptoms are measured. Chemical analytical methods were developed,
allowing the proper identification and quantification of the toxins. However, most groups of toxins can comprise different analogues and
the identity of many of these toxins may still be unknown or proper standards are missing. For this reason the application of the MBA
is still allowed for testing of production areas after 2014. However, the final aim is to replace these tests completely. In vitro bioassays
based on the mode of action (MOA) offer the advantage over analytical methods for being capable to detect known and unknown
compounds with a similar MOA. For other toxic endpoints MOA-based bioassays have already been shown to allow characterization,
selection, purification and identification of unknown compounds in an approach called bioassay-directed identification. The aim of this
project is to develop such in vitro bioassays for the detection of marine biotoxins. We first investigated the effects of three shellfish
poisons on the whole genome mRNA expression of the intestinal cell line in Caco-2 cells exposed to two diarrheic shellfish poisons
(okadaic acid and dinophysistoxin-1) and one azaspiracid shellfish poison (azaspiracid-1). Bioinformatics analysis tools were used to
identify genes potentially useful as marker genes, and generate hypotheses on MOA. Our analysis revealed many genes that were
either toxin type-specific or affected by both types of toxins. Our hypothesis on mode of action and selection of biomarkers to be used
as detection method will be presented in the conference.
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17
Development and validation of a range of lateral flow immunoassays for the rapid screening of marine biotoxins (ASP, DSP, PSP)
in shellfish
Jawaid, W1., Melville, K1.,Meneely, J2.,Campbell, K2.,Rice, J3.,Holmes, S1.,Elliott, C2.,1Research and Development Neogen Europe
Ltd.2School of Biological Sciences Queens University Belfast.3Research and Development Neogen Corporation.
The presence of harmful algal blooms in seawater can result in the accumulation of biotoxins in shellfish such as mussels, scallops,
oysters, clams and cockles. Dependent on the particular toxins present, consumption of contaminated shellfish could lead to illness
in humans including amnesic, diarrheic or paralytic shellfish poisoning (ASP, DSP or PSP respectively). There has been a lack
of suitable rapid screening tools to complement accepted reference methods such as LC-FD, LC-MS/MS and MBA that are used in
national monitoring programs. Our aims were to develop a range of simple and accurate lateral flow immunoassays (LFIA) for the rapid
screening of key phycotoxins from shellfish extracts, which could be performed either in a laboratory or in the field. Three competitive
LFIAs were developed and validated: Reveal® 2.0 ASP (DA), Reveal® 2.0 DSP (OA & DTXs) & Reveal® 2.0 PSP (STX suite). One of the
challenges was to ensure that the thresholds for tests were based at concentrations relevant to regulations, whilst minimizing the risks
of generating positive results from samples deemed compliant to regulations. Qualitative results (negative/positive) were generated
using a portable reader (AccuScan®) to remove interpretation subjectivity. Rapid and simple procedures were devised whereby
samples could be screened in parallel for key phycotoxins (DA, OA, DTX1, DTX2, STX, NEO, GTX1&4, GTX2&3, dcSTX, dcNEO, dcGTX2&3,
C1&C2 and GTX5) in 20 min, including sample extractions. To include detection of DTX3 toxins, a hydrolysis step is incorporated into
the procedure. Validation consisted of evaluating various performance characteristics such as accuracy, sensitivity, cross reactivity,
matrix effects, stability, robustness and ruggedness. Inter-laboratory and intra-laboratory evaluations demonstrated that key toxins
could be detected from a variety of spiked and naturally contaminated shellfish samples and included comparisons with reference
methods. Further data will be presented at the conference. 18
High throughput assays for sensitive screening of marine neurotoxins in seafood
Nicolas, J1., Hendriksen, Peter2.,Rietjens, Ivonne1.,Bovee, Toine2.,1Toxicology Wageningen UR.2Toxicology RIKILT Institute of Food Safety.
The current techniques for the detection of marine biotoxins in seafood are the in vivo mouse bioassay and LC/MS-MS based analysis.
The mouse bioassay is highly unethical and the LC/MS-MS technique is expensive and does not allow for the detection of unknown
marine biotoxins. Therefore, there is an urgent need for the development of in vitro assays with high sensitivity, enabling the detection
of marine biotoxins below current regulatory levels. The present study investigated the suitability of the multielectrode array (MEA)
using rat cortical neurons and the mouse neuroblastoma neuro-2a assay with measurement of changes in membrane potential for
rapid screening of marine neurotoxins in seafood. Model neurotoxic compounds were selected that affect either Na+, Ca2+, K+ channels
or the Na+/K+ ATPase pump. In addition to these model compounds, pure marine neurotoxins commercially available and seafood
extracts were tested as a first proof of principle.
The MEA platform allowed for the detection of most model compounds, pure marine neurotoxins and neurotoxins present in
contaminated extracts. All model compounds (except K+ channel blockers), pure marine neurotoxins and two extracts contaminated
with saxitoxin and tetrodotoxin elicited changes in membrane potential in neuro-2a cells detected by fluorescent probes.
The data obtained with both techniques suggest that these models represent promising alternatives for reducing and ultimately
replacing the unethical in vivo assays currently used for the screening of both known and unknown marine biotoxins in seafood.
15 - 20 March 2015
19
Cell based bioassays for the detection of marine toxins in fish and shell fish for replacing animal testing
Bovee, T1.,1Toxicology and Bioassays & Biosensors RIKILT-Wageningen University and Research Centre. Marine toxins are a worldwide threat to consumers. Most counties rely on in vivo assays and chemical analytical methods to detect
marine toxins.
The main in vivo assays used for the detection of marine biotoxins in seafood are the mouse and rat bioassay, respectively MBA and
RBA. The MBA suffers from false positives and the RBA does not allow the detection of neurotoxic marine biotoxins.At the moment
the LC-MS/MS technique developed by Gerssen et al. in 2010 and the Lawrence method have been validated as an alternative routine
method to the in vivo assays for the detection of lipophilic marine biotoxins in seafood at the European level (European Commission,
2005; 2011). However, there is no LC-MS/MS or other chemical analytical method that is routinely applicable for the broad detection
of marine biotoxins, i.e. ciguatera toxins (CTXs) and neurotoxic brevetoxins (PbTxs) for example are missed.There is thus an urgent
need for the development of cheap animal friendly high-throughputin vitro tests that would allow the detection of marine biotoxins in
seafood products, both toxins that are currently known, but also unknown toxins and those which might emerge, e.g. due to climate
changes.
Recently a broad in vitro test strategy in combination with analytical methods was set-up at our institute. This multi-disciplinary
approach includesseveral bioassays, i.e., biomarker detection in human Caco-2 intestinal cells with qPCR and Luminex technology
for DSPs, the Neuro-2a MTT tests for both DSPs and NSPs, detection of NSPs with beating cardiomyocytes anddetection of NSPs in
neural cells with a multi electrode array (MEA). Over 100 samples from Chile have been analysed by the bioassays, showing very good
agreement with chemical analysis, indicating that the test strategy will allow a replacement of the MBA.
20
Temperature effects on kinetics of paralytic shellfish toxin elimination in the Atlantic surfclam Spisula solidissima
Bricelj, V. Monica1.,Cembella, Allan2.,Laby, David3.,1Institute of Marine and Coastal Sciences Rutgers University.2Ecological Chemistry,
Biological Sciences, Alfred-Wegener-Institut.3Biosciences NRC Institute for Marine Biosciences.
The Atlantic surfclam Spisula solidissimais characterized by accumulation of extremely high levels of paralytic shellfish poisoning
(PSP) toxins, slow toxin elimination rates and an extremely high post-ingestive capacity for toxin bioconversion. Surfclam populations
experience a wide range of temperatures along the NW Atlantic continental shelf, and are undergoing range contraction attributed to
global warming. In this study we determined the influence of temperature on detoxification kinetics of individual PSP toxins in two
tissue compartments of juvenile surfclams under controlled laboratory conditions, over prolonged (2.4 months) depuration. Clams
were toxified with an isolate of the dinoflagellate Alexandriumfundyense from the Gulf of Maine, allowing tracking of toxin composition
and calculated toxicity in surfclam tissues. The viscera detoxified at all temperatures, although toxin loss rate increased with increasing
temperature. In contrast, total toxin content and calculated toxicities in other tissues remained constant or even increased during
depuration, suggesting physiological or biochemical toxin-retention mechanisms and temperature-independent detoxification. In
vivo toxin compositional changes in surfclam tissues provide evidence of specific toxin conversion pathways, involving both reductive
and decarbamoylation pathways. We conclude that such toxin biotransformations, especially in non-visceral tissues, may introduce
a discrepancy in describing kinetics of total toxicity (in saxitoxin equivalents [STXeq]) over prolonged detoxification. Nevertheless,
total toxicity values generated by routine regulatory monitoring based upon mouse bioassays or calculated from chemical analytical
determination of molar toxin concentrations is adequate for first-order modeling of toxin kinetics in this species. Furthermore, the
differential detoxification response of viscera and other tissues in relation to temperature emphasizes the need for two-compartment
modeling to describe the fate of PSP toxins. Finally, we identified key parameters that may prove useful in hindcasting the timing of
toxic blooms or predicting new toxin input to deep offshore waters where routine monitoring of toxic phytoplankton is impractical.
15 - 20 March 2015
21
Overview of recent works of ANSES (the French food safety agency) on marine biotoxins (lipophilic phycotoxins monitoring in
shellfish, ciguatoxins in shark, seawater treatment)
ARNICH, N1.,1Risk Assessment Department ANSES - French Agency for Food, Environmental and Occupational Health & Safety.
ANSES is a public administrative agency and operates under the authority of several Ministries to conduct risk assessment and
provide scientific advice to the competent authority. It is proposed to present an overview of four opinions released recently in the
field of marine biotoxins. 1/ About the official monitoring of lipophilic phycotoxins in shellfish, ANSES assessed the sensitivity and the
specificity of the current definition of high risk periods and suggested a new methodology based on a refined statistical modelling
of the data from 2010 to 2013. 2/ About the vigilance system for lipophilic phycotoxins in shellfish, ANSES reviewed information
generated since 2010 when the official method became the LC-MS/MS analysis (instead of the mouse bioassay). Then, it has been
decided to still use the mouse bioassay on a limited number of sampling points (called reference points) in complement to the LC-MS/
MS analysis, for a systematic monthly sampling, as a vigilance system. 3/ About ciguatoxins in shark, a working group set up by ANSES
made a review of the literature on occurrence, analytical methods, human outbreaks and ethology of the sharks. In complement, a
research project was conducted to analyse samples of a shark involved in a human outbreak in Madagascar in November 2013. 4/
About seawater treatment, another working group set up by ANSES reviewed existing processes used for drinking water and assessed
their applicability to clean contaminated seawater (from a closed area because of phycotoxin or microbiological contamination) for onland shellfish ponds. One of the issues dealt with the bioavailability for shellfish of dissolved toxins.
22
Marine biotoxin risk assessment of Australian wild-caught abalone
Turnbull, A1., Hay, Brenda2.,McLeod, Catherine3.,1Food Safety and Innovation , Seafood Group, South Australian Research and
Development Institute.2Consultant AquaBio Consultants Ltd.3Consultant Seafood Safety Assessment Ltd.
Australia has the world’s largest wild-capture abalone fishery, totalling 4000 tonnes per annum. The Codex Alimentarius Commission
recently recognized marine biotoxins as a potential hazard in abalone. Hence the South Australian Research and Development Institute
conducted a risk assessment for wild-caught Australian abalone according to prescribed Codex Working Principles (CAC/GL 62-2007).
An acute exposure assessment of PSTs (paralytic shellfish toxins) was conducted by examining a variety of scenarios. The potentially
consumed dose of PST (μg toxin kg-1 body weight) was calculated for a range of meal types, serving sizes, processing reductions and
body weights. Dose was calculated by the multiplying the weight of each tissue type in the meal by the maximum concentrations
of PSTs recorded in those tissues during toxic phytoplankton blooms in Australia (0.59 and 2.44 mg saxitoxin eq kg-1 in foot and
viscera respectively) and summing the components. Calculated doses for each scenario were compared to the EFSA provisional Acute
Reference Dose (0.5 μg kg-1 b.w.), and a designated dose derived from the bivalve regulatory maximum level (1.33 μg kg-1 b.w.). The
tissues of the abalone consumed played a significant role in determining the potential ingested dose. The consumption of meals of
processed abalone foot resulted in doses from 0.20 – 1.48 μg kg-1 b.w. for a 60 kg person. Doses from meals consisting of whole
abalone and abalone viscera ranged from 0.58 – 9.15 μg kg-1 b.w. for a 60 kg person. The Acute Reference Dose and designated dose
were exceeded in several scenarios. These findings may be a concern for human health if such doses were found regularly. However,
other data showing that less than 1.6% of abalone are contaminated with PSTs above regulatory levels, and the lack of any confirmed
illness associated with marine biotoxins in abalone support the final conclusion of a low risk of illness associated with Australian wildcaught abalone.
15 - 20 March 2015
23
Pinnatoxin-G and other metabolites of Vulcanodinium rugosum: analysis, toxicity, distribution in Mediterranean lagoons and accumulation
in mussels (Mytilus edulis and Mytilus galloprovincialis)
Mondeguer, F9.,Abadie, E1.,Hervé, F9.,Deschamps, L9.,Séchet, V9.,Raimbault, V9.,Berteaux, T1.,Zendong, Z9.,Palvaudau, H2.,Amzil,
Z9.,Pelissier, F3.,Huguet, A4.,Molgó, J5,6.,Aráoz, R5,6.,Fessard, V4.,Sosa, S7.,Tubaro, A7.,Selwood, A8.,Harwood, T8.,Hess, P9., 1LER-LR,
F-34203 Sète, France, Ifremer.2Laboratoire Sécurisation des Productions en Conchyliculture, Station de Bouin, F-85230 Bouin,
France, Ifremer.3UPR 2301, 91198 Gif sur Yvette, France, Institut de Chimie des Substances Naturelles.4laboratoire de Fougères,
unité Toxicologie des contaminants, 35306 Fougères, France, Anses.5Insitut de Neurosciences Paris-Saclay, UMR9197, 91191 Gif
sur Yvette, France, CNRS.6iBiTecS, Service d?Ingénierie Moléculaire des Protéines, Toxines Récepteur et Canaux, 91191 Gif sur Yvette,
France., CEA.7Trieste, Italy, University of Trieste.8Nelson, New Zealand, Cawthron Institute.9Laboratoire Phycotoxines, 44311 Nantes
Cedex, France, Ifremer.
In a previous study, Pinnatoxin G (PnTX-G) had been identified as a toxin accumulating in mussels and clams in Ingril Lagoon, a small
lagoon on the French Mediterranean coast. The levels found in shellfish from this lagoon were sufficient to explain positive mouse
bioassays (MBA) in the absence of other regulated toxins. The present study was conceived to: (i) determine the distribution of PnTX-G
in other Mediterranean lagoons, (ii) characterise analytical methodology (liquid chromatography coupled to tandem mass spectrometry
(LC-MS/MS), and (iii) understand the accumulation in shellfish of PnTX-G and other bioactive compounds produced by Vulcanodinium
rugosum.
A 1-year study (2013 – 2014) in five Mediterranean lagoons (Prévost, Vic, Ingril, Thau and Leucate) revealed that mussels at Ingril
were the most contaminated, with mussels in the other lagoons accumulating PnTX-G to levels less than those that would cause
positive MBAs. Limits of quantitation in LC-MS/MS analysis were sufficiently low to reliably quantify concentrations below those that
would cause positive MBAs (50 µg PnTX-G / kg whole flesh). Matrix effects differed between clams, mussel whole flesh and mussel
hepatopancreas.
Mussels were exposed to V. rugosum, and accumulated PnTX-G to ca. 20 to 65 µgkg-1whole flesh. Concentrations of portimine were
estimated to exceed those of PnTX-G five-fold in mussels in the laboratory; however, the ratio of portimine over PnTX-G in V. rugosum
was much higher (ca. 20), and hence, portimine is either less accumulated or metabolised or excreted faster in mussels. Both control
and exposed mussels were positive in an assay with the nicotinic acetylcholine receptor, due to contamination of control mussels with
13-desmethyl-spirolide-C. However, extracts from exposed mussels induced a higher cytotoxicity on Neuro2A than control mussel
extracts.
No toxic effects were observed on either Neuro2A or Caco2 cells for pure PnTXs; however, fractions of V. rugosum showed some toxicity,
including apoptosis, DNA breaks and inflammation. PnTX-G moderately absorbed across an intestinal Caco2-cell monolayer. In orally
exposed mice, an LD50of 200 µg/kg was established with only slight alterations of the small intestine from histological examination.
24
VIRUS AND BACTERIA
Extraction, Detection and Quantification of Norovirus in Shellfish Associated with Illnesses
Woods, Jacquelina1., Calci, Kevin1.,Marchant-Tambone, Joey1.,Burkhardt III, William1.,1Microbial Hazard Science Branch US Food and
Drug Administration.
Detection of enteric viral pathogens with molecular based PCR assays has successfully been utilized for many years. For RNA viruses
such as enteroviruses, NoV, and HAV, reverse transcription PCR or the conversion of RNA to cDNA, is necessary. Detection of these
enteric viruses in foods, specifically norovirus can be a challenge due to the low levels typically found in food and the inability to enrich
or propagate these viruses. Quantification of these viruses can also be difficult to the delicate nature of RNA, the uncertainty of reverse
transcription efficiency, and in most instances we are trying to detect target levels that are very low compared to total RNA present
in the sample. Here we report on the extraction, detection, quantification, and characterization of norovirus in shellfish samples that
utilizes ultracentrifugation with the inclusion of non-human Caliciviruses as extraction controls. For outbreak investigations, NoV
GI and NoV GII were detected with average levels ranging from 170- 2981 RT-qPCR U/100 g and 1386-6530 RT-qPCR U/100 g,
respectively. Extraction efficiencies were between 67 to 82%. Phylogenetic analysis of the outbreak sequences for norovirus revealed
strains clustering with NoV GI.8, GI.4, GII.3, GII.4, GII.7, and GII.21. Nucleotide sequencing of the RT-PCR amplicons revealed identical
sequences in the clinical and shellfish samples and multiple strains were also found two outbreaks. Quantification of enteric viruses
can be a challenging task, especially when available methodologies may not provide the sensitivity necessary to detect viruses in low
levels. Here we are able to demonstrate the extraction, detection, quantification, and characterization of norovirus in shellfish. This
information is critical in outbreak investigations and is applicable in establishing NoV dose response and assessing risk of shellfish
consumption.
15 - 20 March 2015
25
Investigation of Norovirus Viability
Kingsley, D1.,1Ag res service, Virologist, USDA.
Due to the lack of cell culture methods or suitable animal models, it is not possible to directly determine whether norovirus detected by
RT-PCR is viable or not. However a new method has been developed that uses a surrogate norovirus receptor (porcine gastric mucin)
coupled to magnetic beads to separate inactivated norovirus from potetnailly infectious norovirus. When combined with real-time
RT-PCR methods, this assay can identify inactivation conditions and treatmants that inactivate human norovirus. Results from high
pressure experiments, UV and chlorine treatments will be presented, as well as a potential means of discriminating infectious from
non-infectius norovirus directly within live shellfish
26
Norovirus detection in oysters: the effect of sample size on the precision of production site virus concentration estimates.
Hunt, Kevin1,2., Doré, Bill1.,Keaveney, Sinéad1.,Butler, Francis2.,1Shellfish Microbiology Unit, , Marine Institute.2School of Biosystems
Engineering University College Dublin.
There is growing interest in the introduction into legislation of quantitative standards for norovirus (NoV) in bivalve shellfish. To support
this introduction, it is important that associated monitoring is based on robust and reliable analysis. The recently published ISO RTqPCR method for NoV in food identifies a minimum sample size of 10 shellfish. However it is unclear whether a universal sample size
of 10 animals is sufficient to provide a representative indication of NoV concentration at a sampling location. This study investigated
the effect that different sample sizes (i.e. individual animal per sample) would have on a single test result from an oyster production
site. 60 oysters from one site were tested individually for norovirus; 30 at a period of high contamination levels, and 30 at a period
when concentrations were near the limit of detection of the test. Bootstrap analysis produced estimates of normality, power, and
precision of sample sizes ranging from 1 to 30 animals. Results indicate that 10 animals approximate normal distributions under both
conditions. Sample sizes beyond 10 did not indicate significant practical increases in the precision of virus load estimates. However,
when virus concentrations are low enough for zero values to be observed in some animals, the increased uncertainty will reduce the
precision for the same sample size. The individual oyster data also allowed additional inference of the range of NoV genome copies
that consumers may be exposed to for a given portion size. This study also investigated the spatial distribution of NoV contamination
across an entire oyster production site. In general, findings indicate the standard ISO method provides a robust and reliable platform
for representative monitoring of oyster production sites when samples contain 10 individual animals. Puerto Varas / Chile
15 - 20 March 2015
27
Results of Three Field Studies Presented at the MSC Information Meeting: Demonstrating Potential Utility of Male-specific Coliphage
in the NSSP
Howell, Thomas1., Stadig, Laura1.,Jones, Steve2.,Marchant, Joey3.,Burkhardt, William3.,1Eliot, Maine, USA Spinney Creek Shellfish
Inc..2Natural Resources and the Environment University of New Hampshire.3Dauphin Island Sea Lab US Food and Drug Administration.
Several studies investigated seasonal persistence and reduction rates of fecal coliform (FC), male-specific coliphage (MSC), norovirus
(NoV) and adenovirus (AdV) in shellfish from Maine and Massachusetts. MSC concentrations in softshell clams showed 2-3 log seasonal
variation, lower in summer and higher in winter, with similar seasonality of NoV GII and AdV. Depuration trials showed consistent
1-log reductions for MSC and NoV GII in 2 days when ambient process waters exceeded 18°C, and increasingly longer times at lower
temperatures. Hence, seasonal depuration works well in the summer months when viral persistence is low and depuration rate is high.
A dye study was conducted in the Royal River, Yarmouth, Maine, USA to determine appropriate growing area classification distances
from a wastewater treatment plant (WTP) outfall and to evaluate a dilution model with field MSC measurements. A subsequent
spatial variation study was conducted at three locations representing 300:1 to 700:1 relative dilution of effluent from the WTP outfall. Shellfish were analyzed for MSC, NoV and AdV. Viruses were at or below detection levels, and the means for MSC at each location were
consistent with the dilution model. Sampling of influent and effluent WTP samples during a 3-inch rainfall showed diminished viral
deactivation rates compared with complete FC deactivation. The magnitude of variability for different factors suggests that viral risk
of shellfish harvested near WTP outfalls is most significantly a function of plant performance, then seasonal persistence and finally
distance from the outfall. Ongoing species-specific bio-accumulation studies of FC and MSC suggest that cold water adapted species:
soft-shelled clams, Pacific oysters and European oysters show strong seasonal MSC patterns, whereas non cold-water adapted
species: American oysters and quahogs stop pumping and can trap MSC for the winter if MSC contamination is present prior to when
growing area waters reach 10°C.
28
Detection of the virus of Hepatitis A (HAV) in marine organisms and other environmental matrices
González Saldía, Rodrigo1., Soto Godoy, Luis1.,Cruzat Cruzat, Fernando1.,1Unidad de Biotecnología Marina, Ciencias Naturales y
Oceanográficas, Universidad De Concepción.
The HAV is a pathogen that infect the human liver been its main pathway for transmission the fecal contamination of water and food.
The virus can remain active in the environment during a month.
The aim of this work was to develop a molecular technique for HAV detection in bivalve mollusk and microplankton samples by
amplifying a segment from 5’NCR region of the virus. To address this task were analyzed 146 samples from the Bío Bío region, using
methods of RNA extraction, reverse transcription (cDNA), conventional PCR amplification, cloning and sequencing of positive samples.
The results indicate that using the method developed was possible to detect the HAV 5’NCR region in hepatopancreas samples of
Perumytilus purpuratus obtained directly from the environment, in contrast, negative results were obtained when bivalve mollusks from
the market and microplankton samples were analyzed. From the positive samples, the fragments sequences of 5’NCR region obtained
showed that the RNA isolated belongs to HAV Ib genotype which it has been reported in South America only for Brazil. The positive
detection of HAV present in samples from Lirquén Harbor is consistent with the subsurface sewage discharges present in the area,
which it is a warning signal about a reservoir existing inside this marine ecosystem able to spread this human pathogen. Finally, this work provides a method for molecular detection of HAV in seafood and corresponds to the first step to understand the
environmental dynamics of this virus on the coast of the Bío Bío region or other sensitive marine ecosystem.
15 - 20 March 2015
29
Evaluation of the characteristics of the wastewater treatment plants for the microbial contamination of bivalve molluscs production
areas in the Italian mid-Adriatic coast
Latini, M1., Gentili, Valentina1.,Barchiesi, Francesca1.,Strubbia, Sofia1.,Parmegiani, Sonia1.,Duranti, Anna1.,1Centro di Referenza
Nazionale per il controllo microbiologico e chimico dei Molluschi Bivalvi Vivi, Sezione di Ancona, Istituto Zooprofialttico Sperimentale
dell´Umbria e delle Marche.
The sanitary survey is the first necessary step required by european legislation in order to approve an area suitable for the bivalve
molluscs production. This activity should find, in a certain area, the main pollution sources and should determine how the contamination
can spread in the sea water where bivalve molluscs grow. The Community Guide for the Classification of Bivalve Mollusc Production
Areas stated that the sewage and wastewater treatment plants must be considered as source of contamination during the sanitary
survey. These plants have different characteristics according to the acceptable maximum load and the discharges after treatment. In
addiction, the percentage of the population served by water treatment plant change considerably from different areas. In the last 4
years the majority of the Italian mid-adriatic coast has been investigated through a proper sanitary survey. The region under study has
short rivers with strong current coming from high mountains situated not far from the coast. Along the year the different seasonal
weather condition may lead to highly difference spread of the contamination, caused by rivers flow, stormwaters, drainage systems
and overflows. The coast investigated is whole classified for the clam Chamelea gallina harvesting as class A or B under the 854/2004
CE Regulation based on the evaluation of fecal indicator germ Escherichia coli. The study compare the Escherichia coli data on Chamelea
gallina with the environmental data. The investigation takes in consideration also the thesis that the inland wastewater treatment
plants can affect the contamination along the coast. The data set for the Escherichia coli parameter is extrapolated by the official
monitoring plan of classified areas of the Chamelea gallina. The microbiological data set used starts from 2007. The data set regarding
the wastewater treatment plans comes from the environment control agency. Comparison between sanitary and environment
data take in consideration seasonality, amount of people served and distance from the coast of the wastewater treatment plants,
percentage of organic load not treated, type of treatment, rainfall, eventually overflowing. The results discussed show interactions
between environment characteristic, water treatment plants characteristics and the contamination of bivalve molluscs. This approach
can be useful for a better understanding on how evaluate the information collected during the sanitary survey for the classification of
shellfish harvesting areas.
30
Assessing the impact of risk management procedures on norovirus concentrations in oysters at a production site over a three year
period
Dore, B1., Hunt, Kevin1.,Keaveney, Sinead1.,1Shellfish Safety Marine Institute.
We used RT-qPCR to monitor the impact of risk management procedures targeting reduction of norovirus (NoV) concentrations
in oysters at a production site between May 2010 and March 2013. The methods used to reduce NoV concentrations in marketed
oysters were a combination of (1) relaying, (2) depuration, (3) use of an alternative harvest site during high risk periods, and (4)
ultimately temporary cessation of harvesting. Regular NoV monitoring, increasing in frequency during the high risk winter periods,
demonstrated that, before risk reduction, 52 of 63 oyster samples (83.0%) from the principal production site contained NoV (GI or GII).
The mean concentration of NoV (GI & GII) in these oysters was 690 genome copies 100g-1 (range <LOD-4642). NoV concentrations
were monitored in seven batches of oysters relayed from the principal production site to one at less risk of NoV contamination. The
mean concentration of NoV in these oysters prior to relaying was 974 genome copies g-1 (range 273-1743) compared with a mean
concentration of 72 (range <LoD-193) following relaying. The average relay period was 30 days (range 14-56). NoV concentrations
before and after depuration were also monitored on 34 occasions. NoV was detected at quantifiable concentrations in pre-depuration
samples on 24 occasions. The extent of NoV reduction during depuration varied between cycles but overall the mean concentration
in oysters prior to depuration was 509.8 genome copies 100g-1 (range 149-1766) compared with 206 genome copies 100g-1 (range
<LoD-838) after depuration. Over the study period, the mean NoV concentration in market ready oysters (i.e. following treatments) was
150.4 genome copies 100g-1 (n=34), approximately 20% of the mean NoV concentration in pre-treatment oysters from the principal
harvest area. Therefore, NoV monitoring demonstrated that risk management procedures used during production at this site were able
to significantly reduce, but not eliminate, consumer exposure to NoV.
15 - 20 March 2015
31
Occurrence of bacterial and viral enteric pathogens and marine bacteria and discrimination of faecal sources in shellfish-harvesting
areas and their catchments in France.
Gourmelon, M1., Balière, C1.,Quenot, E1.,Cozien, J1.,Lozach, S1.,Caprais, M.P1.,Hervio-Heath, D1.,Le Saux, J.C1.,Parnaudeau, S1.,Strubbia,
S1.,Le Guyader, S1.,Balière, Cl2.,Bruey, Q2.,Giard, J.C2.,Rincé, I2.,Jardé, E3.,Rincé, A2.,1LSEM-SG2M-RBE Ifremer, France.2Unité de Recherche
Risques Microbiens Université de Caen, France.3UMR 6118 Géosciences CNRS, France. (Sponsored by The European Regional
Development Fund Interreg IVA Program )
The aim of this study was to evaluate the presence of human norovirus, enteric and marine bacteria in three coastal shellfish beds in
France. Shellfish (oyster, mussel and cockle, n=126), upstream water samples (n=117) and sediment samples (n=39) were collected
monthly from February 2013 to February 2014. Concentrations of E. coli were high in water and cockle samples while oysters and
mussels were less contaminated. Human-, ruminant- and pig-associated Bacteroidales markers were frequently detected in water
samples, but hardly detected in shellfish. Campylobacter spp. were investigated using ISO10272 method and rt-PCR detection of 16S
rRNA genes. They were detected in 74% of water samples and 28% of shellfish. Among the 770 strains, C. jejuni and C. coli were most
frequently isolated in water whereas C. lari was frequently observed in shellfish. Salmonella spp. were detected according to ISO6579
method and detection of invA and ttrBCA genes by rt-PCR. 37.6% of water samples and 10.3% of shellfish were positive for both genes.
Twenty-five strains corresponded to eight different serotypes were isolated. The stx genes were detected from 91.4% and 34.9% of
water and shellfish samples, respectively. 4,560 E. coli strains including 49 Shiga-toxin-producing E. coli and 46 enteropathogenic E.
coli were isolated and corresponded to 36 different serotypes. From May to October 2013, using ISO/DTS 21872 and rt-PCR detection
of specific genes, 143 human pathogenic Vibrio spp. (mainly V. parahaemolyticus) were isolated from shellfish (n=60) and seawater
samples (n=18). Human norovirus (genogroup I and II) were searched in shellfish digestive tissues using the ISO.DIS 15216 modified to
use all the supernatant. After extraction efficiency checking viral RNA were detected by rRT-PCR and 19% were found positive (14/72).
Overall results provide important information for hazard evaluation and on the origin of microbial contamination. 32
Use of historical bivalve monitoring data to inform sanitary survey assessments.
Price-Hayward, M1.,1Food Safety Centre for Environment Fisheries & Aquaculture Science.
In the EU, new bivalve molluscan shellfish production areas have been subject to a requirement to undertake sanitary surveys since
2006. In Scotland, the first sanitary surveys were completed in 2007 and since then, nearly all the Scottish bivalve production areas
have been surveyed. To date, over 140 surveys have been completed, covering both aquaculture production and wild harvest sites.
An assessment of historical bivalve E. coli monitoring data was undertaken for each survey in order to determine whether there was
a relationship between spatial effects, season or other environmental factors (e.g. rainfall, temperature or tidal state), and the E. coli
results.
A review was undertaken of the outcomes of the analyses of E. coli data with respect to spatial distribution and the environmental
factors of season, and rainfall for different bivalve species. The review also considered how the outcomes of these analyses had
contributed to the overall assessments within the sanitary surveys and the consequent recommended sampling plans.
The results of this review provide important insights into some of the factors that affect monitoring results and how assessment of
those effects can be used to improve assessments in future sanitary surveys. Puerto Varas / Chile
15 - 20 March 2015
33
LONG-TERM TRENDS OF PATHOGENIC VIBRIO SPP. POPULATIONS IN NEW ENGLAND, U.S.
Jones, Steve1., Urquhart, Erin2.,Hartwick, Meghan2.,Taylor, Michael2.,Cooper, Vaughn2.,Whistler, Cheryl2.,1Natural Resources and the
Environment University of New Hampshire.2Molecular, Cellular and Biomedical Sciences University of New Hampshire.
The incidence of confirmed vibriosis in the Northeast US has increased over the past decade. Whereas these include some cases
not associated with shellfish consumption, the more recent trend of increasing incidence of regional shellfish-borne Vibrio
parahaemolyticus outbreaks has resulted in harvesting closures and product recalls in some areas. Long-term (2007-2014) research
projects in the Great Bay estuary of New Hampshire and Maine have tracked V. parahaemolyticus, Vibrio vulnificus and Vibrio cholerae
populations in oysters, sediments and overlying water. In 2014 a novel method for separation of water samples into fractions of zoo
and phytoplankton was incorporated. The highest concentrations occurred during 2012 when the Gulf of Maine set record (150 year)
high sea surface temperatures (SST). To generate estimates of vibrio likelihood and abundance, a two-step hybrid approach involving
multivariate classification and regression methods was used. We tested an extensive array of biotic and abiotic predictor variables.
When controlling for site and season, results show that V. parahaemolyticus abundance in oysters can be estimated as a function of
SST, salinity, precipitation, chlorophyll a, and turbidity. These results provide a framework for understanding the recent emergence of
Vibrios as a significant issue for shellfish harvesting in the Northeast U.S.
34
The Mexican Shellfish Sanitation Program (PMSMB) and the sanitary control of the Vibrio parahaemolyticus.
Barreiro, J1., Sanchez, Luis1.,1Executive Direction of Special Programs Ministry of Health, COFEPRIS, Mexico.
The Mexican Shellfish Sanitation Program (PMSMB) is an interinstitutional program presided by the Ministry of Health via the Federal
Commission for Protection Against Sanitary Risks (COFEPRIS), whose main objectives include the safeguardingof public healthand
the supportingof shellfish exports through the regulation of issues such as the sanitary classification of growing areas, the control of
shellfish extraction, the inspection of processing plants and the authorization of testing laboratories. One of the issues handled by
the PMSMB is to keep under control the Vibrio parahaemolyticus, which is a pathogenicorganism that can be found inbivalvemollusks
andrawor undercookedshellfish, causinggastroentericfood poisoning. The PMSBM promotes a variety of regulatory and non-regulatory
actions for the sanitary control of such microorganism. These have allowed to delimit its impact in public health, as well as to decrease
its incidence. Some of the established measures include: setting the level of action of the Mexican Official Standard for fishery products
(104 NMP/g), the implementation of a methodology for detection, and the harmonization of this methodology in the national testing
laboratories. The planification of risk management includes aspects of epidemiological surveillance in growing areas, the investigation
of V. parahaemolyticus outbreaks, and the sanitary dispositions for shellfish harvesting units, processing plants and dealers. The main
purpose of these measures is:
1. Establish the impact of V. parahaemolyticus in public health.
2. Reducesuchimpact through the measures taken bythe health authority.
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35
Risk Management of Vibrio parahaemolyticus in Oysters in Canada from 1997-2014
Buenaventura, Enrico1., Edwards, Robyn2.,1Bureau of Microbial Hazards Health Canada.2Domestic Food Safety Systems Division
Canadian Food Inspection Agency.
In 1997 an unprecedented outbreak of raw oyster - related Vibrio parahaemolyticus (Vp) gastroenteritis occurred throughout the Pacific
Coast of North America that also implicated British Columbia oysters. A risk reduction strategy (RRS) was developed in partnership
with other government departments and industry, and implemented with ongoing refinements each year. Analysis of results of
subsequent field studies and other research projects including information gathered from relevant risk assessments were used to
refine the Vp RRS over the years. Health Canada (HC) interim guidelines limit the Vp to 100 MPN/g in oysters at harvest and various
control measures were put in place to enhance the temperature control from harvest to consumer. The number of Vp related illnesses
dropped and remained relatively low. A study conducted by the B.C. Centre for Disease Control (BCCDC) showed the rate of Vibrio
illness in B.C. at 1.3 per 100,000 in 1997 and an average rate of 0.43 per 100,000 from 2001- 2008.
The East Coast of Canada represented a different picture. Vp did not appear to be a significant concern with no reported outbreaks and
relatively small number of sporadic illnesses reported each year.
Periodic review of the program and broader work under the Safe Food for Canadians Action Plan resulted in adjustments of the CFIA
approach to this pathogen in oysters intended for raw consumption. We report on the transition of the risk management strategy in
Canada and encourage industry innovation to enable more effective controls from harvest to the consumer.
36
Predictive bases of Vibrio parahaemolyticus in shellfish using Air and Water Temperature for Public Health management and production
Cachicas, Viviana1., Vargas, Joaquin2.,Muñoz , Pablo2.,Pinto , Camilo2.,1Microbiologia de Alimentos, Departamento Salud Ambiental ,
Instituto de Salud Pública de Chile.2Carrera de Bioquímica Universidad De Santiago De Chile. (Sponsored by FAO USP Pr 38361 (20082009), CORFO 09 CN 145951 (2010-2012) , US FDA, Universidad De Santiago De Compostela, Universidad De Bath UK & Seremi De
Puerto Montt. )
The southern Chile is an important producer of salmon and bivalve shellfish for domestic and exportation. Since 2004-2008, significant
outbreaks of Vibrio parahaemolyticus, had an annual occurrence even the prohibited consumption of raw or undercooked shellfish. From
2009 to now a decrease of outbreaks to less than 10 people per epidemiological week had occurred, except after a Hot Wave in 2013 where the people sick increased to 160 cases in two weeks. In order to promote the safety of the row consumption using the satellites information of sea and air temperature ( SST & airT) , we
evaluate the concentration of total and pathogenic Vibrio parahaemolyticus per gram of shellfish by Real Time PCR in Most Probable
Number format (qPCR-NMP/g), has recommended by Gulf Coast Seafood Labs of US-FDA. The samples were obtained weekly in
open markets close to shellfish farm from 2008 and in 2013 a specific shellfish farm. The environmental temperatures used were an
average of the area where high density of shellfish farm are located. The results shows than when the concentration less than 1000 qPCR-MPN/g is not able to produce significant outbreaks per
epidemiological week. The specific results obtained in the hot wave event, shown that 3 days of ~30°C of air T, increased the
concentration per gram at least up to 10E5/g. This information can be the base of a predictive model and a good example, of how a pathogen can be potentially harvest manager by
each shellfish farm and decide if consumption will be raw or cooked. Puerto Varas / Chile
15 - 20 March 2015
37
Vibrio parahaemolyticus and fecal pollution in zones of bivalve mollusks at the Mediterranean coast of Egypt. El-Shenawy, Moustafa1., El-Shenawy, Mohammed2., Nasr, Samir3.,Jose , Soriano4.,Mañes, Jordi5.,1Food & Environmental Microbiology.,
Food and Nutrition Science., National Research Center.2Dept. of Microbiology National Institute of Oceanography and Fisheries,
Alexandria, Egypt.3Dept. Environmental Studies, Institute of Graduate Studies and Research, University of Alexandria, Egypt.4Department
of Preventive Medicine and Public Health,., Faculty of Pharmacy., University of Valencia, Spain.5Department of Preventive Medicine and
Public Health, Faculty of Pharmacy, University of Valencia, Spain.
Vibrio parahaemolyticus is commonly found and widely distributed in coastal waters worldwide. Thus, there is a great chance
of being infected after consumption of uncooked or handling sea foods. It causes gastroenteritis, watery diarrhea and septicemia. Shellfish (44samples) and seawater samples (44 surface and 44 bottom samples) were collected seasonally according to the IOS
standers, from eleven stations located along the Mediterranean coast of Egypt, during four seasonal sampling cruises, from March
2013 to February 2014. Vibrio parahaemolyticus density, fecal pollution indicators bacteria (including total coliforms, fecal coliform,
and fecal streptococci ) and total aerobic bacterial plate count were determined using the membrane filtration technique and the
conventional identification tests and kits. Moreover, temperature, salinity, pH and dissolved oxygen of the coastal water samples were
also measured. Mean Counts of Vibrio parahaemolyticus in shellfish were higher 1.5 - 2.5 Log cycle than those reported in
water samples. Counts of coliforms in water and shellfish samples were the highest counts of the fecal pollution parameters followed
by E.coli and fecal streptococci respectively. Counts of coliforms in shellfish were higher about 1.0 - 1.5 Log cycle than those obtained
in water samples. In shellfish, counts of coliforms and Vibrio parahaemolyticus overlap each other. In water samples, Vibrios counts
were less than counts of coliforms but approximately equal to those of E. coli counts. In general, as the temperature gets high, the
higher counts of both coliforms and Vibrios were detected. It could be concluded that there was an association between the fecal
contamination and the presence of the pathogen either in water or in shellfish samples. These results may help to develop sanitary
strategy / strategies for better public health. 38
A miniaturized MPN real-time PCR method for rapid quantification of total and enteropathogenic Vibrio parahaemolyticus in shellfish
HERVIO-HEATH, Dominique1., QUENOT, Emmanuelle1.,VERON, Antoine1.,RICHARD , David1.,CAPRAIS, Marie Paule1.,1RBE/SG2M/
Laboratoire Santé, Environnement et Microbiologie IFREMER, France.
Rapid and reliable methods for detection and quantification of human pathogenic Vibrio spp. are fundamental for monitoring and risk
assessment in regard to the consumption of shellfish. A new method was developed for rapid and specific quantification of total and
enteropathogenic Vibrio parahaemolyticus in oyster and mussel samples. The protocol was based on an enrichment step in alkaline
peptone water using a 96-well plate followed by a MPN-real time PCR method. Pacific oysters (Crassostrea gigas) and mussels (Mytilus
edulis) collected from the Atlantic coast between May and October (2011-2013) were processed within 24h of harvest. Decimal dilutions
of tissue homogenates were prepared in APW and each dilution was inoculated into duplicate sets of 7 wells of a 96-well plate and
incubated at 37°C for 20h. After incubation, DNA from each well was extracted and the presence of total and enteropathogenic V.
parahaemolyticus was detected by real-time PCR using the toxR and tdh/trh genes, respectively. The MPN/g value was estimated
using MPN ver3 calculation program. Quantification of V. parahaemolyticus in oysters and mussels was obtained within 48h. In total,
69% and 38% of the oyster samples and 75% and 25% of the mussel samples examined were positive for total and enteropathogenic V.
parahaemolyticus, respectively. Prevalence were consistent with those determined using ISO 21372 method. Levels of contamination
were variable i.e. Pacific oysters samples ranged from trh positive V. pararahaemolyticus MPN/g and mussels from 0 to 19000 MPN/g
and from 0 to 870 MPN/g, respectively. The miniaturized MPN real-time PCR method is rapid and provides levels of contamination
of V. parahaemolyticus which will be very useful for monitoring and risk assessment program. The comparison of this method to the
BAM-MPN method is in process.
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39
ROUND TABLE
Global bivalve production and issues related to international trade
Iddya, K1.,1Fisheries and Aquaculture FAO.
Bivalves constitute an important commodity contributing to both food security and international trade. Global bivalve production has
been on the rise reaching almost 15 million tonnes and most of the increase comes from aquaculture. Bivalves are traded in different
forms-fresh, chilled, frozen or canned. Global exports in 2011 reached 2.9 billion US$ but for trade in high value commodity- live
bivalves, there are still issues related to market access. Only a very small proportion of bivalve producing countries are able to export
live bivalves and the issues are related to sanitary controls. The Codex Alimentarius Commission has developed standard for live and raw bivalve molluscs and the Code of Practice that needs
to be followed by member countries in order to achieve these standards. But many countries still seem to be having problems in
implementing the Code of Practice or in achieving the standards. FAO has been working with an Expert Group to develop technical
guidelines for implementation of bivalve sanitation programme within the framework of the Codex Code Practice for Fish and Fishery
Products. 40
POSTERS
POSTER 1
EVALUATION OF THE POSSIBLE SOURCE OF DISCREPANCY IN THE RESULTS OBTAINED AFTER THE APPLICATION OF MBA AND LCMS/MS FOR THE ANALYSIS OF LIPOPHILIC TOXINS
Rossignoli , A.E.1., Vilariño-Bermúdez, O.1.,Ben-Gigirey, B.1.,Braña-Magdalena, A.2.,Leão-Martins, J.M.2.,Gago-Martínez, A.1,2.,1EURLMB
European Union Reference Laboratory for Marine Biotoxins-Spanish Consumers Affairs, Food Safety and Nutrition Agency. Ministry of
Health, Social Affairs and Equality.2Analytical Chemistry and Food Department, Faculty of Chemistry, University of Vigo .
The coordination of the application of analytical methods by the National Reference Laboratories (NRLs) in the EU , in particular by
organising comparative testing and by ensuring their appropriate follow-up is one of the key Tasks of the EU Reference Laboratory
for Marine Biotoxins. To accomplish this task, the EURLMB organises yearly a Proficiency Testing (PT) among EU National Reference
laboratories, in which Laboratories from Third Countries could be also involved. These Proficiency Testing have a great potential, not
only to evaluate the performance of the participants on the application of a particular method, but also to evaluate the method itself
taking into account the concordance of the results obtained among the different participants, once their good performance on this
particular method is ensured. The change in the EU Legislation regarding to the replacement of the MBA by the Chemical method LCMS/MS prompted the evaluation in depth of the results of the Proficiency Testing for both methods, in particular during the period
in which both methods coexisted and the aim of this work is to present the data obtained in this evaluation which correspond to the
PTs results for MBA and LC-MS/MS from 2010-2014. The high variability observed for the MBA with a significant number of false
positives, prompted the evaluation of the sources of such discrepancy and with this aim the presence of free fatty acids, as well as
the possible additive effect between okadaic acid and 13-desmethyl-spirolide C were tested. The results obtained in this work do not
allow justify this additive effect as the cause for the mice death, while the presence of free fatty acids in the extracts and their potential synergistic effect on the toxicity of Lipophilic toxins could be suggested.
15 - 20 March 2015
41
POSTER 2
A VALIDATED PP2A METHOD- OKATEST-ADAPTED FOR DETERMINATION OF LIPOPHILIC TOXINS IN COMBINATION WITH LC-MS/
MS
Dominguez, E1., 1MARKETING ZEULAB S.L..}
Domínguez E.*, Calvo L., Razquin P. and Mata L.
ZEULAB. C/Bari 25, dpdo. 50197, Zaragoza, SPAIN.
*edominguez@zeulab.com
Commission Regulation (EU) No. 15/2011 that came into force in July 2011, specifies the LC-MS/MS as the reference method for
lipophilic toxins. Other assays such as the phosphatase inhibition assay can be used for routine testing of official controls or any checks
done by food operators.
OkaTest, a PP2A method that has been inter and intralaboratory validated, complies with the legislation requirements and therefore it
can be used for OA toxins controls. To fully monitor lipophilic toxins this method must be combined with other methodologies.
The commercial PP2A test OkaTest has been adapted to be used together with LC-MS for determination of lipophilic toxins. Two
grams of mollusc sample are extracted with methanol (100% v/v) in a common step for both methods. Then, the sample will be
hydrolysed to be tested with the PP2A method for determination of OA toxins and the non-hydrolysed extract injected in LC-MS/MS
for AZA, PTX and YTX analysis.
Minor volume and weight changes were introduced to the validated PP2A method for a common extraction with LC-MS/MS.
Repeatability, reproducibility and recovery were re-evaluated to confirm that the performance of OkaTest remained unchanged.
Results obtained for OA toxins using the new adapted OkaTest method and LC-MS for around hundred samples showed good
correlation.
Labs with large number of samples or limited access to LC-MS equipment could benefit from using both methodologies in combination.
A shorter time-around time and low cost per sample could be achieved implementing both methods for routine monitoring of lipophilic
toxins.
42
POSTER 3
Biotoxins monitoring program in Brazil
Schramm, Mathias1., Proença, Luis Antonio1.,Alves, Thiago1.,1LAQUA/MPA-Itajaí/SC Instituto Federal de Santa Catarina - Campus
Itajaí.
Before the 80ies, harmful algae did not appeared to be a problem along the Brazilian coast. Taking as reference marine phycotoxins
occurrence maps from the 90ies, we will not see records of DSP, ASP or PSP. The problem started to be spotted after the first monitoring
program for harmful algae and phycotoxins, set as a pilot in 1997 in Santa Catarina, the largest aquaculture mussel producing state
in Brazil. In a few years, the occurrence of PST, DST, AST associated to Gymnodinium catenatum, Dinophysis spp. and Pseudonitzschia
spp. respectively were evidenced. The first official closure on harvesting occurred in january 2007 in Santa Catarina. The alert started
with high counts for D. acuminata and positive mice bioassay tests for DSP. In 2009 also in Santa Catarina a first closure of shellfish
harvesting due to domoic acid occurred. After 15 years from the first toxin analysis, Brazil established in 2012 the National Program
for Mollusks Hygienic and Sanitation Control - PNCMB, which aims to establish minimum requirements for ensuring the safety and
quality of bivalve mollusks for human consumption. In 2012 the program published national regulation limits for four major toxins
groups: okadaic acid, azaspiracids, saxitoxin and domoic acid. The threshold values and analysis protocols were harmonized within
international standards. At the same time, the program created the LAQUA-Itajaí/SC, which is the national reference laboratory
for biotoxin analysis in seafood. Today, a monitoring program is carried out in Santa Catarina enrolling federal and state agencies.
Samples from 44 sampling sites are collected regularly for mussel toxin and microalgae analysis. This presentation will show Santa
Catarina’s monitoring program and the on-going improvements carried out on shellfish control and the efforts to get adapted to the
new methodologies, such as HPLC and LC-MS/MS methods for marine biotoxins.
15 - 20 March 2015
43
POSTER 4
Modelling the spatial and temporal dynamics of paralytic shellfish toxins at different scales: implications for research and management Seguel, Miriam1., Molinet, Carlos2.,Díaz, Manuel2.,Niklitschek, Edwin3., Díaz, Patricio2.,1Centro Regional de Análisis de Recursos y
Medio Ambiente Universidad Austral de Chile.2Programa de Investigación Pesquera & Instituto de Acuicultura Universidad Austral de
Chile.3Centro i~mar Universidad de Los Lagos. (Sponsored by Research Project: FONDEF MR07I1007, 2nd Red Tide Program)
Harmful Algal Blooms, in particular recurrent blooms of Alexandrium catenella, associated to Paralytic Shellfish Poisoning (PSP), pose
a serious socio-economical problem worldwide. Although most affected resources are characterized by very patchy and spatially
heterogeneous distributions, few research and monitoring programs have considered the analysis of scale effects and spatially-explicit
trends in PSP concentrations. Our objective was to study the spatial and temporal dynamic of PSP concentration in specific patches
located within two clam beds (Venus antiqua), affected by an exceptional toxic bloom of A. catenella (1.1x106 cells L-1), during summer
2009, in Southern Chile. Generalized linear models (GLMs) were used to identify the influence of different environmental variables upon
PSP concentrations along 24 months. In order to obtain quantitative descriptors for accumulation and detoxification dynamic of PSP
on clam’s patches, we used a simplified one-box model, defined by two parameters: a proportionality constant between A. catenella
and PSP concentrations and an instantaneous PSP decay rate. Results showed significant variability on the spatial and temporal
dynamics of PSP concentration, within and between patches and beds. Our observations support the relevance of considering different
spatial scales (patch/bed/oceanographic area) to enhance understanding about PSP accumulation and detoxification dynamics and to
improve the efficacy of fisheries management actions after toxic events. 44
POSTER 5
Marine toxins: new technologies and their impact in mussel farming Seguel, Miriam1., Varas , Pia1.,Aros, Javier1.,1Centro Regional de Análisis de Recursos y Medio Ambiente Universidad Austral de Chile.
(Sponsored by Project BIP 30234372-0. Gobierno Regional De Los Lagos, Chile)
Mussel farming is the second most important aquaculture activity in Chile, and is mostly developed in the Los Lagos region. Harvests
have increased from 221.500 tons in 2010 to 255.953 tons in 2013, with a FOB price of M$ 184.652 in 2013. This activity creates
approximately 12.000 direct and indirect jobs. There are currently 817 farming centers operating in the region, with different sizes
and production levels. One of the requirements for exporting to the European Union is the implementation of the European Union
Sanitation Program, which is financed by the mussel farmers.This program demands a weekly marine toxin and noxious phytoplankton
sampling. These analyses are carried out in university laboratories authorized by National Fisheries Service (Sernapesca) and
accredited under the NChISO 17025 norm. Currently, mouse bioassays are used to detect paralytic shellfish poisoning and lipophilic
toxins (including diarrheic shellfish poisoning), and high-performance liquid chromatography (HPLC) is used to detect amnesic
shellfish poisoning. However, beginning in 2015, lipophilic toxins must be analyzed using the LC-MS/MS technique, which will increase
the current analysis cost by three or four times. Research of lipophilic toxins is scarce. Results show that toxins belonging to the
okadaic acids, pectenotoxins, yessotoxins, and azaspiracids groups can be found in bivalve mollusks; however, the annual variation of
toxicological profiles and their concentrations are unknown. Therefore, Chile faces a great challenge in implementing this technique for
certifying exportation produce, and in the economic impact this will have on small- and medium-scale mussel farming. Puerto Varas / Chile
15 - 20 March 2015
45
POSTER 6
Alexandrium catenella cysts in Southern Chile: An overview of fifteen years of studies Seguel, Miriam1., Diaz, Patricio2.,Figueroa, Rosa3,4.,1Centro Regional de Análisis de Recursos y Medio Ambiente Universidad Austral de
Chile.2Programa de Investigación Pesquera & Instituto de Acuicultura Universidad Austral De Chile.3Instituto Español de Oceanografía
(IEO) Centro Oceanográfico de Vigo.4Aquatic Ecology, Department of Biology Lund University. (Sponsored by CIMAR Program. National
Oceanographic Committee (CONA).)
Blooms of Alexandrium catenella are known as producers of Paralytic Shellfish Poisoning (PSP) toxins leading to lengthy shellfish
harvesting bans in Southern Chile. These harmful events are the main threat to human health and the sustainable exploitation of
bivalves in the region. The causes underlying the distribution and timing of A. catenella blooms are poorly understood, but specific
environmental cues such as temperature are known to be involved. Additionally, the accumulation of resting cysts in the sediments,
the so-called “resting cyst beds”, has been cited to explain the onset and recurrence of Alexandrium blooms. Sediment samples were
collected and spatial and temporal distribution and abundance of A. catenella and other dinoflagellates cysts was studied at more than
180 locations in the southern channel and fjord zone from Puerto Mont (42º) to Cabo de Hornos (56º), under the multidisciplinary
research CIMAR Programme of National Oceanographic Committee (CONA). Samplings were carried out mainly during the winters and
spring of the years 1996-2010. Here, we do a retrospective analysis of the available information in an attempt to identify potential
“seed bank cysts” and explain their role in the recurrence of A. catenella blooms in this area. 46
POSTER 7
National Program of surveillance and control of intoxications and Harmful algae blooms (Red tide) in Chile
Vaquero, Alejandra1.,Rivera, Andrea1.,Raymond, Emilia1.,Delgado, Lorena1., 1Salud Ambiental Instituto de Salud Pública de Chile.
Harmful Algae Blooms (HAB) has been observed in Chile since the XVIII century, with cases reported in offshore areas and coasts. Since
1972 until recent years, the presence of toxic dinoflagellates has been increasing in more recurring and in larger areas mainly in the
regions of Los Lagos, Aysén and Magallanes. Marine toxins that are mentioned more frequently, which have a major impact on Public
Health and on Chile’s economy, belong to the groups of paralytic (PSP), diarrheic (DSP) and amnesic (ASP) shellfish poisoning.
The Ministry of Health in Chile is developing and coordinating the National Program of Surveillance and Control of Intoxications from
Harmful Algae Blooms (“Red Tide”), with the objective to verify the presence of marine biotoxins and prevent intoxications in the
population derived from the consumption of marine resources, (bivalve molluscs, echinoderms, tunicates and marine gastropods),
contaminated by Harmful Algae Phenomenon (“Red Tide”). The Secretarías Regionales Ministeriales de Salud (SEREMIs) develop
activities focused in three main roles: Monitoring of the areas of extraction, Product control and Education related to this field. The
analysis are carried out by the regional laboratories with the analytical capacity, establishing the Laboratory of Marine Biotoxins of the
Institute of Public Health of Chile to accomplish the role of National Reference Laboratory, performing confirmation if is needed, settle
results if necessary and and perform the seafood analysis from regions which do not dispose of analytical capacity.
In the year 2013 the network of marine biotoxins laboratories of the country carried out 40.387 analysis, 33.181 for PSP, 4.806 for
DSP and 2.400 for ASP. In the Aysén Region, only 1 case was registered as intoxication by PSP derived from the consumption of
seafood obtained from an area under extraction restriction.
From the analytical point of view, Chile is facing the challenge of implementing the identification, detection and quantification of DSP
through the HPLC MS/MS method with the purpose of determining the presence of chemical compounds belonging to the complex
group DSP. This method has the advantage of confirming each species of lipophilic toxins, which in turn cannot be accomplished with
the mouse bioassay. It will also allow facing and harmonizing the sanitary regulations and changes in international regulations, mainly
from the European Union.
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47
POSTER 8
The first well documented event of Paralytic Shellfish Toxins (PST) in blue mussels (Mytilus edulis) in Sweden
Persson, Malin1.,Karlson, Bengt2.,1Food Control Department National Food Agency.2Research & development, oceanography Swedish
Meteorological and Hydrological institute.
Harvesting of mussels, cockles and oysters has been ongoing along the Swedish Skagerrak coast, North Sea area, for more than 25 years.
Since year 2001 the National Food Agency administers a monitoring program for biotoxins in bivalve molluscs and occurrence of biotoxin
producing algae in production areas. Harmful algae, e.g. biotoxin producing dinoflagellates and diatoms, are observed in production
areas for bivalve molluscs along the Swedish Skagerrak coast. Occurrence of Diarrhetic Shellfish Toxins, produced by Dinophysis spp.,
is a problem for the aqua culture industry with large inter-annual variations of biotoxin content in the bivalves. Alexandrium spp.,
producers of Paralytic Shellfish Toxins (PST) have been observed in low abundances intermittently. Also cyst surveys have revealed
the occurrence of Alexandrium cysts along the Skagerrak coast. However, PST was observed above the regulatory limit (800 µg kg-1)
in mussel samples until 2014. From early April to early June 2014 PST was above the regulatory level in mussels samples from the
Bottne fjord. The maximum concentration was 1300 µg kg-1. During the same time A. ostenfeldii, A. tamarense and A. minutum were
observed with the highest abundances in April. The maximum cell density of A. ostenfeldii and A. tamarense was 26000 cells L-1. The
PST event caused closing of harvesting of farmed and wild mussels in the Bottne fjord and adjacent sea areas. No cases of Shellfish
Poisoning of humans have been reported. The course of toxic events in production areas along the Swedish coast will be described and
possible causes of the PST-event, e.g. local seeding population of Alexandrium cysts, unusual weather etc. will be discussed.
Keywords: Aquaculture, Mussel farming, Blue mussels, Mytilus edulis, Phytoplankton, Harmful Algal Blooms, Dinoflagellates,
Alexandrium, Paralytic Shellfish Toxin, Paralytic Shellfish Poisoning
48
POSTER 9
Analysis of Vibrio parahamolyticus MAM-7 gene polymorphism by RFLP: Proof of concept of a potential pathogenicity gene marker
Esquivel, M1., Beltrán, Sebastián2.,Bergman, Cristian2.,Osorio, Gonzalo3.,Trombert, Annette Nicole2.,1Escuela de Tecnología Médica,
Facultad de Medicina, Universidad Mayor.2Centro de Genómica y Bioinformática, Facultad de Ciencias, Universidad Mayor.3Departamento
de Microbiología Molecular, Facultad de Medicina, Universidad De Chile. (Sponsored by FIDUM Grant 100500 Universidad Mayor)
Vibrio parahemolyticus (VP)is a Gram-negative, halophilic bacterium which is an important foodborne pathogen. Autochthonous to
estuarine, marine and coastal environments, VPcauses acute gastroenteritis through the consumption of raw, undercooked and/or
mishandled seafood. In Chile, there are periodic epidemic outbreaks mainly produced by the pandemic strain RIMD2210633 serotype
O3:K6 (RIMD).
VP can be isolated from environmental samples (ES, mussels, fish and/or contaminated water) or from infected patients (CS, clinic
samples). The strains isolated from the environment can be pathogenic or nonpathogenic.
Currently, the detection of Vibrio spp implies the elimination of all the products potentially contaminated. This brings with it economic
and social repercussions in zones characterized by shellfish production. Thus, a technique that permits to discriminate between
pathogenic and nonpathogenic strains from environmental samples could prevent future outbreaks and/or product loss.
MAM-7 corresponds to a gene that encodes for an adhesin that mediates the pathogen adhesion and cytotoxicity, and is considered as a
virulence factor. In our study, we aimed to find out if the MAM-7 gene sequence could differentiate toxigenic strains from environmental
non-toxigenic strains. To achieve this goal, we implemented a proof of concept using RFLP technique to analyze potential sequence
polymorphisms in the MAM-7 gene and its correlation with pathogenicity.
Thus, 10 Chilean CS (identified as RIMD tdh+ clones) and 10 ES were used to amplify the MAM-7 gene. The products were digested with
seven different endonucleases, and the patterns obtained were compared with RIMD and ATCC17802 (serotype O1:K1). In addition,
tdh gene was detected by PCR in all the samples.
As we expected, 100% of CS showed identical digestion pattern as RIMD and are tdh+. In contrast, approximately 80% of ES presented
ATCC pattern and 20% coincided to neither RIMD nor ATCC. 100% of these were tdh- .
15 - 20 March 2015
49
POSTER 10
Characterization of Vibrio parahaemolyticus in bivalve molluscs from the coast of Buenos Aires, Argentina using pulsed-field gel
electrophoresis (PFGE)
Jurquiza, Verónica1., Pichel, Mariana2.,Costagliola, Marcela1.,González Fraga , Sol3.,1Gabinete de Biología Molecular y Microbiología
INIDEP-Instituto Nacional de Investigación y Desarrollo Pesquero.2Servicio de Enterobacterias INEI-ANLIS “Dr. Carlos G. Malbrán”.
Instituto Nacional de Enfermedades Infecciosas.3Área de Biología Molecular Laboratorio Domecq y Lafage.
The increasing prevalence of Vibrio parahaemolyticus demands an active surveillance of this pathogen, and the need to establish the
relationship among different circulating strains, as well as the origin and divergence of the isolates. In Argentina, there is scarce data
on cases and no information about outbreaks caused by this microorganism. However, its presence in the environment has been
demonstrated in several studies. In addition, there are no molecular epidemiology studies about the distribution of circulating strains
in our country. PFGE typing has been proved to be very effective to evaluate the relationship between bacterial isolates of the same
species. The aim of this study was to characterize the genetic diversity of V. parahemolyticus isolates from bivalve molluscs from the
coast of Buenos Aires, using the standarized PulseNet PFGE protocol. A total of 28 isolates were selected and included in the study,
along with the reference strain ATCC 17802. We found high genetic diversity in the V. parahaemolyticus isolates analyzed by PFGE.
Among the 25 compared isolates, 16 electrophoretic patterns were identified. Four isolates were not typeable by SfiI-PFGE, even after
adding thiourea in the running buffer. Only five genetically similar groups of two to four isolates each were identified. In the different
groups, isolates came from different sites and different samples. Also, the persistence of genetically related isolates was observed in
different periods of time. Interestingly, one electrophoretic pattern was shared by two isolates, C217/09, serotype O4:K18 tdh+/trh+,
and C306/09, serotype O6:K18 tdh-/trh-, Both isolates were recovered from different samples of cockles taken in the same site (Mar
Azul). This study contributed to create the first database of circulating subtypes of V. parahaemolyticus in the Atlantic coast of Buenos
Aires. It highlights the need to pursue further PFGE typing studies to increase this database and therefore facilitate the comparison of
isolates recovered in Argentina and around the world.
50
POSTER 11
Presence of Arcobacter spp. in bivalve molluscs from harvesting areas of the Central Adriatic Sea, Italy
Leoni, F1., Chierichetti, S1.,Rocchegiani, E1.,Ottaviani, D1.,1Sezione di Ancona Istituto Zooprofilattico Sperimentale dell´Umbria e delle
Marche. (Sponsored by Funding From The Italian Ministry Of Health Through Project IZSUM RC 10/2011 Is Acknowledged.)
Arcobacter species have been associated with gastrointestinal human cases and contaminated food and water may represent possible
routes of transmission. Moreover, Arcobacter spp. are present in the intestine of livestock animals, which may act as reservoirs of these
bacteria. A. butzleri is the most important species associated with human infection, followed by A. cryaerophilus. Arcobacter spp. have
been isolated in brackish and marine environments, but only few studies investigated Arcobacter spp. in shellfish. The present study
aimed to investigate the presence of Arcobacter spp. in bivalve molluscs collected from authorised harvesting areas of the central
Adriatic sea. A total of 123 samples of bivalve molluscs (35 mussels and 88 clams) collected between December 2012 and July 2014,
were analysed by a culturing method for detection of Arcobacter spp. Presumptive colonies were tested by PCR for the rpob gene of
Arcobacter spp. Of 123 samples of bivalve molluscs, 71 and 52 samples were from harvesting areas classified as B and A, respectively.
Identification of Arcobacter spp. was performed by DNA sequencing analysis of the rpoB gene. Overall, 51 (41.5%) of the 123 shellfish
samples analyzed were positive for Arcobacter spp. Of these, 39 (76.5%) were from class B areas or closed A areas. Sequencing
analysis of the rpoB gene identified A. butzleri, A. cryaerophilus and A. skirrowii in 32 (68%), 14 (29.8%) and 1 (2.1%) of the 47 samples
analysed, respectively.In conclusion Arcobacter species important for human infections were detected in bivalve molluscs collected
from harvesting areas. However, a higher prevalence of Arcobacter spp. was found in bivalve molluscs harvested from areas which
required post-harvest treatment. In agreement with other studies, A. butzleri was the most prevalent species identified in shellfish. Puerto Varas / Chile
15 - 20 March 2015
51
POSTER 12
Norovirus prevalence in Dutch production area with Class A/B classification. Pol-Hofstad, Irene1., Van Der Meij, Alice1.,Rutjes, Saskia1.,1Laboratory for Zoonoses and Environmental Microbiology RIVM.
In follow-up of the EFSA opinion (EFSA 2012) relating to norovirus in shellfish, The Dutch NRL has set up a project to evaluate the
norovirus status of two national production areas and generate quantitative data to calculate the prevalence of norovirus. Two
production areas have been selected. Lake Grevelingen is a clean area with a stable class A status. Lake Veere is an area with a lot of
recreation and therefore has a class B status. However E.coli numbers measured in shellfish flesh throught the years reflected low
contamination and based on those results this area has been upgraded to class a. To calculated prevalence of Norovirus more than
250 oysters were harvested at 3 locations in these production areas. Oysteres were analysed individually according to ISO/TS 152161. Shellfish from those areas were not implicated in outbreaks as far as known. Norovirus results will be shown and compared to E.coli
numbers from the same area. And the consequences of implementation of certain norovirus criteria for the Dutch situation will be
discussed. 52
POSTER 13
Norovirus from environment samples associated to outbreaks in Chile 2010-2014
Jara, Monica.,Farias , Leonardo1.,Martinez, Maria Cristina1.,Cachicas, Viviana 1.,1Microbiologia de Alimentos, Departamento Salud
Ambiental , Instituto de Salud Pública de Chile. (Sponsored by CORFO 09 CN 145951 & FAO- USP- FDA: PR 38361 , Laboratorios De
Referencia Virología Y Genética Molecular ISP & Unidades De Toma De Muestra De Seremi Regionales De Salud De Chile.)
Norovirus (NoV) is main cause of non-bacterial food-borne gastronentertitis worldwide. The reference clinical laboratories of Chile,
has been studying 1073 clinical samples since 2010. Fifty five percent of the samples were from surveillance and 43 % from outbreaks
studies. NoV was confirmed in 22,2% of the samples and sequenced. Samples from Antofagasta and Ovalle were related to outbreaks.
Others regions evaluated are Región Metropolitana, Valparaiso and Bio-Bio. 87.8 % of the samples were positive for GII and 10.9% for GI. 80.3 % were GII.4 and 10.6% GII.3 genotype. 69.8% of GII.2 were related to strain NewOrleans-2009 and from 2011 , 18 % of
the strains were Sydney-2012. (Boletin ISP Vol.3,N°6, Marzo 2013).
In order to investigate possible environmental suspected sources, we evaluated since 2010 fifty samples from different regions: Antofagasta, Ovalle, O´Higgins, Aysen, Magallanes y Región Metropolitana. The sources of samples were drinking water, irrigation
water, wastewater samples, lettuce and shellfish. Hollow-filtration and ultracentrifugation were used for samples between 2 and 100
Liters. For RNA purification and detection we follow CDC,FDA or ISO recommendation.
Seven samples were positive for GII genotype and two of them were able to be sequenced and related to clinical isolated from specific
outbreak: In 2010 the genotype GII.4 variant New Orleans 2009 were isolated from irrigation water after treatment of wastewater at
Antofagasta outbreak. In 2013, GII. 4 variant Sydney was also isolated irrigation water supply at Ovalle outbreak. Other five positive
GII samples were not able to be sequenced.
This results shows for first time in the environment in the country the presence of NoV related to outbreak and make possible to
management and control it.
15 - 20 March 2015
53
POSTER 14
Detection and quantification of genotypes I and II of Norovirus in bivalve molluscs of the Region of Los Lagos aestival season 2015 Cárcamo, Fernanda1,2., Cachicas, Viviana1.,1Departamento Salud Ambiental, Sección Microbiología de Alimentos, Instituto de Salud
Pública.2Sección Microbiología de Alimentos Instituto de Salud Pública.
Norovirus (NoV) is responsible for a significant number of gastroenteritis and It is associated with the consumption of drinking water or
food, such as vegetables and seafood, contaminated with human feces. Bivalve molluscs have great commercial interest due to their
consumption and economic importance, being the Region of Los Lagos the leading producer and exporter, with more than 1000 farms.
Also the region has significantly improved the treatment of sewage through submarine emissaries and various treatment plants in the
last decades.
In order to characterize the presence and concentrations of enteroviruses in bivalve molluscs in the production area, we started an
annual study of molluscs that arrive weekly to Santiago. The species analyzed are mainly Choro Malton, Choromytilus chorus, and
Cholga, Aulacomya ater. Samples were processed as recommended by the Technical Specification ISO/TS 15216-1: Dissection of
digestive glands, enzymatic treatment with proteinase K, RNA extraction technique with magnetic silica and detection of genotypes
GI and GII Norovirus by real time RT-PCR. The amplification conditions were the manufacturer recommended. For the standard curve,
proficiency testing Cefas 55/2014-2015 and Detection CeeramTools® Kit were used.
he results show in 15 samples between Epidemiological Week N°2 and N°6, absence of genotype G1 and six positive samples for
genotype GII. Virus concentrations ranged between 9,65x103 copies/g to 4,1x104copies/g. The six positive samples are geographically
located in the northeast of Archipelago of Chiloé.
This work, the first one of the country; will allow to georeference the risk areas; compare viral and coliforms indicators, and study
different factors that may affect norovirus presence as: effectiveness of sewage treatment plants, distance from the natural banks
and farms with treatment plants, and the effect of watercourses to finally generate recommendations to producers. Acknowledgements : Inspectors and Laboratorio SEREMI de Salud Región Metropolitana
54
POSTER 15
Harmful algal blooms and Vibrio spp association in fishing areas and marine farm of bivalve mollusks of Sechura and Piscobays, Peru. OROZCO, R1., QUISPE, YESICA2.,LORENZO, ALBERTO3.,1AREA FUNCIONAL DE INVESTIGACION MARINO COSTERO, MICROBIOLOGIA
ACUATICA, INSTITUTO DEL MAR DEL PERU.2LABORATORIO COSTERO DE PAITA, AREA DE OCEANOGRAFIA, INSTITUTO DEL MAR DEL
PERU.3LABORATORIO COSTERO DE PISCO, AREA DE OCEANOGRAFIA, INSTITUTO DEL MAR DEL PERU.
Free-living marine Vibrio species include a wide variety of pathogenic species. The incidence of algal blooms and their consequences
on changes in biological dynamics of marine Vibrio community threaten the development of mariculture and fishing areas of benthic
resources, especially in shallow areas. The bays of Sechura and Pisco are the most important production areas of bivalve molluscs
and mariculture activity. Therefore, between February 2010 to May 2014,the presence of Vibrio in marine sediments and water
was evaluated during the events of algal blooms and normal period. Environmental parameters such as temperature and nutrients
availability were measured. In Sechura, the species Pseudo-nitzschiapungens, P.seriata and Protoperidiniumdepressum, caused algal
blooms and were dominant throughout the evaluation period. The temperatures in this area ranged from 21.8 to 25. 3 °C. In Pisco
bay, the results showed that the species responsiblesof algae bloom were Akashiwosanguineum, Mesodiniumrubrun and Prorocentrum
minimum. In Pisco, the dinoflagellate Cochlodiniumpolikrykoides was dominant and associated with temperatures between 17.1 and
23.3 °C, the ranges of 1.22 -6.85 uM phosphates and 0.15 -7.85 uMnitrates inMay 2011. Moreover, in May 2012 a bloom of algae in
Pisco caused by dinoflagellateAlexandriumperuvianum with temperature ranged 18.0 to 23.2 ° C and phosphate values from 0.73 to
11, 56 mM and nitrates from 0.76 to 9.81 mM. Coliforms were low <2-23 MPN / 100 ml in both areas.During the study period,Vibrio
alginolyticus cosmopolitan species predominated in all areas evaluated. Vibrio vulnificus and V. parahaeolyticus also were registered in
Pisco, when warmer temperatures of sea are common and severe infections cases by eating seafood or fish has been associated with
a pathogen.
15 - 20 March 2015
55
POSTER 16
Tulane virus: a surrogate to study norovirus behavior in oyster?
Le Guyader, Soizick1., Drouaz, Najoua1.,Le Mennec, Cecile1.,Farkas, Tibor2.,Le Pendu, Jacques3.,1Laboratoire de Microbiologie LSEM/
SG2M IFREMER.2College of medecine University of Cincinnati.3UMR 6299 INSERM-CNRS.
Norovirus (NoV) is frequently detected in oyster worldwide and is the most frequent pathogen involved in shellfish-associated
gastroenteritis outbreaks. Since NoV cannot be propagated in cell culture, detection rely on RT-PCR, without providing information on
infectivity. To overcome this problem, different cultivable surrogates have been proposed with no real sucess as they do not replicate the
HBGA binding properties of NoVs, suggested to play an important role in their long lasting persistence in oyster. Recently, recoviruses
represented by the prototype Tulane virus (TV) have been proposed as a promising surrogate as it can be grown in cell culture and
binds to type A or B HBGAs. This work present bioaccumulation experiments using TV, Norwalk virus (NV) and mengovirus (MgV) to
compare bioaccumulation efficiency and tissue distribution after 24 h, persistence over 8 days in aquariums and then over 2 months
in natural conditions (in a controlled area using a scientific farm). Quantifications were performed by real-time RT-PCR (rRT-PCR) with
replicates. All three viruses were bioaccumulated rapidly and were detectable in the digestive tract (DT), gills and mantle after 1-hour
but with up to 2 Log differences of concentration and tissue distributions among the three viruses. At 24 hours, NV and TV had 50 to
100 fold higher copy numbers in DT than in G or M, whereas the accumulation of mengovirus was similar in all three tissues. After 8
days, the concentrations of all 3 viruses remained stable in DT but decrease in other tissues. After 2 months, concentrations, stable for
NV, decreased of about a Log for TV and MgV compared to NV. These data indicate that TV presents a bioaccumulation behavior similar
to that of NV and therefore appears to be a good surrogate for NoV even if further experiments are needed to confirm its stability over
longer periods of time.
56
POSTER 17
Norovirus persistence in oysters after accidental contamination
Parnaudeau, Sylvain1.,Le Saux, Jean-Claude1.,Schaeffer, Julien1.,Ollivier, Joanna1.,Le Guyader, Soizick1., 1Laboratoire de Microbiologie
LSEM/SG2M IFREMER.
Oysters, filtering large volume of water as part of their feeding activities, are able to accumulate and concentrate human enteric virus
such as norovirus (NoV). NoVs are the predominant agents of nonbacterial gastroenteritis in humans and thus may be frequently
detected in sewage. As no regulation has been set up yet in Europe, the French competent authority has decided to close production
area accidentally contaminated by human sewage and implicated in outbreaks. When a contamination has been proved, sampling was
performed for NoV analysis. Using an optimized version of the ISO/CEN TS15216, NoV (genogroups I and II) were searched. Beside
quality controls such as extraction efficiency and inhibitor removal, all samples were extracted 3 times in independent extraction runs
to be more confident in the quantitative approach. This approach was found valuable for naturally contaminated samples showing
variability in the contamination (Ct values may vary up to 2 units). Overall NoV contamination decreased slowly (less than a Log) during
the first month for both sites. The limit of quantification and detection was reached after about 2 2 months, depending of the initial
level of contamination. Some differences were observed depending on the site, presumably associated with tidal or current effects.
This study confirms the long persistence of NoV in oyster tissues. If we cannot exclude new contamination events during the time
period, the quantitative results showed no significant increase, suggesting thus a slow decrease due to NoV elimination presumably by
physiological activity. Further work may be warranted to calculate more accurately NoV elimination, but, once again, this demonstrates
that it will be safer to improve sanitary quality of shellfish growing area.
15 - 20 March 2015
57
Use of historical bivalve monitoring data to inform sanitary survey assessments.
Price-Hayward, M1.,1Food Safety Centre for Environment Fisheries & Aquaculture Science.
In the EU, new bivalve molluscan shellfish production areas have been subject to a requirement to undertake sanitary surveys since
2006. In Scotland, the first sanitary surveys were completed in 2007 and since then, nearly all the Scottish bivalve production areas
have been surveyed. To date, over 140 surveys have been completed, covering both aquaculture production and wild harvest sites.
An assessment of historical bivalve E. coli monitoring data was undertaken for each survey in order to determine whether there was
a relationship between spatial effects, season or other environmental factors (e.g. rainfall, temperature or tidal state), and the E. coli
results.
A review was undertaken of the outcomes of the analyses of E. coli data with respect to spatial distribution and the environmental
factors of season, and rainfall for different bivalve species. The review also considered how the outcomes of these analyses had
contributed to the overall assessments within the sanitary surveys and the consequent recommended sampling plans.
The results of this review provide important insights into some of the factors that affect monitoring results and how assessment of
those effects can be used to improve assessments in future sanitary surveys. 58
Development of a probabilistic risk assessment approach to set the baseline for the comparison of different monitoring/
management strategies for PSP contaminated shellfish THEBAULT, A1., Arnich, Nathalie1.,1Risk Assessment Department ANSES. Quantitative risk assessment for food safety can be described in three steps: exposure assessment, dose-response assessment,
and risk characterization. Exposure assessment is the product of contamination (in our case, oysters contaminated by paralytic
shellfish toxins PSPs) and consumption, giving the ingested dose. Modeling will be use to take into account variability both in oyster
contamination and in human consumption.In the chemical risk assessment approach, the ingested dose is compared to a toxicological
threshold, usually a dose without adverse health effects. In the microbial risk assessment approach, the risk of an adverse event
(percentage of affected persons) is estimated, knowing the dose ingested. We proposed to use probabilistic microbial risk assessment,
separating variability and uncertainty, and different ways to consider the dose-response relationship. Then we will be able to compare
alternative monitoring schemes looking for a more efficient allocation of resources and an improved level of public health protection.
The work presented here started in 2014 and is part of a 3-year research project on PSPs accumulation by oyster Crassostrea gigas
(ANR-13-CESA-0019-05). In this project, our task includes three main actions. The first action is to develop a mathematical model
to provide theoretical distribution of PSP contamination levels in oysters, based on contamination and decontamination kinetics, for
diploid or triploid oysters, in order to describe different situations. The second action is to perform a literature review on human
outbreaks to estimate the dose-response relationship, in particular we will consider the type of symptoms, varying from a slight
tingling sensation or numbness around the lips to fatal respiratory paralysis due to respiratory arrest few hours after ingestion. The
third action is to build the overall model of quantitative risk assessment/risk management. First field data are collected in summer
2014, then we only present preliminary results.
15 - 20 March 2015
59
A dynamic model of norovirus outbreaks accounting for inter-human transmission and consumption of contaminated oysters:
application for shellfish management in coastal areas
THEBAULT, A1., Teunis, Peter2.,Le Guyader, Soizik3.,1Risk Assessment Department ANSES.2Centre for Infectious Disease Control
National Institute of Public Health, RIVM.3Laboratoire Santé, Environnement et Microbiologie IFREMER.
Noroviruses are involved in winter gastroenteritis epidemics but also in foodborne outbreaks, associated with consumption of
contaminated oysters. The aim of this work is to better assess the relative effect of inter-human and oyster transmission in coastal
populations. Quantitative Risk Assessment was used in order to evaluate foodborne transmission. The dose-response relation was
estimated from published foodborne outbreaks, and confirms the high infectivity of those viruses. A dynamic model was built, taking
into account the two main transmission pathways. A contamination incident was simulated, after the beginning of a human epidemic,
as has been reported in actual outbreak situations. Different management options of shellfish in coastal areas were tested: not
any closure of shellfish area, or closure of different durations. First results show the effect of the foodborne pathway on the total
number of cases, in winter epidemics in the coastal population, and numbers of cases attributable to genogroups I and II. The model
demonstrates the potential importance of foodborne transmission, and the need for linking the duration of closure to the environmental
and epidemiologic conditions. The model outcomes emphasize need for increasing the safety of environmental discharge of sewage
in coastal areas. In the future this model should be fitted to observed outbreak data, for better assessing its predictive value in use for
risk management of shellfish culture in coastal areas. 60
Is tetrodotoxin a risk to consumers of European bivalve molluscs? Turner, A1., Powell, Andy1.,Baker-Austin, Craig1.,1Food Safety Cefas.
Tetrodotoxin poisoning is the most commonly-occurring lethal marine poisoning in the world, with the toxin being found in the organs
of fish from the Tetraodontidae family, including most notoriously the Puffer fish. Whilst the exact source of the toxins are unknown,
they are recognised by many authors as being produced by a range of bacteria. These subsequently accumulate through the food
chain and enter the fish as well as in gastropods, crustaceans, amphibians and octopus. Tetrodotoxins are generally associated with
marine species harvested from warm tropical/sub-tropical regions of the world. However, there has been the occasional appearance of
contaminated gastropods outside of these regions, including one episode of human sickness with products originating from European
waters. However, to date there have been no reports of tetrodotoxins found in European bivalve molluscs. This presentation will focus
on the potential risk of tetrodotoxin occurrence to the European shellfish industry. It will present some new data, highlighting that the
risk may potentially be higher than is generally expected.
15 - 20 March 2015
61
An update on the use of alternative testing methodologies for marine biotoxins in Latin American Shellfish Turner, Andrew1., Tarnovius, Sophie1.,Johnson, Sarah1.,Garcia-Mendoza, Ernesto2.,Mancera-Flores, Jennifer3.,Medina, Dinorah4.,SuarezIsla, Benjamin5.,Van Dolah, Frances6.,Broadwater, Margaret6.,Goya, Alejandra7.,1Food Safety Cefas.2Biological Oceanography
Department Centro de Investigación Científica y de Educación Superior de Ensenada.3Biological Oceanography Department Centro
de Investigación Científica y de Educación Superior de Ensenada.4Marine Biotoxins Direccion Nacional de Recursos Acucuticos
(Dinara).5Institute of Biomedical Sciences, Faculty of Medicine, University of Chile.6Marine Biotoxins Program NOAA National Ocean
Service.7Marine Biotoxin Department, Mar del Plata Regional Laboratory, Agri-food Health and Quality National Service (SENASA).
Regions throughout Latin America are under great pressures to valídate and implement marine biotoxin test methods to replace the
use of the mouse bioassay. Alternative methods implemented into official control monitoring programs globally, typically involve
the application of chemical testing methods, most notably liquid chromatography (LC) with fluorescence (FLD) and tandem mass
spectrometric (MS/MS) detection. For PSP testing, options available include two different LC-FLD methods which have been subjected
to interlaboratory validation via the AOAC. Other screening methods are also available for use as end product tests and more recently
a receptor binding assay (RBA) has also been validated formally. A project was set up by Cefas with marine biotoxin laboratories in Latin America to analyse samples from four countries: Argentina,
Chile, Uruguay and Mexico. PSP analysis was conducted using two validated LC-FLD methods, a refined immunochromatography
screen, a new LC-MS/MS method and the PSP RBA. Data enabled a comparison of method performance for species including bivalves,
gastropods and crustacean and provided valuable information regarding toxin profiles. Lipophilic toxins were analysed using LC-MS/
MS, with DSP toxins assessed using a number of rapid screening assays. An overview of the study will be presented, summarising the detection of a range of hydrophilic and lipophilic toxins as well as the
relative performance of each of the replacement methods. The applicability of each method to monitoring in Latin America will be
discussed.

abstract book

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