12th International Conference on Molluscan Shellfish...
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12th International Conference on Molluscan Shellfish Safety September 9-13, 2019 Ensenada, Baja California, México Conference Program & Book Abstracts
Transcript of 12th International Conference on Molluscan Shellfish...
12th International Conference
on Molluscan Shellfish Safety
September 9-13, 2019
Ensenada, Baja California, México
Conference Program
&
Book Abstracts
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INTRODUCTION
The ICMSS is an initiative of researchers from various international
institutions, including the Food and Agriculture Organization of the United
Nations (FAO), related to the safety of molluscan shellfish. It seeks to be a forum
for the diffusion and discussion of the advances and trends in this subject.
Renowned experts from around the world will take part in the conference.
Students, researchers, health authorities, producers of molluscan shellfish and
interested public in this subject are welcome.
Jorge Antonio Vela Díaz Executive Director of Special Programs COFEPRIS, Ministry of Health, Mexico
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TABLE OF CONTENT
INTRODUCTION _______________________________________________________ 2
CICESE MAP _________________________________________________________ 4
COMMITTEES ________________________________________________________ 5
SPONSORS AND EXHIBITORS __________________________________________ 7
CONFERENCE PROGRAM _____________________________________________ 8
SPEAKERS BIOGRAPHY ______________________________________________ 15
BOOK OF ABSTRACTS _______________________________________________ 17
KEYNOTE CONFERENCES __________________________________________ 17
ORAL PRESENTATIONS ____________________________________________ 23
POSTER SESSION _________________________________________________ 57
SOCIAL PROGRAM __________________________________________________ 79
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CICESE MAP
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INTERNATIONAL ADVISORY COMMITTEE
Iddya Karunasagar
William Burkhardt
Stephen Jones
Phillips Hess
Andrew Turner
Jesús Romalde
Enrico Buenaventura
Brian Roughan
Hongsik Yu
Mario Latini
Hajime Toyofuku
NATIONAL COMMITTEE
Erick Julián Núñez Vázquez
Bruno Gómez Gil Rodríguez
Rosalba Alonso Rodríguez
José Fernando Núñez Espinosa
José Luis Sanchez Osorio
Arturo Vargas
José Alejandro Barreiro Isabel
Ernesto García Mendoza
Aramis Olivos Ortiz
Juan Carlos Lapuente Landero
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ORGANIZING COMMITTEE
Michelle Fimbres Martínez
Ernesto García Mendoza
José Alejandro Barreiro Isabel
Aramis Olivos Ortiz
José Luis Peña Manjarrez
José Luis Sanchez Osorio
Mary Carmen Ruíz de la Torre
Alfonsina Eugenia Romo Curiel
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SPONSORS AND EXHIBITORS
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CONFERENCE PROGRAM
MON 9th
8:00 9:00
Registration
9:00 10:00
Opening Ceremony
COFEPRIS, CICESE, INAPESCA, CONAPESCA
Programs for Sanitary Control of Molluscan Shellfish
Chaired By: Jose Luis Sanchez Osorio, Ministry of Health, Baja California
10:00 10:20
Iddya Karunasagar, FAO expert, India: FAO Technical Guidance for the Development of the Growing Area Aspects of Bivalve Mollusc
Sanitation Programmes
10:20 10:40
Rachel Hartnell, Centre for Environment, Fisheries & Aquaculture Science CEFAS, UK: A new FAO Reference Centre for Bivalve Shellfish
Sanitation – Global Food In A Global World
10:40 11:00 Coffee Break
11:00 12:00
Conference Keynote:
Soraya Sandoval Riquelmes, Public Health Institute, Chile: Importance Of The Performance Evaluation Of Laboratories In The Sanitary Control
Programs For Molluscan Shellfish
12:00 12:20
Zaira Ivonne Padrón, Ministery of Agriculture of Tamaulipas: Laguna Madre’s Aquaculture Vocation for Molluscan Shellfish in Tamaulipas,
Mexico
12:20 12:40
Ana María Rivas Montaño, Instituto Tecnológico de Mazatlán: Changes in Aquatic Bacterial Communities, Case: Cyanobacteria in Caimanero
Lagoon, Sinaloa
12:40 13:00
Jorge Cáceres Martínez, CICESE, Mexico: Potential Zoonotic Parasitosis Associated With the Consumption of Bivalve Mollusks in Mexico: Case of
the Clam Chione fluctifraga
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13:00 13:20
Benjamin H Maskrey, CEFAS, UK: Seasonal Occurrence of Pharmaceuticals and Related Compounds in Bivalve Molluscs from
England and Wales
13:20 13:40
Rachel Hartnell. CEFAS, UK: Public Health England’s Shellfish Proficiency Testing Scheme – What Does the Data Tell us?
13:40 15:00
Lunch
Phytoplankton and Biotoxins (I)
Chaired By: Juan Carlos Lapuente,
Deputy Director of INAPESCA
15:00 15:20
Markus Obkircher, Merck, Switzerland: Certification of Marine Toxins by Quantitative NMR (qNMR) and Isotope Dilution MS (IDMS)
15:20 15:40
Casimiro Ramirez, INAPESCA, Mexico: Proposal to Implement a Monitoring Network of Harmful Algal Blooms
Formed by Civil Society People in BCS
15:40 16:00
Coffee Break
16:00 16:20
Lorena Durán-Riveroll, Alfred-Wegener-Institut, Germany: Current State and Perspectives of Harmful Algal Blooms in Mexican Coasts
16:20 16:40
Christine J. Band Schmidt, Centro Interdisciplinario de Ciencias Marinas, Mexico: Paralytic Toxin Producing Dinoflagellates in Latin
America: Ecology and Physiology
16:40 17:00
Jorge I. Mardones, Instituto de Fomento Pesquero, Chile: Unravelling the causes of an exceptional marine fauna-killing red tide by the
toxigenic family Kareniaceae in southern Patagonia
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TUE 10th
Pathogenic Organisms In Molluscan Shellfish Of Interest In Public Health- Virus
Chaired By: Jesus Romalde
9:30 10:30
Conference Keynote:
Hongsik Yu, National Institute of Fisheries Sciences, Republic of Korea: Korean Experience in Mitigation of Viral Contamination in Shellfish
Growing Areas
10:40 11:00
Jesus Romalde, Universidad de Santiago de Compostela, Spain: Prevalence of Sapovirus in Molluscan and Clinical Samples from
Galicia (NW Spain)
11:00 11:20
Sylvain Parnaudeau, IFREMER, France: Prevalence of Norovirus in French Oyster Production Areas and Approved Dispatch Centres
11:20 11:40
Coffee Break
11:40 12:00
Joanne Hewitt, Institute of Environmental Science and Research Limited, New Zealand: Pepper Mild Mottle Virus as an Indicator of Norovirus
Contamination on Shellfish
12:00 12:20
William Burkhardt III, US FDA: Joint US/Canadian Quantitative Risk Assessment Model Addressing the Risk Posed by NoV in
Bivalve Molluscan Shellfish
12:20 13:00
ICMSS2021 location proposal and conference photo
13:00 14:30
Lunch
14:30 16:00
Round Table Virus
(Chair: Hongsik Yu, National Institute of Fisheries Sciences)
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16:30 18:00
Poster Session
THU 12th
Pathogenic Organisms in Molluscan Shellfish of Interest
in Public Health-Bacteria
Chaired By: Arturo Vargas, COFEPRIS
9:30 10:30
Conference Keynote:
Bruno Gil Gómez, CIAD, México: Bacterial Diversity in Oysters
10:40 11:00
Christopher Schillaci, Massachusetts Division of Marine Fisheries, USA: Applying environmental surveillance and epidemiology to manage Vibrio parahaemolyticus risk associated with shellfish
consumption
11:00 11:20 Coffee Break
WED 11th
Technical and Cultural Tours
(Include visit to the growing area of Rincon de Ballenas)
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11:20 11:40
Jessica Jones, US Food and Drug Administration FDA: Management of Vibrio Risk from Oysters
12:00 12:20
Stephen Jones, University of New Hampshire US: Managing Oyster
Aquaculture Practices to Reduce Vibrio Parahaemolyticus Risks in
the Northeast United States
12:20 12:40
Cesare Ciccarelli, Asur Marche, Italy: Review of Shellfish Harvesting Area
Classification: Comparison of Different Frequencies and Minimum Required
Number of Sampling Results
12:40 13:00
Marcial Leonardo Lizarraga-Partida, CICESE Mexico: Vibrio vulnificus in Mexico
13:00 13:20
Arturo Vargas, COFEPRIS, Mexico: Prevalence of Serotypes of Salmonella spp. in Shellfish in Mexico
13:20 13:40
Hajime Toyofuku, Yamaguchi University, Japan:
Norovirus in Oyster in Japan
13:40 15:00
Lunch
15:00 16:30
Round table Vibrios (Chair: Bruno Gomez Gil, CIAD)
16:30 16:40
Coffee Break
Phytoplankton and Biotoxins (II)
Chaired By: Juan Carlos Lapuente, Deputy Director of INAPESCA
16:40 17:00
Ignacio Leyva, Centro Interdisciplinario de Ciencias Marinas, Mexico: Are Lipophilic Toxins a Contamination Risk for Wild Bivalves From
Bahía de La Paz, Baja California Sur Mexico?
17:00 17:20
Krista Thomas, National Research Council, Canada: Reference Materials for Analysis of Tetrodotoxin in Seafood
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17:20 17:40
Erick Núñez Vázquez, CIBNOR, Mexico: Profile of Paralytic Shellfish Toxins of Pyrodinium bahamense and First Detection of Tetrodotoxin
in Mexican Bivalve Mollusks
17:40 18:00
Isabelle Rajotte, National Research Council, Canada: Structure Elucidation and Relative Toxicity of (24R)-24 Hydroxyyessotoxin from
a Namibian Isolate of Gonyaulax spinifera
FRI 13th
9:30 10:30
Conference Keynote:
Andrew Turner, CEFAS UK: International Validation of the Boundy Method
for UHPLC-HILIC-MS/MS Determination of PSP Toxins and
Tetrodotoxins in Bivalve Mollusc Shellfish
10:40 11:00
Erick Núñez Vázquez, CIBNOR, Mexico: Etiology and Epidemiology of
Paralytic Shellfish Poisoning in Latin America
11:00 11:20
Allan Cembella, Alfred-Wegener-Institut, Germany: Metabolic transformation
of paralytic shellfish toxins: Consequences on Toxin Composition and
Toxicity Kinetics in Molluscan Shellfish
11:20 11:40
Ernesto García, CICESE: Marine lipophilic toxins in cultivated mussels (Mytillus galloprovincialis) from Baja California, Mexico, an eight year survey
11:40 12:00
Coffee Break
12:00 12:20
Víctor Mondragón, Agilent México: Analysis of microcystins and
nodularin in drinking water using LC/MS triple quadrupole
12:20 12:40
Christine J. Band Schmidt, Chair of the Organizing Committee:
19th International Conference on Harmful Algae, México, 2020
12:40 14:00
Round Table: Biotoxins
(Chair Andrew Turner, CEFAS)
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14:30 16:00
Lunch
Closing Session
Closing Remarks, Presentation on location of ICMSS 2021
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SPEAKERS BIOGRAPHY
ANDREW D. TURNER
As Principal Chemist in the Cefas Food Safety Group,
Andrew is responsible for the marine biotoxin testing in
molluscs performed on behalf of the UK government
competent authorities. He oversees the development and
implementation of new methods for food safety
surveillance and leads the development of research
activities of the chemistry team. He has over 20 years
postgraduate experience delivering analytical chemistry in
a commercial and government environment. Current
research interests include the development of new
instrumental methods for natural aquatic toxins including the international collaborative
validation of a PSP LC-MS/MS method, the assessment of biomolecular testing methods
and the impact of cyanobacteria on food safety. They also include the development and
production of stable toxin reference materials and risks from new and emerging shellfish
and fin fish toxins to the UK and Europe.
BRUNO GÓMEZ GIL RODRÍGUEZ SALA
Biologist with a doctorate in the University of Stirling,
Scotland. He is of the full time researcher in the biodiversity
of aquatic bacteria and bacterial diseases of aquatic
organisms. He has published more than 102 scientific
articles, several chapters in books and he has trained
undergraduate students, masters and doctoral students. He
is a member of the American Society for Microbiology, the
Association of Vibrio Biologists and the Mexican Academy
of Sciences. He is curator of the Collection of
Microorganisms of Aquatic Importance. He is responsible
for more than 11 research projects and collaborator of several more. In addition to
scientific research, it provides diagnostic and bioassay services for the aquaculture
industry and realize genomic and metagenomic analysis of microorganisms and
bioinformatics.
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HONGSIK YU
Hongsik has been engaged in food science and
microbiology research for 14 years at the university.
After joining the NIFS (National Institute of Fisheries
Science) in 2004, he has been working as a technical
expert in the KSSP (Korean Shellfish Sanitation
Program) and in the implementation of international
agreements. He has provided advice to minimize local
government’s difficulties in implementing the central
government's shellfish sanitation policy. He is pursuing
collaborative research with health authorities and the
industry to find ways to secure public health and
industry benefits. He is currently a microbiology laboratory supervisor and serves as a
coordinator for the general operation of KSSP.
SORAYA SANDOVAL RIQUELME
Graduated from the Universidad de Concepción in the
degree in Pharmaceutical Chemistry Sciences, in 1998
he applied to the Institute of Public Health of Chile for
the Sub department of the Environment, entering in the
Laboratory of Water Analysis. In this laboratory, she
training and supervising to the professionals of the
National Network of Environmental Laboratories in
water analytical and other areas. She worked in the
laboratory carrying out analyzes of marine toxins and
other foods, as HACCP Auditor and ISO 9001, and in
the coordination of the National Subcommittee on
Methods of Analysis and Sampling of the Codex Alimentarius. In 2016 she obtained a
Master's degree in for the Universidad Mayor. She is currently Head of the Sub
department of Metrology and Technological Development of the Department of
Environmental Health, of the Institute of Public Health of Chile.
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BOOK OF ABSTRACTS
KEYNOTE CONFERENCES
Bacterial diversity in oysters
Bruno Gómez-Gil
The bacterial content of cultured oysters has been done largely by culturing it in different
bacteriological media by the Total Viable Count method. This methodology only permits to
quantify the culturable fraction of the microbiota present in a sample, and not the unculturable one,
which often are the larger part of the bacteria community. Since not many years ago, the 16S
amplicon fingerprinting analysis of the metagenome in a sample has considerably expanded our
view of the unculturable microbiota. And more recently, shotgun metagenomics not only permit
us to know who is there in a sample (taxonomy profiling) but also what are they capable of doing
(functional metagenomics), or what actually are they doing (metatranscriptomics).
Traditionally, vibrios have received most of the attention done in oyster bacteriology
because their involvement in human diseases or their pathogenicity to the same oysters. This
presentation will be focused on the microbiota present in cultured oysters in North America by
means of metagenomic approaches. The following question will be tried to be addressed: Who is
inhabiting the oysters? Do oysters have different microbiota depending on the locality or date?
What is the proportion of vibrios?
Sadly, not many metagenomes, either 16S amplicons or shotgun, are available in public
repositories, and these were obtained with different parameters, 16S regions, sequencing
platforms, sources (whole oyster or gut content), that make the comparison between them almost
impossible. Even so, these questions could be answered, at least partially, and thus provide an
insight of the microbiota that inhabit the oyster and how this varies sometimes depending on the
collection date.
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Korean experience in mitigation of viral contamination in shellfish growing
areas
Hongsik Yu
National Institute of Fisheries Science. 216 Gijang-haeanro, Gijang-eup Busan 46083, Korea.
e-mail: [email protected] / [email protected]
Korea operates the Korean Shellfish Sanitation Program to ensure the food safety of shellfish
products shipped to the international market. The program is developing in harmony with
international standards, including shellfish safety regulations of the US, the EU and Japan.
The basics of the shellfish sanitation program are to monitor the concentration of fecal
coliform in the seawater or E. coli in the shellfish meat to check the sanitation status of the growing
areas and assess the suitability of the classification. The Korean authorities have also adhered to
these basic principles, overestimated the dilution effects of seawater, did not actively manage
inland and marine pollution sources, and eventually experienced the norovirus pollution crisis
since the early 2000s.
Although faced with the problem of norovirus contamination, Korean authorities and the
industry rejected the policy to eradicate the sources of pollution due to the high cost and time, the
lack of international standards, and the uncertainty of the test method.
In 2012, however, the U.S. government refused to import filthy Korean oysters, and most
countries rejected Korean shellfish products. Korean consumers avoided the consumption of
seafoods due to disappointment in the pollution of official control growing areas. Korean
authorities made a policy turn to address this issue but this process has been a shameful record in
the history of Korean Shellfish Sanitation Programs.
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The importance of the evaluation of laboratories performance in the sanitary
control programs for molluscan shellfish
Soraya Sandoval Riquelme
Chemist Pharmaceutical, Master in Health Institution Management. Head of the Designated
Metrology Laboratory. Head of the Sub-Department of Metrology and Technological
Development of the Institute of Public Health of Chile. e-mail: [email protected];
The reliability of the results released by the test laboratories that participate in the Sanitary Control
Programs is essential. The Sanitary Control Programs are intended for safety monitoring and
verification of mollusks and other analyses. The validity of the results of each laboratory, which
is shown in a test report, is very important, since it allows deciding on how to proceed in the
Sanitary Control Program. Therefore, the laboratories that participate in sanitary control programs
for shellfish, they must meet the terms with quality regulations, that ensure that they perform with
the minimum requirements to demonstrate their competence.
The standard ISO/IEC 17025:2017 “General requirements for the competence of testing
and calibration laboratories” specifies the general requirements for the competence, impartiality
and consistent operation of laboratories. According to the standard ISO/IEC 17025:2017, the test
laboratory may possibly accomplish comparisons with reference material or certified reference
material to observe its performance. In this way, the laboratory can compare its results with other
test laboratories. The activities can be done across proficiency tests or be part on a interlaboratory
comparisons.
The Ministry of Health of Chile (MINSAL) and the National Fisheries and Aquaculture
Service of Chile (SERNAPESCA) established the External Quality Assessment Program (PEEC)
at the Public Health Institute of Chile (ISP) since 1998. The PEEC is an annual proficiency testing
program focused on the support of the National Surveillance Program of Red Tide (Algal Blooms)
and the Bivalve Mollusc Sanitization Program. The PEEC performs proficiency tests in the
determination of Marine Biotoxins in Bivalve Molluscs, Metals in hydrobiological products and
Enumeration of Escherichia coli.
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Consequently, the PEEC allows to the authorities to annually evaluate the performance of
public and private laboratories, which are part of the Sanitary Control Program. The authorities
may request direct actions in the quality systems of the laboratories evaluated if there is any
unsatisfactory or questionable performance. In this manner, laboratories can improve their
comparability and reliability of their measurements, and they can avoid the risk of erroneous
results.
After years of studies, it is possible to visualize the detection of training needs in
laboratories, as well as the increase in the number of national laboratories that participate in
proficiency tests focused on safety control of hydrobiological products and water quality.
Similarly, there are improvements in the performance of public and private laboratories, which
participate in the programs.
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International validation of the Boundy method for UHPLC-HILIC-MS/MS
determination of PSP toxins and Tetrodotoxins in bivalve mollusc shellfish
Turner D.A.1, Dhanji-Rapkova M.1, Fong T.1, Hungerford J.2, McNabb P.S.3, Boundy M.J.4 and
Harwood D.T.4
1Centre for Environment, Fisheries and Aquaculture Science, Barrack Road, The Nothe,
Weymouth, Dorset, DT4 8UB, United Kingdom. 2United States Food and Drug Administration (USFDA), 22201 23rd Dr, S.E., Bothell, WA
98021, USA. 3Private Consultancy, Elms Street St, Stoke, Nelson 7011, New Zealand. 4Cawthron Institute, 98 Halifax Street East, Nelson 7010, Private Bag 2, Nelson 7042, New
Zealand.
A novel Ultra-High Performance Liquid Chromatography with Tandem Mass Spectrometry
(UHPLC-MS/MS) method for simultaneous determination of paralytic shellfish toxins (PST) and
tetrodotoxin (TTX) was developed in 2014 for the analysis of bivalve molluscs. The Boundy
method, employing a rapid, single-step dispersive extraction with carbon solid-phase extraction
clean-up was single-laboratory validated and shown to be fit-for-purpose for routine monitoring.
In order for the method to be used for regulatory control, however, the method has to be
“internationally accepted”, which required a full collaborative study. Consequently, a programme
of work was developed by Cefas, UK in partnership with the Cawthron Institute, NZ, to design
and deliver a successful collaborative study.
A pre-study was first conducted to enable the refinement of the method protocol and to
provide potential participants the opportunity to test the method and demonstrate acceptable
performance. For the main study, twenty-one laboratories situated in fourteen countries across five
continents participated, including participants from around Europe, North and South America,
Asia, Australasia and the Indian sub-continent. Fifteen different shellfish species were
incorporated into a total of 34 study materials, sourced from around the world and including
mussels, clams, oysters, cockles and scallops. Naturally-contaminated shellfish was used
containing a wide variety of toxin profiles and toxicity levels, having been associated with blooms
of Alexandrium spp., Gymnodinium catenatum and Pyrodinium bahamense. The study was
designed and implemented following AOAC International guidance.
Method trueness for total PST toxicity and individual analytes was determined for all
except three analytes, with mean values showing excellent method performance against expected
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values. No significant difference was found in the trueness results determined using two different
chromatographic column types. The within-laboratory repeatability based on the analysis of blind
duplicates and the between laboratory reproducibility was acceptable, with >99% of valid HorRat
values less than the 2.0 limit of acceptability. With excellent linearity and sensitivity fit-for-
purpose over a range of mass spectrometer instruments, the method compared well against other
internationally accepted PST-detection methods. The LC-MS/MS method includes additional PST
analogues to those incorporated into FLD methods as well as TTX, which to date has not been
incorporated into any other hydrophilic marine toxin official method of analysis.
The results from this study therefore demonstrated that the UHPLC-MS/MS method is
suitable for the analysis of PST analogues and TTX in shellfish tissues and is recommended as an
official alternative method of analysis for regulatory control.
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ORAL PRESENTATIONS
FAO technical guidance for the development of the growing area aspects of
bivalve mollusc sanitation programmes
Iddya Karunasagar1 and Garrido Gamarro Esther
Products, Trade and Marketing Branch (FIAM), Fisheries and Aquaculture Department, Food
and Agriculture Organization, Rome, Italy 1Retired
Bivalves are highly traded and some species like oysters are consumed live and raw in
many countries. Trade in live/fresh/chilled bivalve molluscs have crossed US$ 1.0 billion, but very
few countries are able to access international markets due to stringent sanitary requirements. In
this context, the FAO Committee of Fisheries Sub-Committee on Fish Trade and the Codex
Committee on Fish and Fishery products requested FAO/WHO to develop technical guidance for
implementing bivalve sanitation programme. FAO/WHO constituted an expert committee, which
developed the Technical Guidance document through consultative process. The draft guidance
document was field tested in four South African countries - Namibia, Mozambique, Angola and
Madagascar. The Guidance document has sections on growing area risk profile, growing area risk
assessment, growing area monitoring, and classification of growing areas, growing area
management and growing area review. Developing risk profile of the growing area forms the first
step followed by assessment of the area that includes data collection, shoreline survey, data
analysis, which would lead to decisions on the extent of the growing area and developing plans for
monitoring. Data from primary monitoring would lead to classification of the growing area and
the monitoring would continue to assess the status the classified areas. The section on growing
areas management includes aspects of expected and unexpected event management and
surveillance of the growing area. Guidance for reviewing the growing areas is provided in the last
section.
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A new FAO Reference Centre for bivalve shellfish sanitation – global food in a
global world
Hartnell Rachel, Barreiro Isabel Jose Alejandro, Omar Flores Yhony, Buenaventura Enrico,
Salgado Covadonga, Ellitson Paloma, Ennaffah Btissam, Goblick Gregory, Keerativiriyaporn
Suwimon, Latini Mario, McCoubrey Dorothy-Jean, Bich Nga Tran, Roughan Brian, Masataka
Satomi Claudia Rozas and Garrido Gamarro Esther.
Globally, bivalve molluscs comprise an important proportion of seafood. Increases in
production will contribute to feeding the world population as this expands however bivalves,
especially those consumed live or raw, are recognized as high-risk foods capable of concentrating
contaminants and thereby mediating infectious diseases and intoxications. Food safety
programmes directed at bivalve molluscs are intended to address these risks but have tended to be
confined to developed countries. Application of such programmes in developing countries is
necessary both to protect local consumers and to facilitate international trade, thus raising people
from food poverty and increasing economic benefits to the community or nation. However, of the
approximately 16 million tonnes of bivalves produced annually across the world only around 3%
is traded outside of the country of production. One of the reasons for this restricted trade is the
requirements for exporting nations to satisfy variable, and often very different, food safety
legislation of importing countries. In 2018 FAO and WHO, working through a group of
international experts, produced technical guidance to assist countries in developing and applying
growing area sanitation programmes to help countries step through the complexity of developing
bivalve shellfish programmes www.fao.org/3/CA1213EN/ca1213en.pdf.. The guidance, which is
based on Chapter 7 of the Codex Code of Practice for Fish and Fishery Products has been
welcomed by the Codex Committee on Food Hygiene and recently a new FAO Reference Centre
for bivalve shellfish sanitation has been established to further promote the development of bivalve
shellfish programmes globally. One of the ambitions of the Reference Centre, which is funded by
UK government, is to assist countries wishing to develop, enhance or expand their production of
safe bivalve molluscs through providing scientific technical advice and training through, in
particular, supporting the application of the FAO/WHO Technical Guidance. In this paper we
describe the development, risk-based philosophy and application of the technical guidance, and
further set out the work programme for the FAO Reference Centre to achieve its aim in assisting
nations to achieve their bivalve shellfish food security goals.
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Laguna Madre’s aquaculture vocation for molluscan shellfish in Tamaulipas,
Mexico
Padrón-Cortés, Z.I., Maya-Albarrán, E.C. and Gojon-Báez, H.H.
Secretaría de Pesca y Acuacultura. Gobierno del Estado de Tamaulipas. Ave. Hidalgo No.3309
Col. Guadalupe. C.P. 89120. Tampico, Tamps. e-mail: [email protected]
In terms of quantity, oyster production in Mexico is generated in the littoral zone of the
Gulf of Mexico, with Tabasco being the leader, followed by Veracruz, Tamaulipas and Campeche.
However, it was not until 2007 and until 2013, that Tamaulipas was the first state of this coast to
have classified areas in the southern zone, in accordance with the standards required by the
Mexican Shellfish Sanitation Program (MSSP). However, Tamaulipas has a coastal lagoon of great
importance that is the Laguna Madre, designated as a protected natural area with a management
program since 2015. Due to the extension of this lagoon and wetland of international importance,
it is considered an optimal space to convert the activities of extraction of the american oyster
(Crassostrea virginica), to an aquaculture activity, as an integral policy to confer added value to
the product not only for the care during the development of the organisms to reach ideal sizes and
weights, but for the water quality where they develop. The objective of this work is to establish a
microbiological profile of two polygons in Laguna Madre, to determine the vocation of this body
of water for the American oyster culture based on the criteria of the MSSP, as well as to establish
the bases for the program of management of the specie in this protected natural area based on the
microbiological results obtained from the tissue of the organisms, and the characterization of the
area through the evaluation of meteorological, hydrodynamic and geographic events during 12
consecutive months.
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Changes in aquatic bacterial communities, Case: Cyanobacteria in Caimanero
Lagoon, Sinaloa
Rivas Montaño Ana María1, Méndez-Gómez Evaristo1, Luis-Villaseñor I.E2., Gómez Gil-
Rodríguez Sala B.3 and Lizárraga-Partida M.L4.
1Instituto Tecnológico de Mazatlán. 2Universidad Autónoma de Sinaloa. 3Centro de Investigación en Alimentación y Desarrollo. 4Centro de Investigación Científica y de Educación Superior de Ensenada.
The eutrophication of inland water bodies, including coastal lagoons, has affected the
biodiversity and population relations in time and space, causing loss of productivity, biodiversity
and even public health problems such as poisoning and infections. The microorganisms involved
mainly belong to the Bacteria Domain (vibrios) and toxic microalgae (FAN's), this being the case
in the Caimanero Coastal Lagoon, located in the southern part of the state of Sinaloa in the Mexican
Pacific, a lagoon that has been altered by: overfishing; dredged and discharge of nutrients
discharged by aquaculture and human activities. The evaluation of the population variation of toxic
bacteria and microalgae in this lagoon, from June 2014 to February 2016, was analyzed by
metagenomics; 40 water samples, 40 zooplankton and 40 sediment. The results obtained were
compared with the database of EzBioCloud, finding that all bacterial families were present, but
95% of the records are predominantly in 14 phylums, of which: the proteobacteria that group
pathogenic bacteria and Free-living bacteria accounted for 46.6%; the cyanobacteria that showed
the risk of presence of phytotoxins represented 12.06%; Fimicutes 10.6%; Actinobacteria 8.3%;
Chloroflexi 6.9%; Bacteroides 4.7%; Acidobacteria 1.6%, Planctomycetes 1.0%, Tenericutes
1.0%. Of the 12.06% of cyanobacteria and considering only those that can produce toxins, were:
Merismopediaceae (91.92%); Pseudoanabaenaceae (3.30%); Nostocaceae (2.99%);
Microcystaceae (1.63%); Oscilatoreacea 0.13% and; 0.01% rivulareaceae. The accounting of
presence and relative abundance of cyanobacteria in the studied time, indicates the eutrophication
of the ecosystem, with a tendency towards hypertrophy and saprobity.
27
Potential zoonotic parasitosis associated with the consumption of bivalve
mollusks in Mexico: case of the clam Chione fluctifraga
Cáceres-Martínez, Jorge
Center for Scientific Research and Higher Education of Ensenada (CICESE), Carretera
Ensenada-Tijuana No. 3918, Playitas Zone, Ensenada, Baja California, Mexico, 22860.
e-mail: [email protected]
Until now, the health risk due to the consumption of bivalve mollusks has considered,
fundamentally, the bacteriological quality of the product and the presence of toxins, aspects that
cannot be ignored in terms of public health. However, there is a less known aspect, which refers
to the potential zoonotic parasitosis that can occur due to the consumption of bivalve mollusks,
especially because they are usually eaten raw or undercooked. There is a group of parasitic worms,
belonging to the Echinostomatidae family of the Trematoda class, whose larval stages can be found
parasitizing bivalve mollusks and which can potentially be transmitted to birds and mammals,
including humans, as final hosts. In a study on the parasitic load of the clam Chione fluctifraga,
we detected different parasites, such as the copepod Pseudomyicola spinosus, trematodes and
turbelaria in the lumen of the digestive tract, renal coccidia, trichodine-like ciliates, inclusions of
Rickettsiales-like bacteria, crystal-like formations in the foot muscle and abundant cysts of
trematodes, belonging to the Echinostomatidae family probably of the genus Echinostoma. The
latter represent a potential zoonotic risk, if it is confirmed that their identity corresponds to a
species that can use man as the final host. It is necessary to point out that very little is known of
the "natural" parasitic load of the bivalve mollusk species of commercial importance in Mexico
and, therefore, of the zoonotic risk they represent; In this regard, it is necessary to promote research
in this topic and consider, where appropriate, the relevant health surveillance measures.
28
Seasonal occurrence of pharmaceuticals and related compounds in bivalve
molluscs from England and Wales
Maskrey Benjamin H., Dean Karl, Younger Andrew, Katsiadaki Ioanna
and Turner Andrew D.
Food Safety, Centre for Environment, Fisheries and Aquaculture Science (Cefas), Barrack Rd,
Weymouth, UK DT4 8UB.
The presence of pharmaceutically active compounds in the aquatic environment is well
known, with these compounds able to enter via a number of different sources such as wastewater
treatment plant discharges and sewage outfalls. Due to their presence in the water column, these
compounds have the potential to be bioaccumulated by filter-feeding organisms such as bivalve
molluscs, and as such may pose a threat to the bivalve mollusc consumer. Whilst there is a growing
body of evidence of the presence of these compounds in the environment, data on levels and
profiles in aquatic organisms such as bivalve molluscs is limited. In this study, mussel (Mytilus
edulis) and native oyster (Crassostrea edulis) samples from six monitoring points in England and
Wales were analysed at four time points over a year by UHPLC-MS/MS for a range of
pharmaceuticals and related compounds utilising a stable isotope dilution approach. The results
unequivocally demonstrated the presence of a range of these compounds in these samples with
antidepressants, particularly sertraline, begin present at the highest concentrations. Data will be
presented showing seasonal and site-specific fluxes in the concentrations of these compounds. The
risks in terms of food safety to the consumer, and their potential usage as indicators of water quality
and source tracking are discussed.
29
Public Health England’s shellfish proficiency testing scheme – what do the
data tell us?
Nita Patel, Louise Stockley and Rachel Hartnell
Centre for Environment, Fisheries and Aquaculture Science (Cefas) in collaboration with
the Food and Environmental Proficiency Testing Unit (FEPTU) provides international
microbiology proficiency testing (PT) schemes to laboratories that analyse food, water and
environmental samples. The Shellfish Scheme has been in operation for over twenty years
providing external quality assessment samples for laboratories that examine live bivalve mollusc
flesh (and intervalvular fluid) for the enumeration of Escherichia coli. This scheme is based upon
the use of ISO 16649-3, Microbiology of food and animal feeding stuffs - Horizontal method for
the enumeration of β-glucuronidase-positive E. coli Part 3: Most probable number technique using
5-bromo-4-chloro-3-indolyl-β-D-glucuronide. Although may also be appropriate for alternative
methods used in laboratories for the enumeration of E. coli in bivalve molluscan shellfish flesh.
ISO 16649-3 is the EU reference method stipulated in EU Regulations for enumeration of E. coli
in live bivalve molluscs for classification, monitoring and placing products on the market. Official
Laboratories in the EU and third countries exporting bivalve molluscs to the EU are required to
utilise ISO 16649-3 (or validated alternative) in their Official Control programmes. Regular
participation in PT provides performance data that help to demonstrate compliance with testing
standards and integrity of test results, thereby helping to meet and maintain accreditation
requirements. Again, for EU, and some other global markets, accreditation of test methods to ISO
17025, or equivalent international standards, is an expected legal requirement. Most accreditation
bodies accrediting laboratories to ISO 17025 require evidence of satisfactory performance in PT,
the Shellfish Scheme helps provide laboratories that examine bivalve molluscs this evidence.
Furthermore, PT can assist in trend analysis, performance characterisation of methods, method
implementation, ongoing staff competency and training. Taken together PT supports laboratories
meet internal quality management systems and to demonstrate to Responsible Authorities that test
results are reliable. This in turn helps to ensure that consumer protection from food safety risks
and facilitates trade. In this paper we report on the evolution of the Shellfish Scheme over the last
twenty years, the development of laboratory assessment and trend analysis, focusing on a network
30
of EU laboratories and discuss some of the challenges and benefits inherent in participation in PT
from both the scheme organiser and participant perspectives.
31
Certification of marine toxins by quantitative NMR (qNMR) and Isotope
Dilution MS (IDMS)
Obkircher Markus*, Rueck Alexander, Hellriegel Christine, Koehling Rudolf
*Director, Head of Reference Materials R&D. Industriestrasse 25, 9471 Buchs, Switzerland,
Merck KGaA. e-mail: [email protected]
Since the presence of marine toxins in shell fish and sea food is an emerging worldwide
problem, fast and sensitive LC-MS methods were established for food safety testing.[1] Therefore,
the access to well characterized reference materials for a precise and accurate quantitation of these
different toxins has become an increased need in the market. These reference materials should be
characterized and prepared according to ISO/IEC 17025 and ISO 17034. In order to achieve
certification of such small batches according to this double accreditation at the highest
metrological level, a combined setup of quantitative NMR (qNMR)[2],[3] and Isotope Dilution MS
(IDMS)[4],[5] was successfully established[6]. In a first step, the accurate concentration of a
dissolved toxin is determined by a series of 1H-qNMR measurements. Gravimetric dilution and
ampule filling deliver the final product with a certified concentration and an associated expanded
uncertainty, which can be subsequently applied in an HPLC-IDMS experiment that results in a
concentration for the stable isotope labeled analog. Gravimetric IDMS experiments are also carried
out to determine the homogeneity and stability contribution to the overall uncertainty. These
concepts were successfully adopted for the certification of multiple toxins despite their partial
instability and tendency to undergo rearrangement reactions. Several paralytic shellfish toxins
(PST) were developed, for example the well-known Neosaxitoxin or Saxitoxin and their stabile
isotope labeled analogs 15N7-Neosaxitoxin and 15N7-Saxitoxin. In addition, other toxins like
Okadaic acid, PTX11, GTX-6, Gymnodimine, Pinnatoxin E, F and F as well as several Brevetoxins
could be made available to testing laboratories.
References
[1] M. J. Boundy et al, Journal of Chromatography A, 1387, 1-12, 2015.
[2] M. Weber, Ch. Hellriegel, A. Rueck, R. Sauermoser, J. Wuethrich, Accreditation and Quality Assurance,
18(2), 91- 98, 2013.
[3] M. Weber, Ch. Hellriegel, A. Rueck, J. Wuethrich, P. Jenks, Journal of Pharmaceutical and Biomedical
Analysis, 93, 102-110, 2014.
[4] A. Breidbach, ThOS36-02, IMSC 2104, Geneva, CH.
[5] M. Sargent, Guidelines for Achieving High Accuracy in IDMS, RSC (LGC), Cambridge 2002.
[6] R. Koehling, E. Allenspach, Ch. Hellriegel, A. Rueck, J. Boertz, F. Wahl, M. Weber, M. Obkircher, Poster
DGMS, 2016.
32
Proposal to implement a monitoring network of Harmful Algal Blooms
formed by civil society people in BCS
Ramírez-Camarena Casimiro1, Jiménez-Quiroz María del Carmen1, Barón-Campis Sofía1,
Garate-Lizárraga Ismael2, Núñez-Vázquez Erick3 and Vázquez-Gómez Norberto1
1 Instituto Nacional de Pesca y Acuacultura. Ave. México No. 190. Col. Del Carmen, Deleg.
Coyoacán, CP 04100, CDMX. e-mail:[email protected] 2 Centro Interdisciplinario de Ciencias Marinas. Av. Instituto Politécnico Nacional s/n Col. Playa
Palo de Santa Rita. Apdo. Post. 592. CP 23096. La Paz, BCS, México. 3 Centro de Investigaciones Biológicas del Noroeste. Av. Instituto Politécnico Nacional 195 Col.
Playa Palo de Santa Rita. CP 23096. La Paz, BCS, México.
The frequency of Harmful Algal Blooms (HAB) on the western coast of Baja California
Sur (BCS) has apparently increased in recent years, causing significant damage to wild populations
of important fishery resources (eg, abalone, lobsters and fishes); however, there is not enough
information in this regard, particularly from regions far from the most important urban centers of
BCS One strategy for gathering information is to organize an observer network made up of
fishermen, students and fish farmers to report the occurrence of these events and if possible, to
collect water samples. The samples will be processed by researchers from INAPESCA and other
regional research institutions. With this objective, the first steps have been taken for the formation
of the network by giving talks to members of Cooperative Societies, collaborating with personnel
of companies dedicated to aquaculture and through a campaign through social networks. The first
event that is followed up in this way is the FAN occurred in Magdalena Bay in June 2019, made
up of species with harmful potential (mainly Coscinodiscus cf wailesii), whose information was
provided by members of an aquaculture company installed in this lagoon.
33
Current state and perspectives of harmful algal blooms in Mexican coasts
Durán-Riveroll, L.M.1,2, Band-Schmidt, C.J.3, Okolodkov, Y.B.4, Almazán-Becerril, A.5
1CONACYT-Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de
México, Circuito exterior s/n, Ciudad Universitaria, 04510 Coyoacán, Mexico.
e-mail: [email protected] 2Alfred Wegener Institute, Am Handelshafen 12, 27570, Bremerhaven Germany.
e-mail:[email protected] 3Instituto Politécnico Nacional-Centro Interdisciplinario de Ciencias Marinas, Av. Instituto
Politécnico Nacional s/n, Playa Palo de Santa Rita, 23096 La Paz, B.C.S., Mexico.
e-mail:[email protected] 4Instituto de Ciencias Marinas y Pesquerías, Universidad Veracruzana, Calle Mar Mediterráneo
314, Costa Verde, 9429 Boca del Río, Veracruz, Mexico. e-mail: [email protected] 5Centro de Investigación Científica de Yucatán, A.C. Unidad de Ciencias del Agua, (UCIA),
Calle 8, No. 39, Mz. 29, S. M. 64, Cancún C. P. 77524, Mexico. e-mail: [email protected]
Harmful algal blooms (HABs) are becoming more extensive geographically and in extent
of time, and the causes are not yet fully understood. It has been suggested that apart from natural
causes, there is a relationship with global climate change and anthropogenic pollution in coastal
waters, but the impact of each factor has not been established. A raise in global atmospheric
temperature, with the consequential increase in ocean temperatures has been pointed out as one of
the main reasons for HAB expansion; yet, many coastal changes generated by human activities,
such as land alteration, nutrient enrichment due to agriculture, industry and urban waste also show
a relationship with this expansion. In Mexico, HAB studies are still scarce, including their causes
and consequences, and it is imperative to reinforce research and monitoring strategies since their
impacts are not only reflected in ecosystem damage but also in socio-economic losses in tourism,
public health and monitoring costs. Nevertheless, we have now information about several toxin-
producing species in our coasts, information on recent events, and their plausible relationship with
hydrological conditions and coastal pollution. To better understand this phenomenon and to be
able to act accordingly, inter-institutional and multidisciplinary collaborative work among
researchers, environmental and health authorities, and general population is urgent. To contribute
with this knowledge, we prepared a review of the research held in Mexico on harmful algal blooms
and their probable relationship with coastal pollution and climate change.
34
Paralytic Toxin Producing Dinoflagellates in Latin America:
Ecology and Physiology
Band-Schmidt C.J.1, Durán-Riveroll L.M.2, Bustillos-Guzmán J.J.3, Leyva-Valencia I.4, López-
Cortés D.J.3, Núñez-Vázquez E.J.3, Hernández-Sandoval F.E.3 and Ramírez-Rodríguez D.V.1
1Instituto Politécnico Nacional-Centro Interdisciplinario de Ciencias Marinas, La Paz, B.C.S.,
México. e-mail:[email protected] 2CONACYT, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de
México, Ciudad de México, México. 3Centro de Investigaciones Biológicas del Noroeste, La Paz, B.C.S., México. 4CONACYT-Instituto Politécnico Nacional, Centro Interdisciplinario de Ciencias Marinas, La
Paz, B.C.S., México.
In this review we summarize the current state of knowledge regarding taxonomy, bloom
dynamics, toxicity, autoecology, and trophic interactions, of saxitoxin producing dinoflagellates
in Latin America. The dinoflagellates Gymnodinium catenatum, Pyrodinium bahamense and
several species of Alexandrium are saxitoxin producers, and have been responsible of paralytic
shellfish poisoning in different regions of Latin America, causing intoxications and important
fisheries losses. The species distribution differ; most harmful algal blooms of G. catenatum are
from the northern region, however this species has also been reported in central and southern
regions. Blooms of P. bahamense are mostly reported in North and Central America, while blooms
of Alexandrium species are more common in South America, however this genus is widely spread
in Latin America. Species and regional differences are compared, with the aim to contribute to
future guidelines for an international scientific approach for research and monitoring activities.
Activities that are needed to increase our understanding of paralytic toxin producing
dinoflagellates in this region.
35
Unravelling the causes of an exceptional marine fauna-killing red tide by the
toxigenic family Kareniaceae in southern Patagonia
Mardones Jorge I.
Centro de Estudio de Algas Nocivas (CREAN), Departamento de Medio Ambiente, Instituto de
Fomento Pesquero (IFOP). e-mail:[email protected]
After 20 years, a large dinoflagellate bloom in the southern Chilean coast in summer 2018
impacted salmon farms and wild fauna resulting in the dead of several million animals. Satellite-
derived images of Chl-a (MODIS Aqua) showed bloom initiation in January and dispersal further
to the north in February-March 2018. The bloom was dominated by dinoflagellates of the toxigenic
family Kareniaceae identified by light microscopy. Established cell cultures from the outbreak
allowed further phylogenetic identification (large subunit rDNA) of the toxic dinoflagellate
Karenia selliformis among several other species of Kareniaceae. In vitro analysis of K. selliformis
isolates reveled: (1) high growth rates (max. 1.44 cells d-1), (2) no production of Gymnodimine
toxins, (3) high content of polyunsaturated fatty acids (PUFAs), (4) high production of reactive
oxygen species (ROS) and (5) high cytotoxicity against the RTW1-gill cell line. Hydrodynamic
models (ROMs) showed a significant link between coastal cell aggregations and oceanic fronts
formation. This study represents the first deep insight on the ecology and physiology of the genus
Karenia in the western Patagonian coast.
36
Prevalence of Sapovirus in molluscan and clinical samples from
Galicia (Nw Spain)
Romalde Jesus L. and Varela Miguel F.
Departamento de Microbiologia y Parasitologia, CIBUS-Facultad de Biologia, Universidade de
Santiago de Compostela, 15782, Santiago de Compostela, Spain. e-mail: [email protected]
Human sapovirus (SaV) is considered an emerging enteric virus that causes acute
gastroenteritis worldwide. The aim of the present work was to determine the prevalence and
genetic diversity of SaV in Galicia, Spain. For this purpose, samples of bivalve molluscs from
harvesting areas in the Galician estuaries, as well as clinical samples of feces of patients affected
by acute gastroenteritis in the metropolitan area of A Coruña were collected, analyzed by RT-
qPCR and genotyped. The obtained results showed a significant prevalence of human sapoviruses
in molluscs from Galicia, without any seasonal pattern. The presence of the virus was positive in
60 of the 248 samples of molluscs analyzed (24.2%). Ría de Vigo and Ría do Burgo, under urban
influence, showed higher prevalences (21.8% and 37.5%, respectively) than the Ría de Ares-
Betanzos (14.4%), which is surrounded by rural areas. The incidence of SaV in patients with acute
gastroenteritis (n=2,667) was 15.64 %, being the positive samples detected along the year, with
peaks during autumn and late winter–early spring. The most common genotypes in molluscan
samples were GI.1, GI.2 and GI.3, although sequences belonging to the GII, GIV and GV were
also detected. In clinical samples, the dominant genotypes were GI.1 and GI.2, which coincide
with the results of the bivalve samples. The results obtained indicate a significant presence of this
human pathogen in the Galician population and also in molluscs, and evidence the possible role of
shellfish in SaV transmission for some human age-groups.
37
Prevalence of norovirus in French oyster production areas and approved
dispatch centres
Ollivier J., Parnaudeau S., Wacrenier C., Garry P., and Le Guyader F.S.
Ifremer, Laboratoire de Microbiologie, LSEM/SG2M, Nantes, France.
The coastal shellfish production areas are subjected to increasing human faecal pollution
from different sources (collective and non-collective sanitation, human activities, the agri-food
industry, etc.). During filter-feeding, shellfish can retain and concentrate some microorganisms,
specially norovirus. Oysters contaminated with norovirus pose a particular risk to human health
since they are usually consumed raw. In 2016, DG Santé mandated EFSA to perform an EU
baseline survey to assess the norovirus prevalence in classified production areas (PA) and in oyster
batches in dispatch centres (DC). Present study shows the French results of the project. In France,
74 PA and 167 DC were randomly selected. Samples were collected every two months over two
years and analysed using the ISO 15216-1:2017. A total of 850 samples from PA and 1,793
samples from DC showed valid results. Norovirus were detected in 7% of total samples: 10% from
PA (102 samples) and 6% from DC (112). Twenty-six PA were negatives for norovirus over the
study: 15 of these PA were classified A and 8 classified B and 3 PA changed sanitary status during
the study (2 PA from class A to B and 1 PA from class B to A). In the class A areas, contamination
was detected once in 7 PA, or twice in 2 PA. The norovirus contamination was detected more
frequently in class B PA. Norovirus positive samples were found one time out of 12 samples taken
for 9 PA, twice for 9 PA, 3 times for 9 PA, four times for 2 zones and five times for 3 PA. The
contamination level was low with concentrations of norovirus (genogroupes I + II) lower than 100
genome copies per gram of digestive tissues for 144 out of 214 positive samples (67%). The
contamination occurred during winter months (January – March) with winter 2017 marked with a
higher detection of norovirus GI strains.
38
Pepper mild mottle virus as an indicator of norovirus contamination in
shellfish
Hewitt Joanne and Gyawali Pradip
Institute of Environmental Science and Research Limited (ESR). PO Box 50348, Porirua 5240,
New Zealand. e-mail: [email protected]
Shellfish grown in areas impacted by wastewater/human faecal pollution risk norovirus
contamination. Pepper mild mottle virus (PMMoV) has been proposed as an indicator of human
faecal contamination due to their high concentration in wastewater. However, data are lacking on
suitability of PMMoVs use for shellfish safety and its correlation with norovirus. RT-qPCR was
used to determine the presence and concentration of PMMoV and norovirus (genogroups I and II)
in 73 shellfish samples collected from non-commercial sites around New Zealand. Sample
collection sites ranged from those known to be impacted by human wastewater effluent to areas
with a low anthropogenic impact. The overall occurrence of PMMoV and norovirus in shellfish
was 65/73 (89.0%) and 33/73 (45.2%) respectively. Eight (10.9%) samples were negative for both
norovirus and PMMoV, and 33 (45.2%) samples were positive for both PMMoV and norovirus.
Thirty two (43.8%) samples were positive for PMMoV but norovirus-negative. All norovirus-
positive samples (n=33) were positive for PMMoV. For those samples with quantifiable levels,
PMMoV concentrations ranged from 2.8-5.3 log10 genome copies (GC)/g digestive tissue.
Norovirus (total GI and GII) concentrations ranged from 2.1-4.5 log10 GC/g digestive tissue. While
always detected in norovirus-contaminated shellfish, PMMoV was also frequently detected in
shellfish collected from areas with low anthropogenic impact (and where no norovirus was
detected). The source of PMMoV in norovirus-negative samples and suitability for monitoring
shellfish quality should be further examined.
39
Joint US/Canadian quantitative risk assessment model addressing the risk
posed by NoV in bivalve molluscan shellfish
Burkhardt William
Representing experts the U.S. Food and Drug Administration (FDA), the Canadian Food
Inspection Agency, and Environment and Climate Change Canada.
The model uses Monte-Carlo simulation to predict the concentration of infectious and non-
infectious norovirus (Genotype I and II), and male specific coliphage (MSC) along the oyster
production pathway, when this pathway is impacted by a source of human waste. This includes
raw sewage entering a wastewater treatment plant (WWTP), the WWTP output, the harvest water,
and the shellfish at harvest, post-harvest processing, and consumption (exposure assessment). A
dose-response model (risk characterization) is then applied to estimate the average risk of
norovirus infection and illness following the ingestion of an oyster meal. The model is dynamic,
which means that it is able to capture the impact on contamination and resulting risk to humans
over time. Specifically, this allows the model to represent short-term events (i.e., hours to days)
when higher amounts of human waste contamination enter an estuary upstream or directly in the
shellfish harvest area. Examples of these types of events could include WWTP malfunction, heavy
rainfall resulting in sewage overflows, or a point discharge from a marine vessel in contravention
of legal prohibitions. This presentation will provide an overview of the risk assessment model and
provide some real-world examples that illustrate its practical use.
40
Applying environmental surveillance and epidemiology to manage Vibrio
parahaemolyticus risks associated with shellfish consumption
Schillaci Christopher A.*, Jones Stephen, Whistler Cheryl, Regan Diane
University of New Hampshire, Department of Natural Resources and the Environment, 46
College Rd Durham NH and Massachusetts Division of Marine Fisheries, 251 Causeway St
Boston, MA. e-mail: [email protected]
Vp. is a human pathogenic bacterium that is naturally occurring in coastal waters
worldwide. As a result of the potential for oysters to accumulate significant levels of Vp. through
filter feeding, and consumer trends favoring raw consumption, they are more often associated with
exposure in human Vp. infections than any other seafood product. Despite the implementation of
control measures and illness reporting and response protocols, the number of shellfish related Vp.
cases in the U.S. has not significantly decreased in recent years. The complexity of Vp. illness
source attribution can result in significant challenges when evaluating how the actual risk per
serving in specific areas are changing concurrent with increases in reported illness. These reporting
challenges also impact the ability for managers to apply illness data to the development and
refinement of Vp. control measures and implement harvest area closures in a manner that results
in meaningful consumer protection. We evaluated the potential to use routine surveillance for total
and pathogenic Vp. in oyster samples and epidemiological illness data, in combination with multi-
parameter water quality monitoring, to inform and refine Vp. management strategies in
Massachusetts. We concluded that the most commonly employed genetic markers for total (tlh)
and pathogenic Vp. (trh & tdh) more often than not did not correlate strongly with illness
occurrence and that site to site variability of the environmental conditions associated with historic
illness occurrence and elevated total (tlh) and pathogenic Vp. (trh & tdh) requires a localized
approach to develop accurate risk assessment methodology.
41
Management of Vibrio risk from oysters
Jones, J.L.1*, Miller, J.J.2, Pruente, V.1,3, McGough, M.1,4, Neil, W.A.1, and Walton, W.C.3
1 FDA, Gulf Coast Seafood Laboratory, Dauphin Island, AL USA. 2 FDA, Office of Analytics and Outreach, College Park, MD USA. 3 Auburn University Shellfish Laboratory, Dauphin Island AL USA, 4ORISE, Oak Ridge, TN
USA.
Vibrio parahaemolyticus and Vibrio vulnificus are the leading causes of seafood-associated
morbidity and mortality, respectively, in the United States. Infections most commonly result from
the consumption of raw molluscan shellfish. Risk management of vibrio in shellfish has a multi-
directional approach: understanding the ecology of the organisms in the estuarine environment,
understanding the relative virulence of different strains, and understanding how harvest and
handling practices in the shellfish industry affect the vibrio levels in shellfish. The latter two topics,
strain virulence and shellfish handling practices, will be the focus of this presentation. The
geographical specificity and clinical predominance of certain V. parahaemolyticus strains, as
identified by whole genome sequencing, will be discussed, along with the distribution of potential
virulence markers across clinical and shellfish isolates. Additionally, an overview of recent studies
on the effects of handling practices on V. parahaemolyticus and V. vulnificus levels in cultured
oysters will be presented. The talk will be concluded with a look forward to ongoing and upcoming
research and discussion on how data from multiple areas of study can contribute to more accurate
risk modeling.
42
Managing oyster aquaculture practices to reduce Vibrio parahaemolyticus
risks in the Northeast United States
Jones, Schillaci, Nash, Howell, DeRosia-Banick, Foxall, Whistler
Oyster culture and harvest practices involve handling shellfish out of the water for varying
time periods to reduce fouling and to harvest, cull or grade shellfish. During warm months, these
practices can increase levels of pathogenic Vibrio parahaemolyticus (Vp) populations in oysters
and potentially increase the risk of illness for consumers. A more thorough understanding is needed
of emerging aquaculture practices and appropriate management actions to reduce public health
risks. Field-testing of Vp control measures for pre- and post-harvest husbandry practices have been
conducted at harvest sites in Maine, New Hampshire, Massachusetts and Connecticut where
aquaculture practices, regulations, environmental and seasonal climate conditions, and Vp
populations all vary. Newly developed detection methods for regionally significant pathogenic Vp
were used for evaluating Vp risk reduction. The study provides scientifically sound findings for
each state and the region to use in managing the Vp risk in live shellfish to benefit the shellfish
industry and consumer safety.
43
Review of shellfish harvesting area classification: comparison of different frequencies
and minimum required number of sampling results
Ciccarelli Cesare1*, Semeraro A. Marisa
1, Di Trani Vittoria
1, Leinoudi Melina
2, Ciccarelli Elena
3
1 ASUR MARCHE Area Vasta 5 – San Benedetto del Tronto (Italy)
2 General Chemical State Laboratory – Thessaloniki (Greece) 3 Biologist – Bracknell (UK)
e-mail: [email protected]
Microbial quality of shellfish harvesting areas is assessed by enumeration of the faecal
indicator Escherichia coli because its presence informs of the possible presence of faecal micro-
organism potentially pathogen for humans. In the European Union (EU) the criteria for laying
down the microbiological standard for shellfish harvesting areas are ruled in the Regulation (EC)
854/2004. The reviewing criteria of classified production areas, such as frequency of review and
minimum sampling results, are defined by Competent Authorities of the Member States. Usually
a review lasts three years with required minimum number of 24 sampling results. However these
conditions aren’t always met and it adds another factor of uncertainty. This paper aims to determine
how frequency of review and minimum number of results influence the precision and the
predictable value of classification of harvesting areas. Based on the assumption that the sanitary
classification is a prediction of microbial quality of shellfish harvested in that area, this study
evaluates the degree of belief in that proposition implementing the Brier score, a statistical tool
used to measure the accuracy of a probabilistic prediction. Employing monitoring data of Class A
classified areas with different faecal contamination trends and assuming different frequencies of
classification’s review (three years, one year or sliding window) and different number of results
(24 and 36), we determine which condition is the most reliable. Our findings suggest that, at the
studied faecal contamination trends, annual or sliding window reviews, with 24 or 36 results, show
better performances than three-year reviews.
44
Vibrio vulnificus in México
Lizárraga-Partida Marcial Leonardo
Marine Biotechnology. Center for Scientific Research and Higher Education of Ensenada
(CICESE), Carretera Ensenada-Tijuana No. 3918, Playitas Zone, Ensenada, Baja California,
Mexico, 22860.
Vibrio vulnificus (Vv), is a species that show low tolerance to salinity, a characteristic that
could explain the lack of their detection in oysters farms along the Baja California Peninsula, where
oysters are cultivated in coastal lagoons with high salinity, contrary to the Gulf of Mexico, where
coastal lagoons present low salinity, specially in the rainy season. Vv were constantly isolated from
oysters in provenance from different coastal lagoons of the Gulf of Mexico, selling at the main
seafood market of Mexico City. The species was characterized by PCR with primers directed to
the vvhA toxigenic factor and their clinic or environmental genotype was also identified by PCR.
Since toxicity in Vv seems to be multifaceted and poorly understood, all the Vv positive to vvhA
are considered potential pathogens. Nevertheless, the detection of V. vulnificus by vvhA in
environmental samples, do not necessary correspond with the number of clinical cases registered
in the same area, indicating differences in the pathogenicity of Vv strains. In the CICESE Vv
strain coleccion, we have registered by Pulse Field Gel Electrophoresis, (PFGE), clusters
exclusively presented strains of the clinical (C-vcg) or the environmental (E-vcg) genotype, even
if all of the strains were isolated from the oyster samples. Same results were registered with the
multilocus sequence typing (MLST) and the rtxA analysis, indicating a clear difference between
both genotypes. Selected strains with C-vcg genotype from CICESE collection were used in a
comparative whole genome analysis with reference sequences of C-vcg genotype Vv CMCP6 and
YJ016 strains, reported as acute human pathogens. Results indicate a close genetic similarity
between CICESE and reference strains. We conclude that the PCR to detect the C-vcg genotype
could be used as an indication of the acute pathogenic species of Vv in sanitary studies for
certification of marine organisms, specially molluscs.
45
Prevalence of serotypes of Salmonella spp in shellfish in Mexico
Vargas Arturo, de León Becerril Olivia, Dehmer Mariel Carlos
COFEPRIS/CCAYAC, Comisión de Control Analítico y Ampliación de Cobertura. Dirección
Ejecutiva de Control Analítico. Gerencia de Análisis y Desarrollo de Pruebas Microbiológicas.
Some foodborne diseases such as gastroenteritis caused by Salmonella spp. can be
attributed to the consumption of raw shellfish. To know which serotypes are the most common in
Mexico in this kind of foods, we present a retrospective regarding the common serotypes of this
pathogen in shellfish. The method used was the ISO/TR 6579-3 2014 Guidelines for serotyping of
Salmonella spp. this method use slide agglutination using specific antisera to determine the
somatic factors O and flagellar H to obtain the antigenic formulas and thus determine the serotype
according to the Kauffmann & White scheme. The serological method has been considered for
years as the “gold standard” to determine the serotype of Salmonella spp. although at present many
molecular methods have been developed such as Ribotyping, PFGE, MLST and DNA sequencing:
The serological method is the one that defines the result in case of some discrepancy between the
molecular methods. Given the sanitary importance of the presence of Salmonella spp. in shellfish,
the study of the most common serotypes found in this product is fundamental for risk management
programs, where both serotyping and antimicrobial resistance should be included, as an essential
part of the surveillance programs of Salmonella spp.
46
Norovirus in oyster in Japan
Toyofuku Hajime
Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1, Yoshida, Yamaguchi,
Japan. e-mail: [email protected]
Norovirus is the largest common foodborne diseases in Japan and raw shellfish is
considered as a source of foodborne norovirus infection. To investigate this hypothesis, Food
Safety Commission of Japan compiled a risk profile. Ministry of Agriculture, Fisheries and
Forestry (MAFF) carried out several studies to identify risk management options in Japan,
including the following research in harvested raw oyster: (1) September 2013 to October 2014,
oyster samples from 2 harvest area were tested, and GI genes were detected from 88 samples
(18%), GII gene were detected from 169 samples（35%). (2) From January to March 2015, 89
oyster samples were tested and GI was not detected, but GII were detected from 77 samples (87%).
87 % of detected GII were GII.17and 70% were GII 4. (3) From October 2015 to January 2016,
norovirus copes per gram were estimated from 350 samples (one sample consists from 3 midgut
glands) from 6 harvesting area. Norovirus RNA copies were detected from 83 samples (24%).
Estimated average number of copies varied from 298 to 6618 copies /g. (4) From October 2016 to
February 2017, norovirus copes per gram were estimated from 1092 samples (one sample consists
from one midgut glands) from 6 harvesting area. Norovirus DNA copies were detected from 116
samples (11%). Estimated average number of copies varied from 1235 to 10622 copies / g. In
addition, some oyster harvesting prefectures performed monitoring of norovirus in harvested
oyster. These outcomes will be discussed.
47
Are lipophilic toxins a contamination risk for wild bivalves from Bahía de La
Paz, Baja California Sur Mexico?
Leyva-Valencia Ignacio1*, Hernández-Castro Jesús Ernestina2, Band-Schmidt Christine J.2,
Turner Andrew D.3, O’Neill Alison3, Núñez-Vázquez Erick J.4, López-Cortés David J.4†,
Bustillos-Guzmán José J.4 and Hernández-Sandoval Francisco E.4
1CONACYT- Instituto Politécnico Nacional, Centro Interdisciplinario de Ciencias Marinas, La
Paz, B.C.S., México. e-mail: [email protected] 2Instituto Politécnico Nacional, Centro Interdisciplinario de Ciencias Marinas, La Paz, B.C.S.,
México. 3The Centre for Environment, Fisheries and Aquaculture Science, Weymouth, Dorset, DT4
8UB, United Kingdom. 4Centro de Investigaciones Biológicas del Noroeste, La Paz, B.C.S., México.
In Mexico only okadaic acid (OA) is included in sanitary regulation NOM-242-SSA1-
2009, while YTX and AZA were incorporated in Technical Guide of the Mexican Program of
bivalves molluscs and there is scarce information about cyclic imines (Cls), in our country. Most
of the shellfish fisheries of Mexico occur in the Gulf of California, a region known for its high
primary productivity, that includes dinoflagellates producers of OA, pectentoxins (PTXs),
yessotoxins (YTXs), azaspiracids (AZAs) and Cls. During 25 months from January 2015 to
November 2017, samples of three species of wild bivalves were collected in four sites from
southern of Bahía de La Paz. Pooled tissue extracts were analyzed using LC-MS/MS to detect
lipophilic toxins. Eight groups of toxins were identified (OA, DTX, PTX, YTX, AZA, CIs); these
were clearly lower than the maximum level permitted for human consumption. Interspecific
differences in toxin analogs and their concentrations were observed, even when samples were
collected in the same site. OA was detected in low concentrations, while YTXs and gymnodimines
(GYMs) had the highest concentrations in bivalves. Our results showed that diarrheic toxins were
clearly below the allowed limit according with mexican legislation. The presence of other
lipophilic toxins, mainly YTXs and Cls (GYMs, pinnatoxins and spirolides) suggested a
combinated effect with the OA, however the interactions between Cls with other lipophilic toxins,
and their effects in human health are unknown. To corroborate if Cls are maintained in low
concentrations, we recommend an higher time-frequency for monitoring in this region.
48
Reference materials for analysis of Tetrodotoxin in seafood
Thomas K., Beach D.G., Reeves K., Perez Calderon R.A., Crain S., Kerrin E.S.,
and McCarron P.
Biotoxin Metrology, National Research Council Canada, 1411 Oxford Street, Halifax,
Nova Scotia, B3H 3Z1, Canada. e-mail: [email protected]
Tetrodotoxin (TTX) is a potent neurotoxin most commonly associated with puffer fish
poisoning, but studies have confirmed TTX in bivalves harvested in temperate European waters.
Recent FAO/WHO and EU reports consider establishing regulatory limits for TTX in shellfish,
and regulations have been introduced in the Netherlands. To address analytical needs, calibration
solutions and matrix reference (RM) materials have been developed for TTX analysis. Research
for a calibration solution certified reference material (CRM) included studies on the effects of
temperature and pH on TTX in solution. A CRM was produced for TTX (21.1 µM in 1 mM acetic
acid) and value assigned by quantitative 1H-nuclear magnetic resonance spectroscopy. In a matrix
RM feasibility study, control and DA contaminated mussels (Mytilus edulis) were blended with
TTX contaminated sea slug (Pleurobranchaea maculata) and PST positive dinoflagellate biomass
(Alexandrium tamarense). Stability was assessed over time in equivalent wet and freeze-dried
homogenates. The freeze dried material had no detectable decomposition at temperatures reaching
+40 °C over 28 days. TTX in the wet matrix was only stable at -12 °C, with conversion to epiTTX
at +40 °C over the same time period. These materials are important for development and validation
of analytical methods for analysis of TTX in seafood. The experience gained during the
development of these initial TTX RMs, and the method advances facilitated by their availability,
will establish the necessary capabilities at NRC to produce fully certified shellfish matrix CRMs
for TTX and other classes of polar marine toxins.
49
Profile of paralytic shellfish toxins of Pyrodinium bahamense and first
detection of tetrodotoxin in Mexican bivalve mollusks
Núñez-Vázquez, E.J.1*, 2, Turner, A.D.3, Ramírez-Camarena, C.4, Hernández-Sandoval, F. E.1,
Bustillos-Guzmán, J.1 and Poot-Delgado, C.A.5
1Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Apdo. Postal 128, La Paz, Baja
California Sur. 23000, México. e-mail: [email protected] 2Investigación para la Conservación y el Desarrollo (INCODE), Nayarit 1325 A. Col. Las
Garzas, La Paz, B.C.S. 23079, México. 3Centre for Environment Fisheries and Aquaculture Science (CEFAS). Food Safety Group,
Barrack Road, Weymouth, Dorset, DT4 8UB, United Kingdom. 4Instituto Nacional de la Pesca. Pitagoras No. 1320, Santa Cruz Atoyac 03310. Ciudad de
México. 5Intituto Tecnológico Superior de Champotón, Campeche (ITESCHAM). Carretera Champotón,
Isla Aguada Km 2, Col. El Arenal, C. P. 24400. Champotón, Campeche, México.
Paralytic shellfish poisoning (PSP) is the main public health problem for bivalve
mollusks in Mexico. The HABs of P. bahamense have caused 92.78% of PSP cases, with 476
human intoxications, all occurring along the South Pacific coast. In this work we describe the
chromatographic profiles (HPLC-FLD and UHPLC-MS/MS) of the paralyzing toxins detected in
shellfish during the main PSP events that have had the greatest impact on public health in Mexico.
A concentration of 7,396 μg eq STX/kg was determined in "rock oyster" (Crassostrea iridescens)
from the Bay of Acapulco, Guerrero during the 1995 HAB; between 8,889.3-32,272.5 μg eq
STX/kg in "clams" Donax gracilis del Zapotal and 14,445 μg eq STX /kg in "mussels tichindas"
(Mytella strigata) of Puerto Madero, both in Chiapas during the HAB of 2001. The profiles by
HPLC -FLD were STX, GTX2, dcSTX, dcGTX2, dcGTX3 and B1 in C. iriddescens, STX, Neo-
STX, GTX2, GTX3 and B1 in D. gracilis and STX, GTX2, GTX3 and B1 in M. strigata. In Salina
Cruz, Oaxaca one year later in 2002, 306.3 μg eq STX/kg were still detected in "spiny oyster"
(Spondylus calcifer) with only STX present. Additionally, UHPLC-MS/MS analysis was used to
detect GTX1, GTX5, GTX6, doSTX, dcNEO, C1 and C2 and in a sample of D. gracilis and
tetrodotoxin (TTX) was detected for the first time in bivalve mollusks from Mexico. Recently, in
phytoplankton samples during a bloom of P. bahamense in 2016, NeoSTX and STX were detected
in the southeastern Gulf of Mexico. Overall the results demonstrate both the significant level of
risk associated with shellfish consumption from certain parts of Mexico and the occurrence of
unique PST profiles within certain bivalve species.
50
Structure Elucidation and Relative Toxicity of (24R)-24 Hydroxyyessotoxin
from a Namibian Isolate of Gonyaulax spinifera
Rajotte, I., Rafuse, C., Wright, E. J., Achenbach, J. C., Ellis, L. D., McCarron, P.
National Research Council Canada, Biotoxin Metrology, 1411 Oxford Street, Halifax, Nova
Scotia, Canada, B3H 3Z1. Presenting author e-mail: [email protected]
In 2011 a bloom of dinoflagellates was reported in Walvis Bay, Namibia, from which a
strain of G. spinifera, a known producer of yessotoxins (YTXs), was isolated. Liquid
chromatography-high resolution mass spectrometry (LC-HRMS) analysis of this strain showed the
presence of a number of YTXs. Principal among these were YTX, homoYTX, and a tentatively
identified hydroxylated analogue which did not correspond to any previously confirmed YTX
structures. Culturing of the G. spinifera strain afforded sufficient biomass for purification of the
new analogue through a series of solvent partitioning and chromatographic steps, yielding ~0.9
mg as a solid. NMR spectroscopy, ion-trap mass spectrometry and HRMS identified the new
analogue as (24R)-24-hydroxyYTX. Purified 24-hydroxyYTX was quantitated by NMR and its
relative toxicity evaluated using two embryonic zebrafish toxicity assays. 24-hydroxyYTX
demonstrated reduced toxicity compared to YTX. The confirmation of 24-hydroxyYTX
production by microalgae is of note considering regulations for levels of the 45-hydroxyYTX
metabolite in shellfish. In the absence of standards these compounds can be misidentified using
conventional LC-MS monitoring methods. This work expands knowledge on the occurrence of
toxin-producing harmful algal species in Southern Africa, which is of importance considering the
increased international exploitation of seafood as a commodity for human consumption.
51
Etiology and Epidemiology of Paralytic Shellfish Poisoning in Latin America
Núñez-Vázquez, E.J.1*,2, Band-Schmidt, C.J.3, Bustillos-Guzmán, J.1, Hernández-Sandoval, F.E.1,
López-Cortés, D.J.1†, Leyva-Valencia, I.4, Ramírez-Camarena, C.5, García-Mendoza, E.6, Ruiz
de la Torre, M.C.7, Medina-Elizalde, J. 6 and Peña-Manjarrez, J.L.8
1Centro de Investigaciones Biológicas del Noroeste S. C. (CIBNOR), Av. Instituto Politécnico Nacional
No. 195, Playa Palo de Santa Rita Sur 23096. La Paz, Baja California Sur, México. e-mail:
[email protected] 2Investigación para la Conservación y el Desarrollo (INCODE), La Paz, B.C.S. 23079, México. 3Instituto Politécnico Nacional-Centro Interdisciplinario Ciencias Marinas (IPN-CICIMAR). La Paz, Baja
California Sur, México. 23000. 4CONACYT-Instituto Politécnico Nacional, Centro Interdisciplinario Ciencias Marinas (IPN-CICIMAR).
La Paz, Baja California Sur, México. 23000. 5Instituto Nacional de la Pesca (INAPESCA). Ciudad de México, México 03310. 6Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE). Ensenada, Baja
California 22860. 7Universidad Autónoma de Baja California (UABC). Facultad de Ciencias Marinas. Ensenada, Baja
California, México 22860. 8Centro de Estudios Tecnológicos del Mar (CETMAR). Ensenada, Baja California 22760.
In Latin America (LA) approximately 1,410 people have been reported to be intoxicated
(94 fatalities) by paralytic shellfish poisoning (PSP) from 1970 to 2016. A total of 350 cases have
corresponded to the consumption of seafood associated with blooms caused by Alexandrium spp
in South America. Chile is the country most affected by this genus. HABs of G. catenatum resulted
in 241 cases of PSP (14 fatalities) in Mexican and Venezuelan coasts. During HABs of this
dinoflagellate massive mortalities of fish, larvae and adults of shrimp cultivation have also been
described (affecting the shrimp industry in the Northwest of Mexico) as well as epizootic diseases
with mass mortalities of birds and marine mammals in the Upper Gulf of California. In the Gulf
of California this dinoflagellate has caused continuing sanitary closure by contamination with PSP
toxins in shellfish, affecting their commercialization. PSP cases produced by P. bahamense have
affected several countries in LA, notably the southerrn part of the Mexican Pacific, followed by
Guatemala, Nicaragua, El Salvador and Costa Rica. This species have generated the greatest
number of intoxicated cases by PSP (819) in all of LA. They have also caused massive mortalities
of fish and sea turtles. Other negative impacts by the presence of PSP toxins is the huge economic
costs associated with additional expenditures in the fishery and aquaculture sector because of
sanitary closure, periodic or permanent establishment, closed areas, confiscation, destruction of
products and monitoring of dinoflagellates and their toxins to ensure food safety.
52
Metabolic transformation of paralytic shellfish toxins: consequences on toxin
composition and toxicity kinetics in molluscan shellfish
Cembella, A.D.1, Krock, B.1, Durán-Riveroll, L.M.1,2, Band-Schmidt, C.J.3, Leyva-Valencia, I.3,
and Bustillos-Guzmán, J.4
1Alfred-Wegener-Institut, Helmholtz Zentrum für Polar-und Meeresforschung, Am
Handelshafen 12, 27570, Bremerhaven Germany. e-mail:[email protected];
[email protected] 2CONACYT-Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de
México, Circuito exterior s/n, Ciudad Universitaria, 04510 Coyoacán, Mexico. e-mail:
[email protected] 3Instituto Politécnico Nacional-Centro Interdisciplinario de Ciencias Marinas, Av. Instituto
Politécnico Nacional s/n, Playa Palo de Santa Rita, 23096 La Paz, B.C.S., Mexico. e-
mail:[email protected]; [email protected] 4Centro de Investigaciones Biológicas del Noroeste, Mar Bermejo 195, Col. Playa Palo de Santa
Rita, La Paz, B.C.S. 23096, Mexico. e-mail: [email protected]
About 50 naturally occurring analogs of paralytic shellfish toxins (PSTs) are
biosynthesized among marine dinoflagellates and cyanobacteria, or are created by
biotransformation after ingestion by molluscan shellfish. The biosynthetic pathways for the major
PSTs (e.g., N-sulfocarbamoyl-, N1-OH- and C11-sulfated carbamoyl-derivatives) are well known
for the producing microorganisms, including the structural and functional elements of the
respective gene clusters. The kinetics of toxin biotransformations within molluscan shellfish,
however, are highly species-group specific, and involve an array of enzyme-mediated catabolic
reactions via hydrolases, oxidoreductases and N-aminosulfotransferases, as well as facile
thermodynamic epimerization subject to pH and temperature effects. Among other factors, such
metabolic biotransformations yield profound differences in toxin body burden, net toxicity and
toxin composition even among molluscan shellfish species exposed to the same bloom and
harvested simultaneously from a given site. Here we compare the results of controlled laboratory
feeding studies on the uptake and biotransformation kinetics of PSTs from cultured dinoflagellates
and consequent effects on toxin body burden and composition. Such studies are critical to
hindcasting and simulating the time-course of toxin accumulation for molluscs subjected to natural
toxic blooms. This has implications as well for predicting species-specific toxicity and risk
assessment because many biotransformations, e.g., from N-sulfocarbamoyl analogs, which often
predominate in the dinoflagellate composition, to respective carbamoyl or decarbamoyl derivatives
53
within molluscan shellfish, can increase net toxicity by more than an order of magnitude on a
molar basis. This has implications for designing appropriate toxin monitoring strategies for various
combinations of shellfish species and toxigenic dinoflagellate populations.
54
Marine lipophilic toxins in cultivated mussels (Mytillus galloprovincialis) from
Baja California, Mexico, an eight year survey
Sánchez-Bravo Y.A., García-Mendoza E., Rosas-Campos A.I.,
Departamento de Oceanografía Biológica. Centro de Investigación Científica y de Educación
Superior de Ensenada. Carretera Ensenada-Tijuana 3918, Ensenada, Baja California, México.
e-mail: [email protected]
The presence of lipophilic toxins in bivalves is one of the most important problems related
to economic losses associated with harmful algal blooms. Regardless the importance of the
extraction and consumption of bivalve shellfish mollusk in different regions of Mexico, there is
limited information about the presence of regulated lipophilic toxins in these organisms. The
aquaculture of bivalves is an important coastal economic activity in Baja California, Northwest
Mexico. This activity has been affected since 2010 when sanitary closures were implemented due
to positive results for lipophilic toxins detected by mouse bioassay. The presence of these toxins
was only confirmed by analytical methods until 2012. After this year, FICOTOX laboratory has
maintained a regular monitoring program of lipophilic toxins in phytoplankton samples and
mussels cultivated in the area. These compounds have been detected with liquid chromatography
coupled to mass spectrometry (HPLC-MS/MS) using acidic conditions for the separation 12 toxins
simultaneously. Here, we describe the variation of the concentration of okadaic acid (AO),
dinophysistoxins (DTXs), pectenotoxins (PTX), yessotoxins (YTX), azaspiracids (AZA) and
cyclic imines from 2012 to 2019 in mussels cultivated in Todos Santos Bay. AO+DTX, YTXs
regularly detected and concentrations were higher than the regulation limit in samples collected in
2012. AZAs were also detected in mussels cultivated in the region but the concentration of this
toxins did not reach the regulation limit. We discuss the importance monitoring lipophilic toxins
with reliable methods for risk assessment.
55
Analysis of microcystins and nodularin in drinking water using
LC/MS triple quadrupole
Mondragón Olguín, Víctor Manuel
AGILENT, México, Insurgentes Sur 1602 -801, Crédito Constructor 03940, México, DF.
e-mail: [email protected]
Microcystins and nodularin are potent hepatotoxins produced by various species of
cyanobacteria. During algal blooms, large quantities of microcystins and nodularin can occur in
freshwater systems, threatening livestock and human drinking water sources. Ingesting these
compounds carries health risks; therefore, regulatory agencies such as the US EPA and the World
Health Organization (WHO) recommend limiting microcystins in drinking water to sub-ppb levels.
This study extracted and prepared 500-mL water samples according to the US EPA method 544
for microcystins and nodularin in drinking water. Recovery of the microcystins and nodularin
ranged 82–115 % for both mid-level and low-level demonstrating good efficiency and sensitivity
for the method. The internal standard (MC-LR C2D5) recovery was between 70–130 %, which is
within EPA guidelines. The mid-level spike is at least 10 times lower than the WHO guidelines
for microcystins and nodularin in drinking water. The low-level spike is at least 50 times lower
than the WHO guidelines, well below any individual US state advisory limits for microcystins and
nodularin.
56
19th International Conference on Harmful Algae, Mexico, 2020
Christine J. Band Schmidt
Chair of the Organizing Committee
The 19th International Conference on Harmful Algae (ICHA 2020) of the International
Society for the Study of Harmful Algae (ISSHA), will take place from the 11th to the 16th of
October 2020 in La Paz, BCS, Mexico. The ISSHA was founded in 1997, in response to a request
from the Intergovernmental Oceanographic Commission of UNESCO. This society is affiliated to
the International Council of Science, forming part of the International Union of Biological
Sciences through the International Association of Biological Oceanography. The main objective
of the Society is to promote and encourage research, training programs related with Harmful Algae,
as well as co-sponsor national, regional and international meetings. Since its foundation, the
ISSHA has held international conferences every two years where internationally renowned
researchers attend, in addition to numerous students from various countries, with an attendance
between 500 and 800 attendees to each event. This is the first time this meeting is held in Mexico,
and the second time in a Latin American country. On this occasion, we expect an attendance of
600 people. As the last conferences, ICHA 2020 conference will include topics related with the
understanding of the causes, evolution and impacts of harmful microalgae and cyanobacteria. We
are planning an enjoyable meeting where scientists can present their research, share their ideas,
establish new collaborations, and connect the science on harmful algae with the beneficiaries of
this research. Our reasons for holding this meeting are based on the recurrent presence of Harmful
Algal Blooms that have affected the economy and public health in various coastal regions and
inland waters of Mexico. These events have been associated with the mortality of marine
organisms such has free-living and farmed fish, dolphins, turtles and seabirds; as well as the
presence of diverse phycotoxins in clams, oysters and mussels, which put people´s health and lives
at risk, reason for which it is necessary to establish sanitary closures for periods of weeks or
months. Undoubtedly, this is a problem requiring collaborative efforts to reduce the adverse effects
of these natural phenomena. As the major host of the conference, ISSHA will support the event
with various activities: Travel awards to students and post-docs, ISSHA auction, and several
achievement awards. Looking forward to seeing you in La Paz, Mexico!
57
POSTER SESSION
Poster #1
Novel Vibrio detection method for species and toxigenicity genes identification
using real-time PCR
Skinfill A., Priller F., Grönewald C. and Berghof-Jäger K.,
BIOTECON Diagnostics, Hermannswerder 17, 14473 Potsdam, Germany.
e-mail: [email protected]
Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae are known to be potential
waterborne contaminants of seafood and cause severe health problems worldwide. Traditional
methods for the detection are time consuming and error-prone, while real-time PCR can be done
in less than 24 hours with a high specificity and sensitivity. Our real-time PCR assay can
discriminate between V. parahaemolyticus, V. vulnificus and V. cholerae and simultaneously
detects and individually identifies the pathogenicity factors ctx, tdh, trh1 and trh2 by melting curve
analysis in just one single reaction. By using novel targets, false-positive and false-negative
results, known from other methods using targets like e.g. tlh or hlyA, are avoided. 74 V.
parahaemolyticus, 26 V. vulnificus and 49 V. cholerae were tested for inclusivity: With 100 %
specificity, the assay is superior to other methods for Vibrio detection. There were no false positive
results for all 73 tested samples of 54 closely related species and bacteria of the same habitat. The
sensitivity of the foodproof® Vibrio Detection LyoKit is 1 genomic equivalent (GE) per reaction
for species detection and 10-25 GE per reaction for toxin detection. The assay is compatible with
all tested raw and processed seafood matrices like whole squid, raw oysters or smoked salmon.
The sample preparation includes a live/dead discrimination by using Reagent D, which efficiently
removes DNA of at least 103 cfu/ml dead Vibrio. As seafood often is contaminated with dead
Vibrio, Reagent D treatment prevents false positive results which may be encountered with normal
PCR methods.
58
Poster #2
Statistical assessment of MPN test method and three class sampling plan used
to determine Escherichia coli contamination level in shellfish
Ciccarelli Cesare1, Semeraro A. Marisa
1, Di Trani Vittoria
1, Leinoudi Melina
2, Ciccarelli Elena
3
1ASUR MARCHE Area Vasta 5 – San Benedetto del Tronto, Italy.
2 General Chemical State Laboratory – Thessaloniki, Greece. 3 Biologist – Bracknell, UK.
e-mail: [email protected]
Microbial quality of shellfish is assessed by enumeration of the faecal indicator
Escherichia coli because its presence informs of the possible presence of faecal micro-organism
potentially pathogen for humans. The reference method is the MPN test following ISO 16649-3
and a three class sampling plan plays an international leading role in official food hygiene
monitoring establishing m=230MPN/100g (flesh and intervalvular fluid), M=700MPN/100g, c=1
and sample size n=5. The MPN test, widely used to estimate microbial densities in many matrices,
is a statistical approach based on the probability theory and several factors contribute to variability
of outcomes. Moreover, the three class sampling plan plays a crucial role in the statistical
significance. This paper investigated the performances of MPN test using the Excel spreadsheet
developed for the ISO 16649-3, calculating related sensitivity, specificity and predictive values of
positive or negative test at a wide range of prevalence. The study showed as, focusing on
230MPN/100g, both sensitivity and specificity values are about 0.91 and then, focusing on
700MPN/100g, they are 0.92 and 0.96 respectively. Moreover, using a stochastic model, analysis
method independent, based on the probability theory, we evaluated the accuracy of the mentioned
sampling plan: the study showed as it underrates the compliant sample units when prevalence is
below 0.90 and overrates them when prevalence is higher. These findings allow to better
understand the performances of MPN test and related three class sampling plan which plays a
leading role in official food hygiene international standards such as end product testing.
59
Poster #3
Three sensitive norovirus (GI, GII) plus hepatitis a virus kits:
their validation and all in-process control
Skinfill A., O. Degen, Grönewald C. and Berghof-Jäger K.
BIOTECON Diagnostics, Hermannswerder 17, 14473 Potsdam, Germany.
e-mail: [email protected]
Noroviruses are considered to be the main agent for gastrointestinal diseases in humans
worldwide. Of the five known only genogroups GI and GII are pathogenic to humans. Hepatitis A
virus (HAV) could affect the liver and the disease is characterized by fever, diarrhea and jaundice.
Six HAV genotypes have been defined. HAV and Norovirus are rather stable in the environment
and are resistant to freezing and other food preservation methods. Both have therefore a good
potential to cause food-borne outbreaks which are frequently observed following consumption of
fresh or frozen and thawed fish and seafood. According to EFSA in the European Union and FDA
in the USA foodborne viruses are emerging public health risks. We present three fast and reliable
real-time multiplex PCR test systems to check potentially contaminated food and water. All tests
take into account the norm ISO 15216 which specifies methods for virus concentration and for the
quantification of virus particles. We deliver in our kits a solution of process control as ISO 15216
states that a process control has to be used for each sample from the begin of the procedure of virus
concentration to monitor the recovery of the virus after all protocol steps. Validation data for three
foodproof® Detection Kits applicable on multichannel real-time PCR cyclers are now available:
Norovirus (GI, GII) kit, Hepatitis A Virus kit, and a multiplex Norovirus (GI, GII) plus Hepatitis
A Virus kit.
60
Poster #4
Evaluation of hydrophilic and lipophylic toxins in amerycan oyster
Crassostrea virginica of the coast Campeche: detection of gymnomidin and
spirolides
Núñez-Vázquez, E.J.1,2, Poot-Delgado, C.A.3, Cauich-Sánchez, Y.3, Hernández-Sandoval, F.E.1,
Bustillos-Guzmán, J.1, García-Mendoza, E.4 and Sánchez-Bravo, Y4.
1Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Apdo. Postal 128, La Paz, Baja
California Sur. 23000, México. e-mail: [email protected] 2Investigación para la Conservación y el Desarrollo (INCODE), Nayarit 1325 A. Col. Las Garzas,
La Paz, Baja California Sur. 23079, México. 3Instituto Tecnológico Superior de Champotón, Campeche (ITESCHAM). Carretera Champotón,
Isla Aguada Km 2, Col. El Arenal, C. P. 24400. Champotón, Campeche, México. 4Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE). Carretera
Ensenada-Tijuana 3918, Zona Playitas 23860. Ensenada, Baja California, México 22860.
An evaluation was made of hydrophylic toxins [HT] (paralytic shellfish toxins, PSP and
domoic acid, DA) and lipophylic toxins [LT] (brevetoxins, diarheic shellfish toxins) in "amerycan
oyster" Crassostrea virginica. We analyzed samples of C. virginica from 16 months of the years
2012-2015, obtained from the southeast of the Gulf of Mexico (Cd. Del Carmen, Campeche). The
oysters were processed for the extraction of HT (n=32) and LT (n=32). The detection of HT was
evaluated by mouse bioassay (MBA), lateral flow immunoassay (LFIA), HPLC-FLD and HPLC-
UV. Lipohylic toxins only for MBA and LFIA. All samples of C. virginica were negative for PSP
and DA toxins. In the case of LT, only one sample from 2013 was lethal in the MBA, causing
rapid death (with clinical signs of DSP toxins and Fast Acting Toxins). The presence of OA and
analogs, gymnomidin and spirolides was subsequently detected by LC-MS/MS. Most of the
samples were negative for the toxins evaluated, which is of interest in food safety, however the
presence of LT should continue to be monitored. The presence of LT is confirmed as the OA,
GYM and SPX in Mexican coasts of the Gulf of Mexico.
61
Poster #5
Effect of pH on the bacterial community present in larvae and spat of
Crassostrea gigas
Flores-Higuera Francisco A.1, Luis-Villaseñor Irasema E.2, Rochin-Arenas Jesús A.2, Gómez-Gil
Bruno3, Mazón-Suástegui José Manuel4, Voltolina Domenico5 and
Medina-Hernández Diana4
1Posgrado en Ciencias en Recursos Acuáticos, Facultad de Ciencias del Mar, Universidad
Autónoma de Sinaloa, Mazatlán, Sinaloa, México. 2Facultad de Ciencias del Mar, Universidad Autónoma de Sinaloa, Mazatlán, Sinaloa, México. 3CIAD, Unidad Académica Mazatlán en Acuicultura y Manejo Ambiental, Mazatlán,
Sinaloa, México. 4Centro de Investigaciones Biológicas del Noroeste S.C., La Paz, B.C.S., México 5Centro de Investigaciones Biológicas del Noroeste, Laboratorio UAS-CIBNOR, Mazatlán,
Sinaloa, México.
Changes in marine environments, including pH changes, have been correlated to alterations
in the physiology and disease susceptibility of cultured organisms at the early stages of
development. In this study, high-throughput sequencing of the V3-V4 region of the 16S rRNA
gene was performed to evaluate the bacterial biodiversity of Crassostrea gigas pediveliger larvae
and spat under acidic stress compared to that of larvae at normal pH value. The evaluation was
performed in an experimental system with continuous water flow and pH manipulation by CO2
bubbling to simulate acidification pH 7.384 ± 0.039 (expected for the year 2300), using the current
ocean pH conditions (pH 8.116 ± 0.023) as a reference. The results indicated that the bacterial
communities associated with both pediveliger larvae and spat were modified in response to acidic
conditions. The families Rhodobacteraceae and Campylobacteraceae were the most affected by
the change in pH, with increases in Vibrionaceae in pediveliger larvae and Planctomycetaceae and
Phyllobacteriaceae in spat detected. The results of this study demonstrate that the bacterial
communities associated with C. gigas pediveliger larvae and spat are responsive to changes in
ocean acidification.
62
Poster #6
Comparison of two sampling techniques for quantification of phytoplankton
for sanitation surveillance purposes
Villagrán Lorenzana, Héctor Gustavo
State Commission for the Protection against Health Risks of the Health Services of the State of
Colima. e-mail: [email protected]
The Federal Commission against Sanitary Risks (COFEPRIS) in coordination with the
Federal Entities monitor and regulate the sanitary quality of bivalve molluscs destined for human
consumption; perform a permanent monitoring of toxic phytoplankton concentrations in Mexican
coasts to verify that they do not exceed the maximum established in current sanitary regulations
(1, 2, 3 and 4). The objective is to determine a quantification technique with greater sensitivity
than the observation of direct samples in Sedgewick-Rafter chamber (SR). Ten direct and ten net
samples were taken; the first were gauged to 500 ml and concentrated to a volume of 50 ml; the
network samples were processed according to the Relox technique (5). The readings were made
with an optical microscope composed in SR chamber. Three genera were quantified
(Prorocentrum sp. Protoperidinium sp. and Tripos sp.) The results indicate that the techniques are
reliable in terms of reproducibility, but the quantifications vary significantly between both
techniques. The Relox technique has greater sensitivity and is the most reliable for sanitary
surveillance purposes.
Bibliography:
1. Official Mexican Standard NOM-242-SSA1-2009, Products and services. Fresh fishery products, chilled,
frozen and processed. Health specifications and test methods.
2. Work instruction for phytoplankton sampling and detection of marine biotoxins.
3. Work instruction for Sanitary Control of Molluscs Exposed to Harmful Algal Blooms.
4. Technical Guide for the Sanitary Control of Bivalve Molluscs 2009.
5. Practical guide on paralytic shellfish poisoning monitoring in the Philippines, chap. 7, "Plankton
Analysis", Juan R. Relox, Jr. Gonzalez C. Sakamotos., Furio E. Ogata., Kodama m., and Fukuyo Y. Editors.
63
Poster #7
Variation in the temporal space of bacterial communities and presence of
Vibrio parahaemolyticus toxigenic in Stone oyster (Striostrea prismatica, Gay,
1825) in the Bay of Mazatlán, Sinaloa
Luis Villaseñor, Irasema
Universidad de Sinaloa. e-mail: [email protected]
In Mazatlan Bay, the Rock oyster Striostrea prismatica is a fishing resource of great
commercial importance, which is extracted and consumed raw in the beach directly from the sea.
Due to the risk posed by the consumption of this possible mollusk carrier of toxigenic bacterial
species such as Vibrio parahaemolyticus, it is important to determine the temporal space variation
of the bacterial community and the presence of toxigenic V. parahaemolyticus in the rock oyster
S. prismatica and its relationship with factors environmental present in Mazatlan Bay. For the
analysis of the bacterial and Vibrio community, a combined method of microbiology (NMP/g of
oyster) and molecular biology (detection of toxigenic genes tlh, trh, tdh and orf8 by endpoint PCR,
and 16s DNA Metabarcoding was used. The recording of environmental variables was carried out,
as well as water quality analysis (nitrites, nitrates, phosphates, ammoniacal nitrogen and total
suspended solids TSS). The bacterium V. parahaemolyticus (tlh +) was detected, as well as the
presence of its toxigenic genes tdh, trh and gene orf8 indicative of serotype O3: K6. Metabarcoding
analysis was performed on 27 oyster samples obtained during 4 quarterly samples during 2015-
2016. Results of the sequencing showed 3'093,530 sequences which were assigned to 7,955 OTU's,
with the predominant families Prochlorococcaceae, Vibrionaceae and Planctomycetaceae.
Likewise, NMP/g of oyster was correlated with environmental variables, with temperature,
dissolved oxygen and phosphates being significant positive. It is concluded that V.
parahaemolyticus is present in S. prismatica in Mazatlan Bay, as well as its toxigenic genes which
represents a risk to the health of consumers. The Vibrionaceae family was most abundant at the
site of Playa Cerritos and Luna Palace in December.
64
Poster #8
Detection and genotyping of Hepatitis E virus from molluscan samples
Rivadulla Enrique1, Polo David1, Mesquita João R.2,3, Nascimento Maria S.J.3,4
and Romalde Jesus L.1
1Departamento de Microbiologia y Parasitologia, CIBUS-Facultad de Biologia, Universidade de
Santiago de Compostela, 15782, Santiago de Compostela, Spain.
e-mail: [email protected]. 2Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, 4050-313 Porto,
Portugal. 3Epidemiology Research Unit, Instituto de Saúde Pública, Universidade do Porto, 4050-313 Porto,
Portugal. 4Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia da
Universidade do Porto, Portugal.
Hepatitis E virus (HEV) belongs to the Hepeviridae family within Orthopevirus genus that
includes five genotypes that infect humans mainly through fecal-oral route. A total of 168 samples
obtained from two different Galician estuaries, one in the North (Ría de Ares-Betanzos) and
another in the South (Ría de Vigo), were collected monthly for 18 months (from January 2011 to
June 2012) and they were analyzed by RT-qPCR for HEV detection. These samples included
mussels, clams and cockles, collected at different points of the two estuaries, classified as "class
B" zones (230-4600 CFU Escherichia coli/100 g of shellfish) in accordance to European
legislation. HEV was detected in 41 samples (24.4%) and its prevalence was slightly higher in the
southern harvesting areas. The quantification levels ranged from non-quantifiable (below the limit
of the quantification method) and 1.08x106 copies of RNA/g of tissue. In addition, the phylogenetic
analysis based on the ORF 2 region showed that all the sequenced isolates belonged to the genotype
3 subgenotype e, being closely related to the strains of swine and wild boar origin. The results
obtained suggest that the prevalence of HEV in bivalve molluscs produced in Galicia is significant.
Furthermore, the evidences obtained reinforce the hypothesis of a strong influence of pig farms on
the appearance of HEV contamination. However, more studies are needed to better understand the
importance of the presence of this pathogen, as well as to determine its potential risks to human
health.
65
Poster #9
Occurrence and virulence of Vibrio parahaemolyticus isolated from seawater
and bivalve shellfish of the Gyeongnam coast, Korea, in 2004-2016
Kunbawui Park1, Jong Soo Mok1, A Ra Ryu1, Ji Young Kwon2, In Tae Ham1, Kil Bo Shim1
1Food Safety and Processing Research Division, National Institute of Fisheries Science, Busan
46083, Republic of Korea. 2Southeast Sea Fisheries Research Institute, National Institute of Fisheries Science, Tongyeong
53085, Republic of Korea.
We determined the abundance and virulence of Vibrio parahaemolyticus in seawater and
bivalves from the Gyeongnam coast in Korea, a major area for the seafood industry, during 2004–
2016. V. parahaemolyticus is one of the most common pathogen causing seafood-borne illnesses
in Korea, and increases during the summer. Its occurrence in seawater and bivalve samples was
seasonally dependent, with high levels during the summer to early autumn. There were more
strains in the area of sea continually exposed to inland wastewater. Only 5.1% and 3.5% of V.
parahaemolyticus isolates from seawater and bivalves, respectively, had the trh gene, and only the
bivalve isolates produced the tdh gene at levels below 2%. Continuous monitoring is clearly
needed to reduce seafood-borne outbreaks of disease caused by V. parahaemolyticus, and to reveal
the occurrence patterns and the presence of toxic genes of the strains in different marine
environments.
66
Poster #10
Azadinium spinosum and Alexandrium ostenfeldii as toxigenic species
in Todos Santos Bay
Paredes-Banda Patricia1, García-Mendoza Ernesto2, Ponce-Rivas, Elizabeth1
and Allan Cembella.
1Departamento de Biotecnología Marina, Centro de Investigación Científica y de Educación
Superior de Ensenada. Carretera Ensenada-Tijuana #3918, Baja California, Mexico.
e-mail: [email protected] 2Departamento de Oceanografía Biológica, Centro de Investigación Científica y de Educación
Superior de Ensenada. Carretera Ensenada-Tijuana #3918, Baja California, Mexico 3Alfred-Wegener-Institut, Helmholtz Zentrum für Polar-und Meeresforschung, Am Handelshafen
12, Bremerhaven, Germany
Todos Santos Bay is one of the main regions for mollusk mariculture in Mexico with
certificate areas for the exportation of bivalves. The phytoplankton monitoring and the use of
accurate technics to identify the potentially toxic species and their temporality of appearance are
necessary to protect human health. Azadinium spinosum is a small photosynthetic dinoflagellate
that produces azaspiracids (AZAs). The detection of this species is a challenge due their size. We
use specific primers designed to amplify 18S rDNA to assess the presence of A. spinosum in
phytoplankton samples collected in Todos Santos Bay. The species was identified and detected in
21% of the analyzed samples for the 2013-2014 period (collected close to the mussel cultivation
area) and in 31% in seven sampling stations during 2016-2017. The species showed a clear
seasonal pattern of appearance that is consistent with the detection of the toxin in mollusks that
occurs mainly during the winter. The maximum concentration of AZA-1 was 24 μg kg-1. For
Alexandrium ostenfeldii its detection was achieved with a specific ITS molecular probe. The
species did not present a seasonal pattern of appearance neither the 13-desmethyl spirolide during
the 2013-2014 period. The accumulation in the culture mussels was low with 1.04 μg kg-1. The
species was detected when water temperature was between 17 to 20 ºC. These constitute two new
records of the species on the Northwest Pacific coast.
67
Poster #11
Evaluation of Sanitary Safety for Shellfish in Hansan·Geojeman, Korea
Poong Ho Kim1, Ka Jeong Lee1, Yeon Kye Kim1, Dong Wook Kim1, Yeon Jung Jeong1,
Jong Soo Mok1 and Kwang Soo Ha2
1Food Safety and Processing Research Division, National Institute of Fisheries Science, Busan
46083, Republic of Korea. 2Southeast Sea Fisheries Research Institute, National Institute of
Fisheries Science, Tongyeong 53085, Republic of Korea.
Hansan Geojeman area is the representative oyster hanging culture area in Korea. Based
on the sanitary survey results for the shellfish growing area (5,000 ha) from 1969 to 1971, four
areas (the total of 473 ha) were firstly designated for shellfish production and export on September
6 in 1971. In the Hansan Geojeman area, the 213 cases (998.7 ha) of fishery licenses were
permitted. Among the permitted licenses, there were oyster of 104 cases (580.4 ha), fish of 38
cases (100.3 ha), sea squirt of 42 cases (170.4 ha), short neck clam of 15 cases (77.3 ha), ark shell
of 2 cases (8.9 ha), abalone of 2 cases (4.0 ha), blue mussel of 1 case (3.0 ha), scallop of 2 cases
(7.6 ha), and sea mustard of 7 cases (46.8 ha). The oyster farms were 66 cases with 434 ha in the
designated area of Hansan Geojeman area. From the designated area, 5,421 tons of shucked oysters
were produced in 2013. To evaluate bacteriological and toxicological safety hygienic indicator
bacterium and paralytic and diarrhetic shellfish toxins in the shellfish produced in
Hansan·Geojeman 2013-2017 were investigated. Fecal coliforms were < 18~330 MPN/100 g in
404 oyster samples. But all samples tested, did not exceed 230 E. coli MPN/100 g. Geometric
mean of E. coli for oyster samples collected during major shellfish production period was 24.3
MPN/100 g, considered stable results. Bacteriological quality of oysters collected from
Hansan·Geojeman meets the standard value based on shellfish hygiene of the Food Sanitation Act
of Korea and also meets Grade A, according to classification of shellfish harvesting areas of the
European Union. For toxicological evaluation of Hansan·Geojeman, 532 oyster samples and 268
mussel samples as an indicator, were analyzed. Paralytic shellfish toxins were detected in the range
of 0.42~2.29 mg/kg in eight mussel samples, and exceeded criteria in three samples from early to
late April 2013. Diarrhetic shellfish toxin was detected in three of 120 samples, but it was revealed
to be under regulation value (0.16 mg Okadaic Acid equ./kg). As a result of toxicological
68
evaluation, paralytic and diarrhetic shellfish toxins were not detected in oyster samples, but it was
found that mussel as an indicator species, exceeded the threshold value of paralytic shellfish toxin.
Accordingly, sanitary surveys were continuously requested for food safety management of
shellfish.
69
Poster #12
Initial Analysis of Tetrodotoxin in Canadian Bivalve Shellfish
Gibbs, Ryan1*; Thomas, Krista2; Beach, Daniel2; Rourke, Wade1; Murphy, Cory1; and
McCarron, Pearse2
1Canadian Food Inspection Agency, Dartmouth Laboratory, Dartmouth, Nova Scotia, Canada. 2National Research Council Canada, Biotoxin Metrology, Halifax, Nova Scotia, Canada. *Presenting Author. e-mail: [email protected], 1 (902) 536-1001
Tetrodotoxin (TTX) is a naturally occurring toxin historically found in tropical waters,
commonly associated with pufferfish in the Tetraodontidae family. Recent publications have
reported the detection of TTX in a number of bivalve shellfish species harvested in waters around
European countries, such as the Netherlands and the United Kingdom. This suggests it may be
possible for TTX to be in North American waters. The National Research Council (NRC) and the
Canadian Food Inspection Agency are working together to develop methods and reference
materials for the analysis of TTX in Canadian shellfish samples. Since April 2017, over 300
samples have been collected from both coasts of Canada and stored for TTX analysis. Some
scientific literature suggests a potential link between TTX and Vibrio species, so the presence of
Vibrio was considered when developing the sampling program. A hydrophilic interaction liquid
chromatography (HILIC) mass spectrometry method used at NRC for toxin reference material
characterisation was modified to increase throughput. Using in-house positive and negative
reference materials (RMs), the method was evaluated on three different mass spectrometers. Initial
accuracy and precision experiments using RM TTX-Mus showed an average recovery of 80 ± 10%
for TTX, and RSDs of <15%. Limits of detection (LOD) were in the 10-20 µg/kg range on the
different MS systems. Matrix effects for the RMs were <30%. Using this method, no TTX was
detected in a subset of 39 shellfish samples. Work on the method continues with a focus on
improving the LOD prior to the analysis of the full sample set.
70
Poster #13
Improved Chromatography for Acidic Separation of Lipophilic Shellfish
Toxins
Gibbs Ryan*, Casey Melanie, Rourke Wade, and Murphy Cory.
Canadian Food Inspection Agency, Dartmouth Laboratory, Dartmouth, Nova Scotia, Canada.
e-mail: [email protected], 1 (902) 536-1001
The Canadian Food Inspection Agency’s Dartmouth Laboratory tests ~3400 samples/year
for lipophilic shellfish toxins (LSTs) using the European Union’s standard operating procedure for
LSTs in molluscs by liquid chromatography – tandem mass spectrometry (LC-MS/MS). The
method uses acidic chromatographic conditions and a 2.1x100 mm ACQUITY BEH Shield RP18
column. Since the adoption of this method (2012), numerous samples have had to be re-analyzed
due to the presence of an occasional significant baseline rise in the 803.5>255.1 mass channel. In
2018, an investigation revealed that this was caused by a late eluting compound originating from
the sample three injections prior. A high-flow-rate flush was added to remove the interference, but
this modified gradient resulted in an increased analysis time, and backpressure approaching the
system limit. Three alternative Shield RP18 options were evaluated for use, and the 75 mm version
of the BEH column was chosen for a split-sample verification study. The 75 mm BEH column had
lower backpressure, and resulted in increased sensitivity for most compounds. An in-house quality
control sample demonstrated that the column produced results within the control limits.
Repeatability (%RSDs) for multiple injections of the calibration standards was comparable to the
100 mm BEH column. Spiking experiments showed equivalent accuracy between the columns for
all compounds except the azaspiracids; which were ~20% lower. The gradient on the 75 mm BEH
column restored the laboratory’s daily LST capacity to 41 samples/day from the reduced 28
samples/day using the long-flush method, while minimizing reinjections due to interferences and
system overpressure errors.
71
Poster #14
Helminthological infection by digenic trematode in arms of Octopus
bimaculatus collected in Bahía de Los Angeles, Baja California
Chan-Martin A.J., Martínez-Aquino A., Castellanos-Martínez, S.*
Universidad Autónoma de Baja California. Carretera Ensenada-Tijuana 3917, Fraccionamiento
Playitas, 22860, Ensenada, B.C., México. e-mail: [email protected]
Cephalopods are considered as intermediate or paratenic second hosts of digenean, being
relevant to link and culminate the life cycle of worms in the aquatic environment. To date, little
data is available on the function of these molluscs as hosts of parasitic helminths, in particular
digenean of a potentially zoonotic character. In Baja California, Octopus bimaculatus is the species
that sustains the fishing activity, but there are no records on parasite helminths that infect this
invertebrate. Therefore, the objective of the present work was to detect the presence of digenean
trematodes that infect O. bimaculatus, which could be potentially zoonotic. Six samples were taken
in Bahía de los Angeles, B.C., and a total of 39 octopuses were captured, from which an arm
sample was removed for parasitological review. Twenty-two specimens of a metacercaria stage
digenean (prevalence of 15%, 3.67±3.50 individuals/host) were recorded in O. bimaculatus, these
were fixed and stored for later identification. In addition, samples of infected tissue were collected
for histopathological analysis. According to the present results, we recommend focusing attention
on commercially important organisms to implement strict sanitary measures regarding the
consumption of aquatic organisms without previous apparent records of zoonotic infection.
72
Poster #15
Monitoring of paralytic shellfish poisoning in Striostrea prismatica and Chama
sordida in Bahia de Mazatlan, Sinaloa, Mexico 2003-2015
Delgado-del-Villar, S.J.1, Alonso-Rodríguez-R., Moreno-Hernández L.I., Acevedo-Medina, G.
and Gárate-Lizárraga, I.
Posgrado en Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México,
Ciudad de México, México.
Laboratorio de Biotoxinas Marinas, Instituto de Ciencias del Mar y Limnología, Unidad
Académica Mazatlán, UNAM, Mazatlán, Sinaloa, México.
Instituto Tecnológico de Mazatlán, Tecnológico Nacional de México, Mazatlán, Sinaloa,
México.
Laboratorio de Fitoplancton, Departamento de Plancton y Ecología Marina, Centro
Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional, La Paz, Baja California,
México.
e-mail: [email protected]
The monitoring of paralytic shellfish poisoning (PSP) in Mazatlan, Sinaloa consisted of
determining and comparing the toxicity and profile of PSP in Striostrea prismatica (commercial
species) and Chama sordida (non-commercial species) collected from 2003 to 2015. The PSP
analysis was performed by applying a high-performance liquid chromatography with fluorescence
detector (HPLC-FD) using the official method AOAC (2005.06) and Lawrence et al. (2005). The
method showed high sensitivity to STX, GTX2,3, GTX5, C1,2, dcSTX, dcGTX2,3 and low
sensitivity to GTX1,4 and NEO derivatives. The maximum concentrations of PSP during
monitoring were 13,156.24 µg STX eq kg-1 to S. prismatica and 28,000 µg STX eq kg-1 to C.
sordida. It was determined that C. sordida has high levels of PSP in 70.7% of the data collected.
It has been found that C. sordida exceeds 2.5 times the level of PSP compared to S. prismatica.
There was an increase of PSP in both species from March to May each year. Such seasonality is
related to the presence of the toxic dinoflagellate G. catenatum; it the largest producer of PSP in
the bay. On eight different occasions, (Mar 2003, Apr 2003, May 2003, Nov 2003, Mar 2010, Mar
2011, Mar 2013, Apr 2013), PSP levels exceeded the regulatory action limit set by the Mexican
government (800 µg STX eq kg-1). Under natural conditions, it was determined that no significant
differences (p<0.05) exist in the average concentration of STX, GTX2,3, GTX5 and dcSTX
derivatives between S. prismatica and C. sordida, however, significant differences (p<0.05) were
observed in the concentrations of C1,2 between the two species. C. sordida had a significantly
73
higher concentration of C1, 2. This paper presents evidence of the most vulnerable period’s
toxicity in shellfish (March and April). This data will prove invaluable to proposing measures for
the handling and management of marine sanitation in this area. Furthermore, our research confirms
that monitoring shellfish for PSP in areas with chronic toxic events is an essential tool to prevent
health risks to the local and national population which may inadvertently consume contaminated
shellfish.
74
Poster #16
1989-2019: REMI, 30 years of follow-up for shellfish harvesting area
in France
Piquet J.C., Rocq S., Kaelin G., Parnaudeau S., Le Guyader F.S.
Ifremer, Laboratoire de Microbiologie LSEM/SG2M, Nantes France
Since 1939, it is mandatory in France to classify shellfish production areas according to
their microbiological quality. This national regulation has then been implemented by the European
Commission and today, Chapter II of Annex II to Regulation (EC) No 854/2004 provides for the
classification of all shellfish production areas into three categories according to their
microbiological quality (A, B or C). This classification is based on concentrations of the fecal
bacteria indicator Escherichia coli in shellfish flesh. Following a sanitary survey, the area is
classified based on a regular monitoring of shellfish samples and Escherichia coli detection. Based
on the results of this monitoring the Competent Authority (i.e. the Ministry of Agriculture and
Food) classify this production area. The continuous monitoring allow the Competent Authority to
review the classification and eventually to take the appropriate management measures. In France,
bivalve molluscan shellfish are divided into three groups (burrowing and non-burrowing bivalves,
echinoderms) for the classification of production areas by the decree of November 6, 2013,
according to their physiology and their sensibility to contamination and purification. Since 1989,
REMI (Surveillance network established by Ifremer) has been providing microbiological
surveillance and revision of the ranking of French shellfish growing areas. This surveillance
represents each year about 4200 Escherichia coli results obtained from 410 sampling sites. In 30
years, more than 105 000 results make it possible to identify trends related to the evolution of
anthropic pressures (sanitation, urbanization...). The results of the REMI are also considered by
the Water Framework Directive (2000/60 / EC) to diagnose and implement actions to reclaim the
quality of shellfish waters (investments in wastewater treatment in particular).
75
Poster #17
Algal blooms in Bahía Magdalena, B.C.S., México, 2015-2018
Barón-Campis Sofía1, Jiménez-Quiroz María del Carmen2, Arce-Rocha Graciela1, Vázquez-
Gómez Norberto1, Blasio-Aguilar Selene1, Cervantes-Duarte Rafael3, Ponce-Manjarrez Erick1
and Vega-Juárez Germán1.
1Dirección General Adjunta de Investigación en Acuacultura, Instituto Nacional de Pesca y
Acuacultura. 2Dirección General Adjunta de Investigación Pesquera del Pacífico, Instituto Nacional de Pesca y
Acuacultura. 3Centro Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional
e-mail: [email protected]
Bahía Magdalena, the most important coastal lagoon in Baja California, has presented
significant changes in the environment since 2014, as a result of global warming and the co-
occurrence of “La Mancha” and “El Niño”. Monthly sampling was conducted from January 2015
to August 2018 (except winter 2016), with water collection for quantification of inorganic nutrients
and phytoplankton in nine seasons, at two and three levels deep. Phytoplankton was fixed with
lugol acetate and quantified using the Uthermöhl method. During this period, diatom blooms have
been identified: Eucampia zodiacus (April-2015), Guinardia/Rhizosolenia (September-2016),
Rhizosolenia setigera (August 2017), and Guinardia striata (June-2018), associated with the
increase in nutrients provided by the upwelling (spring) and internal processes of the bay (summer-
autumn). 27 species of toxic and harmful phytoplankton have been identified, among which the
Pseudo-nitzschia delicatissima diatom, and the dinoflagellates: Dinophysis acuminata, D. fortii,
Gymnodinium catenatum, Karlodinium cf. veneficum, Prorocentrum gracile and P. shikokuense,
for their frequency and abundance. Those blooms produced low oxygen production in some areas
of the lagoon, with no reports of deaths or other impacts in aquaculture areas. It is necessary to
continue with the nutrient and phytoplankton evaluations and determine the contribution of these
compounds by the port of San Carlos, the largest population (approximatly 7,000 inhabitants), to
encourage the cultivation of mollusks in the region.
76
Poster #18
Evaluation of the phytoplankton community during 2016-2017 in
Bay of San Quintín, B.C., Mexico
Zumaya L., Ruíz de la Torre M.C., Mejía Trejo A. and Vivanco Aranda M.
Universidad Autónoma de Baja California. Carretera Ensenada-Tijuana 3917, Fraccionamiento
Playitas, 22860, Ensenada, B.C., México. e-mail: [email protected]
The algal blooms and the phytoplankton community are recently studied with greater
interest due to socio-economic impacts and the ecosystem. This study analyzes the phytoplankton
community of San Quintín Bay (SQB) in Baja California, Mexico, as well as its relationship with
hydrographic and its implications in the aquaculture use of the area for two years (2016 and 2017),
at the same time The efficacy of a phytoplankton monitoring implemented by the Mexican Bivalve
Mollusc Health Program (PMSMB) is evaluated. The data used in this work were provided by the
State Committee for Health and Safety of Baja California (CESAIBC), who collaborate in
phytoplankton monitoring implemented by the Federal Commission for Prevention against
Sanitary Risks (COFEPRIS) since 2015 for compliance with one of the guidelines indicated in the
PMSMB for the certification of extraction zones. This monitoring is carried out by network drag
and water intake per bottle at three points within the bay, however, in this study only data from the
network sampling were used. Among the results obtained, the main phytoplankton groups were
diatoms and dinoflagellates constituting 99% of the population during the two years, in 2016 there
were greater abundances during the upwelling season (100,000 cel / L), and in the 2017 there are
more abundances every 4 months (900,000 cel / L). The abundances were greater for 2017 by 80%
which is attributed to the effect of El Niño of 2015. The dominant group was the diatoms (70% in
2016 and 60% in 2017). In addition, it was found that the tide regulates the distribution of
phytoplankton in the three seasons, the upwelling index influences the increase in phytoplankton
abundances and the temperature influences the appearance of diatoms or dinoflagellates, therefore,
it is important that these variables are taken together with phytoplankton monitoring. Some genera
found were Pseudonitzschia sp., Dinophysis sp. and Prorocentrum sp., which have a harmful
potential for biotoxin production.
77
Poster #19
Panopea globosa as a potential source of paralytic shellfish toxins
for certified reference materials
Medina-Elizalde, Jennifer1, García-Mendoza, Ernesto2 and Sánchez-Bravo, Yaireb A.2
1 Doctorado en Biotecnología Marina, Posgrado en Ciencias de la Vida, Centro de Investigación
Científica y de Educación Superior de Ensenada. Carretera Ensenada-Tijuana 3918, Ensenada,
Baja California, México. CP 22890 e-mail: [email protected] 2 Departamento de Oceanografía Biológica, Centro de Investigación Científica y de Educación
Superior de Ensenada.
Paralytic Shellfish Toxins (PSTs) are a group of neurotoxic alkaloids produced by several
species of phytoplankton that can accumulate in shellfish mollusks and filter-feeding fish,
crustaceans and some gastropods. Consumption of contaminated shellfish with PSTs, mainly
bivalves, can lead to paralytic shellfish poisoning (PSP) in humans. This syndrome can cause death
from cardio respiratory arrest. Due the risk for human health, PST are monitoring by the Health
Authorities. The reference method for PSTs determination is Mouse Bioassay (MBA), but analytic
methodologies such as High Performance Liquid Chromatography (HPLC) had been incorporated
into international legislation. These methodologies require Certified Reference Materials (CRMs)
for standardization and validation of protocols. Contaminated shellfish are the source of CRMs.
Panopea globosa is an economically important bivalve in the Gulf of California that accumulates
PSTs. Four harmful algae blooms (HABs) of PSTs produced have occurred in the North of the
Gulf of California between 2015 and 2019. Accumulation of PSTs in geoduck was up 49,000
µgSTXeq Kg-1. The most abundant PSTs analogs detected in clams during those events were
C1&2, GTX5, dcGTX2&3 and STX. M-analogs were also detected at high concentrations. The
structure of this type of toxins has not yet been elucidated. There are no certified reference
standards of these toxins and its quantification and toxicity estimation have been performed based
on structure-activity relations with other saxitoxin analogs. The amount of M analogs present in
P. globosa makes this organism as a potential naturally contaminated source of PSTs for CRMs
production, and particularly, M-analogs.
78
Poster #20
Practical experiences in the culture and use of venus clam from approved
areas by the PMSMB (Mexican Bivalve Mollusk Sanitation Program)
Mesina-Herrera Alfredo, Lizarraga-Velasquez Francia
and Flores-Higuera Francisco Antonio
Sociedad Acuícola Golpac, S.C. De R.L. De C.V. Avenida 55 No. 551 entre callejón Benito
Juárez y calle Juan Aldama Col Nuevo Peñasco CP83553 Puerto Peñasco, Sonora.
The northwest of Mexico offers a large number of species of bivalve mollusks that can be
used for human consumption. The first records of exports of Venus clams to the United States are
from 1997, following the regulations of the Mexican Program of Sanitation of Bivalve Mollusks
(PMSMB), exports of Venus clam through the GOLPAC Certified Plant in 2018 are 750 tons. This
report presents the results of the cultivation of Chionista fructifraga Sonora and Sinaloa, grown in
areas classified as approved by the PMSMB. Juveniles 3–4 mm long of C. fructifraga were planted
in 4 areas classified by the PMSMB Estero Morúa, San Jorge Bay, Santa Bárbara-El Riito in
Sonora and the Bahía Altata-Ensenada Pavilion in Sinaloa. Combined techniques were used with
the sowing and pre-fat suspension (45 days) of 2,000 seeds per basket and the bottom culture. In
Sonora a length of 20 mm was achieved at the time of cultivation, while in Sinaloa a length of 40
mm was achieved during the 8 months of the experiment. The bivalve mollusks cultivation areas
in Sinaloa are considered highly productive coastal systems, favored by the transitional climate
between the template region and the tropical region. Clam growth in Sonora is also considered
adequate, however, more time is required in cultivation to achieve commercial sizes. The
information shown here shows that the marketing and cultivation of Venus clams is important
adding that there are areas, plants and harvesters with sanitary certificates therefore, the cultivation
of the Venus clam is technically and financially viable.
79
SOCIAL PROGRAM
Icebreaker Party
September 8 at 7:00 p.m. (local time)
Boules Restaurant Calle Moctezuma 623, Ensenada, 22800, México
Free transportation from and to the meeting point
Meeting point
Holiday Inn Hotel parking lot
at 6:45 p.m. (local time)
Event sponsoured by
Closing Dinner at Valley of Guadalupe
(Wine Region)
September 13, 7:00 -11:00 p.m. (local time)
Free transportation from and to the meeting point
Meeting point
Holiday Inn Hotel parking lot
at 6:00 p.m. (local time)
