european union reference laboratory for mollusc diseases technical

Transcription

EUROPEAN UNION REFERENCE LABORATORY
FOR MOLLUSC DISEASES
TECHNICAL REPORT
2010
Laboratoire de Génétique et Pathologie,
Av. de Mus de Loup, 17390 La Tremblade, France
Technical Report from the European Union Reference Laboratory for Molluscs Diseases - 2010 - page 1
Technical Report from the European Union Reference Laboratory for Mollusc Diseases
2010
Content:
Introduction
Functions and duties for the European Union Reference Laboratory for mollusc
diseases
Technical report
Organise the 2010 Annual Meeting for the National Reference Laboratories
Produce a report from this meeting
Survey and diagnosis of mollusc diseases
Establish and maintain the library of mollusc pathogens
Establish a panel of histological reference material of pathogens included in the
Council Directive 2006/88/EC for distribution to National Reference
Laboratories
Update the website of the European Union Reference Laboratory
Supply available reference reagents and material to the National Reference
Laboratories in Member States
Identify and characterize mollusc pathogen isolates
Organise an Inter-Laboratory Proficiency test for pathogens included in
Directive 2006/88
Assess alternative methods for the identification of listed pathogens
Provide opportunities of training in laboratory diagnosis of molluscs diseases
Welcome and host visitors to the European Union Reference Laboratory
Attend international meetings and conferences
Prepare a handbook for diagnostic procedures in a CD-ROM format
Promote Quality Assurance in diagnostic laboratories for mollusc diseases
Annex 1: Report from the 2010 Annual Meeting of the National Reference
Laboratories for Mollusc Diseases
Annex 2: Reference Material sent by the EURL during 2010
Annex 3: Characterization and analysis performed by EURL in support of other
laboratories during 2010
Annex 4: Training and scientific collaboration in 2010
Annex 5: Publications relevant to the work of the EURL
Annex 6: Presentations at international conferences and meetings
Cover picture: Oyster ponds ("claires ostréicoles") in Oléron Island, Charente-Maritime, France
(Picture O. Dugornay, Ifremer)
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A. Introduction:
The Ifremer laboratory for Genetics and Pathology in La Tremblade (Laboratoire de
Génétique et Pathologie) has been appointed as the European Reference Laboratory for
mollusc diseases (EURL) since December 1995. Functions and duties of the Reference
Laboratory are established by the Council Directive 2006/88/EC, Annex VI, Part I, and
follow this introduction for your information. These duties of the EURL cover pathogens of
molluscs as listed in the Council Directive 2006/88/EC Annex IV Part II namely Bonamia
ostreae, Bonamia exitiosa, Marteilia refringens, Perkinsus marinus and Mikrocytos mackini.
In 2010, the EURL included eight staff members consisting in three technical assistants and
five scientists. The EURL was accredited in histopathology on the 1st October 2009. The
accreditation was renewed in October 2010 and the EURL is presently building another
quality management system for the organisation of interlaboratory comparison tests.
In March 2010, the Annual Meeting for National Reference Laboratories for Mollusc
Diseases was held in Nantes. Besides the traditional presentation of the current mollusc
disease situation of individual countries, a session focused on abnormal mortality events
affecting Crassostrea gigas in Europe since 2008. The meeting also included a session on the
health situation of molluscs in Japan and presentations on traceability issues, on recent results
from field studies or reseach works.
This report is based on the tentative working programme previously submitted to the
Commission for the year 2010 and describing activities to be undertaken on each item as well
as current status of projects.
La Tremblade, March 2011
Isabelle Arzul, Céline Garcia and Jean-Pierre Joly
B. Functions and duties for the European Union Reference Laboratory for mollusc
diseases Council Directive 2006/88/EC - Annex VI – Part I
The European Union reference laboratories shall:
(a) coordinate, in consultation with the Commission, the methods employed in the
Member States for diagnosing the disease concerned, specifically by:
(i)
typing, storing and, where appropriate, supplying strains of
the pathogen of the relevant disease to facilitate the
diagnostic service in the Community,
(ii)
supplying reference reagents to the national reference
laboratories in order to standardise the tests and reagents
used in each Member State,
(iii)
organising periodic comparative tests (ring tests) of
diagnostic procedures at Community level with the national
reference laboratories designated by the Member States, in
order to provide information on the methods of diagnosis
used and the results of tests carried out in the Community;
(iv)
retaining expertise on the relevant disease pathogen and other
pertinent pathogens to enable rapid differential diagnosis;
(b) assist actively in the diagnosis of outbreaks of the relevant disease in Member
States by receiving pathogen isolates for confirmatory diagnosis,
characterisation and epizootic studies;
(c) facilitate the training or retraining of experts in laboratory diagnosis with a view
to harmonising diagnostic techniques throughout the Community;
(d) collaborate, as regards methods of diagnosing animal diseases falling within
their areas of competence, with the competent laboratories in third countries
where those diseases are prevalent;
(e) collaborate with the relevant OIE reference laboratories with regard to exotic
diseases listed in Part II of Annex IV under their responsibility;
(f) collate and forward information on exotic and endemic diseases, that are
potentially emerging in Community aquaculture.
C. Technical report:
Objectives for
the period
January
December 2010
TECHNICAL REPORT
Organise and
prepare the 2010
Annual Meeting
of the National
Reference
Laboratories
The 2010 Annual Meeting of National Reference Laboratories for Mollusc
Diseases was held in IFREMER in Nantes on the 23rd and 24th of March
2010. In total, 42 participants from 16 European countries (Bulgaria,
Denmark, France, Germany, Greece, Italy, Norway, Poland, Romania,
Slovenia, Spain, Sweden, The Netherlands, United-Kingdom), and experts
from Japan and USA and one representative from DG-SANCO and one
from EFSA attended the meeting.
The Annual Meeting included seven sessions:
1) Current epidemiological situation in the Member States
2) Crassostrea gigas abnormal mortality
3) Mollusc health situation in Japan
4) Traceability
5) Results of recent field studies
6) Studies on interactions between host and pathogens
7) European Reference Laboratory day life activities
1. The first session concerned the epidemiological situation of
mollusc diseases in Europe. Participating countries reported on
their own situation with regards to diseases of molluscs and
abnormal mortality outbreaks, case studies and investigations. In
2009, major epidemiological changes in EU were (1) the detection
of Marteilia refringens (type M) in Sweden (2) the suspicion and
confirmation by the EURL of bonamiosis due to Bonamia ostreae
in flat oysters from Norway (3) the detection of Bonamia exitiosa
in France, once again in flat oysters from Corsica, and in Galicia,
Spain (4) the mass mortalities associated with the presence of
herpesviruses OsHV-1 µvar which affected Crassostrea gigas in
France, Ireland and Jersey.
2. The second session focused on the abnormal mortality events
observed in the Pacific cupped oysters Crassostrea gigas in
Europe and on the Commission Regulation (EU) N° 175/2010.
This new regulation concerns measures to control increased
mortality in C. gigas in connection with the detection of the newly
described OsHV-1 µvar. Recent results obtained on OsHV-1 were
presented during this session including experimental works and a
survey carried out in Tomales Bay in California, U.S.A.
3. The third session was devoted to the health situation of molluscs in
Japan. Decrement of manila clam resources associated with
perkinsosis and mass mortality of pearl oysters potentially due to
Akoya virus are serious concerns currently faced by the bivalve
industries of Japan. The ovarian protozoan parasite Marteilioïdes
chungmuensis, also threatens oyster industry in Japan. In Europe,
where it has never been observed, vigilance should be given to this
parasite by being able to detect it and by avoiding any introduction
of infected animals.
4. The fourth session dealt with traceability. Directive 2006/88/EC
has a set of provisions which aimed at ensuring traceability of
aquaculture animals. Implementation of these provisions for
molluscs is in progress in Europe while for fish it is much
advanced. Example of the implementation of the Directive
2006/88/EC for mollusc and fish issues were presented and
discussed.
5. During the fifth session, results from field studies were presented
including the detection of Bonamia ostreae in larvae of flat
oysters; mussel infection with intracellular ciliates and neoplasia in
Slovenia ; the co detection of Perkinsus chesapeaki and P. olseni
in Ruditapes decussatus in South of France.
6. The sixth session focused on host-pathogen interactions. Some
recent results concerning host-pathogen interactions were
presented included Ostrea edulis - Bonamia ostreae and Ruditapes
decussatus - Perkinsus olseni models. These information are of
central interest in order to propose some alternative measures to
producers like advices for stock management or development of
resistant host strains.
7. In the last session some information related to the EURL activities
were presented like the last inter laboratory comparison tests,
quality management aspects and perspectives for 2010.
A report of the meeting is appended at the end of this document (Annex
1).
Collect material
for and produce
report covering
the 2010 Annual
Meeting of NRLs
As stated in the above section, the 2010 Annual Meeting was held in
March 2010 in IFREMER in Nantes, France. A report from this meeting
was produced providing information on the content of communications
and discussions held during the different sessions.
A report of the meeting is included in annex 1.
Collate material
concerning the
results of
diagnosis and
surveys
regarding
mollusc diseases
Countries attending the Annual Meeting were asked to fill the
questionnaire sent annually by the EURL comprising three sessions: 1)
General data on mollusc production 2) Diagnostic methods used by
laboratories for the detection of mollusc diseases 3) Epidemiological
results for the surveillance of mollusc diseases in 2009.
Establish and
maintain the
library of
mollusc
pathogens.
This task is of central importance in the duties of the EURL. Given the
pivotal role of histology in diagnostic procedures for diseases of molluscs,
a particular emphasis is given to histological blocks, sections and slides.
However more and more requests concern samples for molecular tests. In
that case, tissues collected from infected or non-infected specimens or
DNA extracted from such tissues are provided as positive and negative
controls. Plasmid DNA suspensions (containing PCR fragments) can also
be provided for example as controls for PCR-RFLP for Bonamia species
determination or Marteilia refringens typing.
Establishing and maintaining the library of mollusc pathogens covered the
following items in 2010:
Maintain the existing library :
a library of histological sections was created in 1997 which, now, includes
materials covering the main pathogens of molluscs found in Europe and
more broadly in the World (Bonamia ostreae, B. exitiosa, B. roughleyi, B.
perspora, Bonamia isolates from Chile and from Argentina, Marteilia
refringens, M. sydneyi, Perkinsus olseni, P. marinus, P. chesapeaki, P.
qugwadi, Iridovirus, Herpesvirus, Haplosporidium nelsoni, H. costale,
Mikrocytos mackini, Vibrio tapetis, Vibrio harveyi, Vibrio spp., Nocardia
crassostreae, Marteilioides chungmuensis, Xenohaliotis californiensis,
Minchinia tapetis, Marteilia isolates from Ruditapes philippinarum and
Ensis ensis etc...). Bacterial strains are also conserved in the EURL
collections. Most of them are type strains of Vibrio species or Vibrio
strains isolated from mortality outbreaks.
Acquire new reference material (strains, slides, blocks) :
The collection is continuously enriched with new reference material,
requested on a regular basis from scientists publishing papers in the field
of mollusc pathology.
Give an easy access to the library:
This has been done in the previous years by editing and updating a EURL
catalogue, under a Microsoft Excel format, of the library giving access to
the reference material by key words such as disease, pathogen, host
species, lesion type, geographic origin, etc…
The collection of photos, created in 1999, has been included in a practical
guide for diagnosis in histology established under a html format. This
guide was burnt on CDs and distributed to participants of workshops and
visitors.
A new tool for training in histology and relying on some virtual slides
accessible by NRLs from the mollusc EURL website was put in place in
2009. These slides include the previous ring test slides as well as some
specific slide collections.
Establish a panel
of reference
material of
pathogens of
interest for
distribution to
National
Reference
Laboratories.
As stated above, the library of mollusc pathogens of the EURL includes
histological paraffin blocks as well as ethanol fixed infected tissues of
pathogens listed in the Directive 2006/88/EC which are used to prepare
and distribute, on request, hemalun-eosin (H&E) stained histological
slides and Hemacolor® stained imprints or DNA suspensions for
molecular test controls.
Update the
website of the
European Union
Reference
Laboratory.
The EURL website appearance has been changed due to the reorganisation
of all the Ifremer websites in 2010 (see picture below). It took a few days
to reorganise the pages due to several small bugs occurring during the
transfer of the old pages to the new website.
This reorganisation was the opportunity to change the name and the URL
of the website. The URL is now www.eurl-mollusc.eu to be in accordance
with the EURL for fish diseases (www.eurl-fish.eu).
The website is now organised in seven sections:
• News
• NRL’s pages (if connected)
• Presentation
•
•
•
•
Main activities,
Scientific activities,
SOPs and Quality,
Legislation.
The pages give access to information on legislation, to leaflets about some
pathogens of concern and to some standard operating procedures (SOPs)
useful for the NRLs in the field of histopathology and molecular biology.
Links to other resources of interest for people working in the field of
mollusc health certification and disease survey programmes are also
provided like:
•
•
•
•
OIE Aquatic Animal Diseases Commission,
DFO synopsis of infectious diseases of molluscs,
Aquatic animal pathology database AAPQIS,
FAO database reporting the movements and transfers of aquatic
species.
The website is also a place of advertisements for events in the field of
mollusc health management (workshop, annual meeting, histopathology
and molecular biology proficiency tests).
The website of the EURL has been regularly updated during 2010,
particularly some SOPs (see below).
A new section called “NRL’s pages” has been introduced for the
exclusive use of the European NRLs. This section is presently under
construction and will soon be available. The access to this section will
need to enter a user name and a password in the “login” panel (on the left
of all the pages). This section does not appear in the menu when people
are not connected. When connected, one can access several pages with
specific information like reports of the annual meetings and workshop (see
picture below).
The 10 NRLs pages are organised as follow:
•
•
•
Proficiency test
o Registration
o Results and reports
o Slide viewer
Annual meetings and workshops
o Programmes
o Presentations
o Reports
o Image gallery
EURL annual reports
The “Registration” page gives access to a form to register on line for the
next proficiency test. The “Slide viewer” page is of particular interest for
the European scientists taking the histopathology test. We are presently
organising this page to give access to the scan of all the slides that were
presented during the last histopathology test. When connected people have
access to a special website where the slides scans are stored. By using a
software from this website they are able to browse the entire slides at
several magnifications. They can visualise pathogens they could have not
recognised during the test for example. It can be a perfect training tool for
new scientists positioned in a European Reference Laboratory for
example.
All the information about this new section and the necessary “login” and
“password” will be given to the European NRLs during the 2011 Annual
Meeting.
Some documents have been updated like SOPs on diagnosis of Mikrocytos
sp. by histopathology and diagnosis of Marteilia refringens by PCRRFLP. Some leaflets of the “Tutorial” page have also been updated.
The “News” page is now directly accessible from the website home page
and has been regularly updated, particularly with information on the
mortality events of oysters Crassostrea gigas during summer 2010 in
France.
Supply available
reference
reagents and
material to the
National
Reference
Laboratories in
Member States.
Reference material provided to laboratories working on mollusc diseases
usually consists of H&E stained histological sections and paraffin blocks
when available, bacterial strains as well as DNA suspensions.
In 2010, the EURL answered thirty one requests. Reference material was
provided to the National Reference Laboratories and other laboratories
within EU and from third countries.
A table summarizing material provided to the different laboratories in
2010 is included in Annex 2. Part of this material consisted of plasmidic
DNA suspensions and primers for the detection and characterization of
OsHV-1/OsHV1µvar by PCR.
Identify and
characterize
pathogen
isolates.
The EURL was involved in investigations and diagnosis of different cases
on request of NRLs or diagnostic laboratories from third countries (See
table in Annex 3). Technical assistance and advice were provided by the
EURL to colleagues from :
1. Division of Fish and Shellfish, Department of Animal Health
and Antimicrobial Strategies, National Veterinary Institute, S751 89 Uppsala, Sweden, for histological examination of
Mytilus edulis (4 histological slides, 4 pieces of tissue) and
parasite identification by PCR-RFLP in the context of
suspicion of marteiliosis. Infection with Marteilia refringens
type M was confirmed.
2. Istituto zooprofilattico sperimentale delle venezie, Sezione Di
Adria, Via Leonardo da Vinci 39, 45011 Adria, Italy for
detection and characterization of OsHV-1 / OsHV-1 µvar in
Crassostrea gigas. The oysters (30 pieces of tissue and 16
DNA suspensions) were tested by Real Time PCR, PCR, and
sequencing was performed after cloning. The suspicion of
infection with OsHV-1 and OsHV-1 µvar was confirmed.
3. National Fish Health Research Centre, Fisheries Research
Insitute, 11960 Batu Maung, Penang, Malaysia for histological
examination of mussels Perna canaliculus (3 slides).
Histological examination did not reveal the presence of known
mollusc protozoans.
4. Graduate School of Agricultural Science, Tohoku University,
Tsutsumidori Amamiya-machi, Aoba-ku, Sendai 981-8555
Miyagi, Japan for histological examination, PCR-RFLP and
sequencing for parasite characterization in Ostrea edulis (5
paraffin blocks and 10 pieces of tissue). Results revealed the
absence of Marteilia spp. and the presence of Bonamia ostreae
in the tested animals.
5. Fish Health Unit, Marine Institute, Oranmore Rinville, C.o
Galway, Ireland for histological examination (6 slides) of
Crassostrea gigas. Examination revealed the presence of
lesions that could suggest the presence of viruses.
6. INRH, Casablanca, Morroco to test the presence by histology
and in situ hybridization (13 paraffin blocks) of Marteilia sp.
and then characterize parasites by PCR-RFLP and sequencing
(45 pieces of tissue) in mussels Mytilus galloprovincialis and
Perna perna. Results confirmed the presence of Marteilia
refringens in both mussel species.
7. Institut National Agronomique de Tunisie, Tunis, Tunisie, to
test the presence by histology and in situ hybridization (20
paraffin blocks) of Marteilia sp. and then characterize parasites
by PCR-RFLP and sequencing (15 pieces of tissue) in oysters
Ostrea stentina. Results confirmed the presence of Marteilia
refringens in the oysters.
8. Fisheries Research & Development Institute, 152-1, Haeanro,
Gijang-Gun Busan 619-705, Korea to test the presence of
Perkinsus spp., Bonamia spp and OsHV-1 in oysters
Crassostrea gigas (12 histological slides and 12 pieces of
tissue). Results did not reveal the presence of Bonamia spp.
and OsHV-1 but histological examination revealed the
presence of Perkinsus like parasites in one oyster.
9. Institute for Infectious and Parasitic Diseases -Department of
Aquatic Organisms Pathology, 80, 26th Octovriou str., 546 27
Thessaloniki, Greece to test the presence of Marteilia sp by
histology (22 paraffin blocks) and characterize parasites by
PCR-RFLP and sequencing (12 pieces of tissue) in oysters
Ostrea edulis and mussels Mytilus galloprovincialis. Results
confirmed the presence of Marteilia refringens in the mussels.
10. Centro de Investigaciones Biológicas del Noroeste, S.C. Mar
Bermejo No. 195, Col. Playa Palo de Santa Rita; La Paz, BCS
23090, México to test the presence of Perkinsus spp. by PCR
(92 pieces of tissue) and characterize by RFLP and sequencing
the parasites in oysters Crassostrea corteziensis and C. gigas.
Results confirmed the presence of Perkinsus marinus in some
oysters. In addition some tests were performed by Real Time
PCR to check the presence of OsHV-1, one oyster appeared
positive by this technique with a very low viral load.
11. INSTM, Unité de Pathologie des animaux aquatiques 20025
Salammbô, Tunisia for the double reading of 7 slides.
Formal studies had been scheduled for the year 2010 in the EURL
working plan. The background of these studies, overall goals,
methodology used and main outputs are given in the following sections.
Investigations on Bonamia ostreae and other related parasites
The genus Bonamia includes small-size (2-3 µm) uninucleated
protozoan parasites also called microcells. Two microcells are presently
listed in Annex IV, Part II of the Directive 2006/088/EC: Bonamia ostreae
which affects populations of European flat oyster Ostrea edulis and B.
exitiosa which infects O. chilensis from New Zealand and O. angasi from
Australia. Other known species of the genus are B. roughleyi infecting
Saccostrea commercialis from Australia and B. perspora described in
Ostreola equestris from North Carolina, USA. Besides these four species,
no fully charcaterized Bonamia spp. were reported in O. chilensis in Chile
and in O. puelchana in Argentina.
The notification of Bonamia exitiosa in Spain in October 2007 and its
detection in Adriatic Sea in Italy in Ostrea edulis outside mortality
occurrence supports the apparent wide distribution of the parasite all
around the world and questioned its impact on European flat oyster
populations. The presence of both parasites B. exitiosa and B. ostreae in
same areas and even in same individuals reinforces the need of specific
diagnostic tools.
In this context, in 2010 the EURL has focused its activities on (1) the
characterization of parasites of the genus Bonamia present in Europe and
Mediterranean basin, (2) the characterization of new genes of interest for
taxonomic and diagnostic purposes, (3) some studies to better understand
the cycle of the disease and (4) some study to better understand hostpathogen interactions
(1) Characterisation
Mediterranean basin
of
Bonamia
parasites
in
Europe
and
In October 2007, Spain notified the presence of Bonamia exitiosa in
some flat oysters collected from Ria de Arousa, in Galicia.
Subsequently, the EURL in collaboration with Italian colleagues
detected Bonamia exitiosa in flat oysters Ostrea edulis from Manfredonia
Gulf (Adriatic sea, Italy) (Narcisi et al. 2010).
In 2008, a workplan to characterise Bonamia parasites in Europe has
been proposed by the EURL to NRLs with the support of the European
Commission. The outcome of this study was to determine the spread of B.
exitiosa in the EU.
In 2009, in the context of this survey, Spain detected again the
presence of B. exitiosa in cultivated but also wild flat oysters from
Galicia. In addition to this working programme, some parasites identified
as Bonamia exitiosa were detected in France: during a study carried out
on a natural bed of flat oysters in Corsica, and in association with a
mortality event which affected flat oysters in the Mediterranean Sea near
Thau lagoon.
As conclusions, so far Bonamia exitiosa has been detected and
confirmed in 3 Member States: France, Italy and Spain. However, where
Bonamia exitiosa was not detected, it is not possible to conclude that these
locations are free of Bonamia exitiosa. Mixed-infection in same location
and even in same oyster was observed. The map needs to be completed
and more studies are required to understand why this parasite is there and
to evaluate its impact on flat oyster populations.
In April 2010, Spain detected Bonamia exitiosa in flat oysters collected
from Les cases de Alcanar, in Cataluna. This detection was notified to the
OIE on the 16th of July. In July 2010, Italy detected B. exitiosa in flat
oysters collected from two locations in Veneto region : Delta Po and
Venice Lagoon. This detection was notified to the OIE on the 27th of
October.
In parallel, the EURL gave support to colleagues from Turkey and
Tunisia to test the presence of protozoans in the flat oysters Ostrea edulis
and O. stentina respectively. From tunisian samples, only DNA
suspensions were available. PCR-RFLP tests allowed obtaining B. exitiosa
profiles. Subsequent sequencing work confirmed RFLP results. From
Turkish samples, both histology and molecular tests were carried out and
allowed concluding about the presence of B. exitiosa in some tested
oysters.
Narcisi V., Arzul I., Cargini D., Mosca F., Calzetta A., Traversa D., Robert M.,
Joly J. P., Chollet B., Renault T., Tiscar P. G. (2010) Detection of Bonamia
ostreae and Bonamia exitiosa (Haplosporidia) in Ostrea edulis from the Adriatic
Sea (Italy). Diseases of Aquatic Organisms. 89:79-85
(2) Characterization of new genes of interest for taxonomic and
diagnostic purposes
Bonamia parasites are small no cultivable protozoans. Studies aiming
at improving knowledge of their genomes are thus hampered by the
difficulty of obtaining sufficient amount of pure parasites. Until now, the
only characterized parts of the genome of Bonamia ostreae were some
ribosomal RNA genes and two actin genes. New assays were developed in
order to improve these data and allowed identifying a gene encoding a
Heat Shock protein 90. Further works are needed in order to fully
characterize this gene.
(3) Studies on the parasite cycle
The distribution of the parasite within its host has not been fully
elucidaded, especially its routes of entrance and release. A cohabitation
experiment between infected flat oysters and naive ones was performed
few years ago and challenged oysters were collected in order to follow the
infection using in situ hybridization. First tests allowed observing the
parasites in epithelia of different organs and then infected hameocytes in
different tissues. In 2010, the EURL wanted to completed these results by
measuring parasite load in different organs using Real Time PCR recently
developed in the laboratory (Robert et al. 2009). This work is still in
progress.
On an other hand, the EURL investigated the susceptibility of flat
oyster larvae to bonamiosis. It was commonly admitted that the disease
mainly affect older stages. Considering that flat oysters are larviparous
(they incubate larvae for 8-10 days within the palleal cavity before
releasing them in the water column), we took benefit from a survey on the
reproduction of flat oyster carried out in Quiberon Bay, Brittany in France
to obtain some oysters incubating larvae between 2007 and 2009. We
tested both genitors and larvae by PCR and in situ hybridization and were
able to detect Bonamia ostreae in both stages suggesting that the parasite
can be transmitted from genitor to larvae during this incubation period.
Larvae might thus contribute to spread the disease during their planctonic
life (Arzul et al. Accepted in Veterinary parasitology).
Pictures:In situ hybridization test of Ostrea edulis larvae from Quiberon bay
(Brittany, France) using the probe Bo-Boas (Cochennec et al. 2000). Pictures B.
Chollet
Robert M., Garcia C., Chollet B., Lopez-Flores I., Ferrand S., François C., Joly
J.-P and Arzul I. (2009) Molecular detection and quantification of the protozoan
Bonamia ostreae in the flat oyster, Ostrea edulis. Molecular and Cellular probes,
23: 264–271
Arzul I., Langlade A.; Chollet B.; Robert M.; Ferrand S.; Omnes E.; Lerond S.;
Couraleau Y.; Joly J.-P.; François C. ; Garcia C. (2010) Can the protozoan
parasite Bonamia ostreae infect larvae of flat oysters Ostrea edulis?. Accepted in
Veterinary Parasitology
(4) Studies on interactions between flat oysters and Bonamia ostreae
The means to fight against bonamiosis are relatively limited. They are
mainly based on oyster health surveillance to limit the spread of the
disease. However, the use of predictive models of disease progression in
infected area would help to improve stock management and minimize the
impact pathogens. Moreover the development of resistant animals could
help to revive this production. These different approaches require
appropriate diagnostic tools, a good knowledge of the life cycle of the
pathogen, and of interactions between the parasite and its host. In this
context, studies were carried out to understand the interactions between
the flat oyster Ostrea edulis and the parasite Bonamia ostreae, and
particularly the molecular basis of the resistance to the parasite. In a first
step, a subtractive cDNA bank allowed the identification of ESTs
differentially expressed in haemocytes in response to the parasite.
Expression of some genes, among which a galectin, was measured by Real
Time PCR in the context of in vitro infections. In addition, the cellular
response was investigated by flow cytometry and the infection was
checked by microscopy. These experiments showed a multiplication of the
parasite inside haemocytes associated with a decrease of esterases and
production of ROS. In a second step, a comparative approach was carried
out between a population of oysters resistant to bonamiosis and a natural
population. Results suggest that modulation of apopotosis and decrease of
phagocytosis could be involved in mechanisms related to resistance to
bonamiosis.
Some of these results have been published:
Morga B., Arzul I., Faury N., Renault T. 2010. Identification Of Genes From Flat
Oyster Ostrea Edulis As Suitable Housekeeping Genes For Quantitative Real
Time PCR. Fish and Shellfish Immunology 29(6):937-945.
Morga B., Renault T., Faury N., Chollet B., Arzul I Cellular and molecular
responses of haemocytes from Ostrea edulis during in vitro infection by the
parasite Bonamia ostreae. Accepted in International Journal of Parasitology.
Morga B, Arzul I, Faury N, Segarra A, Chollet B, Renault. T. Molecular
responses of Ostrea edulis haemocytes to an in vitro infection with Bonamia
ostreae. Dev Comp Immunol. doi:10.1016/j.dci.2010.10.005 |
Studies on the life cycle and host range of Marteilia refringens
Marteiliosis due to Marteilia refringens occurs in Europe where it
infects flat oysters, Ostrea edulis, and mussels, Mytilus edulis and M.
galloprovincialis. Two types, O and M were defined on the basis of the
presence or absence of a restriction profile in the ITS-1 region of the
genome. Type M is more often detected in mussels and type O in flat
oysters, however co-infection exists. The event seems to be restricted to
areas where the prevalence of the disease is high. Its life cycle seems to
involve at least one intermediate host and the copepod Paracartia grani is
more than suspected to act as an intermediate host since the parasite could
be transmitted from infected oysters and infected mussels to healthy
copepods.
During the last years, the EURL was involved in studies on (1) the
molecular characterization of Marteilia refringens and the development of
new diagnostic tools and (2) on the life cycle of the parasite.
(1) Molecular characterization of Marteilia refringens and the
development of new diagnostic tools
In 2010, the EURL initiated a study on the variability of Marteilia
refringens isolates originating from different locations and infecting
different hosts. Three regions of the genome were targetted, all belonging
to the ribosomal gene region: the SSU rRNA gene, the ITS-1 and the IGS.
Sequence analyses are in progress.
In 2010, the EURL also collaborated with colleagues from NRLs for
the characterization of M. refringens. Indeed, the parasite was detected for
the first time in Sweden in mussels Mytilus edulis. It is the first detection
of the parasite in a so northern European location. Marteilia refringens
was also detected in flat oysters Ostrea edulis from Greece and in flat
oysters O. stentina from Tunisia.
In addition, in 2010, the EURL have developed a duplex Taqman PCR
assay aiming at detecting and typing Marteilia refringens. This assay relies
on one primer pair and two probes, one detecting M. refringens type O and
the other one type M. Performance of this assay is currently tested at the
population level.
(2) Studies on the life cycle of the parasite.
The life cycle of the Marteilia refringens is not completely known.
However, the copepod Paracartia grani seems to be involved as
intermediate host, at least in the ecosystem of the “claire” in France. A
study performed in Delta del Ebro (Spain) showed the possible
involvement of some other zooplancton species in the parasite cycle. A
similar study involving the EURL was carried out in Diana lagoon in
Corsica, France where flat oysters and mussels cohabit as well as
Marteilia refringens. The dynamics of the infection with the parasite in
both host species has been followed as well as the distribution of parasite
stages within the different organs of infected specimens. Zooplancton was
also collected every two weeks. First analyses performed by PCR suggest
that different species (not only copepod species) might be infected or
carriers. After sorting the zooplancton species present in positive samples,
in situ hybridization tests have been performed and are presently under
progress.
In parallel, some research works have been initiated in Thau lagoon
(South of France) on population dynamics of the copepod Paracartia
grani and its relationships with Marteilia refringens.
Detection of the Ostreid Herpesvirus 1-µvar in Crassostrea gigas
Abnormal mortality events have affected Crassostrea gigas since 2008 in
Europe and especially in France. While the causes of the mortalities still
remain uncertain, the epidemiological investigations undertaken in 2009
suggest that OsHV-1 μvar play a major role in the mortalities.
To ensure a harmonised approach between Member States as regards
measures to control this emerging disease situation Commission
Regulation (EU) No 175/2010 implementing Council Directive
2006/88/EC as regards measures to control increased mortality in oysters
of the species Crassostrea gigas in connection with the detection of
Ostreid herpesvirus 1 μvar (OsHV-1 μvar) was adopted. In this context,
the EURL assisted DGSanco to prepare this regulation especially for the
diagnostic and surveillance issues.
While some Real Time PCR assays were already available for the
detection and quantification of OsHV-1, there was no such sensitive tool
available for the identification of OsHV-1 µvar versus OsHV-1.
Considering the urgent need of a test allowing distinguishing between
both variants, the EURL suggested the use of a conventional PCR assay
available at that time but less sensitive than the Real Time PCR ones. The
EURL provided both positive controls and primer suspensions to all the
concerned NRLs.
In addition the EURL has been involved in a collaboration with colleagues
from the Italian NRL to characterize OsHV-1 in some oysters Crassostrea
gigas originating from France and growing in Italy (Dundon et al.
accepted in Aquaculture)
Dundon W.G., Arzul I., Omnes E., Robert M., Magnabosco C., Zambon M.,
Gennari L., Toffan A., Terregino C., Capua I. Arcangeli G. Detection of Type 1
Ostreid Herpes variant (OsHV-1 µvar) with no associated mortality in Frenchorigin Pacific cupped oyster Crassostrea gigas farmed in Italy. Accepted in
Aquaculture
Use of experimental infection trials to explore Crassostrea gigas oyster
- Vibrio interactions and better characterize vibriosis pathogenesis
Experimental infections of aquatic animals using viruses, bacteria or
parasites can be performed to fulfill different objectives: to estimate the
efficiency of preventive treatments, study routes of infection, test the
resistance of selected lines or different ploidy groups and to assess host
factors linked to susceptibility. For bacteria, experimental infections are
also often used to explore pathogenicity coupled with phenotypic and/or
molecular characterization of virulence factors. We aimed to investigate
the relationships between ploidy, survival and reproductive allocation, in
oysters challenged with pathogenic Vibrio. First, a non-invasive
experimental
infection
protocol
coupled
with
specific
immunohistochemical analysis was used to localize pathogenic Vibrio in
oyster organs and to test for its affinity for gonadic tissue. Second,
susceptibility to vibriosis and gonad development were studied over an
annual reproductive cycle on related diploid and triploid oysters using a
reproducible experimental co-infection protocol and two pathogenic
Vibrio (V. splendidus and V. aestuarianus). Statistical analyses of
mortality kinetics over a 6-day period after injection revealed that active
gametogenesis periods correspond to higher susceptibility to vibriosis and
that there is a significant interaction of this seasonal effect with ploidy.
However, no significant advantage of triploidy was observed. Triploid
oysters even showed lower survival than diploid counterparts in winter
(De Decker et al., 2011).
We also described an original non-invasive experimental infection
technique using cohabitation, designed to explore Crassostrea gigas and
Vibrio sp., whether these be virulent or avirulent. Using real-time PCR
techniques developed for the purpose of this study, we examined the
dynamics of virulent and avirulent Vibrio sp. in oyster hemolymph and
tank seawater, and made a parallel study of the expression of four genes
involved in oyster immune defense: Cg-BPI, Cg-EcSOD, Cg-IkB, CgTIMP. Taken together, our data support (1) the hypothesis that virulent
Vibrio disturbs the immune response of this invertebrate host both rapidly
and significantly, although this occurs specifically during an early and
transient period during the first 6 h of cohabitation challenge, and that (2)
expression of targeted genes is not correlated with vibriosis resistance (De
Decker § Saulnier, 2011).
De Decker S., Normand J., Saulnier D., Pernet F., Castagnet S. & Boudry P.
(2011) Responses of diploid and triploid Pacific oysters Crassostrea gigas to
Vibrio infection in relation to their reproductive status. Journal of Invertebrate
Pathology 106(2):179-191.
De Decker, S. & Saulnier D. (2011) Vibriosis induced by experimental
cohabitation in Crassostrea gigas: Evidence of early infection and downexpression of immune-related genes. Fish and Shellfish Immunology 30:691-699.
Characterization of Perkinsus spp. present in Europe
The genus Perkinsus includes protozoan parasites of marine
molluscs in many different locations around the world. Infection may be
associated with high mortality rates. Two species, Perkinsus marinus and
P. olseni, are given particular attention because of their impact on
aquaculture.
Considering the geographical distribution of Perkinsus olseni,
occurring from Pacific Islands through Australia, Southeast Asia, to
Europe and to Uruguay and its large host range (clams, oysters, cockles,
abalones), one could wonder if this pathogen fulfils OIE listing criteria.
However, within the geographic range of P. marinus, differences in
virulence between isolates were demonstrated, suggesting existence of
several strains of this parasite might exist with differences in genetic
composition, geographic distribution and virulence. Similarly, variability
in the pathogenicity of Perkinsus olseni raises questions on the existence
of types or strains of the parasite or differences in host responses under
different environmental conditions. Since 2008, the EURL has
participated in the characterization of parasites of the genus Perkinsus
detected in different clam production areas in France and in some other
locations from the Mediterranean Basin.
Initially, a study was carried out in France to characterize parasites of the
genus Perkinsus present in clams. Clams were collected in different
locations along the French coasts in 2004 and 2005. Parasites of the genus
Perkinsus were detected and quantified by thioglycolate culture medium.
Molecular characterization was done by PCR-RFLP targeting the ITS
region. PCR products selected according to their restriction profiles were
subsequently cloned and sequenced in order to check potential co
infection within clams. Perkinsus olseni was detected in all the French
investigated producing areas. However, in Leucate lagoon (South of
France), PCR-FRLP results revealed restriction profiles similar to P.
chesapeaki ones in 2 clams. In order to confirm and complete these
results, clams were newly collected in 2008 in Leucate lagoon and tested
by in situ hybridization tests and parasite culture in DMEM:HAM’S F12
medium. Parasite cultures tested by PCR RFLP and sequencing revealed
the presence of Perkinsus olseni and P. chesapeaki–like parasite DNA. In
situ hybridization tests were indicative of the presence of both parasites P.
olseni and P. chesapeaki in the tissue of some clams. These results show
that these two species are sympatric in Leucate Lagoon and raise the
question of the presence of Perkinsus chesapeaki (a parasite only detected
in North America up to now) in France.
a
b
Pictures:In situ hybridization test of Ruditapes decussatus from Leucate lagoon
(South of France) (a) using the probe PchesLSU-485DIG specific of P.
chesapeaki (Reece et al. 2008) (b) and using the probe PolsLSU-464DIG specific
of P. olseni (Moss et al. 2006). Pictures B. Chollet
a
b
Pictures:Trophozoites (a) and zoosporangium (b) of Perkinsus chesapeaki
isolated from Leucate lagoon (South of France) cultivated in DMEM:HAM’S
F12 medium. Pictures B. Chollet
These results were presented at Aquaculture 2010. San Diego, California,
U.S.A., 1-5 March 2010..
Arzul I., Chollet B., Michel J., Robert M., Miossec L., Joly J.-P., François C.,
Garcia C. Perkinsus olseni and a new Perkinsus sp., closed to P. chesapeaki,
sympatric in clams Ruditapes decussatus from Leucate lagoon, France
Organise Inter
Laboratory
Proficiency tests
for pathogens
included in the
Directive
2006/88/EC
Since 1998, seven inter-laboratory proficiency tests have been organised
by the EURL for the detection of some mollusc pathogens by histology
and cytology (imprints). The goal of these proficiency tests is to establish
that the examination of a given sample leads to the same conclusions in
any laboratory within the National Reference Laboratory network.
All these tests included some slides of flat oysters infected or not by
Bonamia ostreae and Marteilia refringens (listed pathogens present in
Europe). In addition, some slides of other bivalve species infected or not
by listed pathogens exotic to Europe were also included and the focus of
each test was adapated according to results previously obtained by NRLs
and according to the evolution of the regulation regarding listed
pathogens.
The four first tests included 30 slides while the three last tests included 60
slides corresponding to two sets of tissue sections: 30 sections for
European pathogens and 30 other sections for other selected pathogens.
The number of participants increased from 10 laboratories in 1998 to 20 in
2007 and 18 in 2009. This increase is partly linked to the enlargement of
EU. Percentages of good answers generally increased suggesting that
participating laboratories improved their ability to detect mollusc
pathogens. However, results generally highlight the difficulty to detect
low levels of infection and also depend on targeted pathogens.
The last inter laboratory proficiency test based on histology/cytology was
organised by the EURL in 2009 and the results were analysed in 2010.
The aim of this test was to evaluate the competency of participants
regarding (1) the detection of EU listed diseases in Directive 2006/88/EC
present in Europe, (2) the detection of exotic pathogens presently
described in oysters from North America. This test included 60 slides
among which 30 corresponded to flat oyster Ostrea edulis sections or
imprint slides and 30 corresponded to cupped oysters Crassostrea gigas
and C. virginica sections. 17 laboratories participated in both collections
and one laboratory only in collection 1.
Participants had 80% of good results for collection 1. Most of the errors
were due to the difficulty of detecting light parasite infection with
Bonamia ostreae. Mean of good answers for collection 2 was 62%. These
results highlighted the difficulty of laboratories to detect exotic pathogens,
specially the parasite Perkinsus marinus.
1
80
0,8
60
0,6
40
0,4
20
0,2
0
Kappa value
% good answers
100
0
2000
2002
2005
Mean (%)
2007
2009
Kappa
Figure:Percentages of good answers and Kappa values for the inter laboratory
proficiency test based on histology/cytology organised by the EURL between
2000 and 2009
a
b
Î
c
Í
Pictures: H&E histological section from Crassostrea virginica. (a)
Stomach epithelium (arrow) (b) Detail of the previous picture (c) Detail of
the previous picture showing few Perkinsus parasites (arrow); Pictures C.
Garcia
In 2008, for the first time the EURL organised an inter laboratory
proficiency test for the detection of Bonamia spp. by PCR. The objective
of this ring test was to test the ability of NRLs to detect Bonamia spp. in
flat oyster Ostrea edulis by PCR, from the DNA extraction up to the PCR
test using reactives and the protocol sent with the samples. Participation in
this ring test was considered optional since PCR for the detection of
Bonamia spp. was not yet used routinely by all the NRLs. Percentages of
good responses were good, above 60% for all participating laboratories.
General Kappa coefficients (statistical measure of inter-rate reliability)
calculated according to Fleiss (1981) were estimated between 0.49
(moderate agreement) and 0.79 (substantial agreement).
In 2010, a new inter laboratory comparison (ILC) based on molecular tool
was organised by the EURL. Its objective was to test the ability of NRLs
to detect Marteilia refringens in flat oysters Ostrea edulis and mussels
Mytilus edulis or M. galloprovincialis by PCR. The test included 12
samples of ethanol fixed piece of digestive gland for each bivalve species,
mussels and flat oysters. The ILC was announced on the 1st of June,
samples were sent on the 1st of July and all the results were sent back to
the EURL on the 31st of August 2010. In total, 13 laboratories participated
in this ILC and obtained between 62 and 100% of good answers with a
mean of 87%.
Maximum discrepancies in the results were observed for three samples
found infected using histology and gave negative results in many
laboratories. Out of these three samples, most of the errors came from a
lack of detection of the parasite in some samples tested positive by
histology which could be attributed to a lack of sensitivity of the assay in
some laboratories conditions.
Assess
alternative
methods for the
identification of
listed pathogens.
Surveillance of mollusc diseases is routinely performed by histology or
cytology and increasingly by PCR. When outbreaks of mortality occur,
histology is indicated as a frontline method if no specific pathogen is
suspected and no presumptive diagnostic methods can be used. When a
pathogen is detected and identified in light microscopy, confirmatory
methods (e.g. electron microscopy and/or molecular probes) are usually
used to overcome the lack of specificity of histology. However, there is no
or very little consensus on how to best apply these different techniques.
The lack of contextual methodological framework certainly hampers the
efficacy and adequacy of investigation and early response to disease
outbreaks.
The efforts in research and development of DNA based diagnostic
methods currently offer a broad panel of probes and tests. These offer the
theoretical advantages of high sensitivity and high specificity, and
possible rapid screening of molluscs for the presence of a targeted
pathogen. However, while they are moving from development in research
laboratories to routine application in disease monitoring programmes,
validation and standardisation of these test are still needed. Combination
of diagnostic methods and selection of tests urgently calls for the
establishment of sensitivity and specificity values.
The further development and use of molecular diagnostic techniques such
as real time PCR, also hold promise for international efforts to control the
introduction of exotic diseases and pests into new geographic areas. One
long recognised caveat to the routine use of molecular diagnostic
techniques is false positive or false negative results. However, efforts
should be put forwards to develop, validate and standardise rapid
diagnostic techniques for major mollusc diseases and pathogens.
The EURL has undertaken comparison and validation studies especially
for some diagnostic techniques targetting Bonamia ostreae and Marteilia
refringens.
A first study consisted in comparing heart imprint and PCR for the
detection of Bonamia sp. Specificity and sensitivity values have been
determined using classical methods (based on a gold standard) and latent
tests showing the interest of latent tests in the case of diagnosis of mollusc
diseases. Comparison between PCR on gills and heart imprints shows a
difference between both techniques, PCR detecting more positive
individuals than heart imprints. Agreement between these techniques
fluctuates according to the tested population and increases in population
where prevalence is lower.
Sensitivity and specificity estimated by latent tests were 0,84 and 1 for
PCR and 0,4 and 1 for heart imprints. These values should be used to draw
a sampling strategy and have an impact on the sampling size.
A second study was initiated in 2009 and is still in progress. It aims at
comparing digestive gland imprint and PCR for the detection of Marteilia
refringens.
Besides these comparison and validation efforts, the EURL is involved in
the development of new methods for the detection of notifiable pathogens.
In 2008, the EUL developed a real-time PCR assay for the detection and
quantification of Bonamia ostreae (Robert et al. 2009). The assay had a
minimum detection limit of 50 gene copies per reaction when plasmid
DNA was used as template. Using infected oyster samples as template, the
assay was at least 10-fold more sensitive than conventional PCR. Oysters
were tested by this quantitative real-time PCR assay and by semi
quantitative approach based on heart imprint examination. A strong
correlation was observed between both techniques. In 2010, some tests
were initiated to quantify the parasite load in different organs of flat
oysters. These first tests need to be completed.
In addition, as mentionned earlier in this report, in 2010, the EURL
developed a duplex Taqman PCR assay aiming at detecting and typing
Marteilia refringens. This assay relies on one primer pair and two probes,
one detecting M. refringens type O and the other one type M. Performance
of this assay is currently tested at the population level.
Robert M., Garcia C., Chollet B., Lopez-Flores I., Ferrand S., François C., Joly
J.-P and Arzul I. (2009) Molecular detection and quantification of the protozoan
Bonamia ostreae in the flat oyster, Ostrea edulis. Molecular and Cellular probes,
23: 264–271
Provide
opportunities of
training in
laboratory
diagnosis of
mollusc diseases.
Several periods were scheduled and proposed in order to give an
opportunity to train technical staff of the National Reference Laboratories
and other laboratories involved in the diagnosis of mollusc pathogens (see
below). During these periods, the staff of the EURL helps trainees to
improve their practice in mollusc disease diagnosis procedures.
Training periods in the laboratory were specifically organised for the
following colleagues:

Tomomasa Matsuyama and Tomokazu Takano from the National
Research Institute of Aquaculture, Fisheries Research Agency
from Japon. This training aimed at presenting surveillance of
mollusc diseases and at detecting pathogens of mollusc by
histology especially those listed in the OIE aquatic Code. 26-28
January 2010

Anders Alfjorden, from the Department of animal health and
antimicrobiologial strategies, National veterinary institute,
Sweden. This training focused on the detection and
characterization of Marteilia refringens in Mytilus edulis. 25
March 2010

Jee Youn Hwang from Pathology Division National Fisheries
Research & Development Institute South Korea : This training
aimed at (1) presenting surveillance of mollusc diseases, (2)
detecting pathogens of mollusc by histology especially those listed
in the OIE aquatic Code (3) detecting Bonamia spp. by PCR and in
situ hybridization. 7-11 June 2010

Refka Elgarsalli, from Institut National Agronomique de Tunisie.
This training aimed at (1) presenting surveillance of mollusc
diseases, (2) detecting pathogens of mollusc by histology
especially those listed in the OIE aquatic Code (3) detecting
Bonamia spp. by PCR (4) detecting and characterizing Marteilia
refringens by PCR-RFLP cloning and sequencing. 25 May - 21
June 2010

Cristina Escobedo Fregoso from Centro de Investigaciones
Biológicas del Noroeste, S.C. Mar Bermejo No. 195, Col. Playa
Palo de Santa Rita; La Paz, BCS 23090, México. This training
focused on the detection and characterization of Perkinsus in
Crassostrea spp. , on the observation of Perkinsus spp. by
histology and on the detection of OsHV-1 using Real Time PCR. 4
september -1 October 2010

Kyung Il Park from the College of Ocean Science and Technology,
Kunsan National University South Korea : This training aimed at
(1) presenting surveillance of mollusc diseases, (2) detecting
pathogens of mollusc by histology especially those listed in the
OIE aquatic Code 13-21 Dcember 2010
Visitors of the
EURL in 2010
The EURL also welcomed the following visitors in 2010:
Anna Pampushchenko & Oxana Balas from the « Université
Nationale Agraire de Bila Tcherkv” , Ukraine
Dorsaf Essabai Elarmi, from INSTM, Tunisia

Attend
international
meetings and
conferences.
Prepare a
handbook for
diagnostic
procedures in a
CD-Rom format.
The EURL was also involved in a project of collaboration with
institutes of research from Mexico. In this context, the EURL
participated in welcoming nine colleagues from four institutes
(CIAD, UNISON, CIBNOR and IAES) between the 17th and the
21st of May 2010
Meetings and conferences provide opportunities for contacts and
collaboration with colleagues as well as a way to keep abreast of new
development in the field of mollusc pathology. Members of the EURL
attended the following meetings in 2010:

Aquaculture 2010. San Diego, California, U.S.A., 1-5 March 2010.

European Association of Aquaculture. Porto, Portugal, 5-8 October 2010

OIE working group on the surveillance of diseases of aquatic animals.
Paris 9&10 February

Second Global Conference of OIE Reference Laboratories and
Collaborating Centres, Paris, France, 21-23 June 2010
The European Reference Laboratory hands a high number of materials and
documents that constitute a significant interest for diagnostic laboratories
in the Member Countries.
It was proposed to organize these elements in order to set a handbook for
diagnostic procedures. A first version of a CD-ROM presenting EURL’s
collections has also been developed since 2002. The objective of this
material is to provide diagnostic laboratories with a practical guide in
histology of main mollusc species of commercial interest. The material is
devoted to diagnostic laboratories for mollusc diseases, National
Reference Laboratories for mollusc diseases, and scientists, veterinarians,
or technicians, involved in diagnosis of molluscs diseases. It is available
for potential users, free from charge, on request to the European Reference
Laboratory. The last edition (version 1.3) edited in 2007 includes oyster,
mussels, pearl oysters, abalone and clam species.
The EURL started two years ago testing new tools for training in
histology. These tools rely on some virtual slides which could be easily
accessible from the mollusc EURL website. These slides could include the
previous ring test slides and some specific slide collections. For example,
in 2009, the EURL tested the NanoZoomer System developed by
Hamamatsu Photonics which can scan tissue glass slides at magnification
20 or 40 and convert them to high resolution digital slides. Through
computer connected to the internet, digital slides can be observed with
“NDP serve” ® software. In 2009 and 2010, NRLs were given access to
slides from the interlaboratory comparison test organised by the EURL in
2007 using the following link http://www.ndpserve.com (Login:
IFREMER1, password: chollet).
Quality
Management
Accreditation:
The laboratory is accredited against the International Standard ISO 17025
for the following diagnosis techniques:
-
Diagnosis of Bonamia sp. by histopathology
Diagnosis of Marteilia sp. by histopathology
Diagnosis of Perkinsus sp. by histopathology
Diagnosis of Mikrocytos sp. by histopathology
The accreditation was renewed in October 2010 after a successful audit in
August. Again the French accreditation body (Comité français
d’accréditation: Cofrac) had to hire a technical auditor from a European
country because such competency was not available in France.
The EURL has continued to work in 2010 on the quality management
system for the organisation of interlaboratory comparison (ILC) tests.
Many documents are now available and were used for the organisation of
the 2010 European ILC in molecular biology.
The main change regarding the organisation of ILC tests is the issue of a
new standard in 2010: “ISO/CEI 17043, Conformity assessment – General
requirements for proficiency testing”. The EURL is now waiting for this
new standard to be transposed in particular requirements by Cofrac to
adapt its documents.
The EURL is seeking accreditation for the organisation of ILC by the end
of 2011- beginning of 2012.
Standard operating procedures:
One of the main objectives of the International Standard 17025 «General
requirements for the competence of testing and calibration laboratories» is
to facilitate cooperation between laboratories particularly for the
harmonisation of standard operating procedures (SOPs). Some European
laboratories need help in writing the documentation of their Quality
Management system, and the EURL proposes some SOPs that can help
them to write their own documents to be used to diagnose some of the
most important diseases to report in Europe and the world. These
documents are available on the EURL website on the “SOPs and Quality”
page and can be downloaded as PDF files:
http://www.eurl-mollusc.eu/SOPs-quality
In the field of histopathology diagnosis:
•
Diagnosis by histo-cytopathology of Bonamia spp. in the flat
oyster Ostrea edulis (4th edition, 2009)
•
Diagnosis by histo-cytopathology of Marteilia spp. in the flat
oyster Ostrea edulis and the mussels Mytilus edulis and M.
galloprovincialis (2nd edition, 2009)
•
Diagnosis by histopathology of Perkinsus sp. in molluscs (1st
edition, 2009)
•
Diagnosis by histo-cytopathology of Mikrocytos sp. in oysters (1st
edition, 2009)
•
Diagnosis by histopathology of Haplosporidium sp. in oysters (1st
edition, 2009)
•
Molluscs processing for diagnosis by histology (2nd edition, 2006)
•
Koehler illumination system for the microscope (1st edition, 2009)
In the field of molecular biology diagnosis:
•
Bonamia spp detection by Polymerase Chain Reaction and species
characterisation by Restriction Fragment Length Polymorphism
(1st edition, 2008)
•
Marteilia refringens detection and characterisation by Polymerase
Chain Reaction- Restriction Fragment Length Polymorphism (2nd
edition, 2010)
•
Marteilia refringens detection and characterization by in situ
hybridization (ISH) (1st edition, 2009)
These SOPs are regularly updated following technical improvement or
regulation evolution. The next SOPs being currently prepared and soon
available on the website are: “Quantification of Perkinsus sp. infection
intensity using Ray’s Fluid Thioglycolate Medium (RFTM) method” and
“Detection and quantification of OsHV-1 by Real Time PCR”..
Annex 1: Report from the 2010 Annual Meeting of the National Reference Laboratories
for Mollusc Diseases
Executive summary
Follow highlights of the discussions, expert opinion and recommendations formulated during
the 2010 Annual Meeting of National Reference Laboratories (NRLs) for Diseases of
Molluscs.
¾ In 2009, major epidemiological changes in EU were (1) the detection of Marteilia
refringens (type M) in Sweden (2) the suspicion and confirmation by the EURL of
bonamiosis due to Bonamia ostreae in flat oysters from Norway (3) the detection of
Bonamia exitiosa in France, once again in flat oysters from Corsica, and in Galicia,
Spain (4) the mass mortalities associated with the presence of herpesviruses OsHV-1
µvar which affected Crassostrea gigas in France, Ireland and Jersey.

The detection of Marteilia refringens in mussels for the first time in Northern
Europe led to interesting discussions notably about the parasite life cycle and the
distribution of its intermediate host(s). It also highlights the need of including mussels
in programmes for demonstrating free status regarding marteiliosis.
¾ A synthesis of data collected through the epidemiological reports between 2005 and
2008 was presented and showed the interest of filling in carefully the Excel® file sent
annually by the EURL. The difficulty to obtain production data was discussed
reminding that they should be provided by competent authorities. The lack of
information for the sections related to laboratory accreditation was underlined. This
synthesis showed an evolution of the techniques used for the detection of listed
pathogen.
¾ Following the abnormal Crassostrea gigas mortality events observed in France,
Ireland and Jersey in 2009, and the detection of a new genotype of the virus OsHV-1,
called OsHV-1 µvar, a new regulation has been proposed at the European level. The
implementation of this regulation will lead to a high number of analyses to test the
presence of OsHV-1 µvar in some Member States. France has started to build a
network of recognised laboratories in order to absorb this increased amount of
diagnostic activity. In parallel, the European Commission has requested EFSA about
the role and possible importance of infectious agents and environmental factors on the
occurrence of the oyster mortality but also on determining if other mollusc species are
of epidemiological relevance and differences between life stages.
¾ Experimental work is important to study the pathogenicity of pathogens. Recently
some cohabitation trials succeeded in transmitting OsHV-1µvar from infected to
healthy Crassostrea gigas juveniles. In addition real time quantitative PCR allowed
investigating the kinetics of OsHV-1 µvar detection in different oyster organs and in
seawater. In Tomales Bay, California, seed mortalities have occurred in Pacific
oysters since 1993, and the oyster herpesvirus (OsHV-1) was first detected in 2002.
Elevated temperatures appear consistently related to mortalities. Viral DNA was
detected by qPCR in multiple species of bivalves grown in Tomales Bay including
mussels; qPCR copy numbers were low in each species tested but were significantly
greater in C. gigas. Such investigations should be carried out in other infected areas in
order to evaluate the role of multiple bivalve species as reservoirs of the virus.
¾ Japanese mollusc production mainly relies on scallop and oysters. Decrement of
manila clam resources potentially associated with perkinsosis and mass mortality of
pearl oysters potentially due to Akoya virus are serious concerns which bivalve
industries of Japan currently face. Abnormal enlargement of the ovaries in the Pacific
oyster, caused by an ovarian protozoan parasite Marteilioïdes chungmuensis, also
potentially threatens oyster industries in Japan. This protozoan is presently not
reported in Europe and is not notifiable. However, considering its impact on oyster
production and marketability, vigilance should be given to this parasite by being able
to detect it and by avoiding any introduction of infected animals.
¾ Directive 2006/88/EC has a set of provisions aimed at ensuring traceability of
aquaculture animals. These provisions would provide the ability to trace back possible
contact farms in an epidemiological investigation. Implementation of these provisions
for molluscs is in progress in Europe while for fish it is much advanced. Key factors
for these provisions are (1) the proper authorisation and registration of all farms and
certain processing establishments and (2) their categorisation according to their status
regarding listed diseases. A WebGIS tool has been developed in Veneto, Italy, with
the aim to analyse data related to mollusc farms and laboratory results arising from
monitoring programmes, to integrate them with the pollution sources data and to asses
the sanitary and epidemiological situation of bivalve shellfish production. This
exercise is an excellent example of the implementation of the Directive 2006/88/EC
for mollusc issues.
¾ No reliable data is presently available concerning oysters transfers. This lack of data
has considerably hindered the epidemiological investigation of increased mortality
events which affected Crassostrea gigas in some Member States in 2008 and 2009. A
project of modelling the network of oyster populations movements between oyster
farms in an important French production area was presented. Movement data collected
through a questionnaire will be analyzed using social network analysis, to map and
measure the oyster flows among oyster farms. Such model could also help to
characterize oyster farms according to the risk their husbandry practices may represent
for the introduction or spread of an infectious disease.
¾ A world wide fish pathogens database www.fishpathogens.eu was created by the
EURL for fish diseases giving open and semi-open access to detailed information on a
number of pathogens causing diseases in fish. When new outbreaks arise, isolates can
rapidly be sequenced and aligned to other known isolations giving hints to the
possible origin of the outbreaks. Using the same model for creating a mollusc
pathogen database would be a very useful tool for the EURL.
¾ Results from several recent field studies were presented. The detection of Bonamia
ostreae in larvae of flat oyster raise new questions notably about their involvement in
the spread of the parasite during their planctonic stage. Mussel infection with
intracellular ciliates in Slovenia seems to be negatively correlated with salinity. In
the same study, few mussels showed neoplastic neoplasia. One mortality event
affected cockles in Germany. These cockles were imported from Ireland and exhibited
dark spots which could not be yet interpreted. A study carried out in France to
characterize species of Perkinsus showed that P. olseni was present in all investigated
clam production areas. However, in Leucate lagoon, P. chesapeaki appeared
sympatric with P. olseni. It is the first description of this parasite species in Europe
and in Ruditapes decussatus. Its distribution in Europe should be assessed especially
in areas where species known to be susceptible to P. chesapeaki are present (e.g.
Mercenaria mercenaria, Mya arenaria, Macoma baltica). This result highlights the
need of molecular characterization of pathogens for which routine diagnosis is based
on non specific techniques like histology, cytology or culture in thioglycolate medium.
¾ Given that in an infected area, eradication is most of the time not possible, knowledge
of host-pathogen interactions is of central interest in order to propose some
alternative measures to producers like advices for stock management or development
of resistant host strains. Some recent results concerning host-pathogen interactions
were presented included Ostrea edulis - Bonamia ostreae and Ruditapes decussatusPerkinsus olseni models.
¾ In 2009, the EURL for mollusc diseases organised the seventh histo-cytology inter
laboratory comparison (ILC) test. This test was organized according to the
recommendations given in the ILAC guide (ILAC*-G13:2007 – Guidelines for the
requirements for the competence of providers of proficiency testing schemes). The
ILC test aimed at testing ability of NRLs (1) to detect Bonamia sp. and Marteilia sp.
in the European flat oyster Ostrea edulis and (2) to detect exotic pathogens presently
described in cupped oysters (Crassostrea virginica or C. gigas) from North America.
Results highlighted difficulties of some laboratories to detect low level infections of
Bonamia sp. and to detect exotic pathogens especially Perkinsus marinus. Training is
needed for laboratories which had 60% or less of good answers. The EURL suggests
to organised a special workshop for these laboratories in March 2011. In addition,
considering the increase in use of molecular techniques in NRLs, an ILC to test the
ability of participants to detect Marteilia refringens by PCR will be proposed in May
2010.
¾ The EURL website http://wwz.ifremer.fr/EURLmollusc/ is continuously updated.
During the following months, it is proposed to create a section devoted and restricted
to the network of NRLs. This section will host reports presentations of the annual
meeting, technical reports, ILC reports… In parallel some electronic questionnaires
will be accessible through the website and will need to be filled for any material,
support or training request.
¾ The EURL proposes to plan the next annual meeting for the beginning of 2011
(March) in association with a Technical Workshop in La Tremblade, France. NRLs
are invited to propose topics on which they are working or for which they need
information and/or training.
ANNUAL MEETING SESSION I : DIAGNOSIS AND SURVEY OF MOLLUSC
DISEASES
(Secretary : I. Arzul)
Current epidemiological situation in the Member States
National delegates participating in the Annual Meeting briefly presented outstanding issues
and major changes with regards to the mollusc health status in their country. The detailed
reports received from NRLs are given in annex of this report (Annex D). A summary of the
reports is given here country by country followed by questions and discussion which took
place at the end of each presentation. The session ended by a synthesis of the data collected
through the questionnaires between 2005 and 2008
United Kingdom- M. Gubbins: During 2009 the 2006/88/EC Directive on Aquatic Animal
Health was implemented in the UK, replacing the previous controls governed by Directive
91/67/EEC. The new legislation has required the authorisation of all aquaculture production
businesses, so that the new rules on aquatic animal health can be applied.
The main species cultivated in the UK are mussels, Pacific oysters, native oysters and clams,
Pacific oysters and clams are supplied from hatchery production, whilst native oysters and
mussels are both from harvested wild stock. There are a total of 479 aquaculture production
businesses in the UK, producing a over 40,000 tonnes of shellfish. Again, mussels made up
the majority of this production with over 38,000 tonnes being recorded.
Surveillance and monitoring for Bonamia and Marteilia has continued in England, Scotland,
Wales and Northern Ireland with samples of Ostrea edulis from all zones being tested for the
disease. In 2009, there were no new confirmations for Bonamia and the continued absence of
Marteilia refringens was demonstrated.
In Scotland, Spring sampling of 8 aquaculture/wild sites, holding Ostrea edulis, continued.
All results proved negative for Bonamia and Marteila. The Loch Sunart infected site was re
surveyed and 13 adult O. edulis found in shallow water, they tested negative for Bonamia.
The site is considered as fallow and is to be seeded with sentinel animals and be tested over a
two year period together with a control area. The long term aim is to apply to the Commission
to remove controls on Loch Sunart and for the loch to regain approved zone status. Scotland
remains Marteilia free.
Abnormal mortalities were reported at two Pacific oyster sites in Scotland, although no listed
pathogens were detected during the follow up investigations. At one site the farmer noted a
5% loss in bags as being unusual for the site. A force 10 gale and a recent occurrence of DSP
were noted. 2 oyster samples were taken (1+30) for diagnostic investigation. No pathogen
was identified by histology/bacteriology, mortality returned to normal. At the second site,
Loch Fyne Oysters more than a 20% loss was observed in one stock. Animals there had been
moved from the depuration area to another part of facility due to DSP, mortality was
considered to be associated with spawning stress due to high temperatures. Ten C. gigas were
sampled for diagnostic investigation. Results showed no sign of notifiable disease or
suspicion of any other pathogen. Mortalities returned to normal. The 2010 August
surveillance will target the site, amongst others in Scotland, for OHV.
In England and Wales, abnormal mortalities were reported at 4 sites: one in cockles in the
Wash; one in native oysters in Chichester Harbour; the other two in Pacific oysters. No listed
disease was identified during the investigations. A combination of environmental conditions
and presence of other parasites were linked with the cockle mortality, which exhibited a
similar profile to mortality events reported in previous years in the Burry Inlet in Wales. One
of the mortality events in Pacific oysters was considered to be as a result of an unusual red
tide event that affected the SW coast of England, the other in the Camel estuary appeared to
be associated with a viral infection (identified by histological examination), although the
oysters tested negative for the presence of OsHV-1 by PCR. This mortality is the subject of
further investigations, and more samples are to be gathered during the proposed surveillance
programme for OsHV-1 which is to be implemented in 2010.
Other than this, a small survey was carried out at the sites which had received Pacific oysters
from areas experiencing unusual mortalities. None of these samples tested positive for any
listed disease, and the PCR screen for OsHV-1 was negative in all these cases. Again theses
sites are to be included in the upcoming surveillance programme for OsHV-1.
A sample of Pacific oysters was submitted to The Cefas laboratory from a farm site in Jersey
as a result of an on-going mortality in a number of stocks. This when tested was found to be
positive for OsHV-1, which when sequenced was found to be similar to the virus reported in
France in 2009.
In summary, no significant change to the disease status of the UK was identified in 2009; and
the Directive 2006/88/EC has been implemented. However, OsHV-1 was found in Pacific
oysters from Jersey in the Channel Islands. Future work includes the instigation of a
surveillance programme for OsHV-1, which will take place in 2010.
JF Pépin asked about the primers used for the detection of OsHV-1. M. Gubbins answered
that they used C2-C6 primer pair. T. Renault asked if some transmission electron microscopy
observation was done on cockles suspected to be infected by viruses. M. Gubbins said that
TEM analyses are presently under progress. I. Arzul asked if mortality was later observed in
juveniles closed to batches showing mortality. M Gubbins explained that mortality was
reported about 2 months after import from France to Jersey. These batches were further
moved to other bays and also displayed mortality.
Sweeden – A. Alfjorden: most of Swedish mollusc farming industry, (mainly blue mussel
farms) is concentrated in the northern parts of the Swedish west coast from Gothenburg up to
Strömstad (boarder towards Norway). This year nearly 2200 tons of blue mussels were
produced. There is still no farms producing oysters for consumption even though a flat oyster
hatchery/nursery has been active in one year at the northern part of the west coast (Koster
islands). Swedens has during 2009 followed up the application of freedom for Marteilioses
2008 by sampling 150 blue mussels in a targeted program for this disease. Two farms and two
wild mussel populations (30 specimens/ site) were investigated and found not infected. The
third farm of these investigated farms (situated between Orust and Tjörn) were on the other
hand found infected with Marteilia refringens typ M. This infection was diagnosed in five
specimens out of the 30 investigated blue mussels and this is the first recording of the disease
in the northern parts of Europe. Discussion are ongoing how to proceed with the investigation
due to this finding. No mortality events have been reported in farms or wild populations
during this year.
L. Madsen asked about the consequences of the detection of Marteilia refringens on the
zoning and on the status of the other Swedish zones. A. Alfjorden answered that the zone is
presently declared infected and transfers of all bivalves from and within the zone are
restricted (mussel seeds were also coming from this area). I. Arzul asked if other bivalves
species are present in the zone. A. Alfjorden replied that there are wild cockles and clams. I.
Arzul suggested to test them regarding the presence of Marteilia refringens since these
species have previously been reported infected by the parasite. T. Renault asked about the
type of the parasite. A. Alfjorden said that it was M. refringens type M. L. Madsen wondered
why Sweden decided to perform such an effort on mussels. A. Alfjorden explained that the
present legislation lists mussels as susceptible species to M. refringens and it is now
necessary to include mussels in the surveillance programme to be recognised free of the
parasite. D. Fraser wondered about water temperatures in Sweden. A. Alfjorden replied that
samples were collected when the water temperature was between 17 and 22°C. I. Arzul added
that the correlation between prevalence and temperature seems to change according to the
different ecosystems. If 17°C appears as a threshold above which the parasite can be
transmitted in the claire system and in other areas along the Atlantic ocean, it seems different
in Adriatic Sea according to data from G. Ceschia. I. Arzul explained that Paracartia grani,
suggested to be an intermediate host of M. refringens, was reported for the first time in 1904
off Western Norway and has then been observed in shelf and coastal water of the Northeastern Atlantic, North Sea and more recently in the Mediterranean Sea. I. Arzul suggested to
include a session on Marteiliosis during next annual meeting.
Spain – R. Aranguren: Most of the Spanish production of shellfish is located in Galicia and
it is based mainly in mussel (Mytilus galloprovincialis) aquaculture. In 2009, 630 flat oysters,
Ostrea edulis, were sampled from different production areas in order to determine the
distribution of marteiliosis and bonamiosis in Spain. Oysters were tested in spring and autumn
in the Atlantic coast (Galicia, Asturias) and in autumn in the Mediterranean coast (Cataluña,
Comunidad Valenciana and Islas Baleares). Since the detection of Bonamia exitiosa in
Galicia in 2007, PCR analyses are also being conducted in all sampled oysters in order to
discriminate between Bonamia especies. Bonamia ostreae and Bonamia exitiosa was only
detected in the Atlantic coast (Galicia) both in wild and cultured flat oyster. Although PCR
analyses are not yet finished, the prevalence of Bonamia exitiosa in wild oysters is very low
(0,8%). Coinfection of both Bonamia especies was also detected. Marteilia refringens was
detected on the Mediterranean coast (Cataluña and Comunidad Valenciana). 630 mussels
were collected from 21 different sampling sites (wild and cultured) in Galicia and 150
mussels were collected from 1 site in Ceuta in order to study the distribution of Marteilia
refringens in Mytilus galloprovincialis. Mussels from Galicia were infected by the parasite.
Perkinsus sp. was detected in Ruditapes decussatus, R. philippinarum, Venerupis pullastra
and Cerastoderma edule collected in Galicia, in R. decussatus from Asturias and in R.
phillipinarum from Cantabria. Molecular analyses were carried out in stocks of Crassostrea
gigas harvested from Asturias and Andalucía in order to detect the presence of the oyster
herpes virus. No positive results were obtained.
I. Arzul asked about the species of Perkinsus detected during this working programme. R.
Aranguren explained that the detection of Perkinsus was performed by histology which does
not allow species identification. I. Arzul asked about the evolution of Bonamia exitiosa and B.
ostreae prevalence among Galician flat oyster populations. R Aranguren said that
prevalence was variable according to the investigated sites and no special evolution has yet
been observed since 2007 in infected areas.
Slovenia - M. Gombac: The Slovene Sea is part of the Gulf of Trieste, the northernmost end
of The Adriatic Sea, where the Mediterranean pushes furthest into the European continent. It
is bordered by the line between Grado and Savudrija and the coastline. The entire Slovene
coast is 46.6 km long and is made of Eocene sedimentary flysch. The average depth of the sea
is only about 17 metres and the deepest point is 37.25 metres deep. The sea temperature and
salinity vary considerably: during the summer the shallows can heat up to 30o C and the
coastline can even freeze during very cold winters. The average temperature is 15.8o C. The
average salinity is between 37 and 38‰. Many large and small rivers, groundwater and
underwater springs have a strong influence on salinity, which fluctuates from 20‰ after
abundant rainfall to 38‰ during the late summer and winter. The oxygen concentration varies
accordingly to the sea temperature - the average oxygen concentration at the sea bottom is 6
mg/l in summer and 9 mg/l during winter.
Only Mediterranean mussels (Mytilus galloprovincialis) are bred on three locations i.e., Seča,
Strunjan and Debeli rtič in natural fisheries - shellfish farms. In 2004, the Slovene National
reference laboratory for mollusc diseases established two official sampling sites, i. e. in Seča
and Strunjan, are situated in semi-closed small bays, the first in Piran bay and the second in
Strunjan bay. The farms are quite small and composed of 5 fields, each measuring 20 000
square meters. In each field there are 20 lines with 18 to 20 butts, which are lashed to each
other with two ropes. Twenty clusters of mussels in nylon socks, up to 3 m long, hang from
ropes approximately 1 m under the sea surface. The seeds of 1 to 1.5 cm shell length are
collected manually from buoys and ropes in the shellfish farm or from buoys and ropes in
neighbouring a fish farm, but often the so-called “seeds trap” are used - old, worn and semitorn ropes approximately 1.5 m in length are fixed in shellfish the farm and used for caching
the seeds. Collection of the seeds is conditioned with the autumnal arrival of sea breams
(Sparus aurata) – if the seeds are put in socks before their arrival, sea breams usually eat all
seeds and completely ruin the shellfish farm. After collection, small clusters of seeds are
tightly filled in the dense white cotton socks which are put in white nylon mesh sleeves or
socks. These are fastened on ropes, which hang from rafts and submerged in the water. In a
few days, the mussels attach themselves by byssus. After approximately 6 months, the cotton
socks decompose, which enables the mussels to grow through the nylon socks. When they are
half-grown and they over-grow the nets, they are removed, cleaned and redistributed in few
bigger socks, where they reach market size (Fonda, 2009; personal communication).
During their growth, the mussels are exposed to direct sunlight once or twice for a whole day.
This procedure kills all the organisms living attached to the shells. Mussels reach commercial
size in approximately 18 months and at that time, they are 5 to 7 cm long. The annual
production of each field is from 25 to 30 tons, in all 251 tons of mussels are produced in the
Slovene sea every year.
In 2009 shellfish farms in both official sampling sites were monthly inspected for eventual
mortality. No mortality was detected in 2009, so no sampling was performed. Seča and
Strunjan areas will be monitored in 2010 for the presence of Bonamia sp. in the oyster Ostrea
edulis.
Romania – M. Costea: In the year 2009, Romania carried out sanitary veterinary surveillance
of farmed and wild population of molluscs. Molluscs samples were represented by M.
galloprovincialis (cultured and wild) and C. gigas (cultured). The main aim of surveillance
was Marteilia refringens in M. galloprovincialis and M. mackini, P. marinus and H. nelsoni
in C. gigas. No abnormal mortality was reported during the year. The position of sampling
was located in zone 4 (between Agigea and Mangalia places) for cultured molluscs and in
zone 3 for wild molluscs (between Navodari and Constanta Harbor). The sampling time was
in spring (May). Negative results for Marteilia refringens, M. mackini, P. marinus and H.
nelsoni were found by means of histology. By RTFM culture, for detection of Perkinsus sp,
the results were negative too. Only a passive surveillance is planned in 2010.
I. Arzul asked about the origin of Crassostrea gigas in Romania. M. Costea explained that
these oysters were imported from France three years ago. No mortality was reported among
this stock.
Norway – A. Medhus: The Norwegian mollusc production mainly relies on mussels Mytilus
edulis (2000 tons), and in a lesser concern on flat oysters Ostrea edulis (3 tons) and scallops
Pecten maximus (4 tons). In 2009, there were 397 locations. Some flat oysters were moved
from Arendal area to Sunnhordland before established control-zone. No B. ostreae was
identified from these samples. Norway is designed as one zone.
Arendal – B.ostreae was confirmed by the EURL in 2008 autumn samples from Arendal (2/9
individuals positive for B. ostreae by histology and PCR with sequencing) Actions taken upon
confirmation by EURL were : the establishment of a control-zone in summer 2009 , an
increased surveillance programme, an epizootic investigation. These actions included the
observations of four sites by skin diving, and the collection of 50 flat oysters from each site
for macroscopic evaluation, histopathological ecamination and PCR-analyses: C-primers,
BO-BOAS-primers. There was no evidence of a clinical out-break: oysters looked normal,
though variable condition of live oysters. Mortality was very low ( < 5 %), there were few
empty shells corresponding to previous mortalities, juvenile recruitment was normal and
regular (several generations). Histopathology and PCR tests did not reveal the presence of
parasites. Considering these results, the Competent authority plans to abolish control-zone
spring 2010.
In 2010 the surveillance of infection with B. ostreae in flat oyster populations will be
continued. The increased level of surveillance in Arendal will be maintained, Mytilus edulis
will be included in the surveillance regarding infection with Marteilia refringens. Lastly,
Crassostrea gigas is a new invasive species and the importance may increase.
S. Cabot added that the decision of removing the control zone for bonamiosis in Norway is
under the authority of EFTA Surveillance Authority (ESA). D. Fraser asked how the control
zone can be abolished in the absence of a programme to prove the absence of the susceptible
species and the eradication of Bonamia from the area concerned. A. Medhus explained that
it is a suggestion of the Norwegian Food Authority. T. Renault suggested to develop some cell
culture from haemocytes collected from flat oysters exhibiting neoplasia.
The Netherlands – M. Engelsma: Mytilus edulis is the most important shellfish produced in
the Netherlands, followed by the Pacific oyster Crassostrea gigas and a small production of
the flat oyster Ostrea edulis. Annually a surveillance is carried out in spring and autumn for
detection of shellfish diseases in these three species. Since 1988 Bonamia ostreae has been
present in O. edulis from Lake Grevelingen. In 2009 Bonamia ostreae was observed in 12%
and 5% of the investigated flat oysters in spring and autumn respectively. The bacterium
Nocardia crassostreae was observed in a limited number of flat oysters (n=3 in total). No
Matreilia refringens was observed in the investigated oysters and mussels. Furthermore, no
abnormal mortality events were reported in 2009. The same surveillance as 2009 will be
conducted in 2010.
I. Arzul asked about the reason of importing some bivalves from U.S.A. M Engelsma replied
that people who imported these animals did not know about transfer legislation and wanted to
test a new market.
Italy - G. Arcangelli: During 2009, it’s to remark, for the first time, the presence of Marteilia
in mussels (M. galloprovincialis) in Lazio and Campania regions. The prevalence was less
than 1% in Lazio and around 10% in Campania. No evidence of mortality was notified by
farmers.
The positive samples was determined with cytological and histological method. The PCR
confirmation wasn’t applied for the lack of material. Further sampling will be carried out
during next months in the same areas. Bonamia ostreae and B. exitiosa wasn’t confirmed but
only few samples were examined. Perkinsus olseni is confirmed the most spread disease in
manila clams with a prevalence from 30 to 60% in lagoons. Some mortality events were
notified: in lagoon manila clams for sudden change in salinity and temperature. Mortality in
baby clams (Chamelea gallina) and smooth clams (Callista chione) was notified in the sea.
Bacteriological and virological investigation were negative. The presence of some parasites
like Nematopsis in baby clams and Porospora, Coccidia and Rickettsia like was found, but
probably they wasn’t the main cause of the event for the presence also in healthy animals. The
sea chemical-physical parameters were normal. Further investigations have to be done.
Analytical implementations was applied to assess the PCR assay for Ostreid Herpesvirus 1
µvar in Crassostrea gigas: an investigation is foreseen during the next months. In July 2009
the NRL in IZSVe -Udine was transferred to IZSVE-Adria (Rovigo): during 2010 the
cytological and histological procedures will be re-accredited in the new lab. according the
ISO/IEC 17025:2005 standard.
Ireland (not represented during the annual meeting): In 2009 there was a reduction in the
monitoring for Bonamia and Marteilia in Ireland with the implementation of 2006/88/EC.
The main issues for the country related to increased mortality in C. gigas. Mortality was
reported from 16 bays in predominantly seed and juvenile oysters, although in a small number
of bays mortalities were also observed in adults. Mortlaity levels reported ranged from 15 –
100% depending on location and affected stock. In 15 bays OsHV-1 µVar was detected. In
the 16th bay extensive testing ruled out the presence of the virus. In addition a consignment
of larvae that was received into the country following the closure of bays in France was
quarantined and subsequently screened for the virus though no mortality was detected in the
stock. This batch also tested positive for the virus. In all cases a link to oysters imported from
France in either 2008 or 2009 could be established. Transmission to stocks from other origins
was seen in several bays. Several samples were received late in the year the latest of which
was received on 11/5/09 3 months after mortality had ceased. The virus was still easily
detectable using the C2-C4 and C2-C6 primer sets. A study of bays which had received seed
from only the English or Channel Island hatcheries was conducted. In all cases no OsHV-1
µVar could be detected. Whilst mortality was reported from a single site in 2 of these areas
the pattern of mortality was not consistent with that observed in the bays where OsHV-1 was
detected and in 1/2 cases husbandry issues were clearly evident.
Greece – N. Ziogas: In Greece, there are 197 farms which are systematically cultivating
Mytilus galloprovincialis, and 1 new farm which cultivates Ostrea edulis. Total production for
2009 amounted to 23.237,6 tonnes of mussels, but there is no oyster production yet (new
farm). About 80% of the annual production is exported for consumption to Italy, France and
Spain. The Department of Aquatic Organisms Pathology routinely performs Histology as a
Screening method for surveillance purpose. Epidemiological data for 2009 show high
infection by Marteilia sp, since the parasite was found in 12 mussel samples taken from 12
different zones from the 13 investigated. The rate of the infection was over 60% in every
case. In 2009 there were no increased mortality outbreaks. Finally, we must mention that we
have located in both of the samples of Ostrea edulis (rate 80%), a protozoan parasite (in the
digestive gland parenchyma and especially in the epithelium of the digestive tubes), but we
cannot identify its species. The NRL has a new laboratory but the personnel needs training
Germany- S. Ramdohr made a presentation entitled “Histopathological detection of
Marteilia refringens in blue mussels (Mytilus edulis). Preliminary examinations in the Lower
Saxony Wadden Sea, Germany, 2009” (Authors : S. Ramdohr & S. Bergman)
In accordance with the EU Directive 2006/88 on “animal health requirements for aquaculture
animals and products thereof, and on the prevention and control of certain diseases in aquatic
animals”, and the “German Regulation on Fish Diseases” (FischseuchenVO), preliminary
examinations of Blue mussel, (Mytilus edulis) on Marteilia refringens were carried out in
2009 in Lower Saxony, that covers the East Frisian Wadden Sea part of the German coastal
waters. The analysis was done in the Institute for Fish and Fishery Products (IFF) Cuxhaven
of the Lower Saxony State Office for Consumer Protection and Food Safety (LAVES). The
results obtained are preliminary as the proficiency testing procedure due to ISO 17025:2005
is pending for the method used in the due laboratory of IFF.
A total number of 28 samples from 10 individuals of blue mussel each were prepared for
imprint and dissection slides, thus a total number of 560 slides were examined histologically.
The laboratory method used meets the Standard Operation Procedures (SOPs) of the
Community Reference Laboratory (EURL) for Mussel diseases at IFREMER, La Tremblade,
France, and the SOPs of the German National Reference Laboratory (NRL) for Mollusc
Diseases at Friedrich-Loeffler-Institut (FLI), Insel Riems, Germany, which also performs the
laboratory training for personals employed by the State Institute in Lower Saxony in
Germany. The area monitored covers the rear part of the Lower Saxony Wadden Sea between
the firm land and the East Frisian Islands. In this area the five production areas for Blue
mussels are located. Their geographical division was used also as zones for the disease
monitoring described. No signs of increased mortality in Blue mussel and no Marteiliosis in
those were detected during the monitoring time and area. Suspicious results in samples were
additionally examined and confirmed by the NRL-Laboratory.
M. Engelsma asked if other bivalve species including Crassostrea gigas and Ostrea edulis are
present in the zone targeted by the survey (between The Netherlands and Cuxhaven). S.
Ramdohr answered that C. gigas invades mussel beds and hinders mussel spat collection. I.
Arzul questioned the period of sampling. S. Ramdohr replied that they sampled mussels
during winter-spring also for public health purpose. I. Arzul noted that it is not the most
recommended period for the detection of Marteilia refringens. She also asked about the
perspectives of this survey. S. Ramdohr said that they will continue to investigate the potential
presence of Marteilia refringens in mussels by PCR and in extended number of samples if
possible. The surveillance of other diseases depends on further discussion with the competent
authority.
France – C. François: The mollusc production remains constant with an annual production
of 214390 tons. The main productions are Pacific oyster, Crassostrea gigas and mussels,
Mytilus edulis and M. galloprovincialis. In 2009, France continued to participate at the
programme settled by the EURL in 2008 in order to characterize the parasite Bonamia present
in Europe. In 2008, only the main production areas of flat oysters, Ostrea edulis (zone III =
Brittany) were sampled; in 2009, the different wild flat oysters bed were sampled. Eleven
samples accounting 890 individuals were analysed in histology and PCR; 159 individuals
were infected by Bonamia ostreae (confirmation by PCR-RFLP, cloning and sequencing) and
5 individuals with Marteilia refringens. One individual was co-infected with B. ostreae and
B. exitiosa and this individual came from Diana lagoon in Corsica. B. exitiosa was already
detected in this area the precedent years.
In 2008, during a mortality event in Mediterranean Sea, Bonamia exitiosa was detected in flat
oyster Ostrea edulis. This flat oyster stock came from 2 different French locations (Brest bay
and Bourgneuf bay); a sampling was done in these areas and B. exitosa was detected in
Bourgneuf bay (no detection in Brest bay). In 2009, all these flat oysters stocks (from
Mediterranean Sea, Brest bay and Bourgneuf bay) were destroyed in order to eradicate the
parasite B. exitiosa. A sample of these oysters was done before their eradication and B.
exitiosa was only detected in Mediterranean Sea on 45 individuals and in Bourgneuf bay on
only one individual like last year.
In 2009 as in 2008, important increased mortality events occurred in most Pacific oyster
producing areas in France during spring and summer. The mortalities were sudden and mainly
affected 6 to 18 month old Crassostrea gigas juveniles. OsHV-1 and bacteria belonging to
Vibrionaceae family were frequently detected in affected populations. Contrary to 2008, only
OsHV-1 genotype microvar was detected in 2009.
Few other mortality events occurred in 2009 and concerned scallops (2 cases), clams (1 case)
and mussels (2 cases). In one case of mussel mortality, there is an atypical detection of
Marteilia refringens; this parasite was mainly observed in histology in connective tissues of
mantle, labial palps and gills whereas M. refringens is classically a parasite of the mollusc
digestive gland.
For 2010, France will maintain its efforts on the study of mortality and particularly on wild
oyster bed and will continue the surveillance of the parasite Bonamia exitiosa particularly in
Corsica and in Mediterranean Sea.
A. Afonso asked about the context of collection of the 61 samples tested regarding presence of
OsHV-1. C. Francois explained that these samples were only collected during mortality
event. D. Fraser asked about the duration period of the mortality in France. C. Francois
replied that mortality reports lasted from March to November 2009 with a peak between May
and July. D. Fraser wondered about the possibility of OsHV-1 detection after the end of the
mortality outbreak with the potential for asymptomatic carriers after movement restrictions
were lifted. C. François explained that two samplings were performed after the end of the
mortality event in Arcachon Bay and in Charente Martime and the virus could be detected. T.
Renault added that some surveys were carried out on wild stocks before, during and after
mortality. The viral load is maximum during the mortality event. M. Gubbins would like to
know which virus type was detected in 2009. C. Francois explained that in 2008, both OsHV1 and OsHV1 µvar were detected while in 2009 only OsHV-1 µvar was detected in French
tested samples. M. Gubbins asked about the presence of OsHV-1 µvar in other bivalve
species. Some detection tests were carried out on clams, scallops and flat oysters during
mortality events but the virus was not detected.
Denmark- L. Madsen: The Mytilus edulis production in Denmark of 37,427 tons for 2009
was on the same level as the year before. The production of Ostrea edulis, located in
Limfjorden, has been between 1,200 to 1,500 tons per year for the last three years, the amount
being 1,170 tons for 2009. The fishing is regulated by a quoting system as well as the fishing
is only allowed in certain time periods during a year. Around 30 individuals were collected in
spring and autumn 2009 in three sites from Limfjorden. Investigations for 2009 were not
finished at meeting dates. Denmark is officially recognized Bonamia ostreae and Marteilia
refringens free in the Limfjorden area since December 2004.
I. Arzul wondered about the reason of the decrease of flat oyster production in Denmark
between 2008 and 2009. L. Madsen explained that it was not due to mortality but was related
to harvesting quota measures.
Croatia (no represented during this annual meeting): In the year 2009. Croatian molluscs’
farmers and fishermen put on the market 3696 tons of bivalve molluscs. Cultivated species
were represented with 3631t while it is assumed that approximately 100 t were collected from
natural beds. There are 126 farms cultivating European flat oysters (Ostrea edulis) and
mussels (Mytilus galloprovincialis) and one experimental hatchery for oysters in Zone III.
New sampling points were included in the surveillance program mainly in Zone II.
Surveillance is performed according the National Laws and mainly oysters and mussels were
monitored by means of cytology and histology to the presence of Bonamia ostreae and
Marteilia refringens as well as some other species from natural beds to listed pathogens.
Sampling was carried out during April and from August to October. Totally 43 samples
consisting 30 to 50 oysters of mussels, while 3 samples of collected species (Pecten Jacobeus,
Chlamys varia and Venus verrucosa) were submitted to laboratory examination. Results of
the analysis revealed presence of Marteilia refringens in mussels from all zones with
prevalence of 13,27%. The suspicion to presence of Perkinsus sp in wart venus (Venus
verrucosa) was not confirmed nor rejected.
Bulgaria – V. Chikova: In Bulgaria in 2009, there were 8 mollusc farms producing Mytilus
galloprovincialis. Histological and cytological examinations performed on 60 individuaals
did not reveal the presence of Marteilia refringens.
I. Arzul asked about the period of mussel sampling. V. Chikova said that mussels were
collected in summer and autumn. As previously mentioned some difference can be observed
between marteiliosis prevalence according to the ecosystems. Black Sea could present a
different environmental system that could influence the parasite cycle. A sampling at another
period could be suggested.
Synthesis of epidemiological data in Europe on the period 2005-2008. C. François
The EURL for Mollusc Diseases adopted a new form of report template which was presented
at the Annual Meeting of NRLs for Mollusc Diseases in 2005. This new report template in
excel® form deals with : i) General data (number of farms and hatcheries-nurseries,
production data), ii) Laboratory data (diagnostic tests for screening, presumptive and
confirmatory purposes), iii) Epidemiological data (surveillance of Bonamia ostreae and
Marteilia refringens, study of increased mortality, surveillance of other pathogens).
The report templates collected during the period 2005-2008 were studied and this synthesis
summarizes the information of 16 States that have completed the template each year:
Belgium, Croatia, Denmark, Estonia, France, Ireland, Italy, Netherlands, Norway, Poland,
Portugal, Romania, Slovenia, Spain, Sweden and United Kingdom.
Concerning the general data, the synthesis underlines the lack of official production data. The
number of farms and hatcheries-nurseries remains uncertain, as this data are not fully
completed each year.
Concerning the laboratory data, NRLs considered in this study use various diagnostic
methods for the detection of UE listed non-exotic and exotic pathogens (Directive 06/88/EC).
Some NRLs have also at their disposal diagnostic tools for the detection of exotic pathogens
previously listed in UE regulation (ex Directive 95/70/EC) or in Aquatic Code of OIE and
few NRL have developed tests for the detection of non-listed agents that cause or are
suspected to cause economic and health impact on mollusc population. The number of NRL
accredited in accordance with the European Standards (EN ISO/IEC 17025 on ‘General
requirements for the competence of testing and calibration laboratories) remains uncertain.
Concerning epidemiological data, the surveillance of Bonamia ostreae and Marteilia
refringens is well documented by NRLs. Based on questionnaire information, 12/16 States
have population of mollusc susceptible to B. ostreae and 11/16 States perform B. ostreae
surveillance, 14/16 States have population of mollusc susceptible to B. ostreae and 14/16
States perform M. refringens surveillance. The definition of zone and compartment is
different from one State to another and few States have an official zoning for the surveillance
of these listed pathogens.
The study of increased mortality of mollusc is reported by NRLs. The number of States in
which occur at least one mortality event per year fluctuates between 4 and 6/16 on the period
2005-2008. For those States, it represents between one and more than 60 mortality events per
year. Investigation carried out during and after mortality event show that cause of mortality
are often multifactorial. When diagnostic results reveal pathogen(s), co-detection of different
agents is possible. Some pathogen are frequently described in context of mortality event
(OsHV-1, Vibrio aestuarianus, Vibrio splendidus, Marteilia refringens, Nocardia
crassostreae, etc…) and new agent or strain are also mentionned (Picorna-like virus in Manila
clams for example). The surveillance of others pathogens is considered in a different way
from one State to another : 4/16 States accomplished general program for the detection of all
pathogen of mollusc on the period 2005-2008, some States develop specific program targeting
one or two agents in their sensible species (Candidatus Xenohaliotis californiensis in Haliotis
sp., Perkinsus olseni in Ruditapes phillipinarum, Nocardia crassostreae in Crassostrea gigas,
Mytilicola intestinalis in Mytilus edulis for examples).
S. Cabot pointed out different things: (1) the lack of official data concerning production
figures, number of farms, hatcheries, the zoning, imports, exports should be solved once the
Directive 2006/088 will be implemented in Member States; (2) if an exotic pathogen is
detected, this detection must be notified to the European Commission; (3) the surveillance
targets a list of notifiable pathogens, however, if other pathogens are posing a serious threat
to the mollusc production at national levels, Member States may according to Article 43 of
the Directive implement a control programme. If that programme would include restrictions
on intra Union trade, the programme must be approved by the Commission; (4) the notion of
compartment is more adapted to fresh water than to mollusc production. I. Arzul highlighted
the difficulty to know which laboratories are accredited and for which technique/ which
pathogen.
ANNUAL MEETING SESSION
MORTALITY
(Secretary : C. François)
II
:
CRASSOSTREA
GIGAS
ABNORMAL
Commission Regulation (EU) No 175/2010 – EU harmonisation of measures to control
increased mortality in Crassostrea gigas in connection with the detection of OsHV-1
μvar. S. Cabot
Increased mortality in Pacific oysters (Crassostrea gigas) was detected in several areas in
France, Ireland in 2008 and 2009 and in 2009 also on Jersey (UK). The Member States
concerned took measures to control the emerging disease situation. While the causes of the
mortalities still remain uncertain, the epidemiological investigations undertaken in 2009
suggest that OsHV-1 μvar play a major role in the mortalities.
To ensure a harmonised approach between Member States as regards measures to control this
emerging disease situation Commission Regulation (EU) No 175/2010 implementing Council
Directive 2006/88/EC as regards measures to control increased mortality in oysters of the
species Crassostrea gigas in connection with the detection of Ostreid herpesvirus 1 μvar
(OsHV-1 μvar) was adopted.
The Regulation only applies to Pacific oysters (Crassostrea gigas) and regulates increased
mortalities in connection with the detection of OsHV-1 μvar. Increased mortalities not related
to OsHV-1 μvar is not covered by these specific measures and the general requirements of
Council Directive 2006/88EC will apply.
The Regulation lays down:
• Criteria for the establishment and lifting of containment areas
• Movements restrictions for Pacific oysters originating from containment areas and
areas previously under containment measures
• Requirements for programmes for the early detection of OsHV-1 μvar
• Sampling and diagnostic methods
The temporary measures provided for in the Regulation apply until the end of 2010. The
disease situation and the measures taken will be re-assessed in Autumn 2010, taking into
account the knowledge and experience gained in 2010 and the EFSA scientific opinion on the
matter which is requested by the Commission.
More information on the Regulation and the measures taken by relevant Member States can
be
found
on:
http://ec.europa.eu/food/animal/liveanimals/aquaculture/oyster_mortalities_en.htm
S. Cabot suggested to have a direct link to the regulation text from the EURL website. N.J.
Olesen asked explanation about the containment measures. S. Cabot explained that after
identifying zones affected by mortality, transfer restrictions shall be applied as well as
diagnostic tests for the detection of OsHV-1µvar. If the virus is detected, restriction
measures are maintained until the end of the mortality. In addition, to be able to export
towards zones which have implemented an early detection programme, diagnostic tests
showing the absence of OsHV-1µvar are necessary, tests must follow the procedure detailed
in the regulation. Results must be available through internet. The Member States web pages
should have: 1) the containment areas, 2) the compartments with absence of OsHV-1 (in
previous containment areas), 3) areas previously subjected to containment measures and 4)
areas under programmes for early detection of OsHV-1. S. Ramdohr wanted to know if
mortality of Crassostrea gigas is observed in association with the presence of OsHV-1µvar,
what should be done for mussels present in a same area. S. Cabot replied that the regulation
does not concern other species than Pacific cupped oysters. S. Bergman asked why the primer
sequences are not given in the regulation. I. Arzul explained that colleagues from Ifremer
who have developed the PCR method would like to protect their results by applying for a
patent. In order to respect this wish and not to block NRL, the EURL has decided to send
primers CF and CR to NRLs. D. Fraser worried about the case of hatcheries where there is
no mortality but in which OsHV-1µvar is present and which could export their spat. I. Arzul
answered that hatcheries should be regarded as compartment within zones eventually
affected by mortality. If hatcheries want to export spat to zones which have implemented early
programmes, they would first need to demonstrate the lack of OsHV-1µvar in their facilities.
A Afonso asked about the definition of containment area based on increased mortality and
wondered about the definition of increased mortality. The Directive 2006/088 considers that
an increased mortality means unexplained mortalities significantly above the level of what is
considered to be normal for the farm or mollusc farming area in question under the
prevailing conditions. What is considered to be increased mortality shall be decided in
cooperation between the farmer and the competent authority. A. Afonso would like to know
which are the differences between containment areas 1 and 3 as defined in the presentation of
S. Cabot. S. Cabot explained that containment area 1 corresponds to currently containment
area while case 3 corresponds to area previously subjected to containment (in 2008 and
2009). S. Bergman asked if OsHV-1 is concerned by this regulation. S. Cabot said no. D.
Fraser pointed out that the surveillance covered by this regulation is time consuming and
expensive and asked about the possibility to use pools instead of individual testing. I. Arzul
answered that at the moment the approach by pool has not been validated and thus it is not
possible to recommend it. C. François said that the regulation is an interesting approach for
emerging disease and it appears different from the Directive 2006/088 which targets more
endemic/exotic diseases. S. Cabot said that all this regulation works within the scope of the
Directive. M. Gubbins said that transfer restrictions are lifted after a negative result
regarding OsHV-1µvar detection and would be applied again if mortality occurs in the area.
S. Cabot replied that OsHV-1µvar infection is treated as an emerging disease and at the
moment the question about the risk represented by this disease is asked to EFSA.
Transfer of diagnostic techniques and inter-laboratory comparison assays for some
bivalve mollusc diseases: Implementation of a French network of designated
laboratories for the diagnosis of mollusc pathogens. T. Renault
The French Rural Code (Articles R. 202-1 to R. 202-34) defines 3 different categories of
laboratories involved in diagnosis services for animal health purpose and gives their functions
and duties: national reference laboratories, designed laboratories (‘agréés’) and recognised
laboratories (‘reconnus’). The national reference laboratories coordinate the methods at a
national level for diagnosing the diseases concerned under their responsibility, assist in the
diagnosis of outbreaks of relevant diseases by receiving samples for confirmatory diagnosis,
develop epizootic studies, organise periodic comparative tests of diagnostic procedures at
national level and ensure a regular and open dialogue with the French competent authorities
(Ministry of Agriculture). Designated laboratories are in charge of analyses for the competent
authorities and recognized laboratories carry out analyses for shellfish farmers (self-testing).
The Genetics and Pathology Laboratory (Ifremer, La Tremblade) was designed as the French
national reference laboratory in December 2009.
No designated laboratories and no recognized laboratories have been designed at present for
mollusc bivalve diseases. Within this context, the Genetics and Pathology Laboratory
(Ifremer, La Tremblade) carry out all official analyses for the French Ministry of Agriculture.
However, in the case of massive mortality outbreaks or an emerging disease situation this
laboratory may be in the incapacity to realize all the requested analyses.
OsHV-1 and bacteria belonging to the genus Vibrio were associated with mortality outbreaks
among Pacific oysters Crassostrea gigas in France since several years. Increased mortality
events related to the detection of these pathogens including a newly reported genotype of
OsHV-1 (OsHV-1 µvar), were reported in 2008 and 2009 in France, Ireland and Channel
Islands. Within this context, a new EU regulation (175/2010) related to specific measures to
be taken in case of increased mortality in association with the detection of OsHV-1 µVar was
implemented in March 2010. Moreover, French shellfish farmers request more and more
frequently analyses (self testing) in absence of disease or mortality outbreaks in order to
search pathogens (Vibrios and OsHV-1).
The Ministry of Agriculture (General Directorate of Food, Section of laboratories and
coordination of official controls) is defining a specific legislation framework through a
specific call for designated laboratories defining their functions and duties and a forthcoming
decree for recognized laboratories.
Transfer of diagnostic techniques for OsHV-1 and Vibrio detection was carried out by Ifremer
(Genetics and Pathology Laboratory, La Tremblade) and two sessions have already been
realized, the first one in January 2009 (4 laboratories), another in November 2009 (4 other
laboratories). Two comparative tests of transferred diagnosis procedures using reference
biological samples have already been organized, one in April/May 2009, another in
February/March 2010.
D. Fraser asked who funds such a network. T. Renault replied that the French administration
supports the development of the network and takes in charge the diagnostic tests. D. Fraser
would like to know which types of laboratories would be included in the network. T. Renault
answered that the administration will recognise only public laboratories and not private
ones. D. Fraser wondered which diagnostic approach will be followed by this network for the
detection of OsHV-1µvar. T. Renault explained that screening will be done using the Real
Time PCR and positive samples will be sent to the NRL for genotype characterization. M.
Engelsma asked about the diagnostic assays used by the laboratories. T. Renault said that
they use the Real Time PCR published by Pépin et al. 2008. M. Engelsma asked if
laboratories are accredited for the diagnostic methods they use. T. Renault answered that
laboratories work under quality management but it is presently not possible to be accredited
for any molecular assays in France.
Experimental infection of the Pacific oyster Crassostrea gigas by the Ostreid Herpes
Virus-1 (OsHV-1): monitoring of the viral kinetics of detection in oyster tissues and
seawater. D. Schikorski
Herpes and herpes-like viruses are known to infect a wide range of bivalve mollusc species
throughout the world. Abnormal summer mortalities associated to the detection of Ostreid
herpesvirus 1 (OsHV-1) have been currently reported in France among larvae and spat of the
Pacific cupped oyster Crassostrea gigas. In order to explore the causes of massive mortality
outbreaks affecting French C. gigas spat, experimental infections were carried out through
experimental trials. The disease was firstly reproduced experimentally thanks to the
preparation of infective virus suspensions from naturally infected fresh oysters collected on
the field during mortality outbreaks occurring on French coasts during summer 2008. Indeed,
the intramuscular injection of these virus suspensions induced high mortality rates reaching
up to 80% in 3 days in healthy spat oysters. Moreover, the availability of OsHV-1
experimentally infected oysters allowed to develop a protocol of horizontal transmission
through cohabitation between healthy and experimentally infected oysters by intramuscular
injection of virus suspensions. At the view of our present results, a short time of contact (2
days) appears sufficient for virus transmission from infected oysters to healthy ones.
Mortality rates recorded in initially healthy oysters during the cohabitation experiment were
lower than those obtained after intramuscular infection of the viral suspension and required a
longer period (50% of mortality after 8 days). This longer period could be related to the time
needed by the virus to be convoyed in seawater to attain naïve individuals and to infect them
by natural means, which is more representative of what happens in natural conditions. These
experimental cohabitation trials allowed us to investigate and characterize for the first time
the kinetics of OsHV-1 detection in different oyster organs and in seawater. Virus DNA
quantification was performed by real time quantitative PCR (qPCR) in the time course of the
experiment in seawater and in different oyster tissues including haemocytes, gills, mantle,
adductor muscle, and digestive gland. Taken together, results suggested that infective virus
particles could first enter thought the digestive gland and the haemolymphatic system. In a
second step, viral particles could be transported by haemolymph to the different target organs
before to finally engage an intense replication phase conducting to the development of the
disease in target tissues. Thus, our results contributed to the set up of a reproducible model for
studying the pathogenicity of OsHV-1 on C. gigas oysters in experimental conditions. This
work constitutes the first study on the kinetic of OsHV-1 DNA detection in experimentally
infected C. gigas by cohabitation.
C. Burge would like to know which was the water temperature used during the experiment. D.
Schikorski said that the experiment was performed at 22°C. C. Magnabosco asked about the
negative control used in the experiment. D. Schikorski explained that negative controls were
injected using material from non infected oysters. E. Harrang wondered how water samples
were done. D. Schikorski said that water was collected only during the cohabitation
experiment. I. Arzul asked if the virus tested during this experiment was OsHV-1 µvar. D.
Schikorski said yes and future experiment will be done in order to test OsHV-1 infection. S.
Bergman asked if in situ hybridization was performed. D. Schikorski said that paraffin blocks
are available and ISH tests will be soon performed.
Examining the roles of environment, host, and pathogen in the host-pathogen
relationship between the oyster herpesvirus and the Pacific oyster in the USA. C. Burge
The Pacific oyster, Crassostrea gigas, an oyster species indigenous to Japan, has been
introduced globally becoming the primary species of oyster cultured in many areas of the
world. In Tomales Bay, California, seed mortalities have occurred in Pacific oysters nearly
annually since 1993, and the oyster herpesvirus (OsHV), a virulent pathogen of larval and
juvenile bivalves (particularly known in Pacific oysters) was first detected in 2002. Sentinel
field studies (2000-2003) were conducted in Tomales Bay in order to better understand the
role of environmental factors (temperature, phytoplankton, and salinity) and oyster health
(measured using histology and/or OsHV-specific Polymerase Chain Reaction (PCR)) on
Pacific oyster survival. Elevated temperatures were the only environmental factor
consistently related to mortalities (2000-2003), and in 2003, elevated temperature means
predicted OsHV presence (p < 0.005); OsHV presence predicted mortality (p=0.01). A
separate survey conducted in 2003 detected OsHV in multiple species of bivalves grown in
Tomales Bay (C. gigas, Ostrea edulis, C. virginica, C. sikamea, Mytilus galloprovincialis,
and Venerupis phillipinarum) and C. gigas grown in nearby Drakes Bay using OsHV-specific
PCR and/or quantitative PCR (qPCR); qPCR copy numbers were low in each species tested
but were significantly greater in C. gigas (p < 0.0001) the only species that appear to be
impacted by mortalities. To confirm the infectious etiology of OsHV detected in Tomales
Bay, Pacific oyster larvae were exposed to either filtered homogenates from OsHV-infected
Pacific oysters in Tomales Bay or un-infected oyster tissue. OsHV was detected and
quantified only in oyster larvae exposed to OsHV using qPCR and reverse transcriptase
qPCR, and confirmed using transmission electron microscopy. Taken together, data from
field and lab-based experiments indicates an infectious disease (OsHV) acts in synergy with
temperature to kill Pacific oysters in Tomales Bay, California.
Preliminary gene
identification of both upregulated host and OsHV genes in larvae exposed to OsHV was
conducted using SOLiD sequencing and GeneFishing PCR. Genes identified may provide a
foundation to better understand both host response and virus infection, ultimately better
defining the host-pathogen relationship between OsHV and Pacific oysters.
S. Cabot would like to know if Crassostrea gigas in Tomales Bay are further moved. C. Burge
explained that usually C. gigas is imported there for growth and will not be moved except for
human consumption. S. Bergman wondered if the virus under its latent form is integrated in
host chromosomes. C. Burge said that the host genome is likely an extrachromasomal
plasmid. S. Bergman asked if it is known for sure and C. Burge answered that we do not
know. C. Francois asked if the virus was detected in mussels. C. Burge confirmed that yes
OsHV-1 was detected in mussels Mytilus galloprovincialis by qPCR. It was also detected in
Manila clam with qPCR as well as O. edulis and Kumamoto oyster but not in Olympic oyster.
I. Arzul asked which tissues were tested for the detection of OsHV-1. C. Burge answered that
they used gills and mantle. I. Arzul also asked if in situ hybridization tests were performed. C.
Burge said that these tests have not yet been realised and obtaining positive signals would
probably be difficult regarding the low infection levels.
EFSA- Scientific opinion on the increased mortality events in Crassostrea gigas. A.
Afonso
The European Food Safety Authority has received a request to issue a scientific opinion
concerning the increased mortality events of Pacific Oysters that occurred in 2008-2009. The
terms of reference will focus on the role and possible importance of infectious agents and
environmental factors on the occurrence of the oyster mortality but also on determining if
other mollusc species are of epidemiological relevance and if there are differences between
life stages. The mandate was attributed to the Animal Health and Welfare. The panel has
nominated a Working group of ad-hoc experts to develop the risk assessment, the deadline for
the opinion is November 2010.
S. Cabot encouraged Member States to collaborate with EFSA in its work by providing any
relevant information to EFSA.
ANNUAL MEETING SESSION III : FOCUS ON THE MOLLUSC HEALTH
SITUATION IN JAPAN (Secretary : C. Garcia)
Mollusc health situation in Japan. N. Itoh
In Japan, annual yield of aquatic molluscs is approximately 120 million tons, of which 81
million tons is derived from bivalves species. The main bivalve products are scallop (287,000
tons by catch and 203,000 tons by aquaculture) and oysters (219,000 tons only by
aquaculture). Production of these important aquaculture bivalves are stable with little
fluctuation. On the other hand, decrement of manila clam resources and mass mortality of
pearl oysters are serious concerns which bivalve industries of Japan currently face. In addition
to these recent topics, this presentation will focus on our activity against abnormal
enlargement of the ovaries in the Pacific oyster.
In recent years, manila clam production has declined to only 20% of the maximum
recorded in 1983. Environmental damage, introduction of invasive predators and several
parasitic organisms were suspected of causing this reduction, but recent studies strongly
suggest that infections by protozoan parasites, Perkinsus spp., are the main cause for the
decrease of manila clams. Now several intensive studies are conducted to understand the
influence of Perkinsus spp. on manila clams in various locations of Japan.
Destruction of pearl oyster culture is also a serious concern in the fishing industry of
Japan. After 1970, pearl oyster industries grew along with economy development, however,
in the mid-1990s, mass mortality of pearl oysters occurred in various farms. Moribund oysters
were characterized with unusual reddening in the soft parts. While environmental damage,
parasitic infections or stress by high-density culturing were considered, the establishment of
experimental infection was later demonstrated, and currently it is broadly accepted that this
disease is caused by a virus. Thus far, any efficient control method has not been established
and a few hundred pearl oyster farms and hatcheries have been closed since the outbreaks.
I. Arzul asked which scallop species is reared in Japan and if a surveillance programme is in
place on this species and on oyster species. N. Itoh answered that the scallop species is the
yesso scallop, Patinopecten yessoensis and at present no official surveillance programme
exists on scallops or oysters; some non official studies can be carried out but sampling is not
systematic. I. Arzul wondered if differential mortality was observed between clams infected
with Perkinsus olseni and P. honshuensis in Japan. N. Itoh said that before the description of
this new Perkinsus species in 2006, no molecular characterization was performed on
Perkinsus and it was considered that only Perkinsus olseni was present in Japan on clams. At
present, the researchers working on this subject are interested in comparing pathogenicity of
these Perkinsus species on clams.
Marteilioïdes chungmuensis and its impact on Crassostrea gigas. N. Itoh
Abnormal enlargement of the ovaries in the Pacific oyster, caused by an ovarian
protozoan parasite Marteilioides chungmuensis, also potentially threatens oyster industries in
Japan, since diseased oysters show an unusual appearance, and eventually lose their
marketability. Since the first report in 1934, the knowledge and information for the parasite
had been quite limited. Recently, molecular biological techniques were developed for this
parasite, and intensive field studies were conducted. Based on these studies, several important
pieces of information, such as life cycles, infection periods, pathogenicity and host ranges,
have been obtained. Moreover, it was found that disease progression was strongly related to
host’s sexual maturation. Based on these findings, we manipulated the sexuality of oysters by
changing culture environmental conditions, resulting in the establishment of a control method
to decrease prevalence in the experimental stage.
I. Arzul asked which the geographical distribution of Marteilioïdes chungmuensis is. N. Itoh
said that Marteilioïdes chungmuensis is found in Southern East of Japan, In Korea and in
Australia. M. Engelsma asked Marteilioïdes infection is correlated with water temperature.
N. Itoh said that the lack of temperature differences between infected areas does not allow to
establish such link. Meanwhile, generally, infection occurs when temperature is superior to
18°C. I. Arzul wondered if the infection is variable according to the oyster age. N. Itoh
answered that all oysters are sensitive but Marteilioïdes chungmuensis is more detected in
old oysters.
ANNUAL MEETING SESSION IV : TRACEABILITY
(Secretary: J.P. Joly)
Traceability: What to do in the context of mollusc production – requirements under
Directive 2006/88/EC. S. Cabot
Traceability – defined in ISO (8402) as the ability to trace the history (…) of an entity by
means of recorded identifications – is crucial the management of disease outbreaks and food
safety incidents.
Directive 2006/88/EC on animal health requirements for aquaculture animals and products
thereof, and on the prevention and control of certain diseases in aquatic animals 1 has a set of
provisions aimed at ensuring traceability of aquaculture animals:
• A prerequisite for traceability is the authorisation/registration of all aquaculture
production businesses. The Directive also requires that the Member States establish a
register containing information on the disease status of all farms and mollusc farming
areas.
• Each farm/mollusc farming area must keep records of all movements into and out of
the farm/mollusc farming area.
• Transporters of aquaculture animals must keep a record of farms being visited.
• Certain movements of aquaculture animals must be accompanied with an animal
health certificate, to demonstrate that the animal health status of the place of
destination is not jeopardised.
• All movements between Member States of aquaculture animals must be notified in
TRACES, regardless whether an animal health certificate is required or not.
In total these provisions would provide the ability to trace back possible contact farms in an
epidemiological investigation. However, the Directive does not require tracing of individual
animals or batches of animals. Whether more detailed traceability is feasible or desirable
would be up to the MS and industry itself to decide. In this context it must be noted that
whilst the requirements for placing on the market and import are EU harmonised, Member
States may establish more ambitious systems as regards the record keeping by aquaculture
production businesses.
I. Arzul asked how far the member states are in implementing this requirement. Italy and
Denmark started traceability on fish. H. Hellberg explained that it’s hardly done in Norway.
However she added that you get information only if you ask people. I. Arzul added that
hatcheries tend to mix different batches of seeds so it’s hard to trace. S. Cabot reminded that
the Directive asks only to trace the origin of molluscs and the place of relaying. D. Fraser
explained that farmers associations in Scotland have introduced and follow an industry code
of
good practice, following consultation with authorities and scientific advisors
(http://assg.org.uk/cgi-bin/download.cgi). A bar code attached to each batch would be a good
way to trace animals (particularly for public health aspect). S. Cabot concluded by reminding
that implementation is the competent authority responsibility (not NRL responsibility).
Fish farm categorisation in some EU Member states. N.J. Olesen
Council Directive 2006/88/EC lay down the animal health requirements for aquaculture
animals and products thereof and contain provisions on the prevention and control of certain
1
OJ L324, 24.11.2006, p. 14.
diseases in aquatic animals. A key factor for these provisions is the proper authorisation and
registration of all farms and certain processing establishments. Part of this process includes
the placement of all farms in one of 5 categories for each of the 4 listed non-exotic fish
diseases.
Examples of how this categorisation were implemented in fish farming in Europe will be
given with focus on the how it was implemented in Denmark.
In addition risk based approach to surveillance in fish farming will be discussed.
I. Arzul asked if hatcheries are classed in high risk categories. N. Olesen explained that only
3 categories of risk is simplified. S. Cabot added that there are few eradication programs in
EU (in Shetland and Norway). N. Olesen wondered if categorisation of mollusc farms is
already in place. The answer was no. I. Arzul suggested to include such data in the annual
epidemiological reports. S. Cabot reminded that each Member State is given quite some
flexibility in deciding on which approach to take in the categorisation.
When geographic information meets molecular data. N. J. Olesen
When aquaculture farms and locations of pathogen isolations are inserted in digital maps it
creates a possibility for overview on e.g. where the infected farms are situated? It can be used
as an efficient tool for epidemiological analysis of disease spread, with pictures of status
regular intervals. It is valuable for risk-assessment, which farms are in nearby locations or
situated at the same stream? And for risk-management for creation of protection zones.
Digital maps are available in many different forms. Since August 2008 all EU Member states
have been obliged, through Council Directive 2006/88/EC (Anon. 2006), to provide
electronically available information on authorisation status including geographical
coordinates of all farms and the Commission shall facilitate the interoperability of
information systems between Member States. These demands thus provide fish health
managers, epidemiologists and pathologists a new and very useful tool in their work.
As sequencing of pathogen genes have become a common and easily accessible tool unique
possibilities for molecular tracing with efficient visualisation has risen in the recent years. A
world wide fish pathogens database www.fishpathogens.eu was created by the EURL fish
giving open and semi-open access to detailed information on a number of pathogens causing
diseases in fish (Jonstrup, Gray, Kahns, Skall, Snow & Olesen 2009). The database was
initially focusing on VHSV as model, and has since expanded to include IHNV. Databases on
the pathogens causing the diseases SVC and ISA are in process. Within the following years
KHV, IPNV, Rana viruses, perch rhabdoviruses etc. will follow. Using the same model for
creating a mollusc pathogen database would be a realistic tool for the EURL.
For VHSV the G-gene encoding the viral glycoprotein has been chosen as the best sequence
to use for molecular tracing due to the high polymorphism of this gene. It has also been
shown that the best resolution for genotyping the viruses are by sequencing the full length Ggenes (Einer-Jensen, Ahrens & Lorenzen 2005). VHSV is stable within the same clinical
outbreak but the G-gene of VHSV will often vary from one outbreak to another. Thus when
new outbreaks arise, isolates can rapidly be sequenced and aligned to other known isolations
giving hints to the possible origin of the outbreaks.
Examples of molecular tracing from Switzerland, Denmark, and USA will be given.
References
Anon. (2006) Council Directive 2006/88/EC of 24 October 2006 on animal health requirements for aquaculture
animals and products thereof, and on the prevention and control of certain diseases in aquatic animals. Official
Journal of the European Communities L 328, 14-56.
Einer-Jensen K., Ahrens P. & Lorenzen N. (2005) Parallel phylogenetic analyses using the N, G or Nv gene
from a fixed group of VHSV isolates reveal the same overall genetic typing. Diseases of Aquatic Organisms 67,
39-45.
Jonstrup S.P., Gray T., Kahns S., Skall H.F., Snow M. & Olesen N.J. (2009) FishPathogens.eu/vhsv: a userfriendly viral haemorrhagic septicaemia virus isolate and sequence database. Journal of Fish Diseases 32, 925929.
I. Arzul asked if competent authorities are interested by such tool (molecular databases for
pathogens traceability). N. Olesen said not yet. Mostly scientists are interested. H. Hellberg
highlighted that input in the database requires effort and wondered if Competent authorities
are ready to pay for it and to update it. N. Olsen reminded that they have to do it by EU
regulation. The problem is that scientists want to publish before giving their data. We must
convince our colleagues to input data in the database.
The management information system implemented in Veneto region (north eastern
Italy) to support shellfish sanitary surveys. M. Dalla Pozza
The European Union (EU)sets out a strategy for the sustainable development of European
aquaculture with the aim of creating a long-term employment in the aquaculture sector,
including promoting high animal health and welfare standards, and environmental actions to
ensure a sound industry. Among the EU countries Italy has a significant aquaculture
production. More than 80% of the whole Italian production of clam (Tapes filipinarum) is
yielding in the Venice Lagoon (Veneto Region, north eastern part of Italy). Moreover in the
same area an important production of Mytilus galloprovincialis,and Chamelea gallina, is
present. The breeding, rearing and the placing on the market of shellfish animals and products
thereof constitutes an important source of income for persons working in this sector.
Monitoring programs are in force in Veneto region, with the aim of verifying the levels of
microbiological an chemical contamination of shellfish, together with the health status related
to notifiable infectious diseases . In fact outbreaks of diseases in aquaculture animals could
cause severe losses to the industry concerned and all disease control measures have an
economic impact. Inadequate controls may lead to a spread of pathogens, which may cause
major losses and compromise the animal health status of molluscs.
In order to adequately monitor the epidemiological situation related to microbiological an
chemical contamination of shellfish and related to diseases that may affect them, the Veneto
is developing and applying official monitoring programs for the classification of bivalve
mollusc harvesting areas and for the surveillance of their health status, according to the EU
and national legislation. In order to collect, store and manage the data related to farms,
harvesting areas, and laboratory results arising from these monitoring programmes, a regional
shellfish database has been set up. It contains information concerning farm characteristics
and their geographical position. A WebGIS tool has been implemented with the aim to
analyse all the above mentioned data, to integrate them with the pollution sources data and to
asses the sanitary and epidemiological situation of bivalve shellfish production. A procedure
of interoperability between this web-gis database of farms and harvesting area and the
Laboratory Management information System (LIMS) of the Animal Health Institute is going
to be implemented in order to integrate all these information and produce updated reports on
the diagnostic activity and the results of the monitoring programme. The Web-GIS tool will
be used to disseminate information generated by the monitoring program, allowing
Competent Veterinary Authority to have an overview of the disease situation, to facilitate, a
rapid reaction in the case of a suspicion of disease and to protect farms or mollusc farming
areas having a high animal health standard.
I. Arzul wondered if this information system is planned at a national level. M. Della Pozza
answered that this tool will be presented to the Italian concerned ministry and added that
Veneto has a long experience of managing these data. S. Ramdorh worried about data
confidentiality. M. Della Pozza explained that the system is dedicated to the competent
authority, the veterinarians and laboratories, not to the public and highlighted the need of
sharing production data with everyone.
S. Ramdorh noticed that showing only Salmonella results and not Norwalk results would
advantage the farm. M. Della Pozza approved but said that it’s better than nothing. S. Cabot
asked who can access the database. M. Della Pozza said that the competent authority is
presently thinking about this question.
A forthcoming modelling of oysters transfers
C. Lupo1, I. Arzul1, C. François1, C. Garcia1, T. Renault1 et N. Bareille2
1
: IFREMER, National reference laboratory for mollusc disease, Avenue de Mus de loup, 17390 La Tremblade,
France
2
: ONIRIS & INRA, Atlanpôle - La chantrerie, BP 40706, 44307 Nantes Cedex 03, France
Since two years, mass mortality occurred on Pacific oysters (Crassostrea gigas) in France.
According to current EU regulation (Directive (CE) n°2006/88), these abnormal mortalities
were notified to competent authority. A previous study described an atypical pattern of these
declared mortality cases in time and space. In addition, the national surveillance network of
molluscs diseases (REPAMO) detected pathogenic agents in samples of these mortality cases.
An infectious implication in the mass mortality was thus suspected. In veterinary medicine,
animal movements are one of the main ways to introduce or spread an infectious animal
disease. In French oyster farming, many stakeholders and premises are heterogeneously
spread over the country and a highly dynamic flow of oysters exists among them. However,
no reliable data is publicly available concerning oysters transfers.
Thus, a field study is about to be conducted to model the network of oyster populations
movements between oyster farms, to map this network and to take inventory of husbandry
practices as regards oyster transfers. Fifty to 100 oyster producers will be randomly selected
in Charente Maritime bay between May and July 2010, according to a stratified sampling
design based on the farm category towards oyster flows. Data related to the history of
movements of the flocks in production at the time of the survey will be collected during a
face-to-face interview of the oyster producer, using a standardized questionnaire and a map of
the exploitation. Movement data will be analyzed using social network analysis, to map and
measure the oyster flows among oyster farms.
The results of this study are expected to draw a theoretical network of the oyster flows, to
obtain maps of the incoming and outgoing flows. The consistency of these maps with the
notified mortality cased pattern in space and time will be assessed. The results of this study
could also help to characterize oyster farms according to the risk their husbandry practices
may represent for the introduction or spread of an infectious disease.
ANNUAL MEETING SESSION V : RESULTS OF RECENT FIELD STUDIES
(Secretary: J.P. Joly)
Detection of Bonamia ostreae in larvae of Ostrea edulis. I. Arzul, M. Robert, C. Garcia, B.
Chollet and A. Langlade
Bonamia ostreae is an intracellular protistan parasite affecting flat oysters Ostrea edulis. It
can be detected in juveniles but mortalities mainly affect oysters which are more than 2 year
old. The parasite is usually observed inside haemocytes and sometimes free in gill epithelia
suggesting a parasite release through these organs. However, the infective form and ways of
entry and release remain undetermined. A controversial description proposed that B. ostreae
was an ovarian tissue parasite for part of its life cycle but this hypothesis could never be later
confirmed.
Flat oysters breed their larvae in their mantles for about one week before releasing them in
the water. Consequently larvae are in strong contact with their mother for one week and one
can wonder if larvae are susceptible to Bonamia ostreae and might become infected during
this period We benefited from a survey carried out in Quiberon bay (France) known to be
infected with Bonamia ostreae and the most important area in France regarding Ostrea
edulis spat collection. This survey aimed at controlling the presence of larvae within flat
oysters in order to advice farmers about the most suitable period for spat collection.
Flat oysters exhibiting larvae during this suvey were selected for our study and were treated
in order to perform some in situ hybridization (ISH) and PCR tests on both spawners and
larvae.
PCR tests revealed the presence of parasite DNA in some adults and larvae. Specific
labelling could be detected by ISH in connective tissue of gills, mantle and in one case of
gonad from spawners and in stomach epitehlium of some larvae.
Our results suggest that larvae might present some risks in terms of Bonamia ostreae spread
and their movements should thus be restricted when they are exported from infected zones.
M. Gubbins asked about the incidence of Bonamiosis in Quiberon Bay. I. Arzul answered that
prevalence usually ranges between 10 and 15%. M. Gubbins remarked that in UK,
prevalence is lower, around 2% and incubating oysters could constitute a good target for
Bonamia survey.
M. Engelsma wondered if there are differences between the techniques regarding sensitivity.
I. Arzul explained that in the context of this study it is not easy to compare these techniques.
Moreover, entry and release of parasites are uncertain.
M. Engelsma pointed out that usually there is a low level of infection in the gonad even in
high infected oysters.
I. Arzul added that considering the low number of larvae infected (as observed by in situ
hybridization), spawning is probably not the preferred way to release Bonamia.
Protozoan infestation dynamics and occurrence of neoplasias in digestive gland of
Mediterranean mussels (Mytilus galloprovincialis) in the Slovene sea in correlation with
sea temperature, salinity and oxygenation. Mitja Gombač, Vlasta Jenčič, Veterinary
Faculty of Ljubljana, Slovenia
Introduction The health status of cultured and wild Mediterranean mussels (Mytilus
galloprovincialis) in the Slovene sea, which due to its position, shallowness and many other
factors, highly influencing the temperature, oxygenation and salinity which varie considerably
througout the year, represents a very specific habitat (Lipej, 2004; Richter, 2005), was till
date unknown. To find out which protozoa and neoplasia affect the digestive gland of Slovene
Mediterranean mussels an annual study was performed.
Material and methods In all 1280 adult Mediterranean mussels were monthly collected and
included in our study. Water temperature, oxygenation and salinity were measured at each
sampling. All mussels were macroscopically inspected and measured. The flesh condition
index was calculated for each mussel by weighting the fresh mussel. A standard section
through the digestive gland of each mussel was performed, tissue section was routinely
paraffin embedded and cut at 4µm. One slide per mussel containing digestive gland was
stained with haematoxylin and eosin and examined by light microscopy for the presence of
protozoa and neoplasia.
Results and Conclusions Microscopic examination of digestive glands revealed intracellular
ciliates of mussels in 293 mussels (22.9% prevalence) and haemocytic neoplasia of mussels in
the connective tissue of 14 mussels (1.1% prevalence). Intracellular ciliates were pear or
spindle-shaped with a polymorphic oval to globular basophilic, fragmented macronucleus.
They were found inside the digestive tubule epithelia or were in the lumina of digestive
tubules. A slight enlargement of epithelial cells that carried ciliates of a large size was the
only alteration of digestive glands tubules observed. A mild diffuse haemocytic infiltration in
digestive gland connective tissue was noticed in 2% and haemocytic neoplasia of mussels in
1% of mussels infected with intracellular ciliates of mussels. The highest average prevalence
of infection was detected in spring with the average sea temperature 15.2oC, the average
oxygenation 9.3 mg/l and the average salinity 29.6‰. The lowest prevalence was in summer
with the average sea temperature 24.1oC, the average oxygenation 7.6 mg/l and the average
salinity 38.1‰. We concluded that salinity had influence on infection with intracellular
ciliates, whereas temperature and oxygenation had not - the higher was the salinity the lower
was the infection. The average condition index of infected mussel was slightly higher than in
healthy ones, ciliates were most often noticed in longer and heavier mussels. Haemocytic
neoplasia was characterised by highly pleomorphic and anysocitotic neoplastic cells with
large, hyperchromatic rounded or pleomorphic nucleus with finely dispersed or dense
chromatin without nucleolus. Bi- and tri-nucleated cells were also noticed. Nucleus to
cytoplasm ratio was high and the number of mitosis was 2 mitoses per HPF. A diffuse
infiltration of neoplastic cells was noticed in four mussels and small number of single
neoplastic cells or small foci of neoplastic cells in connective tissue of digestive glands were
noticed in 10 mussels. Necrosis and multifocal atrophy of digestive tubules were noticed in
mussels with diffuse neoplasia whereas severe haemocytic infiltration of connective tissue
was seen in mussels with single neoplastic cells. In mussels with multifocal form of
haemocytic neoplasia no alterations were seen. Haemocytic neoplasia was more frequently
observed in spring, when the sea temperature was between 11oC and 20.2oC, the oxygenation
below 9 mg/l and the salinity between 28‰ and 32‰ and in autumn, when the sea
temperature was between 17.8oC and 20.3oC, the oxygenation below 7.1 mg/l and the salinity
between 26‰ and 39‰. Only one affected mussel was detected in winter with the sea
temperature 9.5oC, the oxygenation 10.95 mg/l and the salinity 35‰. The average condition
index of mussels with haemocytic neoplasia was higher than in healthy ones.
References
Lipej L, Orlando Bonaca M, Makovec T. Obravnavano območje. In: “Raziskovanje biodiverzitete v slovenskem
morju” (Nacionalni inštitut za biologijo, Morska biološka postaja, Ed.). pp.11-24. Piran. 2004. ISBN 96190363-5-2.
Richter M. The Gulf of Trieste and our sea. In: “Our sea: The Environmental and Living World of the Gulf of
Trieste” (Narodna in univerzitetna knjižnica, Ed.), pp. 20-63. Piran. 2005. ISBN 961-90692-8-5.
S. Bergman asked about the cause of neoplasia. M. Gombac said that the cause is unknown
but Hg, Cd, pesticides in Slovenia sea will be tested together with the distribution of
phytoplankton (DSP, PSP, ASP). S. Bergman asked if neoplasia could be caused by a virus.
C. Burge added that some neoplasia cases are due to retroviruses. I. Arzul asked if
transmission experiments are planned, M. Gombac said yes. Finally, T. Renault suggested to
develop some cell culture from haemocytes collected from mussels exhibiting neoplasia.
Investigations on cockle mortality with samples obtained from Irish coasts. S. Bergman
I. Arzul explained that in Bonamia tests by PCR, several non specific bands can be obtained
without clear explanation. By repeating DNA extraction, these non specific PCR products
generally disappear. N. Itoh said that black spots have already been observed in digestive
gland of molluscs in Japan.
Detection of Perkinsus chesapeaki like parasite in Ruditapes decussatus from Leucate
lagoon France. I. Arzul*, B. Chollet, J. Michel, M. Robert, L. Miossec, J.P. Joly, C. François
and C. Garcia
A study was carried out in France to characterize parasites of the genus Perkinsus present in
clams. Clams were collected in different locations along the French coasts in 2004 and 2005.
Parasites of the genus Perkinsus were detected and quantified by thioglycollate culture
medium. Molecular characterization was done by PCR-RFLP targeting the ITS region. PCR
products selected according to their restriction profiles were subsequently cloned and
sequenced in order to check potential co infection within clams. Perkinsus olseni was
detected in all the French investigated producing areas. However, in Leucate lagoon (Figure
1), PCR-FRLP results revealed restriction profiles similar to P. chesapeaki ones in 2 clams.
In order to confirm and complete these results, clams were newly collected in 2008 in Leucate
lagoon. 60 specimens were processed for in situ hybridization tests and parasite culture in
DMEM:HAM’S F12 medium. Parasite cultures from 16 clams could finally be obtained and
tested by PCR RFLP. 10 individuals showed P. olseni restriction profiles, 2 individuals
showed P. chesapeaki restriction profiles and two clams revealed co infection profiles. The
two other ones were not interpretable. Sequencing confirmed the PCR-RFLP results:
sequences displaying 99 to 100% of homology with Perkinsus olseni could be obtained as
well as sequences showing a maximum identity with P. chesapeaki (96-97%). Parasite
cultures characterized as P. olseni and “P. chesapeaki –like” were examined daily in order to
compare the different parasite stages. The 60 clams were examined by histology and
specimens detected infected by Perkinsus sp. were tested by in situ hybridization using probes
specific to P. olseni and specific to P. chesapeaki (Figure 2). Results are indicative of the
presence of both parasites P. olseni and Perkinsus sp. closed to if not P. chesapeaki. We thus
conclude about the sympatry of these two parasites in Leucate Lagoon and discuss the
presence of “Perkinsus chesapeaki-like” in France.
I. Arzul added that molecular characterization of Perkinsus species present in Europe is not
systematic. Some results indicate that Perkinsus olseni is also reported in Spain an Italy
G. Arcangeli noted that Mercenaria mercenaria is present in Italy and should be checked for
P. chesapeaki.
SESSION VI : STUDIES ON INTERACTION BETWEEN HOST AND PATHOGENS
(Secretary I. Arzul)
Host responses to infection with Bonamia ostreae : comparison between resistant and
wild flat oysters. B. Morga*, I. Arzul, N. Faury, C. Garcia, S. Lerond, B. Chollet, M. Robert,
S. Lapegue and T. Renault.
Bonamiosis due to the parasite Bonamia ostreae is a disease affecting the flat oyster Ostrea
edulis. Bonamia ostreae is a protozoan, affiliated to the order of haplosporidia and to the
phylum of cercozoan. This parasite is mainly intracellular, infecting haemocytes; cells notably
involved in oyster defence mechanisms. Recently it was shown that in vitro infection with
Bonamia ostreae modifies haemocyte activities as well as expression of genes including SOD
(Superoxide Dismutase), OGST (omega gluthatione-s-transferase).
In the present study two groups of flat oysters, a group selected for resistance to bonamiosis
and a second group composed of oysters from the wild, were maintained for 6 months in
cohabitation with infected oysters. After 4 and 6 months of cohabitation, parasite presence
was monitored by heart smears. Haemolymph was also collected individually. Pools of
haemolymph were then prepared according to the infectious status (presence or absence of the
parasite) and the origin of the oysters (selected versus wild). Non specific esterases, ROS
(Reactive Oxygen Species) and phagocytosis were measured by flow cytometry. RNA
extraction from haemocytes were performed in order to quantify the expression level of
candidate genes including SOD (Superoxide Dismutase), EcSOD (extracellular S uperoxide
Dismutase), OGST (omega gluthatione-s-transferase), metallothionein, IAP (Inhibitor of
apoptosis) and Fas-ligand.
Differences could be observed between selected and wild flat oysters. Flow cytometry
analysis showed that the phagocytosis capacity for infected selected oysters was lower than
for infected wild oysters. Similar results were observed for non specific esterases activities
while ROS measures did not show significant difference between both tested types of flat
oyster. Real time PCR assays were developed to study the relative expression of candidate
genes.
Infection with Bonamia ostreae in resistant population is related to an increased expression of
metallothionein and Fas-ligand. Infection with Bonamia ostreae in wild population is related
to an increased expression of IAP (Inhibitor of apoptosis) and Fas-ligand.
This study contributes to improve our understanding of the defence mechanisms developed by
the host against the parasite. Moreover, the targeted genes will be tested regarding their
potential interest a genetic markers of resistance to bonamiosis.
S. Bergman said that the term “resistance” should be taken with caution and in the present
case it is a resistance to mortality and not to infection . S. Bergman would like to know if
there is any suspicion about some genes involved in the resistance. B. Morga said that it is
difficult to answer this question since there is no close model to rely on and compare with.
An eQTL approach to detect parts of the genome involved in the expression of candidate
genes for resistance to bonamiosis in the european flat oyster, ostrea edulis. E. Harrang,
B. Chollet, N. Faury, B. Morga, I. Arzul, S. Heurtebise, S. Lapègue.
The subtidal area of aquacultural production of the european flat oyster, Ostrea edulis, has
been dramatically affected, since the early 1980s, by the emergence of the intrahaemocytic
parasite responsible for the bonamiosis disease, Bonamia ostrea. In the present study, we
propose to use a new approach to characterize O. edulis resistance to B. ostreae; an eQTL
approach (expression Quantitative Trait Loci). This approach will combine informations from
different research areas and will be a breakthrough in the study of the relationships between a
marine bivalve and one of his parasite. In a previous study, we have sequenced 41 EST
portions of candidate genes for bonamiosis tolerance/sensibility on 22 oysters from different
origins (natural populations or selected lineages for surival to bonamiosis). The screening of
the sequence alignments had enabled the detection of 511 SNPs (Single Nucleotide
Polymorphism) on a total sequencing lenght of 19,926 bp. The frequence of 1 SNP every 39
bp found on the european flat oyster is quite similar to the frequence previously found on
Crassostrea gigas (1 SNP every 40-60 bp). The further experiment will be conduct on
individuals from the 3rd generation of a selected lineage for survival to bonamiosis
(knowledge of the genotype of the 1st and 2nd generations), by injection of purified parasite
into the adductor muscle. The eQTL mapping will associate genotyping of new molecular
markers (the SNPs previously identified) to a phenotypical criteria (the parasite load) with a
potential differential of expression of some candidate genes.
S. Bergman asked if SNPS observed every 39bp in average induced changes at the amino acid
level. E. Harrang said that they did not investigate this issue. M. Engelsma asked how the
difference between true SNP and PCR sequencing error was done. E. Harrang answered that
sequencing was done in both ways and then sequences were further tested by genotyping to
confirm if it was true SNP or not.
Differentially expressed genes of the carpet shell clam Ruditapes decussatus against
Perkinsus olseni. Prado-Alvarez M, Gestal C, Novoa B, Figueras A.
Suppression-Subtractive Hybridization (SSH) was used to identify differentially expressed
Ruditapes decussatus genes against the protozoan Perkinsus olseni infection. A forward and a
reverse subtraction were carried out to identify up- and down-regulated genes in both
haemocytes and gills of clams naturally infected with P. olseni. New genes, candidates for
further investigation into the functional basis of resistance to pathogens, have been detected
for the first time in the clam (R. decussatus). A total of 305 differentially expressed sequences
were obtained, 221 of them in haemocytes and 84 in gills of infected clams. The number of
ESTs with potential similarity with known genes was 97, 42 among them were related with
immunity and stress related functions. The pattern of expression of the immune selected
genes was studied by quantitative PCR with samples of naturally Perkinsus infected clams
and compared with samples from an in vitro infection of clam haemocytes with Perkinsus
zoospores. The maximum expression was found 1h post infection. The complete open reading
frames of selected sequences adiponectin-C1q and Defender Against Death were determined
and named Rd adiponectin-C1q and Rd DAD-1, respectively. The Rd adiponectin-C1q
showed the highest expression values. Since the DAD-1 is related with apoptosis inhibition,
the TUNEL assay (Terminal eoxynucleotidyl transferase Nick End Labelling) was developed
in natural infected samples. A decrease in the number of apoptotic haemocytes in highly
Perkinsus infected samples was observed showing the importance of this process during a
Perkinsus infection. Our results provide new insights into the molecular basis of hostpathogen interactions in R. decussatus
I. Arzul asked if the work was also done on lightly infected clams. M. Prado said that they
only used no infected and highly infected clams.
SESSION VII: EURL DAY LIFE ACTIVITIES
(Secretary: C. Francois)
Proficiency test 2009 for National Reference Laboratories for mollusc diseases
C. Garcia, B. Chollet, J.P. Joly, I. Arzul
A proficiency test aims at establishing that the examination of a given sample leads to the
same conclusions in any laboratory within the National Reference Laboratory network. It also
permits (1) to determine a laboratory’s capability to conduct specific diagnostic tests, (2) to
check or certify the performance of individual operators, (3) to harmonise existing test
methods (4) to resolve interlaboratory differences and (5) to evaluate new test methods.
In 2009, it was the seventh histo-cytology proficiency test organised by the Community
Reference Laboratory and it was composed of two slide collections (30 slides per collection).
These objectives were:
- For collection 1, the detection of EU listed diseases in Directive 2006/88/EC,
bonamiosis due to Bonamia ostreae and marteiliosis due to Marteilia refringens in the
European flat oyster Ostrea edulis by histology and cytology,
- For collection 2, the detection of listed or not listed exotic pathogens presently
described in cupped oysters (Crassostrea virginica or C. gigas) from North America by
histology.
17 laboratories participated in both collections and 1 laboratory only in collection 1.
The laboratory results for collection 1 were similar to those of the previous proficiency tests;
the mean of all laboratories was 80.37%, lightly inferior to this of 2007 proficiency test. The
main difficulties were encountered for the detection of light parasite infestations particularly
for the parasite Bonamia ostreae.
The results of collection 2 highlighted the difficulty of laboratories to detect exotic pathogens,
specially the parasite Perkinsus marinus. Mikrocytos mackini was relatively well detected but
generally light parasite infections are misdiagnosed.
A disparity was observed between laboratories for the second slide collection (exotic
pathogens). This disparity emphasizes the need for training in diagnosis by means of
histology notably for the detection of Perkinsus marinus.
So, a new training session will be organized in 2011 during the next workshop and will focus
on this parasite and also on the PCR detection of the oyster herpesvirus, OsHV-1. A particular
training session on bonamiosis and marteiliosis diagnosis will also occur in 2011 for some
laboratories.
Moreover, as histology and cytology techniques are often not sufficient to demonstrate free
status and molecular tools, including PCR, in diagnostic procedures are more and more used,
a new proficiency test was implemented in 2008 and was based on the detection of Bonamia
ostreae by PCR. So, a new PCR proficiency test will occur in 2010 and will concern the
detection of Marteilia refringens in flat oysters and mussels.
A. Turnbull asked if it would be possible to come back on low infection levels. I. Arzul
explained that ring test slides have been scanned and will be available quite soon through the
EURL website. Slides from the first collection will be accessible for one month and then slides
from collection 2 will be accessible for several months. As mentioned by B. Chollet there is
presently no possibility of slide annotation. S. Ramdohr asked how the infection levels are
defined. I. Arzul reminded that for the analysis of ring test results, level of infection is not
taken into account. However, the levels of infection are generally based on scales used in the
laboratory. S. Ramdohr aksed if his laboratory could be included in the next interlaboratory
comparison test. I. Arzul explained that the priority is given to the NRLs’ network but other
laboratories could receive the slides after the NRLs. If too many laboratories from a same
country ask to participate in the ILC the EURL suggests that the NRL of this country
organises a national test with material provided by the EURL. However in the case of
Germany it seems that only one laboratory performs histological detection of mollusc
pathogens.
Quality management and the EURL website. J.P. Joly
The “new” website
During 2009 the EURL website was revised. Ifremer communication managers decided to
standardise the software used to manage the different Ifremer’s websites and they chose the
Eziweb software developed by a French company from the eZpublish freeware. Eziweb is
managed like a big database of texts, pictures, PDF files, etc., and each page seen on internet
is a “virtual” page built instantly from several scattered sub-units downloaded from the
database. It took several days to move each page one by one from the previous website to the
new one but finally the presentation is quite similar and should not disturb the usual users of
the EURL website. For more convenience it has been decided to move the most important
pages in the homepage menu and so to skip the usual “Presentation” page. A new section has
been created and will be soon available. It’s a section devoted to European National
Laboratories for molluscs diseases with a limited access to the public. Each NRL will receive
a code and a password from the EURL to access these pages. The NRL’s pages will give
information on several subjects on annual meetings and workshops (programmes, list of
participants, reports from the meetings, photo gallery), organisation of proficiency tests
(information on the tests, registrations forms, data recording forms, tests final reports, slide
viewer of scanned slides from the tests). In parallel the free access section will have new
pages on organisation on training sessions (information on the next training session,
registration forms, request forms for special trainings) and pages about request forms for
reference material or for assistance (e.g. for confirmatory diagnosis). All these forms will be
referenced and handled according to the EURL quality management system for traceability
reasons.
The new standard ISO 17043:2010 on general requirements for proficiency testing
The EURL is presently building a system meeting the French accreditation body requirements
(Cofrac document N° LAB-CIL-REF-02:2007). These requirements are based on the ILAC
guide (ILAC*-G13:2007 – Guidelines for the requirements for the competence of providers of
proficiency testing schemes). The documents presently used for the interlaboratory
comparison (ILC) tests organized by the EURL (information document, registration form,
records form and report) are written according to the Cofrac requirements. These documents
will be available on the EURL website each time a proficiency test is organized.
The new international standard ISO 17043:2010 “Conformity assessment - General
requirements for proficiency testing” has been issued in February 2010 and will soon be
adopted as a National standard by European countries.
The new standard is mainly based on the two previous guides and on an ISO standard:
• ILAC-G13:2007 guide – Guidelines for the requirements for the competence of
providers of proficiency testing schemes
• ISO guide 43-1 guide (1997) – Proficiency testing by interlaboratory comparison. Part
1: development and operation of proficiency testing schemes.
• ISO/CEI 17025 standard – General requirements for the competence of testing and
calibration laboratories
This new standard will probably apply very soon to European providers of ILC tests seeking
accreditation.
S. Cabot asked if there is a link from the EURL website to DG Sanco. JP Joly said that
presently no but it could be easily done. S. Bergman suggested to have email addresses of
contact persons from the different NRLs for Mollusc Diseases. N.J. Olesen explained that the
EURL for fish diseases had similar discussion regarding pages restricted to NRLs. Most of
NRLs had lost their password and generally did not access this restricted section. They finally
decided to have all the documents in open access.
Working prospects of the European Union Reference Laboratory for mollusc diseases
and concluding remarks. I. Arzul
In 2009, the NRLs network for mollusc diseases included 20 NRLs for mollusc diseases.
Functions and duties of the EURL for mollusc diseases are given in the Annex VI of the
Directive 2006/088/EC.
One of the main aims of the EURL is to harmonize diagnosis within the EU. For this purpose
the EURL has created and maintains a collection of pathogens available for laboratories in
Member States. A CD-ROM on histology and anatomo-pathology has also been developed
since 2002. The CD-ROM proposes illustrations and comments believed to be valuable for
mollusc diseases diagnostic, especially diseases notifiable to the EU and OIE. It is subject to
permanent reviewing and updating. Last update was done in 2007.
Reference material is sent on request to NRLs. In 2009, 72 slides (histological and cytological
slides), 5 paraffin blocks, 2 bacteriological strains and 65 plasmidic DNA suspensions were
distributed. In 2010, the EURL would like NRLs to send their request through an electronic
questionnaire available from the EURL website.
Inter-laboratory proficiency tests are regularly organised to test the ability of laboratories to
identify listed and important pathogens by histology. In 2009, the EURL organised a ring test
based on histology and cytology for the detection of endemic and exotic pathogens. In 2010 a
ring test will be organised to test the ability of NRLs to detect Marteilia refringens by PCR.
In 2011, the EURL plans to organise a new inter laboratory comparison test based on histo
cytopathology for the detection of Bonamia sp., Marteilia sp. in Ostrea edulis and Mytilus
spp. as well as Mikrocytos mackini and Perkinsus marinus in Crassostrea spp.
An advisory group in Quality Assurance was created in 2004. Exchange of information
between NRLs during annual meetings or by e-mails allowed the EURL to write quality
assurance documents that could be used by NRLs wishing to build their Quality System.
These Standard Operating Procedures are available on the EURL website
(http://wwz.ifremer.fr/EURLmollusc/). Several SOPs were prepared in 209 including the
detection of Marteilia refringens by in situ hybridization.
The EURL collect and collate epidemiological data every year. These reports are included in
the report of the annual meeting and the EURL proposes to give access to the xls format
through the EURL website under the NRLs’ section.
The EURL assists the NRLs in the diagnosis of disease outbreaks in Member States. In 2009,
the EURL assisted several European and non European laboratories including Norwegian,
Turkish and Tunisian laboratories in the context of some suspicion cases of bonamiosis. In
2010, the EURL would like NRLs to send their request through an electronic questionnaire
available from the EURL website. This questionnaire will include some fields concerning the
origin and nature of the samples, the context of the request and will give recommendations
about the sending of the material.
The EURL provides opportunities of training and retraining through trainees, technical
workshop and annual meetings. In 209, the EURL welcomed two colleagues from Korea.
Again in 2010, the EURL would like NRLs to send their training request through an
electronic questionnaire available from the EURL website. The EURL plans additional
opportunities of training especially in histopathology. The first one should be organised in
October 2010 for a restricted number of participants and will not be free. The second one is
dedicated to NRLs who had 60% or less of good answers for one and or both of slide
collections included in the 2009 ring test. This workshop will focus on European listed
pathogens and on listed exotic pathogens of cupped oysters.
The EURL develops some works for validating and improving some methods of identification
of listed pathogens. During the last couple of years the EURL carried out comparison between
heart imprints and PCR for the detection of Bonamia sp.. In 2009, the EURL have initiated
such a comparison between digestive gland imprints and PCR for the detection of Marteilia
refringens. These studies are necessary to establish sensitivity and specificity of the
diagnostic methods and finally to establish sampling size. and improvement. The EURL has
also started to evaluate the impact of sample pooling on the detection of Bonamia sp. by PCR
and Real Time PCR.
In addition the EURL carries out several studies on listed pathogens. In 2009, works have
been done to characterize new genes in the genome of Bonamia ostreae and also to
characterize Bonamia parasites present in Europe. In 2009, the EURL was also involved in
studies on the life cycle and host range of Marteilia refringens as well as in the
characterization of parasites belonging to the genus Perkinsus present in France.
All these activities are presented on the EURL website: www.eurl-mollusc.eu
Next annual Meeting should take place in Ifremer in La Tremblade in March 2011. It should
be organised jointly with a technical workshop including two sessions: the detection of
OsHV-1µvar by PCR and the detection of Perkinsus marinus by histology.
Technical Report from the European Reference Laboratory for Molluscs Diseases – 2010 - page 60
Annex 2: Reference Material sent by the EURL during 2010
Date
17/02/2010
Type of material Number of samples
Plasmidic DNA
Ethanol fixed
tissue
Host species
Pathogens
2 DNA suspensions pCR®II-TOPO vector SS2-SAS1 from Marteilia
refringens
Bo-Boas from Bonamia
ostreae
Ostrea edulis
Bonamia ostreae
One sample of gills
Marteilia refringens
One sample of
digestive gland
Histological
slides
3 histological slides
Crassostrea gigas
Ostrea edulis
Imprint slides
2 slides
Ostrea edulis
17/02/2010
Histological
slides
Ostrea edulis
Mytilus
galloprovincialis
22/03/2010
Plasmidic DNA
and primers
01/04/2010
Paraffin block
01/04/2010
Plasmidic DNA
Country
Recipient
Japan
National Research Institute of Aquaculture, Fisheries
Research Agency, from Japan
Norway
National Veterinary Institute Bergen, PO Box 1263,
Sentrum, N5811, Bergen, Norway
Marteilioides chungmuensis
Bonamia ostreae
Bonamia ostreae
Bonamia ostreae
Healthy animals
19 laboratories: Sweden, Spain (2 laboratories), Italy,
19 sets of the
Germany, Ireland, Romania, The Netherlands,
following material:
2 primer suspensions CF and CR primers 500 µl of primers (100µM) European Slovenia, Northern Ireland, Norway (2 laboratories),
CF -CR from OsHV-1
Laboratories
England, Denmark, Scotalnd, Poland, Portugal,
2 DNA suspensions pCR®II-TOPO vector
CF -CR from OsHV-1 µvar
Croatia
12 paraffin blocks
Ostrea edulis
Mytilus edulis
Bonamia ostreae
Norway
National Veterinary Institute Bergen, PO Box 1263,
Sentrum, N5811, Bergen, Norway
M2A-M3AS from M.
refringens type M
M2A-M3AS from M.
refringens type O
United
KingdomScotland
Marine Laboratory, PO Box 101 Victoria Road,
Aberdeen AB11 9DB, Scotland, United Kingdom
Date
Host species
Pathogens
pCR®II-TOPO vector Bo-Boas from B. ostreae
Bonamia ostreae
Ostrea edulis
Country
Recipient
Croatia
Croatian Veterinary Institute Dept for Fish Diseases
and Aquaculture Savska 143, 10000 Zagreb Croatia
M2A-M3AS from M.
refringens type M
M2A-M3AS from M.
refringens type O
U.S.A.
Aqua Technics Inc., 455 West Bell Street
Sequim, WA 98382 ,U. S. A.
Crassostrea gigas
Mikrocytos mackini,
Turkey
20 imprint slides
Ostrea edulis
Bonamia ostreae
Healthy animals
Germany
Friedrich-Loeffler-Institut
Federal Research Institute for Animal Health,
Institute for Infectology, Boddenblick 5a, 17493
Greifswald – Insel Riems, Germany
Histological
slides
2 histological slide
Crassostrea gigas
Crassostrea virginica
Haplosporidium nelsoni
Perkinsus marinus
Sweden
Avdelning för vilt, fisk och miljö, Fisksetionen, S-751
89 Uppsala, Sweden
01/04/2010
Histological
slides
Ostrea edulis
Marteilia refringens,
Bonamia ostreae,
Slovenia
Institute of Pathology Forensic and Administrative
Veterinary Medicine, University of Ljubljana,
Veterinary Faculty, Gerbiceva 60, SL - Ljubljana,
Slovenia
30/04/2010
Histological
slides, Imprints
slides,
CDRom
4 histological slides,
4 imprint slides,
1 CDRom
Ostrea edulis
Mytilus edulis
Bonamia ostreae,
Healthy animals
Greece
Institute for Infectious and Parasitic Diseases Department of Aquatic Organisms Pathology, 80,
26th Octovriou str., 546 27 Thessaloniki, Greece
01/04/2010
Plasmidic DNA
Ethanol fixed
tissue
1 DNA suspension
1 sample of gills
01/04/2010
Plasmidic DNA
01/04/2010
Histological
slide
01/04/2010
Imprint slides
01/04/2010
Bornova Veterinary Control and Research Institute
Erzene Mah Ankara Cad No 172/155, 35010
Bornova-Izmir-Turkey
Date
15/06/2010
Histological
slides
24/06/2010
Plasmidic DNA
Host species
Pathogens
Country
Recipient
Ostrea edulis
Bonamia ostreae,
United
KingdomScotland
2 DNA suspensions pCR®II-TOPO vector M2A-M3AS from Marteilia South Korea Fisheries Research & Development Institute, 152-1,
refringens type O
Haeanro, Gijang-Gun Busan 619-705, KOREA.
ostreae
Histological
slides
Ostrea edulis
Bonamia ostreae
Imprint slides
2 slides
Ostrea edulis
Bonamia ostreae
24/06/2010
Plasmidic DNA 2 primer suspensions CF and CR primers 500 µl of primers (100µM)
CF -CR from OsHV-1
and primers
26/06/2010
DNA extracted
from tissues
2 DNA suspensions
12/07/2010
primers
2 primer suspensions
28/07/2010
Paraffin blocks
2 paraffin blocks
28/07/2010
CDRom,
1 CDrom
Ostrea edulis
Mytilus edulis
CF and CR primers 500 µl of primers (100µM)
Ostrea edulis
Bonamia ostreae
Mexico
Centro de Investigaciones Biológicas del Noroeste,
S.C. Mar Bermejo No. 195, Col. Playa Palo de Santa
Rita; La Paz, BCS 23090, México.
The
Central Veterinary Institute CVI, P.O. Box 65, 8200
Netherlands AB Lelystad, The Netherlands
United
KingdomScotland
South Korea Fisheries Research & Development Institute, 152-1,
Haeanro, Gijang-Gun Busan 619-705, KOREA.
U.S.A.
Fish and Wildlife Research Institute, Molluscan
Fisheries, St Petersburg, Florida, U.S.A.
Date
Host species
Pathogens
20/08/2010
Primers
2 primer suspensions
CF and CR primers 500 µl of primers (100µM)
06/09/2010
Paraffin blocks
DNA extracted
from tissues
2 paraffin blocks
2 DNA suspensions
Crassostrea gigas OsHV-1 µvar
08/09/2010
CDRom,
1 CDrom
Ethanol fixed 10 ethanol fixed pieces Crassostrea gigas
tissue
of mantle
DNA extracted 6 DNA suspensions
from tissues
08/09/2010
Plasmidic DNA 2 primer suspensions CF and CR primers 500 µl of primers (100µM)
CF -CR from OsHV-1
and primers
15/10/2010
09/11/2010
Ireland
Australia
Spain
08/09/2010
16/09/2010
Country
OsHV-1µvar
Recipient
Fish Health Unit, Marine Institute, Oranmore
Rinville, C.o Galway, Ireland
CSIRO, Livestock Industries Australian Animal
Health, Laboratory 5, Portarlington Road, Geelong,
Esat Victoria, 3220, Australia
INTECMAR, Peirao de Vilaxoan, 36611 Villagarcia
de Arousa, Pontevedra, Spain.
The
Central Veterinary Institute CVI, P.O. Box 65, 8200
Netherlands AB Lelystad, The Netherlands
Italy
Instituto Zoprofilattico Sperimentale del Piemonte
Liguria e Valle d’Aosta via Degli Stagnoni n.96 –
19136 La Spezia, Italy
Histological
slides
5 Histological slides
Plasmidic DNA
1 DNA suspension
Bonamia ostreae
Perkinsus olseni Ruditapes decussatus
Crassostrea virginica Perkinsus marinus
pCR®II-TOPO vector CF -CR from OsHV-1
Histological
slide
Crassostrea virginica Perkinsus marinus
Tunisia
INSTM 20025 Salammbô Tunisia
MT1, MT2, MT1B 100 µl of primers (100µM)
and MT2B primers
M2A-M3AS from M.
pCR®II-TOPO vector
refringens type M
and type O
France
ECOLAG, Université de Montpellier, Place Eugene
Bataillon, 34095 Montpellier, France
Plasmidic DNA 4 primer suspensions
and primers
2 DNA suspensions
Ostrea edulis
South Africa Marine Aquaculture Disease Department of
Agriculture, Forest and Fisheries, Roggebaai 8012,
Cape Town, South Africa
Date
Host species
Pathogens
Country
Recipient
M2A-M3AS from M.
refringens type M
ostreae
CF -CR from OsHV-1
Norway
Institute of Marine Research, PO Box 1870 , Nordnes,
5817 Bergen, Norway
Canada
Pacific Biological Station, Aquatic Animal Health
Program, 3190 Hammond Bay Road
Nanaimo, BC V9T 6N7, Canada
15/11/2010
Plasmidic DNA
26/11/2010
Paraffin blocks
2 paraffin blocks
Ethanol fixed 3 ethanol fixed pieces
tissue
of mantle
30/11/2010
tissue
of digestive gland
Ostrea edulis
Mytilus edulis
Healthy animals
Italy
Istituto zooprofilattico sperimentale delle venezie
Sezione Di Adria
30/11/2010
tissue
of digestive gland
Ostrea edulis
Mytilus edulis
Healthy animals
Spain
Instituto de Investigaciones Marinas, CSIC
C/ Eduardo Cabello, n°6, 36208 Vigo, Spain
Crassostrea gigas OsHV-1 µvar
Annex 3: Characterization and analysis performed by EURL in support of other laboratories during 2010
Date
13/01/2010 Histological slides
Ethanol fixed
tissue
14/04/2010
Ethanol fixed
tissue
Extracted DNA
4
4
30
Host species
Pathogens
Mytilus edulis
Marteilia
refringens
Crassostrea gigas OsHV-1/ OsHV1µvar
Country
Sweden
Italy
16
Sender
Division of Fish and Shellfish,
Department of Animal Health and
Antimicrobial Strategies, National
Veterinary Institute, S-751 89
Uppsala, Sweden
Istituto zooprofilattico
sperimentale delle venezie,
Sezione Di Adria, Via Leonardo da
Vinci 39, 45011 Adria, Italy
Performed tests
Histology
PCR-RFLP
Real Time PCR
PCR
Cloning and sequencing
Histology
3
Perna canaliculus
Suspicion of
marteiliosis
Malaysia
23/03/2010
Ethanol fixed
tissue
Paraffin blocks
10
Ostrea edulis
Bonamia ostreae
Japan
Paraffin Blocks
Ethanol fixed
tissue
13
45
Mytilus
galloprovincialis
Perna perna
Marteilia
refringens
Morocco
6
Crassostrea gigas
Presence of
lesions
Ireland
20 Paraffin blocks,
15 ethanol fixed
tissues
Ostrea stentina
Marteilia
refringens
Tunisia
16/07/2010
03/05/2010
25/05/2010
Paraffin blocks,
ethanol fixed
tissue
5
National Fish Health Research
Centre, Fisheries Research Insitute,
11960 Batu Maung, Penang,
Malaysia
Histology,
Graduate School of Agricultural
PCR-RFLP
Science, Tohoku University,
Tsutsumidori Amamiya-machi,
Aoba-ku, Sendai 981-8555
Miyagi, Japan
Histology
INRH, 2 rue de Tiznit, Casablanca, In situ hybridization
Morocco
PCR-RFLP
Histology
Fish Health Unit, Marine Institute,
Oranmore Rinville, C.o Galway,
Ireland
Institut National Agronomique de
Histology
Tunisie, Departement des
PCR-RFLP
ressources animales, halieutiques Cloning and sequencing
et des technologies alimentaires
Date
Host species
07/06/2010 Histological slides 12 histological slides Crassostrea gigas
Ethanol fixed
12 ethanol fixed
tissue
tissue
08/07/2010
02/09/2010
Paraffin blocks, 22 blocks, 12 ethanol Ostrea edulis and
Mytilus
and ethanol fixed fixed pieces of tissue
galloprovincialis
tissue
Ethanol fixed
tissue
Pathogens
Suspicion of
infection with
protozoans
Marteilia
refringens.
Country
Sender
Performed tests
South Korea Fisheries Research & Development
Histology, in situ
Institute, 152-1, Haeanro, Gijang- hybridization, PCR and
Gun Busan 619-705, Korea
real time PCR
Greece
Institute for Infectious and
Histology,
Parasitic Diseases -Department of
PCR-RFLP
Aquatic Organisms Pathology, 80, Cloning and sequencing
26th Octovriou str., 546 27
Thessaloniki, Greece
92 pieces of tissue
Crassostrea
Perkinsus spp.
corteziensis
OsHV-1
Crassostrea gigas
Mexico
Centro de Investigaciones
PCR –
Biológicas del Noroeste, S.C. Mar
PCR RFLP
Bermejo No. 195, Col. Playa Palo
Real Time PCR
de Santa Rita; La Paz, BCS 23090, Cloning and sequening
México
7
Crassostrea gigas Double reading
Tunisia
INSTM 20025 Salammbô Tunisia
Histology
Annex 4: Training and scientific collaboration in 2010
Name
Institute
Dates
Country
26-28/01/2010
Japan
25/03/2010
Sweden
Anders Alfjorden
Department of animal health and
Detection and characterization of
antimicrobiologial strategies,
Marteilia refringens in Mytilus edulis
National veterinary institute, Sweden
7-11/06/2010
South Korea
Jee Youn Hwang
Pathology Division National
(1) presenting surveillance of mollusc
Fisheries Research & Development diseases, (2) detecting pathogens of
Institute South Korea
mollusc by histology especially those
listed in the OIE aquatic Code (3)
detection Bonamia spp. by PCR and in
situ hybridization.
25/05/2010 – 21/06/2010
Tunisia
Refka Elgarsalli
Institut National Agronomique de
Tunisie
4/09/2010-1/10/2010
Mexico
13-21/12/2010
South Korea
Tomomasa Matsuyama andNational Research Institute of
Tomokazu Takano,
Aquaculture, Fisheries Research
Agency from Japon.
Training
Surveillance of mollusc diseases and
detection of pathogens of mollusc by
histology especially those listed in the
OIE aquatic Code.
diseases, (2) detecting pathogens of
listed in the OIE aquatic Code (3)
detection Bonamia spp. by PCR (4)
detection and characterization of
Marteilia refringens by PCR-RFLP
cloning and sequencing.
Cristina Escobedo Fregoso Centro de Investigaciones Biológicas Detection and characterization of
del Noroeste, S.C. Mar Bermejo No. Perkinsus in Crassostrea spp. ,
195, Col. Playa Palo de Santa Rita; observation of Perkinsus spp. by
La Paz, BCS 23090, México.
histology and detection of OsHV-1 using
Real Time PCR
Kyung Il Park
College of Ocean Science and
Technology, Kunsan National
University South Korea
diseases, (2) detecting pathogens of
listed in the OIE aquatic Code
Annex 5: Publications relevant to the work of the EURL.
Arzul I., Langlade A.; Chollet B.; Robert M.; Ferrand S.; Omnes E.; Lerond S.; Couraleau Y.; Joly J.P.; François C. ; Garcia C. (2010) Can the protozoan parasite Bonamia ostreae infect larvae of
flat oysters Ostrea edulis?. Accepted in Veterinary Parasitology
Dundon W.G., Arzul I., Omnes E., Robert M., Magnabosco C., Zambon M., Gennari L., Toffan A.,
Terregino C., Capua I. Arcangeli G. Detection of Type 1 Ostreid Herpes variant (OsHV-1
µvar) with no associated mortality in French-origin Pacific cupped oyster Crassostrea gigas
farmed in Italy. Accepted in Aquaculture
Morga B., Arzul I., Faury N., Renault T. (2010). Identification of genes from flat oyster Ostrea edulis
as suitable housekeeping genes for quantitative real time PCR. Fish & Shellfish Immunology,
29(6), 937-945.
Morga B., Renault T., Faury N., Chollet B., Arzul I Cellular and molecular responses of haemocytes
from Ostrea edulis during in vitro infection by the parasite Bonamia ostreae. Accepted in
International Journal of Parasitology.
Morga B, Arzul I, Faury N, Segarra A, Chollet B, Renault. T. Molecular responses of Ostrea edulis
haemocytes to an in vitro infection with Bonamia ostreae.Dev Comp Immunol.
doi:10.1016/j.dci.2010.10.005 |
Narcisi V., Arzul I., Cargini D., Mosca F., Calzetta A., Traversa D., Robert M., Joly J-P., Chollet B.,
Renault T., Tiscar P. G. (2010). Detection of Bonamia ostreae and B. exitiosa (Haplosporidia)
in Ostrea edulis from the Adriatic Sea (Italy). Diseases Of Aquatic Organisms, 89(1), 79-85.
Saulnier D., De Decker S., Haffner P., Cobret L., Robert M., Garcia C. (2010). A Large-Scale
Epidemiological Study to Identify Bacteria Pathogenic to Pacific Oyster Crassostrea gigas
and Correlation Between Virulence and Metalloprotease-like Activity. Microbial Ecology,
59(4), 787-798.
Sauvage C., Boudry P., De Koning D. -J., Haley C. S., Heurtebise S., Lapegue S. (2010). QTL for
resistance to summer mortality and OsHV-1 load in the Pacific oyster (Crassostrea gigas).
Animal Genetics, 41(4), 390-399.
Segarra A., Pepin J-F., Arzul I., Morga B., Faury N., Renault T. (2010). Detection and description of a
particular Ostreid herpesvirus 1 genotype associated with massive mortality outbreaks of
Pacific oysters, Crassostrea gigas, in France in 2008. Virus Research, 153(1), 92-99.
Annex 6: Presentations at international conferences and meetings
Arzul I., Omnes E., Robert M., Chollet B., Joly J.-P., Miossec L., François C., Garcia C. (2010).
Distribution of Bonamia exitiosa in flat oyster Ostrea edulis populations in France.
Communication orale. Aquaculture 2010. San Diego, California, U.S.A., 1-5 March 2010.
Arzul I., Chollet B., Michel J., Robert M., Miossec L., Joly J.-P., François C., Garcia C. (2010).
Perkinsus olseni and a new Perkinsus sp., closed to P. Chesapeaki, sympatric in clams
Ruditapes decussatus from Leucate lagoon, France. Communication orale. Aquaculture 2010.
San Diego, California, U.S.A., 1-5 March 2010.
Morga B., Arzul I., Faury N., Garcia C., Lerond S., Robert M., Chollet C., Lapegue S., Renault T.
(2010). Host responses to infection with Bonamia ostreae : comparison between resistant and
wild flat oysters. Communication orale. Aquaculture 2010. San Diego, California, U.S.A., 1-5
March 2010.

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