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CAWTHRON INSTITUTE | REPORT NO. 2093 AUGUST 2012
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REPORT NO. 2093
STUDIES ON OSTREID HERPES VIRUS – 1:
A CAUSAL AGENT IMPLICATED IN SUMMER MORTALITY IN THE OYSTER CRASSOSTREA GIGAS BY PCR OF ARCHIVAL HISTOLOGY SPECIMENS
CAWTHRON INSTITUTE | REPORT NO. 2093 AUGUST 2012
STUDIES ON OSTREID HERPES VIRUS – 1: A CAUSAL AGENT IMPLICATED IN SUMMER MORTALITY IN THE OYSTER CRASSOSTREA GIGAS BY PCR OF ARCHIVAL HISTOLOGY SPECIMENS
STEVE WEBB
Final report: Year 2 – this report is available for public release International Mobility Fund Activity Report on IMF Programme Dd’U11-05
Commencement date 17 December 2010 Duration of funded collaboration Until 10 June 2012; extended to 1 August 2012 Funding provided: Year 1 $4,320 15 August 2011 Year 2 $4,320 10 June 2012; extended to 1 August 2012
NZ Principal Investigator: Dr Steve Webb Senior Scientist, Aquaculture Pathology Cawthron Institute, Nelson, New Zealand
Principal Collaborator: Dr Tristan C Renault Head of the Genetics and Pathology Laboratory
Laboratoire de Génétique et Pathologie (LGP) Institut Français de Recherche pour l’Exploitation de la Mer (IFREMER) 17390 La Tremblade, France
CAWTHRON INSTITUTE 98 Halifax Street East, Nelson 7010 | Private Bag 2, Nelson 7042 | New Zealand Ph. +64 3 548 2319 | Fax. +64 3 546 9464 www.cawthron.org.nz
REVIEWED BY: Kevin Heasman
APPROVED FOR RELEASE BY: Robert Matheson
ISSUE DATE: 31 July 2012
RECOMMENDED CITATION: Webb S 2012. Studies on ostreid herpes virus-1: a causal agent implicated in summer mortality in the oyster Crassostrea gigas by PCR of archival histological specimens. Prepared for Royal Society of New Zealand. Cawthron Report No. 2093. 5 p
© COPYRIGHT: Apart from any fair dealing for the purpose of study, research, criticism, or review, as permitted under the Copyright Act, this publication must not be reproduced in whole or in part without the written permission of the Copyright Holder, who, unless other authorship is cited in the text or acknowledgements, is the commissioner of the report.
CAWTHRON INSTITUTE | REPORT NO. 2093 AUGUST 2012
I confirm that the grant provided was expended only on the costs
associated with my IMF contract Dd’U11-05.
Signed: Steve Webb Date: 25 July 2012
CAWTHRON INSTITUTE | REPORT NO. 2093 AUGUST 2012
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TABLE OF CONTENTS
1. INTRODUCTION ........................................................................................................... 1
2. PROJECT AND ACTIVITY REPORT ............................................................................. 1
3. SEMINARS/ WORKSHOPS ........................................................................................... 2
4. BENEFITS ..................................................................................................................... 2
5. PUBLICATION INTENTIONS ......................................................................................... 3
6. INTERNATIONAL OPPORTUNITIES............................................................................. 3
7. INTERNATIONAL RECOGNITION ................................................................................ 4
8. MAINSTREAM FUNDING .............................................................................................. 4
9. FUTURE COLLABORATION ......................................................................................... 4
10. HIGHLIGHTS ................................................................................................................. 5
11. APPENDIX ..................................................................................................................... 5
CAWTHRON INSTITUTE | REPORT NO. 2093 AUGUST 2012
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1. INTRODUCTION
The Dumont d’Urville International Mobility Fund has, for the last two years, supported
joint New Zealand/French work on the ostreid herpes virus-1 (OsHV-1). Details of the
projected work are provided in Webb (2011). Full citations to this and any subsequent
works mentioned here in the text are cited in the current report under Section 5
Publication Intentions. The project focused on the development of assays and the
detection and characterisation of the ostreid herpes virus–1. End-point PCR and
QPCR methods have been developed and employed to detect OsHV-1 infections in
fresh and paraffin-embedded archival histology specimens of the Pacific oyster
Crassostrea gigas and other bivalve species. The specimens were from French
mortality events with known involvement of OsHV-1 and oysters sampled from
mortality events in New Zealand to which no cause has yet been ascribed.
2. PROJECT AND ACTIVITY REPORT
As previously reported (Webb 2011), we successfully adapted PCR methods from
end-point PCR to QPCR and used them in our study of a range of oyster specimens
including fresh, alcohol-preserved and paraffin-embedded tissue samples of the
Pacific oyster Crassostrea gigas. In addition, we also used the PCR primers C2/C6,
ORF37, IA1/IA2 CF/CR, ORF38, 37 TER F/R, ORF 36, ORF 37. Successful detection
of OsHV-1 and differential results with these primers and sequencing allowed us to
infer genetic differences in geographic strains of the virus – these are reported in
Renault et al (2012).
Further work on paraffin-embedded oyster tissue samples was restricted to New
Zealand material as (in contrast to New Zealand) the French principal collaborator
already had an extensive archive of DNA previously extracted from fresh material. It
also became clear in the course of the work that when there is a choice between
paraffin and fresh material for DNA extraction, the latter is always preferred as DNA
quality and quantity is generally better. Nevertheless, being able to use paraffin
specimens is a great advantage, as this makes available a large resource of archival
material in New Zealand for which no previous DNA analysis has been done.
The results of last year’s work are summarised in Renault et al (2012). We found that
the New Zealand virus appears to be a single strain with close affinities to the so-
called OsHV-1 µvar - a particular strain of the ostreid herpes virus that has been
implicated in severe mortalities in several countries. Preliminary PCR assays on 2012
New Zealand oysters indicate that they too have a virus closely related to OsHV-1
µvar. Further sequencing is required before firm conclusions can be drawn about the
fine-scale affinities of the New Zealand virus. Full details of these findings will be
published in the proposed paper mentioned in Section 5.
AUGUST 2012 REPORT NO. 2093 | CAWTHRON INSTITUTE
The second visit by the New Zealand principal investigator (PI) to IFREMER in June
2012 extended previous work on DNA purified from fresh and archival fixed paraffin
embedded material - OsHV-1 positives were detected in both types of sample.
A consequence of the raised profile gained by Cawthron was the visit by a European
Union (EU)-funded researcher who set up the method for In situ hybridisation (ISH).
Results are detailed in publications mentioned in Section 5 of this report. In this year’s
assays of fresh Crassostrea gigas material, 16 samples out of 47 were OsHV-1
positive. In paraffin Crassostrea gigas archival samples 3 out of 9 were OsHV-1
positive. Proposed work mentioned in the last report (Webb 2011) was completed in
2012 - samples of sand clams Paphies subtriangulata, Spisula aequilatera and Mactra
sp. were assayed for OsHV-1 and found negative. Work is continuing to sequence the
successful amplifications and we intend to publish the results as mentioned in
Section 5.
3. SEMINARS/ WORKSHOPS
A seminar titled Molluscan pathology in New Zealand) was delivered on Wednesday 5th July 2012 at Centro de Investigaciones Marinas (CIMA), Spain.
4. BENEFITS
Advantage to French researcher
The French researcher has further access to New Zealand oyster specimens from
routine and mortality samplings. In addition, the New Zealand PI has again
demonstrated the efficacy of assaying DNA extracted from formalin-fixed paraffin-
embedded archival material. The French researcher has benefited from the Cawthron-
devised OHVDFor-OHVDRev OsHV-1 primers and has used them in internationally
published studies. A further extension of the current French study on geographic
variation of the OsHV-1 was facilitated by the New Zealand PI’s subsequent visit to a
Swedish oyster hatchery where specimens were taken and sent to IFREMER for
analysis.
Advantages to New Zealand
This visit and the subsidiary trips made possible by the Dumont d’Urville International
Mobility Fund has, by conferring greater New Zealand pathology capability, enhanced
our ability to support exporters of New Zealand aquaculture products to overseas
markets. We now have a better diagnostic capability in the event of disease outbreaks
and have improved methods to assess and establish the health status of our shellfish.
Direct access to expertise in the latest methods will benefit the New Zealand industry
– IFREMER can help us in these aims, not least because it is a European reference
laboratory for molluscan pathogens, and it has expressed interest in collaborating with
CAWTHRON INSTITUTE | REPORT NO. 2093 AUGUST 2012
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a New Zealand-based investigator in continuing to work on relevant parasite species
in New Zealand molluscs.
Other benefits include:
Raised international profile ( See Section 7)
Scientific publications (See Section 5)
Greater understanding about the OsHV-1 oyster disease
New collaborators (See Section 6)
5. PUBLICATION INTENTIONS
In addition to the works below, we also expect to publish on the OsHV-1 detection
and characterisation findings this year.
Renault T, Moreau P, Faury N, Pepin J-F, Segarra A, Webb S 2012.
Phylogenetic analysis of clinical ostreid herpesvirus 1 (Malacoherpesviridae)
isolates using amplified fragments from three virus genome areas. Journal of
Virology 86:5942-5947.
López Sanmartín M, Webb S 2012 (In Preparation). In-situ hybridation for detection and localisation of ostreid herpes virus (OsHV-1) in oyster histological sections. Cawthron Report, 9pp.
Webb SC 2011. Studies on ostreid herpes virus – 1: a causal agent implicated in summer mortality in the oyster Crassostrea gigas, by PCR of archival histology specimens. Prepared for the Royal Society of New Zealand. Cawthron Report No. 1956. 4pp.
6. INTERNATIONAL OPPORTUNITIES
New collaborators
Dr Antonio Villalba, (CIMA), Spain. Exchange of shellfish pathology information
and study materials, supply of EU-funded post-doctoral researchers to work on
pathology issues of mutual interest such as bivalve with infections Chlamydia,
rickettsiae and Perkinsus.
Dr. Alyssa Joyce, Tjärnö Marine Biological Laboratory, Sweden. Joint applicant
for Swedish-funded research on oyster pathology.
Karljohan Smedman and Kent Berntsson Ostrea Sverige AB, Sweden. Further
work for this hatchery to address disease issues.
Monserrat López Sanmartín IFAPA, Spain. Setting up ISH at Cawthron. Further
work could include the localisation of OsHV-1 activity in infected oyster tissues.
AUGUST 2012 REPORT NO. 2093 | CAWTHRON INSTITUTE
Dr Susana Darriba Couñgo, Spain. Exchange of shellfish pathology information
and study materials, laboratory harmonisation and establishment of Quality
Assurance protocols.
7. INTERNATIONAL RECOGNITION
Three examples below show that the collaboration has contributed to the promotion of
New Zealand as a centre for innovation:
Publications coming from this project.
By being available in Europe and by having a raised scientific profile I was
invited to an expenses-paid visit to Ostrea Sverige AB, Sydkoster, West
Sweden to advise about disease investigation.
The seminar delivered on Wednesday 5rd July at Centro de Investigaciones
Marinas (CIMA), Spain generated interest and has shown that we, in New
Zealand, have a significant pathology resource that can make a useful
contribution to their work.
8. MAINSTREAM FUNDING
The internationally-published results from the work will provide further leverage
when bidding for funding.
Funding bid with Dr. Alyssa Joyce (in preparation) for research on oyster pathology. The Royal Swedish Academy of Sciences, funding to support internationalisation and scientific renewal at the Sven Lovén Centre for Marine Sciences.
Ongoing collaboration with IFREMER will enhance our scientific quality and
therefore facilitate publication of work done at Cawthron.
The support of expertise available at IFREMER will add credibility to future bids
in the field of shellfish pathology.
9. FUTURE COLLABORATION
Ongoing work with Dr Tristan Renault and his institute to chart the spatial and
temporal variation in OsHV-1, its host specificity and virulence. During the visit,
opportunity was taken to discuss other fields of study with a view to extending
collaboration to work on other important molluscan pathogens.
Please see Section 6 for details of prospective future collaborations.
CAWTHRON INSTITUTE | REPORT NO. 2093 AUGUST 2012
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10. HIGHLIGHTS
The project has led to several new collaborations: see Section 6.
Support from the Dumont d’Urville International Mobility Fund, and previous similar New Zealand government funds, have allowed the development of expertise in a New Zealand researcher such that this expertise is recognised internationally, resulting in an invitation to visit and provide pathology advice to a Swedish oyster hatchery.
My raised profile also resulted in an EU-funded researcher requesting to work in Cawthron under my guidance. The results are described in López San Martín M, Webb S (2012, In Preparation).
11. APPENDIX
Collaborators’ Contact Details
Dr Antonio Villalba Centro de Investigaciones Marinas (CIMA) Conselleria do Medio Rural e do Mar Xunta de Galicia Aptdo.13 Vilanova de Arousa, 36620 Spain Dr. Alyssa Joyce Tjärnö Marine Biological Laboratory Department of Marine Ecology Gothenburg University Strömstad 452 96, Sweden Karljohan Smedman (Managing Director and Member of the Board) Dr Kent Berntsson (Hatchery Manager) Ostrea Sverige AB Sydkoster, West Sweden Monserrat López Sanmartín IFAPA, Centro Agua del Pino Crta. El Rompido-Punta Umbría, Km 3.8 21459 Cartaya, Huelva Spain Dr Susana Darriba Couñgo Head of pathology unit (INTECMAR) Xunta de Galicia Vilanova de Arousa 36611, Spain
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