studies on ostreid herpes virus - cawthron · pdf filecawthron institute | report no. 2093...

CAWTHRON INSTITUTE | REPORT NO. 2093 AUGUST 2012

1

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


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.


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


vii

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


1

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


3

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.


5

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

studies on ostreid herpes virus - cawthron · pdf filecawthron institute | report no. 2093...

Documents