Working Group on Pathology and Diseases of Marine Organisms Reports/Expert Group...Working Group on Pathology and Diseases of Marine Organisms Aberdeen, UK 11–15 March 2003 This

International Council for the Exploration of the Sea

Conseil International pour l’Exploration de la Mer

Palægade 2–4 DK–1261 Copenhagen K Denmark

Mariculture Committee ICES CM 2003/F:03 Ref. ACME

REPORT OF THE

Working Group on Pathology and Diseases of Marine Organisms

Aberdeen, UK 11–15 March 2003

This report is not to be quoted without prior consultation with the General Secretary. The document is a report of an expert group under the auspices of the International Council for the Exploration of the Sea and does not necessarily represent the views of the Council.

TABLE OF CONTENTS

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EXECUTIVE SUMMARY ................................................................................................................................................ 1 1 OPENING AND STRUCTURE OF THE MEETING............................................................................................... 3 2 ICES: ITEMS OF RELEVANCE TO WGPDMO..................................................................................................... 3

2.1 ICES Annual Science Conference 2002.......................................................................................................... 3 2.2 ICES Mariculture Committee (MARC) .......................................................................................................... 4 2.3 ICES Advisory Committee on the Marine Environment (ACME) ................................................................. 4 2.4 Baltic Sea Regional Project (BSRP) ............................................................................................................... 4

3 TERMS OF REFERENCE, ADOPTION OF AGENDA, SELECTION OF RAPPORTEURS................................ 5 3.1 Terms of Reference......................................................................................................................................... 5 3.2 Adoption of the Agenda.................................................................................................................................. 5 3.3 Selection of Rapporteurs................................................................................................................................. 5

4 OTHER RELEVANT INFORMATION.................................................................................................................... 5 5 ANALYSE NATIONAL REPORTS ON NEW DISEASE TRENDS IN WILD AND CULTURED FISH,

MOLLUSCS AND CRUSTACEANS ....................................................................................................................... 6 5.1 Wild Fish......................................................................................................................................................... 6

5.1.1 Conclusions....................................................................................................................................... 8 5.1.2 Recommendations............................................................................................................................. 9

5.2 Farmed fish ..................................................................................................................................................... 9 5.2.1 Conclusions..................................................................................................................................... 12 5.2.2 Recommendations........................................................................................................................... 12

5.3 Wild and Farmed Shellfish ........................................................................................................................... 12 5.3.1 Conclusions..................................................................................................................................... 16 5.3.2 Recommendations........................................................................................................................... 16

6 REPORT ON PROGRESS IN THE ONGOING INVESTIGATIONS OF THE EFFECT OF TEMPERATURE ON BONAMIA INFECTION DYNAMICS AND REPORT ON THE CONFIRMATION OF THE AGENT OF CRASSOSTREA ANGULATA GILL DISEASE AND ITS INFECTIVITY TO CRASSOSTREA GIGAS AND OTHER OYSTERS SPECIES ................................................................................................................................. 17 6.1 Conclusions................................................................................................................................................... 18 6.2 Recommendations......................................................................................................................................... 18

7 REVIEW THE EXISTING STRATEGIES TO ASSESS THE PREVALENCE OF SHELLFISH DISEASES IN PARALLEL TO FISH DISEASES AND CHEMICAL CONTAMINATION LEVELS IN ENVIRONMENTAL MONITORING PROGRAMMES. .......................................................................................................................... 18

8 REVIEW OF MOLECULAR TECHNIQUES USED TO DIFFERENTIATE THE VARIOUS SPECIES/ISOLATES OF PERKINSUS SPP. .......................................................................................................... 19

9 OBTAIN INFORMATION ON THE EU PROJECT “DIAGNOSIS OF OYSTER HERPES-LIKE VIRUS: DEVELOPMENT AND VALIDATION OF MOLECULAR, IMMUNOLOGICAL AND CELLULAR TOOLS” (FAIR-PL98-4334) AND REVIEW THE RESULTS.............................................................................................. 20

10 REVIEW AND REPORT ON PROGRESS MADE IN THE “FISH DISEASES AND LIVER HISTOPATHOLOGY” COMPONENT OF THE BEQUALM SELF-FUNDING SCHEME. ............................... 21

11 REVIEW AND ASSESS THE IMPACT OF DISEASES OF FARMED FISH ON WILD FISH STOCKS .......... 22 11.1 Conclusions................................................................................................................................................... 22 11.2 Recommendations......................................................................................................................................... 23

12 MAINTAIN AN OVERVIEW OF THE SPREAD OF ICHTHYOPHONUS IN HERRING STOCKS AND THE DISTRIBUTION AND POSSIBLE CAUSE(S) OF THE M74 SYNDROME........................................................ 23 12.1 Conclusions................................................................................................................................................... 24 12.2 Recommendations......................................................................................................................................... 24

13 ASSESS AND REPORT ON THE EFFECTIVENESS OF SALMON FARMING MANAGEMENT CONTROL METHODS FOR SEA LICE ................................................................................................................................... 24 13.1 Conclusions................................................................................................................................................... 25 13.2 Recommendations......................................................................................................................................... 25

14 REVIEW PROGRESS MADE IN THE MODIFICATIONS TO THE ICES DATABASES AND REVIEW AND APPROVE THE REVISED ICES ENVIRONMENTAL DATA REPORTING FORMAT (VERSION 3.2) ........ 25

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14.1 Conclusion .................................................................................................................................................... 26 14.2 Recommendations......................................................................................................................................... 26

15 REVIEW THE CRITERIA FOR THE INCORPORATION OF EXTERNALLY VISIBLE FISH DISEASES INTO MONITORING PROGRAMMES ON BIOLOGICAL EFFECTS OF CONTAMINANTS – AND PROVIDE FEEDBACK TO WGBEC..................................................................................................................... 26 15.1 Conclusions................................................................................................................................................... 27 15.2 Recommendations......................................................................................................................................... 27

16 REVIEW PROGRESS MADE WITH REGARD TO THE UPDATE OF ICES PUBLICATIONS ON PATHOLOGY AND DISEASES OF MARINE ORGANISMS ............................................................................. 27 16.1 Web-based report on diseases and parasites of wild and farmed marine fish and shellfish as part of the ICES

environmental status report ........................................................................................................................... 27 16.1.1 Conclusions..................................................................................................................................... 27 16.1.2 Recommendations........................................................................................................................... 27

16.2 Manuscript on methods for the statistical analysis of fish disease data for submission to the ICES TIMES series ............................................................................................................................................................. 27 16.2.1 Conclusion ...................................................................................................................................... 28 16.2.2 Recommendations........................................................................................................................... 28

16.3 Current status of the ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish ............ 28 16.3.1 Conclusions..................................................................................................................................... 28

16.4 Review the status of the cooperative research report “Report on important trends in diseases occurring in finfish and shellfish culture in the ICES area in the period 1997–2001” ...................................................... 29 16.4.1 Conclusion ...................................................................................................................................... 29 16.4.2 Recommendations........................................................................................................................... 29

17 ANY OTHER BUSINESS ....................................................................................................................................... 29 17.1 WGPDMO Chair .......................................................................................................................................... 29 17.2 WGPDMO web site ...................................................................................................................................... 29 17.3 Template for national reports on new disease trends in wild and farmed fish, molluscs and crustaceans .... 30 17.4 “Prestige” oil spill ......................................................................................................................................... 30

18 PROGRESS WITH TASKS..................................................................................................................................... 30 19 FUTURE ACTIVITIES OF WGPDMO .................................................................................................................. 30 20 APPROVAL OF RECOMMENDATIONS ............................................................................................................. 30 21 APPROVAL OF THE DRAFT WGPDMO REPORT............................................................................................. 30 22 CLOSING OF THE MEETING............................................................................................................................... 31 ANNEX 1: LIST OF PARTICIPANTS............................................................................................................................ 32 ANNEX 2: TERMS OF REFERENCE ............................................................................................................................ 34 ANNEX 3: AGENDA ...................................................................................................................................................... 36 ANNEX 4: RAPPORTEURS ........................................................................................................................................... 37 ANNEX 5: SUMMARY OF STATUS OF ISA IN MAINE, US, IN 2002...................................................................... 39 ANNEX 6: REVIEW OF PATHOLOGIES IN DEMERSAL FISHES OF THE BARENTS SEA AND ADJACENT

WATERS ................................................................................................................................................................. 40 ANNEX 7: REPORT ON ISA IN IRELAND .................................................................................................................. 47 ANNEX 8: MSX AND SSO IN CANADA ..................................................................................................................... 48 ANNEX 9: REVIEW THE EXISTING STRATEGIES TO ASSESS THE PREVALENCE OF SHELLFISH

DISEASES IN PARALLEL TO FISH DISEASES AND CHEMICAL CONTAMINANT LEVELS IN ENVIRONMENTAL MONITORING PROGRAMMES........................................................................................ 49

ANNEX 10: REVIEW OF MOLECULAR TECHNIQUES USED TO DIFFERENTIATE THE VARIOUS SPECIES/ISOLATES OF PERKINSUS................................................................................................................... 54

ANNEX 11: OBTAIN INFORMATION ON THE EU PROJECT “DIAGNOSIS OF OYSTER HERPES-LIKE VIRUS: DEVELOPMENT AND VALIDATION OF MOLECULAR, IMMUNOLOGICAL AND CELLULAR TOOLS” (FAIR-PL98-4334) AND REVIEW THE RESULTS.............................................................................................. 61

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ANNEX 12: REVIEW AND REPORT ON PROGRESS MADE IN THE 'FISH DISEASES AND LIVER HISTOPATHOLOGY' COMPONENT OF THE BEQUALM SELF-FUNDING SCHEME ................................. 66

ANNEX 13: REVIEW AND ASSESS THE IMPACT OF DISEASES OF FARMED FISH ON WILD FISH STOCKS74 ANNEX 14: STRUCTURE OF THE INTEGRATED ICES DATA BASE .................................................................... 80 ANNEX 15: RAW DATA SUBMISSION TABLE FOR TEST OF THE INTEGRATED REPORTING FORMAT

VERSION 3.2 .......................................................................................................................................................... 81 ANNEX 16: REVIEW THE CRITERIA FOR THE INCORPORATION OF EXTERNALLY VISIBLE FISH

DISEASES INTO MONITORING PROGRAMMES ON BIOLOGICAL EFFECTS OF CONTAMINANTS..... 82 ANNEX 17: TENTATIVE TABLE OF CONTENTS OF THE MANUSCRIPT ENTITLED “STATISTICAL

METHODS FOR THE ANALYSIS OF FISH DISEASE DATA”.......................................................................... 92 ANNEX 18: ANALYSIS OF PROGRESS WITH TASKS ............................................................................................. 93 ANNEX 19: RECOMMENDATIONS TO THE ICES COUNCIL.................................................................................. 94 @#

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EXECUTIVE SUMMARY

WGPDMO met from 11–15 March 2003 at the Marine Laboratory, Aberdeen, UK, with thirteen participants from nine ICES Member Countries, chaired by T. Lang (Germany). In order to consider all eleven Terms of Reference in an appropriate way, intersessional work was carried out by WGPDMO members who provided written background documents subsequently reviewed at the meeting.

A number of new disease trends were reported by Member Countries: In the USA, ten new isolates of the Viral Haemorrhagic Septicaemia Virus (VHSV) have been isolated in wild marine fish species. Sea lice (Lepeophtheirus salmonis) have been implicated in unusually low returns of pink salmon (Oncorhynchus gorbuscha) in western Canada and continues to be a major problem for wild Atlantic salmon (Salmo salar) and sea trout (Salmo trutta) in Norway. For the first time, diseases of wild fish in the Barents Sea and adjacent waters have been studied systematically in the period 1999–2001. The results were considered as important baseline data and WGPDMO recommends continuing these studies. In farmed fish, the Infectious Salmon Anaemia Virus (ISAV) has been found for the first time in Ireland. The parasite Parvicapsula sp. was recorded for the first time in farmed salmon in Norway, causing considerable mortality in affected farms. Losses of farmed salmon due to algae or jellyfish remained a problem in Scotland and Norway. Haplosporidium nelsoni (MSX) and H. costale (SSO) were recorded for the first time in Atlantic Canada, the former causing heavy localised mortality. Bonamia ostreae was observed in flat oyster (Ostrea edulis) from an EU-approved zone in Ireland. Viruses have been isolated from Crustacea (bacilliform virus in wild brown shrimp, Crangon crangon, in the UK; herpes-like virus causing high mortalities in wild spiny lobsters, Panulirus argus, in Florida, USA) (Report Section 5).

Results from experimental studies carried out in the framework of the European programme DISENV reveal that water temperature plays an important role regarding the infection and mortality associated with Bonamia ostreae in flat oysters, with lower temperatures favouring the infection, and thus, causing higher mortalities (Report Section 6).

A review of existing monitoring and research programmes indicated that there are only few examples where fish and shellfish pathology have been studied/monitored in parallel (Report Section 7).

A review has been carried out summarising information on techniques used to differentiate between the various species/isolates of Perkinsus spp., a group of parasites that is lethal to many commercially important mollusc species on a worldwide scale. So far, 7 species have been named; however, the use of current molecular tools provided evidence that only four of these are valid species. It was suggested that a set of clear criteria should be established for species identification and naming (Report Section 8).

Based on results obtained in the EU-funded project VINO, of which the objective was to develop molecular, immunological and cellular tools for the diagnosis and study of bivalve herpesvirus, there is evidence that the Oyster herpesvirus type 1 (OsHV-1) found to affect various bivalve species is not related to vertebrate herpesviruses (Report Section 9).

The previously EU-funded project BEQUALM, developing a framework for quality assurance in biological effects monitoring, came to an end in 2002 and will be continued as a self-funding scheme beginning in April 2003. The progress made in the “Fish diseases and liver histopathology” component and future activities of the programme were reviewed and Member Countries were encouraged to sign up to the programme (Report Section 10).

Based on a background document, WGPDMO reviewed information on the impact of diseases of farmed fish on wild fish stocks. It was noted that disease interactions are a complex issue and that there are many areas that require further studies. It was emphasised that diseases can be transmitted in both ways, from wild to farmed fish and vice versa. Examples for a disease transfer from farmed to wild fish are sea lice and furunculosis in salmonids (Report Section 11).

Ichthyophonus hoferi infection persists at low prevalences in herring (Clupea harengus) in the northern North Sea, the Baltic Sea and the Barents Sea. In North America, the parasite has been recorded in a variety of fish species, partly associated with mortalities. There is indication that there are increasing problems with the M74 Syndrome in Baltic salmon. Preliminary data suggest that the percentage of female salmon producing offspring with M74 was 10–12 % higher in 2002 than in 2001. There is still no conclusive information as to the causes of the syndrome. WGPDMO, therefore, recommends that sufficient resources for studies on the aetiology of the disease be provided by relevant ICES Member Countries (Report Section 12).

A review was conducted on the effectiveness of salmon farming management control methods for sea lice. Based on a comparison of the situation in Scotland and Norway, it was concluded that the most successful approach to reduce the

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number of lice is “integrated pest control”, involving a farm management regime that includes both preventative and treatment strategies (Report Section 13).

WGPDMO reviewed progress made in the modification of the ICES Databases and the Environmental Data Reporting Format (new version 3.2). The modifications achieved so far were endorsed, because the new structure facilitates a future, more holistic analysis of data from different sources. Furthermore, the new structure offers the possibility to produce more easily data summaries and products for information of the broader public (Report Section 14).

Based on a request from the Council, WGPDMO provided input to WGBEC regarding the review of criteria for the incorporation of externally visible fish diseases into monitoring programmes on biological effects of contaminants. It was emphasised that there is evidence from long-term field data for a relationship between contaminants in sediments as well as contaminant body burdens and externally visible diseases. Furthermore, it was concluded that externally visible fish diseases meet the criteria for the use of biological effects techniques established by WGBEC and that disease studies should, therefore, be retained as a method for biological effects monitoring. However, WGPDMO also recommends that WGBEC should revise the criteria, taking more into account the various purposes of monitoring (Report Section 15).

Progress has been made in the preparation of ICES publications on pathology and diseases of marine organisms: 1) Web-based report on diseases and parasites of wild and marine fish and shellfish as part of the ICES Environmental Status Report; 2) Manuscript on statistical methods for the analysis of fish disease data for publication in the ICES TIMES Series; 3) ICES Leaflets for diseases and parasites of fish and shellfish; and 4) Manuscript on trends in important diseases affecting the culture of fish and molluscs in the ICES Area 1998–2002 for publication in the ICES CRR Series (Report Section 16).

Any other business: WGPDMO approved T. Lang, who served as acting Chair for the 2003 meeting, as new Chair. Ways will be explored before the 2004 meeting to establish a WGPDMO website. An oral report by the Spanish WGPDMO member on the “Prestige” oil spill was received and suggestions were made for techniques that can be applied in order to provide information on the health status of affected shellfish stocks (Report Section 17).

WGPDMO concluded that all Terms of Reference for the 2003 meeting were considered in a comprehensive manner. Since there are several important issues in the field of pathology and diseases of marine organisms requiring further consideration, it was agreed that a further WGPDMO meeting is required in 2004. An invitation was received and acknowledged to organise the 2004 meeting at the Åbo Akademi University, Åbo, Finland. The proposed dates are 9–13 March 2004.

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1 OPENING AND STRUCTURE OF THE MEETING

The ICES Working Group on Pathology and Diseases of Marine Organisms (WGPDMO) met at the Fisheries Research Services, Marine Laboratory, Aberdeen, UK, with T. Lang as new Chair. The meeting was opened at 10:00 hrs on Tuesday, 11 March 2003, with the Chair welcoming the participants, particularly the new members who have not previously attended WGPDMO meetings.

A list of participants is appended in Annex 1.

Apologies were received from S. Bower, C. Couillard and S. McGladdery (Canada), T. Bezgachina, A. Karasev, G. Rodjuk (Russia), G. Díez (Spain), O. Haenen (The Netherlands), B. Hjeltnes (Norway), A. Köhler and S. Klimpel (Germany), R. Medne and I. Savecka (Latvia), J. Rokicki and W. Piasecki (Poland).

Two former WGPDMO members have retired in 2002: V. Kadakas (Estonia) and G. Bylund (Finland). The Chair acknowledged their valuable input to the WG and agreed to send best wishes on behalf of WGPDMO.

The Chair informed the members that the previous WGPDMO Chair, S. Mellergaard, had to resign from his term after the 2002 WGPDMO meeting because he changed affiliation. T. Lang was intersessionally nominated by WGPDMO as acting Chair for the 2003 WGPDMO meeting and was approved by the Mariculture Committee and the Council at the 2002 ICES Statutory Meeting.

The meeting was held as a series of plenary sessions with ad-hoc specialist subgroups established as appropriate in order to consider some agenda items in detail before reporting conclusions back to the plenum for further consideration and endorsement.

2 ICES: ITEMS OF RELEVANCE TO WGPDMO

The Chair highlighted items of relevance to WGPDMO.

2.1 ICES Annual Science Conference 2002

The ICES Annual Science Conference (ASC) took place in Copenhagen, Denmark, 1–5 October 2002. Because of the ICES centenary, the meeting was accompanied by a number of celebration activities, including the signature of the new ICES Copenhagen Declaration by representatives of all 19 ICES Member Countries, highlighting the goals of ICES and expressing a strong commitment of the ICES Member Countries to support the work of ICES. Furthermore, a flotilla of 10 research vessels from ICES Member Countries visited the inner harbour in the centre of Copenhagen and were open to the public. ICES published two major books as part of the Centenary activities, reflecting the significant contributions of ICES to marine science:

• The Sea Knows No Boundaries – A Century of Marine Science under ICES (by Helen M. Rozwadowski) • 100 Years of Science under ICES (ICES Marine Science Symposia 215). A number of Theme Sessions with issues relevant for WGPDMO were held:

Mariculture Issues

• Session R – Immuno-modulators and Probiotics: Alternatives to Chemotherapeutics ? • Session S – Juvenile Fish Cultivation: Improvements in Quality • Session T – Salmon Aquaculture, Enhancement, and Ranching: Are they a Threat to Wild Salmonid Stocks ? • Session U – New Developments and Applications of Genetics in Fisheries Management and Aquaculture

Environmental Issues

• Session X – Biological Effects of Contaminants in Marine Pelagic Ecosystems

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2.2 ICES Mariculture Committee (MARC)

The Chair presented the 2002 WGPDMO Report to the MARC at its meeting during the ICES ASC. The report and its recommendations were accepted. A new Term of Reference (k) was added, based on a request from the ICES Advisory Committee on the Marine Environment (ACME) (see Annex 2).

The MARC emphasised that there is a need to strengthen the ICES activities in the field of marine shellfish culture. Therefore, it recommended to establish a new ICES Working Group on Marine Shellfish Culture, a recommendation that was later approved by the ICES Council.

Furthermore, the MARC recommended to the ICES Consultative Committee that a Theme Session, entitled “Shellfish Culture in the ICES Area: Perspectives and Limitations”, be organised for the ICES ASC 2004 in Vigo, Spain. Since issues considered in the Theme Session will include disease aspects, the WGPDMO was asked to contribute to the session and nominate a co-convener. Other co-conveners already identified are A. Smaal (The Netherlands) and A. Bodoy (France). It was agreed that the Chair, supported by the WGPDMO shellfish disease specialists, would make the necessary contacts in order to identify an appropriate co-convener.

The MARC Chair mentioned that efforts are under way to determine whether some of the MARC Working Groups might be able to meet in conjunction with meetings of the European Aquaculture Society (EAS) or the World Aquaculture Society, as the EAS has approached ICES to establish a more formal working arrangement.

The MARC suggested that an executive summary should be included in all Working Group reports emphasising the main highlights of the report and, thus, providing the reader with a brief and concise overview. The WGPDMO endorsed this suggestion and the Chair volunteered to prepare the executive summary for this year’s meeting.

The three-year term of the MARC Chair, Tony Calabrese, ended in December 2002, so an election was held for a new Committee Chair. Thomas Sephton (Canada) was elected new MARC Chair.

2.3 ICES Advisory Committee on the Marine Environment (ACME)

The Chair informed the WGPDMO of topics considered by WGPDMO at its 2002 meeting that were subsequently reviewed by the ACME at its annual meeting in June 2002 and incorporated in the 2002 ACME Report:

a) Trends in diseases of wild and farmed fish and shellfish; b) Status of the M74 Syndrome in Baltic salmon and status of Ichthyophonus in herring; c) Nodavirus in aquaculture fish species; d) Infectious Pancreatic Necrosis Virus in salmonid fish farming; e) Studies on the relationship between environmental contaminants and shellfish pathology; f) Quality assurance procedures for biological effects techniques, including fish diseases; g) Web-based report on trends in fish disease prevalence and mariculture-relevant diseases.

2.4 Baltic Sea Regional Project (BSRP)

The BSRP will officially start on 15 March 2003 and is a major international effort to develop a holistic, integrated management of the Baltic Sea ecosystem in order to ensure its long-term sustainability. The project is designed to improve the research capacity of the eastern Baltic countries (Estonia, Latvia, Lithuania, Poland and Russia) so that they can contribute fully to ecosystem monitoring activities, including fisheries, within the frameworks of ICES, the Helsinki Commission (HELCOM) and the International Baltic Sea Fisheries Commission (IBSFC). The BSRP is part of the worldwide Global Environment Facility (GEF) Programme that is being financed by the World Bank. More information can be found at http://www.ices.dk/projects/balticsea.asp.

Since one component of the BSRP will be to foster biological effects monitoring, including studies on fish diseases and histopathology in the Baltic Sea, the WGPDMO Chair was asked, together with Chairs of other relevant ICES WGs, to contribute to a new ICES planning group for the implementation of the BSRP. The Chair will keep WGPDMO informed of any new developments in this direction. In the discussion it was pointed out that Baltic countries/institutes carrying out biological effects monitoring by applying standard techniques, such as those recommended by ICES, will have to participate in the self-funding BEQUALM programme (see Section 10).

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3 TERMS OF REFERENCE, ADOPTION OF AGENDA, SELECTION OF RAPPORTEURS

3.1 Terms of Reference

The WGPDMO took note of the Terms of Reference published as C. Res. 2002/2F03 (Annex 2). The agenda once again demanded extensive intersessional work by the members of the WGPDMO. Some members were requested to produce written working/discussion documents to be reviewed at the meeting and to be included in the WGPDMO report as Annexes, as appropriate. As agreed in WGPDMO, all working documents were to be prepared two weeks before the meeting and distributed by e-mail. As a result, the majority of the national reports and the remaining working documents were sent to the participants prior to the meeting. The Chair thanked the members for preparing these reports in advance, a task that ensures that the Terms of Reference can be treated efficiently.

3.2 Adoption of the Agenda

A draft agenda was circulated and accepted with an addition to Agenda Item 16 regarding the progress made in the report on trends in important diseases affecting the culture of fish and molluscs in the ICES area 1998–2002 to be published in the ICES Cooperative Research Report Series (Annex 3).

3.3 Selection of Rapporteurs

Rapporteurs were accepted as indicated in Annex 4.

4 OTHER RELEVANT INFORMATION

Information was given on scientific conferences/workshops with relevance to the work of WGPDMO to be held in 2003:

• Northeast Farmed Fish Health Workshop, 3 April 2003, University of Maine, Machias, ME, USA; • Fish Vaccinology Symposium, 9–11 April 2003, Bergen, Norway; • American Fisheries Society Northeastern Division and the Northeast Fish and Wildlife Conference, 13–16 April

2003, Newport, RI, USA; • National Shellfisheries Association Meeting, 13–17 April 2003, New Orleans, LA, USA; • Fish Immunology Workshop, 21–25 April 2003, Wageningen Institute of Animal Sciences, The Netherlands; • Eastern Fish Health Workshop, 21–25 April 2003, Gettysburg, PA, USA; • SETAC (Society of Environmental Toxicology and Chemistry) Europe 13th Annual Meeting, 27 April–1 May

2003, Hamburg, Germany; • PRIMO (Pollution Responses in Marine Organisms) 12th International Symposium, 9–13 May 2003, Tampa Bay,

Florida, USA; • Offshore Oil and Gas Environmental Effects Monitoring Workshop, 27–30 May 2003, Dartmouth, Nova Scotia,

Canada; • American Fisheries Society/Fish Health Section (AFS/FHS) and the Western Fish Diseases Workshop, 15–17 July

2003, Seattle, WA, USA; • American Fisheries Society Annual Meeting, 10–14 August 2003, Quebec City, Quebec, Canada • EAFP (European Association of Fish Pathologists) 11th International Conference, 21–26 September 2003, St.

Julians, Malta; • 6th International Parasitology Symposium, 21–26 September 2003, Bloemfontein, South Africa; • ICES Annual Science Conference, 24–27 September 2003, Tallinn, Estonia.

WGPDMO endorsed a proposal to include under this item information on major international research projects/programmes in the report beginning next year.

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5 ANALYSE NATIONAL REPORTS ON NEW DISEASE TRENDS IN WILD AND CULTURED FISH, MOLLUSCS AND CRUSTACEANS

5.1 Wild Fish

VIRUSES

Lymphocystis - The virus was isolated from wild striped bass (Morone saxatilis) from 3 estuaries in New Brunswick, Canada. While the overall prevalence of lymphocystis in North Sea dab (Limanda limanda) shows no new trend, slight increases were found at two locations in the Irish Sea and at the Flamborough off Ground and Dogger Bank. The prevalence of this viral disease continues to decrease in dab from the German Bight, but the prevalence was up to 29.2 % in Baltic flounder (Platichthys flesus) off the Polish coast. In Scotland, lymphocystis decreased in dab from the previous year (4.9 % to 2.6 %).

Infectious Haematopoietic Necrosis Virus (IHNV) - As in previous years, IHNV was isolated from sockeye salmon (Oncorhynchus nerka) newly emerging fry and spawning and post-spawned adults in British Columbia, Canada. No mortalities were associated with the isolations.

Infectious Pancreatic Necrosis Virus (IPNV) - IPNV was isolated from wild brook trout (Salvelinus fontinalis) in Nova Scotia, Canada, with no signs of disease. A wild fish survey in Scotland indicated that the presence of IPNV is very low (occasional positive findings in Atlantic salmon, Salmo salar, and haddock, Melanogrammus aeglefinus) in contrast to the steady increase in IPN in farmed salmonids.

Birnavirus serogroup II was isolated from plaice (Pleuronectes platessa) and flounder in Denmark.

Infectious Salmon Anaemia Virus (ISAV) - ISAV was not detected in nearly 3000 non-salmonid marine fishes surveyed in the USA, nor in 377 wild caught Atlantic salmon broodfish brought into a federal hatchery in Maine, USA. The only positive results for ISAV were obtained from a few non-salmonids, e.g., cod (Gadus morhua) and pollock (Pollachius virens) entrained in ISAV-infected Atlantic salmon cages (see Annex 5). Experimental studies on the susceptibility of Pacific salmonids to the Norwegian and American strains of ISAV showed a general resistance of Oncorhynchus spp. to ISAV. However, chum salmon (Oncorhynchus keta) was able to sustain significant titres of viable virus for at least 2 weeks.

Nodavirus - Nodavirus was isolated from wild caught cod after it was held and spawned in captivity in New Brunswick, Canada.

Viral Haemorrhagic Septicaemia Virus (VHSV) - High losses of sardines (Sardinops sagax) were reported at two locations in northern Vancouver Island (Canada) in the winter as in previous years. High titres of the North American strain of the virus were isolated from fish showing external lesions. Ten new isolates of the North American strain were isolated from various species of fishes in California and Washington. Nine of the isolates may represent a genotypic subgroup distinct from the previously known North American strains found in Washington, Alaska (USA) and British Columbia (Canada). VHSV was isolated from wild striped bass found dead in an estuary in New Brunswick, Canada.

In a Danish survey of over 4800 fish, VHSV was isolated from herring (Clupea harengus), sprat (Sprattus sprattus), cod, flounder and sandeel (Ammodytes sp.). Ammodytes sp. represents a new host for the European strain of the virus. Genotype I of VHSV was isolated from a wild Atlantic salmon in Spain diseased with Aeromonas salmonicida.

No viruses were isolated from 90 cod and 235 returning Atlantic salmon broodfish in Ireland.

BACTERIA

Aeromonas salmonicida - The bacterium was isolated from Atlantic salmon mortalities collected from the Miramichi, Restigouche and Saint John rivers in New Brunswick, Canada.

Renibacterium salmoninarum - The aetiological agent of bacterial kidney disease was not found in nearly 3000 non-salmonid marine fishes collected from the Atlantic coast of the northeastern USA.

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Mycobacterium - Two new mycobacterial species (Mycobacterium shottsii and M. chesapeaki) have been identified from striped bass in the Chesapeake Bay, USA. The predominant organism in the on-going epizootic is M. shottsii, found in 76 % of striped bass with and without external signs of disease.

Acute/healing skin ulcerations - No new general trends were found in the prevalence of these lesions in North Sea dab, although a few locations in the Irish Sea showed increased prevalences from 2001. Highest levels in Baltic flounder were reported off the Lithuanian coast (9.6 %). Baltic cod show much higher prevalences, up to 36.3 % near Gdansk Bay, but in the western Baltic, high prevalences reported in the late 1990s have decreased significantly. The prevalence of skin ulcerations in cod from the southeastern Baltic Sea was less than 0.1 %. In the Barents Sea prevalences of skin ulcerations were 0.1 % in cod, 0.2 % in long rough dab (Hippoglossoides platessoides) and 0.3 % in wolffish (Anarhichias sp.).

FUNGI

Information on Ichthyophonus is provided in Section 12.

ALGAE

Karlodinium micrum - K. micrum was responsible for a kill of 1700 menhaden (Brevoortia tyrannus) in the Chesapeake Bay, USA. Although K. micrum is fairly common at low levels in the Bay, this is the first reported fish kill associated with it.

Pfiesteria - Experiments with P. shumwayae demonstrated that this dinoflagellate does not produce a toxin but physically attacks larval fish, digesting and consuming epithelial tissue.

PARASITES

X-cell lesions - The number of North Sea dab with gill X-cell lesions was very low in most areas sampled in Scottish waters, except at Wee Bankie where the prevalence exceeded 1 %.

Pleistophora sp. - Pleistophora is suspected of having a role in reproductive disturbances of roach (Rutilis rutilis) in southern Finland.

Stephanostomum baccatum - Very high prevalences of this parasite continue to occur in dab from the northwestern North Sea and the Dogger Bank. Long-term data indicate a spread of the parasite in dab during the 1990s from the southern North Sea progressively into the northern North Sea.

Gyrodactylus salaris - This trematode remains a major threat to Atlantic salmon in Norway as two additional river systems became infested in 2002.

Anguillicola crassus - This swimbladder parasite was found at 40 % to 100 % prevalence in eels (Anguilla anguilla) collected from 15 sites in the southeastern Baltic Sea and Curonean and Vistula Lagoons.

Pseudodactylogyrus sp. - Pseudodactylogyrus was found for the first time on eel gills collected from the southeastern Baltic Sea.

Sea Lice - Lepeophtheirus salmonis is implicated in the unusually low returns of pink salmon (Oncorhynchus gorbuscha) in the Broughton archipelago in British Columbia, Canada. Observations of lice on salmon returning to rivers in Maine, USA, indicate the highest prevalences reported in July, with an increasing trend from 1998 to 2002 (40 % to 58 %).

Lice continue to be a problem for Atlantic salmon and sea trout (Oncorhynchus mykiss) in Norway. The extent of the problem varies with geographical areas, with some areas having more severe infestations than in 2001.

L. pectoralis prevalence in dab from inner Cardigan Bay was 45.5 %, confirming long-term data that demonstrate high prevalences in the central and southern North Sea.

Anchorworms - An unusual mortality event occurred in a Massachusetts, USA, bay with adult menhaden infested with large numbers (typically 50) of Lernaeid-like copepod parasites.

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Sphyrion lumpi - Long-term data (1982–2002) on the prevalence of Sphyrion lumpi on redfish (Sebastes mentella) from the Barents Sea show a minimum prevalence of 10 % during 1982–1984 and a maximum of 70 % in 1993. For 2002, the prevalence was 25 %.

OTHER DISEASES

Epidermal hyperplasia/papilloma – The prevalence of epidermal hyperplasia/papilloma in North Sea dab showed little change, maintaining overall prevalences between 2 % and 4 %.

Hyperpigmentation - Hyperpigmentation recorded over the past 10 years in North Sea dab indicates consistently high prevalence, but there is some indication of a decrease in prevalence at the Dogger Bank and Firth of Forth areas. In contrast, the prevalence appears to be increasing in one area off Scotland.

Liver nodules - The pronounced differences in prevalence of liver nodules observed until the mid-1990s in dab from various areas of the North Sea have almost disappeared due to significant decreases in prevalence of nodules at the former hot spots (German Bight and Dogger Bank).

Hepatocellular lesions - Hepatocellular fibrillar inclusions were found in flounder at several contaminated UK estuaries at prevalences ranging from 16 % to 63.3 %, while these lesions were not found in flounder from the reference site. Foci of hepatocellular alteration also were detected in flounder from these impacted estuaries, the highest prevalence (18.4 %) being reported from the Mersey.

Ocular necrosis - Necrosis of the eyes was observed mainly in cod and haddock of the Barents Sea. Prevalence was 0.15 % in cod and 0.21 % in haddock. Ocular necrosis accounted for 5 % of the overall pathologic conditions recorded in Barents Sea fishes in 2002, a decrease from 13 % in 2001.

Skeletal deformities - Skeletal deformities in Baltic cod were lower than at the end of the 1990s. New data acquired in the southeastern Baltic confirm the low prevalence. In the Barents Sea, prevalences of deformities were comparable for cod and haddock at around 0.15 %.

Ovotestis - Up to 20 % prevalence of ovotestis was reported for flounder in the Mersey estuary (UK), comparable to previous years.

Toxoplasma gondii - An unexplained decline in the federally protected sea otter population in California may be linked to infection with Toxoplasma gondii, as indicated by antibodies to this cat pathogen in 42 % of live and dead otters tested.

REVIEW OF PATHOLOGIES IN DEMERSAL FISHES OF THE BARENTS SEA AND ADJACENT WATERS

V. Donetskov presented results from a study carried out in the years 1999–2002 in the Barents Sea and adjoining areas. Although these are important areas for commercial fisheries for several countries, there has been little information on diseases of fish prior to 1999 when Russia (PINRO) began investigations into pathologic conditions and parasites of fishes. Data currently exist for over 89,000 specimens of 8 species of fishes collected over a four-year period. Ulcers, skeletal deformities, tumours and necrosis of the eye were the dominant pathologic conditions observed. The prevalences recorded are considered to be low compared to other areas, possibly indicating that the Barents Sea is a rather pristine ocean. These data establish an important baseline of information on the pathologic conditions of demersal marine fishes (Annex 6).

5.1.1 Conclusions

1) Ten new isolates of the North American strain of VHSV were isolated from several species of fishes from the USA, most of which may represent a new genotypic subgroup. In Denmark, Ammodytes sp. represents a new host for the European strain of the virus;

2) Lymphocystis continues to be widely detected at low levels in North Sea dab, however a significantly high prevalence was reported in Baltic flounder near the Polish coast;

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3) Two new species of Mycobacterium have been isolated from skin ulcers on striped bass in the USA, one of which is the predominant organism in the on-going epizootic;

4) Prevalences of acute/healing skin ulcerations in Baltic cod are high near Gdansk Bay but have decreased in the western Baltic;

5) The first fish kill due to Karlodinium micrum was reported in the Chesapeake Bay, USA, in 2002;

6) Gyrodactylus salaris remains a threat to Atlantic salmon in Norway as two additional river systems became infested in 2002;

7) Lepeophtheirus salmonis has been implicated in the unusually low returns of pink salmon in western Canada, and continues to be a major problem for salmon and sea trout in Norway;

8) There has been a significant decline in the prevalence of liver nodules in North Sea dab at the historical hot spots suggesting the prevalences are approaching natural background levels;

9) The report on diseases of fish species in the Barents Sea and adjacent waters is the first comprehensive report of a large number of specimens comprising several species of fishes from the Barents Sea and adjacent areas. These data establish an important baseline, particularly as concerns the health of the environment and quality of commercial fishes.

5.1.2 Recommendations

i) WGPDMO recommends that member countries ensure adequate funding is made available to continue health surveillance of wild fish stocks. Continued disease monitoring is necessary to:

a) be used as an indicator of environmental conditions; b) assess the impact of disease in wild fish stocks; c) assess the potential for disease interactions between wild and farmed fish; d) recognise emerging diseases caused by infectious agents and/or contaminants; e) understand the mechanisms of disease transmission and epizootic events.

ii) WGPDMO strongly recommends that fish disease studies in the Barents, Greenland and eastern Norwegian Seas continue, and that possibilities should be explored by Russian scientists for intercalibration of data collection and analysis with scientists conducting fish disease monitoring programmes in other areas.

5.2 Farmed fish

VIRUSES

Infectious Haematopoietic Necrosis Virus (IHNV) - Atlantic salmon (Salmo salar) are particularly susceptible to IHNV. Several farms in British Columbia (Canada) experienced significant losses in the winter of 2001/2002. The source of the virus is suspected to be wild stocks in the vicinity of the salmon farms since all fish are health-tested prior to transfer to the net cage sites.

Infectious Pancreatic Necrosis Virus (IPNV) - Infectious Pancreatic Necrosis Virus is an important disease problem in Norway and Scotland. In the former, exceptionally high water temperatures in late summer and early autumn resulted in reduced growth and health-related problems in farming of salmonids. Some regions (Trøndelag, Norway) have experienced a significant increase in the number of reported cases associated with first-feeding salmon fry. The mortality is variable, but in some cases, the losses have been very high. There are indications that high density, increased use of oxygenation and low water flow may increase losses in post smolts. Furthermore, there is increasing evidence that IPN isolates belonging to the same serotype show variations in virulence.

In Scotland, IPNV showed strong regional variation in marine waters between 1996 and 2001. An annual increase in the prevalence of this virus was found in seawater (10 %) and freshwater sites (2 % to 3 %), with a greater increase (6.5 %) at freshwater sites in the Shetland Isles. Trends in clinical IPN in Scotland over the last 5 years have increased from

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1.1 % to 12.5 %, but with prevalence changes between years. Similarly, in the Shetland Isles, IPN prevalence has increased from 5.2 % to 14.4 %.

Nodaviruses and nodavirus-like viruses - In the summer of 2002, large-scale mortality of cultured haddock (Melanogrammus aeglefinus) occurred in a facility in New Brunswick (Canada) due to an infection by a nodavirus. Significant mortalities have also been reported for juvenile Atlantic cod (Gadus morhua) in Newfoundland (Canada) in 2002 and previously for cultured juvenile cod in Nova Scotia (Canada).

In the USA, nodavirus was responsible for the loss of 160,000 juvenile cod (3–20 g) over a period of 30 days at a private hatchery. RT-PCR-positive results were obtained from brood cod and haddock at federal and academic facilities, but the virus was not isolated on these occasions.

Nodavirus continues to be a problem in sea bass (Dicentrarchus labrax) farms in France. A new isolate was found in this species that was pathogenic at temperatures <18 °C and was similar to the Atlantic halibut (Hippoglossus hippoglossus) isolate.

Aquareovirus - Aquareovirus has been isolated from haddock and Atlantic halibut in eastern Canada. In some cases, infections were associated with liver pathology. In Scotland, moribund Atlantic halibut larvae were sampled for a suspect nodavirus infection in August. At present, the aetiology is a suspect Aquareovirus.

Viral Haemorrhagic Septicaemia virus (VHSV) - A single outbreak occurred at one rainbow trout (Oncorhynchus mykiss) farm following transfer to marine waters in Denmark. In Finland, VHS has spread to several new rainbow trout farms, and 12 new farms were reported affected in 2002. In Spain, a number of VHSV strains have been isolated from turbot (Scophthalmus maximus), seabream (Sparus auratus) and sole (Solea sp.). The turbot isolate belongs to genotype 3, the sole isolate to the freshwater genotype from salmonids, and the seabream isolate to the genotype 3. In Spain, VHSV was not isolated after 2 weeks’ incubation. However, a further 2 weeks resulted in isolation of VHSV.

Infectious Salmon Anaemia Virus (ISAV) - A decreasing number of new outbreaks of ISA were reported in Norway and a national plan for the control of ISA has been worked out. No ISAV was recorded in Scotland. In July 2002, ISAV was isolated from rainbow trout on two isolated marine sites in the west of Ireland. All other farms in the country have been tested for ISA (using virus isolation), and found to be negative (see Annex 7). Following the outbreak of ISA in the Maine, USA, Atlantic salmon farming industry in 2001, USDA implemented an ISA programme with the State of Maine to eradicate the disease (Annex 5). ISAV was not detected in U.S.-farmed salmon in 2002.

Salmon Pancreas Disease Virus (SPDV) - Pancreas disease caused enormous mortality in Atlantic salmon on many sites in Ireland in 2002. The disease has been observed at very low levels in Ireland during the past 8–9 years and its re-emergence in 2002 cannot be readily explained. The disease has now been confirmed on all salmon farms north of Galway Bay (this area includes approximately ¾ of the Irish Industry) and one site in the southwest of the country.

Pancreas Disease was diagnosed at 12 sea sites in Norway. Two of these cases were in rainbow trout. The losses varied but may be prolonged and substantial. All reported cases so far have been in western areas. However, infection appears to be spreading.

Salmon pancreas disease prevalence is low in farmed salmon in Scotland and the virus has not been detected in wild fish.

BACTERIA

Aeromonas salmonicida - Atypical Aeromonas salmonicida was isolated following mortality in adult haddock from a culture facility in eastern Canada.

In Denmark, rainbow trout vaccinated against furunculosis and vibriosis (Vibrio anguillarum) suffered several outbreaks of furunculosis although 60 % of the fish had been vaccinated. Photobacterium damselae was isolated as the dominant bacterium in a mixed culture with A. salmonicida from some of the outbreaks, probably due to high water temperatures. Furunculosis is still present in several rainbow trout farms in Finland in spite of a comprehensive vaccination programme. Significant losses due to A. salmonicida are reported in turbot in France and Spain.

In Scotland A. salmonicida is isolated from fish in fresh water and sea water, with prevalence ranging from 0 % to 1.1 % over the last 5 years, whereas A. salmonicida subspecies achromogenes has only been isolated once during the last 5 years. A. salmonicida has a low prevalence in salt and fresh water (ranging from 0–1.1 % between 1998–2001).

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Renibacterium salmoninarum - For the first time R. salmoninarum caused high mortality in a Danish marine rainbow trout farm. In Finland, BKD, however, shows an increasing trend and some new infected rainbow trout farms were reported. The disease seems to cause mortality in connection with vaccination (against vibriosis and furunculosis) and transport. BKD remains low in Norway in farmed salmon. There is no change in BKD in Scotland, with a prevalence of < 0.4 % in sea water since 1998.

Piscirickettsia salmonis - In Norway during 2002, 18 new cases were reported in Atlantic salmon from sea water. Some of these may have the same origin. The summer of 2002 was exceptionally warm and this may have influenced the increased incidence. During 2002, P. salmonis was confirmed in Scottish Atlantic salmon from tissue sections and ELISA after the first records of Piscirickettsia-like organisms reported in 1996 from Scottish salmon.

Flavobacterium psychrophilum and Flavobacterium columnare - In Finland, Flavobacterium psychrophilum and F. columnare show an increasing trend in losses of rainbow trout in fresh water as well as in brackish water.

Vibriosis - Vibriosis is decreasing from year to year in Finland in rainbow trout due to the vaccination programme. In France, the incidence of Listonella (Vibrio) anguillarum has increased in sea bass and turbot. A vaccine for treatment of vibriosis has been developed in Russia.

Pseudomonas anguilliseptica - In Finland, P. anguilliseptica infections cause problems in some farms in sea trout (Salmo trutta) hatcheries in sea water and to a lesser degree in rainbow trout farms but the trend is decreasing. P. anguilliseptica was isolated for the first time in a few turbot farms in Spain.

In Spain, the first experimental vaccination trial was carried out in seabream. The vaccine was prepared with a non-mineral oil adjuvant and inactivated P. anguilliseptica, isolated frequently in the brain of fish affected by the winter disease syndrome. According to the farmer, the trial was a success, and no mortality occurred in the vaccinated fish.

Tenacibaculum maritimum (Flexibacter maritimus)- T. maritimum caused significant losses in cultured sea bass, seabream and turbot in France.

Edwardsiella tarda - E. tarda was reported from a turbot farm in France and represents the first isolation since 1991.

PARASITES

Myxidium leei - Myxidium leei (Enteromyxum leei) in seabream caused variable losses resulting in emaciation, decrease in growth and chronic mortality up to 15 %. The loss in commercial value was 10 % to 20 % of total value. This is an increasing trend.

Amoebic gill disease - Amoebic gill disease was reported in brown trout (Salmo trutta) from France. The clinical disease is associated with temperature exceeding 14 °C. The fish are lethargic, with open opercula and appear anorexic. Mortality up to 20 % occurs after 4 weeks. The transfer of the fish in fresh water for 1 hour seems effective against the parasite. However, re-infection can occur within a month.

Sea lice - The mean number of sea lice per salmon has been reduced in Norway over the period 1998 to 2002.

Parvicapsula sp.- Clinical disease outbreaks caused by Parvicapsula sp. occurred in Norway. This myxosporean was diagnosed in five Atlantic salmon farms in northern Norway. A large number of parasites were found especially in the pseudobranch, but also in liver and kidney. The mortality ranged from 3 % to 50 %. This is the first known outbreak of disease caused by this parasite in Norwegian farmed fish.

P. minibicornis was also suspected to cause high mortality in pre-spawning sockeye salmon (Oncorhynchus nerka) in the Fraser River in British Columbia, Canada.

Gyrodactylus salaries - G. salaris was detected in three salmon hatcheries in Norway.

Spironucleus barkhanus - S. barkhanus was detected from two sea sites in Norway, one for salmon and one for Arctic charr (Salvelinus alpinus).

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OTHER DISEASES

Skin ulcerations - Ulcerations causing significant losses remain a major problem in both salmon and rainbow trout farming in Norway. There appears to be an increasing trend in disease prevalence.

Skeletal muscle inflammation - A condition associated with heart and skeletal muscle inflammation was first described in Norway in 1999. In 2002 there has been an increase in diagnosed cases of this condition in Norway.

There is a growing concern regarding diseases that might be production-related such as cataract, skeletal deformities and heart abnormalities.

Algal blooms - Six notifications of fish losses were reported in Scotland with a total of 42,000 salmon of various sizes. One outbreak occurred on Isle of Skye in July. The other five farms were located in Shetland Isles and were affected by a bloom occurring in September.

Jellyfish - In Scotland, 20 farms rearing Atlantic salmon reported losses due to jellyfish swarming against the cages, with a total loss of 1,815,800 fish. Several fish farms in Norway experienced losses due to jellyfish (Muggiaea atlantica). The reason for this may be the exceptional, high water temperatures in late summer and early autumn.

5.2.1 Conclusions

1) ISAV has been found in Ireland for the first time and the planned epidemiological report will be of interest; 2) Aquareovirus was reported for the first time in Atlantic halibut in Canada; 3) Edwardsiella tarda was reported from a turbot farm in France and the first isolation since 1991; 4) A Parvicapsula sp. was recorded for the first time in Norway; 5) Contact with jellyfish swarms can have a significant impact with high mortality recorded in farmed fish.


WGPDMO recommends that:

i) The impact and risk of Aquareovirus for farmed fish be determined; ii) Further work be conducted to clarify the observation that extended incubation during VHSV testing will show

positive results, when negative results would have been reported with the normal incubation time; iii) Further studies be undertaken to identify whether wild fish are reservoirs of Parvicapsula sp. and to undertake

transmission trials to determine whether horizontal transmission is possible between salmon; iv) The impact of jellyfish on farmed fish be monitored.

5.3 Wild and Farmed Shellfish

VIRUSES

Herpesvirus in bivalves - Some PCR-confirmed cases of herpesvirus infections of larvae and spat were observed in the Pacific oyster (Crassostrea gigas) in the summer of 2002 in France. Positive spat samples originated from natural beds and from hatcheries. PCR-confirmed herpesvirus were also found in C. gigas in Tomales Bay, California, USA, where summer oyster mortalities have been observed; however, a relationship between the oyster deaths and herpes infection has yet to be established. Hatcheries in Oregon and Washington (USA) that furnished the Tomales Bay seed oysters have tested negative. To date, limited geographic examination has found the virus only in this embayment.

Unusually high mortality of juvenile Pacific oysters (over 80 % in some batches) was experienced at two farm sites in the UK during August. Oysters at one site were confirmed as being infected by Oyster Herpesvirus (OsHV-1) and co-infected with Vibrio splendidus.

Shrimp viruses - A novel intranuclear bacilliform virus (IBV) (Baculo-like virus) was reported in wild-caught brown shrimp (Crangon crangon) from UK estuaries. The virus causes necrosis of the hepatopancreas and midgut epithelial cells. A prevalence of up to 100 % was found at sites in the Clyde estuary and high levels (up to 85 %) at sites in the Forth and Mersey estuaries. Other sites are currently being assessed.

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Herpes-like virus in spiny lobster - A herpes-like virus has been discovered in wild populations of the Caribbean spiny lobster (Panulirus argus) and appears to be the first reported viral disease of lobsters. The HLV-PA occurs in as many as 16 % (N = 850) of the juvenile spiny lobster populations sampled from the mid- to lower Florida Keys in the Gulf of Mexico, USA. The virus attacks haemocytes causing the blood to become milky white, and appears to affect juveniles only. The virus is highly lethal in experimental conditions. Molecular and biophysical studies are under way to accurately identify this organism and its geographical distribution is being investigated. Further experiments are planned to determine the susceptibility of spiny lobster adults and other species of crustaceans to the virus.

BACTERIA

Nocardiosis - No new trends in Canada; no new information from the USA.

Withering syndrome of abalone - No new information.

Vibrio tapetis - No new information.

Vibrio spp. - In France, some isolates of V. splendidus, V. lentus and V. aestuarianus were reported in association with summer mortality among cultured C. gigas spat. Moreover, challenge by two different isolates of V. splendidus produced greater (60–80 %) mortality than either isolate injected alone (20–30 %). A batch of Pecten maximus juveniles presenting abnormal mortality (20–50 %) in a French nursery was found to be infected by a Vibrio pectinicida-like bacterium. In Spain, some mortalities were reported in Manila clams (Ruditapes philippinarum) associated with the presence of Vibrio sp., but without the brown ring symptom.

Necrotizing hepatopancreatitis (NHP) - Continues to be a problem on shrimp (Litopenaeus vannamei) farms in Texas, USA.

FUNGI

Yeast in bivalves - The appearance of an unidentified organism, interpreted as a yeast, was reported in the cockle, Cerastodema edul, in the Croisic area (Loire Atlantique, France) after the “Erika” oil spill. The presence of the “yeast” was associated with a massive cellular reaction. A causative role of the pollution in the appearance of the infection is suspected, but has not been demonstrated.

Yeast in crustaceans - An unidentified yeast was found in virus-infected wild brown shrimp (Crangon crangon) from UK estuaries.

ALGAE

Abnormal mortality (more than 15 % in 2 weeks) was reported among C. gigas in association with the detection of a dinoflagellate, Karenia mikimotoï, in France.

PARASITES

Bonamia ostreae in Ostrea edulis - B. ostreae was detected for the first time since 1994 (when one of 450 oysters was found to be Bonamia-positive) in a bed of wild oysters in Achill Sound, Co. Mayo, Ireland. Prevalence of infection was 13 % (of 150 oysters) in this bed, but remained unchanged in other affected zones in Ireland (<20 %). Achill Sound thereby lost its EU-approved zone status (2002/300/EC) with respect to B. ostreae.

The overall level of infection of B. ostreae in oysters in England (2.9 %) was lower than in previous years. Four samples were taken from the wild fishery in the River Blackwater, England, in spring 2002 following the finding of 60 % (15 of 25 sampled) prevalence in O. edulis from one farm in the same river in autumn 2001. There were concerns of a spread into the wild fishery, although no unusual mortalities had been noticed. Prevalences of B. ostreae in the spring 2002 samples were 0 %, 3 %, 3 % , and 23 %.

The ongoing 2002 investigation for the presence of B. ostreae in the native flat oyster population in the Limfjord, Denmark, has not found evidence of the parasite.

Investigations in France, Spain and Scotland show no change in status.

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Marteilia refringens in O. edulis and M. edulis - No new trends reported.

Paramarteilia-like parasite in Cancer pagurus - The parasite was found in connective tissues and tissue sinuses of a single crab collected from a site on the southern English coast.

Mikrocytos mackini in C. gigas - No new trends in Canada, but the parasite was reported for the first time in the USA in Washington State in May 2002. The organism was detected in two feral populations of C. gigas at a prevalence of 6 % (mild infections) in each population as determined by both histology and PCR. More than 15 other populations of wild and farmed brood stock Pacific oysters were then surveyed throughout Washington with no positive results. The disease agent requires temperatures below about 15 oC and closer to 10 oC to manifest itself, which may limit its southern distribution. There are two incident reports of a similar organism occurring in the native Olympia oyster (Ostrea conchaphila) which is known to be susceptible to the disease. The disease agent has probably not moved southward. More likely, it has been present in feral old oysters at very low prevalence and intensity for some time and was never detected because such oysters were never examined.

P. marinus (Dermo) in Crassostrea virginica - Severe drought and a warm winter resulted in exceptionally high prevalence and intensity in the mid-Atlantic and northeastern USA. Harvest of C. virginica in Chesapeake Bay was low at all times due to mortalities caused by P. marinus (and H. nelsoni), which has intensified pressure to introduce a non-native species, C. ariakensis. Triploid individuals of this Asian species are currently being evaluated for performance in Chesapeake Bay.

Perkinsus atlanticus in Venerupis spp. and Ruditapes spp. - No new trends reported in France and Spain.

Perkinsus chesapeaki/andrewsi in Mya arenaria and Macoma balthica - Continuing analysis supports the contention that P. chesapeaki and P. andrewsi are likely to be the same species (see Section 8 and Annex 10).

Haplosporidium nelsoni (MSX) in Crassostrea virginica - Was reported for the first time in Canada in October 2002, when it was confirmed (by histology and PCR) as the cause of mass mortalities (> 90 %) on several leases in Cape Breton, Nova Scotia (see map in Annex 8). To date, infections appear to have a tight distribution focus related to the St. Patricks Channel area of Bras d’Or Lakes. The parasite became more widely distributed than ever before in the Virginia portion of Chesapeake Bay (USA), being observed in the upper James and Rappahannock Rivers for the first time. The spread is probably associated with a severe drought and concomitant salinity increases. No change was observed elsewhere in its reported range along the entire Atlantic coast of the USA.

Haplosporidium costale (SSO) in Crassostrea virginica - Was reported for the first time in Canada, at prevalence less than 5 % (by PCR), during the intense surveillance effort mounted to map the distribution of MSX (see map in Annex 8). Several samples that showed “light” “background” plasmodial infections but no indication of pathology were found on the north shore of Cape Breton, as well as at several sites within the Gulf of St. Lawrence. No change was reported in the USA.

Haplosporidium sp. in Mytilus edulis - A haplosporidian was identified in a single blue mussel (Mytilus edulis) in Canada, which was heavily infected by spores. EM and histological comparison with MSX and SSO reference material showed differences in size and tissue location, and PCR examination confirmed this to be a different species.

Haplosporidium sp. in Carcinus maenas - A novel haplosporidian infection was detected in approximately 10 % of shore crabs (Carcinus maenas) captured from a southern England site. The parasite causes lethargy and loss of aggressiveness in infected crabs. Haemolymph appears opaque and creamy and coats internal organs.

Quahog Parasite X (QPX) in Mercenaria mercenaria - Abnormal mortality (10–25 %) of clams was reported from a localized area of Raritan Bay, south of New York City, USA. 33 % of clams from this site were found with patent QPX infections and another 20 %, with lesions possibly associated with the parasite. Subsequent sampling of wild populations in the area, including those adjacent to the site where some of the infected clams had been transplanted, showed only very low prevalences (≤ 5 %). The heavily infected clams were part of an unusually dense set that had occurred 2–3 years earlier and the high densities probably facilitated disease outbreaks.

Paramoeba in Callinectes spp. - Data on haemolymph samples from over 7300 blue crabs (Callinectes sapidus) collected over a 9-year period revealed 0.5 % of crabs to be infected with Paramoeba perniciosa. Infections were found in crabs collected only from the New Jersey to Virginia region of the US Atlantic coast; no infections were detected in crabs from the Gulf of Mexico. Infections were found only in the months of June, July, September and October with the highest prevalences (1 %) being in June and July. A Paramoeba sp., having a secondary body dissimilar to that of P.

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perniciosa, was detected in one of 139 lesser blue crabs (Callinectes similis) sampled in Maryland and may represent a variant of P. perniciosa or a new species.

Hematodinium sp. in Chionoecetes tanneri - A population study on Chionoecetes tanneri off the west coast of Vancouver Island, Canada, revealed the presence of Hematodinium sp. in up to 4 % of the captured Tanner crabs. The nucleotide sequences of part of the small subunit (SSU) rDNA gene were nearly identical to the sequences in GenBank of Hematodinium sp. from blue crabs on the Atlantic coast of the USA.

Hematodinium sp. in Callinectes sapidus – Unusually heavy and widespread mortalities of blue crabs were reported in Georgia, USA, and attributed to Hematodinium spp. Drought-caused high salinities are thought to have favoured the parasite.

Blue mussel (Mytilus edulis) parasitic digenean Prosorhynchus squamatus - No new information.

Johanssonia arctica in the Kamchatka crab (Paralithodes camchaticus) - Increased prevalence of this leech in Kamchatka crabs was noticed in Russia. Year-to-year variation in prevalence of the parasite was negatively correlated with variation in temperature, and positively correlated with variation in oxygen in the near-bottom layer of the Murman Current of the Barents Sea.

DISEASES OF UNCONFIRMED AETIOLOGY

Juvenile Oyster Disease (JOD) of Crassostrea virginica - JOD was virtually non-existent throughout the Northeast, including Maine (the only place in the USA where it was reported in 2001). The Roseobacter that has been implicated in JOD outbreaks in Maine was found in a few oysters obtained from two New York culture sites.

Shell deposits in clams - Samples of wild clams fished from the River Roach (UK) exhibited shell deposits similar to Brown Ring Disease (BRD), but showed no evidence of Vibrio tapetis, the aetiological agent of BRD.

Histopathological disorders in Mytilus edulis - In Germany, a study on histopathological disorders in wild blue mussel (Mytilus edulis) populations during the period 1994–2000 revealed an elevated prevalence of haemocyte infiltration, granulocytomas and pathological disorders of the gonads (atrophy and atresia), possibly linked to higher levels of TBT and TPT in animals in harbour areas.

Haemic neoplasia - Haemic neoplasia in soft-shell clams (Mya arenaria) persists in wild stocks throughout Prince Edward Island (Canada), and surveillance has demonstrated high prevalences in neighbouring waters along the Gulf shore of New Brunswick (Canada). Repeat transmission experiments to investigate preliminary results in 2001 suggesting an infectious agent, failed to confirm an infectious aetiology. An eight year (1989 to 1997) survey of Mya arenaria in northeastern North America found positive samples in Maine (USA) and three Canadian provinces (New Brunswick, Nova Scotia, and Prince Edward Island) at prevalences of 3–50 %. A field experiment in which naïve clams were placed in close proximity to diseased clams showed no evidence of transmission after 6 months.

Unexplained mortalities of giant sea scallop (Placopecten magellanicus) in Canada - No new information.

Unexplained mortalities of mussels (Mytilus edulis) - No pathogens were found in mussels sampled from Swansea Bay (UK), where some mortalities had been experienced.

Unexplained mortalities of adult Pacific oysters (Crassostrea gigas) – Low levels of late summer mortality in Pacific oysters are regularly noticed at a farm site in North Cornwall (UK). No disease agents have been found in samples taken in previous years. Temperature data show wide daily fluctuations, consistent with the idea that this is inducing spawning and weakening the animals such that any handling stress is giving some mortality. Reduced handling at this time has improved the situation.

Mortalities of adult European oysters (Ostrea edulis) - Mortalities along southwest Nova Scotia, Canada, reported in 2001 persisted in 2002. Oysters from the same populations sent to France as part of a genetic study were destroyed following detection of a microcell-like parasite. PCR studies indicated a Mikrocytos-like parasite that does not have the same sequence profile as M. mackini. The same PCR results were obtained from European oyster samples collected in Atlantic Canada, but neither samples yielded levels of infection that could be directly attributed to the mortalities. High summer temperatures are believed to have played a role.

Withering syndrome of abalone in USA - No new information.

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Black spot disease of Crangon crangon - No new trends reported.

Shell disease of Homarus americanus in USA - No new information.

Summer mortality of Homarus americanus - Sustained elevated temperatures combined with hypoxic conditions were associated with lobster mortalities during the summer of 2002 in Long Island Sound, USA. These are the same conditions that accompanied heavy lobster mortalities in the summer of 1999, but in contrast to the paramoeba linked to the 1999 deaths, no pathogen was associated with the 2002 mortalities. A new disease syndrome has been identified. It is described as an excretory calcinosis, characterized by diffuse, severe mineralised granulomatous inflammation of the antennal glands and, in severe cases, the gills. The calculi can block the gills, causing coagulative necrosis and presumably respiratory failure at the high temperatures and hypoxic conditions observed in 2002. Calcinosis was observed in 30–40 % of lobsters in the autumn.

5.3.1 Conclusions

1) Bonamia ostreae was reported in 2002 in an EU-approved zone in Ireland; 2) No important new trends were reported for Marteilia refringens, Nocardia crassostreae, Withering Syndrome, or

Perkinsus spp.; 3) Haplosporidium nelsoni (MSX) and H. costale (SSO) were reported for the first time in Canada (Cape Breton,

Nova Scotia). H. nelsoni infections caused heavy, although localized mortalities, in an area of the Bras d’Or Lakes. H. costale was found at low prevalence and in light infections on the north shore of Cape Breton;

4) Mikrocytos mackini was reported for the first time in USA, although it is not certain whether this represents a new introduction;

5) A new microcell parasite was detected in Ostrea edulis from Atlantic Canada, but it is genetically different from M. mackini;

6) A new intranuclear bacilliform virus was reported at high prevalence for the first time in the brown shrimp, Crangon crangon, in the UK;

7) A new and highly lethal (causing 100 % mortality in laboratory challenges) herpes-like virus was reported in juvenile wild spiny lobsters (Panulirus argus) off the Florida coast (USA);

8) A new haplosporidian was reported to infect about 10 % of Carcinus maenas from the south shore of England, causing lethargy and loss of aggressiveness;

9) A new disease syndrome, described as an excretory calcinosis, was observed in Homarus americanus in Long Island Sound, USA, during the summer of 2002, when sustained elevated temperatures and hypoxic conditions were associated with lobster mortality. The paramoeba linked to mortalities under similar environmental conditions in 1999 was not found in 2002;

10) Yeasts were found to co-infect both molluscs and crustaceans in conjunction with viruses or Hematodinium spp. and were also discovered in cockles affected by the “Erika” oil spill in France.



i) Studies continue in Canada to attempt to determine whether the finding of H. nelsoni and H. costale in the Cape Breton area represents new introductions or whether the parasites were present earlier and, if the former, how the introduction occurred;

ii) Studies be initiated or expanded on viral diseases in crustaceans, including the herpes-like virus in Panulirus argus, to specifically identify agents and their effects on various crustacean species;

iii) The haplosporidian infecting Carcinus maenas be identified using molecular techniques and that its effects on crab populations be determined;

iv) Studies continue to determine the causes of the summer mortality syndrome in the Pacific oyster, Crassostrea gigas;

v) Studies be initiated to examine the impact of the “Prestige” oil spill on the prevalence of diseases; vi) The existing information on viral diseases of crustaceans with emphasis on commercially important species be

reviewed by the WGPDMO at its 2004 meeting; vii) The ICES Working Group on Introductions and Transfers of Marine Organisms (WGITMO) review

information related to the current efforts to introduce a non-native oyster species, Crassostrea ariakensis, into Chesapeake Bay, USA, where the native C. virginica has suffered very heavy disease-caused mortalities over

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the past decade. The entire effort is being conducted with considerable public debate, scientific input, and governmental oversight. Trials of C. ariakensis are currently under way using ICES protocols to avoid the introduction of pests and disease agents, and any larger-scale introduction will also follow ICES recommendations. The WGPDMO requests that it be kept informed of the findings of the WGITMO.

6 REPORT ON PROGRESS IN THE ONGOING INVESTIGATIONS OF THE EFFECT OF TEMPERATURE ON BONAMIA INFECTION DYNAMICS AND REPORT ON THE CONFIRMATION OF THE AGENT OF CRASSOSTREA ANGULATA GILL DISEASE AND ITS INFECTIVITY TO CRASSOSTREA GIGAS AND OTHER OYSTER SPECIES

T. Renault presented results of the European programme DISENV.

Investigations of the effect of temperature on Bonamia ostreae infection dynamics

A major focus of the European Programme DISENV (Environmental Factors and Shellfish Diseases, FAIR-CT98-4129, involving three participants: UBO, Brest, France; IFREMER La Tremblade, France and University of Glasgow, UK) was to study the effects of acclimation at low (10 °C) or high (20 °C) temperature on Bonamia ostreae infection and on defence mechanisms in the host, the European flat oyster, Ostrea edulis. Oysters were acclimated at 10 °C or 20 °C for one month. One set remained at the acclimation temperature and another set was transferred to the other temperature (Table 6.1). In each treatment, some individuals were injected with purified parasites. The development of the disease was monitored up to 5 months post-inoculation. Results showed an effect of temperature on B. ostreae infection prevalence and on certain haemocyte parameters (Table 6.1).

Table 6.1. Temperature-associated mortality, infection prevalence in survivors, and haemocyte parameters in Ostrea edulis after inoculation with Bonamia ostreae.

Acclimation oC Test oC Percent Mortality

Prevalence in Survivors

Haemocyte Parameters

20 20 ~25 5

20 10 ~50* 18 Reduction in circulating granulocytes compared to 20 oC

10 10 ~25 38 Fewer circulating haemocytes and lower free radical production than at 20 oC.

10 20 ~25 20 Relatively small effect * examination of dead oysters indicated involvement of B. ostreae in the mortalities.

Thus, contrary to expectations based on field observations of the apparent disappearance of B. ostreae from the Limfjord, Denmark, after the importation of infected O. edulis in the 1980s, the lower temperature tested appeared to be related to higher, not lower, parasite prevalences. Since granulocytes are the most phagocytic haemocyte and contain the most lysosomal enzymes, their temperature-associated decrease might be linked to the temperature effects on infection prevalence.

Investigate the causative agent of gill disease in Crassostrea angulata in southern Europe

Crassostrea angulata was the species of major economic importance in Europe up to the early 1970s, when iridoviral infections led to its near total disappearance. A few populations persisted in southern Spain and Portugal and it is known that the virus also affected these stocks. Moreover, symptoms of iridoviral infection were also observed in C. gigas originating from Japan, Korea and Canada, and the presence of iridoviruses was later confirmed by transmission electron microscopy in French C. gigas. Reported mortalities were always lower in C. gigas than in C. angulata although it is clear that both taxa may be infected. Therefore, it is important that the health status of C. angulata populations should be closely followed for the security of both cultured and wild stocks.

Low prevalence of gill disease was reported in 1999 in C. angulata in Portugal. Since, at the light microscopic level, these resemble the lesions associated with mass mortalities of the same species in the 1970s, it is important to confirm the aetiological agent, particularly given the known sensitivity of C. angulata to iridoviral infections. A study was conducted in 2001 and 2002 to assess, using conventional techniques such histology and transmission electron microscopy, the health status of certain wild C. angulata populations from Portugal and to determine whether the iridoviruses were present in C. angulata produced in France under hatchery controlled conditions. These studies failed

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to detect iridoviral infections in Portuguese and French oysters. In 2003, a new study will be carried out on adult oysters originating from natural beds located in south of Portugal and larvae produced in a Portuguese hatchery.

6.1 Conclusions

1) Experimentally induced B. ostreae infections developed more readily at 10 oC than at 20 oC. Temperature also affected certain haemocyte parameters that may be involved in defence. However, it remains unclear whether temperature has a direct influence on the parasite;

2) No infectious agent has been identified in C. angulata from Portugal that had gill lesions similar to those associated with mass mortalities of this species in France in the 1970s.

6.2 Recommendations


i) Further studies on the effects of temperature on host defence mechanisms and on pathogen development and virulence be carried out in different bivalve models;

ii) Investigations continue to determine the cause of gill lesions reported in C. angulata from Portugal and Spain, and that any new information be integrated into national reports.

7 REVIEW THE EXISTING STRATEGIES TO ASSESS THE PREVALENCE OF SHELLFISH DISEASES IN PARALLEL TO FISH DISEASES AND CHEMICAL CONTAMINATION LEVELS IN ENVIRONMENTAL MONITORING PROGRAMMES

A working document was presented by S.W. Feist, which had been prepared intersessionally on the use of fish and shellfish diseases in environmental monitoring programmes (Annex 9). Advantages and disadvantages of using fish or shellfish, a review of case studies and an outline of the current status of studies in Europe and North America were provided. It was recognised that fish diseases had long been used by several ICES Member Countries as part of the biological effects suite of techniques but in addition, that only few studies had incorporated molluscan shellfish or crustacean pathology/disease in combination with fish disease and contaminant data.

In reviewing existing monitoring strategies it was concluded that practically none incorporated shellfish. The multi-year National Status and Trends Program covered a large number of sites on all coasts of the USA and included the assessment of fish and shellfish species, using an integrated sampling approach including contaminant levels. It now continues with shellfish and contaminants. No such integrated programmes currently exist in other ICES Member Countries although there are research projects which incorporate both fish and shellfish diseases in assessments. It was stressed that clear strategic objectives for the monitoring were crucial in deciding whether to include several target species, including shellfish. For assessment of environmental impact of pollution incidents such as oil spills, it is desirable to examine the disease status of a broad range of species appropriate for the region concerned and these would include shellfish species. For longer-term monitoring, sentinal species for detecting the impact of contaminants on health should be used, this being the current strategy employed by a number of national monitoring programmes. However, these do not routinely utilise shellfish species. It was recognised that there were limitations concerning the proposed use of shellfish as target organisms for monitoring. These included the lack of baseline data on normal tissue and organ structure, immunological parameters and host responses to contaminants. However, the use of histopathology to detect host tissue responses was thought to be valuable in providing information on the overall health status of target shellfish species because more specific tools will not be available. Regarding the observation of parasite pathogens and associated host responses, it was considered that these may provide important data on individual and population health status. The use of such data for assessment of contaminant effect is more problematic unless the life cycles, including requirements of intermediate hosts of the parasites are known.

It was recognised that it would be possible to use material collected during national monitoring programmes, e.g., for EU-listed notifiable diseases (e.g., Bonamiosis and Marteiliosis) and for assessment of algal toxins. Since diagnosis of notifiable diseases is mainly based upon examination of histological sections, these can readily be assessed for provision of additional data on more general pathological effects as well as disease intensity and incidence, which could be used in the context of environmental assessment.

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7.1 Conclusions

1) Apart from the U.S. National Status and Trends Program there are no integrated environmental monitoring programmes in ICES Member Countries, which have utilised shellfish disease in combination with fish disease and contaminant levels;

2) There is a general lack of coordination between monitoring programmes for notifiable diseases, presence of algal toxins, and for biological effects of contaminants in shellfish.

7.2 Recommendations

The WGPDMO recommends that:

i) ICES Member Countries should endeavour to ensure that monitoring efforts in coastal and marine environments are more integrated to maximise the data obtained from samples taken and to avoid duplication in sampling effort;

ii) the results of the U.S. National Status and Trends Program and of other environmental monitoring programmes incorporating studies on diseases/pathology of marine organisms are reviewed by WGPDMO at its 2004 meeting.

8 REVIEW OF MOLECULAR TECHNIQUES USED TO DIFFERENTIATE THE VARIOUS SPECIES/ISOLATES OF PERKINSUS SPP.

The WGPDMO reviewed a report entitled “Review of molecular techniques used to differentiate the various species/isolates of Perkinsus” prepared by S. Bower, in collaboration with G. Burreson and K. Reece at the Virginia Institute of Marine Science, outlining the criteria used for species identification in the genus Perkinsus (Annex 10).

Perkinsus spp. are pathogens that have been detected in over 67 species of marine molluscs found in many locations around the world. From the identification of the first species, Perkinsus marinus, in the late 1940s to the present, there have been 7 named species. These have been identified on the basis of their ability to enlarge in Fluid Thioglycollate Medium (FTM) as well as on morphological characteristics seen in histological section and electron microscopy, and more recently, in in vitro culture. The development of molecular tools has added a new dimension to species identification criteria, and as a consequence it now seems likely that two of the named species are the same as two others: P. atlanticus = P. olseni and P. andrewsi = P. chesapeaki. The validity of another named species, P. karlssoni, has been refuted because the description is likely to have been based partly on a contaminating organism. Thus, of the original 7 named species, only 4 are now thought to be valid: P. marinus, a parasite that causes widespread mortalities of the eastern oyster, Crassostrea virginica, in the United States; P. olseni, which infects a wide variety of bivalves and gastropods from Japan to Europe; P. qugwadi, which infects the introduced scallop, Patinopecten yessoensis, in western Canada; and P. chesapeaki, a parasite of Mya arenaria and Macoma balthica in Chesapeake Bay, USA.

Further, molecular characterisation of the genus Perkinsus indicates that it should be removed from its present position in the Phylum Apicomplexa because it is genetically more similar to the dinoflagellates. A new classification scheme has been proposed in which the Perkinsus spp. are placed in a newly created phylum with the name Perkinsozoa.

8.1 Conclusions

1) Members of the genus Perkinsus are lethal parasites of many important commercial molluscs and have a world-wide distribution;

2) It is difficult to distinguish among Perkinsus spp. based solely on morphological features or enlargement in FTM and molecular sequencing has become an important tool for species identification;

3) Improved methods of distinguishing among species that may or may not be pathogenic to specific hosts will aid in the design of disease control and prevention programmes;

4) A set of criteria should be developed to aid in the identification and naming of new species of Perkinsus that includes non-specific assays such as FTM and histology, morphological and physiological characteristics in vitro, and molecular analysis.

8.2 Recommendations


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i) The document “Review of molecular techniques used to differentiate the various species/isolates of Perkinsus” prepared by Bower et al. be circulated widely (i.e., through the internet on sites such as the “PerkID” list hosted by Franck Berthe (IFREMER) and an Asian molluscan health list hosted by FAO) so as to solicit international comments and contributions on the information and ideas presented;

ii) An updated report on techniques used to differentiate among Perkinsus spp., incorporating input received as a result of the above solicitation, should be prepared for, and reviewed at, the 2004 WGPDMO meeting.

9 OBTAIN INFORMATION ON THE EU PROJECT “DIAGNOSIS OF OYSTER HERPES-LIKE VIRUS: DEVELOPMENT AND VALIDATION OF MOLECULAR, IMMUNOLOGICAL AND CELLULAR TOOLS” (FAIR-PL98-4334) AND REVIEW THE RESULTS

T. Renault presented a review of the results obtained during the course of the VINO project (Annex 11).

The aim of the VINO project (involving 7 participants from France (IFREMER and UBO), Scotland, Belgium, Ireland, Spain, and England) was the development and validation of molecular, immunological and cellular tools for the diagnosis of, and studies on, the bivalve herpesviruses. An initial step of the VINO programme involved cloning of virus DNA. The sequence data demonstrate that the Oyster herpesvirus type 1 (OsHV-1) is not closely related to herpesviruses in vertebrate hosts (including fish), and the data support the view that herpesviruses found in mammals and birds, fish and amphibians, and invertebrates form three major lineages of the herpesvirus group. Currently, OsHV-1 is the single representative of what may be a large number of invertebrate herpesviruses.

The work done during the VINO project also provided cloned viral DNA fragments suitable for characterising the viral genome and preparing specific diagnostic probes (PCR primers, labelled DNA probes and specific antibodies). Polymerase chain reaction (PCR) assays have been developed which allow the rapid, specific and sensitive diagnosis of herpesviruses in bivalve samples. Another technique developed is in situ hybridisation (ISH). VINO partners have conducted trials using PCR and ISH techniques in order to standardise these techniques. Specific rabbit antisera and monoclonal antibodies have been obtained and may be used for diagnostics.

The ability of the virus to replicate in vitro was tested in different cell lines. The viral replication was not successful in fish cell lines or mammalian cell lines. Promising results have been observed in primary cultures of embryonic oyster cells, but it is not at this time a reliable tool to detect OsHV-1.

The development of molecular, immunological and cellular tools for OsHV-1 diagnosis may facilitate virus detection in infected material, and the developed tools have been used by four European laboratories to analyse a wide range of bivalve samples (spat, larvae and adults from hatcheries and farms in France, Spain, UK and Ireland) and to confirm the usefulness of the diagnostic methods. Herpes viral infections were confirmed in France in 1999, 2000 and 2001 and some positive samples were also reported in Spain and UK. Positive samples were observed in four bivalve species: Crassostrea gigas, Ostrea edulis, Ruditapes decussatus and R. philippinarum.

9.1 Conclusions

1) The results of the EU project “Diagnosis of oyster herpes-like virus: development and validation of molecular, immunological and cellular tools” provide significant information on herpesviruses infecting bivalves;

2) The Oyster herpesvirus type 1 (OsHV-1) infecting bivalves is not closely related to vertebrate herpesviruses, and no viral replication is reported in tested mammalian cell lines or fish cell lines;

3) Molecular diagnostic techniques such as PCR and in situ hybridisation are available, as are specific polyclonal and monoclonal antibodies;

4) Promising results were observed using primary cultures of embryonic oyster cells in order to obtain virus replication in vitro;

5) OsHV-1 has been found in several bivalve species: Crassostrea gigas, Ostrea edulis, Ruditapes decussatus and R. philippinarum, and infections are reported in France, Spain and UK using molecular techniques.

9.2 Recommendations


i) Further studies be carried out to compare and validate the different diagnostic techniques for oyster herpes-like virus;

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ii) Tools developed in the VINO project should be used to search for Oyster herpesvirus type 1 (OsHV-1) in different geographical areas, especially in cases of outbreaks of summer mortality syndrome in Pacific oysters.

10 REVIEW AND REPORT ON PROGRESS MADE IN THE “FISH DISEASES AND LIVER HISTOPATHOLOGY” COMPONENT OF THE BEQUALM SELF-FUNDING SCHEME

The WGPDMO reviewed the “External fish diseases and liver histopathology” section of the BEQUALM final report presented by S.W. Feist (Annex 12). The purpose and scope of BEQUALM were described and the activities of the original EU-funded programme were summarised. These were:

• To develop appropriate reference materials or type collections; • To develop an infrastructure for assessing the comparability of data from individual laboratories; • To demonstrate that biological effects analyses are under statistical control and are of known quality.

Specific aims of the fish disease and liver histopathology component were:

a) identification of participating laboratories; b) to hold practical workshops to establish robust protocols and diagnostic criteria for the assessment of fish diseases

during field monitoring cruises and for the diagnosis of liver histopathology in dab (Limanda limanda) and flounder (Platichthys flesus).

In addition, provision of teaching material in the form of histological sections had also been undertaken. The main output from the programme was a CD-ROM containing all of the information relating to sampling and assessment of external diseases and liver histopathology, including laboratory protocols and images of all diseases currently used for monitoring purposes by ICES Member Countries. A comprehensive section on liver histopathology is also included which incorporates additional material from North American flatfish species kindly donated by M. Myers (NOAA, USA). The CD-ROM was demonstrated and ways in which it is intended to expand the information contained were described. In particular, the inclusion of additional examples of histopathological lesions from dab and flounder, as well as from other fish species, as well as improvements for highlighting specific features of external disease and histopathological lesions already contained on the CD-ROM were highlighted. A manuscript entitled “The use of fish liver histopathology for the assessment of contaminant effects in monitoring”, containing more comprehensive descriptions of liver histopathology and a key of the diagnostic criteria, has been submitted for publication in the ICES Techniques in Marine Environmental Sciences (TIMES) series.

The self-funded scheme is to be launched in April 2003 with details for registration being available on the BEQUALM website (www.bequalm.org). The programme of activities planned for this component during the first year of the BEQUALM self-funded scheme was outlined to the meeting. These include preparation and distribution of reference materials for training purposes, updating the CD-ROM as described above, undertaking ring-tests using a limited number of histological slides as well as smaller exercises using e-mailed images. A workshop is not planned for 2003 but will be a key activity for subsequent years.

The importance for all institutes engaged in fish disease monitoring and/or utilising fish liver histopathology in environmental quality assessments to subscribe to BEQUALM was stressed. This is important because the ICES Marine Data Centre will accept only data that have been obtained conforming to the quality assurance standards established under BEQUALM. WGPDMO members were encouraged to disseminate information on the BEQUALM programme to colleagues who might be interested in joining.

10.1 Conclusion

1) The WGPDMO acknowledged the progress made during the BEQUALM programme and emphasised that the programme had been successful in the establishment of the basic quality assurance procedures required for monitoring programmes.

10.2 Recommendations


i) Laboratories engaged in environmental monitoring programmes using fish diseases and/or liver histopathology subscribe to the BEQUALM quality assurance scheme;

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ii) Progress made in the BEQUALM self-funding scheme be reviewed by the WGPDMO at its 2004 meeting.

11 REVIEW AND ASSESS THE IMPACT OF DISEASES OF FARMED FISH ON WILD FISH STOCKS

D.W. Bruno presented a working document prepared intersessionally (Annex 13).

The aquaculture industry worldwide is assessing new species for aquaculture. As the number of fish species increases, the dynamics for disease interaction change. However, there are practical difficulties in measuring prevalence, incidence and the pathogenicity of diseases in wild stock and overall these have not been widely investigated. Most bacterial and viral diseases have a broad host range and there is also potential transfer of infection from wild to farmed fish, and similarly from farmed to wild fish.

As a result of diversification there is an increase in infectious disease among farmed animals and their significance and impact are outlined. IPN virus (IPNV) is widespread in farmed fish particularly in Norway and Scotland and an increasing number of species are found carrying the virus. As this virus is stable for long periods in marine and brackish waters, this could also have an impact on seawater outbreaks of IPNV and transmission from wild to farmed stock, and vice versa.

The Nodaviridae have been reported as the causative agents of disease throughout the world, referred to as viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER), and are economically important emerging pathogens. The increasing number of susceptible species identified worldwide will increase the chance of infection moving from wild to farmed stock and vice versa.

Infectious Salmon Anaemia virus (ISAV) has been the cause of ISA in salmonids, and has been a serious problem in farmed fish in Norway, Scotland, Canada, Maine, USA and the Faroe Islands, Denmark. The limited host range for ISAV suggests that outbreaks will be infrequent in new fish groups. A wild host for the virus has not been identified.

Viral haemorrhagic septicaemia (VHS) is traditionally considered a disease of rainbow trout resulting in extensive losses to freshwater culture operations across continental Europe. Recently, a number of genotypes have been identified in wild fish and consequently the risk of infection and transfer is dependent on the species and genotype.

Infectious haematopoetic necrosis virus (IHNV) has occurred in British Columbia among Atlantic salmon. The source of the virus is suspected to be wild stocks in the vicinity of the salmon farms.

In Norway, Gyrodactylus salaris has occurred in three hatcheries. At one site, this was the first occurrence since the 1970s. One hatchery is located close to an infected river system that could represent a wild source of this infection. Evidence suggests that fish movements between hatcheries account for the other outbreaks.

In Norway, furunculosis has been a problem for farmed fish since its first occurrence in 1964, following an introduction of rainbow trout from Denmark. The disease spread to several farms and also to wild stocks thereafter. However, eradication programmes were successful. In 1985, the disease was again recorded in marine salmon farms, following importation of salmon smolts from Scotland. By the end of 1992, furunculosis had spread to 550 farms and concurrently to 74 natural watercourses, where it affected wild salmon, sea trout and brown trout. Although the ecological consequences are not known, there is concern that the disease may affect populations if a significant number of the broodstock die before spawning.

Sea lice are a significant problem for salmon aquaculture, and a report from Norway provides evidence for a negative impact on wild populations resulting from the transfer of a parasite from farmed to wild fish. Returning wild salmon may carry adult female lice that could represent a local source of lice eggs. Observations show the fish are also infected by juvenile lice settling on the fish as they pass through coastal zones. The latter aspect may be a function of salmon farming.

11.1 Conclusions

1) The impact of diseases of farmed fish on wild fish stocks is a complex issue and there are many areas that require further study;

2) Viral, bacterial and parasitic diseases are a significant threat to aquaculture animals, but in the wild, the impacts are largely unknown;

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3) Information is available on the pattern and number of sea lice in farmed and wild fish. Long-term experiments and monitoring are required to assess the regulatory effect of lice in these populations;

4) Evidence for the transfer of disease agents from farmed to wild fish include the recent observation of sea lice and furunculosis.



i) Further work be done to evaluate the risk of disease transmission from farmed fish to wild fish, and equally the risk from wild fish infecting farmed stock, by applying appropriate epidemiological methods;

ii) Emerging viral diseases be assessed for risk to aquaculture animals and the risk to wild fish; iii) The significance of parasitic diseases (e.g., Gyrodactylus salaris) on hatchery production be assessed; iv) Further studies be conducted to assess the impact of sea lice and furunculosis on wild salmonid fish stocks; v) WGPDMO review current information on disease/parasite interactions between wild and farmed fish and on

related management control methods at its 2004 meeting.

12 MAINTAIN AN OVERVIEW OF THE SPREAD OF ICHTHYOPHONUS IN HERRING STOCKS AND THE DISTRIBUTION AND POSSIBLE CAUSE(S) OF THE M74 SYNDROME

Ichthyophonus hoferi

Information was extracted from the national reports on diseases of wild fish.

Ichthyophonus hoferi was observed in Atlantic herring (Clupea harengus harengus) in the northern North Sea east of Shetland Islands in German stock assessments in 2002, but no specific studies on the prevalence of the parasite were conducted. No new trends were observed in I. hoferi infection in Baltic herring (Clupea harengus membras) in the Baltic Sea. In trawl catches in the Barents Sea, Atlantic herring were observed infected by I. hoferi.

I. hoferi was isolated from migrating Atlantic salmon taken from rivers flowing into the Barents Sea. Histological examination revealed spores and hyphae in testes, kidneys and in muscle underlying skin haemorrhage and ulceration.

I. hoferi was also detected in wild yellowtail flounder (Platichthys ferrugineus) caught off Sable Island (Atlantic coast of Canada). In Puget Sound, Washington, USA, four new hosts (Pacific tomcod (Microgadus proximus), speckled sanddab (Cihtarichthys stigmaeus), Puget Sound rockfish (Sebastes emphaeus) and surf smelt (Hypomesus pretiosus pretiosus)) of I. hoferi were identified by gross observations of different organs and laboratory cultures. Most infections were subclinical. In Pacific herring (Clupea pallasii) infection prevalence ranged from 12 % among juveniles to 58 % among age 6 and older fish. This high prevalence in adult herring may contribute to the increase in mean estimated annual mortality rate of Pacific herring, which has increased from 20 % in the late 1970s to 87 % in 1999. I. hoferi was also found in four rockfish species (Sebastes alutus, S. flavidus, S. pinniger and S. reedi) collected from the Vancouver (BC), Oregon and Washington, USA coastal waters. In contrast, in a study done in 1972, no I. hoferi infections were observed in S. flavidus. In laboratory studies it was demonstrated that tidepool sculpin (Oligocottus maculosus) and starry flounder (Platichthys stellatus) can become infected after consuming I. hoferi-infected tissue.

M74 Syndrome

T. Wiklund presented a report prepared intersessionally by the former WGPDMO member G. Bylund.

In accordance with the prognosis presented to ICES last year, the prevalence of M74 continued to increase compared to the two preceding years. The final prevalence figures for all rivers are not available yet but seem to vary between 16–55 %. Preliminary data suggest the M74 prevalences in Finnish and Swedish rivers to be 10–12 % higher compared to the previous year (2001). For example, in the River Tornionjoki and River Simonjoki, the most important rivers for salmon smolt recruitment on the Finnish coast, 30.1 % of the salmon females produced offspring with 100 % mortality while 52.9 % of the females produced offspring with varying levels of mortality. There are presently no data available for a reliable prognosis for the disease prevalence in the 2003 hatch.

Research on M74 continued, however, no conclusive information on the aetiology of the M74 syndrome has been obtained so far. Recent research in Sweden has focused on the physiological and biochemical processes in salmon and

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salmon fry suffering from thiamine deficiency, i.e., effects of thiamine deficiency on liver enzyme activity and on neurotransmitters in the brain. These studies were aimed at explaining behavioural and other symptoms characterising the M74 syndrome. Studies in Finland currently focus on the thiaminase activity in the food chain of Baltic salmon. All data thus far suggest that the high thiaminase activity of salmon prey species, Baltic herring, but not of sprat (Sprattus sprattus), is the immediate cause behind the thiamine deficiency syndrome of salmon. The herring material examined, including herring samples collected in spring 2002 from the southern and middle parts of the Baltic area, indicates that there is no pronounced geographical variation in the thiaminase activity of Baltic herring. But there is seasonal variation with the highest thiaminase activity in late summer and autumn and there is a very pronounced variation even between individual fish specimens within the same catch. So far there is no plausible explanation for the high thiaminase activity in herring. Analysis on the thiaminase activity of a considerable number of strains of blue-green algae has not supported the hypothesis that algal blooms are a main factor in the aetiology of the M74 syndrome.

Research is currently also focused on the role that coplanar PCBs and similar compounds may play in the aetiology of M74. The concentrations of these compounds were previously found to have increased in Baltic salmon coincidentally with the outbreak of M74 but still a direct link between accumulation of these compounds and M74 remains to be shown. It has been demonstrated in rats that exposure to PCBs induced reduction in the thiamine levels, possibly due to free radical oxidation of the phenol ring of thiamine. The possibility for similar reaction patterns in salmon is currently being explored.

12.1 Conclusions

1) Ichthyophonus hoferi was isolated from four new hosts in Puget Sound, USA and from Atlantic salmon from Russian rivers flowing into the Barents Sea. High prevalences of I. hoferi have been reported for Pacific herring and this fungus may play a role in the unexplained decline of the adult herring population. I. hoferi is still present in North Sea and Barents Sea herring populations although no new trends in infection prevalence were observed in the examined herring stocks;

2) The prevalence of the M74 syndrome in Atlantic salmon in the Baltic Sea showed an increasing trend, compared to the two preceding years. So far, no obvious cause of the M74 syndrome has been established.



i) ICES Member Countries are encouraged to continue monitoring the prevalence of Ichthyophonus hoferi, if appropriate in connection with herring stock assessments, so as to be aware of changes in status that may forecast an epizootic disease outbreak;

ii) Because of the continuous significance of the M74 Syndrome in Baltic salmon, relevant ICES Member Countries are urged to provide sufficient resources for continued studies into the aetiology of M74, specifically addressing the role of food organisms and contaminants.

13 ASSESS AND REPORT ON THE EFFECTIVENESS OF SALMON FARMING MANAGEMENT CONTROL METHODS FOR SEA LICE

D.W. Bruno presented a summary of two submitted manuscripts that focus on management measures and epidemiological patterns of salmon lice (for references see below).

Salmon lice cost the Scottish salmon farming industry about £30 M per year in treatment costs and production losses. Current chemical control methods are sometimes inadequate and are contentious in terms of environmental and human health. However, despite intensive research on sea lice biology and control on salmon farms, sea lice remain the most economically significant parasite in salmon farming in sea cages. Grazing on the mucus stresses the fish, and leads to epidermal erosion and eventually death of the fish. Furthermore, sea lice may transmit other pathogens and may infect wild fish.

A growing list of therapeutics is used to control lice abundance but does not necessarily prevent their occurrence. “Fallowing” is considered an effective method of prevention on cage sites, although there is evidence that new infections from wild fish or escapees can occur. Alternatively, tidal transport of the larval stages cannot be discounted.

In Norway, a “National Action Plan” against sea lice was developed in 1997. This plan contained the following objectives:

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• to establish regional working groups to plan and coordinate regional efforts; • to document strategic lice treatments; • compulsory reporting of lice numbers; • compulsory reporting of lice treatments; and • monitoring salmon lice infections on sea trout and wild salmon smolts.

Differences exist in lice numbers per fish between Norway and Scotland. In the latter, fallowing is not routinely practised, and the availability of particular medicines, shallower cages and less stringent discharge consent regulations are factors that help to explain differences in lice levels between these countries. Biological control methods such as improving the host immune response and developing a vaccine may also be preventative if they are successful. The use of wrasse as cleaner-fish can be effective and has been widely used in Norway. These biological approaches may significantly reduce the need for medicines. However, no one method of prevention or control is likely to be universally successful.

13.1 Conclusions

1) The factors influencing re-infestation rates from other farms and wild fish remain poorly understood; 2) The most successful approach to reduce the number of lice is “integrated pest control” involving a farm

management regime that includes both preventative and treatment strategies.



i) Studies be carried out on the behaviour and dispersal of infective larval stages of sea lice; ii) Mathematical models be developed to predict population growth of lice and dispersal and that they be

integrated with local models of hydrodynamics.

References

Heuch, P.A., Bjørn, P.A., Finstad, B., Holst, J.C., Asplin, A., and Nilsen, F. Relationships between salmon lice on wild and farmed salmonids: A review of population dynamics, management measures and effects on wild salmonid fish stocks in Norway. In press.

Heuch, P.A., Revie, C.W., and Gettinby, G. A comparison of epidemiological patterns of salmon lice (Lepeophtheirus salmonis) infections in Norway and Scotland. In press.

14 REVIEW PROGRESS MADE IN THE MODIFICATIONS TO THE ICES DATABASES AND REVIEW AND APPROVE THE REVISED ICES ENVIRONMENTAL DATA REPORTING FORMAT (VERSION 3.2)

Using the documents provided by ICES, including recent updates of the database components as available on the Internet, W. Wosniok provided an overview of the present structure of the Integrated Environmental Data Reporting Formats (Version 3.2) (Annex 14). Besides providing an easier and more comprehensive data access, ICES needs the new structure for its official activities, e.g., related to OSPAR and HELCOM, and also for its plans to produce information summaries derived from the integrated database. This facility could support the activity of WGPDMO to provide fish disease and related information on the web.

In the new integrated structure, fish disease and biological effects data belong to different parameter groups on the same hierarchy level and are stored in the same database. The new database joins all the information that was formerly stored in technically different and separate environmental databases, thus facilitating the future analysis of data from different sources. Records and fields for new parameters, particularly biological effects, were introduced according to the input already provided by various ICES Working Groups. The new structure will facilitate additions to the set of permissible codes for a record field, however, the introduction of new fields within a record should be avoided for cost and time reasons once the database structure is approved and technically realised. Amendments and corrections to the structure should reach ICES prior to the programming activities, which are scheduled to begin in May 2003.

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Additionally, members of ICES Working Groups are requested to provide ICES with samples of existing or prospective new data in order to allow ICES to check whether the present database design allows the entry of all desired types of data.

WGPDMO discussed the new structure and appreciated the new possibilities emerging from the new structures. No problems with entering standard fish disease data were revealed. Also the option to have future data summaries for a broader public, including web-based presentations, generated by ICES in collaboration with relevant ICES Working Groups, was considered as a promising new perspective.

However, the ICES request for raw data was considered liable to generate unnecessary work for ICES as well as for WGPDMO members, as the present lab-specific files would need a specific explanation for ICES, and ICES would have to convert a large variety of different files. It seemed more efficient to have WGPDMO members convert files of their data to the new integrated format and to report success or deficiencies to ICES. D.W. Bruno, T. Lang and S.W. Feist were nominated to carry out this task for data sets of the types that should be incorporated into the new database. The WGPDMO Chair will contact members from other countries to check whether they would contribute to this process. However, if there is a need to supply raw data to ICES, persons listed in Annex 15 will provide ICES with it. ICES is requested to contact the WGPMO Chair in this case.

14.1 Conclusion

1) The WGPDMO approved the proposed database structure and appreciated the introduction of the new structure as well as the new possibilities associated with it. The prospect to have data summaries for a broader public produced by ICES facilities is welcomed as a valuable development.



i) ICES contact the WGPDMO Chair to clarify whether a raw data transfer to ICES is required; ii) WGPDMO review the progress made in the modifications of the ICES Databanks and the ICES Environmental

Data Format at its 2004 meeting.

15 REVIEW THE CRITERIA FOR THE INCORPORATION OF EXTERNALLY VISIBLE FISH DISEASES INTO MONITORING PROGRAMMES ON BIOLOGICAL EFFECTS OF CONTAMINANTS – AND PROVIDE FEEDBACK TO WGBEC

The ICES Working Group on Biological Effects of Contaminants (WGBEC) regularly reviews the list of techniques recommended for biological effects monitoring. Based on discussions at the 2002 WGBEC meeting, it was suggested to exclude two techniques for biological effects monitoring, externally visible fish diseases and benthic community analysis. At the 2002 meeting of the ICES Advisory Committee on the Marine Environment, there was some disagreement about the removal of these two methods. Consequently, WGBEC was tasked to review the criteria for methods for biological effects monitoring, taking into account the input from the Working Group on Pathology and Diseases of Marine Organisms (WGPDMO) on the issue of externally visible fish diseases.

WGPDMO reviewed and discussed a document prepared intersessionally by T. Lang, W. Wosniok and C. Couillard (Annex 16) and statistical data presentations by W. Wosniok. It was recognised that ICES has spent a considerable number of years establishing standard methods for gathering externally visible fish disease data and analysis, and those analyses suggest that contaminants may affect fish health. Data presented in the reviewed document demonstrate significant correlation between sediment loads of heavy metals and disease prevalence in North Sea dab (Limanda limanda). Further, univariate and multivariate statistical analyses of current data show a strong correlation between body burdens of organochlorines and multiple externally visible disease outcome.

WGBEC’s consideration that externally visible fish diseases do not meet the criteria for the biological effects monitoring appears to be unfounded. The current ICES fish disease studies do meet the criteria in that they are adequately tested in the field over many years, are standardised and intercalibrated, and have been demonstrated to have as good sensitivity to contaminants as other techniques accepted for biological effects monitoring.

Monitoring has various purposes and, naturally, the methods involved in a monitoring programme would vary depending on the purpose. WGPDMO considers that these different purposes of monitoring have not been considered adequately by WGBEC when establishing criteria for incorporation of biological effects techniques in monitoring

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programmes. Direct cause and effect relationships between contaminants and disease are difficult to establish due to the multiplicity of factors involved in the disease process. However, fish diseases are an integrated endpoint of the effects of the environment (anthropogenic and natural) on fish and therefore are valuable in establishing the health of the environment.

15.1 Conclusions

1) Long-term field data on fish diseases exist that demonstrate significant correlation between sediment contaminant loads and body burdens and externally visible lesions;

2) Externally visible fish diseases do meet the criteria established by the ICES WGBEC for the use of biological effects techniques and should be retained as a method for biological effects monitoring.

3) The criteria should be revised according to the various purposes of monitoring.


i) WGPDMO recommends that WGBEC revise the criteria for biological effects monitoring based on the various purposes of the monitoring programmes;

ii) WGPDMO recommends that monitoring of externally visible fish diseases should be retained as a component of an integrated monitoring programme.

16 REVIEW PROGRESS MADE WITH REGARD TO THE UPDATE OF ICES PUBLICATIONS ON PATHOLOGY AND DISEASES OF MARINE ORGANISMS

16.1 Web-based report on diseases and parasites of wild and farmed marine fish and shellfish as part of the ICES Environmental Status Report

WGPDMO reviewed the information presented on the web-based report on wild and farmed marine fish and shellfish diseases. The presentation included criteria for inclusion in trend maps and the updates made on the colour coding for disease prevalence levels. It was suggested that in addition to the trend maps that are generated, maps on the relative prevalence levels be generated also. The group discussed separating out the data gathered in winter and spring, however, it was pointed out that this would leave too few data for good trend estimates. Adjustments to the trend estimation procedure can be made that are expected to solve this problem. The problem of major gaps in recent data and consequently of inadequate number of data points make it nearly impossible to establish trend figures for the various diseases recorded. The inadequacy of available data in the database contributed to limiting progress on this ToR that could be accomplished in the past year.

16.1.1 Conclusions

1) There is not an adequate amount of recent data in the ICES fish diseases database to develop current trends for most fish diseases;

2) There is a need to revise the calculations and illustrations of disease trends and to calculate and illustrate the relative disease prevalence levels in the ICES areas.


i) WGPDMO encourages all ICES Member Countries gathering fish disease data to submit these data to the ICES fish diseases data bank;

ii) WGPDMO recommends that work on this project, including the application of revised methods, continue for an additional year with a progress report to be made at the next WGPDMO meeting.

16.2 Manuscript on methods for the statistical analysis of fish disease data for submission to the ICES TIMES series

W. Wosniok gave an overview of the present manuscript. Progress has been made with the main sections as agreed upon during the WGPDMO 2002 meeting. However, some structural changes were found to be necessary. For the sake of brevity, some introductory parts, some sections on basic statistical techniques and some theoretical sections were deleted or shortened. Additional sections were introduced to deal with techniques, the importance of which have become apparent while working on actual prevalence data recently. This refers to methods dealing with:

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• time series containing observations irregularly distributed over seasons; • multivariate methods accounting for the interaction among several factors and their simultaneous impact on

several effects; • variable selection techniques; • the treatment of correlated explaining factors.

Several sections were reorganised from a method-oriented style towards a problem-oriented style by introducing a directory of problems which guides the reader to method descriptions and/or examples. The tentative table of contents is given in Annex 17. At present, a manuscript size of 30–40 pages is to be expected. The final version of the manuscript is expected prior to the 2004 meeting of WGPDMO.

16.2.1 Conclusion

1) WGPDMO acknowledged the progress made in preparing the manuscript on the statistical analysis of fish disease data to be submitted to the ICES TIMES series and agreed to review the final version of the manuscript at the 2004 meeting of WGPDMO.



i) The final version of the manuscript on the statistical analysis of fish disease data should be reviewed and adopted by the WGPDMO at its 2004 meeting;

ii) The adopted manuscript should be submitted for publication in the ICES TIMES series.

16.3 Current status of the ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish

ICES publishes a series of Identification Leaflets, for which S. McGladdery is the editor on the subject Diseases and Parasites of Fish and Shellfish.

WGPDMO reviewed the editor’s report of 2002. One of last year’s recommendations was the evaluation of the possibility of putting revised and new ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish onto the ICES website. The WGPDMO once more endorsed this recommendation. The Chair informed WGPDMO that all Identification Leaflets on Zooplankton have been scanned by ICES to be compiled in an electronical library and that ICES indicated that this might also be possible for the ICES Disease Leaflets. WGPDMO appreciated this and agreed on doing this with all old disease leaflets, however only as a first step in the process of publishing the leaflets on the ICES website. It was suggested that new leaflets and revised ones will be formatted so they can be incorporated in a future web site for the Identification Leaflets directly. WGPDMO agreed that the editor of the ICES Disease Leaflets will contact ICES in order to discuss ways how this can be achieved without a major delay.

It was decided that a possible overlap with OIE publications would not to be a problem, as it is considered that the ICES Disease Leaflets should remain a complete series and that other web-accessible disease information (OIE, NACA, FAO and AusVet) does not provide comparative identification for non-reportable infectious agents and rarely provides information on diseases of non/infectious aetiology.

The sub-group established last year in order to support the editor of the ICES Disease Leaflets in her work was expanded to two people per area (wild fish: S. Feist and S. MacLean; aquaculture: D. Bruno and L. Madsen; shellfish: S. Bower and M. Lyons). This subgroup will review the list of proposed ICES Disease Leaflets suggested by S. McGladdery for revision and review existing titles proposed by the WGPDMO.

16.3.1 Conclusions

1) A more structured procedure has to be developed concerning publication of the ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish , including a time schedule;

2) Old ICES Disease Leaflets should be scanned by ICES as soon as possible; 3) New and updated ICES Disease Leaflets should be formatted in a way that they can be published directly on the

ICES website;

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4) Efforts should be continued to publish the ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish on the ICES website.



i) the ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish (old, revised and new leaflets) are being published on the ICES website;

ii) the old ICES Disease Leaflets be scanned by ICES and that new or revised leaflets be formatted in a way that they can directly be used for publication on the ICES website;

iii) the editor S. McGladdery contact ICES to make arrangements for the scanning of the old ICES Disease Leaflets and the publication of the ICES Disease Leaflets on the ICES website;

iv) a more structured procedure (including time table) for publishing the leaflets be developed; v) the sub-group established to support the editor of the ICES Disease Leaflets in her work review the existing and

proposed leaflets with S. McGladdery, review the proposed titles by the WGPDMO and make recommendations to WGPDMO regarding the need for new leaflets;

vi) the editor of the ICES Disease Leaflets, after consultation with the sub-group, contact all authors to advise them of any steps they need to take (e.g., submit colour photos, update) before the document can be published and that finalised versions be translated and submitted to ICES as soon as possible.

16.4 Review the status of the Cooperative Research Report “Report on important trends in diseases occurring in finfish and shellfish culture in the ICES area in the period 1997–2001”

The WGPDMO reviewed the status of the manuscript for submission to the ICES Cooperative Research Report Series. The Chair reported that the ICES Mariculture Committee had approved the report for publication after some modifications, which have been made. Some contributors pointed out, however, that there is a lack of consistency among sections, especially for diseases that affect more than one species.

The Chair agreed to assume responsibility for contacting contributors with a list of items that they should check to ensure consistency among sections. It was agreed that contributors would also update sections with any available new information and that the original title is modified accordingly: “Trends in Important Diseases Affecting the Culture of Fish and Molluscs in the ICES Area 1998–2002”.

16.4.1 Conclusion

1) The manuscript “Trends in Important Diseases Affecting the Culture of Fish and Molluscs in the ICES Area 1998–2002” will be ready for publication after contributors make revisions to ensure that sections are consistent and updated.


The WGPDMO recommends that

i) The WGPDMO Chair returns the manuscript “Trends in Important Diseases Affecting the Culture of Fish and Molluscs in the ICES Area 1998–2002” to contributors with a list of items to check to ensure consistency among sections. Final revisions, including any changes required for updating or consistency, should be returned by contributors to the Chair by 17 April 2003;

ii) The WGPDMO Chair submit the manuscript to the ICES Secretariat for publication by 30 April 2003.

17 ANY OTHER BUSINESS

17.1 WGPDMO Chair

WGPDMO approved T. Lang, who has served as acting Chair for the 2003 meeting, as new WGPDMO Chair.

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17.2 WGPDMO website

A proposal was made that WGPDMO should establish its own website that could be used for the exchange of information amongst WGPDMO members and as a means to facilitate the dissemination of information on the WGPDMO’s activities to the wider scientific community and to other relevant organisations.

The Chair explained that he had contacted the ICES Secretariat before in order to explore possibilities of how this can be achieved. Due to staff limitations, ICES is not in a position to establish and maintain such a website. This would have to be done by WGPDMO members and their institutes, respectively. The Chair agreed to find out before the 2004 WGPDMO meeting who would be able to offer an appropriate possibility.

17.3 Template for national reports on new disease trends in wild and farmed fish, molluscs and crustaceans

A template was used for the first time for submission of national reports on new disease trends by ICES Member Countries for review by WGPDMO. This facilitates the extraction of relevant information to be included in the WGPDMO Report. WGPDMO considered the template as useful, but it was suggested that the national reports could be made more informative if those new trends are highlighted that constitute significant changes compared to previous years. It was agreed that the template will be modified accordingly by the Chair.

17.4 “Prestige” oil spill

A summary of the events surrounding the sinking of the oil tanker “Prestige” in November 2002 and of the subsequent impact of the oil spill was presented to the WGPDMO by J. Barja. The main coastline affected has been the northern Galician coast but also affected are the Spanish Atlantic islands national park and to a lesser degree the north Atlantic coast and part of the French coast. The dramatic impact on affected beaches was shown and the use of techniques for the assessment of biological effects in shellfish resident in affected areas was discussed. It was noted that the biological status of the impacted areas was well known based on previous monitoring and that more intensive investigations have been implemented after the incident, concentrating on chemical analyses.

Techniques, which could provide important information on the biological responses and health status of shellfish stocks, were recommended by WGPDMO for inclusion in the long-term monitoring programme. These should include:

1. Histopathology to detect toxicopathic lesions and infectious disease conditions; 2. Contaminant-specific and general biological effects methods (as recommended by the ICES Working Group

on Biological Effects of Contaminants (WGBEC) in its 2002 report, ICES CM2002/E:02); 3. Use of indices which reflect the physiological state of the organisms such as lipid content and condition

factor; 4. Measures of population performance such as density, growth, reproductive success.

18 PROGRESS WITH TASKS

An analysis of progress of tasks in the Terms of Reference was conducted and presented in Annex 18. All items had been dealt with in a comprehensive manner. Several intersessional tasks were identified during the meeting.

19 FUTURE ACTIVITIES OF WGPDMO

Since there are several issues of importance in the field of pathology and diseases of marine organisms requiring further consideration, it was agreed that a further meeting of WGPDMO is required in 2004 to consider the results of intersessional work and to discuss outstanding items.

It was agreed that the invitation to host the next meeting of the WGPDMO from T. Wiklund, Laboratory of Aquatic Pathobiology, Department of Biology, Åbo Akademi University, Åbo, Finland be accepted. The proposed dates are 9–13 March 2004. The Chair thanked T. Wiklund on behalf of WGPDMO for the kind invitation.

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20 APPROVAL OF RECOMMENDATIONS

The recommendations to the ICES Council contained in this report were discussed by the WGPDMO and approved. The recommendations and justifications for recommendations to the Council are appended in Annex 19.

21 APPROVAL OF THE DRAFT WGPDMO REPORT

The report of the 2003 meeting was approved before the end of the meeting and the draft report was circulated to the participants on 28 March 2003. The conclusions on the Terms of Reference and associated Annexes where advice was specifically sought by other ICES bodies would be extracted and sent separately to ICES.

22 CLOSING OF THE MEETING

The Chair thanked the local host, D.W. Bruno, for providing excellent meeting facilities and arrangements and the participants for their efforts and input during and in preparation of the meeting. The 2003 WGPDMO meeting was closed at 14.00 hrs on 15 March 2003.

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ANNEX 1: LIST OF PARTICIPANTS

Name Address Telephone Fax E-mail

Maria Lyons Alcantara Fish Health Unit Marine Institute Snugboro Road Abbotstown Dublin 15 Ireland

+353 1 8228280 +353 8228303 [email protected]

Juan Barja University of Santiago Departamento de Microbiologia Instituto de Acuicultura Fac. Biologia 15782 Santiago de CompostelaSpain

+34 981 56 31 00 +34 981 56 69 04 [email protected]

David Bruno FRS Marine Laboratory PO Box 101 375 Victoria Road Aberdeen AB11 9DB Scotland

+44 1224 295615

+44 1224 295667 [email protected]

Vladimir Donetskov Knipovich Polar Research Institute of Marine Fisheries and Oceanography 6, Knipovich Street 183763 Murmansk Russia

+7 8152 473362 +4778910-518 [email protected]

Steve Feist CEFAS Weymouth LaboratoryThe Nothe Weymouth Dorset DT4 8UB United Kingdom

+44 1305 206600 +44 1305 206601 [email protected]

Susan E. Ford Rutgers University Haskin Shellfish Research Laboratory 6959 Miller Avenue Port Norris, NJ 08349 USA

+1 856 785 0074 ext. 105

+1 856 785 1544 [email protected]

Thomas Lang (Chair) Bundesforschungsanstalt für Fischerei Institut für Fischereiökologie Deichstrasse 12 27472 Cuxhaven Germany

+49 4721 38034 +49 4721 53583 [email protected]

Sharon Maclean NOAA/NMFS Narragansett Laboratory 28 Tarzwell Drive Narragansett, RI 02882-1199 USA

+1 401 782 3258 +1 401 782 3201 [email protected]

Lone Madsen Danish Institute for Fisheries Research Fish Disease Laboratory Stigbøjlen 4 DK-1870 Frederiksberg Denmark

+45 35282763 +45 35282711 [email protected]

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Tristan Renault IFREMER Laboratoire de Génétique, Aquaculture et Patologie La Tremblade 17390 France

+33 5 4636 9841 +33 5 4636 3751 [email protected]

Martine Vigneulle AFSSA Brest BP 70 29280 Plouzané France

+33 2 98 22 44 61 +33 2 98 05 51 65 [email protected]

Tom Wiklund Laboratory of Aquatic Pathobiology Department of Biology BioCity, Artillerig. 6 Åbo Akademi University 20520 Åbo Finland

+358 2 2154301

+358 400 758957

+358 2 2154748 [email protected]

Werner Wosniok

University of Bremen Institute of Statistics P.O.Box 330 440 D-28334 Bremen Germany

+49 42 12 18 34 71 +49 42 12 18 40 20 [email protected]

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ANNEX 2: TERMS OF REFERENCE

2F03 The Working Group on Pathology and Diseases of Marine Organisms [WGPDMO] (Chair: T. Lang, Germany) will meet in Aberdeen, UK from 11–15 March 2003 to:

a) analyse national reports on new disease trends in wild and cultured fish, molluscs and crustaceans;

b) report on progress in the ongoing investigations of the effect of temperature on Bonamia infection dynamics and report on the confirmation of the agent of Crassostrea angulata gill disease and its infectivity to Crassostrea gigas and other oyster species;

c) review the existing strategies to assess the prevalence of shellfish diseases in parallel to fish diseases and chemical contaminant levels in environmental monitoring programmes;

d) review and assess a report prepared intersessionally on investigations into the molecular comparisons among the various species/isolates of Perkinsus in collaboration with the OIE reference laboratory for Perkinsus at the Virginia Institute of Marine Science (E. Burreson);

e) obtain information on the EU project “Diagnosis of oyster herpes-like virus: development and validation of molecular, immunological and cellular tools” (FAIR-PL98-4334) and review the results;

f) review and report on progress made in the “fish diseases and liver histopathology” component of the BEQUALM self-funding scheme;

g) review and assess the impact of diseases of farmed fish on wild fish stocks;

h) maintain an overview of the spread of Ichthyophonus in herring stocks and the distribution and possible cause(s) of the M74 syndrome;

i) assess and report on the effectiveness of salmon farming management control methods for sea lice in the different ICES Member Countries;

j) review progress made in the modifications to the ICES Databases and review and approve the revised ICES Environmental Data Reporting Format (Version 3.2);

k) review the criteria for the incorporation of externally visible fish diseases into monitoring programmes on biological effects of contaminants;

l) review progress made with regard to the update of ICES publications on pathology and diseases of marine organisms:

i) web-based report on diseases and parasites of wild and farmed marine fish and shellfish as part of the ICES Environmental Status Report,

ii) manuscript on methods for the statistical analysis of fish disease data for submission to the ICES TIMES series,

iii) ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish;

WGPDMO will report by 31 March 2003 for the attention of the Mariculture and Diadromous Fish

Committees and ACME. Supporting Information

Priority: WGPDMO is of fundamental importance to the ICES science and advisory process.

Scientific Justification: a) New disease conditions and trends in diseases of wild and cultured marine organisms continue to appear and an assessment of these should be maintained.

b) Experimental work is required to confirm field observations and the hypothesis of Bonamia suppression vs. destruction over long periods of low temperatures. This question is important for accurately assessing climate effects on Bonamiasis and European oyster culture. There are historic records of an iridoviral infection of Crassostrea gigas gills, associated with low/transient pathology. This suggests that the gill disease agent may have multi-host infection potential, which needs to be addressed for like-to-like C. gigas and Ostrea edulis transfers.

c) In recent years, an increasing effort in ICES Member Countries has been given to studies on shellfish diseases and pathology in relation to environmental contaminants. WGPDMO considered it timely to obtain an overview of existing

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strategies within this field. d) Confusion occurs in the taxonomy of Perkinsus spp., which cause significant

disease and mortalities in molluscs in many locations around the world. New species are being named based on differences in molecular sequences. The current status of species identification should be reviewed to document acceptable criteria for species identification.

e) Herpesvirus infections have been reported in different bivalve species around the world. Specific diagnostic tools are needed in order to assess the causative role of these viruses in shellfish mortalities. Within the ongoing EU-funded project VINO, specific tools for diagnosing these viral infections have been developed and validated. WGPDMO will assess the most recent knowledge within this field.

f) Within the EU-funded project BEQUALM, a quality assurance programme for fish diseases and liver pathology has been developed. In future, it is intended to be a self-funding scheme, which will form an essential part of wild fish disease monitoring programmes and be important for implementing data on liver pathology in the ICES Fish Disease Database.

g) The impact of the increasing development of aquaculture on diseases in wild fish populations is an issue of concern. WGPDMO considers it important to be updated on the most recent knowledge within this field.

h) ICES C.Res. 1993/2:23(m) requested that WGPDMO maintain an overview of the M74 syndrome and the Ichthyophonus issue as part of its regular agenda.

i) In the process of assessing the effectiveness of salmon farming management control methods for the control of sea lice, WGPDMO will assess the most recent knowledge within this field, which will be published in a report submitted by the Norwegian National Committee for sea lice.

j) WGPDMO considers it necessary to follow the process of modification of the ICES Databank structure in order to assist, if required, and to obtain an overview of changes suggested or introduced by other ICES Working Groups. In addition, a revised version of the ICES Environmental Data Reporting Format will need to be reviewed by WGPDMO and approved, with amendment if necessary, before its use by the ICES Marine Data Centre.

k) WGBEC is revising the list of techniques recommended for biological effects monitoring and has been requested to take into account input from WGPDMO.

l) A number of ICES publications, either web-based or in ICES publication series, are being prepared or updated at present, the progress of which has to be reviewed by WGPDMO at its next meeting. It will be necessary to consider ways by which these can be linked to each other.

Relation to Strategic Plan: Responds to Objectives

Resource Requirements: None required, other than those provided by the host institute.

Participants: Representatives of all Member Countries with expertise relevant to pathology and disease of wild and cultured finfish and shellfish.

Secretariat Facilities: None required

Financial: None required

Linkages to Advisory Committees:

ACME

Linkages to other Committees or Groups:

MARC

Linkages to other Organisations:

BEQUALM, OIE, EU

Cost share ICES:100 %

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ANNEX 3: AGENDA

1. Opening of the meeting

2. Terms of reference, adoption of agenda, selection of rapporteurs

3. ICES Annual Science Conference 2002, items of relevance to WGPDMO

4. Other relevant reports/activities for information

5. Analyse national reports on new disease trends in wild and cultured fish, molluscs and crustaceans

6. Report on progress in the ongoing investigations of the effect of temperature on Bonamia infection dynamics and report on the confirmation of the agent of Crassostrea angulata gill disease and its infectivity to Crassostrea gigas and other oyster species

7. Review the existing strategies to assess the prevalence of shellfish diseases in parallel to fish diseases and chemical contaminant levels in environmental monitoring programmes

8. Review and assess a report prepared intersessionally on investigations into the molecular comparisons among the various species/isolates of Perkinsus in collaboration with the OIE reference laboratory for Perkinsus at the Virginia Institute of Marine Science (E. Burreson)

9. Obtain information on the EU project “Diagnosis of oyster herpes-like virus: development and validation of molecular, immunological and cellular tools” (FAIR-PL98-4334) and review the results

10. Review and report on progress made in the “fish diseases and liver histopathology” component of the BEQUALM self-funding scheme

11. Review and assess the impact of diseases of farmed fish on wild fish stocks

12. Maintain an overview of the spread of Ichthyophonus in herring stocks and the distribution and possible cause(s) of the M74 Syndrome

13. Assess and report on the effectiveness of salmon farming management control methods for sea lice in the different ICES Member Countries

14. Review progress made in the modifications to the ICES Databases and review and approve the revised ICES Environmental Data Reporting Format (Version 3.2)

15. Review the criteria for the incorporation of externally visible fish diseases into monitoring programmes on biological effects of contaminants – and provide feedback to WGBEC

16. Review progress made with regard to the update of ICES publications on pathology and diseases of marine organisms:

a. web-based report on diseases and parasites of wild and farmed marine fish and shellfish as part of the ICES Environmental Status Report

b. manuscript on methods for the statistical analysis of fish disease data for submission to the ICES TIMES series

c. ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish

d. report on important trends in diseases occurring in finfish and shellfish culture in the ICES area in the period 1997–2001

17. Any other business

18. Analysis of progress with tasks

19. Future activities of WGPDMO

20. Approval of recommendations

21. Approval of draft WGPDMO Report

22. Closing of meeting

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ANNEX 4: RAPPORTEURS

Agenda

Item(s)

Session(s) Rapporteurs

1–4 Introductory sessions T. Lang

5 ToR a): Analyse national reports on new disease trends in wild and cultured fish, molluscs and crustaceans

Wild fish

Farmed fish

Wild and farmed shellfish and crustaceans

S. Feist/S. MacLean/V. Donetskov

D. Bruno/M. Vigneulle

M. Lyons Alcantara/S. Ford/ L. Madsen/ T. Renault

6 ToR b): Report on progress in the ongoing investigations of the effect of temperature on Bonamia infection dynamics and report on the confirmation of the agent of Crassostrea angulata gill disease and its infectivity to Crassostrea gigas and other oysters species

S. Ford/T. Renault

7 ToR c): Review the existing strategies to assess the prevalence of shellfish diseases in parallel to fish diseases and chemical contaminant levels in environmental monitoring programmes

S. Ford/S. Feist

8 ToR d): Review and assess a report prepared intersessionally on investigations into the molecular comparisons among the various species/isolates of Perkinsus in collaboration with the OIE reference laboratory for Perkinsus at the Virginia Institute of Marine Science

S. Ford/M. Lyons Alcantara

9 ToR e) Obtain information on the EU project “Diagnosis of oyster herpes-like virus: development and validation of molecular, immunological and cellular tools” (FAIR-PL98-4334) and review the results

L. Madsen/T. Renault

10 ToR f) Review and report on progress made in the “fish diseases and liver histopathology” component of the BEQUALM self-funding scheme

S. MacLean/S. Feist

11 ToR g): Review and assess the impact of diseases of farmed fish on wild fish stocks

M. Vigneulle/D. Bruno

12 ToR h): Maintain an overview of the spread of Ichthyophonus in herring stocks and the distribution and possible cause(s) of the M74 Syndrome

T. Wiklund/D. Bruno

13 ToR i): Assess and report on the effectiveness of salmon farming management control methods for sea lice in the different ICES Member Countries

T. Wiklund/D. Bruno

14 ToR j): Review progress made in the modifications to the ICES Databases and review and approve the revised ICES Environmental Data Reporting Format (Version 3.2)

S. Feist/W. Wosniok

15 ToR k): Review the criteria for the incorporation of externally visible fish diseases into monitoring programmes on biological effects of contaminants – and provide feedback to WGBEC

S. MacLean/S. Feist/V. Donetskov

16 ToR l): Review progress made with regard to the update of ICES publications on pathology and diseases of marine organisms

a) web-based report on diseases and parasites of wild and farmed marine fish and shellfish as part of the ICES Environmental Status Report

b) manuscript on methods for the statistical analysis of fish disease data for submission to the ICES TIMES series

c) ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish

d) Report on important trends in diseases occurring in finfish and shellfish culture in the ICES area in the period 1997–2001

S.MacLean/W. Wosniok

T. Wiklund./W. Wosniok

S. Ford/L. Madsen

S. Ford/ D. Bruno

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17–19 Any other business, Analysis of progress with tasks, Future activities of WGPDMO, Recommendations

D. Bruno/S. Feist/T. Wiklund

20–22 Approval of draft report, Closing of the meeting T. Lang

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ANNEX 5: SUMMARY OF STATUS OF ISA IN MAINE, US, IN 2002

S. MacLean, NOAA/NMFS, Narragansett Laboratory, Narragansett, USA

(See Annex 5 of the previous year’s WGPDMO Report (ICES CM 2002/F:02) for background information on the ISA outbreak in the US and the management actions taken to control the disease)

Following the outbreak of ISA in the Maine Atlantic salmon farming industry in 2001, USDA implemented an ISA program with the state of Maine to eradicate the disease. This included eradication of 1.5 million salmon in Cobscook Bay that were infected with or exposed to ISAV, disinfection of gear, boats and equipment, a 100-day fallowing period, and designation of two zones for restocking of single year class fishes. The State of Maine mandated monthly surveillance for ISA at all Atlantic salmon farms in Maine coastal waters. Surveillance at farms outside Cobscook Bay began in January 2002; surveillance began in Cobscook Bay in May after fallowing and re-introduction of fish in April.

1.8 million fish were re-stocked at 6 of 14 sites into one zone of Cobscook Bay in April after 100 days of fallowing, during which time nets, barges, boats, equipment and gear were disinfected by steam heat sterilisation. All fish had been vaccinated for ISAV and all sites are involved in an INAD for integrated sea lice management. Surveillance at all sites was conducted monthly (and continues) by onsite veterinarians and consisted of 10 moribunds or morts that were tested for ISA using IFAT and RT-PCR. Suspect or unconfirmed positive results raise the level of surveillance to two times per month. At present there are no confirmed positives but 13 suspect IFAT results and one unconfirmed RT-PCR have been reported.

Assays conducted on nearly 3000 wild non-salmonid fishes have not detected ISAV-positive fishes by RT-PCR or viral isolation. Fishes tested include alewife (Alosa pseudoharengus), American eel (Anguilla rostrata), Atlantic herring (Clupea harengus harengus), Atlantic mackerel (Scomber scombrus), cod (Gadus morhua), pollack (Pollachius virens), and winter flounder (Pseudopleuronectes americanus). As reported last year, positive assays resulted only from non-salmonids entrapped in ISA-diseased salmon cages. Screening of 377 Atlantic salmon broodstock taken from the Penobscot River, Maine did not detect ISAV by RT-PCR or viral isolation.

Through cooperation with the industry, environmental studies are underway to monitor water, nets, surfaces of cages and boats at previously infected but fallowed sites in Maine and infected sites in New Brunswick to better understand the role of the aquaculture system in the epidemiology of ISAV. Samples are taken from harvest boat hulls, pontoons adjacent to infected and uninfected cages, nets from infected and uninfected cages, invertebrates (mussels and sealice) associated with infected and uninfected cages, and from water at the culture sites.

Susceptibility of Pacific salmonids to ISAV has been experimentally assessed using both the European and North American strains of the virus. In controlled experiments, steelhead trout (Oncorhynchus mykiss), and chum (O. keta), chinook (O. tsawytscha), coho (O. kisutch) and Atlantic salmon (Salmo salar) were injected intraperitoneally with high, moderate or low doses of the Norwegian or North American strain of ISAV. No signs of disease or mortality due to ISAV infection occurred in the Oncorhynchus species. At 13 days post-inoculation, virus was isolated from steelhead, chum, coho, and chinook, and was not recovered from these species 28 days post-inoculation. Titers in chum salmon were 104 and 105, and 103 or less in the other oncorhynchids. These results indicate Oncorhynchus species are quite resistant to ISAV but the potential for ISAV to adapt to these species should not be ignored.

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ANNEX 6: REVIEW OF PATHOLOGIES IN DEMERSAL FISHES OF THE BARENTS SEA AND ADJACENT WATERS

By T. A. Karaseva and V. V. Donetskov Polar Research Institute of Marine Fisheries and Oceanography (PINRO), Russia, Murmansk

INTRODUCTION

The Barents Sea and areas adjoining to it from the north and west are zones of the active international fisheries. Fishery for demersal fishes in those areas is carried out by Russia, Norway, Great Britain and many other states. At present, the Barents Sea is studied intensively. However, among the abundant data on biology and ecology of marine organisms there is no information on pathology and diseases of fishes.

In connection with this, in 1999 the Polar Research Institute of Marine Fisheries and Oceanography (PINRO) began the investigation of epizootic situation in the Barents Sea and formation of a database on pathologies of marine demersal fishes. Their main purpose is the development of an actual database necessary for working out of standard biological tests of estimation of health of commercial fishes population and quality of their environment.

Results of diagnostic and statistical processing of materials on pathology of cod, haddock, plaice, halibut, wolffish obtained in 1999–2002 are presented.

MATERIALS AND METHODS

Investigations were carried out in 1999–2002 in the Barents Sea (Div. I), the Greenland Sea (Div. IIb) and in the eastern part of the Norwegian Sea (Div. II a) (Figure A6.1). Sampling of material was performed during the biological analysis of fish by scientists and observers of PINRO on board RV “Nansen” and fishing vessels equipped with bottom trawls or longlines. All specialists participating in sampling of fish pathology material passed special training at PINRO. During sampling all fish species found in catches were analysed. For the given paper, we have selected and analysed materials on 89,516 individuals of fish of 8 species: Pleuronectes platessa, Hippoglossoides platessoides limandoides, Reinhardtius hippoglossoides, Gadus morhua, Melanogrammus aeglefinus and 3 species of Anarhichas. As for fish with pathology, their biological characteristics were recorded, external pathology and morphological changes in the internal organs were described and fish tissues and organs were fixed for the further hystopathological examination. Laws of the alternative distribution were used for the statistical processing and a comparative analysis of data. This distribution takes into account the presence or absence of a sign, as well as discreteness of distribution of fish with pathology. Discrepancies mentioned in the paper are statistically reliable at the confidence level not lower than 95 %. Results and Discussion Brief characteristics of types of fish pathology. Pathologies of four main types have been found in the analysed fish: ulcers, skeletal deformations, tumors and necroses. We considered ulcers as skin defects caused by inflammations in the organism. Three main variants can be marked among the revealed ulcers that were observed most frequently:

1. The surface ulcers that do not touch stratum spongiosum, ulcers in flounders. 2. Deep ulcers, hystologically presented tissue complexes, in wolffishes.

3. Specific ulcers (ulcerous syndrome) in cod.

Other ulcers were found in fish periodically; therefore they cannot be systematised. The skeletal deformations are as follows: S-shaped and oriented in the horizontal or vertical planes curvature of the spine, shift of vertebrae, anomalous development of cranium without disturbance of skin integument and skeletal

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muscles. In the sampled material, annually not less than 80 % of deformations consisted of various forms of spinal curvatures in cod, haddock and long rough dab. In these fish species, defects of spine development were observed with equal frequency not exceeding 0.2 %. Deformations of the cranium were peculiar for cod; but sometimes they were found in flounders and halibut. At the pathology in cod called conditionally “the head of a dolphin” the exhaustion of fish was recorded almost always. In flounders the curvatures or underdevelopment of jaws were observed. We registered pathologies characterised by excessive and morphologically atypical growth of various tissues as tumors. The most distributed tumors were epithelial and of connective tissue. At present, some of them are still unidentified, for example, tumors of violet colour localised on head of cod and haddock. Necrosis was registered in cases when putrificating, degenerating or dead tissues were observed in fishes alive. We have observed necrosis of skin and fins, erosion and breakdown of jaws, as well as a complex of pathologies stipulated by necrobiosis of eyes. The most often observed variety of necrosis was putrification of the caudal fin with gradual breakdown of interradial tissue, skin, muscles and osseous elements of the caudal stem. The necrobiosis of eyes in wolffishes and cods were of one type and affected mainly only one eye. One-sided exophthalmoses, running out of vitreous body through a hole, cataract, leukoma, deformation or prolapse of eye were recorded in diseased fish. As Figure A6.2 shows, in 1999 the ulcers were predominant among recorded pathologies, accounting for 66 %, on average. In 2000–2001, they were not found so often. In 2002 the portion of ulcers decreased to 6 % (Figure A6.3). As for the frequency of occurrence, the skeletal deformations are attributed to a group of relatively stable and constantly present pathologies of fish. In 1999–2002, they accounted for 7–23 % of pathologies. In 1999–2001 tumors were 11–19 % of all pathologies, mainly occurring in wolffishes, cod, haddock and long rough dab. In 2002, the portion of tumors decreased to 3 % of all pathologies Increase of a portion of necroses from 4 % in 1999 to 80 % in 2002, including necrosis of eyes, was recorded. Wolffishes and cod with diseased eyes were found for the first time in 2000. Maximal number of eye pathologies was registered in 2001 when their portion among others constituted 13 %. In 2002 a portion of eye necrobiosis decreased to the level registered in 2000, i.e., 5 %. Frequency of occurrence of pathologies in fish of different species Data reflecting the frequency of occurrence of demersal fish with pathologies by fish species, areas and years of investigations are in Table A6.1. As it is seen from these materials, pathologies in wolffishes were observed most often. Compared to the other fish species, a level of morbidity of wolffishes in 1999–2002 was constantly high – 2.8–8.2 %. A range of their pathologies was not big and a very specific. It is numerous epithelium tumors (papillomas) and ulcers. These ulcers were probably dependent on tumors as well and arose as the result of papillomas rejection. In 2001, necrosis of skin and fins and diseases of eyes (mainly two-sided cataract and deformation of eyes) have appeared in wolffishes. During recent two years the frequency of occurrence of necroses in wolffishes constituted 5.3–5.5 %. Of the flounders sampled about 1 % of individuals, on the average, have various pathologies. Range of pathologies in flounders was much greater than in halibut. All main pathologies were recorded in them but ulcers absolutely dominated. In 1999–2000, pathologies of halibut were very rare. However, since 2001 individuals of this species have been found with erosion of caudal fin, caudal stem and jaws. A tendency to the increase of occurrence of halibut with necrosis continued in 2002, since the frequency of occurrence of necroses increased 2.5 times compared to 2001 and constituted 1.9 %. The next group of fish consisted of cod and haddock. In 1999–2001, the frequency of occurrence of pathologies in these fishes was less than 1 %. The mostly often-observed pathology of cod was specific ulcers associated with the ulcerous syndrome. The disease was predominantly registered at the stage of non-healing ulcers. Most of cod with ulcers were found in 1999. Frequency of occurrence of diseased fish constituted 0.9 %. In the next years, their number decreased almost 5 times. As for haddock, ulcers were observed in some individuals only. The number of fish with ulcers was 57 times less than, for example, in wolffishes.

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Necrobiosis has been registered in cod since the year of 2000. Changes in cod eyes were almost identical to those pathologies, which are described above for wolffishes. Differences are as follows: in cod the eye changes were observed together with a tumor on the head or with a strong inflammation of head tissues. In 2000, tumors were recorded in cod and haddock more than other years. The frequency of their occurrence in surveyed areas constituted 0.16 %, on the average. They predominantly were tumors of connective tissue with different stages of maturity and development in skin, muscles, gonads and in head tissues. Distribution of fish with pathologies in the surveyed areas Statistical analysis of our data, carried out for 95 % confidence level, has shown that in 1999–2000 in Divs. I and II b, the frequency of occurrence of fish with pathologies was similar. However, since 2001 Div. IIb has stood apart. During 2000–2002, the least number of fish with pathologies – 0.5 % - were registered there, whereas in Div. I in 2001 the frequency of occurrence of fish with pathologies constituted 0.9% and in 2002 – 2.2 %. In 2002, differences in the number of fish with pathologies between Divs. II a and II b were revealed for the first time – 2.3 % and 0.5 %, correspondingly. One can assume that this difference existed before. Apparently, these differences were not discovered earlier just because of the lack of data for Div. II a. The most completed data were obtained for the ICES Div. I (the Barents Sea). Result of analysis showed an increasing tendency in the number of fish with pathologies in this area. The frequency of pathology occurrence varied from 0.5 % in 1999 to 2.2 % in 2002. Conclusions Thus, ulcers, tumors, necroses and skeletal deformations were revealed in wolffishes, flounders and cods dwelling in the Barents Sea. Morphological diversity within these groups of pathologies let us assume that their appearance could be connected with the influence of various endogenous and exogenous factors of the biological, physical or chemical origin. We were fortunate to make during a short period of observations a fairly complete view of the situation in populations of commercial fishes of the Barents Sea and adjacent areas. However, at present it is impossible to state the significance of the results. The reason is that there are no standards regulating the occurrence of fish with these pathologies in the natural populations of fish. In spite of the fact that the basis of our investigations on pathologies was the methodology of PINRO the experience has shown that it is necessary to adapt the method to the peculiar conditions in the areas of investigations considering species composition of ichthyofauna, specific features of distribution and fishery for fish. A reliable estimation of increases in pathology conditions and, correspondingly, judgements of quality of fish environment by means of visual observation of “external” pathologies without determination of aetiology for at least dominating pathologies seems to be problematic. However, the results we obtained have shown that monitoring of pathologies in demersal fishes is undoubtedly effective, and in a short time using simple methods, the main changes in epizootic situation over the vast area can be followed. It is necessary to continue and expand these investigations in order to increase the time series of observations.

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Figure A6.1. Sites of sampling on pathologies of fish of different species in 1999–2002.

10° 15° 20° 25° 30° 35° 40° 45° 50°68°

69°

70°

71°

72°

73°

74°

75°

76°

77°

78°

79°

80°

- Pleuronectes platessa- Hippoglossoides platessoides limandoides- Reinhardtius hippoglossoides- Gadus morhua

- Melanogrammus aeglefinus- Anarhichas sp.

I

II b

II a

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Figure A6.2. Qualitative and quantitative composition of pathologies in demersal fish in ICES Divisions I and II b in 1999–2000.

1999

66%4%4%

7%

19%UlcersNecrosis of fins, skin and jawsPathologies of liverSkeletal deformationsTumors

2000

36%

10%3%15%

22%

1%13% Ulcers

Necrosis of fins, skin and jawsNecrosis of eyesPathologies of liverSkeletal deformationsCranium deformationTumors

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Figure A6.3. Qualitative and quantitative composition of pathologies in demersal fish in ICES Divisions I, II b, II a in 2001–2002.

2001

30%

23%13%

22%

1% 11%UlcersNecrosis of fins, skin and jawsNecrosis of eyesSkeletal deformationsCranium deformationTumors

2002

6%

75%

5%9% 2% 3%

UlcersNecrosis of fins, skin and jawsNecrosis of eyesSkeletal deformationsCranium deformationTumors

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Table A6.1. Frequency of occurrence of demersal fish with pathologies in ICES Divs. I, II a and II b in 1999–2002.

Fish with pathologies, %

Pleuro-nectes

platessa

Hippoglossoides

platessoides

limandoides

Reinhardtius

hippoglossoides

Gadus morhua Melanogram-mus aeglefinus

Anarhichas sp.

Average

ICES Divis-

ion

No. of analy-

zed fish

n=2809 n=4766 n=8092 n=62916 n=7644 n=3289 n=89516

1999 г.

I 4983 0.36+0,14 0.45+0.17 - 0.87+0,43 0.53+0.24 2.78+2.74 0.48+0.10

II b 319 - * 0.94+0.54 - - - - 0.94+0.54

2000 г.

I 9656 - 1.22+0.49 - 0.40+0,08 0.39+0.14 5.75+2.50 0.49+0.07

II b 15531 - 1.29+0.45 5.36+3.01 0.46+0,06 0.22+0.11 5.49+2.39 0.51+0.06

2001 г.

I 2016 0.37+0.21 1.00+0.99 - 1.85+0.92 1.13+0.36 - 0.89+0.21

II b 12102 - - 0.76+0.25 0.36+0.06 0.50+0.50 8.22+3.21 0.45+0.06

II a 820 - - - 0.73+0.30 - - 0.73+0.30

2002 г.

I 24734 3.23+3.18 1.44+0.34 2.25+0.22 1.44+0.10 4.01+0.52 5.19+0.41 2.17+0.09

II b 17953 - 0.72+0.41 1.45+0.36 0.41+0.05 0.29+0.29 8.33+3.57 0.51+0.05

II a 1402 - - 2.23+0.40 - - 8.33+7.98 2.28+0.40

Average

89516

0.39+0.12 0.97+0.14 1.94+0.15 0.66+0.03 1.11+0.12 5.32+0.39 1.00+0.03

* - Data are absent

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ANNEX 7: REPORT ON ISA IN IRELAND

F. Geoghegan, The Marine Institute, Fisheries Research Centre, Dublin, Ireland In July 2002, ISAV was isolated from rainbow trout on two isolated marine sites in the west of Ireland. Both sites were owned by the same company and shared personnel, equipment and a common land base. The virus was isolated as a result of an Industry approved National Screening Programme, which has been running since 2000 and was combined with annual inspections under Directive 91/67/EEC. Clinical disease was not observed at any stage on either site. By virtue of the fact that ISA is a List 1 disease, Council Directive 93/53/EEC requires that all fish must be withdrawn immediately from a site on which ISA is found. However, Commission Decision 2001/27/EC, which was published as a result of the experiences gained during the ISA outbreaks in Scotland in 1998 and 1999, allows for the submission to the EU Commission of a “Withdrawal Plan” which provides for the implementation of a more risk based regime in the event of an outbreak. Ireland’s Withdrawal Plan was approved by the EU Commission in December 2001 and was used as the protocol by which both cases were dealt with. However, although the Withdrawal Plan allows for the adoption of a more flexible withdrawal of fish over time, the strict definitions outlined in the Plan also state that isolation of ISAV, even in the absence of clinical disease, must result in the confirmation of the site as ISA positive. Following a Risk Assessment, fish may be on-grown to market size but the site must then be fallowed for a six-month period. One of the infected sites was in the process of harvesting at the time the virus was isolated. The harvesting schedule was accelerated and full bio-security measures were put in place both at the site itself and at the company’s processing plant. Once empty, the site was fully cleaned and disinfected under official supervision. The site is currently undergoing a six- month fallowing period prior to restocking. The second site had smaller (500 g) fish which were of no commercial value at the time the virus was isolated. A Risk Assessment Group was convened to determine what degree of on-growing might be considered under the circumstances. For the reasons outlined in the First Risk Assessment Report, which included the very isolated geographical location of the infected site and the fact that the fish continued to remain clinically healthy, it was decided that provided mortality levels did not exceed 0.05 % per day attributable to ISA, then the fish should be allowed to remain on site at least until the end of March 2003. This date was chosen because it would allow all fish to be removed from the bay prior to the spring/summer wild salmonid runs and would thus minimise the chance of the virus being carried to other aquaculture facilities in the area by wild fish. However, hard information as to the exact prevalence of wild fish in the bay was unavailable to the Group at the time when the first report was written. Industry responded to the first Risk Assessment with a request to on-grow the fish past the end of March and onto the end of June. A Second Risk Assessment Report was then prepared which concluded that the fish could only be on-grown past the end of March, provided the level of risk which was previously attributed to leaving them on site did not increase. This assessment could only be made if solid information could be gathered with respect to the number of wild salmonids (particularly sea trout) present in the bay. It has now been decided to carry out netting experiments during the months March, April and May. The information gathered during these experiments will be fed into a population model which exists for an adjacent bay, with the aim of determining whether in fact wild salmonids are present in the bay in biologically significant numbers. It has also been decided that the Risk Assessment Group will meet monthly from March to June to assess results both from the wild fish netting experiments and the results of ongoing virological testing of the rainbow trout. These fish continue to be tested virologically and to date; the virus has not been isolated since summer 2002. The conclusions of the Risk Assessment are that the fish may remain on-site until the end of March. After that date, a combination of biologically significant numbers of wild fish in the bay, plus the shedding of ISAV by the rainbow trout, will trigger the implementation of an Emergency Harvesting Plan which will empty the site in the shortest possible time frame. If the numbers of wild fish are found to be small, the fish may remain on-site until the end of June, 2003. Once empty of fish, the site will be cleaned, disinfected and fallowed for 6 months. As required by Article 4(h) of Council Directive 93/53/EEC, an Epidemiological Study to determine the possible origin of the virus and the length of time it may have been on site prior to detection, is currently underway. Results will be reported to the EU Commission as they become available. All other farms in the country have been tested for ISA (using virus isolation), and found to be negative.

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ANNEX 8: MSX AND SSO IN CANADA

ort

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ANNEX 9: REVIEW THE EXISTING STRATEGIES TO ASSESS THE PREVALENCE OF SHELLFISH DISEASES IN PARALLEL TO FISH DISEASES AND CHEMICAL CONTAMINANT LEVELS IN

ENVIRONMENTAL MONITORING PROGRAMMES

C. Couillard (Institute Maurice-Lamontagne, Mont-Joli, Canada), S. Feist (CEFAS, Weymouth, UK),

T. Renault (IFREMER, La Tremblade, France) Introduction Diseases in fish and shellfish may be used as indicators of marine pollution. The ICES WGPDMO has developed standardised methodologies for monitoring marine fish external diseases and flatfish liver pathology. These methods are used by the ICES Member Countries to investigate the relationship between changes in the marine ecosystem and the prevalence of fish diseases. In parallel, standardised methodologies have been developed to evaluate the prevalence of imposex/intersex in marine gastropods as an indicator of TBT contamination. Currently, within the ICES monitoring programmes, surveys of fish and shellfish diseases are not integrated: they are conducted at different times and sites, and have different objectives. The objective of this short paper is to evaluate the potential usefulness of integrating the monitoring of fish and shellfish diseases to evaluate marine ecosystem health. The advantages and disadvantages of using fish or shellfish for monitoring the effects of contaminants on the health of marine organisms Several criteria are used to select sentinel species used in marine monitoring programmes. They should be abundant and widespread, easy to sample, non-migratory and preferably not very mobile, sediment-dwelling. They should have a high potential for exposure and a measurable response to contaminants. Species with a higher fat content, a longer lifespan, located at a high trophic level are more likely to accumulate persistent pollutants. Filter feeders, such as bivalves, may also accumulate high concentrations of contaminants in their tissues. Several fish and shellfish species fulfil these criteria. Generally, no single species is an ideal sentinel for all contaminant groups and vulnerability varies markedly among groups of contaminants. Fish have stronger metabolic capacities (e.g., cytochrome P4501A1) than shellfish. Thus, compared to shellfish, they may be more sensitive to contaminants that are bioactivated by the cytochrome P450 system (such as carcinogenic PAHs) but may be less sensitive to toxicants that are detoxified by cytochrome P450. Toxicology, pathology, physiology, and endocrinology have been more studied in fish than in shellfish. Cause-effect relationships between exposure to contaminants and histological changes have been more extensively documented in fish than in shellfish. In the 1970s, most environmental programmes focused on persistent organic pollutants. Top-predator fish were useful sentinel species for these studies. In the last ten years, the focus has been on new classes of contaminants, including endocrine disruptive compounds (EDCs), pesticides, pharmaceutical compounds, many of which are not accumulating in fish tissues and are more concentrated in the coastal areas. Because they are generally sedentary, shellfish species may be particularly useful to localise point sources of contamination and to evaluate their effects. Cage studies may be easier with shellfish (particularly with bivalves) than with fish and are very useful for conducting experiments in the field to demonstrate cause-effect relationships. Invertebrates, particularly the psodobranch snails, are sensitive to endocrine disruption at environmentally relevant concentrations. In freshwater and marine (Nucella lapillus, Nassarius reticulatus) psodobranch species exposed in the laboratory, xeno-estrogens (e.g., bisphenol A, octylphenol) caused induction of superfemales and reduction in the size of the male sex organs. Xeno-androgens (triphenyltin, tributyltin) caused virilization of females (imposex). Anti-androgens (cyproterone, acetate, vinclozolin) caused reduced male sexual organs and suppression of imposex development (Oehlman et al., 2000; Schulte-Oehlman et al., 2000; Tillmann et al., 2001). The relative simplicity of invertebrate immune functions offers a good model to study the complex interactions between exposure to environmental contaminants and immune dysfunction (Galloway and Depledge, 2001). In 2001, IFREMER (Institut Français de Recherche pour l’Exploitation de la Mer) funded a multi-year study called MOREST (2001–2005) to investigate summer mortality in Pacific oysters, Crassostrea gigas, that may be caused by multiple factors including elevated temperatures, the physiological stress associated with maturation, aquaculture practices, pathogens and pollutants. Recent studies have shown that oyster defenses may be influenced by pollutants (Fournier et al., 2002; Sauvé et al., 2002). However possible relations between pollutants, the physiological state of oysters, their immune system and pathogens need further investigations. In France, B. Gagnaire, a doctoral fellow working with T. Renault (IFREMER, Laboratoire de Génétique et Pathologie, La Tremblade), investigates hemocyte responses (ratio between granulocytes and hyalinocytes, phagocytosis, percentages of cells possessing hydrolytic enzymes and reactive oxygen

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species production) in vitro, in vivo and in situ in the presence of pollutants (heavy metals, pesticides, herbicides such as atrazine) (Gagnaire et al., 2003). Several fish and shellfish diseases, with the potential to cause deleterious impacts on fish and shellfish populations, have been associated with exposure to environmental contaminants. Most of these conditions have a multifactorial aetiology and may be triggered by a variety of natural and anthropogenic factors. Imposex in gastropods is used successfully as a biomarker of exposure to tributyltin in the marine environment in several ICES countries. Haemic and gonadal neoplasia in bivalves have been associated with environmental contamination by herbicides but a cause-effect relationship has not yet been fully demonstrated. Hepatic pre-neoplastic and neoplastic lesions in fish are used successfully as a biomarker of exposure to environmental carcinogens, particularly PAHs. Prevalence of intersex in fish is used as an indicator of contamination of the marine environment by oestrogenic compounds. Thus, histopathological lesions in fish and shellfish are presently used to detect different groups of contaminants and are complementary. A review of the information published in the literature: cases studies where the prevalence of diseases was assessed in both fish and shellfish species as an indicator of environmental contamination. While numerous studies are published on the association between fish diseases or shellfish diseases and environmental contamination, very few studies have looked simultaneously at the prevalence of fish and shellfish diseases at contaminated sites. Carcinogens The eastern oyster (Crassostrea virginica) developed neoplastic disorders when experimentally exposed both in the laboratory and field to chemically contaminated sediment from Black Rock Harbor (BRH), Bridgeport, Connecticut, US. High concentrations of polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), and chlorinated pesticides were measured in BRH sediment and in oysters exposed to the sediment in the laboratory and in the field. Neoplasia was observed in oysters after 30 and 60 days of continuous exposure in a laboratory flow-through system to a 20 mg/L suspension of BRH sediment plus post-exposure periods of 0, 30 or 60 days. Tumour occurrence was highest in the renal excretory epithelium, followed in order by gill, gonad, gastrointestinal, heart, and embryonic neural tissue. In field experiments, gill neoplasms developed in oysters deployed in cages for 30 days at BRH. The uptake of PAH and PCBs from BRH sediment observed in oysters also occurs in blue mussels (Mytilus edulis). Winter flounder (Pseudopleuronectes americanus) fed BRH-contaminated blue mussels contained xenobiotic chemicals analysed in mussels. The flounder developed renal and pancreatic neoplasms and pre-neoplastic hepatic lesions, demonstrating trophic transfer of sediment-bound carcinogens up the food chain (Gardner and Yevich, 1988; Gardner et al., 1991). Petroleum hydrocarbons Field studies conducted following the “Amoco Cadiz” oil spill have shown pathological changes in several shellfish and fish species. In April–May 1978, cockles (Cerastoderma edule) and mussels (Mytilus edulis) were deployed in cages for 25 days in the Baie de Morlaix, a site heavily oiled by the “Amoco Cadiz” and in the Rade de Brest, a control site. Mussels and cockles from the contaminated site had much higher accumulation of lipid and lysosomal granules in their digestive diverticula compared to mussels from the control site. Plaice (Pleuronectes platessa) were sampled in two estuaries heavily oiled by the “Amoco Cadiz”, the Aber Wrac’h and the Aber Benoit, and in reference sites located on the western and southern coasts of Brittany. Several histopathological changes were found at oil-contaminated sites including fin and tail necrosis, hyperplasia and hypertrophy of gill lamellar mucous cells, increased density of pigmented macrophage aggregates in the liver, delayed maturation of the oocytes (Haensley et al., 1982; Stott et al., 1983). In both of these studies, fish and shellfish were exposed to complex mixtures of contaminants and developed different types of histopathological changes. It is not known if they responded to the same group or to different groups of chemicals. The demonstration of toxic-induced pathological changes in both fish and shellfish species has contributed to assess the ecological impact of the contaminants and has suggested the possibility of trophic transfer of contaminants. In a study on biomarkers of pollution stress in marine molluscs and fish in littoral regions in the Red Sea, Mediterranean Sea and the North Sea, researchers found elevated frequency of DNA lesions (alkaline and acidic DNA unwinding), increased prevalence of micronucleus-containing haemocytes and organ pathologies in samples from contaminated sites (Bresler et al., 1999).

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A review of existing environmental monitoring programmes assessing the prevalence of shellfish diseases in parallel to fish diseases (at the same sites and times) Few environmental monitoring programmes integrate the evaluation of fish and shellfish pathological and histopathological changes. In Canada, Sylvie St. Jean, a post-doctoral fellow working with Simon Courtenay (Department of Fisheries and Oceans, Gulf Fisheries Center, Moncton, NB), has been looking at histopathology in mummichogs (Fundulus heteroclitus) at the same sites as she has been looking at immunological biomarkers and incidence of haemic neoplasia (with Carol Reinisch from Woods Hole) in caged mussels. Fish and mussels were collected in the areas within and around Pictou Harbour, Nova Scotia receiving the effluent of a pulp and paper mill. Analyses of the results and preparation of publication are under way. In the UK, the research programme “Endocrine Disruption in the Marine Environment” (EDMAR) (1998–2001) (Allen et al., 2002) investigated the effects of endocrine disrupters (ED) in three fish species, European flounder (Platichthys flesus), viviparous blenny (Zoarces viviparus) and the sand goby (Pomatoschistus minutus) and in crustacean species (Cancer pagurus) from estuarine environments from around the UK. Flounder and blenny were found to exhibit increased vitellogenin (VTG) induction and presence of intersex in estuaries contaminated with endocrine disrupting chemicals. Sand gobies were not found to be susceptible to either elevated VTG levels or intersex induction. However, morphologically intermediate papilla syndrome (MIPS) (Kirby et al., 2002) was described in gobies from several locations. Increased induction of vitellin (crustacean analogue of VTG) was not found to occur in crabs exposed to EDs either naturally or experimentally. Firm evidence of changes to the gonads and secondary sexual characteristics was also lacking. Histological evaluation of gonad tissue and other organs was not undertaken. Brown shrimp (Crangon crangon) sampled in the same estuaries were also examined but did not show evidence of endocrine disruption. In a current UK DEFRA-funded programme, investigations based at CEFAS Weymouth Laboratory are evaluating biological effects of contaminants in estuarine organisms. The main aim of this work is to provide a more holistic assessment of effects of contaminants in estuarine environments by integrating biological effects measurements from several organisms. The study includes the same fish species as were targeted in the EDMAR programme, but extends this to incorporate other species found to be abundant at particular locations. In addition, crustacean species are also sampled at the same time to provide an insight into the pathological responses exhibited by resident shellfish. Molluscs are also under investigation but samples are difficult to obtain using small trawls used to collect fish and crustacean samples. The study utilised external disease (following ICES guidelines) and multi-organ histopathological biomarkers in all species sampled. In addition, concurrent samples are analysed for gene mutation analysis, DNA adduct formation and micronuclei analysis (in fish). Data on parasite prevalence and abundance are used to augment the assessment of individual and population health and appear to be a powerful tool to discriminate between impacted and reference sites. Results to date indicate that liver histopathology is effective in identifying end-point effects of contaminants in estuarine environments (Stentiford et al., 2003) and intersex provides a good end-point marker for ED exposure. Preliminary data on crustacean pathology have identified several types of lesions associated with infectious agents that may be of use for health assessment in this group. Analysis of mollusc samples and the extended observation of fish and crustacean samples are ongoing. In 2001, a practical workshop was held to evaluate biological effects of contaminants in pelagic ecosystems: a coastal area (German Bight) and an area in the vicinity of an oil rig (Statfjord). Pathological changes are evaluated in blue mussels (Mytilus edulis) and in Atlantic cod (Gadus morhua), exposed in cages at each site. In the Biological Effects of Environmental Pollution in Marine Coastal Ecosystems (BEEP-2000–2004), coordinated by P. Garrigues, University of Bordeaux, France (http://beep.lptc.u-bordeaux.fr/_summary.asp), gonad alterations are assessed in fish and mussels and are used as an index of stress and of alteration of organism performance. One objective of the programme is to identify new sensitive species and species with different life strategies in the ecosystem that can be monitored. Multiple biomarker suites will be developed to be applied in different biota (including both fish and shellfish species) with different environmental toxins. Conclusions Pathological changes in fish and shellfish species may be used to indicate the presence of toxic contaminants in the marine environment. The combined used of fish and shellfish species monitoring studies is advantageous since fish and shellfish species differ in terms of their vulnerability to various groups of contaminants. Very few studies have

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investigated pathological responses of fish and shellfish species collected at the same study sites. In these studies, a variety of histopathological changes were observed in fish and shellfish species exposed to complex mixtures of contaminants and it is not known if they responded to the same group or different groups of chemicals. Recent interest in the effect of endocrine disrupting compounds on the marine biota has triggered research in the comparative toxicopathology in various fish and shellfish species. These studies will contribute to identify additional sensitive species and histopathological biomarkers. Recommendation: the WGPDMO should evaluate a range of pathological and histopathological biomarkers for use in a variety of sensitive fish and shellfish species exposed to different groups of contaminants and incorporate these biomarkers into the ICES monitoring programme. References Allen, Y., Balaam, J., Bamber, S., Bates, H., Best, G., Bignell, J., Brown, E., Craft, J., Davies, I.M., Depledge, M.,

Dyer, R., Feist, S., Hurst, M., Hutchinson, T., Jones, G., Jones, M., Katsiadaki, I., Kirby, M., Leah, R., Matthiessen, P., Megginson, C., Moffat, C.F., Moore, A., Pirie, D., Robertson, F., Robinson, C.D., Scott, A.P., Simpson, M., Smith, A., Stagg, R.M., Struthers, S., Thain, J., Thomas, K., Tolhurst, L., Waldock, M. and Walker, P. 2002. Endocrine disruption in the marine environment (EDMAR). 67 pp.

Bresler, V., Bissinger, V., Abelson, A., Dizer, L., and Hansen, P.D. 1999. Marine molluscs and fish as biomarkers of pollution stress in littoral regions of the Red Sea, Mediterranean Sea and North Sea. Helgol. Mar. Res., 53: 219–243.

Fournier, M., Pellerin, J., Lebeuf, M., Brousseau, P., Morin, Y., and Cyr, D. 2002. Effects of exposure of Mya arenaria and Mactromeris polynyma to contaminated marine sediments on phagocytic activity of hemocytes. Aquatic Toxicol., 59(1-2): 83–92.

Gagnaire, B., Renault, T., Bouilly, K., Lapègue, S., and Thomas-Guyon, H. 2003. Study of atrazine effects on Pacific oyster, Crassostrea gigas, haemocytes. Current Pharmaceutical Design, 8: 99–110.

Galloway, T.S., and Depledge, M.H. 2001. Immunotoxicity in invertebrates: measurement and ecotoxicological relevance. Ecotoxicology, 10: 5–23.

Gardner, G.R., Yevich, P.P., Harshbarger, J.C., and Malcolm, A.R. 1991. Carcinogenicity of Black Rock Harbor sediment to the eastern oyster and trophic transfer of Black Rock Harbor carcinogens from the blue mussel to the winter flounder. Environ Health Perspect., 90: 53–66.

Gardner, G.R., and Yevich, P.P. 1988. Comparative histopathological effects of chemically contaminated sediment on marine organisms. Mar. Environ. Res., 24: 311–316.

Haensly, W.E., Neff, J.M., Sharp, J.R., Morris, A.C., Bedgood, M.F., and Boem, P.D. 1982, Histopathology of Pleuronectes platessa L. from Aber Wrac’h and Aber Benoit, Brittany, France: long-term effects of the Amoco Cadiz crude oil spill. J. Fish Dis., 5: 365–391.

Kirby, M.F., Bignell, J., Brown, E., Craft, J., Davies, I., Dyer, R. A., Feist, S. W., Jones, G., Matthiessen, P., Robertson, F.E. and Robinson, C. 2002. The presence of morphologically intermediate papilla syndrome (MIPS) in UK populations of sand goby (Pomatoschistus spp.): Endocrine Disruption? Environmental Toxicology and Chemistry, 22(2): 239–251.

Oehlman, J., Schulte-Oehlman, U., Tillman, M., and Markert, B. 2000. Effects of endocrine disruptors on psodobranch snails (mollusca: Gastropoda) in the laboratory. Part I: bisphenol A and octylphenol as xeno-estrogens. Ecotoxicology, 9: 399–412.

Sauve, S., Brousseau, P., Pellerin, J., Morin, Y., Senecal, L., Goudreau, P., and Fournier, M. 2002. Phagocytic activity of marine and freshwater bivalves: in vitro exposure of hemocytes to metals (Ag, Cd, Hg and Zn). Aquat Toxicol., 58(3-4): 189–200.

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Schulte-Oehlman, U., Tillman, M., Markert, B., Oehlman, J., Watermann, B., and Scherf, S. 2000. Effects of endocrine disruptors on psodobranch snails (mollusca: Gastropoda) in the laboratory. Part II: triphenyltin as a xeno-androgen. Ecotoxicology, 9: 399–412.

Stentiford, G.D., Longshaw, M., Lyons, B.P. and Feist, S.W. 2003. Histopathological biomarkers in estuarine fish species for the assessment of biological effects of contaminants. Marine Environmental Research, 55: 137–159.

Stott, G.G., Haensly,W.E., Neff, J.M., and Sharp, J.R. 1983. Histopathologic survey of ovaries in plaice, Pleuonectes platessa L., from Aber Wrac’h and Aber Benoit, Brittany, France: long-term effects of the Amoco Cadiz crude oil spill. J. Fis Dis., 6: 429–437.

Tillmann, M, Schulte-Oehlmann, U, Duft, M, Marert, B, and Oehlmann, J. 2001. Effects of endocrine disruptors on psodobranch snails (Mollusca: Gastropoda) in the laboratory. Part III: Cyproterone acetate and vinclozolin as antiandrogens.

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ANNEX 10: REVIEW OF MOLECULAR TECHNIQUES USED TO DIFFERENTIATE THE VARIOUS SPECIES/ISOLATES OF PERKINSUS

S. Bowern( Dept. of Fisheries & Oceans, Pacific Biological Station, Nanaimo, Canada), E. Burreson, K. Reece (Virginia Institute of Marine Science, USA)

Species in the genus Perkinsus are pathogenic parasites of marine molluscs that have been detected in many locations around the world. Members of this genus have been detected in over 67 species of molluscs (primarily bivalves) from temperate to tropical regions of the Atlantic and Pacific oceans (Perkins, 1996). Because some of these parasites cause significant disease in commercially important shellfish, tools to specifically identify the species are vital to international trade (introductions and transfer concerns) and to understanding the biology in order to identify disease control/prevention programmes. Species have been discriminated on the basis of morphological differences in the various developmental stages, in vitro characteristics, host species, and host response to infection. However, several named species are currently facing taxonomic controversy pertaining to specific identity. The current major limitations to identifying the various species of Perkinsus are the absence of significant morphological differences among known species (Coss et al., 2001a) and the broad host range encountered for isolates tested in the laboratory and assayed from the field (Goggin and Lester, 1995; Perkins, 1996; Coss et al., 2001b). Sequences of some regions of the genomes (primarily within the RNA gene complex region) of most Perkinsus species, including several isolates of some species, have been used to distinguish among Perkinsus species (Casas et al., 2002; Dungan et al., 2002; Murrell et al., 2002). The following review will present the seven named species and outline criteria that were used for specific identification. In addition, a working list that suggests approaches to finding acceptable criteria for the identification of species within Perkinsus is presented. All species have two stages within the living host: 1) a trophozoite (aplanospore) stage, often observed within host haemocytes; and 2) a multinucleate schizont (tomont or “rosette”) stage. Immature trophozoites (1–10 µm in diameter), developed from an infective zoospore or released from a schizont, grow into mature trophozoite (10–20 µm in diameter) characterised by a large eccentric vacuole containing a vaculoplast that displaces the parasite’s nucleus peripherally (“signet-ring” stage). In a living host, the mature tropozoite develops into a schizont. If the infected host dies, mature trophozoites become prezoosporangia (Perkins, 1996). The most diagnostic feature of all Perkinsus spp. (except P. qugwadi, see below) is the development of prezoosporangia (hypnospores that may attain up to 250 µm diameter) when infected host tissue is placed in Fluid Thioglycollate Medium (FTM) at room temperature for approximately 7 days. Characteristically, these prezoosporangia stain blue/black with Lugol's iodine, a feature developed into a sensitive diagnostic assay by Ray (1966) (also see Bushek et al., 1994). Depending on the isolate involved, zoosporulation can occur in the host tissues (only known for P. qugwadi), in nutrient culture medium or in sea-water, especially after anaerobic incubation in FTM. The zoosporangium has a thick cell wall and is usually flask-shaped with a discharge tube (Perkins, 1996; Coss et al. 2001a). Numerous biflagellated, uninucleated ovoid zoospores (about 3 to 5 µm long) form within the zoosporangium, emerge from the discharge tube and are believed to be the normal transmission stage. The main characteristic feature of all Perkinsus zoospores is an elaborate organelle at the anterior end that is very reminiscent of the apical complex of the Apicomplexa, and was used to affiliate Perkinsus spp. with that group of protistans (Perkins, 1976). The best known and first named species, Perkinsus marinus, is one of the prime challenges to the productivity of the eastern (American) oyster (Crassostrea virginica) along the Atlantic and Gulf of Mexico coasts of the United States, including the devastation of the once profitable oyster industry in Chesapeake Bay (Andrews, 1988; Burreson et al., 1994; Ford, 1996; Burreson and Ragone Calvo, 1996; Cook et al., 1998). Because of variations observed in the virulence of P. marinus to genotypically different stocks of C. virginica, Bushek and Allen (1996a, 1996b) proposed that different strains of P. marinus that vary in virulence may exist or different oyster stocks may vary in resistance to infection. Similar differences in apparent susceptibility to infection and resulting pathology have been a recurring problem in attempts to identify species of Perkinsus encountered during mollusc disease surveillance studies (Hine and Thorne, 2000). Understanding of the taxonomic affinity of P. marinus has evolved since this parasite was first encountered in association with mass mortalites of oysters in the Gulf of Mexico in the 1940s (Mackin et al., 1950) and then in Chesapeake Bay in the 1950s (Andrews, 1996). Originally named Dermocystidium marinum, this protistan parasite was thought to be affiliated with fungi (Mackin et al., 1950; Mackin, 1951). This original scientific name resulted in the nickname “Dermo” disease, that has persisted throughout several subsequent taxonomic revisions. Ultrastructural studies placed it within the protistan fungal-like Labyrinthomorpha, and it was renamed Labyrinthomyxa marina (Mackin and Ray, 1966). Further examination of the zoospore ultrastructure led to the erection of a new class within the Apicomplexa: Perkinsasida, and reclassification to Perkinsus marinus within the Family Perkinsidae (Levine, 1978).

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Recent phylogenetic analyses based on the small subunit ribosomal RNA gene and actin gene, however, indicate that the Perkinsasida may not belong in the Phylum Apicomplexa but have a close affiliation with the Dinoflagellida (Goggin and Barker, 1993; Perkins, 1996; Siddall et al., 1997; Reece et al., 1997b; de la Herrán et al., 2000; Murrell et al., 2002). Norén et al. (1999) proposed that perkinsids, which share features with both dinoflagellates and apicomplexans, be described as a taxon on level with other alveolate phyla, with the phylum name of Perkinsozoa. In addition to P. marinus in C. virginica, six other Perkinsus spp. have been named. The most distinctive species is Perkinsus qugwadi, considered enzootic in British Columbia, Canada, but only known from Japanese scallops (Patinopecten yessoensis) that were introduced into Canada from Japan for culture purposes (Blackbourn et al., 1998). Scallops native to enzootic areas (Chlamys rubida and Chlamys hastata) were resistant to infection (Bower et al., 1999). Unlike all other Perkinsus spp., P. qugwadi: 1) proliferated and was pathogenic at cool temperatures (8–15 oC); 2) developed zoospores within interstitial tissues of living hosts (in some heavily infected juvenile scallops less than 50 mm in valve height) instead of outside the host; and 3) had no prezoosporangia development in fluid thioglycollate medium (FTM) nor subsequent dark staining with Lugol’s iodine (Ray, 1966) used to diagnose all other known Perkinsus spp. (Bower et al., 1998). In addition to these differences, phylogenetic analyses based on the internal transcribed spacer (ITS) regions of ribosomal RNA of P. qugwadi consistently place this species at the base of a clade containing the other Perkinsus spp. (Coss et al., 2001b, 2002 ; Dungan et al., 2002). In addition to P. marinus and P. qugwadi, five other species have been named but the taxonomic validity of some species requires substantiation. Perkinsus olseni, first reported as a pathogen of abalone (Haliotis rubra) in Australia, was the second-named species of Perkinsus (Lester and Davis, 1981). This species is now reported from three other species of abalone (Haliotis laevigata, Haliotis cyclobates and Haliotis scalaris) along the southern coast of Australia. It is also believed to occur in a wide variety of molluscan species from the Great Barrier Reef but was not detected in abalone from that area (Goggin and Lester, 1995). Perkinsus olseni was experimentally transmitted and highly infectious to a range of molluscs under laboratory conditions (Goggin et al. 1989). The third species of Perkinsus to be named was Perkinsus atlanticus, a pathogen of native clams (Ruditapes (=Tapes, =Venerupis) decussatus, Ruditapes (=Tapes) semidecussatus, Ruditapes pullastra, Venerupis aurea, Venerupis pullastra) and the introduced Manila clam (Venerupis (=Tapes, =Ruditapes) philippinarum) along the coasts of Portugal, Spain (Galicia and Huelva areas), and the Mediterranean Sea (Azevedo, 1989; Figueras et al., 1992; Navas et al., 1992; Rodríguez et al., 1994; Sagristà et al., 1995; Cigarría et al., 1997; Ordás et al., 2001; Casas et al., 2002). De la Herrán et al. (2000) published a molecular characterisation of the ribosomal RNA gene region of P. atlanticus isolated in FTM from the gills of Ruditapes decussatus. Perkinsus atlanticus was also tentatively identified from Macoma balthica in a tributary of Chesapeake Bay, USA (Kleinschuster et al., 1994). In the late 1990s, a Perkinsus sp. was associated with significant mortalities of native stocks of Manila clams in Korea, Japan, and China (Choi and Park, 1997; Maeno et al., 1999; Lee et al., 2001; Liang et al., 2001; Park and Choi, 2001; Choi et al., 2002). Hamaguchi et al. (1998) found that the nucleotide sequence of two internal transcribed spacers (ITS1 and ITS2) and the 5.8 S region of the rRNA of the Perkinsus sp. from Manila clams in Japan were almost identical to those of P. atlanticus and P. olseni and suggested that the parasite in Japan may be P. atlanticus. Genetic analysis of the same regions of the genome by Goggin (1994) could not differentiate between P. atlanticus (from the carpet-shell clam, R. decussatus, from Portugal) or P. olseni (from the abalone H. laevigata from Australia). Robledo et al. (2000) found high sequence homology in the ITS2 and rRNA non-transcribed spacers (NTS) regions of P. atlanticus from Portuguese clams and P. olseni and an isolate from the Australian clam Anadara trapezia. Casas et al. (2002) also reported that the ITS sequence from 13 isolates of Perkinsus sp. from Ruditapes decussatus in Galicia (NW Spain) were closely matched with equivalent sequences from P. atlanticus, P. olesni, and Perkinsus sp. from Chama pacificus and A. trapezia. However, in all these publications, the gene sequences of the P. atlanticus and P. olseni isolates were distinct from those of P. marinus (from the oyster C. virginica from Virginia, USA). Based on similarity (98–99%) in the sequences of the NTS, Murrell et al. (2002) proposed that P. olseni and P. atlanticus be synonymised with the name P. olseni having priority. If this synonymy is upheld, P. olseni will have a wide host range (infecting gastropods as well as bivalves) and a wide geographic range (including the coasts of Australia, New Zealand, Japan, Korea and Europe). The wide variability in the pathogenicity of this parasite may be attributed to either differences in strains of the parasite or differences in host responses. The validity of another named species of Perkinsus has been refuted. Perkinsus karlssoni was detected as a pathogen of cultured bay scallops, Argopecten irradians, being conditioned for spawning under hatchery conditions in Atlantic Canada (McGladdery et al. 1991; Whyte et al. 1993). This parasite was described because a Lugol-positive organism in FTM cultures from diseased scallops was indicative of a perkinsiid with characteristics consistent with FTM positive results from P. marinus infected oysters. However, diagnosis by FTM alone is controversial and Goggin et al. (1996) surmised that the description was based on a contaminant biflagellate organism, which grew in the FTM cultures from

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diseased scallop tissues. This species may be reinstated if further FTM positive prezoosporangia are obtained and phylogenetic analyses of sequence data place the species within the Perkinsus clade. Another proposed species, initially identified as culture G117 and later named Perkinsus chesapeaki, has been isolated from the gills of soft-shell clams (Mya arenaria) from the same location (Chesapeake Bay, USA) as P. marinus (McLaughlin and Faisal 1998a, 1998b, 2001; McLaughlin et al., 2000). Although the life cycle stages and zoosporulation process were similar to those described for other Perkinsus spp., P. chesapeaki was identified based on minor differences in the morphology of the zoospore and molecular differences to P. marinus (McLaughlin et al., 2000). The genetic sequence of the small subunit ribosomal rRNA of this isolate was reported as distinct from that of P. marinus (Kotob et al., 1999a, 1999b) and this conclusion has been supported by subsequent studies (Casas et al., 2002, Dungan et al., 2002). McLaughlin and Faisal (2001) reported a difference in the production of extracellular proteins by P. chesapeaki and P. marinus that may help to explain the difference in pathology observed in infected Mya arenaria and Crassostrea gigas, respectively. More recently, Perkinsus andrewsi was described from the clam Macoma balthica from the east coast of the United States. The species identification was based on sequence data from the rRNA locus that differed from that of P. marinus, P. atlanticus, P. olseni and P. qugwadi (Coss et al., 2001b). DNA analysis (using polymerase chain reaction (PCR) assays on regions of the ribosomal RNA SSU loci (mainly ITS1 and ITS2)) indicated that P. andrewsi can coexist with P. marinus in M. balthica and other sympatric clams (Macoma mitchelli and Mercenaria mercenaria) and the eastern oyster (C. virginica) (Coss et al., 2001b). Subsequent analysis of the ITS regions of several species of Perkinsus (including several isolates of some species) consistently grouped P. chesapeaki and P. andrewsi (Murrell et al., 2002, Casas et al., 2002). Analysis of the ITS sequence from cloned isolates of Perkinsus sp. from Mya arenaria and Tagelus plebeius from Chesapeake Bay suggested that the variations among ITS sequences of P. chesapeaki and P. andrewsi indicate true polymorphism within a single parasite species (Dungan et al., 2002). If these species are synonymous, P. chesapeaki would have precedence over P. andrewsi. Conclusions Over 67 species of molluscs from around the world are known to serve as hosts for species in the genus Perkinsus. From some of these hosts, seven species have been named. However, a conservative perspective suggests that only four of these names may be valid (i.e., P. marinus, P. olseni, P. qugwadi and P. chesapeaki). If this perspective is correct, then the host and geographic range is very broad for at least one of these species (i.e., P. olseni) and at least two other species can infect the same hosts in the same geographic area (i.e., P. marinus and P. chesapeaki). These characteristics and the lack of distinctive morphological features render the identity of Perkinsus spp. encountered in the field and the specific identity of all new isolates open to question. Because of the significant negative economic impact caused by some of these parasites, it is important to be able to differentiate between pathogenic and supposed non-pathogenic species or to determine which species are pathogenic for which hosts. In hopes of avoiding future confusion, it is incumbent upon scientists working on this group of parasites to develop a set of criteria for species identification. Following is an initial attempt at listing research areas that should begin to address the identification of required criteria:

• Implementation of non-specific assays such as FTM and histopathology are suitable for the initial detection of perkinsosis. However, these assays should not be used for the specific identification of any isolate regardless of host identity.

• Describe morphological differences between species by the accurate comparison of the morphology of various developmental stages in vitro of several isolates of named species under similar incubation conditions (e.g., media, parasite density, time in culture, incubation temperature, etc.). Studies conducted in parallel with the same isolates in several laboratories would be ideal.

• If morphological characteristics prove too variable and overlapping for species identification, implementation of molecular tools for diagnosis as suggested by Berthe et al. (1999) should be considered. Possibly all molecular assays described to date (e.g., Robledo et al., 2000, Yarnall et al., 2000) should be tested in parallel and validated.

• Verify if multiple polymorphic sequences of the ITS region of the rRNA gene complex as document by Dungan et al. (2002) for isolates from two species of clams in Chesapeake Bay occurs in all Perkinsus spp. If these parasites are diploid in their molluscan host as indicated by Reece et al. (1997a), the development of specific molecular diagnostic assays will have to be adjusted and validated accordingly.

• Assess if the NTS region of the genome is a good discriminator of species and strains of Perkinsus as suggested by Murrell et al. (2002). Apparently, this region of the genome has low similarity between species and high similarity within species, but has not been adequately assessed for intraspecific variation. It was used by

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Robledo et al. (2000) to develop a specific and sensitive PCR based diagnostic assay for “P. atlanticus” from several species of clams in Spain.

• Identify other regions of the genome that may prove useful for species differentiation. • Develop diagnostic assays that will clearly discriminate between all “valid” species. • Other suggestions?

References

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Figueras, A., Robledo, J.A.F., and Novoa, B. 1992. Occurrence of haplosporidian and Perkinsus-like infections in carpet-shell clams, Ruditapes decussatus (Linnaeus, 1758), of the Ria de Vigo (Galicia, NM Spain). Journal of Shellfish Research, 11: 377–382.

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Hamaguchi, M., Suzuki, N., Usuki, H., and Ishioka, H. 1998. Perkinsus protozoan infection in short-necked clam Tapes (=Ruditapes) philippinarum in Japan. Fish Pathology, (Tokyo) 33: 473–480.

Hine, P.M., and Thorne, T. 2000. A survey of some parasites and diseases of several species of bivalve mollusc in northern Western Australia. Diseases of Aquatic Organisms, 40: 67–78.

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Kleinschuster, S.J., Perkins, F.O., Dykstra, M.J., and Swink, S.L. 1994. The in vitro life cycle of a Perkinsus species (Apicomplexa, Perkinsidae) isolated from Macoma balthica (Linneaus, 1758). Journal of Shellfish Research, 13: 461–465.

Kotob, S.I., McLaughlin, S.M., van Berkum, P., and Faisal, M. 1999a. Characterization of two Perkinsus spp. from the softshell clam, Mya arenaria using the small subunit ribosomal RNA gene. Journal of Eukaryotic Microbiology, 46: 439–444.

Kotob, S.I., McLaughlin, S.M., VanBerkum, P., and Faisal, M. 1999b. Discrimination between two Perkinsus spp. isolated from the softshell clam, Mya arenaria, by sequence analysis of two internal transcribed spacer regions and the 5.8S ribosomal RNA gene. Parasitology, 119: 363–368.

Lee, M.-K., Cho, B.-Y., Lee, S.-J., Kang, J.-Y., Jeong, H.D., Huh, S.H., and Huh, M.-D. 2001. Histopathologucal lesions of Manila clam, Tapes philippinarum, from Hadong and Namhae coastal areas of Korea. Aquaculture, 201: 199–209.

Lester, R.J.G., and Davis, G.H.G. 1981. A new Perkinsus species (Apicomplexa, Perkinsea) from the abalone Haliotis ruber. Journal of Invertebrate Pathology, 37: 181–187.

Levine, N.D. 1978. Perkinsus gen. n. and other new taxa in the protozoan phylum Apicomplexa. Journal of Parasitology, 64: 549.

Liang, Y.-B., Zhang, X.-C., Wang, L.-J., Yang, B., Zhang, Y., and Cai, C.-L. 2001. Prevalence of Perkinsus sp. in the Manila clam Ruditapes philippinarum along northern coast of Yellow Sea in China. Oceanologia et Limnologia Sinica, 32: 502–511. (Chinese with English abstract.).

Mackin, J.G. 1951. Histopathology of infection of Crassostrea virginica (Gmelin) by Dermocystidium marinum Mackin, Owen, and Collier. Bulletin of Marine Science of the Gulf and Caribbean, 1: 72–87.

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Mackin, J.G., Owen, H.M., and Collier, A. 1950. Preliminary note on the occurrence of a new protistan parasite, Dermocystidium marinum n.sp. in Crassostrea virginica (Gmelin). Science, (Washington D C) 111: 328–329.

Maeno, Y., Yoshinaga, T., and Nakajima, K. 1999. Occurrence of Perkinsus species (Protozoa, Apicomplexa) from Manila clam Tapes philippinarum in Japan. Fish Pathology, (Tokyo) 34: 127–131.

McGladdery, S.E., Cawthorn, R.J., and Bradford, B.C. 1991. Perkinsus karlssoni n. sp. (Apicomplexa) in bay scallops Argopecten irradians. Diseases of Aquatic Organisms, 10: 127–137.

McLaughlin, S.M., and Faisal, M. 1998a. In vitro propagation of two Perkinsus species from the softshell clam Mya arenaria. Parasite, 5: 341–348.

McLaughlin, S.M., and Faisal, M. 1998b. Histopathological alterations associated with Perkinsus spp. infection in the softshell clam Mya arenaria. Parasite, 5: 263–271.

McLaughlin, S.M., and Faisal, M. 2001. Pathogenesis of Perkinsus spp. in bivalve molluscs. Bulletin of the National Research Institute of Aquaculture Supplement, 5: 111–117.

McLaughlin, S.M., Tall, B.D., Shaheen, A., Elsayed, E.E., and Faisal, M. 2000. Zoosporulation of a new Perkinsus species isolated from the gills of the softshell clam Mya arenaria. Parasite, 7: 115–122.

Murrell, A., Kleeman, S.N., Barker, S.C., and Lester, R.J.G. 2002. Synonymy of Perkinsus olseni Lester and Davis, 1981 and Perkinsus atlanticus Azevedo, 1989 and an update on the phylogenetic position of the genus Prekinsus. Bulletin of the European Association of Fish Pathologists, 22: 258–265.

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Navas, J.I., Castillo, M.C., Vera, P., and Ruiz-Rico, M. 1992. Principal parasites observed in clams, Ruditapes decussatus (L.), Ruditapes philippinarum (Adam et Reeve), Venerupis pullastra (Montagu) and Venerupis aureus (Gmelin), from the Huelva coast (S.W. Spain). Aquaculture, 107: 193–199.

Norén, F., Moestrup, O., and Rehnstam-Holm, A.-S. 1999. Parvilucifera infectans Norén et Moestrup gen. et sp. nov. (Perkinsozoa phylum nov.): a parasitic flagellate capable of killing toxic microalgae. European Journal of Protistology, 35: 233–245.

Ordás, M.C., Gomez-Leon, J., and Figueras, A. 2001. Histopathology of the infection by Perkinsus atlanticus in three clam species (Ruditapes decussatus, R. philippinarum and R. pullastra) from Galicia (NW Spain). Journal of Shellfish Research, 20: 1019–1024.

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Perkins, F.O. 1976. Zoospores of the oyster pathogen, Dermocystidium marinum. I. Fine structure of the conoid and other sporozoan-like organelles. Journal of Parasitology, 62: 959–974.

Perkins, F.O. 1996. The structure of Perkinsus marinus (Mackin, Owen and Collier, 1950) Levine, 1978 with comments on taxonomy and phylogeny of Perkinsus spp. Journal of Shellfish Research, 15: 67–87.

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Reece, K.S., Siddall, M.E., Burreson, E.M., and Graves, J.E. 1997b. Phylogenetic analysis of Perkinsus based on actin gene sequences. Journal of Parasitology, 83: 417–423.

Robledo, J.A.F., Coss, C.A., and Vasta, G.R. 2000. Characterization of the ribosomal RNA locus of Perkinsus atlanticus and development of a polymerase chain reaction-based diagnostic assay. Journal of Parasitology, 86: 972–978.

Rodriguez, F., Godoy, T., and Navas, J.I. 1994. Cross-infection with Perkinsus atlanticus in Ruditapes decussatus, Ruditapes philippinarum and Venerupis pullastra. Bulletin of the European Association of Fish Pathologists, 14: 24–27.

Sagristà, E., Durfort, M., and Azevedo, C. 1995. Perkinsus sp. (Phylum Apicomplexa) in Mediterranean clam Ruditapes semidecussatus: ultrastructural observations of the cellular response of the host. Aquaculture, 132: 153–160.

Siddall, M.E., Reece, K.S., Graves, J.E., and Burreson, E.M. 1997. “Total evidence” refutes the inclusion of Perkinsus species in the phylum Apicomplexa. Parasitology, 115: 165–176.

Whyte, S.K., Cawthorn, R.J., MacMillan, R.J., and Despres, B. 1993. Isolation and purification of developmental stages of Perkinsus karlssoni (Apicomplexa: Perkinsea), a parasite affecting bay scallops Argopecten irradians. Diseases of Aquatic Organisms, 15: 199–205.

Yarnall, H.A., Reece, K.S., Stokes, N.A., and Burreson, E.M. 2000. A quantitative competitive polymerase chain reaction assay for the oyster pathogen Perkinsus marinus. Journal of Parasitology, 86: 827–837.

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ANNEX 11: OBTAIN INFORMATION ON THE EU PROJECT “DIAGNOSIS OF OYSTER HERPES-LIKE VIRUS: DEVELOPMENT AND VALIDATION OF MOLECULAR, IMMUNOLOGICAL AND

CELLULAR TOOLS” (FAIR-PL98-4334) AND REVIEW THE RESULTS

T. Renault, IFREMER, Laboratoire de Génétique, Aquaculture et Patologie, La Tremblade, France Introduction Little information is available on viral infections that affect bivalve molluscs. Such a lack of data is due to a certain inadequacy of the diagnosis methods that are employed when massive mortality events occur. Most laboratories involved in mollusc pathology still analyse samples through light microscopy. Viral detection in bivalves may be performed on two kinds of biological material. When mortality events occur, moribund animals may be collected in the affected farms. This fresh material may be immediately used for nucleic acid extraction and further analysis. On the other hand, collected infected organisms can be frozen or fixed and remain archived for long periods of time, constituting a bank of reference material. Worldwide, there is thus currently a lack of information concerning the occurrence of bivalve herpesviruses. This is probably due to the lack of suitable diagnostic tools. The basic method for identification and examination of suspect samples is predominantly histopathology. This enables the identification of any cellular changes, but is not conclusive identification of bivalve herpesviruses. This technique does not allow, by itself, to detect viruses unless it is completed by other methods, such as transmission electron microscopy, the study of cytopathogenic effects in cell cultures or the detection through specific reactives. At present, no bivalve cell line is available: the detection of cytopathogenic effects in a homologous system is thus impossible. Since invertebrates lack antibody-producing cells, the direct detection of viral agents remains the only possible tool. In these conditions, the use of transmission electron microscopy is a necessity for visual confirmation. However, histology and transmision electron microscopy are time consuming and inadequate for epidemiological studies. The aim of the VINO project (involving 7 participants: 1. Institut Français de Recherche pour l’Exploitation de la Mer (IFREMER), La Tremblade, France; 2. Medical Research Council (MRC), Glasgow, United Kingdom; 3. Eurogentec S. A., Seraing, Belgium; 4. Université de Bretagne Occidentale (UBO), Brest, France; 5. University College Cork, Cork, Ireland; 6. Instituto de Investigaciones Marinas (CSIC), Vigo, Spain; 7. Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Weymouth, United Kingdom) was therefore the development and validation of molecular, immunological and cellular tools for the diagnosis of, and studies on, the bivalve herpesviruses. The main objective was to develop these “state of the art” diagnostic techniques. They should be applicable for identification of viruses during disease outbreaks. In addition, these techniques must also be suitable for the detection of subclinical infections and latent virus. The specific objectives of the VINO programme were:

1. Obtaining the complete oyster herpesvirus (OsHV-1) DNA sequence with determination of the genome structure.

2. Comparing OsHV-1 with viruses belonging to the Herpesviridae family on the basis of sequence data and genome structure.

3. Developing molecular tools for OsHV-1 detection. 4. Developing immunological tools for OsHV-1 detection. 5. Developing cellular tools for OsHV-1 detection using oyster primary cell cultures and vertebrate cell lines. 6. Application of developed diagnostic tools for OsHV-1 detection in oyster samples from different geographical

locations. An initial step of the VINO programme involved cloning of virus DNA in cosmids and plasmids. This work provided cloned viral DNA fragments suitable for characterising the virus genome and preparing specific diagnostic probes (PCR primers, labeled DNA probes and specific antibodies). Tests of oyster primary cell cultures and vertebrate cell lines were planned in order to study the ability of the virus to replicate in vitro. The development of molecular, immunological and cellular tools for OsHV-1 diagnosis may facilitate virus detection in infected material. Developed reagents have been used by four European laboratories to analyse a wide range of bivalve samples and to confirm the usefulness of the diagnostic tests. The entire virus genome has been cloned and sequenced. All viral sequences were analysed and the relationship of the OsHV to other members of the Herpesviridae family was determined.

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To date, polymerase chain reaction (PCR) assays have been developed, which allow the rapid, specific and sensitive diagnosis of herpesviruses in bivalve samples. Another technique that has also been developed is in situ hybridisation (ISH). VINO partners have conducted trials using PCR and ISH techniques in order to standardise and further develop the techniques in their respective laboratories. In addition to continuing the calibration of PCR and ISH, a main target was obtaining viral replication in oyster primary tissue cultures or different fish cell lines. However, all assays failed. The production of antibodies against bivalve herpesviruses appeared also a necessity for the development of any serological diagnostic/research technique. The development of immunoenzymatic tests and ELISA (Enzyme Linked Immunosorbent Assay) is now possible because of the availability of antibodies specific for bivalve herpesviruses. Indeed, specific rabbit antisera were obtained and may be used for diagnosis development. The best experimental conditions have been defined for their use in ELISA and Western blot analysis. A good immunization of mice was also obtained with two recombinant viral antigens and clones producing specific monoclonal antibodies have been isolated and characterized by ELISA, Western blot analysis and immunochemistry analysis. Applied to field samples, this calibration/standardisation step has provided an opportunity to carry out a preliminary epidemiological study. This was currently being achieved by the invaluable provision of bivalve spat, larvae and adults from private hatcheries and shellfish farms in France, Spain, the United Kingdom and Ireland. Herpesviral infections were confirmed in France in 1999, 2000 and 2001 and some positive samples were also reported in Spain and in The United Kingdom. Obtaining a complete virus genomic library and DNA sequence The genome sequence has been completed and analysed. Virus particles have been purified from fresh infected Crassostrea gigas larvae and viral DNA extracted from purified virions. At completion, each nucleotide was determined an average of 10.8 times and 96.1 % of the sequence was determined. The overall genome structure is: TRL - UL - IRL - X - IRS - US - TRS with a 207439 bp total genome size. TRL and IRL (7584 bp) are inverted repeats flanking a unique region (UL, 167843 bp). TRS and IRS (9774 bp) are inverted repeats flanking a unique region (US, 3370 bp), and X (1510 bp) is located between IRL and IRS. A similar genome structure has evolved independently in certain vertebrate herpesviruses (e.g., herpes simplex virus and human cytomegalovirus). The sequences of the genome termini were determined. They are not located uniquely, but a predominant form is apparent for each. The nature of the sequence between IRL and IRS was also determined. As with the termini, the IRL - IRS junction is not located uniquely, but the predominant form corresponds to a fusion of the two termini if each possesses two unpaired nucleotides at the 3' end. Unpaired nucleotides are characteristic of herpesvirus genome termini. Southern blot hybridisation experiments using PCR-generated probes from the ends of UL and US showed that the two orientiations of UL and US are present in approximately equimolar amounts in viral DNA, giving rise to four genome isomers. This is also a feature of the vertebrate herpesvirus genomes with similar structures. Both the database and restriction endonuclease digests indicated that a minor proportion (approximately 20–25 %) of genomes contain a 4.8 kbp region in UL in inverse orientiation. These data indicate that the virus contains a mixture of genome forms. In light of the fact that the virion DNA that was sequenced originated from a virus that had not been clonally purified, this was not unexpected. A detailed analysis of the coding potential of the genome sequence indicated the presence of 132 unique protein-coding open reading frames (ORFs). Owing to the presence of inverted repeats, 13 ORFs are duplicated, resulting in a total of 145 ORFs. This is an approximation of the gene number, chiefly because of the presence of fragmented genes that might not encode functional proteins. Seven genes encode enzymes (DNA polymerase, deoxyuridine triphosphatase, two subunits of ribonucleotide reductase, helicase, a putative primase and the ATPase subunit of terminase). Seven proteins bear sequence similarities with viral or cellular inhibitors of apoptosis proteins. IAPs are also encoded by baculoviruses and entomopoxviruses (both of which have insect hosts), underscoring the importance of the apoptotic responses of invertebrates against viral infections. Ten ORFs encode class I membrane proteins. An additional 17 proteins contain a hydrophobic domain indicating a possible assocation with membranes. A total of 39 proteins share sequence similarities with other proteins encoded by the virus, defining 13 multigene families in addition to the IAPs. An additional notable feature, located between ORFs 50 and 51, is a large palindrome. By analogy with certain vertebrate herpesviruses, this palindrome is a candidate origin of DNA replication. The sequence data demonstrate that the oyster herpes-like virus type 1 (OsHV-1) in not closely related to herpesviruses with vertebrate hosts (including fish). Amino acid sequence comparisons failed to identify a single protein which has homologues only in other herpesviruses. Several OsHV-1 proteins have homologues that are distributed widely in nature (e.g., DNA polymerase), but these are no more closely related to homologues in other herpesviruses than to homologues in other organisms. In this context, phylogenetic analyses are not of great utility in determining whether OsHV-1 and vertebrate herpesviruses have a common origin. However, a genetic indication of a common origin between OsHV-1 and vertebrate herpesviruses resides with the ATPase subunit of the terminase. Homologous genes are present in all herpesviruses, and the only non-herpesvirus counterparts are specified by T4 and related bacteriophages. The T4 and OsHV-1 genes are unspliced, whereas those in herpesviruses of mammals and birds contains one intron and

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those in herpesviruses of fish and amphibians contains two introns. Moreover, a similar genome structure was observed in certain vertebrate herpesviruses. The presence of several isomers described in the OsHV-1 genome is also a feature reported in vertebrate herpesvirus genomes. The available data support the view that herpesviruses of mammals and birds, herpesviruses of fish and amphibians and herpesviruses of invertebrates form three major lineages of the herpesviruses. OsHV-1 would have established a separate lineage about a billion years ago, and the fish viruses about 400 million years ago. OsHV-1 is currently the single representative of what may be a large number of invertebrate herpesviruses. Morover, recent data shown that OsHV-1 can infect several bivalve species. This contrasts with vertebrate herpesviruses, which are generally confined to a single species in nature. Consequently, the true host of OsHV-1 is unknown. The apparent loss of several gene functions in OsHV-1 prompts the speculation that this may have promoted interspecies transmission in the context of introduction of non-native bivalve species and use of modern aquaculture techniques. It is possible that the parental virus still resides in its natural host. Development of diagnosis tools To diagnose herpes-like virus infections, the basic method for examination of suspect samples is still light microscopy. This method appears poorly adapted to viral diseases and needs to be improved upon by other techniques such as transmission electron microscopy. Both techniques are time consuming and inadequate for epidemiological surveys. In addition, research into virus cytopathogenic effects in cell cultures is impossible because the lack of bivalve cell lines. A breakthrough was achieved recently in the development of a protocol, based on sucrose gradient centrifugation, for purifying oyster herpes-like virus particles from fresh infected larval Crassostrea gigas (Le Deuff and Renault, 1999). This advance has served as an appropriate platform for generating molecular biological reagents to diagnose virus infections. A procedure to detect herpes-like virus in French oysters using the polymerase chain reaction (PCR) was developed (Renault et al., 2000). PCR offers many advantages for disease diagnosis. With regard to herpes-like viruses from oysters, important advantages include its extreme sensitivity, pathogen specificity, ease of sample processing, and availability of reagents. Another technique that has also been developed is in situ hybridisation (ISH) (Lipart and Renault, 2002). In addition to continuing the calibration of PCR and ISH, a main target was the production of antibodies to the virus. The development of immunochemistry and ELISA tests became possible because of the availability of cloned sequences of an oyster herpes-like virus which enables the synthesis of recombinant virus proteins. Developing molecular tools and techniques A PCR-based procedure for detecting a herpes-like virus that infects the Pacific oyster, Crassostrea gigas, in France was developed. Two primer primers (A3/A4 and A5/A6) were designed to provide specific amplification products ranging in size 917 and 1001 bp when performed on oyster herpes-like virus DNA (Renault et al., 2000). No amplification was observed on oyster genomic DNA nor on the DNA from vertebrate herpesviruses. Crude samples were prepared and submitted to nested PCR, allowing the amplification of DNA fragments of the expected size when performed on infected larval and spat samples. The procedure used to prepare the sample for PCR was found to be critical because of the presence of unidentified substances in oyster tissues that inhibit the PCR reaction. A quick and convenient sample preparation using ground tissues allowed a sensitive detection of the herpes-like virus infected oysters. The ability of the defined PCR protocol to diagnose herpes-like virus infections in oysters was compared to the transmission electron microscopy technique from 15 C. gigas larval batches presenting or not mortalities. PCR amplification is as sensitive diagnosis assay for herpes-like virus as the transmission electron microscopy. However, the nested PCR protocol is more convenient and less time consuming. The relationship between reported mortalities among C. gigas oyster spat and herpes-like virus DNA detection by PCR was also investigated. Stastitical analysis showed that virus detection and mortalities are correlated. A competitive PCR method has also been developed using previously designed primers in order to detect and quantitate herpes-like virus DNA. The method is based on the use of oyster herpesvirus specific primer pairs and an internal standard competitor that differs from the target DNA by a deletion of 76 base pairs (Arzul et al., 2002). The internal standard DNA molecule was generated by PCR and then co-amplified with the target DNA. The resulting PCR products which were different in size were separated on agarose gels. The assay was found to be specific and sensitive, allowing the detection of 1 fg of viral DNA among 0.5 mg of oyster tissues. The method was used to demonstrate the absence of PCR inhibitors in oyster spat ground tissues. PCR inhibition was observed in adult oyster samples when the same tissue preparation procedure was used. On the contrary, classical phenol/chloroform DNA extraction from adult oyster tissues allowed amplification of the internal standard competitor and the viral DNA. The method was successfully used to demonstrate the presence of viral DNA in asymptomatic adult oysters indicating that oyster herpes-like virus infects animals presenting no anomalous mortality. Quantitations of herpes-like virus DNA in infected spat and asymptomatic adult oysters were also carried out. Although between 1.5 pg and 325 pg of viral DNA per 0.5 mg of oyster tissues were detected in adults, amounts of viral DNA in infected oyster spat varied from 750 pg to 35 ng per 0.5 mg of ground tissues.

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Two primer pairs were also developed in order to amplify small DNA fragments from OsHV-1 DNA. The first primer pair, called OH1/OH4, yielded 196 bp amplicons when genomic viral DNA was used as template. The size of PCR products obtained with the second primer pair (IAP1/IAP2) was 207 bp. Both primer pairs have been designed in order to obtain PCR amplification when DNA extracted from histological blocks was used as template. Several primer pairs previously designed (Renault et al., 2000; Arzul et al., 2001a and b) have already been tested using this type of DNA. They failed producing amplicons. These results could be explained by DNA fragmentation. Both primer pairs OH1/OH4 and IAP1/IAP2 allowed the production of amplicons when DNA extracted from wax blocks was used. A classical technique was choosen for DNA extraction from histological sections using dewaxing in xylene and treatment with proteinase K. Archived material has been used. PCR analysis using DNA extracted from this material showed clear bands presenting expected sizes when both primer pairs were used. This suggested that both primer combinations were reliable tools to detect viral DNA in archived material. Moreover, the primer pairs have been designed in two different areas of the genomic viral DNA enhancing the specificity of the detection. The OH1/OH4 primer pair recognises a gene coding for a protein of unknown function and the IAP1/IAP2 primer combination amplifies a fragment of a gene corresponding to a putative inhibitor of apoptosis (IAP). Developing immunological tools and techniques The immunoscreening with specific anti-OsHV-1 antibodies and the results of sequencing of the virus genome allowed to identify two open reading frames (ORFs) encoding for putative immunogenic viral proteins. The first ORF codes for a protein of 748 amino acids. This protein contains a highly hydrophobic C-terminal domain, potential N-glycosylation sites (Asn-X-Ser/Thr) and a potential signal peptide at the N-terminal end. This ORF could code for a membrane glycoprotein, the typical profile of surface viral antigens. The second identified ORF codes for a protein of which different regions are recognized by anti-OsHV-1 ascitis. This protein of 364 amino acids doesn’t present the characteristics of membrane proteins but seems to be a potential immunogenic protein. This ORF codes for a protein presenting homologies with baculovirus, insect and mammal IAPs (Inhibitor of Apoptosis). The two ORFs encoding for putative immunogenic viral proteins have been cloned in baculovirus expression system in order to prepare recombinant proteins and antibodies for diagnosis use. The prodcuction of polyclonal and monoclonal antibodies specific for the two selected viral proteins furnished specific immunological reagents. Developing and testing cell cultures for virus replication Several assays of herpes-like virus cultivation in oyster primary cultures and fish cell lines have been carried out. No cytopathic effect has been observed in tested fish cell lines. OsHV-1 may be not able to multiply in fish cell lines or under cultivation conditions used viral replication doesn’t occur. Tested fish cell lines cannot be used for the herpes-like virus infection diagnosis. Preliminary assays performed in primary cultures of embryonic oyster cells showed the presence of viral DNA in infected cultures using PCR and in situ hybridisation. However, experiments must be reiterated and other techniques as transmission electron microscopy used in order to demonstrate the presence of the virus in embryonic cells. Although promising results have been observed, primary cultures of embryonic oyster cells are not at this time a reliable tool to detect OsHV-1. Herpes-like infection surveys Periodic losses in bivalve hatcheries are regularly reported in Europe. Current practise in the private shellfish hatcheries takes account of basic research findings about food provision and avoidance of Vibrio infection but uncontrolled variables are still damaging the industry, particularly since 1991. Among these uncontrolled variables, herpes-like virus infections seem to play a key role. The observed association between oyster mortality and herpes-like virus infections provides an imperative to determine the extent to which the virus is involved as a causative agent of massive larval mortalities in different European countries. PCR was use to investigate the presence of herpes-like virus DNA in bivalve samples belonging to different bivalve species from different geographical origins (Renault and Arzul, 2001). The laboratories involved in mollusc epidemiological surveys have collected bivalve samples in 1999, 2000 and 2001 to search viral infections using the developed tools. In 2001, both PCR and in situ hybridization were used to diagnose herpes-like virus infections in bivalves. Positive samples were reported France, Spain and the United Kingdom using molecular techniques. These results confirm previous data indicating that herpes-like virus infections may be observed in France in the fied and in hatcheries. Morover, some PCR positive results were also obtained for bivalve samples originating from Spain and United Kingdom. Positive samples were observed in four bivalve species: Crassostrea gigas, Ostrea edulis, Ruditapes decussatus and R. philippinarum.

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References Arzul, I., Renault, T., and Lipart, C. 2001a. Experimental herpes-like viral infections in marine bivalves: demonstration

of interspecies transmission. Dis aqua Org, 46: 1–6.

Arzul, I., Renault, T., Lipart, C., and Davison, A.J. 2001b. Evidence for inter species transmission of oyster herpesvirus in marines bivalves. J Gen Virol, 82: 865–870.

Arzul, I., Renault, T., Thébault, A., and Gérard, A. 2002. Detection of oyster herpesvirus DNA and proteins in asymptomatic Crassostrea gigas adults. Virus Research, 84: 151–160.

Le Deuff, R.-M., and Renault, T. 1999. Purification and partial genome characterization of a herpes-like virus infecting the Japanese oyster, Crassostrea gigas. J. Gen. Virol. 80, 1317–1322.

Lipart, C., and Renault, T. 2002. Herpes-like virus detection in Crassostrea gigas spat using DIG-labelled probes. J. Virol. Meth, 101: 1–10.

Renault, T., and Arzul, I. 2001. Herpes-like virus infections in hatchery-reared bivalve larvae in Europe: specific viral DNA detection by PCR. J. Fish Dis., 24: 161–167.

Renault, T., Le Deuff, R.-M., Lipart C., and Delsert C. 2000a. Development of a PCR procedure for the detection of a herpes-like virus infecting oysters in France. J. Virol. Meth. 88, 41–50.

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ANNEX 12: REVIEW AND REPORT ON PROGRESS MADE IN THE “FISH DISEASES AND LIVER HISTOPATHOLOGY” COMPONENT OF THE BEQUALM SELF-FUNDING SCHEME

S.W. Feist, CEFAS, Weymouth, UK The following has been taken from the BEQUALM final report and summarises the activities undertaken during the project and includes the work programme established for 2003. Outline of objectives The overall objective of this work package was to produce a formal quality assurance infrastructure to validate the data obtained from field studies on the occurrence of externally visible diseases and liver histopathologies in flatfish that shall involve the establishment of protocols for assessments of data submitted from Participating Laboratories.

The following were the stated objectives for this work package. An explanation of how these were achieved is provided below.

• The establishment of appropriate reagents whose suitability has been assessed by Partner 6;

This was achieved. Reagents required for this work package relate to those required for the histological assessment of liver histopathology. The methods selected for use in biological effects monitoring are those used routinely in experienced histology laboratories and are readily available. It should be noted that for histology, analytical grade reagents are not required. These aspects of the contract formed an important component of the activities of the first workshop and are discussed below under “summary of workshops”.

• The establishment of agreed descriptions of neoplastic and non-neoplastic liver lesions;

This was achieved. This was the most important component of the work undertaken under this contract and formed the focus of the activities of the workshops and specifically of the ring test. In formulating the best possible criteria, contact was maintained with experts from the US who also employ fish liver histopathology in environmental quality assessments. The work undertaken during this contract has established diagnostic criteria that have taken into account the most up-to-date knowledge available. Under BEQUALM these criteria were primarily developed for use in marine environmental monitoring programmes targeting flatfish species; however, they are equally applicable for any fish species in freshwater or marine environments.

• The establishment of reproducibility between participating laboratories by the development of standard operating procedures (SOPs) for the various methodologies utilised including preparation of liver tissue for each of the analyses required, fixation and preservation, processing, quantification of histopathological/histochemical changes, archiving and reporting of data;

This was achieved. Agreed technical protocols for pathological and histological techniques have been provided in the workshop reports and submitted to the BEQUALM website for wider dissemination. Comprehensive information is included in the CD-ROM. • Distribution of laboratory reference materials consisting of sets of approximately ten stained tissue sections

representing the major categories of liver lesion encountered in flatfish of importance for biological effects monitoring. The reference sets shall be accompanied by supplementary notes on the specific interpretation of each lesion and may include colour print-outs of representative features of each specimen to assist in identification of specific diagnostic features;

This was achieved. Reference material in the form of a set of histological slides containing a variety of liver lesions was provided to the participants of the first workshop. In addition, a large number of images and supporting diagnostic information has been collected during the contract. Many of these were sent to participating laboratories as part of the developmental process for the CD ROM which forms one of the major outputs of this contract. The provision of reference material remains an ongoing process, which was discussed in detail at the second workshop (see below). In addition, the specific requirements for “The use of liver histopathology for the assessment of the biological effects of contaminants” have been prepared for publication in the ICES Techniques in Marine Environmental Sciences (TIMES) series. This includes figures of the major lesion types with full descriptions and key diagnostic features.

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• The design and implementation of intercalibration programmes for diagnosis of liver lesions. Material collected during national monitoring programmes shall be tested, with material to be supplied by the lead laboratory and at least one other. This shall incorporate liver material for the confirmation of gross pathology and randomly selected livers from specific size groups of fish. Twelve participating laboratories from across Europe shall be involved in the evaluation of the test materials which will be sent by post;

This was achieved. A ring test for the diagnosis of histological liver lesions was designed and implemented. A summary of the ring test report is included below.

• Two workshops, in years 1 and 3 shall be held. The first shall involve the establishment of protocols and practical exercises on diagnostic criteria. These shall determine the degree of agreement in the interpretation of pathology between participants and enable agreed limits of acceptable variation in diagnostic reporting to be set. The main aim of the year 3 shall be to assess the results obtained from earlier intercalibration exercises and also data obtained from national monitoring programmes;

This was achieved. Two workshops were held at the Centre for Environment, Fisheries and Aquaculture Science (CEFAS) Weymouth Laboratory, Barrack Road, The Nothe, Weymouth Dorset. DT4 8UB. UK. The summary of the workshops is provided below. • Establishment of procedures for appropriate action in the event of consistent disagreements in diagnoses of

particular lesions between participating laboratories;

Three main procedures have been adopted to resolve disagreements in the diagnosis of liver lesions. The most important has been to use an independent expert for assessment of “difficult” specimens. In this role and since the beginning of the BEQUALM programme, Dr M. Myers (Environmental Conservation Division, Northwest Fisheries Centre, National Marine Fisheries Service/NOAA, Seattle, USA) has kindly undertaken this task. His expertise has been extremely valuable in assisting BEQUALM in resolving specific questions relating to the discrimination between key lesion categories. Secondly, the freely offered material from North American fish species has proved invaluable for our familiarisation with lesion types rarely or not recorded from the main European species used in monitoring programmes (dab and European flounder). Some of this material has been included in the CD-ROM and has been included in the set of reference images available for BEQUALM participants. Finally, a diagnostic key providing detailed information and cut-off points for accurately determining lesion features to allow placement in specific lesion categories was prepared.

• The production of a colour atlas of common histopathological liver changes, possibly using CD-ROM technology.

This was achieved. A CD ROM containing a comprehensive methodology, protocols and reporting procedures for all aspects of environmental monitoring using fish diseases and liver pathology was produced. Several BEQUALM participants contributed material for the CD. This was incorporated and the final version of the CD and will be distributed to participants. The appearance of the front page and an example of a teaching sequence section are shown below.

6

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Summary of the workshops

First workshop held at CEFAS Weymouth Laboratory, 21–23 October, 1999

The first workshop aimed at establishing protocols for fish disease monitoring and studies on liver histopathology. Existing sampling strategies developed through ICES activities were reviewed. Training and intercalibration of diagnostic criteria formed an important component of the workshop. The full report is available on the BEQUALM website.

Several key points and recommendations arose from the workshop. In addition, the main aims of the meeting were achieved. These are summarised below:

• There was agreement for the protocols for fixation, histological processing and staining of livers for general diagnosis;

• Agreement was reached on the diagnostic criteria to be used for histological assessment of liver histopathology; • Fish disease monitoring programmes were generally were well integrated with other biomarker and analytical

measurements; • Research projects involving laboratory studies into the use of biomarkers were being investigated in some

countries; • There were reduced national programmes for monitoring fish diseases in the North Sea and the Baltic Sea reported

by most participants; • There appeared to be a reducing prevalence of some external diseases in some areas; • There appeared to be a reducing prevalence of liver changes in the target fish species, dab and flounder; • Where there was a reduction of programmes, it was reported that it was not always possible to adhere to

standardised sampling strategies; • The reason for the reduced efforts were related to reduction in project budgets; • It was agreed that there was a need for continued development of techniques of potential value for liver pathology

assessment; • It was recommended that for quality assurance purposes a CD-ROM should be made as a training guide for

sampling and examining fish in field studies; • This CD-ROM would also incorporate the proposed “Atlas of flatfish liver histopathology”; • It was recommended that a ring test for standardisation of interpretation of liver pathology be organised by

CEFAS Weymouth; • It was recommended that outside experts should be engaged as impartial judges in the event of disagreements of

interpretation.

Second workshop held at CEFAS Weymouth Laboratory, 8-11 November, 2001

All of the recommendations from the first workshop were taken up and developed during the second year of the contract. The second workshop addressed the continuing need for training in liver histopathology diagnosis, the results of the ring test and the finalisation of the CD-ROM. The full report is available on the BEQUALM website.

Several key points and recommendations arose from the workshop. These are summarised below:

• The integration of existing routine disease studies with other biomarkers, environmental and contaminant measurements continues to improve.

• A number of successful research projects aiming to correlate biomarker responses during contaminant exposure have been carried out.

• The BEQUALM workshop was successful in establishing criteria for external disease assessment and liver pathology diagnosis and has identified areas that require further refinement.

• The ring test for the diagnosis of flatfish liver histopathology was completed successfully.

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• It was recommended that additional reference material should be provided to participating laboratories and that further ring tests should be instigated on a regular basis.

• It was agreed that the CD-ROM (v.1) provides the necessary information to undertake field studies using externally visible diseases and laboratory-based assessment of liver histopathology.

• It was recommended that the incorporation of disease assessment quality assurance criteria (including multi-organ histopathology) for other fish species be investigated, thereby broadening the interest for this component of BEQUALM to other potential participants in Europe and further afield.

Summary of ring test

This task was completed. The main objective of the ring test was to assess the diagnostic accuracy of different laboratories for the main potentially toxicopathic lesion types encountered in the liver of fish. The results obtained allowed for an assessment of problem areas and possible weak points in diagnostic criteria. It was suggested that for future trials of this type, a nucleus of twelve to fifteen laboratories should be involved. It was also stated that ring tests carried out on a continuous basis are vital in ensuring that all participating laboratories are tied into a quality assurance programme that allows for accurate and transferable reporting of data to international databases.

A number of important points were highlighted during the meeting:

• Considerable variation in diagnostic proficiency was observed between different participating laboratories. The highest level of proficiency for diagnostic scoring was ~ 90 %, while the lowest score was ~ 30 %, comparative to the master score sheet.

• Participating laboratories generally over-scored for the presence of foci of cellular alteration (FCA).

• Participating laboratories generally under-scored on the presence of malignant neoplasms (such as hepatocellular carcinoma).

• There appeared to be two major ‘cut-off’ areas that caused diagnostic problems for participating laboratories. The first one was in the differentiation between the pre-neoplastic lesions (FCA) and the benign neoplasms (such as adenoma) and the second one was between the benign neoplasms and the malignant neoplasms (carcinoma). In light of this information, a flow diagram was designed to show which characteristics each group of lesions possess and how these criteria should be used to make a diagnosis. The flow chart had been designed by incorporating the results of the ring trial and Dr Myers’ description of the lesion types from these slides. All of the details required for the successful diagnosis of pre-neoplastic lesions are in place. However, for diagnosis of FCA types, a further flow diagram is required to describe characteristic cellular staining. The flow diagram can be seen below.

All workshop participants agreed that re-sending of the original ring test slides along with Dr Myers’ descriptions and electronic images of the principal lesion to all ring test participants would be beneficial for training purposes. Furthermore, it was suggested that a liver histopathology training workshop should be held, possibly in conjunction with the next EAFP meeting in 2003.

Agreed protocols

As stated above, from the initiation of the BEQUALM programme, emphasis was given to the establishment of agreed protocols. These encompass every procedure from collection of fish samples, handling, external disease assessment and recording, sampling for organ pathology, laboratory procedures, diagnostic criteria and reporting. All of these are fully explained in the CD-ROM and in the workshop reports. Reporting forms for external disease assessments and macroscopic liver pathology are provided in a downloadable format for use in field situations.

Acceptable limits of variation

As indicated above, ICES has developed requirements for the international reporting of fish diseases over many years in order to minimise variation between laboratories regarding the accuracy and reproducibility of data generated. These have been reviewed by BEQUALM and produced in CD-ROM format. Each externally visible disease (lymphocystis, acute and healing skin ulcerations, epidermal hyperplasis/papilloma and liver tumours) has a minimum requirement for reporting and severity is assessed according to criteria allocated to three stages (lymphocystis, ulcerations and epidermal hyperplasia/papilloma only). Macroscopic liver nodules are only recorded if the minimum diameter exceeds 2 mm. Each case has to be verified histologically to exclude the possibility that lesions are the response to parasites, cysts, necrotic or inflammatory foci. As such the acceptable limits of variation for disease recording are well established.

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Submission of fish disease data to the ICES Marine Data Centre has been formalised by the introduction of the ICES Environmental Reporting Format designed specifically for the purpose. This is used for fish disease, contaminant and biological effects data. The programme includes internal screening procedures for the validation of the data submitted providing further quality assurance.

With regard to the application of liver histopathology as a tool in biological effects monitoring, the activities undertaken within BEQUALM have been successful in the establishment of the methodology and diagnostic criteria. The diagnostic key (see below) provides clear criteria to discriminate between the lesion types, thus minimising the possibility of mis-diagnosis.

However, the results of the ring test on the diagnosis of liver pathologies demonstrated that there was considerable variation in proficiency between participating laboratories. Therefore, it was emphasised that further intercalibration exercises are required in order to minimise inter-observer variation and to establish acceptable limits of variation. These should be carried out as an ongoing process in order to ensure continuous quality assurance of data obtained.

.

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Presence of nodule*

Lack of compression**

Presence of compression**

Little disruption to normal architecture. Tubules blend with

surrounding parenchyma

Disruption/thickening of tubular architecture, presence of enlarged cells,

clear separation from surrounding parenchyma

Lacking cellular atypia*atypia not significantly

surrounding par

Level of cellular asignificantly above that

parenchym

* Nodule: A discrete accumulation of hepatocytes (more than 10 cells). These lesions have usually a decreased concentration of pigmented

** Compression: Complete compression around the circumference of a lesion is rare. More commonly, compression is seen at certainrelatively compressed. The lesion periphery may be marked by the presence of MMC’s and flattened blood vessels.

*** Atypia: Most livers will contain a certain degree of nuclear and cellular atypia (though proportions of atypical nuclei and cells wiimportant to consider the degree of nuclear (size, shape, chromatin pattern) and cellular (size, shape, cytoplasmic content) atypia relativsitu, the degree of cellular and nuclear atypia within the suspect carcinoma should be greater than that of the surrounding nodule parench

Diagnostic key: Hepatocellular nodules (after Couillard, Stentiford, Feist, Myers2003 WG

PDM

O Report

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Hepatocellular carcinoma

** or a level of above that of

enchyma

FCA

Hepatocellular adenoma

typia*** of surrounding

a

macrophage aggregates relative to the surrounding parenchyma.

sections of the lesion periphery. Adjacent parenchyma will appear

ll be very small in most cases). In terms of lesion diagnostics, it is e to the surrounding parenchyma. For the diagnosis of carcinoma in yma.

& Fournie)

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Conclusions

Largely through activities of the International Council for the Exploration of the Sea (ICES), standardised methodologies for surveys on the occurrence of externally visible diseases of flatfish species from the North Sea and adjacent areas have been developed and intercalibrated repeatedly. Practical guidelines have been established for all methodologies involved, including sampling of fish, diagnosis of diseases, reporting of data to ICES and statistical data analysis.

As part of the work carried out in BEQUALM Work Package 6, these guidelines were reviewed and, where necessary, additional details and methodologies for the collection, diagnosis and reporting of fish disease data were provided. This information can be found on the CD-ROM produced.

For the reasons detailed above, the participants of BEQUALM Work Package 6 consider that methodologies developed for surveys on externally visible fish diseases of flatfish have sufficiently been tested and intercalibrated and are readily applicable in international European monitoring programmes. Disease data generated by institutes actively participating in the above quality assurance procedures can be regarded to be of consistently high quality and can be utilised as scientific baseline data for future monitoring programmes.

The need to formalise and provide quality assurance for the application of liver histopathology as a tool to detect biological end points of contaminant exposure formed the major emphasis for the activities undertaken in Work Package 6.

Preliminary guidelines for macroscopic and microscopic inspection of flatfish livers for the occurrence of neoplastic lesions have also been provided by ICES. Within BEQUALM Work Package 6, these guidelines were reviewed and, based on this assessment, a strategy for the implementation of quality assurance procedures was developed and successfully applied. This comprised the definition of toxicopathic histopathological liver lesions useful for monitoring purposes, intercalibration programmes on the diagnosis of these lesion types (including a ring test), the provision of reference materials, diagnostic keys and a colour atlas of common lesions in marine flatfish species for supporting the diagnosis of histological slides, and technical guidelines for standard operating procedures for all histological techniques involved, from tissue sampling to staining procedures. All relevant information was included on the CD-ROM produced as part of the tasks of Work Package 6.

The partipants of the work carried out within Work Package 6 emphasised that quality assurance for externally visible diseases and liver histopathology of flatfish species has to be considered as an ongoing process and that, therefore, activities initiated through BEQUALM have to be continued. Ways how this can be accomplished should be explored.

Work programme for 2003

The work programme for the first year of the self-funded scheme is as follows:

1. Send letters of invitation to prospective new participants. 2. Prepare and distribute reference materials. These will consist of images of histopathological lesions at different

magnifications with explanatory text. In addition, material from the last ring test will be sent again with the full diagnosis for training purposes.

3. The CD will be updated with material supplied by participants as appropriate. 4. The reference collection will be supplemented by submission of material from monitoring programmes and ad hoc

investigations. 5. A ring test will be undertaken as before using a single set of histological slides. This will involve distribution,

collation of results and production of results. 6. Since the full ring test is time consuming, smaller trials will be instigated using e-mailed images of specific

pathology for diagnosis. These will form an important part of the on-going training required. 7. Produce section for the BEQUALM Newsletter. 8. Update BEQUALM website (www.bequalm.org).

It is not intended to organise a workshop in 2002/03 but this will obviously form a key activity in future years.


ANNEX 13: REVIEW AND ASSESS THE IMPACT OF DISEASES OF FARMED FISH ON WILD FISH STOCKS

D.W. Bruno, FRS Marine Laboratory, Aberdeen, UK Introduction

Atlantic salmon, Salmo salar and rainbow trout are traditional species for aquaculture, but the decline in catches of wild Atlantic cod, Gadus morhua has led to an increase in the market value and stimulated interest in farming of this and other species. For example, Atlantic halibut, Hippoglossus hippoglossus has been farmed for several years in Norway, the UK, Iceland and Canada and annual production is increasing. Haddock, Melanogrammus aeglefinus and Dover sole, Microstomus pacificus are likely to be farmed in increasing numbers in the near future. Several RNA and DNA viruses infecting fish have been studied intensively and many induce diseases resulting in economic losses especially in aquaculture. The importance of several important RNA viruses such as orthomyxo-, paramyxo-, rhabdo-, toga-, picorna -, noda- and birnaviruses, and DNA viruses such as parvo-, irido-, and adeno-viruses are reviewed by Essbauer and Ahne (2001). There are examples of carrier status and equally examples where mass mortality has been attributed to virus infections. Clearly any increase in the diversity of fish species increases the opportunity for disease interaction, however there are practical difficulties in measuring the prevalence, incidence and the pathogenicity of diseases in wild stock and the factors associated with the host and environment have not been widely investigated. As most bacterial, parasite and viral diseases have a broad host range there is also potential transfer of infection from wild to farmed fish, and equally from farmed to wild fish. It should be emphasised that knowledge of disease transfer and the impact of disease on wild fish is limited.

Viral infections Infectious pancreatic necrosis virus (IPNV)

Infectious pancreatic necrosis (IPN) is a highly contagious viral disease of young fish particularly of salmonid species held under intensive rearing conditions. IPN virus (IPNV), or viruses showing serological relatedness to IPNV, have been reported to cause disease in farmed marine fish species, including yellowtail (Seriola quinqueradiata), turbot (Scophthalmus maximus), halibut (Wood et al., 1996), Atlantic salmon (Sousa et al., 1996), sea trout and Atlantic cod (Rimstad et al., 1990; Crane et al., 2000). The first report of IPNV in farmed Atlantic cod occurred in fry on the west coast of Denmark during 1991 with almost total loss of the stock. Between 1992–1994, Lorenzen et al. (1995) also reported a similar high mortality among farmed fry from the Faroe Islands, Denmark. Likewise, IPNV has been detected in wild caught chinook salmon in New Zealand after an estimated 2–3 years in marine waters (Tisdall and Phipps, 1987) and in wild trout from Nova Scotia, Canada. The virus is widespread in farmed fish particularly in Norway and Scotland and an increasing number of species is found carrying the virus. Hatcheries and smolt mortality particularly in the Shetland Isles, Scotland is increasing, for example, in Shetland the rise in prevalence of IPNV in marine waters among farmed fish has increased from 44 % to 75–80 % in 1999 and 2000. In Shetland’s freshwater sites prevalence rose from 16 % to 30–40 %. Clinical IPN in Scotland has also increased from a prevalence of 1.1 % to 12.5 % for the whole of Scotland between 1988–2002. The source of the virus is speculative, and could include the transfer of infected fish from fresh water, failure of fish to adapt to sea water, strain differences emerging in certain areas and expression of low level virus or a wild reservoir. (Bruno and Woo, 2002). IPNV is stable for long periods in sea and brackish waters and this could also have an impact on seawater outbreaks of IPNV and transmission from wild to farmed stock, and visa versa.

Wild fish surveys carried out within Scotland to determine the prevalence of IPNV in wild marine and freshwater fish are detailed below.

1. A survey of Atlantic salmon and brown trout Salmo trutta captured by electrofishing at 11 freshwater river sites from 11 areas in Scotland was carried out. Juvenile salmon were taken in preference to brown trout and in total 330 fish were examined, comprising 222 Atlantic salmon parr and 118 brown trout. Two out of three pools from salmon were positive for IPNV from the River Arnisdale (NG 860 098, area 41, case number 20020955). This site had been stocked with salmon fingerlings obtained from a supplier specialising in producing salmon and sea trout from wild origin. This survey area was also located nearby a large farm site that had also tested positive for IPNV.

2. A demersal trawl survey at locations in the North Sea, namely offshore from the Firth of Forth, Moray Firth and Fair Isle where a total 2,753 fish consisting of 15 species were sampled. One haddock was virus positive and additional work is required to identify the isolate. A further ten locations were sampled on the West Coast of Scotland in the Kyle of Lochalsh area comprising 3,765 fish (18 species). All these samples were tested by tissue culture and were virus negative.


3. A survey in the Firth of Clyde comprised 779 fish (14 species). One non-IPN virus positive sample is currently being identified.

4. Freshwater fish were obtained from 15 locations throughout Scotland using electrofishing and gillnetting. In total 923 fish from 7 species were sampled, primarily juvenile Atlantic salmon and brown trout. One salmonid was found to be IPN positive by tissue culture.

Nodaviridae

Nodavirdae are considered the most economically important emerging viral pathogen and halibut, cod and salmon are considered susceptible (Bruno and Woo, 2002). The Nodaviridae have been reported as the causative agents of disease throughout the world and referred to as viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER). Lesions show the classic signs of VNN and the brain and retinal tissues from moribund fish showed diffuse degenerative vacuolative encephalopathy and degenerative histiocytic retinitis. Significant losses are reported in juvenile and adult fish. In Norway, mortality reaching 100 % has occurred in juvenile farmed Atlantic halibut (Grotmol et al., 1997; Johans et al., 2002), and Aspehaug et al. (1999) reported viral nervous necrosis in adult and mature halibut. In addition, mortality between 11–60 % of farmed sea bass at different locations in Greece has been reported (Le Breton et al., 1997). Disease outbreaks have also occurred in juvenile cod (Starkey et al., 2001), hence hatcheries must reduce the risk of this virus entering if reliable larval fish production is to be maintained; however, vertical transmission of the virus may hinder control measures. Recently RT-PCR in broodstock cod were found in the USA, although no virus was isolated and large scale mortality of cultured haddock occurred in a facility in New Brunswick, Canada. VER continues to be a problem in sea bass farms in France and a new isolate was found in sea bass. It was pathogenic at temperatures < 18°C and was similar to the isolate of Atlantic halibut. There were no isolations of nodavirus from wild or farmed fish in Scotland during 2002 but the increasing number of susceptible species identified worldwide will increase the chance of infection moving from wild to farmed stock and visa versa.

Infectious salmon anaemia virus (ISAV)

Infectious Salmon Anaemia virus (ISAV) is the cause of ISA in salmonids, and is a serious problem in farmed fish in Norway, Scotland, Canada, Maine, USA and the Faroe Islands, Denmark. The virus has been shown experimentally to be transmitted between fish in fresh water, and experimentally and clinically in sea water. The infection is spread by management activity such as well-boat traffic, but also possibly through contact with wild fish (Rimstad and Mjaaland 2002).

The susceptibility of cod and halibut to ISAV infection is unknown, although experimentally it has not been possible to infect Atlantic halibut or Atlantic cod indicating that they are resistant to this virus. In addition wild-caught saithe, Pollachius virens appear to be resistant to this Norwegian isolate of ISAV and incapable of supporting its replication (Snow et al., 2002). Hence, saithe that co-exist with salmon in and around aquaculture facilities are unlikely to have a significant impact on the epizootiology of this virus. In addition the apparent limited host range for ISAV would suggest that outbreaks will be infrequent in new fish groups. A wild host for the virus has not been identified.

Infectious Haematopoietic Necrosis Virus (IHNV)

Atlantic salmon (Salmo salar) are the primary species cultured in British Columbia and are particularly susceptible to IHNV. Several farms experienced significant losses in the winter of 2001/2002. The source of the virus is suspected to be wild stocks in the vicinity of the Atlantic salmon farms since all fish are health-tested prior to transfer to the net cage sites.

Viral haemorrhagic septicaemia virus (VHSV)

Viral haemorrhagic septicaemia (VHS) is traditionally considered a disease of rainbow trout resulting in extensive losses to freshwater culture operations across continental Europe. An outbreak of VHS in farmed turbot in Scotland in 1994 prompted the initiation of a widespread VHS virus (VHSV) surveillance programme across the European marine environment. This resulted in the recovery of over 150 isolates of VHSV from a wide range of wild marine fish species in the North Sea, Baltic Sea and eastern Atlantic (Mortensen et al., 1999; King et al., 2001). Most isolates are from herring (Clupea harengus) and sprat (Sprattus sprattus). It is likely that the Baltic Sea, the Kattegat and Skagerrak are the most prevalent areas for marine VHS in Northern Europe. Studies show that marine VHSV isolates are of low risk to rainbow trout and Atlantic salmon and marine VHSV isolates appear to be of low risk to marine aquaculture species other than turbot. VHSV has been reported to infect wild cod, haddock and many other marine fish species (Mortensen et al., 1999; Smail 2000; King et al., 2001), although epizootics resulting in morbidity have not been observed in wild fish in Europe.


In 1988 adult chinook (Oncorhynchuus tshawytscha) and coho (O. kisutch) salmon returning to coastal hatcheries in Washington State, USA were examined and found to harbour VHSV. These resulted in the mandated destruction of all fish (approximately 5 million eggs, fry, and juveniles) and complete disinfection of the two facilities (Winton et al. 1991). In subsequent years, VHSV was recovered from other stocks of salmon in Washington State and from Pacific cod in Prince William Sound, Alaska (Stewart et al., 1990; Meyers et al., 1992). Research comparing the North American and European isolates of VHSV revealed differences at the biological level, such as growth in cell culture (Batts et al., 1991) and virulence (Winton et al., 1991). Additional differences were found at the genetic level (Oshima et al., 1993). A European strain of viral haemorrhagic septicaemia virus (VHSV) isolated from wild-caught cod was shown to cause clinical disease and mortality in excess of 80 % in juvenile Atlantic cod when administered by the intra-peritoneal (i.p.) route (Snow et al., 2000). Cod were not susceptible to VHSV following waterborne exposure and this raises important questions surrounding the propagation, maintenance and impact of a naturally occurring reservoir of virus in the marine environment. Furthermore, Atlantic halibut appear to be resistant. Recently high mortality has been reported in sardines (Sardinops sagax) around Vancouver Island, Canada with isolation of the European strain. The sandeel was also recently identified as a carrier of VHS in Denmark. VHSV is now divided into several genotypes and the risk of infection and transfer will depend on the species and genotype.

Aquareovirus

An Aquareovirus was demonstrated in macrophages of both naturally and experimentally infected halibut in Canada (Cusack et al., 2001). The virus survived and replicated in the phagocytic cells, as demonstrated by electron microscopy. In Scotland farmed moribund Atlantic halibut larvae were sampled for a suspect nodavirus infection. An examination of the tissue sections showed moderate degeneration of hepatocytes with slight haemorrhage or capillary dilation, irregular clear vacuoles, cell rounding and early necrosis with cell fragments. Kidney tubule dilation was recorded and an increased faint eosinophilic staining within the trabeculae layer. The samples were negative for IPN, VHS, ISA, nodavirus and bacterial infection and currently the aetiology of the mortality in Scotland is a suspect Aquareovirus.

Sleeping disease virus (SDV)

An outbreak of sleeping disease was confirmed at a trout fish farm on the River Earn, Scotland system in June. Wild fish were obtained from three locations within the catchment area, using a combination of electrofishing and gillnetting. In total 176 fish consisting of juvenile Atlantic salmon, juvenile and adult brown trout, escapee rainbow trout, stone loach and minnow were sampled. Tissue culture, PCR, histology and blood samples were obtained and the tests were negative. No isolations have been found in the wild fish samples.

Salmon pancreas disease virus (SPDV)

Salmon pancreas disease prevalence is low in farmed salmon and the virus has not been detected in wild fish in Scotland. Weston et al. (2002) reported minor biological differences between SPDV and SDV and concluded on the basis of the close genetic similarity that they are closely related isolates of the same virus species for which the name salmonid alphavirus was proposed. However, there is increasing mortality on many sites in Ireland. The disease has been observed at low levels during the past 8–9 years and its re-emergence in 2002 cannot be readily explained. Certain sites have suffered in excess of 30 % stock losses whilst others have experienced lower but more protracted losses. A nationwide epidemiological study has just been launched to determine the factors which may have contributed to the re-emergence of this economically significant disease. Pancreas Disease was diagnosed on 12 sea sites in Norway. Two of these cases were on rainbow trout. The losses vary, but may last over a long time period and become substantial. All reported cases so far have been in Western areas, however, there is a trend that the disease is spreading. Diseases cycle and incidence will change over time and at the present time this disease is restricted to farmed fish.

Bacterial infections

Among the major bacterial pathogens affecting farmed halibut, salmon, cod and trout resistance and susceptibility varies. For example, halibut are resistant to Aeromonas salmonicida, whereas Renibacterium salmoninarum is confined to trout and salmon. Bergh et al. (2001) concluded that Vibrio anguillarum, Flexibacter ovolyticus and atypical Aeromonas salmonicida were the major bacterial pathogens affecting halibut production.

Wild fish have been recorded dying of furunculosis during high water temperatures following transfer.

Between 1998–2002 there is no major trend in bacterial infections. It is noteworthy that P. salmonis was confirmed in Scottish Atlantic salmon from tissue sections and ELISA in 2002.


Parasite infections

Protozoan and metazoan parasites have been recorded from halibut and include Ichthyobodo sp., the microsporidium Enterocytozoon sp., Trichodina hippoglossi, Entobdella hippoglossi and Lepeophtheirus hippoglossi (Bergh et al., 2001). In general the impact of these parasites on farmed and wild fish is unknown.

Sea lice

Norway started a National Action Plan against “Salmon lice on Salmonids” in 1997 (NA). The legal limit for the maximum mean number of lice per farmed fish, strategic regional treatments against lice and compulsory reporting of lice numbers were included (Heuch et al., in press). Bag net catches show that the homing salmon are infected by juvenile lice as the fish pass through coastal zones and this appears to be related to salmon farming. Homing salmon that stay in fjords and coastal areas for prolonged periods may have a high number of mature female lice. Work by Karasev et al. (1997) shows that salmon lice infestations in the White Sea basin show low salmon lice in this farm-free area. Data show high salmon louse infestation levels in populations of sea trout and sea charr, and predictions of negative effects on fish populations. Long-term experiments and monitoring are required to assess the regulatory effect of lice in these populations. Sea trout and Arctic charr over-winter in fresh or brackish waters and the lice generally die, resulting in a low prevalence and intensity of adult females; the infection pressure is low and does not constitute a threat to running salmon smolts. Heuch et al (in press) has looked at national data sets from Scotland and Norway and concluded that the salmon in Scotland are exposed to higher levels of mobile and chalimus infestations than Norwegian farmed salmon. In Norway production takes place in mixed year classes and single year classes in Scotland. Furthermore, there are differences in cage size and stocking densities, however, there are still differences even after adjustment for these management practices.

Gyrodactylus salaris

Gyrodactylus salaris has occurred in three hatcheries in Norway. One of the hatcheries was infected in the 1970s and remained clear until now. The hatchery is located close to an infected river system and could represent the source of this infection. Infections in the other hatcheries could have resulted from fish movements.

References

Aspehaug, V., Devold, M., and Nylund, A., 1999. The phylogenetic relationship of nervous necrosis virus from halibut (Hipploglossus hipploglossus). Bull. Euro Assoc. Fish Pathol,. 19: 196–202.

Batts, W.N., Traxler, G.S., and Winton, J.R. 1991. Factors affecting the efficiency of plating for selected fish rhabdoviruses. In Proceedings of the Second International Symposium on Viruses of Lower Vertebrates, p. 17–24. Oregon State Univ. Press, Corvallis.

Bergh, O., Nilsen, F., and Samuelsen, O.B., 2001. Diseases, prophylaxis and treatment of the Atlantic halibut Hippoglossus hippoglossus: a review. Dis. Aquat. Org., 48: 57–74.

Bernard, J., Lecocq-Xhonneux, F., Rossius, M., Thiry, M.E., and de Kinkelin., P., 1990. Cloning and sequencing the messenger RNA of the N gene of viral haemorrhagic septicaemia virus. J. Gen. Virol., 71 : 1669–1674.

Bruno D.W., and Woo, P.T.K., 2002. Sporadic and Emerging diseases. In Diseases and Disorders of Finfish in Cage Culture. Eds., Woo P.T.K. Bruno D.W., and Lim S.H.L. CABI Publishing, Oxon, England.

Crane, M.S., Hardy-Smith, P., Williams, L. M., Hyatt, A.D., Eaton, L.M., Gould, A., Handlinger, J., Kattenbelt, J., and Gudkovs, N. 2000. First isolation of an aquatic birnavirus from farmed and wild fish species in Australia. Dis. Aquat. Org., 43 : 1–14.

Cusack, R.R., Groman, D.B., MacKinnon, A.M., Kibenge, F.S., Wadowska, D., and Brown, N. 2001. Pathology associated with an aquareovirus in captive juvenile Atlantic halibut Hippoglossus hippoglossus and an experimental treatment strategy for a concurrent bacterial infection. Diseases of Aquatic Organisms, 44: 7–16.

Essbauer, S., and Ahne, W., 2001. Viruses of lower vertebrates. J Vet. Med. B Infect. Dis. Vet Public Health, 48: 403–475.


Grotmol, S., Totland, G.K., Thorud, K., and Hjeltnes, B.K., 1997. Vacuolating encephalopathy and retinopathy associated with a nodavirus-like agent: a probable cause of mass mortality of cultured larval and juvenile Atlantic halibut Hippoglossus hippoglossus L. Dis.Aquat. Org., 29: 85–97.

Heuch, P.A., Bjørn, P.A., Finstad, B., Holst, J.C., Asplin, A., and Nilsen, F. Relationships between salmon lice on wild and farmed salmonids: A review of population dynamics, management measures and effects on wild salmonid fish stocks in Norway, In press.

Heuch, P.A., Revie, C.W., and Gettinby, G. A comparison of epidemiological patterns of salmon lice (Lepeophtheirus salmonis) infections in Norway and Scotland. In press.

Johans, R., Ranheim, T., Hansen, M.K., Taksdal, T., and Totland, G.K., 2002. Pathological changes in juvenile Atlantic halibut Hippoglossus hippoglossus persistently infected with nodavirus. Dis.Aquat. Org., 50: 161–169.

Karasev, A.B., Kuzmin, O.G., Finstad, B., and Nilsen, S.T. 1997. Occurrence and levels of infestation of salmon lice, Lepeophtheirus salmonis on wild salmon of the White Sea. ICES CM 1997/M:4, Ref: F.

King, J.A., Snow, M., Smail, D.A., and Raynard, R.S., 2001. Distribution of viral haemorrhagic septicaemia virus in wild fish species of the North Sea, north east Atlantic Ocean and Irish Sea. Dis.Aquat. Org, 47: 81–86.

Le Breton, A., Grisez, L., Sweetman, J., and Ollevier, F., 1997. Viral nervous necrosis (VNN) associated with mass mortalities in cage-reared sea bass, Dicentrarchus labrax (L.). J. Fish Dis., 20: 145-151.

Meyers, T.R., Sullivan, J. Emmenegger, E., Follett, J. Short, S Batts, W.N., and Winton, J.R., 1992. Identification of viral hemorrhagic septicemia virus isolated from Pacific cod Gadus macrocephalus in Prince William Sound, Alaska, USA. Dis. Aquat. Org., 12 : 167–175.

Mortensen, H.F., Heuer, O.E., Lørenzen, N., Otte, L., and Olesen, N.J., 1999. Isolation of viral haemorrhagic septicaemia virus from wild marine fish species in the Baltic sea, Kattegat, Skagerrak and the North Sea. Virus Research, 63: 95–106.

Oshima, K.H., Higman, K.H., Arakawa, C.K., de Kinkelin, P., Vestergard Jorgensen, P.E., Meyers, T.R., and Winton. J.R. 1993. Genetic comparison of viral hemorrhagic septicemia isolates from North America and Europe. Dis. Aquat. Org., 17 : 73–80.

Rimstad, E., Krona, R., Hornes, E., Olsvik, O., and Hyllseth, B. 1990. Detection of infectious pancreatic necrosis virus (IPNV) RNA by hybridization with an oligonucleotide DNA probe. Vet Microbiol, 23: 211–9.

Rimstad, E., and Mjaaland, S. 2002. Infectious salmon anaemia virus. Apmis., 110: 273–282.

Smail, D.A. 2000. Isolation and identification of viral haemorrhagic septicaemia (VHS) virus from North Sea cod (Gadus morhua L.). Diseases of Aquatic Organisms, 41: 231–235.

Snow, M, Cunningham, C.O., and Bricknell, I.R., 2000. Susceptibility of juvenile Atlantic cod Gadus morhua to viral haemorrhagic septicaemia virus isolated from wild-caught Atlantic cod. Dis. Aquat. Org., 41 : 225–229.

Snow, M., Raynard, R., Bruno, D.W., Van Nieuwstadt, A.P., Olesen, N., Løvold, T., and Wallace, C. 2002. An investigation into the susceptibility of saithe Pollachius virens to infectious salmon anaemia virus (ISAV) and their potential role as a vector for viral transmission. Diseases of Aquatic Organisms, 50: 13–18.

Sousa, J.A., Romalde, J.L., Ledo, A., Eiras, J.C., Barja, J.L., and Toranzo, A.E. 1996. Health status of two salmonid aquaculture facilities in North Portugal: Characterization of the bacterial and viral pathogens causing notifiable diseases. J. Fish Dis, 19: 83–89.

Starkey, W.G., Ireland, J.H., Muir, K.F., Jenkins, M.E., Roy, W.J., Richards, R.H., and Ferguson, H.W. 2001. Nodavirus infection in Atlantic cod and Dover sole in the UK. Vet Rec., 149: 179–181.

Stewart, B.C., Olson, C., and Lutz, S., 1990. VHS virus detected at Lummi Bay Sea Ponds, Bellingham, Washington. Am. Fish. Soc. Fish Health Newsletter, 18: 2–3.


Tisdall, D., and Phipps, J. 1987. Isolation and characterisation of a marine birnavirus from returning quinnat salmon (Oncorhynchus tshawtscha) in the South Island of New Zealand. New Zealand Vet. J., 35: 217–218.

Weston, J., Villoing, S., Bremont, M., Castric, J., Pfeffer, M., Jewhurst, V., McLoughlin, M., Rodseth, O., Christie, K. E., Koumans, J., and Todd, D. 2002. Comparison of two aquatic alphaviruses, salmon pancreas disease virus and sleeping disease virus, by using genome sequence analysis, monoclonal reactivity, and cross-infection. J. Virol., 76: 6155–6153.

Winton, J.R., Batts, W.N., Deering, R.E., Brunson, R., Hopper, K., Nishizawa, T., and Stehr, C. 1991. Characteristics of the first North American isolates of viral hemorrhagic septicemia virus. In Proceedings of the Second International Symposium on Viruses of Lower Vertebrates, pp. 43–50. Oregon State Univ. Press, Corvallis.

Wood, B.P., Bruno, D.W., and Ross, K. 1996. Infectious pancreatic necrosis virus (IPNV) mortalities among farmed Atlantic halibut, Hippoglossus hippoglossus L. Bull. Euro. Assoc. Fish Pathol., 16: 214–216.


ANNEX 14: STRUCTURE OF THE INTEGRATED ICES DATABASE

(Record types marked by an arrow are relevant for fish disease information)

Seawater, Sediment and Biological Community dataBiota data

33Phytobenthos Transect Species Record

32Phytobenthos Transect Substrate Record

31Phytobenthos Transect Extent Record

20Sampling method

10Parameter measurement

07Tissue

04Specimen

93QA-information

27Storage Method

Analytical Methods

10Parameter measurement

38Abundance/Biomass measurement

||

||

03Sample

92Site/Haul

91Station/Area

90Platform/Cruise

00Header record


ANNEX 15: RAW DATA SUBMISSION TABLE FOR TEST OF THE INTEGRATED REPORTING FORMAT VERSION 3.2

Name of expert Area of expertise Raw data submitted: data types

Date sent

S.W. Feist Fish diseases, pathology Individual fish data, summaries (Excel spreadsheet)

3 March 2003

D. Bruno Fish diseases, pathology --- ---

T.Lang Fish diseases, pathology --- ---


ANNEX 16: REVIEW THE CRITERIA FOR THE INCORPORATION OF EXTERNALLY VISIBLE FISH DISEASES INTO MONITORING PROGRAMMES ON BIOLOGICAL EFFECTS OF

CONTAMINANTS

T. Lang (Bundesforschungsanstalt für Fischerei, Cuxhaven, Germany), W. Wosniok (Univ. Bremen, Bremen, Germany), C. Couillard (Institute Maurice-Lamontagne, Mont-Joli, Canada), S. MacLean, NOAA/NMFS, Narragansett,

USA)

Introduction

At its 2002 meeting, the ICES Working Group on Biological Effects of Contaminants (WGBEC) discussed the incorporation of externally visible fish diseases into the list of techniques recommended for biological effects monitoring regularly updated by WGBEC. The tables were first compiled in 1991 and updated subsequently in 1994, 1995, 1997, and 1999. Membership over this time had changed, so WGBEC revisited the criteria used to admit methods to the “promising” and “recommended” lists.

For recommended methods, criteria were defined as (from WGBEC reports 1994 and 1995):

1) Methods should have been adequately tested in both the laboratory and the field; 2) Satisfactory dose–response or concentration–response relationships should be known for a reasonable number of

chemicals; 3) They should be practical methods that have been approved by the WGBEC. It is desirable that as many methods as

possible should be published in the ICES Techniques in Marine Environmental Sciences (TIMES) series; 4) Their sensitivity compared with existing equivalent monitoring techniques should be known, if appropriate.

For promising techniques, members should (from WGBEC 1997 report):

1) Provide information, preferably by a presentation at WGBEC, on the method. 2) Provide information on sensitivity, repeatability and dose response. 3) Provide adequate documentation on methodology, standard procedures and references in the scientific literature.

Based on the discussions during the 2002 WGBEC meeting, two methods were removed from the recommended list: externally visible fish diseases and benthic community analysis. In both instances it was felt that they did not meet the criteria of exhibiting contaminant dose–response or concentration–response relationships. According to the WGBEC report, relationships between contaminant exposure and either external fish diseases or benthic community changes need to be investigated. WGBEC emphasised that it would welcome collaboration with other relevant ICES working groups, specifically WGPDMO and BEWG, on this issue.

This issue was taken up later by the ICES Advisory Committee on the Marine Environment (ACME) and there was some disagreement about the removal of the two methods mentioned above. WGBEC was, therefore, tasked for its 2003 meeting to:

ToR e): Review the criteria for the preparation of the tables on existing, recommended, and promising methods for biological effects monitoring, taking into account input from WGPDMO and BEWG, and thereafter revise the tables according to the various purposes of these tables;

Justification e): The criteria for including methods on these tables should be reviewed with the various purposes of monitoring in mind, e.g., general biological effects, specific contaminants (dose/response) relationship;

As input to the 2003 WGBEC meeting, the present working document prepared by WGPDMO members provides some background information on the present status of fish disease monitoring and on the role of ICES. It highlights the strengths and limitations of studies on externally visible diseases in the context of monitoring programmes on biological effects of contaminants and discusses the concepts for biological effects monitoring and the criteria used to select appropriate monitoring techniques.

Short history of fish disease monitoring under ICES

Diseases of wild marine fish have been studied on a regular basis by many ICES Member Countries for more than two decades. Disease surveys are often integrated with other types of biological and chemical investigations as part of


national and international monitoring programmes aiming at an assessment of the health of the marine environment, in particular in relation to the impact of human activities. Countries conducting more or less regular fish disease monitoring in the North Sea and/or adjacent waters, such as the English Channel, the Irish Sea, and the Baltic Sea are Germany, The Netherlands, UK, Poland, Russia and Sweden. Fish disease studies are also being carried out in the USA and Canada, and by Russia in the Barents Sea.

Since the early 1980s, ICES has played an active role in the initiation and coordination of fish disease surveys and has contributed considerably to the development of standardised methodologies. A fish disease data bank has been established within the ICES Marine Data Centre, consisting of disease prevalence data of key fish species and diseases and accompanying information submitted by ICES Member Countries. Quality assurance procedures have been implemented at all stages, from sampling of fish to submission of data to ICES (ICES, 1989; Bucke et al., 1996; Lang and Mellergaard, 1999; Wosniok et al., 1999, 2000; Lang and Wosniok, 2000; Lang, 2002).

Current ICES activities, through the work of the ICES Working Group on Pathology and Diseases of Marine Organisms (WGPDMO), have focused on the development and application of statistical techniques for an assessment of disease data with regard to the presence of spatial and temporal trends in the North Sea and western Baltic Sea. In a more holistic approach, analyses have been carried out combining the disease data with oceanographic, nutrient, contaminant and fishery data extracted from the ICES data banks in order to improve the knowledge about the complex cause-effect relationships. The results have shown that there are relationships between environmental factors, including contaminants, and the prevalence of the fish diseases considered. However, from this and other analyses there is clear indication that the relationship is not a simple one and that a variety of environmental and host-specific factors is involved in the aetiology and pathogenesis of diseases (Lang, 2002).

Types of externally visible diseases monitored

Based on the monitoring practice in the North Sea and the Baltic Sea, Table A16.1 provides an overview of externally visible diseases and parasites of common fish species that are quantified on a regular basis.

In combination with the quantification of externally visible diseases, many of the programmes include studies on neoplastic, pre-neoplastic and non-neoplastic macroscopic and microscopic liver lesions in flatfish (dab and flounder).


Table A16.1: Externally visible diseases and parasites of fish species in the North Sea and Baltic Sea that are monitored on a regular basis.

Disease/Parasite Aetiology Host Species

Lymphocystis Viral Dab (L. limanda)

Flounder (P. flesus)

Epidermal Hyperplasia/Papilloma Viral Dab (L. limanda)

Flounder (P. flesus) (Baltic Sea)

Skin Ulceration Bacterial Dab (L. limanda)


Cod (Gadus morhua)

Fin Rot Bacterial

Dab (L. limanda)


X-Cell Gill Lesion Probably Parasitic Dab (L. limanda)

Pseudobranchial Swelling Probably Parasitic Cod (G. morhua)

Acanthochondria cornuta Parasitic Dab (L. limanda)


Lepeophtheirus pectoralis Parasitic Dab (L. limanda)

Stephanostomum baccatum Parasitic Dab (L. limanda)

Lernaeocera branchialis Parasitic Cod (Gadus morhua)

Cryptocotyle lingua Parasitic Cod (Gadus morhua)

Hyperpigmentation Unknown Dab (L. limanda)

Flounder (P. flesus) (Baltic Sea)

Skeletal deformities

Multifactorial Dab (L. limanda)


Cod (Gadus morhua)

Relationship of externally visible diseases to environmental contaminants

It is generally accepted today that most diseases have a complex multifactorial aetiology. Most wild fish diseases monitored are caused by pathogens (see Table A16.1). However, other endogenous or exogenous factors may be required before the disease develops. One of these factors can be environmental pollution which either may affect the immune system of the fish in a way that increases its susceptibility to disease, or may alter the number and virulence of pathogens (including parasites). Contaminants may also cause specific and/or non-specific changes at various levels of biological organisation (molecule, subcellular units, cells, tissues, organs) leading to disease without involving pathogens (Lang, 2002).

There is manifold information in the literature from studies carried out in the past 20 years clearly indicating that marine pollution can induce significant changes in the prevalence of externally visible infectious and non-infectious diseases. Actually, the finding of elevated disease prevalences was the starting point for biological effects monitoring in the late 1970s.

In many instances, it is the observation of external lesions on fish and shellfish that has led to the identification of new groups of toxic environmental contaminants. For example, it is the observation of high prevalence of hermaphrodite fish (with grossly visible gonad abnormalities) that led to the identification of estrogenic chemicals in municipal effluents. The observation of oysters with abnormal shells and of gastropods with imposex led to the identification of tributyltin, as an agent causing endocrine disruption at very low environmental concentrations. Thus, monitoring of external abnormalities in fish and shellfish is an important way to identify the presence of new toxic contaminants in the


environment. It has also been used successfully to look at spatial gradients of contamination and to monitor the recovery of contaminated sites after the application of remediation actions (liver tumors, fin erosions, imposex).

Several fish and shellfish external diseases have been produced experimentally in fish exposed to contaminants. Imposex has been produced experimentally in gastropods exposed to tributyltin. Prevalence of imposex in feral gastropods has decreased following a reduction of the release of tributyltin to the marine environment (e.g., Svavarsson, 2000). Spinal deformities have been induced in fish exposed experimentally to a variety of contaminants including heavy metals, organochlorine pesticides such as toxaphene and kepone, and organophosphate pesticides. Vertebral deformities were generated by exposing fish to bleached kraft mill effluent or to isolated chlorinated compounds and were observed in feral fourhorn sculpin (Myoxocephalus quadricornis) caught near bleached kraft pulp mills in Sweden Bengtsson et al., 1988). Fin erosions have been produced experimentally in fish exposed to petroleum oil hydrocarbons and to pulp and paper mill effluent.

The prevalence of fin erosion declined following changes in the bleaching process in a Swedish pulp mill (Lindesjöö and Thulin, 1994) and reduction in the chemical discharges in sewage effluents in Boston Harbor (Moore et al., 1996), indicating that changes in the prevalence of fin erosion can be used as an indicator of changes in environmental contamination. According to Ziskowski and Murchelano (1975), the prevalence of fin erosion in winter flounder was markedly higher at a dump site in New York Bay compared to pristine offshore and nearshore sites away from the dumping ground.

Contrary to imposex, fin erosion and spinal deformities are not specific to contaminants. These lesions may occur in fish captured at non-contaminated sites and may result from various factors such as low dissolved oxygen, changes in temperature, bacterial infections, nutritional deficiencies.

During the ICES/IOC Bremerhaven Workshop, a correlation was found between prevalences of epidermal hyperplasia/papilloma in North Sea dab and a contaminant gradient from the inner German Bight to the Dogger Bank and also with contaminant levels in dab liver (Vethaak et al., 1992).

The immune system is a sensitive target of environmental pollutants and several groups of contaminants have been shown experimentally to alter the immune response of fish: PAHs, halogenated hydrocarbons, heavy metals, pesticides, bleached kraft pulp mill effluents, and several endocrine disruptor compounds. Juvenile chinook salmon collected from an urban estuary were more susceptible to a laboratory challenge to Vibrio anguillarum than salmon collected from non-urban estuaries and from a hatchery (Arkoosh et al., 1998).

In the last ten years, the Ohio EPA’s surface water program has used the occurrence of external deformities, erosions, lesions (defined as ulcers and tumours (DELT anomalies)) as an indicator of environmental quality for water resources and fish health. DELT anomalies have been proven an effective IBI metric (Index of Biotic Integrity) (Sanders et al. 1998). This indicator has been very helpful in identifying sites affected by multiple stresses and is more responsive to chemical factors than to physical factors (habitat modifications). Generally, low levels of anomalies indicate good chemical quality of water and sediments. Consistently, low prevalences of these anomalies have been reported at non-polluted sites and high prevalences at polluted site, particularly associated with municipal or industrial discharges. Photographs of fish with external lesions are very effective communicators of degraded aquatic quality to managers or to the public (the public is much more concerned about elevated prevalence of deformed fish than of a change in a biochemical response, for example).

The analysis of the ICES fish disease data in combination with data on contaminants in water, sediments and biota (Wosniok et al., 1999, 2000; Lang and Wosniok, 2000) and more recent results from a German project (EFFSTAT) revealed some significant relationships between contaminants and the prevalence of some of the diseases monitored. In particular, the comparison of trends in levels of sediment trace metals and persistent organochlorines in North Sea dab and changes in the disease prevalence in the period 1987–1998 showed some significant relationships (Lang, Wosniok and Dethlefsen, unpublished results). These results clearly indicate that such relationships exist and that it is possible to demonstrate these effects, provided that an appropriate monitoring strategy is the basis of the studies. As an example of such relationships, Figure A16.1 shows results of a correlation analysis comparing levels of chromium and copper in sediments with the prevalence of lymphocystis in dab at various sampling sites in the North Sea (N stations), western British waters (G stations) and the English Channel (F stations) in 1998 (Dethlefsen et al., submitted). In Figure A16.2, results from a long-term study in the German Bight are provided, indicating a significant relationship between temporal changes in sediment levels of cadmium and the prevalence of lymphocysts over the period 1985–1998. In Figure A16.3, the results from a statistical analysis (partial least squares technique, PLS) are depicted, relating data on diseases and parasites of dab with organochlorine levels in livers of dab from four North Sea areas. The plots indicate a close relationship between the contaminant levels and the prevalences, since they demonstrate a close agreement between the observed and estimated prevalences, where the estimates were based solely on the contaminant levels. However, in


contrast to other approaches, the PLS method allows to account for combined effects of compounds and also for the (realistic) possibility of multiple effects caused by one compound. (Lang et al., unpublished data).

Apart from such contaminant effects, the results of these studies also provide evidence that the disease prevalence is influenced by biological population parameters (age, length, gender, physiological condition, population density) and hydrographic/climatic factors (temperature, salinity). This implies that, in order to asses the contribution of these factors to the disease prevalence, all of these parameters have to be monitored in parallel to the fish diseases/parasites and the contaminant levels. Some of the present and more integrated monitoring programmes are taking account of this and, therefore, there is an increasing amount of data indicating effects of contaminants on the prevalence of externally visible diseases.

Monitoring biological effects of contaminants: Why should studies on externally visible fish diseases be included?

The selection of biological effects techniques to be used for monitoring purposes clearly has to be based on the purpose of the monitoring programme. If the monitoring is aiming at identifying and quantifying effects of specific contaminants (or groups of contaminants), e.g., at a point source of contamination, biological responses have to be measured that react to these contaminants. Furthermore, it has to be decided whether contaminant-specific short-term or long-term effects are to be measured. And it has to be decided whether the responses shall be indicators of exposure or of effects.

In this context, those externally visible diseases, for which relationships with contaminants in question have been demonstrated, could be used as biological responses of long-term effects. An increased prevalence of externally visible fish diseases clearly indicates a potential deleterious impact of contaminants on fish health while the relevance of the responses of other biomarkers with respect to fish health is less clear. Being non-specific, externally visible fish diseases may help to detect the presence in the environment of new groups of contaminants or of complex interactions among contaminants that may not be detected by other more specific biomarkers. Thus, if externally visible lesions are not used as a biomarker, we may not detect significant impacts of contaminants on fish health that could lead to detrimental effects on fish populations.

If the objective of the monitoring, however, is more general and aims at the identification of anthropogenic deviances from normal ecosystem functions or ecosystem health, other biological responses have to be chosen. Contaminants would only be one of the factors that would have to be considered in this context. This is particularly the case in a heavily impacted area such as the North Sea, where there are many anthropogenic pressures on the ecosystem, the effects of which definitely have to be assessed applying an appropriate monitoring programme. Ideally, for such a programme, biological responses would be selected that provide information on disturbances at several levels of biological organisation, from the molecule to the community and from short-term to long-term effects (with emphasis on long-term effects of chronic disturbance). There can be no doubt that for assessing the health of ecosystems, the health status of its inhabitants has to be monitored.

In the environment, there are multiple interactions between the pathogens, the host and the environment, and it is difficult to identify which stressor is responsible for a disease outbreak (see above). Hot spots of external lesions caused by infectious agents may be used to locate areas with high environmental stress. Chemical analyses, a battery of biomarkers including immunological biomarkers, and possibly cage-exposures may be used to investigate the potential role of contaminants at these sites. Because the immune system is sensitive to a wide range of contaminants, external diseases may indicate areas where there are significant fish health problems induced by contaminants which may not be detected by the other, more specific biomarkers. Immune impairment and development of disease outbreaks are an integrative response that detects the combined effects of multiple contaminants.

A weakness of the list of techniques recommended by WGBEC is that it does not clearly enough take into account the above deliberations. There is no doubt that the list could be improved if it were based on the different objectives of monitoring programmes and if the criteria used are adapted accordingly.

If one wants to assess the usefulness and responsiveness of different biological responses used for monitoring, Table A16.2 of the present working document might be of use, because it highlights some strengths and limitations of monitoring externally visible fish diseases. It has often been stressed that the major limitation of externally visible diseases and parasites is the fact that many natural and anthropogenic factors, not only contaminants, potentially have an impact on their prevalence, and that they, therefore, are too unspecific an indicator. However, the same is true for many other biological responses of organisms used or recommended as biomarkers in biological effects monitoring programmes, simply because there are only very few responses (if at all) that are contaminant-specific. Experience gained over the last decade or so has shown that many of the well-established biomarkers are influenced by natural physical or chemical factors. The impact of water temperature on EROD levels is a good example, and there can be no


doubt that other techniques recommended for monitoring are influenced in the same way (e.g., reproductive success, scope for growth, lysosomal stability). Therefore, there is no principal difference at all between externally visible diseases/parasites and other types of biological responses recommended for monitoring.

WGBEC decided to remove externally visible diseases from the list of recommended techniques because of the lack of information on dose-response or concentration-response relationships. This argument can be disputed for various reasons. Such relationships are mainly derived from laboratory experiments where test animals are exposed to a single chemical or groups of chemicals that are applied over relatively short periods at high doses, normally far above the in situ conditions (except around point sources of contamination). There is agreement that it is not possible to extrapolate from the results of such experiments to the situation in the field. Firstly, because the responsiveness of a technique in an experiment does not exclude the possibility that there are other factors that cause the same response. Secondly, the situation in the field is much more complex and involves, e.g., thousands of chemicals that act in concert and may exert cumulative, synergistic or antagonistic effects that cannot be simulated in experiments. Therefore, the use of this criterium for approving or rejecting techniques for monitoring purposes should again be assessed. Apart from this, it should be mentioned that there are, of course, results of many experimental studies on effects of contaminants on the immune system of fish which clearly provided evidence for immunosuppressive effects, a prerequisite for an increase in disease prevalence (see above).

Criteria established by WGBEC for the approval of biological effects techniques that are being perfectly met by studies on externally visible fish diseases are:

• Initiated by ICES, studies on fish diseases are a technique that has been adequately tested in the field (for much longer than any of the other techniques considered). Testing in the field is preferable compared to testing in the laboratory for the reasons given above.

• It is a practical method that has been intercalibrated between laboratories repeatedly under the auspices of ICES and standardised methods have been published (in the ICES TIMES Series and elsewhere) (ICES 1989; Bucke et al., 1996; Lang and Mellergaard, 1999).

• Based on long-term experience and extensive data analysis (which are essential in this context), its sensitivity compared to other techniques is well-known.

Conclusions

There can be no doubt that there is a continuous need to monitor the state of the marine environment in order to assess the extent of anthropogenic ecological change. Besides other factors, pollution still is of major significance because large amounts of contaminants continue to enter the seas, despite national and international efforts to protect the marine environment from inputs. ICES has played a major role in defining appropriate monitoring concepts and strategies and in coordinating environmental monitoring programmes. The ICES WGBEC has always been the leading ICES Working Group with regard to the development and establishment of guidelines for monitoring biological effects of contaminants. In this context, the regular assessment of monitoring techniques and the update of the list of recommended techniques based on new information are of great importance. It is felt that this work can be improved by considering to a greater extent the various purposes of monitoring programmes and by reassessing the criteria established for approval or disapproval of techniques accordingly.

Based on the long-term experience gathered in the monitoring of externally visible diseases/parasites in the ICES area, there is sufficient evidence to recommend that such studies should be part of an integrated monitoring programme assessing anthropogenic effects on ecosystem health, including effects of contaminants. Due to the complexity of biological systems and the variety of natural and anthropogenic factors potentially influencing the well-being of organisms, biological effects monitoring should be based on an integrated multidisciplinary approach, involving physical, chemical and biological measurements.

References

Arkoosch, M.R., Casillas, E., Huffman, P., Clemons, E., Evered, J., Stein, J.E., and Varanasi, U. 1998. Increased susceptibility of juvenile chinook salmon from a contaminated estuary to Vibrio anguillarum. Trans. Amer. Fish. Soc., 127: 360–374.

Bengtsson, B.E., Larsson, A., Bengtsson, A., and Renberg, L. 1988. Sublethal effects of tetrachloro-1,2-benzoquinone—a component in bleachery effluents from pulp mills on vertebral quality and physiological parameters in fourhorn sculpin. Ecotoxicol Environ Saf., 15(1): 62–71.


Bucke, D., Vethaak, A.D., Lang, T., and Mellergaard, S. 1996. Common diseases and parasites of fish in the North Atlantic: Training guide for identification. ICES Techniques in Marine Environmental Sciences, 19. 27 pp.

Dethlefsen, V., Albrecht, H., Bade, T., and Wosniok, W. Heavy metals in sediment and diseases of dab (Limanda limanda) (submitted to Archive of Fishery and Marine Research).

ICES. 1989. Methodology of fish disease surveys. Report of an ICES Sea-going Workshop held on RV U/F “Argos” 16–23 April 1988. ICES Cooperative Research Report, 166, 33 pp.

Lang, T. 2002. Fish disease surveys in environmental monitoring: the role of ICES. ICES Marine Science Symposia, 215: 202–212.

Lang, T., and Mellergaard, S. 1999. The BMB/ICES Sea-Going Workshop “Fish Diseases and Parasites in the Baltic Sea” – introduction and conclusions. ICES Journal of Marine Science, 56: 129–133.

Lang, T., and Wosniok, W. 2000. ICES data for an holistic analysis of fish disease prevalence. In Report of the ICES Advisory Committee on the Marine Environment, 1999. ICES Coop. Res. Rep., 239: 193–209.

Lindesjoo, E., and Thulin, J. 1994. Histopathology of skin and gills of fish in pulp mill effluents. Dis. Aquat. Org., 18: 81–93.

Moore, M.J., Shea, D., Hillman, R.E., and Stegeman, J.J. 1996. Trends in hepatic tumors and hydropic vacuolation, fin erosion, organic chemicals and stable isotope ratios in winter flounder from Massachusetts, USA. Mar. Poll. Bull., 32: 458–470.

Sanders, R.E., Miltner, R.J., Yoder, C.O., and Rankin, E.T. 1998. The use of external deformities, erosions, lesions, and tumors (DELT anomalies) in fish assemblages for characterizing aquatic resources: a case study of seven Ohio streams. Chapter 9 In Assessing the sustainability and biological integrity of water resources using fish communities. Ed. by T. P. Simon, CRC Press, Boca Raton, Florida, US. pp.225–246.

Svavarsson, J. 2000. Imposex in the Dogwhelk (Nucella lapillus) due to TBT Contamination: Improvement at High Latitudes. Marine Pollution Bulletin, 40(11): 893–897.

Vethaak, A.D., Bucke, D., Lang, T., Wester, P.W., Jol, J., and Carr, M. 1992. Fish disease monitoring along a pollution transect: a case study using dab (Limanda limanda) in the German Bight, North Sea. Mar. Ecol. Prog. Ser., 91: 173–192.

Wosniok, W., Lang, T., Dethlefsen, V., Feist, S.W., McVicar, A.H., Mellergaard, S., and Vethaak, A.D. 2000. Analysis of ICES long-term data on diseases of North Sea dab (Limanda limanda) in relation to contaminants and other environmental factors. ICES CM 2000/S:12. 15 pp.

Wosniok, W., Lang, T., Vethaak, A.D., des Clers, S., and Mellergaard, S. 1999. Statistical analysis of fish disease prevalence data from the ICES Environmental Data Centre. In Report of the ICES Advisory Committee on the Marine Environment, 1998, pp. 297–327. ICES Cooperative Research Report, 233. 375 pp.

Ziskowski, J., and Murchelano., R. 1975. Fin erosion in winter flounder. Marine Pollution Bulletin, 6: 26–29.


Table A16.2. Strengths and limitations of fish disease monitoring (Lang, 2002).

Strengths Limitations

Diseases are an overt and integrative biological endpoint of physiological changes at different levels of biological organisation affecting the organism’s homeostasis that are associated with environmental change.

In concert with more specific early-warning biological effects techniques, e.g., biomarkers of contaminant exposure and contaminant-induced damage, fish diseases can be used more specifically as an indicator of effects of contaminants.

Significant changes in disease prevalence are a biologically and ecologically relevant warning sign for adverse environmental changes, since diseases may affect growth, reproduction, and survival of affected individuals and may, therefore, have implications on the population level.

Data on the prevalence and spatial distribution of diseases (including parasites) of commercial fish species are of direct use for quality controls of fish as a food resource for human consumption.

Fish disease monitoring is cost-effective since it can be carried out directly on board research or even commercial vessels, possibly in combination with stock assessment surveys, without involving subsequent laboratory work (except histopathology).

Externally visible target diseases identified are, with a certain degree of training, easy to recognise.

A large number of fish and large geographical areas can be screened and results are immediately available.

Methodologies for fish disease surveys have been established and repeatedly intercalibrated.

Standard procedures for data submission and validation, statistical analysis, and data presentation have recently been developed by ICES.

A large ICES database with long-term fish disease data from the North Sea and adjacent areas submitted by ICES Member Countries has been built up and can be used as baseline information for future monitoring programmes.

Most diseases (particularly infectious) have a complex multifactorial aetiology, potentially involving the impact of anthropogenic and/or natural variations of host, pathogens and environmental characteristics.

Changes in prevalence of wild fish diseases are often a non-specific indicator of environmental change, the causes of which are difficult to identify.

The elucidation of cause-effect relationships between contaminants and changes in disease prevalence requires a multidisciplinary monitoring strategy, involving the measurement of a wide range of potentially explanatory host-specific (e.g., age, length, gender, population density, status of the immune system, migration patterns), disease-specific (aetiology, pathogenesis, transmission pathways, seasonal effects, natural background levels, effects on hosts) and site-specific factors (e.g., community structure, contaminant levels in different compartments, bioavailability of contaminants, fishing pressure, hydrography).

Due to the complexity of the relationship between the environment and the pathogenesis of diseases and due to the large effort needed to identify cause-effect relationships related to contaminant effects, results from wild fish disease monitoring can, at best, provide circumstantial evidence for the existence of such a relationship, rather than indisputable scientific proof.


Cr vs Lymphocystis, 1998

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Figure A16.1. Relationship between chromium (upper figure) and copper (lower figure) concentrations in sediments (fraction < 20 µm) and the prevalence of lymphocystis in dab (Limanda limanda) from sampling sites in the North Sea (N), western British waters (G) and the English Channel (F) in spring/summer 1998 (Dethlefsen et al., submitted).

Figure A16.2. Relationship between concentrations of cadmium in surface sediments (fraction < 20 µm) from the German Bight and the prevalence of lymphocystis in dab (Limanda limanda) in the period 1985–1998 (black dots: from interpolation curves, open dots: annual means) (Dethlefsen et al., submitted).


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Figure A16.3. Observed disease prevalence versus prevalence predicted from the body burden of organochlorine compounds in dab (Limanda limanda) from four different areas in the North Sea (N01: German Bight; N04: Dogger Bank; N06: Firth of Forth; N22: off Humber estuary) (predicted prevalence estimated by Partial Least Squares Technique) (“+”: lymphocystis, “x”: epidermal hyperplasia/papilloma, square: acute/healing ulcerations, triangle: hyperpigmentation, circle: liver nodules > 2 mm, diamond: Acanthochondria cornuta, star: Stephanostum baccatum). A point position close to the 45° diagonal indicates good agreement between observed and predicted value.


ANNEX 17: TENTATIVE TABLE OF CONTENTS OF THE MANUSCRIPT ENTITLED “STATISTICAL METHODS FOR THE ANALYSIS OF FISH DISEASE DATA”

W. Wosniok, T. Lang, A.D. Vethaak, S. desClers, S. Mellergaard, S.W. Feist, A.H. McVicar, V. Dethlefsen

1 Introduction 1.1 Objectives 1.2 Typical data 1.3 Typical data problems 1.4 Notation 1.5 The data set used in the examples 1.6 Guide to the tasks addressed

2 Comparison of fish disease prevalences 2.1 Objectives 2.2 Prevalence estimation and the precision of estimates 2.3 The comparison of prevalences 2.4 Detection of monotone spatial gradients or monotone temporal trends 2.5 Required sample sizes

3 Fish disease prevalence and explanatory quantities 3.1 Objectives 3.2 Exploratory versus confirmatory analysis 3.3 Exploring the data: classification/ regression trees and neural networks 3.4 The basic modelling approach for prevalences: logistic regression 3.5 Generalizing logistic regression: the Generalized Additive Model 3.6 Model selection techniques 3.7 Dealing with correlated explanatory variables 3.8 Example XXX: Analysing the effect of length and gender on prevalence 3.9 Example XXX: Analysing general spatial patterns of prevalence 3.10 Example XXX: Checking for a prevalence trend along a transect 3.11 Example XXX: Checking for a temporal prevalence trend in an area, regular observations 3.12 Joint spatial and temporal analysis 3.13 Example XXX: Checking for a temporal prevalence trend in an area, irregular observations 3.14 Modelling the relation between the prevalences of a set of diseases and a set of explanatory variables 3.15 Comparing prevalences in size-structured populations: Standardisation techniques 3.16 Epidemiological methods: case-control studies 3.17 Display 3.18 Missing data

4 Software 5 References 6 Glossary of terms and notation


ANNEX 18: ANALYSIS OF PROGRESS WITH TASKS

a) analyse national reports on new disease trends in wild and cultured fish, molluscs and crustaceans; Reports on new diseases and trends of diseases were evaluated from national reports presented at the meeting and conclusions were drawn.

b) report on progress in the ongoing investigations of the effect of temperature on Bonamia infection dynamics and report on the confirmation of the agent of Crassostrea angulata gill disease and its infectivity to Crassostrea gigas and other oysters species; A status of the present knowledge was presented and further progress made will be reviewed at the 2004 WGPDMO meeting as part of the presentation of the national reports on new disease trends.

c) review the existing strategies to assess the prevalence of shellfish diseases in parallel to fish diseases and chemical contaminant levels in environmental monitoring programmes; Available published information was assessed and a summary report was presented. Based on the outcome of the deliberations, a recommendation for a new Term of Reference for the 2004 WGPDMO meeting was drafted.

d) review and assess a report prepared intersessionally on investigations into the molecular comparisons among the various species/isolates of Perkinsus in collaboration with the OIE reference laboratory for Perkinsus at the Virginia Institute of Marine Science (E. Burreson); Available information was assessed and a summary report was presented. A recommendation for a Term of Reference for the 2004 WGPDMO meeting continuing the work regarding this issue was drafted.

e) obtain information on the EU project “Diagnosis of oyster herpes-like virus: development and validation of molecular, immunological and cellular tools” (FAIR-PL98-4334) and review the results; Available information was assessed and a summary report was presented.

f) review and report on progress made in the “fish diseases and liver histopathology” component of the BEQUALM self-funding scheme; A summary progress report was provided with an outline of the future prospects of the project. Further progress made will regularly be reviewed at the next WGPDMO meetings.

g) review and assess the impact of diseases of farmed fish on wild fish stocks; A status of the present knowledge was reviewed and, due to the importance of this topic, WGPDMO recommended to revisit this issue at its 2004 meeting.

h) maintain an overview of the spread of Ichthyophonus in herring stocks and the distribution and possible cause(s) of the M74 syndrome; A report summarising available information on M74 and information on Ichthyophonus extracted from the national reports were presented.

i) assess and report on the effectiveness of salmon farming management control methods for sea lice in the different ICES Member Countries; Available information was assessed and a summary report was presented.

j) review progress made in the modifications to the ICES Databases and review and approve the revised ICES Environmental Data Reporting Format (Version 3.2); The new Reporting Format was presented and discussed and, as requested, WGPDMO provided information on its viewpoints to ICES.

k) review the criteria for the incorporation of externally visible fish diseases into monitoring programmes on biological effects of contaminants; A background document produced intersessionally was reviewed and, as requested, information was prepared as WGPDMO's input to discussions in the WGBEC.

l) review progress made with regard to the update of ICES publications on pathology and diseases of marine organisms:

• web-based report on diseases and parasites of wild and farmed marine fish and shellfish as part of the ICES Environmental Status Report; A proposal for improvements of the WGPDMO contribution to the ICES Environmental Status Report was presented and will be submitted to ICES for inclusion in the ICES website.

• Manuscript on methods for the statistical analysis of fish disease data for submission to the ICES TIMES series; The content of the updated version of the manuscript was presented and agreed on.

• ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish; A progress report was presented and a strategy for the production of the Leaflets was discussed and a plan for revisions was made.

• Report on important trends in diseases occurring in finfish and shellfish culture in the ICES area in the period 1997–2001; A progress report was presented and plans were made for further amendments and submission to ICES for publication in the CRR Series.


ANNEX 19: RECOMMENDATIONS TO THE ICES COUNCIL

The Working Group on Pathology and Diseases of Marine Organisms [WGPDMO] (Chair: T. Lang) will meet in Åbo, Finland, from 9–13 March 2004 to: a) analyse national reports on new disease trends in wild and cultured fish, molluscs and crustaceans; (all members)

b) review and report on environmental monitoring programmes and associated quality assurance activities

incorporating studies on pathology and diseases of marine organisms; (C. Couillard, S. Feist, S. MacLean)

c) review an intersessionally prepared updated report on techniques used to differentiate among Perkinsus spp., incorporating input received from web-based international solicitation of comments; (S. Bower, S. McGladdery)

d) review the existing information on viral diseases of crustaceans with emphasis on commercially important species;

(S.W. Feist, S. MacLean)

e) review the use of epidemiological methods for the assessment of diseases and population effects risk; (W. Wosniok, S.W. Feist, T. Lang)

f) review current information on disease/parasite interactions between wild and farmed fish and on related

management control methods; (D. Bruno, B. Hjeltnes)

g) maintain an overview of the spread of Ichthyophonus in herring stocks and the distribution and possible cause(s) of the M74 syndrome; (T. Wiklund, D. Bruno)

h) review progress made in the modifications to the ICES Databases and the revised ICES Environmental Data

Reporting Format (Version 3.2); (W. Wosniok, T. Lang)

i) review progress made with regard to the update of ICES publications on pathology and diseases of marine organisms:

a. web-based report on diseases and parasites of wild and farmed marine fish and shellfish as part of the ICES Environmental Status Report, (W. Wosniok, T. Lang)

b. manuscript on methods for the statistical analysis of fish disease data for submission to the ICES TIMES series, (W. Wosniok, T. Lang)

c. ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish; (S. McGladdery) d. report on important trends in diseases occurring in finfish and shellfish culture in the ICES area in the

period 1997–2001 (T. Lang)

Supporting Information

Priority: WGPDMO is of fundamental importance to the ICES science and advisory process.

Scientific Justification: a) New disease conditions and trends in diseases of wild and cultured marine organisms continue to appear and an assessment of them should be maintained.

b) Many ICES Member Countries are conducting marine environmental monitoring

programmes incorporating studies on pathology and diseases of fish and shellfish species. The WGPDMO considers it important for its work to review the progress made, including the implementation of quality assurance programmes.

c) Since the first identification of a Perkinsus sp. in the late 1940s, several methods

have been used to identify and characterise new species. The use of different criteria has caused confusion in the taxonomy of the genus, although newly developed genetic methods are helping to sort out the taxonomy. A set of identification criteria needs to be developed and agreed upon by the widest possible group of molluscan disease investigators.

d) Several crustacean species are commercially highly valuable with many wild

stocks being heavily exploited. Considerable information is available for cultured species. For instance, penaeid shrimp culture is a major aquaculture industry in several countries around the world and is seriously affected by the viral disease “White Spot Syndrome”. In contrast to knowledge of cultured crustaceans, very little is known about viral diseases of wild-stock crustaceans. Recent studies


indicate these infections to be more prevalent than previously thought and it is timely to review the available information on the types and pathogenicity of viruses in wild crustaceans.

e) Risk assessment aims to quantify the risk of (usually adverse) effects and serves as

a basis for many regulatory decisions. Epidemiological methods are tools to calculate risk quantifications and, given a particular data structure, are effective also for small samples. The applicability of epidemiological methods to existing or obtainable data and potential gain by future use should be reviewed.

f) The impact of the increasing development of aquaculture on diseases in wild fish

populations is an issue of concern. WGPDMO considers it important to be updated on the most recent knowledge within this field.

g) ICES C.Res. 1993/2:23(m) requested that WGPDMO maintain an overview of the M74 syndrome and the Ichthyophonus issue as part of its regular agenda.

h) WGPDMO considers it necessary to follow the process of the modification of the

ICES Databank structure in order to assist, if required, and to obtain an overview of changes suggested or introduced by other ICES Working Groups.

i) A number of ICES publications, either web-based or in ICES publication series, are being prepared or updated at present, the progress of which has to be reviewed by WGPDMO at its next meeting. It will be necessary to consider ways by which these can be linked to each other.

Relation to Strategic Plan: Responds to Objectives

Resource Requirements: None required, other than those provided by the host institute.

Participants: Representatives of all Member Countries with expertise relevant to pathology and disease of wild and cultured finfish and shellfish.

Secretariat Facilities: None required

Financial: None required

Linkages to Advisory Committees:

ACME

Linkages to other Committees or Groups:

MARC

Linkages to other Organisations:

BEQUALM, OIE, EU, OSPAR

Cost share ICES:100 %

Working Group on Pathology and Diseases of Marine Organisms Reports/Expert Group...Working Group on Pathology and Diseases of Marine Organisms Aberdeen, UK 11–15 March 2003 This

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