Annual Meeting of the National Reference Laboratories for Mollusc Diseases

Transcription

1 REPORT OF THE Annual Meeting of the National Reference Laboratories for Mollusc Diseases Nantes, 2223 March 2006 Organised by the Community Reference Laboratory for Mollusc Diseases IFREMER, La Tremblade, France

2 Report of the 2006 Annual Meeting of the National Reference Laboratories for Mollusc Diseases Nantes, 2223 March 2006 Table of content Executive summary p. 2 Introduction p. 3 Session I : Diagnosis and Survey of Mollusc Diseases p. 4 Session II : Examples of mollusc disease surveys p. 7 Session III : Surveillance of wild stocks p. 10 Session IV : Methods for disinfection p. 12 Session V : Legislation Matters p. 14 Session VI : EU projects in support of CRLs / NRLs activities p. 16 Session VII : News from the Community Reference Laboratory p. 18 Annex A: Agenda of the meeting p. 22 Annex B: List of participants p. 24 Annex C: Annual reports from the NRLs on National epidemiological situation p. 28 Annex D : New epidemiological template p. 85 Annex E : Quick guide to complete the epidemiological template p. 91 1

3 Executive summary Follow highlights of the discussions, expert opinion and recommendations formulated during the 2006 Annual Meeting of National Reference Laboratories (NRLs) for Diseases of Molluscs. In 2005, major epidemiological changes in EU were (1) the detection of Candidatus Xenohaliotis californiensis and the characterization of a new Haplosporidian, Haplosporidium montforti in association with high mortality rates of Haliotis tuberculata in Spain and (2) the spread of bonamiosis in a previously free area, Lough Foyle bay located between Republic of Ireland and Northern Ireland. A new form of the epidemiological report template was submitted to NRLs aiming at improving the quality of collected data and at collating homogeneous data. This new version will be used for the first time in 2007 and will be distributed to NRLs in association with a quick guide explaining how to fulfil data. Examples of surveillance studies have been presented stressing the importance of collecting environmental data in association with pathological data. Such studies are interesting notably for a better disease control, and even for disease modelisation. Modelisation can be very useful to estimate stock evolution and advice producers by predicting evolution of diseases. The interest in including wild stock in surveillance programme has been discussed. Indeed, unlike farmed stocks, wild stocks are not submitted to transfers and thus are good representatives of a zone. Considering potential exchanges between farmed and wild stocks, risk based surveillance approaches must include wild stocks as possible pathogen source or pathogen carriers. Disinfection is needed for several purposes including good laboratory practices and production practices, and also, in case of urgency when an epizootic outbreak occurs or when a farm wants to be approved in a non approved zone. However, very few information is available concerning disinfection against mollusc pathogens. The future fish and shellfish health Directive has been presented. The new requirements regarding surveillance programmes and reference laboratories functions and duties have been developed. This new directive should start to be applied in January A possible future project on Perkinsus olseni was exposed. The goal of this project is to bring together industry, research community and administration in a workshop to address the threat of perkinsosis to European shellfish industry. News from the EU projects PANDA and DIPNET were given during the annual meeting and if not already done NRLs delegates were asked to register on PANDA and DIPNET websites. In the context of PANDA Workpackage 4, a table was prepared summing up for the most serious aquatic diseases: disease status; susceptible species in Europe; available diagnostic tests (histology, PCR, ISH and other techniques). PANDA WP4 aims at identifying the optimal diagnostic methods currently available for the most serious aquatic diseases and providing recommendations for their standardization and harmonization procedures throughout Europe 2

4 and for any needs to improve their accuracy, rapidity and applicability. A collection of bacterial pathogens isolated from aquatic organisms and belonging mainly to Vibrio genus has been constituted. This collection is now accessible on a web site at the following address For each bacterial strains, information is available concerning their taxonomical position and their virulence. The working group on Quality Assurance (QA) which had been set during the 2002 Annual Meeting continues to provide advice and assistance to the NRLs in the implementation process of QA in diagnosis of mollusc diseases. SOPs about bonamiosis, marteiliosis and perkinsosis diagnosis by histology.are now available and other documents being prepared for 2006 will concern perkinsosis diagnosis by RFTM, SOPs for histology and cytology processing and SOP for opening bivalves. SOP about histology process is almost ready and will be submitted to all NRLs comments very soon. A new website of the CRL has been created. It is especially dedicated to all NRLs looking for information regarding mollusc diseases ( The website is organised in five sessions: main activities, scientific activities, legislation, Network of NRLs and Tutorials. NRLs delegate are asked to go and visit this website and to send us their comments and/or corrections. Results of the last interproficiency test organised fro the NRLs for Mollusc diseases were presented highlighting the difficulty to detect low infection level of some diseases especially bonamiosis. Next ring test should start at the end of 2006 and should include at least histological slides of Ostrea edulis. The CRL proposes to plan the next annual meeting for the beginning of 2007 (MarchApril) in La Tremblade combined to a new Technical workshop on the detection of bonamiosis by PCR and on the detection of Perkinsus olseni using the thioglycolate medium. NRLs are invited to propose topics on which they are working or for which they need information and/or training. Introduction The 2006 Annual Meeting of National Reference Laboratories for Mollusc Diseases was held in Nantes on the 22 nd and the 23 rd of March participants from 12 countries (Belgium, Spain, France, Germany, Denmark, Italy, Romania, The Netherlands, UnitedKingdom, Slovenia, Norway and Ireland) attended the meeting. The Annual Meeting was planned on two days and included seven sessions: 1) Diagnosis and survey of mollusc diseases 2) Examples of mollusc disease surveys 3) Surveillance of wild stocks 4) Methods for disinfection 5) Legislation matters 6) EU projects in support of CRLs / NRLs activities 7) News from the Community Reference Laboratory. 3

5 This report provides summaries of the questions discussed during the meeting and outstanding facts for followup activities. It contains collective expert opinion and recommendations made during the meeting. SESSION I: DIAGNOSIS AND SURVEY OF MOLLUSC DISEASES (Chair: C. Garcia) Current epidemiological situation in the Member States National delegates participating in the Annual Meeting briefly presented outstanding issues and major changes with regards to the mollusc health status in their country. The detailed reports received from NRLs are given in annex of this report (Annex C). Croatia: In 2005, 75 farms produced 80 t of flat oysters, Ostrea edulis and 2400 t of mussels. No Marteilia refringens or Bonamia ostreae was detected in flat oysters in Marteilia sp. was reported in all the mussel samples analysed from the three main production areas. Denmark: The production of flat oysters tends to stabilize in 2005 with a production of 942 tons. The surveillance mainly targets Limfjorden area. Investigations for 2005 were not finished at the meeting dates. Denmark is officially recognised Bonamia ostreae and Marteilia refringens free on the Limfjorden area since December France: No major change was recorded. In 2005, 5 hatcheries produce abalones, Haliotis tuberculata. The official recognition of the free status of French zones is still under progress. However, Bonamia ostreae was detected in oysters collected closed to the limits of Granville bed and this result highlights the importance of good geographic delimitation of zones. Abnormal mortality outbreaks of Pacific oysters, Crassostrea gigas, were mainly observed during summer. Some of these outbreaks could be associated with the detection of herpesvirus, OsHV1. Moreover, in 2005, Haplosporidium nelsoni was detected in 6 Pacific oysters by histology and in situ hybrisization. Detection frequency of this parasite in France was still low in 2005 but increased compared to previous years (1 % o to 1% in 2005) and for the first time, H. nelsoni spores could be observed in Crassostrea gigas. Vibrio harveyi was detected in abalones, Haliotis tuberculata, presenting mortalities. A two yearstudy on the presence of Perkinsus olseni in main production sites of Ruditapes species was initiated in Results show that the parasite is present in all investigated sites with various detection frequencies and various infection intensities according to clam ages and biomass. Germany: In 2005, one mortality event was reported on Crassostrea gigas in lower Saxony Germany. Oyters and also mussels bred near oysters were sampled for analysis by histology and using cell lines (CHSC). No pathogen could be observed by histology but gills were destroyed and infiltration of cells could be observed between ovocytes. Based on this observation, a viral infection was suspected and analysis was performed using cell lines. 4

6 Italy: No Marteilia sp. was detected in mussel samples analysed in Perkinsus olseni is present with high prevalence and infestation rate in manila clams Ruditapes philippinarum from FriuliVenezia Giulia and Veneto areas with 75% and 66% of detection frequencies respectively. The detection of P. olseni is performed by histology and culture using thioglycollate. Culture using thioglycolate is more sensitive than histology but both techniques present concordant results when infection levels are high. Ireland: In 2005 there are 5 hatcheries producing abalones in Ireland, three of them produces both Haliotis tuberculata and the exotic species H. discus hannai. The distribution of bonamiosis due to Bonamia ostreae enlarges year after year. In 2005, a previously free area has been detected infected with the parasite. At this moment, no clear explanation has been found to understand how the disease emerged in this area. High shellfish mortalities have been reported in different parts of Ireland. These mortalities were attributed to prolonged algal bloom of Karenia mitimotoi. The Netherlands: Bonamia ostreae is still detected in flat oysters, Ostrea edulis, from Grevelingen and from Oosterschelde with a prevalence of 10.4 and 3.7 % respectively in Bacteria similar to Nocardia were reported in one Pacific oyster from Grevelingen. Two mortality cases were reported in 2005 affecting Crassostrea gigas in Grevelingen and Cerastoderma edule in Westerschelde. These events could not be attributed to a specific pathogen. However, bacteria similar to Nocardia were reported in one Pacific oyster from Grevelingen. Norway: Norway was officially recognized as a free zone in Norway is considered as one zone. No mortality was reported in Portugal: Portugal mainly produces clams but has also a small production of flat oysters, Ostrea edulis. Analyses on this species revealed in Algavre the presence of Marteilia refringens while no Bonamia ostreae was actually detected. One case of clam abnormal mortality was reported in summer Romania: In 2005, no change compared to Slovenia: The Slovenian mollusc production is represented by mussels, Mytilus galloprovincialis, (130 tons) and 2 farms. In 2005, 320 mussels were analysed by histology. No pathogen was detected and no mortality was recorded. Nevertheless, G. Ceschia mentionned that the best period to detect Marteilia maurini in mussels from Adriatic is May instead of June or later. Thus, countries planning to detect this pathogen should adapt their sampling dates. Spain: Most of the Spanish production of shellfish is located in Galicia. In this area, Bonamia ostreae was reported in 82 % of flat oysters analysed in Marteilia refringens was not reported in Galicia but could be detected in Alicante. Perkinsus presumably P. olseni was detected in Ruditapes decussatus, R. philippinarum and Venerupis pullastra. Two mortality events were reported in 2005 in Haliotis tuberculata from Galicia and in Chamelea gallina in las Islas Baleares. Histological analysis revealed the presence of rickettsia like organisms identified as Candidatus Xenohaliotis californiensis by PCR and the presence of haplosporidian like organisms later identified as a new Haplosporidium species namely H. montforti. United Kingdom: Consequently to the detection of Marteilia maurini in mussels in June 2003 and 5

7 2004, analyses were performed on oysters sampled in this area in 2005 and did not reveal the presence of the parasite. Consequently to the detection of Bonamia ostreae in Lough Foyle in 2005 on the Republic of Ireland side, sampling was performed in the same area on the Northern Irish side. Analyses were performed by histology and PCR. Histological examination did not reveal the presence of the parasite while 6 oysters were found infected by PCR. One Crassostrea gigas mortality event was reported in summer 2005 and was apparently due to high stocking densities. One of the most new information is the detection of Candidatus Xenohaliotis californiensis in Spain. This listed pathogen was reported in association with high mortality rates of Haliotis tuberculata. However, it seems that mortality was attributed to another pathogen, a new characterized Haplosporidian, Haplosporidium montforti. Both pathogens were detected by histology and the diagnosis was confirmed by a PCR conducted with specific primers. Initially, mortality affected abalones introduced from Ireland. Retrospectively, molecular analyses have been performed on the imported seeds and revealed the presence of both pathogens (Candidatus X. californiensis and H. montforti). Both pathogens were also detected by molecular analyses on abalone seeds imported from a french hatchery although they were not used for aquaculture purposes. The distribution of bonamiosis is still increasing and in 2005 the disease has affected a previously free area: Lough Foyle bay located between Republic of Ireland and Northern Ireland. No clear information is available at the moment to explain the emergence of the disease in this place. During these presentations several points were tackled including the importance of zone limitations. Indeed, the parasite Bonamia ostreae has been detected in a French natural bed under agreement process but apparently outside the official zone limits. Sampling size applied for freedom demonstration has been discussed notably through the case of Norway which is considered as one and unique zone but has a very long coast. Similarly to last year, another point of discussion focused on the way for a country to be approved free from bonamiosis and marteiliosis when this country doesn t produce flat oysters. Y. Torgersen explained that the future directive will propose a list of several susceptible species with regards to listed diseases. If the most susceptible host species is not present in a country, the surveillance programme should target another species, while if none of these host species is present, the country has to prove it. Lastly, the risk linked to the fauna associated to shellfish when transferred for aquaculture purposes has been reminded. Indeed, transfers of molluscs usually include all the fouling and fauna fixed on the shells which can represent a risk for the health of endemic species. Presentation of the new epidemiological report template. Cyrille François Last year, the CRL proposed to develop a new form of the epidemiological report template in order to improve the quality of the data collected. This easytouse questionnaire gives a homogeneous format of production, laboratory and epidemiological data for each state, and makes statistics possible as it is based on a.xls format (Microsoft Excel). Each state will receive this year an 6

8 individual questionnaire completed with data of 2005 as example and will be invited to comment the form and the content. A quick guide has been also developed which explain the correct way to fulfil quantitative and qualitative information. These two documents will also be submitted to the Commission before their application in French questionnaire (draft) is available for consultation in annex D as well as a quick guide explaining how to fulfil data in annex E. Little remarks or requests have been expressed during the meeting like: in the tittle: include country code ISO and the year in production data: include fishing and cultured data (given by the FAO) in epidemiological data, study of increased mortality: specify affected population SESSION II: EXAMPLES OF MOLLUSC DISEASE SURVEYS (Chair: I. Arzul) Results of the perkinsosis survey ( ) in France Laurence Miossec A 2year survey was performed in France in 2004 and 2005 in order to investigate the health status of 2 clam species Ruditapes philippinarum and Ruditapes decussatus regarding the presence of Perkinsus olseni in farming and natural harvested areas. This survey takes part of the French surveillance and monitoring programme developed in farming and natural harvested beds with the aims to observe whether there is abnormal mortality and to keep track of the health situation of the stocks. Perkinsus olseni causes perkinsosis in clams and abalones which is an OIE notifiable disease. This pathogen is listed in the Directive 95/70/EC modified by the Decision 2003/83/EC. Five areas with wild and cultured clams were investigated in 2004; two additional clam areas were selected the following year according to production data published in Sampling strategy was designed in order to detect infected animals at a 95% confidence level and minimum prevalence of 2%, assuming that sensitivity and specificity of the diagonsitic technique were 1. The first year, in each monitored area, 150 individuals were collected once a year allocated in 5 points of 30 individuals. The second year, the sampling was adjusted according to the first results. It was systematic in each area with a random subsampling on each sampling point. Clams were collected in SeptemberOctober according to the life cycle of the pathogen. Perkinsus olseni was detected and quantified using the Ray s Fluid Thioglycolate Method (RFTM). Perkinsus olseni was present in all investigated areas. The prevalence varied between 3 and 100% according to years and areas. High prevalences were specially observed in Arcachon bay and along the Mediterranean Sea. Lower prevalences were registered in other areas along the Atlantic coast and English Channel. The less infected area was situated in far west of Brittany where sea water temperatures are very low. Detection frequencies were slightly reduced in 2005 compared to No abnormal mortality was registred during the 2year survey. G. Ceschia suggested to check which type of substratum can be found in all investigated areas. Indeed, it seems that Perkinsus olseni does not appreciate sandy sites and prefers muddy areas. Moreover, perkinsosis prevalence increases when water renewal is low. 7

9 Herpesvirus and oyster mortalities in France Céline Garcia Since the seventies, Crassostrea gigas mortalities have been regularly observed in France. These mortalities mainly affect both larvae and spat produced in hatchery or in the wild. In , massive oyster spat mortalities were reported in France and a herpesvirus OsHV1 was detected in C. gigas spat. Although the pathogen effect of OsHV1 was demonstrated on larval stages, its impact on oyster juveniles is not also well documented. To better understand its real impact, a systematic research of OsHV1 has been done by the network for survey of mollusc diseases (Repamo) when oyster farmers reported spat mortalities. The network data analysis between 1997 and 2005 showed two most results: (1) a typology of oyster spat mortalities in which OsHV1 was detected. The detection of OsHV1 mainly occurred in summer and in the same time as the beginning of summer mortality of spat. OsHV1 detection follows the mortalities but at an inferior level. Moreover, the virus appearance seems to link to the temperature; it is detected first in the south of France in MayJune and in JulyAugust in the north. These mortalities are rapid, massive and uniform in hatchery and nursery; in the wild, they are generally in spots and principally occurred in a little brewed waters like lagoons, estuaries. (2) an optimization of sampling procedures. Between 1997 and 2002, OsHV1 was found in 20% of oyster spat mortality cases; since 2003, it was detected in 40% of mortality cases. This increase in detection frequency could be explained by a change in sample collection procedure. Indeed, OsHV1 is more detected if samples contain moribund oysters and if time between mortality and sample is less than one week. To improve the OsHV1 detection, it is recommended to collect samples with more close to mortality and to take both alive and moribund oysters. A long term study on bonamiosis and marteiliosis in France Isabelle Arzul Marteiliosis was first reported in 1968 in association with flat oyster mortalities in Aber Wrach, North Brittany, France. About ten years later, in June 1979, a new disease, bonamiosis was also reported in association with mass mortality of flat oysters, Ostrea edulis, in L Ile Tudy, South Brittany, France. These both diseases rapidly spread to the entire oyster farming areas in France but also in other European countries. The French flat oyster production, because of these parasitosis, decreased from nearly mt in 1968 to less than 2000 mt after In 2001, about 350 French farms sell 1650 mt of flat oysters. This production mainly relies on natural spat collection which specially occurs in Quiberon bay. One third of this spat is transferred from South to North Brittany for further growth. Quiberon bay constitutes an interesting site regarding the surveillance of bonamiosis because of the simultaneous presence of spat and adult flat oysters. We analyze and present herein the long term series of data collected in this area. On the period more than oysters were sampled for different purposes in Quiberon bay and were tested by histology and/or heart imprints for the detection of Bonamia ostreae. Detection frequencies presented some fluctuations including two peaks in and Disease frequencies recorded during the first period of this study ( ) were lower than those reported later ( ), probably related to the flat oyster mass mortality. Differences could be noted according to animal age and sampling season. 8

10 Although the disease is enzootic in Quiberon bay since 1980, the production of flat oyster is still present in this area. One can wonder how environment and cultural practices can influence the evolution of the disease and the oyster defense mechanisms while the parasite appears to inescapably lead to the dead of the infected oysters. From data collected on the period through the REPAMO, the French network for the surveillance of mollusc diseases, the distribution of marteiliosis appears dependent on the site nature, the parasite prefers bays and estuaries than open sea. Three production areas appear particularly interesting regarding the presence of the parasite and the number of collected data: Brest bay (North Brittany); Quiberon rivers (South Brittany); Thau lagoon (Mediterranean sea). The evolution of detection frequencies shows some fluctuations on the studied period. However, Thau lagoon presents less variation compared to Brest bay. The study of detection frequencies according to the oyster age shows some differences between spat and adults, these last appear generally more infected than young stages. Epidemiological surveys of both diseases in sites of interest are still needed in order to improve our knowledge of hostparasite interactions in a given ecosystem according to environmental parameters. Such studies present interest notably for a better disease control. Data collected for bonamiosis in Brittany should allow modelising the disease. Modelisation can be very useful for predicting evolution of disease. Haplosporidium infections in flat oysters in The Netherlands Marc Engelsma During the shellfish disease surveillance program in the spring of 2004 a high prevalence of a Haplosporidian parasite was found in one sample of European flat oysters (Ostrea edulis) from a single location in the Oosterschelde, The Netherlands. No increased mortality of flat oysters was observed at the site. By light microscopy plasmodial stages were observed in 4 out of 37 flat oysters. In addition to this one oyster contained a mass sporulation in connective tissue of a Haplosporidian parasite. In order to further identify the Haplosporidian parasite, small subunit rrna of the parasite was amplified by PCR with conserved primers for Haplosporidia and subsequently sequenced. The near fulllength SSU rrna sequence was obtained and revealed that the parasite indeed belonged to the phylum Haplosporidia. Closest relative was shown to be Haplosporidium costale. A PCR was developed to rapidly discriminate plasmodial stages of this Haplosporidium species from other Haplosporidia as H. costale and H. nelsoni. Most likely candidate is Haplosporidium armoricanum which was previously described to be present at very low prevalence in the Netherlands. Plasmodial stages of Bonamia ostreae can also be observed in some flat oysters and may be confused with Haplosporidium armoricanum plasmodial stages. The PCR technique presented here may be very useful to differentiate both parasites. 9

11 Pathological events of abalones in Europe Céline Garcia Since these last years, the abalone production has regularly increased and the risk of disease emergence too. Recently, different outbreaks in the production of Haliotis tuberculata occurred in Europe, mainly in France. Two bacteria were involved in these outbreaks: Vibrio harveyi and V. splendidus. These two bacteria are ubiquitous, naturally present in marine animals and were found in many fish, crustacean and shellfish including abalones in different parts of the world (Australia, Japan). In France, V. harveyi was responsible of two outbreaks in and in on wild and cultured abalones and V. splendidus was incriminated in one outbreak in one farm in Both induced white spots on the foot, a weakness of abalones followed by high mortalities. In histology, a necrosis of muscle fibres and the presence of rodshaped bacteria in the abalone foot were observed. The diagnosis of these bacterial diseases is actually difficult due to the absence of diagnostic tools to identify the bacteria in routine. At present, it is necessary (1) to use sequencing to identify the bacteria (2) to do experimental infections to know if it is a pathogenic bacterial strain or not. In the world, other abalone diseases are emerging such haplosporidiosis, withering syndrome, herpesviruses and sabellid polychaetes. This talk was followed by a presentation on the case of abnormal mortality reported in an experimental abalone farm in Galicia, Spain in July Abalones, Haliotis tuberculata were introduced from Ireland in First histological examinations performed in the imported seeds in June 2003, August 2003 and later, in April 2004 did not reveal any lesion or pathogen. Moribund and dead abalones showed darkness of foot and loss of surface adherence. Histological analyses performed on affected animals revealed the presence Candidatus Xenohaliotis californiensis in lumen of digestive tract and a new haplosporidialike organisms named H. montforti in several organs including gills, digestive tract, muscle and kidney. This diagnosis was confirmed by a PCR conducted with specific primers. Retrospective molecular analyses were performed on animals fixed just after the introduction from Ireland. Results showed presence of both parasites in these seed. Both pathogens were also detected by PCR on abalones imported from a french hatchery used as an alternative source of abalone seeds. SESSION III: SURVEILLANCE OF WILD STOCKS (Chair: L. Miossec) News from DIPNET Laurence Miossec The European project DIPNET, started on October 2004, aims to integrate current knowledge on the transfer of pathogens between wild and cultured aquatic animal populations. It addresses key issues needed to ensure sustainability and responsible exploitation of aquatic environments. A consortium of 5 participants, including IFREMER (Institut Français de la Recherche pour l Exploitation de la Mer) as a coordinator of the project, VESO (Centre for Veterinary Contract and Commercial Services), FRS (Fisheries Research Services), CEFAS (Centre for Envrironment, 10

12 Fisheries and Aquaculture Science) and University of Zaragoza, forms the coordinating team to facilitate the establishment and operation of the project, decide on the organisation of scientific workshops and efficient management of the project funds. The project s specific tasks focus on (i) a review of disease interactions and pathogen exchanges, (ii) risk assessment and modelling of pathogen exchanges, (iii) epidemiology and surveillance of infectious diseases in wild fish and shellfish, and (iv) network building and knowledge dissemination ( ). Findings and recommendations will be disseminated to all stakeholders via the web site and will also be collated into reports to the European Commission. The European project DIPNET organizes a workshop on April 34 (2006) in Prague. The main objectives are to provide a scientific overview of pathogen transmission between wild and farmed fish and shellfish and to review the evidence for disease interactions and population impacts. The first draft of this bibliographic review will be presented and commented for improvement. A seminar for user groups and stakeholders on Risk Assessment and Modelling, planned on May 2223th 2006 in Weymouth (UK). An epidemiology seminar will be organised on May 30th June 1st 2006 in Zaragoza (Spain). Bibliographical synthesis of pathogen exchanges between wild and cultivated molluscs Stein Mortensen The name of the European DIPNET is an acronym for Disease Interactions and Pathogen exchange between farmed and wild animal populations a European NETwork. The output from the project includes a review on pathogen exchanges between wild and farmed molluscs, crustaceans, as well as a review on possible pathogen exchanges between fish and shellfish. The shellfish subgroups consists of partners from France, UK and Norway. Draft reports have been written, as a background for the upcoming second DIPNET workshop in Prague in April. Examples of the bibliographic synthesis will be presented, in order to illustrate the focus on pathogenic agents causing severe disease problems, their ways of spreading and how the reviews present available literature both scientific, "grey" literature and unpublished findings. A main challenge is to make the extensive reviewed information accessible to the users. This will be done through the DIPNET webpage ( both as newsletters as well as an access to the reports and reviews produced. Additionally, Inter Research has offered us a special issue of Diseases of Aquatic Organisms, with selected, focused review papers on disease transmission in European aquaculture. This issue may be used as a synopsis of a major part of the knowledge collected during the DIPNET project. A table including a list of mollusc pathogens and data concerning their detection in different host species, different age classes and breeding conditions has been presented and discussed just after the presentation and is also available in Annex F. Synthesis of epidemiological surveillance programmes in Europe regarding zoosanitary status of wild stocks Laurence Miossec One of the objectives of the WP3 of the European DIPNET project is to review the epidemiological methodology used in surveillance in aquatic animal populations including farms and wild 11

13 populations of fish and molluscs in order to point out areas of potential epidemiological improvements. A questionnaire was designed to collect information on epidemiological methodology developed in the routine monitoring and surveillance programmes and was sent by mail to the 23 EU National Reference Laboratories for Mollusc Diseases (including 4 with no mollusc production) in July From the 19 relevant NRLs, we received 11 answers (57.9%) which gave a good view of the epidemiological methods currently used for monitoring and surveillance of mollusc disease and pathogens in Europe. The ongoing surveillance activities on wild and farmed shellfish are carried out according to the current legislation and mainly focused on pathogen detection. The epidemiological methodology is in accordance with OIE Manual recommendations, but could be improved (ie sampling strategy). Moreover this analysis emphasizes the necessity to develop specific studies to evaluate specificity and sensitivity of diagnostic methods. The interest in including wild stock in surveillance programme has been discussed. Indeed, unlike farmed stocks, wild stocks are not submitted to transfers and thus are good representatives of a zone. If biosecurity measures are well applied in a natural bed free of a listed disease, sampling size and frequency can eventually be reduced. Buffer zone around an infected area can contribute to avoid spread of diseases. Buffer zone around free area can also be imagined to protect it and avoid introduction of new pathogens. SESSION IV: METHODS FOR DISINFECTION (Chair: C. François) When disinfection is needed? Cyrille François This presentation introduces the session concerning methods of disinfection in shellfish farming and research. To avoid any confusion about the terms used, disinfectant is defined and distinguished from other antiinfectives: A disinfectant is a substance which inhibits or destroys various microorganisms, by means of an unspecific mechanism, working at high concentrations, characterized by a quick and short action, displaying a general toxicity that allows this substance for an application on inert materials only. In the European regulation of mollusc health, disinfectants are mentioned for specific goals, related to transport (disinfection of water and means of transport) and restoration of the approval of a zone or a farm (disinfection of equipment and facilities after an outbreak of a listed disease). As disinfectants are meant to be used only on inert materials, an approval for agricultural and hygiene use is sufficient. In the case of disinfection after an outbreak of a listed disease, these substances should usually belong to an official list of disinfectants approved by the Member State. The scope of disinfectants is very large. It concerns both aquaculture establishments and experimental structures, in a day to day approach but also during health crisis. Considering the great number of parameters that should be considered to make an efficient and safe use of these products, it is recommended to write detailed guidelines. However it raises a question: is there enough information at our disposal for a reasonable utilization of disinfectants in shellfish farming and research? 12

14 There is generally very few information available for molluscs. S. Bergman said that a list of disinfectant products in Gemany is available against aquatic diseases. Y. Torgersen mentioned that there are European standards to test efficacy of products against pathogens. However, it is difficult to realize tests because all the pathogen development stages must be considered. G. Ceschia said that in Italy ozone is the only disinfectant used against mollusc pathogens. Which are the general methods for disinfection? Yngve Torgersen Disinfection is employed as a common disease management tool in aquaculture. Disinfection procedures should be part of a disinfection programme designed for a specific purpose. Disinfection may be used in biosecurity programmes to eradicate or exclude specific diseases from aquaculture establishments, as well as a routine sanitary measure to reduce disease incidence within aquaculture establishments. Physical means of disinfectants are often not recognised and utilised in aquaculture. In many situations, they would be a useful supplement or even replacement of chemical disinfectants. The efficacy of chemical disinfection is affected by various factors, including temperature, ph, and the presence of organic matter. At high temperatures, the disinfecting action is faster as long as the decomposition of the disinfectant does not occur. At low temperatures the biocidal efficacy of most disinfectants decreases. Many disinfectants have an optimum ph range/level, and product choice should depend on the ph of the diluents (water). For example, quaternary ammonia is more efficient at alkaline ph while iodine and iodophores are more efficient at neutral or acid ph. The presence of organic material and greasy substances may significantly reduce the efficacy of a disinfectant. Therefore, surfaces should be cleaned thoroughly before applying disinfectants. The use of disinfectants may require measures to protect personnel, aquatic animals and the environment. The manufacturer s instructions for safe and effective use and for disposal of a chemical disinfectant under aquaculture conditions should be followed. Chemical disinfectants to be used in aquaculture should be evaluated or tested against relevant aquatic pathogens under relevant conditions. I. Arzul questioned the possibility to approve a farm in a non approved zone. Considering the lack of data concerning disinfectant against mollusc pathogens, it is impossible to be sure that intake water does not bring pathogens. According to Y. Torgersen, the future legislation will allow free establishment in non approved zone but EU standards indicating minimum disinfectant concentration will be required. 13

15 Biosecurity and disinfection procedures in mollusc aquaculture facilities David Fraser Emerging diseases have a significant impact on shellfish aquaculture worldwide. This highlights the importance of preventing the introduction of pathogens and minimising transmission of disease. Good husbandry and biosecurity practice on farms are important to the health of stocks, as is development of standard operation procedures and compliance with codes of practice. Staff training and cooperation is essential to ensure high standards are achieved and maintained. Identification and management of the risks associated with the movement of live shellfish, grading and harvesting equipment, processing, effluent discharge and disposal of waste are considered. Cleaning and disinfection procedures, based upon current scientific knowledge and practical experience during outbreaks of serious disease and routine farm surveillance, are presented. Effective disinfectant treatments are required as best practice for each task, to reduce the potential for introduction and spread of shellfish disease. The development of standards of disinfection for shellfish facilities is discussed. The aim should be to identify health risks, improve communication at all levels, and develop measures to protect shellfish aquaculture interests. Regarding the need of standards and minimum measures in using disinfectants, there will be a call in next FP7 in autumn under priority 1 start aquatic animal health in support to Policy. SESSION V: LEGISLATION MATTERS (Chair : Y. Torgersen) European legislation for the mollusc diseases: which survey/diagnosis for which diseases? Yngve Torgersen The new aquatic animal health Directive introduces a general risk based animal health surveillance which shall be applied in all farms or farming areas. This is an extension of the requirements in the present Council Directive 95/70/EC, where all Member States must have a monitoring and sampling program to detect abnormal mortality in farmed mollusc stocks and harvested natural beds. It is proposed to make the surveillance risk based. It is therefore impossible to lay down in the primary legislation specific requirements on inspections frequency and sample size taking into account the diversity of the industry in the Member States. The details (sample frequency, sample size etc) will be laid down by secondary legislation in accordance with Article 10.3 of the proposal, taking into account the guidelines and recommendations in Annex IV. The level of surveillance will be related to both the recognised health status and the risk of contracting and spreading of disease into/out of the farm or farming area. The list of exotic diseases covered by the proposed directive has been discussed. This list should be be based on the OIE list but could differwhere this was considered appropriate for the Community. Some diseases were dropped because their impact is negligible. Future directive should also include a list of susceptible and carrier host species which will be established on the basis of scientific results. 14

16 The impact of the new Directive for laboratories Yngve Torgersen The main part of the aquaculture industry is covered by the general Feed and Food Law (Regulation (EC) No178/2002. In addition to the general Food Law, a number of more detailed and specific Regulations have been adopted (the Hygiene package ). Regulation (EC) No 882/2004 on the official controls performed to ensure the verification of compliance with the feed and food law, animal health and animal welfare rules, in particular Article 11 Methods of sampling and analysis Article 12 Official laboratories Article 32 Community reference laboratories Article 33 National reference laboratories These provisions have an impact on the proposal from the Commission for a Council Directive on animal health requirements for aquaculture animals and products thereof, and on the prevention and control of certain diseases in aquatic animals. The Commission proposes that any laboratory examination for the purpose of the Directive shall take place only in designated national laboratories authorised for such purpose by the competent authority, and that the laboratory examinations to confirm the presence of listed diseases shall be carried out according to standardised methods laid down in secondary Community legislation (where possible according to the OIE Manual). The function and duties of the designated national laboratories are laid down in Annex VI part III to the Directive The designation of Community reference laboratories shall be carried out in accordance with secondary Community legislation, including the period for which the designations shall apply, and the function and duties of the Community reference laboratories are laid down in Annex VI, Part I to the Directive. The Member States must nominate a national reference laboratory for each of the Community reference laboratories designated. The function and duties of the national reference laboratories are laid down in Annex VI part II to the Directive. As laid down in Regulation (EC) No 882/2004, minimum requirements for quality assurance and/or accreditation will apply, and the participation in comparative ring testing will be compulsory. The new directive is planned to be applied on the 1 st of January Reference laboratories can be accreditated for a system and not neccesarily for a list of diseases. Extension of time may be proposed for laboratories to obtain accreditation once the directive will be applied. CRL will be designed for a fixed periode of time. 15

17 SESSION VI: EU PROJECTS IN SUPPORT OF CRLs / NRLs ACTIVITIES (Chair : J.P. Joly) EU project on Perkinsus olseni: Workshop for the analysis of the impact of perkinsosis on the European shellfish industry Acronym: WOPER Philippe Soudant Perkinsosis affects molluscs in the five continents, resulting in high mortality of some commercial species and huge economic impact. Some species of the genus Perkinsus (P. olseni and P. mediterraneus) are known to affect bivalve molluscs of the European coasts and other species (P. marinus and P. chesapeaki) are exotic to Europe. The goal of this project is to bring together industry, research community and administration in a workshop to address the threat of perkinsosis to European shellfish industry. The different aspects that have to be considered in the analysis of the disease and its implications for the shellfish industry will be distributed in 5 groups (the genus Perkinsus, epizootiology, hostpathogen interaction, effects on industry and control and fighting strategies), each of them representing a task and a session in the workshop. Preparation of the workshop will involve the production of a document for each task that will be the basis for discussion in the workshop. Special emphasis will deserve looking for the involvement of European industry and regulators, and experts from other countries whose shellfish industries are deeply concerned with perkinsosis, such as USA, South Korea and Australia. Outcomes of the workshop will be state of the art knowledge, current and future research priorities and recommendations for industry and for policy makers, all of which will be included in a specific publication to enhance its broad dissemination. Coordinator: Dr. Antonio Villalba (CIMA, Spain) Partners: Dr. Rosa Fernández (CETMAR, Spain), Ms. Marisa Fernández (CETMAR, Spain) Dr. Philippe Soudant (UBOCNRS, France), Dr. Isabelle Arzul (IFREMER, France) Dr. Leonor Cancela (UALG, Portugal) Dr. Giuseppe Ceschia (IZSV, Italy) Dr. Antonio Figueras (IIMCSIC, Spain) Dr. Steve Feist (CEFAS, UK). Y. Torgersen raised possible conflicts of interest between scientists, industry and policy makers. The listed pathogens constitute a good example of such conflict: Mikrocytos mackini will be withdrawn from the OIE list and Perkinsus olseni will be maintained while it is the contrary for the E.U. list. Indeed, Perkinsus olseni is considered present in most of EU countries producing susceptible species and the impact of this parasite on European shellfish industry is not clear (but needs to be determined). Thus in the present future directive version, Perkinsus olseni is not listed while it is included in the OIE list. 16

18 PANDA year 2 Isabelle Arzul The PANDA project aims to establish a permanent network of aquatic animal health specialists (researchers and diagnosticians) to provide a forum for the debate of issues concerning diseases in European aquaculture, and to communicate the results of these discussions to the European Commission with provision of advice on EU policy and legislation for aquatic animal health. PANDA consists of five scientific work packages: risk analysis, epidemiology, diagnosis, environmentally safe strategies for disease control, training in research and diagnostics. These workpackages are lead by PANDA consortium members, supported by small task force of specialists, with wider input from experts registered in the PANDA network. Focused workshops and ongoing online discussions on subjects related to aquatic infectious diseases are being held for exchange of scientific information and opinion. Progress and outputs A project website has established, and 280 worldwide experts have joined the network. A simple hazard scoring system was used to identify and prioritise the most significant exotic, emerging and reemerging disease hazards for European aquatic animals. A prototype database of epidemiological characteristics of the identified disease hazards has been developed, information is being collated and entered onto it. An assessment of the current best methods for rapid and accurate detection was undertaken for the main aquatic disease hazards and will help to identify requirements for improvements, standardisation and validation. Disease treatment strategies and their known or likely impact on the environment were reviewed and discussed. A links based database of training opportunities for aquatic animal disease research and diagnosis is under development, and can be viewed on the website. Further details are available on the website ( where you can register on the expert database to keep abreast of progress, contact other experts in your field, and contribute to the discussions and debates. 17

19 SESSION VII: NEWS FROM THE COMMUNITY REFERENCE LABORATORY (Chair : I. Arzul) Synthesis on PANDA WP4 regarding diagnostic of mollusc diseases JeanPierre Joly Work of WP4 PANDA members started in September 2004 by using the initial WP2 list of emerging, reemerging and exotic diseases characterised by their risk and uncertainty scores. A questionnaire was elaborated with a list of the most important viral, bacterial and parasitic mollusc diseases. The questionnaire was then filled with information collected from the scientific literature regarding the sensitivity and specificity of available diagnostic tools compared to histology. The questionnaire was complemented by a bibliography of the most important papers about the considered diseases. Each disease was also characterised using this classification: 1 Nonexotic disease with diagnostic tools available 2 Exotic disease to be considered 3 Nonexotic disease of second interest for WP4 This questionnaire was submitted to the European experts during the 2005 annual meeting of European National Reference Laboratories (NRLs). Working groups during this meeting showed that some diagnostic techniques still need validation. From this work a new table evolved including the infectious agent to be considered, the susceptible species in Europe and the diagnostic tools available. Each diagnostic tool is characterised as: 1 Screening or confirmatory technique, validated or not 2 Technique used by most NRLs or technique that should be used by NRLs or techniques that could be adopted by NRLs Comments on this table are needed from the participants of the meeting. From these comments WP4 can make suggestions to the EU commission about techniques needing training among the EU NRLs. S. Bergman remarked that the validation is well defined by the OIE and should be used according to this definition. I. Arzul replied that in theory, validation in the context of this workpackage was considered according to the OIE definition but from a practical point of view, very few diagnostic tools are well validated. If scientific publications and experience show that a diagnostic technique can be used routinely in laboratories, PANDA workpackage 4 has considered it validated (e.g. PCR for the detection of OsHV1). Elaboration of a collection of bacterial pathogens for aquatic organisms Denis Saulnier, Nicole Faury, JeanClause Masson, AnneGeneviève Martin and Frédérique Le Roux. A collection of bacterial pathogens isolated from aquatic organisms and belonging mainly to Vibrio genus has been constituted. This collection is now accessible on a web site at the following address It was elaborated thanks to a grant of the Ifremer and CNRS institutes and the French Research Department. Several French laboratories specialized in disease of aquatic organisms were involved in this project: LGP and LPI of Ifremer in La Tremblade and Brest respectively, LEMAR of IUEMUBO in Brest, LAC and LAT of Ifremer in New Caledonia and Tahiti respectively, in French Polynesia. The aims were firstly to characterize 18

20 taxonomically bacterial strains, mainly isolated from shrimp and molluscs, by classical phenotypic tests and molecular techniques. Secondly to evaluate their virulence potential by standardized experimental infection, thirdly to constitute a cryobank duplicated in two implantations to assure the sustainability of the collection, and finally to create a web site as a management tool allowing also to share information about each bacterial strain with the scientific community. This presentation gives a glimpse of the work which has been done to characterize each bacterial strains of the collection and give details to how navigate in this newly created web site Interlaboratory proficiency test Isabelle Arzul Interlaboratory proficiency tests aim at establishing that the examination of a given sample lead to the same results in all the laboratories of the NRL network. Ring tests are of upmost importance for accreditation and for harmonisation of diagnostic within the EU. The Community Reference Laboratory for Mollusc Diseases has already organised 4 interproficiency tests since These tests were all based on an itinerant collection of histological sections including European but also exotic pathogens. In 2004, a special test was organised for NRLs from candidate countries and focused on endemic diseases: bonamiosis and marteiliosis in flat oysters, Ostrea edulis. After the contents of the test being decided, slides are prepared from blocks registered in the collection of reference material. New slides are checked according to their quality and their status before being included in the ring test. Announcement is sent through the reflabnet list and the collection can start travelling from a laboratory to another. Participants have one week to read one collection of 30 to 40 slides and to send their results to the CRL. Expected results are only qualitative (presence or absence of pathogens). In , a fifth ring test was realized. It included two itinerant slide collections: the first one targetted Ostrea edulis listed diseases, bonamiosis and marteiliosis, and the second one concerned Crassostrea gigas listed or not listed pathogens, Mikrocytos mackini, Haplosporidium nelsoni and Marteilioides chungmuensis. 19 laboratories agreed to read collection 1 and 14 laboratories read collection 2. Concerning collection 1, 50% of participants obtained 71, 43%, 100 % and 80% or more of good responses for heart imprints, digestive gland imprints and histological sections respectively. Low levels of infection with Bonamia ostreae constituted main errors. Concerning collection 2, 50% of participants obtained 83,83% or more of good responses. Most of errors corresponded to confusion between microsporidia in ovocytes and Marteilioide chungmuensis or to mis diagnosis of low levels of infection with Haplosporidium nelsoni. Compared to previous ring tests, results obtained by NRLs for Mollusc diseases are a bit worse for Ostrea edulis diseases and better for Crassostrea gigas diseases. However, training is still needed for histological examination. Y. Torgersen proposed to identify NRLs needing training from the ring test results. E.U. Commission can provide funds for such training. D. Fraser asked how NRLs could have feedback from the CRL after misinterpretation of slides. I. Arzul suggested to put some pictures corresponding to certain slides on the website. I. Arzul also suggested to send two sets of identical slides in the same time in order to save time. Indeed, in the case of mollusc diseases and considering the number of participants, ring tests usually take around 12 months between the first to the last participants. 19

21 Progress in harmonisation of QA procedures in National Reference Laboratories JeanPierre Joly Several National Reference Laboratories and Pathology Laboratories are currently building their Quality Management System based on ISO Standard (General requirements for the competence of testing and calibration laboratories). Some of them are already accredited but most of them are seeking for accreditation by their respective accreditation bodies. Some of these laboratories, and more particularly those in the last countries joining the European Union, are asking for standardised procedures in histopathology. The CRL proposes to write standard operating procedures (SOP) about histopathology techniques. These SOPs can be used as templates by European laboratories and can help them when writing down their own documentation for Quality Management. As a first step we wrote SOPs about bonamiosis, marteiliosis and perkinsosis diagnosis by histology. These documents were prepared by the CRL and proposed in They were reviewed by six European pathologists from five different European National Laboratories before being accepted. These SOPs are now available on the CRL website ( They will be updated regularly. Other documents being prepared for 2006 will concern perkinsosis diagnosis by RFTM, SOPs for histology and cytology processing and SOP for opening bivalves. SOP about histology process is almost ready and will be submitted to all NRLs comments very soon. G. Ceschia and A. Turnbull said that accreditation bodies now ask for risk estimation to be added to the procedures. The new CRL website JeanPierre Joly A new website of the CRL has been created. It is especially dedicated to all NRLs looking for information regarding mollusc diseases ( The website is organised in five sessions: main activities, scientific activities, legislation, Network of NRLs and Tutorials. Pages make available general information, events, legislation, reference photos, links to other resources of interest for people working in the field of mollusc health certification and disease survey programmes (OIE Aquatic Animal Diseases Commission, DFO synopsis of infectious diseases of molluscs, aquatic animal pathology database AAPQIS and a database reporting the movements and transfers of aquatic species from FAO ). The web site is also a place of advertisements of events in the field of mollusc health management. Working prospects of the Community Reference Laboratory for molluscs diseases Isabelle Arzul 20

22 In 2005, the NRLs network for mollusc diseases included 18 NRLs from Member States and 4 NRLs from other countries (Norway, Croatia, Romania, and Turkey). Functions and duties of the CRL for mollusc diseases are given by the Directive 95/70/EC. One of the main aims of the CRL is to harmonize diagnosis within the EU. For this purpose the CRL has created and maintains a collection of pathogens available for laboratories in Member States. A CD ROM on histology and anatomopathology has also been developed since The CDROM proposes illustrations and comments believed to be valuable for mollusc diseases diagnostic, especially diseases notifiable to the EU and OIE. The first edition was focused on the two oyster species Ostrea edulis and Crassostrea gigas and is subject to permanent reviewing and updating. The second edition (version 1.1), available in 2003, includes oyster species and, now mussels, pearl oysters and abalone. It will be updated in Interlaboratory proficiency tests are regularly organised to test the ability of laboratories to identify listed and important pathogens by histology. In a new ring test will be proposed to NRLs including at least Ostrea edulis histological sections. An advisory group in Quality Assurance was created in Exchange of information between NRLs during annual meetings or by s allowed the CRL to write drafts of quality assurance documents that could be used by NRLs wishing to build their Quality System. The CRL also aims at collecting and collating information on the mollusc disease situation in Member States and worldwide. A new template for the 2006 epidemiological report was presented during the present Annual Meeting and will circulate through NRLs network for further comments. Information collected through the questionnaire help to draw a map presenting the situation of European Countries regarding bonamiosis and marteiliosis. However, this map is subjected to several comments since it represents data included in the previous epidemiological report and not necessarily the actual situation. The CRL assists the NRLs in the diagnosis of disease outbreaks in Member States and provides opportunities of training and retraining through trainees, technical workshop and annual meetings. It is proposed to have the 2007 annual meeting combined with a technical workshop in MarchApril 2007 in La Tremblade. 21

23 Annex A : Agenda of the Annual Meeting of the National Reference Laboratories for Mollusc Diseases, Nantes 2223 March 2006 Wednesday 22 March Registration for the annual meeting Welcome address and announcements Y. Torgersen (European Commission) I. Arzul (Community Reference Laboratory for Mollusc Diseases) SESSION I: DIAGNOSIS AND SURVEY OF MOLLUSC DISEASES Chairman C. Garcia Representatives from each Member State present the disease situation in their respective country in 2005, emphasising mollusc diseases listed in the EU Directives 91/67 and 95/70, and/or the diagnosis of pathogens of special interest ( Coffee Break) Lunch Presentation of the new epidemiological report template. C. François SESSION II: EXAMPLES OF MOLLUSC DISEASE SURVEYS Chairman I. Arzul Results of the perkinsosis survey ( ) in France. L. Miossec Herpesvirus and oyster mortalities in France. C. Garcia A long term study on bonamiosis and marteiliosis in France. I. Arzul Haplosporidium infections in flat oysters in The Netherlands. M. Engelsma Pathological events o abalones in Europe. C. Garcia Coffee Break SESSION III: SURVEILLANCE OF WILD STOCKS Chairman L. Miossec News from DIPNET. L. Miossec Bibliographical synthesis of pathogen exchanges between wild and cultivated molluscs. S. Mortensen Synthesis of epidemiological surveillance programmes in Europe regarding zoosanitary status of wild stocks. L. Miossec Discussion Dinner downtown 22

24 Thursday 23 March SESSION IV: METHODS FOR DISINFECTION Chairman C. François When disinfection is needed? D. Fraser Which are the general methods for disinfection? Y. Torgersen Disinfection means in mollusc establishments. C. François Coffee break SESSION V: LEGISLATION MATTERS Chairman Y. Torgersen European legislation for the mollusc diseases: which survey/diagnosis for which diseases? Y. Torgersen The impact of the new Directive for laboratories. Y. Torgersen SESSION VI: EU PROJECTS IN SUPPORT OF CRLs / NRLs ACTIVITIES Chairman J.P. Joly EU project on Perkinsus olseni P. Soudant PANDA year 2 I. Arzul Lunch SESSION VII: NEWS FROM THE COMMUNITY REFERENCE LABORATORY Chairman I. Arzul Synthesis on PANDA WP4 regarding diagnostic of mollusc diseases J.P. Joly Elaboration of a collection of bacterial pathogens for aquatic organisms. D. Saulnier Interlaboratory proficiency test. I. Arzul Progress in harmonisation of QA procedures in National Reference Laboratories. J.P. Joly The new CRL website. J.P. Joly Working prospects of the Community Reference Laboratory for molluscs diseases. I. Arzul 23

25 Annex B: List of Raquel Aranguren Instituto de Investigaciones Marinas CSIC Eduardo Cabello, Vigo Spain arangur@iim.csic.es Isabelle Arzul Community Reference Laboratory for molluscs Ifremer LGP RonceLesBains La Tremblade France (33) (33) iarzul@ifremer.fr MarieAthénaïs De Schaetzen Laboratoire National de Référence des mollusques bivalves vivants, Service de microbiologie, Département des Sciences des Denrées Alimentaires, Bât B43Bis, n 20 Boulevard de Colonster, 4000 Liège, Belgique madeschaetzen@ulg.ac.be Sven M Bergmann FriedrichLoefflerInstitut Federal Research Institute for Animal Health Institute for Infectology Boddenblick 5a Greifswald Insel Riems Germany sven.bergmann@fli.bund.de Giuseppe Ceschia Dipartimento di Ittiopatologia Instituto Zooprofilattico Sperimentale delle Venezie Via della Roggia, Basaldella di Campoformido UD Italy gceschia@izsvenezie.it 24

26 Deborah Cheslett Marine Institute, Abbotstown, Snugboro Road, Dublin 15, Republic of Ireland ( deborah.cheslett@marine.ie Mihaela Costea Institute for Diagnosis and Animal Health 63 Dr Staicovici Street Sector 5, cod RO Bucharest Romania costea.mihaela@idah.ro office@idah.ro Marc Engelsma CIDCLelystad, Institute for Animal Disease Control P.O. Box AA Lelystad The Netherlands marc.engelsma@wur.nl Sylvie Ferrand Community Reference Laboratory for molluscs Ifremer LGP Ronce Les Bains La Tremblade France (33) (33) Sylvie.Ferrand@ifremer.fr Cyrille François Community Reference Laboratory for molluscs Ifremer LGP Ronce Les Bains La Tremblade France (33) (33) Cyrille.francois@ifremer.fr David Fraser FRS Aberdeen Scotland PO Box 101 Victoria Road Aberdeen AB11 9DB Scotland UnitedKingdom fraserdi@marlab.ac.uk 25

27 Céline Garcia Community Reference Laboratory for molluscs Ifremer LGP Ronce Les Bains La Tremblade France (33) (33) Celine.garcia@ifremer.fr Mitja Gombac Institute of Pathology Forensic and Administrative veterinary medicine University of Ljubljana, Veterinary Faculty Gerbiceva 60 SI 1000 Ljubljana Slovenia mitja.gombac@vf.unilj.si JeanPierre Joly Community Reference Laboratory for molluscs Ifremer LGP RonceLesBains La Tremblade France Jean.pierre.joly@ifremer.fr Ian Laing CEFAS Weymouth laboratory Barrack road The Nothe Weymouth, Dorset DT4 8UB England, United Kingdom i.laing@cefas.co.uk Lone Madsen Danish Institute for Fisheries ResearchDepartment of Marine Ecology and Aquaculture Fish Disease Laboratory Stigbøjlen Frederiksberg C Danmark lm@dfu.min.dk lm@difres.dk Laurence Miossec Community Reference Laboratory for molluscs Ifremer LGP La Tremblade France (33) (33) lmiossec@ifremer.fr 26

28 Stein Mortensen Institute of Marine Research PO Box 1870, Nordnes 5817 Bergen Norway Maeva Robert Community Reference Laboratory for molluscs Ifremer LGP RonceLesBains La Tremblade France (33) (33) Maeva.robert@ifremer.fr Denis Saulnier Ifremer Genetic and Pathology Laboratory Ronce Les Bains La Tremblade France (33) (33) Denis.saulnier@ifremer.fr Philippe Soudant Université de Bretagne Occidentale ; Institut Universitaire Européen de la Mer LEMAR Laboratoire des sciences de l'environnement marin (UMR 6539) Technopole Brest Iroise, Place Nicolas Copernic, Plouzané France (33) (33) Philippe.Soudant@univbre st.fr Yngve Torgersen European Commission Office FR Rue Froissant 101 B1049 Brussels Belgium Yngve.torgersen@cec.eu.in t Anna Turnbull FRS Marine Laboratory, Victoria Road, Torry, Aberdeen Scotland a.turnbull@marlab.ac.uk 27

29 Annex C : Annual reports from the NRLs on epidemiological situation in Member States in General data Production of the country Zoning Production of zones in the country Number of farms within the country Main exports for aquaculture purpose towards other Member States Main imports for aquaculture purpose from other Member States Main exports for aquaculture purpose towards Third Countries Main imports for aquaculture purpose from Third countries Number of hatcheries in the different zones with production Flat oyster Mussel Fishermen s catch: Flat oyster Mussel Other (Pectinidae, clams, etc.) O. edulis M. galloprovincialis O. edulis M. galloprovincialis Name Zone I Istra (Limski Bay, Vabriga, Catching area Zub) Zone II River Krka Estuary Zone III Malostonski Bay Zone I Istra Zone II River Krka Estuary Zone III Malostonski Bay Zone I Istra Zone II River Krka Zone III Malostonski Although Croatia is on the list of the Third Countries until now there are no exports of live molluscs. There are no imports of live molluscs. Zone III Malostonski Bay There is one experimental hatchery O. edulis M. galloprovincialis Other species O. edulis M. galloprovincialis Other species O. edulis M. galloprovincialis Other species Ostrea eulis CROATIA 80 t 2400 t 8 t 35 t 50 t 9 t 200 t 35 t 1 t 435 t 10 t 78 t 1800 t 5 t

30 2. Laboratory data Diagnostic methods used for Bonamia ostreae Screening Methods Presumptive Diag. Meths Histology/smears Histology/smears Confirmatory Methods Diagnostic methods used for Marteilia refringens Screening Methods Presumptive Diag. Meths Histology/smears Histology/smears Confirmatory Methods Diagnostic methods used for exotic pathogens Pathogen A Pathogen B Presumptive Diagnostic Confirmatory Presumptive Diagnostic There is no monitoring for exotic diseases. Confirmatory Etc Diagnostic methods used for other diseases or pathogens monitored in the country Marteilia spp. in mussels (M.galloprovi ncialis) Screening Methods Presumptive Diagnostic Confirmatory Histology/smears Histology/smears Pathogen B Screening Methods Presumptive Diagnostic Confirmatory Etc 29

31 3. Epidemiological data SURVEILLANCE AND MONITORING OF BONAMIA / MARTEILIA : Sampling size (number of samples and individuals per sample) and sampling dates for each zone SURVEILLANCE AND MONITORING OF OTHER PATHOGENS : Sampling size (number of samples and individuals per sample) and sampling dates for each zone and each targeted pathogen STUDY OF ABNORMAL MORTALITY Mortality rate and sampling date for each declared mortality case per bivalve species and per zone Number of positive results for Bonamia / Marteilia Number of positive results for other diseases / pathogen Results regarding cases of abnormal mortality outbreaks Number of samples examined in total Zone I Zone II Zone III Marteilia maurini/m. galloprovincialis Zone I Zone II Zone III Other There were no report about abnormal mortalities Zone I Zone II Zone III M. maurini Zone I Zone II Zone III Other April/ August March/ September March, April/ September September/October September August/September September 3 samples (150 ind)/ 3samples (150 ind.)) 1sample (30 ind.) /1sample (30 ind.) 5 samples (250 ind.) / 4 samples (200 ind.) 3samples /90 ind. 1 sample/30 ind. 3 samples/90 ind. 2 samples/60 ind. Bonamia ostreae :0 positive / 150 analysis Marteilia refringens : 0 positive/150analysis Bonamia ostreae :0 positive / 30 analysis Marteilia refringens : 0 positive/30analysis Bonamia ostreae :0 positive / 250 analysis Marteilia refringens : 0 positive/200analysis M.galloprovinci alis Zone (name) Bivalve species Pathogen Pathogen 5 positive /90 analysis 2 positive /30 analysis 8 positive /90 analysis 7 positive /60 analysis x/x 26 samples (1080 individuals) Additional comments: Accreditation process in the frame of the CARDS 2002 project and under the supervision of SINAL experts (Italy) has started and it is planned to be finished at the end of y/y 30

32 1. General data Production of the country Zoning Production of zones in the country Number of farms within the country Main exports for aquaculture purpose towards other Member States Main imports for aquaculture purpose from other Member States Main exports for aquaculture purpose towards Third Countries Main imports for aquaculture purpose from Third countries Number of hatcheries in the different zones with production 2. Laboratory data Diagnostic methods used for Bonamia ostreae Diagnostic methods used for Marteilia refringens Diagnostic methods used for exotic pathogens Diagnostic methods used for other diseases or pathogens monitored in the country Flat oyster Mussel Cockles Mini clams Name of the zone / status O. edulis M. edulis Cardium spp Spisola.solida Limfjorden/Bonamia and Marteilia approved Limfjorden Flat oyster Mussel Cockles DENMARK 942 t 69,155 t 1,002 t 30 t 942 t 45,043 t 675 t Limfjorden 3 Species: Species: Species: Species: Origin / Destination: Origin / Destination: Origin / Destination: Origin / Destination: Limfjorden Flat oyster 1 Screening Methods Presumptive Diag. Meths Confirmatory Methods Screening Methods Presumptive Diag. Meths Confirmatory Methods Histology (imprints) Histology Histology Histology 31

33 3. Epidemiological data SURVEILLANCE AND MONITORING OF BONAMIA / MARTEILIA : Sampling size (number of samples and individuals per sample) and sampling dates for each zone Limfjorden May 9th June 21st October 31st 2 samples of 30 individuals 1 sample of 30 individuals 3 samples of 30 individuals SURVEILLANCE AND MONITORING OF OTHER PATHOGENS : Sampling size (number of samples and individuals per sample) and sampling dates for each zone and each targeted pathogen Pathogen/Bivalve species (names) 0 STUDY OF ABNORMAL MORTALITY Mortality rate and sampling date for each declared mortality case per bivalve species and per zone Number of positive results for Bonamia / Marteilia Number of positive results for other diseases / pathogen Results regarding cases of abnormal mortality outbreaks Number of samples examined in total Limfjorden Bonamia ostreae: x positive* /90 analyses Marteilia refringens: y positive*/90 analyses 0 6 (180 individuals) Additional comments: *Investigations for 2005 not finished 32

34 1. General data Production of the country Zoning Production of zones in the country Clam Pacific oyster Flat oyster Cockles Mussels Scallops Abalones R. decussates/r. philippinarum C. gigas O. edulis Cerastoderma edule M. edulis/m. galloprovincialis Pecten maximus Haliotis tuberculata Name of the zone / status: I, II, III, IV, V, VI, VII, VIII, IX and X Zones I, II, III, IV Mussels Pacific oysters Clams FRANCE 3000 t t 1500 t 2000 t t No data No data t t No data Zone V Pacific oysters Clams 9000 t No data Zone VI Pacific oysters Mussels Clams t 1480 t No data Zone VII Pacific oysters Mussels Cockles 9000 t t No data Zone VIII Pacific oysters Flat oysters Mussels Clams t 1500 t t No data Number of farms within the country Zones IX, X Zone III Zone IIIIV Zone V Zone VI Zone VII Zone VIII Zone IX Zone X Pacific oysters Mussels Clams t t No data

35 Main exports for aquaculture purpose towards other Member States Pacific oysters (<40g): Total: Italy Belgium Germany Spain Switzerland Russia Ireland Luxemburg Netherlands 6320 t 4169 t 640 t 386 t 245 t 233 t 133 t 35 t 85 t 76 t Flat oysters (<40g) : Total: Spain Italy Kuwait Germany Belgium 252 t 127 t 87 t 9 t 8 t 11 t Mussels (alive or fresh) Total: Spain Switzerland Italy Germany Belgium Luxemburg Netherlands 4173 t 2581 t 290 t 538 t 328 t 228 t 61 t 48 t Scallops (alive or fresh) Total: Italy Spain Belgium Germany Luxemburg Austria Switzerland United Kingdom 1622 t 720 t 475 t 223 t 71 t 55 t 20 t 15 t 12 t 34

36 Main imports for aquaculture purpose from other Member States Pacific oysters (<40g): Total: Ireland United Kingdom Spain Portugal Denmark Netherlands 2812 t 1472 t 514 t 434 t 224 t 59 t 82 t Flat oysters (<40g) : Total: Ireland United Kingdom Spain 65 t 36 t 23 t 6 t Mussels (alive or fresh) Total: Netherlands Spain Ireland United Kingdom Greece Italy Norway Belgium Denmark t t 9330 t 8601 t 3345 t 3925 t 2659 t 1195 t 284 t 486 t Scallops (alive or fresh) Total: United Kingdom Ireland Norway Netherlands Belgium 3548 t 2780 t 188 t 45 t 60 t 13 t Main exports for aquaculture purpose towards Third Countries Main imports for aquaculture purpose from Third countries No data No data No data Scallops (alive or fresh) USA Chili 387 t 103 t 35

37 Number of hatcheries in the different zones with production Zone I to IV total 1 hatchery 1 Zone VI: total 3 hatchery 1 hatchery 2 hatchery 3 Zone VII : total 4 hatchery 1 hatchery 2 hatchery 3 hatchery 4 Zone VIII : total 4 hatchery 1 hatchery 2 hatchery 3 hatchery 4 Zone IX total 4 hatchery 1 hatchery 2 hatchery 3 hatchery 4 Pacific oysters/clams Pacific oysters Pacific oysters Clams Pacific oysters Pacific oysters Abalones Pacific oysters Scallops Abalones Pacific oysters Cams Pacific oysters, clams Abalones Abalones Abalones No data 36

38 2. Laboratory data Diagnostic methods used for Bonamia ostreae Screening Methods Presumptive Diag. Meths Histology, tissue imprints, PCR (in development) Histology, tissue imprints Diagnostic methods used for Marteilia refringens Confirmatory Methods Screening Methods Presumptive Diag. Meths ISH, TEM, sequencing Histology, tissue imprints Histology, tissue imprints Diagnostic methods used for exotic pathogens B exitiosa Confirmatory Methods Presumptive Diagnostic Confirmatory ISH, TEM, PCRRFLP, sequencing Histology, tissue imprints ISH, PCR, TEM, sequencing B. roughleyi M. sydneyi M. mackini Presumptive Diagnostic Confirmatory Presumptive Diagnostic Confirmatory Presumptive Diagnostic Confirmatory Histology, tissue imprints PCR, ISH, TEM, sequencing Histology, tissue imprints ISH, PCR, sequencing Histology, tissue imprints ISH, PCR, TEM, sequencing Histology TEM, ISH, PCR, sequencing H. costale Presumptive Diagnostic Confirmatory Histology, thioglycolate culture TEM, PCR, sequencing P. marinus Presumptive Diagnostic Confirmatory Histology TEM, PCR, ISH, sequencing Xenohaliotis californiensis Presumptive Diagnostic Confirmatory 37

39 Diagnostic methods used for other diseases or pathogens monitored in the country Haplosporidium nelsoni Perkinsus. olseni OsHV1 Vibrio infection Vibrio tapetis Screening Methods Presumptive Diagnostic Confirmatory Screening Methods Presumptive Diagnostic Confirmatory Screening Methods Presumptive Diagnostic Confirmatory Screening Methods Presumptive Diagnostic Confirmatory Histology Histology ISH, PCR Histology, thioglycolate culture Histology, thioglycolate culture PCR, sequencing PCR PCR, histology ISH, PCR Culture, biochemical identification Culture, biochemical identification Sequencing Vibrio harveyi Screening Methods Presumptive Diagnostic Confirmatory Macroscopy, PCR, culture, biochemical identification PCR, culture, biochemical identification Immunological assay, PCR, sequencing Marteilia maurini Screening Methods Presumptive Diagnostic Confirmatory Screening Methods Presumptive Diagnostic Confirmatory Culture, biochemical identification Culture, biochemical identification Sequencing Histology, tissue imprints Histology, tissue imprints PCRRFLP 38

40 3. Epidemiological data SURVEILLANCE AND MONITORING OF BONAMIA / MARTEILIA : Sampling size (number of samples and individuals per sample) and sampling dates for each zone Bonamia ostreae Zone I 03/30/05 10/10/05 3 samples of 30 individuals, 1 of 28, 1 of 25 and 1 of 26 2 samples of 30 individuals Zone VIII 01/17/05 1 sample of 50 individuals 03/16/05 1 sample of 150 individuals 04/12/05 1 sample of 150 individuals 06/14/05 1 sample of 50 individuals and 1 sample of 39 individuals 07/05/05 1 sample of 50 individuals 10/14/05 1 sample of 50 individuals, 1 of 40 and 1 of 38 11/02/05 1 sample of 30 individuals 11/02/05 1 sample of 150 individuals Zone IX 08/04/05 1 sample of 49 individuals 09/14/05 1 sample of 101 individuals 11/23/05 1 sample of 125 individuals 39

41 Marteilia refringens Zone I Zone VIII Zone IX 03/30/05 10/10/05 01/17/05 03/16/05 04/12/05 11/02/05 11/02/05 08/04/05 09/14/05 11/23/05 3 samples of 30 individuals, 1 of 25, 1 of 27 and 1 of 26 2 samples of 30 individuals 1 sample of 50 individuals 1 sample of 150 individuals 1 sample of 150 individuals 1 sample of 150 individuals 1 sample of 30 individuals 1 sample of 49 individuals 1 sample of 101 individuals 1 sample of 125 individuals 40

42 SURVEILLANCE AND MONITORING OF OTHER PATHOGENS : Sampling size (number of samples and individuals per sample) and sampling dates for each zone and each targeted pathogen Perkinsus olseni in Ruditapes philippinarum and R. decussatus Zone III Zone IV Zone V Zone VI Zone VIII Zone IX 09/22/05 10/28/05 10/04/05 09/07/05 09/20/05 10/07/05 09/23/05 10/07/05 10/20/05 09/07/05 10/20/05 2 samples of 30 individuals and 1 of 22 2 samples of 30 individuals and 1 of 9 2 samples of 30 individuals 3 samples of 30 individuals 1 sample of 30 individuals 1 sample of 29 individuals 3 samples of 30 individuals 2 samples of 30 individuals 6 samples of 30 individuals and 1 of 26 2 samples of 30 individuals 2 samples of 30 individuals 41

43 STUDY OF ABNORMAL MORTALITY Mortality rate and sampling date for each declared mortality case per bivalve species and per zone Zone IV Zone VI Crassostrea gigas Crassostrea gigas 06/02/05 06/22/05 75% 29% Crassostrea gigas 06/27/05 25% Crassostrea gigas 06/27/05 10% Crassostrea gigas 06/27/05 90% Crassostrea gigas 07/01/05 44% Crassostrea gigas 10/25/05 20% Crassostrea gigas 10/25/05 15% Crassostrea gigas 11/02/05 20% Zone VII Crassostrea gigas 04/05/05 10% Crassostrea gigas 06/23/05 15% Crassostrea gigas 08/09/05 30% Crassostrea gigas 11/02/05 20% Zone VIII Crassostrea gigas 06/28/05 15% Crassostrea gigas 07/07/05 59% Crassostrea gigas 03/15/05 40% Crassostrea gigas 07/22/05 25% 42

44 Crassostrea gigas 07/22/05 35% Crassostrea gigas 08/08/05 35% Pecten maximus 11/22/05 43% Pecten maximus 22/11/05 35% Crassostrea gigas 11/30/05 18% Zone IX Crassostrea gigas 07/29/05 67% Crassostrea gigas 09/06/05 4% Haliotis tuberculata 09/07/05 10% Haliotis tuberculata 10/10/05 55% Zone X Mytilus edulis 06/01/05 5% Donax vittatus 07/11/05 20% 43

45 Number of positive results for Bonamia / Marteilia Number of positive results for other diseases / pathogen Zone I Zone VIII Zone IX Perkinsus olseni Bonamia ostreae :0 positive /229 analysis Marteilia refringens : 10 positives /228 analysis Bonamia ostreae: 88 positives /797 analysis Marteilia refringens: 15 positives /530 analysis Bonamia ostreae :2 positives /275 analysis Marteilia refringens : 0 positive/275 analysis Zone III Zone IV Zone V Zone VI Zone VIII Zone IX R. decussatus R. decussatus R. philippinarum R. philippinarum R. philippinarum R. philippinarum 62 positives /82 analysis 66 positives /69 analysis 55 positives /60 analysis 18 positives /149 analysis 85 positives /356 analysis 77 positives /120 analysis 44

46 Results regarding cases of abnormal mortality outbreaks Zone IV Zone VI Crassostrea gigas Crassostrea gigas Crassostrea gigas OsHV1 Myicola sp Vibrio sp OsHV1 Mytilicola sp Rickettsialike Haplosporidium nelsoni Ciliates VGH Vibrio sp Copepod 3 pools/6 2/30 1/3 19 pools/35 2/120 3/120 1/120 9/120 2/120 4/10 3/120 Zone VII Crassostrea gigas OsHV1 Vibrio sp. Ciliates 3 pools/24 9/13 2/60 Zone VIII Pecten maximus OsHV1 Ciliates Myicola sp. Mytilicola sp. Haplosporidium nelsoni Microsporidia Copepod Trichodina sp. Rickettsialike 7 pools/29 18/180 3/180 8/180 5/180 1/180 1/180 6/180 1/180 Crassostrea gigas Vibrio sp Rickettsialike Coccidia Ciliates Trichodina sp. 4/10 35/60 12/60 13/60 19/60 Zone IX Haliotis tuberculata Mytilus edulis OsHV1 Ciliates Trichodina sp. VGH 1 pool/6 4/60 2/60 1/60 Zone X Donax vittatus Vibrio harveyi Trematode Ciliates Mytilicola sp. Rickettsialike Trematode 5/9 2/47 5/30 1/30 3/30 3/30 Number of samples examined in total Gregarine 15/30 Coccidia 1/30 Trematode 14/30 79 samples 3299 individuals 45

47 Additional comments: VGH = Viral Gametocytic Hypertrophy The laboratory operates under quality assurance. The programme for surveillance and monitoring concerns (1) the areas under agreement for Bonamia ostreae and Marteilia refringens (2) the infection of clams by the parasite Perkinsus olseni in 2004 and 2005 (3) the mollusc mortalities declared. X VIII IX VII VI V III II IV I 46

48 1. General data ITALY Production of the country Clam Mussel Pacific oyster Flat oyster Ruditapes spp. M. galloprovincialis C. gigas O. edulis t t (farm) t (capture) no official data Zoning Name of the zone / status there are no approved zones FriuliVenezia Giulia mussel (M. galloprovincialis) clam (R. philippinarum) clam (R. decussatus) t t Veneto mussel (M. galloprovincialis clam (R. philippinarum) flat oyster (O. edulis) t (2004) > t (2004) Production of zones in the country Number of farms within the country EmiliaRomagna Marche Abruzzo Molise Puglia Liguria Toscana Lazio Campania Sicilia Sardegna FriuliVenezia Giulia mussel (M. galloprovincialis) clam (R. philippinarum) mussel (M. galloprovincialis) mussel (M. galloprovincialis) mussel (M. galloprovincialis) mussel (M. galloprovincialis) clam (R. decussatus) mussel (M. galloprovincialis) mussel (M. galloprovincialis) oyster (C. gigas) mussel (M. galloprovincialis) mussel (M. galloprovincialis) mussel (M. galloprovincialis) mussel (M. galloprovincialis) clam (R. decussatus) mussel (M. galloprovincialis) clam (R. philippinarum/r. decussatus) no official data no official data no official data no official data no official data no official data no official data no official data no official data no official data no official data no official data no official data no official data no official data Veneto clam (R. philippinarum) mussel (M. galloprovincialis) flat oyster (O. edulis) (experimentally) EmiliaRomagna Marche Abruzzo Molise Puglia Liguria Toscana Lazio Campania Sicilia Sardegna clam (R. philippinarum) mussel (M. galloprovincialis) mussel (M. galloprovincialis) mussel (M. galloprovincialis) mussel (M. galloprovincialis) mussel (M. galloprovincialis) clam (R. decussatus) mussel (M. galloprovincialis) mussel (M. galloprovincialis) oyster (C. gigas) mussel (M. galloprovincialis) mussel (M. galloprovincialis) mussel (M. galloprovincialis) mussel (M. galloprovincialis) clam (R. decussatus) no official data no official data no official data no official data no official data no official data no official data no official data no official data no official data no official data no official data no official data no official data no official data 47

49 Main exports for aquaculture purpose towards other Member States Main imports for aquaculture purpose from other Member States Main exports for aquaculture purpose towards Third Countries Main imports for aquaculture purpose from Third countries Number of hatcheries in the different zones with production Species Origin / Destination no official data Species Origin / Destination no official data Species Origin / Destination no official data Species Origin / Destination no official data FriuliVenezia Giulia Veneto Clam (R. philippinarum) hatchery nursery Clam (R. philippinarum, R. decussatus) hatchery nursery Liguria M. galloprovincialis 1. FriuliVenezia Giulia Ruditapes spp. M. galloprovincialis 9. Toscana M. galloprovincialis C. gigas Veneto Ruditapes spp. M. galloprovincialis O. edulis 10. Lazio M. galloprovincialis 11. Campania M. galloprovincialis 12. Sardegna M. galloprovincialis R. decussatus Tirrenia 5 Adriatic Emilia Romagna Ruditapes spp. M. galloprovincialis 4. Marche M. galloprovincialis 5. Abruzzo M. galloprovincialis 13. Sicilia M. galloprovincialis 6. Molise M. galloprovincialis Puglia M. galloprovincialis R. decussatus Italy: map of regions with mollusc farms 48

50 2. Laboratory data Diagnostic methods used for Bonamia ostreae Diagnostic methods used for Marteilia refringens Diagnostic methods used for exotic pathogens Perkinsus sp. other pathogens Screening Methods Presumptive Diag. Meths Confirmatory Methods Screening Methods Presumptive Diag. Meths Confirmatory Methods Screening Methods Presumptive Diag. Meths Confirmatory Methods Screening Methods Presumptive Diagnostic histology/cytological histology/cytological histology histology/cytological culture/histology culture PCR histology histology Diagnostic methods used for other diseases or pathogens monitored in the country Marteilia sp. other pathogens Confirmatory Screening Methods Presumptive Diagnostic Confirmatory Screening Methods Presumptive Diagnostic histology histology/cytological histology histology Confirmatory 49

51 3. Epidemiological data SURVEILLANCE AND MONITORING OF BONAMIA / MARTEILIA : Sampling size (number of samples and individuals per sample) and sampling dates for each zone SURVEILLANCE AND MONITORING OF OTHER PATHOGENS : Sampling size (number of samples and individuals per sample) and sampling dates for each zone and each targeted pathogen Veneto O. edulis 2 samples 3080 individuals/sample FriuliVenezia Giulia R. philippinarum R. decussatus 12 samples 50 individuals/sample 3 samples 50 individuals/samples April/October May/October May/October M. galloprovincialis Veneto R. philippinarum R. decussatus C. gallina (natural bank) M. galloprovincialis Solen marginatus (natural bank) 12 samples 150 individuals/sample 125 samples 50 individuals/sample 2 samples 30 individuals/sample 16 samples 30 individuals/sample 26 samples 150 individuals/sample 3 samples 1550 individuals/sample4 May/September JanuaryDecember July/November January/March/May/June/ September/October/ November/December JuneAugust February/November/ December STUDY OF ABNORMAL MORTALITY Mortality rate and sampling date for each declared mortality case per bivalve species and per zone Number of positive results for Bonamia / Marteilia Italy Veneto (O. edulis) no official data Bonamia ostreae 0/110 Marteilia refringens 0/110 50

52 Number of positive results for other diseases / pathogen FriuliVenezia Giulia M. galloprovincialis Marteilia sp. 0/1200 (cytological) 0/600 (histology) Ciliata 12/600 (histology) M. intestinalis 6/600 (histology) Cnidaria 21/600(histology) Trematoda 1/600 (histology) Neoplasia 8/600 (histology) R. philippinarum Perkinsus olseni 179/240 (culture) 160/360 (histology) Trematoda 9/360 (histology) Rickettsialike 24/360 (histology) Nematopsis sp. 1/360 (histology) Veneto R. decussatus M. galloprovincialis Perkinsus sp. 2/40 (culture) 0/60 (histology) Marteilia sp. 0/2300 (cytological) 0/1300 (histology) Ciliata 3/1300 (histology) Cnidaria 13/1300 (histology) Trematoda 3/1300 (histology) M. intestinalis 2/1300 (histology) R. philippinarum Perkinsus sp. 1380/2080 (culture) 1439/3729 (histology) Trematoda 13/3729 (histology) Rickettsialike 80/3729(histology) Turbellaria 2/3729 (histology) R. deccussatus Perkinsus sp. Ciliata 0/40 (culture) 0/60 (histology) 7/60 (histology) C. gallina S. marginatus Perkinsus sp. 0/140 (culture) 0/475 (histology) Rickettsialike 8/475 (histology) Trematoda 1/475 (histology) Nematopsis sp.135/475 (histology) Ciliata (1/475 (histology) Nematopsis sp. 10/125 (histology) Trematoda 10/125 (histology) Rickettsialike 6/125 (histology) Results regarding cases of abnormal mortality outbreaks No official data Number of samples examined in total 201 samples ( individuals) Additional comments: Laboratory operates under QA standards (ISO 17025) 51

53 IRELAND 1. General data Production of the country Zoning Clam R. decussatus R. philippinarum C. gigas O. edulis X t Y t Z t, etc Pacific oyster Flat oyster Bonamiosis: Approved Zone status for the whole coast of Ireland except Cork Harbour, Galway Bay, Ballinakill Harbour, Clew Bay, Belmullet, Achill Sound and Lough Foyle Commission Decision 2002/300/EC and ammendements Marteiliosis: Approved Zone Status for the whole coast of Ireland (2002/300/EC) Production of zones in the country Number of farms within the country Main exports for aquaculture purpose towards other Member States Not available. The inspection and sampling programme of the mollusc producing waters is based on discrete areas of production Co Louth Co. Wexford Co. Waterford Co. Cork Co. Kerry Co. Limerick Co. Clare Co. Galway Co. Mayo Co. Sligo Co. Donegal Species: M. Edulis seed Origin / Destination: Irish Sea / Northern Ireland Co. Kerry & Carlingford Lough / Jersey See figures for production of the country Total: 473 5,260T 32.5T C. gigas Carlingford Lough / Scotland 16T H. tuberculata Galway / Spain & Scotland 4,900 individuals 52

54 Main imports for aquaculture purpose from other Member States Species: C. gigas Main exports for None aquaculture purpose towards Third Countries Main imports for None aquaculture purpose from Third countries Number of hatcheries Cartron point, Shellfish in the different zones Ltd, New Quay, Burrin, with production Co. Clare. Lisadell Shellfish Ltd, Lisadell, Co. Sligo Bere Island Aquaculture Productsa, Ballinakilla, Bere Island, Co. Cork Boet Mor Seafoods Ltd, Cushastrough, Claddaghduff, Cliften, Co. Galway Deegagh Point Shellfish. Mulroy Bay, Cranford, Co. Donegal. Sea Lyons, Sea food Pier Road, Carrigaholt, Kilrush, Co. Clare Brandon Bay Seafoods Ltd, The Ponds, Kilshannig, CastleGregory, Co. Kerry. Origin / Destination: Seasalter, UK / Various France / various Guernsey / various Oysters, Scallops, abalone, clams Clams (T. decasata), (T. semidecasata) Pacific oysters, Abalone (H. discus hannai and H,. tuberculata) Abalone ( H. tuberculata) Abalone (H. discus hannai and H,. tuberculata) Pacific oysters (nursery only) Abalone (H. Discus Hannai H. tuberculata) 33million individuals & 14.2T 57 million individuals & 26T million individuals Production not available 14 million 42 million 2.5 million Production not available 250,000 individuals Production not available 1.5 million seed 500,000 individuals 53

55 2. Laboratory data Diagnostic methods used for Bonamia ostreae Diagnostic methods used for Marteilia refringens Diagnostic methods used for exotic pathogens Diagnostic methods used for other diseases or pathogens monitored in the country Routine screening for exotic pathogens was not carried out V. tapetis (BRD) Screening Methods Presumptive Diag. Meths Confirmatory Methods Screening Methods Presumptive Diag. Meths Confirmatory Methods Screening Methods Presumptive Diag. Meths Confirmatory Methods Blood smears Histology PCR Digestive gland smears Histology Histology is the initial diagnostic method used when abnormal mortalities are reported Macroscopical/microscop ical examination. Bacteriological analysis. Identification of vibrio using standard morphological, biochemical and physiological test. Herpes virus Others Histology and PCR in collaboration with University College Cork Histological sections stained with haematoxylineosin, other staining methods available 54

56 3. Epidemiological data SURVEILLANCE AND MONITORING OF BONAMIA / MARTEILIA : Sampling size (number of samples and individuals per sample) and sampling dates for each zone SURVEILLANCE AND MONITORING OF OTHER PATHOGENS : Sampling size (number of samples and individuals per sample) and sampling dates for each zone and each targeted pathogen STUDY OF ABNORMAL MORTALITY Mortality rate and sampling date for each declared mortality case per bivalve species and per zone Number of positive results for Bonamia / Marteilia Cork Harbour Tralee Bay Galway Bay Kilkieran Bay Clew Bay Achill Sound Bellmullet Lisadell Lough Swilly Lough Foyle Pathogen/Bivalve species (names) Co Cork Co Donegal Co Cork Co Donegal Cork Harbour Tralee Bay Galway Bay Kilkieran Bay Clew Bay Achill Sound Bellmullet Lisadell Lough Swilly Lough Foyle Spring / Autumn 3/11/ /6/2005 C. gigas 30/bed in Spring for Bonamai testing. 30 / bed for Bonamaia and Marteilia in the Autumn.. In areas with multiple beds a representaive section of the beds are examined. 30 x C. gigas 30 x C. gigas 3/11/ % C. gigas 30/6/2005 Bonamia (Spring / Autumn) 33.3% / 10% 3.94% / 14.6% 15.3% / 23.3% 1.4% / 3.45% 43.3% in one bed 50% Number of positive results for other diseases / pathogen Results regarding cases of abnormal mortality outbreaks Number of samples examined in total na Algal blooms Primary cause All areas negative for Marteilia 99/2099 positive for bonamia (42 samples) 55

57 1. General data Production of the country Zoning Production of zones in the country Number of farms within the country Main exports for aquaculture purpose towards other Member States Main imports for aquaculture purpose from other Member States Main exports for aquaculture purpose towards Third Countries Main imports for aquaculture purpose from Third countries Number of hatcheries in the different zones with production 2. Laboratory data Diagnostic methods used for Bonamia ostreae Diagnostic methods used for Marteilia refringens Diagnostic methods used for exotic pathogens Diagnostic methods used for other diseases or pathogens monitored in the country Mussel Pacific oyster Flat oyster Cockle No approved zones or farms Waddenzee Grevelingen Oosterschelde Whole territory Species: Species: Species: Species: Oosterschelde Experimental hatchery Pathogens annex D directive 95/70/EC Haplosporidium sp. M. edulis C. gigas O. edulis C. edule Mussel Pacific oyster, Flat oyster Mussel, Pacific oyster, Cockle Mussel Oyster Cockle Origin / Destination: Origin / Destination: Origin / Destination: Origin / Destination: THE NETHERLANDS No data yet No data yet No data yet No data yet X Y Z X t Y t Z t, etc X t Y t Z t, etc X t Y t Z t, etc X t Y t M. edulis 1 Screening Methods Presumptive Diag. Meths Confirmatory Methods Screening Methods Presumptive Diag. Meths Confirmatory Methods Presumptive Diagnostic Confirmatory Screening Methods Presumptive Diagnostic Confirmatory Histology (H&E) Smear PCR, ISH Histology (H&E) Smear ISH Histology (H&E) EM available, ISH for H. costale and H. nelsoni Histology (H&E) PCR PCR, ISH, EM 56

58 3. Epidemiological data SURVEILLANCE AND MONITORING OF BONAMIA / MARTEILIA : Sampling size (number of samples and individuals per sample) and sampling dates for each zone Grevelingen x 25 O. edulis 1 x 25 C. gigas 6 x 25 O. edulis 1 x 25 C. gigas Oosterschelde x 27 O. edulis 3 x 25 C. gigas 1 x 25 M. edulis x 1 O. edulis 3 x 25 C. gigas 1 x 25 M. edulis SURVEILLANCE AND MONITORING OF OTHER PATHOGENS : Sampling size (number of samples and individuals per sample) and sampling dates for each zone and each targeted pathogen Pathogen/Bivalve species (names) Zone (name) Sampling date X samples of x individuals STUDY OF ABNORMAL MORTALITY Mortality rate and sampling date for each declared mortality case per bivalve species and per zone Grevelingen Westerschelde Pacific oyster Cockle % > 80% Number of positive results for Bonamia / Marteilia Grevelingen Oosterschelde Bonamia ostreae :29/275 Marteilia refringens : 0/226 Bonamia ostreae :1/27 Marteilia refringens : 0/52 57

59 Number of positive results for other diseases / pathogen Rickettsialike Grevelingen Oosterschelde O.edulis O.edulis 4/226 1/27 Ciliates Grevelingen Oosterschelde O.edulis C.gigas O.edulis C.gigas M.edulis 3/226 7/25 2/27 6/96 1/25 Copepods Grevelingen Oosterschelde O.edulis O.edulis C.gigas M.edulis 1/226 1/27 2/96 3/25 Results regarding cases of abnormal mortality outbreaks Grevelingen C. gigas Nocardia Ciliates Neoplasia 1/30 7/30 1/30 Westerschelde C. edule Trematods Copepods 3/25 6/25 Number of samples examined in total 26 (635 individuals) 58

60 1. General data Production of the country Mussels Flat oysters Scallops Zoning Norway /free Production of zones in the country Number of farms within the country Main exports for aquaculture purpose towards other Member States Main imports for aquaculture purpose from other Member States Main exports for aquaculture purpose towards Third Countries Main imports for aquaculture purpose from Third countries Number of hatcheries hatchery 1 in the different zones with production hatchery 2 M. edulis O. edulis P. maximus (harvested from wild populations) P. maximus O. edullis NORWAY 2921 t? t 673 t See total production None None None None

61 2. Laboratory data Diagnostic methods used for Bonamia ostreae Screening Methods Presumptive Diag. Meths Confirmatory Methods Histology Histology Refer to CRL Diagnostic methods used for Marteilia refringens Screening Methods Presumptive Diag. Meths Confirmatory Methods Histology Histology Refer to CRL Diagnostic methods used for exotic pathogens Presumptive Diagnostic Confirmatory Histology Refer to CRL Diagnostic methods used for other diseases or pathogens monitored in the country Presumptive Diagnostic Confirmatory Histology Refer to CRL 60

62 3. Epidemiological data SURVEILLANCE AND MONITORING OF BONAMIA / MARTEILIA : Sampling size (number of samples and individuals per sample) and sampling dates for each zone Zone (name) Spring 2005 Autumn samples of 30 individuals 6 samples of 30 individuals SURVEILLANCE AND MONITORING OF OTHER PATHOGENS : Sampling size (number of samples and individuals per sample) and sampling dates for each zone and each targeted pathogen Pathogen/Bivalve species (names) None STUDY OF ABNORMAL MORTALITY Mortality rate and sampling date for each declared mortality case per bivalve species and per zone Number of positive results for Bonamia / Marteilia Number of positive results for other diseases / pathogen Results regarding cases of abnormal mortality outbreaks Number of samples examined in total Norway None Bonamia ostreae :0 positive /390 analysis Marteilia refringens : 0 positive/390 analysis None None 13 (390 individuals) Additional comments: The Norwegian National Reference Laboratory for mollusc diseases is now accredited by the Norwegian Board of Accreditation for histological detection of Bonamia/Marteilia. The 2005 analyses were done according to the accredited method. 61

63 Geographical location of the sample sites in the surveillance and control programme for bonamiosis and marteiliosis in European flat oysters (Ostrea edulis L.) in

64 1. General data Production of the country Zoning Production of zones in the country Clam Pacific oyster and Portuguese oyster Flat oyster (not aquaculture) Mussels Cockles R. decussatus R. philippinarum C. gigas/c. angulata O. edulis PORTUGAL t 415 t 50 t? Mytilus edulis 279 t Cerastoderma edule 23 t Norte, Centro, Lisboa, Alentejo, Algarve (I Faro Lagoon; II Alvor Lagoon and III Sagres Off shore Long lines) Zone (Algarve I, II,III) Clam Pacific oyster Flat oyster Cockle Number of farms within the country Main exports for aquaculture purpose towards other Member States Main imports for aquaculture purpose from other Member States Main exports for aquaculture purpose towards Third Countries Main imports for aquaculture purpose from Third countries Number of hatcheries in the different zones with production Zone (Lisboa) Zone (Centro) Zone (Algarve I,II, III) Zone (Lisboa) Zone (Centro) Species: Clams and oysters Species: Oyster seed Species: Species: None commercial unit. 2 pilot units of IPIMAR Clam Portuguese oyster Mussels Pacific Oyster Mussels Destination: Spain and France Origin : France Origin / Destination: Origin / Destination: ± 1500 ± 25 ± 20 nd Nd Nd Nd 63

65 2. Laboratory data Diagnostic methods used for Bonamia ostreae Screening Methods Presumptive Diag. Meths Histology Smears Confirmatory Methods Diagnostic methods used for Marteilia refringens Screening Methods Presumptive Diag. Meths Histology Smears Confirmatory Methods Diagnostic methods used for exotic pathogens Haplosporidiosis Mikrocytosis Perkinsiosis Screening Methods Presumptive Diag. Meths Histology Histology Histology and Makin&Ray Test in FTM Diagnostic methods used for other diseases or pathogens monitored in the country Pathogen Perkinsus atlanticus Screening Methods Presumptive Diagnostic Confirmatory Makin&Ray Test in FTM Histology 64

66 3. Epidemiological data SURVEILLANCE AND MONITORING OF BONAMIA / MARTEILIA : Sampling size (number of samples and individuals per sample) and sampling dates for each zone Algarve I Sampling date: March to Nov. 9 x 40 individuals SURVEILLANCE AND MONITORING OF OTHER PATHOGENS : Sampling size (number of samples and individuals per sample) and sampling dates for each zone and each targeted pathogen Pathogen/Bivalve species (names) Perkinsiosis/Clam Ruditapes decussatua Zone:Algarve I Algarve II March, April, May, June, July, August May, June, July 6 samples x 30 individuals 3 samples x 30 individuals Pathogen Haplosporidiosis, microcytosis an Perkinsisosis/ Bivalvespecies Oysters Crassostrea gigas and C. angulata Zone: Algarve I Algarve II Algarve III Lisboa Jan, Feb, Jun, Jan, Jun Jun May, Oct. 3x 30 individuals 2x 30 1x30 2x30 STUDY OF ABNORMAL MORTALITY Mortality rate and sampling date for each declared mortality case per bivalve species and per zone Zone Algarve I, II Bivalve species Clam Ruditapes decussatus Sampling date June, July, August mortality rate 60 to 70% 65

67 Number of positive results for Bonamia / Marteilia Number of positive results for other diseases / pathogen Zone (name) Algarve I Pathogen (name) Perkinsus atlanticus Zone Algarve I, II Bonamia ostreae :?? / 30 analysis Marteilia refringens : 20 positive/30 analysis. Final results are not totaly avaiable. Bivalve species Clam Ruditapes decussatus 25 analysis /14 positive 25/18 25/19 25/16 Results regarding cases of abnormal mortality outbreaks Zone (name) Zone (name) Bivalve species Pathogen Pathogen x/x y/y Number of samples examined in total 26 (650 individuals) 66

68 1. General data Production of the country Zoning Production of zones in the country SLOVENIA Mediteranian Mussel Mytillus galoprovincialis 130 t Name of the zone / status Name / status, etc Zone (name) Zone (name) Number of farms Zone (name) within the country Zone (name) Zone (name) Main exports for Species: aquaculture purpose towards other Member States Main imports for Species: aquaculture purpose from other Member States Main exports for aquaculture purpose towards Third Countries Main imports for aquaculture purpose from Third countries Number of hatcheries in the different zones with production Species: Species: Zone (name) hatchery 1 Origin / Destination: Origin / Destination: Origin / Destination: Origin / Destination: 130 t 2 No No No No No 67

69 2. Laboratory data Diagnostic methods used for Bonamia ostreae Diagnostic methods used for Marteilia refringens Diagnostic methods used Pathogen A for exotic pathogens Diagnostic methods used Pathogen A for other diseases or pathogens monitored in the country 3. Epidemiological data SURVEILLANCE AND MONITORING OF BONAMIA / MARTEILIA : Sampling size (number of samples and individuals per sample) and sampling dates for each zone SURVEILLANCE AND MONITORING OF OTHER PATHOGENS : Sampling size (number of samples and individuals per sample) and sampling dates for each zone and each targeted pathogen STUDY OF ABNORMAL MORTALITY Mortality rate and sampling date for each declared mortality case per bivalve species and per zone Number of positive results for Bonamia / Marteilia Number of positive results for other diseases / pathogen Results regarding cases of abnormal mortality outbreaks Number of samples examined in total Zone (name) Zone (name) Screening Methods Presumptive Diag. Meths Confirmatory Methods Screening Methods Presumptive Diag. Meths Confirmatory Methods Presumptive Diagnostic Confirmatory Screening Methods Presumptive Diagnostic Confirmatory Pathogen/Bivalve species (names) Zone (name) Zone (name) Zone (name) Pathogen (name) Zone (name) Zone (name) 320 samples of Mytillus galloprovincialis Sampling date: June 15 th Sampling date: September 29 th Sampling date Sampling date Bivalve species Sampling date No Yes No 150 samples 170 samples X samples of x individuals Y samples of y individuals No Marteilia refringens : four samples were suspecious Bivalve species Bivalve species Pathogen Pathogen 68

70 1. General data Production of the country Clams R.. philippinarum V. pullastra R. decussatus Donax trunculus Chamelea gallina Callista chione SPAIN 1.964,5 t 1.393,3 t 1.062,9 t 217,1 t 28,04 t 6,6 t Oysters O.edulis C. gigas 2.099,58 t 1211,5 t Mussels M. galloprovincialis M. edulis ,2 t 1.701,8 t Cockles Cerastoderma edule Acantocardia tuberculata Cerastoderma glaucum 5.162,9 t 22,8 t 13,9 t Scallops Pecten jacobaeus 1,4 t Razors S. marginatus 2,2 t Abalones Haliotis tuberculata experimental Others 2,9 t Total production ,62 t Zoning Region I: Galicia Region II: Asturias Region III: Cantabria Region IV: Cataluña Region V: Islas Baleares Region VI: Alicante I II III IV IV VI V VI V 69

71 Production of zones in the country Region I: Galicia Clams: R. decussatus R. phillipinarum V. pullastra Oysters: O. edulis C. gigas Mussels: M. galloprovincialis Cockle: C. edule Abalones: H. tuberculata 951,7 t 1.964,1 t 1.393,3 t 2.099,3 t 127 t ,1 t 5.162,9 t experimental Region II: Asturias Clams: R.decussatus Oysters: C. gigas Razors: S. marginatus 2,6 t 230 t 2,2 t Region III: Cantabria Clams: R. decussatus R. phillipinarum V. pullastra Oysters: O. edulis C. gigas Not available Not available Not available Not available Not available Region IV: Cataluña Clams: R. decussatus R. phillipinarum D. trunculus Chamelea gallina Callista chione Oysters : C. gigas O.edulis Mussels : M. edulis Cockles : C. glaucum Scallops : Acantocardia tuberculata Pecten jacobaeus Others 108,02 t 0,42 t 217,1 t 28,04 t 6,6 t 854,5 t 0,28 t 1.701,8 t 13,9 t 22,8 t 1,4 t 1,038 t 70

72 Production of zones in the country Region V : Islas Baleares Clams : R. decussatus Mussels : M. galloprovincialis Others 0,6 t 67,1 t 1,84 t Region VI : Alicante Oysters O. edulis Not available Number of farms within the country Region I : Galicia Aquaculture : 3537 rafts : mussels, oysters and clams 1281 concession culture areas : clams and cockles Extractive fishing : 24 infaunal zones : clams, cockles and flat oysters. Region II : Asturias Aquaculture: 3 farms in area AST 101 (Ría Eo) : clams Extractive fishing: Area AST 101 (Ría Eo) : clams, oysters Area AST 102 (Ría Villaviciosa) : clams, razors Region III : Cantabria Aquaculture: 1 farm in area I (San Vicente) Extractive fishing: Area I: San Vicente Area II: (Santoña/Santander) Región IV: Cataluña Aquaculture: CAT 12: 90 farms: mussels, oysters, clams CAT 13: 3 concession culture areas: Clams CAT 15: 55 farms: mussles, oysters; 5 concession culture areas: clams. 71

73 Región IV: Cataluña Extractive fishing: CAT 101: clams CAT 102/03: clams, cockles, scallops, mussels, oysters CAT 104: clams, cockles, scallops CAT 108: clams CAT 109: clams CAT 113: clams CAT 116: clams CAT 117: clams CAT 120: clams CAT 121: clams Region V: Islas Baleares Aquaculture: BAL 1/01: 3 farms of M. galloprovincialis Extractive fishing: BAL 1/01: mussels, clams BAL 1/02: Warty venus, banded murex. BAL 1/03: Warty venus, Banded murex, Imperial purple Region VI: Alicante Aquaculture 1 farms of O.edulis Main exports for aquaculture purpose towards other Member States Species: Crassostrea gigas Origin: Región II (Asturias) Destination: France 230 t Main imports for aquaculture purpose from other Member States Species: Haliotis tuberculata Origin: Ireland, Destination: Region I (Galicia) No data Crassostrea gigas Origin: France, Destination: Region II (Asturias) 20 t 72

74 Main exports for aquaculture purpose towards Third Countries Main imports for aquaculture purpose from Third countries Number of hatcheries in the different zones with production No data Destination: No data No data No data Origin : No data No data Region I: Galicia Hatchery 1: Bivalve species: O. edulis R. decussatus R. phillipinarum V. pullastra Production Not available Not available Not available Not available Hatchery 2: Hatchery 3: Hatchery 4: Hatchery 5: R. decussatus R. phillipinarum V. pullastra O. edulis C. gigas R. phillipinarum R. phillipinarum R. phillipinarum V. pullastra Not available Not available Not available Not available Not available Not available Not available Not available Not available Region III: Cantabria Hatchery 1: C. gigas R. decussatus R. phillipinarum V. pullastra Not available Not available Not available Not available 73

75 2. Laboratory data Diagnostic methods used for Bonamia ostreae Diag. Meths Histology, smears, PCR Diagnostic methods used for Marteilia refringens Diag. Meths Histology, smears Diagnostic methods used for exotic pathogens M. mackini P. marinus Candidatus Xenohaliotis californiensis Diagnostic methods Diagnostic methods Diagnostic methods Histology Histology Histology, PCR Diagnostic methods used for other diseases or pathogens monitored in the country H. nelsoni P. olseni/atlanticus BRD H.montforti n.sp Diagnostic method Diagnostic method Diagnostic method Diagnostic method Histology Histology, RFTM, PCR Gross observation Histology, PCR 74

76 3. Epidemiological data SURVEILLANCE AND MONITORING OF BONAMIA / MARTEILIA : Sampling size (number of samples and individuals per sample) and sampling dates for each zone Bonamia ostreae Region I: Galicia Sampling date February March April May June July August September October November Number of individuals 8 samples of 30 individuals 9 samples of 30 individuals 3 samples of 30 individuals 1 samples of 30 individuals; 1 sample of 14 individuals 9 samples of 30 individuals 1 sample of 30 individuals 2 samples of 30 individuals 3 samples of 30 individuals; 1 sample of 25 individuals 4 samples of 30 individuals 1 sample of 30 individuals Marteilia refringens Region I: Galicia February March April May June July August September October November 8 samples of 30 individuals 9 samples of 30 individuals 3 samples of 30 individuals 1 samples of 30 individuals; 1 sample of 14 individuals 9 samples of 30 individuals 1 sample of 30 individuals 2 samples of 30 individuals 3 samples of 30 individuals; 1 sample of 25 individuals 4 samples of 30 individuals 1 sample of 30 individual Region VI: Alicante November 1 sample of 30 individuals 75

77 SURVEILLANCE AND MONITORING OF OTHER PATHOGENS : Sampling size (number of samples and individuals per sample) and sampling dates for each zone and each targeted pathogen Pathogen/Bivalve species (names) Region I: Galicia P. olseni/atlanticus P. marinus H. nelsoni M. mackini Candidatus Xenohaliotis californiensis H. montforti n.sp Sampling date January April May June July August Sept. October Nov. June May May Number of individuals 2 samples of 30 individuals of R. decussatus 1 sample of 30 individuals of R. decussates 3 samples of 30 individuals of R. decussatus 4 samples of 30 individuals of V. pullastra 3 samples of 30 individuals of R. phillipinarum 1 sample of 30 individuals of R. decussatus. 2 samples of 30 individuals of R. decussates. 1 sample of 30 individuals of V. pullastra 3 samples of 30 individuals of R. decussatus 2 samples of 30 individuals of R. decussatus 1 sample of 30 individuals of R. decussates 2 samples of 30 individuals of V. pullastra 5 samples of 30 individuals of C. gigas 2 samples of 20 individuals of H. tuberculata 2 samples of 20 individuals of H. tuberculata Region II: Asturias P. Not available 76 2 samples of 100 individuals of R. decussatus

78 STUDY OF ABNORMAL MORTALITY Mortality rate and sampling date for each declared mortality case per bivalve species and per zone Region I: Galicia Region V: Islas Baleares Specie H.tuberculata Chamelea gallina Sampling date May April, August, Sept Mortality rate % 95 % Number of positive results for Bonamia / Marteilia Region I: Galicia Region VI: Alicante Bonamia ostreae :36 positive /44 analysis Marteilia refringens : 0 positive/34 analysis Marteilia refringens : 1 positive/1 analysis Number of positive results for other diseases / pathogen Region I: Galicia P. olseni/atlanticus R. decussatus V. pullastra R. phillipinarum 7 positive /15 analysis 5 positive/ 7 analysis 1 positive/3 analysis P. marinus H. nelsoni M.mackini C.gigas 0 positive/1 analysis Candidatus Xenohaliotis californiensis H. tuberculata 2 positive/2 analysis H. montforti n.sp H. tuberculata 2 positive/2 analysis Results regarding cases of abnormal mortality outbreaks Number of samples examined in total Region V: Islas Baleares Chamelea gallina Rickettsia 3 positive/3 analysis 2379 individuals 77

79 Additional comments A new species of a haplosporidian parasite, H. montforti has been described infecting Haliotis tuberculata. Azevedo C., Balseiro P., Casal G., Gestal C., Aranguren R., Stokes N.A., Carnegie R.B., Novoa B., Burreson E.M., Figueras A Ultrastructural and molecular characterization of Haplosporidium montforti, parasite of the European abalone Haliotis tuberculata. Journal of Invertebrate Pathology. In press Candidatus Xenohaliotis californinesis has been detected in Haliotis tuberculata for the first time in Europe. Balseiro P., Aranguren R., Gestal C., Novoa B., Figueras A Candidatus Xenohaliotis californiensis and Haplosporidium montforti associated to mortalities of abalone Haliotis tuberculata cultured in Europe. Aquaculture, Accepted. 78

80 UNITED KINGDOM 1. General data Production of the country This is farmed shellfish produced for the table. In addition, there are substantial landings of cockles, scallops and native oysters from managed fisheries. Zoning (See Map for Zones A, B, C) Production of zones in the country Farmed shellfish produced for the table. Native oyster production from wild fisheries is about 1,000 tonnes per year, the majority of which is from Zones A & B. Clam Pacific oyster Flat oyster Mussels Cockles Scallops Queens M. mercenaria and R. philippinarum C. gigas O. edulis M. edulis C. edule P.maximus C.opercularis 18 t 1,019 t 118 t 28,900 t 10 t 10 t 45 t Zone A. The Lizard to Start Point / Marteilia Approved Zone B. Portland Bill to Selsey Bill / Marteilia Approved Zone C. Shoeburyness to Landguard Point / Marteilia Approved Zone D. Remainder of coast of Great Britain/ Bonamia and Marteilia Approved. Northern Ireland. Whole coast / Marteilia Approved. Whole coast apart from Lough Foyle / Bonamia Approved. Zone A Zone B Zone C Zone D (rest of England, Wales and Scotland) Northern Ireland Pacific oyster Flat oyster Mussels Clams Pacific oyster Flat oyster Mussels Cockles Clams Pacific oyster Flat oyster Clams Mussels Pacific oyster Flat oyster Mussels Scallops Queens Clams Pacific oyster Flat oyster Musssels 22.1 t 2.3 t 209 t 1.4 t t 9.5 t t 10 t 2.6 t 46.2 t 90.5 t 3 t 185 t t 11.7 t 21,289 t 10 t 45 t 11 t 278 t 4 t 6,600 t 79

81 Number of farms within the country Main exports for aquaculture purpose towards other Member States Zone A Zone B Zone C Zone D Northern Ireland Pacific oysters * Average weight of consignments = 1.4 t England to Eire England to Guernsey England to Jersey Northern Ireland to Great Britain consignments * 1 consignment * 2 consignments * 205 t Main imports for aquaculture purpose from other Member States Mussels * Average weight of consignments = 0.3 t Pacific oysters England to Jersey England to Guernsey Northern Ireland to France and Holland Guernsey to Great Britain Guernsey to Northern Ireland Republic of Ireland to Northern Ireland Republic of Ireland to Great Britain No information 31 consignments * 1 consignment * 5,100 t 2.8 million 0.9 t 1,160 t 187,000 Main exports for aquaculture purpose towards Third Countries Main imports for All Species: All UK NONE aquaculture purpose from Third countries Number of hatcheries Zone D in the different zones Hatchery 1 O. edulis with production C.gigas R. philippinarum 125 thousand 329 million 340 million 80

82 2. Laboratory data Diagnostic methods used for Bonamia ostreae Diagnostic methods used for Marteilia refringens Screening Methods Presumptive Diag. Meths Confirmatory Methods Screening Methods Presumptive Diag. Meths Confirmatory Methods Histological sections stained with Haematoxylin and eosin (or Gomori) Histological sections stained with Haematoxylin and eosin with a variety of additional staining methods available, such as Gomori trichrome, Gram stain, Giemsa and PAS. Electron microscopy is also available As above, for Bonamia ostreae Diagnostic methods used for exotic pathogens MSX/SSO Mikrocytosis Perkinsosis Histological sections stained with Haematoxylin and eosin with a variety of additional staining methods available, such as Gomori trichrome and Giemsa. Electron microscopy is also available and may be used when histological examination detects haplosporean stages. Histological sections stained with Haematoxylin and eosin with a variety of additional staining methods available, such as Gomori trichrome, Gram stain, Giemsa and PAS. Impression smears stained with a Giemsa technique may be used. Electron microscopy is also available. As above. Diagnostic methods used for other diseases or pathogens monitored in the country No routine monitoring. Samples from reported unusual mortalities would also be tested for oyster herpesvirus (OHV). Method: Light microscopy using resin sections prepared for electron microscopy. Ultrastructural examination to visualise viral particles. PCR technique in regular use. 81

83 3. Epidemiological data SURVEILLANCE AND MONITORING OF BONAMIA / MARTEILIA : Sampling size (number of samples and individuals per sample) and sampling dates for each zone SURVEILLANCE AND MONITORING OF OTHER PATHOGENS : Sampling size (number of samples and individuals per sample) and sampling dates for each zone and each targeted pathogen STUDY OF ABNORMAL MORTALITY Mortality rate and sampling date for each declared mortality case per bivalve species and per zone Number of positive results for Bonamia / Marteilia Zone A Zone B Zone C Zone D Northern Ireland Pathogen/Bivalve species (names) Zone D (Hatchery) Annex D pathogens/ Clams Flat oyster Pacific oyster Strangford Lough Larne Lough Lough Foyle Lough Foyle Lough Foyle Strangford Lough Larne Lough Lough Foyle Spring: 7 th March 11 th May Autumn: 24 th Aug 5 th Dec 10/05/ /12/ March March May July August October October November 2005 Zone D Pacific oysters 11/07/2005 and 07/09/2005 Zone A Zone B Zone C Zone D Northern Ireland excluding Lough Foyle Bonamia ostreae 0/120 51/660 70/510 0/660 0/ samples x 30 oysters (Zone D only) 52 samples x 30 oysters (all Zones) samples (Flat oysters, Pacific oysters, Manila clams) x 30 individuals 3 samples (Pacific oysters, Manila clams, Palourdes) x 30 individuals <30% in adults Marteilia refringens 0/120 0/660 0/510 0/660 0/106 Number of positive results for other diseases / pathogen Results regarding cases of abnormal mortality outbreaks Number of samples examined in total Northern Ireland Lough Foyle (see comments) Pathogen (name) Zone D Hatchery 6/480 Clams Pacific oysters Flat oysters 0/480 No positive results from 6 samples Annex D Pathogens Zone D Pacific oysters No pathogens. Occasional ciliates and RLOs 79 (3,302 individuals) 82

84 Additional comments Shellfish farm site visiting programmes are in place throughout the UK, when samples are taken, stocks are examined and movement and mortality records checked. Farmers are obliged to report any incidents of abnomally high mortality to the official services. Lough Foyle lies between Northern Ireland (UK) and the Republic of Ireland (RoI). Bonamia ostreae was first detected in flat oysters collected on the RoI side and tested by the Marine Institute in spring Following this positive result, two further rounds of testing were performed in collaboration with RoI. Three sets of 150 shell fish were sampled from the RoI side, the NI side and the middle of the Lough, with each lab testing the shellfish from their own side and and half of the shellfish from the middle. This was repeated again late in Testing was conducted in NI by histological examination and also by PCR. No positive samples were found by histology. However 6 samples, collected during summer 2005 were found positive by PCR and (for some) confirmed by sequencing. Results from RoI will be reported independently. There has been no spread of Bonamia to new areas in the rest of Great Britain. Approved Zone status for Marteilia has been maintained for the whole of the UK. Samples of bivalves from the UK hatchery continue to test free of all OIE listed diseases. Native oyster samples collected from beds in Southampton Water, near to where mussels have tested positive for Marteilia maurini, continue to test negative for Marteilia sp. It is noted with concern that OIE propose to consider M.maurini and M.refringens as one species. A shellfish farmer in the River Camel, Cornwall continues to experience occasional late summer mortality in adult (23 year old) Pacific oysters. Samples of oysters were collected on two occasions (July and September). No significant pathogens were detected on either occasion. Some ciliates and RLOs were present. Readings from a continuous temperature recorder loaned to him in order to assist him in investigating this problem indicated that there were no episodes of unusually high temperature in It is thought that these mortalities may be due to excessive stocking densities. Some native oyster shells were submitted from the small fishery in the estuary of the river Stour (SE England) as there was concern that the animals may have Dutch Shell Disease. The shells did not show any symptoms of this, but rather there were signs of delayed hardening of new shell growth, possibly due to local environmental factors (temperature and salinity). The samples were negative for Bonamia and Marteilia. Mussels in one Scottish sea loch suffered from poor meat yields and thin shells, affecting marketing. No pathology was detected by histology. In England and Wales, the prevalence of infection of Bonamia ostreae in native oysters at farm sites increased slightly and in fisheries decreased slightly, compared with Bonamia was not detected in any of the samples taken from the Fal wild fishery for the first time ever. There was no evidence that higher than usual mortalities at some sites in Essex were as a result of disease. Adverse water quality is a suspected cause of this problem. FRS is accredited for testing for Bonamia and Marteilia. Cefas successfully achieved accreditation to for the screening and identification of VHSV, IHNV and BKD and for screening for SVCV in Work is continuing towards accreditation to this standard for the testing of shellfish for Bonamia and Marteilia and for the confirmation of SVCV by PCR, it is anticipated that the initial assessment for this will be late 2006 or early The Fish Health Inspectorate is working towards accrediting the statuary inspection work carried out by the Fish Health Inspectorate on registered farm sites (including shellfish farm sites) to ISO standard. The initial assessment for this is scheduled for June

85 MAP OF ZONES in ENGLAND AND WALES 84

86 Annex D : New epidemiological report template (example of France) 85

87 86