Carp Edema Virus CEV WORKSHOP FINAL AGENDA

Carp Edema Virus – CEV WORKSHOP FINAL AGENDA Monday 12th of January 2015 12.15 - 13.00

Arrival and lunch (facultative)

13:00 – 13:15

Welcome address: Niccolò, Niels and Olga Chair: Olga, and Minutes: Niccolò

13:15 – 15:00 15.00 - 15.30 15.30 - 17.30

19.00

Representative of each laboratory present their diagnostic findings and relation to CEV, with their experience (approx.10 min each) Tea break • Diagnostic techniques currently available for detection of CEV, recommendations for harmonized diagnostic method • Pathogen characterization focusing of differences between viral strains detected in carp and koi • Exchange of materials between labs •

•

Joint dinner at Cassiopeia

Tuesday 13th of January 2015 8.30 – 10.00 ANIHWA and other calls: discussion, followed by active writing of proposal Chair: Olga, and Minutes: Anna 10:00 – 10.30

Coffee break

10.30 – 12:30

• • • •

12.30 - 13.30

Proposal writing continued Appointments on a paper on urgency concerning CEV for EAFP Bulletin EAFP CEV workshop Las Palmas Sept 2015 (Haenen, Way and Waltzek organizers). Miscellaneous...

Lunch and goodbyes

CEV WORKSHOP 2015 Participants list Number

Name

1 2 3 4

Sven Bergmann Heike Schütze Laurent Bigarré Mikolaj Adamek

5

Verena Jung-Schroers

6

Tomáš VESELÝ

7 8 9

Olga Haenen Thomas Waltzek Mansour El Matbouli

10

Veronika Piačková

11 12 13 14 15 16 17

Keith Way David Stone Marek Matras Mona Gjessing Ole B. Dale Anna Toffan Miriam Abbadi

Institution

FLI-Germany FLI-Germany Anses Fish Disease Research Unit, University of Veterinary Medicine in Hanover Fish Disease Research Unit, University of Veterinary Medicine in Hanover Veterinary Research Institute, Brno, Czech Republic CVI, part of WUR, Lelystad, University of Florida University of Veterinary Medicine Vienna University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters CEFAS CEFAS Piwet_Poland NVI NVI IZSVE IZSVE

E-mail

[email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected]

[email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected]

‘Koi Sleepy Disease’ voor het eerst in Nederland aangetoond in koikarpers OLGA HAENEN1, KEITH WAY2, DAVID STONE2 EN MARC ENGELSMA1

Samenvatting praktisch inzicht

Eind september 2013 is de koikarperziekte ‘Koi Sleepy Disease’ (KSD) voor het eerst aangetoond in Nederland door het Vis-, schaal- en schelpdierziektelaboratorium van Central Veterinary Institute, onderdeel van Wageningen UR. De klinische verdenking werd bevestigd door het zusterlab CEFAS in Engeland. KSD wordt veroorzaakt door Carp Edema Virus (CEV), een pokkenvirus. Ziekteverschijnselen lijken erg op die van de al meerdere jaren aanwezige koi herpesvirusziekte (KHVD), veroorzaakt door koi herpesvirus (KHV). De klinische verschijnselen van beide ziekten zijn: enophthalmus, overmatige slijmproductie en kieuwnecrose. Een verschil vormt echter het onmiskenbare slaapgedrag waaraan de ziekte z’n naam dankt. Sinds de zeventiger jaren van de vorige eeuw wordt KSD in Japan regelmatig gevonden bij oudere koikarpers. De ziekte veroorzaakt daar sterfte tot 76 procent. Ondanks vele importen van Japanse koikarpers is de ziekte pas in 2011 voor het eerst aangetoond in Europa. Dit zal onder andere komen doordat de diagnostiek voor deze ziekte slechts op een

1 Vis-, schaal- en schelpdierziektelaboratorium, Central Veterinary Institute, onderdeel van Wageningen UR, Edelhertweg 15, 8219 PH Lelystad, Nederland. Email: [email protected], tel.: +31320238352. 2 NRL for Fish Diseases, Centre for Environment, Fisheries & Aquaculture Science (CEFAS), Weymouth, Engeland.

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Tijdschrift voor Diergeneeskunde | nr 4 | april 2014

enkel visziektelaboratorium wordt uitgevoerd. Bij CVI is in februari 2014 een diagnostische PCR-test geïmplementeerd voor KSD. Omdat Nederland vele hobbyvijvers telt en koikarpereigenaren hun dierenarts consulteren in verband met ziekte bij hun vissen is het belangrijk dat dierenartsen KSD en KHVD in het veld kunnen herkennen. Bij het visziektelaboratorium kan vervolgens bevestigingsdiagnostiek plaatsvinden.

Ziekten van de koikarper De koikarper, een kleurvariant van de karper (Cyprinus carpio) kent vele kleurvariaties en geniet in Nederland al jaren een toenemende populariteit als huisdier. De vaak kostbare koikarpers worden als huisdier gehouden in hobbyvijvers. In Nederland zwommen begin 2012 6,6 miljoen vissen als huisdier rond, zowel in aquaria als in hobbyvijvers, en een deel daarvan betreft koikarpers (2). Van oudsher kunnen (koi) karpers in het vroege voorjaar sporadisch ziek worden door het ‘Spring Viremia of Carp’-virus (SVC-virus) (1). In 2002 werd echter een serieuze nieuwe bedreiging van de (koi)karper gevonden in Europa: het koi herpesvirus, dat grote sterfte veroorzaakt onder met name koikarpers (4,5,7). De meeste importen van koikarpers komen uit twee landen: Israël, waar de koikarpers vóór export tegen KHV-ziekte worden gevaccineerd (8), en Japan, dat vrij is van KHV (5). Herfst 2013 is een derde virale ziekte van koikarpers voor het eerst aangetoond in Nederland, de ‘Koi Sleepy Disease’ (6).

Koikarpers

Foto 1: Koi herpesvirusziekte in koikarper: enophthalmus, kieuwnecrose en overmatige slijmproductie. KSD lijkt hierop, maar de koi vertoont bij KSD tevens een sterk slaapgedrag. Foto uit: Haenen et al., 2011.

Casus: eerste geval van ‘Koi Sleepy Disease’ in Nederland Twee zieke koikarpers uit een grote hobbybuitenvijver in het midden van Nederland werden naar CVI gebracht voor diagnostiek (6). Enkele weken daarvoor had de eigenaar in Nederland gekweekte koikarpers van Japanse oudervissen aan zijn bestand toegevoegd. De nieuwe koikarpers en een deel van de eigen koikarpers werden ziek. Ze vertoonden al gauw apathisch gedrag en hingen aan het wateroppervlak. Badbehandelingen met een breed spectrum antiparasiticum en antibiotica uit een dierenspeciaalzaak gaven geen verbetering. De zieke koikarpers vertoonden een sterk ‘slaapgedrag’: ze lagen op de bodem en konden door aanraking wel weer tijdelijk tot activiteit worden gebracht, maar zakten daarna weer terug, waarna ze overleden. De watertemperatuur was circa 20 graden Celsius. Bij aankomst op CVI bleek dat de nog levende zieke koikarper apathisch was en ademde aan het oppervlak van de emmer, anorexia, enophthalmus en kieuwnecrose vertoonde, en een zware kieuwworminfectie had door trematode wormen, Gyrodactylus spp. De parasitaire infectie werd echter niet causaal geacht voor de problemen. De kieuwlamellen waren sterk oedemateus. Inwendig werden geen afwijkingen vastgesteld. Het totale ziektebeeld deed vanwege het slaapgedrag van de koikarper ‘koi sleepy

disease’ vermoeden. Nadat bij CVI de PCR-test voor KHV negatief was gebleken, werd kieuwmateriaal en inwendig orgaanmateriaal van de koikarper gestuurd naar het zusterlaboratorium CEFAS (Centre for Environment, Fisheries & Aquaculture Science), Weymouth, Engeland. De PCR-test van CEFAS op het Carp Edema Virus (CEV), dat KSD veroorzaakt, was positief. Daarmee werd het eerste geval van KSD in Nederland bevestigd (6).

De ziekte KSD Zo’n negen jaar geleden is CEV in Japan beschreven als veroorzaker van KSD in oudere koikarpers (9). KSD staat voor ‘koi sleepy disease’, een ziekte van volwassen koikarpers, die wordt veroorzaakt door een pokkenvirus met sterfte tot 76 procent (9). Het gaat om ‘carp edema virus’ (CEV), een groot DNA-virus van circa 333 tot 400 bij 400 tot 413 nanometer (9). Recent is het virus ook in Europa aangetroffen: in Engeland is CEV in 2011 aangetoond in zieke koikarpers en ook in Frankrijk is het in koikarpers gedetecteerd (12). De watertemperatuur is cruciaal: tussen de 13 en 23 graden Celsius treedt deze ziekte op bij koikarpers. Bij KSD ontstaat zuurstofgebrek door de kieuwaantasting en dit tast de inwendige organen aan (9). Een overlevende koikarper vertoont iets opgezette kieuwen, maar geen andere van de bovengenoemde verschijnselen. In het midden van de jaren zeventig werd CEV al


27

beschreven als ‘carp edema virus’ bij juveniele koikarpers in de Niigata Prefecture in Japan, waar het elk jaar in de periode van juni tot juli ziekte veroorzaakt. Bij jonge koikarpers gaat het echter om een totaal ander ziektebeeld dan bij KSD. Het ‘slaapgedrag’ wordt dan niet waargenomen, maar er is wel sterke oedeemvorming, gevolgd door een significante sterfte van 80 tot 100 procent (11). Het KSD is nog geen aangifteplichtige ziekte voor de EU of voor de OIE (World Organization for Animal Health), terwijl KHVD dat wel is, zowel voor de EU als voor de OIE (3, 10). De virussen CEV en KHV zijn overigens onschadelijk voor de mens.

Ziekteverschijnselen KSD ten opzichte van die van KHVD

praktisch inzicht

De klinische verschijnselen van ‘Koi Sleepy Disease ‘(KSD) zijn grotendeels vergelijkbaar met die van Koi herpesvirusziekte (KHVD) (foto 1): Bij beide ziekten ziet men: 1. Apathisch gedrag, koikarper hangt onder het wateroppervlak; 2. Ingevallen ogen; 3. Verlies van de slijmhuid; 4. Kieuwnecrose; 5. Acute sterfte na enkele dagen, die sterk kan oplopen. Bij KSD valt verder op: 6. Oudere koikarper gaat op de bodem liggen en is door aanraking slechts kortstondig aan het zwemmen te krijgen; 7. Bloedingen; 8. Treedt op bij watertemperaturen van 13 tot 23 graden Celsius, terwijl KHVD tussen de 17 en 27 graden Celsius optreedt. Je kunt KSD bij ons dus verwachten in het voor- en najaar en KHVD in het late voorjaar en in de zomer.

‘CEV-like virus’ in zieke karper In maart en november 2012 werd bij een watertemperatuur van 6 tot 9 graden Celsius een verwant (CEV-achtig) virus gedetecteerd in karpers in sportvisserijvijvers in respectievelijk Zuidoost-Engeland en de Midlands, waarbij een hoge sterfte werd gezien (12). Het virus werd ‘CEV-like’ genoemd, omdat het niet helemaal overeenkwam met de virusstam uit koikarpers. Het is de vraag of het ‘CEV-like’ karpervirus ook al in Nederland aanwezig is. Er is tot op heden niet op getest.

onderdrukken: bij koikarpers in Japan bleek blootstelling aan een 0,5 procent zoutbad, direct na stressvolle handelingen zoals sorteren of transport, te helpen een KSD-uitbraak te voorkomen. Deze ‘behandeling’ werd ook geadviseerd gedurende minimaal vier weken na introductie van koikarpers vanuit andere wateren, om KSD te voorkomen (9). Eventueel aanwezig virus wordt hiermee echter niet geïnactiveerd. Een andere maatregel om de symptomen te bestrijden, is de watertemperatuur te verhogen naar boven de 23 graden, zodat de virusreplicatie wordt geremd. De koikarper kan dan zelf weerstand opbouwen tegen het virus. Om uitbraken te voorkomen, is het belangrijk symptoomloze koikarpers aan te schaffen en partijen koikarper van verschillende herkomst niet te mengen.

Toekomst van KSD in Nederland? De verspreiding in Europa en de impact van KSD voor koikarpers is nog onduidelijk. Het is opvallend dat deze ziekte al ruim veertig jaar bekend is in Japan, maar dat Europa ondanks de vele koikarpertransporten nog maar weinig gevallen kent. Hierbij moet worden vermeld dat de KSD-diagnostiek tot nu toe maar op een enkel laboratorium in Europa is geïmplementeerd. Op basis van diagnostische bevindingen bij verdenking op KSD kan mettertijd een beeld worden verkregen van waar de ziekte aanwezig is. De dierenarts speelt hierin een belangrijke rol als eerstelijns expert die het slapende gedrag van de koikarper traceert en diagnostiek laat uitvoeren. Zo kan men een beeld krijgen van de impact van KSD.

Diagnostiek en inzenden Het ‘carp edema virus’ (CEV-virus) is door middel van PCR-testen aan te tonen in kieuwweefsel en huid van koikarpers (9). Bij CVI is nu een kwantitatieve PCR in ontwikkeling. Analoog aan bemonstering voor de koi herpesvirustest kan een levende of verse, bevroren koikarper naar CVI in Lelystad worden gebracht, of kunnen bemonsterde kieuwen en nieren van een koikarper in een lekvrij buisje met >70 procent ethanol gekoeld worden opgestuurd naar CVI. Nadere info en contact over diagnostiek visziekten: (0320) 23 83 73 en visdiagnostiek.cvi@wur. nl, www.wageningenUR.nl/cvi.

Literatuur Transmissie, therapie en preventie Transmissie van de ziekte KSD loopt via het water. Er bestaat geen geneesmiddel tegen KSD. Er is wel een methode om de symptomen te

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1. Ahne, W, Bjorklund, HV, Essbauer, S, et al. (2002). Spring viremia of carp (SVC). Dis. aquat. Org., 52, 261–272. 2. DSZ (2012). Feiten & cijfers in de dierensector

Koikarpers

3.

4.

5.

6.

– update, 12 nov 2012. http://www.dsz-actueel. nl/nieuws/feiten-cijfers-in-de-dierensector-update/. European Commission (2006). Council Directive 2006/88/EC of 24 October 2006. http://app.vlex.com/#vid/36463860/ graphical_version Haenen, OLM, Engelsma, MY, Van Beurden, SJ (2011). Ziekten van vissen, schaal-, en schelpdieren, van belang voor de Nederlandse aquacultuur. Central Veterinary Institute, Lelystad. pp.164 ISBN 978-94-6190-101-9. http://www.wageningenur.nl/upload_mm/4/a/ c/9448d4c4-198d-4339-8583-22b45573c8ac_ Ziektenvisschaalschelpdierwebversie.pdf geopend 24 jan 2014. Haenen, OLM, Way, K, Bergmann, SM and Ariel, E, (2004). The emergence of Koi herpesvirus and its signiﬁcance to European aquaculture. Bull. Eur. Ass. Fish Pathol. 24(6): 293-307. Haenen, O, Way, K, Stone, D, Engelsma, M, (2013). Koi Sleepy Disease (KSD) door ‘Carp Edema Virus’: eerste detectie in Nederlandse koi. Aquacultuur 5: 27-29.

7.

8.

9.

10.

11.

12.

Hedrick, RP, Gilad, O, Yun, S, et al. (2000). A herpesvirus associated with mass mortality of juvenile and adult koi, a strain of common carp. J. Aquat. Anim. Health, 12, 44–57. Ilouze, M, Davidovich, M, Diamant, A, Kotler, M, and Dishon, A, (2011), The outbreak of carp disease caused by CyHV-3 as a model for new emerging viral diseases in aquaculture: a Review. Ecol. Res. 26: 885–892. Miyazaki, T, Isshiki, T, Katsuyuki, H (2005). Histopathological and electron microscopy studies on sleepy disease of Cyprinus carpio koi in Japan. Dis. Aquat. Org. 65:197-207. OIE, 2013. Office International des Epizooties. Aquatic Animal Health Code. OIE, Paris. http:// www.oie.int/en/international-standard-setting/ aquatic-code/access-online/, 554 pp. Oyamatsu, T, Hata, N, Yamada, K, et al. (1997). An etiological study on mass mortality of cultured colorcarp juveniles showing edemas. Fish Pathol. 32:81–88. Way, K and Stone, D (2014). Emergence of Carp edema virus-like (CEV-like) disease in the UK. Fin Fish News (in press).

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Application form. Proposal CoVetLab (Club 5) joint research 2014 Maximum 4 pages; Acronym: CEVirE (CEV Epidemiology) Title: Carp Edema Virus (CEV), a novel fish virus for Europe: Epidemiology, impact and optimization of diagnostic methods. Participating partners (institutes): P1. Central Veterinary Institute of Wageningen University and Research center (CVI), Netherlands P2. National Veterinary Institute, Technical University of Denmark (DTU.VET), Denmark P3. National Veterinary Institute (SVA), Sweden P4. French Agency for Food, Environmental & Occupational Health & Safety (ANSES),Brest, France Associated partners (AP): AP1. Centre for Environment, Fisheries & Aquaculture Science (CEFAS), Weymouth, England AP2. Friedrich-Loeffler-Institut (FLI), Insel Riems, Germany AP3. National Veterinary Research Institute (NVRI), Pulawy, Poland

Proposed project leader: Name: Olga Haenen (CVI) Email address and telephone numbers: [email protected] Proposal Summary (no more than 100 words) CEV, Carp Edema Virus, is a novel detected pathogenic virus of carp and koi in Europe, and the disease is known from Japan. In Europe only 4 NRL’s have a test for this virus. NRLs for Fish Diseases need reliable and fast tests for CEV. Diagnostic methods will be assessed with respect to sensitivity, specificity, reproducibility and robustness and comprehensive SOPs will be produced with recommendations on sampling and diagnostic methods for detection of the virus and the disease. CEV will be incorporated in a multiplex PCR assay for detection of CyHV 1-3, covering most important DNA viruses in carp. It will be investigated if CEV is a putative emerging disease in Europe. Eventual presence of CEV in Europe will be assessed through testing of carp in surveillance programs and in archive material. Spreading mechanisms for CEV will be investigated as will control and containment strategies. A European CEV network will be established. Validated diagnostic tests and dissemination of information on CEV will favour all laboratories involved in surveillance and diagnosis of carp diseases.

Length of project Months Proposed start Date 12 1st October 2014 in months Introduction and Background. ( including implications of not funding the project) Viral diseases in freshwater fish have been threatening wild and cultured stocks of fish since decades. In carp and koi (Cyprinus carpio), Spring Viraemia of Carp (SVC) and Koi Herpesvirus Disease are well known in Europe. A third virus is CEV (Carp Edema Virus): this virus was first detected and described in Japan in the 70ies, where it caused viral oedema of juvenile carp resulting in high mortalities (Murakami et al. 1976, Ono et al. 1986). It was shown to be a poxvirus by electron microscopy (Oyamatsu et al. 1997). CEV causes severe oedema in young koi and carp, and more recently the so-called “Koi Sleepy Disease” (KSD) in older koi was described (Oyamatsu et al., 1997; Miyazaki et al., 2005). The lethargy manifests as sleepy behaviour, where the affected fish lie on the bottom of the pond and eventually die of anoxia (Miyazaki et al. 2005). Losses from KSD/CEV occur in spring and autumn in Japan, at a temperature range of 15 – 25°C, and mortalities may reach 80%. The Japanese manage or prevent outbreaks of KSD by holding the koi in 0.5% salt water following exposure to stress, such as grading or transportation (Way & Stone, 2014). The virus is not harmful to warm blooded animals and man. In 2009 CEV was detected for the first time in Europe, in England in imported diseased koi, and again in 2011-2013. Later it was also detected in diseased koi from hobbyist ponds. Low levels of CEV-like virus were also detected in healthy koi imports from Israel and Japan at ornamental fish wholesalers during 2013 in England (Way & Stone, 2014). In March 2012 a CEV-like virus was detected for the first time in diseased common carp undergoing mortality, from a cluster of fishery sites in south-east England. In November 2012 a fishery site in the Midlands reported mortalities in common carp showing clinical disease and a CEV-like virus was again detected. Further

Application form joint research CoVetLab (Club 5) 2014

detections in carp were made in 2013 at two other fishery sites in the Midlands. Cefas currently screens all unexplained carp mortalities for CEV and frequently detects CEV. An important feature of the CEV-like virus detected in common carp is that the detections were made from disease outbreaks that occurred during periods of low water temperatures (6-9°C) in winter and early spring (Way & Stone, 2014). CEV was also detected in Germany in imported koi from Japan in 2008 (Bergmann, unpublished), and in France (Bigarré et al., unpublished; Way & Stone, 2014) in 2013 in imported koi. In September 2013, the first Koi Sleepy Disease case was diagnosed by CVI in the Netherlands in diseased koi with a high mortality rate (Haenen et al., 2014). In addition, in March 2014, CVI detected CEV in clinically healthy wild carp sampled from a carp mortality case. CEFAS, UK confirmed by PCR this first detection of CEV in koi in NL. CVI also detected CEV in 2 archive samples from carp mortalities, these cases were also confirmed by CEFAS. CEFAS has developed a range of diagnostics assays for CEV, including a conventional nested PCR, a TaqMan qPCR assay, and in-situ hybridisation. In parallel, CVI has recently developed a Sybr green qPCR based on sequences received from CEFAS, which is currently use at the CVI. Prior to 2013, in Europe, only 3 NRL’s (CEFAS, ANSES and FLI) tested for CEV. Therefore, the presence and distribution of CEV in Europe is still unknown and many carp mortalities associated with CEV may not have been diagnosed in the past. Despite its apparent spread, only few data are published about this disease and consequently, most stakeholders in Europe do not have access to diagnostic tests and are not even aware about the existence of CEV. For instance, most NRLs for Fish Diseases and regional laboratories in Europe do not have a CEV test yet. Eastern European countries which produce a significant amount of carp (e.g. 20,000 tons of carp/year in Poland) as edible fish should have access to CEV tests. Apart from this, many carps are kept for angling purposes or are wild carp, and many, often expensive koi are kept in private ponds. Apart from the devastating viruses Koi Herpesvirus (KHV) and Spring Viremia of Carp Virus (SVCV), we need to investigate the spread and impact of CEV. As only very few laboratories conducted diagnostic testing for this virus, the spread and molecular epidemiology of this virus is unknown. Possibilities to detect CEV need to be improved. References 1. Haenen,O, Way K, Stone D, Engelsma M (2014). Koi Sleepy Disease voor het eerst in Nederland aangetoond in koikarpers. Tijdschrift voor Diergeneeskunde 4: 26-29. 2. Miyazaki, T, Isshiki, T, Katsuyuki, H (2005). Histopathological and electron microscopy studies on sleepy disease of koi Cyprinus carpio koi in Japan. Dis.Aquat.Org. 65:197-207. 3. Murakami Y, Shitanaka M, Toshida S, Matsuzato T (1976) Studies on mass mortality of juvenile carp: about mass mortality showing edema. Bull Hiroshima Fresh Water Fish Exp Station 19–33 (in Japanese) 4. Ono S, Nagai A, Sugai N (1986) A histopathological study on juvenile colorcarp, Cyprinus carpio, showing edema. Fish Pathol 21:167–175 5. Oyamatsu, T, Hata, N, Yamada, K, et al. (1997). An etiological study on mass mortality of cultured colorcarp juveniles showing edemas. Fish Pathol. 32:81–88. 6. Way, K and Stone, D (2014). Emergence of Carp edema virus-like (CEV-like) disease in the UK. Fin Fish News (in press).

Research Questions     

Is CEV a putative emerging disease in Europe? What is the distribution of the disease? How is the disease and presence of the causative virus best diagnosed? What are the spreading mechanisms for CEV? (horizontal and vertical transmission) How can the disease be controlled and contained?

Key Objectives:      

To develop SOPs for sampling, detection and typing of CEV for use by the NRLs and regional laboratories for Fish Diseases in Europe. To establish a multiplex PCR assay for simultaneous detection of CyHV 1-3 and CEV. To exchange materials used in diagnostics To validate the tests To assess the possible presence and spread of CEV in the partner countries To assess the possible impact of CEV for cultured carp and koi and wild carp in Europe, with a draft research project proposal for a future call.




To disseminate results to NRL and regional labs of Europe, to write a joint paper, and give presentations on this subject at (inter)national meetings.

Brief workplan, including key milestones (M) 

Initialy the plans will be discussed in a teleconference, (M1). Each partner (P) and associated partner (AP) will investigate its collection of samples of carp and koi and of reagents for diagnostic testing.  Month 1-2: current methods and reagents are exchanged between partners (M2).  Month 3: methods are implemented and standardized and all P’s and AP’s start testing (M3)(PCR, possibly also sequencing) fresh and archive samples of carp and koi (Cyprinus carpio) from their own diagnostic lab/storage. Furthermore,  P1 will: coordinate the project, analyse results of all P’s and AP’s, organize the workshop (M6), coordinate writing a draft research proposal (D3), and coordinate writing the final report (D4), a.o..  P2 will: aim to establish a multiplex PCR for simultaneously detection of CEV, CyHV-1, CyHV-2 and CyHV-3 using the newly established technology platform at the institute; Organize an inter-laboratory proficiency test in cooperation with AP1 and P1 (M4), conduct a survey on CEV in carp in Denmark, and disseminate results and SOPs through its EURL Fish Diseases function (D1).  P3 will: extend sampling of carp in Sweden for KHV and CEV detection P4 will: potentially test a duplex Taqman CEV / internal housekeeping gene (to ensure DNA quality) as part of SOPs.  AP1 will: advise on best diagnostic materials and methods, provide positive control material for the PCR-based assays and help with analysis results with P1 and P2.  AP2 will: advise on CEV from a German point of view with its numerous carp ponds.  AP3 will: extend sampling of carp in Poland for KHV and CEV detection. All P’s and AP’s: will participate in inter-laboratory proficiency test to validate their tests, participate in the discussions on impact of CEV in teleconferences (M1), participate in communications, presentations etc. (M5, D2), and in the workshop (M6), and in writing up a draft research proposal, publications, (M7) etc.

Milestone No 1 2 3 4 5 6 7

Milestone title

Month

Start: teleconference of max. 1 day, and a second one

1, 6

Exchange of current methods and biologicals

2

Each lab implements and standardises the test and starts testing fresh and archive samples Organized inter-laboratory proficiency test

3-11

Presentations at international conferences

1-12

Workshop at CVI, to discuss the the objectives, the results of the project, and to complete and deliverables SOPs, project report, and draft project proposals ready, peer reviewed paper being made

11

9

10-12

Expected value for CoVetLab institutes   

Standardized validated diagnostic procedures (SOPs) for CEV Tools to test archive and fresh samples of carp and koi for CEV A multiplex PCR for concominant detection of CEV and cyprinid herpesviruses (CyHV13) in carp  A network on CEV and its effects on koi and carp  An overview of the spread of CEV in the participating countries  A publication, and a draft international research proposal as output Output.( deliverables, to include possible publications and IP issues) 1. SOPs for sampling, detection and typing of CEV 2. A draft international research proposal 3. Joint abstracts, publication(s) on methods and epidemiology of CEV in Europe 4. Final project report

Deliverable (D) No.

Deliverable title

Month

1

SOPs for sampling, detection and typing of CEV

11


2 3 4

Joint abstracts, publication(s) on methods and epidemiology of CEV in Europe A draft international research proposal Final report

12 12 12

Project Team (at least 3 CoVetLab partners are preferred) and contribution per partner Name and role Institute Email address Telephone number(s) Olga Haenen, project leader Marc Engelsma Niels Jørgen Olesen Susie Sommer Mikkelsen Charlotte Axén Laurent Bigarré Keith Way David Stone Sven Bergmann Michal Reichert

CVI CVI DTUVet DTU Vet SVA ANSES CEFAS CEFAS FLI NVRI Pulawy

[email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected]

Proposed total budget € 80,000.Indicative contribution per partner Institute Indicative contribution P1: CVI

€ 20,000.-

P2: VetDTU

€ 20,000.-

P3: SVA

€ 20,000.-

P4: ANSES

€ 20,000.-

AP1: CEFAS

€ 0,-

AP2: FLI

€ 0,-

AP3: NVRI Pulawy

€ 0,-

+31 +31 +45 +45 +46 +33 +44 +44 +49 +32

320 238352 320 238729 29 24 43 10 61 70 26 65 18 67 43 76 2 98 22 49 82 1305 206639 1305 206600 3835 17103 81 8893060

Comments costs AP’s* will be shared between Club 5 partners costs AP’s* will be shared between Club 5 partners costs AP’s* will be shared between Club 5 partners costs AP’s* will be shared between Club 5 partners *Costs paid for travel and subsistence only; Own budget for hours *Costs paid for travel and subsistence only; Own budget for hours *Costs paid for travel and subsistence only; Own budget for hours

Will any part of the project be co-funded from other financial sources The budget from Club 5 covers max 50% of the real costs. All further costs, including sample testing will be funded from own internal budgets outside this extra budget.


Tampere 2013

08/01/2015

CARP EDEMA-LIKE VIRUS DETECTED DURING DISEASE OUTBREAKS IN KOI AND COMMON CARP (Cyprinus carpio) IN THE UK Keith Way & David Stone Centre for Environment, Fisheries and Aquaculture Science, Weymouth, Dorset, UK

CEV workshop Copenhagen 2015

Detection of CEV-like virus in England & Wales  At the Cefas laboratory a CEV-like virus was first detected by PCR in imported Israeli koi, showing signs of KSD, at ornamental fish wholesalers in 2009 and again in 2011.  Further detections were then made from KSD-affected koi in hobbyist’s ponds in June 2012, May 2013 and June 2013.  Low levels of CEV-like virus also detected in healthy koi imported from Israel and Japan at ornamental fish wholesalers during 2013  In March 2012, a CEV-like virus was detected for the first time in common carp, displaying KSD-signs, obtained from a cluster of 4 fishery sites in south- east England.  In November 2012, CEV-like virus was detected at a fishery site in the English midlands.  Further detections were made in 2013 at 2 other fishery sites in the English midlands and at one new fishery site in SE England in 2014. CEV workshop Copenhagen 2015

Locations of CEV-like detections & disease outbreaks in England

Gross clinical signs

Common carp

 Extreme lethargy  Lesions & ulcers on body, around mouth & base of fins  Gill necrosis

 CEV-like virus detected at  6 Ornamental fish wholesalers - 2009, 2011, 2013 & 2014

Birmingham

●

 3 Hobbyist ponds in 2012 & 2013

●● London

●

Gill necrosis

 8 Fisheries / Angling waters in 2012, 2013 & 2014

Weymouth

- Important towns/cities

Outbreaks :

- in common carp fisheries


● - in koi ponds


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Tampere 2013

08/01/2015

Further investigations

Issues

Collection of live, disease affected, carp from a Midlands fishery site after a second outbreak of disease in February 2013 allowed more extensive virology investigations to be carried out.  Attempts to purify virus : From infected gill tissue (on Sucrose & Caesium Chloride gradients) - Unsuccessful Concentration of PCR primer target sequence was seen to increase at each purification step - but no virus visualised on EM Inability to visualise virus - tissue sampled too late in infection when virus levels have decreased and/or virus closely associated with host tissue  Virus transmission studies: Bath infection of juvenile common carp with infected gill tissue - Unsuccessful

 Gross clinical signs are very similar to koi herpesvirus disease (KHVD)  Appear to be similar issues to the in-apparent spread of KHVD in carp populations  Widespread release (& stocking) of ornamental fish species (including koi carp) into fisheries has occurred – as a result this disease is likely to be more widespread in England & Wales !  In England & Wales, CEV-like disease outbreaks have been reported to occur across a wide temperature range including water temperatures below 10°C  Could this disease be a major contributor to Spring Carp Mortality Syndrome (SCMS) – unexplained mortalities, reported to occur in carp fisheries since 1980s ?


EAFP Tampere 2013

Acknowledgements

 Defra [for funding under contracts FA001 & FC1202]

 The Environment Agency [Brampton, UK]

 The Fish Health Inspectorate at Cefas

F.H.I.


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CEV References, Version 6 January 2015

CEV References Amita, K. Oe, M., Matoyama, H., Yamagushi, N., Fukuda, H. 2002. A survey of Koi herpes virus and carp edema virus in colorcarp cultured in Niigata Prefecture, Japan. Fish. Pathol. 37:197-8. Haenen, O., Way, K., Stone, D., Engelsma, M. 2013. Koi Sleepy Disease found for the first time in koi carps in the Netherlands.[in Dutch]. Tijdschr. Diergeneeskd. 5: 26–29. Hartman K., Yanong, R., Pouder, D.B., Petty B.D., Francis-Floyd, R., Riggs, A.C., Waltzek T.B. 2013. Koi herpesvirus (KHV, CyHV3) Disease. University of Florida EDIS Publication VM149. http://edis.ifas.ufl.edu/vm149. Hedrick, R.P., Antonio, D.B., Munn, R. J. 1997. Poxvirus like agent associated with epizootic mortality in juvenile koi (Cyprinus carpio). FHS Newsletter..........???.. Hiroshima Prefectual Freshwater Fish Experiment Station, 1976. Studies on a considerable mortality of juvenile carp. –On a considerable mortality of carp having edema in juvenile stage. Suisancho Shitekenkyu Byogaikenkyu Houkokusho 19-33 (In Japanese). Hiroshima Prefectual Freshwater Fish Experiment Station, 1977. Studies on a considerable mortality of juvenile carp. –On a considerable mortality of carp having edema in juvenile stage. Ibid. 13-25 (In Japanese). Hiroshima Prefectual Freshwater Fish Experiment Station, 1978. Studies on a considerable mortality of juvenile carp. Ibid. 9-17 (In Japanese). Hosoya, H., Suzuki, M. 1976. Effect of NaCl solution bath on a disease of juvenile colorcarp accompanied by mass mortality, arising at the rainy season. Report of Niigata Prefectual Inland Fisheries Experimental Station 4: 69-70 (In Japanese). Lewisch, E., Gorgoglione, B., Way, K., El-Matbouli, M. 2014. Carp edema virus/koi sleepy disease: An emerging disease in central-east Europe. Transbound Emerg Dis. http://onlinelibrary.wiley.com/doi/10.1111/tbed.12293/full. Miyazaki, T., Isshiki T, Katsuyuki H. 2005. Histopathological and electron microscopy studies on sleepy disease of koi Cyprinus carpio koi in Japan. Dis Aquat Organ. 65: 197207. Murakami, Y., Shitanaka, M., Toshida, S., Matsuzato, T. 1976. Studies on mass mortality of juvenile carp: about mass mortality showing edema. Additional publication of Bull Hiroshima Fresh Water Fish Exp Station 19–33 (in Japanese). Murakami, Y., Shitanaka, M., Toshida, S., Matsuzato, T. 1977. Studies on mass mortality of juvenile carp-II: About mass mortality showing edema. Additional publication of Bull Hiroshima Fresh Water Fish Exp Station 19–36 (in Japanese). Murakami, Y., Tanaka, J., Hayashi, J., Toshida, S., Matsuzato, T. 1978. Studies on mass mortality of juvenile carp.- about mass mortality showing edema. Report of Research on Fish disease. Additional publication of Bull Hiroshima Fresh Water Fish Exp Station 18–37 (in Japanese).

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CEV References, Version 6 January 2015

Murukami, Y. and Nemoto, N. 1982. Disease and pesticidal injury, in “Recent status on the technic of the fancy-carp culture”(ed. By M. Suzuki). Japan Fisjeries Resource Conservation Association, Tokyo, 103-124 (In Japanese). Ono, S. I., Nagai, A., Sugai, N. 1986. A histopathological study on juvenile colorcarp Cyprinus carpio, showing edema. Fish Pathol. 21: 167-175. Oyamatsu, T., Hata, N., Yamada, K., Sano, T., Fukuda, H. 1997a. An etiological study on mass mortality of cultured colorcarp juveniles showing edema. Fish Pathol. 32:81–88. Oyamatsu, T., Matoyama, H., N., Yamamoto , K., Fukuda, H. 1997b. A trail for the detection of Carp Edema Virus by using polymerase chain reaction. Suisanzoshoku 45: 247–251. Seno, R., Hata, N., Oyamatsu, T., Fukuda, H., 2003. Curative effect of 0.5% salt water treatment on carp, Cyprinus carpio, infected with Carp Edema Virus (CEV) – Results mainly from reviving the physiological condition of the host. (Short Paper). Suisanzoshoku 51: 123-124. Sugai, N. and Koike, T. 1980. Some findings on epidemics among juvenile nishikigoi (Cyprinus carpio) during rainy season. – Test on the infectiousness. Report of Niigata Prefectual Inland Water Fisheries Experimental Station 8: 58-63. (In Japanese). Suzuki, S. and Fukuda, K. 1987. Effect of NaCl solution bath and experimental infection on mass mortality of juvenile colorcarp. Report of Saitama Prefectural Fisheries Experimental Station 46: 49-55 (In Japanese). Waltzek TB, Weber ES, Groff JM, Nordhausen RW, LaPatra SE, Goodwin AE, Hedrick RP (2011). Double-stranded DNA viruses of poikilothermic viruses. 36th Eastern Fish Health Workshop, Charleston, NC. Presentation and abstract. Waltzek, T. B., Gotesman, M., Steckler, N., Spears, S., Shelley, J., Yanong, R. 2014. Overview of DNA Viruses Impacting Ornamental Aquaculture. ISAAH . Portland, OR. Presentation and Abstract. Way, K., Stone, D. 2013. Emergence of Carp edema virus-like (CEV-like) disease in the UK. Finfish News.15: 32-34.

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Carp Edema Virus CEV WORKSHOP FINAL AGENDA

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