Koi herpesvirus disease
Activities in 2009
Koi herpesvirus disease
Keith Way
Centre for Environment, Fisheries and Aquaculture Science (Cefas)
Weymouth laboratory, The Nothe, Weymouth, DT4 8UB, UK
Tel.: (+44-1305) 206639, Fax: (+44-1305) 206601
, www.cefas.co.uk
Summary of general activities related to the disease
1. Test(s) in use/or available for the specified disease at your laboratory
Test / For / Specificity / Total+ (Number positive for KHV)
PCR-KHV / Viral DNA detection / KHV / 71 (19)
PCR-HPV / Viral DNA detection / Cyprinid herpesviruses / 71 (21)
CCB cell culture at 20°C / Virus isolation
(Diagnosis only) / Broad / 30* (5)
* - virus isolation on CCB cells only carried out from July to October, inclusive.
2. Production and distribution of diagnostic reagents
The laboratory produced :
· details of PCR primer sequences and protocols for the detection and confirmation of KHV.
· reference cyprinid herpesvirus-1 (CyHV-1, carp pox) and CyHV-3 (KHV) isolates.
· CyHV-1 and CyHV-3 (KHV) DNA.
· koi fin (KF-1) cell cultures.
· Carp serum containing antibodies to KHV and antibody negative carp serum.
Diagnostic reagents were supplied for internal use but there were no requests in 2009 from national (UK) laboratories to supply reagents.
Requests were received from the following OIE member countries for supply of diagnostic reagents:
Growing cultures of KHV reference virus were supplied to the Marine Institute, Galway, Republic of Ireland and Bornova Veterinary Control & Research Institute, Izmir, Turkey.
Purified preparation of KHV reference virus was supplied to the National Fish Health (NAFish) Management Centre, Pulau Pinang, Malaysia.
Activities specifically related to the mandate
of OIE Reference Laboratories
3. International harmonisation and standardisation of methods for diagnostic testing or the production and testing of vaccines
The Cefas Weymouth laboratory compiled and distributed a report of the 2008 KHV PCR methods ring trial to all 44 ring-trial participants in June 2009. This was the third annual ring-trial of KHV PCR assay protocols to be co-ordinated by the Cefas laboratory. The ring trial included 26 laboratories in 19 EU member states and 18 laboratories in 13 other countries around the world.
The 2008 ring-trial was of a similar format to the 2007 trial. Participants were requested to use their preferred DNA extraction method and to trial the two PCR protocols recommended in the OIE diagnostic manual chapter on KHV disease. They were also expected to use a real-time PCR assay or a nested-PCR assay to analyse the samples. The main difference for 2008 was that one of the samples was spiked with a cyprinid herpesvirus (CyHV) that was not KHV and laboratories were then asked to test the samples using a protocol that included generic primers that target the CyHV polymerase gene. All of the sample vials contained lower amounts of virus DNA and total DNA than in previous samples making the 2008 ring trial more technically demanding than the previous trials. As a result, a much larger proportion of participants reported incorrect results (false positives and negatives) than in previous ring-trials. False positive results, with one or more of the assays used, were reported by 18 laboratories. This suggests that they may have cross-contamination and sample handling issues in the laboratories they use for their molecular virology analyses.
In 2009 the designated expert provided comments on the methods for diagnosis of KHVD to be included in the draft EC Diagnostic manual for certain aquatic animal diseases. Also, information and advice was provided to the EPIZONE working group on ‘KHV diagnosis and surveillance’.
4. Preparation and supply of international reference standards for diagnostic tests or vaccines
No internationally recognised standard reference materials were supplied to other OIE Member Countries.
5. Research and development of new procedures for diagnosis and control
The following research is being carried out as part of Department of Environment, Food & Rural Affairs (Defra) funded projects at Cefas:
1] To determine the conditions for koi herpesvirus (KHV) survival and persistence in the aquatic environment.
The project in 2007-08 to determine the distribution of KHV in farms and fishery sites in England and Wales identified a number of carp populations that had suffered KHV outbreaks in 2006 and maintained a high prevalence of KHV-antibody positive fish in each of the following three years. Virus transmission studies have been conducted with carp obtained from two of these antibody-positive carp populations. Further transmission studies will also be undertaken to assess the potential for disease spread via angling equipment.
2] To determine prevalence of KHV in susceptible species and identify KHV-vector species.
To determine under what conditions virus prevalence may increase in goldfish and to determine how long the virus persists and under what conditions goldfish spread KHVD to carp. Also, to confirm the susceptibility of goldfish or crucian carp x common carp hybrids to KHV. Hybrids are stocked into many fisheries in the UK and could present a high risk of spreading KHV.
3] To improve tests to provide accurate identification of KHV and to detect latent KHV and improve surveillance tools.
Understanding virus latency and reactivation is essential to developing detection and control strategies for diseases caused by herpesviruses. Studies of KHV latency will be enhanced by collaboration on a post-graduate project at the Marine Institute, Galway, Republic of Ireland. Results from this project will be used to improve methods for detecting latent virus. Further studies on variant KHV and vaccine strains are needed to determine their impact on the reliability of detection methods and surveillance tools for KHV.
4] Studies on the molecular epidemiology of KHVD.
To further develop the tools required to screen regions of polymorphism in the KHV genome and identify an alternative marker to discriminate between vaccine and wt KHV infections. Also, to assess the suitability of the KHV genome length polymorphisms to future epidemiological investigations.
6. Collection, analysis and dissemination of epizootiological data relevant to international disease control
Only one potential new strain (variant) of KHV was detected in 2009 in koi carp imported from Singapore. All of the koi were healthy and no KHV disease was observed during a six week period of quarantine at 23°C. Sequence analysis revealed that the partial DNA polymerase gene of the variant shared 98% nucleotide identity with KHV and 94% nucleotide identity with the KHV variants detected in the UK in 2006, 2007 & 2008. The generic cyprinid herpesvirus polymerase primer sets, developed at Cefas were used to detect the variant in the Singapore koi. Details of these primer sets were made available to all of the participants in the 2008 KHV PCR methods ring trial.
Studies in France to develop a duplex PCR for differentiation of CyHV-3 (KHV) lineages confirmed the USA/Israel (U/I) and Japan (J) lineages identified in earlier studies by Japanese researchers. [Aoki T, Hirono I, Kurokawa K & 7 others (2007) J Virol 81 5058-5065] However, the French study also identified a third genotype intermediate between the U/I and J lineages and demonstrated that the three lineages of CyHV-3 have been introduced into Europe since 2001 via imported koi carp. [Bigarré L, Baud M, Cabon J, Antychowicz J, Bergmann SM, Engelsma M, Pozet F, Reichert M, Castric J, 2009. Differentiation between Cyprinid herpesvirus type-3 lineages using duplex PCR. J. Virol. Methods 158, 51-57.]
Outbreaks of KHVD associated with large mortalities of common carp (Cyprinus carpio carpio) were reported, in June, to have occurred at Lake Mohave, Arizona, USA and, in August, at Lake Kaweah, California, USA. A report was received in July of two outbreaks of KHVD in koi populations in ornamental ponds in Belgium. A report was received in August of an outbreak of KHVD in a koi population in an ornamental pond in Skanelan, Sweden. No other member countries reported KHV outbreaks to the OIE in 2009.
No other new epizootiological data on KHV (occurrence by country and by host species) arose from the laboratory’s diagnostic work.
In studies conducted by the Friederich Loeffler Institute in Germany, KHV has been detected by nested PCR in several different varieties of goldfish as well as grass carp, ide and ornamental catfish (Ancistrus sp.) collected from ornamental fish wholesalers in Germany. The detection in the goldfish and grass carp has been confirmed by in situ hybridisation using different primers to those used in the PCR [Bergmann SM., Schütze H., Fischer U., Fichtner D., Riechardt M., Meyer K., Schrudde D. and Kempter J. (2009), Bull. Eur. Ass. Fish Pathol., 29(5),145-152]. A study conducted by the West Pomeranian University of Technology, Division of Aquaculture in Poland reported the detection of KHV by PCR in Russian sturgeon (Acipenser gueldenstaedtii) and Atlantic sturgeon (A. oxyrinchus) from fish farms in Northern Poland. The presence, in gill and kidney tissue, of KHV protein and viral genome were confirmed by indirect fluorescent antibody test and in situ hybridisation, respectively [Kempter J., Sadowski J., Schutze H., Fischer U., Dauber M., Fichtner D., Panicz R. and Bergmann S.M. (2009), Acta Ichthyologica et Piscatoria 39 (2): 119–126].
These reports do not demonstrate the presence of viable virus but do imply that other fish species, apart from goldfish, are potential vectors of KHV.
All data on KHV occurrence published in 2009 by OIE or in the scientific literature, was entered into the International Database on Aquatic Animal Diseases to add to the historical data. Access to the database is available on-line to OIE member countries via the Aquatic Animals Commission pages on the OIE web site, and the web site of the OIE Collaborating Centre for Information on Aquatic Animal Diseases at www.collabcen.net.
7. Provision of consultant expertise to OIE or to OIE Members
Requests were received from the following OIE Members for provision of advice:
Information and advice on export testing requirements for KHV was provided to USDA, DVL-BP, Ames, Iowa, USA.
Advice was provided in reviewing a preliminary report of a KHV project at Oregon State University funded by the Associated Koi Clubs of America.
Advice on sourcing of CCB cells for KHV culture was provided to National Veterinary Research and Quarantine Service (NVRQS), Republic of Korea and Bornova Veterinary Control & Research Institute, Turkey. Information on source of carp for KHVD studies was provided to the Institute of Aquaculture, Stirling, Scotland, UK.
Information on incidence of KHV outbreaks in England and Wales and suitable references for KHV in the UK provided to Ornamental Aquatic Trade Association (OATA), Trowbridge, UK and Dept. of Fisheries and Oceans Canada, Pacific Biological Station, Nanaimo, Canada.
Information and advice on CyHV-3 (KHV) in goldfish to the National Fish Health (NAFish) Management Centre, Pulau Pinang, Malaysia and Department of Aquatic Animals - Animal Biosecurity, Canberra, Australia. General information on hosts/vectors of KHVD was provided to a Private Aquatic Veterinarian in The Netherlands.
Advice/clarification of sampling and diagnostic methods in the OIE manual of diagnostic tests for aquatic animal diseases was provided to Frederich Loeffler Institute, Griefswald, Germany.
Information and advice on KHV PCR methods suitable for diagnostic and surveillance tests was provided to the Department of Fisheries and Oceans Canada, Pacific Biological Station, Nanaimo, Canada ; Marine Institute, Galway, Republic of Ireland ; University of Arkansas at Pine Bluff, USA ; ‘KHV-info’, Nijmegen, The Netherlands ; Animal & Plant Health Laboratories, Agri-Food & Veterinary Authority of Singapore and to a Private Aquatic Veterinarian, Vienna, Austria.
Information/advice on sampling and sample collection for KHV diagnostic testing and surveillance was provided to the National Veterinary Institute, Uppsala, Sweden; State Veterinary & Food Institute, Dolny Kubin, Slovakia and Molecular Diagnostic Services, Durban, South Africa.
Advice and information on ELISA for detection of antibody to KHV and/or protocols were provided to Fisheries Biosecurity Unit, Penang, Malaysia; AFBI, Northern Ireland, UK; Fish Health Service for Saxony, Konigswartha, Germany ; National Fish Health (NAFish) Management Centre, Pulau Pinang, Malaysia; Barford Lakes, Norfolk, UK.;‘KHV-info’, Nijmegen, The Netherlands and Bavarian Fish Health Service, Weiden, Germany.
General information on cyprinid herpesviruses and other fish herpesviruses was provided to Frederich Loeffler Institute, Griefswald, Germany; Associated Koi Clubs of America; Henderson Morley, Birmingham, UK and Marine Institute, Galway, Republic of Ireland. Information and advice on koi sleepy disease was provided to Colors Aquaculture R&D, Israel.
Research materials were provided to the following laboratories:
Animal & Plant Health Laboratories, Agri-Food and Veterinary Authority, Singapore (Carp serum containing antibodies to KHV and antibody negative carp serum).
8. Provision of scientific and technical training to personnel from other OIE Members
Training, testing and longer term capacity building for the Malaysian competent authority for Aquatic Health & Hygiene. Involved the training of four technicians from three laboratories in Malaysia and included KHV diagnostic methods and testing for KHV antibodies (March 2009).
9. Provision of diagnostic testing facilities to other OIE Members
No requests were received.
10. Organisation of international scientific meetings on behalf of OIE or other international bodies
No requests were received from OIE or other bodies to organise scientific meetings.
11. Participation in international scientific collaborative studies
Studies on KHV latency (PhD project) with Marine Institute, Galway, Republic of Ireland.
Collaboration with National Fish Health (NAFish) Management Centre, Pulau Pinang, Malaysia to set-up and trial the KHV antibody ELISA [Part of longer term capacity building for the Malaysian competent authority for Aquatic Health & Hygiene].
12. Publication and dissemination of information relevant to the work of OIE (including list of scientific publications, internet publishing activities, presentations at international conferences)
¡ Presentations at international conferences and meetings
Keith Way (2009) The 2008 KHV PCR methods ring trial. Oral presentation to the 13th Annual meeting of E.U. National Reference Laboratories for Fish Diseases, Copenhagen, Denmark May 26-28, 2009.
Keith Way (2009) Optimisation and validation of an ELISA for detection of antibody to cyprinid herpesvirus-3 (CyHV-3, KHV). Oral presentation to the 13th Annual meeting of E.U. National Reference Laboratories for Fish Diseases, Copenhagen, Denmark May 26-28, 2009.
P.F. Dixon, C.L. Joiner, K. Way, R.A. Reese, G. Jeney & Z. Jeney (2009). Studies on the resistance of selected families of common carp Cyprinus carpio (L.), to koi herpesvirus. Oral presentation - 14th International EAFP Conference on Diseases of Fish and Shellfish, Prague, Czech Republic, 14-19 September 2009.