OIE Reference Laboratory Reports
Activities in 2011
Name of disease (or topic) for Koi herpesvirus disease
which you are a designated OIE
Address of laboratory: Centre for Environment, Fisheries
and Aquaculture Science (Cefas)
The Nothe, Weymouth, DT4 8UB,
Tel.: (+44-1305 ) 206639
Fax: (+44-1305 ) 206601
e-mail address: firstname.lastname@example.org
Name of Head of Laboratory Stephen Irving
Name of OIE Reference Expert: Keith Way
Name of writer of this report
(if different from above):
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010 1
Koi herpesvirus disease
Part I: Summary of general activities related to the disease
1. Test(s) in use/or available for the specified disease/topic at your laboratory
Test For Specificity Total + (Number
positive for KHV)
PCR-KHV Viral DNA detection KHV 62 (14)
PCR-HPV Viral DNA detection Cyprinid herpesviruses 53 (15)
CCB cell culture at 20°C Virus isolation Broad 25* (1∞)
* - virus isolation on CCB cells only carried out from July to October, inclusive.
- a large proportion of samples were received frozen and unsuitable for inoculation onto CCB cells, hence the low
number of virus isolations.
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.
Carp serum containing antibodies to KHV and antibody negative carp serum.
Diagnostic reagents were supplied for internal use but there were no requests in 2011 from national (UK)
laboratories to supply reagents.
Requests were received from the following OIE member countries for supply of diagnostic reagents:
Growing culture of KHV in CCB cells to Phyterra Bio. Inc., Prince Edward Island, Canada.
KHV PCR protocols to Department of Fisheries and Oceans, Winnipeg, Canada, and Institute of Food Safety,
Animal Health & Environment, Riga, Latvia and to National Fisheries Research & Development Institute in Busan
and Incheon, Republic of Korea.
Samples of DNA from CyHV-1 detections and isolations to School of Veterinary Medicine, University of
California, Davis, USA.
Part II: 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
a) Establishment and maintenance of a network with other OIE Reference Laboratories
designated for the same pathogen or disease and organisation of regular inter-laboratory
proficiency testing to ensure comparability of results
2 Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
Koi herpesvirus disease
b) Organisation of inter-laboratory proficiency testing with laboratories other than OIE
Reference Laboratories for the same pathogens and diseases to ensure equivalence of
The Cefas laboratory participated in the Annual Inter Laboratory Proficiency Test, distributed to over
30 laboratories, by the EU Reference laboratory (EURL) for fish diseases in Arhus, Denmark. The
proficiency test consisted of 10 ampoules containing lyophilised, virus-infected, cell culture supernatant and
again some ampoules contained KHV. Participating laboratories returned results to the EURL in December
2011 and a report of the results has not yet been distributed to the participants.
The Cefas Weymouth laboratory has participated in two distributions of a KHV PCR Proficiency test (PT),
sent out in May and October 2011, and provided expert comments on the results reported by all participants
to the QA unit (VETQAS) at the Animal Health & Veterinary Laboratories Agency in Sutton Bonington,
A total of 24 laboratories returned results from the first distribution of the KHV PCR proficiency test (PT)
sent out in April and May 2011. Of the 24 participants, 22 laboratories used a traditional PCR and produced
clean, correct results for all five samples tested. Real-time qPCR was used by 15 of the 24 participants and all
15 laboratories achieved the correct results. No false positives or false negatives were reported by any of the
laboratories and no problems were encountered with the on-line result submission.
A total of 23 laboratories returned results from the second distribution of the KHV PCR-PT sent out in
October 2011. Of the 23 participants, 18 laboratories used a traditional PCR and 16 of these produced clean,
correct results for all five samples tested. Real-time qPCR was used by 13 of the 23 participants and
12 laboratories achieved the correct results. False negatives and positives were reported by two of the
laboratories but these were found, in one instance, to result from mislabelling and from submission of the
wrong set of results in the second instance. Again, no problems were encountered with the on-line result
Results were reported in 2011 of an EPIZONE KHV serology proficiency test, in which the Cefas Weymouth
laboratory participated in 2010. In the test a panel of carp sera was analysed by seroneutralisation (SNT),
ELISA and indirect immunofluorescence (IFAT) and the participants included laboratories in France,
Denmark, Germany, The Netherlands, Italy, Poland, Sweden, UK (Cefas) and China. The proficiency test
demonstrated that antibodies against KHV can be detected by ELISA, SNT and IFAT, although not all
plasma / sera were found to be positive or negative in all tests in all laboratories. Diagnostic sensitivities
between 74 and 97 % for ELISA, between 91 and 95 % for SNT and 90 – 91 % for IFAT were achieved. The
diagnostic specificities calculated from the results with antisera against CCV, HVA, carp pox virus and
negative sera reached 93 % for ELISA and IFAT and 94 % for SNT. It was concluded, that serology of KHV
is a very useful tool for surveillance of KHV infections in common carp and koi carp populations.
An updated KHV chapter, in the revised format, for the draft 7 th edition of the OIE manual of diagnostic tests
for aquatic animal diseases was submitted to the OIE on 24th May 2011 for circulation to the OIE member
states for comments. In the latter part of 2011 the designated expert was active in responding to OIE member
comments on the new KHV chapter for the OIE diagnostic manual. These comments and the revised chapter
are to be submitted to the OIE in early January 2012.
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 define and quantify the risk pathway associated with transmitting KHV on anglers nets.
Following on from a previous study that showed that, during a disease outbreak, KHV can be transmitted on
contaminated keep-nets, further studies were designed to provide data for a risk assessment. These studies
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010 3
Koi herpesvirus disease
demonstrated that anglers nets may have played a role in the rapid spread of KHV in recreational fisheries. In an
experimental study, it was shown that carp infected with, and shedding KHV continue feeding and therefore have
the potential to be caught by anglers. The previous study had shown that, infected carp, if held, are capable of
contaminating nets to a level that allows transmission of KHV to naive fish. Further in vitro studies showed that
drying and exposing nets to sunlight can substantially reduce the risk of transferring KHV to other waters. A high
proportion of UK carp fisheries allow fish to be held in keepnets and allow the use of landing nets. Compared to
live fish movements the risk of transmitting KHV on anglers nets is low, however the high frequency at which
such contacts occur provide the potential for nets to have a substantial influence on the spread of the pathogen.
2] To determine prevalence of KHV in susceptible species and identify KHV-vector species.
Further information was collected on KHV-susceptible and vector species. Further evidence, from studies in
Germany, for the susceptibility of goldfish was published in 2011.
3] Other research is being conducted to improve tests to provide accurate identification of KHV and to detect
latent KHV and improve surveillance tools and studies are continuing on the molecular epidemiology of KHVD.
6. Collection, analysis and dissemination of epizootiological data relevant to international disease
A previously identified new strain (variant) of KHV was detected at a further 5 fishery sites in the UK in 2011 by
PCR. This variant was first detected in the UK in 2006 with further detections, at a handful of fishery sites, in
subsequent years. The variant shares 96% nucleotide identity with wild-type KHV (CyHV-3) and was detected
using the generic Cyprinid herpesvirus (CyHV) DNA polymerase primer set developed at Cefas.
A first occurrence of KHVD in koi carp at a site in Limoges, Haute-Vienne, France was notified to the OIE in
June 2011. A first occurrence of KHVD in wild common carp in the Beznar Reservoir, Granada, Andalucía, Spain
was notified to the OIE in July 2011. A re-occurrence of KHVD in koi carp at a site in Svartsjo, Sweden was
notified to the OIE in August 2011. No other member countries reported KHVD outbreaks to the OIE in 2011.
The WAHID interface ( OIE World Animal Health Information Database) lists in 2011, clinical KHVD observed
in domestic carp in Poland (3 locations), Thailand (Pathum Thani), Malaysia (Perak) and Singapore. This
information was included in 6 monthly reports in 2011and not in Immediate Notification reports or follow-ups.
Large mortalities of common carp (Cyprinus carpio carpio), suspected to be caused by KHV, were reported on
ProMed Mail to have occurred in the USA, in Kent Lake, Michigan in June and in the St Josephs River in Indiana
All data on KHV occurrence published in 2011 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. Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the
pathogen and the disease concerned
Following assessments by inspectors from the Department of Health and the United Kingdom Accreditation
Service (UKAS) we maintain accredited laboratory status and certification as follows:
The Aquatic Health and Hygiene inspection service (field inspections etc.) is accredited to ISO 17020. The
diagnostic service, including some research laboratories, is accredited to ISO 17025. The laboratory has biosecure
aquarium facilities with additional security containment and complies fully with the UK Good Laboratory Practice
All laboratories are built and operate to UK Biosecurity level 2. All live pathogens are dispatched according to the
International Air Transport Association requirements. Live pathogens are not dispatched to a country where the
4 Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
Koi herpesvirus disease
disease does not occur without approval from the relevant authorities in that country.
8. Provision of consultant expertise to OIE or to OIE Members
Information on KHV disease and diagnostic methods to National Fisheries Research & Development Institute in
Busan and Incheon, Republic of Korea.
Advice on recommended PCR assays for screening carp for KHV and on generic primers suitable for detection of
Cypriniviruses (Cyprinid herpesviruses) to Istituto Zooprofilatico Sperimentale delle Venezie, Padova, Italy.
Further advice on sampling for KHV surveillance and interpretation of results to the Marine Institute, Galway,
Republic of Ireland.
Advice on KHV cell culture and information on source of cell lines to AFBINI, Northern Ireland, UK and
information on transportation of fish cell lines to Shenzhen Exit & Entry Inspection and Quarantine Bureau,
Advice on propagation of CyHV-1 and CyHV-2 in cell culture to Central Taiwan University of Science &
Technology, Taichung City, Taiwan.
Information on KHV disease progression to School of Life Sciences, Keele University, UK, on diagnostic clinical
signs in koi to Atlantic Veterinary College, Prince Edward Island, Canada and KHV disease images to Marine
Scotland, Aberdeen, UK.
Information on recent detections of KHV in England and Wales to Ornamental Aquatic Trade Association
(OATA), Westbury, UK and information and advice on KHV detection in sturgeon to a UK Ornamental Fish
Importer. Advice on fish species susceptibility to KHV to Marine Scotland, Aberdeen, UK and advice on KHV
vectors to Water Lane Fish Farm, Dorset, UK.
Advice on potential KHV disease disinfection methods to a UK Fishing Tackle Company.
9. Provision of scientific and technical training to personnel from other OIE Member Countries
Training in fish disease inspection, investigation and diagnosis (including KHVD) to personnel from Shenzhen
Exit & Entry Inspection and Quarantine Bureau, Futuan, Shenzhen, The People’s Republic of China. Similar
training (including diagnosis of KHVD) also given to personnel from National Fisheries Research & Development
Institute in Busan and Incheon, Republic of Korea.
10. Provision of diagnostic testing facilities to other OIE Member Countries
Provision of diagnostic testing facilities to Marine Institute, Galway, Republic of Ireland. Included, quarantine of
common carp at elevated temperatures and serum antibody testing using an ELISA for detection of antibody to
11. 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.
12. Participation in international scientific collaborative studies
Studies on KHV latency (PhD project) with Marine Institute, Galway, Republic of Ireland (on-going).
13. 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
O. Donohoe, K. Henshilwood, K. Way, D. Walls & D. Stone (2011) Investigation into the existence of koi
herpesvirus derived micro-RNAs. Oral presentation - 15th International EAFP Conference on Diseases of Fish and
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010 5
Koi herpesvirus disease
Shellfish, Split, Croatia, 12-16 September 2011.
V.Baldwin, C.Joiner, R.Paley, N.Stinton, N.Taylor, & K.Way (2011) Transmission of koi herpesvirus via angling
equipment. Poster presentation - 15th International EAFP Conference on Diseases of Fish and Shellfish, Split,
Croatia, 12-16 September 2011.
M. Engelsma, M.Voorbergen-Laarman, K.Way & D.Stone (2011) Cyprinid herpesvirus 3: to be, or not to be?
Poster presentation - 15th International EAFP Conference on Diseases of Fish and Shellfish, Split, Croatia, 12-16
J. Castric, S.M.Bergmann, E. Jansson, O.Haenen, G.Bovo, M.Matras, J. Kempter, K. Way, D. Hong-an & N.J.
Olesen (2011) Development and assessment of 3 methods for detection of carp antibodies against KHV. Poster
presentation - 15th International EAFP Conference on Diseases of Fish and Shellfish, Split, Croatia, 12-16
Scientific publications in peer-reviewed journals
Taylor NGH, Norman RA, Way K and Peeler EJ (2011) Modelling the koi herpesvirus (KHV) epidemic highlights
the importance of active surveillance within a national control policy. Journal of Applied Ecology 48 348-355.
Annual report of reference laboratory activities on KHVD in 2010 submitted to OIE.
O. Donohoe., K. Henshilwood, K. Way, D. Stone & D. Walls (2010) Investigation of latency associated with koi
herpesvirus infection in carp (Cyprinus carpio). Poster presentation at Biotech Research Day 2011, Dublin City
University, February 2011.
6 Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010