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					                    OIE Reference Laboratory Reports
                                           Activities in 2011

   Name of disease (or topic) for                                         Koi herpesvirus disease
 which you are a designated OIE
         Reference Laboratory:

                             Address of laboratory:                        Centre for Environment, Fisheries
                                                                            and Aquaculture Science (Cefas)
                                                                                 Weymouth laboratory,
                                                                           The Nothe, Weymouth, DT4 8UB,
                                                                                 UNITED KINGDOM

                                                 Tel.:                           (+44-1305 ) 206639

                                                 Fax:                            (+44-1305 ) 206601

                                     e-mail address:                  


     Name of Head of Laboratory                                                 Stephen Irving
          (Responsible Official):

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∞)
                                                 (Diagnosis only)

                          * - 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

               No activity

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
     in October.

     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

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
     compliance programme.

     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,
     Shenzhen, China.
     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
    September 2011.

    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
    September 2011.

       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.

       Other communications

    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

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