Koi herpesvirus disease

OIE Reference Laboratory Reports

Activities in 2011

Name of disease (or topic) for which you are a designated OIE Reference Laboratory: / Koi herpesvirus disease
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: /
website: / www.cefas.co.uk
Name of Head of Laboratory (Responsible Official): / Stephen Irving
Name of OIE Reference Expert: / Keith Way
Name of writer of this report
(if different from above):


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
(Diagnosis only) / 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

No activity

b)  Organisation of inter-laboratory proficiency testing with laboratories other than OIE Reference Laboratories for the same pathogens and diseases to ensure equivalence of results

The Cefas laboratory participated in the Annual Inter Laboratory Proficiency Test, distributed to over 30laboratories, 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, UK.

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 12laboratories 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 submission.

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 7th 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 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 control

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 www.collabcen.net.

7. Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the pathogen and the disease concerned

Accreditation

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.

Biosecurity

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 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 KHV.

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 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.