Division II.1 Preparation of samples from fish

If possible, fish that have been kept for a prolonged time period at the virus permissive temperature range (namely, 2 to 3 weeks at 15 °C to 26 °C) shall be sampled. If possible, samples shall be collected 24 hours but not later than 72 hours after management practices that may reactivate the virus in fish with a carrier status, such as netting or transport, in order to enhance the chance of KHV detection.

For the purpose of the surveillance of KHVD, the fish may be sent alive or killed and packed separately in sealed aseptic containers or alternatively frozen organs or organ pieces preserved in 80 % to 100 % alcohol or viral transport medium (to be processed within 48 hours after collection) may be used for testing PCR based methods. For the surveillance of KHVD, gill and kidney tissue shall be collected.

For the purpose of surveillance of KHVD, pooling shall be avoided where possible. If pooling is necessary, tissue material from a maximum of two fish may be pooled. Larger samples shall be homogenised in mortar and pestle or stomacher, and subsamples retrieved for DNA extraction before clarification. Alternatively, subsamples may be collected from each tissue included in the sample and placed in ‘lysis-tubes’.