Division I.4.3 Centrifugation of homogenate

The homogenate shall be centrifuged in a refrigerated centrifuge at 2 °C to 5 °C at 2 000 to 4 000 × g for 15 minutes and the supernatant collected and may be treated for either four hours at 15 °C or overnight at 4 to 8 °C with antibiotics. If the sample has been shipped in a transport medium, the treatment of the supernatant with antibiotics may be omitted.

Where practical difficulties arise, such as incubator breakdown or problems with cell cultures, which make it impossible to inoculate cells within 48 hours after the collection of the fish tissue samples, the supernatant may be frozen at – 80 °C and virological examination may be carried out within 14 days.

If the collected supernatant is stored at – 80 °C within 48 hours after the sampling, it may be reused only once for virological examination.

Prior to the inoculation of the cells, the supernatant shall be mixed with equal parts of a suitably diluted pool of antisera to the indigenous serotypes of Infectious pancreatic necrosis (IPN) virus and incubated with this for a minimum of one hour at 15 °C or a maximum of 18 hours at 4 °C. The titre of the antiserum shall be at least 1/2 000 in a 50 % plaque neutralisation test.

Treatment of all inocula with antiserum to IPN virus aims at preventing cytopathic effect (CPE) due to IPN virus from developing in inoculated cell cultures. This will reduce the duration of the virological examinations as well as the number of cases in which occurrence of CPE would have to be considered potentially indicative of VHSV or IHNV.

When samples come from production units, which are considered free from IPN, the treatment of inocula with antiserum to IPN virus may be omitted.