# Commission Implementing Decision (EU) 2015/1554 of 11 September 2015 laying down rules for the application of Directive 2006/88/EC as regards requirements for surveillance and diagnostic methods (notified under document C(2015) 6188) (Text with EEA relevance), Division II.2.2: Inoculation on cell cultures

Extent: E+W+S+N.I.

Atlantic salmon kidney (ASK) cells shall be used for primary ISAV isolation. Other cell lines of proven efficacy and sensitivity in isolating ISAV may be used, taking into consideration strain variability and the ability of different strains to replicate in different cell lines. The ASK cells seem to support isolation and growth of the hitherto known virus isolates, as long as a low passage level is used. A more distinct cytopathic effect (CPE) may appear in ASK cells than in other susceptible cell lines like SHK-1 (Salmon head kidney-1).
ASK (pass 65 or lower) cells shall be grown in L-15 medium containing 10 % foetal bovine serum, 2 % (vol:vol) 200 mM L-glutamine, and 0,08 % (vol:vol) 50 mM 2-mercaptoethanol in multi-well plates. Antiserum-treated organ suspension shall be inoculated into young actively growing cell cultures to give a final dilution of tissue material to culture medium of 1:1 000. For each organ, suspension 40 μl of inoculum shall be added to one well containing 2 ml of culture medium. To minimise the risk of cross-contamination, separate 12- or 24-well plates shall be used for samples from different fish farm sites.
One plate shall be left uninoculated to serve as a negative control. A separate plate shall be inoculated with a reference isolate of ISAV as a positive control, as follows. One hundred μl of a stock preparation of ISAV (minimum titre 107 Tissue culture infective dose at the 50 % end point (TCID50 ml– 1)) shall be inoculated into the first well and mixed. A volume of this material shall be transferred from the first well to the second well to make a 1:10 dilution and mixed. This shall be repeated across the plate to make six 10-fold dilutions. Stock ISAV may be stored at – 80 °C for at least 2 years but once thawed must be used within 3 days. Care shall be taken to prevent cross-contamination of test plates with positive control material. To avoid that risk, positive controls shall be set up and handled separately from test plates. A sensitivity test every 6 months of ASK cells towards ISAV isolates may replace the use of including a positive control at each inoculation.
Samples shall be incubated at 15 ± 2 °C for up to 15 days. Using a microscope, cell cultures shall be examined for CPE twice, between 5 to 7 and 12 to 14 days following inoculation. If any pool shows CPE, virus identification procedures shall be initiated immediately in accordance with point II.2.4. If no CPE is observed by day 14, an Indirect fluorescent antibody test (IFAT), haemadsorption or RT-PCR shall be performed.

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Source: https://www.legislation.gov.uk/eudn/2015/1554/annex/II/part/3/division/II/division/II.2/division/II.2.2 (the official version)
Page: https://openlegislation.uk/eu-decision-2015-1554/annex-II/part-3/division-II/division-II.2/division-II.2.2
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.