ALTEX :: Alternatives to Animal Experiments

1998, Supplement

Testing Fish Vaccines with an In Vitro Lymphocyte Blastogenesis Assay

Ralf Peter Pund, Katrin Nold, and Elke Henrion

Bundesamt für Verbraucherschutz und Veterinaermedizin (BgVV), D-Berlin

SUMMARY

Lethal-challenge experiments are provided by German law till now for testing fish vaccines. Finding an in vitro assay which could replace these experiments was the subject of our work.

Two questions must have been answered to develop the lymphocyte blastogenesis assay:

  1. Do lymphocytes of immunised fish react the same way as lymphocytes of naive fish, being incubated with antigen; and
  2. Is there a coherence between the lymphocyte's in vitro response and the immune status of challenged fish.

Methods to take blood samples from anaesthetised fish and to obtain lymphocytes by centrifugation on gradients were worked out and the lymphocyte's demands on culture requirements were evaluated thoroughly because the ability to proliferate depends on many exogenous parameters. Proliferation was represented and measured by a tetrazolium based colorimetric assay (MTT).

Keywords: fish vaccines, cellular immune reactivity, lymphocyte proliferation, MTT