A new cell line designated TO which provides a high yield of infectious salmon anaemia virus (ISAV) has been established. The cells originate from head kidney leukocytes isolated from Atlantic salmon and grow well at 20°C in EMEM with 5% CO 2 and without CO 2 supplement in HMEM. The cells have at present been passed more than 150 times and no changes in morphology, growth or virus production have been observed. The virus infection results in cytopathic effects (CPE) within 9 d, and the virus titre obtained from centrifuged and filtrated cell lysates, measured as TCID 50 , was about 10 9.1 ml -1 . The virus isolated from lysates of infected cells by a sucrose gradient provided purified ISAV when examined by silver stained SDS-PAGE. Salmon injected with diluted virus supernatant showed mortalities, hematocrit values and clinical signs in accordance with infectious salmon anaemia. KEY WORDS: Cell line · Salmon · Infectious salmon anaemia virus, ISAV · Fish virus Resale or republication not permitted without written consent of the publisherDis Aquat Org 44: [183][184][185][186][187][188][189][190] 2001 MATERIALS AND METHODS Primary cell culture. A head kidney was obtained from unvaccinated Atlantic salmon Salmo salar L. weighting 3 kg reared in the facilities of The Industrial and Aquatic Laboratory, Bergen, Norway. The fish were kept in 6500 l tanks at a temperature of 8°C and with a constant flow rate of saline water of 1.0 l kg fish -1 min -1 . The fish were fed commercial salmon pelleted food dispensed from an automatic feeder 8 h a day. The fish had no history of previous infectious diseases. The head kidney was removed aseptically, placed in 10 ml of holding medium containing RPMI 1640 (BioWhittaker), 100 µg ml -1 gentamicin sulphate (BioWhittaker), 2 mM L-glutamine (BioWhittaker) and 10% foetal calf serum (FCS) (Gibco BRL) and homogenized. The cell suspension was applied on ficoll gradient (Pharmacia Biotech AB) and centrifuged at 1000 × g for 30 min at 4°C. The isolated leukocytes were suspended in 30 ml of the holding medium and mixed well with a vortex mixer before centrifugation at 900 × g for 10 min at 4°C. The cell pellet was suspended in culture medium containing Eagle's MEM with Earle's BSS, without L-glutamine (EMEM) (BioWhittaker), 200 µg ml -1 gentamicin sulphate, 1 µg ml -1 fungizone (BioWhittaker), 292 µg ml -1 L-glutamine, 50 mM mercaptoethanol (Gibco BRL), 1% MEM Eagle Non Essential Amino Acid (NEAA) (100 ×) (BioWhittaker) and 10% FCS (BioWhittaker). Cells (10 7.9 ml -1 ) were cultured in 25 cm 2 tissue culture flasks (Nunc) at 20°C in air with 5% CO 2 . Non-adherent cells were removed the next day and fresh culture medium was added. Thereafter, the cells were observed daily and half of the medium was changed approximately every second week. After about 3 mo growing cell layers were observed and these were further cultivated. Cell layers were trypsinated (Trypsin Versene, BioWhittaker) and non-adherent cells were transferred to a new tissue culture flask, where some adherent cells...

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In the present study, we have isolated leucocytes from peripheral blood, head kidney and spleen from lumpsucker (Cyclopterus lumpus L.), and performed functional studies like phagocytosis and respiratory burst, as well as morphological and cytochemical analyses. Different leucocytes were identified, such as lymphocytes, monocytes/macrophages and polymorphonuclear cells with bean shaped or bilobed nuclei. In addition, cells with similar morphology as described for dendritic cells in trout were abundant among the isolated leucocytes. Flow cytometry was successfully used for measuring phagocytosis and respiratory burst activity. The phagocytic capacity and ability were very high, and cells with different morphology in all three leucocyte preparations phagocytised beads rapidly. Due to lack of available cell markers, the identity of the phagocytic cells could not be determined. The potent non-specific phagocytosis was in accordance with a high number of cells positive for myeloperoxidase, an enzyme involved in oxygen-dependent killing mechanism present in phagocytic cells. Further, high respiratory burst activity was present in the leucocytes samples, verifying a potent oxygen- dependent degradation. At present, the specific antibody immune response could not be measured, as immunoglobulin or B-cells have not yet been isolated. Therefore, analyses of the specific immune response in this fish species await further clarification. The present study presents the first analyses of lumpsucker immunity and also the first within the order Scopaeniformes.

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