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.

A total of 30 isolates of Francisella noatunensis subsp. noatunensis isolated from Atlantic cod Gadus morhua L. were tested for susceptibility, in the form of minimal inhibitory concentration (MIC) values, against the following antibacterial agents: flumequine, oxolinic acid, ciprofloxacin, florfenicol, oxytetracycline, erythromycin, streptomycin sulphate, trimetoprim/ sulphadiazine and rifampin. All the isolates had a low susceptibility to oxytetracycline, trimetoprim/sulphadiazine (Tribrissen ® ), erythromycin, ciprofloxacin and streptomycin with MIC values of 64, 64 to 128, 16, 8 and 32 to 128 µg ml Dis Aquat Org 98: [57][58][59][60][61][62] 2012 markedly more active against oxolinic-acid-resistant isolates of Aeromonas salmonicida (Lewin & Hastings 1990). F. noatunensis subsp. noatunensis is a slowgrowing bacterium, and rifampin, erythromycin and streptomycin sulphate are all antibacterial agents that are used in human medicine in the treatment of Mycobacterium tuberculosis, another slow-growing bacterium. Erythromycin is also occasionally used against bacterial kidney disease (BKD) in salmonids.The aim of the present study was to test the susceptibility, in the form of MIC values, to a number of isolates of Francisella noatunensis subsp. noatunensis against the selected group of antibacterial agents. MATERIALS AND METHODS Bacterial strainsA total of 30 isolates of Francisella noatunensis subsp. noatunensis from Atlantic cod Gadus morhua L. were analysed. Of these, 28 isolates were provided by the Norwegian Veterinary Institute (Norway) and 2 isolates, GM2212/LMG 24256 (Nylund et al. 2006, Ottem et al. 2007) and EK-4b, were provided by the Institute of Marine Research (Norway). All isolates originate from farmed cod, except EK-4b, which was isolated from a wild cod. The strains were previously identified as F. noatunensis subsp. noatunensis using the methods described by Ottem et al. (2008). ChemicalsFlumequine, oxolinic acid, oxytetracycline, florfenicol, Tribrissen (trimethoprim/sulfadiazine), erythromycin, ciprofloxacin, streptomycin sulphate and rifampin were all obtained from Norwegian Medical Depot (Bergen, Norway). Stock solutions of antibacterial agents were prepared at a concentration of 1.0 mg ml −1 in methanol (florfenicol, Tribrissen, rifampin), in water (oxytetracycline, erythromycin, streptomycin sulphate), 0.03 M NaOH (flumequine, oxolic acid) and 0.03 M NaOH/methanol (1:1) (ciprofloxacin). MIC determinationIn order to meet the demands of Francisella noatunensis subsp. noatunensis for specific growth factors, the method of choice in the present work was a modified agar dilution test for the determinations of MIC values (Alderman & Smith 2001). Briefly, F. noatunensis subsp. noatunensis isolates were grown on modified Mueller-Hinton agar (MMHA) supplemented with 3% foetal bovine serum (FBS) (PAA Laboratories), 0.8% glucose (Merck), 0.4% L-cystein (Sigma) and 4% Yeastolate Ultrafiltrate (Gibco) and containing 2-fold dilutions of the antibacterial agents tested. All supplements and a...

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The RIG-I receptors RIG-I, MDA5 and LGP2 are involved in viral recognition, and they have different ligand specificity and recognize different viruses. Activation of RIG-I-like receptors (RLRs) leads to production of cytokines essential for antiviral immunity. In fish, most research has focused on interferons, and less is known about the production of proinflammatory cytokines during viral infections. In this study, we have cloned the full-length MDA5 sequence in Atlantic salmon, and compared it with RIG-I and LGP2. Further, the salmonid cell line TO was infected with three fish pathogenic viruses, infectious pancreatic necrosis virus (IPNV), infectious salmon anaemia virus (ISAV) and salmonid alphavirus (SAV), and differential gene expression (DEG) analyses of RLRs, interferons (IFNa-d) and proinflammatory cytokines (TNF-α1, TNF-α2, IL-1β, IL-6, IL-12 p40s) were performed. The DEG analyses showed that the responses of proinflammatory cytokines in TO cells infected with IPNV and ISAV were profoundly different from SAV-infected cells. In the two aforementioned, TNF-α1 and TNF-α2 were highly upregulated, while in SAV-infected cells these cytokines were downregulated. Knowledge of virus recognition by the host and the immune responses during infection may help elucidate why and how some viruses can escape the immune system. Such knowledge is useful for the development of immune prophylactic measures.