Phaeobacter gallaeciensis can antagonize fish-pathogenic bacteria in vitro, and the purpose of this study was to evaluate the organism as a probiont for marine fish larvae and their feed cultures. An in vivo mechanism of action of the antagonistic probiotic bacterium is suggested using a non-antagonistic mutant. P. gallaeciensis was readily established in axenic cultures of the two microalgae Tetraselmis suecica and Nannochloropsis oculata, and of the rotifer Brachionus plicatilis. P. gallaeciensis reached densities of 107 cfu/ml and did not adversely affect growth of algae or rotifers. Vibrio anguillarum was significantly reduced by wild-type P. gallaeciensis, when introduced into these cultures. A P. gallaeciensis mutant that did not produce the antibacterial compound tropodithietic acid (TDA) did not reduce V. anguillarum numbers, suggesting that production of the antibacterial compound is important for the antagonistic properties of P. gallaeciensis. The ability of P. gallaeciensis to protect fish larvae from vibriosis was determined in a bath challenge experiment using a multidish system with 1 larva per well. Unchallenged larvae reached 40% accumulated mortality which increased to 100% when infected with V. anguillarum. P. gallaeciensis reduced the mortality of challenged cod larvae (Gadus morhua) to 10%, significantly below the levels of both the challenged and the unchallenged larvae. The TDA mutant reduced mortality of the cod larvae in some of the replicates, although to a much lesser extent than the wild type. It is concluded that P. gallaeciensis is a promising probiont in marine larviculture and that TDA production likely contributes to its probiotic effect.

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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Vibrionaceae infections are a major obstacle for marine larviculture; however, little is known about virulence differences of Vibrio strains. The virulence of Vibrio strains, mostly isolated from vibriosis outbreaks in farmed fish, was tested in larval challenge trials with cod (Gadus morhua), turbot (Scophthalmus maximus) and halibut (Hippoglossus hippoglossus) using a multiwell dish assays with single-egg/larvae cultures. The strains differed significantly in virulence as some caused a high mortality of larva reaching 100% mortality after a few days, while others had no or only marginal effects on survival. Some Vibrio strains were pathogenic in all of the larva species, while some caused disease only in one of the species. Twenty-nine of the Vibrio anguillarum strains increased the mortality of larvae from at least one fish species; however, pathogenicity of the strains differed markedly. Other Vibrio species had no or less pronounced effects on larval mortalities. Iron uptake has been related to V. anguillarum virulence; however, the presence or absence of the plasmid pJM1 encoding anguibactin did not correlate with virulence. The genomes of V. anguillarum were compared (D. Castillo, P.W. D'Alvise, M. Middelboe & L. Gram, unpublished data) and most of the high-virulent strains had acquired virulence genes from other pathogenic Vibrio.

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