Phaeobacter gallaeciensis Reduces Vibrio anguillarum in Cultures of Microalgae and Rotifers, and Prevents Vibriosis in Cod Larvae

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

Competition of pathogens and probionts in cod yolk sac larvae measured in vivo, and the impact of increased temperature

Aquaculture are today one of the biggest food-producing sectors in the world. Over the recent decades there has been a great increase in intensity and commercialization of aquaculture production, which has led to an unavoidable growth in disease problems. This has again led to a global over consumption of antibiotics and other pharmaceuticals which have caused problems as pollution, resistance and enormous losses for the industry. Farming of Atlantic cod, Gadus morhua, was anticipated to be the new success in Norwegian aquaculture after salmon, but partly due to the global financial crisis, and partly to high mortality - including the early life stages - the success have not become as large as expected. The high density of marine larvae and biological waste during rearing might contribute to high growth of opportunistic pathogenic bacteria, which could result in high larval mortality. Due to the fact that treatments with antibacterial agents are not favourable, and since vaccination is not possible due to the immature immune system of larvae, there has been carried out various studies to find new alternative treatments for the early life stages of cod and other marine species. The aim of this thesis is to enhance the knowledge of probiotics and test the possibility to use probiotics as an alternative for antibiotics in cod larval rearing facilities. In the present work a multi-dish system was used as a model for bath challenge experiment, and the species challenged with Vibrio anguillarum HI610 and different types of probiotics were cod egg/larvae. Cod eggs delivered from a commercial hatchery were randomly selected and placed separately in wells in a multi-dish system. Each well is seen as an independent unit and contained 2 ml of 80% aerated sterile seawater. After the eggs were placed in the wells, the wells were challenged with high dose (approximately 106 CFU ml-1) of different probiotic strains alone, and together with high dose (approximately 106 CFU ml-1) Vibrio anguillarum HI610. There were done experiments at 7°C and 13°C (15°C). The experiments did also include a negative control group consisting of unchallenged larvae and a positive control with only high dose Vibrio anguillarum HI610. The day hatching reached 50% was defined as day 0, and every day from day 0 and for as long as the experiment carried on, the mortality was registered. In the present work there were used one pathogen Vibrio anguillarum HI610 and there were tested eight probiotics: Phaeobacter 27-4, the mutant JBB1001, Phaeobacter M23-3.1, Ruegeria F1926, Ruegeria M43-2.1, Phaeobacter gallaeciensis BS107-wt, the mutant Phaeobacter gallaeciensis BS107-Pda8 and AQ10 a Pseudoalteromonas citrea. The results are introduced in graphs made in Microsoft Office Excel 2007 showing cumulative mortality in percent (%) per days post hatch (dph) for every challenge group. The mortality data showed that the pathogenic bacteria Vibrio anguillarum HI610 gives a high and rapidly mortality soon post hatch. The probiotics alone did not harm the larvae and could show a slightly positive effect on the normal mortality. The use of the probiotics together with V.anguillarum HI610 enhanced an inhibitory effect against the pathogenic bacteria Vibrio anguillarum HI610 and/or almost eradication of the effect of the pathogenic bacteria when added at the same time or when the probiotics were added 48hours prior to the addition of the..

Mortality of cod larvae during the challenge trials.

<p>Mean values of two independent triplicate experiments. The single-larvae cultures were simultaneously inoculated with <i>P. gallaeciensis</i> wild type and <i>V. anguillarum</i> (T5, •), or with the TDA-negative mutant of <i>P. gallaeciensis</i> and <i>V. anguillarum</i> (T6, □). Unexposed larvae and larvae exposed to single bacterial strains acted as controls: Negative Control (T1, ▪), only <i>V. anguillarum</i> (T2, ▴), only <i>P. gallaeciensis</i> wild type (T3, ▾), and only <i>P. gallaeciensis</i> TDA-negative mutant (T4, ♦).</p

Expression of <i>tdaC</i> in co-culture with <i>Tetraselmis suecica</i>.

<p>Phase contrast (A) and fluorescence (B) micrographs of <i>P. gallaeciensis</i> pPDA11 (<i>tdaCp::gfp</i>) in co-culture with <i>T. suecica</i>. The two panels show the same seven algal cells of which some are dividing, and a marine snow-like particle which is colonized by <i>P. gallaeciensis</i> carrying the promoter-fusion on a plasmid. The green fluorescence of <i>P. gallaeciensis</i> on the particle shows that the gfp gene is expressed from the tdaC promoter, indicating production of TDA.</p

Reduction of <i>V. anguillarum</i> in cultures of <i>Tetraselmis suecica</i> by <i>Phaeobacter gallaeciensis</i>.

<p>Colony-forming units of <i>V. anguillarum</i> inoculated at 10¹ cfu/ml (A) and at 10⁴ cfu/ml (B) in presence of <i>P. gallaeciensis</i> wild type (▪), in presence of the <i>P. gallaeciensis</i> TDA-negative mutant (▴), and in the monoxenic control (▾).</p

Reduction of <i>Vibrio anguillarum</i> by <i>Phaeobacter gallaeciensis</i> in rotifer cultures.

<p>Mean values of two duplicate experiments: colony-forming units of <i>V. anguillarum</i> in co-culture with <i>P. gallaeciensis</i> wild type (▴), with the TDA-negative mutant of <i>P. gallaeciensis</i> (▾), and in the monoxenic control (▪).</p

Concentrations of <i>Tetraselmis suecica</i> and <i>Phaeobacter gallaeciensis</i> in the co-cultures.

<p>Means and standard deviations of eight experiments: colony-forming units of <i>P. gallaeciensis</i> wild type (♦) and the TDA-negative mutant (•), and concentrations of <i>T. suecica</i> with <i>V. anguillarum</i> (▾), <i>T. suecica</i> with <i>P. gallaeciensis</i> wild type (▪), <i>T. suecica</i> with <i>P. gallaeciensis</i> TDA-negative mutant (▴), and axenic <i>T. suecica</i> (□). The <i>P. gallaeciensis</i> strains were inoculated at 10⁷ cfu/ml and remained as a steady population, while the algae went from late log into stationary phase.</p

Localization of bacteria in cultures of <i>Tetraselmis suecica</i>.

<p>Phase-contrast (A,C) and fluorescence (B,D,E) micrographs. Co-culture of <i>Tetraselmis suecica</i> with <i>Phaeobacter gallaeciensis dsRed</i> (A,B), axenic <i>T. suecica</i> (C,D), co-culture of <i>T. suecica</i> with <i>V. anguillarum gfp</i> (E). Panel A and B show two single (left) and one dividing algal cell (right side), and a marine snow-like particle consisting of algae-debris which is colonized by red-fluorescent <i>P. gallaeciensis</i>. Red fluorescence of algae is due to chlorophyll. Panels C and D show an algal cell and particles from an axenic culture, recorded using the same settings as for the panels above. Panel E shows red-fluorescent algae cells and green-fluorescent <i>V. anguillarum</i>, which do not colonize particles, but remain in suspension as single, motile cells.</p

Group numbers and treatments in the challenge trial.

<p>Group numbers and treatments in the challenge trial.</p