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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 The genomes of V. anguillarum were compared (D. Castillo, P.W. D'Alvise, M. Middelboe & L. Gram, unpublished data) and most of the high-virulent strains had acquired virulence genes from other pathogenic Vibrio., (© 2017 John Wiley & Sons Ltd.)

The granuloma disease caused by Francisella noatunensis subsp. noatunensis in farmed Atlantic cod has not been successfully treated by use of antibacterials, even when antibacterial resistance testing indicates a sufficient effect. The reason for this treatment failure may be the intracellular existence of the bacteria within immune cells, mainly macrophages. To investigate the effect of antibacterials on intracellular Francisella replication, we established a protocol for the detection of drugs within Atlantic cod immune cells using high-performance liquid chromatography (HPLC). When the uptake and intracellular concentrations of oxolinic acid and flumequine were analysed in isolated adherent head kidney leucocytes (HKLs) by HPLC, we found that uptake was rapid and the intracellular concentrations reflected the extracellular exposure concentrations. To investigate the effect of the antibacterial compounds on intracellular bacterial replication, adherent HKLs experimentally infected with the bacteria were analysed using flow cytometry and intracellular labelling of bacteria by specific antibodies. We found that flumequine did not inhibit intracellular bacterial replication. Unexpectedly, the results indicated that the intracellularly effiacy of the drug was reduced. The HPLC method used proved to be highly applicable for accurate determination of intracellular drug concentrations. When combined with sensitive and specific flow cytometry analyses for identification and measurement of intracellular bacterial replication, we suggest that this approach can be very valuable for the design of antibacterial treatments of intracellular pathogens.

Francisella noatunensis subsp. noatunensis, causing granulomatosis in cod, has been shown to reside within cod immune cells, mainly within monocytes and macrophages. In the present study, we analysed the ability of the bacterium to replicate within adherent cells isolated from head kidney by in vitro infection of leucocytes. Two different technical approaches for flow cytometry analyses were performed for detection of intracellular bacteria. The presence of the wild type was assessed after identification by intracellular binding of specific antibodies to the pathogen. The other way was to use green fluorescent protein (GFP) transformed bacterium for infection studies allowing direct measurements of fluorescence from infected cells. By both methods we found an increase in fluorescence in infected cells, verifying bacterial replication, both after 4 and 28 h post infection in leucocytes isolated from head kidney (HKL). The GFP transformed bacterium was similar to the wild type in growth and infectivity pattern, showing that it can be a valuable tool for further studies of infection routes and pathology. Further, F. noatunensis subsp. noatunensis was found to inhibit respiratory burst activity, a potent pathogen killing mechanism, in cod leucocytes, but not in such cells from salmon. Our findings may indicate that inhibition of respiratory burst during Francisella infection is a key to its intracellular existence. This strategy seems to be conserved through evolution as it is also observed during infections in higher vertebrates caused by bacteria within the Francisella genus. The results presented here, showing the intracellular existence of Francisella, its replication within leucocytes and the inhibitory effect on respiratory burst, strongly support that these factors contribute to disease and pathology in infected cod. The intracellular replication shown in the present study might contribute to explain the problems of obtaining protective vaccines against Francisella and effective antibiotic treatment of infected fish., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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 10(7) 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.

The oxidation of dihydrorhodamine 123 (DHR) to the fluorescent rhodamine 123 (RHO) was detected using flow cytometry. This assay for detection of respiratory burst activity was established in peripheral blood leucocytes (PBL) and head kidney leucocytes (HKL) of Atlantic salmon and Atlantic cod. The leucocytes were stimulated by phorbol 12-myristate 13-acetate (PMA). For cod cells 10 times lower concentration of PMA had to be used compared to salmon cells, as higher concentrations were toxic and resulted in considerable cell death. The cells found to be RHO-positive were monocytes/macrophages and neutrophils based on the scatter dot plots, but for salmon also some small cells were found to have high fluorescence intensity both in the flow cytometry analyses and by fluorescence microscopy of cytospin preparations. The nature of these cells is not known. For cod leucocytes, such cells were not obvious. The instrument settings are a bit more demanding for cod, as cod cells die more easily compared to salmon cells. In both assays the limit between negative and positive cells has to be carefully considered. The presented flow cytometry protocols for measurements of respiratory burst in salmon and cod leucocytes can be applied in various studies where respiratory burst functions are involved, such as to verify if it is activated or suppressed in connection with infections and immunostimulation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Francisella noatunensis causes the systemic granulomatous inflammatory disease, francisellosis in cod. Little is known about the lifestyle of this facultative intracellular bacterium within cod leucocytes. We have examined the interaction of this bacterium with phagocytic cells isolated from cod with emphasis on monocytes, macrophages, neutrophils and phagocytic B-cells. It is clear from confocal microscopy sections through adherent cell preparations that numerous bacteria were located intracellularly following in vitro infection in monocytes and macrophages. In these sections bacteria were immunostained and cell actin was stained using Alexa Fluor® 488 phalloidin. Bacteria were observed in close association with neutrophils and intracellularly (low numbers) in B-cells. Bacteria were observed more frequently in head kidney- than in peripheral blood- and spleen- leucocytes. Following infection, bacteria were initially observed grouped together and located close to the nucleus. Later they were found spread within the cytoplasm. This indicates regression of F. noatunensis from the phagosome to the cytoplasm where replication possibly takes place. It may be hypothesised that the bacteria may alter maturation of the phagosome and thus, avoid the potent intracellular killing mechanisms of phagocytic cells. The intracellular lifestyle involving escape to cytoplasm prior to fusion with the lysosome may have consequences for vaccine development as well as antibiotic treatment of infected cod., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Phagocytosis by fish cells has mostly been studied using adherent leucocytes, excluding suspended cells such as the majority of B-cells and neutrophils, but a recent study describes professional phagocytosis of latex beads and bacteria by B-cells from rainbow trout. In the present study, phagocytosis by B-cells and neutrophils from salmon and cod was studied. Leucocytes were isolated from peripheral blood (PBL) and head kidney (HKL). By flow cytometry analyses, proportions of MAb labelled cell populations with internalized fluorescent beads, as well as the number of beads within each cell, could be determined. Phagocytic capacity and ability were demonstrated in B-cells and neutrophils from salmon and cod. In salmon, B-cells had higher phagocytic ability than neutrophils in HKL, but not in PBL. For cod the phagocytic ability of B-cells were lower than for neutrophils in both HKL and PBL, but the phagocytic capacity of cod B-cells were higher than for neutrophils in both HKL and PBL. For salmon B-cells the phagocytic capacity was lower than or similar to neutrophils in HKL and PBL. The total phagocytic ability of leucocytes was different in the species studied. The highest phagocytic ability was observed in cod, showing similar values for PBL and HKL. Salmon PBL displayed about twice the phagocytic ability of cod PBL. There seemed to be some major differences between the two fish species concerning phagocytosis. In salmon, a rather large proportion of phagocytic leucocytes were phagocytic B-cells, indicating that B-cells may have an important function in particle clearance in this species. In cod, phagocytic leucocytes in HKL and PBL were mostly neutrophils, and only a small proportion of B-cells were phagocytic, supporting the more prominent role of innate immune functions in cod neutrophils., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Proportions of leucocytes from head kidney, blood and spleen were identified as B-cells and neutrophils using a polyclonal antibody to cod IgM and a monoclonal antibody which previously has been shown to bind specifically to salmon and trout neutrophils. The cell specific binding of the antibodies was supported by double immunostaining. The morphology of isolated leucocytes was examined on Diff Quick stained slide preparations, and myeloperoxidase positive neutrophils were identified by diaminobenzidine staining. The antibodies clearly identified distinct cell populations. Using flow cytometry, high proportions of neutrophils were observed in peripheral blood leucocytes and high proportions of B-cells were found in head kidney leucocytes when compared to proportions of these cells in Atlantic salmon (Salmo salar L.). The spleen contained the highest proportion of B-cells. Cytoplasmic staining of immunoglobulin positive cells in slide preparations indicated that plasma cells were present, but not strikingly abundant, in head kidney, spleen and peripheral blood. Staining for myeloperoxidase identified, in accordance with the flow cytometry results, a large number of neutrophils, especially in peripheral blood leucocytes. The neutrophil nucleus was not clearly segmented, but appeared more irregular than rounded. The findings of high proportions of neutrophils in peripheral blood suggest that these cells of the innate immune system might have a central role in defence and protection against infections in cod.