Seasonal incidence of autochthonous antagonistic Roseobacter spp. and Vibrionaceae strains in a turbot larva (Scophthalmus maximus) rearing system

Bacteria inhibitory to fish larval pathogenic bacteria were isolated from two turbot larva rearing farms over a 1-year period. Samples were taken from the rearing site, e.g., tank walls, water, and feed for larvae, and bacteria with antagonistic activity against Vibrio anguillarum were isolated using a replica plating assay. Approximately 19,000 colonies were replica plated from marine agar plates, and 341 strains were isolated from colonies causing clearing zones in a layer of V. anguillarum. When tested in a well diffusion agar assay, 173 strains retained the antibacterial activity against V. anguillarum and Vibrio splendidus. Biochemical tests identified 132 strains as Roseobacter spp. and 31 as Vibrionaceae strains. Partial sequencing of the 16S rRNA gene of three strains confirmed the identification as Roseobacter gallaeciensis. Roseobacter spp. were especially isolated in the spring and early summer months. Subtyping of the 132 Roseobacter spp. strains by randomly amplified polymorphic DNA with two primers revealed that the strains formed a very homogeneous group. Hence, it appears that the same subtype was present at both fish farms and persisted during the 1-year survey. This indicates either a common, regular source of the subtype or the possibility that a particular subtype has established itself in some areas of the fish farm. Thirty-one antagonists were identified as Vibrio spp., and 18 of these were V. anguillarum but not serotype O1 or O2. Roseobacter spp. strains were, in particular, isolated from the larval tank walls, and it may be possible to establish an antagonistic, beneficial microflora in the rearing environment of turbot larvae and thereby limit survival of pathogenic bacteria.     

Quantitative measurement of endotoxin in rainbow trout (Salmo gairdneri) serum by the chromogenic substrate method

The amount of endotoxin in serum collected from normal rainbow trout ( Salmo gairdneri) and trout inoculated with viable Vibrio anguillarum or lipopolysaccharide (LPS) extracted from bacteria was determined by the chromogenic substrate method. The mean values of endotoxin in four different groups of normal rainbow trout sera ranged from 31.9 to 65.3 pg/ml. When fish were inoculated with viable bacteria (1 × 10⁸), they became septicaemic and a large amount of endotoxin (> 14 ng/ml) was detected in the sera. In fish inoculated with a smaller number of bacteria the amount of endotoxin was several times higher than that of normal fish in spite of failure of bacterial isolation. Although the endotoxin level in serum increased rapidly (> 100 ng/ml) after intraperitoneal inoculation with purified V. anguillarum LPS (540 μg), no fish died during the experiment. The high level of endotoxin in normal rainbow trout and the resistance of trout to endotoxin are in striking contrast to those of mammalian and avian species.     

Quantitative measurement of endotoxin in rainbow trout (Salmo gairdneri) serum by the chromogenic substrate method

The amount of endotoxin in serum collected from normal rainbow trout (Salmo gairdneri) and trout inoculated with viable Vibrio anguillarum or lipopolysaccharide (LPS) extracted from bacteria was determined by the chromogenic substrate method. The mean values of endotoxin in four different groups of normal rainbow trout sera ranged from 31.9 to 65.3 pg/ml. When fish were inoculated with viable bacteria (1 x 10(8], they became septicaemic and a large amount of endotoxin ng/ml) was detected in the sera. In fish inoculated with a smaller number of bacteria the amount of endotoxin was several times higher than that of normal fish in spite of failure of bacterial isolation. Although the endotoxin level in serum increased rapidly (greater than 100 ng/ml) after intraperitoneal inoculation with purified V. anguillarum LPS (540 micrograms), no fish died during the experiment. The high level of endotoxin in normal rainbow trout and the resistance of trout to endotoxin are in striking contrast to those of mammalian and avian species.     

Lipopolysaccharide O-antigen prevents phagocytosis of Vibrio anguillarum by rainbow trout (Oncorhynchus mykiss) skin epithelial cells

Colonization of host tissues is a first step taken by many pathogens during the initial stages of infection. Despite the impact of bacterial disease on wild and farmed fish, only a few direct studies have characterized bacterial factors required for colonization of fish tissues. In this study, using live-cell and confocal microscopy, rainbow trout skin epithelial cells, the main structural component of the skin epidermis, were demonstrated to phagocytize bacteria. Mutant analyses showed that the fish pathogen Vibrio anguillarum required the lipopolysaccharide O-antigen to evade phagocytosis and that O-antigen transport required the putative wzm-wzt-wbhA operon, which encodes two ABC polysaccharide transporter proteins and a methyltransferase. Pretreatment of the epithelial cells with mannose prevented phagocytosis of V. anguillarum suggesting that a mannose receptor is involved in the uptake process. In addition, the O-antigen transport mutants could not colonize the skin but they did colonize the intestines of rainbow trout. The O-antigen polysaccharides were also shown to aid resistance to the antimicrobial factors, lysozyme and polymyxin B. In summary, rainbow trout skin epithelial cells play a role in the fish innate immunity by clearing bacteria from the skin epidermis. In defense, V. anguillarum utilizes O-antigen polysaccharides to evade phagocytosis by the epithelial cells allowing it to colonize rapidly fish skin tissues.     

Study of Vibrio anguillarum strains from different sources with emphasis on ecological and pathobiological properties.

A total of 317 Vibrio anguillarum strains were isolated from water, sediment, and diseased as well as healthy rainbow trout at a Danish mariculture farm and from feral fish caught close to the farm. All strains were examined serologically. Ten sera permitted determination of the O group in 66.7% of the strains from diseased rainbow trout. Furthermore, the O group could be determined in 45.1 to 65.4% of the strains from mucus, gills, and intestinal contents of healthy rainbow trout, while only 22.2 to 28.8% of the isolates from water, sediment, and gills or mucus of feral fish were groupable. Serogroup O1 and to some extent O2 appeared to be associated with trout. Strains from these serogroups were selected for analyses of hemagglutinating activity and surface hydrophobicity. Serogroup O1 comprised hemagglutinating as well as nonhemagglutinating strains; from cases of vibriosis, all O1 strains were nonhemagglutinating. The strains belonging to serogroup O2 were generally hemagglutinating. Examinations of surface hydrophobicity by salt aggregation and hydrophobic interaction chromatography suggested that the O1 strains were more hydrophobic than the O2 strains. In pathogenicity tests, O1 strains isolated from gills and mucus of healthy rainbow trout killed all trout in the test groups. A strain from the intestinal contents of healthy rainbow trout did not produce significant mortality. This strain could, however, be frequently reisolated from the pronephros of fish in the test group concerned. After challenge with strains from eel mucus and seawater, mortality was not produced, and furthermore, these strains could not be reisolated from the pronephros.     

Effects of the antimicrobic tiamulin on seven gram-negative bacterial fish pathogens

In vitro and in vivo test were carried out with tiamulin and gram-negative bacterial pathogens of fish. Determination of minimum inhibitory concentration for 51 strains of seven species of gram-negative bacterial pathogens showed that only strains of Vibrio anguillarum were sensitive at 1.6-6.25 ppm, while the rest of test strains required 25- greater than 100 ppm. Control of infection was not achieved when tiamulin was fed for 14 days at 5 or 50 mg/kg to rainbow trout (Salmo gairdneri) experimentally infected with Yersinia ruckeri.     

Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum

The fish pathogen Vibrio anguillarum causes a lethal infection in rainbow trout (Salmo gairdneri). Three different avirulent mutants, constructed by transposon insertion mutagenesis (VAN20 and VAN70) or as antibiotic-resistant mutants (VAN1000), were isolated by screening 200 individual isolated mutants for avirulence. When used as live vaccines, all three avirulent mutants were able to induce protective immunity against the homologous as well as a heterologous strain of V. anguillarum. When VAN1000 was used, protective immunity could be recorded 1 week after bath vaccination with 10(7) bacteria per ml of water for 30 min. A single-dose immunization was effective for at least 12 weeks. Western immunoblotting showed that strains of V. anguillarum have antigenic determinants in common with Aeromonas strains. Therefore, we tested and confirmed that VAN1000 also was able to induce protective immunity against challenge with Aeromonas salmonicida.     

Selection and identification of autochthonous potential probiotic bacteria from turbot larvae (Scophthalmus maximus) rearing units

The purpose of this study was to select, identify and characterise bacteria as a disease control measure in the rearing of marine fish larvae (turbot, Scophthalmus maximus). Thirty-four out of 400 marine bacterial strains exhibited in vitro anti-bacterial activity against three fish larval pathogens. Two strains originated from culture collections and thirty two strains were isolated directly from turbot larvae rearing units using a pre-selection procedure to facilitate detection of antagonists. Approximately 8,500 colonies from colony-count plates were replica-plated on agar seeded with Vibrio anguillarum, and 196 of them caused zones of clearing in the V. anguillarum agar layer. Of these, 32 strains exhibited reproducible antibacterial properties in vitro when tested against the fish pathogens V. anguillarum 90-11-287, V. splendidus DMC-1 and a Pseudoalteromonas HQ. Seventeen antagonists were identified as Vibrio spp. and four of twelve tested were lethal to yolk-sac larvae. The 15 remaining strains were identified as Roseobacter spp. based on phenotypic criteria and 16S rDNA gene sequence analysis of two strains representing the two major RAPD groups. Most of the remaining 164 strains selected in the initial replica plating were identified as Vibrionaceae or Pseudoalteromonas. Roseobacter spp. were not lethal to egg yolk sac turbot larvae and in two of three trials, the mortality of larvae decreased (p > 0.001) in treatments where 10(7) cfu/ml Roseobacter sp. strain 27-4 was added, indicating a probiotic potential.     

Study of Vibrio anguillarum strains from different sources with emphasis on ecological and pathobiological properties

A total of 317 Vibrio anguillarum strains were isolated from water, sediment, and diseased as well as healthy rainbow trout at a Danish mariculture farm and from feral fish caught close to the farm. All strains were examined serologically. Ten sera permitted determination of the O group in 66.7% of the strains from diseased rainbow trout. Furthermore, the O group could be determined in 45.1 to 65.4% of the strains from mucus, gills, and intestinal contents of healthy rainbow trout, while only 22.2 to 28.8% of the isolates from water, sediment, and gills or mucus of feral fish were groupable. Serogroup O1 and to some extent O2 appeared to be associated with trout. Strains from these serogroups were selected for analyses of hemagglutinating activity and surface hydrophobicity. Serogroup O1 comprised hemagglutinating as well as nonhemagglutinating strains; from cases of vibriosis, all O1 strains were nonhemagglutinating. The strains belonging to serogroup O2 were generally hemagglutinating. Examinations of surface hydrophobicity by salt aggregation and hydrophobic interaction chromatography suggested that the O1 strains were more hydrophobic than the O2 strains. In pathogenicity tests, O1 strains isolated from gills and mucus of healthy rainbow trout killed all trout in the test groups. A strain from the intestinal contents of healthy rainbow trout did not produce significant mortality. This strain could, however, be frequently reisolated from the pronephros of fish in the test group concerned. After challenge with strains from eel mucus and seawater, mortality was not produced, and furthermore, these strains could not be reisolated from the pronephros.     

Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum.

The fish pathogen Vibrio anguillarum causes a lethal infection in rainbow trout (Salmo gairdneri). Three different avirulent mutants, constructed by transposon insertion mutagenesis (VAN20 and VAN70) or as antibiotic-resistant mutants (VAN1000), were isolated by screening 200 individual isolated mutants for avirulence. When used as live vaccines, all three avirulent mutants were able to induce protective immunity against the homologous as well as a heterologous strain of V. anguillarum. When VAN1000 was used, protective immunity could be recorded 1 week after bath vaccination with 10(7) bacteria per ml of water for 30 min. A single-dose immunization was effective for at least 12 weeks. Western immunoblotting showed that strains of V. anguillarum have antigenic determinants in common with Aeromonas strains. Therefore, we tested and confirmed that VAN1000 also was able to induce protective immunity against challenge with Aeromonas salmonicida.     

Serotyping of Vibrio anguillarum.

A serotyping scheme based on the detection of O antigens by slide agglutination in fish-pathogenic strains of Vibrio anguillarum is presented. Over a period of 5 years 270 Vibrio strains from feral and cultured fish, 189 strains from the environment, and 36 strains from invertebrates were collected. The strains were divided into 10 distinct serotypes (O1 through O10). More than 90% of the fish-pathogenic strains, but only 40% of the environmental strains, were typable; 71% of the strains isolated from cultured rainbow trout were serotype O1, whereas 78% of the strains isolated from feral fish were serotype O2. No dominating environmental serotype was found. A serotyping system for V. anguillarum is proposed. A total of 90 strains received from culture collections and laboratories in different countries were typed according to the present system.     

Serotyping of Vibrio anguillarum

A serotyping scheme based on the detection of O antigens by slide agglutination in fish-pathogenic strains of Vibrio anguillarum is presented. Over a period of 5 years 270 Vibrio strains from feral and cultured fish, 189 strains from the environment, and 36 strains from invertebrates were collected. The strains were divided into 10 distinct serotypes (O1 through O10). More than 90% of the fish-pathogenic strains, but only 40% of the environmental strains, were typable; 71% of the strains isolated from cultured rainbow trout were serotype O1, whereas 78% of the strains isolated from feral fish were serotype O2. No dominating environmental serotype was found. A serotyping system for V. anguillarum is proposed. A total of 90 strains received from culture collections and laboratories in different countries were typed according to the present system.     

Seasonal Incidence of Autochthonous Antagonistic Roseobacter spp. and Vibrionaceae Strains in a Turbot Larva (Scophthalmus maximus) Rearing System

Bacteria inhibitory to fish larval pathogenic bacteria were isolated from two turbot larva rearing farms over a 1-year period. Samples were taken from the rearing site, e.g., tank walls, water, and feed for larvae, and bacteria with antagonistic activity against Vibrio anguillarum were isolated using a replica plating assay. Approximately 19,000 colonies were replica plated from marine agar plates, and 341 strains were isolated from colonies causing clearing zones in a layer of V. anguillarum. When tested in a well diffusion agar assay, 173 strains retained the antibacterial activity against V. anguillarum and Vibrio splendidus. Biochemical tests identified 132 strains as Roseobacter spp. and 31 as Vibrionaceae strains. Partial sequencing of the 16S rRNA gene of three strains confirmed the identification as Roseobacter gallaeciensis. Roseobacter spp. were especially isolated in the spring and early summer months. Subtyping of the 132 Roseobacter spp. strains by randomly amplified polymorphic DNA with two primers revealed that the strains formed a very homogeneous group. Hence, it appears that the same subtype was present at both fish farms and persisted during the 1-year survey. This indicates either a common, regular source of the subtype or the possibility that a particular subtype has established itself in some areas of the fish farm. Thirty-one antagonists were identified as Vibrio spp., and 18 of these were V. anguillarum but not serotype O1 or O2. Roseobacter spp. strains were, in particular, isolated from the larval tank walls, and it may be possible to establish an antagonistic, beneficial microflora in the rearing environment of turbot larvae and thereby limit survival of pathogenic bacteria.     

Relationships between plasmids and phenotypes of presumptive strains of Vibrio anguillarum isolated from different fish species

On the basis of plasmid composition as well as serological and biochemical properties, 26 strains identified as Vibrio anguillarum isolated from diseased fish could be assigned to two different groups. Except for three reference strains, these++ strains were isolated from Norwegian fish. The four strains isolated from rainbow trout (Salmo gairdneri), the only strain isolated from char (Salvelinus alpinus), and three of six strains isolated from Atlantic salmon (Salmo salar) harbored a plasmid of 47 megadaltons (MDa). Restriction endonuclease analysis showed that this plasmid and the virulence plasmid pJM1, carried by V. anguillarum strain 775, were very similar but not identical. Strains harboring the 47-MDa plasmid had nearly identical biochemical properties and were serotype O1. Strains isolated from reared coastal cod (Gadus morhua), turbot (Scophthalmus maximus), halibut (Hippoglossus hippoglossus), free-living saithe (Pollachius virens), and partly from reared Atlantic salmon differed from strains harboring the 47-MDa virulence plasmid by not containing this plasmid, by having different biochemical traits, and by being serotype O2. Rainbow trout which were experimentally infected with a strain isolated from cod suffering from vibriosis developed clinical symptoms similar to those in cod but quite different from those usually seen in rainbow trout.     

An inhibitor of bacterial quorum sensing reduces mortalities caused by Vibriosis in rainbow trout (Oncorhynchus mykiss, Walbaum)

The fish pathogen Vibrio anguillarum produces quorum sensing signal molecules, N-acyl homoserine lactones (AHLs), which in several Gram-negative human and plant pathogenic bacteria regulate virulence factors. Expression of these factors can be blocked using specific quorum-sensing inhibitors (QSIs). The purpose of this study was to investigate the effect of a QSI, furanone C-30, on mortality of rainbow trout during challenge with V. anguillarum. Addition of 0.01 or 0.1 microM furanone C-30 to rainbow trout infected by cohabitation caused a significant reduction in accumulated mortality from 80-100% in challenge controls to 4-40% in treated groups. Furanone C-30 had no effect in an immersion challenge system, probably due to a very high water exchange and a rapid dilution of furanone C-30. Growth and survival of V. anguillarum were not affected by the concentrations of furanone C-30 used in the challenge experiments, thus avoiding selection for resistance. To elucidate the mechanism of disease control by furanone C-30, we determined its effect on the bacterial proteome, motility, and respiration. No effects were seen of furanone C-30 in any of these experiments. Although no cytotoxic effect on HeLa cells were observed, exposure to 1 microM (or higher) concentrations of furanone C-30 had detrimental effects on the rainbow trout. Our results indicate that QSIs can be used in non-antibiotic based control of fish diseases. However, they also underline the need for development of novel, less toxic QSI compounds and the need for understanding the exact mechanism(s) of action.     

Relationships between plasmids and phenotypes of presumptive strains of Vibrio anguillarum isolated from different fish species.

On the basis of plasmid composition as well as serological and biochemical properties, 26 strains identified as Vibrio anguillarum isolated from diseased fish could be assigned to two different groups. Except for three reference strains, these++ strains were isolated from Norwegian fish. The four strains isolated from rainbow trout (Salmo gairdneri), the only strain isolated from char (Salvelinus alpinus), and three of six strains isolated from Atlantic salmon (Salmo salar) harbored a plasmid of 47 megadaltons (MDa). Restriction endonuclease analysis showed that this plasmid and the virulence plasmid pJM1, carried by V. anguillarum strain 775, were very similar but not identical. Strains harboring the 47-MDa plasmid had nearly identical biochemical properties and were serotype O1. Strains isolated from reared coastal cod (Gadus morhua), turbot (Scophthalmus maximus), halibut (Hippoglossus hippoglossus), free-living saithe (Pollachius virens), and partly from reared Atlantic salmon differed from strains harboring the 47-MDa virulence plasmid by not containing this plasmid, by having different biochemical traits, and by being serotype O2. Rainbow trout which were experimentally infected with a strain isolated from cod suffering from vibriosis developed clinical symptoms similar to those in cod but quite different from those usually seen in rainbow trout.     

In vitro interactions between rainbow trout (Oncorhynchus mykiss) macrophages and Vibrio anguillarum serogroup O2a

The sensitivity of Vibrio anguillarum serogroup O2a to killing by rainbow trout macrophages in the presence or absence of specific antibodies and complement components was evaluated using an in vitro assay. Fluorescence microscopy revealed that V. anguillarum serogroup O2a was phagocytosed by rainbow trout macrophages. In the absence of specific antibodies and complement components the bacteria were killed to a limited extent by the macrophages and there was no increased killing if the bacteria were opsonised with either antibodies or antibodies and complement. Furthermore, activated macrophages did not show enhanced ability to kill the bacteria. Vibrio anguillarum serogroup O2a were susceptible to both cell-free superoxide anion (O2-) and hydrogen peroxide (H2O2), which might be generated during the macrophage respiratory burst and the bacteria did not quench cell-free O2-. However, the production of O2- by macrophages was undetectable during the first 30 min following infection and no respiratory burst was inducible by phorbol myristate acetate (PMA) 4 h after infection with V. anguillarum. This suggests that the bacteria were able to inhibit the production of O2- by the infected macrophages. Naive fish were protected when passively immunised with anti-V. anguillarum serogroup O2a antiserum. However, previous results suggest that antibodies are unlikely to provide the fish with protective immunity directly through activation of the complement system and lysis of the bacterial cells. The present in vitro findings suggest that the protective mechanisms of antibody against V. anguillarum serogroup O2a may not involve the opsonising effect of antibodies for enhanced killing by macrophages. However, the possibility exists that such antibodies may prevent the attachment of the pathogen to the host's tissues.     

Characterization of probiotic carnobacteria isolated from rainbow trout (Oncorhynchus mykiss) intestine

Aims:  To characterize two probiotic carnobacterial isolates, Carnobacterium maltaromaticum (B26) and C. divergens (B33), derived from rainbow trout ( Oncorhynchus mykiss ) intestine.
Methods and Results:  Both cultures, which were able to colonize the fish gut mucosal layer, comprised nonsporogenous, nonmotile, Gram-positive, catalase and oxidase-negative rods. The growth of both carnobacteria occurred between 0 and 37°C, in 0–10% (w/v) NaCl and at pH 5–10. Specifically, strain B26 grew in nutrient broth supplemented with 15% (w/v) NaCl. The most abundant cellular fatty acid of both cultures was 9-octadecenoic acid (18 : 1 n -9) (B26 = 52·6%; B33 = 40·6%), which was characteristic of Carnobacterium . Both cultures were inhibitory to Aeromonas salmonicida , Aer. hydrophila , Streptococcus iniae and Vibrio anguillarum , and strain B33 inhibited Listeria monocytogenes . Both carnobacteria, which did not contain plasmids, produced inhibitory compounds against Gram-positive and Gram-negative bacteria.
Conclusions:  Both probiotic cultures, B26 and B33, had unique phenotypic characteristics and showed a broad spectrum of antibiotic resistance against varying pathogenic bacteria.
Significance and Impact of the Study:  The results of this study contribute to new information and significance of carnobacterial species.     

Bacterial skin flora variation and in vitro inhibitory activity against Saprolegnia parasitica in brown and rainbow trout

Variations in the number and diversity of bacteria from the skin of brown trout Salmo trutta L. and rainbow trout Oncorhynchus mykiss Walbaum were surveyed from different rivers and fish farms in northern Spain. In addition to determining bacterial populations in skin samples of healthy fish, bacterial populations were determined from skin lesions (of brown trout only) infected with Saprolegnia parasitica, the causal agent of saprolegniosis. Mean bacterial counts from skin lesions of brown trout suffering from saprolegniosis were nearly 1000 times greater than from the skin of uninfected brown and rainbow trout. More than 20 different genera of bacteria were identified, with isolates of Aeromonas and Iodobacter being the predominant genera associated with saprolegniosis lesions. The in vitro inhibitory activity of 72 of these skin isolates was tested against S. parasitica using 3 different assays. These included (1) assessing the inhibition by bacteria of colony growth on agar media, (2) the inhibition of colony growth from colonized hemp seeds in liquid media and (3) the inhibition of cyst germination in liquid media. Finally, the fungicidal effect of the 24 most inhibitory bacterial species, and the inhibitory activity of their culture supernatants, was tested in the same way. Isolates identified as Aeromonas piscicola, A. sobria, Pantoea agglomerans and Pseudomonas fluorescens achieved the highest inhibition against S. parasitica. Many of these inhibitory isolates were obtained primarily from skin lesions of fish with saprolegniosis. It is suggested that some of these isolates might be useful in the biological control of saprolegniosis.