Phenotypic Characteristics and Virulence of Vibrio anguillarum-Related Organisms

The phenotypic, molecular, and virulence properties of 46 Vibrio anguillarum-related (VAR) strains isolated from diseased fish and shellfish and from the environment were investigated. Twelve reference strains belonging to the 10 serotypes of V. anguillarum and the Vibrio splendidus type strain were included for comparison. Numerical taxonomy studies allowed us to group the isolates into four phena. The main phenotypic traits to differentiate VAR strains from V. anguillarum were fermentation of arabinose and mannitol, indole and Voges-Proskauer reactions, gelatin and casein hydrolysis, hemolytic activity, growth at 37 and 4°C, and resistance to ampicillin. Serological analysis confirmed that phena I and II were composed mainly of strains of V. anguillarum, while phena III and IV included VAR strains. Excluding the reference strains, the typeable isolates belonged to serotypes O3 (15 strains), O4 (3 strains), and O5 (2 strains) of V. anguillarum. The infectivity trials showed that only 9 of a total of 24 strains tested displayed virulence for rainbow trout. Virulent strains (50% lethal dose ranging from 10² to 10⁶ cells) included V. anguillarum strains belonging to serotypes O1 (one strain), O2 (one strain), O3 (three isolates), and O4 (one isolate) and only three strains of the VAR group. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of lipopolysaccharide and outer membrane proteins showed heterogeneity not only among the 10 V. anguillarum serotypes but also within the VAR group. Immunoblot assays demonstrated a close relationship among V. anguillarum strains from the same serotype, while strains from different serotypes were not antigenically related. The VAR strains did not share antigenic components with the serotypes of V. anguillarum tested (serotypes O1 to O5). Plasmids were detected in only 19 of the total of 59 strains. The majority of the strains carrying plasmids were grouped within phenon IV, in which plasmid bands of 27 and 36 MDa were found in all the isolates. No correlation between the plasmid content of VAR microorganisms and their phenotypic or virulence characteristics was observed. From these results it can be concluded that VAR strains associated with disease should be included together with V. anguillarum in the formulation of vaccines against vibriosis.     

Vibriophages and Their Interactions with the Fish Pathogen Vibrio anguillarum

Vibrio anguillarum is an important pathogen in aquaculture, responsible for the disease vibriosis in many fish and invertebrate species. Disease control by antibiotics is a concern due to potential development and spread of antibiotic resistance. The use of bacteriophages to control the pathogen may offer a non-antibiotic-based approach to reduce vibriosis. A detailed understanding of the phage-host interaction is needed to evaluate the potential of phages to control the pathogen. In this study, we examined the diversity and interactions of 11 vibriophages, 24 V. anguillarum strains, and 13 Vibrio species strains. Together, the host ranges of the 11 phages covered all of the tested 37 Vibrio sp. host strains, which represented considerable temporal (20 years) and geographical (9 countries) differences in their origins of isolation. Thus, despite the occurrence of unique susceptibility patterns of the individual host isolates, key phenotypic properties related to phage susceptibility are distributed worldwide and maintained in the global Vibrio community for decades. The phage susceptibility pattern of the isolates did not show any relation to the physiological relationships obtained from Biolog GN2 profiles, demonstrating that similar phage susceptibility patterns occur across broad phylogenetic and physiological differences in Vibrio strains. Subsequent culture experiments with two phages and two V. anguillarum hosts demonstrated an initial strong lytic potential of the phages. However, rapid regrowth of both phage-resistant and phage-sensitive cells following the initial lysis suggested that several mechanisms of protection against phage infection had developed in the host populations.     

Development of a DNA probe using differential hybridization to detect the fish pathogenVibrio anguillarum

The fish pathogenVibrio anguillarum causes significant economic losses in commercially cultured fish species worldwide. At present, identification ofV. anguillarum requires conventional isolation and culturing techniques. Using differential hybridization, a 310 base pairV. anguillarum-specific DNA fragment was isolated for use as a probe. In specificity studies against 19 different bacterial species, including twoVibrio sp. and fish pathogens, and 223 marine bacterial isolates, the probe hybridized exclusively toV. anguillarum strains. The probe also strongly hybridizes to 7 of 9 serotypes tested, with serotype 09 giving a weak probe reaction and serotype O7 negative. The probe allows rapid and accurate detection of both pathogenic and environmental strains ofV. anguillarum.     

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.     

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.     

Kocuria SM1 controls vibriosis in rainbow trout (Oncorhynchus mykiss,Walbaum)

Aims: To develop probiotics for the control of vibriosis caused by Vibrio anguillarum and Vibrio ordalii in finfish. Methods and Results: Kocuria SM1, isolated from the digestive tract of rainbow trout, was administered orally to rainbow trout (Oncorhynchus mykiss) for 2 weeks at a dose equivalent to c. 10⁸ cells per g of feed and then challenged intraperitoneally with V. anguillarum and V. ordalii. Use of SM1 led to a reduction in mortalities to 15–20% compared to 74–80% mortalities in the controls. SM1 stimulated both cellular and humoral immune responses in rainbow trout, by elevation of leucocytes (5·5 ± 0·8 × 10⁶ ml^?1 from 3·7 ± 0·8 × 10⁶ ml^?1), erythrocytes (1·2 ± 0·1 × 10⁸ ml^?1 from 0·8 ± 0·1 × 10⁸ ml^?1), protein (23 ± 4·4 mg ml^?1 from 16 ± 1·3 mg ml^?1), globulin (15·7 ± 0·2 mg ml^?1 from 9·9 ± 0·1 mg ml^?1) and albumin (7·3 ± 0·2 mg ml^?1 from 6·1 ± 0·1 mg ml^?1) levels, upregulation of respiratory burst (0·05 ± 0·01 from 0·02 ± 0·01), complement (56 ± 7·2 units ml^?1 from 40 ± 8·0 units ml^?1), lysozyme (920 ± 128·8 units ml^?1 from 760 ± 115·3 units ml^?1) and bacterial killing activities. Conclusions: Kocuria SM1 successfully controlled vibriosis in rainbow trout, and the mode of action reflected stimulation of the host innate immune system. Significance and Impact of the Study: Probiotics can contribute a significant role in fish disease control strategies, and their use may replace some of the inhibitory chemicals currently used in fish farms.     

Proteases production by two Vibrio species on residuals marine media

A comparative study was carried out on the growth and production of alkaline proteases by two Vibrio species using different marine peptones from fish viscera residues. The bacteria tested, Vibrio anguillarum and Vibrio splendidus, are producers of high levels of proteolytic enzymes which act as factors of virulence in fish cultures, causing high mortality rates. The kinetic assays and subsequent comparison with the parameters obtained from the adjustment to various mathematical models, highlighted the potential interest of the media formulated, for their possible production on an industrial scale, particularly the production of proteases by V. anguillarum growing in rainbow trout and squid peptones.     

Rapid identification of Vibrio anguillarum by Colony hybridization

A 562 base pair fragment of DNA from a serotype A strain of Vibrio anguillarum was cloned into pUC9 and used as a hybridization probe for the rapid identification of Vibrio anguillarum by colony hybridization. The probe was tested on nine different fish pathogens, 15 Vibrio isolates, 2 organisms closely related to Vibrio, and 9 serotypes of V. anguillarum. The probe hybridized only with the DNA of V. anguillarum serotypes A and H. The sequence of the 562 nucleotides have been determined. This probe allows rapid, reliable, and specific detection of V. anguillarum in freshwater ayu, Plecoglossus altivelis.     

Vibrio diseases of marine fish populations

SeveralVibrio spp. cause disease in marine fish populations, both wild and cultured. The most common disease, vibriosis, is caused byV. anguillarum. However, increase in the intensity of mariculture, combined with continuing improvements in bacterial systematics, expands the list ofVibrio spp. that cause fish disease. The bacterial pathogens, species of fish affected, virulence mechanisms, and disease treatment and prevention are included as topics of emphasis in this review.     

Pathogenic activities of live cells and extracellular products of the fish pathogen Pasteurella piscicida.

The pathobiological activities in vivo and in vitro of live cells and extracellular products (ECP) of eleven Pasteurella piscicida strains of different origin were examined. Infectivity trials showed that P. piscicida did not possess strict host specificity since the majority of the isolates were virulent for gilthead seabream, rainbow trout and turbot, with LD50 values ranging between 10(3) and 10(6) live cells. However, none of the strains tested were pathogenic for mice (LD50 > 10(8) cells)). In addition, the ECP were strongly toxic for fish (LD50 ranging from 1.0 to 4.6 micrograms protein per g fish), which clearly demonstrates their important role in the pathogenesis of pasteurellosis. All the ECP samples were cytotoxic for fish and homoiothermic cell lines, possessed notable phospholipase activity and displayed haemolytic activity for sheep, salmon and turbot erythrocytes (but not for trout erythrocytes). However, the production of proteolytic enzymes differed among the P. piscicida strains. Although no strain displayed elastase activity, five isolates (the Japanese and Italian strains) hydrolysed casein and gelatin. All these biological activities in vivo and in vitro were lost after heat treatment (100 degrees C for 10 min). The general enzymic patterns of both live cells and ECP evaluated by the API-ZYM system also revealed some variation among the P. piscicida isolates. Generally, whole cells showed a wider range of enzymic activities than ECP. The results presented here are important for the selection of strains in the development of effective polyvalent pasteurellosis vaccines containing both whole cells and ECP.     

Mitogenicity and Adjuvanticity of a Marine Bacterium,Vibrio anguillarum,in Mice

The effects of whole cells of three different O serotypes of Vibrio anguillarum on the murine immune response were studied. The addition of different doses (1–100/ig/ml) of V. anguillarum cells, as well as Salmonella typhimurium lipopolysaccharide, markedly increased the incorporation of [³H] thymidine into in vitro cultured spleen cells of C57BL/6 mice. All three serotype strains of V. anguillarum were able to induce the mitogenic effect at 10 μg/ml and 100 μg/ml, but serotype I strains were more potent than the others. Since pretreatment of spleen cells with rabbit anti-mouse thymocyte antiserum did not affect the mitogenic activity of V. anguillarum, Vibrio cells may be a B-lymphocyte mitogen. When sheep or horse erythrocytes and Vibrio cells were injected intraperitoneally into ddY mice, Vibrio cells exhibited an enhancing effect on antibody response in vivo, regardless of the different serotypes. Vibrio cells, when injected intraperitoneally into mice before the antigen, markedly suppressed the antibody response. Several days after the injection of Vibrio cells, these mice showed an enhanced carbon clearance activity. Acid phosphatase activity in their peritoneal cells was also augmented, suggesting that Vibrio cells activated macrophages in the mice.     

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.     

Immune response of rainbow trout (Oncorhynchus mykiss) to antigenic preparations fromVibrio anguillarumserogroup O1

The humoral and cellular immune responses of rainbow trout were investigated following injection with formalin-killedVibrio anguillarumin Freund's incomplete adjuvant (FIA) in terms of reactivity towards different antigen preparations of the bacterium. Vaccinated fish were compared with control fish that had been injected only with FIA. The antigen preparations used for the comparative studies were formalin-killed bacteria, extracellular products (ECP), outer membrane proteins (OMP) and cytoplasmic membrane proteins (CMP). Humoral antibody as measured by ELISA was detected with all antigen preparations. As evaluated by ELISPOT and by proliferation assays, leucocytes isolated from vaccinated fish reacted most strongly with the OMP preparation. This observation suggests the existence of undefined potent antigenic components among these proteins. In proliferation assays, the tested antigen preparations contained components that were mitogenic to cell cultures from unvaccinated fish. However, in terms of antibodies measured by ELISA and ELISPOT techniques, only vaccinated fish reacted with theV. anguillarumpreparations.     

Determining Vaccination Frequency in Farmed Rainbow Trout Using Vibrio anguillarum O1 Specific Serum Antibody Measurements

Background

Despite vaccination with a commercial vaccine with a documented protective effect against Vibrio anguillarum O1 disease outbreaks caused by this bacterium have been registered among rainbow trout at Danish fish farms. The present study examined specific serum antibody levels as a valid marker for assessing vaccination status in a fish population. For this purpose a highly sensitive enzyme-linked immunosorbent assay (ELISA) was developed and used to evaluate sera from farmed rainbow trout vaccinated against V. anguillarum O1.

Study Design

Immune sera from rainbow trout immunised with an experimental vaccine based on inactivated V. anguillarum O1 bacterin in Freund’s incomplete adjuvant were used for ELISA optimisation. Subsequently, sera from farmed rainbow trout vaccinated with a commercial vaccine against V. anguillarum were analysed with the ELISA. The measured serum antibody levels were compared with the vaccine status of the fish (vaccinated/unvaccinated) as evaluated through visual examination.

Results

Repeated immunisation with the experimental vaccine lead to increasing levels of specific serum antibodies in the vaccinated rainbow trout. The farmed rainbow trout responded with high antibody levels to a single injection with the commercial vaccine. However, the diversity in responses was more pronounced in the farmed fish. Primary visual examinations for vaccine status in rainbow trout from the commercial farm revealed a large pool of unvaccinated specimens (vaccination failure rate = 20%) among the otherwise vaccinated fish. Through serum analyses using the ELISA in a blinded set-up it was possible to separate samples collected from the farmed rainbow trout into vaccinated and unvaccinated fish.

Conclusions

Much attention has been devoted to development of new and more effective vaccines. Here we present a case from a Danish rainbow trout farm indicating that attention should also be directed to the vaccination procedure in order to secure high vaccination frequencies necessary for optimal protection with a reported effective vaccine.     

Humoral and peritoneal cell responses of rainbow trout (Oncorhynchus mykiss) to ovalbumin, Vibrio anguillarum and Freund's complete adjuvant following intraperitoneal and bath immunisation

ELISA methods were used to evaluate the humoral immune responses of rainbow trout (Oncorhynchus mykiss) to ovalbumin and Vibrio anguillarum. Antibody responses to ovalbumin administered intraperitoneally (i.p.) were inconsistent even when Freund's complete adjuvant (FCA) was used and the induction phase (4-6 weeks) of the response was longer compared with the response to V. anguillarum (<4 weeks). Significant elevation in antibody level was noted 3 weeks after bath vaccination with V. anguillarum but levels decreased thereafter. Humoral responses of greatest magnitude occurred where V. anguillarum was given in an emulsion with Freund's complete adjuvant (FCA). However, i.p. administration of FCA alone 3 weeks prior to i.p. immunisation with non-adjuvanted V. anguillarum resulted at 5 weeks in similar elevations in antibody to those in fish given V. anguillarum and FCA concurrently, suggesting that the effects of FCA were not limited to creation of an antigen depot. Proliferation of peritoneal inflammatory cell populations which included macrophages and plasma cells was detected histologically within 3 weeks of administration of FCA, but changes were not detected in the spleen or haematopoietic kidney.     

The importance of escaped farmed rainbow trout (Oncorhynchus mykiss) as a vector for the salmon louse (Lepeophtheirus salmonis) depends on the hydrological conditions in the fjord

The objectives of the study were to see if escaped rainbow trout (Oncorhynchus mykiss) spread rapidly or not from fish farms, and to test whether the hydrological conditions in a fjord influence their vertical distribution and importance as vector for the salmon lice (Lepeophtheirus salmonis). Fifty farmed rainbow trout were tagged with acoustic transmitters including depth sensors and released from two of 11 fish farms in the fjord system. In addition, unintentionally escaped rainbow trout were recaptured for analysis of salmon lice and stomach content. Dispersal out of the fjord system was limited. Most fish stayed in the vicinity of and moved between the fish farms but fed primarily on a variety of indigestible items. They moved in the warm relatively fresh surface layer from late spring until early autumn where the risk of being infested with salmon lice was low. They swam gradually deeper and became much more infested with salmon lice as the surface layers cooled and salinity and temperature gradients became less distinct over the course of the winter. The observed post-escapement behavior may challenge the control of the spread of diseases and parasites between neighboring farms and to wild fish, but also increases opportunities for recapture.     

Varying effects of anadromous sockeye salmon on the trophic ecology of two species of resident salmonids in southwest Alaska

1. Anadromous salmon transport marine‐derived nutrients and carbon to freshwater and riparian ecosystems upon their return to natal spawning systems. The ecological implications of these subsidies on the trophic ecology of resident fish remain poorly understood despite broad recognition of their potential importance. 2. We studied the within‐year changes in the ration size, composition and stable isotope signature of the diets of two resident salmonids (rainbow trout, Oncorhynchus mykiss; Arctic grayling, Thymallus arcticus) before and after the arrival of sockeye salmon (Oncorhynchus nerka) to their spawning grounds in the Bristol Bay region of southwest Alaska. 3. Ration size and energy intake increased by 480–620% for both species after salmon arrived. However, the cause of the increases differed between species such that rainbow trout switched to consuming salmon eggs, salmon flesh and blowflies that colonized salmon carcasses, whereas grayling primarily ate more benthic invertebrates that were presumably made available because of physical disturbances by spawning salmon. 4. We also observed an increase in the δ¹⁵N of rainbow trout diets post‐salmon, but not for grayling. This presumably led to the observed increase in the δ¹⁵N of rainbow trout with increasing body mass, but not for grayling. 5. Using a bioenergetics model, we predicted that salmon‐derived resources contributed a large majority of the energy necessary for growth in this resident fish community. Furthermore, the bioenergetics model also showed how seasonal changes in diet affected the stable isotope ratios of both species. These results expand upon a growing body of literature that highlights the different pathways whereby anadromous salmon influence coastal ecosystems, particularly resident fish.     

Yeast colonizing the intestine of rainbow trout (Salmo gairdneri) and turbot (Scophtalmus maximus)

Yeast were isolated from the intestine of farmed rainbow trout (Salmo gairdneri), turbot (Scophtalmus maximus), and free-living flat-fish (Pleuronectes platessa and P. flesus). The average number of viable yeasts recovered from farmed rainbow trout was 3.0 × 10³ and 0.5 × 10² cells per gram homogenized intestine for white and red-pigmented yeasts, respectively. The dominant species were Debaryomyces hansenii, Saccharomyces cerevisiae, Rhodotorula rubra, and R. glutinis. In 5 of 10 free-lving marine fish, > 100 viable yeast cells per gram intestinal mucus were recovered. Red-pigmented yeasts dominated and composed >90% of the isolates. Colonization experiments were performed by inoculating rainbow trout and turbot with fish-specific, isolated yeast strains and by examining the microbial intestinal colonization at intervals. Inoculation of experimental fish with pure cultures of R. glutinis and D. hansenii HF1 yielded colonization at a level several orders of magnitude higher than before the inoculation. Up to 3.8 × 10⁴, 3.1 × 10⁶, and 2.3 × 10⁹ viable yeast cells per gram intestine or feces were recovered in three separate colonization experiments. The high level of colonizing yeasts persisted for several weeks. The concentrations of yeasts in the tank water never exceeded 10³ viable cells per milliliter. No traces of fish sickness as a result of high yeast colonization were recorded during any of the colonization experiments. For periods of the experiments, the concentration of aerobic bacteria in the fish intestine was lower than the intestinal yeast concentration. Scanning electron microscopy studies demonstrated a close association of the yeasts with the intestinal mucosa. The mucosal colonization was further demonstrated by separating intestinal content, mucus, and tissue. All compartments were colonized by >10³ viable yeast cells per gram. No bacteria were detected on the micrographs, indicating that their affinity for the intestinal mucosa was less than that of the yeasts. Correspondence to: Thomas Andtid