Deoxyribonucleic Acid Relationships Among Marine Vibrios

The deoxyribonucleic acid (DNA) relationships of 80 strains identified either as Vibrio parahaemolyticus, V. alginolyticus, and V. anguillarum, or as allied marine vibrios were delineated by DNA-DNA competition experiments as well as by measuring the thermal stabilities of the DNA-DNA duplexes formed in direct binding studies. The tested strains included isolates from Japan, Europe, and the United States. The V. parahaemolyticus and V. alginolyticus groups showed an average of 67% homology to one another and 30% to strains of V. anguillarum. Significantly, a number of the isolates from the Pacific Northwest which had been previously identified as V. parahaemolyticus based on morphological, biochemical, and serological evidence were shown either to be strains of V. anguillarum or to belong to as yet unnamed groups. Most strains isolated from diseased salmon in the Pacific Northwest proved to be virtually identical with V. anguillarum type C by DNA homology experiments, thereby differentiating them from similar strains isolated from diseased herring and occasionally from salmon. The latter Pacific Northwest isolates fell into two distinct genotypic groups. A plot of the per cent homology by competition versus the difference in the thermal stabilities of heterologous and homologous duplexes (DeltaT(m,e)) between the same DNA species shows a linear decline in homology of 4.25% per degree of DeltaT(m,e). The use of this relationship for estimating the percentage of the mispaired bases distinguishing DNA preparations directly from competition experiments is discussed.     

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.     

A medium for presumptive identification of Vibrio anguillarum.

A medium (VAM) for differentiation of Vibrio anguillarum is described. The presence of bile salts, the high pH, and the high NaCl concentration select mainly for Vibrio species. The high salinity and the ampicillin select for a fraction of Vibrio species, and sorbitol fermentation differentiates among those vibrios still able to grow. One hundred ninety-seven of 227 strains of V. anguillarum were identified with this medium. Only 3 of 66 strains of Vibrio that were not V. anguillarum or V. anguillarum-like were recognized with this medium, and any of 7 non-Vibrio strains related to fish diseases or Escherichia coli grew on the medium. It is our contention that the medium described here constitutes an efficient instrument for presumptive detection of V. anguillarum in pathological and environmental samples.     

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.     

Monoclonal antibodies against Vibrio anguillarum O2 and Vibrio ordalii identify antigenic differences in lipopolysaccharide O-antigens

Abstract Monoclonal antibodies (mAbs) that recognize distinct species-specific antigenic epitopes in O-antigens from Vibrio anguillarum O2, O2a and certain O2b strains (mAb 7B4) and from Vibrio ordalii strains (mAbs A16 and 7D11) were generated. Western immunoblot analysis using these mAbs revealed that vibrio strains grown in the presence of fresh rainbow trout blood expressed lipopolysaccharide (LPS) with longer (high molecular mass) O-antigens and extracellular capsular layers when compared to strains grown without rainbow trout blood. We also generated mAbs that react with O-antigens from V. anguillarum serotype O1 (mAbs 7B8, 7B5 and 1C3) and serotype O3 (mAbs 13A1 and 14C5) strains. These mAbs provide rapid and accurate diagnostic reagents for serological differentiation of V. ordalii from serotype O2 strains of V. anguillarum , and for serotyping of these pathogenic vibrios.     

Conjugation of drug-resistance plasmids from vibrio anguillarum to Vibrio parahaemolyticus

Drug-resistant strains of Vibrio anguillarum, a fish pathogen, were isolated from diseased fish in culture ponds. In investigations of these strains, the transfer of resistance to chloramphenicol, tetracycline, and sulfanilamide from multiple resistant organisms to laboratory recipients was observed. The plasmid from V. anguillarum was stably maintained in both recipient strains of Vibrio parahaemolyticus and Escherichia coli. The plasmids isolated from Vibrio anguillarum belong to incompatibility group C. The molecular weight of these plasmids determined by electron microscopic observation was about 103 to 113 megadaltons.     

Characterization of Vibrio anguillarum and closely related species isolated from farmed fish in Norway.

A total of 264 bacterial strains tentatively or definitely classified as Vibrio anguillarum were examined. The strains were isolated from diseased or healthy Norwegian fish after routine autopsy. With the exception of five isolates from wild saithe (Pollachius virens), the strains originated from nine different species of farmed fish. The bacteria were subjected to morphological, physiological, and biochemical studies, numerical taxonomical analyses, serotyping by slide agglutination and enzyme-linked immunosorbent assay, DNA-plasmid profiling, and in vitro antimicrobial drug susceptibility testing. The results of the microbiological studies were correlated to anamnestic information. The bacterial strains were identified as V. anguillarum serovar O1 (n = 132), serovar O2 (n = 89), serovar O4 (n = 2), serovar O8 (n = 1), and not typeable (n = 1) as well as Vibrio splendidus biovar I (n = 36) and biovar II (n = 1), Vibrio tubiashii (n = 1), and Vibrio fischerii (n = 1). V. anguillarum serovar O1 or O2 was isolated in 176 out of 179 cases of clinical vibriosis in Atlantic salmon (Salmo salar). V. anguillarum serovar O1 was the only serovar isolated from salmonid fish species other than Atlantic salmon, while V. anguillarum serovar O2 was isolated from all marine fish suffering from vibriosis. A 48-Mda plasmid was isolated from all V. anguillarum serovar O1 isolates examined. Serovar O2 isolates did not harbor any plasmids. Resistance against commonly used antibiotic compounds was not demonstrated among V. anguillarum isolates. Neither V. splendidus biovar I nor other V. anguillarum-related species appeared to be of clinical importance among salmonid fish.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Characterization of toxigenic vibrios isolated from the freshwater environment of Hiroshima, Japan.

The occurrence and characterization of toxigenic vibrios in surface water and sediment samples of the fresh water environment of the Ohta River were studied. The membrane filter, pad preenrichment technique, followed by the placement of membranes onto thiosulfate citrate-bile salt-sucrose agar, was used for the enumeration of total vibrios. Qualitative examination of pathogenic vibrios was also attempted. In addition, a survey was conducted to determine the incidence of Clostridium botulinum in sediment samples of the Ohta River and the Hiroshima coast. In the identification of 361 strains, 12 species of Vibrio and two species of Listonella were observed. Non-01 Vibrio cholerae was prevalent among the members of the genus Vibrio. Vibrio parahaemolyticus (serotype 04:K34), isolated in fresh water, is significant and suggests that some still unknown conditions promote the survival of these organisms in fresh water. An estimated 132 strains were hemolytic by a simple agar method, and further characterization revealed that 82% of the hemolytic vibrios (107 strains) produced various toxins. About 71% (93 strains) elaborated cytotoxin, 55% (72 strains) produced hemolysin, and 44% (58 strains) responded for both cytotoxin and hemolysin in the crude toxin extracts. All the non-01 V. cholerae showed cytotoxic activity, and the virulent strains of Vibrio fluvialis and Vibrio spp. showed cytotonic responses in RK-13 cells. Of 36 sediment samples tested, 10 harbored C. botulinum spores (28%) and were isolated invariably in all the regions of the Hiroshima coast and in the Ohta River, except the upper region of the Ohta River.     

Characterization of toxigenic vibrios isolated from the freshwater environment of Hiroshima, Japan

The occurrence and characterization of toxigenic vibrios in surface water and sediment samples of the fresh water environment of the Ohta River were studied. The membrane filter, pad preenrichment technique, followed by the placement of membranes onto thiosulfate citrate-bile salt-sucrose agar, was used for the enumeration of total vibrios. Qualitative examination of pathogenic vibrios was also attempted. In addition, a survey was conducted to determine the incidence of Clostridium botulinum in sediment samples of the Ohta River and the Hiroshima coast. In the identification of 361 strains, 12 species of Vibrio and two species of Listonella were observed. Non-01 Vibrio cholerae was prevalent among the members of the genus Vibrio. Vibrio parahaemolyticus (serotype 04:K34), isolated in fresh water, is significant and suggests that some still unknown conditions promote the survival of these organisms in fresh water. An estimated 132 strains were hemolytic by a simple agar method, and further characterization revealed that 82% of the hemolytic vibrios (107 strains) produced various toxins. About 71% (93 strains) elaborated cytotoxin, 55% (72 strains) produced hemolysin, and 44% (58 strains) responded for both cytotoxin and hemolysin in the crude toxin extracts. All the non-01 V. cholerae showed cytotoxic activity, and the virulent strains of Vibrio fluvialis and Vibrio spp. showed cytotonic responses in RK-13 cells. Of 36 sediment samples tested, 10 harbored C. botulinum spores (28%) and were isolated invariably in all the regions of the Hiroshima coast and in the Ohta River, except the upper region of the Ohta River.     

Analysis of antigens present in the extracellular products and cell surface of Vibrio anguillarum serotypes O1, O2, and O3.

Antigens present in the extracellular products (ECP) and cell walls of strains of Vibrio anguillarum of serotypes O1, O2, and O3 isolated from different fish species in distinct geographic areas were characterized. The usefulness of slide agglutination, dot blot assay, and quantitative agglutination for subtyping V. anguillarum serovars was also evaluated. The three serological assays used to establish the serogroups within V. anguillarum isolates demonstrated that serotype O1 constitutes a homogeneous group, whereas within serotypes O2 and O3, two different patterns of serological reactions were detected. Among the three serological methods used, only dot blot and quantitative agglutination assays differentiated subgroups within serotypes O2 and O3 with unabsorbed sera. Electrophoretic analysis and immunoblot assays of cell envelope and ECP components showed that strains belonging to serotype O1 possessed immunologically related lipopolysaccharide (LPS) and proteins, while V. anguillarum isolates grouped in serotypes O2 and O3 exhibited internal heterogeneity in their LPS and protein banding patterns. On the other hand, although the LPS present in the ECP and those obtained from cell envelopes of V. anguillarum strains showed apparently different gel patterns, a strong relationship between both types of LPS was seen by immunoblot assay. From these results, it can be concluded that V. anguillarum strains representative of each of the antigenic groups (O1, O2 alpha, O2 beta, O3A, and O3B) and their ECPs should be included in the formulation of vaccines against vibriosis in areas where the three serotypes coexist.     

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.     

Significance of Na+ in the fish pathogen, Vibrio anguillarum, under energy depleted condition

Vibrio anguillarum kills various kinds of fish over salinities ranging from seawater to freshwater. In this study, we investigated the role of Na(+) in V. anguillarum, especially under energy-depleted conditions such as in natural seawater. V. angustum S14, which is a typical marine vibrio, was used for comparison. V. anguillarum only required Na(+) for starvation-survival, but in contrast, V. angustum S14 always required Na(+) for both growth and starvation-survival. In marine vibrios, Na(+) is used in the Na(+)-dependent respiratory chain that produces the sodium motive force (SMF) across the cell membrane. It has been considered that marine vibrios always need a SMF produced by Na(+), however in the case of V. anguillarum, the SMF is not required for growth, but becomes more important for starvation-survival.     

Plasmids mediating iron uptake in Vibrio anguillarum strains isolated from turbot in Spain

Vibrio strains isolated from diseased turbot in an experimental fish farm on the Atlantic coast of northwest Spain were identified as Vibrio anguillarum. The isolates shared many biochemical characteristics with V. anguillarum strains obtained from other sources, and harboured a plasmid species that showed extensive homology with plasmid pJM1, carried by V. anguillarum strain 775 isolated from an epizootic in North America. Restriction endonuclease analysis showed that the two plasmids were very similar albeit not identical. The presence of the plasmid in the turbot isolates was associated with their ability to cause disease in fish. Plasmid-carrying bacteria could also grow under conditions of iron limitation. Two outer membrane proteins, of 86 and 79 kDal, were induced, and a similar siderophore activity to that produced by V. anguillarum 775 was also detected under these conditions. The 86 kDal outer membrane protein cross-reacted immunologically with antiserum raised against the outer membrane protein OM2 produced by strain 775. Nonvirulent plasmidless derivatives were unable to grow under iron-limiting conditions, and were also unable to produce either siderophore activity or the 86 kDal outer membrane protein, suggesting the plasmid-mediated nature of these components.     

Antigenic and molecular characterization of Vibrio ordalii strains isolated from Atlantic salmon Salmo salar in Chile

Biochemical, serological and molecular properties of a group of 14 Vibrio ordalii strains isolated from cultured Atlantic salmon Salmo salar in Chile in recent years were studied. The characteristics of isolates were compared with the type strain V. ordalii ATCC 33509T. The Chilean V. ordalii represented a biochemically homogenous group; however, some minor differences with the type strain were observed. The serological relationships among isolates, as well as the study of their antigenic determinant (LPS) revealed a strong reaction with antisera raised against Atlantic salmon strains and the antiserum raised against Listonella anguillarum serotype O2. However, LPS electrophoretic patterns were completely different from the V. ordalii type strain, regardless of the serum employed, suggesting the possibility that the Chilean strains constitute a new serological subgroup within this bacterial species. Genetic analyses by PFGE, RAPD, REP-PCR and ERIC-PCR demonstrated that all V. ordalii strains were genetically homogenous, displaying similar DNA patterns, regardless of the techniques used. Moreover, the analysis of DNA banding patterns generated by ERIC-PCR and REP-PCR also clearly separated the type strain from the Chilean strains. This is the first report of characterization of V. ordalii strains from the Southeastern Pacific area, the results of which should facilitate the development of vaccines for protecting cultured Atlantic salmon against vibriosis in this area.     

Agglutination Typing of Vibrio anguillarum Isolates from Diseased Fish and from the Environment

Agglutinating activity was widely distributed among 101 Vibrio anguillarum strains of different origin and three Vibrio ordalii strains from salmonids. The spectrum of cells which were agglutinated comprised yeast cells and human (type O), poultry, guinea pig, and trout erythrocytes, whereas ovine, bovine, and tanned bovine erythrocytes were not affected. Mannose-sensitive hemagglutination, mannose-resistant hemagglutination, and non-agglutinating strains were recognized. The three V. ordalii strains showed mannose-resistant hemagglutination, whereas V. anguillarum exhibited either mannose-sensitive hemagglutination or was non-agglutinating. Among V. anguillarum, sensitivity to d-galactose and l-fucose occurred sporadically. An agglutination typing scheme was developed for strains of V. anguillarum based on the agglutination pattern of human, poultry, guinea pig, and trout erythrocytes and yeast cells. Eight different agglutination types (A through H) were defined. The distribution of these types among fish pathogenic and environmental V. anguillarum strains were studied. The application of the typing scheme in ecological and epidemiological studies and for preventive medical purposes is discussed.     

Vibrio anguillarum serogroup O3 and V. anguillarum-like serogroup O3 cross-reactive species--comparison and characterization

Forty-five Vibrio anguillarum-like isolates reacting with V. anguillarum serogroup O3 antiserum were examined in 30 characters to clarify their phenotypical properties, while their genotype was examined by ribotyping. The strains were isolated from diseased and dead fish or from environmental sources such as water, sediment, plankton, and faeces and gills of healthy fish. Phenotypically, the similarity of all the strains was more than 90%. However, significant differences between the fish-associated and environmental strains were detected. Biochemically, deviations were found in the Voges-Proskauer test and lysine decarboxylase reaction. Clustering analysis of the ribotypes showed two distinct clusters with a similarity of only 32%. Two strains representing each of these groups were used in a LD50 study, which showed some difference also in the pathogenicity between environmental and fish strains. It is suggested that the environmental strains belong to another species than V. anguillarum, but serologically cross-reacting with the V. anguillarum serogroup O3. The ribotyping as well as biochemical results indicated that the environmental strains possibly belong to Vibrio aestuarianus. The bona fide V. anguillarum serogroup O3 strains proved to be very homogeneous both phenotypically and genotypically, and the similarity of ribotypes was more than 96%. The V. anguillarum-like, serogroup O3-reactive strains from the environment were more heterogeneous in their biochemical behaviour, and showed an approximately 70% similarity in ribotypes.     

Intestinal colonization potential of turbot (Scophthalmus maximus)- and dab (Limanda limanda)-associated bacteria with inhibitory effects against Vibrio anguillarum.

Of more than 400 bacteria isolated from turbot (Scophthalmus maximus), 89 have previously been shown to inhibit the in vitro growth of the fish pathogen Vibrio anguillarum. The aim of the present study was to investigate the potential of seven of these strains, as well as of intestinal isolates (four strains) from a closely related fish, dab (Limanda limanda), for colonizing farmed turbot as a means of protecting the host from infection by V. anguillarum. In addition, the inhibitory effect of these strains on the pathogen was further studied. Colonization potential was measured by the capacity of the strains to adhere to and grow in turbot intestinal mucus. These parameters were also used to investigate the potential of V. anguillarum to amplify in the turbot intestinal tract. Because of the observed rapid growth of V. anguillarum in intestinal mucus, it can be proposed that the intestinal tract is a site for V. anguillarum multiplication. Strains isolated from the intestine showed greater capacity for adhesion to and growth in fish intestinal mucus than did the pathogen and the skin mucus isolates. All of the isolates released metabolites into the culture medium that had inhibitory effects against V. anguillarum. The results are discussed with emphasis on administering bacteria of host origin to farmed turbot in order to control V. anguillarum-induced disease.     

Distribution of plasmid- and chromosome-mediated iron uptake systems in Vibrio anguillarum strains of different origins

We investigated the incidence of plasmid-mediated and chromosome-mediated iron uptake systems in strains of Vibrio anguillarum that belong to serotypes O1 and O2 and were isolated from different fish species and in different geographic areas. All of the strains gave positive reactions in CAS agar medium and in the Arnow test, which indicated that catechol types of siderophores were produced. The majority of V. anguillarum serotype O1 strains harbored a 65-kb plasmid similar to plasmid pJM1 from strain 775, which encodes the siderophore anguibactin and its outer membrane receptor, protein OM2. All of the isolates harboring this plasmid promoted the growth of an anguibactin-deficient receptor-proficient mutant derived from strain 775, but none of these isolates promoted the growth of mutants lacking receptor OM2. Furthermore, under iron-limiting conditions all of these strains induced outer membrane proteins that were identical in size to protein OM2 of strain 775. In contrast, none of the serotype O2 strains contained a high-molecular-weight plasmid, but all of them induced the growth of mutants defective in the anguibactin-mediated system regardless of the presence or absence of receptor OM2. The serotype O2 strains, but not the plasmid-bearing serotype O1 strains, also induced the growth of Salmonella typhimurium enb-1 which utilizes only enterobactin as a siderophore.(ABSTRACT TRUNCATED AT 250 WORDS)