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.     

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.     

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.     

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.     

Real-time PCR detection and quantification of fish probiotic Phaeobacter strain 27-4 and fish pathogenic Vibrio in microalgae, rotifer, Artemia and first feeding turbot (Psetta maxima) larvae

Aims:  To develop a SYBR Green quantitative real-time PCR protocol enabling detection and quantification of a fish probiotic and two turbot pathogenic Vibrio spp. in microcosms.
Methods and Results:  Phaeobacter 27-4, Vibrio anguillarum 90-11-287 and Vibrio splendidus DMC-1 were quantified as pure and mixed cultures and in presence of microalgae ( Isochrysis galbana ), rotifers ( Brachionus plicatilis ), Artemia nauplii or turbot ( Psetta maxima ) larvae by real-time PCR based on primers directed at genetic loci coding for antagonistic and virulence-related functions respectively. The optimized protocol was used to study bioencapsulation and maintenance of the probiont and pathogens in rotifers and for the detection and quantification of Phaeobacter and V. anguillarum in turbot larvae fed rotifers loaded with the different bacteria in a challenge trial.
Conclusions:  Our real-time PCR protocol is reproducible and specific. The method requires separate standard curve for each host organism and can be used to detect and quantify probiotic Phaeobacter and pathogenic Vibrio bioencapsulated in rotifers and in turbot larvae.
Significance and Impact of the Study:  Our method allows monitoring and quantification of a turbot larvae probiotic bacteria and turbot pathogenic vibrios in in vivo trials and will be useful tools for detecting the bacteria in industrial rearing units.     

The probiotic potential against vibriosis of the indigenous microflora of rainbow trout

The antibacterial properties of the indigenous microflora of rainbow trout ( Oncorhynchus mykiss Walbaum) and the potential use of inhibitory bacteria as fish probiotics were investigated. A total of 1018 bacteria and yeasts were isolated on tryptone soy agar (TSA) from skin, gills and intestine. Forty-five of these inhibited growth of the fish pathogenic bacterium Vibrio anguillarum in a well diffusion assay. The antagonism was most prominent among Pseudomonas spp., as 28 (66%) of the antagonistic bacteria belonged to this genus, despite constituting only 15% of the total tested flora. As pseudomonads are typically siderophore producers, chrome azurol S (CAS) agar was used as a semi-selective medium for isolation of antagonistic bacteria. On this medium, 75% of the iron-chelating strains were inhibitory to V. anguillarum . Eight strains out of a subset of 11 antagonists caused a 3–6 log unit reduction in the density of V. anguillarum [measured by polymerase chain reaction (PCR) detection in a most probable number (MPN) regimen] in a broth co-culture assay. Survival of rainbow trout infected with vibriosis was improved 13–43% by six out of nine antagonistic strains tested in vivo. All disease-protecting strains were pseudomonads, isolated from CAS plates, whereas two Carnobacterium spp. that were antagonistic in in vitro well diffusion assays did not alter the accumulated mortality of rainbow trout. The addition of live bacterial cultures to fish-rearing water may thus improve survival of the fish; however, in vitro antagonism could not completely predict an in vivo effect. Further studies on the underlying mechanism of activity are required to design appropriate selection criteria for fish probiotic bacteria.     

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.     

Determination of Vibrio scophthalmi and its phenotypic diversity in turbot larvae

The association of Vibrio scophthalmi with turbot larvae was assessed, by molecular methods with a species-specific probe, in the rearing stages of turbot (Scophthalmus maximus) larvae using a routine batch of production at a fish farm. The phenotypic diversity of this bacterial species was also studied to identify predominant phenotypes at successive stages of larval development. Vibrio scophthalmi was detected in all turbot larvae samples except in the sample from day 0 after hatching. The percentage of V. scophthalmi in the intestinal microbiota increased throughout larval development. Vibrio scophthalmi was also detected in live food (brine shrimps) and water from the tanks, but not in the sediment. All turbot larvae, 15-57 day old, showed several V. scophthalmi phenotypes, and a pattern of successive waves of phenotypes was observed during successive larval stages. This indicates that certain strains may colonize the intestine more efficiently and thus maintain their population for longer than other strains. Vibrio scophthalmi populations from turbots of different origin were very similar, suggesting that irrespective of geographical area, turbot populations share similar V. scophthalmi strains. Vibrio scophthalmi strain was not isolated from other cultured fish, only turbot larvae, at the same hatchery receiving water from the same supply.     

Vibrio splendidus biotype 1 as a cause of mortalities in hatchery-reared larval turbot, Scophthalmus maximus (L.)

AIMS: To characterize bacteria associated with turbot larvae feeding on Artemia and identify pathogens causing mortalities in larvae. METHODS AND RESULTS: To identify bacteria associated with mortalities in larval turbot rearing, bacteria were isolated from homogenates of Artemia or from several batches of well-performing or poorly performing turbot larvae. Samples were plated onto marine agar and were characterized using biochemical tests and BIOLOG GN plates. Total culturable aerobic bacteria ranged from 1.9 x 10(5) to 1.8 x 10(6) CFU per larva and >96% of bacteria identified were vibrios. Almost all bacteria were haemolytic and clustered into two phenons represented by Vibrio alginolyticus and Vibrio splendidus. The bacterial flora of Artemia was almost entirely V. alginolyticus, whereas V. splendidus biotype 1 dominated the larval turbot gut flora (69/115 isolates in seven experiments) and formed four different groups based on BIOLOG GN reactions. Of 16 isolates tested for virulence towards turbot larvae, four of the 11 V. splendidus biotype 1 isolates were lethal and all belonged to the same group of V. splendidus biotype 1 isolates. CONCLUSIONS: In a commercial turbot hatchery, the microbial flora of the larval gut was dominated by V. splendidus biotype 1. Four of the 11 V. splendidus biotype 1 isolates caused mortalities in larval turbot and all belonged to one group of the biotype 1 strains identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of four isolates of V. splendidus that are pathogenic for turbot larvae from three separate batches of larval turbot will allow these to be compared with avirulent isolates to define how V. splendidus causes mortalities in larval turbot.     

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)     

Stimulation or inhibition of growth of the unicellular alga Pavlova lutheri by bacteria isolated from larval turbot culture systems

During growth of larval turbot in aquaculture the first food supplied is usually the rotifer, Brachionus plicatilis and algae are commonly included in the system as food for the rotifers, thereby maintaining their nutrient quality. As bacteria are known to influence markedly the survival of larval turbot, the effect of bacteria, isolated from larval turbot, on growth of Pavlova lutheri was measured over a 3-d period. Of 41 bacteria tested, 23 inhibited growth to various degrees, eight had no effect and 10 were weak growth stimulants. Four bacteria, identified as a Flavobacterium, Vibrio fluvialis, Vibrio natrigens and a Vibrio sp., were strongly inhibitory and the Flavobacterium inhibited growth of Pavlova lutheri from an inoculum of 10³ colony-forming units per ml. Inhibition was due to a heat-labile factor released by the Flavobacterium into the culture medium. The Flavobacterium also produced bacteriocin(s) which inhibited the growth of a range of vibrios. Bacteria antagonistic towards algae would be undesirable in larval rearing and if bacteria are to be selected which are beneficial (probiotics) in larval rearing systems their possible interaction with algae must be considered.     

Numerical taxonomy of gram-negative, facultative anaerobic bacteria isolated from skin of turbot (Scophthalmus maximus) and surrounding water

A numerical taxonomic study of 473 gram negative heterotrophic facultative anaerobic bacteria isolated from skin of turbot (Scophthalmus maximus) and its culture water was performed. The study included 53 type and reference strains belonging to the genera Vibrio, Aeromonas and Listonella. The strains were characterized using 90 tests and data were examined by Simple Matching coefficient (S(SM)) and Jaccard coefficient (S(J)). UPGMA (unweighted pair group method, arithmetic average) defined 66 phena at S(SM) values > or = 84% and 27 groups at S(SM) > or = 80%. Six phena were defined as Vibrio albensis, V. (Listonella) anguillarum, V. splendidus biotype I, V. fischeri, V. ordalii and V. scophthalmi including reference strains. Some groups clustered different phena for one species, although others as the V. anguillarum related strains and inactive Vibro group required S(SM) > or = 84% to define species. More studies are necessary to identify the Vibrio spp. strains and to confirm some species identifications.     

Ecology, inhibitory activity, and morphogenesis of a marine antagonistic bacterium belonging to the Roseobacter clade

Roseobacter strain 27-4 has been isolated from a turbot larval rearing unit and is capable of reducing mortality in turbot egg yolk sac larvae. Here, we demonstrate that the supernatant of Roseobacter 27-4 is lethal to the larval pathogens Vibrio anguillarum and Vibrio splendidus in a buffer system and inhibited their growth in marine broth. Liquid chromatography (LC) with both UV spectral detection and high-resolution mass spectrometry (HR-MS) identified the known antibacterial compound thiotropocin or its closely related precursor tropodithietic acid in the bioactive fractions. Antibacterial activity correlated with the appearance of a brownish pigment and was only formed in marine broth under static growth conditions. A thick biofilm of multicellular star-shaped aggregated cells formed at the air-liquid interface under static growth conditions. Here, the bioactive compound was the base peak in the LC-UV chromatograms of the extracts where it constituted 15% of the total peak area. Aerated conditions results in 10-fold-higher cell yield, however, cultures were nonpigmented, did not produce antibacterial activity, and grew as single cells. Production of antibacterial compounds may be quorum regulated, and we identified the acylated homoserine lactone (3-hydroxy-decanoyl homoserine lactone) from cultures of Roseobacter 27-4 using LC-HR-MS. The signal molecule was primarily detected in stagnant cultures. Roseobacter 27-4 grew between 10 and 30 degrees C but died rapidly at 37 degrees C. Also, the antibacterial compounds was sensitive to heat and was inactivated at 37 degrees C in less than 2 days and at 25 degrees C in 8 days. Using Roseobacter 27-4 as a probiotic culture will require that is be established in stagnant or adhered conditions and, due to the temperature sensitivity of the active compound, constant production must be ensured.     

Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot (Scophthalmus maximus L.) larvae

Polyclonal antisera made in rabbits against whole washed cells of Vibrio pelagius and Aeromonas caviae were used for detection of these bacterial species in the rearing water and gastrointestinal tract of healthy turbot ( Scophthalmus maximus ) larvae exposed to V. pelagius and/or Aer. caviae . The results demonstrated that this method is suitable for detection of V. pelagius and Aer. caviae in water samples and larvae at population levels higher than 10³ ml⁻¹ and 10³ larva⁻¹. Populations of aerobic heterotrophic bacteria present in the gastrointestinal tract of turbot larvae, estimated using the dilution plate technique, increased from approximately 4 × 10² bacteria larva⁻¹ on day 3 post-hatching to approximately 10⁵ bacteria fish⁻¹ 16 days post-hatching. Sixteen days after hatching, Vibrio spp. accounted for approximately 3 × 10⁴ cfu larva⁻¹ exposed to V. pelagius on days 2, 5 and 8 post-hatching. However, only 10³ of the Vibrio spp. belonged to V. pelagius . When larvae were exposed to Aer. caviae on day 2 post-hatching, the gut microbiota of 5-day old larvae was mainly colonized by Aeromonas spp. (10⁴ larva⁻¹), of which 9 × 10³ belonged to Aer. caviae . Later in the experiment, at the time when high mortality occurred, 9 × 10⁵ Aer. caviae were detected. Introduction of V. pelagius to the rearing water seemed to improve larval survival compared with fish exposed to Aer. caviae and with the control group. It was therefore concluded that it is beneficial with regard to larval survival to introduce bacteria ( V. pelagius ) to the rearing water.     

Bacterial influences on Atlantic halibut Hippoglossus hippoglossus yolk-sac larval survival and start-feed response

A bacteria-free halibut larval rearing system was used to test 20 bacterial isolates, from British halibut hatcheries, for their toxicity towards halibut yolk-sac larvae under microbially controlled conditions. The isolates tested spanned a range of genera and species (Pseudoalteromonas, Halomonas marina, Vibrio salmonicida-like, Photobacterium phosphoreum and V. splendidus species). A pathogen of turbot, Scophthalmus maximus, V. anguillarum 91079, and 2 isolates from adult halibut were also included. Isolates were inoculated, at a concentration of 5 x 10(2) cfu ml(-1), into flasks containing 25 recently hatched axenic halibut larvae, using a minimum of 3 flasks for each treatment. Control survivals to 38 d post-hatch for the 3 experiments averaged 84, 51.5 and 49%, respectively. With the exception of V. anguillarum 91079, which was highly pathogenic towards halibut yolk-sac larvae, there was no statistically significant difference in survival between the controls and the different treatments. This suggests that most of the bacteria routinely isolated from halibut hatcheries are not harmful to yolk-sac larvae, even though most flasks contained in excess of 5 x 10(6) cfu m(-1) of the inoculated organism when the experiments were terminated. Three organisms previously shown to inhibit growth of bacteria in vitro were tested for their ability to protect halibut yolk-sac larvae against invasion by V. anguillarum. In 4 separate challenge experiments none of the test isolates, a Pseudoalteromonas strain and 2 Carnobacterium-like organisms, showed any protective effect. To investigate how particular bacteria influence their start-feed response, larvae were fed axenic and gnotobiotic Artemia colonized with a range of different Vibrio spp., and examined after 8 d. There were no statistically significant between-treatment differences in the proportion of Artemia-containing larvae, indicating that bacterial contamination of the live food does not appear to influence initiation of the feeding response.     

Rapid detection and identification of Vibrio anguillarum by using a specific oligonucleotide probe complementary to 16S rRNA.

Partial 16S rDNA from Vibrio collection type strains and recent isolates of Vibrio-related strains were sequenced and compared with previously published sequences. A 24-base DNA oligonucleotide (VaV3) was designed and used as a specific probe for detection and identification of Vibrio anguillarum. Its specificity was tested against collection type strains and environmental isolates and no cross-reaction was found. The probe detected 8 of the 10 V. anguillarum serovars. It was applied to screen different Vibrio-related strains isolated from marine hatcheries and fish farms. The detection limit in DNA-DNA slot blot hybridization was 150 pg.     

Phaeobacter and Ruegeria Species of the Roseobacter Clade Colonize Separate Niches in a Danish Turbot (Scophthalmus maximus)-Rearing Farm and Antagonize Vibrio anguillarum under Different Growth Conditions

Members of the Roseobacter clade colonize a Spanish turbot larval unit, and one isolate (Phaeobacter strain 27-4) is capable of disease suppression in in vivo challenge trials. Here, we demonstrate that roseobacters with antagonistic activity against Vibrio anguillarum also colonize a Danish turbot larval farm that relies on a very different water source (the Danish fiord Limfjorden as opposed to the Galician Atlantic Ocean). Phylogenetic analyses based on 16S rRNA and gyrase B gene sequences revealed that different species colonized different niches in the larval unit. Phaeobacter inhibens- and Phaeobacter gallaeciensis-like strains were primarily found in the production sites, whereas strains identified as Ruegeria mobilis or Ruegeria pelagia were found only in the algal cultures. Phaeobacter spp. were more inhibitory against the general microbiota from the Danish turbot larval unit than were the Ruegeria spp. Phaeobacter spp. produced tropodithietic acid (TDA) and brown pigment and antagonized V. anguillarum when grown under shaking (200 rpm) and stagnant (0 rpm) conditions, whereas Ruegeria spp. behaved similarly to Phaeobacter strain 27-4 and expressed these three phenotypes only during stagnant growth. Both genera attached to an inert surface and grew in multicellular rosettes after stagnant growth, whereas shaking conditions led to single cells with low attachment capacity. Bacteria from the Roseobacter clade appear to be universal colonizers of marine larval rearing units, and since the Danish Phaeobacter spp. displayed antibacterial activity under a broader range of growth conditions than did Phaeobacter strain 27-4, these organisms may hold greater promise as fish probiotic organisms.     

A selective and differential medium for Vibrio harveyi.

A new medium, termed Vibrio harveyi agar, has been developed for the isolation and enumeration of V. harveyi. It is possible to differentiate V. harveyi colonies from the colonies of strains representing 15 other Vibrio species with this medium. This medium has been shown to inhibit the growth of two strains of marine Pseudomonas spp. and two strains of marine Flavobacterium spp. but to allow the growth of Photobacterium strains. Colonies displaying typical V. harveyi morphology were isolated from the larval rearing water of a commercial prawn hatchery with V. harveyi agar as a primary isolation medium and were positively identified, by conventional tests, as V. harveyi. This agar displays great potential as a primary isolation medium and offers significant advantages over thiosulfate-citrate-bile salts-sucrose agar as a medium for differentiating V. harveyi from other marine and estuarine Vibrio species.     

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 and Relatedness of Marine Vibrios Pathogenic to Fish: Physiology, Serology, and Epidemiology

Cultural characteristics and serological relationships of pathogenic marine vibrios isolated from fish in the Pacific Northwest were studied. These organisms were compared with cultures of Vibrio anguillarum, a known fish pathogen. On the basis of morphological and cultural characteristics, the Pacific Northwest strains of Vibrio were found to be closely related to V. anguillarum. Serological analyses of thermostable antigens served to distinguish three serotypes among the vibrios. Serotype 1 was composed of organisms isolated from Northwest salmonids; serotype 2 of strains of V. anguillarum from European waters; and serotype 3 of organisms isolated from Pacific herring. The epidemiology of vibrio disease among populations of fish in the Pacific Northwest is discussed.