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.     

Isolation and characterization of turbot (Scophtalmus maximus)-associated bacteria with inhibitory effects against Vibrio anguillarum.

More than 400 isolates from the intestine and the external surface of farmed Scophtalmus maximus as well as from fish food and hatchery water were screened for inhibitory effects against the fish pathogen Vibrio anguillarum HI 11345 and seven other fish pathogens. The bacteria with inhibitory effects were then characterized with regard to their sites of colonization, especially the intestinal regions and sites within each region. Of the total number of bacterial isolates from the intestine, 28% were inhibitory against V. anguillarum HI 11345. A marine biochemical assay was used to order the inhibitory strains into different phena. Most inhibitory bacteria were found in the rinse and mucus fractions of the gastrointestinal tract. No correlations among the different phena, site of colonization, and inhibitory effect could be found; however, a biochemical diversity was noted in the strains with an inhibitory effect. Of the isolates with an inhibitory effect against V. anguillarum HI 11345, 60% had an inhibitory effect on five other fish-pathogenic serotypes of V. anguillarum. Inhibitory effects of the isolates were also shown against Aeromonas salmonicida and Aeromonas hydrophila.     

Isolation and characterization of turbot (Scophtalmus maximus)-associated bacteria with inhibitory effects against Vibrio anguillarum.

More than 400 isolates from the intestine and the external surface of farmed Scophtalmus maximus as well as from fish food and hatchery water were screened for inhibitory effects against the fish pathogen Vibrio anguillarum HI 11345 and seven other fish pathogens. The bacteria with inhibitory effects were then characterized with regard to their sites of colonization, especially the intestinal regions and sites within each region. Of the total number of bacterial isolates from the intestine, 28% were inhibitory against V. anguillarum HI 11345. A marine biochemical assay was used to order the inhibitory strains into different phena. Most inhibitory bacteria were found in the rinse and mucus fractions of the gastrointestinal tract. No correlations among the different phena, site of colonization, and inhibitory effect could be found; however, a biochemical diversity was noted in the strains with an inhibitory effect. Of the isolates with an inhibitory effect against V. anguillarum HI 11345, 60% had an inhibitory effect on five other fish-pathogenic serotypes of V. anguillarum. Inhibitory effects of the isolates were also shown against Aeromonas salmonicida and Aeromonas hydrophila.     

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.     

不同大菱鲆(Scophthalmus maximus)个体肠道菌群结构差异研究

目的:肠道微生物能够帮助宿主完成多种生理生化功能,对宿主的健康生长也起到非常重要的作用。大菱鲆(Scophthalmus maximus)是我国北方最重要的海水养殖品种之一。然而,细菌性疾病的频发造成大规模的鱼类死亡。此外,许多大菱鲆致病菌也被证实是人类潜在的致病菌。因此鉴定成年养殖大菱鲆肠道中所包含的核心菌群,并筛选可能与大菱鲆或人类健康密切相关的细菌尤为重要。方法:构建三个大菱鲆个体肠道16S rRNA文库,利用限制性片段长度多态性分析(RFLP)及生物信息学技术研究三个大菱鲆个体肠道菌群结构及多样性。结果:共得到758个阳性克隆,16个OTU类型。分类分析显示,变形菌门在大菱鲆肠道中占优势地位。进一步将细菌按属来分类显示,弧菌属所占的比例最高,约为95.3%。韦恩图显示大菱鲆三个个体共有的OTU类型数量为3,分别占三个大菱鲆个体肠道文库中阳性克隆总数的94.9%、96.7%以及93.5%。分类结果显示它们分别属于条件致病菌哈氏弧菌、副溶血弧菌和创伤弧菌。结论:大菱鲆的核心肠道菌群为弧菌属,它的主要成员哈氏弧菌、副溶血弧菌和创伤弧菌对大菱鲆及人类的健康起到关键的作用。在水产养殖过程中,通过监控这些弧菌种类的浓度变化能够及时对养殖环境进行评价,并及时调整温度、水质等因素,这为预防大菱鲆细菌性疾病,保证人类健康提供重要的理论依据。     

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.     

The Chemotactic Response of Vibrio anguillarum to Fish Intestinal Mucus Is Mediated by a Combination of Multiple Mucus Components

Chemotactic motility has previously been shown to be essential for the virulence of Vibrio anguillarum in waterborne infections of fish. To investigate the mechanisms by which chemotaxis may function during infection, mucus was isolated from the intestinal and skin epithelial surfaces of rainbow trout. Chemotaxis assays revealed that V. anguillarum swims towards both types of mucus, with a higher chemotactic response being observed for intestinal mucus. Work was performed to examine the basis, in terms of mucus composition, of this chemotactic response. Intestinal mucus was analyzed by using chromatographic and mass spectrometric techniques, and the compounds identified were tested in a chemotaxis assay to determine the attractants present. A number of mucus-associated components, in particular, amino acids and carbohydrates, acted as chemoattractants for V. anguillarum. Importantly, only upon combination of these attractants into a single mixture were levels of chemotactic activity similar to those of intestinal mucus generated. A comparative analysis of skin mucus revealed its free amino acid and carbohydrate content to be considerably lower than that of the more chemotactically active intestinal mucus. To study whether host specificity exists in relation to vibrio chemotaxis towards mucus, comparisons with a human Vibrio pathogen were made. A cheR mutant of a Vibrio cholerae El Tor strain was constructed, and it was found that V. cholerae and V. anguillarum exhibit a chemotactic response to mucus from several animal sources in addition to that from the human jejunum and fish epithelium, respectively.     

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.     

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)     

Characterization of the properties of human- and dairy-derived probiotics for prevention of infectious diseases in fish

The present study aimed to investigate the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei Shirota, Lactobacillus bulgaricus, L. rhamnosus LC 705, Bifidobacterium lactis Bb12, and Lactobacillus johnsonii La1, and one for animal use, Enterococcus faecium Tehobak, for use as a fish probiotic. The strains for human use were specifically chosen since they are known to be safe for human use, which is of major importance because the fish are meant for human consumption. The selection was carried out by five different methods: mucosal adhesion, mucosal penetration, inhibition of pathogen growth and adhesion, and resistance to fish bile. The adhesion abilities of the seven LAB and three fish pathogens, Vibrio anguillarum, Aeromonas salmonicida, and Flavobacterium psychrophilum, were determined to mucus from five different sites on the surface or in the gut of rainbow trout. Five of the tested LAB strains showed considerable adhesion to different fish mucus types (14 to 26% of the added bacteria). Despite their adhesive character, the LAB strains were not able to inhibit the mucus binding of A. salmonicida. Coculture experiments showed significant inhibition of growth of A. salmonicida, which was mediated by competition for nutrients rather than secretion of inhibitory substances by the probiotic bacteria as measured in spent culture liquid. All LAB except L. casei Shirota showed tolerance against fish bile. L. rhamnosus ATCC 53103 and L. bulgaricus were found to penetrate fish mucus better than other probiotic bacteria. Based on bile resistance, mucus adhesion, mucus penetration, and suppression of fish pathogen growth, L. rhamnosus ATCC 53103 and L. bulgaricus can be considered for future in vivo challenge studies in fish as a novel and safe treatment in aquaculture.     

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     

Host range susceptibility of Enterococcus sp. strains isolated from diseased turbot: possible routes of infection.

Experiments were conducted to assess the pathogenicity of Enterococcus sp. strains isolated from diseased turbot for several fish species (turbot, salmon, trout, and seabream), as well as for mice. The intraperitoneal injection assays indicated that the tested strains showed host specificity for turbot, with a high degree of virulence (50% lethal dose of 10(4) cells per g of fish). The Spanish Enterococcus sp. isolates were nonpathogenic for the other fish species studied and for mice. The possible routes of infection were determined by bath exposure (with and without prior abrasion of the skin) and by intragastric inoculations with food and feces contaminated with the pathogen. The bath challenges indicated that the Enterococcus isolates were able to overcome the defense mechanisms present on the surface of the turbot only if the skin was abraded prior to the exposure. The antibacterial activities of components of a glycoprotein nature present in the turbot skin mucus are probably responsible in part for the resistance in noninjured fish to infection. On the other hand, we demonstrated the capacity of this pathogen to overcome adverse conditions in the stomachs of fish when associated with food or fecal material, since it is able to establish an infective state and to produce mortalities after 16 to 20 days postingestion. From all of these findings, we can conclude that horizontal transmissions through water and the fecal-oral route are the main avenues of infection of turbot streptococcosis.     

Lactic acid bacteria associated with the digestive tract of Atlantic salmon (Salmo salar L.)

The present study reports the effect of excessive handling stress and starvation on the lactic acid bacteria associated with the digestive tract of Atlantic salmon (Salmo salar L.). A relatively low population level (approximately 2 x 103 bacteria per gram wet tissue) of viable adherent heterotrophic bacteria was associated with the digestive tract (foregut, midgut and hindgut). Of the 752 bacterial isolates isolated from diet, water and the digestive tract, 201 isolates belonged to the carnobacteria. Of these isolates, one from the diet, one from the rearing water and 80 from the gastrointestinal tract, were further identified on the basis of 16S rDNA sequence analysis. All these isolates were identified as being Carnobacterium piscicola-like. Daily repeated stress and starvation of the fish over 11 d had no influence on the total culturable bacterial numbers or population level of C. piscicola associated with the digestive tract. C. piscicola-like isolates colonizing the various intestinal regions (foregut, midgut and hindgut) were also screened for their ability to produce growth inhibitory compounds active against the fish pathogen Aeromonas salmonicida. Of the 199 C. piscicola isolates tested, 139 inhibited growth of the pathogen.     

Evidence for the existence of distinct populations of Vibrio anguillarum serogroup O1 based on plasmid contents and ribotypes.

A total of 103 Vibrio anguillarum serogroup O1 strains displaying 15 different plasmid profiles were characterized with respect to biochemical properties and ribotypes. The results confirmed that V. anguillarum O1 is a biochemically homogeneous group. The 103 strains could be allocated to three main clusters with high similarity coefficients. None of the biochemical properties were connected with the presence of plasmids. In total, 12 different ribotypes were demonstrated, with HindIII being used as the restriction enzyme. Forty of the strains were isolated from the same Danish fish farm, some from the kidneys of diseased fish and some from the environment, and some strains were isolated from the mucus, gills, and feces of healthy fish. Nineteen of these isolates possessed the 67-kb virulence plasmid alone or in combination with other plasmids, while 21 had no plasmids. All strains isolated from the kidneys of diseased fish on this farm had plasmids. Irrespective of their origin (kidneys, gills, or mucus), all 19 strains carrying the 67-kb virulence plasmid had the same ribotype, profile 1, while isolates without plasmids belonged to five different profiles, all different from profile 1. These results suggest that pathogenic V. anguillarum O1 strains possessing a virulence plasmid and nonpathogenic strains without plasmids from a small geographical area and even from the same fish may constitute two essentially distinct populations. Thus, it may be suggested that an exchange of virulence plasmids among strains is unlikely to occur in vivo.     

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.     

Characterization of the Properties of Human- and Dairy-Derived Probiotics for Prevention of Infectious Diseases in Fish

The present study aimed to investigate the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei Shirota, Lactobacillus bulgaricus, L. rhamnosus LC 705, Bifidobacterium lactis Bb12, and Lactobacillus johnsonii La1, and one for animal use, Enterococcus faecium Tehobak, for use as a fish probiotic. The strains for human use were specifically chosen since they are known to be safe for human use, which is of major importance because the fish are meant for human consumption. The selection was carried out by five different methods: mucosal adhesion, mucosal penetration, inhibition of pathogen growth and adhesion, and resistance to fish bile. The adhesion abilities of the seven LAB and three fish pathogens, Vibrio anguillarum, Aeromonas salmonicida, and Flavobacterium psychrophilum, were determined to mucus from five different sites on the surface or in the gut of rainbow trout. Five of the tested LAB strains showed considerable adhesion to different fish mucus types (14 to 26% of the added bacteria). Despite their adhesive character, the LAB strains were not able to inhibit the mucus binding of A. salmonicida. Coculture experiments showed significant inhibition of growth of A. salmonicida, which was mediated by competition for nutrients rather than secretion of inhibitory substances by the probiotic bacteria as measured in spent culture liquid. All LAB except L. casei Shirota showed tolerance against fish bile. L. rhamnosus ATCC 53103 and L. bulgaricus were found to penetrate fish mucus better than other probiotic bacteria. Based on bile resistance, mucus adhesion, mucus penetration, and suppression of fish pathogen growth, L. rhamnosus ATCC 53103 and L. bulgaricus can be considered for future in vivo challenge studies in fish as a novel and safe treatment in aquaculture.     

Occurrence of plasmids in Danish isolates of Vibrio anguillarum serovars O1 and O2 and association of plasmids with phenotypic characteristics.

Two hundred and twenty-eight isolates of Vibrio anguillarum serovar O1 (125 isolates) and serovar O2 (103 isolates) have been characterized with regard to plasmid contents, biochemical properties, and in vitro hemagglutination and hydrophobic properties. Among 74 V. anguillarum isolates from diseased fish, 63 carried only a 67-kb plasmid (pJM1), 9 carried an additional 98-kb plasmid, and 1 isolate carried only the 98-kb plasmid. Only one isolate was without plasmids. In V. anguillarum serovar O1 from nondiseased fish (mucus and gills), plasmids of the same sizes were present in 29 isolates (58%), whereas 21 isolates (42%) were plasmid free. Based on hemagglutination and biochemical properties, V. anguillarum serovar O1 isolates were divided into eight biovars. The plasmid-carrying strains (102 isolates) all fell within biovars 1 and 2, whereas the 23 strains of biovars 3 to 8 were without plasmids. It was tentatively concluded there are two populations of V. anguillarum serovar O1. One population contains plasmid(s), is hemagglutination negative and trehalose negative, and does not form pellicles in broth cultures, whereas the other population is plasmid free and has the opposite characteristics. The former group is the one related to disease in fish. All 20 V. anguillarum serovar O2 isolates from the environment were without plasmids, whereas 54 (65%) of the isolates from fish (trout and cod) carried plasmids. The biochemical diversity within serovar O2 was pronounced; 13 different biovars were demonstrated. No correlation between the presence of plasmids and biochemical properties was observed.     

Occurrence of plasmids in Danish isolates of Vibrio anguillarum serovars O1 and O2 and association of plasmids with phenotypic characteristics.

Two hundred and twenty-eight isolates of Vibrio anguillarum serovar O1 (125 isolates) and serovar O2 (103 isolates) have been characterized with regard to plasmid contents, biochemical properties, and in vitro hemagglutination and hydrophobic properties. Among 74 V. anguillarum isolates from diseased fish, 63 carried only a 67-kb plasmid (pJM1), 9 carried an additional 98-kb plasmid, and 1 isolate carried only the 98-kb plasmid. Only one isolate was without plasmids. In V. anguillarum serovar O1 from nondiseased fish (mucus and gills), plasmids of the same sizes were present in 29 isolates (58%), whereas 21 isolates (42%) were plasmid free. Based on hemagglutination and biochemical properties, V. anguillarum serovar O1 isolates were divided into eight biovars. The plasmid-carrying strains (102 isolates) all fell within biovars 1 and 2, whereas the 23 strains of biovars 3 to 8 were without plasmids. It was tentatively concluded there are two populations of V. anguillarum serovar O1. One population contains plasmid(s), is hemagglutination negative and trehalose negative, and does not form pellicles in broth cultures, whereas the other population is plasmid free and has the opposite characteristics. The former group is the one related to disease in fish. All 20 V. anguillarum serovar O2 isolates from the environment were without plasmids, whereas 54 (65%) of the isolates from fish (trout and cod) carried plasmids. The biochemical diversity within serovar O2 was pronounced; 13 different biovars were demonstrated. No correlation between the presence of plasmids and biochemical properties was observed.     

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.     

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.