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

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

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.     

An attenuated plasmid-cured strain of Aeromonas hydrophila elicits protective immunity in Clarias batrachus L

Wild type Aeromonas hydrophila (Strain AO1) isolated from the lesions of ulcerative disease syndrome (UDS) affected fish bears a 21 kb virulence plasmid. With plasmid curing the isolates became attenuated and failed to induce fatal haemorrhagic ulcers in fish. The objective of the present work was to check the immunogenicity of these plasmid-cured derivatives and determine whether such strains could be used as candidate antigens for eliciting protective immunity to A. hydrophila infections in the Indian catfish Clarias batrachus L. It was observed that the plasmid-cured strains were immunogenic since infection with live plasmid-cured AO1 isolates generated effective T cell responses and led to increase in serum antibacterial agglutinin titres in C. batrachus. Plasmid-cured AO1 strains injected into C. batrachus could disseminate into head kidney (HK) and spleen but never attained the same bacterial loads obtained with wild type AO1 and were cleared rapidly from the host. Immunisation with plasmid-cured bacteria prevented systemic spread and conferred protection against lethal challenge (10 x LD(50)) with wild type A. hydrophila as well as other pathogenic strains of Aeromonas sp. These results demonstrate the potentials of plasmid-cured A. hydrophila derivatives as candidate antigens for eliciting protective immunity in fish and the possibility of using such isolates as shuttle vectors in aquaculture.     

A numerical taxonomic study of species of Vibrio isolated from the aquatic environment and birds in Kent, England

A numerical taxonomic study has been carried out to confirm the identity of strains of the family Vibrionaceae isolated during an ecological study. A total of 237 strains were studied including 148 from the aquatic environment, 6 from estuarine birds, 1 from sheep faeces, and 61 control cultures. Duplicates of 21 of the strains were randomly selected and included to estimate test and operator error. Taxonomic resemblance was estimated on the basis of 148 characters using Euclidean distance. The taxonomic position of some strains was reevaluated using the pattern different coefficient. Strains were clustered by three methods, all of which gave similar results. The estimated average probability of test error was 1.5%. Strains previously identified as Vibrio anguillarum fell into four distinct phenons corresponding to V. anguillarum biovar I, 'V. anguillarum biovar II', V. diazotrophicus, and strains pathogenic to oyster larvae. The latter group characteristically degraded xanthine and probably represents a new species. The phenon corresponding to V. cholerae included the type strain, strains of human origin, and strains isolated in the United Kingdom from birds and the aquatic environment. Some strains of V. cholerae were luminous. Other phenons were identified as V. metschnikovii, V. fluvialis, and Aeromonas spp.     

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.     

A numerical taxonomic study of species of Vibrio isolated from the aquatic environment and birds in Kent, England

A numerical taxonomic study has been carried out to confirm the identity of strains of the family Vibrionaceae isolated during an ecological study. A total of 237 strains were studied including 148 from the aquatic environment, 6 from estuarine birds, 1 from sheep faeces, and 61 control cultures. Duplicates of 21 of the strains were randomly selected and included to estimate test and operator error. Taxonomic resemblance was estimated on the basis of 148 characters using Euclidean distance. The taxonomic position of some strains was reevaluated using the pattern difference coefficient. Strains were clustered by three methods, all of which gave similar results. The estimated average probability of test error was 1.5%. Strains previously identified as Vibrio anguillarum fell into four distinct phenons corresponding to V. anguillarum biovar I, ' V. anguillarum biovar II', V. diazotrophicus , and strains pathogenic to oyster larvae. The latter group characteristically degraded xanthine and probably represents a new species. The phenon corresponding to V. cholerae included the type strain, strains of human origin, and strains isolated in the United Kingdom from birds and the aquatic environment. Some strains of V. cholerae were luminous. Other phenons were identified as V. metschnikovii, V. fluvialis , and Aeromonas spp.     

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.     

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.     

An investigation of aquatic bacteria capable of utilizing chitin as the sole source of nutrients

R. OSAWA AND T. KOGA. 1995. A total of 48 bacterial strains capable of utilizing chitin (a polymer of N -acetyl-D-glucosamine [NAG]) as a sole source of nutrients were isolated from river and marine waters in Tokushima. These bacteria were identified as Vibrio fluvialis, V. parahaemolyticus, V. alginolyticus, V. mimicus, Listonella anguillarum and Aeromonas hydrophila . All strains were found positive for chitinase and chitobiase activities, and capable of utilizing NAG as a sole source of carbon and nitrogen.     

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.     

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)     

Enzymatic characterization of Vibrionaceae strains isolated from environment and cold-blooded animals.

Enzymatic profiles were determined by the API ZYM system for 15 strains of non 01 Vibrio cholerae, 4 strains of V. metschnikovii, 9 strains of V. anguillarum, 6 strains of Plesiomonas shigelloides and 115 strains motile Aeromonas sp. All of the tested strains produced alkaline phosphatase, leucine aminopeptidase and did not possess alpha-fucosidase and alpha-mannosidase. Some differences in enzymatic activities among the tested Vibrionaceae strains were noted. The strains of non 01 V. cholerae, V. metschnikovii, V. anguillarum and P. shigelloides did not produce trypsin, whereas all of the tested Vibrio sp. strains appeared to be positive for this enzyme. Only the strains of P. shigelloides produced BI-Phospho-hydrolase. The lack of acid phosphatase activity was observed among the strains of V. anguillarum.     

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.     

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

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

Proteomic approach for acute-phase proteins of hemolymph and muscles in Scylla serrata challenged by a pathogenic bactedum

Acute-phase response is documented to be a significant mechanism of innate immunity in vertebrates and invertebrates.In this study,proteomic methodologies were applied for different protein expressions in hemolymph of Scylla serrata challenged by Vibrio parahaemolyticus after immunization,and in muscles of the crabs separately challenged by V.parahaemolyticus.V.anguillarum and Aeromonas hydrophila.Up-regulated cryptocyanin is documented in the hemolymph and up-regulated calexcitin,wingless(fragment)and tachykinin-related peptide in the muscle as acute-phase proteins.All the four altered proteins were responsible for bacterial stress,but cryptocyanin seemed to be a memory response protein against the challenge by a live bacterium after immunization of the live cells.These up-regulated proteins can be indicative of an understanding of immunity of a crab.     

Aeromonas proteolytica--a halophilic aeromonad?

Aeromonas proteolytica, a halophilic bacterium originally isolated from the intestine of a marine isopod, was compared with 93 strains of Aeromonas hydrophila and representative strains of five similar genera in a computer and serological analysis. Aeromonas proteolytica was peritrichate and non-reactive with six A. hydrohila antisera, and possessed a percentage guanine + cytosine ratio (49.5) outside the range for other aeromonads (55-62%). Moreover, in the computer analysis it shared a lower level of similarity with other aeromonads than with both strains of Vibrio anguillarum tested.     

利用抑制性消减杂交（SSH）技术研究不同毒力的鳗弧菌菌株的基因多样性

[目的和方法]鳗弧菌是一种嗜盐的革兰氏阴性细菌,也是鱼类弧菌病的主要病原.对斑马鱼的半数致死量研究结果表明,鳗弧菌菌株VIB72具有较高的毒力,而菌株CW1的毒力较低.本文利用抑制性消减杂交(SSH)技术对这两个菌株的遗传差异进行了研究.[结果]通过对差减文库筛选,分离到59个对菌株VIB72的阳性克隆,并对这些克隆的DNA序列进行了测定.17个基因片断与其它细菌的已知功能的基因有较高的同源性,其中包括可溶性溶胞壁质转糖基酶、转移蛋白MobA和MobC、转座子IS66、抑制相关蛋白(金属β-内酰胺酶和乙酰转移酶家族)、毒素蛋白(DT-201和alveicin A免疫蛋白)、与OLD家族相似的ATP依赖性核酸内切酶以及SocE和GTP结合蛋白HflX(有高频率的溶原化).这些基因片断有可能是鳗弧菌毒力岛的一部分.其他的基因片断与其它的已知基因没有明显的相关性.[结论]这些结果表明,SSH技术成功地鉴定了不同致病性的鳗弧菌菌株的基因差异及潜在的毒力基因.     

Chemotactic motility is required for invasion of the host by the fish pathogen Vibrio anguillarum

The role of the flagellum and motility in the virulence of the marine fish pathogen Vibrio anguillarum was examined. Non-motile mutants were generated by transposon mutagenesis. Infectivity studies revealed that disruption of the flagellum and subsequent loss of motility correlated with an approximate 500-fold decrease in virulence when fish were inoculated by immersion in bacteria-containing water. However, the flagellar filament and motility were not required for pathogenicity following intraperitoneal injection of fish. The transposon-insertion site for six mutants was determined by cloning and sequencing of the Vibrio DNA flanking the transposon. V. anguillarum genes whose products showed strong homology to proteins with an established role in flagellum biosynthesis were identified. One of the aflagellate mutants had a transposon insertion in the rpoN gene of V. anguillarum . This rpoN mutant failed to grow at low concentrations of available iron and was avirulent by both the immersion and intraperitoneal modes of inoculation. A chemotaxis gene, cheR , was located upstream of one transposon insertion and an in-frame deletion was constructed in the coding region of this gene. The resulting non-chemotactic mutant exhibited wild-type pathogenicity when injected intraperitoneally into fish but showed a decrease in virulence similar to that seen for the non-motile aflagellate mutants following immersion infection. Hence, chemotactic motility is a required function of the flagellum for the virulence of V. anguillarum