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1.
Biological activities of cell-free culture filtrate of 3 virulent strains of fish Vibrio were examined to determine the relationship to the pathogenesis of fish vibriosis. Among the 3 strains examined, V anguillarum strains NCMB6 and NCMB571 produced hemolysin and protease, whereas V ordalii strain N7802 did not. Culture filtrate of strain NCMB571 were lethal to rainbow trout and produced a cytotoxic effect on fish cell line. Results revealed that the extracellular products may be involved in the pathogenesis of fish vibriosis.  相似文献   

2.
Hemolytic activity of 3 pathogenic strains, of fish Vibrio commonly associated with vibriosis (V anguillarum NCMB6 and NCMB571 strains, and Vibrio sp N7802 strain) was examined, using chicken and mammalian erythrocytes. Vibrio strains NCMB6 and NCMB571 and their culture filtrates had hemolytic activity against 8 kinds of erythrocytes tested, whereas Vibrio strain N7802 produced only a little amount of hemolysin. Strain NCMB571 culture filtrate and its material partially purified by column chromatography were lethal in mice. From 2 peaks of protein, hemolysin was recovered from the 1st peak, which coincided with toxicity in mice. Heat-inactivation of culture filtrate indicated that hemolytic materials may be thermolabile proteins, but that toxic material may be comparatively thermostable.  相似文献   

3.
Healthy Atlantic salmon and salmon with a history of chronic natural Aeromonas salmonicida subsp. achromogenes infection were compared with respect to total serum protein and the concentration and specificity of serum immunoglobulin. The immunoglobulin level was measured using competitive ELISA and the specific antibody activity against Aeromonas salmonicida subsp. achromogenes was measured using double sandwich ELISA. Significant elevation of serum protein and immunoglobulin concentration was observed in the infected salmon compared with the healthy fish. This was accompanied by weak anti-A. salmonicida activity in the infected fish which seemed to contribute to the raised immunoglobulin level to only a limited degree.  相似文献   

4.
Survival problems are encountered at early stages of intensive fish rearing and antibiotics are widely used to remedy the situation. Probiotics may provide a potential alternative method to protect larvae from opportunistic and pathogenic bacteria and promote a balanced environment. This study was designed to search for new probiotics to target this critical period in cod rearing. Potential probionts were selected from the natural microbiota of cod aquacultural environment. The selection was based on several criteria: pathogen inhibition potential, growth characteristics, strain identification, metabolite production and adhesion to fish cell lines. Our study demonstrated that 14% of screened bacteria (n=188) had antagonistic properties towards fish pathogens. The majority of these isolates were Gram-positive (81%), belonging to Firmicutes (69.2%) and Actinobacteria (11.5%) phyla based on 16S rRNA gene sequencing. Only 6 (3.2%) of 188 isolates could inhibit all three pathogens tested: Vibrio anguillarum, Aeromonas salmonicida subsp. achromogenes and Vibrio salmonicida. Differences observed in activity intensity and spectrum among inhibitory isolates emphasise the need to develop probiotic mixtures for efficient prophylactic methods. Comparison of growth behaviour of inhibitory isolates and pathogens at cod rearing temperatures, metabolite production and adhesion capacity were considered for final probiont selection. Four promising isolates that could be used as a mixed supplement to rearing water were identified as putative probiotic bacteria. This study emphasises the importance and potential of lactic acid bacteria in aquaculture.  相似文献   

5.
The aim of this study was to evaluate the use of two molecular techniques, repetitive extragenic palindromic polymerase chain reaction (REP-PCR) and repetitive intergenic consensus PCR (ERIC-PCR), as epidemiological tools with which to discriminate among genetically distinct strains within two bacterial fish pathogens, Pseudomonas anguilliseptica and Aeromonas salmonicida. A total of 30 A. salmonicida and 52 P. anguilliseptica were analyzed. For P. anguilliseptica, three different major fingerprints were obtained with both techniques, which defined three genomic groups: one was composed of strains isolated from eels Anguilla spp., the second of strains from turbot Scophthalmus maximus and blackspot seabream (also known as red seabream) Pagellus bogaraveo, and the third of strains from other fish species, such as gilthead seabream (also known as gilthead bream) Sparus auratus, sea bass Dicentrarchus labrax (also known as European bass Morone labrax), and salmonids. In the case ofA. salmonicida, promising results were obtained with both techniques for subspecies differentiation. Thus, two genomic profiles were obtained by ERIC-PCR. The first profile consisted of A. salmonicida subsp. salmonicida strains isolated from the different hosts. The second profile was composed of two A. salmonicida subsp. masoucida and one A. salmonicida subsp. achromogenes. Using REP-PCR, three genotypes were obtained within this pathogen that were related to the diverse subspecies analyzed. In summary, both methodologies are useful for typing distinct strains associated with different host species and therefore are helpful in epidemiological studies of P. anguilliseptica. In contrast, in the case of A. salmonicida, more studies are needed to determine their utility in discriminating the subspecies salmonicida from the other two subspecies.  相似文献   

6.
The potential of two candidate probiotic bacteria (GP21 and GP12), isolated from the gut of Atlantic cod, to adhere to primary cultures of the epithelial cells from the different regions of the intestine and to interfere with the adhesion of two pathogens, Vibrio anguillarum and Aeromonas salmonicida subsp. salmonicida were investigated. The intestinal isolates showed clear preference in adhering to the cells from the different intestine segments. GP12 adhered strongly to the fore- and mid intestine cells. The adherence of GP21 was most to the cells from the hind intestine followed by those from the mid-segment. The adhesion of V. anguillarum was affected by both GP21 and GP12; GP12 interfered through competition, but a specific mode of action was not observed for GP21. In the case of A. salmonicida, competition was the principal mechanism by which GP21 interfered with their adhesion, while exclusion mechanism was favoured by GP12. In addition, GP21 was more auto-aggregative than GP12, but the latter was more co-aggregative with both the pathogens. The isolates were also capable of lowering lactate dehydrogenase activity compared to that by the pathogen and they reduced the caspase-3 activity in the epithelial cells from the hind intestine, to which the pathogens adhered the most. Thus it could be concluded that the adhesion of the candidate probiotics is segment-specific and their interference with the adhesion of pathogens is dependent on both source of the epithelial cells and the mechanism adopted by the isolates. This information is novel in the case of fish and the manner in which potential probiotic organisms interfere with the pathogen adhesion provides supportive information for disease control.  相似文献   

7.
An examination of 41 fish pathogenic and 51 environmental strains of Vibrio anguillarum from Danish coastal areas, and of 8 reference strains of Vibrio anguillarum, biotype 1, showed that these strains shared rather broad-spectered activities towards many carbohydrates, amino acids, proteins and lipids.They also exhibited rather uniform growth characteristics, including parameters as salinity (NaCl), temperature, antibiotic resistance, and growth on specific media.A computer analysis of these strains and 3 strains of Vibrio anguillarum, biotype 2 (Vibrio ordalii), showed that the Danish strains all belonged to Vibrio anguillarum, biotype 1, and that no basis existed for setting up new biotypes.Mean values of the G+C content in DNA of strains of the various categories of Vibrio anguillarum, biotype 1, ranged from 45.5 to 46, while the mean value for the Vibrio ordalii strains was 48.9.The biochemically active Vibrio anguillarum seems more suitable for environmental studies than the more fastidious Vibrio ordalii.Key words: Vibrio anguillarum, Vibrio ordalii, biochemical activities, environmental, fish pathogenic, reference strains  相似文献   

8.
The fish pathogen Vibrio anguillarum causes a lethal infection in farmed fish characterized by hemorrhagic septicemia. There are no reports of experimental laboratory infections in warm-blooded animals. We investigated the effects of an intraperitoneal infection with different doses of a V. anguillarum suspension in mice and guinea pigs. The infection caused a 95-100% of mortality in 24-48 h. Hemorrhagic septicemia was observed at necropsy and confirmed by histological and hematological examination. Immunohistochemically positive bacterial clumps were detected exclusively in vessel lumen in all examined organs, including brain, and V. anguillarum was reisolated in pure culture from all organs, particularly from the kidney. Blood analysis showed erythropenia and leukopenia with granulocytosis in mice, platelet reduction and leukopenia with lymphocytosis in guinea pigs.  相似文献   

9.
The aims of the study were to evaluate a new PCR protocol designed to detect Aeromonas salmonicida in fish tissues and to develop a non-destructive method for the diagnosis of furunculosis. A set of primers (Fer3, Fer4), flanking a fragment of the fstA gene (coding for the ferric-siderophore receptor) was designed, showing to be sensitive and specific. When compared to PCR methods previously reported, the new protocol recognized all the 69 A. salmonicida strains evaluated, with no cross-reactions with the other bacterial species analysed. Sensitivity assays were performed in fish tissues seeded with serial dilutions of pure cultures of A. salmonicida and mixed cultures of this bacterium with Vibrio anguillarum and Aeromonas hydrophila. Detection limits obtained were of 60 and 450 bacterial cells 100 mg(-1) of tissue, respectively. Mucus and blood were evaluated in order to develop a non-destructive tool to detect the pathogen. The detection limits in seeded mucus and blood samples were 2.5 x 10(2) and 1 x 10(5) bacterial cells mL(-1), respectively. When the method was used to detect A. salmonicida in asymptomatic wild salmon, four samples of mucus and six of blood were positive, corresponding to 6 out of the 31 fish examined, whereas only one of the samples resulted positive by culture methods. It is concluded that the PCR protocol evaluated is fast, specific and sensitive to detect A. salmonicida in infected and asymptomatic fish, and will be helpful for the control of the disease through the prompt detection of carriers within fish populations.  相似文献   

10.
Balb/c mice, injected i.p. with extracellular products (ECP) of Aeromonas salmonicida subsp. achromogenes (Asa), displayed symptoms similar to toxic shock syndrome. The LD(50) observed was between 1.5 and 2.0 microg g(-1) and the mice died within 19 h. Four inflammatory cytokines were measured in mice receiving sublethal ECP doses. TNF-alpha and IL-6 showed a sharp peak in the serum while IL-1 beta and IL-2 were not detected. When peritoneal macrophages were cultivated in the presence of ECP, AsaP1 (a toxic caseinolytic metallo-protease purified from ECP) or LPS, all cultures produced TNF-alpha, IL-6 and IL-1 beta. The same antigens were mitogenic in spleen cell cultures. Furthermore, IL-2 production, which is a normal T-cell response to ConA stimulation, was downregulated in spleen cell cultures from mice injected with ECP.  相似文献   

11.
The purification, characterisation and lethal effect of an extracellular protease present in the extracellular products (ECPs) of a pathogenic Vibrio pelagius (7P) strain are described. The extracellular protease was purified by size-exclusion high-performance liquid chromatography and characterised by enzymatic assays. The lethal effect was evaluated by injection into fish. The native protease had a molecular mass of 39 kDa, was active on casein and L-leucyl-beta-naphthylamide (LNA) and its metalloprotease nature was shown by the LNA inhibition profile. Kinetic studies on the hydrolysis of casein and LNA confirmed a competitive inhibition of one substrate with respect to the other. The temperature assays showed that both aminopeptidolytic and caseinolytic activities were labile at 70 degrees C for 3 min. The N-terminal amino acid sequence of 7P protease revealed a high degree of homology with other metalloproteases of Vibrio species that are implicated in virulence. The purified 7P protease showed an LD(50) of 1.77 microgprotein/g fish for turbot. The quick lethal effect (<24h) and the macroscopic damage (external haemorrhagic areas, principally on fins and mouth, petechial haemorrhages in internal organs, but with no external or internal apparent necrotic areas) detected in the host were similar to those obtained by injection of total ECP and live cells of 7P strain.An extracellular protease with endopeptidolytic and exopeptidolytic activities, responsible for the lethal effect of ECP and clinical signs of vibriosis in turbot was purified from a pathogenic V. pelagius (7P) strain.  相似文献   

12.
From 1995 to 1997 several defined species like V. alginolyticus, V. anguillarum, V. cholerae (non O1 and non O139), V. mimicus, V. parahaemolyticus and V. vulnificus were isolated during a survey to determine the presence of V. vulnificus in the brackish water of the Baltic Sea in Germany. Moreover atypical Vibrio isolates were detected. Four isolates belonging to a group of atypical Vibrio and possibly representing a new species in the genus Vibrio were characterized in detail. All four strains were isolated from surface costal waters. Based on 16S rDNA sequence analysis they showed the highest relatedness to the species V. navarrensis and V. vulnificus. The strains did not harbor the species specific hemolysin gene vvhA from V. vulnificus as shown by PCR and hybridization experiments. Moreover, they differed in at least two biochemical parameters tested from the hitherto described Vibrio species. All these strains induced hemolysis on washed blood agar dishes and showed phase variations on Luria Bertani agar dishes. Because of the similarity to the eel pathogen V. vulnificus, we infected eels with one of the four atypical strains (CH-291), but no pathogenicity for eels could be detected. Furthermore, Vero cell tests with supernatants of bacterial cultures did not reveal secreted Vero cell cytotoxic compounds. This indicates a nonpathogenic nature of these strains.  相似文献   

13.
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14.
The phagocytosis of Vibrio anguillarum by peritoneal macrophages from normal rainbow trout was enhanced by antibody and complement. Treatment of either macrophages or the bacteria by antibody also enhanced opsonisation. Five weeks after immunisation with V anguillarum, the phagocytic activity of macrophages from rainbow trout was increased significantly compared with the activity of those from unvaccinated fish. Although agglutinin titres did not increase until three weeks after immunisation, seven out of 10 fish challenged one week after immunisation survived, indicating that immunised fish had developed resistance to vibrio infection before significant levels of antibody or phagocytic activity became detectable.  相似文献   

15.
A specific DNA probe for the identification of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), was developed from one of 3 clones pRS47, pRS49, and pRS26 of 5.1 kb, 5.3 kb, and 11.3 kb, respectively. The biotinylated pRS47/BamHI insert probe was tested on 3 dilutions of DNA extracted from 3 strains of R. salmoninarum and from 1 strain each of Arthrobacter protophormiae, Aeromonas salmonicida, Corynebacterium aquaticum, Carnobacterium piscicola, Listonella anguillarum, Micrococcus luteus, Pseudomonas fluorescens, Vibrio ordalii, and Yersinia ruckeri. In a dot blot assay, this probe hybridized only with the DNA from the R. salmoninarum strains. When used on kidney samples from fish challenged with R. salmoninarum, the dot blot hybridization assay with the probe was found to be as sensitive as culture. In a fluorescent antibody test, samples that were negative in culture and dot blot hybridization showed no more than one fluorescing cell in 50 microscopic fields examined. This DNA probe, therefore, has the potential for use in the diagnosis of BKD of fish.  相似文献   

16.
Thirty-one Aeromonas hydrophila, 13 A. sobria and two A. salmonicida strains of diverse sources were tested for enterotoxigenicity, hemagglutination and cell surface hydrophobicity. Although 93% of the culture supernatant fluids of the Aeromonas strains exhibited cytotoxic effects on Y1 adrenal and Chinese hamster ovary (CHO) cells, typical rounding of Y1 adrenal cells was reproducibly observed before cytotoxicity for 80% of the isolates within 1 h of exposure.Twenty-eight strains were positive for delayed permeability factor (DPF) activity in rabbit skin. Culture filtrates of 16 of 20 strains that were positive both in the Y1 adrenal cell test and for DPF activity elicited fluid accumulation in rabbit ileal loops. The DPF and ileal loop activities were neutralizable by cholera antitoxin. All, except two strains each of A. sobria and A. hydrophila, produced a heat-stable, rapid permeability factor (RPF) detected in rabbit skin. Heat-treated culture supernatant fluids of two A. hydrophila and one A. sobria isolate gave positive responses in the infant mouse assay. Nine other strains gave borderline reactions.When A. hydrophila and A. sobria isolates were grown in broth, approximately 90% agglutinated bovine, chicken, human group A and guinea-pig erythrocytes in the presence of mannose at 4°C and/or 20°C. The two A. salmonicida isolates produced mannose resistant hemagglutination (MRHA) of these four blood types.Hydrophobic interaction chromatography indicated adhesive potential in 61% A. hydrophila and 100% A. sobria strains expressing weak to strong hydrophobic cell surface properties. The results of these investigations strongly imply that the Aeromonas strains produce a cytotonic enterotoxin immunologically related to cholera toxin. Adhesive characteristics were commonly found in both clinical and routine isolates.  相似文献   

17.
ABSTRACT: Bacterial taxonomy has progressed from reliance on highly artificial culture-dependent techniques involving the study of phenotype (including morphological, biochemical and physiological data) to the modern applications of molecular biology, most recently 16S rRNA gene sequencing, which gives an insight into evolutionary pathways (= phylogenetics). The latter is applicable to culture-independent approaches, and has led directly to the recognition of new uncultured bacterial groups, i.e. "Candidatus", which have been associated as the cause of some fish diseases, including rainbow trout summer enteritic syndrome. One immediate benefit is that 16S rRNA gene sequencing has led to increased confidence in the accuracy of names allocated to bacterial pathogens. This is in marked contrast to the previous dominance of phenotyping, and identifications, which have been subsequently challenged in the light of 16S rRNA gene sequencing. To date, there has been some fluidity over the names of bacterial fish pathogens, with some, for example Vibrio anguillarum, being divided into two separate entities (V. anguillarum and V. ordalii). Others have been combined, for example V. carchariae, V. harveyi and V. trachuri as V. harveyi. Confusion may result with some organisms recognized by more than one name; V. anguillarum was reclassified as Beneckea and Listonella, with Vibrio and Listonella persisting in the scientific literature. Notwithstanding, modern methods have permitted real progress in the understanding of the taxonomic relationships of many bacterial fish pathogens.  相似文献   

18.
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19.
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20.
We have examined the cytotoxic responses produced in HeLa and Vero cell cultures by sonicates from 15 non-enterotoxigenic (STa-, LT-) strains of E. coli, highly lethal for mice parenterally LD50 less than 3 X 10(7) CFU), which had been isolated from feces of diarrheic calves. Three types of cytotoxic responses were observed. Type 1 (five strains) consisted of enlargement, rounding and polynucleation of HeLa cells, an effect previously reported with cytotoxic necrotizing factor (CNF) in E. coli from infant and piglet enteritis. Type 2 toxicity (three strains and the control Vir strain S5) was also characterized by enlargement and polynucleation of HeLa cells, but in contrast to Type 2 effect, cells were elongated. Sonicates from the latter strains were lethal for chickens, producing the lesions previously described with Vir strains. Type 3 toxicity (two strains and the control VT strain H19), produced an extensive destruction of both Vero and HeLa cell cultures. Cytotoxic effects were completely abolished upon heating for 1 h at 60 degrees C for Type 1 and 2 extracts and at 80 degrees C for Type 3 extracts. Seroneutralization assays showed that cytotoxins of the same type were closely related antigenically. In addition, a slight cross-neutralization was observed between Type 1 (CNF) and Type 2 (Vir) toxins.  相似文献   

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