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PubMed1984

Vitamin B6 biosynthesis and glycolate reductase in Flavobacterium sp. 238-7.

An enzyme which reduces glycolate to glycolaldehyde as the first step of the biosynthesis of vitamin B6 was studied using the particulate fraction of Flavobacterium sp. 238-7, a vitamin B6 producer. The enzyme catalyzes the reduction of glycolate in the presence of NADPH, but not the oxidation of glycolaldehyde, and is designated as glycolate reductase. There is a positive correlation between the activity of the enzyme and vitamin B6 biosynthesis. The activity and the formation of the enzyme were not affected by vitamin B6. This enzyme probably plays a role in the extraordinary accumulation of vitamin B6 by the bacterium.

Alcohol Oxidoreductases↗

PubMed1986

Menaquinone (vitamin K2)-6 production by mutants of Flavobacterium meningosepticum.

Flavobacterium meningosepticum IFO 12535, a menaquinone (MK) producer, was mutagenized to improve productivity. A mutant, which was resistant to 1-hydroxy-2-naphthoate (HNA), was found to produce MK more abundantly: 34 mg/liter of culture broth and 5.5 mg/g of dry cells. The mutant was less sensitive to inhibition by HNA on MK biosynthesis than the wild-type strain. MK was isolated from cells of the mutant and identified as MK-6 based on its physicochemical characteristics. Mutants, which were given each KCN resistance, aromatic amino acid auxotrophy and no carotenoid productivity, did not show further increase of productivity.

Amino Acids↗

PubMed1998

Fingerprinting of Flavobacterium psychrophilum isolates by ribotyping and plasmid profiling.

Flavobacterium psychrophilum is the agent of cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. Ribosomal RNA gene restriction patterns (ribotypes) and plasmid profiles were determined on a collection of 85 strains isolated from different countries and fish species. Several ribotypes were obtained by using the restriction endonucleases Hinc II and Pvu II. Computer analysis of the ribotypes revealed that some of them were clearly associated with the fish species from which the strains were isolated, whereas no correlation with the geographical origin was found. Most of the strains harboured at least one plasmid and several different plasmid profiles were observed, even among strains sharing the same ribotype. These methods, used alone or in combination with other typing techniques, can be considered powerful tools for the epidemiological tracing of F. psychrophilum infections.

Animals↗

PubMed1999

Reproducible methods for experimental infection with Flavobacterium psychrophilum in rainbow trout Oncorhynchus mykiss.

Experiments were done in order to achieve a reproducible method that can be used to infect rainbow trout Oncorhynchus mykiss with Flavobacterium psychrophilum, the causal agent of coldwater disease and rainbow trout fry syndrome. The main method investigated was intraperitoneal injection, and this method was tested using isolates with different elastin-degrading profiles and representing different serotypes. Injecting trout, average weight 1 g, with 10(4) CFU (colony-forming units) per fish caused cumulative mortalities around 60 to 70%. The virulent strains belonged to certain serotypes and degraded elastin. The intraperitoneal injection challenge method could be used on larger fish, but the infection dose was 10(7) CFU per fish before mortalities occurred. Bath infection and bath infection in combination with formalin treatment (stress) seemed to be reproducible methods that could be used as alternatives to the intraperitoneal method, although the mortalities among infected trout were lower. The results of investigated methods were influenced by parameters such as the challenge isolate, number of fish in the tank affecting the infection pressure, origin of fish and weight of fish.

Animals↗

PubMed1999

Flavobacterium psychrophilum in Baltic salmon Salmo salar brood fish and their offspring.

Baltic salmon brood fish were investigated for the presence of Flavobacterium psychrophilum in the kidney, spleen, brain and sexual products (ovarian fluid, unfertilised eggs and milt). Samples for bacteriology were taken at capture, when the fish were ascending their native river to spawn, and after a period of captivity in indoor pools, at stripping. During captivity, abnormal wiggling behaviour was recorded in some of the fish. Bacterial samples were taken to determine if F. psychrophilum had any role in the aetiology of the condition. Furthermore, the presence of F. psychrophilum on egg surfaces during incubation was investigated. F. psychrophilum was isolated from internal organs and/or sexual products in 7 out of 50 (14.0%) fish sampled at capture and 63 out of 272 (23.2%) fish sampled at stripping. The bacteria was isolated from either spleen or gonads in 2 out of 19 (10.5%) fish with abnormal wiggling behaviour but no bacteria was isolated from the brain. No F. psychrophilum was isolated from eggs at the eyed stage. Just before hatching, the bacterium was isolated from 5 out of 15 (33.3%) family groups. The present study shows that Baltic salmon brood fish are carriers of F. psychrophilum during their spawning migration. The presence of the bacteria in sexual products from both females and males indicates that transmission from the brood fish to the offspring should be considered an important route of infection.

Animals↗

PubMed2000

Effect of Flavobacterium psychrophilum strains and their metabolites on the oxidative activity of rainbow trout oncorhynchus mykiss phagocytes.

The oxidative activity of rainbow trout phagocytes was studied using a chemiluminescence technique using 12 different Flavobacterium psychrophilum strains and their metabolites. Phagocytes were obtained from the head kidney of rainbow trout Oncorhynchus mykiss. The addition of viable F. psychrophilum or their metabolites to the phagocytes resulted in an immediate chemiluminescence response. The stimulating effects of both the F. psychrophilum and their metabolites on the phagocytes were found to be heat stable. No significant differences in stimulation capacity were found between the strains tested. To investigate the nature of the stimulating agent, both the bacteria and the supernatant were treated with either sodium metaperiodate or polymyxin B. Adding polymyxin B to the bacterial cells and supernatant did not change the chemiluminescence pattern, suggesting that the capacity of F. psychrophilum to stimulate the phagocytes probably is not due to lipopolysaccharides (LPS). However, following incubation of the bacteria and their metabolites with sodium metaperiodate, the capacity to stimulate phagocytes was significantly impaired. This suggests that a carbohydrate component most likely plays an important role in the ability of F. psychrophilum to stimulate phagocytes. Opsonisation of the bacteria with native trout serum or with rabbit anti-F. psychrophilum serum resulted in an additional chemiluminescence peak which was significantly higher than the first peak. This extra peak disappeared following heat treatment of the trout serum and the rabbit anti-F. psychrophilum serum, pointing towards the involvement of heat labile complement in opsonisation.

Animals↗

PubMed2000

Standardization of experimental infection with Flavobacterium psychrophilum, the agent of rainbow trout Oncorhynchus mykiss fry syndrome.

Rainbow trout fry syndrome (RTFS) is a septicaemic infection of young rainbow trout Oncorhynchus mykiss occurring at low temperatures and responsible for severe economic losses in European fish farming. The causative agent, Flavobacterium psychrophilum, is a gliding bacterium, and difficulties in culturing it have long been an impediment to investigations on pathogenesis and immunity. Successful attempts at experimentally inducing the disease have been reported, but no experimental model resulting in well-controlled and quantitatively reproducible effects has been described. Recent improvements in F. psychrophilum cultivation made it possible to produce bacterial suspensions with nearly constant viability and to complete challenge injections in rainbow trout fingerlings, using accurately adjusted infective doses. Parenteral injection resulted in significant mortality, which was higher when administered intramuscularly (IM) than intraperitoneally (IP). Lethal doses 50 % lower than 10(3) colony forming units were consistently obtained in trout weighing 3 to 5 g, and the regular shape of the cumulative mortality curves appeared to lend itself to quantitative analyses. Bath experiments produced milder effects, although mortality ranging between 45 and 60 % was obtained in 6 g trout when skin lesions or stressors were induced along with bacterial exposure. Temperature, salinity and the process of preserving isolates (at least over short periods of time) did not seem to be associated with the severity of infection. Nevertheless, infection trials performed at 2 different locations differing both in water quality and in the system of fish maintenance resulted in different mortalities. These findings notwithstanding, the proposed IM model appears easy to apply under standardized experimental conditions and should contribute to effective advances in the study of the disease.

Animals↗

PubMed2000

Flavobacterium psychrophilum, invasion into and shedding by rainbow trout Oncorhynchus mykiss.

The infection route of Flavobacterium psychrophilum into rainbow trout Oncorhynchus mykiss was studied using bath and cohabitation challenges as well as oral challenge with live feed as a vector. Additionally, the number of bacterial cells shed by infected fish into the surrounding water was determined in the cohabitation experiment and in challenge experiments at 3 different water temperatures. The experiments showed that skin and skin mucus abrasion dramatically enhanced the invasion of F. psychrophilum into the affected fish in bath and cohabitation challenges. Disruption of the skin is discussed as an important invasion route for F. psychrophilum into the fish. The shedding rate of F. psychrophilum by infected fish was associated with water temperature and the mortality of the infected fish. High numbers of F. psychrophilum cells were released into the water by dead rainbow trout during a long time period compared to the numbers of cells shed by live fish. The results emphasise the importance of removing dead and moribund fish from rearing tanks in order to diminish the infection pressure against uninfected fish in commercial fish farms. In immunohistochemical examinations of organs and tissues of orally infected fish, F. psychrophilum cells were detected in only 1 fish out of 31 studied. Mortality of the orally challenged fish was not observed in the experiment.

Animals↗

PubMed2002

Occurrence of Flavobacterium psychrophilum in fish-farming environments.

Occurrence of Flavobacterium psychrophilum in fish farms and fish-farming environments was studied using agar plate cultivation, the immunoflourescence antibody technique (IFAT) and nested PCR. Characteristics of 64 F. psychrophilum isolates from rainbow trout Oncorhynchus mykiss, fish farm rearing water, ovarian fluid and wild fish were serotyped, ribotyped and compared biochemically. Virulence of F. psychrophilum isolates from different sources was compared by injection into rainbow trout. Additionally, the number of F. psychrophilum cells shed by naturally infected rainbow trout was determined. F. psychrophilum was detected and isolated from skin mucus, skin lesions and internal organs of diseased rainbow trout and from fish without clinical disease. The pathogen was also present in wild perch Perca fluviatilis, roach Rutilus rutilus, and ovarian fluids of farmed rainbow trout brood fish. Isolates were biochemically homogenous, excluding the capability to degrade elastin. Five different agglutination patterns with different antisera against F. psychrophilum were found among the isolates studied. Although several different ribopatterns were found (ClaI: 12 ribopatterns and HaeIII: 9 ribopatterns), ribotype A was the most dominant. Farmed rainbow trout brood fish carried a broad-spectrum of serologically and genetically different F. psychrophilum in ovarian fluids. Virulence of the tested isolates in rainbow trout varied and naturally infected rainbow trout shed 10(4) to 10(8) cells fish(-1) h(-1) of F. psychrophilum into the surrounding water.

Animals↗

PubMed2003

Nanoinjection as a tool to mimic vertical transmission of Flavobacterium psychrophilum in rainbow trout Oncorhynchus mykiss.

Newly fertilised eggs of rainbow trout Oncorhynchus mykiss were nanoinjected with Flavobacterium psychrophilum in order to mimic vertical transmission. Two bacterial isolates with different elastin-degrading capacity were used. All infected groups (10, 100 and 1000 colony forming units egg(-1)) showed significantly higher cumulative mortalities than the control groups at the end of the experiment, 70 d post-hatching. The total mortalities in the control groups were below 2.5%. In the high-dose groups, 95 to 100% of the eggs died during the eyed stage. In the intermediate group infected with the elastin-negative isolate, the major mortality occurred during the eyed stage of the egg, with a total cumulative mortality of 83% at the end of the experiment. In the intermediate group infected with the elastin-positive isolate, a total mortality of 63% was recorded. In this group, diseased fry showed clinical signs of disease and morphological changes similar to those described in connection with rainbow trout fry syndrome (RTFS) shortly after the beginning of feeding. In the low dose groups, the mortality in the elastin-negative group was 14% and in the elastin-positive group 11%. The bacterium was isolated from dead eggs and fry in infected groups and demonstrated in internal organs of dead and moribund fry by immunohistochemistry. The nanoinjection method used in this study may be a useful method to study pathogens, like F. psychrophilum, that can be vertically transmitted.

Animals↗

PubMed2003

Adhesion of high and low virulence Flavobacterium psychrophilum strains to isolated gill arches of rainbow trout Oncorhynchus mykiss.

The ability of Flavobacterium psychrophilum to adhere to the gill tissue of rainbow trout Oncorhynchus mykiss was evaluated. A gill perfusion model was adopted, offering a number of advantages compared to other in vitro as well as in vivo models. A comparison between the adhesion capacity of a high and low virulence F. psychrophilum strain was made. Experiments were additionally carried out to assess the influence of water quality (organic material, nitrite) and temperature on the adhesion process of the bacterial cells. The high virulence strain attached more readily to the gill tissue than did the low virulence strain. Moreover, the adherence of the high virulence strain of F. psychrophilum was influenced by a number of factors. These were immersion of the gill arches in water to which organic material or nitrite were added, and elevated temperature. The former 2 increased the adhesion ability, while the latter had a negative influence on the adherence process.

Animals↗

PubMed2003

Starvation of Flavobacterium psychrophilum in broth, stream water and distilled water.

Physical changes in Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome (RTFS), were examined over a 19 wk period of starvation. Bacteria were maintained in either Cytophaga broth, filtered stream water, or filtered distilled water, or were maintained in broth after disinfection as a negative control for dead bacteria. Culturability and viability of the bacterium were assessed using colony-forming units (CFUs) and a commercially available live/dead kit. Antigenic profiles and general morphology of the bacterium were also examined using Western blot analysis and electron microscopy, respectively. The bacterium appeared to stop multiplying and became smaller and rounded when maintained in stream water. Its culturability declined until it was no longer possible to obtain colonies on agar plates at the end of the trial at 19 wk, and results from the live/dead kit did not correspond with the viability obtained as CFUs in culture. However, it was still possible to obtain growth of the bacterium after 36 wk with a resuscitation step in Cytophaga broth. Bacteria maintained in distilled water or treated with a disinfectant appeared non-viable using the live/dead kit and were unable to grow on agar 1 h after setting up the experiment; no morphological changes were observed in the bacteria maintained under these conditions. Bacteria maintained in broth were present as long, slim rods, some of which developed into 'ring' formations. Small differences were observed in the antigen profiles of the bacteria maintained under the different treatments, possibly due to a reduction in the size and metabolism of the bacteria. There was also a marked decline in the sensitivity of the PCR with bacteria maintained under the different treatments 14 wk from the onset of the study.

Analysis of Variance↗

PubMed2003

Genotyping of Flavobacterium psychrophilum using PCR-RFLP analysis.

Genetic variability among 242 strains of Flavobacterium psychrophilum was characterized using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis. Universal Primers GYR-1 and GYR-1R, which were designed to amplify the gyrase subunit B gene (gyrB), yielded a 1178 bp PCR product encoding gyrB and a 290 bp PCR product of anonymous DNA from all F. psychrophilum strains tested. In the RFLP analysis of the anonymous 290 bp DNA marker, the restriction enzyme HinfI generated 2 cleavage patterns (Genotypes A and B). Genotype A was found only in isolates from ayu (n = 109), while Genotype B was found in isolates from coho salmon (n = 11), ayu (n = 35), rainbow trout (n = 43) and other fishes (n = 44). In the second experiment, Primers PSY-G1F and PSY-G1R specific for F. psychrophilum, were used to amplify gyrB. The specific primer pair amplified the expected size (1017 bp) PCR product from all F. psychrophilum strains. In the RFLP analysis of the gyrB, the restriction enzyme RsaI produced 2 genotypes, R and S. Genotype R was found in isolates from coho salmon (n = 6), ayu (n = 27), rainbow trout (n = 39) and other fishes (n = 4). Genotype S was found in isolates from coho salmon (n = 5), ayu (n = 117), rainbow trout (n = 4) and other fishes (n = 40).

Animals↗

PubMed2004

Protective immunity in rainbow trout Oncorhynchus mykiss following immunization with distinct molecular mass fractions isolated from Flavobacterium psychrophilum.

Vaccine development for coldwater disease (CWD), also known as rainbow trout fry syndrome (RTFS), has been based primarily on whole-cell bacterins or outer membrane fractions of Flavobacterium psychrophilum. In the present study, immunogenic regions of the bacterium corresponding to 18-28, 41-49, and 70-100 kDa were identified by western blot analysis using rainbow trout Oncorhynchus mykiss immune sera. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), antigens within these regions were isolated by electro-elution and used in immunization trials. Groups of rainbow trout fry were immunized with these regions emulsified with Freund's complete adjuvant (FCA) and a formalin-killed bacterin emulsified with FCA. It was demonstrated that the 70-100 and 41-49 kDa regions and F. psychrophilum treatments elicited significant protection when compared to the saline control following subcutaneous challenge with 2 doses of a virulent strain of F. psychrophilum. Immunization with the 70-100 kDa region resulted in near complete protection in fish with mean cumulative percent mortality (CPM) of 6% and mean relative percent survival (RPS) of 94% at the lower challenge dose (6.25 x 10(6) colony forming units fish(-1)). This preparation also stimulated a high level of specific antibody to F. psychrophilum, as detected by an enzyme-linked immunosorbent assay (ELISA). Western blot analysis using sera from fish immunized with the 70-100 kDa region demonstrated that high molecular weight proteins and the O-polysaccharide component of lipopolysaccharide (LPS) are recognized by serum antibodies. This suggests that these antigens may be involved in eliciting a highly protective immune response, and could serve as vaccine candidates.

Analysis of Variance↗

PubMed2004

Experimental infection of Flavobacterium psychrophilum in fins of Atlantic salmon Salmo salar revealed by scanning electron microscopy.

Infections caused by Flavobacterium psychrophilum include 'bacterial coldwater disease' (BCWD) and 'rainbow trout fry syndrome' (RTFS), which are severe diseases that can cause high mortality and significant losses in hatchery-reared salmonids worldwide. Usually, these conditions start with necrosis along the edge of the fins. As the infection progresses, both the fish surface and the internal organs can be involved. The aetiological agent produces a Ca-dependent protease that can be responsible for some of the pathogenic responses, although the precise nature of the response remains to be elucidated. Atlantic salmon Salmo salar were experimentally infected by F. psychrophilum in order to investigate the bacterial invasion in the fin tissues by scanning electron microscopy. The images showed numerous bacteria embedded in the mucous layer when this remained on the tegument. In other zones without mucus, it was observed that bacteria were present on the axis of fin rays, but not on the epidermal surface. The material on these axes was largely eroded by tubular boreholes, and bacterial rods could be seen in these perforations. EDX (Energy Dispersive X-ray) microanalysis of the axis of the fin rays showed significant amounts of P and Ca, revealing the ossification of the ray axis. The protease activity could explain the formation of the tubular boreholes, allowing the bacteria the necessary Ca for the activation of the enzyme. The erosion pattern suggests that the gliding motility of F. psychrophilum could be involved in this burrowing ability.

Animals↗

PubMed2004

Impact of PCB on resistance to Flavobacterium psychrophilum after experimental infection of rainbow trout Oncorhynchus mykiss eggs by nanoinjection.

The effects of sublethal exposure of a commercial blend of polychlorinated biphenyls (PCB), i.e. Clophen A50, on disease resistance to the aetiological agent of rainbow trout fry syndrome, Flavobacterium psychrophilum, were investigated. Newly fertilised rainbow trout Oncorhynchus mykiss eggs were nanoinjected with 2 doses of Clophen A50 (0.4 or 2 microg egg(-1)) and/or 100 colony forming units of F. psychrophilum. The mean cumulative mortality in control groups, and groups exposed to the lower dose of Clophen A50 (0.4 microg egg(-1)) was below 5.0%. The mean cumulative mortality in groups exposed to the higher dose of Clophen A50 (2.0 microg egg(-1)) was 5.8%, which was not significantly different from the control groups. In all groups infected with F. psychrophilum, with or without exposure to Clophen A50, significantly higher cumulative mortalities compared with control groups were recorded. No differences in mortality were recorded between groups exposed to bacteria alone or bacteria in combination with the higher dose of Clophen A50 (21.6 and 20.4%, respectively). Decreased disease resistance was recorded in groups exposed to F. psychrophilum and the lower dose of Clophen A50, with a mean cumulative mortality of 56.0%. These results could be due to non dose-dependent effects on the immune system, or toxic effects of PCB or their metabolites on the bacteria in groups exposed to the higher dose of Clophen A50. The present study indicates that maternal transfer of PCB might affect disease resistance to vertically transmitted F. psychrophilum.

Animals↗

PubMed2005

Effect of Pseudomonas sp. MT5 baths on Flavobacterium columnare infection of rainbow trout and on microbial diversity on fish skin and gills.

Use of Pseudomonas sp. strain MT5 to prevent and treat Flavobacterium columnare infection was studied in 2 experiments with fingerling rainbow trout Oncorhynchus mykiss. In the first experiment, length heterogeneity analysis of PCR-amplified DNA fragments (LH-PCR) was used to assess the effect of antagonistic baths on the microbial diversity of healthy and experimentally infected fish. In the 148 samples studied, no difference was found between bathed and unbathed fish, and 3 fragment lengths were detected most frequently: 500 (in 75.7% of the samples), 523 (62.2%) and 517 bp (40.5%). The species contributing to these fragment sizes were Pseudomonas sp., Rhodococcus sp. and F. columnare, respectively. A specific PCR for detection of Pseudomonas sp. MT5 was designed, but none of the tissue samples were found to be positive, most likely indicating poor adhesion of the strain during bathing. LH-PCR was found to be a more powerful tool for detecting F. columnare in fish tissue than traditional culture methods (chi2 = 3.9, df = 1, p < 0.05). Antagonistic baths had no effect on the outbreak of infection or on fish mortality. F. columnare was also detected in healthy fish prior to and after experimental infection, indicating that these fish were carriers of the disease. In the second experiment, intensive Pseudomonas sp. MT5 antagonistic baths were given daily to rainbow trout suffering from a natural columnaris infection. Again, the antagonistic bacteria had no effect on fish mortality, which reached 95 % in both control and antagonist-treated groups in 7 d.

Animals↗

PubMed2005

Early interactions of Flavobacterium psychrophilum with macrophages of rainbow trout Oncorhynchus mykiss.

The early interactions of a low and a highly virulent Flavobacterium psychrophilum strain with head kidney and spleen macrophages of rainbow trout Oncorhynchus mykiss were characterized. The highly virulent strain was killed 5.8 to 11 times less frequently than the low virulent strain. The head kidney macrophages showed a microbicidal activity approximately twice as high as that of the spleen macrophages. A 2- to 3-fold higher production of reactive oxygen species (ROS) was induced by the highly virulent strain than by the low virulent one. The head kidney macrophages produced approximately twice as much ROS as the spleen macrophages. The low virulent strain was killed approximately 10 times more frequently by H2O2 than was the highly virulent strain. In spleen macrophages, the highly virulent strain caused twice as much cytotoxic effects compared to the low virulent strain. In conclusion, virulence in F. psychrophilum appears to be correlated with higher O. mykiss macrophage cytotoxicity and resistance to ROS and, therefore, with enhanced resistance to bacterial killing. Moreover, due to lower ROS production, spleen macrophages have a lower antimicrobial action against F. psychrophilum, compared to head kidney macrophages and, thus, might form a 'safe site' in which bacteria can reside.

Animals↗