Association of Flavobacterium psychrophilum with rainbow trout (Oncorhynchus mykiss) kidney phagocytes in vitro

The capacity of virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes to associate with isolated rainbow trout (Oncorhynchus mykiss, 300-500 g) kidney phagocytes was evaluated in vitro. The results showed that F. psychrophilum was associated with the phagocytes but large differences in association were observed between the different bacterial strains examined. These differences in association with the phagocytes was not clearly related to the serotype or virulence of the bacteria, although all strains tested of the non-virulent serotype FpT showed strong association with the isolated phagocytes. A competitive association assay with treatment of the phagocytes with seven different carbohydrates, suggested a role for N-acetylneuraminic acid (sialic acid) in the binding of F. psychrophilum to phagocytes. A significant dose dependent inhibition of the association was observed with sialic acid. Treatment of F. psychrophilum with sodium-metaperiodate showed that carbohydrate components play a role in the adhesion of the bacteria to the phagocytes. The results indicate that the binding of F. psychrophilum to rainbow trout kidney phagocytes can be mediated by opsonin independent cell-receptor adhesion. All tested strains seemed to be non-cytotoxic for rainbow trout kidney phagocytes in vitro suggesting that a phagocyte toxin is not necessary for the virulence of F. psychrophilum     

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.     

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.     

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.     

Survival of Flavobacterium psychrophilum in rainbow trout (Oncorhynchus mykiss) serum in vitro

Virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes were examined for survival and growth in non-immune and immune rainbow trout serum, in vitro. A majority of the examined strains consumed complement of non-immune serum, but the complement cascade was not able to cause an immediate (after 3 h incubation) notable reduction in viability of the inoculated cells. After 24 h incubation a more pronounced reduction in the number of viable bacteria was observed in untreated serum as well as in serum heated at 45 degrees C. In serum heated at 56 degrees C this reduction in cell number was not observed, but an increase in cell number did not occur either. The serum survival of one of the examined strains was different from the others in showing cell multiplication after 24 h incubation in normal as well as heat-treated (45 and 56 degrees C) serum. In immune serum no immediate reduction in viability of inoculated cells, of all tested strains, was observed. The number of viable cells showed a slow decrease or remained almost unchanged for up to 72 h post-inoculation in untreated serum, at 5 degrees C as well as 15 degrees C. In heat-treated serum (45 degrees C) the number of viable cells decreased slowly at 5 degrees C and 15 degrees C for up to 72 h. The results suggest that the examined strains were unaffected by the alternative complement reaction present in fish serum as well as by antibodies against F. psychrophilum. However, some unknown component(s) in the fish sera, or lack of nutrients or essential growth factors, inhibited the growth of most of the examined strains in the tested fish sera.     

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.     

Experimental bath infection with Flavobacterium psychrophilum, inducing typical signs of rainbow trout Oncorhynchus mykiss fry syndrome

Flavobacterium psychrophilum infection in salmonid fish, known as rainbow trout fry syndrome (RTFS) or bacterial coldwater disease (BCWD), is widespread in fish farms and natural waters. Despite many studies in which attempts at infection were made, an adequate method of infection has not yet been established. In this study, we evaluated a bath infection method in which we used bacteria at different stages of growth in the infection of rainbow trout Oncorhynchus mykiss. Rainbow trout with a mean body weight of 1.3 or 5.6 g, respectively, were infected by immersion in a bacterial suspension at different stages of growth (18 to 66 h shaking culture at 15 degrees C). The fish immersed in a logarithmic phase culture showed higher mortality than those in other culture phases. Indeed, 1.3 and 5.6 g fish showed typical clinical signs including ulcerative tissue of the trunk and lack of caudal fin edge. F. psychrophilum was detected by immunohistochemistry (IHC) in these tissue samples. These results indicate that experimental bath infection using a logarithmic phase bacterial solution is the most appropriate method for studies of infectious mechanisms.     

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.     

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.     

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.     

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.     

Adhesion of the fish pathogen Flavobacterium psychrophilum to unfertilized eggs of rainbow trout (Oncorhynchus mykiss) and n-hexadecane

AIMS: The ability of Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome (RTFS) in fish, to attach to unfertilized rainbow trout (Oncorhynchus mykiss) eggs and to hydrocarbon n-hexadecane was examined in the present study. METHODS AND RESULTS: Five different isolates of Fl. psychrophilum obtained from a variety of origins were compared. The effect of the age of the bacterium and conditions of starvation on the ability of the bacterium to adhere, were also evaluated. CONCLUSION: The different isolates were found to exhibit a similar ability to attach to both substrates. Increased surface hydrophobicity and a greater ability to attach to the surface of the eggs were observed with bacteria aged for one month, compared to bacteria cultured in Cytophaga agar for only three days. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide useful information regarding the pathogenicity of RTFS, especially during the initial steps of infection.     

Molecular analysis of intestinal microbiota of rainbow trout (Oncorhynchus mykiss)

The aim of this study was to evaluate different molecular tools based on the 16S rRNA gene, internal transcribed spacer, and the rpo B gene to examine the bacterial populations present in juvenile rainbow trout intestines. DNA was extracted from both pooled intestinal samples and bacterial strains. Genes were PCR-amplified and analysed using both temporal temperature gradient gel electrophoresis (TTGE) and restriction fragment length polymorphism methods. Because of the high cultivability of the samples, representative bacterial strains were retrieved and we compared the profiles obtained from isolated bacteria with the profile of total bacteria from intestinal contents. Direct analysis based on rpo B-TTGE revealed a simple bacterial composition with two to four bands per sample, while the 16S rRNA gene-TTGE showed multiple bands and comigration for a few species. Sequencing of the 16S rRNA gene- and rpo B-TTGE bands revealed that the intestinal microbiota was dominated by Lactococcus lactis, Citrobacter gillenii, Kluyvera intermedia, Obesumbacterium proteus , and Shewanella marinus . In contrast to 16S rRNA gene-TTGE, rpo B-TTGE profiles derived from bacterial strains produced one band per species. Because the single-copy state of rpo B leads to a single band in TTGE, the rpo B gene is a promising molecular marker for investigating the bacterial community of the rainbow trout intestinal microbiota.     

Spleen size predicts resistance of rainbow trout to Flavobacterium psychrophilum challenge

Selective breeding of animals for increased innate resistance offers an attractive strategy to control disease in agriculture. However, this approach is limited by an incomplete knowledge of the heritability, duration, and mechanism(s) of resistance, as well as the impact of selection on the immune response to unrelated pathogens. Herein, as part of a rainbow trout broodstock improvement program, we evaluated factors involved in resistance against a bacterial disease agent, Flavobacterium psychrophilum. In 2005, 71 full-sibling crosses, weighing an average of 2.4 g, were screened, and resistant and susceptible crosses were identified. Naive cohorts were evaluated at 10 and 800 g in size, and most maintained their original relative resistant or susceptible phenotypes, indicating that these traits were stable as size increased >300-fold. During the course of these studies, we observed that the normalized spleen weights of the resistant fish crosses were greater than those of the susceptible fish crosses. To test for direct association, we determined the spleen-somatic index of 103 fish crosses; created high, medium, and low spleen-index groups; and determined survival following challenge with F. psychrophilum or Yersinia ruckeri. Consistent with our previous observations, trout with larger spleen indices were significantly more resistant to F. psychrophilum challenge; however, this result was pathogen-specific, as there was no correlation of spleen size with survival following Y. ruckeri challenge. To our knowledge, this is the first report of a positive association between spleen size and disease resistance in a teleost fish. Further evaluation of spleen index as an indirect measure of disease resistance is warranted.     

Immersion exposure of rainbow trout (Oncorhynchus mykiss) fry to wildtype Flavobacterium psychrophilum induces no mortality,but protects against later intraperitoneal challenge

Flavobacterium psychrophilum, the causative agent of RTFS or rainbow trout fry syndrome, causes high mortality among hatchery reared rainbow trout (Oncorhynchus mykiss) fry in Europe and the USA. Despite several attempts, no efficient vaccines have yet been developed, the main obstacle being that the fry have to be vaccinated very early, i.e. around 0.2–0.5 g, where RTFS usually starts to give problems in the fish farms. Consequently, only oral or bath vaccines are relevant. Immersion of fry in inactivated or attenuated bacteria has resulted in RPS values of less than 50%. However, the results are biased by the fact that the fish have been challenged by intraperitoneal (ip) or subcutaneous (sc) injection against which an immersion/oral vaccine may not protect. Therefore, the present study was undertaken in order to investigate whether the presumably most potent immersion immunization, i.e. bathing in high titres of non-attenuated isolates of F. psychrophilum, was able to induce immunity to a subsequent ip challenge. Immersion in live bacteria for 30 or 50 min caused no mortality and protected a major fraction of the fry against challenges 26 and 47 days later with RPS values of 88.2 and 60.3%, respectively. Increased specific antibody titres suggested that adaptive immune mechanisms were involved in the protection.     

虹鳟对时间和地点的学习记忆(英文)

为确定虹鳟(Oncorhynchus mykiss)是否能将日投放两次食物的不同时间和地点联系起来,在30天内,于09:00-10:00将食饵投放在水族箱一侧,于15:00-16:00将食饵投放在水族箱另一侧.在第21天和28天,不放食饵以确定虹鳟对时间-地点的学习记忆.结果表明,虹鳟将不同时间和地点联系起来以获取食物,并且在水族箱两个投放食饵之处均表现出预取食活动,提示该物种具有控制时间-地点学习任务的内在机制.     

Microbial diversity of intestinal contents and mucus in rainbow trout (Oncorhynchus mykiss)

AIMS: The aim of this study was to understand the microbial community of intestinal contents and mucosal layer in the intestine of rainbow trout by means of culture-dependent conventional and independent molecular techniques. METHODS AND RESULTS: Forty-one culturable microbial phylotypes, and 39 sequences from 16S rRNA and two from 18S rRNA genes, were retrieved. Aeromonadaceae, Enterobacteriaceae and Pseudomonadaceae representatives were the dominant cultured bacteria. Genomic DNA isolated from intestinal contents and mucus was used to generate 104 random clones, which were grouped into 32 phylotypes at 99% minimum similarity, most of which were affiliated with Proteobacteria (>70% of the total). However, unlike library C (intestinal contents), the phyla Bacteroidetes and Fusobacteria were not found in intestinal mucus (library M), indicating that the microbiota in the gut mucus was different from that of the intestinal contents. Twelve sequences were retrieved from denaturing gradient gel electrophoresis analysis, and dominant bands were mostly related to Clostridium. CONCLUSIONS: Many novel sequences that have not been previously recognized as part of the intestinal flora of rainbow trout were retrieved. SIGNIFICANCE AND IMPACT OF THE STUDY: The fish gut harbours a larger bacterial diversity than previously recognized, and the diversity of gut mucus is different from that of intestinal contents.     

Flavobacterium oncorhynchi sp. nov., a new species isolated from rainbow trout (Oncorhynchus mykiss)

Eighteen isolates of a Gram-negative, catalase and oxidase-positive, rod-shaped bacterium, recovered from diseased rainbow trout (Oncorhynchus mykiss), were characterized, using a polyphasic taxonomic approach. Studies based on comparative 16S rRNA gene sequence analysis showed that that the eighteen new isolates shared 99.2-100% sequence similarities. Phylogenetic analysis revealed that isolates from trout belonged to the genus Flavobacterium, showing the highest sequence similarities to F. chungangense (98.6%), F. frigidimaris (98.1%), F. hercynium (97.9%) and F. aquidurense (97.8%). DNA-DNA reassociation values between the trout isolates (exemplified by strain 631-08(T)) and five type strains of the most closely related Flavobacterium species exhibited less than 27% similarity. The G+C content of the genomic DNA was 33.0 mol%. The major respiratory quinone was observed to be menaquinone 6 (MK-6) and iso-C(15:0), C(15:0) and C(16:1) ω7c the predominant fatty acids. The polar lipid profile of strain 631-08(T) consisted of phosphatidylethanolamine, unknown aminolipids AL1 and AL3, lipids L1, L2, L3 and L4 and phospholipid PL1. The novel isolates were differentiated from related Flavobacterium species by physiological and biochemical tests. On the basis of the evidence from this polyphasic study, it is proposed that the isolates from rainbow trout be classified as a new species of the genus Flavobacterium, Flavobacterium oncorhynchi sp. nov. The type strain is 631-08(T) (= CECT 7678(T) = CCUG 59446(T)).     

Brain distribution of myosin Va in rainbow trout Oncorhynchus mykiss

This study presents data on myosin Va localization in the central nervous system of rainbow trout. We demonstrate, via immunoblots and immunocytochemistry, the expression of myosin Va in several neuronal populations of forebrain, midbrain, hindbrain and spinal cord. The neuronal populations that express myosin Va in trout constitute a very diverse group that do not seem to have many specific similarities such as neurotransmitters used, cellular size or length of their processes. The intensity of the immunoreactivity and the number of immunoreactive cells differ from region to region. Although there is a broad distribution of myosin Va, it is not present in all neuronal populations. This result is in agreement with a previous report, which indicated that myosin Va is approximately as abundant as conventional myosin II and kinesin, and it is broadly involved in neuronal motility events such as axoplasmatic transport. Furthermore, this distribution pattern is in accordance with what was shown in rats and mice; it indicates phylogenetic maintenance of the myosin Va main functions.     

Differences in MHC class I genes between strains of rainbow trout (Oncorhynchus mykiss)

In rainbow trout there is only one dominant classical MHC class I locus, Onmy-UBA, for which four very different allelic lineages have been described. The purpose of the present study was to determine if Onmy-UBA polymorphism could be used for strain characterisation. This was performed by lineage-specific PCR investigation of 30 fish, each of the Nikko and Donaldson strains, and by sequence analysis of 25 of the amplified DNA fragments. Two new MHC class I lineages were detected in addition to the four previously described lineages, thus six distinct lineages were observed within the fish examined (Sal-MHCIa*A-F). The distribution of lineages appeared to be strain-specific. For example, the lineage Sal-MHCIa*A was very common in the Nikko strain but could not be detected in the Donaldson strain. Analysis of MHC class I variation may help to elucidate relationships between strains and the roles of MHC alleles in disease resistance.