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.     

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.     

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     

Identification of potential vaccine target antigens by immunoproteomic analysis of a virulent and a non-virulent strain of the fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS). To identify antigens associated with virulence or host immunity, we compared total and immunogenic proteins of cellular and extracellular products (ECP) between a virulent (CSF-259-93) and non-virulent (ATCC 49418) strain of F. psychrophilum. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis of total cellular proteins revealed only minor differences between the strains; however, separation of ECP showed that proteins were differentially expressed. Western blot analysis using rainbow trout (Oncorhynchus mykiss) anti-CSF-259-93 sera showed greater reactivity to proteins of the virulent strain, including many > 50 kDa. Further analysis by 2-dimensional electrophoresis (2DE) identified numerous differences between the strains. Western blot analysis combined with 2DE identified several immunogenic proteins that reacted with the antisera and were shared between the 2 strains. However, at least 15 immunogenic proteins appeared to be unique to the virulent strain, while 4 such proteins were identified in the non-virulent strain; 8 proteins unique to the virulent strain and 6 shared proteins were further analyzed for identification by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Of these, 3 immunogenic proteins (heat shock proteins HSP 60 and HSP 70) and 2 other proteins (ATP synthase and thermolysin) were conclusively identified. The 2 highly immunogenic heat shock proteins were shown to share extensive homology with heat shock proteins of related bacteria. This approach for antigen identification may provide a basis for targeted vaccine development against CWD and RTFS.     

The outer membrane fraction of Flavobacterium psychrophilum induces protective immunity in rainbow trout and ayu

Flavobacterium psychrophilum is the causative agent of coldwater disease, which is responsible for serious losses in fish aquaculture in several parts of the world. No commercial vaccines are currently available for the prevention of coldwater disease. The present study sought to assess the efficacy of a F. psychrophilum vaccine based on the antigenic outer membrane fraction (OMF). This fraction induced significantly higher protection against coldwater disease in both rainbow trout (Oncorhynchus mykiss) and ayu (Plecoglossus altivelis) compared to inactivated whole cell F. psychrophilum bacterin. Coincident with higher protection, sera of fish immunised with the OMF vaccine had higher agglutination titres than those of fish immunised with inactivated whole cell F. psychrophilum.     

Isolation and characterization of bacteriophages infecting the fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum is a serious pathogen in trout aquaculture, responsible for the diseases rainbow trout fry syndrome (RTFS) and cold water disease (CWD). Bacteriophage control of F. psychrophilum may constitute a realistic approach in the treatment of these diseases; however, a detailed understanding of the phage-host interactions is needed to evaluate the potential of F. psychrophilum bacteriophages for that purpose. Twenty-two F. psychrophilum phages from Danish rainbow trout farms were isolated and characterized. The phage genome sizes differed considerably and fell into three major size classes (8.5 to 12 kb, 48 kb, and 90 kb). The phage host ranges comprised from 5 to 23 of the 28 tested F. psychrophilum strains, and 18 of the phage isolates showed unique host ranges. Each bacterial strain had a unique pattern of susceptibility to the 22 phages, and individual strains also showed large variations (up to 10(7)-fold differences) in susceptibility to specific phages. Phage burst size (7 to 162 phages infected cell(-1)) and latency period (4 to 6 h) also showed pronounced differences both between phages and, for a specific phage, between host strains. In general, the characterization documented the presence of diverse F. psychrophilum phage communities in Danish trout farms, with highly variable patterns of infectivity. The discovery and characterization of broad-host-range phages with strong lytic potential against numerous pathogenic F. psychrophilum host strains thus provided the foundation for future exploration of the potential of phages in the treatment of RTFS and CWD.     

Involvement of a sialic acid-binding lectin with hemagglutination and hydrophobicity of Flavobacterium psychrophilum

Strains of Flavobacterium psychrophilum were studied for their ability to adhere and cause agglutination of erythrocytes and yeast cells. Strains of the serotype Th showed low or no hemagglutinating (HA) properties toward human, avian, bovine, and rainbow trout erythrocytes, whereas strains of serotype Fd and Fp(T) exhibited distinct HA properties. None of the strains was able to cause agglutination of yeast cells. Greater adherence specificity toward rainbow trout blood cells was seen for the HA-positive strains. Growth at 5 degrees C, compared to that at 15 degrees C, induced an increase in the hemagglutination of some strains. HA activities of F. psychrophilum were inhibited only by sialic acid (N-acetyl-neuraminic acid), heat treatment at 65 degrees C, and proteinase K treatment and not by any of seven other carbohydrates, periodate oxidation, or treatment with trypsin. The supernatant from washed bacterial cells also showed some HA properties. All strains were shown to be highly hydrophobic by the hydrophobic interaction chromatography test, although some contradictions to the results of the salt aggregation test (showing some strains as less hydrophobic) were seen. These results indicate that the aggregation of F. psychrophilum and erythrocytes depend on a lectin present on the surface of HA-positive F. psychrophilum strains and absent on HA-negative strains. This lectin reacts specifically with sialic acid. The adhesion differences observed for F. psychrophilum strains do not appear to correlate with the virulence but still provide insights into the interaction of F. psychrophilum and rainbow trout.     

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.     

Detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification

Rainbow trout fry syndrome and cold-water disease, caused by Flavobacterium psychrophilum, are important diseases in farmed salmonids. Some of the presently available techniques for the detection of Fl. psychrophilum are either time consuming or lack sufficient sensitivity. In the present investigation, the possible detection of Fl. psychrophilum from fish tissue and water samples was examined using nested PCR with DNA probes against a sequence of the 16S rRNA genes. The DNA was extracted using Chelex(R) 100 chelating resin. The primers, which were tested against strains isolated from diseased fish, healthy fish, fish farm environments and reference strains, proved to be specific for Fl. psychrophilum. The obtained detection limit of Fl. psychrophilum seeded into rainbow trout brain tissue was 0.4 cfu in the PCR tube, corresponding to 17 cfu mg-1 brain tissue. The PCR-assay proved to be more sensitive than agar cultivation of tissue samples from the brain of rainbow trout injected with Fl. psychrophilum. In non-sterile fresh water seeded with Fl. psychrophilum the detection limit of the PCR-assay was 1.7 cfu in the PCR tube, corresponding to 110 cfu ml-1 water. The PCR-assay detected Fl. psychrophilum in water samples taken from a rainbow trout farm, but Fl. psychrophilum could not be isolated using inoculation on selective agar. The method presented here has the potential to detect low levels of Fl. psychrophilum in fish tissue and in water samples, and the technique can be a useful tool for understanding the epidemiology of Fl. psychrophilum.     

Association of Flavobacterium psychrophilum strains with intestinal explants of rainbow trout Oncorhynchus mykiss

Flavobacterium psychrophilum is an important pathogen in rainbow trout Oncorhynchus mykiss. The portal of entry for F. psychrophilum is not well known. In this study, the role of the intestine as a colonization site for F. psychrophilum was determined. For this purpose, the ability of a high (Dubois) and a low (99/10A) virulence strain of F. psychrophilum to adhere to intestinal explants of rainbow trout was evaluated. After incubation, samples of the gut were examined bacteriologically, histologically and by electron microscopy. The number of gut-associated F. psychrophilum bacteria was significantly higher for the Dubois than for the 99/10A strain. Histological samples clearly showed numerous bacteria of the high virulence strain associated with the intestinal tissue as opposed to only a few bacteria of the low virulence strain. Additionally, extensive exfoliation of intestinal epithelium was noted after incubation with the high virulence strain, but less with the low virulence strain. These findings were confirmed using scanning electron microscopy and suggest that the intestinal epithelium might represent an important site for colonization of the F. psychrophilum strain and may act as a portal of entry for high virulence F. psychrophilum.     

Electrophoretic and Western blot analyses of the lipopolysaccharide and glycocalyx of Flavobacterium psychrophilum

Flavobacterium psychrophilum is the aetiological agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS) and it has emerged as one of the most significant bacterial pathogens in salmonid aquaculture worldwide. Previous studies have suggested that the O-polysaccharide (O-PS) component of the lipopolysaccharide (LPS) of F. psychrophilum is highly immunogenic and may be involved in eliciting a protective immune response in rainbow trout (Oncorhynchus mykiss Walbaum). In the present study, SDS-PAGE and Western blotting techniques were used to analyse the carbohydrate antigens of F. psychrophilum. Our analysis identified two distinct carbohydrate-banding patterns. One banding pattern corresponds with LPS, and we hypothesise that the other carbohydrate-banding pattern is that of the loosely associated glycocalyx of F. psychrophilum. Electron microscopy of F. psychrophilum cells immunogold labelled with a monoclonal antibody specific for this banding pattern supports this hypothesis as the outermost layer of the bacterium was heavily labelled. This is a significant finding because the immunogenic antigens that have been referred to as the O-PS of LPS, and implicated as potential vaccine candidate antigens, appear to be components of the glycocalyx of F. psychrophilum. This research suggests that the glycocalyx of F. psychrophilum may be an important antigen to consider for the development of a vaccine to control CWD and RTFS.     

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.     

Phenotypic and genotypic analysis of Flavobacterium psychrophilum isolates from Ontario salmonids with bacterial coldwater disease

Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome. BCWD has a considerable economic impact on aquaculture operations in Ontario, Canada, and our limited understanding of the population structure and epidemiology of F. psychrophilum isolates is an impediment to the development of improved management strategies. Seventy-five 16S rRNA gene and gyr polymerase chain reaction positive isolates of F. psychrophilum that had been collected over a 16-year period from farmed salmonids with tail rot, necrotic myositis, and osteochondrosis were characterized morphologically, biochemically, and genotypically. Although the isolates were homogeneous by preliminary biochemical and phenotypic characterization, two distinct biovars were found by API ZYM testing. As well, four restriction pattern types were detected by 16S rRNA polymerase chain reaction - restriction fragment length polymorphism analysis and there was a significant (P < 0.001) correlation between biovar I and digestion with MaeIII and between biovar II and digestion with MnlI or no site (P < 0.05). Further heterogenity was detected by sequence analysis of a 194 bp stem loop 3 region of rRNA. Nine sequence types were identified; 40/46 biovar I isolates were sequence type "a", while 21/32 biovar II isolates belonged to either sequence type "c" or "d". More than one biovar and genotype was identified among the strains recovered from separate fish sampled from three groups of rainbow trout (Oncorhynchus mykiss) experiencing BCWD mortality events. No association was found between genotype or biovar and type of disease. Taken together, these data suggest that F. psychrophilum from Ontario can be grouped into two major lineages based on biovar and 16S rRNA polymorphisms, and although three major strain types were most frequently isolated in this study, it appears that the population of F. psychrophilum with pathogenic potential is quite heterogeneous.     

Relationship between gyrA mutations and quinolone resistance in Flavobacterium psychrophilum isolates

Flavobacterium psychrophilum is the causative agent of the fish diseases called bacterial cold-water disease and rainbow trout fry syndrome. It has been reported that some isolates of F. psychrophilum are resistant to quinolones; however, the mechanism of this quinolone resistance has been unexplained. In this study, we examined the quinolone susceptibility patterns of 27 F. psychrophilum strains isolated in Japan and the United States. Out of 27 isolates, 14 were resistant to both nalidixic acid (NA) and oxolinic acid (OXA), and the others were susceptible to NA and OXA. When amino acid sequences deduced from gyrA nucleotide sequences of all isolates tested were analyzed, two amino acid substitutions (a threonine residue replaced by an alanine or isoleucine residue in position 83 of GyrA [Escherichia coli numbering] and an aspartic acid residue replaced by a tyrosine residue in position 87) were observed in the 14 quinolone-resistant isolates. These results strongly suggest that, as in other gram-negative bacteria, DNA gyrase is an important target for quinolones in F. psychrophilum.     

Development and application of a nested PCR to monitor brood stock salmonid ovarian fluid and spleen for detection of the fish pathogen Flavobacterium psychrophilum

AIMS: To develop a nested PCR to detect Flavobacterium psychrophilum based on the intergenic spacer region 16S-23S rRNA and in 16S rRNA for analysis of brood stock salmonid fish samples. METHODS AND RESULTS: The sensitivity and specificity of the test was evaluated using pure cultures, spiked and naturally contaminated samples. Samples were internal organs (spleen and kidney), eggs and ovarian fluid from rainbow trout and coho salmon from European fish farms (France, Spain). This nested PCR was more specific and sensitive that the nested PCR based on 16S rRNA sequences primers only. The detection limit of this PCR assay was one bacterium per PCR tube corresponding to 10 bacteria/mg of spleen and 5 bacteria/ml from ovarian fluid. Analysis of mixed ovarian fluid samples from reproductive salmonids in various French hatcheries demonstrated that 69% of hatcheries were contaminated with Fl. psychrophilum. The analysis of individual samples demonstrated that 39% of rainbow trout (Oncorhynchus mykiss) and 62.5% of coho salmon (O. kisutch) samples were contaminated. CONCLUSIONS: The results demonstrated a very sensitive and specific detection of this fish pathogen and that most of the female rainbow trout and coho salmon breeders analysed carry Fl. psychrophilum in the ovarian fluid. SIGNIFICANCE AND IMPACT OF THE STUDY: The understanding of Fl. psychrophilum dissemination and transmission and the detection of asymptomatic carriers is important for the development of free breeders stock and for significantly decreasing Flavobacteriose.     

Specificity of DNA vaccines against the U and M genogroups of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss)

Infectious hematopoietic necrosis virus (IHNV) is a fish rhabdovirus that causes significant mortality in salmonid species. In North America IHNV has three major genogroups designated U, M, and L. Host-specificity of the M and U genogroups of IHNV has been established both in the field and in experimental challenges, with M isolates being more prevalent and more virulent in rainbow trout (Oncorhynchus mykiss), and U isolates being more prevalent and highly virulent in sockeye salmon (Oncorhynchus nerka). In this study, efficacy of DNA vaccines containing either M (pM) or U (pU) virus glycoprotein genes was investigated during intra- and cross-genogroup challenges in rainbow trout. In virus challenges at 7 days post-vaccination (early antiviral response), both pM and pU were highly protective against either M or U IHNV. In challenges at 28 days post-vaccination (specific antiviral response), both pM and pU were protective against M IHNV but the homologous pM vaccine was significantly more protective than pU in one of two experiments. At this stage both pM and pU induced comparably high protection against U IHNV challenge. Correlates of protection were also investigated by assessing the expression of the interferon-stimulated gene Mx-1 and the production of neutralizing antibodies (NAbs) following pM or pU DNA vaccination. Mx-1 gene expression, measured at 4 and 7 days post-vaccination as an indicator of the host innate immune response, was found to be significantly higher after pM than pU vaccination in some cases. Neutralizing antibody was produced in response to the two vaccines, but antibody titers did not show consistent correlation with protection. The results show that the rainbow trout innate and adaptive immune responses have some ability to distinguish between the U and M genogroup IHNV, but overall the pM and pU vaccines were protective against both homologous and cross-genogroup challenges.     

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).     

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.