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.     

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.     

Lipopolysaccharide O-antigen antibody-based detection of the fish pathogen Flavobacterium psychrophilum

Several yellow-pigmented species within the family Flavobacteriaceae are commonly associated with diseases in fish and are difficult to speciate due to their fastidious, slow-growing nature and cross-reactive antigens. Here we report the development of specific, antibody-diagnostic tests for Flavobacterium psychrophilum, the aetiological agent of rainbow trout fry syndrome and bacterial cold water disease. A unique antigen from F. psychrophilum, the lipopolysaccharide (LPS) O-polysaccharide (O-PS), formed the basis for the antibody test. LPS O-PS was purified and conjugated to keyhole limpet haemocyanin and bovine serum albumin for the generation of rabbit immune sera and the development of antibody-based diagnostic tests. Rabbit polyclonal anti-O-PS serum was highly specific for F. psychrophilum, without the need for prior cross-absorption with related bacteria and was the basis of an effective ELISA diagnostic test. Antibodies were purified from rabbit anti-O-PS serum and adsorbed onto coloured latex beads for the development of a specific, bead agglutination assay for F. psychrophilum.     

Identification of a ribosomal L10-like protein from Flavobacterium psychrophilum as a recombinant vaccine candidate for rainbow trout fry syndrome

The psychrophilic bacterium Flavobacterium psychrophilum is a rapidly emerging, virulent pathogen of a variety of commercially important finfish species, including salmonids. No vaccines against F. psychrophilum are currently available, partly due to its recalcitrant growth in vitro. Consequently, we explored the possibility of constructing recombinant vaccines in Escherichia coli as a prophylactic biotechnological strategy to counter F. psychrophilum infections. An immunoreactive clone from a F. psychrophilum expression library was found to express a approximately 16 kDa protein antigen. A proteomics approach was taken to identify the ORF encoding the approximately 16 kDa protein. Tryptic fragments of the approximately 16 kDa protein were analyzed by MALDI-TOF mass spectrometry and compared to theoretical (in silico) tryptic fragments of translated ORFs predicted within the cloned DNA. The target protein was identified as a 166 amino acid protein (named 7-166) with homology to rplJ which encodes bacterial ribosomal protein L10. Whenhighly expressed in E. coli as an N-terminal fusion protein, this chimera reacted with convalescent rainbow trout serum. When adjuvanted and administered intraperitoneally to immature rainbow trout a high level of protection (82% RPS) was afforded against virulent F. psychrophilum challenge; thus establishing F. psychrophilumrplJ homologue 7-166 as a promising vaccine candidate for RTFS.     

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.     

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.     

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     

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.     

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.     

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.     

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.     

The structure of the lipopolysaccharide O-antigen produced by Flavobacterium psychrophilum (259-93).

Flavobacterium psychrophilum, a Gram-negative bacterium, is the etiological agent of rainbow trout fry syndrome and bacterial cold water disease, septicemic infections in reared salmonids. In humans Flavobacterium spp. have been associated with neonatal meningitis and septicemia, catheter-associated bacteremia, and pneumonia. Recently, several F. psychrophilum surface molecules, including lipopolysaccharide (LPS), have been implicated in its pathogenesis and identified as potential vaccine and diagnostic candidate macromolecules. Studies on the LPS produced by the bacterium are reported herein. The structure of the antigenic O-polysaccharide contained in the LPS of F. psychrophilum was deduced by the application of analytical NMR spectroscopy, mass spectrometry, glycose and methylation analysis, and partial hydrolysis degradations, and was found to be an unbranched polymer of trisaccharide repeating units composed of L-rhamnose (L-Rhap), 2-acetamido-2-deoxy-L-fucose (L-FucpNAc) and 2-acetamido-4-((3S,5S)-3,5-dihydroxyhexanamido)-2,4-dideoxy-D-quinovose (D-Quip2NAc4NR, 2-N-acetyl-4-N-((3S,5S)-3,5-dihydroxyhexanoyl)-D-bacillosamine) (1 : 1 : 1) and having the structure: -->4)-alpha-L-FucpNAc-(1-->3)-alpha-D-Quip2NAc4NR-(1-->2)- alpha-L-Rhap-(1--> where R is (3S,5S)-CH3CH(OH)CH2CH(OH)CH2CO-.     

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.     

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.     

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.     

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.     

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.     

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.