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.     

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.     

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     

The Flavobacterium psychrophilum OmpA, an outer membrane glycoprotein, induces a humoral response in rainbow trout

AIMS: The purpose of this study was to characterize OmpA, a major glycoprotein isolated from the membrane fraction of Flavobacterium psychrophilum, and to evaluate its potential as antigenic unit in a possible vaccine. METHODS AND RESULTS: The expression product of ompA is a 465-amino-acid protein precursor that contains a 21-amino acid signal peptide and has overall homology (up to 60% identity) with similarly sized proteins of some bacteria belonging to the Flavobacteriaceae family. The carboxy-terminal region contains the 'OmpA/MotB' domain/signature and five putative 'Thrombospondin type 3 repeats' domains have been identified in the central region. OmpA was clearly detected in the outer membrane fraction and its surface exposure was demonstrated. OmpA is one of the immunodominant antigens and binding of specific anti-OmpA antibodies lead to cell lysis in the presence of complement. Fish immunized with OmpA emulsified with Freund's adjuvant developed a high antibody titter. CONCLUSIONS: Collectively, the data obtained here indicate that OmpA may be involved in Fl. psychrophilum/host cell interactions and appears to be a potential immunogen for a vaccine. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is one step in the direction of understanding pathogenesis of Fl. psychrophilum and development of future vaccine.     

A Protective Immune Response Is Generated in Rainbow Trout by an OmpH-Like Surface Antigen (P18) of Flavobacterium psychrophilum

Investigations of the surface characteristics of Flavobacterium psychrophilum, an important pathogen of fish, assisted us in identifying a surface protein termed P18. In the current study, we developed a simple and efficient procedure for the purification of this protein by a two-step method. First, P18 was selectively released from flavobacteria by a heat-HEPES treatment of the cells and then subjected to anion-exchange high-performance liquid chromatography. De novo sequencing was used to generate a fragmented peptide spectrum from purified P18. Comparison of two obtained peptide sequences with a partial genome sequence of F. psychrophilum (INRA, Jouy-en-Josas, France) identified one gene encoding a 166-amino-acid OmpH-like protein that mostly likely undergoes N-terminal cleavage of the 23-residue signal peptide. The susceptibility of the OmpH-like protein to proteinase K treatment and the bacteriostatic/bactericidal activities of anti-OmpH-like protein antibodies indicated that this protein is actually exposed on the surface of F. psychrophilum. Vaccination trials showed that the OmpH-like protein can induce a high titer of anti-OmpH-like protein antibodies which are protective. Taken together, these results suggest that this surface protein produced by F. psychrophilum could be used in future vaccine development as a promising candidate antigen.     

Identification of P18, a surface protein produced by the fish pathogen Flavobacterium psychrophilum

AIMS: This study was focused on the identification of associated outer membrane proteins which may play a role in the specific interactions between Flavobacterium psychrophilum (the aetiological agent of cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide) and the fish tissues. METHODS AND RESULTS: The surface protein interactions with the outer membrane being mainly ionic, different methods were used for the detachment of proteins from the cell surface of Fl. psychrophilum involving detergent-free buffers or solutions known to perturb the ionic interactions. Such treatments led to the isolation of a surface protein, named P18 in accordance with its relative molecular mass. The expression of P18 was not related to the growth conditions (liquid or solid medium, temperature and aeration) or the strains of Fl. psychrophilum tested here. CONCLUSIONS: Preliminary characterization indicated that P18 is a surface antigen which is not sugar-modified and might be a subunit of a surface layer (i.e. S-layer), one of the most common surface structures on bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Data reported here should be used as the basis for further works involving the purification and characterization of P18 to identify the specific roles of such a surface protein, especially the interaction between this protein and the host surface.     

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.     

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.     

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.     

DNA vaccines as a tool for analysing the protective immune response against rhabdoviruses in rainbow trout

DNA vaccines based on the glycoprotein genes of the salmonid rhabdoviruses VHSV and IHNV have been demonstrated to be very efficient in inducing a protective immune response against the respective diseases in rainbow trout. Nanogram doses of plasmid DNA delivered by intramuscular injection are sufficient to induce high levels of immunity in fingerling-size fish, whereas larger fish require more vaccine for protection. The protection is long lasting and, more surprisingly, is partly established already 4 days post vaccination. The early protection involves cross-protective anti-viral defence mechanisms, while the long duration immunity is highly specific. The nature of these immune response mechanisms is discussed and it is suggested that the efficacy of the vaccines is related to their ability to activate the innate immune system as it is activated by live virus.     

Effects of dietary Ergosan on cutaneous mucosal immune response in rainbow trout (Oncorhynchus mykiss)

The effects of dietary Ergosan on the growth performance and mucosal immunity in rainbow trout skin were investigated. 60 rainbow trout (100-110 g) were randomly assigned to 2 groups in triplicates and fed one of the experimental diet formulated with 5 g kg⁻¹ Ergosan or control diet for 50 days. Results showed that on the 45th day of feeding trial, Ergosan supplementation significantly enhanced the growth performance compared to control group. Various enzyme activities, namely lysozyme, protease, alkaline phosphatase and esterase in treatment group were also enhanced on the 45th and 50th day. Skin mucus in Ergosan-fed fish showed the agglutination of erythrocytes while in control group, no visible agglutination was shown. In addition, skin mucus in treatment group showed strong antibacterial activity against Yersinia ruckeri. In conclusion, the major immune components of rainbow trout mucus that are involved in the non-specific immunity were enhanced by administration of Ergosan in 5 g kg⁻¹.     

Cercariae and diplostomules of Diplostomum spathaceum (Digenea) elicit an immune response in rainbow trout, Salmo gairdneri Richardson

The immune response of rainbow trout to cercariae and diplostomules of Diplostomum spathaceum was investigated. Following immunization, serum was tested for the presence of antibody, using an enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. Circulating antibody was detected to both cercariae and diplostomules by 6–7 weeks postimmunization and the sera were tested for their cross-reactivity with the other larval stage, using an ELISA.     

Characterisation of mucosal and systemic immune responses in rainbow trout (Oncorhynchus mykiss) using surface plasmon resonance

Mucosal and systemic antibody production in rainbow trout, Oncorhynchus mykiss (Walbaum), was evaluated following different antigen delivery routes. A BIAcore instrument (Pharmacia) allowed direct detection of antibody-antigen interactions by surface plasmon resonance changes. These interactions were measured in real-time without secondary reagents or extraneous labels. Groups of rainbow trout were immunised with a hapten-carrier antigen consisting of fluorescein isothiocyanate (FITC) conjugated to keyhole limpet haemocyanin (KLH) or phosphate buffered saline (PBS) pH 7.2. Antigens were administered intraperitoneally (i.p.) with or without Freund's complete adjuvant (FCA) or peranally (p.a.) directly to the gastrointestinal (GI) tract. Serum and mucosal anti-FITC responses were significantly (P<0.05) higher in FITC-KLH/FCA groups, clearly showing that adjuvant incorporation enhances mucosal as well as sytemic immunity. Antigen uptake and processing in fish immunised p.a. and i.p. without adjuvant was much less efficient and resulted in relatively low levels of serum and mucosal antibody production. Interestingly, mucosal responses in these groups peaked prior to serum responses suggesting possible early stimulation of mucosal defences. Mucosal antibody production in fish receiving FITC-KLH/FCA correlated more closely with serum responses, indicating possible transfer of serum derived antibody to mucosal sites. Mucosal and serum responses were confirmed as immunoglobulin (Ig) by subsequent reactivity with an anti-trout serum IgM monoclonal antibody (1.14) passed over flow cells containing anti-FITC antibodies. Further analysis showed significantly lower (P<0.05) reactivity of early mucus anti-FITC components (4 weeks post-immunisation) to 1.14. Purified serum and mucus Ig from non-immunised fish showed different protein banding patterns by SDS-PAGE under reducing conditions. Immunoblotting with 1.14 also showed weak reactivity to mucus Ig in control fish while reacting strongly to mucus Ig from immunised fish. These data suggest that early mucosal responses in trout may consist of heterogeneous forms of Ig differing in characteristics to serum Ig. BIAcore analysis in this context and as a means of measuring antibody response proved useful, and has the potential to become a valuable new tool in the study of fish immunology.     

Innate immune responses in rainbow trout (Oncorhynchus mykiss, Walbaum) induced by probiotics

Carnobacterium maltaromaticum B26 and Carnobacterium divergens B33, which were isolated from the intestine of healthy rainbow trout (Oncorhynchus mykiss, Walbaum), were selected as being potentially useful as probiotics with effectiveness against Aeromonas salmonicida and Yersinia ruckeri. Thus, rainbow trout administered with feed supplemented with B26 or B33 dosed at >10(7) cells g(-1) feed conferred protection against challenge with virulent cultures of the pathogens. Moreover, both cultures persisted in the gut for up to 3 weeks after administration. The cultures enhanced the cellular and humoral immune responses. Specifically, fish fed with B26 demonstrated significantly increased phagocytic activity of the head kidney macrophages, whereas the use of B33 led to significant increases in respiratory burst and serum lysozyme activity. Also, the gut mucosal lysozyme activity for fish fed with both cultures was statistically higher than the controls.     

Enhancement of protective immune response to recombinant Toxoplasma gondii ROP18 antigen by ginsenoside Re

Toxoplasma gondii, the etiological agent of toxoplasmosis, is an obligate intracellular protozoan parasite that infects a variety of mammals including humans. In an attempt to find new antigen-adjuvant combinations that enhance the immunogenicity of antigen candidates for toxoplasma vaccines, we analyzed the potent protection in mice immunized with recombinant protein ROP18 when co-administered with ginsenoside Re, a most important component isolated from Panax ginseng. All immunized mice produced specific anti-rROP18 immunoglobulins, with high levels of IgG antibody and a mixed IgG1/IgG2a response, with predominance of IgG1 production. The cellular and humoral immune responses were associated with the production of IFN-γ and IL-4 cytokines respectively. Vaccinated mice displayed a significantly increased survival time compared with control mice which died within 6 days of challenge with RH strain. Our data demonstrate that by addition of ginsenoside Re, the rROP18 triggered a stronger humoral and cellular response against T. gondii, and that Re is a promising vaccine adjuvant against toxoplasmosis, deserves further evaluation and development.     

Dietary organic chromium supplementation and its effect on the immune response of rainbow trout (Oncorhynchus mykiss)

Recently, there has been an increasing interest in the potential effect of dietary chromium on the health of fish, particularly with respect to their metabolism and growth. Information as to the role of this mineral on their immune response, is limited however, so the aim of this study was to assess the effects of dietary chromium yeast supplementation on the immune response of rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout (56 g average weight) were fed three semipurified diets containing different levels of chromium (1540, 2340 and 4110 ppb), obtained by supplementing a basal diet with 800 or 2570 ppb chromium yeast, for 6 weeks. After this, time differences in their immune response were examined. A positive influence was observed on serum lysozyme activity at this time in fish maintained on the high chromium diet. The respiratory burst of head-kidney macrophages was also examined, and statistical differences were found in the level of respiratory burst elicited by macrophages from both groups of fish fed supplemented chromium after 3 and 6 weeks of feeding (absorbance at 3 weeks: 0.118, 0.166. 0.151 and 6 weeks 0.114, 0.168, 0.151 for the 1540, 2340 and 4110 ppb groups). Macrophages of fish receiving diets supplemented with chromium also had a greater ability to phagocytose yeast after 6 weeks than the control fish (40.5, 48 and 48.5% macrophages phagocytic in the 1540, 2340 and 4110 ppb groups, respectively). The results of the study show that chromium yeast is able to modulate the immune response of rainbow trout, and this effect appears to be both dose- and time-dependent.     

Enhancement of the immune response and protection induced by probiotic lactic acid bacteria against furunculosis in rainbow trout (Oncorhynchus mykiss)

We analysed the effect of probiotic strains on the cellular and humoral immune responses of rainbow trout (Oncorhynchus mykiss), and their capacity to prevent furunculosis during a challenge trial. Probiotic strains (Lactococcus lactis ssp. lactis CLFP 100, Leuconostoc mesenteroides CLFP 196, and Lactobacillus sakei CLFP 202) were administered orally to fish for 2 weeks at 10(6) CFU g(-1) of feed. In comparison to untreated control fish, the phagocytic activity of head kidney leukocytes and the alternative complement activity in serum were significantly greater in all probiotic groups at the end of the second week. With the exception of the group fed with Lactobacillus sakei, superoxide anion production was also significantly increased in the probiotic groups. Analysis of lysozyme activity did not exhibit any significant difference in the probiotic and control groups. Fifteen days after the start of the probiotic feeding, fish were challenged with Aeromonas salmonicida ssp. salmonicida. The fish supplemented with probiotics exhibited survival rates ranging from 97.8% to 100%, whereas survival was 65.6% in fish not treated with the probiotics. These results demonstrate that probiotic supplementation to fish can reduce the severity of furunculosis, and suggest that this reduction may be associated with enhanced humoral and cellular immune response.     

Oxidative stress generated by dietary Zn-deficiency: studies in rainbow trout (Oncorhynchus mykiss).

To date, there is scarce information on the metabolic and biochemical repercussions of Zn-deficiency in fish. In this work, the effect of dietary Zn-deficiency on the diet utilization and the metabolism of activated oxygen species in rainbow trout (Oncorhynchus mykiss) has been studied. Fish were randomly separated in different lots according to their Zn-starvation and diet intake. In crude extracts of liver, gut and muscle, total and isoenzymatic superoxide dismutase and catalase activities were analysed. Lipid peroxidation was also determined in the same tissues. Western blotting was performed using antibodies against manganese- and copperzinc-containing superoxide dismutase. Lots fed on the Zn-deficient diet and with low intake showed significantly lower weight gain and feed-conversion efficiency indexes than control trouts. However, these parameters returned to control values when trouts were recovered by feeding them a control diet ad libitum. In control trouts, three independent copperzinc superoxide dismutase isozymes were detected in liver, whereas only one isozyme was present in the other lots. However, by Western blotting analysis the presence of a manganese superoxide dismutase was found in liver from all lots except in control trouts. Catalase activity and lipid peroxidation values were mainly detected in liver and gut, respectively, and both parameters increased in all lots with respect to the control group. Our results thus suggest that in rainbow trout an oxidative stress appears to occur as a consequence of Zn-deficiency.