Immunostimulation of antibody-producing cells and humoral antibody to fish bacterins by a biological response modifier

Numbers of splenic antibody-producing cells and humoral antibody titres were elevated during immunization regimes in rainbow trout when the bacterins Yersinia ruckeri or Aeromonas salmonicida O-antigen preparations were mixed with the immunostimulator FK-565. Fish sampled 14 days after injection showed a marked increase in the immune response when doses of 5, 10 or 100 μg of antigen were used. The immunostimulator may aid initial antigen uptake and processing.     

Electron microscopic examination of antigen uptake by salmonid gill cells after bath immunization with a bacterin

Atlantic salmon, Salmo salar , were given 2-min bath immunization with Yersinia ruckeri O-antigen bacterins at doses of 10, 100, and 1000 μg ml⁻¹. The uptake of the antigen was followed by light and electron microscopy of samples taken immediately and periodically after immunization, and the immune response monitored by the passive haemolytic plaque assay. The particulate antigen was observed in the gill mucus, adhering to and within the pavement cells covering the gill filaments, and in mononuclear phagocytes below the epidermal gill cells. There was a direct doseresponse correlation in the observed immune response according to the numbers of splenic antibody-producing cells 14 days after immunization. The cells involved in the recognition and uptake of a bacterin are initial important steps in the immune response, and these studies may aid in the immunopotentiation of fish vaccines and bacterins.     

Cadmium exposure of rainbow trout, Salmo gairdneri Richardson: effects on immune functions

Differential leucocyte counts, phagocytosis, humoral antibody response and the in vitro blasto-genetic response to mitogens (lipopolysaccharide and Concanavalin A) and to an antigen ( Vibrio anguillarum ) were studied in rainbow trout exposed to 0,0.7 or 3.6 μg Cd 1⁻¹ for 12 weeks.
Although the fish did not exhibit any clinical or histological changes, cadmium exposure was found to affect two of the immune parameters measured. The cellular response of fish immunized with V. anguillarum to the homologous antigen was significantly lower for splenocytes obtained from fish exposed to cadmium for 9 weeks (3.6 μg Cd 1⁻¹ group) than for splenocytes obtained from non-exposed fish. Conversely, the humoral antibody response to V. anguillarum O-antigen was higher in the 3.6 μg Cd 1⁻¹ group than in the non-exposed group. Protective immunity of fish vaccinated against V. anguillarum was equally as good in the cadmium-exposed group as in the non-exposed group. No cadmium-induced changes in differential leucocyte counts or in the proportions of phagocytic cells were observed.     

The effect of sublethal endrin exposure on rainbow trout, Salmo gairdneri Richardson. II. The effect of altering serum cortisol concentrations on the immune response

The effect of altering serum cortisol concentrations on the immune response was elucidated in endrin- and non-endrin-exposed rainbow trout, Salmo gairdneri. Fish were immunized with 10 μg of Yersinia ruckeri O-antigen following 30 days of treatment. The migration inhibition factor assay (MIF), plaque-forming cell assay (PFC) and serum agglutination titres (SAG) were performed 2, 14 and 30 days post-antigen inoculation. Endrin exposure was continued subsequent to antigen inoculation. Control fish were fed 20 and 35 mg kg⁻¹ body weight day⁻¹ of cortisol and metyrapone, respectively. Endrin-exposed fish received 35 mg kg⁻¹ body weight day⁻¹ of metyrapone in their diet. Control fish receiving cortisol had significantly reduced MIF, PFC and SAG responses. The MIF response was completely restored in endrin-exposed fish receiving dietary metyrapone. The PFC response and SAG titres were partially restored, 61 and 69% respectively, in endrin-exposed fish receiving metyrapone. The results indicate that elevated serum cortisol concentration obtained in endrin-exposed fish has a central role in repression of the immune response.     

Denaturing gradient gel electrophoresis for nonlethal detection of Aeromonas salmonicida in salmonid mucus and its potential for other bacterial fish pathogens

Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA was used to nonlethally detect Aeromonas salmonicida and other bacteria in salmonid skin mucus. Mucus samples from wild spawning coho salmon (Oncorhynchus kisutch) with endemic A. salmonicida and from cultured lake trout (Salvelinus namaycush) were tested by PCR-DGGE and were compared with mucus culture on Coomassie brilliant blue agar and internal organ culture. PCR-DGGE gave a highly reproducible 4-band pattern for 9 strains of typical A. salmonicida, which was different from other Aeromonas spp. Aeromonas salmonicida presence in mucus was evident as a band that comigrated with the bottom band of the A. salmonicida 4-band pattern and was verified by sequencing. PCR-DGGE found 36 of 52 coho salmon positive for A. salmonicida, compared with 31 positive by mucus culture and 16 by organ culture. Numerous other bacteria were detected in salmonid mucus, including Pseudomonas spp., Shewanella putrefaciens, Aeromonas hydrophila and other aeromonads. However, Yersinia ruckeri was not detected in mucus from 27 lake trout, but 1 fish had a sorbitol-positive Y. ruckeri isolated from organ culture. Yersinia ruckeri seeded into a mucus sample suggested that PCR-DGGE detection of this bacterium from mucus was possible. PCR-DGGE allows nonlethal detection of A. salmonicida in mucus and differentiation of some Aeromonas spp. and has the potential to allow simultaneous detection of other pathogens present in fish mucus.     

The effect of sublethal endrin exposure on rainbow trout, Salmo gairdneri Richardson. I. Evaluation of serum cortisol concentrations and immune responsiveness

Two-hundred-and-forty rainbow trout, Salmo gairdneri , were exposed for 30 days to sublethal concentrations of the pesticide endrin. On day 30, ten fish from each treatment group were sacrificed and examined for the ability of peritoneal macrophages to phagocytize latex beads. The remaining fish were immunized with 10 μg of Yersinia ruckeri O-antigen, and exposure to endrin continued. The migration inhibition factor assay (MIF), plaque forming cell assay (PFC), and serum agglutination titres (SAG) were performed 2, 14, and 30 days post-antigen inoculation. Serum was collected from all fish for serum cortisol concentrations. Endrin exposure had no effect on the phagocytic ability of peritoneal macrophages. However, the MIF, PFC and SAG responses were significantly reduced from control values. Serum cortisol concentrations were found to be significantly elevated in endrin-exposed fish. Serum cortisol concentrations were found to be significantly higher on days 44 and 60 (192 and 194 ng ml⁻¹, respectively) when compared to days 30 and 32 (159 and 141 ng ml⁻¹, respectively). Cortisol values for days 30 and 32 did not differ significantly, nor did those of days 44 and 60. The relationship between elevated serum cortisol concentrations and endrin exposure on the immune response is discussed.     

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.     

Detection of Yersinia ruckeri in rainbow trout blood by use of the polymerase chain reaction

We evaluated a polymerase chain reaction (PCR) method for detecting Yersinia ruckeri, the bacterial pathogen causing enteric redmouth disease (ERM), in blood of rainbow trout Oncorhynchus mykiss. Identification of the PCR product was confirmed by Southern blot hybridization with a 32P-labeled oligonucleotide probe matching a sequence within the small subunit ribosomal RNA gene of Y. ruckeri. Following a 1 h immersion of rainbow trout in water with 4.5 x 10(6) colony-forming units of Y. ruckeri l(-1), the PCR was positive for all blood samples from 1 h (first sample) to 5 d and was negative from 9 to 30 d (last sample). Fish in this experiment did not show signs of disease, probably because they had been vaccinated against Y. ruckeri. To test this method with naturally infected fish, 42 rainbow trout from hatcheries were examined. Four of these fish had clinical signs of ERM and were infected with Y. ruckeri based on bacteriological culture. The PCR method detected Y. ruckeri in blood, intestine, liver, and trunk kidney from the 4 fish with ERM and from 5 additional rainbow trout that were bacteriologically negative for Y. ruckeri. Three of 5 rainbow trout from streams receiving effluent from hatcheries were positive for Y. ruckeri when tested with PCR, although there was no growth of Y. ruckeri on culture plates inoculated with the same samples. Samples were successfully stored for 1 wk in lysis buffer at 25 degrees C. This study demonstrated that a non-lethal blood sample can be used with PCR to detect Y. ruckeri.     

Direct and circulatory paths of Permethrin (NRDC-143) causing histopathological changes in the gills of rainbow trout, Salmo gairdneri Richardson

Rainbow trout were exposed to sublethal concentrations of Permethrin in water (0.09, 0.18 and 0.35 μg l⁻¹) and in food (85, 180 and 350 μg kg⁻¹ dry diet) in 20–0 day experiments. Histological changes in the gills included epithelial separation or necrosis, mucus cell hyperplasia, clubbing of epithelial cells or hyperplasia and fusion of adjacent secondary lamellae. These changes were noted in all fish, suggesting that the pesticide or its metabolites reached the gills not only directly through the water, but also indirectly via the circulation.     

Catecholamine release and interrenal response of brown trout, Salmo trutta, exposed to aluminium in acidic water

Cannulated brown trout, Salmo trutta , were exposed for 36 h to synthetic water with a low calcium content of pH 5 and similar synthetic water dosed with aluminium to raise the filterable A1 from 5 to 290 μg 1⁻¹ over the 36-h period. There were no significant disturbances of plasma concentrations of glucose, cortisol or catecholamine (adrenaline and noradrenaline) in fish held in water of pH 5. The addition of aluminium to this acidic synthetic water resulted in a generalized endocrine stress response with a four-fold increase in plasma glucose concentration after 18 h and a significant increase in plasma cortisol concentration from 24 h onwards when filterable A1 exceeded 200 μg 1⁻¹. Plasma catecholamine concentration indicated an adrenergic stress response in aluminium-exposed brown trout. A transient doubling in noradrenaline after 6 h in A1 was followed by a larger increase in both plasma adrenaline and noradrenaline concentrations in fish surviving the 36-h exposure to A1.     

Dietary administration of beta-mercapto-ethanol treated Saccharomyces cerevisiae enhanced the growth, innate immune response and disease resistance of the rainbow trout, Oncorhynchus mykiss

The effects of dietary whole cell yeast (Saccharomyces cerevisiae), n-3 HUFA-enriched yeast and treated yeast cells with beta-mercapto-ethanol (2ME) on immunity, growth performance and disease resistance to Yersinia ruckeri were investigated in Oncorhynchus mykiss. During 30 days, juvenile rainbow trout were fed diets supplemented with different forms of yeast at 5 × 10(7) CFU g(-1) or a control diet. After the feeding trial, remaining fish of each treatment were challenged by pathogenic Yersinia ruckeri and kept under observation for 14 days to record clinical signs and daily mortality rate. Yeast supplementation in all treatment groups significantly promoted the growth performance compared to control group. A significantly increase was also observed in immune responses in juvenile fish fed 2ME-treated yeast diet. More ever, the lowest fish mortality was obtained in this treatment group. The present results show that a diet supplemented with 2ME-treated yeast stimulates the immune system and growth of juvenile rainbow trout thus enhancing their resistance against Y. ruckeri.     

Bacterial-binding activity and plasma concentration of ladderlectin in rainbow trout (Oncorhynchus mykiss)

Soluble, defense lectins bind conserved microbial patterns leading to pathogen opsonization, enhanced phagocytosis and activation of complement. These immune functions, however, vary widely among individuals due to genetic and acquired differences affecting binding capacity or plasma concentration. Most evidence for the defensive function of soluble lectins is based on mammals, but several functionally homologous, but less well-characterized, lectins have been identified in fish. In this study, we compared binding of rainbow trout plasma ladderlectin to relevant, intact bacterial targets. A polyclonal antiserum raised against a synthetic peptide identical to the 20 N-terminal amino acids of the reduced 16 kDa rainbow trout ladderlectin subunit was used to detect plasma ladderlectin in immunoblots and indirect enzyme-linked immunosorbent assay (ELISA). Ladderlectin binding to Aeromonas salmonicida subsp. salmonicida, Aeromonas hydrophila, Yersinia ruckeri and Pseudomonas sp. was detected by PAGE and immunoblots of saccharide elutions from intact bacteria incubated in the presence of normal trout plasma. Although plasma concentrations of immunoreactive ladderlectin were low in the majority of trout, significant (P < 0.0001) variation between individual fish was observed in two separate populations. In addition, one population demonstrated a subset of individuals whose ladderlectin levels were approximately seven-fold higher than the population median. These findings indicate that rainbow trout have variable amounts of plasma ladderlectin capable of binding to the surfaces of several relevant bacterial targets.     

The interaction of trimethoprim and quinolones against Gram-negative fish pathogens

The effect of combination of trimethoprim with other non-sulphonamide antibacterial agents, in particular oxolinic acid and nalidixic acid, was evaluated against Gram-negative fish pathogens. The species included Aeromonas salmonicida, Yersinia ruckeri , some Vibrio spp. and Escherichia coli as a reference. The extent of synergy found by other workers with these substances against human Gram-negative bacteria was not apparent here. Some positive interaction between trimethoprim and oxolinic acid was found with Aer. salmonicida, Y. ruckeri and E. coli and between trimethoprim and nalidixic acid with Y. ruckeri in double disc diffusion tests but was not supported by fractional inhibitory concentration indices. The combinations were not effective in preventing emergence of resistance in passage on a drug gradient. Trimethoprim-resistant isolates of Aer. salmonicida were inhibited by low levels of oxolinic acid but the converse did not apply.     

The tissue localization of Aeromonas salmonicida in rainbow trout, Salmo gairdneri Richardson, following three methods of administration

The localization of a live, and a formalized vaccine preparation of Aeromonas salmonicida within the tissues of rainbow trout, Salmo gairdneri , was followed over a 5 day period. When presented by intraperitoneal injection, both the live bacteria and the vaccine localized in the spleen, liver, kidney and gut. When presented orally, the bacteria and the vaccine were confined almost exclusively to the gut region. Direct immersion resulted in low detectable levels within the tissues, with the spleen and kidney localizing the Aeromonas after its initial attachment to the outer surfaces of the fish.     

Characterization of an ADP-ribosyltransferase toxin (AexT) from Aeromonas salmonicida subsp. salmonicida

An ADP-ribosylating toxin named Aeromonas salmonicida exoenzyme T (AexT) in A. salmonicida subsp. salmonicida, the etiological agent of furunculosis in fish, was characterized. Gene aexT, encoding toxin AexT, was cloned and characterized by sequence analysis. AexT shows significant sequence similarity to the ExoS and ExoT exotoxins of Pseudomonas aeruginosa and to the YopE cytotoxin of different Yersinia species. The aexT gene was detected in all of the 12 A. salmonicida subsp. salmonicida strains tested but was absent from all other Aeromonas species. Recombinant AexT produced in Escherichia coli possesses enzymatic ADP-ribosyltransferase activity. Monospecific polyclonal antibodies directed against purified recombinant AexT detected the toxin produced by A. salmonicida subsp. salmonicida and cross-reacted with ExoS and ExoT of P. aeruginosa. AexT toxin could be detected in a wild type (wt) strain of A. salmonicida subsp. salmonicida freshly isolated from a fish with furunculosis; however, its expression required contact with RTG-2 rainbow trout gonad cells. Under these conditions, the AexT protein was found to be intracellular or tightly cell associated. No AexT was found when A. salmonicida subsp. salmonicida was incubated in cell culture medium in the absence of RTG-2 cells. Upon infection with wt A. salmonicida subsp. salmonicida, the fish gonad RTG-2 cells rapidly underwent significant morphological changes. These changes were demonstrated to constitute cell rounding, which accompanied induction of production of AexT and which led to cell lysis after extended incubation. An aexT mutant which was constructed from the wt strain with an insertionally inactivated aexT gene by allelic exchange had no toxic effect on RTG-2 cells and was devoid of AexT production. Hence AexT is directly involved in the toxicity of A. salmonicida subsp. salmonicida for RTG-2 fish cells.     

In vitro suppression of the phagocytic response of fish macrophages by tetracyclines

Tetracycline (TC) and oxytetracycline (OTC) caused a dose-dependent suppression of the chemiluminescence (CL) emitted by phagocytes from the kidney of rainbow trout, Salmo gairdneri , using zymosan or latex beads as the stimulus. Compared to the control response without antibiotics, partial but significant suppression was found after exposing the cells to OTC concentrations of 0.1–50.0 μg ml⁻¹. Cells exposed to 100 or 500 μg ml⁻¹ OTC showed CL responses below the base levels elicited by control cells. Comparable results were obtained with cells exposed to TC and stimulated by the same stimuli. The kinetics of the CL response and the suppressive effects of the antibiotics were similar in cells from individual fish but the magnitude of responses varied. No acclimation occurred following extended exposure to the drugs.     

Lipopolysaccharide O-antigen prevents phagocytosis of Vibrio anguillarum by rainbow trout (Oncorhynchus mykiss) skin epithelial cells

Colonization of host tissues is a first step taken by many pathogens during the initial stages of infection. Despite the impact of bacterial disease on wild and farmed fish, only a few direct studies have characterized bacterial factors required for colonization of fish tissues. In this study, using live-cell and confocal microscopy, rainbow trout skin epithelial cells, the main structural component of the skin epidermis, were demonstrated to phagocytize bacteria. Mutant analyses showed that the fish pathogen Vibrio anguillarum required the lipopolysaccharide O-antigen to evade phagocytosis and that O-antigen transport required the putative wzm-wzt-wbhA operon, which encodes two ABC polysaccharide transporter proteins and a methyltransferase. Pretreatment of the epithelial cells with mannose prevented phagocytosis of V. anguillarum suggesting that a mannose receptor is involved in the uptake process. In addition, the O-antigen transport mutants could not colonize the skin but they did colonize the intestines of rainbow trout. The O-antigen polysaccharides were also shown to aid resistance to the antimicrobial factors, lysozyme and polymyxin B. In summary, rainbow trout skin epithelial cells play a role in the fish innate immunity by clearing bacteria from the skin epidermis. In defense, V. anguillarum utilizes O-antigen polysaccharides to evade phagocytosis by the epithelial cells allowing it to colonize rapidly fish skin tissues.     

Recovery of a new biogroup of Yersinia ruckeri from diseased rainbow trout (Oncorhynchus mykiss,Walbaum)

Cultures of a new biogroup of Yersinia ruckeri, the causal agent of enteric redmouth (ERM), were recovered in England from diseased rainbow trout (Oncorhynchus mykiss, Walbaum), which had been previously vaccinated with a commercial ERM vaccine. The bacterial isolates were confirmed as Y. ruckeri by the results of sequencing the 16S rRNA, but differed from the characteristics of the taxon by positivity for the Voges Proskauer reaction and a general lack of motility, and could not be equated with any of the existing serovars. Cultures were pathogenic in laboratory-based infectivity experiments with 100% mortalities occurring in juvenile rainbow trout (average weight = 10 g) within 4-days of intraperitoneal or intramuscular injection with 10(5) cells/fish. Protection against disease was achieved using a formalin-inactivated whole vaccine prepared against a representative isolate.     

Simultaneous detection of Aeromonas salmonicida,Flavobacterium psychrophilum,and Yersinia ruckeri,three major fish pathogens,by multiplex PCR

A multiplex PCR assay based on the 16S rRNA genes was developed for the simultaneous detection of three major fish pathogens, Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri. The assay proved to be specific and as sensitive as each single PCR assay, with detection limits in the range of 6, 0.6, and 27 CFU for A. salmonicida, F. psychrophilum, and Y. ruckeri, respectively. The assay was useful for the detection of the bacteria in artificially infected fish as well as in fish farm outbreaks. Results revealed that this multiplex PCR system permits a specific, sensitive, reproducible, and rapid method for the routine laboratory diagnosis of infections produced by these three bacteria.