Immune enhancement in rainbow trout (Oncorhynchus mykiss) by potential probiotic bacteria (Lactobacillus rhamnosus)

The present study assessed the immune enhancement of fish by a lactic acid bacterium (LAB) Lactobacillus rhamnosus (ATCC 53103). The bacterium was administered orally at five different doses 7.9 x 10(4) (LAB4), 2.1 x 10(6) (LAB6), 2.8 x 10(8) (LAB8), 1.9 x 10(10) (LAB10) and 9.7 x 10(10) (LAB11) CFU/g feed to rainbow trout for two weeks and the feed was changed to un-supplemented diet. From the onset of feeding supplemented diets at 1, 2, 3 and 4 weeks, blood and mucus samples were taken. During the LAB feeding period L. rhamnosus persisted in the fish intestine and in the tank water in high numbers. However, L. rhamnosus disappeared from the intestine, skin mucus and tank water within one week after the change to the non-supplemented feed. In comparison to untreated control fish, respiratory burst activity of blood cells was raised significantly in the LAB4 treated group on week 2. Serum-mediated killing of Escherichia coli was increased significantly in group LAB6 on week 2. Serum immunoglobulin levels were significantly raised only in LAB8 group on week 1 and in LAB4 and LAB8 at the end of the trial. The results show that rainbow trout immune parameters were enhanced by using probiotic bacteria.     

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.     

Characterization of probiotic carnobacteria isolated from rainbow trout (Oncorhynchus mykiss) intestine

Aims:  To characterize two probiotic carnobacterial isolates, Carnobacterium maltaromaticum (B26) and C. divergens (B33), derived from rainbow trout ( Oncorhynchus mykiss ) intestine.
Methods and Results:  Both cultures, which were able to colonize the fish gut mucosal layer, comprised nonsporogenous, nonmotile, Gram-positive, catalase and oxidase-negative rods. The growth of both carnobacteria occurred between 0 and 37°C, in 0–10% (w/v) NaCl and at pH 5–10. Specifically, strain B26 grew in nutrient broth supplemented with 15% (w/v) NaCl. The most abundant cellular fatty acid of both cultures was 9-octadecenoic acid (18 : 1 n -9) (B26 = 52·6%; B33 = 40·6%), which was characteristic of Carnobacterium . Both cultures were inhibitory to Aeromonas salmonicida , Aer. hydrophila , Streptococcus iniae and Vibrio anguillarum , and strain B33 inhibited Listeria monocytogenes . Both carnobacteria, which did not contain plasmids, produced inhibitory compounds against Gram-positive and Gram-negative bacteria.
Conclusions:  Both probiotic cultures, B26 and B33, had unique phenotypic characteristics and showed a broad spectrum of antibiotic resistance against varying pathogenic bacteria.
Significance and Impact of the Study:  The results of this study contribute to new information and significance of carnobacterial species.     

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.     

Immunostimulant effects of Cotinus coggyria on rainbow trout (Oncorhynchus mykiss)

In this study, non-specific immune effects of tetra (Cotinus coggyria) on rainbow trout (Oncorhynchus mykiss) by dietary intake were investigated. Fish were fed daily ad libitum with diets containing 0.5% and 1.0% tetra for 3 weeks. After this period, fish were switched back to the basal diet for additional 6 weeks. Extracellular and intracellular respiratory burst activities, phagocytosis in blood leukocytes, lysozyme activities, and total plasma protein levels were evaluated at the end of the tetra feeding period and every 3 weeks during the basal diet period. Extracellular and intracellular respiratory burst activities, phagocytic activity, lysozyme activity and total protein level parameters of the groups containing 0.5% and 1.0% tetra were higher than the control group at the end of the 3rd, 6th and 9th weeks, respectively (P < 0.05). The highest values of the non-specific immune parameters were observed in the group fed with 1.0% tetra. Tetra groups did not show any significant difference (P > 0.05) in terms of specific growth rate and average weight of the fish.     

Expression of immune-related genes in rainbow trout (Oncorhynchus mykiss) induced by probiotic bacteria during Lactococcus garvieae infection

The aim of the present study was to investigate the effect of lactic acid bacteria (LAB) on the control of lactococcosis as well as to assess the impact of probiotics on the expression of immune-related genes in the head kidney and intestine of rainbow trout (Oncorhynchus mykiss). Lactobacillus plantarum, Lactococcus lactis and Leuconostoc mesenteroides, were administered orally at 10? CFU g?1 feed to fish for 36 days. Twenty-one days after the start of the feeding period, fish were challenged with Lactococcus garvieae. Only the fish fed the diet containing Lb. plantarum showed significantly (P < 0.05) improved protection against L. garvieae compared to the control. Subsequently, real-time PCR was employed to determine the mRNA levels of IL-1β, IL-8, IL-10 and TNF-α in the head kidney, and IL-8, Tlr5 and IgT in the intestine of the control and Lb. plantarum groups. IL-1β, IL-10 and TNF-α gene expression were significantly up-regulated by Lb. plantarum. Moreover, the mRNA levels of IL-10, IL-8 and IgT were significantly higher in the Lb. plantarum group after L. garvieae infection, suggesting that Lb. plantarum can stimulate the immune response of rainbow trout. PCR-DGGE revealed no detectable levels of the probiotics or the pathogen present on the distal intestinal mucosa. These findings demonstrate that direct probiotic-host interactions with the intestine are not always necessary to induce host stimulatory responses which ultimately enhance disease resistance. Furthermore, as L. garvieae did not colonise the intestinal tract, and therefore likely did not infect via this route, the antagonistic properties of the probiotic candidate towards L. garvieae were likely of little influence in mediating the improved disease resistance which could be attributed to the elevated immunological response.     

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.     

Cloning and characterization of glucose transporter in teleost fish rainbow trout (Oncorhynchus mykiss)

The facilitated diffusion of monosaccharides across the plasma membrane is mediated by glucose transporters (GLUTs). In contrast to mammals, the glucose transport system of lower vertebrates remains unexplored. We detected glucose transport activity in rainbow trout embryos. Two GLUTs sharing 83% amino acid identity were cloned from juvenile fish, these have been denoted OnmyGLUT1A and OnmyGLUT1B. In adult trout OnmyGLUT1A is predominantly expressed in the heart with low expression in other tissues. An inverse terminal repeat of a Tc1-like transposable element was found in the 3'-untranslated region of OnmyGLUT1B. Phylogenetic analysis suggested that rainbow trout genes share a common ancestor with higher vertebrate GLUT1. We also found GLUT genes in several salmonid species.     

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.     

Immunomodulatory effects of decaffeinated green tea (Camellia sinensis) on the immune system of rainbow trout (Oncorhynchus mykiss)

In order to study the immunomodulatory effects of decaffeinated green tea extract on rainbow trout, a study with a 30-day feeding trial was conducted. Commercial diets with graded levels of decaffeinated green tea extract, 20 mg (T1), 100 mg (T2), 500 mg (T3) per kg feed were prepared. 120 rainbow trout (35 ± 3 g) were randomly assigned to 4 groups in triplicates and fed one of the 3 experimental diets formulated or control diet. After feeding trial, 12 fish from each group were sampled for analysis of some immunological parameters. Remaining fish were injected with 0.5 ml of chicken red blood cell (C-RBC) suspension (2%) intraperitoneally on days 5 and 15 after feeding trial. Results of the current study showed that the inclusion of 20 mg kg-1 green tea (T1) in fish diet enhanced the serum bactericidal activity against Yersinia ruckeri, while significant elevation of lysozyme activity was shown in T2 group. Anti-trypsin activity due to α1-antiprotease was significantly higher in T1 and T2 groups while peroxidase content showed significant increase in all treatment groups compared to control group. Hemagglutination antibody titer against C-RBC was significantly higher in fish administered with 100 mg kg(-1) green tea (T2). Our findings showed that decaffeinated green tea in lower doses of administration could be optimum to enhance the immunity of rainbow trout.     

Intra-strain dioxin sensitivity and morphometric effects in swim-up rainbow trout (Oncorhynchus mykiss)

Inter and intra-specific differences in sensitivity of early life stage salmonids to 2,3,7,8-TCDD exposure have been reported, but intra-strain differences have not been found in the literature. Our results indicate that intra-strain variability in terms of embryo mortality (LD50) is small in Eagle Lake strain of rainbow trout, LD50 values ranging from 285 to 457 pg TCDD egg g(-1). These results confirm Eagle Lake as a less sensitive strain within rainbow trout, and do not indicate overlap with reported LD50 values for brook or lake trout. Our results also demonstrate that although generalized edema in regions including the yolk-sac are frequently associated with mortality following dioxin exposure, not all edematous fish die. We detected dose-dependent decreases in cranial length, eye diameter, mass, and total length (P < 0.05) in viable swim-up rainbow trout. These effects are presumed to indicate more subtle dose-dependent disruptions of the viteline vein vasculature and, therefore, in access to energy sources. A tendency for dose-dependent decrease in liver glycogen reserves concurred with previous results on salmonids and with the well described TCDD-induced alterations in intermediate metabolism of rats and chicken embryos (wasting syndrome). This syndrome could be contributing to the reduced growth that we observed.     

Early biochemical effects of Microcystis aeruginosa extracts on juvenile rainbow trout (Oncorhynchus mykiss)

Microcystins (MC) are usually the predominant cyanotoxins associated with cyanobacterial blooms in natural surface waters. These toxins are well-known hepatotoxic agents that proceed by inhibiting protein phosphatase in aquatic biota; recent studies have also reported oxidative stress and disruption of ion regulation in aquatic organisms. In the present study, young trout (Oncorhynchus mykiss) were exposed to crude extracts of Microsystis aeruginosa for four days at 15 °C. The level of microcystins was calculated to confirm the presence of toxins in these crude extracts: 0, 0.75, 1.8 and 5 μg/L. Protein phosphatase measured in the liver increased by at least 3-fold and is significantly as a result of exposure to these sublethal concentrations of crude extract, his indicates an early defense response against protein phosphatase inhibition from cyanotoxins. This was corroborated by the decreased phosphate content in proteins found in the liver and brain. No increase in glutathione-S transferase (GST) activity was observed and lipid peroxidation was unaffected in both liver and brain tissue exposed to the cyanobacterial extracts. The data revealed that the proportion of the reduced (metal-binding) form of metallothionein (MT) decreased by two-fold relative to the control group (with a concomitant increase in the proportion of the oxidized form). The level of phosphate associated with MT increased by 1.5-fold at the highest concentration of crude extract. Acetylcholinesterase (AChE) activity in brain tissue was decreased after exposure to the highest concentration of crude extract, suggesting a slowdown in neural activity. However, no biotransformation processes or detoxification of GST was triggered. Our findings show early sign of biochemical effects of MC-LR in young trout.     

Effects of nonylphenol on hematological parameters and immune responses in immature rainbow trout (Oncorhynchus mykiss)

ABSTRACT

This study investigated the effects of nonylphenol (NP) on hematological and immunological parameters in both male and female rainbow trout (Oncorhynchus mykiss). Fish were randomly distributed into six groups and administered with NP (10, 50 and 100 μg g^-1 week^-1 BW) and a single dose of 17-β estradiol (E2; 2 μg g^-1 week^-1 BW, positive control). The solvent controls received ethanol and coconut oil as a vehicle, while the controls were not injected. Red blood cells (RBCs) count, hematocrit (Hct), hemoglobin (Hb), white blood cells (WBCs), and lymphocytes demonstrated a NP dose-dependent decrease, whereas mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), monocytes, and neutrophils showed an increasing trend in both male and female fish 21 days post-treatment. Also, RBCs, Hb, MCHC, WBCs, and lymphocytes were significantly reduced (p<0.05) in E2 treated fish. Lysozyme, complement components (C3 and C4) and immunoglobulin M (IgM) were increased in fish sera subjected to 10 and 50 μg g^-1 NP, while these decreased in groups administered with 100 μg g^-1 NP and 2 μg g^-1 E2. Except for C4 level at 10 μg g^-1 NP, no significant differences were observed in hematological and immunological parameters of male and female in each treatment. Overall, a frequent exposure to NP could lead to adverse effects on fish immune-physiological functions which may cause serious ecological threats of fish natural population sustainability.     

Endocrine and orexigenic actions of growth hormone secretagogues in rainbow trout (Oncorhynchus mykiss)

The effects of growth hormone secretagogues (GHSs) on the teleost somatotropic axis are poorly understood, particularly with respect to insulin-like growth factor-I (IGF-I) and the IGF-binding proteins (IGFBPs). To assess the endocrine and orexigenic responses of rainbow trout (Oncorhynchus mykiss) to GHS treatment, animals were injected with human GHRH(1-29)-amide, KP-102 or rat ghrelin at 0, 1 or 10 pmol/g body mass. Feed intake was tested at 2 and 5 h post-injection and plasma levels of growth hormone (GH), IGF-I and the IGFBPs were determined at 3, 6 and 12 h post-injection. Feed intake was significantly elevated by all of the GHSs tested at both post-injection time points. All GHSs elevated plasma GH levels in a time-dependent manner. Plasma IGF-I levels were elevated by all GHSs at 3 h post-injection, whereas those animals treated with KP-102 and ghrelin exhibited depressions at 6 h. Four IGFBPs were identified in the plasma by western blotting. Levels of the 20 kDa IGFBP decreased over the sampling time. Levels of the 32 kDa IGFBP were significantly depressed by all GHSs tested. Levels of the 42 kDa IGFBP were significantly elevated by all GHSs tested. Plasma levels of the 50 kDa IGFBP was decreased in some treatment groups at 3 h, but elevated by 6 h in the ghrelin-treated groups and elevated in all treatment groups by 12 h post-injection. The endocrine and orexigenic responses demonstrate that GHSs influence the teleost neuroendocrine system beyond short-term actions (<3 h post-injection) on GH release and the responses of the IGFBPs to GHS treatment support this notion and clarify their identification as functional homologues to mammalian IGFBPs.     

Two novel muramidases from skin mucosa of rainbow trout (Oncorhynchus mykiss)

Two novel antibacterial muramidases were purified to homogeneity from skin exudates of rainbow trout (Oncorhynchus mykiss). Unusually, one has an acidic isoelectric point and it is the first anionic muramidase to be reported for fish. Its molecular mass is 14,268 Da, as determined by mass spectrometry. The other muramidase is cationic with a mass of 14,252 Da. Partial N-terminal amino acid sequencing and peptide mapping strongly point to it being a c-type lysozyme, the first to be purified and characterised from skin of a salmonid. Its optimum pH ranges from 4.5 to 5.5 and its optimum temperature, at pH 5.0, is 33-49 degrees C, although it still exhibits activity at 5 degrees C. It is strongly bactericidal to the Gram-(+) bacterium Planococcus citreus, with a minimum bactericidal concentration of 100 U ml(-1), but is neither chitinolytic nor haemolytic. These two muramidases probably contribute to epithelial defence of the fish against microbes, either alone or in synergism with antibacterial peptides.     

Effect of salinity on flavin-containing monooxygenase expression and activity in rainbow trout (Oncorhynchus mykiss)

In order to obtain more information about the physiological role(s) of flavin-containing monooxygenases (FMOs) in euryhaline teleost fishes, two experimental series were performed using adult and juvenile rainbow trout (Oncorhynchus mykiss). Cannulated adult trout were exposed to freshwater or 21% seawater for 48 h, whereas juvenile trout were acclimated to one of four different salinities: freshwater, 7%, 14%, or 21% during a 2-week period. FMO expression and activity were determined in red blood cells (RBC), liver, gill, kidney, gut, heart and brain. Furthermore, the content of trimethylamine oxide (TMAO; an FMO metabolite and an osmolyte) as well as urea were determined in various tissues. FMO expression and activity increased significantly and in a salinity dependent manner in osmoregulatory organs (gills, kidney and gut) in both juveniles and adult trout and, furthermore, in RBC in adults. No significant changes were observed in liver or heart. Urea content increased significantly and in a salinity dependent manner in all tissues, whereas TMAO was accumulated primarily in muscle tissue. Salinity dependent adjustment of FMO expression and activity primarily in osmoregulatory organs as well as regulation of TMAO content in muscle is consistent with previous studies showing an association of FMO with osmoregulation in euryhaline teleosts. However, the lack of a parallel increase of TMAO with urea in other tissues of fish at high salinity indicates other mechanisms of protection from intracellular urea may exist in non-muscular tissues.     

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.