In vitro modulation of fish phagocytic cells by beta-endorphin

The activation of rainbow trout, Oncorhynchus mykiss, and carp, Cyprinus carpio, phagocytic cells by synthetic chum salmon, O. keta, beta-endorphin was analysed in vitro. Rainbow trout head kidney leukocytes were cultured in RPMI 1640 medium containing 1, 10, 50 or 100 ng ml-1 of chum salmon beta-endorphin and the production of superoxide anion was measured via the reduction of nitroblue tetrazolium (NBT) in vitro. Macrophages incubated with 10 ng ml-1 up to 100 ng ml-1 of beta-endorphin showed an increase in their production of superoxide anion in comparison with control macrophages which were cultured without hormone. beta-endorphin also increased the production of superoxide anion in phagocytic cells prepared from kidney of carp. This stimulation was inhibited by naloxone. Phagocytic cells treated with beta-endorphin also displayed increased phagocytic activity and phagocytic index. These results showed that beta-endorphin in lower vertebrates activates the function of phagocytic cells in vitro.     

Effects of estradiol, progesterone and testosterone on the function of carp, Cyprinus carpio, phagocytes in vitro

The modulation of phagocytic cells by beta-estradiol, 11-ketotestosterone and progesterone was analyzed in common carp Cyprinus carpio. Carp kidney leukocytes were cultured in RPMI 1640 medium containing 0.1, 1, 10, 100 or 1000 nM concentration of each hormone. The production of superoxide anion, nitric oxide (NO) and phagocytosis were measured in vitro. Similar concentrations of cortisol were used as control. Phagocytic activities of carp macrophages was suppressed by treatment with beta-estradiol, progesterone and 11-ketotestosterone. The production of NO in carp macrophages was suppressed by progesterone and 11-ketotestosterone. However, carp macrophages incubated with beta-estradiol, progesterone and 11-ketotestosterone did not show any difference in the production of superoxide anion in comparison with control macrophages in the absence of hormones. Carp macrophages treated with cortisol suppressed phagocytosis and the production of nitric oxide and superoxide anion.     

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.     

Effect of gonadotropin-releasing hormone on phagocytic leucocytes of rainbow trout

To clarify the role of gonadotropin-releasing hormone (GnRH) in the fish immune system, in vitro effect of GnRH was examined in phagocytic leucocytes of rainbow trout (Oncorhynchus mykiss). Gene expression of GnRH-receptor was detected by RT-PCR in leucocytes from head kidney. Administration of sGnRH increased proliferation and mRNA levels of a proinflammatory cytokine, tumor necrosis factor (TNF)-α, in trout leucocytes. Superoxide production in zymosan-stimulated phagocytic leucocytes was also increased by sGnRH in a dose-related manner from 0.01 to 100 nM. There was no significant effect of sGnRH on mRNA levels of growth hormone (GH) expressed in trout phagocytic leucocytes. Immunoneutralization of GH by addition of anti-salmon GH serum into the medium could not block the stimulatory effect of sGnRH on superoxide production. These results indicate that GnRH stimulates phagocytosis in fish leucocytes through a GnRH-receptor-dependent pathway, and that the effect of GnRH is not mediated through paracrine GH in leucocytes.     

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.     

Dietary vitamin E and rainbow trout (Oncorhynchus mykiss) phagocyte functions: effect on gut and on head kidney leucocytes

The effects of vitamin E (deficiency or supplementation) on the non-specific immune system in rainbow trout, Oncorhynchus mykiss, were evaluated. Rainbow trout were fed daily a semi-purified diet supplemented with vitamin E at 0, 28 and 295 mg x kg(-1) of diet. After 80 days of experimental feeding, the phagocytic function (respiratory burst evaluated by the CL response, phagocytosis) from gut leucocytes and head kidney enriched macrophages was measured; head kidney cell pinocytosis and serum lysozyme activity were also analysed. The results showed that some phagocyte functions were influenced by dietary vitamin E. When fish were fed the high dietary dose of vitamin E an enhancement of phagocytosis was found, but only significantly for the leucocytes isolated from the gut of rainbow trout; moreover, an impaired response was also observed in the fish fed no vitamin E for 80 days. However, no significant differences were noticed on the oxidative burst (CL) response of both gut and head kidney cells according to the dietary dose of vitamin E. Pinocytosis evaluated on head kidney cells was not influenced by dietary vitamin E. Fish fed vitamin E at 295 mg x kg(-1) had a lower serum lysozyme activity than those fed with vitamin E at 28 mg x kg(-1) and the fish fed no vitamin E for 80 days had an impaired activity. Thus, the present results demonstrate that altered dietary levels of vitamin E modulates the phagocytic functions of gut leucocytes in rainbow trout; moreover, the vitamin E diet effect seems to be greater on the local intestinal response as compared to systemic (head kidney). Taken together, this study confirms the crucial role of gut phagocytes in mucosal non-lymphoid defences in fish.     

Enhancement of innate immunity in rainbow trout (Oncorhynchus mykiss Walbaum) associated with dietary intake of carotenoids from natural products

The effects of orally administered carotenoids from natural sources on the non-specific defense mechanisms of rainbow trout were evaluated in a nine-week feeding trial. Fish were fed four diets containing either beta-carotene or astaxanthin at 100 and 200 mg kg-1 from the marine algae Dunaliella salina and red yeast Phaffia rhodozyma, respectively, and a control diet containing no supplemented carotenoids. Specific growth rate and feed:gain ratio were not affected by dietary carotenoid supplementation. Among the humoral factors, serum alternative complement activity increased significantly in all carotenoid supplemented groups when compared to the control. On the other hand, serum lysozyme activity increased in the Dunaliella group but not in the Phaffia group, whereas plasma total immunoglobulin levels were not altered by the feeding treatments. As for the cellular responses, the superoxide anion production from the head kidney remained unchanged while the phagocytic rate and index in all supplemented groups were significantly higher than those of the control. These findings demonstrate that dietary carotenoids from both D. salina and P. rhodozyma can modulate some of the innate defense mechanisms in rainbow trout.     

Suppression in function of phagocytic cells in common carp Cyprinus carpio L. injected with estradiol,progesterone or 11-ketotestosterone

The modulation of phagocytic cells by beta-estradiol, 11-ketotestosterone, progesterone and cortisol was analysed in common carp (Cyprinus carpio L.). Carp were injected intraperitoneally (1 and 5 microg/kg fish) with each steroid and the activity of kidney macrophages was recorded 1 and 3 days post-injection. Bovine serum albumin-injected fish served as controls. Phagocytosis, superoxide anion and nitric oxide production were suppressed in all sex steroid treatments, and the suppression was in a dose-dependent manner. Cortisol treatment also inhibited the immune parameters to an extent similar to each of the steroid hormone. These results show that sex steroids can modulate the function of the phagocytic cells.     

Stimulation of the phagocytic function in guinea pig peritoneal macrophages by physical activity stress.

A study was made of all the different stages of the phagocytic function in peritoneal macrophages from male guinea pigs [3 (SD 1) months old] before, immediately after, and 24 h after being subjected to stress from physical activity (swimming until exhaustion). The early (10 min) and late (40 min) adherence to tissue substrates, chemotaxis, attachment and phagocytosis of Candida albicans, ingestion of inert particles (latex beads), and basal oxidative metabolism [measured by nitroblue tetrazolium (NBT) reduction] were significantly stimulated by the physical activity. After 24 h, late adherence, attachment capacities, and basal oxidative metabolism returned to basal values, whereas early adherence, chemotaxis, phagocytosis of cells and inert particles, and microbicidal capacity (production of superoxide anion measured by NBT reduction in presence of ingested material) remained significantly increased. The stress produced by physical activity, reflected in increased serum corticosterone values, led to a global stimulation of the phagocytic function.     

Evaluation of in vitro endocytosis and antibody synthesis by rainbow trout head kidney cells treated with bovine lactoferrin

Bovine lactoferrin (LF) was evaluated for its capacity to modulate the in vitro endocytosis (phagocytosis and pinocytosis) and antibody synthesis by head kidney cells of rainbow trout Oncorhynchus mykiss . Phagocytic activity and phagocytic index of head kidney macrophages, determined by measurement of ingested yeast, were influenced by bovine LF starting from the LF concentration of 1 and 0·1 μg ml⁻¹, respectively. Endocytosis, determined by the evaluation of droplet uptake of neutral red dye solution, was significantly enhanced by 10 μg ml⁻¹ of LF. In contrast, antibody synthesis by head kidney cells, evaluated by immunoenzymatic assay, from fish immunized against human‐γ‐globulins (HGG) in vivo was not affected by bovine LF. Although these results showed that bovine LF had no effect on specific immunoglobulin production in vitro , an enhancement of the acquired immune response may be assumed in LF‐treated fish in vivo , as observed in higher vertebrates.     

Studies on the role of exogenous proteolytic enzymes in digestion processes in fish

Studies were carried out on the proteolytic activity of the fry of common carp, rainbow trout, grass carp, and whitefish, as well as on the activity of digestive organs of adult common carp and rainbow trout. Activity of exogenous enzymes in relation to endogenous ones was assessed on the basis of the proteolytic value of fish food and the activity of digestive organs. It was found that the share of proteolytic enzymes of natural food in the digestion process in fish was high. Beginning from a weight of 50–100 g for common carp and 10 g for rainbow trout, the relation between the daily enzymatic ration and the weight of fish indicates the cooperation of an approximately constant amount of exogenous enzymes.     

Effect of angiotensin II and losartan on the phagocytic activity of peritoneal macrophages from Balb/C mice

Angiotensin II (AII), a product of rennin-angiotensin system, exerts an important role on the function of immune system cells. In this study, the effect of AII on the phagocytic activity of mouse peritoneal macrophages was assessed. Mice peritoneal macrophages were cultured for 48 h and the influence of different concentrations of AII (10(-14) to 10(-7) M) and/or losartan, 10(-16) to 10(-6) M), an AT1 angiotensin receptor antagonist, on phagocytic activity and superoxide anion production was determined. Dimethylthiazoldiphenyltetrazolium bromide reduction and the nucleic acid content were used to assess the cvtotoxicity of losartan. A stimulatory effect on phagocytic activity (P < 0.05) was observed with 10(-13) M and 10(-12 M) AII concentrations. The addition of losartan (up to10(-14) M) to the cell cultures blocked (P < 0.001) the phagocytosis indicating the involvement of AT1 receptors. In contrast, superoxide anion production was not affected by AII or losartan. The existence of AT1 and AT2 receptors in peritoneal macrophages was demonstrated by immunofluorescence microscopy. These results support the hypothesis that AII receptors can modulate murine macrophage activity and phagocytosis, and suggest that AII may have a therapeutic role as an immunomodulatory agent in modifying the host resistance to infection.     

Activation of rainbow trout, Oncorhynchus mykiss, phagocytic cells by administration of bovine lactoferrin

The chemiluminescent response of kidney phagocytic cells was significantly increased by the oral administration of bovine lactoferrin to rainbow trout, Oncorhynchus mykiss. The phagocytic activity of kidney cells was also increased by administration of bovine lactoferrin to the fish. Activation of kidney cells was also observed in vitro with cells cultured in the presence of bovine lactoferrin.     

Isolation of leucocytes from the anterior kidney and spleen of rainbow trout in a self-generating density gradient

Separation of leucocytes from tissues and enrichment of specific types of leucocytes are essential first steps in studies of leucocyte function. We describe a simple and rapid method for separating and enriching leucocytes from the anterior kidney and spleen of rainbow trout. Leucocytes were separated on self-generating density gradients of Sepracell-MN, a colloidal silica-based medium. Recovery of leucocytes from the gradients was near 100% and was not affected by procedural variables such as cell suspension: Sepracell-MN ratio (separation ratio), initial temperature, centrifugation time, or number of cells per gradient. Two bands of leucocytes formed at separation ratios of 1 : 1, 1:1.5, 1:2 and 1 : 3. Recovery of selected leucocyte types could be maximized, and contamination by other cell types reduced, by selection of the appropriate separation ratio and cell band. Recovered leucocytes were responsive in assays of functional capability (adherence, phagocytosis, superoxide anion production). Leucocyte populations that adhered to glass were enriched for macrophages, but some neutrophils and lymphocytes (paricularly spleen lymphocytes) were also adherent. The most abundant leucocytes in the anterior kidney and spleen of the experimental fish were lymphocytes (respectively, 47 and 88%, including thrombocytes), neutrophils (35 and 7%), and macrophages (11 and 3%).     

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.     

Immunomodulation by dietary beta-1, 3-glucan in the brooders of the black tiger shrimp Penaeus monodon

The present study evaluated the effectiveness of beta-1,3-glucan derived from Schizophyllum commune in enhancing shrimp survival as well as haemocyte phagocytosis and superoxide anion production in brooder Penaeus monodon. Pond-reared P. monodon adults (135 +/- 25 g) stocked in outdoor or indoor tanks were fed either a test diet containing beta-1,3-glucan (2.0 g kg(-1) or a glucan-free control diet for 40 days. Their survival was compared. The brooders reared in indoor tanks were analysed at days 0, 1, 3, 6, 12, 24, 30 and 40 for their haemocyte phagocytic activity and superoxide anion production. The results showed that regardless of indoor or outdoor rearing the survival rate of shrimp fed the glucan diet was significantly higher (P<0.001) than that of the control group. The brooders showed enhanced haemocyte phagocytic activity, cell adhesion and superoxide anion production when glucan was administered in their diets. The immunostimulatory enhancement peaked at day 24 after starting the dietary exposure and subsequently decreased to the pre-feeding level at the end of the 40 days feeding trial.     

Changes in several functions of murine peritoneal macrophages by N-acetylcysteine and thioproline ingestion. Comparative effect between two strains of mice

The administration of the thiol compounds, N-acetylcysteine (NAC) and in particular thioproline (thiazolidine-4-carboxylic acid) at 0.1% w/w concentration in the diet, improves lymphocyte functions in old female Swiss mice, as has been shown in our previous studies. In the present work, adult mice from two different strains, namely BALB/c (an inbred strain) and OF1-Swiss (noninbred strain), were fed a diet supplemented with the above dose of each thiol compound jointly for five weeks. At 28 weeks of age, peritoneal cell suspensions were obtained and different steps of the phagocytic process, the most representative activity of macrophages, as well as interleukin-1beta (IL-1beta) production, were studied. Thus, adherence to substrate, mobility directed to a chemoattractant gradient (chemotaxis), ingestion of inert particles and superoxide anion production were analysed. The results show that diet supplementation with NAC plus thioproline increased all macrophage functions studied with the exception of superoxide anion production, which was decreased. These effects were more evident in macrophages from Swiss mice, whereas in BALB/c mice the stimulation of phagocytosis and IL-1beta production was lower and no differences were seen after treatment in adherence and superoxide anion production. These data suggest that immune function can be improved in adult mice by administration of the above thiol compounds, especially in the noninbred strain of OF1-Swiss mice.     

Hepatic glucokinase is induced by dietary carbohydrates in rainbow trout, gilthead seabream, and common carp

Glucokinase (GK) plays a central role in glucose homeostasis in mammals. The absence of an inducible GK has been suggested to explain the poor utilization of dietary carbohydrates in rainbow trout. In this context, we analyzed GK expression in three fish species (rainbow trout, gilthead seabream, and common carp) known to differ in regard to their dietary carbohydrate tolerance. Fish were fed for 10 wk with either a diet containing a high level of digestible starch (>20%) or a diet totally deprived of starch. Our data demonstrate an induction of GK gene expression and GK activity by dietary carbohydrates in all three species. These studies strongly suggest that low dietary carbohydrate utilization in rainbow trout is not due to the absence of inducible hepatic GK as previously suggested. Interestingly, we also observed a significantly lower GK expression in common carp (a glucose-tolerant fish) than in rainbow trout and gilthead seabream, which are generally considered as glucose intolerant. These data suggest that other biochemical mechanisms are implicated in the inability of rainbow trout and gilthead seabream to control blood glucose closely.     

In vitro effect of diacetoxyscirpenol and deoxynivalenol on microbicidal activity of murine peritoneal macrophages

Diacetoxyscirpenol and deoxynivalenol, two trichothecene mycotoxins shown previously to exert immunosuppressive effects on the immune system were examined for their in vitro effects on some functions of murine peritoneal macrophages. The cells were pre-incubated for 4 hr with the mycotoxin concentrations of 0.1 ng/ml–1 g/ml. At concentrations that did not affect the cell viability (Specific Lactate Dehydrogenase test), diacetoxyscirpenol and deoxynivalenol suppress microbicidal activity of phagocytic cells. The diacetoxyscirpenol concentrations, which reduce phagocytosis (2ng/ml), microbicidal activity (1 ng/ml), superoxide anion production (1 ng/ml) and phagosome-lysosome fusion (0.1 ng/ml), indicate that the inhibition of killing mechanism arise from both oxidative and non-oxidative pathways. Phagocytosis, microbicidal activity and superoxide anion production are inhibited by deoxynivalenol at 1 ng/ml whereas phagosome-lysosome fusion is reduce above 100 ng/ml. These results suggest that microbicidal activity inhibition by deoxynivalenol did not depend on non-oxidative pathway (phagosome-lysosome fusion) impairment.     

Bacillus subtilis AB1 controls Aeromonas infection in rainbow trout (Oncorhynchus mykiss, Walbaum)

AIM: To develop a probiotic with effectiveness against Aeromonas sp., which was pathogenic to rainbow trout. METHODS AND RESULTS: When Bacillus subtilis AB1, which was obtained from fish intestine, was administered for 14 days to rainbow trout in feed at a concentration of 10(7) cells per gram either as viable, formalized or sonicated cells or as cell-free supernatant, the fish survived challenge with the pathogen. AB1 stimulated immune parameters, specifically stimulating respiratory burst, serum and gut lysozyme, peroxidase, phagocytic killing, total and alpha1-antiprotease and lymphocyte populations. CONCLUSIONS: Bacillus subtilis AB1 was effective as a probiotic at controlling infections by a fish-pathogenic Aeromonas sp. in rainbow trout. SIGNIFICANCE AND IMPACT OF THE STUDY: Disease control in fish is possible by means of the oral application of live and inactivated cells and their subcellular components with the mode of action reflecting stimulation of the innate immune response.