Endocytosis via the scavenger- and the mannose-receptor in rainbow trout (Salmo gairdneri) pronephros is carried out by nonphagocytic cells

Endocytosis of modified human serum albumin (HSA) and mannose-terminated glycoproteins was studied in pronephros cells from rainbow trout (Salmo gairdneri). Blood clearance and tissue uptake of dinitrophenylated human serum albumin (DNP-HSA) was dependent on the number of DNP-groups conjugated to the albumin molecule. Uptake of DNP₃₅-HSA in isolated pronephros cells was saturable. Pronephros cells also internalized the mannose-terminated glycoprotein invertase by a receptor-mediated process. DNP-HSA and invertase were recovered in the same cell fractions when pronephros cells containing in vivo endocytosed ligands were separated by density gradient centrifugation. The cells endocytosing these ligands were apparently not macrophages. The macrophages were recovered in cell fractions with higher densities. They were identified by their ability to adhere to glass and to carry out phagocytosis. Cultured macrophages did not endocytose chemically modified albumin (DNP-HSA and formaldehyde-treated albumin) and mannose-terminated glycoproteins (ovalbumin) in vitro. These ligands were not recovered in glass adherent pronephros cells after in vivo endocytosis of the proteins. The present data suggest that macrophages in rainbow trout pronephros possess neither the receptor for chemically modified albumin, the scavenger receptor, nor the mannose-specific receptor.     

Stress responses and disease resistance in salmonid fish: Effects of chronic elevation of plasma cortisol

Basal levels of plasma cortisol in unstressed salmonid fish are normally in the range 0–5 ng ml⁻¹. An acute stress such as handling or 1 h confinement caused a temporary elevation of the plasma cortisol levels of both brown trout,Salmo trutta L., and rainbow trout,Salmo gairdneri Richardson, in the range 40–200 ng ml⁻¹ with a return to basal levels within 24–48 h. The extent of the cortisol elevation in response to an acute stress was dependent upon both the species and strain of trout. Chronic stresses, such as prolonged confinement or crowding, resulted in an elevation of plasma cortisol levels to approximately 10 ng ml⁻¹. Under these circumstances, blood cortisol levels remained elevated for periods of up to 4 weeks before acclimation finally occurred. It is shown, by means of intraperitoneal implantation of cortisol, that chronic elevation of plasma cortisol levels in the brown trout results in a dose-dependent increase in mortality due to common bacterial and fungal diseases. This effect is apparent at plasma cortisol levels as low as 10 ng ml⁻¹, levels below those often reported as being representative of ‘unstressed’ fish. These findings are discussed in relation to the known immunosuppressive effects of corticosteroids in teleost fish.     

Hematological response in fish: pronephric and splenic involvements in the goldfish,Carassius auratus L.

Pronephric and splenic involvements in erythropoiesis and in stress-induced hematological response by goldfish,Carassius auratus, were examined under conditions of minimal stress and following transient (3h) temperature-induced elevation of O₂ demand and transient (3h) exposure to hypoxia. Although hemoglobin content and red cell numbers were little affected, the ontogenic composition of the circulating erythrocyte population was significantly altered by both types of respiratory challenge and also by the stresses associated with capture, air exposure during transfer and intraperitoneal administration of label. Juvenile cell numbers increased sharply while mature erythrocyte abundances declined. Consistent with this, [³H] methylthymidine-labeled cell numbers rose in both spleen and pronephros following imposition of stress. Splenic erythropoietic activity was modest relative to that of the pronephros. Our observations point to a readily triggered response involving: [1] release of cells reservoired in the spleen and pronephros, [2] increased erythropoiesis and [3] karyorrhexis. We suggest that while the magnitude of response may depend on the quality and magnitude of the imposed stress, its central features are essentially constant.     

Salmonid macrophages: separation, in vitro culture and characterization

Abstract. A method is described for the separation of fish leucocytes and the establishment of pure monolayers of fish macrophages in vitro. The method makes it possible to study the important role of cellular immunity in fish. Fish leucocytes were obtained from the pronephros of rainbow trout, Salmo gairdneri Richardson, and compared to those obtained from the pronephros of Atlantic salmon, Salmo salar L. Single cell suspensions were separated by density gradient centrifugation and seeded on glass cover slips for maintenance in culture. After 20 h in culture a subpopulation of the cells had adhered and spread out on the cover slips and were macrophage-like by morphological criteria. About 90–99% of these cells had the ability to phagocytose a variety of particles, including fixed sheep erythrocytes, latex, carbon particles, yeast and Vibrio anguillarum. Opsonization of particles with mammalian immunoglobulins and mammalian complement did not enhance the phagocytic activity.     

Effect of Dietary Lipids on Macrophage Function,Stress Susceptibility and Disease Esistance in Atlantic Salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>)

As the supply of marine fish oil is becoming a limiting factor in the production of Atlantic salmon (Salmo salar), new diets and alternative sources of energy are being tested. Plant oils are natural potential candidates to replace fish oil, but the different levels of essential polyunsaturated fatty acids may influence the health and growth of salmon. In this study, we have investigated the resistance to transport stress and bacterial infection, phagocytic activity in head kidney macrophages and eicosanoid metabolism in salmon fed three different diets. In high-energy fishmeal based diets, 50% and 100% of the supplementary fish oil (FO) was replaced with soybean oil (SO). The three dietary groups were fed for 950 day-degrees at 5 °C (27 weeks) and 12 °C (11 weeks) before challenging the fish with Aeromonas salmonicida, analyzing the lipid composition of head kidney and examining macrophage function in vivo and in vitro. Dietary fatty acids affected the lipid composition of the kidney. The level of eicosanoid precursor’s 20:4n-6 and 20:3n-6 were 3 and 7-fold higher in the 100% SO group compared with the FO group. The total fraction of n-3 lipids in kidney was 19% in the SO group, compared to 16% and 12% in the 50% or 100% SO groups, respectively. However, the production of leucotriene B₄ (LTB) and prostaglandin E₂ (PGE) immunoreactive materiel from exogenously added arachidonic acid in head kidney macrophages was only affected by the composite diet (increased) at 5 °C. In addition, the phagocytic activity of kidney macrophages in vivo and in vitro was not affected by diet. No effect of diet was observed on transport stress or susceptibility to a bacterial infection with Aeromonas salmonicida. Atlantic salmon therefore seems to tolerate a diet solely based on soybean oil as lipid source, without any detrimental effects on growth, health and immune functions.     

Factors influencing in vitro respiratory burst assays with head kidney leucocytes from rainbow trout,Oncorhynchus mykiss (Walbaum)

Head kidney leucocytes are central elements in a number of in vivo and in vitro assays elucidating innate and adaptive immune mechanisms in teleosts following stimulation with various antigens. These systems are sensitive to several factors affecting the outcome of the assays. The present work describes the importance of temperature, cell concentration, exposure time and immune-modulatory molecules on the respiratory burst activity (RBA) of rainbow trout head kidney leucocytes in vitro. Some variation in RBA was observed among individual fish. However, use of cells pooled from four individuals produced satisfactory results following exposure to phorbol 12-myristate 13-acetate, zymosan and β-glucan. Temperature was shown to have a significant effect on production of reactive radicals as illustrated by a high activity in cells maintained at 15–20 °C and a reduced activity at temperature extremes (1, 4 and 30 °C). Highest activity was found at a cell concentration of 1 × 10⁷ cells mL⁻¹. Reactivity showed a clear decline when cells were exposed for more than 4 h. Moreover, incubation of cells with inhibitory substances viz., DiMePE2, cortisol and superoxide dismutase decreased the RBA. It is concluded that several biotic and abiotic factors should be taken into account when conducting RBA assays with head kidney leucocytes for elucidation of rainbow trout immune responses.     

Modelling the effects of capture and sea lice [Lepeophtheirus salmonis (Krøyer)] infestation on the cortisol stress response in trout

Measuring baseline cortisol in wild salmonids is problematic because of the stress induced by most capture methods. The cortisol response to different periods of confinement stress was modelled in a laboratory population of rainbow trout [Oncorhynchus mykiss (Walbaum)]. A quadratic model was used to predict baseline (precapture) cortisol levels in these fish. A similar quadratic response pattern of cortisol was observed in wild sea trout (Salmo trutta L.). These were captured in timed sequences on four dates by seine netting in Clew Bay, west coast of Ireland. The estimated precapture cortisol for these fish indicated higher levels soon after migration to sea and a positive correlation with the level of infestation by Lepeophtheirus salmonis (Krøyer). Curvilinearity of the quadratic model was correlated with levels of lice infestation, indicating an enhanced cortisol response to confinement stress during capture when parasitic abundance was high. Estimated precapture cortisol for individual sea trout was positively correlated with numbers of lice although the linear regression had low predictive power. The method provides, for the first time, a means to estimate baseline cortisol levels in wild salmonids, and addresses one of the most difficult problems in working with wild fish. Keywords: Cortisol, trout, Lepeophtheirus salmonis (Krøyer), capture, quadratic model     

Bacteriophage‐based cocktail modulates selected immunological parameters and post‐challenge survival of rainbow trout (Oncorhynchus mykiss)

Recently, a rapid increase in the resistance of pathogenic bacteria to antibiotics and chemotherapeutics admitted for use in aquaculture has been observed. This happens especially often in intensive breeding. The use of drugs in closed circuits is problematic because it can damage biological filters. Therefore, in recent years, there has been a growing interest in natural methods of combating pathogens. These include bacteriophages. The aim of the study was to determine the safety of the new BAFADOR^® bacteriophage‐based preparation, its effect on selected immunological parameters and the effectiveness of prophylactic and therapeutic use after experimental infections with pathogenic bacteria Aeromonas hydrophila and Pseudomonas fluorescens. The use of BAFADOR^® increased the activity of lysozyme, total protein level and immunoglobulin level. The level of ceruloplasmin in the rainbow trout serum remained unchanged regardless of the route of administration of the preparation. Potential killing activity and metabolic activity of spleen phagocytes and proliferation of pronephros lymphocytes were higher compared to the control group. Both therapeutic and prophylactic application of the preparation after mixed experimental infection of A. hydrophila and P. fluorescens limited the mortality of rainbow trout.     

Ultrastructural alteration of lymphocytes in spleen and pronephros of grass carp (Ctenopharyngodon idella) experimentally exposed to microcystin-LR

Relatively little is known in relation to pathological changes of immune organs in fish when exposed to MC-LR. The ultrastructural alteration of lymphocytes was examined in the spleen and pronephros of grass carp Ctenopharyngodon idella injected experimentally with microcystin-LR. The fish were intraperitoneally injected with MC-LR at a dose of 50 µg/kg body weight, and the spleen and pronephros were dissected out at 1, 2, 7, 14 and 21 days post intraperitoneal injection (dpi). Pathological changes were then examined by transmission electron microscopy. Apoptosis was detected only in lymphocytes in the spleen, with obvious apoptotic features observed at 2 dpi; pathological changes of lymphocytes in the pronephros were also serious with mitochondria being highly edematous. However, damaged lymphocytes were almost un-observed in the spleen and pronephros at 21 dpi. These findings suggest that MC-LR can induce toxic effect on immune organs in grass carp, and the spleen may be much more sensitive to MC-LR stimulation.     

Effect of Oyster Mushroom,Pleurotus ostreatus,Extract on Hemato‐Immunological Parameters of Rainbow Trout,Oncorhynchus mykiss

The current study investigated the effects of oyster mushroom Pleurotus ostreatus, extract on immunological and hematological parameters of rainbow trout, Oncorhynchus mykiss. The fish were fed diets containing 0 (control), 1, and 2% supplementation of the oyster mushroom extract for 6 wk. Blood samples were collected weekly for the first 6 weeks. After 6 wk of feeding, the fish were challenged with Lactococcus garvieae and mortality was recorded. The results of this study showed that feeding rainbow trout an oyster mushroom extract–supplemented diet stimulated phagocytic activity of phagocytic cells, lysozyme activity, and myeloperoxidase activity in serum, but it did not significantly affect the total serum immunoglobulin level. At the same time, a significant increase was found in the number of neutrophils, monocytes, and total white blood cells. Fish fed the diet supplemented with oyster mushroom extract showed reduced mortalities following L. garvieae infection compared with controls. These results show that supplementation of fish diets with oyster mushroom extract at 1 and 2% concentrations significantly imrpoves hematological parameters and modulates the immune response against L. garvieae in rainbow trout.     

Phenotypical parameters as a tool to evaluate the immunostimulatory effects of laminarin in Oncorhynchus mykiss

Laminarin is a β‐glucan from the brown algae Laminaria digitata (J.V. Lamour), which can activate innate immune responses. With the aim of developing a strategy to evaluate specific phenotypical parameters of the effects of laminarin in trout innate immunity, we (i) fed fish with laminarin‐supplemented diet (0.2 g kg^?1 day^?1) for 21 days and (ii) treated fish with a single dose of intraperitoneal injected laminarin (0.08 g kg^?1 fish). The evaluation of cellular and humoral immune parameters was established at phenotypic level by the phagocytic activity of headkidney macrophages and detection of inflammatory cytokines in head kidney and gill tissue by indirect ELISA. Results showed that both delivery methods of laminarin produce an increase in the phagocytic activity in head kidney macrophages and a significant increase in the production of TNFα and IL‐8 in gill tissue at day 21. Additionally, some of these parameters were significantly correlated (P < 0.025), which places them as new potential combined markers to detect activation of trout defense mechanisms by laminarin. These results highlight the importance of developing new protocols to quantitate immune parameters, in order to evaluate immunostimulants in fish farming.     

Enhanced hypoosmoregulatory response to growth hormone after cortisol treatment in immature rainbow trout,Salmo gairdneri

The growth-independent effect of ovine growth hormone (oGH) and oGH + cortisol treatment on seawater (SW) adaptation in immature rainbow trout, Salmo gairdneri was investigated. Fish were injected every second day with saline, 2.0 μg oGH/g or 2.0 μg oGH + 8.0 μg cortisol/g for a maximum of 8 injections in freshwater (FW). Subgroups were transferred to 28‰ SW after 4 or 8 injections, and changes in plasma Na⁺ and Cl⁻, muscle water content and gill Na⁺/K⁺-ATPase activity were measured. In both of the hormone-treated groups retained in FW, gill Na⁺/K⁺-ATPase activity and interlamellar chloride cell density increased. The effects were most pronounced in the oGH + cortisol group after 2 weeks of treatment. After transfer to SW most of the control fish died due to the osmotic stress, whereas in the hormone-treated groups, mortality was low and there was a positive correlation between pretransfer gill Na⁺/K⁺-ATPase and the ability to maintain ionic-osmotic homeostasis after SW transfer. After two weeks of oGH + cortisol treatment, gill Na⁺/K⁺-ATPase activity was maximal. In contrast, after SW transfer, Na⁺/K⁺-ATPase activity increased further in the oGH-treated group. This group regulated ionic-osmotic parameters less effectively than the oGH + cortisol-treated group. The data indicate that GH and cortisol are important hormones in the regulation of hypoosmoregulatory mechanisms in S. gairdneri.     

In vitro effect of levamisole on the cell viability,phagocytosis and respiratory burst of Barbel chub (Squaliobarbus curriculus) macrophages

The present study was to determine in vitro effect of levamisole on the immune functions of Barbel chub (Squaliobarbus curriculus), head–kidney‐derived (HKM), spleen‐derived (SM) and peripheral blood monocyte‐derived macrophage (PBM). Macrophages were incubated with levamisole 10³, 10¹, 10⁻¹ and 10⁻³ ng mL⁻¹ to assay the cell viability, respiratory burst and phagocytosis. The results showed that macrophages treated with levamisole 10⁻³ ng mL⁻¹ gave a maximum respiratory burst response, whereas levamisole 10³ ng mL⁻¹ had no effect. Phagocytosis activity of the macrophages treated with levamisole enhanced significantly when compared to control, maximum response being at 10⁻³ ng mL⁻¹. While using the methylthiazoletetrazolium method to measure the cell activity for 12, 24, 36, 48, 60 h, there was no significant role on proliferation and the cell viability began to decline after 24 h. In conclusion, levamisole is a potent enhancer of Barbel chub macrophage activity with low concentration.     

Proliferative kidney disease: cellular aspects of the rainbow trout, Oncorhynchus mykiss (Walbaum), response to parasitic infection

Proliferative kidney disease (PKD) is an immunopathological condition of salmonid fish, caused by the hyperplastic response of their principal lymphoid tissues to infection with the spores of Tetracapsula bryosalmonae , a myxozoan parasite formerly designated proliferative kidney organism – unknown (PKX). In order to investigate the nature of cells involved in this host response and possible alterations of their functions during parasitic infection the course of PKD was studied by flow cytometry (FCM) techniques, using blood, pronephros and spleen leucocyte populations from rainbow trout infected by intraperitoneal (i.p.) injection with parasitic cells from infected donor fish. The parameters of the cellullar response studied were: cytogram of cell population, lymphoproliferation, phagocytosis, oxidative burst, and non-specific cytotoxicity. The modifications of cell population distribution and function in the PKD-infected fish mainly affected the pronephros cell populations and were coincident with the clinical phase of disease. During this phase, the lymphocytes constituted the major leucocyte cell population and underwent proliferation and were thus responsible for the renal tissue hyperplasia. Meanwhile, phagocytosis and oxidative burst were depressed. These data are in agreement with the patho-epidemiological background of PKD where the enhancement of the fish sensitivity to bacterial infections reflects the impairment of certain cellular defence mechanisms of innate immunity.     

The liver parenchymal cells of rainbow trout (Salmo gairdneri) endocytose mannose-terminated glycoproteins

A mannose-terminated glycoprotein,¹²⁵I-invertase, was taken up and degraded by isolated rainbow trout liver cells at 12°C. The uptake was inhibited by EGTA and no degradation occurred in the presence of ammonium ions. The liver cell suspension was fractionated by differential centrifugation in parenchymal and nonparenchymal cells, respectively. The parenchymal liver cells seemed to be the most active cells in uptake of labelled invertase bothin vitro andin vivo. Only negligible amounts of ligand were recovered in the nonparenchymal cells. Internalization of¹²⁵I-invertase at different temperatures was demonstrated indirectly by releasing surface-bound ligand with EGTA. Ligand was internalized even at 0°C in trout liver cells.In vitro uptake of¹²⁵I-invertase was inhibited by excess unlabelled invertase, by mannan and by N-acetylglucosamine.These data suggest that invertase is endocytosed by a mannose-specific pathway by the parenchymal liver cells of rainbow trout.     

Antimicrobial peptide CAP18 and its effect on Yersinia ruckeri infections in rainbow trout Oncorhynchus mykiss (Walbaum): comparing administration by injection and oral routes

The antimicrobial peptide CAP18 has been demonstrated to have a strong in vitro bactericidal effect on Yersinia ruckeri, but its activity in vivo has not been described. In this work, we investigated whether CAP18 protects rainbow trout Oncorhynchus mykiss (Walbaum) against enteric red mouth disease caused by this pathogen either following i.p. injection or by oral administration (in feed). It was found that injection of CAP18 into juvenile rainbow trout before exposure to Y. ruckeri was associated with lowered mortality compared to non‐medicated fish although it was less effective than the conventional antibiotic oxolinic acid. Oral administration of CAP18 to trout did not prevent infection. The proteolytic effect of secretions on the peptide CAP18 in the fish gastrointestinal tract is suggested to account for the inferior effect of oral administration.     

Enhancement of resistance to bacterial infection in rainbow trout, Oncorhynchus mykiss (Walbaum), by oral administration of bovine lactoferrin

Abstract Bovine lactoferrin was evaluated for its ability to enhance resistance of rainbow trout, Oncorhynchus mykiss (Walbaum), to infection with Vibrio anguillarum. Oral administration of lactoferrin (100 mg kg⁻¹) to fish daily for 3 days prior to an intraperitoneal challenge with V. anguillarum resulted in increased survival rates, and enhanced resistance against Streptococcus sp., although to a lesser extent. In lactoferrin-treated fish, an increase in phagocytic and chemiluminescent (CL) activities of pronephros cells against V. anguillarum was observed. The phagocytic and CL activities of cells against Streptococcus sp. were also significantly increased. However, no in vitro bactericidal activities of lactoferrin against V. anguillarum or Streptococcus sp. were observed. This suggests that the lactoferrin enhanced the resistance of the rainbow trout against bacterial infection through the activation of phagocytes.     

A specific inhibitor of mammalian kallikrein,Phe-Phe-Arg-chloromethyl ketone,inhibits the production of vasoactive substances from trout plasma by kallikrein and blocks endogenous kallikrein-like activity in trout gills

The cardiovascular effects of the kallikrein-kinin system (KKS) have not been completely characterized in lower vertebrates. In the present study, a specific, irreversible kallikrein inhibitor, Phe-Phe-Arg-chloromethyl ketone (PPACMK) was used to examine: 1. the role of the KKS in blood pressure regulation in vivo; 2. the nature of the pressor substance formed by the action of kallikrein on trout plasma in vivo and in vitro; and 3. the presence of kallikrein in trout gills and kidney. Dorsal aortic cannulated rainbow trout were used for in vivo blood pressure assays and two colorimetric serine-protease assays were used to examine tissue kallikrein activity. PPACMK alone had no effect on blood pressure in vivo. Pretreatment of porcine kallikrein with PPACMK inhibited the enzyme's pressor effect in trout by 80% and significantly attenuated the synthesis of vasopressor substance(s) from heat-treated trout plasma in vitro. Approximately 30% of gill serine protease activity was inhibited by pretreatment with PPACMK; no PPACMK-sensitive kallikrein activity was observed in the kidney. Salt water adaptation did not affect kallikrein-specific activity in the gill. These results show that the salmonid KKS does not appear to be involved in the regulation of systemic blood pressure. Further, the formation of pressor substances from trout plasma in vivo and in vitro is due to kallikrein activity and the peptides generated are similar. A kallikrein, similar to the mammalian enzyme, is found in trout gills, suggesting that the salmonid KKS may be a local mediator of gill function.     

Pro‐inflammatory caspase‐1 activation during the immune response in cells from rainbow trout Oncorhynchus mykiss (Walbaum 1792) challenged with pathogen‐associated molecular patterns

In response to pathogens, the higher vertebrate innate immune system activates pro‐inflammatory caspase‐1 which is responsible for the processing and secretion of several important cytokines involved in the host's defence against infection. To date, caspase‐1 has been described in few teleost fish, and its activity has been demonstrated through substrate cleavage and inhibition by pharmacological agents. In this study, the detection of the active form of caspase‐1 during the immune response in salmonid fish is described, where two antibodies were produced. These antibodies differentially recognize the structural epitopes of the inactive pro‐caspase‐1 and the processed active form of the caspase. Firstly, caspase‐1 activation was demonstrated in vitro by ELISA, Western blotting and immunocytochemistry in rainbow trout macrophages exposed to different pathogen‐associated molecular patterns plus the pathogen Aeromonas hydrophila. This activity was clearly abrogated by a caspase inhibitor and seems to be unrelated to IL‐1β secretion. Caspase‐1 activation was then validated in vivo in gill cells from fish challenged with Aeromonas salmonicida. These results represent the first demonstration of caspase‐1 activation in salmonids, and the first evidence of the putative regulatory role which this protease plays in inflammatory response in this fish group, as described for some other teleosts and mammals.     

Cortisol-induced changes in oxygen consumption and ionic regulation in coastal cutthroat trout (Oncorhynchus clarki clarki) parr

The influence of cortisol on oxygen consumption and osmoregulatory variables was examined in coastal cutthroat trout (Oncorhynchus clarki clarki) parr kept in fresh water (FW) and transferred to seawater (SW). Intraperitoneal implants containing cortisol (50 g g^–1) in vegetable oil resulted in elevated plasma cortisol titres similar to those observed in fish following a 24h SW exposure. Cortisol treatment significantly increased the oxygen consumption and plasma glucose levels of trout in FW, consistent with the glucocorticoid role of cortisol. Cortisol treatment did not cause any changes in plasma ion concentrations or gill Na⁺,K⁺-ATPase activity in FW after 10 days. Cortisol-implanted fish exposed to SW for 24h showed slightly improved ion regulatory ability compare to non-implanted controls. The results of this study suggest that during SW transfer in juvenile salmonids, increases in cortisol may act as both a mineralocorticoid and a glucocorticoid, depending on the developmental state of the fish (e.g., smolt versus parr). Furthermore, the relative energetic costs of osmoregulation and that of the stress associated SW transfer cannot be discerned using whole-animal oxygen consumption rates.