Spleen Of Rainbow Trout Research Articles

Effects of cypermethrin on rainbow trout (Oncorhynchus mykiss)

The aim of this study was to assess the effect of cypermethrin [(R,S)-α-cyano-3-phenoxybenzyl (1RS)-cis,tra-3-(2,2-dichlorovinyl)-2,2-dimethyl cyclopropane carboxylate] on rainbow trout (Oncorhynchus mykiss). The effect was assessed on the basis of the results of acute toxicity tests and on the comparison of results of haematological, biochemical and histopathological tissue examinations of a control and experimental group exposed to Alimetrine 10 EM pesticide preparation (active substance 100 g/l of cypermethrin). The acute semistatical toxicity test lasting 96 h was performed on rainbow trout juveniles. The 96hLC50 value of Alimethrine 10 EM was 31.4 µg/l. Examination of erythrocyte, leukocyte and biochemical profile and histopathological tissue examination was performed on 15 control and 15 experimental specimens of one-to-two-year-old rainbow trout after 96 h of exposure to Alimetrine 10 EM in the concentration of 31.4 µg/l. The experimental group showed significantly higher values (P < 0.01) of plasma ammonia (NH<sub>3</sub>), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK), lactate (LACT) and significantly lower (P < 0.01) values of alkaline phosphatase (ALP) compared to the control group. Also, a significant decrease in count of developmental forms of myeloid sequence, and segmented neutrophile granulocytes in the experimental group were found. Teleangioectasiae of secondary gill lamellae and degeneration of hepatocytes were observed with histopathological examination. No histopathological changes were demonstrated in tissues (skin, spleen, cranial and caudal kidney) of rainbow trout following exposure to cypermethrine. The cypermethrine-based Alimethrinee 10 EM pesticide preparation was classified among substances strongly toxic for fish.

Effects of moderate and substantial hypoxia on erythropoietin levels in rainbow trout kidney and spleen

Erythropoietin (EPO) is a glycoprotein hormone that regulates the proliferation and differentiation of erythroid progenitor cells in mammals. Although EPO has been identified in fish, the specific function and effects of hypoxia have not been investigated previously. In this study, we have demonstrated a relationship between increases in renal EPO levels and decreases in spleen EPO levels and spleen-somatic index (SSI), with increases in haemoglobin (Hb) concentration in the blood during hypoxia exposure in rainbow trout. Splenic contraction and the subsequent red blood cell release accounts for the initial increase in Hb concentration in the blood, whereas EPO action probably accounts for the later increases in hemoglobin concentration in the blood. Our data indicate that fish and mammalian erythropoietic systems are similar in response to hypoxia, in that erythropoiesis in fish is influenced by EPO.

Quantitative expression profiling of immune response genes in rainbow trout following infectious haematopoietic necrosis virus (IHNV) infection or DNA vaccination

Infectious haematopoietic necrosis virus (IHNV) is a well-studied virus of salmonid fishes. A highly efficacious DNA vaccine has been developed against this virus and studies have demonstrated that this vaccine induces both an early and transient non-specific anti-viral phase as well as long-term specific protection. The mechanisms of the early anti-viral phase are not known, but previous studies noted changes in Mx gene expression, suggesting a role for type I interferon. This study used quantitative real-time reverse transcriptase PCR methodology to compare expression changes over time of a number of cytokine or cytokine-related genes in the spleen of rainbow trout following injection with poly I:C, live IHNV, the IHNV DNA vaccine or a control plasmid encoding the non-antigenic luciferase gene. The target genes included Mx-1, viral haemorrhagic septicaemia virus induced gene 8 (Vig-8), TNF-α1, TNF-α2, IL-1β1, IL-8, TGF-β1 and Hsp70. Poly I:C stimulation induced several genes but the strongest and significant response was observed in the Mx-1 and Vig-8 genes. The live IHN virus induced a significant response in all genes examined except TGF-β1. The control plasmid construct and the IHNV DNA vaccine marginally induced a number of genes, but the main difference between these two groups was a statistically significant induction of the Mx-1 and Vig-8 genes by the IHNV vaccine only. The gene expression profiles elicited by the live virus and the IHNV DNA vaccine differed in a number of aspects but this study confirms the clear role for a type I interferon-like response in early anti-viral defence.

Production of zebrafish germ-line chimeras from embryo cell cultures.

Although the zebrafish possesses many characteristics that make it a valuable model for genetic studies of vertebrate development, one deficiency of this model system is the absence of methods for cell-mediated gene transfer and targeted gene inactivation. In mice, embryonic stem cell cultures are routinely used for gene transfer and provide the advantage of in vitro selection for rare events such as homologous recombination and targeted mutation. Transgenic animals possessing a mutated copy of the targeted gene are generated when the selected cells contribute to the germ line of a chimeric embryo. Although zebrafish embryo cell cultures that exhibit characteristics of embryonic stem cells have been described, successful contribution of the cells to the germ-cell lineage of a host embryo has not been reported. In this study, we demonstrate that short-term zebrafish embryo cell cultures maintained in the presence of cells from a rainbow trout spleen cell line (RTS34st) are able to produce germ-line chimeras when introduced into a host embryo. Messenger RNA encoding the primordial germ-cell marker, vasa, was present for more than 30 days in embryo cells cocultured with RTS34st cells or their conditioned medium and disappeared by 5 days in the absence of the spleen cells. The RTS34st cells also inhibited melanocyte and neuronal cell differentiation in the embryo cell cultures. These results suggest that the RTS34st splenic-stromal cell line will be a valuable tool in the development of a cell-based gene transfer approach to targeted gene inactivation in zebrafish.

A monoclonal antibody recognising a surface marker on rainbow trout (Oncorhynchus mykiss) monocytes

The characterisation of a monoclonal antibody (mab 45) reacting with phagocytic leucocytes isolated from blood and spleen of rainbow trout (Oncorhynchus mykiss, Walbaum) is described. The surface marker labelled by this mab is expressed at relative low levels on the membrane of large, nearly non-granulated trout leucocytes, and having the typical morphology of monocytes in flow cytometry (Kfoury et al., 1999, Fish Pathology, 34, 1–6). No reaction of mab 45 with granulocytes, lymphocytes or thrombocytes was detected. In spleen and head kidney, large, polymorphonuclear leucocytes were immunostained. The mab most strongly recognised an antigen of 48kDa prepared from trout leucocytes of different organs, but not in trout plasma. In an in vitro phagocytosis assay trout monocytes were stained with mab 45 after phagocytosis of Aeromonas salmonicida labelled with the lipophilic fluorescent cell surface linker PKH26. However, previous binding of mab 45 on trout leucocytes did not inhibit the phagocytosis of A. salmonicida particles.Using mab 45, the dynamics of monocytes in blood, spleen and peritoneal cavity could be demonstrated after intraperitoneal injection of trout with inactivated A. salmonicida. The described mab serves as a useful tool to investigate the involvement of monocytes/macrophages in immune reactions of trout to a variety of pathogens.

Constitutive and LPS-induced gene expression in a macrophage-like cell line from the rainbow trout ( Oncorhynchus mykiss)

The lack of cell lines from mononuclear phagocytes of salmonid fish has impeded the study of immune function at a cellular level in these economically and ecologically important animals. Here, we report on the further characterization of RTS11, a previously described macrophage-like cell line from the rainbow trout spleen, with regard to its expression of a number of immunologically relevant genes. Analysis of gene expression by reverse transcription-polymerase chain reaction, using rainbow-trout-specific primers demonstrated that RTS11 cells express the β chain of the class II major histocompatibility complex, the cytokines transforming growth factor-β (TGF-β) and interleukin-1β (IL-1β), and cyclo-oxygenase-2 (COX-2). Inducing the cells with lipopolysaccharide led to increased expression of IL-1β and COX-2, as determined by Northern blotting. These results together suggest that RTS11 retains many of the characteristics expected of mature macrophages, and should be a valuable tool for further study of the expression and function of these immunomodulatory proteins in fish.

A stromal cell line from rainbow trout spleen, RTS34ST, that supports the growth of rainbow trout macrophages and produces conditioned medium with mitogenic effects on leukocytes.

A rainbow trout spleen cell line, RTS34, was developed from a long-term hemopoietic culture. This cell line consisted of a mixed stromal cell layer with an associated cell population of macrophage-like cells that formed proliferative foci and released nonadherent progeny cells into the culture medium. A stromal cell line, RTS34st, was isolated from the RTS34 cell line. RTS34st cultures contained cells with fibroblast-like and epithelial-like morphologies and showed enhanced [3H]thymidine incorporation in response to either FBS or rainbow trout serum. The combination of FBS and trout serum was synergistic. Conditioned medium from RTS34st stimulated thymidine incorporation by peripheral blood and head kidney leukocytes, but not by leukocytes from the spleen. In addition, RTS34st provided a hemopoietic inductive microenvironment for immature precursor cells, selectively supporting the growth of macrophage-like cells. Therefore, RTS34st appears useful for studying the different roles of the stroma in regulating hemopoiesis in fish.

Response of fish immune cells to in vitro organotin exposures

The presence of contaminants in aquatic environments may compromise the health and survival of fish. Many of these compounds are known immunotoxins in mammals; however, relatively little information is available on the immunotoxic responses of fish to these pollutants. Organotins are examples of widespread industrial and agricultural compounds which persist in aquatic environments. In this study, we investigate the effects of tributyltin (TBT) and its dealkylated metabolite dibutyltin (DBT) on fish immune responses. Immune cells were isolated from the spleen and head kidney of juvenile rainbow trout ( Oncorhynchus mykiss) and exposed to 0, 2.5, 50 and 500 ppb of either TBT or DBT. Mitogenesis was quantified by tritiated thymidine incorporation into cells cultured with the mitogens concanavalin A (Con A) or lipopolysaccharide (LPS). No changes in immune function occurred at the lowest organotin dose of 2.5 ppb. Con A-stimulated mitogenesis was significantly suppressed by 85% in spleen cells on exposure to 50 ppb DBT. LPS-stimulated mitogenesis was significantly suppressed by 96% in spleen cells and by 58% in head kidney cells with 50 ppb DBT. The highest concentration of 500 ppb of TBT and DBT inhibited both Con A- and LPS-stimulated mitogenesis by more than 95% in both head kidney and spleen cells. Flow cytometric analysis revealed dose-dependent changes in the cell population profile which correlated with the inhibition of mitogen-stimulated lymphoproliferation. In contrast, natural cytotoxic cell activity was not inhibited by in vitro exposure to either compound, as determined by the lysis of chromium-51-labelled K562 human erythroleukaemia cells and P815 mouse mastocytoma cells. These results show that organotins have both functional and tissue-specific effects on the fish immune system, i.e. spleen ⪢ head kidney tissue and, in general. LPS-responsive ⪢ Con A-responsive leukocytes. In aquatic systems, TBT is considered to be the most toxic organotin compound, and this toxicity decreases with progressive dealkylation to diand mono-organotins. However, our results indicate that DBT is a more potent immunotoxin than TBT, and suggest a need for the reassessment of the potential toxicity of DBT to aquatic organisms.

Vimentin in a cold-water fish, the rainbow trout: highly conserved primary structure but unique assembly properties.

We have isolated from a rainbow trout (Oncorhynchus mykiss) spleen cDNA library a clone coding for vimentin. The deduced amino acid sequence reveals a high degree of identity with vimentin from carp (81%), frog (71%), chick and human (73% each). Large stretches in the central alpha-helical rod are identical within all four classes of vertebrates, but in 17 residues spread over the entire rod, the two fish differ distinctly from the tetrapod species. In addition, in the more diverged non-helical head domain, a nonapeptide motif previously shown to be important for regular filament formation is conserved. Recombinant trout vimentin assembles into bona fide filaments in vitro, with a temperature optimum between 18 and 24 degrees C. Above 27 degrees C, however, filament assembly is abruptly abolished and short filaments with thickened ends as well as structures without typical intermediate filament appearance are formed. This distinguishes its assembly properties significantly from amphibian, avian and mammalian vimentin. Also in vivo, after cDNA transfection into vimentin-free mammalian epithelial cells, trout vimentin does not form typical intermediate filament arrays at 37 degrees C. At 28 degrees C, and even more pronounced at 22 degrees C, the vimentin-positive material in the transfected cells is reorganized in the perinuclear region with a partial fibrillar appearance, but typical intermediate filament arrays are not formed. Together with immunoblotting and immunolocalization data from trout tissues, where vimentin is predominantly found in glial and white blood cells, we conclude that vimentin is indeed important in its filamentous form in fish and other vertebrates, possibly fulfilling cellular functions not directly evident in gene targeting experiments carried out in mice.

Development of long-term rainbow trout spleen cultures that are haemopoietic and produce dendritic cells

Different tissue dissociation protocols and serum supplements resulted in a variety of primary cultures from rainbow trout spleens. These were classified as either haemopoietic or non-haemopoietic. In haemopoietic cultures, a complex stromal layer developed on the cell culture surface; round cells appeared on the stromal layer; and ultimately some round cells were released into the medium as a non-adherent population. Many non-adherent cells were able to reattach in new culture vessels. Most of these had the characteristic shape and unique motility of dendritic cells. Cultures of these cells were subcultivated 2–4 times but continuous cell lines did not arise. Haemopoietic cultures resulted with collagenase dissociation and maintenance in high foetal bovine serum (FBS) concentrations. Non-haemopoietic cultures contained adherent cells, including macrophages and fibroblasts, but did not develop a non-adherent population. These cultures resulted with 10% FBS or horse serum and after explant outgrowth or dissociation with trypsin. Both culture types should be useful for studying the regulation of haemopoiesis and the functions of specific immune cells in fish.

Pronephros and spleen cells from rainbow trout (Oncorhynchus mykiss) in vitro: Growth factors produced under the influence of mitogens

Supernatants or conditioned media (CM) were produced by rainbow trout pronephros cells (PNC) and spleen cell cultures (1×106/ml), by addition of 20 μg/ml phythaemagglutinin (PHA), 5 ng/ml 12-O-tetradecanoyl-phorbol-13-acetate (PHA) (for 2h), PHA together with PMA, 10% horse serum (HS), or 100 μg/ml concanavalin A (ConA) after a culture of 7 days. Only PNC were effective growth factor producers. The effect of different concentrations of CM (5%–28%) on cell number was tested after a cultivation period of 14 days. PNC at a concentration of 1×106/ml were cultured with various concentrations of CM and the proliferation was tested by the XTT-test (testing the dehydrogenase activity of the cells by formation of a formazan) after 10 days. CM produced by cells with PHA, PHA and PMA and HS increased the proliferation in a concentration-dependent manner. CM produced with PMA alone was effective in the XTT-test. There was no synergistic or additive effect of PMA with PHA. CM produced with ConA had no effect, although in the XTT-test a strong proliferation of PNC in presence of 100 μg/ml ConA was observed. In a semisolid culture with collagen, CM treatment resulted in prevention of cell death and an increase in cell size but did not induce proliferation. All CM obtained from spleen cells had no effect. Stimulation of spleen cells by CM could be seen only in the XTT-test. PHA and HS trigger the PNC to release growth factors in vitro which stimulate cell growth and/or prevent cell death.

Androgens and immunocompetence in salmonids: Specific binding in and reduced immunocompetence of salmonid lymphocytes exposed to natural and synthetic androgens

Leukocytes from spleen and anterior kidney of both juvenile chinook salmon ( Oncorhynchus tshawytscha) and rainbow trout ( O. mykiss) demonstrated specific binding of 3H-testosterone and the synthetic androgen 3H-mibolerone. Saturation analysis of leukocytes from anterior kidney of rainbow trout using 3H-mibolerone indicated a single class of high affinity binding sites with K d = 1.04 nM ± 0.31 nM and B max = 0.11 nM ± 0.01 nM. Specific binding of 3H-mibolerone could be displaced by the natural androgens testosterone and 11-ketotestosterone and by the synthetic androgens mibolerone and 17 α-methyltestosterone, while even 500-fold excess of the known immunosuppressive steroid cortisol did not displace specifically bound 3H-mibolerone. A link between these steroid binding sites and androgen induced immunosuppression is indicated by the fact that the androgens which demonstrated specificity for the binding sites (testosterone, mibolerone and 17α-methyltestosterone) all significantly reduced the number of antibody producing cells in in vitro cultures of chinook salmon anterior kidney leukocytes.

Investigation of the structural and functional features of splenic ellipsoids in rainbow trout (Oncorhynchus mykiss)

The ultrastructure of ellipsoids in the spleen of rainbow trout (Oncorhynchus mykiss) is described. The endothelium of terminations of arterioles bulged into the lumen, and gaps between the endothelial cells were evident. A continuous basal lamina was not present, and there were extensive interdigitations between the endothelial cells and surrounding reticular cells. The interdigitating processes were rich in microfilaments. Intravenously injected colloidal carbon, approximately 0.03 μm in diameter, was held in the reticular matrix of the ellipsoidal wall and taken up by macrophages that extended cellular processes among the reticular and endothelial cells. The intravenous injection of fluorescent polystyrene microspheres of known diameter showed that microspheres with a diameter of 0.5 or 1.0 μm were localised in the red pulp, whereas microspheres with a diameter of 0.15 μm were retained in ellipsoidal walls. Thus, the terminations of splenic arterioles in rainbow trout were found to be consistent with descriptions of ellipsoids in other vertebrates in that they possessed a speciallised cuboidal endothelium, lacked a continuous basal lamina, were surrounded by a sheath of macrophages and reticular cells, and had a sheath of macrophages and reticular cells, and had a role in the selective filtration and retention of bloodborne particles.

A Comparison of Pathological Changes Caused by Vibrio anguillarum and Its Extracellular Products in Rainbow Trout(Oncorhynchus mykiss).

Some hematological parameters and histopathological changes in the liver, spleen, kidney, gills, gut, skin, muscle and heart of rainbow trout (Oncorhynchus mykiss) injected intraperitoneally with virulent Vibrio anguillarum or with V. anguillarum extracellular products (ECPs) were compared.The number of circulating red blood cells, the hematocrit and hemoglobin values decreased in the fish injected with either bacteria or ECPs compared with controls. The erythrocyte sedimentation rate increased in both groups of injected fish; however, the serum protein levels were similar to those observed in control fish.Light and electron microscopic observations of samples obtained from fish injected with V. anguillarum cells or its ECPs revealed similar histopathological changes in most tissues. Both groups of fish showed degeneration and necrosis of hepatocytes, vasodilation of the glomerular capillaries, cloudy swelling and hyaline droplet degeneration of the epithelial tubular cells, edematous gills, desquamation of the intestinal mucosa and necrosis of the muscle fibers at the site of injection. However, the bacterium multiplied extensively in the kidney and spleen where it caused more acute alterations than ECPs.Based on these results, it was concluded that the extracellular products play an important role in the virulence mechanisms of V. anguillarum.

Long-term liquid culture of haematopoietic precursor cells from the head kidney and spleen of the rainbow trout (Oncorhynchus mykiss)

Cells from the spleen and head kidney of rainbow trout (Oncorhynchus mykiss) were cultured in vitro. The cells were held in culture for more than three months. Spleen cells lived only a few days if cultured between October and May, but long-term cultures were possible in late spring and summer. This variation is assumed to be due to seasonal variations in the content of precursor cells.

In vitro stimulation of salmonid leucocytes with mitogens and with Aeromonas salmonicida

Leucocytes from peripheral blood (PBL), spleen and head kidney of Atlantic salmon ( Salmo salar ) and rainbow trout ( Oncorhynchus mykiss ) responded well when stimulated with lipopolysaccharide from Escherichia coli and phytohaemagglutinin (PHA). Thymocytes responded to PHA only. PBL from Atlantic salmon vaccinated against furunculosis showed significantly higher responses as compared to non-vaccinated fish when stimulated with an antigen preparation of whole cells of Aeromonas salmonicida . In leucocyte cultures from rainbow trout vaccinated against furunculosis, specific antibodies to A. salmonicida were produced in vitro . None or very low antibody levels were detected in cultures from non-immunised fish during the first 7 days in culture. After 9 days, a primary antibody response to A. salmonicida could, however, be detected in these cultures. Good correlation ( r =0·88) between antibody production by blood leucocytes and splenocytes was seen.

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%).

Effects of adrenaline on the blood flow through the spleen of rainbow trout ( Salmo gairdneri)

1. 1. Effects of adrenaline on the blood flow through the spleen were examined with rainbow trout, Salmo gairdneri. 2. 2. Administration of adrenaline induced an initial increase and an ensuing decrease in the flow rate. 3. 3. The dose producing 50% response of the maximum one was 1.2 ± 0.7 μmol/l. 4. 4. Observation of the intrasplenic vascular system by a corrosion casting method showed blind ended arteries and dramatic constriction of the arteries. 5. 5. The splenic arteries are an important site for control of blood flow and cause decreased flow rate after adrenaline administration. 6. 6. The possible effector unit causing rapid expulsion of stored blood cells is discussed.

Establishment of a Cell Line with Reticulo-endothelial Characteristics from a Rainbow Trout Spleen Explant

A cell line from rainbow trout (Oncorhynchus mykiss) with phagocytic-like characteristics was established. First we found that it was necessary to inject the fish with antibiotics (penicillinstreptomycin) in order to clear tissue of bacterial contamination. Two days later, the spleen was excised and minced with scissors in MEM10. The large number of erythrocytes in the suspension were removed by low centrifugation, then the tissue fragments were incubated on tissue culture flasks at 18°C for 30 days with complete changes of media every other day.After 30 days in culture, primary explants from spleen were composed of three types of cells. The majority were large mononucleate, plecmorphic cells with thinly spread cytoplasm. These cells were trypsinized and subcultured. Another cell types was simple, round cells, somewhat similar to mature lymphocytes, while the third type appeared to be similar to fibroblastic cells.After 9 months with the culture in 17 to 20 passages, the predominate cell type was showing similarities to phagocytic cell. These cells had a marked reticular cytoplasm and grew individually without parallel orientations. By enzyme-specific stains, the cells were observed to have abundant acid phosphatase and β-glucuronidase activities of the cytoplasm. They were negative for peroxidase. The cells were also found to have highly adherent and phagocytic properties when incubated with latex particles.The morphological, enzymatic, and functional findings suggested the possibility that these cells in culture have some properties similar to the cells which are composed of reticulo-endothelial system.

Distribution of omega-3 and omega-6 fatty acids in the ether- and ester-linked phosphoglycerides from tissues of the rainbow trout, Salmo gairdneri

1. 1. Data presented here demonstrate that polyunsaturated fatty acids in the phospholipids of rainbow trout tissues are compartmentalized differently than in mammalian tissues. 2. 2. We have determined the distribution of omega-3 (n-3) and omega-6 (n-6) fatty acids in the alkyl-, alk-1-enyl-, and diacyl- subclasses of phosphatidylcholines (PC), phosphatidyl-ethanolamines (PE), phosphatidylinositols (PI), and phosphatidylserines (PS) from gill, kidney and spleen of rainbow trout. 3. 3. Alkyl-linked PC and alk-1-enyl-linked PE were the most abundant ether-containing phospholipids, amounting to 10–15% of each class; no ether-linked PI or PS was detected. 4. 4. C20:4 n-6 was found in high concentrations only in PI; the n-3 fatty acids were found in highest concentration in the ether-linked phospholipids as compared with the diacyl subclasses and C20:5 n-3 was especially prevalent in 1-O-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamine and C22:6 n-3 was prevalent in PS.