Atlantic Salmon Head Kidney Research Articles

Serum amyloid A transcription in Atlantic salmon ( Salmo salar L.) hepatocytes is enhanced by stimulation with macrophage factors, recombinant human IL-1β, IL-6 and TNFα or bacterial lipopolysaccharide

Serum amyloid A (A-SAA) has previously been reported to be an acute-phase protein in salmonids. Hepatocytes represent a major source of A-SAA in salmonids, but nothing is known about hepatocyte SAA synthesis in fish. In the present work, the expression of A-SAA transcripts in primary cultures of Atlantic salmon hepatocytes in response to macrophage derived cytokines, human recombinant cytokines and bacterial lipopolysaccharide (LPS) was studied by Northern blot analysis. The macrophage supernatants were prepared by stimulating Atlantic salmon head kidney macrophages with LPS, yeast glucan or a leukocyte derived macrophage activating factor (MAF). The supernatants from glucan- or MAF-stimulated macrophages had no effect on A-SAA expression of the hepatocytes, while supernatants from LPS-stimulated macrophages gave about a 2-fold increase in expression. The combination of either glucan and MAF, or LPS and MAF were more effective and these supernatants gave a 3.4- and 5.2-fold increase in A-SAA expression, respectively. The hepatocytes were also treated with the human recombinant cytokines TNFα, IL-1β and IL-6, alone or in combination. The A-SAA response to each of them alone was modest, but TNFα and IL-6 or IL-1β and IL-6 in combination gave a higher response than each cytokine alone. These data suggest that the expression of A-SAA by hepatocytes from Atlantic salmon is induced by cytokine-like molecules. Interestingly, hepatocytes treated directly with LPS gave a more than 10-fold increase in SAA mRNA expression, but it is not known if this is a direct effect of LPS on the hepatocytes or if it is mediated by other contaminating cell types.

Localization of purified surface molecules (lipopolysaccharide and A‐protein) from Aeromonas salmonicida in the kidney and spleen from Atlantic salmon, Salmo salar L.

Lipopolysaccharide (LPS) and A‐layer protein purified from Aeromonas salmonicida were administered intravenously in Atlantic salmon, Salmo salar L., either alone or in combination. Tissues from each organ were examined by immunohistochemical techniques, using a polyclonal antiserum against A‐protein and a monoclonal antibody against LPS. When given simultaneously, the antigens seemed to be taken up by different cells in both the head and trunk kidney. The most striking finding was that A‐protein was located in epithelial cells in renal proximal tubules, in contrast to LPS, which was not detected in this location. The amount of A‐protein in the tissue increased with time until 2 h after injection. Autoradiography of SDS polyacrylamide gel electrophoresis of head kidney homogenates showed that in vivo processing of A‐protein coupled to iodinated tyramine cellobiose (125I‐TC‐A‐protein) was completed within 24 h, in contrast to LPS, which was maintained in tissues. The findings of the present study suggest that cells of the head kidney of Atlantic salmon are capable of taking up and processing the A‐protein.

Nutritional impacts on the chemiluminescent response of Atlantic salmon (Salmo salar L.) head kidney phagocytes, in vitro

Measurement of an induced chemiluminescent response of Atlantic salmon (Salmo salar L.) head kidney phagocytes preincubated in plasma preparations varying in vitamin E, vitamin C, astaxanthin, copper (Cu), iron (Fe), manganese (Mn), zinc (Zn), cholesterol, triglycerides (TAG), glucose, and total protein concentrations were performed in order to determine nutritional in vitro effects on the amount of reactive oxygen species (ROS) produced during respiratory burst activity of these cells. Phorbol myristate acetate was used to stimulate the respiratory burst, and luminol was used as substrate. These plasma preparations were obtained from Atlantic salmon fed diets containing high or low levels of selected dietary pro-and antioxidants using a reduced factorial design. Plasma vitamin C and glucose significantly inhibited the generation of chemiluminescence measured as response curve area. There was also a tendency to an inhibitory effect of plasma Zn. A negative correlation between plasma Mn and chemiluminescent response was observed only at high concentrations of plasma vitamin C. Plasma concentrations of vitamin E, astaxanthin, Cu, Fe, cholesterol, TAG, and total protein showed no effects on the generation of ROS.

Expression of Mhc class I and II mRNA in a macrophage-like cell line (SHK-1) derived from Atlantic salmon, Salmo salar L., head kidney

A cell-line derived from Atlantic salmon (Salmo salar L.) head kidney (SHK-1) consisting of macrophage-like cells was investigated for major histocompatibility complex (Mhc) class I and class II messenger ribonucleic acid (mRNA) expression following stimulation with lipopolysaccaride (LPS), human recombinant insulin-like growth factor-I (IGF-I), and virus infection. A reverse transcriptase-polymerase chain reaction (RT-PCR) was used to provide a semi-quantitative comparison of specific mRNA levels. Incubation with LPS from Escherichia coli (100μg ml−1) resulted in a decrease in the expression of Mhc class I heavy chain mRNA after 24h followed by a weak increase or slightly reduced expression. Incubation with LPS from Aeromonas salmonicida subsp. salmonicida (10μg ml−1and 100μg ml−1) induced a more marked reduction in expression after 24h than LPS from E. coli, and a greater increase in expression after 72h. Similar observations were also recorded with respect to Mhc class II mRNA expression. Following infection with two different viruses that are pathogenic for Atlantic salmon (infectious salmon anaemia (ISA) virus, and infectious pancreatic necrosis (IPN) virus), the level of expressed mRNA for both Mhc classes increased at 24h post-infection, whereas the level was markedly reduced or absent 72h post-infection. IGF-I (10μg ml−1and 100μg ml−1) induced either a reduction or absence of Mhc transcripts following 3h and 48h incubation. The present study shows that a cell-line consisting of macrophage-like cells expresses Mhc class I and class II, and that the expression can be modified in response to bacterial components, infective viruses, and hormonal stimulation. The study demonstrates a link in expression between the two Mhc classes, indicating common regulatory mechanisms.

Characterisation of a long-term cell line (SHK-1) developed from the head kidney of Atlantic salmon (Salmo salarL.)

A recently established cell line from Atlantic salmon head kidney, SHK-1, was characterised with respect to phenotypic and functional properties. The cell line was derived from a macrophage-enriched cell culture. Proliferating cells appeared after several weeks in culture, but did not resemble macrophages. The cells were flattened and elongated with a variable morphology. Subcultured cells had a more homogeneous morphology. No reactivity for alkaline phosphatase was observed in cytocentrifuge preparations of SHK-1 cells, while strong to moderate reactivity was detected for acid phosphatase, non-specific esterase, Mg2+-dependent adenosine triphosphatase and 5′ nucleotidase. The SHK-1 cells reacted with monoclonal antibodies (MAbs) directed against human cytokeratin and porcine vimentin. No reaction was observed after staining with MAbs against Atlantic salmon and rainbow trout serum immunoglobulins (Ig). The cells reacted strongly with MAbs directed against Atlantic salmon peripheral blood leucocytes but not with a MAb specific for Atlantic salmon polymorphonuclear leucocytes. The SHK-1 cells did not endocytose protein ligands which are recognised by the scavenger, mannose- and galactose-specific receptors. Specific uptake of transferrin or Atlantic salmon Ig could not be demonstrated. The SHK-1 cells did phagocytose the fish pathogenAeromonas salmonicidato some extent, but no bactericidal activity was observed over a period of 72h. Thus, from this profile of phenotypic and functional properties, it is concluded that the SHK-1 cell line is derived from leucocytes that have some of the properties of a macrophage.

Microsilica as antigen carrier and adjuvant in the Atlantic salmon ( Salmo salarL.)

Bovine serum albumin and a glycine/HCl extract from Aeromonas salmonicidawere most effectively adsorbed to microsilica particles at pH 5·0 compared with pHs of 6·0, 7·0, 8·0 and 9·0. Atlantic salmon were immunised by intraperitoneal injection of the glycine/HCl extract from A. salmonicidaadsorbed to microsilica. Antibody titres against A. salmonicidaantigens demonstrated a prominent adjuvant effect of the microsilica 8 weeks after immunisation compared with soluble antigens. The duration of the antibody response suggests that microsilica may have some depot effect. The antibody response was, however, lower than the response obtained with Freund's complete adjuvant. Microsilica stimulated the respiratory burst activity in cultured Atlantic salmon head kidney macrophages. Some side effects of the microsilica were observed. Adhesions between abdominal organs and between the organs and the peritoneum were distinct. In addition, a dark greyish pigmentation at the sites of adhesion was evident. Cultured macrophages in the presence of high concentrations of microsilica resulted in toxic effects on the cells.

Isolation of acid peptide fractions from a fish protein hydrolysate with strong stimulatory effect on atlantic salmon (Salmo salar) head kidney leucocytes

Medium size (3000 d > Mw > 500 d) peptides from a hydrolysate of emptied stomachs from Atlantic cod (Gadus morhua) were fractionated on an S-Sepharose cation exchange chromatography column. Four distinctly separated acid peptide fractions were used in in vitro stimulatory experiments with head kidney leucocytes from Atlantic salmon (Salmo salar). All four acid peptide fractions promoted strongly elevated oxidative burst reactions in the leucocytes after 2 and 7 days of incubation at concentrations from 1 to 25 μg/ml. The stimulation was equally good, and in most cases better than the stimulation achieved with similar concentrations of lipopolysaccharides from the fish pathogen Aeromonas salmonicida. Visual inspection and pictures of peptide stimulated cells showed strongly enhanced vacuolisation and formation of long stretched out pseudopodes after 7 days of incubation. Acid peptide fractions from fish protein hydrolysate may be useful as adjuvants in fish vaccine and as an immune stimulant in fish feed.

Detection of Aeromonas salmonicidaby polymerase chain reaction in Atlantic salmon vaccinated against furunculosis

Furunculosis vaccine DNA was demonstrated by polymerase chain reaction (PCR) in head kidney and spleen cell preparations of Atlantic salmon. DNA primers transcribed from sequences of 16SrRNA were used, and the PCR products were verified by an internal, radioactively labelled 16SrDNA oligonucleotide probe. Intraperitoneally, single and double vaccinated pre-smolts were sampled 8 and 16 weeks after vaccination and found to contain vaccine DNA. Unvaccinated fish acted as negative controls. The spleen cell preparation of the double vaccinated fish were negative after 16 weeks.

A sequential light microscopic and ultrastructural study on the uptake and handling of Vibrio salmonicida in phagocytes of the head kidney in experimentally infected Atlantic salmon (Salmo salar L.).

The uptake and handling of Vibrio salmonicida in phagocytes of the head kidney of Atlantic salmon (Salmo salar L.) were evaluated by light and electron microscopy, including in situ identification of the bacterium by immunolabeling at the light microscopical and the ultrastructural level. Fish were injected with live bacteria, and 4, 24, 48, and 72 hours after inoculation, samples were collected after perfusion fixation. Morphologically, the most prominent change in the course of the experiment was an increasing number of intrasinusoidal, endothelial cell-adherent phagocytes and the elevated number of interstitial melanomacrophages. Immunohistochemically, bacterial antigens were initially identified in intrasinusoidal phagocytes, and at 24 hours postinfection in endothelium-adherent phagocytes and intrasinusoidal melanomacrophages. Later, (48 and 72 hours postinfection), the interstitial melanomacrophages were also found to harbor bacterial antigen. Ultrastructurally, bacteria were identified in phagosomes in intrasinusoidal phagocytes, and morphological findings also indicated an increased cellular degradation, including autophagocytosis. Immunoelectron microscopy indicated that bacterial antigens were associated with melanomacrophages, specifically in their electron-dense cytoplasmic granules. These findings indicate that intrasinusoidal phagocytes and melanomacrophages participate in the rapid and active clearance of particulate material from the circulation, i.e., pathogenic microorganisms, and in the scavenging of cellular degradation products. The process of formation of melanomacrophages and their possible function is discussed.

The stimulatory effect of a muscle protein hydrolysate from Atlantic cod, Gadus morhuaL., on Atlantic salmon, Salmo salarL., head kidney leucocytes

This study shows the stimulatory capacity of a cod muscle protein hydrolysate on Atlantic salmon head kidney leucocytes both in vivoand in vitro. Increased respiratory burst activities were demonstrated in leucocytes after stimulation with a peptide fraction in the molecular weight range 500–3000 Da. The respiratory burst activity was quantified using reduction of nitroblue tetrazolium (NBT) as a measure of superoxide anion production following stimulation with phorbol myristate acetate (PMA) as the trigger. In vitrodose-response studies demonstrated an increased stimulation with increasing concentrations of the peptide fraction (30–1000 μg ml −1). Intraperitoneal injection of the peptide fraction in Atlantic salmon (100 mg kg −1) resulted in enhanced levels of superoxide anion production in head kidney leucocytes 2 and 4 days post-injection when assayed immediately after isolation. Culturing the cells for 18 or 41 h abolished the stimulatory capacity of the peptides. Also, increased levels of the lysosomal enzyme acid phosphatase were measured in the isolated head kidney leucocytes after intraperitoneal injection. Microscopic examination of tissue slices two days after i.p. administration revealed accumulation of FITC-labelled muscle hydrolysate (500–3000 Da) mainly in macrophages and endothelial cells of the kidney.

Spleen and kidney of Atlantic salmon ( Salmo salar L.) show histochemical changes early in the course of experimentally induced infectious salmon anaemia (ISA)

Infectious salmon anaemia (ISA) is a disease of farmed Atlantic salmon ( Salmo salar L.) in Norway that affects both erythrocytic and leucocytic cells. Both cell types are possible target cells for the aetiological ISA agent, which is probably a virus. In the present study the distribution and phenotype of leucocyte populations in the spleen and head kidney of Atlantic salmon that were developing ISA have been examined. Frozen tissues were collected from fish at various times after inoculation with ISA-infective material. Immune and enzyme histochemical techniques were used to characterise the response of leucocyte populations. Acid phosphatase positive macrophages predominantly in the red pulp of the spleen appeared to have engulfed erythrocytes at day 4 after infection. Evidence of degradation products of phagocytosed erythrocytes was present in macrophages in red pulp of the spleen at day 7 after infection, in addition to the usual site of erythrophagocytosis in melanomacrophage accumulations. Signs of erythrophagocytosis were not found in the head or body portions of the kidney. The activation of macrophages in the spleen at day 7 was suggested by decreased reactivity for the enzyme 5′ nucleotidase. From day 7, clusters of immunoglobulin positive (Ig + ) cells were present in the head kidney, while from day 11, the ellipsoids of the spleen showed reactivity for Ig and complement factor C3. These observations are discussed in relation to early immunoglobulin production and possible immune complex trapping. The present results suggest that the leucocyte populations in Atlantic salmon respond to ISA infection through macrophage activation and the initiation of an immune response.

Effects of antigens from Aeromonas salmonicida ssp. achromogenes on leukocytes from primed and unprimed Atlantic salmon ( Salmo salar L.)

The effects of extracellular antigens isolated from Aeromonas salmonicida ssp. achromogenes, were tested on leukocyte cultures from the head-kidney of Atlantic salmon ( Salmo salar L.) fingerlings. Samples from vaccinated fingerlings were compared to samples from control fingerlings that were injected with incomplete Freunds adjuvant (IFA) or phosphate buffered saline (PBS). The antigen preparations used for the comparative studies were extracellular products (ECP) and a 20 kDa toxic metallo-caseinase (AsaP1). Lipopolysaccharide (LPS) isolated from the bacterium was tested on unvaccinated fingerlings only. All antigen preparations were mitogenic to cultures from unvaccinated fish. In cultures from vaccinated fish there were difficulties in discerning between mitogenic effects, adjuvant induced cell activation and possible specific responses to the antigens.

In vitrodegradation of Aeromonas salmonicidaand Limulus polyphemushemocyanin by Atlantic salmon, Salmo salarL. macrophages

Cultivated Atlantic salmon head kidney macrophages endocytosed and digested iodinated formaldehyde-fixed Aeromonas salmonicidaantigens effectively. The degradation was time-dependent as demonstrated by increased acid soluble radioactivity obtained in macrophage media with time after the addition of antigens. Gel chromatography of the degraded material revealed a peak of radioactivity corresponding to molecular weights between 1000 and 6500 Da. In vitrodegradation of Limulus polyphemushemocyanin (LPH) adsorbed to mineral particles was less efficient compared to the degradation of whole bacterial cells, whereas degradation of soluble LPH was negligible. There were no significant differences in the rate of processing at 4 (6), 12 and 18°C. Inhibition studies using ammonium chloride, a substance that raises the pH of endosomes and lysosomes, showed decreased intracellular degradation of A. salmonicidain a dose-dependent manner. Monensin, a substance reported to block receptor-mediated phagocytosis, very effectively reduced the extent of processing whereas leupeptin and pepstatin, specific inhibitors of the major classes of cysteine and aspartic proteinases respectively, only slightly affected the processing.

Isolation of the causal virus of infectious salmon anaemia (ISA) in a long-term cell line from Atlantic salmon head kidney.

A long-term cell line (SHK-1) supporting replication of the causal virus of infectious salmon anaemia (ISA) has been established. The cell line was developed from a culture of Atlantic salmon (Salmo salar L.) head kidney cells. CPE was observed in SHK-1 cells 12-14 days after inoculation with ISA-infective tissue material. The time for CPE to develop decreased after repeated passages of medium from infected cell cultures to new cultures. Transmission trials demonstrated that Atlantic salmon parr developed ISA after intraperitoneal injection of preparations made from infected cells and growth medium. The ISA infectivity of the cell preparations increased with incubation time of inoculated cells. Cell cultures in a second passage were found to have a higher infectivity than the primary inoculated cultures. Virus particles with a diameter of approximately 100-120 nm, and which contained an external envelope and granules were seen in electron micrographs of thin sections of infected cells. Most of the virus particles were located extracellularly close to the cell surface, and in some cases, a connection between virus and plasma membrane could be observed. This indicates that virus particles were released by budding. Enveloped virus particles of 45-140 nm in diameter were seen in abundance in electron micrographs of a negatively stained purified virus preparation. Large, highly pleomorphic particles up to 700 nm in the longest dimension were occasionally observed in unpurified preparations. The evidence is therefore strong that the virus isolated in SHK-1 cells is the aetiological agent of ISA.

In vitro studies of infectious pancreatic necrosis virus infections in leucocytes isolated from Atlantic salmon ( Salmo salar L.)

Leucocytes were isolated from blood and head kidney of Atlantic salmon carrying infectious pancreatic necrosis virus (IPNV). Results that indicated an IPNV replication were only obtained from adherent head kidney leucocytes (mainly macrophages). IPNV specific fluorescence was detected in both carrier and in vitro infected macrophages, and the number of fluorescent cells increased during a week in culture. However, the release of infectious IPNV was low. Preliminary results also indicated that in vitro IPNV infections of macrophages seemed to reduce the respiratory burst activity in these cells.

Yeast β-glucan stimulates respiratory burst activity of Atlantic salmon ( Salmo salar L.) macrophages

Previous studies have shown that head kidney macrophages isolated from glucan injected rainbow trout ( Oncorhychus mykiss) and Atlantic salmon ( Salmo salar L.) have increased ability to kill Aeromonas salmonicida. The present work was aimed at investigating the in vitro effects of glucan on the respiratory burst and bactericidal potential of Atlantic salmon head kidney macrophages. Salmon macrophages were incubated for 1–7 days with various concentrations of yeast β-glucan (MacroGard ®) and tested for respiratory burst activity by the reduction of nitroblue-tetrazolium (NBT) after exposure to phorbol myristate acetate (PMA) or opsonized zymosan. The macrophages showed a marked increase in respiratory burst activity 4 to 7 days after addition of glucan. Macrophages treated with 0.1–1 pg mL −1 gave a maximum respiratory burst response, whereas 10 μg mL −1 had no effect and 50 μg mL −1 was inhibitory. The glucan also triggered respiratory burst activity directly, but this occurred only at relative high concentrations with a maximal effect at ⩾200 μg mL −1. The validity of using the NBT-assay as a measure of respiratory burst activity was confirmed by using inhibitors of O 2 − production (superoxide dismutase, trifluoperazine and diphenylene iodonium). Despite the stimulatory effect of glucan on the respiratory burst activity of salmon macrophages, these cells did not show increased bactericidal activity against the avirulent and virulent strain of A. salmonicida. Upregulation of burst activity alone is thus apparently not sufficient to enhance bactericidal activity against this pathogen by Atlantic salmon macrophages.

Immune and enzyme histochemical phenotypes of lymphoid and nonlymphoid cells within the spleen and head kidney of Atlantic salmon ( Salmo salar L.)

A panel of immune and enzyme histochemical techniques was used to characterise the phenotypes of lymphoid and nonlymphoid cells in the spleen and head kidney of Atlantic salmon. Emphasis was placed on the identification of nonlymphoid cells and the definition of compartments within lymphoid organs. Reticular cells were characterised by reactivities for 5′nucleotidase and alkaline phosphatase and macrophages by reactivities for acid phosphatase and non-specific esterase. These nonlymphoid cell populations were found among the melanomacrophage accumulations of the head kidney and spleen and within the periellipsoidal macrophage sheaths of the spleen. Mg 2+ dependent adenosine triphosphatase-positive cells in the spleen had a more prominent dendritic morphology than cells with similar enzyme reactivity in the head kidney. In the spleen there was a clear association between immunoglobulin-positive cells, melanomacrophages and alkaline phosphatase-positive arterioles and non-specific esterase-positive venous sinusoids. In the head kidney the large population of immunoglobulin-positive cells was scattered through the parenchyma as individual cells or in small groups. Patterns of immune or enzyme histochemical reactivities that would be consistent with a germinal centre reaction were not observed in any of the fish examined in this study. The absence of such specialised microenvironments within the major secondary lymphoid organs of the Atlantic salmon agrees with the current understanding of the immune response and immunological memory in salmonids.

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.