Flow cytometry assays of respiratory burst in Atlantic salmon (Salmo salar L.) and in Atlantic cod (Gadus morhua L.) leucocytes

The oxidation of dihydrorhodamine 123 (DHR) to the fluorescent rhodamine 123 (RHO) was detected using flow cytometry. This assay for detection of respiratory burst activity was established in peripheral blood leucocytes (PBL) and head kidney leucocytes (HKL) of Atlantic salmon and Atlantic cod. The leucocytes were stimulated by phorbol 12-myristate 13-acetate (PMA). For cod cells 10 times lower concentration of PMA had to be used compared to salmon cells, as higher concentrations were toxic and resulted in considerable cell death. The cells found to be RHO-positive were monocytes/macrophages and neutrophils based on the scatter dot plots, but for salmon also some small cells were found to have high fluorescence intensity both in the flow cytometry analyses and by fluorescence microscopy of cytospin preparations. The nature of these cells is not known. For cod leucocytes, such cells were not obvious. The instrument settings are a bit more demanding for cod, as cod cells die more easily compared to salmon cells. In both assays the limit between negative and positive cells has to be carefully considered. The presented flow cytometry protocols for measurements of respiratory burst in salmon and cod leucocytes can be applied in various studies where respiratory burst functions are involved, such as to verify if it is activated or suppressed in connection with infections and immunostimulation.     

Experimental study on the effect of low molecular DNA from salmon milt on hemopoiesis]

Biological activity of low molecular DNA isolated from salmon milt was studied. When administered subcutaneously to mice with with acute experimental radiation disease in a course dose of 10 mg/kg, it showed a therapeutic effect, stimulated hemopoiesis, increased the survival rate and the average life span of the animals. Moreover, its marked effect on the humoral immunity was observed.     

Induction of Mx protein in Atlantic cod with poly I:C: immuno-cross reactive studies of antibodies to Atlantic salmon Mx with Atlantic cod

A polyclonal rabbit antiserum directed against the conserved region of the Atlantic salmon antiviral Mx1 protein was used to detect the putative Atlantic cod Mx protein using Western and dot blotting. A doublet band at about 75kDa and 65kDa was detected by Western blotting in kidney and spleen extracts of cod 3 and 4 days after i.p. injection with poly I:C but not in control fish injected with PBS. In blood leucocyte lysates, similar immunostaining could also be detected in Atlantic cod weakly after injection with PBS and more intensely after injection with poly I:C, suggesting some constitutive expression of Mx protein by leucocytes. Dot blot analysis showed that the Mx protein level was significantly higher in spleen, kidney, liver and gill of cod at least up to 4 days after injection with poly I:C when compared with the PBS-injected controls.     

Experimental study on the effect of low molecular DNA from salmon milt in pseudotuberculosis infection]

The effect of low molecular DNA from salmon milt (nDNA) in experimental pseudotuberculosis in mice was studied. When nDNA was admiministered orally, dissemination of the organs by Yersinia pseudotuberculosis lowered and the survival of the animals infected with 100-percent lethal dose of the bacteria increased. nDNA decreased contamination of the epithelial cells by the microbe in vitro and prevented the lethal effect of the Y. pseudotuberculosis toxins on the mice.     

The effect of stress on reproduction in Atlantic cod

This study imposed stress upon spawning cod Gadus morhua in the laboratory to evaluate the potential effects of trawl avoidance on their reproductive physiology and spawning behaviour. Plasma cortisol levels of stressed fish were significantly higher than controls. Stressed cod initiated fewer courtships and often performed an altered courtship sequence. Both control and stressed fish spawned and there was little difference in the production of eggs, fertilization rate, hatching success or time to starvation of larvae. However, stressed fish produced abnormal larvae more frequently. Cod exposed to a chronic stressor are able to spawn successfully, but there appears to be a negative impact of this stress on their reproductive output, particularly through the production of abnormal larvae.     

In vivo and in vitro insulin and fasting control of the transmembrane fatty acid transport proteins in Atlantic salmon (Salmo salar)

We have examined the nutritional and insulin regulation of the mRNA expression of transmembrane fatty acid (FA) transporters [FA transport protein-1 (FATP1) and CD36] together with the lipoprotein lipase (LPL), the cytosolic FA carrier FA binding protein (FABP3), and mitochondrial FA-CoA and -carnitine palmitoyl transferase carriers (CPT)1 and -2 in Atlantic salmon tissues and myocyte cell culture. Two weeks of fasting diminished FATP1, CD36, and LPL in adipose tissue, suggesting a reduction in FA uptake, while FABP3 increased in liver, probably enhancing the transport of FA to the mitochondria. Insulin injection decreased FATP1 and CD36 in white and red muscles, while both transporters were upregulated in the adipose tissue in agreement with the role of insulin-inhibiting muscle FA oxidation and stimulating adipose fat stores. Serum deprivation of 48 h in Atlantic salmon myotubes increased FATP1, FABP3, and CPT-2, while CPT-1 was diminished. In myotubes, insulin induced FATP1 expression but decreased CD36, FABP3, and LPL, suggesting that FATP1 could be more involved in the insulin-stimulated FA uptake. Insulin increased the FA uptake in myotubes mediated, at least in part, through the relocation of FATP1 protein to the plasma membrane. Overall, Atlantic salmon FA transporters are regulated by fasting and insulin on in vivo and in vitro models.     

Catecholamine biosynthesis in vitro and in vivo in the chromaffin tissue of the Atlantic cod, Gadus morhua

1. The biosynthesis of [3H]catecholamines from [3H]L-tyrosine in the intact chromaffin tissue of cod posterior cardinal veins was studied in vitro and in vivo at 10 degrees C. 2. The tritiated products dopamine, noradrenaline and adrenaline were separated from the [3H]tyrosine by paper chromatography of tissue extracts and the radioactivity of 1 cm strips of the chromatogram was determined by liquid scintillation spectrophotometry. DOPA could never be demonstrated in the tissue extracts from any of the experiments performed. 3. The content of [3H]noradrenaline in pieces of the cardinal veins incubated in vitro was found to increase rapidly. The tissue content of dopamine and adrenaline remained at lower levels which were reached during the first few hours of the incubation. A similar pattern could be demonstrated in the chromaffin tissue in vivo after infusion of [3H]tyrosine, but the total content of the [3H]catecholamines was lower than in the in vitro experiments. 4. The results are consistent with the view that the methylation of noradrenaline is the rate-limiting step in the biosynthesis of adrenaline in cod chromaffin tissue.     

Chum salmon (Oncorhynchus keta) stanniocalcin inhibitis in vitro intestinal calcium uptake in Atlantic cod (Gadus morhua)

Summary Chum salmon (Oncorhynchus keta) stanniocalcin was purified, partially identified and tested for bioactivity in an assay on the intestinal calcium uptake in a marine teleost (Gadus morhua). Basic ethanol extraction, ion exchange chromatography, gel filtration and reverse-phase high-performance liquid chromatography resulted in the isolation of a homogenous glycoprotein that appears as a 46-kDa product under non-reducing conditions and as a 23-kDa product under reducing conditions after sodium dodecylsulphate-polyacrylamide gel electrophoresis. The glycoprotein is likely to be a homodimer composed of two subunits of 23 kDa each. Further characterization indicates homology to Australian eel, sockeye salmon, coho salmon and rainbow trout stanniocalcin, and the glycoprotein is thus concluded to be stanniocalcin. Stanniocalcin-like immunoreactivity was demonstrated in the corpuscles of Stannius of the Atlantic cod, with a specific antiserum raised against purified chum salmon stanniocalcin. The physiological importance and the biological activity of chum salmon stanniocalcin was tested by evaluating its effect on intestinal calcium uptake by the Atlantic cod in vitro. The intestine was perfused, both vascularly and through the intestinal lumen, and the calcium mucosa-to-serosa flux was measured using ⁴⁵Ca²⁺ as a tracer. Stanniocalcin decreased the intestinal calcium uptake in a dose-related manner by 13.5% and 22.4% at doses of 2.2 and 10.9 nM stanniocalcin, respectively. The results establish the intestine as a target organ for stanniocalcin in marine teleosts.Abbreviations BIS balanced intestinal solution - CS corpuscles of Stannius - dpm disintegrations per minute - FW freshwater - J _in ^Ca influx of calcium across the intestinal mucosa - MW molecular weight - NRS normal rabbit serum - PBS phosphate buffered saline - PBST phosphate buffered saline containing 0.05% Tween-20 - PITC phenyl isothiocyanate - rp-HPLC reverse phase - SW seawater - STC phenyl isothiocyanate - rp-HPLC reverse phase - SW seawater - STC stanniocalcin - TFA Trifluoroacetic acid - Tris Tris(hydroxymethyl) aminomethan - V volume per fraction     

Transport-associated proteins in Atlantic salmon (Salmo salar)

 The major histocompatibility complex (Mhc) regions of mice, rats, and humans all contain a pair of related genes, TAP1 and TAP2, which encode members of a large superfamily of proteins of similar structure and function. A functional TAP1/TAP2 heterodimer is probably required for efficient presentation of antigens to CD8⁺ T cells. This heterodimer resides in the membrane of the endoplasmic reticulum, and transports peptides from the cytoplasm into the endoplasmic reticulum lumen for binding to Mhc class I molecules. The TAP transporter demonstrates specificity for both peptide sequence and length, and in rats, allelic variation in the sequence of the transporter molecules results in differential ability to transport particular peptides. Here we report two expressed Sasa-TAP2 loci, both of which are polymorphic, as well as an expressed Sasa-TAP1 locus from Atlantic salmon. The Sasa-TAP2A locus has a genomic organization similar to the human TAP2 equivalent. Received: 23 December 1996 / Revised: 26 February 1997     

Gorgoderina vitelliloba: interaction with frog leucocytes in vivo and in vitro

Ultrastructural observations were made on the response in vivo of adult Rana temporaria to Gorgoderina vitelliloba and the interaction between host cells and the parasite in vitro. In both cases, the cellular attack on the fluke was most intense 21-23 days postinfection. Host leucocytes (believed to be eosinophils) lay flat against the surface of the parasite. Degranulation of their electron-dense, peroxidase-positive granules occurred and vacuoles formed. The outer plasmalemma of the tegument was breached and eosinophils migrated over the basal lamina, stripping the tegument from the surface of the fluke. The cellular response was so vigorous in vitro that the tegument was sometimes completely lost. In vivo, damage was slight, and the majority of the parasites successfully completed their migration to the bladder, where there was no further cellular response, and tegumental repair occurred. The fact that the migration of the fluke from the kidney to the bladder occurred at the same time as the peak of the cellular response may not be coincidental, and it is suggested that the cellular attack may initiate migration from the kidneys.     

The intracellular lifestyle of Francisella noatunensis in Atlantic cod (Gadus morhua L.) leucocytes

Francisella noatunensis causes the systemic granulomatous inflammatory disease, francisellosis in cod. Little is known about the lifestyle of this facultative intracellular bacterium within cod leucocytes. We have examined the interaction of this bacterium with phagocytic cells isolated from cod with emphasis on monocytes, macrophages, neutrophils and phagocytic B-cells. It is clear from confocal microscopy sections through adherent cell preparations that numerous bacteria were located intracellularly following in vitro infection in monocytes and macrophages. In these sections bacteria were immunostained and cell actin was stained using Alexa Fluor^® 488 phalloidin. Bacteria were observed in close association with neutrophils and intracellularly (low numbers) in B-cells. Bacteria were observed more frequently in head kidney- than in peripheral blood- and spleen- leucocytes. Following infection, bacteria were initially observed grouped together and located close to the nucleus. Later they were found spread within the cytoplasm. This indicates egression of F. noatunensis from the phagosome to the cytoplasm where replication possibly takes place. It may be hypothesised that the bacteria may alter maturation of the phagosome and thus, avoid the potent intracellular killing mechanisms of phagocytic cells. The intracellular lifestyle involving escape to cytoplasm prior to fusion with the lysosome may have consequences for vaccine development as well as antibiotic treatment of infected cod.     

Phagocytosis by B-cells and neutrophils in Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.)

Phagocytosis by fish cells has mostly been studied using adherent leucocytes, excluding suspended cells such as the majority of B-cells and neutrophils, but a recent study describes professional phagocytosis of latex beads and bacteria by B-cells from rainbow trout. In the present study, phagocytosis by B-cells and neutrophils from salmon and cod was studied. Leucocytes were isolated from peripheral blood (PBL) and head kidney (HKL). By flow cytometry analyses, proportions of MAb labelled cell populations with internalized fluorescent beads, as well as the number of beads within each cell, could be determined. Phagocytic capacity and ability were demonstrated in B-cells and neutrophils from salmon and cod. In salmon, B-cells had higher phagocytic ability than neutrophils in HKL, but not in PBL. For cod the phagocytic ability of B-cells were lower than for neutrophils in both HKL and PBL, but the phagocytic capacity of cod B-cells were higher than for neutrophils in both HKL and PBL. For salmon B-cells the phagocytic capacity was lower than or similar to neutrophils in HKL and PBL. The total phagocytic ability of leucocytes was different in the species studied. The highest phagocytic ability was observed in cod, showing similar values for PBL and HKL. Salmon PBL displayed about twice the phagocytic ability of cod PBL. There seemed to be some major differences between the two fish species concerning phagocytosis. In salmon, a rather large proportion of phagocytic leucocytes were phagocytic B-cells, indicating that B-cells may have an important function in particle clearance in this species. In cod, phagocytic leucocytes in HKL and PBL were mostly neutrophils, and only a small proportion of B-cells were phagocytic, supporting the more prominent role of innate immune functions in cod neutrophils.     

Size-dependent mortality of juvenile Atlantic cod, estimated from recaptures of released reared cod and tagged wild cod

The recapture rate and survival of reared cod Gadus morhua L. (15–45 cm) released in fjords and open coastal areas of Norway increased with increasing size at release, while release area and season also had a significant influence on the results. High mortality soon after release reduced the number of released fish by more than two–thirds within 6 months, and reduced the potential commercial yield to a low level. In most cases the recapture yield was lower than the released biomass and the net yield increased little or not at all with increasing size at release. In western Norway high fishing mortality of young fish reduced the numbers of surviving fish rapidly and the growth overfishing reduced the potential yield still further. Natural mortality decreased with increasing size at release, and became low when the fish approached their size refuge, i.e. when they could not be eaten by their usual predators.     

Miltpain, a cysteine proteinase, from milt of Pacific cod (Gadus macrocephalus): purification and characterization

Miltpain (EC.3.4.22.-) is a cysteine proteinase that preferentially hydrolyzes basic proteins, previously found in the milt of chum salmon. Here we report a similar cysteine proteinase in the milt of the marine Pacific cod. The enzyme was isolated and purified 6900-fold and with an estimated mass of 63 kDa by gel filtration chromatography and 72 kDa by SDS/PAGE. Cod miltpain has an optimum pH of 6.0 for Z-Arg-Arg-MCA hydrolysis, and K_m of 11.5 μM and k_cat of 19.0 s⁻¹ with Z-Arg-Arg-MCA. It requires a thiol-inducing reagent for activation and is inhibited by E-64, iodoacetamide, CA-074, PCMB, NEM, TLCK, TPCK, ZPCK and o-phenanthroline. This proteinase strongly hydrolyzes basic proteins such as salmine, clupeine and histone, and exhibits unique substrate specificity toward paired basic residues such as Lys-Arg, Arg-Arg on the substrates of P2-P1. The isoelectric point is 5.2 by isoelectric focusing. N-Terminal sequencing gave a sequence of <EVPVEVVRXYVTSAPEK. The cysteine proteinase from Pacific cod very closely matches the previously reported miltpain from chum salmon.