Immune parameters of plaice, Pleuronectes platessa, L. along a sewage sludge gradient in the Firth of Clyde, Scotland.

: Plaice were caught at five stations varying in distance from the sewage sludge dumping site at Garroch Head, in the Firth of Clyde, Scotland and a number of physical and immunological parameters monitored. Significant intergroup differences were apparent in condition factor, hepatosomatic index, serum protein concentration, serum lysozyme, serum immunoglobulin (Ig), liver vitamin E, kidney leucocyte respiratory burst activity and kidney leucocyte bactericidal activity. Of these parameters, the hepatosomatic index, serum lysozyme, serum Ig and kidney leucocyte bactericidal activity showed a negative correlation with distance from the dump site (that is, were highest at the dump site); serum protein and liver vitamin E showed a positive correlation. Factors found not to vary between the groups included the spleen index, serum antiprotease activity and kidney leucocyte phagocytic activity. These findings are discussed in relation to recent experimental data on the effects of sewage sludge exposure on the immune system of fish.

Effect of temperature on macrophage activation and the production of macrophage activating factor by rainbow trout (Oncorhynchus mykiss) leucocytes.

Production of macrophage activating factor (MAF) by rainbow trout leucocytes has been shown to be temperature dependent in vivo and in vitro. Cells from fish held at 14 degrees C and stimulated to produce MAF immediately after isolation were capable of secreting MAF down to 6 degrees C (the lowest temperature tested). However, after 48 h at 6 degrees C, these leucocytes show impaired MAF secretion. Acclimation of fish to low temperatures (7 degrees C) did not recover the inhibitory effects of low in vitro temperatures on MAF production, but if these leucocytes were preincubated at 10 or 18 degrees C for 48 h, MAF was produced from these cells. Interestingly, macrophages isolated from fish kept at 7 or 14 degrees C and cultured at low temperatures (6 degrees C) were responsive to MAF-containing supernatants, and showed a higher relative increase in respiratory burst activity compared with their counterparts cultured at 10 and 18 degrees C. Such observations clearly demonstrate that a major impairment of bactericidal activity at low temperatures resides within the specific immune compartment of fish. The implications for fish health are discussed.

The effect of dietary vitamin A on the immunocompetence of Atlantic salmon (Salmo salar L.).

Atlantic salmon, Salmo salar L. were maintained on diets containing low (0.37 mg kg(-1) diet), normal (1.95 mg kg(-1) diet) and high (15 mg kg(-1) diet) levels of vitamin A fed at 1.5% body weight per day. After 4 months, liver vitamin A levels reflected dietary intake and growth rates of all three groups were similar. Kidney leucocyte migration and serum bactericidal activity were found to be significantly reduced in fish fed low levels of vitamin A. On the other hand, high levels of vitamin A in the diet were found to augment serum antiprotease activity relative to the levels found in the other dietary groups. However, phagocyte respiratory burst activity, bactericidal activity and eicosanoid production were unaffected by the dietary vitamin A regime, as were lymphocyte functions (lymphokine and antibody production) and both serum lysozyme and classical complement activity. That the overall immunomodulatory effect of vitamin A was small was reflected in the resistance to Aeromonas salmonicida. No significant differences were found between the different vitamin A intake groups despite a trend to decreased resistance in the low vitamin A diet group.

Agglutinin activity in Pacific oyster (Crassostrea gigas) hemolymph following in vivo Vibrio anguillarum challenge.

Hemolymph from the Pacific oyster (Crassostrea gigas) contains lectins that agglutinate horse (Gigalin E) and human (Gigalin H) erythrocytes. The gigalins also agglutinate bacteria, including Vibrio anguillarum, and were adsorbed from oyster hemolymph at different temperatures by living, heat-killed, and freeze-dried V. anguillarum cells. Baseline activities of the two gigalins were established by measuring their activities in oyster hemolymph over a period of 4 years. A normal distribution of Gigalin H activity (mean titer 139) was found, whereas the distribution of Gigalin E activity in the same samples was skew (mean titer 512). No covariance was observed between the two agglutinin activities. Increased lectin activity above this baseline was found in oysters exposed for varying time intervals to V. anguillarum at different seasons and temperatures over a period of 2 years. Such exposure resulted in an increase in activity (titer) of four- to nine-fold for Gigalin E and three- to seven-fold for Gigalin H when compared with controls, and in augmentation in the hemolymph of a protein with the same electrophoretic mobility as affinity-purified oyster lectins (gigalins). Challenge with either living or heat-killed bacteria resulted in a significant increase of Gigalin E activity, whereas results for Gigalin H were variable. Oysters challenged with bacteria were observed to filter normally with open shells during the experiments. Also, no increase was found in hemolymph calcium that could indicate anoxia following bacterial challenge (0.49 +/- 0.004 mg mL-1) compared to unexposed oysters (0.50 +/- 0.001 mg mL-1). Increase in the concentration of free amino acids in oyster hemolymph was observed following exposure to bacteria (15.05 mM) and anaerobiosis (13.51 mM) compared to controls (9.06 mM), and changes (in mol %) of individual amino acids differed considerably between hemolymph from animals challenged with bacteria and animals kept anaerobic. The augmented lectin activity in oyster hemolymph, following in vivo exposure to increased bacteria in the seawater, suggests their involvement in enhancing bacterial clearance and defense in the oyster.

Immunocompetence as a measure of the biological effects of sewage sludge pollution in fish.

1. Dab, Limanda limanda, exposed to nominal concentrations of 0 (control), 0.0032% (low) and 0.032% (high) sewage sludge in seawater for 12 weeks, were assessed for their immunological competence. 2. No effect upon total blood leucocyte and erythrocyte numbers was found, although significantly fewer thrombocytes were seen in the high-exposure group. 3. A decreased serum protein level was found in the high exposure group, but lysozyme and immunoglobulin levels showed non-significant differences between the groups. 4. Melano-macrophage centres were also affected in the high-exposure dab, which had increased numbers in the spleen and kidney. No effect upon spleen weights or oxygen free radical production by splenocytes was noted. However, oxygen free radical production by kidney leucocytes was inhibited in the low-exposure dab.

The enhanced induction of metallothionein by zinc, its half-life in the marine fish Pleuronectes platessa, and the influence of stress factors on metallothionein levels.

Intraperitoneal injection of zinc raised levels of a hepatic metallothionein-like species. Assuming that this species was metallothionein (MT) then levels were raised from approximately 20 micrograms/g to 300 micrograms/g in 7 days, and levels thereafter remained high for the next 4 weeks. The half-lives of the protein in liver and kidney from starved fish, measured using in vivo incorporation of 35S cysteine at 11 degrees C, were approximately 27 days and 32 days respectively. The following agents failed to stimulate synthesis of MT in plaice: stress (due to catching), endotoxin, dexamethasone, cortisol and turpentine.

Effect of opsonization on oxidative metabolism of plaice (Pleuronectes platessa L.) neutrophils.

The effect of serum opsonization on Vibrio alginolyticus (heat-killed)-stimulated chemiluminescence (CL) by plaice kidney- and peritoneal exudate-derived neutrophils was investigated. Peritoneal neutrophils only recognized heat-labile and kidney neutrophils only heat-stable opsonic activity in normal serum. Specific antibody did not show opsonic activity nor any synergism with the normal serum opsonins for either neutrophil population. Evidence was found for the production, by plaice neutrophils, of H2O2, O2-, OH. and two or more, as yet unidentified, reactive oxygen species (ROS).

Polar and neutral lipid composition of the gonads and serum of the plaice,Pleuronectes platessa L.

The individual polar and neutral lipids of plaice (Pleuronectes platessa L.) ovaries, testes and serum were determined, just before spawning, in February 1984 and March 1985. Serum was also assayed in June and September 1984. Phosphatidylcholine (PC) was found to be the major polar lipid in both male and female plaice serum throughout the year and in the ovaries. The testes, however, contained almost equal amounts of PC and phosphatidylethanolamine. The polar lipid levels in plaice gonads, expressed as a percentage of the total lipid present, were higher in the ovaries than in the testes, but in male plaice serum, the percentage of polar lipid was consistently higher than in the female. In plaice testes, 86% of the total neutral lipid occurred as cholesterol, but this represented only 49% of the total neutral lipid in the ovaries, with triacylglycerol as the other major ovarian neutral lipid.

Modulation of nonspecific host defenses in fish.

Seasonal, nutritional and hormonal effects on fish nonspecific protective mechanisms have been reviewed, together with the influence of some environmental pollutants. In the plaice (Pleuronectes platessa L.), the serum concentration of lysozyme and the acute phase reactant, C-reactive protein (CRP) and in vitro neutrophil migration have been used as indicators of the ability of test substances to modulate nonspecific defense mechanisms. Cortisol and adrenalin appear to be implicated in the production of CRP, and cortisol, at concentrations found in naturally stressed plaice, significantly reduces the migration of peritoneal neutrophils. Exposure to sub-lethal concentrations of inorganic mercury reduce circulating levels of lysozyme. The results provide further support for an association between environmental stressors and the disease susceptibility of fish populations. Phagocytic cell function, as measured by chemiluminescence, migration or phagocytic index, emerges as a useful test system.

Migration of fish leucocytes in vitro: the effect of factors which may be involved in mediating inflammation.

Plaice (Pleuronectes platessa L.) neutrophils were isolated from the kidney on a discontinuous Percoll gradient and from the peritoneal cavity at the peak of a glycogen-elicited inflammatory response. The migratory ability of neutrophils was assessed using a 48-well microchemotaxis chamber, with an incubation of 1.5 h at 12 degrees C. The two neutrophil populations showed different responses to N-formylmethionyl-leucyl-phenylalanine (FMLP). Whereas kidney neutrophils only showed a significant enhancement of migration at 10(-7) M, inflammatory neutrophils exhibited a bimodal response, with one peak of migratory activity at 10(-9) M and a second at greater than 10(-6) M. Kidney neutrophils showed a consistent response with various concentrations of a 24 h culture supernatant of Vibrio alginolyticus. In every case increased migration was observed with 5-, 10- and 100-fold dilutions, with the latter two conditions producing a significant enhancement (p less than 0.01 and p less than 0.05 respectively). The undiluted and 2-fold diluted supernatant caused a decreased cell migration compared with control values. The supernatant from kidney neutrophils cultured with serum-opsonized, heat-killed V. alginolyticus produced greater migratory activity than neutrophils or the treated bacteria incubated alone (the controls). In each case, the enhanced activity of the supernatant was detectable by 1 h of incubation. By 4 h, the activity of the neutrophil/bacteria supernatant was significantly higher than that of the controls (p less than 0.01), but by 24 h had fallen to control levels. There was no evidence for a chemotactic response with FMLP, the bacterial supernatant or the neutrophil-derived factor and the responses were therefore assumed to be chemokinetic.

Serum cortisol, glucose and lipids in plaice (Pleuronectes platessa L.) exposed to starvation and aquarium stress.

Plaice were maintained in the aquarium (11-12 degrees C) during May for 15 days without feeding. Within 48 hr, there was a decline in serum total lipids (P less than 0.001), phospholipids (P less than 0.01), triglycerides (P less than 0.001), cortisol (P less than 0.01) and glucose (P less than 0.001), but an increase in nonesterified fatty acids (NEFA; P less than 0.01). There was a significant inverse correlation between NEFA and glucose over 15 days (P less than 0.001) and between NEFA and cortisol over the first 5 days (P less than 0.01). Cortisol and glucose showed a significant correlation over 15 days (P less than 0.01). Serum cortisol and glucose were not apparently affected by starvation. Only cortisol provided a sensitive indicator of aquarium disturbance. Exposure of the fish to agitation or reduced O2 for 1 hr significantly elevated cortisol (P less than 0.001) but only the latter treatment elevated glucose (P less than 0.01); neither treatment affected the lipids.

Isolation of substances with vascular permeability-increasing activity from the skin of the plaice (Pleuronectes platessa L.).

A methanolic extract of plaice skin, from which lipids had been removed, was chromatographed on alumina, eluted with decreasing concentrations of ethanol. Only the 60% ethanol fraction exhibited smooth muscle activity, with bradykinin-like properties. The 20% ethanol fraction increased vascular permeability in rat skin, as measured by dye-leakage. This was not due to the degranulation of mast cells. Intradermal injection of either fraction into the plaice caused localized erythema.

The influence of hormones and inflammatory agents on C-reactive protein, cortisol and alanine aminotransferase in the plaice (Pleuronectes platessa L.).

Endotoxin stimulates production of both C-reactive protein (CRP) and cortisol in the plaice within 24 hr. Cortisol alone (optimum dose i.p. 500 micrograms/300 g wt fish) also stimulates CRP production and the possibility that endotoxin acts through cortisol was examined. Dexamethasone suppresses cortisol production but elevates CRP. Cortisol levels are restored to normal within 24 hr of endotoxin injection. Turpentine and ACTH which stimulate cortisol do not affect CRP. Endotoxin and cortisol have no significant effect on alanine aminotransferase activity in the serum and liver although it is elevated in the serum within 24 hr of the administration of adrenalin or turpentine.

Radioimmunoassay of serum cortisol in the plaice (Pleuronectes platessa L.).

The validity of a radioimmunoassay kit for direct measurement of cortisol in the plaice is described. This technique was used to determine the cortisol concentration in adult male and female plaice in 100 microliter of either plasma or serum. There was no significant difference in the serum cortisol values determined by radioimmunoassay either with or without chromatography. Storage of whole blood at 4 degrees for varying periods up to 24 hr, before removal of the serum, did not affect the cortisol concentration. Decreases also did not occur in serum samples kept at room temperature for up to 5 days, although losses of 1-12% occurred after 7 days and 11-39% after 8 days. The daily variations in serum cortisol levels were examined in fish exposed at 11 degrees to 12 hr light: 12 hr dark over a 32-hr period and the existence of a 24-hr cortisol rhythm was observed. Blood samples were taken from the same six fish every 4 hr, each fish being bled nine times during the experiment. Blood samples were also taken every 4 hr from groups of seven fish, each fish during this experiment being bled only once. Measurement of monthly serum samples throughout 1 year showed cortisol concentrations at a maximum in April, during the peak spawning period. There was no significant difference between cortisol levels in male and female plaice, except in January, and no difference between serum and plasma values in either sex.

Distribution of endotoxin and its effect on serum concentrations of C-reactive protein and cortisol in the plaice (Pleuronectes Platessa L.).

Bacterial endotoxins cause a significant increase in the serum concentration of both C-reactive protein (CRP) and cortisol in the plaice. The time course of the CRP elevation varies with the bacterial source, method of isolation of the endotoxin and the route of administration. Injection of the prostaglandin synthetase inhibitor, indomethacin, 2 hr before endotoxin, abolishes the increases in both CRP and cortisol, whereas it has no effect when given 6 hr after. Using [51Cr]labeled endotoxin, 40% of the total injected dose was localized in the kidney and 36% in the spleen, 24 hr after i.v. injection. Uptake was five times greater per g of spleen than of kidney.

Distribution of radiolabeled erythrocytes and the effect of temperature on clearance in the plaice (Pleuronectes platessa L.).

The removal of carbon and turbot erythrocytes (TRBC) from the circulation of plaice, acclimated for 7 days at temperatures between 5 and 19 degrees C, revealed similar biphasic clearance patterns with up to 90% of particles removed over the first 30 min. There was no statistically significant difference in the rate of clearance over this wide temperature range. Organ localization of 51Cr-labeled TRBC at 12 degrees C revealed the kidney and spleen as the main phagocytic organs. Carbon-blockade experiments resulted in a significant depression of subsequent 51Cr-TRBC uptake by the kidney, although the spleen was unaffected. In the plaice there was no compensatory organ uptake, such as by the spleen in blockaded mammals, and particles persisted in the circulation.

Serum concentrations of C-reactive protein and serum amyloid P component in plaice (Pleuronectes platessa L.) in relation to season and injected lipopolysaccharide.

1. Mean monthly serum levels of total protein, C-reactive protein (CRP) and serum amyloid P component (SAP) in plaice, showed no significant difference between the sexes. 2. Highest values for CRP and protein were found between June and September, with no significant seasonal variation in SAP. 3. There was no change in CRP concentration in plaice maintained for 7 days at a higher temperature of 18.5 degrees C. 4. Injection of lipopolysaccharide caused the highest value for CRP on day 1 and for the spleen index on day 5 after injection. 5. Phagocytic stimulation with carbon had no significant effect on the CRP response to endotoxin.

Evolution of alpha 2-macroglobulin. The purification and characterization of a protein homologous with human alpha 2-macroglobulin from plaice (Pleuronectes platessa L.) plasma.

A papain-binding protein (PB-protein) was purified to homogeneity from the plasma of plaice (Pleuronectes platessa L.). PB-protein inhibited the activity of trypsin and pancreatic elastase (serine proteinases), thermolysin (a metalloproteinase) and papain (a cysteine proteinase). Presaturation of PB-protein with trypsin prevented the subsequent inhibition of thermolysin, and vice versa. Only catalytically active endopeptidases were bound by PB-protein. The catalytic activity of trypsin bound by PB-protein was inhibited by 95% against an insoluble protein substrate, but only by 38% against a low-molecular-weight synthetic substrate. The remaining activity of the bound trypsin was partially protected against further inhibition by soya-bean trypsin inhibitor. Trypsin bound by PB-protein showed a decrease of 67% in its reactivity with antibodies. The inhibitory activity of PB-protein was inactivated at pH 8.0 by methylamine (0.2M) or dithiothreitol (1 mM). The inhibition of proteinases by plaice PB-protein shows the distinctive characteristics of inhibition by human alpha 2-macroglobulin, and it is concluded that the plaice protein is a homologue of the human macroglobulin.

C-reactive protein and serum amyloid P component in the plaice (Pleuronectes platessa L.), a marine teleost, are homologous with their human counterparts.

C-reactive protein and serum amyloid P component were isolated from serum of the plaice (Pleuronectes platessa L.), a murine teleost. The isolation was based on their calcium-dependent binding affinity for pneumococcal C-polysaccharide and for agarose, respectively. These specificities are the same as those of human C-reactive protein and serum amyloid P component, respectively, and we have previously reported that the plaice molecules resemble human C-reactive protein and serum amyloid P component in their electron microscopic appearance. We describe here estimation of the molecular weights of plaice C-reactive protein and serum amyloid P component and their subunits, and analysis of their amino acid composition, glycosylation and partial amino-terminal amino acid sequences. The results establish that plaice C-reactive protein and serum amyloid P component are homologous with each other and with their human counterparts and indicate that there has been stable conservation of this protein family throughout vertebrate evolution.