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The large yellow croakers (Larimichthys crocea) are mainly present in the Chinese coast and near seas with high economic importance, but vulnerable to many diseases, especially in the breeding and aquaculture. The purpose of this research was to boost the innate immune system of the large yellow croaker by administering bitter peptides into their peritoneal cavity. Total 120 Juvenile of large yellow croakers in very even weight of 60 g were divided into 4 different groups in 200/300 L of water tank, respectively. Fish growth were observed for 3 months before and after different treatments. The bitter peptides from pepsin hydrolysis were applied because they possess the highest bitter sensory scores. The blood of fish from the different groups was collected and tested for different immune parameters to evaluate the effectiveness of bitter peptides as immune stimulants after administration for 8 weeks. The average ratio of leukocytes/total blood cells (%) for control was found at 14.6%, for the low dose of bitter peptides 0.6 mg/fish was at 29.3%, for middle dose of 1.2 mg/fish was at 35%, and high dose of 2.4 mg/fish was at 30%. The lysozyme assay showed that the OD (optical density) units of relative progress lysis activity at 60 min were 0.17, 0.101, 0.307 and 0.198, respectively. Similarly in the same order as in phagocyte assay, most importantly the middle dose (1.2mg/fish) gave the highest survival rate throughout the assay. The results showed that bitter peptides can be used as immune boosters for the yellow croakers and the optimum dose was 1.2 mg/fish due to both leukocytes and lysozyme activity in the treated samples increased significantly compared with the control group. According to the results obtained, we suggest that the incorporation of middle dose of bitter peptides into fish feeds may reduce the fish diseases in aquaculture, at least for large yellow croakers.

Phagocyte recognition of lipopolysaccharide (LPS) is an early key event for triggering the host innate immune response necessary for clearance of invading bacteria. The ability of fishes to recognise LPS has been questioned as contradictory results have been presented. We show here that monocyte/macrophage cultures from Atlantic cod (Gadus morhua) and Atlantic salmon (Salmo salar) respond with an increased expression of inflammatory and antibacterial genes to both crude and ultrapure Escherichia coli LPS. Crude LPS produces higher induction than the ultrapure LPS type in both species in vitro as well as in vivo in cod injected with LPS. Crude LPS gave, in contrast to ultrapure LPS, an additional weak up-regulation of antiviral genes in salmon macrophages, most likely because of contaminants in the LPS preparation. Increased levels of chicken (c)-type lysozyme transcripts and enzyme activity were measured in salmon macrophages following ultrapure LPS stimulation demonstrating not only increased transcription but also translation. Simultaneous use and even pre-treatment with bovine sera suppressed the LPS-induced expression thereby reflecting the presence of transcription inhibitory components in sera. Together, these findings show that both cod and salmon recognise LPS per se and that the observed induction is highly dependent on the absence of sera.

Sea bass (Dicentrarchus labrax L.) were injected intraperitoneally with monovalent (Photobacterium damselae subsp. piscicida or Vibrio anguillarum) and divalent (Ph. damselae subsp. piscicida and V. anguillarum) vaccine formulations, with or without adjuvants (mineral oil, liposome or alginate), to evaluate the short and long-term pathological effects. Eight animals from each group were sampled one, two, four and 11 months after intraperitoneal injection. The acute peritoneal response and the progression to a chronic status were evaluated by analysing peritoneal leucocytes collected during the first days post-injection. To evaluate the chronic response, the late peritoneal leucocyte response was analysed and the peritoneal cavity was examined and the intra-abdominal lesion level scored based on a pre-defined scale. Correlation between leucocyte exudative response, tissue inflammatory response and the development of granuloma were sought. The acute leucocyte response was characterized by an early (24-48 h) mobilization of neutrophils and macrophages, with phagocyte numbers dependent on the formulation, but no significant variations were observed in lymphocytes/small cells and EGCs. Later on, a steady increase occurred in lymphocytes/small cells and EGCs and a high concentration in neutrophils and macrophages was maintained up to 30-60 days in groups i.p. injected with oil adjuvanted formulations with antigen. All the lesions observed were moderate, indicating that in sea bass, the pathological effects due to intraperitoneally injected vaccines are less severe than in other fish species. The divalent oil adjuvanted vaccine induced the most severe side effects, with macroscopic granulomas consistently present up to 11 months.

Interleukin 6 (IL-6) is a protein secreted by T cells and macrophages and plays an important role in immune responses. Include regulated inflammatory reactions, proliferation and differentiation lymphocyte, regulation in both innate and adaptive immune responses; these responses will help to eliminate the invaded pathogens in mammal. In our previous research, the grouper (Epinephelus coioides) IL-6 is proven to enhance the production of immunoglobulin, antimicrobial peptide, acute phase protein, increasing phagocyte phagocytosis and lysozyme activity; that was also increase the survival rate of recombinant IL-6 injected fish under bacterial or viral pathogen challenge. These results may represent the IL-6 might be a good immune immunestimulatant candidate for fish to prevent or treatment disease. However, an optimal administrated route of the recombinant IL-6 is important for its efficacy and used friendly. In this study, we try to evaluated minimal effective dose in the various administration routes include injection, oral, immersion and jet injection by detect immune gene expression and survival rate in the challenge test. The results shown injection administration with the lowest dose can achieve the protection; even with the higher dose, the oral administration can also achieve the same protection level; but it is not achieve the protection level in immersion or jet injection administration method. These tentative results shown the injection administration route was still the most efficient method, however oral application was the potential to be the most convenience method if we can overcome the protein digestion problem in the GI tract.

Fish have many non-specific and specific, humoral and cellular mechanisms to resist bacterial diseases. Non-specific humoral factors include growth inhibiting substances, e.g. transferrin and antiproteases; lysins, e.g. lysozyme, C-reactive protein (CRP), bactericidal peptides and most importantly, complement which has lytic, proinflammatory, chemotactic and opsonic activities thus making a link with non-specific phagocyte responses. These are primarily executed by neutrophils and macrophages. The phagocytes contain many hydrolytic enzymes and when stimulated by bacteria produce reactive oxygen species (ROS), especially the hydroxyl radical, via generation of superoxide anions and nitric oxide. ROS are highly bactericidal. In addition, the neutrophils possess large quantities of myeloperoxidase (MPO) which generates bactericidal hypohalite ions from halide ions and H2O2. Macrophages can receive MPO from neutrophils and this may enhance their bactericidal activity. Antibody constitutes a specific humoral defence by inhibiting bacterial adherence or invasion of non-phagocytic host cells and neutralising bacterial toxins. Antibody can act as an opsonin and by activating complement by the classical pathway may lyse the bacterial cell, activate inflammation, phagocyte influx and further enhance phagocytosis. Macrophage bactericidal activity can be markedly increased by cytokines released by T lymphocytes on contact with their specific antigen. Pathogenic bacteria have evolved many ways of subverting these defence mechanisms of fish. These include the in vivo production of high affinity iron uptake systems (e.g. siderophores and their surface receptors, in Aeromonas salmonicida, Vibrio anguillarum). Some bacteria e.g. V. anguillarum strains of serotype 01, while being resistant to the alternative pathway of complement activation in normal serum, are killed in immune serum. In other bacteria, surface layers, capsules and elongated lipopolysaccharide (LPS) 0-antigens protect against classical complement-mediated killing (e.g. A. salmonicida, Photobacterium piscicida, V. anguillarum serotype 02 respectively). While some important pathogens (e.g. P. piscicida) are readily killed by macrophages, others (A. salmonicida) possess specialised superoxide dismutase and catalase to resist ROS and are toxic themselves to macrophages. Renibacterium salmoninarum uses antibody, complement and other serum components to activate the respiratory burst of macrophages on immediate contact thus exhausting this macrophage bactericidal mechanism before the bacterium is phagocytosed. Subsequently, R, salmoninarum is taken up by the macrophage where it multiplies as an intracellular pathogen. However, these two highly adapted pathogens, A. salmonicida and R. salmoninarum are both killed by activated macrophages indicating that the whole concerted efforts of specific and non-specific, humoral and cell-mediated immune responses are required for immunity against these two important pathogens.

To address the dearth of techniques for medium term primary culture of shellfish blood cells, a simple method has been devised for the maintenance of crab,Liocarcinus depurator(L) andCarcinus maenas(L), hyaline haemocytes in monolayer culturein vitrofor a minimum of 14 days. This is based on L15 medium supplemented with 0·4 m NaCl, 10% foetal calf serum and antibiotics. Separated hyaline cells kept in this medium remain ca. 90% viable after 2 days, >80% after 7 days and >70% after 14 days. More importantly, the cells retain defence functionality, as measured by phagocytic uptake of the marine bacterium,Psychrobacter immobilis, over the full 14 day incubation period. This method has potential value in a number of applications, particularly for fundamental studies of crustacean cellular immune processes, pathology or ecotoxicology.

As part of ongoing efforts to determine the role of reactive oxygen species (ROS) in eastern oyster ( Crassostrea virginica ) immune reactions, the contribution of ROS to bactericidal activity was evaluated. By using striped bass ( Morone saxatilis ) macrophages as a positive comparative model, it was shown previously that the ROS-generating capability of eastern oyster haemocytes is comparatively low and may be unable to exceed bacterial antioxidant capability. The current study tested the hypothesis that eastern oyster haemocytes do not rely on ROS-dependent bactericidal mechanisms by evaluating the ability of haemocytes to kill the non-pathogenic bacteria Bacillus megaterium and Pseudomonas fluorescens in the presence and absence of an NADPH oxidase inhibitor. Striped bass macrophages again served as a positive comparative model. The inhibition of zymosan-stimulated ROS production by eastern oyster haemocytes and striped bass macrophages by the inhibitor diphenyleneiodonium (DPI) suggests that an NADPH oxidase-like activity is associated with these phagocytes. The eastern oyster haemocytes were considerably more sensitive to the effect of DPI on ROS generation than were the striped bass macrophages: elimination of superoxide production by the oyster and fish phagocytes occurred at 0·5 μm and 5·0 μm DPI, respectively. DPI at these respective concentrations did not affect phagocytic capability. A DPI concentration of 5·0 μm resulted in an approximate 50% reduction in the ability of striped bass macrophages to kill B. megaterium and completely abolished bactericidal activity towards P. fluorescens . A contribution of ROS to the bactericidal activity of oyster haemocytes was not evident, as 0·5 μm DPI did not affect the killing of B. megaterium and reduced bactericidal activity towards P. fluorescens by only 4%. Although significant stimulation of striped bass macrophage ROS production occurred in response to the test bacteria, exposure of oyster haemocytes to the bacteria did not result in enhanced ROS production. These data suggest that, in contrast to striped bass macrophages, eastern oyster haemocytes do not rely on ROS-dependent bacterial killing mechanisms. This conclusion does not preclude the possibility of alternative (e.g., regulatory) functions of phagocyte-generated ROS in eastern oyster and striped bass immunology.

Superoxide anion (O − 2 ) generation by Eastern oyster haemocytes before and after phagocytosis of living or dead Perkinsus marinus was quantified by lucigenin-augmented chemiluminescence (CL). Superoxide is the initial product formed during the respiratory burst; this radical and its more cytotoxic metabolites are thought to play roles in the oxygen-dependent defence mechanisms available to the oyster. The haemocytes, in the absence of added particles, produced a constant, very low-level CL activity. Phagocytosis of osmotically-killed P. marinus triggered a rapid CL response that peaked at a value 5–7-fold greater than the CL level of the untreated cells. However, phagocytosis of viable P. marinus was associated with minor, short-lived CL stimulation followed by a period (>2 h) of sustained inhibition of O − 2 gener-ation. Phagocytosis of zymosan produced an immediate CL response, which was maintained for the >2 h course of the experiment. Haemocytes simultaneously exposed to zymosan and osmotically-killed P. marinus produced higher levels of O − 2 than those receiving zymosan alone. Cells similarly exposed to both zymosan and living P. marinus produced significantly lower levels of O − 2 than the zymosan-stimulated haemocytes. The stimulatory and inhibitory effects of killed and living P. marinus on CL were apparently both dose-dependent. Thedata suggest that both living and dead P. marinus are avidly phagocytosed, but only live P. marinus cells produce excretory/secretory products that scavenge O − 2 and/or suppress aspects of the oxygen-dependent immune system. In this fashion the parasites can survive within the haemocytes, multiply in the haemolymph, and eventually produce lethal infections.

The effects of copper and recovery from sublethal (100 μg/l) copper exposure were studied in three groups of goldfish (Carassius auratus) (1) control (2) copper (4–25 d) and (3) recovery (4 d exposure followed by 3–21 d recovery). The mean concentration of copper measured in the liver of the continuously exposed group was significantly higher than in the control and recovery groups by 11 and 18 days of exposure, respectively. In the posterior kidney, copper accumulation in the copper group was not significantly greater than the control and recovery groups until 18 days of exposure. The liver contained 4–5-fold higher concentrations of copper than the posterior kidney in all three treatment groups. Levels of copper in the posterior kidney correlated with those of the anterior kidney. There were no measurable differences in liver, kidney, or serum copper levels within or between the control and recovery groups at any time point. The production of superoxide (O2−) was compared between groups by measuring the reduction of NBT in anterior kidney phagocytes. In the copper group, production of O2−increased compared to controls in response to exposure periods lasting up to 11 days. O2−production in the recovery group also increased in response to 4 days of copper exposure. However, after 3 and 7 days of recovery, O2−production decreased below then increased above the control levels, respectively. By 18 days of copper exposure or 14 days of recovery, O2−production was no longer significantly different from control values. To determine if the decreased O2−production measured on recovery day 3 was mediated by a humoral factor, phagocytes from untreated fish were incubated with serum from control or 3 d recovery fish. Compared to the control serum,in vitroexposure to serum from copper-recovered animals resulted in a 29–34% decrease in O2−production. This was consistent with the 25% decrease measured in phagocytes from copper-recovered animals. It is concluded that the decrease in O2−production demonstrated in goldfish recovering 3 days from acute (4 d) sublethal copper exposure is not due to a direct effect of copper but to a factor(s) present in the serum of the recovering animals.

Cells isolated from lymphomyeloid tissues and peripheral blood from two species of elasmobranch, the nurse shark ( Ginglymostoma cirratum Bonaterre) and the clearnose skate ( Raja eglanteria Bosc) were assayed for in vitro phagocytic and pinocytic activities at various times during 24 h in culture. The purpose of this research was to begin to characterise functional activities of cells isolated from unique lymphomyeloid tissues found in elasmobranchs. Phagocytosis was evaluated by assessing relative abilities of immune cells to ingest Congo Red stained yeast, and included determination of percentage of active cells and numbers of yeast per cell. Pinocytosis was assayed by spectrophotometric measurement of neutral red dye accumulation by immune cells. Phagocytic activity was greatest in cells isolated from shark epigonal and skate epigonal and Leydig organs, with over 60% of skate Leydig cells containing yeast after 24 h in culture. Phagocytic activity was 2·5–6 times lower in cells from peripheral circulation or spleen. Pinocytosis was 1·5–3-fold greater in cells isolated from Leydig and epigonal organs compared to peripheral blood leucocytes or splenocytes. Documentation of phagocytic and pinocytic activities by cells isolated from epigonal and Leydig organs suggests significant immune functional roles for these unique tissues in the elasmobranch immune system.

Relatively little is known about factors that modulate in vivo lower vertebrate phagocyte responses, particularly in fish. In this study we report that head-kidney leucocytes from the marine fish gilthead seabream ( Sparus aurata L.) release soluble macrophage-activating factor/s (MAF) after stimulation with concanavalin A (Con A) and phorbol myristate acetate (PMA). Migration, phagocytosis, reactive oxygen intermediates (ROI) production and bactericidal activity were enhanced after incubation of target phagocytes with the MAF-containing supernatants. In order to ascertain the effect of MAF and bacterial lipopolysaccharide (LPS) on ROI and reactive nitrogen intermediates (RNI) production in gilthead seabream macrophages, macrophages were incubated with MAF in the presence of LPS. The results revealed that co-stimulation of macrophages with MAF and LPS inhibited O 2 − production but synergistically induced release of nitric oxide. These data suggest that the main activities of fish phagocytes are regulated by cytokines, which can interact positively or negatively with pathogen-derived molecules to determine the final activation state of the cells.

The immunoregulatory role of GH in sea bass (Dicentrarchus labrax), in relation to the intensity of infection by the myxosporean parasite Sphaerospora dicentrarchi, was investigated. The in vitro detection of superoxide anion (O2 -) in head kidney phagocytes was optimised using the nitroblue tetrazolium (NBT) assay. O2 - production was proportional to the quantity of total protein per well, and 15 and 30 min of PMA triggering gave the highest stimulation index. Cells adhered overnight produced more O2 - than cells adhered for 3h. Trifluoperazine abolished the reduction of NBT almost completely, whereas superoxide dismutase only caused 374% inhibition. Cells from fish parasitised by S. dicentrarchi, produced higher levels of O2 - compared with non-parasitised fish. Overnight priming with 5 nM recombinant rainbow trout growth hormone (rtGH) produced a significant increase in the respiratory burst of phagocytes from non-parasitised fish. Phagocytes from fish with medium intensity of infection attained the maximum response at 50 nM and 150 nM rtGH. LPS (20 μg ml-1) priming did not induce an increase in O2 - production, but it significantly increased cell adherence in non-parasitised fish., This work was supported by a researchgrant from the Spanish CICYT no. AGF95-0058. J. A. Calduch-Giner was recipient of aresearch grant from the Generalitat Valenciana, and P. Muñoz received a grant fromthe Ministerio de Educación y Ciencia of Spain.

In order to determine the immunomodulatory effect of the dietary intake of levamisole in the seawater teleost gilthead seabream (Sparus aurataL.), specimens were fed diets containing 0 (control), 125, 250 and 500 mg levamisole kg−1dry diet for 10 days. Growth, haematocrit, and phagocyte and lymphocyte responses were examined 0, 5 and 10 weeks after last administration of levamisole. The results demonstrate that 10 weeks after last administration of levamisole the specimens from the treated groups were larger and heavier than those of the control group. The experimental groups showed significantly lower haematocrit values 0 and 5 weeks after levamisole administration, although no differences were observed at 10 weeks. Leucocyte functions including phagocytosis and respiratory burst activity by phagocytes, and lymphokine (MAF) production by lymphocytes were enhanced by the dietary intake of levamisole. All of these responses peaked 5 weeks post-treatment. Serum complement activity was also found to be enhanced by treatment after 10 weeks. Fish fed 0 (control) and 500 mg levamisole kg−1dry diet were challenged with a virulent strain ofVibrio anguillarum4 weeks post-treatment, and mortalities were recorded over a 30-day period. The levamisole-treated fish were the more resistant. The present results suggest that the introduction of levamisole into the diet of fish grown in farms could increase their resistance to infection, reduce fish mortality rates and offer economic benefits.

Carp which receive intraperitoneal injections of sodium alginate show a high survival rate after being challenged with Edwardsiella tarda . In order to elucidate the immunoenhancement by sodium alginate, its effects on the non-specific defence system of carp were investigated. Sodium alginate had little influence either on the activity of the alternative complement pathway or on the phagocytic and respiratory burst activities of head kidney phagocytes (HKP), yet it greatly enhanced the migration of HKP to the peritoneal cavity (the site of injection) and concurrently elevated their phagocytic activity. The number of phagocytes mobilised by sodium alginate was 2 to 50 times greater than that by the well-known peritoneal exudate cell-eliciting agents when injected at the same dose. Accordingly, it is highly probable that the early elimination of challenge bacteria by such mobilised and activated phagocytes was responsible for the high survival rate of the alginate-injected fish. In chemotaxis assays, it was revealed that sodium alginate stimulated some leucocyte subpopulation(s) within the peritoneal cavity to produce and/or secrete chemotactic factor(s), whilst concurrently enhancing the sensitivity of HKP to the factor(s).

Protocols have been successfully developed for collecting kidney phagocytes from dab (Limanda limanda L.), based on the adherence of cells to plastic microtitre plates. For comparative purposes, cell adherence was quantified using both total protein measurements (the more rapid method) and counts of lysed adherent cell nuclei. There was a positive correlation (r2=0·67, P

The effects of chronic copper exposure on the macrophage chemiluminescent response and gill histology in goldfish ( Carassius auratus L.) were determined. Fish were exposed to sublethal concentrations (30, 65, 100, and 175 ppb) of copper sulphate for 96 h. Pronephric (head kidney) cells were collected and were comprised on lymphocytes, thrombocytes, monocytes/macrophages neutrophils and eosinophils/basophils (cell types grouped due to difficulty in identification). There were no significant differences in population profiles of cells used for chemiluminescence studies after exposure to 100 ppb copper. Density gradient-separated cells were stimulated with zymosan, the luminol-enhanced chemiluminescence (CL) response was measured and the results were compared to non-copper exposed controls. There was a significant correlation between copper concentration and reduction in the time at which the peak CL response [time to peak (TTP) CL] occurred. In addition, there was a significant parabolic dose response curve for peak CL response, which suggests increased production of reactive oxygen intermediates by phagocytes from fish exposed to concentrations up to 100 ppb, followed by inhibition at 175 ppb. These results indicate that copper affects phagocyte function in a complex manner, stimulating or inhibiting production of reactive oxygen intermediates depending on copper concentration. Exposure to concentrations of copper also resulted in varying degrees of gill hyperplasia. The relative median severity score (a score assigned to each gill based upon the amount of hyperplasia seen in a histological section) correlated significantly with the copper concentration. In addition, there was a very high correlation between the degree of hyperplasia and the TTP CL response. Levels of copper in the head kidney of goldfish exposed to 100 ppb were similar to controls as measured by atomic absorption spectroscopy. Thus, changes in the CL response of head kidney phagocytes of fish exposed to copper may be caused indirectly, via effects on other immunoregulatory cells or other physiological mechanisms. We conclude that addition of heavy metals such as copper to water can modify the CL responses of phagocytes and alter the histopathology of the gills in C. auratus .

A procedure was developed to culture isolated head kidney macrophages fromsalmonid fish and to study their phagocytic activity. The method was used to compare in vitro macrophage phagocytic activity against Vibrio salmonicida in rainbow trout and Atlantic salmon. These two species of salmonid fish differ in their natural resistance to V. salmonicida , with rainbow trout being more resistant. In both species, the isolated cell population from head kidney had morphological and functional characteristics typical of macrophages. The cells' functional activities were evaluated by studying their ability to grow and adhere to glass, to phagocytise uncoated monodispersed polymer beads, their acid phosphatase activity, and their uptake and degradation of bacteria in a phagocytic assay. To distinguish between extra cellular/cell-adherent bacteria and intracellular bacteria, a paired immunofluorescence technique was developed, based on monoclonal antibodies identifying epitopes on the lipopolysaccharide component of the bacterium. A unique identification of bacterial localisation was achieved by sequential incubation steps with intermittent cell membrane permeabilisation with absolute ethanol. At initial time points macrophages from rainbow trout exhibited a significantly higher phagocytosis of unopsonised V. salmonicida , compared to Atlantic salmon macrophages. Phagocytosed bacteria were easily degraded intracellularly, but fragments of the bacterium were identified by the two monoclonal antibodies at 72 h after inoculation.

Human tumor necrosis factor a (hTNFa) was found to increase rainbow trout macrophage respiratory burst activity and neutrophil migration. The increase in respiratory burst activity was inhibited by preincubation of the hTNFa with a soluble 55 kDa hTNFa receptor (TNF-Rl). Preincubation of the phagocytes with monoclonal antibodies (MAbs) to the TNF-R1 was found to ablate their responsiveness to hTNFa, dependent on the concentration of MAbs used. These findings are discussed with a view towards using such MAbs to elucidate the possible secretion of TNF by fish leucocytes.

This work studies the capacity of blood granulocytes from a cyprinid, the tench (Tinca tinca, L), to adapt to low winter temperatures of 12°C with regard to phagocytosis and antigen destruction. The capacity to ingest inert particles (latex beads) by means of an in vitro test at two incubation temperatures, 12°C (the actual temperature of the natural habitat of these fish during the winter), and 22°C (a commonly used temperature within the optimum range) was studied. The capacity to destroy the inert particles indirectly was evaluated by measuring the production of the superoxide anion (O2-), using the nitroblue tetrazolium (NBT) reduction technique. The results indicated that tench blood granulocytes presented an increased capacity to ingest inert particles at 12°C, whether measured after 30 min or 60 min of incubation in both sexes. The females, however, showed a greater ingestion capacity than the males at 12°C, and at 30 min of incubation. The higher capacity for ingestion at 12°C was due both to a greater number of granulocytes with phagocytic capacity and to a greater effectiveness of the phagocytosis at this temperature. This increased particle ingestion capacity corresponded in both males and females to a greater production of O2- indicating a greater capacity for destruction of the antigen at 12°C.

Phagocytic stimulation produced a marked chemiluminescent response by cells isolated from the anterior kidney of the Japanese medaka ( Oryzias latipes ). This response was due to the production of reactive oxygen intermediates which are thought to be involved in cell-mediated antimicrobial activity. Exposure of the cells to analytical grade pentachlorophenol (PCP) inhibited chemiluminescence (CL) in a dose-dependent fashion; the effect was evident after 1h and became progressively more pronounced with increasing length of exposure. Technical.grade PCP, containing contaminants including various dioxins, elicited slightly greater suppression of CL than did pure PCP. Pentachlorophenol-induced inhibition of CL was reversible after removal of the toxicant from the medium. The data suggest that PCP, a common estuarine and marine pollutant, can exert a direct inhibitory effect on this component of non-specific immunity in fish.

The opsonic effect of carp complement on the phagocytosis by carp peritoneal neutrophils was investigated using antibody-sensitised sheep erythrocytes (EA) and baker's yeast (Y), which are the activators of the classical complement pathway (CCP) and the alternative complement pathway (ACP), respectively. Carp antibody (A) exerted little opsonic effect on the phagocytosis of sheep erythrocytes (E). The treatment of EA and Y with normal carp serum (C) greatly enhanced phagocytosis. Such opsonic effect is apparently due to the complement being activated via CCP and ACP, because the treatment of EA and Y with C3-depleted carp serum produced no effect. It was also found that the treatment of EAC and YC with the Fab fragment of anti-carp C3 (rabbit IgG) abolished the opsonising effect of carp complement deposited on the target cells. These results indicate that C3 is the major phagocytosis-promoting factor (opsonin). In addition, it was also demonstrated that pretreatment of the neutrophil surface with trypsin greatly decreased the phagocytosis of EAC, suggesting the presence of C3 receptors on the neutrophil surface.