Accumulation of immunomodulatory laminaran [β(1,3)-D-glucan] in the spleen and kidney of Atlantic salmon, Salmo solar L.

Abstract. The tissue distribution of ³H- and FITC-laminaran in Atlantic salmon, Salmo salar L., was investigated after intravenous administration. Liquid scintillation counting and whole-body autoradiography revealed that the concentration of ³H-labelled laminaran in the spleen and anterior kidney was higher than in blood throughout the experimental period (1 h to 12 days). Renal excretion and intestinal exsorption were the main elimination pathways for laminaran. Microscopic examination revealed accumulation of FITC-labelled laminaran in macrophages in kidney and spleen. In addition, endothelial cells in the kidney, spleen, liver and intestine contained the immunomodulator. Thus, these experiments show that laminaran accumulates and is retained in immunologically relevant cells in the kidney and spleen.     

Tissue distribution and cellular uptake of Aeromonas salmonicida lipopolysaccharide (LPS) in some marine fish species

Τhe uptake and distribution of lipopolysaccharide (LPS), isolated from Aeromonas salmonicida, was investigated in Atlantic cod, Gadus morhua L., turbot, Scophthalmus maximus L., and Atlantic halibut, Hippoglossus hippoglossus L. LPS was radiolabelled by bromine oxidation and subsequent sodium borotritide reduction (³H-LPS), and fluorescence-labelled by introducing a fluorescein isothiocyanate derivative (FITC-LPS). After intravenous and intraperitoneal injections in cod, high amounts of radioactive LPS (³H-LPS) were present in heart, spleen and kidney throughout the experimental period (1–168 h). After peroral administration, a high amount of ³H-LPS was observed in intestinal tissues, whereas internal organs and tissues contained considerably lower amounts. Following intravenous administration of ³H-LPS in turbot, high contents of radioactivity were revealed in spleen, liver and kidney, whereas the content in heart was lower than in blood at the sampling times (1–24 h). The same pattern was observed after intraperitoneal administration. The spleen and liver contained high amounts of radioactivity when the turbots were intubated perorally with ³H-LPS. The spleen, kidney and heart were the main scavenging organs following intravenous administration of ³H-LPS in Atlantic halibut. A minor amount of radioactivity was present in the liver. The same pattern emerged after intraperitoneal injection in halibut. As observed for turbot, the spleen was the main accumulation site for ³H-LPS following peroral administration. Fluorescence microscopy of sections of organs and tissues from cod, intravenously and intraperitoneally injected with FITC-LPS, revealed that endocardial cells of both atrium and ventricle contained large amounts of the fluorochrome, whereas in turbot and halibut only atrial endothelial cells accumulated the substance. In all species, macrophages in kidney and spleen contained FITC-LPS and in the spleen the fluorochrome was trapped in the ellipsoidal walls. At later time points (e.g. 48 h) in the turbot spleen, FITC-LPS was located in cells adjacent to the ellipsoidal walls. Halibut endothelial cells that were located in the connective tissue of the intestine and gills also contained FITC-LPS. After peroral administration to the different fish species, specific fluorescence was found only in intestinal epithelial cells of halibut and in cells located in the lamina propria. Fluorescence was not detected in internal organs such as the kidney, spleen and liver after peroral administration of FITC-LPS. Gel chromatographic analysis of plasma samples from cod, turbot and halibut after intravenous and intraperitoneal injections showed that high molecular weight radioactivity was present. A minor amount of radioactivity that corresponded to low molecular weight substances was also observed. In conclusion, there is a high degree of variation with respect to the site of accumulation and some variation in the type of cells involved in the uptake of purified LPS in cod, turbot and halibut.     

The immunomodulatory effect of laminaran [β(1,3)-D-glucan] on Atlantic salmon, Salmo salar L., anterior kidney leucocytes after intraperitoneal, peroral and peranal administration

Anterior kidney leucocytes obtained from Atlantic salmon, Salmo salar L., 2 days after administration of laminaran, were assayed for their capacity to reduce nitroblue tetrazolium to formazan, and for their activity of lysosomal acid phosphatase after intraperitoneal (15 mgkg^?1), peroral (150 mg kg^?1) or peranal (150 mg kg^?1) administration. Leucocytes obtained from salmon treated by an intraperitoneal injection of laminaran produced significantly more superoxide anion than cells obtained from fish treated with dextran or sodium chloride immediately after cell isolation. Immediately after extraction, the activity of acid phosphatase in anterior kidney leucocytes obtained from salmon injected with laminaran was significantly higher than in cells harvested from fish treated with dextran or sodium chloride. Furthermore, cells obtained from salmon treated by peroral instillation of laminaran showed significantly enhanced production of superoxide anion compared with leucocytes from fish treated with either sodium chloride or dextran. The acid phosphatase activity in anterior kidney leucocytes from salmon treated by peroral and peranal instillation of laminaran was significantly higher than in cells from fish treated either with sodium chloride or dextran. Finally, fluorescence microscopic examination of tissue sections from fish treated peranally by intubation with fluorescein labelled laminaran revealed fluorescent vesicles in intestinal epithelial cells and in anterior kidney macrophages.     

The immunomodulatory effect of LPS, laminaran and sulphated laminaran [β(l,3)-D-glucan] on Atlantic salmon, Salmo salar L., macrophages in vitro

Abstract. The stimulatory effect of LPS ( Vibrio anguillarum ), laminaran and sulphated laminaran, aqueous soluble β(1,6)-branched β(l,3)-D-glucan obtained from Laminaria hyperborea , on head kidney macrophages of Atlantic salmon, Salmo salar L., is reported. The macrophages, after stimulation with LPS or laminaran, showed pronounced spreading, membrane ruffling and increased organellc content when examined by light microscopy. LPS stimulation induced enhanced phagocytic and pinocytic activity, higher intraccllular production of superoxide anion, and higher activity of acid phosphatase compared to control cells. Native laminaran stimulated the cells to pinocytose more fluid phase, increase the intracellular production of superoxide anion and to elevate the activity of acid phosphatase. Sulphated laminaran induced higher production of superoxide anion and higher activity of acid phosphatase by macrophages than in control cells.     

Optimising of culture conditions and stimulation of head kidney macrophages from Atlantic cod, Gadus morhua L.

The cultivation of Atlantic cod, Gadus morhua L., head kidney macrophages was optimal at 4–6 °C. The viability of cells in culture was examined by the trypan blue exclusion test 2, 5 and 7 days after seeding. Simultaneously, the number of adherent cells at the various times was checked. The morphology of the cells was also evaluated during the cultivation period. Enhanced respiratory burst activities were demonstrated in macrophages after stimulation with known immunostimulants like lipopolysaccharide, tuftsin and an acid peptide fraction previously shown to stimulate Atlantic salmon leucocytes efficiently. Generally, the Atlantic cod macrophages seemed to respond to a lesser extent compared with macrophages obtained from both mammalian and other fish species.     

Intestinal absorption of immunomodulatory laminaran and derivatives in Atlantic salmon, Salmo salar L.

Abstract. Radiolabelled (¹²⁵I, ³H) immunomodulatory laminaran (isolated from Laminaria hyperborea ), a β(1,6)-branched β(1,3)-D-glucan and different radiolabelled sulphated analogues of laminaran were administered peranally to Atlantic salmon, Salmo salar L. Intestinal absorption and tissue distribution were examined by means of radioactive tracer techniques. The intestinal uptake was highest with native laminaran and laminaran with a low degree of sulphatation, while highly sulphated laminarans were poorly absorbed. The tissue distribution analysis revealed high amounts of radiolabelled compound in the liver, and anterior and posterior kidney, whereas the spleen contained low amounts. Peak serum and organ concentrations were reached about 30 min after administration. The results were confirmed by autoradiography of tissue sections from salmon after peranal administration of radiolabelled laminaran. Finally, the peranal administration of fluorescence labelled laminaran revealed epithelial supranuclear fluorescent vacuoles containing laminaran. It is concluded that native laminaran and slightly sulphated laminaran are absorbed from the posterior intestine and that they are distributed to tissues rich in immunocompetent cells. Thus, these compounds may have potential as immunomodulatory feed additives.     

Tissue tropism of nervous necrosis virus (NNV) in Atlantic cod, Gadus morhua L., after intraperitoneal challenge with a virus isolate from diseased Atlantic halibut, Hippoglossus hippoglossus (L.)

Atlantic cod, Gadus morhua , averaging 100 g, were experimentally challenged by intraperitoneal injection of nervous necrosis virus (NNV) originating from Atlantic halibut. Cod tissues, including blood, gill, pectoral fin, barbel, ventricle, atrium, spleen, liver, lateral line (including muscle tissue), eye (retina) and brain, were sampled at day 25 and 130 and investigated by real-time RT-PCR for the presence of NNV. Relative quantifications at day 130 were calculated using the 2^−ΔΔCt method. Immunosuppression by injection of prednisolone-acetate was introduced for a 30-day period, and tissue sampled at day 180 and relative quantification estimated. No mortality or clinical signs of disease were observed in the challenged group. The challenge resulted in detection of NNV in blood, spleen, kidney, liver, heart atrium and heart ventricle at day 25, and by the end of the experiment NNV showed a clear increase in brain and retina, suggesting these to be the primary tissues for viral replication. There was no increase in the relative amount of NNV in blood, atrium, ventricle, spleen, liver and kidney. Corticosteroid implants resulted in a weak increase in virus RNA in spleen, kidney, liver and brain. These findings suggest that Atlantic cod is susceptible to infection with NNV from halibut. The observed tissue tropism patterns suggest an initial viraemic phase, followed by neurotrophy. Head-kidney is the best tissue identified for possible NNV detection by non-lethal biopsy, but detection was not possible in all injected fish.     

Evaluation of Electrical Stunning of Atlantic Cod (Gadus morhua) and Turbot (Psetta maxima) in Seawater

The aim of this study was to assess electrical stunning of Atlantic cod and turbot in seawater to develop a protocol for the process of stunning and killing. An induced general epileptiform insult (unconscious) had a duration of 40 ± 27 s (n =14) in cod (2.6 ± 0.5 kg) and 34 ± 18 s (n = 19) in turbot (520 ± 65 g). Seven cod and 3 turbot displayed a physical reaction, and 11 turbot registered an electroencephalogram (EEG) response to pain stimuli administered 30 s post-stun. The heart rate was 32 ± 6 beats/min in cod and 25 ± 7 beats/min in turbot prior to stunning. Post-stunning, the electrocardiogram (ECG) revealed fibrillation and reduced activity post-stun. EEG, ECG recordings, and behavioral observations indicate that when a bipolar square wave current was applied with a frequency of 133 Hz and 43% duty cycle side to side (turbot) and at 170 Hz and 33% duty cycle (cod) head to tail, both species were stunned in seawater at current densities of 3.2 A/dm² and 2.5 A/dm², respectively. For turbot, a 5 s exposure to electricity followed by chilling in ice water for 15 min is sufficient to prevent recovery. For cod, a killing method needs to be established.     

Effects of Loma morhua (Microsporidia) infection on the cardiorespiratory performance of Atlantic cod Gadus morhua (L).

The microsporidian Loma morhua infects Atlantic cod (Gadus morhua) in the wild and in culture and results in the formation of xenomas within the gill filaments, heart and spleen. Given the importance of the two former organs to metabolic capacity and thermal tolerance, the cardiorespiratory performance of cod with a naturally acquired infection of Loma was measured during an acute temperature increase (2 °C h^?1) from 10 °C to the fish's critical thermal maximum (CT_Max). In addition, oxygen consumption and swimming performance were measured during two successive critical swimming speed (U_crit) tests at 10 °C. While Loma infection had a negative impact on cod cardiac function at warm temperatures, and on metabolic capacity in both the CT_Max and U_crit tests (i.e. a reduction of 30–40%), it appears that the Atlantic cod can largely compensate for these Loma‐induced cardiorespiratory limitations. For example, (i) CT_Max (21.0 ± 0.3 °C) and U_crit (~1.75 BL s^?1) were very comparable to those reported in previous studies using uninfected fish from the same founder population; and (ii) our data suggest that tissue oxygen extraction, and potentially the capacity for anaerobic metabolism, is enhanced in fish infected with this microsporidian.     

Blood cleansing cells in head kidney and spleen in Buenos Aires tetra, Hyphessobrycon anisitsi (Eigenmann), (Characidae: Teleostei)

The general structure and cell types in kidney and spleen in Buenos Aires tetra, Hyphessobrycon anisitsi, family Characidae, are described. The capability and capacity of these organs to clean foreign ferritin from the blood stream are analysed and compared. Head kidney was mainly composed of neutrophils, macrophages, lymphocytes and other white blood cells, whereas unmatured and matured red blood cells were few in number. Spleen often contained much red pulp, that is mainly matured red blood cells between splenic cords, often with some macrophages and neutrophils in the latter. Occasionally, this pulp contained large volumes of unmatured red blood cells, particularly in the periphery of the spleen. The splenic white pulp consisted of ellipsoids composed of an inner endothelial layer covered by a thick sheet of white blood cells, which in the periphery consisted mainly of macrophages. Erythrocytes occupied nearly the entire splenic volume in some specimens, whereas up to half of this volume was filled by ellipsoid macrophages, neutrophils, lymphocytes and other white blood cells in other specimens. The macrophages and sinusoidal endothelial cells in kidney and spleen from ferritin-injected specimens were tightly packed by yellow-brown granules or Prussian blue precipitations, in tissue treated with Mallory stain or acid ferrocyanide, respectively, suggesting a large uptake of foreign ferritin. In the present tetra large amounts of white blood cells are developed in head kidney, where macrophages and sinusoidal endothelial cells play important roles in the cleansing of scavenger and foreign molecules and particles from the blood stream. The spleen seems primarily to be a site for iron recycling and production and storage of red blood cells. Sometimes, however, it was rich in macrophages, neutrophils, lymphocytes and other white blood cells, suggesting functions like blood cleansing and non-specific and specific defence in such specimens.     

Absorption of immunomodulating β(1,3)-glucan in yolk sac larvae of Atlantic halibut, Hippoglossus hippoglossus (L.)

A soluble immunomodulating β(1,3)-glucan, laminaran, was given in drinking water to yolk-sac larvae of Atlantic halibut, Hippoglossus hippoglossus L. The larvae absorbed laminaran when the concentration was 25 mg l^–1. After 5 days exposure to laminaran, each larva contained 2.3 ng and 46 ng of ³H- and ¹²⁵I-labelled laminaran, respectively. After 10 days exposure, each larva contained 4.5 ng and 47 ng of ³H- and ¹²⁵I-laminaran, respectively. The absorption was confirmed by fluorescence microscopy of larvae exposed to fluorescein-labelled laminaran (FITC-laminaran). The fluorescence was localized to cells in the intestinal epithelial layer and the skin epithelial layer.     

Infectivity of internal tissues of Atlantic salmon, Salmo salar L., experimentally infected with the aetiological agent of infectious salmon anaemia (ISA)

Abstract. Infectivity of internal organs and cells of Atlantic salmon, Salmo salar L., was studied at various times after infection with the aetiological agent of infectious salmon anaemia (ISA). Experimentally infected salmon smolts developed anaemia just prior to the onset of ISA-mortality. ISA-infectivity of preparations made from liver, kidney, spleen, plasma, red blood cells (RBC) and head kidney leucocytes was determined by inoculating Atlantic salmon parr with the respective preparations. ISA-mortality was observed after inoculation of salmon parr with preparations of kidney and liver, and to a minor degree, with spleen and a fraction of head kidney leucocytes (WBC1) collected 7 days post-infection. At 11 days post-infection, the infectivity of these preparations increased and ISA-infectivity was also observed with a second fraction of head kidney leucocytes (WBC2), red blood cells (RBC) and blood plasma. At this time, the ISA-infectivity of kidney was not significantly higher (P > 0.05) than that of liver but was significantly higher than all other preparations as judged by Cox-regression analysis. At days 14 and 18 post-infection, the infectivity of kidney was significantly higher (P < 0.05) than that of liver, but not at 21 and 25 days post-infection. Generally, the ISA-infectivity of kidney was higher than spleen, head kidney leucocytes (WBC1 and WBC2), RBC and plasma, although the difference was not significant at all time points. For example, at day 25 post-infection, the infectivity of kidney was only significantly higher than that of spleen and plasma. On a per gram basis, head kidney leucocytes proved to contain higher amounts of infectious matter than RBC. ISA-infective leucocytes present in the kidney tissue may have contributed to a major part of the infectivity recognized in the kidney preparations. Thus, head kidney leucocytes and other kidney leucocytes also may be considered to be among the most important target cells of the aetiological agent of ISA.     

A comparison among differently enriched rotifers (Brachionus plicatilis) and their effect on Atlantic cod (Gadus morhua) larvae early growth, survival and lipid composition

We evaluated the effect of differently enriched rotifers on the early growth, survival and lipid composition of Atlantic cod larvae (Gadus morhua). The enrichments tested were: (i) AlgaMac 2000^®; (ii) AquaGrow^® Advantage; and (iii) a combination of Pavlova sp. paste and AlgaMac 2000^®. Larvae from treatment 3 [1.50 ± 0.11 mg dry weight (dw) and 7.10 ± 0.14 dw specific growth rate (SGR)] were heavier (P = 0.006) and grew faster (P = 0.004) than larvae from treatment 2 (1.03 ± 0.04 mg dw and 6.29 ± 0.04 dw SGR). No significant differences were found in the final weight and SGR among larvae from treatment 1 (1.21 ± 0.07 mg dw and 6.58 ± 0.20 dw SGR) and larvae from treatments 2 and 3. The treatment 3 also resulted in the best survival at the end of the experimental period, estimated to be 3 on a scale from 1 to 5, whereas the survival estimates for the two other groups were 1–2. Larvae from the treatment 3 reached 37 days posthatch with levels of ω6DPA 32‐fold higher than newly hatched larvae. Differences in the larval enrichment of ω6DPA may explain the differences in growth and survival of the Atlantic cod larvae.     

Pre-anaesthetic metomidate sedation delays the stress response after caudal artery cannulation in Atlantic cod (Gadus morhua)

Recovery from caudal artery cannulation with and without pre-anaesthesia metomidate sedation was assessed in Atlantic cod (Gadus morhua). The levels of plasma cortisol, glucose, electrolytes and acid–base parameters were compared between sedated and unsedated cod and to those in uncannulated individuals, where the samples were obtained by sacrificial sampling (reference level). Metomidate sedation delayed the stress response, causing sedated cod plasma cortisol to return to the reference level more slowly [day 4 post surgery (PS)] than in unsedated cod (day 2 PS). Plasma glucose was elevated in both sedated and unsedated cod up to and including day 5 PS. Plasma K⁺ was lower and pH was higher in cannulated cod than in the reference from 24 h PS until the end of experimentation, indicating a stress effect of sacrificial sampling on plasma K⁺ and pH that was likely caused by an acute stress response. Metomidate sedation delayed the stress response following CA cannulation and should therefore not be used as a pre-anaesthetic sedation in Atlantic cod. The caudal artery cannulation can be a useful tool in obtaining repeated blood samples from Atlantic cod given an adequate recovery time, which was determined to be 6 days irrespective of pre-anaesthesia sedation status.     

Yersiniosis in Atlantic cod,Gadus morhua (L.), characterization of the infective strain and host reactions

A disease outbreak in farmed Atlantic cod caused by Yersinia ruckeri is reported. Mortality started following vaccination of cod reared in two tanks (A and B). The accumulated mortality reached 1.9% in A and 4.8% in B in the following 30 days when treatment with oxytetracycline was applied. Biochemical and molecular analysis of Y. ruckeri isolates from the cod and other fish species from fresh and marine waters in Iceland revealed a high salinity‐tolerant subgroup of Y. ruckeri serotype O1. Infected fish showed clinical signs comparable with those of Y. ruckeri ‐infected salmonids, with the exception of granuloma formations in infected cod tissues, which is a known response of cod to bacterial infections. Immunohistological examination showed Y. ruckeri antigens in the core of granulomas and the involvement of immune parameters that indicates a strong association between complement and lysozyme killing of bacteria. Experimental infection of cod with a cod isolate induced disease, and the calculated LD₅₀ was 1.7 × 10⁴ CFU per fish. The results suggest that yersiniosis can be spread between populations of freshwater and marine fish. Treatment of infected cod with antibiotic did not eliminate the infection, which can be explained by the immune response of cod producing prolonged granulomatous infection.     

Isolation, cultivation and characterization of head kidney macrophages from Atlantic cod, Gadus morhua L.

Head kidney macrophages from Atlantic cod, Gadus morhua L., were isolated by density sedimentation and maintained under serum-free conditions for up to one week. The cells adhered and spread well on glass and plastic, were highly phagocytic, and had typical macrophage morphology as shown by phase contrast and scanning and transmission electron microscopy. Histochemical studies with the light microscope showed that the macrophages were acid phosphatase and non-specific esterase positive, and alkaline phosphatase and peroxidase negative. Compared with unstimulated control cells, LPS-treated cells showed enhanced superoxide anion formation, as measured by the reduction of nitroblue tetrazolium, and increased levels of acid phosphatase activity.     

太平洋鳕RAG1和IgM基因荧光定量PCR方法的建立及初步应用

为研究太平洋鳕发育早期特异免疫系统形成的机制,通过RAG1和IgM基因的转录水平衡量特异免疫系统的发育特点.根据GenBank中RAG1和IgM的序列信息,分别设计1对特异引物,从太平洋鳕头肾中扩增得到RAG1和IgM的基因片段.将所获基因片段分别插入到克隆载体pMD18-T中,从而构建太平洋鳕RAG1和IgM基因的质粒标准品.建立并优化太平洋鳕RAG1和IgM基因绝对荧光定量PCR方法.为进一步验证该方法的可靠性,分别利用绝对定量和相对定量检验目的基因在太平洋鳕早期发育过程不同组织内的表达差异.以优化后的绝对荧光定量PCR方法检测不同发育时期太平洋鳕RAG1和IgM的表达情况.结果显示,RAG1的回归方程为y=-3.266x+33.77,回归系数R2=0.996;IgM的回归方程为y=-3.119x +27.61,回归系数R2 =0.998.绝对定量和相对定量结果在基因转录趋势上显现出一致性,即RAG1基因在胸腺和头肾中表达,且在胸腺中的表达量显著高于头肾中的表达量,在肝脏和脾脏中无表达;IgM基因在胸腺、头肾、肝脏和脾脏中均有表达,其中脾脏中表达量最高,其次是头肾.RAG1基因在太平洋鳕发育早期的表达水平很低,到61日龄(days posthatching,dph)至95 dph表达量显著提高;IgM基因在早期表达水平同样很低,到33 dph至61 dph才有明显表达,在95 dph时表达量显著提高.研究表明,本实验方法可靠,特异性较强,可成功对目标基因转录水平进行检测.     

In vitro effect of levamisole on the cell viability,phagocytosis and respiratory burst of Barbel chub (Squaliobarbus curriculus) macrophages

The present study was to determine in vitro effect of levamisole on the immune functions of Barbel chub (Squaliobarbus curriculus), head–kidney‐derived (HKM), spleen‐derived (SM) and peripheral blood monocyte‐derived macrophage (PBM). Macrophages were incubated with levamisole 10³, 10¹, 10⁻¹ and 10⁻³ ng mL⁻¹ to assay the cell viability, respiratory burst and phagocytosis. The results showed that macrophages treated with levamisole 10⁻³ ng mL⁻¹ gave a maximum respiratory burst response, whereas levamisole 10³ ng mL⁻¹ had no effect. Phagocytosis activity of the macrophages treated with levamisole enhanced significantly when compared to control, maximum response being at 10⁻³ ng mL⁻¹. While using the methylthiazoletetrazolium method to measure the cell activity for 12, 24, 36, 48, 60 h, there was no significant role on proliferation and the cell viability began to decline after 24 h. In conclusion, levamisole is a potent enhancer of Barbel chub macrophage activity with low concentration.     

Histopathology of atypical Aeromonas salmonicida infection in Atlantic cod, Gadus morhua L.

Abstract. The histopathology of an atypical Aeromonas salmonicida found in Atlantic cod, Gadus morhua L., is described. Unlike members of the Salmonidae, the cod showed a well-developed host reaction to A. salmonicida involving encystment of the bacteria. When Atlantic salmon, Salmo salar L., were given an intramuscular injection with suspensions of cultures of this strain of A. salmonicida no cellular host reaction was observed. When cod were given a similar injection the bacteria showed degenerative changes, leucocytes accumulated, and cyst formation was seen in the spleen and kidney. Since cod can be infected by this organism the possibility exists that they could act as carriers, a source of infection for salmonids in saltwater cage culture or in the wild.