Unmethylated CpG oligodeoxynucleotides activate head kidney leukocytes of Atlantic cod, <Emphasis Type="Italic">Gadus morhua</Emphasis>

Bacterial DNA and synthetic oligodeoxynucleotides (ODNs) that contain unmethylated CpG motifs are strong inducers of immune response in most mammalian organisms. The use of these synthetic CpG motifs in fish, particularly in salmonids and carp, resulted in the modulation of their immune system. However, much less is known in other species of fish such as gadoids including Atlantic cod, Gadus morhua. Using head kidney (HK) leukocytes of cod in an in vitro study, we determined the effects of some established CpG-ODNs on the cellular responses of the fish immunocytes. Incubation of the HK leukocytes with 2 μM concentration of the CpG-ODNs resulted in enhanced respiratory burst. There were differential effects on the activities of acid phosphatase and cellular myeloperoxidase. Only CpG-ODN 1826 triggered a significant increase in the level of both enzymes. On the other hand, the supernatants derived from the HK leukocytes after incubation with different CpG-ODNs did not possess bactericidal activity against Vibrio anguillarum and Aeromonas salmonicida. This study has shown that CpG-ODNs at low concentrations are able to stimulate respiratory burst in cod but have minimal effects on cellular enzymatic activities and antibacterial action.     

Short-term handling stress affects the humoral immune responses of juvenile Atlantic cod,Gadus morhua

Juveniles of Atlantic cod, Gadus morhua L., weighing 30–50 g were subjected to short-term handling stress by exposing them to air for 3 min and immediately placing in the rearing tank for recovery. Indices for the primary stress response as well as selected immune- and metabolism-related parameters were measured in the plasma before the application of the stressor and at 1-h, 24-h and 7-day post-stress. Plasma cortisol significantly increased at 1-h post-stress then returned to the prestress levels at 24-h post-exposure. Plasma glucose and total antioxidant capacity had two peaks at 1 h and 7 days after handling stress. Alkaline phosphatase significantly increased at 24-h post-exposure, while no significant changes in myeloperoxidase activity was observed. Lysozyme and antiprotease activities were significantly downregulated at 1- and 24-h post-exposure, respectively. These findings showed that short-term handling stress in Atlantic cod juveniles resulted in temporal elevation of the primary stress response and metabolism with a corresponding decrease in its defense against bacterial pathogens.     

Responses of Atlantic cod <Emphasis Type="Italic">Gadus morhua</Emphasis> head kidney leukocytes to phytase produced by gastrointestinal-derived bacteria

This study identified phytase-producing bacteria that were previously isolated from the gastrointestinal tract of Atlantic cod, Gadus morhua and determined its effect on head kidney leukocytes. Out of the 216 bacterial strains tested, the two phytase producers were identified as Pseudomonas sp. and Psychrobacter sp. based on their 16S rDNA sequence. Crude phytase from these two bacterial strains was produced employing the shake flask method. Even though the total protein of the crude phytase was not significantly different for the two bacteria, the phytase activity of the crude enzyme produced by Pseudomonas sp. (97.1 ± 16.7 U) was significantly higher than that of the enzyme from Psychrobacter sp. (75.9 ± 2.4 U). When cod head kidney leukocytes were incubated with the crude phytase (50 μg ml⁻¹), it resulted in enhanced cell proliferation, higher myeloperoxidase, and acid phosphatase activities. Extracellular responses—respiratory burst activity and hydrogen peroxide production were not enhanced by the crude enzyme. As a consequence, the growth of two pathogenic bacteria Aeromonas salmonicida and Vibrio anguillarum was not suppressed by the supernatants obtained from head kidney leukocytes incubated with the crude bacterial phytase. Thus, the enzyme from phytase-producing intestinal bacteria of Atlantic cod can stimulate intracellular head kidney leukocyte activities but not the production of extracellular substances that are involved in antibacterial response. These have implications on the potential use of bacterial phytase as feed supplement to boost cellular immune response of the fish and could be employed as a health management strategy in culture systems.     

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.     

瓦氏黄颡鱼头肾组织学观察与先天性免疫屏障研究

为探明瓦氏黄颡鱼(Pelteobagrus vachelli)头肾组织结构和先天性免疫屏障,该研究用苏木素-伊红(HE)染色法和改良James染色法对瓦氏黄颡鱼头肾的显微结构进行了观察,并使用台盼蓝活体染色法对头肾免疫屏障部位进行了定位.结果显示,瓦氏黄颡鱼头肾由被膜、淋巴细胞聚集区、粒细胞聚集区、前肾间组织、网状纤维...     

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.     

褐藻酸降解菌的筛选及其生长条件

采用安藤芳明选择性培养基从自然发病明显的海带幼苗上分离出8株褐藻酸降解菌。研究表明，8株菌能够不同程度地降解褐藻酸钠，菌株A1和A2表现出较强的降解能力，接种1d后液体培养基即变清。温度是该菌大量繁殖的决定性因子，其生长的最适温度为20℃，最适pH7.5。褐藻酸钠质量分数为0.5％-0.6％时，不同氮源和碳源对菌体生长的影响不同，其中有机氮和铵态氮有利于菌体生长，尿素和硝酸钠抑制菌体生长；只有在培养基中含有褐藻酸钠时，菌体才能正常生长。     

小球藻固定化培养的初步研究

一氧化氮 (NO) 是一种具有高细菌抑制率的气体信号分子,被广泛用于生物医学治疗当中,但是目前还存在如NO供体材料生物相容性差、NO负载量低以及控释缓释效果不佳等问题。以我国盛产的海藻多糖海藻酸钠 (SA) 为原料,制备了巯基化海藻酸钠 (SA-SH) 和NO供体S-亚硝基海藻酸钠 (SA-SNO),并以不同比例复合SA-SH与SA-SNO制备了原位成型水凝胶。结果表明随着SA-SNO含量的增加,NO负载量显著增加;释放曲线表明该凝胶具有较长的释放周期,5 d后NO释放量高达30.12~44.32 μmol·g⁻¹,总释放量为152~264 µmol·mg⁻¹。抗菌实验表明水凝胶对金黄色葡萄球菌 (Staphylococcus aureus)、大肠杆菌 (Escherichia coli) 具有显著抑制作用。综上所述,研究制备的水凝胶具有作为伤口抗菌敷料的应用潜力。     

Vaccination experiments and studies of the humoral immune responses in cod, Gadus morhua L., to four strains of monoclonal-defined Vibrio anguillarum

Abstract. Vibrio anguillarum isolated from diseased cod, Gadus morhua L., were serotyped by use of a panel of monoclonal antibodies. Four serotypes could be distinguished, having different lipopolysaccharide determinants. These phenotypic differences were also reflected in the genetic map, as revealed by fingerprinting of bacterial DNA. Antisera were raised in cod after immunization with the V. anguillarum serotypes, and Western blot techniques demonstrated production of specific antibodies mainly to LPS-antigens. The immune system in cod discriminates to a eertain degree between the four serotypes as shown by crossreactions of the immune sera in elisa . Moreover, it was also shown that natural antibodies to bacterial antigens are present in non-immune sera, but these specificities are non-LPS in nature. As a consequence of the heterogeneity of the V. anguillarum strains, vaccination experiments were performed under laboratory conditions to compare the effectiveness of bacterins based on either single vaccines or polyvaccines. The results from these experiments were promising since challenge with one strain demonstrated 100% protection both in fish vaccinated with the homologous serotype as well as a mixture of all the four serotypes.     

褐藻酸降解菌诱发海带病烂的组织细胞学观察

用褐藻酸降解菌对海带进行人为感染，经培养使其产生烂斑，通过组织显微切片观察表明：1、褐藻酸降解菌首先侵入内皮层，然后再逐渐侵入到外皮层、髓部和表皮，最终导致海带病烂。2．感染后内皮层伤害最重，其次是外皮层和髓部，表皮的伤害最轻。3、侵染海皮层的细胞中，有大量颗粒状储藏物质，其直径范围在1．5－4．0μm之间，平均直径为2．7μm。形状为椭圆或圆形。     

褐藻酸降解茵诱发海带病烂的组织细胞学观察

用褐藻酸降解菌对海带进行人为感染,经培养使其产生烂斑,通过组织显微切片观察表明1、褐藻酸降解菌首先侵入内皮层,然后再逐渐侵入到外皮层、髓部和表皮,最终导致海带病烂.2、感染后内皮层伤害最重,其次是外皮层和髓部,表皮的伤害最轻.3、浸染海带内皮层的细胞中,有大量颗粒状储藏物质,其直径范围在1.5～4.0μm之间,平均直径为2.7 μm.形状为椭圆或圆形.     

褐藻酸降解菌对海带配子体克隆的作用及其药物防治研究

通过回染实验，确认褐藻酸降解菌是海带配子体克隆病害的主要致病菌；观察了对其海带配子体克隆细胞侵染的现象，并研究了抗生素对海带配子体克隆细胞和褐藻酸降解菌的作用，发现0．5－100mg/L青霉素对配子体克隆细胞的生长有一定促进作用，实验范围内各浓度青霉素对褐藻酸降解菌均有较好的抑菌效果，且抑菌效果随浓度的提高而加强。其中100mg/L青霉素10d内可将褐藻酸降解菌浓度降低到初始浓度的1／20以下，可作为海带配子体克隆保存中有效的抑菌药物。     

褐藻酸降解菌引起海带内皮层细胞储藏颗粒变化的初步研究

实验对正常海带用褐藻酸降解菌进行了人工感染，经培养使其产生烂斑，通过组织显微切片进行比较观察，发现褐藻酸降解菌首先侵入内皮层，然后再逐渐侵入到外皮层、髓部，最终导致海带病烂。切片中发现在侵染海带切片内皮层的细胞中，有大量储藏颗粒，形成椭圆或圆形。其直径范围在1．5－4．0μm之间，平均直径为2．7μm。该颗粒主要存在内皮层，从其产生到消亡的过程分析，与褐藻酸降解菌的侵入有关，并作为营养起到促进菌增殖的作用。     

活性氧在海带抗褐藻酸降解菌感染中不同时期的抗氧化能力

以海带为材料,研究了活性氧(ROS)在褐藻酸降解菌感染中的产生及其作用.结果表明①褐藻酸降解菌感染引起海带活性氧的大量产生,且海带No.1活性氧的产生速率始终明显大于海带901(P<0.05).②在褐藻酸降解菌感染的早期阶段,海带维持着较高的抗氧化能力和对褐藻酸降解菌感染较高的抗性,而且海带的抗感染能力与活性氧产生速率呈一定的正相关性;2个品系相比,海带No.1的抗性又明显大于海带901(P<0.01).③在褐藻酸降解菌感染的后期,海带的抗氧化能力显著降低,膜脂过氧化和脱酯化伤害作用加剧,同时对褐藻酸降解菌感染的抗性显著降低,海带的抗感染能力与活性氧产生速率呈一定的负相关性,其中海带 No.1活性氧的产生速率、膜脂过氧化作用和脱酯化作用都远远高于海带901;相反,海带No.1对褐藻酸降解菌的抗性明显低于海带901(P<0.05).指示在感染的早期阶段,活性氧的产生在海带抵抗褐藻酸降解菌的感染中起着重要作用;而在后期阶段,活性氧的产生又大大降低了海带对褐藻酸降解菌感染的抗性.海带在感染的早期维持较高水平的抗氧化能力,而在感染的后期抗氧化能力显著下降是活性氧表现出双重作用的重要原因之一.     

Early ontogeny-related changes of the fatty acid composition in the Percichthyid fishes trout cod,Maccullochella macquariensis and Murray cod,M. peelii peelii

Changes in the fatty acid profiles of the Percichthyid fish trout cod, Maccullochella macquariensis (Cuvier), and Murray cod, M. peelii peelii (Mitchell), two Australian native freshwater fish species, were investigated during early development from egg to yolk-sac-resorbed larval stage. In the two Percichthyid fishes polyunsaturated fatty acids (PUFA) accounted for more than 50 % of the 19 quantified fatty acids in total lipid. The fatty acids that occurred in the highest abundance in both trout cod and Murray cod, in all developmental stages, in order, were docosahexaenoic acid [DHA 22:6(n-3)], arachidonic acid [AA 20:4(n-6)], oleic acid [18:1(n-9)] and palmitic acid (16:0), all of which exceeded 100 μg per mg total lipid in most instances. The ratio of 22:6(n-3) to eicosapentaenoic acid- 20:5(n-3) in eggs of trout cod and Murray cod was 5.4:1 and 7.3:1, respectively, and remained almost unchanged through development, and was considerably higher than the 2:1 ratio generally reported for fish eggs. In trout cod, 11 of the 19 fatty acids in total lipid decreased during the transformation from egg to yolk-sac-resorbed larva. In Murray cod, only 16:1(n-7) showed a significant decrease whilst 20:4(n-6) increased significantly with development. Overall, there was a tendency in both species to conserve n-3 and n-6 series highly unsaturated fatty acids (HUFA), suggesting their essentiality in first feeding larvae. These observations are discussed in relation to the feeding habits of trout cod and Murray cod, which are top order, freshwater carnivores.     

Influence of dietary glucan and vitamin C on non-specific and specific immune responses of rainbow trout (Oncorhynchus mykiss)

A trial was conducted to determine the effect of a combination of dietary glucan and vitamin C on the immune response of rainbow trout. After 3 weeks of adaptation on a control diet (without glucan but containing 150 ppm vitamin C), rainbow trout were fed the experimental diets containing yeast glucan and vitamin C at 150, 1000 and 4000 ppm for 2 weeks and then switched back to the control diet for the following 4 weeks. Macrophage activity (chemiluminescence response), complement activation and lysozyme levels were monitored just after feeding the experimental diets (week 0) and 2 and 4 weeks later. The kinetics of antibody response after vaccination against enteric redmouth disease were determined. The fish were immunised at week 0, at the end of the experimental feeding.Tissue ascorbic acid contents were monitored every 2 weeks and reflected the dietary treatments. No differences were observed in complement levels activated via the classical pathway. Two weeks after feeding the experimental diets, alternative pathway of complement activation and chemiluminescence response were enhanced by high doses of vitamin C. Significant enhancement of macrophage activity was still evident at week 2. No significant differences were observed in lysozyme levels. The specific immune antibody response was enhanced following vaccination, when glucan was also present in the diet.     

Voluntary responses and limits of tolerance to pressure reduction and swimbladder expansion in farmed Atlantic cod

Atlantic cod and other teleosts with closed swimbladders have slow gas resorption rates and are therefore vulnerable to sudden pressure reductions that lead to swimbladder expansion and possible loss of behavioural control. This problem is of particular relevance to fish farmers, who require safe protocols for lifting of cages containing cod that account for swimbladder expansion. To recommend a limit for pressure reduction, we experimentally determined the maximum pressure reduction, relative to neutral buoyancy pressure, free-swimming farmed cod would expose themselves to. Classical reward conditioning was used to motivate cod that were neutrally buoyant at 2?C3.3 ATM (10?C23 m depth) to respond to a sound signal by swimming rapidly upwards to a feeding station positioned at 1.5 ATM (5 m depth). Sixteen cod were implanted with data storage tags (DSTs) that registered their ambient pressure throughout the experiment. The DST data showed that cod voluntarily stopped rapid ascents before they reached the feeding station at an average maximum pressure reduction of about 41% relative to their neutral buoyancy pressure. This was equivalent to a 70% swimbladder expansion relative to neutral volume. During the experiment, cod seldom remained above their neutral buoyancy depths, except when feeding, indicating a behavioural aversion to positive buoyancy. To avoid the loss of buoyancy control in cod, we conclude that single lifting events of sea-cages, which forces swimbladder expansion, should never exceed a 70% expansion of the swimbladder above the pre-existing neutral buoyancy volume.     

褐藻糖胶的提取纯化方法

褐藻糖胶是存在于褐藻中的硫酸酯多糖，具有多种生物活性。本文综述了褐藻糖胶的提取、纯化和分级等方面的研究工作。     

褐藻酸降解菌感染下海带活性氧产生的研究

以海带为材料，选用5株感染能力较强的褐藻酸降解菌作为致病菌，研究了活性氧（ROS）在海带抗褐藻酸降解菌感染中的产生及其作用。结果表明：（1）海带在褐藻酸降解菌感染下活性氧的产生具有普遍性。（2）活性氧的大量产生只发生在褐藻酸降解菌感染的早期阶段，随着感染时间的不断延长，活性氧的产生速率逐渐降低。说明活性氧的产生是海带在褐藻酸降解感染早期启动的反应特征之一。     

The amino acid profiles in developing eggs and larvae of the freshwater Percichthyid fishes,trout cod,Maccullochella macquariensis and Murray cod,Maccullochella peelii peelii

Results on changes in the total amino acids (protein bound + free) and the free amino acids (FAA) in relation to development, from egg (unfertilised and/or fertilised) to yolk-sac resorbed larva, before first feeding, in two Percichthyid fish, trout cod, Maccullochella macquariensis and Murray cod, Maccullochella peelii peelii, which lay demersal, adhesive eggs, are presented. Throughout development, the FAA accounted for only a small proportion (0.19 % in fertilised eggs of both species) of the total amino acid pool. Nine essential amino acids (EAA) and eight non-essential amino acids (NEAA) were quantified in the amino acid pool at all stages of development. In both species, the total amino acid content decreased during the transformation (at 20 ± 1 °C) from newly hatched larva to yolk-sac resorbed larva. Overall, the changes in the TEAA and TNEAA reflected that of the amino acid pool. In trout cod, all but one EAA (lysine) and two NEAA (cysteine and glycine) decreased with ontogeny, from fertilised egg to yolk-sac resorbed larva. In Murray cod, however, the exceptions to the general decline were two NEAA (aspartic acid and glycine). In contrast, the FAA increased with development, the changes being reflected in both FEAA and FNEAA. Qualitatively, the predominant free amino acids in trout cod and Murray cod eggs were alanine, lysine, leucine and serine. Because the egg protein and the total amino acid contents declined with development, it is concluded that the rate of breakdown of yolk protein was higher than the anabolic and catabolic processes during embryogenesis. Data also suggest that in freshwater fish FAA are an unlikely primary energy substrate during embryogenesis.