Dietary vitamin E and rainbow trout (Oncorhynchus mykiss) phagocyte functions: effect on gut and on head kidney leucocytes

The effects of vitamin E (deficiency or supplementation) on the non-specific immune system in rainbow trout, Oncorhynchus mykiss, were evaluated. Rainbow trout were fed daily a semi-purified diet supplemented with vitamin E at 0, 28 and 295 mg x kg(-1) of diet. After 80 days of experimental feeding, the phagocytic function (respiratory burst evaluated by the CL response, phagocytosis) from gut leucocytes and head kidney enriched macrophages was measured; head kidney cell pinocytosis and serum lysozyme activity were also analysed. The results showed that some phagocyte functions were influenced by dietary vitamin E. When fish were fed the high dietary dose of vitamin E an enhancement of phagocytosis was found, but only significantly for the leucocytes isolated from the gut of rainbow trout; moreover, an impaired response was also observed in the fish fed no vitamin E for 80 days. However, no significant differences were noticed on the oxidative burst (CL) response of both gut and head kidney cells according to the dietary dose of vitamin E. Pinocytosis evaluated on head kidney cells was not influenced by dietary vitamin E. Fish fed vitamin E at 295 mg x kg(-1) had a lower serum lysozyme activity than those fed with vitamin E at 28 mg x kg(-1) and the fish fed no vitamin E for 80 days had an impaired activity. Thus, the present results demonstrate that altered dietary levels of vitamin E modulates the phagocytic functions of gut leucocytes in rainbow trout; moreover, the vitamin E diet effect seems to be greater on the local intestinal response as compared to systemic (head kidney). Taken together, this study confirms the crucial role of gut phagocytes in mucosal non-lymphoid defences in fish.     

Interrelationship of the serum lysozyme level to the leukocyte count in the blood]

The leucocyte counts in the peripheral blood and titers of the serum lysozyme under conditions of various experimental models were compared. Albino rats, mice, guinea pigs and rabbits exposed to the effect of various factors of a biological and chemical nature were used in the experiments. In addition, observations on humans immunized with bacterial and viral vaccines were analyzed. Development of 3 reaction types was shown to be possible. In the 1st type the dynamics of the lysosome activity and blood leucocyte counts changed in the same way (immunization with smallpox vaccine, listeria infection). Decreased counts of the leucocytes due to the use of cytostatic drugs were accompanied by a parallel decrease in the lysozyme activity. Such variant of the reaction was predominating. The 2nd type of the shifts characterized by an impaired synchronous pattern of the dynamics of the indices was not so frequent (the use of high doses of cytostatics). The 3rd type of the reactions with an exactly opposite character of the shifts in the leucocyte counts and lysozyme activity was observed only as an exclusion. It is concluded that the quantitative dynamics of the leucocytes is not enough by itself for creating an opinion on the nature of the changes in the serum lysozyme. The possible role of macrophages in the above processes is discussed.     

Change in the alkaline phosphatase activity and glycogen content in the leukocytes of white rat blood under the influence of penicillin preparations]

The contents of alkaline phosphatase and glycogen in the neutrophils of the peripheral blood of unbred albino rats weighing 170-200 gm were determined. Benzylpenicillin and oxacillin in doses of 50000 and 20000 Units/kg respectively were administered intramuscularly once a day over a long period of time. Two-phase changes in the contents of alkaline phosphatase and glycogen under the effect of penicillin were found. An increase in their contents was observed on the 5-10th days then it was followed by a decrease. Increase in the contents of the metabolites proceeded simultaneously with enhancing of the digestive capacity of the leucocytes and activity of lysozyme in them.     

The effect of temperature on non-specific defence parameters of three strains of juvenile Atlantic halibut (Hippoglossus hippoglossus L.)

The effect of temperature (8, 12, 15 and 18 degrees C) on a variety of non-specific defence and haematological parameters was examined in three geographically distinct reared strains (Canadian, Icelandic, Norwegian) of Atlantic halibut. The results indicate that temperature exerts a considerable influence on some blood parameters (packed cell volume and the percentage population of leucocytes in peripheral blood) and on some humoral parameters (serum lysozyme activity and serum protein levels) of halibut. A high temperature of 18 degrees C caused a decrease in the number of circulating blood cells and an increase in serum lysozyme levels; effects consistent with those reported within the literature for stress. The different strains of halibut exhibited differing responses with respect to differential counts of peripheral blood lymphocytes and thrombocytes, and to serum protein concentrations, serum lysozyme activity, serum iron content, unsaturated iron binding capacity of serum and O2- production by kidney macrophages.     

饲料中添加溶菌酶对吉富罗非鱼生长、免疫-抗氧化功能及血清抗菌性能的影响

为研究在饲料中添加不同水平的溶菌酶制品对吉富罗非鱼(GIFT, Oreochromis niloticus)生长性能、免疫-抗氧化功能和血清抗菌性能的影响, 选用平均体重为(11.350.08)g的吉富罗非鱼960尾, 随机分为6组(每组4个重复, 每个重复40尾), 分别投喂基础饲料(对照组)和5种添加水平分别为18、36、54、72和90 mg/kg溶菌酶制品的试验饲料, 养殖周期为60d。结果表明: (1) 54 mg/kg溶菌酶添加组鱼的生长性能和饲料利用情况最优, 增重率和蛋白质效率均显著高于对照组, 饲料系数显著低于对照组(P0.05); 肝体比随溶菌酶添加水平的增加呈现下降趋势, 90 mg/kg添加组显著低于对照组(P0.05); 脾脏指数在36和54mg/kg添加组显著低于对照组(P0.05); 全鱼粗蛋白和粗灰分含量在54 mg/kg添加组均呈现较高水平, 显著高于对照组(P0.05)。(2)溶菌酶添加水平对罗非鱼的免疫-抗氧化能力产生影响, 54和72 mg/kg添加水平能显著提高鱼体血清和肝脏的超氧化物歧化酶、过氧化氢酶活性, 降低丙二醛含量(P0.05); 肝脏溶菌酶活性在54和72 mg/kg添加组均显著高于对照组(P0.05), 而血清溶菌酶活性随溶菌酶添加水平的增加呈现下降趋势(L90组除外), 显著低于对照组(P0.05)。(3)血清抗菌试验显示, 54和72 mg/kg溶菌酶添加组罗非鱼对大肠杆菌、金黄色葡萄球菌、嗜水气单胞菌和溶藻弧菌的抑制能力显著高于对照组(P0.05), 而对枯草芽孢杆菌的抵抗能力最低, 比对照组分别低34.71%和42.21% (P0.05)。结果表明, 在本试验条件下, 在吉富罗非鱼饲料中添加54 mg/kg溶菌酶制品可以改善其生长性能; 当添加水平为54和72 mg/kg时, 罗非鱼的免疫-抗氧化能力和血清抗菌性能均得到了显著提高。     

Enhancement of nonspecific immunity and disease resistance in Oreochromis mossambicus by Solanum trilobatum leaf fractions

The purpose of this study was to determine the effect of water and hexane soluble fractions of the Indian medicinal plant, Solanum trilobatum on the nonspecific immune mechanisms and disease resistance in Oreochromis mossambicus. Fish were intraperitoneally injected with different doses of 0, 4, 40 or 400 mg kg(-1) body weight of water or hexane soluble fraction. The nonspecific immune mechanisms were assessed in terms of serum lysozyme activity, reactive oxygen species production and reactive nitrogen species production by peripheral blood leucocytes. The functional immunity in terms of percentage mortality and Relative Percent Survival (RPS) was assessed by a challenge with live Aeromonas hydrophila. Almost all the doses of both water and hexane soluble fractions enhanced the serum lysozyme activity. All the doses of water soluble fraction significantly enhanced the ROS production on most of the days tested. In hexane soluble fraction treated groups, the enhancement in the ROS production was observed at least on 2 days. All the doses of water soluble fraction significantly enhanced the production of RNS only on one day. The RNS production was enhanced significantly only in the group treated with 40 mg kg(-1) of hexane fraction. The leaf fraction administration preceding the challenge with live A. hydrophila, decreased the percentage mortality in the experimental group with the consequent increase in RPS values. This preliminary study indicates that S. trilobatum could be used to promote the health status of fish in intensive finfish aquaculture.     

Effects of prolactin and growth hormone on plasma levels of lysozyme and ceruloplasmin in rainbow trout

In vivo and in vitro effects of prolactin (PRL) and growth hormone (GH) on plasma levels of lysozyme and ceruloplasmin were examined in the rainbow trout (Oncorhynchus mykiss). Hypophysectomy had no effect on the plasma lysozyme level. Implantation of PRL- or GH-containing cholesterol pellets increased the lysozyme level in a dose-related manner. After hypophysectomy and sham operation, plasma ceruloplasmin was elevated above the level in intact fish, suggesting inflammation caused by the surgery. PRL or GH treatment significantly attenuated the increased level of ceruloplasmin in the operated fish. Expression of lysozyme mRNA was detected in the leucocytes isolated from the peripheral blood by RT-PCR. In vitro administration of PRL or GH showed no effect on the proliferation of isolated leucocytes or on the total protein content; however, lysozyme activity in the medium increased in a dose-related manner. These results suggest that PRL and GH directly stimulate lysozyme production without affecting the proliferation of leucocytes, and the attenuated ceruloplasmin level increased in response to inflammation.     

Change in the activity of specific and nonspecific immunity mechanisms in vitamin B1 deficiency]

The effect of thiamin deficiency on the immune response and activity of some mechanisms of natural immunity was studied in experimental mature rats. Thiamin deficiency was simulated by a single injection of hydroxythiamin (thiamin antagonists). Hydroxythiamin markedly decreased the complement activity, phagocytic activity of peripheral blood leucocytes, serum bactericidal activity as well as antibody production in response to sheep red blood cells. On the contrary, lysozyme activity increased. Vitamin B1 deficiency reduced 14C-leucine incorporation activity in the liver proteins.     

A genetic association between bovine serum and colostrum lysozyme levels

Serum and colostrum lysozyme activity was determined in primiparous cows employing a lysoplate method described elsewhere. Samples from 336 animals were collected over a 5-year period from a research station. The animals were sired by 20 elite bulls, one of which (1893) is probably heterozygous for a dominant high lysozyme level gene. The resulting two-population split of high and low level offspring from 1893 is also consistent for the present cow material both in serum and colostrum. A highly significant overall correlation (P less than 0.01) between serum and colostrum lysozyme was estimated. Positive correlation was also found within families and this was particularly high for 1893. No association between serum and colostrum lysozyme activity could be detected when high level animals were excluded. This means that the genetic association between lysozyme activity in the two body fluids is solely dependent upon the major gene described. Consequently, selection of bulls for serum lysozyme activity will influence the colostrum or milk lysozyme activity in the cow population, provided that the major gene is present in the population.     

Effects of short-term crowding stress on the gilthead seabream (Sparus aurata L) innate immune response

Gilthead seabream specimens were subjected to an intense short-term crowding stress of 100 kg m(-3) for 2 h. After 0, 1, 2, 3 and 4 days, blood glucose and serum cortisol levels, serum complement activity, phagocytic and respiratory burst activities of head-kidney leucocytes, and the percentage of monocyte/ macrophages and granulocytes in head-kidney and circulating blood were determined. An immediate effect of the stress was a depression in complement and phagocytic activities, both of which recovered after 3 or 2 days, respectively, while respiratory burst remained unaffected. The depression of phagocytosis in head-kidney leucocytes seemed to correlate with stress-induced migration of active cells from the organ to circulating blood. The present results point to the importance of minimising intense short-term crowding stress in order to reduce possible states of immunodepression in farmed fish.     

Activity of some lysosomal enzymes in plasma and leucocytes of rabbits exposed to effect of retinol and hydrocortisone.

Observing activity of some lysosomal enzymes in blood serum and leucocytes of rabbits subjected to injection of 200,000 units of retinol and 25 mg of hydrocortisone/kg of body weight it was found that: 1. In the effect of retinol administration there was an increase in the activity AP, BGAL, BGLU, AspAT and lipase in blood serum after 72 hours and NAGL after 168 hours while in leucocytes BGAL and NAGL after 72 hours and AGAL after 168 hours. 2. As a result of hydrocortisone injection the activity of all the enzymes examined (except Ala-Na) in blood serum increased markedly already after 24-48 hours. 3. In leucocytes hydrocortisone caused a significant increase in the activity of AP, BGRD, NAGL, BGAL, AGAL and cathepsin D. 4. The glucose level in blood plasma decreased after 48 hours and 120 hours after hydrocortisone injection and 168 hours after retinol injection.     

Effect of benzylpenicillin and tetracycline on the phagocytic, nonspecific humoral and biochemical activity of uninfected animals

The effect of benzylpenicillin and tetracycline on the activity, intensity and completion of phagocytosis with the leucocytes of the peritoneal exudate, on the activity of lysozyme and beta-lysins of the blood serum, on the content of nucleic acids, the activity of succinate dehydrogenase and total and acid phosphatase in the liver and kidney cells was studied experimentally on noninfected albino mice. The study showed that administration of benzylpenicillin and tetracycline to the animals for 5 days induced a decrease in the activity of the cell and humoral defence and the activity of the above enzymes in the liver and kidney cells. The content of the nucleic acids did not change under the effect of the antibiotics.     

The activity of several components of the innate immune system in diploid and triploid turbot

The use of triploid fish may be of interest in research, e.g. study of how this condition affects the size and activity of cells. In addition, triploid fish are sterile and production of triploids in fish species that are marketed after reaching sexual maturity may be of economic interest. In the present study, the effects of triploidy on the activity of several components of the innate immune system of turbot (Psetta maxima L) were determined. Triploid turbot had bigger cells (erythrocytes and neutrophils) but the number of blood erythrocytes, leucocytes and thrombocytes was lower than in diploid fish. The differential cell count was similar in both types of fish. The respiratory burst and the phagocytic activities were higher in neutrophils of triploid turbot. However, because the number of neutrophils was higher in diploids, the total respiratory burst activity and the phagocytosis per microliter of blood was similar in both types of fish. No differences were found in serum complement, lysozyme or bactericidal activities. The results indicate that the activities of the humoral components of the innate immune system tested are similar in diploid and triploid fish and that the lower leucocyte number found in triploids is compensated for by higher cell activity.     

Changes in some innate defence parameters of seabream (Sparus aurata L.) induced by retinol acetate

The effects of high doses of dietary or intraperitoneally (i.p.) injected retinol acetate on the gilthead seabream (Sparus aurata L.) innate immune system were studied. Gilthead seabream specimens were fed a commercial non-supplemented diet containing 1.75 mg of vitamin A kg(-1) (as control) or the same diet supplemented with 50, 150 or 300 mg of retinol acetate kg(-1) (as vitamin A source). After 1, 2, 4 or 6 weeks, serum samples and head-kidney leucocytes were obtained from each fish. Serum lysozyme activity and myeloperoxidase (MPO) content were unaffected by the vitamin A diet content. The phagocytic and respiratory burst activities of head-kidney leucocytes were established, as well as their myeloperoxidase content. While phagocytosis was not enhanced by dietary vitamin A intake and was even slightly decreased after 2 weeks, respiratory burst activity was enhanced in specimens fed supplements of 150 and 300 mg retinol acetate kg(-1) diet for 1 or 2 weeks. Leucocyte MPO content was also enhanced when seabream were fed the highest vitamin A dose for 2 or 4 weeks and after being fed the 150 or 50 mg supplemented diets for 4 or 6 weeks, respectively. Three different groups of seabream were i.p. injected with 1 ml of phosphate buffer containing an amount of retinol acetate equivalent to the daily dietary supplements from the first experiment (0-control-, 0.05 or 0.30 mg 100 g(-1) biomass). Both injection doses of retinol acetate were toxic for the gilthead seabream which showed hypervitaminic effects. These data show that retinol acetate plays an important role in the gilthead seabream nonspecific cellular immune system due to its antioxidant properties. They also point to the importance of the way in which it is administered, by dietary uptake or intraperitoneal injection.     

Experimental effect of lysozyme on macrophage metabolism and resistance to infection

The peritoneal macrophages of mice treated with lysozyme were studied by cytochemical assay. In single and repeated doses of 0.5-5 mg/kg lysozyme induced an increase in macrophage metabolism. This was evident from an increased activity of succinate dehydrogenase, NADP X N-DH and the enzymes catalyzing glycolysis typical of these cells (lactate dehydrogenase and alpha-glycerophosphate). The changes in the activity of the enzymatic systems were most pronounced in minute and less mature macrophages after repeated administrations of the drugs. In a dose of 50 mg/kg lysozyme somewhat decreased the activity of a number of the enzymes. In the doses optimal for the macrophage activity lysozyme had a low effect on the infection resistance and slightly increased the cephotaxim efficiency in experimental staphylococcal infection. This may be mainly due to the immunomodulating effect of lysozyme and its low effect on the large macrophages having the bactericidal effect.     

Lysozyme activity in rainbow trout, Oncorhynchus mykiss (Walbaum), stressed by handling, transport and water pollution

Blood lysozyme activity was determined in rainbow trout that were stressed by handling and transport, or suffered from acute ammonia poisoning. To optimize the lysoplate assay, the pH dependence of the lysozyme activity was studied. For serum and kidney lysozyme, the pH optimum was 5.5 to 6.0. Ventilation frequency and plasma glucoseconcentration of the fish were used as stress indicators. Less severely stressful situations, such as 30 min of handling, brought about either a stimulation or a suppression of the lysozyme activity. Strong stressors, such as transport lasting 2 h or acute water pollution, reduced the lysozyme level significantly. The decrease of lysozyme activity due to handling and transport persisted for 24 h, but the activity returned to normal within 2 weeks following confrontation with the stressor.     

Effect of lysozyme on hepatocyte resistance

Antitoxic effect of lysozyme was shown on a model of experimental acute toxic hepatitis of rats and mice. Administration of lysozyme to the animals in a dose of 5 mg/kg 24 hours before administration of carbon tetrachloride markedly decreased the level of morphological damages in the liver tissue and promoted a decrease in increased levels of alanine aminotransferase in blood serum. Higher levels of lysozyme in blood serum and cells of mouse peritoneal exudate 3 hours after administration of lysozyme were observed. The role of lysozyme as one of the main products secreted by activated macrophages in providing the general and antitoxic resistance of hepatocytes is discussed. Possible use of lysozyme as a hepatoprotective agent is suggested.     

Localization of chitinolytic enzymes in blood of turbot, Scophthalmus maximus, and their possible roles in defence

The intracellular localizations ofchitinase and β-N-acetylglucosaminidase were detected in turbot blood smears, using a novel method employing fluorogenic substrates. The two enzymes showed different distributions, with chitinase being more generally distributed and N-acetylglucosaminidase being strongly associated with distinct intracellular bodies, probably lysosomes. The fluorogenic substrates were used to analyse soluble and membrane fractions of homogenates of red and white blood cells prepared on Percoll gradients. In the leucocytes, the chitinase and N-acetylglucosaminidase activities were mostly in the soluble fraction. In the erythrocytes the activities were lower, at about one-hundredth and one-tenth specific activities, respectively, and were distributed between soluble and membrane-bound fractions at about 2 : 1 and 3 : 1, respectively. In contrast, lysozyme had a soluble distribution in leucocytes and was not detected in erythrocytes. Plasma was rich in chitinase and lysozyme activities but had no detectable N-acetylglucosaminidase. Two possible roles for the chitinolytic enzymes are discussed: defence against pathogens and processing of glycoproteins or glucosaminoglycans. Evidence for a defence role for the chitinase and lysozyme is provided by demonstrating that they had inhibitory activity against the chitinous fungus Mucor mucedo .     

牛磺酸对虎纹蛙非特异性免疫及血清氧化还原状态的影响

为探讨牛磺酸在无尾两栖类免疫调节和抗氧化能力方面的功能和作用,通过灌胃方式研究了不同浓度牛磺酸对虎纹蛙(Hoplobatrachus rugulosus)脾巨噬细胞呼吸爆发、外周血吞噬活力、胃溶菌酶活力以及血清丙二醛(MDA)和谷胱甘肽(GSH)含量的影响。结果表明:连续7 d的牛磺酸灌胃可显著提高脾巨噬细胞的呼吸爆发强度和外周血细胞的吞噬活力并呈现出明显的剂量效应。当灌胃浓度达到0.8 g/L时两者均达到峰值,但高于0.8 g/L时则表现出条件毒性。胃溶菌酶活力在各浓度下无明显变化。此外,血清MDA含量随牛磺酸浓度的升高而降低,GSH含量在一定的浓度范围内呈现出明显的剂量效应,在1 g/L时达到峰值。研究结果证明牛磺酸可以明显提高虎纹蛙的非特异性免疫功能和机体氧化防御能力,但摄入过量则表现出条件毒性。基于上述指标的评估,虎纹蛙对牛磺酸的适宜需求量按体重计算约为16~20 mg/kg。     

Effects of mannan oligosaccharide on the physiological responses,HSP70 gene expression and disease resistance of Allogynogenetic crucian carp (Carassius auratus gibelio) under Aeromonas hydrophila infection

We evaluated the effect of dietary supplementation with mannan oligosaccharide (MOS) on the resistance to Aeromonas hydrophi1a infection in Allogynogenetic crucian carp. The fish were randomly divided into five groups: a control group was fed with basal diet, and four treatment groups fed with basal diet supplemented with 60, 120, 240, 480 mg/kg MOS for 10 weeks, respectively. We then challenged the fish with A. hydrophi1a and recorded the mortality and the changes in serum cortisol, T3, T4, lysozyme, alkaline phosphatase (ALP), globin and hepatic total anti-oxidative capacity, superoxide dismutase (SOD), malondialdehyde (MDA) and the relative expression of heat shock protein 70 (HSP70) mRNA for a period of 7 d. Supplementation with 240 mg/kg MOS significantly increased serum ALP activity before infection, 1d and 2d after infection, serum globin concentration prior to infection, 1d and 7d after infection, serum lysozyme activity at 2d after infection, T3 concentration at 2d after infection, hepatic total anti-oxidative capacity prior to infection, hepatic SOD activity at 7d after infection and reduced serum cortisol concentration at 2d after infection, hepatic malondialdehyde content at 1d and 2d after infection. Supplementation with 480 mg/kg MOS significantly increased serum ALP activity before infection, 1d and 2d after infection, T3 content 1d after infection, T4 content prior infection and 7d after infection, serum globin concentration prior to infection, 1d and 7d after infection, serum lysozyme activity prior infection and 1d after infection, serum total anti-oxidative capacity prior to infection and 7d after infection, hepatic SOD activity at 7d after infection and the relative level of hepatic HSP70 mRNA at 2d and 7d after infection, had decreased levels of serum cortisol concentration before the infection, at 2d after infection, T4 concentration at 1d and 2d after infection, hepatic malondialdehyde content at 1d and 2d after infection. Mortality was significantly lower in the group of 240 and 480 mg/kg MOS than the control. Our results suggest that ingestion of a basal diet supplemented with 240–480 mg/kg MOS can enhance resistance against pathogenic infections in Allogynogenetic crucian carp.