Natural Haemolytic Complement Activity Research Articles

Immune defence mechanisms presented in liver homogenates and bile of gilthead seabream (Sparus aurata).

Because the role of the liver of fishes in providing possible immunity remains largely unknown, the aim of this work was to identify and characterize different humoral defence mechanisms in the liver homogenates and bile of gilthead seabream (Sparus aurata) for the first time. Total protein levels and several immune parameters (complement activity, lysozyme and immunoglobulin M level) were studied. Furthermore, the activity of some lytic (proteases, antiproteases, esterase, alkaline phosphatase) and antioxidant (superoxide dismutase, catalase and peroxidase) enzymes was determined. Finally, bacteriostatic activity on three opportunist fish pathogens (Vibrio harveyi, Vibrio angillarum and Photobacterium damselae) was measured. Lysozyme and antiprotease activity were undetected in liver and bile, while natural haemolytic complement activity was only detected in bile, and immunoglobulin M was detected in both samples. The levels of proteases, esterase and antioxidant enzymes were greater in bile than in liver homogenates, while the level of alkaline phosphatase was very low in both samples. In addition, while no bacteriostatic activity was detected on liver homogenates, the bile revealed a very potent bacteriostatic activity against all the tested pathogenic bacteria. These results corroborate that fish liver - especially fish bile - contains many factors involved in innate immunity that could be useful for better understanding the role of the liver as an organ involved in fish immune functions as well as the possible contribution of bile to gut mucosal immunity.

Probiotic properties of Debaryomyces hansenii BCS004 and their immunostimulatory effect in supplemented diets for gilthead seabream ( Sparus aurata )

Debaryomyces hansenii BCS004 strain is a non-pathogenic, polyamine and superoxide dismutase secreting marine yeast. The objective of this study was to investigate the marine yeast DhBCS004 as probiotics and evaluate its antimicrobial properties and dietary immunostimulant effect on gilthead seabream specimens when administered at 106 colony-forming unit/g−1 or 1.1% for 4 weeks. This yeast strain showed potential probiotic properties, including stability in gastrointestinal survival (pH, bile conditions) and attachment to intestinal cells. The DhBCS004 had antimicrobial activity against important marine pathogens. Interestingly, this yeast has a high amount of n−3 polyunsaturated fatty acids with high concentrations of oleic and linoleic acids, which may confer nutritional and therapeutic values. The in vivo immunological assay revealed that dietary DhBCS004 significantly increased phagocytosis capacity, peroxidase and respiratory burst activities in leucocytes. Furthermore, the natural haemolytic complement activity and total IgM levels increased in serum after weeks 2 or 4 of trial. Finally, DhBCS004 significantly increased the transcriptions of TNF-α, C3 and IgM in intestine samples. This work reinforces the biotechnological and immunological potential of D. hansenii BCS004 in the field of fish aquaculture.

Changes in natural haemolytic complement activity induced by stress in gilthead seabream (Sparus aurata L.)

In aquaculture, animals can be continually exposed to environmental stress factors that put their health and even survival at risk. Two experiments were performed to evaluate the impact of different stress conditions (acute crowding and anaesthetic) on the natural haemolytic complement activity in serum and skin mucus of gilthead seabream (Sparus aurata L.). In the first experiment, fish were subjected to 10 kg m−3 (low density, control group) and 50 kg m−3 (high density, crowding group) during 2, 24 and 48 h. In the second experiment, fish were unexposed (control) or exposed to 40 ppm of MS-222 or 5 ppm or 10 ppm of clove oil for 1 h. In fish maintained in acute crowding conditions only an increase of the haemolytic complement activity was observed in the skin mucus after 24 h of exposure. However, a similar statistically significant increase was observed in serum and skin mucus of fish exposed for 1 h to the lowest concentration of clove oil (5 ppm) tested. The results point to a new and alternative way to assess stress in farmed fish by using skin mucus instead of blood serum and confirm that the measurement of natural haemolytic complement activity serves as an indicator of stress in fish.

Enrichment of gilthead seabream (Sparus aurata L.) diet with microalgae: effects on the immune system

The present study assessed the effects of three orally administered microalgae (Nannochloropsis gaditana, Tetraselmis chuii and Phaeodactylum tricornutum) on different immune parameters and immune-related gene expression of gilthead seabream (Sparus aurata L.). Fish were fed a control or one of six experimental diets and sampled at 2 and 4 weeks of treatment. At the end of the trial, growth performance and different systemic and local immune activities were measured (natural haemolytic complement activity, IgM levels, phagocytosis and respiratory burst). The expression levels of different immune-associated genes (EF-1α, IgM(H), TCR-β, MHCIα, MHCIIα, CSF-1R and β-defensin) were analysed in head-kidney and gut. Administration of N. gaditana and T. chuii resulted in a significant increase in haemolytic complement activity, phagocytic capacity, as well expression level of β-defensin, as well as MHCIIα and CSF-1R, respectively. The P. tricornutum-supplemented diet provoked immunostimulation, and very little effect on gene expression was observed. These results suggest that dietary microalgae enhance gilthead seabream defence activity, which could be very important in order to consider such microalgae as a possible additive in fish diets.

Effects of dietary inulin and heat-inactivated Bacillus subtilis on gilthead seabream (Sparus aurata L.) innate immune parameters

In the present study, a feeding trial was conducted to evaluate the effect of inulin and heat-inactivated Bacillus subtilis, single or combined, on several innate immune activities of gilthead seabream (Sparus aurata). Forty-eight specimens were randomly assigned to four dietary treatments: 0 (control), inulin (10 g/kg, prebiotic group), B. subtilis (10(7) cfu/g, probiotic group), or B. subtilis + inulin (10(7) cfu/g + 10 g/kg, synbiotic group). After two and four weeks, six fish of each group were sampled, with the main innate immune parameters (natural haemolytic complement activity, serum and leucocyte peroxidase, phagocytosis, respiratory burst, and cytotoxic activities) being determined. Inulin or heat-inactivated B. subtilis failed to significantly stimulate the innate immune parameters assayed, although some activities showed no significant increase through these treatments. A combination of inulin and B. subtilis resulted in an increase of such parameters, with the haemolytic complement activity being the only one significantly stimulated. To conclude, inulin and B. subtilis, when administered as a synbiotic, have a synergistic effect and enhance some innate immune parameters of gilthead seabream.

Modulation of the immune parameters and expression of genes of gilthead seabream (Sparus aurata L.) by dietary administration of oxytetracycline

The oxytetracycline (OTC) is an antibiotic widely used in the aquaculture, principally in the treatment of infections caused by the pathogenic Gram-negative bacteria. In the present study, the consequences of the oral administration of OTC on the main humoral (natural haemolytic complement activity, IgM levels and seric peroxidase level) and cellular (respiratory burst, intracellular peroxidase level and phagocytosis) immune parameters of gilthead seabream (Sparus aurata, L) were evaluated after 7, 14 and 21days of treatment. Furthermore, the expression levels of immune-associated genes (MHC-IIα, C3, IL-1β, COX-2 and β-defensin) were analysed by real-time polymerase chain reaction in head-kidney and gut. Three experimental groups were established: control group (fed with non-supplemented OTC food) and experimental groups (4 and 8mg OTC g−1 feed, respectively) (N=30). The results reveal a decrease of the natural haemolytic complement activity to the higher dose of OTC administered. By contrast, after 7days of treatment, fish fed the lowest concentration of OTC were observed to have a statistically significant increase in the percentage of phagocytic cells, while the phagocytic capacity and respiratory burst increased after 14days of treatment for both concentrations of OTC. No significant effects were observed after 21days of treatment for both concentrations of OTC with regard to the values obtained in the control group. In head-kidney and gut, the dietary administration of OTC not caused statistically significant variation, only in C3 gene expression at 7days in gut. The present results suggest that after dietary administration of OTC, the humoral and cellular innate immune parameters of gilthead seabream are depressed and stimulated respectively, and induce up- or down-regulation of the immune-relevant genes analysed depending on the concentration of OTC supplied in diet.

Propolis and Herba Epimedii extracts enhance the non-specific immune response and disease resistance of Chinese sucker, Myxocyprinus asiaticus

The effect of traditional Chinese medicine (TCM) formulated from propolis and Herba Epimedii extracts at the ratio of 3:1 (w/w) on non-specific immune response of Chinese sucker ( Myxocyprinus asiaticus) was investigated. Fish were fed diets containing 0 (control), 0.1%, 0.5% or 1.0% TCM extracts for five weeks. The respiratory burst and phagocytic activities of blood leukocytes, lysozyme and natural haemolytic complement activities in plasma were measured weekly. After five weeks of feeding, fish were infected with Aeromonas hydrophila and mortalities were recorded. Results of this study showed that feeding Chinese sucker with different dosage of TCM extracts stimulated respiratory burst activity, phagocytosis of phagocytic cells in blood and lysozyme activity in plasma. They had no effect on plasma natural haemolytic complement activity. All dosage of treated groups showed reduced mortality following A. hydrophila infection. Feed containing 0.5% TCM extracts was the most effective with the mortality of the fish significantly reduced by 35% compared to the control. The results indicate that propolis and Herba Epimedii extracts in combination enhances the non-specific immune response and disease resistance of Chinese sucker against A. hydrophila.

Non-specific Immune Response of Rainbow Trout (Oncorhynchus Mykiss) by Dietary Heat-inactivated Potential Probiotics

Background: The effects of the dietary administration of two heat-inactivated whole bacteria from the Vibrionaceae family, singly or combined, on innate immune response of the rainbow trout were studied. The two bacteria (Pdp11 and 51M6), which were obtained from the skin of rainbow trout, showed in vitro characteristics that suggested they could be considered as potential fish probiotics. Methods: The fish were fed four different diets: control (non-supplemented), or diets supplemented with heat-inactivated bacteria at 10 8 cfu/g Pdp11, 10 8 cfu/g 51M6 or with 0.5×10 8 cfu/g Pdp11 plus 0.5×10 8 cfu/g 51M6 for 4 weeks. Six fish were sampled at weeks 1, 2, 3 and 4, and then the main humoral (natural haemolytic complement activity and serum peroxidase content) and cellular innate immune responses (leucocyte peroxidase content, phagocytosis, respiratory burst and cytotoxicity) were evaluated. Results: The serum peroxidase content and the natural haemolytic complement activity increased with time, reaching the highest values in the third and fourth weeks of feeding, respectively. The phagocytic ability of specimens fed the mixture of the two inactivated bacteria was significantly higher than in the controls after 2 and 3 weeks of treatment. The same activity increased significantly in rainbow trout fed the Pdp11 diet for 2 weeks or the 51M6 diet for 3 weeks. Respiratory burst activity was unaffected by all the experimental diets at all times assayed. Cytotoxic activity had significantly increased after 3 weeks in fish fed the 51M6 diet. Conclusion: Our results demonstrated the usefulness of incorporating inactivated probiotic bacteria into fish diets. [Immune Network 2008;8(3):67-74]

Dietary levamisole modulates the immune response and disease resistance of Asian catfish Clarias batrachus (Linnaeus)

In order to determine the immunomodulatory effect of dietary levamisole in Asian catfish (Clarias batrachus), fish were fed four different diets for 10 days: a formulated diet as control and the same diet supplemented with 50, 150 or 450 mg levamisole kg−1 feed. The serum bacterial agglutination titre against Aeromonas hydrophila as a measure of specific immunity, serum haemagglutination titre, natural haemolytic complement activity (ACH50), myeloperoxidase and lysozyme activities, total protein level and oxidative radical production by neutrophils as a measure of non-specific immunity as well as disease resistance against A. hydrophila challenge to separate vaccinated and non-vaccinated groups were evaluated at 0, 1, 2 and 3 weeks after last administration of levamisole. Levamisole supplement at the lowest level (50 mg kg−1) significantly enhanced oxidative radical production and serum myeloperoxidase (MPO) content immediately after 10 days of feeding, which reached peak values after 3 and 2 weeks of feeding respectively. Haemolytic complement and haemagglutination titre were significantly enhanced after 3 and 1 weeks respectively. Haemolytic complement activity and MPO activities were significantly raised to 150 mg kg−1 after 3 and 2 weeks, respectively. At the highest level of levamisole feeding (450 mg kg−1) significant decreases in superoxide production and complement activity were measured immediately after levamisole feeding, which returned to the normal level after 1 week post- feeding. Fish were challenged with a virulent strain of A. hydrophila at 0, 1, 2 and 3 weeks after levamisole feeding, and the cumulative per cent survival was recorded over 10 days. Feeding levamisole at 50, 150 or 450 mg kg−1 increased per cent survival in vaccinated fish immediately after levamisole feeding, and survival was significantly higher at 450 mg kg−1. There was no difference in mortality patterns in non-vaccinated fish. The results support the use of levamisole at 50 mg kg−1 feed for 10 days as an immunostimulant in Asian catfish farming.

Gilthead seabream ( Sparus aurata L.) innate immune response after dietary administration of heat-inactivated potential probiotics

The effects of the dietary administration of two heat-inactivated whole bacteria from the Vibrionaceae family, singly or combined, on innate immune response of the seabream were studied. The two bacteria (Pdp11 and 51M6), which were obtained from the skin of gilthead seabream, showed in vitro characteristics that suggested they could be considered as potential fish probiotics. The fish were fed four different diets: control (non-supplemented), or diets supplemented with heat-inactivated bacteria at 10 8 cfu g −1 Pdp11, 10 8 cfu g −1 51M6 or with 0.5 × 10 8 cfu g −1 Pdp11 plus 0.5 × 10 8 cfu g −1 51M6 for 4 weeks. Six fish were sampled at weeks 1, 2, 3 and 4, when the main humoral (natural haemolytic complement activity and serum peroxidase content) and cellular innate immune responses (leucocyte peroxidase content, phagocytosis, respiratory burst and cytotoxicity) were evaluated. The serum peroxidase content and the natural haemolytic complement activity increased with time, reaching the highest values in the third and fourth weeks of feeding, respectively. The phagocytic ability of specimens fed the mixture of the two inactivated bacteria was significantly higher than in the controls after 2 and 3 weeks of treatment. The same activity increased significantly in seabream fed the Pdp11 diet for 2 weeks or the 51M6 diet for 3 weeks. Respiratory burst activity was unaffected by all the experimental diets at all times assayed. Cytotoxic activity had significantly increased after 3 weeks in fish fed the 51M6 diet. These results are discussed in terms of the usefulness of incorporating inactivated probiotic bacteria into fish diets.

The effect of dietary intake of vitamins C and E on the stress response of gilthead seabream (Sparus aurata L.)

High dietary doses of the antioxidant vitamins C and E were administered to gilthead seabream (Sparus aurata L.) in an attempt to reduce the stress response in specimens exposed to a multiple stress situation. Fish were fed four different diets for 6 weeks: a commercial feed containing 0·1g vitamin C and 0·1g vitamin E kg−1 acted as control diet, while experimental diets consisted of the same feed supplemented with 3g vitamin C kg−1, 1·2g vitamin E kg−1 or both 3g vitamin C and 1·2g vitamin E kg−1. After 2, 4 and 6 weeks fish were exposed to stressors typical of aquacultural practices, and serum cortisol levels, complement activity (measured by the alternative pathway), blood glucose level and respiratory burst activity of head-kidney leucocytes were evaluated. The results showed that all stress-induced increases in blood glucose concentration were lower in fish fed the vitamin C and/or E-supplemented diet than in fish fed the control diet after 2 weeks of treatment, although no other differences were found at the rest of the times. Cortisol levels increased in stressed fish and did not suffer depletion as a consequence of administering vitamins C and/or E as a supplement. The natural haemolytic complement activity was not affected by the stressors but enhanced in specimens fed vitamin-supplemented diets at week 6. The respiratory burst activity was depressed by the stressors in fish fed the control diet, although only after 6 weeks of treatment were the differences statistically significant. These results suggest that vitamins C and E are involved in the hypothalamic-sympathetic-chromaffin cell axis and also interfere in tertiary stress responses such as immunodepression, where they protect the leucocyte functions.

Immunomodulatory effects of dietary intake of chitin on gilthead seabream (Sparus aurata L.) innate immune system

To determine the effects of chitin (poly [1→4]-β-N-acetyl-D-glucosamine) on the innate immune response of gilthead seabream (Sparus aurata L.), fish were fed diets containing 0mg (control), 25, 50 or 100mg kg−1chitin for 2, 4 and 6 weeks. Lysozyme and natural haemolytic complement activities, together with head-kidney leucocyte respiratory burst, phagocytic and cytotoxic activities, were studied at each of the assayed times. Lysozyme activity was unaffected by the administration of chitin. The innate humoral and cellular immune response activities assayed were enhanced by the dietary intake of chitin, each increasing at a different time: natural haemolytic complement activity and cytotoxic activity after 2 weeks of treatment, respiratory burst activity from 4 weeks and phagocytic activity after 6 weeks, although, unlike the other activities, no statistically significant differences were observed in the first. The results indicate that chitin increases the activity of the seabream innate immune system, and its use as an immunostimulant is discussed, especially with regards to the protective role.

Effects of injecting chitin particles on the innate immune response of gilthead seabream (Sparus aurataL.)

To determine the effects of chitin (poly [1→4]-β-N-acetyl-D—glucosamine) particles on the innate immune response of gilthead seabream (Sparus aurata L.), specimens were injected intravenously or intraperitoneally with the substance. Natural haemolytic complement activity, head-kidney leucocyte respiratory burst activity and phagocytic and cytotoxic activities were analysed in vitro 3, 5 or 10 days post-injection. All the immune parameters assayed remained unaffected when chitin was intravenously administered. However, the fish that had been intraperitoneally injected showed increased humoral and cellular immune responses. Natural haemolytic complement activity increased from 5 days post-injection although no statistically significant differences were observed. Respiratory burst and phagocytic activities peaked at 3 and 5 days post-injection, respectively, while cytotoxic activity had increased by 3 days post-injection and remained high until 10 days post-injection.

High dietary intake of α-tocopherol acetate enhances the non-specific immune response of gilthead seabream (Sparus aurata L.)

To determine the effects of three high levels of dietary intake of α-tocopherol acetate (vitamin E) on the non-specific immune response of gilthead seabream (Sparus aurata L.), specimens were fed a commercial diet (100mg α-tocopherol kg−1) as control, or vitamin E supplemented diets (600, 1200 or 1800mg α-tocopherol acetate kg−1) for 15, 30 or 45 days. Growth, serum α-tocopherol levels, natural haemolytic complement activity and head–kidney leucocyte migratory, respiratory burst and phagocytic activities were studied at each of the assay times. A positive correlation between α-tocopherol acetate intake and serum α-tocopherol levels was observed, the increase being linked to both the dosage and length of treatment. Specimens fed the diet supplemented with 600mg vitamin E kg−1showed no enhancement in any of their immune parameters, while those fed the diet supplemented with 1200mg vitamin E kg−1presented a slightly higher (but not statistically significant) specific growth rate than fish fed the other diets. In addition, serum haemolytic activity and the phagocytosis of head–kidney leucocytes were enhanced by the dietary intake of 1200mg vitamin E kg−1after 30 and 45 days of treatment, although leucocyte migration and respiratory burst activity remained unaffected. The highest vitamin E dietary dose used, 1800mg kg−1, unexpectedly provoked no immunostimulation. These results indicate that a moderate level of vitamin E in the diet (1200mg kg−1) stimulates the seabream's non-specific immune system after 30 days of administration. Lower or higher vitamin E concentrations may not be so effective, because of an imbalance in the vitamin E ratio with other antioxidants. The proposed dietary levels of vitamin together with the indicated administration time could be useful for reducing the susceptibility of farmed fish to infectious diseases.

Effect of high dietary intake of vitamin C on non-specific immune response of gilthead seabream (Sparus aurata L.)

To determine the effect of a high level of dietary intake of ascorbic acid (vitamin C) on the non-specific immune response of gilthead seabream (Sparus aurata L.), specimens were fed diets containing 500mg kg−1(control) and 3,000mg kg−1ascorbic acid for 2, 4, 6, 8 or 10 weeks. Growth, serum level of vitamin C, natural haemolytic complement activity and head-kidney leucocyte migratory, respiratory burst and phagocytic activities were studied at each one of the assayed times. A positive relationship between vitamin C intake and serum ascorbic acid was observed, the enhancement being evident after 2 weeks of administration and maintained until 10 weeks. Specimens fed a vitamin C supplemented diet showed a slightly higher (but no statistically significant) specific growth rate than fish fed the control diet. The non-specific immune response parameters assayed were enhanced by the dietary intake of vitamin C although, each one peaked at a different time: phagocytic activity after 2 weeks of administration, natural haemolytic complement activity after 6 weeks, respiratory burst after 8 weeks. However, leucocyte migration was not affected. The results indicated that the serum ascorbate concentration of specimens fed a high level of vitamin C reach a plateau. The non-specific immune response parameters assayed increased as a consequence of a high vitamin C supply, although the increase was not maintained but returned to the initial levels. This suggests that the immune response accomodates to the new high vitamin C plasma availability after a transient enhancement.