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Chinese herbs (Astragalus membranaceus and Lonicera japonica) and boron enhance the non-specific immune response of Nile tilapia (Oreochromis niloticus) and resistance against Aeromonas hydrophila
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Aquaculture 275 (2008) 26 – 33 www.elsevier.com/locate/aqua-online
Chinese herbs (Astragalus membranaceus and Lonicera japonica) and boron enhance the non-specific immune response of Nile tilapia (Oreochromis niloticus) and resistance against Aeromonas hydrophila László Ardó a , Guojun Yin b , Pao Xu b , László Váradi a , Gábor Szigeti c , Zsigmond Jeney a , Galina Jeney a,⁎ b
a Research Institute for Fisheries, Aquaculture and Irrigation, Anna liget 8, H-5540, Szarvas, Hungary Freshwater Fisheries Research Center of Chinese Academy of Fishery Sciences, Wuxi, 214081, PR China c Central Veterinarian Institute, Tábornok u. 2, Budapest XIV, Hungary
Received 30 August 2007; received in revised form 13 December 2007; accepted 19 December 2007
Abstract The effect of two Chinese medicinal herbs (Astragalus membranaceus and Lonicera japonica) and boron on non-specific immune response of Nile tilapia (Oreochromis niloticus) was investigated. Five diet variations in addition to a control diet (without herbs or boron) were used. These contained 0,1% Astragalus (with and without 0,05% boron), 0,1% Lonicera (with or without 0,05% boron) and a mixture of the two herbs with 0,05% boron. The diets were fed for four weeks, and respiratory burst and phagocytic activities of blood leukocytes; plasma lysozyme, total protein and total immunoglobulin level were measured weekly. After four weeks of feeding, fish were infected with Aeromonas hydrophila and mortalities were recorded. Results of this study showed that feeding tilapia with two herbs alone or in combination significantly enhanced phagocytic and respiratory burst activity of blood phagocytic cells. They had a moderate effect on the plasma lysozyme level and no effect on plasma total protein and total immunoglobulin level. Both herbs reduced the mortality following A. hydrophila infection. The lowest mortality was observed in the group fed with the combination of both herbs and boron. Combination of the herbs and boron could even more increase the survival rate of infected fish. It can be concluded that Astragalus and Lonicera extracts and boron supplementation added to fish feed can act as immunostimulants and enhance the immune response and disease resistance of cultured fish. © 2007 Elsevier B.V. All rights reserved. Keywords: Astragalus membranaceus; Lonicera japonica; Boron; Oreochromis niloticus; Non-specific immunity; Disease resistance; Aeromonas hydrophila
1. Introduction Enhancement of the immune system seems to be the most promising method of preventing fish diseases. This modulation can be achieved with vaccines, which enhance the acquired (or specific) immune response of the fish and are considered to be the most effective agents, but a single vaccine is effective against only one type of pathogens, and in the case of many pathogens (like the bacterium Aeromonas hydrophila) no effective vaccine is available due to the complex antigenic structure.
⁎ Corresponding author. Tel.: +36 66 515317; fax: +36 66 312142. E-mail address: [email protected] (L. Ardó). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.12.022
In contrast to vaccines, immunostimulants enhance the innate (or non-specific) immune response (Galeotti, 1998; Sakai, 1999). The major components of the innate immune system are macrophages, monocytes, granulocytes and humoral elements, like lysozyme or complement system (Secombes and Fletcher, 1992; Magnadóttir, 2006). Immunostimulants can be applied via injection, bathing or oral administration, the latter seems to be the most practicable (Jeney and Anderson, 1993; Sakai, 1999; Yin et al., 2006). Many types of biological and synthetic compounds have been shown to enhance non-specific immune system of cultivated fish (Sakai, 1999). Best-known immunostimulants are components of bacterial cell wall, like lipopolysaccharide (LPS) (Goetz et al., 2004) or glucans (Engstad et al., 1992), but synthetic compounds, polysaccharides, animal and plant extracts or
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vitamins can enhance the non-specific immune response of fish (Siwicki, 1987, 1989; Hardie et al., 1991; Thompson, I. et al., 1993; Thompson, K.D. et al., 1995). However, the effect is dosage-dependent and there is a chance to overdose (Kajita et al., 1990) neither are they always effective in early developmental stages of fish (Huttenhuis et al., 2006). In traditional Chinese human medicine, herbs have been used as immunostimulants for thousands of years (Tan and Vanitha, 2004). These herbs contain many types of active components, like polysaccharides, alkaloids or flavonoids. The immunostimulating activity of herbal components have been most widely studied in mice, chickens or human cell lines (Shan et al., 1999; Cao and Lin, 2003; Lin and Zhang, 2004), and in the case of some herbs (e.g. Astragalus membranaceus or Nelumbo nucifera) even the molecular mechanisms of their effect is known (Shao et al., 2004; Liu et al., 2004). There is a growing interest in using medicinal herbs as immunostimulants in aquaculture. In common carp (Cyprinus carpio) and large yellow croaker (Pseudosciena crocea) respiratory burst activity of phagocytic cells and plasma lysozyme activity has been significantly increased after feeding with a ration containing a mixture of A. membranaceus and Angelica sinensis (Jian and Wu, 2003, 2004). Similar results have been reported about experiments on various fish species, e.g. rainbow trout (Oncorhynchus mykiss), Indian major carp (Catla catla) and Mozambican tilapia (Oreochromis mossambicus) (Dügenci et al., 2003; Dey and Chandra, 1995; Logambal and Michael, 2000). Trace elements or micronutrients like selenium, boron or zinc are essential for growth, development and health of the fish (Watanabe et al., 1997). In our previous experiments we found that boron can enhance the immunomodulatory effect of some medicinal herbs (G. Jeney, unpublished results). In this study, two Chinese medicinal herbs, A. membranaceus and Lonicera japonica were chosen for the experiments. Various extracts of Astragalus root have been used as immunostimulants in traditional Chinese human medicine (Wang et al., 1999) and there are many reports supporting its immunomodulating effects (Song et al., 2000; Lee et al., 2003). L. japonica is used as traditional human medicine to treat fever and headache (Kumar et al., 2005) and as an anti-inflammatory agent (Lee et al., 2001). However, one of its active components, chlorogenic acid has a multiple immunomodulatory effect. (Koethe et al., 1995; Boon et al., 2002; Wu et al., 2004). In the study reported here, Nile tilapia were fed with fish feed containing Astragalus or Lonicera extracts or their combination and with or without boron supplementation. Key factors of the non-specific immune response were measured during the experiment and at the end fish were challenged with A. hydrophila to determine disease resistance in fish fed with these substances.
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2.2. Herbal extracts Powdered Astragalus extract containing 90% of Astragalus polysaccharide and powdered Lonicera extract containing 25% of chlorogenic acid were commercial products of Nantong Sihai Plant Extracts Co., Ltd., China.
2.3. Experimental design Experiments were performed in the recirculation system of the HAKI, Szarvas, Hungary. Fish were kept in fibreglass tanks with a constant water temperature and pH (22–23 °C; pH 8.5). Dissolved oxygen was maintained at 80–90% of saturation, and water flow was 7 l/min during the experimental period. Fish were allocated into 6 groups (50 fish/group) and fed diets containing different medicinal herbs and boron. The compositions of the feeds were the following: Group 1: 0.1% Astragalus extract Group 2: 0.1% Astragalus extract and 0.05% boron Group 3: 0.1% Lonicera extract Group 4: 0.1% Lonicera extract and 0.05% boron Group 5: 0.1% Astragalus extract, 0.1% Lonicera extract and 0.05% boron Group 6: Control (without medicinal herbs or boron) Cornmeal oil (10 ml/kg feed) was used to bind the powdered herbal extracts and boron to the fish feed. The prepared feed was maintained at room temperature.
2.4. Blood sampling and separation of leukocytes Blood samples (6 fish/group) were collected from the caudal vein 1, 2, 3 and 4 weeks after start of feeding. Heparin was used as an anticoagulant. Individual fish were sampled only once to avoid the influence on the assays due to multiple bleeding and handling stress on the fish. Leukocytes for assay were separated from each blood sample by densitygradient centrifugation. One ml of histopaque 1.119 (Sigma) containing 100 µl of Bacto haemagglutination buffer, pH 7.3 (Difco, USA) was dispensed into siliconised tubes. One ml of a mixture of histopaque 1.077 and haemagglutination buffer was layered on the top. One ml of blood sample was then layered carefully on the top of the gradient. Sample preparations were centrifuged at 700 G for 30 min at 4 °C. After centrifugation, plasma was collected and stored at −20 °C for future analysis. Separated leukocytes were gently removed and dispensed into siliconised tubes, containing phenol red free Hank's Balanced Salt Solution (HBSS). Cells were then washed in HBSS and adjusted to 107 viable cells/ml.
2.5. Respiratory burst activity Respiratory burst activity of isolated leukocytes was quantified by the nitroblue tetrazolium (NBT) assay (Secombes, 1990), which measures the quantity of intracellular oxidative free radicals. This method was slightly changed, the concentration of NBT solution was 2 mg/ml.
2.6. Phagocytosis assay Phagocytosis activity of blood leukocytes was determined spectrophotometrically by the method of Seeley et al. (1990).
2.7. Lysozyme assay 2. Materials and methods 2.1. Fish Tilapia (O. niloticus) propagated and reared by the Research Institute for Fisheries, Aquaculture and Irrigation (HAKI, Szarvas, Hungary) were used for the experiment. Fish were fed with a dry feed, produced in the experimental milling facility of the institute and kept in 2000 l fibreglass tanks in the recirculation system of the institute at water temperature of 22–23 °C.
Plasma lysozyme activity was measured spectrophotometrically by the method of Sankaran and Gurnani (1972).
2.8. Plasma total protein assay The total protein concentration of the plasma was determined by a colorimetric assay based on the Biuret reaction, using a protein diagnostic reagent kit (Reanal, Hungary). The assay was performed in 96-well microtiter plates. 10 µl
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L. Ardó et al. / Aquaculture 275 (2008) 26–33 United Kingdom. Bacteria were inoculated into 10 ml of liquid tryptic soy broth (TSB, Sigma) medium and were grown overnight at 28 °C. Cultures were centrifuged at 850 G for 15 min. Supernatant was removed and the pelleted bacteria were washed twice in sterile phosphate buffered saline (PBS) solution. The concentration of bacteria was adjusted to 5 × 107 by the optical density of suspension. 0.1 ml of suspended bacteria was injected into the peritoneal cavity of fish. Mortality was recorded during the ten days following infection.
2.11. Statistics Results of the assays, except for the challenge test are presented as average (± standard error) for six fish and were compared at each time point using one way analysis of variance (ANOVA) and Student–Newman–Keuls multiple range tests (SigmaStat 2.3, SPSS, Inc.). Significant differences between experimental groups were expressed at a significance level of P b 0.05. Feeding trials were executed once, without repeats.
3. Results 3.1. Phagocytic activity Feeding the fish with 0.1% of Astragalus extract significantly increased the phagocytic activity of tilapia leukocytes, even after one week (Fig. 1). The enhanced phagocytic activity remained until the
Fig. 1. Phagocytic activity of isolated phagocytic cells in control group and groups fed diets containing different herbal extracts. A: groups fed diets without boron supplementation, B: groups fed diets with boron supplementation. Data is expressed as the mean of six fish ± SEM. Significant differences (P b 0.05) from the control group are indicated by asterisks. plasma, reagent blank or standard solution and 300 µl diluted Biuret solution was added to the wells in triplicates. After 20 min of incubation at room temperature, absorbance was measured with a multiscan spectrophotometer at 550 nm. The protein concentrations of the samples were calculated by the following formula: y = Am / Ast * x, where y is the protein concentration of the sample, Am is the absorbance of the sample, Ast is the absorbance of the standard and x is the standard's known protein concentration.
2.9. Plasma total immunoglobulin assay This assay was similar to the previous one. 50 µl plasma and 50 µl polyethylene glycol (PEG) was added to each well of a 96-well microtiter plate. After 2 h of incubation at room temperature, plates were centrifuged at 1000 G for 15 min. The protein content of the supernatant was determined by the assay described above. This value was subtracted from the total protein level, and the result was equal to the total immunoglobulin concentration of the plasma.
2.10. Challenge tests with A. hydrophila At the end of the experiment, a challenge test was performed on each experimental group with A. hydrophila. B2/12 strain of A. hydrophila, which had originally been isolated in Bangladesh, was used for the test. This strain was chosen because in our preliminary experiments it proved to be the most virulent of our strains (data not shown). A. hydrophila strains were obtained from Bacteriology Unit, Institute of Aquaculture, University of Stirling, Stirling,
Fig. 2. Respiratory burst activity of phagocytic cells isolated from blood of fish in control group and in groups fed different kinds of herbal extracts, without (A) or with (B) boron supplementation. Legends are the same as on Fig. 1.
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end of the experiment. Fish fed diets supplemented with 0.1% Astragalus extract and 75 mg/kg boron showed enhanced phagocytic activity for the first three weeks. Feeding the fish with mixtures supplemented with Lonicera extract, Lonicera extract and boron or both herbs and boron had a weaker effect. A significant increase was measured on the second, second and third, and first week, respectively for these combinations. 3.2. Respiratory burst activity The production of intracellular oxidative radicals showed a statistically significant increase on the fourth week in all treated groups (Fig. 2), compared to the control. In addition, a significant difference was measured on the first week in the group fed feed containing Lonicera extract and on the second week in the group fed feed containing Lonicera and boron. 3.3. Plasma lysozyme activity A significant increasing of plasma lysozyme activity was found in groups receiving feed containing Astragalus, Lonicera, Astragalus and boron or both herbs and boron after one week (Fig. 3). There was a significant enhancement on the third week in all three groups treated with feed containing herbal extracts and boron, but not in the groups fed with herbs alone.
Fig. 4. Changes in plasma total protein levels in control group and in groups fed diets containing different kinds of herbal extracts, without (A) or with (B) boron supplementation. Legends are the same as on Fig. 1.
3.4. Plasma total protein and immunoglobulin levels The similar results were observed in the case of both humoral parameters. A significant increase of plasma total protein level was measured only on the first week and only in the group treated with Astragalus extract and boron. There were no significant changes in plasma total immunoglobulin levels (Figs. 4 and 5). 3.5. Challenge test with A. hydrophila After four weeks of feeding fish were challenged with A. hydrophila and cumulative mortality was registered during one week (Fig. 6). All five treated groups showed reduced mortality compared to the control. Feeds containing Astragalus reduced the mortality by 5% (Astragalus only) and 35% (Astragalus and boron). Feeds with Lonicera were more effective. Lower mortalities were observed in groups fed with Lonicera and boron and Lonicera only. Feed supplemented with both herbs and boron was the most effective with the mortality of the fish reduced by 55%. All these data are in comparison to the control group.
4. Discussion Fig. 3. Changes in plasma lysozyme activities in control group and in groups fed diets containing different kinds of herbal extracts, without (A) or with (B) boron supplementation. Legends are the same as on Fig. 1.
The immunostimulating effect of Astragalus and Lonicera extracts and boron supplementation were investigated in this
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Fig. 5. Changes in plasma total immunoglobulin levels in control group and in groups fed different kinds of herbal extracts without (A) or with (B) boron supplementation. Legends are the same as on Fig. 1.
study. Both herbal extracts were able to enhance the nonspecific immune response of Nile tilapia. Phagocytic cells are the most important cellular components of the innate immune system of fish (MacArthur and Fletcher, 1985). Their phagocytic activity is a primitive defence mechanism (Neumann et al., 2001) and an important characteristic of the non-specific immune system (Seeley et al., 1990). This parameter usually shows an increase after oral administration of immunostimulants (Ainsworth, 1994; Siwicki et al., 1994; Jeney et al., 1997). Herbal medicine extracts can also enhance phagocytosis in various fish species (Dügenci et al., 2003; Chen et al., 2003) as well as probiotics (Salinas et al., 2005; Kim and Austin, 2006), which are microbial cell preparations that have a beneficial effect on the health and well-being of the host (reviewed by Fuller, 1989). In our experiments, Astragalus extract significantly enhanced the phagocytic activity of leukocytes isolated from Nile tilapia, even one week after the start of feeding. This elevated activity was maintained during the entire experiment (in the group fed with feed containing Astragalus extract only) or three weeks (group fed with feed containing Astragalus extract and boron). The main active component of Astragalus extract is a
polysaccharide. This Astragalus polysaccharide (APS) is a well-studied immunostimulant (Tan and Vanitha, 2004). In murine macrophage-like cells it could enhance the expression of cytokine genes, e.g. IL-1, IL-6 or TNF-α (Song et al., 2000), the nitric oxide production of these cells and the expression of the inducible nitric oxide synthase (iNOS) gene (Lee et al., 2005). The Lonicera extract had a less marked effect on the phagocytic activities in our experiment. Lonicera flower extract contain many different active components. One of them, chlorogenic acid could activate macrophages through the calcineurin pathway in human cell line (Wu et al., 2004). Based on this observation we considered that chlorogenic acid could act as an in vivo activator of macrophages. The determination of the correct dose requires more in vivo experiments. Phagocytes also produce toxic oxygen forms during a process called respiratory burst (Neumann et al., 2001). This activity can be measured photometrically by detecting the amount of superoxide (O2− ) anion. We used the nitroblue tetrazolium (NBT) assay, which measures the amount of intracellular superoxide anions (Siwicki and Studnicka, 1987). Feeding of yeast glucan could enhance intracellular superoxide production of large yellow croaker macrophages (Ai et al., 2007). Probiotics increased this parameter in rainbow trout (Kim and Austin, 2006), but not in gilthead seabream (Sparus aurata) (Salinas et al., 2005). A. hydrophila bacterins significantly enhanced the intracellular respiratory burst activity in leukocytes of Indian major carps (C. catla, Labeo rohita and Cirrhinus mrigala) (John et al., 2002). Herbal medicine extracts can also increase the intracellular respiratory burst activity of fish phagocytic cells. This effect was observed in large yellow croaker and common carp after feeding them with a diet containing a mixture of A. membranaceus and A. sinensis extracts (Jian and Wu,
Fig. 6. Cumulative mortalities (%) of fish in control group and in groups fed diets containing different kinds of herbal extracts with or without boron supplementation. The figure shows a six-day period following an artificial infection with Aeromonas hydrophila. Twenty fish from each group were used for the challenge test.
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2003, 2004). We observed a significant increase in the respiratory burst activities in all treated groups on the fourth week after start of feeding, and a significant increase was measured on the first and second weeks in two different groups, therefore both Astragalus and Lonicera extracts had a positive effect on the intracellular superoxide anion production of isolated tilapia leukocytes. The innate immune system has also humoral elements: the complement system, lysozyme, transferrin, agglutinins and precipitins (reviewed by Magnadóttir, 2006). It was observed several times that immunostimulants, vaccines and probiotics can enhance the plasma lysozyme activity (Siwicki et al., 1994; Hanif et al., 2005; Kim and Austin, 2006). Elevated lysozyme level was measured in crucian carp (Chen et al., 2003), large yellow croaker (Jian and Wu, 2003) and common carp (Jian and Wu, 2004) after feeding the fish with various Chinese herbal extracts. In our experiment, a significantly elevated activity was measured on the first week after start of feeding in four treated groups and a significant increase was measured on the third week in the groups fed with diets containing herbal extract and boron. Plasma proteins include the humoral elements of the nonspecific immune system, e.g. immunoglobulins, transferrin, agglutinins or precipitins (Magnadóttir, 2006). In our experiment, total protein and total immunoglobulin levels of plasma were measured, but neither of these humoral parameters was affected by herbal medicine extracts. Immunostimulants can have a contradictory effect on these parameters. A decrease of total protein level in Russian sturgeon (Acipenser güldenstadti) plasma was measured after treatment with glucan, chitosan and finnstim (Kolman et al., 1998). Treatment with epin and glucan significantly increased plasma immunoglobulin levels in Siberian sturgeon (Acipenser baeri), whereas application of chitosan and finnstim decreased this parameter in Russian sturgeon (Kolman et al., 1998; Kolman, 2001). In rainbow trout, plasma total protein level was significantly increased after feeding the fish with various herbal extracts (Dügenci et al., 2003). After challenge with A. hydrophila, all treated groups showed a reduced mortality compared to the control group. The best survival rate was observed in the group treated with both herbs and boron. Among the other groups, fish treated with Lonicera extract had a lower mortality than the fish treated with Astragalus. Boron supplementation reduced the mortality even more in the group treated with Astragalus. It had some negative effect on survival in the group treated with Lonicera, however, the differences between the two groups (with or without boron supplementation) was not big. Survival rates of infected fish are usually increased after treatment with various immunostimulants (Anderson, 1992; Sakai, 1999), vaccines (Gudmundsdóttir and Magnadóttir, 1997; Bakopoulos et al., 2003) and probiotics (Brunt et al., 2007). Feeding carp with chitosan and levamisole reduced mortality of common carp after challenge with A. hydrophila (Gopalakkanan and Arul, 2006). A similar result was reported after feeding large yellow croaker with glucan and challenging with Vibrio harveyi (Ai et al., 2007). Results of our study showed that both Astragalus and Lonicera extracts alone or in combination could significantly
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enhance respiratory burst and phagocytic activity of blood phagocytes and plasma lysozyme activity. Herbal extracts had no effect on plasma total protein or total immunoglobulin levels. Both herbs alone or in combination enhanced the survival rate after challenge with A. hydrophila. Survival rates could be even more enhanced by supplementing fish feed with boron. Thus, it can be concluded that both herbal extracts and boron can be used as immunostimulants to enhance immune response and disease resistance of cultured fish species. Acknowledgements This research was supported by the Bilateral Science and Technology Cooperation between China and Hungary (Project number CHN-13/04) and by the Hungarian National Office for Research and Technology (Project number OMFB-00220/ 2005). References Ai, Q., Mai, K., Zhang, L., Tan, B., Zhang, W., Xu, W., Li, H., 2007. Effects of dietary β-1,3-glucan on innate immune response of large yellow croaker, Pseudosciena crocea. Fish & Shellfish Immunology 22, 394–402. Ainsworth, A.J., 1994. A beta-glucan inhibitable zymosan receptor on channel catfish neutrophils. Veterinary Immunology and Immunopathology 41, 141–152. Anderson, D.P., 1992. Immunostimulants, adjuvants and vaccine carriers in fish: applications to aquaculture. Annual Review of Fish Diseases 2, 281–307. Bakopoulos, V., Volpatti, D., Gusmani, L., Galeotti, M., Adams, A., Dimitriadis, G.J., 2003. Vaccination trials of sea bass, Dicentrarchus labrax (L.) against Photobacterium damsela subsp. piscicida using novel vaccine mixtures. Journal of Fish Diseases 26/2, 77. Boon, A.C., Vos, A.P., Graus, Y.M., Rimmelzwaan, G.F., Osterhaus, A.D., 2002. In vitro effect of bioactive compounds on influenza virus specific B- and T-cell responses. Scandinavian Journal of Immunology 55 (1), 24–32. Brunt, J., Newaj-Fizul, A., Austin, B., 2007. The development of probiotics for the control of multiple bacterial diseases of rainbow trout, Oncorhynchus mykiss (Walbaum). Journal of Fish Diseases 30 (10), 573–579. Cao, L.Z., Lin, Z.B., 2003. Regulatory effect of Ganoderma lucidum polysaccharides on cytotoxic T-lymphocytes induced by dendritic cells in vitro. Acta Pharmacologica Sinica 24 (4), 312–326. Chen, X., Wu, Z., Yin, J., Li, L., 2003. Effects of four species of herbs on immune function of Carassius auratus gibelio. Journal of Fish Sciences of China 10, 36–40 (China). Dey, R.K., Chandra, S., 1995. Preliminary studies to raise disease resistant seed (fry) of Indian major carp Catla catla (Ham.) through herbal treatment of spawn. Fish Chimes 14, 23–25. Dügenci, S.K., Arda, N., Candan, A., 2003. Some medicinal plants as immunostimulant for fish. Journal of Ethnopharmacology 88, 99–106. Engstad, R.E., Robertsen, B., Frivold, E., 1992. Yeast glucan induces increase in activity of lysozyme and complement-mediated haemolytic activity in Atlantic salmon blood. Fish & Shellfish Immunology 2, 287–297. Fuller, R., 1989. Probiotics in man and animals. Journal of Applied Bacteriology 66, 365–378. Galeotti, M., 1998. Some aspects of the application of immunostimulants and a critical review of methods for their evaluation. Journal of Applied Ichtiology 14, 189–199. Goetz, F.W., Iliev, D.B., McCauley, L.A.R., Liarte, C.Q., Tort, L.B., Planas, J.V., MacKenzie, S., 2004. Analysis of genes isolated from lipopolysaccharidestimulated rainbow trout (Oncorhynchus mykiss) macrophages. Molecular Immunology 41, 1199–1210.
32
L. Ardó et al. / Aquaculture 275 (2008) 26–33
Gopalakkanan, A., Arul, V., 2006. Immunomodulatory effects of dietary intake of chitin, chitosan and levamisole on the immune system of Cyprinus carpio and control of Aeromonas hydrophila infection in ponds. Aquaculture 225, 179–187. Gudmundsdóttir, B.K., Magnadóttir, B., 1997. Protection of Atlantic salmon (Salmo salar L.) against an experimental infection of Aeromonas salmonicida ssp. achromogenes. Fish & Shellfish Immunology 7, 55–69. Hanif, A., Bakopoulos, V., Leonardos, I., Dimitriadis, G.J., 2005. The effect of sea bream (Sparus aurata) broodstock and larval vaccination on the susceptibility by Photobacterium damsela subsp. piscicida and on the humoral immune parameters. Fish & Shellfish Immunology 19, 345–361. Hardie, L.J., Fletcher, T.C., Secombes, C.J., 1991. The effect of dietary Vitamin C on the immune response of Atlantic salmon (Salmo salar). Aquaculture 95, 201–214. Huttenhuis, H.B.T., Ribeiro, A.S.P., Bowden, T.J., Van Bavel, C., TaverneThiele, A.J., Rombout, J.H.W.M., 2006. The effect of oral immunostimulation in juvenile carp (Cyprinus carpio L.). Fish & Shellfish Immunology 21, 261–271. Jeney, G., Anderson, D.P., 1993. Enhanced immune response and protection in rainbow trout to Aeromonas salmonicida bacterin following prior immersion in immunostimulants. Fish and Shellfish Immunology 3, 51–58. Jeney, G., Galeotti, M., Volpatti, D., Jeney, Z., Anderson, D.P., 1997. Prevention of stress in rainbow trout (Oncorhynchus mykiss) fed diets containing different doses of glucan. Aquaculture 154, 1–15. Jian, J., Wu, Z., 2003. Effects of traditional Chinese medicine on nonspecific immunity and disease resistance of large yellow croaker, Pseudosciaena crocea (Richardson). Aquaculture 218, 1–9. Jian, J., Wu, Z., 2004. Influences of traditional Chinese medicine on nonspecific immunity of Jian carp (Cyprinus carpio var. Jian). Fish & Shellfish Immunology 16, 185–191. John, M.B., Chandran, M.R., Aruna, B.V., Anbarasu, K., 2002. Production of superoxide anion by head-kidney leukocytes of Indian major carps immunised with bacterins of Aeromonas hydrophila. Fish & Shellfish Immunology 12 (3), 201–207. Kajita, Y., Sakai, M., Atsuta, S., Kobayashi, M., 1990. The immunomodulatory effect of levamisole on rainbow trout, Oncorhynchus mykiss. Fish pathology 25, 93–98. Kim, D., Austin, B., 2006. Innate immune responses in rainbow trout (Oncorhynchus mykiss, Walbaum) induced by probiotics. Fish & Shellfish Immunology 21, 513–524. Koethe, S.M., Nelson, K.E., Becker, C.G., 1995. Activation of the classical pathway of complement by tobacco glycoprotein. Journal of Immunology 155 (2), 826–835. Kolman, H., 2001. The humoral effects of epin in Siberian sturgeon (Acipenser baeri Brandt). Archives of Polish Fisheries 9 (1), 61–69 (Poland). Kolman, R., Kolman, H., Siwicki, R., 1998. The effect of some immunomodulators on the growth rate of sturgeon fry (Acipenseridae). Archives of Polish Fisheries 6 (2), 383–390 (Poland). Kumar, N., Singh, B., Bhandari, P., Gupta, A.P., Uniyal, S.K., Kaul, V.K., 2005. Biflavonoids from Lonicera japonica. Phytochemistry 66, 2740–2744. Lee, J.H., Ko, W.S., Kim, Y.H., Kang, H.S., Kim, H.D., Choi, B.T., 2001. Antiinflammatory effect of the aquaeous extract from Lonicera japonica flower is related to inhibition of NF-kappaB activation through reducing IkappaBalpha degradation in rat liver. International Journal of Molecular Medicine 2001. 7 (1), 79–83. Lee, Y.S., Han, O.K., Park, C.W., Suh, S.I., Shin, S.W., Yang, C.H., Yeon, T.W., Lee, E.S., Kim, K.J., Kim, S.H., Yoo, W.K., Kim, H.J., 2003. Immunomodulatory effects of aqueous-extracted Astragali-radix in methothrexate-treated mouse spleen cells. Journal of Ethnopharmacology 84 (2–3), 193–198. Lee, Y.S., Han, O.K., Park, C.W., Yang, C.H., Jeon, T.W., Yoo, W.K., Kim, S.H., Kim, H.J., 2005. Pro-inflammatory gene expression and nitric oxide regulation of aquaeous extracted Astragali radix in RAW 264.7 macrophage cells. Journal of Ethnopharmacology 100 (3), 289–294. Lin, Z.B., Zhang, H.N., 2004. Anti-tumor and immunoregulatory activities of Ganoderma lucidum and its possible mechanisms. Acta Pharmacologica Sinica 25 (11), 1387–1395.
Liu, C., Tsai, W., Lin, Y., Liao, J., Chen, C., Kuo, Y., 2004. The extracts from Nelumbo nucifera suppress cell cycle progression, cytokine genes expression, and cell proliferation in human peripheral blood mononuclear cells. Life Sciences 75, 699–716. Logambal, S.M., Michael, R.D., 2000. Immunostimulatory effect of azadirachtin in Oreochromis mossambicus (Peters). Indian Journal of Experimental Biology 38, 1092–1096. MacArthur, J.I., Fletcher, T.C., 1985. Phagocytosis in fish. In: Manning, M.J., Tatner, M.F. (Eds.), Fish Immunology. Academic Press, London, pp. 29–46. Magnadóttir, B., 2006. Innate immunity of fish (overview). Fish and Shellfish Immunology 20, 137–151. Neumann, N.F., Stafford, J.L., Barreda, D., Ainsworth, A.J., Belosevic, M., 2001. Antimicrobial mechanisms of fish phagocytes and their role in host defence. Developmental and Comparative Immunology 25, 807–825. Sakai, M., 1999. Current research status of fish immunostimulants. Aquaculture 172, 63–92. Salinas, I., Cuesta, A., Esteban, M.A., Meseguer, J., 2005. Dietary administration of Lactobacillus delbrüecki and Bacillus subtilis, single or combined, on gilthead seabream cellular innate immune responses. Fish & Shellfish Immunology 19, 67–77. Sankaran, K., Gurnani, S., 1972. On the variation in the catalytic activity of lysozyme in fishes. Indian Journal Biochemistry and Biophysics 9, 62–165. Secombes, C.J., 1990. Isolation of salmonid macrophages and analysis of their killing activity. In: Stolen, J.S., Anderson, D.P., Robertson, B.S., van Miuswinkell, W.B. (Eds.), Techniques in Fish Immunology. SOS Publications, Fair Haven, pp. 137–154. Secombes, C.J., Fletcher, T.C., 1992. The role of phagocytes in the protective mechanisms of fish. Annual Review of Fish Diseases 2, 58–71. Seeley, K.R., Gillespie, P.D., Weeks, B.A., 1990. A simple technique for the rapid spectrophotometric determination of phagocytosis by fish macrophages. Marine Environmental Research 30, 123–128. Shan, B.E., Yoshida, Y., Sugiura, T., Yamashita, U., 1999. Stimulating activity of Chinese medicinal herbs on human lymphocytes in vitro. International Journal of Immunopharmacology 21, 149–159. Shao, B., Xu, W., Dai, H., Tu, P., Li, Z., Gao, X., 2004. A study on the immune receptors for polysaccharides from the roots of Astragalus membranaceus, a Chinese medicinal herb. Biochemical and Biophysical Research Communications 320, 1103–1111. Siwicki, A.K., 1987. Immunomodulating activity of levamisole in spawners carp (Cyprinus carpio L.). Journal of Fish biology 31, 245–246. Siwicki, A.K., 1989. Immunomodulating influence of levamisole on nonspecific immunity of carp (Cyprinus carpio). Developmental and Comparative Immunology 14, 231–237. Siwicki, A., Studnicka, M., 1987. The phagocytic ability of neutrophils and serum lysozyme activity in experimentally infected carp, Cyprinus carpio L. Journal of Fish Biology 31 (Suppl. A), 57–60. Siwicki, A.K., Anderson, D.P., Rumsey, G.L., 1994. Dietary intake of immunostimulants by rainbow trout affects non-specific immunity and protection against furunculosis. Veterinary Immunology and Immunopathology 41, 125–139. Song, Q.H., Kobayashi, T., Xiu, L.M., Tie, H., Cyong, J.C., 2000. Effects of Astragali root and Hedysari root on the murine B and T cell differentiation. Journal of Ethnopharmacology 73 (1–2), 111–119. Tan, B.K.H., Vanitha, J., 2004. Immunomodulatory and antimicrobial effect of some traditional Chinese medicinal herbs. Current Medical Chemistry 11, 1423–1430. Thompson, I., White, A., Fletcher, T.C., Houlihan, D.F., Secombes, C.J., 1993. The effect of stress on the immune responses of Atlantic salmon (Salmo salar L.) fed diets containing different amounts of vitamin-C. Aquaculture 114, 1–18. Thompson, K.D., Cachos, A., Inglis, V., 1995. Immunostimulating effect of glucans and oxytetracycline in rainbow trout, Oncorhynchus mykiss, on serum lysozyme and protection. In: Shariff, M., Subashige, R.P., Arthur, J.R. (Eds.), Fish Health Section. Diseases in Asian Aquaculture, vol 11. Asian Fisheries Society, Manila, Philippines, pp. 433–439.
L. Ardó et al. / Aquaculture 275 (2008) 26–33 Wang, R., Li, D., Bourne, S., 1999. Can 2000 years of herbal medicine history help us to solve problems in the year 2000? Biotechnology in the feed industry. Proceedings of Alltech's 14th Annual Symposium, pp. 273–291. Watanabe, T., Kiron, W., Satoh, S., 1997. Trace minerals in fish nutrition. Aquaculture 151, 185–207.
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Wu, H., Luo, J., Yin, Y., Wei, Q., 2004. Effects of chlorogenic acid, an active compound activating calcineurin, purified from Flos Lonicerae on macrophage. Acta Pharmacologica Sinica 12, 1685–1689. Yin, G., Jeney, G., Rácz, T., Pao, X., Jeney, Z., 2006. Effect of two Chinese herbs (Astragalus radix and Scutellaria radix) on non–specific immune response of tilapia, Oreochromis niloticus. Aquaculture 253, 39–47.
Aquaculture 275 (2008) 26 – 33 www.elsevier.com/locate/aqua-online
Chinese herbs (Astragalus membranaceus and Lonicera japonica) and boron enhance the non-specific immune response of Nile tilapia (Oreochromis niloticus) and resistance against Aeromonas hydrophila László Ardó a , Guojun Yin b , Pao Xu b , László Váradi a , Gábor Szigeti c , Zsigmond Jeney a , Galina Jeney a,⁎ b
a Research Institute for Fisheries, Aquaculture and Irrigation, Anna liget 8, H-5540, Szarvas, Hungary Freshwater Fisheries Research Center of Chinese Academy of Fishery Sciences, Wuxi, 214081, PR China c Central Veterinarian Institute, Tábornok u. 2, Budapest XIV, Hungary
Received 30 August 2007; received in revised form 13 December 2007; accepted 19 December 2007
Abstract The effect of two Chinese medicinal herbs (Astragalus membranaceus and Lonicera japonica) and boron on non-specific immune response of Nile tilapia (Oreochromis niloticus) was investigated. Five diet variations in addition to a control diet (without herbs or boron) were used. These contained 0,1% Astragalus (with and without 0,05% boron), 0,1% Lonicera (with or without 0,05% boron) and a mixture of the two herbs with 0,05% boron. The diets were fed for four weeks, and respiratory burst and phagocytic activities of blood leukocytes; plasma lysozyme, total protein and total immunoglobulin level were measured weekly. After four weeks of feeding, fish were infected with Aeromonas hydrophila and mortalities were recorded. Results of this study showed that feeding tilapia with two herbs alone or in combination significantly enhanced phagocytic and respiratory burst activity of blood phagocytic cells. They had a moderate effect on the plasma lysozyme level and no effect on plasma total protein and total immunoglobulin level. Both herbs reduced the mortality following A. hydrophila infection. The lowest mortality was observed in the group fed with the combination of both herbs and boron. Combination of the herbs and boron could even more increase the survival rate of infected fish. It can be concluded that Astragalus and Lonicera extracts and boron supplementation added to fish feed can act as immunostimulants and enhance the immune response and disease resistance of cultured fish. © 2007 Elsevier B.V. All rights reserved. Keywords: Astragalus membranaceus; Lonicera japonica; Boron; Oreochromis niloticus; Non-specific immunity; Disease resistance; Aeromonas hydrophila
1. Introduction Enhancement of the immune system seems to be the most promising method of preventing fish diseases. This modulation can be achieved with vaccines, which enhance the acquired (or specific) immune response of the fish and are considered to be the most effective agents, but a single vaccine is effective against only one type of pathogens, and in the case of many pathogens (like the bacterium Aeromonas hydrophila) no effective vaccine is available due to the complex antigenic structure.
⁎ Corresponding author. Tel.: +36 66 515317; fax: +36 66 312142. E-mail address: [email protected] (L. Ardó). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.12.022
In contrast to vaccines, immunostimulants enhance the innate (or non-specific) immune response (Galeotti, 1998; Sakai, 1999). The major components of the innate immune system are macrophages, monocytes, granulocytes and humoral elements, like lysozyme or complement system (Secombes and Fletcher, 1992; Magnadóttir, 2006). Immunostimulants can be applied via injection, bathing or oral administration, the latter seems to be the most practicable (Jeney and Anderson, 1993; Sakai, 1999; Yin et al., 2006). Many types of biological and synthetic compounds have been shown to enhance non-specific immune system of cultivated fish (Sakai, 1999). Best-known immunostimulants are components of bacterial cell wall, like lipopolysaccharide (LPS) (Goetz et al., 2004) or glucans (Engstad et al., 1992), but synthetic compounds, polysaccharides, animal and plant extracts or
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vitamins can enhance the non-specific immune response of fish (Siwicki, 1987, 1989; Hardie et al., 1991; Thompson, I. et al., 1993; Thompson, K.D. et al., 1995). However, the effect is dosage-dependent and there is a chance to overdose (Kajita et al., 1990) neither are they always effective in early developmental stages of fish (Huttenhuis et al., 2006). In traditional Chinese human medicine, herbs have been used as immunostimulants for thousands of years (Tan and Vanitha, 2004). These herbs contain many types of active components, like polysaccharides, alkaloids or flavonoids. The immunostimulating activity of herbal components have been most widely studied in mice, chickens or human cell lines (Shan et al., 1999; Cao and Lin, 2003; Lin and Zhang, 2004), and in the case of some herbs (e.g. Astragalus membranaceus or Nelumbo nucifera) even the molecular mechanisms of their effect is known (Shao et al., 2004; Liu et al., 2004). There is a growing interest in using medicinal herbs as immunostimulants in aquaculture. In common carp (Cyprinus carpio) and large yellow croaker (Pseudosciena crocea) respiratory burst activity of phagocytic cells and plasma lysozyme activity has been significantly increased after feeding with a ration containing a mixture of A. membranaceus and Angelica sinensis (Jian and Wu, 2003, 2004). Similar results have been reported about experiments on various fish species, e.g. rainbow trout (Oncorhynchus mykiss), Indian major carp (Catla catla) and Mozambican tilapia (Oreochromis mossambicus) (Dügenci et al., 2003; Dey and Chandra, 1995; Logambal and Michael, 2000). Trace elements or micronutrients like selenium, boron or zinc are essential for growth, development and health of the fish (Watanabe et al., 1997). In our previous experiments we found that boron can enhance the immunomodulatory effect of some medicinal herbs (G. Jeney, unpublished results). In this study, two Chinese medicinal herbs, A. membranaceus and Lonicera japonica were chosen for the experiments. Various extracts of Astragalus root have been used as immunostimulants in traditional Chinese human medicine (Wang et al., 1999) and there are many reports supporting its immunomodulating effects (Song et al., 2000; Lee et al., 2003). L. japonica is used as traditional human medicine to treat fever and headache (Kumar et al., 2005) and as an anti-inflammatory agent (Lee et al., 2001). However, one of its active components, chlorogenic acid has a multiple immunomodulatory effect. (Koethe et al., 1995; Boon et al., 2002; Wu et al., 2004). In the study reported here, Nile tilapia were fed with fish feed containing Astragalus or Lonicera extracts or their combination and with or without boron supplementation. Key factors of the non-specific immune response were measured during the experiment and at the end fish were challenged with A. hydrophila to determine disease resistance in fish fed with these substances.
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2.2. Herbal extracts Powdered Astragalus extract containing 90% of Astragalus polysaccharide and powdered Lonicera extract containing 25% of chlorogenic acid were commercial products of Nantong Sihai Plant Extracts Co., Ltd., China.
2.3. Experimental design Experiments were performed in the recirculation system of the HAKI, Szarvas, Hungary. Fish were kept in fibreglass tanks with a constant water temperature and pH (22–23 °C; pH 8.5). Dissolved oxygen was maintained at 80–90% of saturation, and water flow was 7 l/min during the experimental period. Fish were allocated into 6 groups (50 fish/group) and fed diets containing different medicinal herbs and boron. The compositions of the feeds were the following: Group 1: 0.1% Astragalus extract Group 2: 0.1% Astragalus extract and 0.05% boron Group 3: 0.1% Lonicera extract Group 4: 0.1% Lonicera extract and 0.05% boron Group 5: 0.1% Astragalus extract, 0.1% Lonicera extract and 0.05% boron Group 6: Control (without medicinal herbs or boron) Cornmeal oil (10 ml/kg feed) was used to bind the powdered herbal extracts and boron to the fish feed. The prepared feed was maintained at room temperature.
2.4. Blood sampling and separation of leukocytes Blood samples (6 fish/group) were collected from the caudal vein 1, 2, 3 and 4 weeks after start of feeding. Heparin was used as an anticoagulant. Individual fish were sampled only once to avoid the influence on the assays due to multiple bleeding and handling stress on the fish. Leukocytes for assay were separated from each blood sample by densitygradient centrifugation. One ml of histopaque 1.119 (Sigma) containing 100 µl of Bacto haemagglutination buffer, pH 7.3 (Difco, USA) was dispensed into siliconised tubes. One ml of a mixture of histopaque 1.077 and haemagglutination buffer was layered on the top. One ml of blood sample was then layered carefully on the top of the gradient. Sample preparations were centrifuged at 700 G for 30 min at 4 °C. After centrifugation, plasma was collected and stored at −20 °C for future analysis. Separated leukocytes were gently removed and dispensed into siliconised tubes, containing phenol red free Hank's Balanced Salt Solution (HBSS). Cells were then washed in HBSS and adjusted to 107 viable cells/ml.
2.5. Respiratory burst activity Respiratory burst activity of isolated leukocytes was quantified by the nitroblue tetrazolium (NBT) assay (Secombes, 1990), which measures the quantity of intracellular oxidative free radicals. This method was slightly changed, the concentration of NBT solution was 2 mg/ml.
2.6. Phagocytosis assay Phagocytosis activity of blood leukocytes was determined spectrophotometrically by the method of Seeley et al. (1990).
2.7. Lysozyme assay 2. Materials and methods 2.1. Fish Tilapia (O. niloticus) propagated and reared by the Research Institute for Fisheries, Aquaculture and Irrigation (HAKI, Szarvas, Hungary) were used for the experiment. Fish were fed with a dry feed, produced in the experimental milling facility of the institute and kept in 2000 l fibreglass tanks in the recirculation system of the institute at water temperature of 22–23 °C.
Plasma lysozyme activity was measured spectrophotometrically by the method of Sankaran and Gurnani (1972).
2.8. Plasma total protein assay The total protein concentration of the plasma was determined by a colorimetric assay based on the Biuret reaction, using a protein diagnostic reagent kit (Reanal, Hungary). The assay was performed in 96-well microtiter plates. 10 µl
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L. Ardó et al. / Aquaculture 275 (2008) 26–33 United Kingdom. Bacteria were inoculated into 10 ml of liquid tryptic soy broth (TSB, Sigma) medium and were grown overnight at 28 °C. Cultures were centrifuged at 850 G for 15 min. Supernatant was removed and the pelleted bacteria were washed twice in sterile phosphate buffered saline (PBS) solution. The concentration of bacteria was adjusted to 5 × 107 by the optical density of suspension. 0.1 ml of suspended bacteria was injected into the peritoneal cavity of fish. Mortality was recorded during the ten days following infection.
2.11. Statistics Results of the assays, except for the challenge test are presented as average (± standard error) for six fish and were compared at each time point using one way analysis of variance (ANOVA) and Student–Newman–Keuls multiple range tests (SigmaStat 2.3, SPSS, Inc.). Significant differences between experimental groups were expressed at a significance level of P b 0.05. Feeding trials were executed once, without repeats.
3. Results 3.1. Phagocytic activity Feeding the fish with 0.1% of Astragalus extract significantly increased the phagocytic activity of tilapia leukocytes, even after one week (Fig. 1). The enhanced phagocytic activity remained until the
Fig. 1. Phagocytic activity of isolated phagocytic cells in control group and groups fed diets containing different herbal extracts. A: groups fed diets without boron supplementation, B: groups fed diets with boron supplementation. Data is expressed as the mean of six fish ± SEM. Significant differences (P b 0.05) from the control group are indicated by asterisks. plasma, reagent blank or standard solution and 300 µl diluted Biuret solution was added to the wells in triplicates. After 20 min of incubation at room temperature, absorbance was measured with a multiscan spectrophotometer at 550 nm. The protein concentrations of the samples were calculated by the following formula: y = Am / Ast * x, where y is the protein concentration of the sample, Am is the absorbance of the sample, Ast is the absorbance of the standard and x is the standard's known protein concentration.
2.9. Plasma total immunoglobulin assay This assay was similar to the previous one. 50 µl plasma and 50 µl polyethylene glycol (PEG) was added to each well of a 96-well microtiter plate. After 2 h of incubation at room temperature, plates were centrifuged at 1000 G for 15 min. The protein content of the supernatant was determined by the assay described above. This value was subtracted from the total protein level, and the result was equal to the total immunoglobulin concentration of the plasma.
2.10. Challenge tests with A. hydrophila At the end of the experiment, a challenge test was performed on each experimental group with A. hydrophila. B2/12 strain of A. hydrophila, which had originally been isolated in Bangladesh, was used for the test. This strain was chosen because in our preliminary experiments it proved to be the most virulent of our strains (data not shown). A. hydrophila strains were obtained from Bacteriology Unit, Institute of Aquaculture, University of Stirling, Stirling,
Fig. 2. Respiratory burst activity of phagocytic cells isolated from blood of fish in control group and in groups fed different kinds of herbal extracts, without (A) or with (B) boron supplementation. Legends are the same as on Fig. 1.
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end of the experiment. Fish fed diets supplemented with 0.1% Astragalus extract and 75 mg/kg boron showed enhanced phagocytic activity for the first three weeks. Feeding the fish with mixtures supplemented with Lonicera extract, Lonicera extract and boron or both herbs and boron had a weaker effect. A significant increase was measured on the second, second and third, and first week, respectively for these combinations. 3.2. Respiratory burst activity The production of intracellular oxidative radicals showed a statistically significant increase on the fourth week in all treated groups (Fig. 2), compared to the control. In addition, a significant difference was measured on the first week in the group fed feed containing Lonicera extract and on the second week in the group fed feed containing Lonicera and boron. 3.3. Plasma lysozyme activity A significant increasing of plasma lysozyme activity was found in groups receiving feed containing Astragalus, Lonicera, Astragalus and boron or both herbs and boron after one week (Fig. 3). There was a significant enhancement on the third week in all three groups treated with feed containing herbal extracts and boron, but not in the groups fed with herbs alone.
Fig. 4. Changes in plasma total protein levels in control group and in groups fed diets containing different kinds of herbal extracts, without (A) or with (B) boron supplementation. Legends are the same as on Fig. 1.
3.4. Plasma total protein and immunoglobulin levels The similar results were observed in the case of both humoral parameters. A significant increase of plasma total protein level was measured only on the first week and only in the group treated with Astragalus extract and boron. There were no significant changes in plasma total immunoglobulin levels (Figs. 4 and 5). 3.5. Challenge test with A. hydrophila After four weeks of feeding fish were challenged with A. hydrophila and cumulative mortality was registered during one week (Fig. 6). All five treated groups showed reduced mortality compared to the control. Feeds containing Astragalus reduced the mortality by 5% (Astragalus only) and 35% (Astragalus and boron). Feeds with Lonicera were more effective. Lower mortalities were observed in groups fed with Lonicera and boron and Lonicera only. Feed supplemented with both herbs and boron was the most effective with the mortality of the fish reduced by 55%. All these data are in comparison to the control group.
4. Discussion Fig. 3. Changes in plasma lysozyme activities in control group and in groups fed diets containing different kinds of herbal extracts, without (A) or with (B) boron supplementation. Legends are the same as on Fig. 1.
The immunostimulating effect of Astragalus and Lonicera extracts and boron supplementation were investigated in this
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Fig. 5. Changes in plasma total immunoglobulin levels in control group and in groups fed different kinds of herbal extracts without (A) or with (B) boron supplementation. Legends are the same as on Fig. 1.
study. Both herbal extracts were able to enhance the nonspecific immune response of Nile tilapia. Phagocytic cells are the most important cellular components of the innate immune system of fish (MacArthur and Fletcher, 1985). Their phagocytic activity is a primitive defence mechanism (Neumann et al., 2001) and an important characteristic of the non-specific immune system (Seeley et al., 1990). This parameter usually shows an increase after oral administration of immunostimulants (Ainsworth, 1994; Siwicki et al., 1994; Jeney et al., 1997). Herbal medicine extracts can also enhance phagocytosis in various fish species (Dügenci et al., 2003; Chen et al., 2003) as well as probiotics (Salinas et al., 2005; Kim and Austin, 2006), which are microbial cell preparations that have a beneficial effect on the health and well-being of the host (reviewed by Fuller, 1989). In our experiments, Astragalus extract significantly enhanced the phagocytic activity of leukocytes isolated from Nile tilapia, even one week after the start of feeding. This elevated activity was maintained during the entire experiment (in the group fed with feed containing Astragalus extract only) or three weeks (group fed with feed containing Astragalus extract and boron). The main active component of Astragalus extract is a
polysaccharide. This Astragalus polysaccharide (APS) is a well-studied immunostimulant (Tan and Vanitha, 2004). In murine macrophage-like cells it could enhance the expression of cytokine genes, e.g. IL-1, IL-6 or TNF-α (Song et al., 2000), the nitric oxide production of these cells and the expression of the inducible nitric oxide synthase (iNOS) gene (Lee et al., 2005). The Lonicera extract had a less marked effect on the phagocytic activities in our experiment. Lonicera flower extract contain many different active components. One of them, chlorogenic acid could activate macrophages through the calcineurin pathway in human cell line (Wu et al., 2004). Based on this observation we considered that chlorogenic acid could act as an in vivo activator of macrophages. The determination of the correct dose requires more in vivo experiments. Phagocytes also produce toxic oxygen forms during a process called respiratory burst (Neumann et al., 2001). This activity can be measured photometrically by detecting the amount of superoxide (O2− ) anion. We used the nitroblue tetrazolium (NBT) assay, which measures the amount of intracellular superoxide anions (Siwicki and Studnicka, 1987). Feeding of yeast glucan could enhance intracellular superoxide production of large yellow croaker macrophages (Ai et al., 2007). Probiotics increased this parameter in rainbow trout (Kim and Austin, 2006), but not in gilthead seabream (Sparus aurata) (Salinas et al., 2005). A. hydrophila bacterins significantly enhanced the intracellular respiratory burst activity in leukocytes of Indian major carps (C. catla, Labeo rohita and Cirrhinus mrigala) (John et al., 2002). Herbal medicine extracts can also increase the intracellular respiratory burst activity of fish phagocytic cells. This effect was observed in large yellow croaker and common carp after feeding them with a diet containing a mixture of A. membranaceus and A. sinensis extracts (Jian and Wu,
Fig. 6. Cumulative mortalities (%) of fish in control group and in groups fed diets containing different kinds of herbal extracts with or without boron supplementation. The figure shows a six-day period following an artificial infection with Aeromonas hydrophila. Twenty fish from each group were used for the challenge test.
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2003, 2004). We observed a significant increase in the respiratory burst activities in all treated groups on the fourth week after start of feeding, and a significant increase was measured on the first and second weeks in two different groups, therefore both Astragalus and Lonicera extracts had a positive effect on the intracellular superoxide anion production of isolated tilapia leukocytes. The innate immune system has also humoral elements: the complement system, lysozyme, transferrin, agglutinins and precipitins (reviewed by Magnadóttir, 2006). It was observed several times that immunostimulants, vaccines and probiotics can enhance the plasma lysozyme activity (Siwicki et al., 1994; Hanif et al., 2005; Kim and Austin, 2006). Elevated lysozyme level was measured in crucian carp (Chen et al., 2003), large yellow croaker (Jian and Wu, 2003) and common carp (Jian and Wu, 2004) after feeding the fish with various Chinese herbal extracts. In our experiment, a significantly elevated activity was measured on the first week after start of feeding in four treated groups and a significant increase was measured on the third week in the groups fed with diets containing herbal extract and boron. Plasma proteins include the humoral elements of the nonspecific immune system, e.g. immunoglobulins, transferrin, agglutinins or precipitins (Magnadóttir, 2006). In our experiment, total protein and total immunoglobulin levels of plasma were measured, but neither of these humoral parameters was affected by herbal medicine extracts. Immunostimulants can have a contradictory effect on these parameters. A decrease of total protein level in Russian sturgeon (Acipenser güldenstadti) plasma was measured after treatment with glucan, chitosan and finnstim (Kolman et al., 1998). Treatment with epin and glucan significantly increased plasma immunoglobulin levels in Siberian sturgeon (Acipenser baeri), whereas application of chitosan and finnstim decreased this parameter in Russian sturgeon (Kolman et al., 1998; Kolman, 2001). In rainbow trout, plasma total protein level was significantly increased after feeding the fish with various herbal extracts (Dügenci et al., 2003). After challenge with A. hydrophila, all treated groups showed a reduced mortality compared to the control group. The best survival rate was observed in the group treated with both herbs and boron. Among the other groups, fish treated with Lonicera extract had a lower mortality than the fish treated with Astragalus. Boron supplementation reduced the mortality even more in the group treated with Astragalus. It had some negative effect on survival in the group treated with Lonicera, however, the differences between the two groups (with or without boron supplementation) was not big. Survival rates of infected fish are usually increased after treatment with various immunostimulants (Anderson, 1992; Sakai, 1999), vaccines (Gudmundsdóttir and Magnadóttir, 1997; Bakopoulos et al., 2003) and probiotics (Brunt et al., 2007). Feeding carp with chitosan and levamisole reduced mortality of common carp after challenge with A. hydrophila (Gopalakkanan and Arul, 2006). A similar result was reported after feeding large yellow croaker with glucan and challenging with Vibrio harveyi (Ai et al., 2007). Results of our study showed that both Astragalus and Lonicera extracts alone or in combination could significantly
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enhance respiratory burst and phagocytic activity of blood phagocytes and plasma lysozyme activity. Herbal extracts had no effect on plasma total protein or total immunoglobulin levels. Both herbs alone or in combination enhanced the survival rate after challenge with A. hydrophila. Survival rates could be even more enhanced by supplementing fish feed with boron. Thus, it can be concluded that both herbal extracts and boron can be used as immunostimulants to enhance immune response and disease resistance of cultured fish species. Acknowledgements This research was supported by the Bilateral Science and Technology Cooperation between China and Hungary (Project number CHN-13/04) and by the Hungarian National Office for Research and Technology (Project number OMFB-00220/ 2005). References Ai, Q., Mai, K., Zhang, L., Tan, B., Zhang, W., Xu, W., Li, H., 2007. Effects of dietary β-1,3-glucan on innate immune response of large yellow croaker, Pseudosciena crocea. Fish & Shellfish Immunology 22, 394–402. Ainsworth, A.J., 1994. A beta-glucan inhibitable zymosan receptor on channel catfish neutrophils. Veterinary Immunology and Immunopathology 41, 141–152. Anderson, D.P., 1992. Immunostimulants, adjuvants and vaccine carriers in fish: applications to aquaculture. Annual Review of Fish Diseases 2, 281–307. Bakopoulos, V., Volpatti, D., Gusmani, L., Galeotti, M., Adams, A., Dimitriadis, G.J., 2003. Vaccination trials of sea bass, Dicentrarchus labrax (L.) against Photobacterium damsela subsp. piscicida using novel vaccine mixtures. Journal of Fish Diseases 26/2, 77. Boon, A.C., Vos, A.P., Graus, Y.M., Rimmelzwaan, G.F., Osterhaus, A.D., 2002. In vitro effect of bioactive compounds on influenza virus specific B- and T-cell responses. Scandinavian Journal of Immunology 55 (1), 24–32. Brunt, J., Newaj-Fizul, A., Austin, B., 2007. The development of probiotics for the control of multiple bacterial diseases of rainbow trout, Oncorhynchus mykiss (Walbaum). Journal of Fish Diseases 30 (10), 573–579. Cao, L.Z., Lin, Z.B., 2003. Regulatory effect of Ganoderma lucidum polysaccharides on cytotoxic T-lymphocytes induced by dendritic cells in vitro. Acta Pharmacologica Sinica 24 (4), 312–326. Chen, X., Wu, Z., Yin, J., Li, L., 2003. Effects of four species of herbs on immune function of Carassius auratus gibelio. Journal of Fish Sciences of China 10, 36–40 (China). Dey, R.K., Chandra, S., 1995. Preliminary studies to raise disease resistant seed (fry) of Indian major carp Catla catla (Ham.) through herbal treatment of spawn. Fish Chimes 14, 23–25. Dügenci, S.K., Arda, N., Candan, A., 2003. Some medicinal plants as immunostimulant for fish. Journal of Ethnopharmacology 88, 99–106. Engstad, R.E., Robertsen, B., Frivold, E., 1992. Yeast glucan induces increase in activity of lysozyme and complement-mediated haemolytic activity in Atlantic salmon blood. Fish & Shellfish Immunology 2, 287–297. Fuller, R., 1989. Probiotics in man and animals. Journal of Applied Bacteriology 66, 365–378. Galeotti, M., 1998. Some aspects of the application of immunostimulants and a critical review of methods for their evaluation. Journal of Applied Ichtiology 14, 189–199. Goetz, F.W., Iliev, D.B., McCauley, L.A.R., Liarte, C.Q., Tort, L.B., Planas, J.V., MacKenzie, S., 2004. Analysis of genes isolated from lipopolysaccharidestimulated rainbow trout (Oncorhynchus mykiss) macrophages. Molecular Immunology 41, 1199–1210.
32
L. Ardó et al. / Aquaculture 275 (2008) 26–33
Gopalakkanan, A., Arul, V., 2006. Immunomodulatory effects of dietary intake of chitin, chitosan and levamisole on the immune system of Cyprinus carpio and control of Aeromonas hydrophila infection in ponds. Aquaculture 225, 179–187. Gudmundsdóttir, B.K., Magnadóttir, B., 1997. Protection of Atlantic salmon (Salmo salar L.) against an experimental infection of Aeromonas salmonicida ssp. achromogenes. Fish & Shellfish Immunology 7, 55–69. Hanif, A., Bakopoulos, V., Leonardos, I., Dimitriadis, G.J., 2005. The effect of sea bream (Sparus aurata) broodstock and larval vaccination on the susceptibility by Photobacterium damsela subsp. piscicida and on the humoral immune parameters. Fish & Shellfish Immunology 19, 345–361. Hardie, L.J., Fletcher, T.C., Secombes, C.J., 1991. The effect of dietary Vitamin C on the immune response of Atlantic salmon (Salmo salar). Aquaculture 95, 201–214. Huttenhuis, H.B.T., Ribeiro, A.S.P., Bowden, T.J., Van Bavel, C., TaverneThiele, A.J., Rombout, J.H.W.M., 2006. The effect of oral immunostimulation in juvenile carp (Cyprinus carpio L.). Fish & Shellfish Immunology 21, 261–271. Jeney, G., Anderson, D.P., 1993. Enhanced immune response and protection in rainbow trout to Aeromonas salmonicida bacterin following prior immersion in immunostimulants. Fish and Shellfish Immunology 3, 51–58. Jeney, G., Galeotti, M., Volpatti, D., Jeney, Z., Anderson, D.P., 1997. Prevention of stress in rainbow trout (Oncorhynchus mykiss) fed diets containing different doses of glucan. Aquaculture 154, 1–15. Jian, J., Wu, Z., 2003. Effects of traditional Chinese medicine on nonspecific immunity and disease resistance of large yellow croaker, Pseudosciaena crocea (Richardson). Aquaculture 218, 1–9. Jian, J., Wu, Z., 2004. Influences of traditional Chinese medicine on nonspecific immunity of Jian carp (Cyprinus carpio var. Jian). Fish & Shellfish Immunology 16, 185–191. John, M.B., Chandran, M.R., Aruna, B.V., Anbarasu, K., 2002. Production of superoxide anion by head-kidney leukocytes of Indian major carps immunised with bacterins of Aeromonas hydrophila. Fish & Shellfish Immunology 12 (3), 201–207. Kajita, Y., Sakai, M., Atsuta, S., Kobayashi, M., 1990. The immunomodulatory effect of levamisole on rainbow trout, Oncorhynchus mykiss. Fish pathology 25, 93–98. Kim, D., Austin, B., 2006. Innate immune responses in rainbow trout (Oncorhynchus mykiss, Walbaum) induced by probiotics. Fish & Shellfish Immunology 21, 513–524. Koethe, S.M., Nelson, K.E., Becker, C.G., 1995. Activation of the classical pathway of complement by tobacco glycoprotein. Journal of Immunology 155 (2), 826–835. Kolman, H., 2001. The humoral effects of epin in Siberian sturgeon (Acipenser baeri Brandt). Archives of Polish Fisheries 9 (1), 61–69 (Poland). Kolman, R., Kolman, H., Siwicki, R., 1998. The effect of some immunomodulators on the growth rate of sturgeon fry (Acipenseridae). Archives of Polish Fisheries 6 (2), 383–390 (Poland). Kumar, N., Singh, B., Bhandari, P., Gupta, A.P., Uniyal, S.K., Kaul, V.K., 2005. Biflavonoids from Lonicera japonica. Phytochemistry 66, 2740–2744. Lee, J.H., Ko, W.S., Kim, Y.H., Kang, H.S., Kim, H.D., Choi, B.T., 2001. Antiinflammatory effect of the aquaeous extract from Lonicera japonica flower is related to inhibition of NF-kappaB activation through reducing IkappaBalpha degradation in rat liver. International Journal of Molecular Medicine 2001. 7 (1), 79–83. Lee, Y.S., Han, O.K., Park, C.W., Suh, S.I., Shin, S.W., Yang, C.H., Yeon, T.W., Lee, E.S., Kim, K.J., Kim, S.H., Yoo, W.K., Kim, H.J., 2003. Immunomodulatory effects of aqueous-extracted Astragali-radix in methothrexate-treated mouse spleen cells. Journal of Ethnopharmacology 84 (2–3), 193–198. Lee, Y.S., Han, O.K., Park, C.W., Yang, C.H., Jeon, T.W., Yoo, W.K., Kim, S.H., Kim, H.J., 2005. Pro-inflammatory gene expression and nitric oxide regulation of aquaeous extracted Astragali radix in RAW 264.7 macrophage cells. Journal of Ethnopharmacology 100 (3), 289–294. Lin, Z.B., Zhang, H.N., 2004. Anti-tumor and immunoregulatory activities of Ganoderma lucidum and its possible mechanisms. Acta Pharmacologica Sinica 25 (11), 1387–1395.
Liu, C., Tsai, W., Lin, Y., Liao, J., Chen, C., Kuo, Y., 2004. The extracts from Nelumbo nucifera suppress cell cycle progression, cytokine genes expression, and cell proliferation in human peripheral blood mononuclear cells. Life Sciences 75, 699–716. Logambal, S.M., Michael, R.D., 2000. Immunostimulatory effect of azadirachtin in Oreochromis mossambicus (Peters). Indian Journal of Experimental Biology 38, 1092–1096. MacArthur, J.I., Fletcher, T.C., 1985. Phagocytosis in fish. In: Manning, M.J., Tatner, M.F. (Eds.), Fish Immunology. Academic Press, London, pp. 29–46. Magnadóttir, B., 2006. Innate immunity of fish (overview). Fish and Shellfish Immunology 20, 137–151. Neumann, N.F., Stafford, J.L., Barreda, D., Ainsworth, A.J., Belosevic, M., 2001. Antimicrobial mechanisms of fish phagocytes and their role in host defence. Developmental and Comparative Immunology 25, 807–825. Sakai, M., 1999. Current research status of fish immunostimulants. Aquaculture 172, 63–92. Salinas, I., Cuesta, A., Esteban, M.A., Meseguer, J., 2005. Dietary administration of Lactobacillus delbrüecki and Bacillus subtilis, single or combined, on gilthead seabream cellular innate immune responses. Fish & Shellfish Immunology 19, 67–77. Sankaran, K., Gurnani, S., 1972. On the variation in the catalytic activity of lysozyme in fishes. Indian Journal Biochemistry and Biophysics 9, 62–165. Secombes, C.J., 1990. Isolation of salmonid macrophages and analysis of their killing activity. In: Stolen, J.S., Anderson, D.P., Robertson, B.S., van Miuswinkell, W.B. (Eds.), Techniques in Fish Immunology. SOS Publications, Fair Haven, pp. 137–154. Secombes, C.J., Fletcher, T.C., 1992. The role of phagocytes in the protective mechanisms of fish. Annual Review of Fish Diseases 2, 58–71. Seeley, K.R., Gillespie, P.D., Weeks, B.A., 1990. A simple technique for the rapid spectrophotometric determination of phagocytosis by fish macrophages. Marine Environmental Research 30, 123–128. Shan, B.E., Yoshida, Y., Sugiura, T., Yamashita, U., 1999. Stimulating activity of Chinese medicinal herbs on human lymphocytes in vitro. International Journal of Immunopharmacology 21, 149–159. Shao, B., Xu, W., Dai, H., Tu, P., Li, Z., Gao, X., 2004. A study on the immune receptors for polysaccharides from the roots of Astragalus membranaceus, a Chinese medicinal herb. Biochemical and Biophysical Research Communications 320, 1103–1111. Siwicki, A.K., 1987. Immunomodulating activity of levamisole in spawners carp (Cyprinus carpio L.). Journal of Fish biology 31, 245–246. Siwicki, A.K., 1989. Immunomodulating influence of levamisole on nonspecific immunity of carp (Cyprinus carpio). Developmental and Comparative Immunology 14, 231–237. Siwicki, A., Studnicka, M., 1987. The phagocytic ability of neutrophils and serum lysozyme activity in experimentally infected carp, Cyprinus carpio L. Journal of Fish Biology 31 (Suppl. A), 57–60. Siwicki, A.K., Anderson, D.P., Rumsey, G.L., 1994. Dietary intake of immunostimulants by rainbow trout affects non-specific immunity and protection against furunculosis. Veterinary Immunology and Immunopathology 41, 125–139. Song, Q.H., Kobayashi, T., Xiu, L.M., Tie, H., Cyong, J.C., 2000. Effects of Astragali root and Hedysari root on the murine B and T cell differentiation. Journal of Ethnopharmacology 73 (1–2), 111–119. Tan, B.K.H., Vanitha, J., 2004. Immunomodulatory and antimicrobial effect of some traditional Chinese medicinal herbs. Current Medical Chemistry 11, 1423–1430. Thompson, I., White, A., Fletcher, T.C., Houlihan, D.F., Secombes, C.J., 1993. The effect of stress on the immune responses of Atlantic salmon (Salmo salar L.) fed diets containing different amounts of vitamin-C. Aquaculture 114, 1–18. Thompson, K.D., Cachos, A., Inglis, V., 1995. Immunostimulating effect of glucans and oxytetracycline in rainbow trout, Oncorhynchus mykiss, on serum lysozyme and protection. In: Shariff, M., Subashige, R.P., Arthur, J.R. (Eds.), Fish Health Section. Diseases in Asian Aquaculture, vol 11. Asian Fisheries Society, Manila, Philippines, pp. 433–439.
L. Ardó et al. / Aquaculture 275 (2008) 26–33 Wang, R., Li, D., Bourne, S., 1999. Can 2000 years of herbal medicine history help us to solve problems in the year 2000? Biotechnology in the feed industry. Proceedings of Alltech's 14th Annual Symposium, pp. 273–291. Watanabe, T., Kiron, W., Satoh, S., 1997. Trace minerals in fish nutrition. Aquaculture 151, 185–207.
33
Wu, H., Luo, J., Yin, Y., Wei, Q., 2004. Effects of chlorogenic acid, an active compound activating calcineurin, purified from Flos Lonicerae on macrophage. Acta Pharmacologica Sinica 12, 1685–1689. Yin, G., Jeney, G., Rácz, T., Pao, X., Jeney, Z., 2006. Effect of two Chinese herbs (Astragalus radix and Scutellaria radix) on non–specific immune response of tilapia, Oreochromis niloticus. Aquaculture 253, 39–47.