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Chinese herbs (Astragalus radix and Ganoderma lucidum) enhance immune response of carp, Cyprinus carpio, and protection against Aeromonas hydrophila
Fish & Shellfish Immunology 26 (2009) 140–145
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Chinese herbs (Astragalus radix and Ganoderma lucidum) enhance immune response of carp, Cyprinus carpio, and protection against Aeromonas hydrophila Guojun Yin a, L. Ardo´ b, K.D. Thompson c, A. Adams c, Z. Jeney b, G. Jeney b, * a
Key Open Laboratory for Genetic Breeding of Aquatic Animals and Aquaculture Biology, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Quitang 1, Wuxi 214081, China b Research Institute for Fisheries, Aquaculture and Irrigation, Anna liget 8, Szarvas H-4440, Hungary c Institute of Aquaculture, University of Stirling, Stirling, Scotland FK9 4LA, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 June 2008 Received in revised form 19 August 2008 Accepted 22 August 2008 Available online 6 September 2008
The effect of Chinese herbs (Astragalus radix and Ganoderma lucidum) on immune response of carp was investigated. Fish were fed diets containing Astragalus (0.5%), Ganoderma (0.5%) and combination of two herbs (Astragalus 0.5% and Ganoderma 0.5%) for 5 weeks. Other groups of fish were vaccinated (i.p.) against Aeromonas hydrophila/Aeromonas salmonicida (Shering Plough, Essex, U.K.) at the beginning of the experiment and fed the same diets as described above. Control fish (negative control) and fish vaccinated only (positive control) were fed basal diets without supplements of herbs. The respiratory burst activity, phagocytosis, lysozyme activity and circulatory antibody titres in plasma were monitored. Following 5 weeks after feeding, fish were infected with A. hydrophila and mortalities were recorded. The results of this study showed that feeding non-vaccinated and vaccinated carp with combination of Astragalus and Ganoderma stimulated respiratory burst activity, phagocytosis of phagocytic cells in blood and lysozyme and circulatory antibody titres in plasma in vaccinated carp. Fish challenged with A. hydrophila had variable survival. The best survival (60%) was in vaccinated group fed with both herbs, while almost 90% of control fish (negative control) and 60% of fish vaccinated only (positive control) died. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Immune response Astragalus radix Ganoderma lucidum Aeromonas hydrophila Common carp Cyprinus carpio Vaccine Immunostimulant
1. Introduction Several Aeromonads are noted as causing major problems for carp aquaculture. Although motile Aeromonads are fish pathogens it is important to note that these bacteria also compose part of the normal intestinal microflora of healthy fish [1] and consequently stress is often considered to be a contributing factor in disease outbreaks caused by these bacteria. Aeromonas hydrophila is more abundant in waters with a high organic load than in relatively unpolluted water [2]. Vaccines are being developed against A. hydrophila but these are not yet commercially available. A. hydrophila is such a heterogeneous species, having variable antigens, that vaccine development is extremely complex. Using immunostimulants in combination with fish vaccine is an attractive method for increasing the protective capabilities of fish, and by boosting the potency of the vaccine smaller doses can be given [3]. In our research we used herbs to give early activation to
* Corresponding author. Tel.: þ86 66515317; fax: þ86 66312142. E-mail address: [email protected] (G. Jeney). 1050-4648/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2008.08.015
the non-specific defence mechanisms and to elevate the specific immune response. Chinese herbs have been used as traditional medicine and immune booster for human beings for thousands of years in China. Recently, growing interest has been paid to the immune stimulating function of some herbs in aquaculture, non-specific immunity such as bacteriolytic activity and leucocyte function was improved by mixtures of Chinese herbs in shrimp (Penaeus chinensis) and tilapia [4,5]. Phagocytosis by white blood cells and lysozyme activity in the serum of crucian carp were both increased by feeding four different herbs: Rheum officinale, Andrographis paniculata, Isatis indigotica, Lonicera japonica [6], NBT positive cells and lysozyme activities were increased in jian carp (Cyprinus carpio var. Jian) by feeding herbal mixture of traditional Chinese medicine [7]. In this study extracts of two Chinese herbs from Astragalus root (Astragalus radix extracted from Astragalus membranaceus) and from Ganoderma mushroom (Ganoderma lucidum) were chosen because of their recorded ability to enhance the immune system. Analysis shows that A. radix contains polysaccharides, monosaccharides, flavonoid and alkaloid, together with choline, betaine, folic acid, various amino acids, mucoitin, gum, cellulose, and 14 trace minerals, including selenium, zinc, and iron, which are
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essential micronutrients for man and animals. Recently published research has found that some components such as: polysaccharides, organic acids, alkaloids, glucosides and volatile oil can enhance immune function [8,9]. In previously reported experiments with tilapia we showed that A. radix had a positive influence on the immune system by acting as a booster [10]. G. lucidum is an important medicinal herb containing polysaccharides. Ganoderma polysaccharides have been reported to be effective in modulating immune functions, inhibiting tumour growth [11], preventing oxidative damage [12], protecting liver, reducing serum glucose levels, while producing no toxic effects [13]. In the study reported here, carp (C. carpio) were fed with different doses of two extracts of Chinese herbs: A. radix and G. lucidum to investigate the effect of these substances on the nonspecific and specific immune response of non-vaccinated and vaccinated carp and to determine resistance against A. hydrophila. After feeding the herbs, increased activity of the non-specific defence mechanism was monitored. Parameters measured included, neutrophil oxidative activity by reduction of ferricytochrome c and phagocytic activity. These non-specific parameters and the specific immune response were also observed when fish were injected with vaccine. Specific immune response activity was demonstrated by increased circulating antibody titres. Furthermore we determined resistance to A. hydrophila by challenge the fish with virulent pathogen. 2. Materials and methods 2.1. Fish Carp (C. carpio) (62.8 5.40 g) were held in a recirculation system of the Research Institute for Fisheries, Aquaculture and Irrigation (HAKI, Szarvas, Hungary). Fish were fed with a dry feed, produced in the experimental milling facility of the Institute and kept in 2000 l fibreglass tanks at water temperature of 22–23 C.
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2.4. Separation of leucocytes from the blood Leucocytes for assay were separated from each blood sample by density-gradient centrifugation. One millilitre of histopaque 1.119 (Sigma) containing 100 ml of bacto hemagglutination buffer, pH 7.3 (Difco, USA) was dispensed into siliconised tubes. One millilitre of a mixture of 1.077 density histopaque and hemagglutination buffer and 1 ml of blood was carefully layered on the top. The sample preparations were centrifuged at 700 g for 15 min at 4 C. After centrifugation, plasma was collected and stored at 80 C for future analysis. Separated leucocytes were gently removed and dispensed into siliconised tubes, containing phenol red free Hanks Balanced Salt Solution (HBSS, Sigma). Cells were then washed twice in HBSS and adjusted to 1 107 viable cells ml1.
2.5. Respiratory burst activity Respiratory burst activity of isolated leucocytes was quantified by reduction of ferricytochrome c [14]. Briefly, 100 ml of leucocyte suspension and an equal volume of cytochrome c (2 mg l1 in phenol red free HBSS) containing phorbol 12-myristate 13-acetate (PMA, Sigma) at 1 mg ml1 were placed in triplicate in microtitre plates. In order to test specificity, another 100 ml of leucocyte suspensions and solutions of cytochrome c containing PMA and superoxide dismutase (SOD, Sigma) at 300 U ml1 were prepared in duplicate in microtitre plates. Samples were then mixed and incubated at room temperature for 15 min. Extinctions were measured at 550 nm against a cytochrome c blank in a multiscan spectrophotometer. Readings were converted to nmol O 2 by subtracting the O.D. of the PMA/SOD treated supernatant from that treated with PMA given alone for each fish, and converting O.D. to nmol O 2 by multiplying 15.87. Final results were expressed as 5 nmol O 2 produced per 10 blood leucocytes.
2.2. Herbal extracts
2.6. Phagocytosis assay
Astragalus extract containing 40% Astragalus polysaccharide and Ganoderma extract containing 30% polysaccharides were commercial products from Xuancheng Baicao Plants Industry and Trade Ltd., China.
Phagocytic activity of blood leucocytes was determined spectrophotometrically by the method as described in Ref. [15]. This assay involves the measurement of congo red-stained yeast cells that have been phagocytised by cells. To perform the assay, 1000 ml of the leucocyte solution was mixed with 2000 ml of the congo redstained and autoclaved yeast cell suspension (providing a yeast cell: leucocyte ratio of 20:1). The mixtures were incubated at room temperature for 60 min. Following incubation, 1 ml ice-cold HBSS was added and 1 ml of histopaque (1.077) was injected into the bottom of each sample tube. The samples were centrifuged at 850 g for 5 min to separate leucocytes from free yeast cells. Leucocytes were harvested and washed twice in HBSS. The cells were then resuspended in 1 ml trypsin–EDTA solution (5.0 g l1 trypsin and 2.0 g l1 EDTA, Sigma) and incubated at 37 C overnight. The absorbance of the samples was measured at 510 nm using trypsin– EDTA as a blank.
2.3. Experimental design and sampling procedure All experiments were carried out in the recirculation system of the HAKI Institute, Szarvas, Hungary. Batches (eight groups) of 60 five-month old carp with an average initial weight of 62.8 5.40 g were held in 100 l fibreglass tanks. Water temperature and pH were constant (22–23 C; pH 8.5) during the experimental period, and dissolved oxygen was maintained at 80–90% of saturation. Water flow was maintained at 7 l/ min. Fish were fed ad libitum 6 times a day with a pelleted feed containing either A. radix (0.5%), or Ganoderma (0.5%), or combination of Astragalus (0.5%) and Ganoderma (0.5%) for 5 weeks. At the beginning of experiment four groups of fish were vaccinated (i.p. 0.1 ml/fish) using vaccine developed against A. hydrophila/A. salmonicida (Shering Plough, Essex, U.K.) and fed the same diets as described above. Control fish (negative control) and fish vaccinated only (positive control) were fed basal diets without supplements of herbs. Blood samples (five fish/group) were collected from caudal vein 1, 2, 3, 4 and 5 weeks after the 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.
2.7. Lysozyme assay Plasma lysozyme activity was measured spectrophotometrically according to the method as described in Ref. [16]. The lysozyme substrate was a 0.02% (w/v) suspension of Micrococcus lysodeikticus made up in phosphate buffer (0.05 M, pH 6.2). Lyophilised hen egg white lysozyme was used as a standard. A new standard curve was prepared for each assay. Standard solutions as well as samples were added to the substrate at 25 C. The results were expressed as mg ml1 equivalent of hen egg white enzyme activity.
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2.8. ELISA assay for determining antibody titres Antibody titres were measured against A. hydrophila. Serum antibody titres were determined using an indirect ELISA [17]. Plates were coated with suspensions of A. hydrophila adjusted to an OD610 of 1.0. Serum samples from vaccinated fish were serially diluted (twofold dilutions) in PBS, and a monoclonal antibody (anti-carp IgM monoclonal antibody, Aquatic diagnostic Ltd, Stirling, Scotland) was used as the second antibody. 2.9. Challenges with virulent pathogen The susceptibility of the fish fed herbs to a bacterial challenge was examined in vivo A. hydrophila (strain OB 212 Bacteriology Unit, Institute of Aquaculture University of Stirling, Scotland) was used as the challenge strain. Groups of 30 fish fed with herbs alone or in combination of herbs and vaccine were challenged 5 weeks after the start of treatments. Carp were injected intraperitoneally with a 1-day growth of virulent pathogen adjusted to 1 106 cells per fish. The fish were observed regularly at 4 h intervals for behavioural changes and mortalities. All the dead fish were removed and bacterial swabs were taken from the kidneys, and cultured on tryptone soya agar (TSA) plates. Bacteria isolated from the fish were confirmed as A. hydrophila using conventional methods. 2.10. Statistics Results are presented as the average (standard error) for five fish, and were compared at each time point using one-way ANOVA and Dunn’s multiple range tests (SigmaStat 3.2). Significant differences between experimental groups were expressed at a significant level of P < 0.05. Mortality (obtained at the end of experiment) in each treated group was compared statistically with control group using Chi-square test (SigmaStat 3.2). Significant differences between control and treated groups were expressed at a significant level of P < 0.001 and P < 0.05. 3. Results The effect of herbs and combination of herbs and vaccination on respiratory burst activities of isolated phagocytic cells is shown in Fig. 1. Respiratory burst activities were elevated in the group fed with Ganoderma on week 1 and in fish fed with Astragalus on week 3 compared to the negative control (Fig. 1a). Otherwise there was
no effect on respiratory burst activities in fish fed with herbs only during the whole experiment, although in some cases fish fed with Astragalus had lower values of respiratory burst activities, the differences were not significant comparing to control. When using the combination of herbs and vaccine in fish, elevation of respiratory burst activities was noticed on weeks 3 and 4 compared to control (Fig. 1b), whereas significant inhibition of respiratory burst activities was observed on week 3 in vaccinated groups fed with herbs compared to positive control (vaccinated only). At the end of experiment, vaccinated fish fed with Astragalus and combination of Astragalus and Ganoderma showed significantly higher values of respiratory burst activities compared to both controls – negative and positive (Fig. 1b). Elevated phagocytic activity was noted after 3 weeks in fish fed with herbs (Fig. 2a), while in vaccinated groups fed with Ganoderma and the combination of two herbs, elevated levels were noted on week 1 and at the end of experiment compared to both controls (Fig. 2b). On the second and the third weeks vaccinated fish fed with herbs phagocytosis was significantly lower when compared to both controls. There were no significant differences among groups fed with different herbs and fish treated with the combination of vaccine and herbs. Plasma lysozyme activities were significantly higher after 1 week in groups fed with herbs compared to control (Fig. 3a), whereas in vaccinated fish significant differences were measured only in groups fed with Ganoderma (second week), Astragalus (third week) and in fish fed with the combination of herbs a significant difference was observed at the end of experiment (Fig. 3b). Following vaccination all the vaccinated fish were found to have circulatory antibody 1 week post-vaccination (Fig. 4). Antibodies were not detected in control fish and fish fed with herbs only, whereas all fish immunised with the vaccine were responding well. Significant differences (P < 0.05) were noted between immunised fish and control throughout the whole experiment, the highest antibody titres were measured at the end of experiment. However, there were no significant differences among fish vaccinated only and fish treated with the combination of vaccine and herbs. Fish infected with A. hydrophila started to die after 36 h after injection. Mortalities due to A. hydrophila first occurred in control fish reaching almost 50% after 2 days post-infection and a cumulative mortality over the 6-day experimental period was 87% (Fig. 5a). Cumulative mortalities in fish fed with herbs were at significantly lower level comparing to control, reaching 58% and 60%. Fish treated with vaccine only started to die after 2 days post-infection,
Fig. 1. Respiratory burst activity of isolated blood cells in carp fed diets containing Astragalus radix, Ganoderma lucidum and combination of them (a) and vaccinated carp fed the same diets (b). Data is expressed as the mean of five fish SEM. Significant differences (P < 0.05) from the untreated control are indicated by asterisks. Significant differences (P < 0.05) from the vaccinated group only (positive control) are indicated by letter a.
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Fig. 2. Phagocytic activity of isolated blood cells in carp fed diets containing Astragalus radix, Ganoderma lucidum and combination of them (a) and vaccinated carp fed the same diets (b). Legends are the same as on Fig. 1.
cumulative mortality reached 50% at 4-day experimental period. The same tendency was observed in vaccinated fish fed with Astragalus. The significantly lowest mortality comparing to control was noted in vaccinated fish fed with combination of two herbs with a cumulative mortality 38%. 4. Discussion Both herb extract used alone or in combination with vaccine can modulate the non-specific defence mechanism. The present results showed that fish fed with Chinese herbs alone or in combination with vaccine significantly enhanced respiratory burst activity of phagocytic cells, phagocytosis and lysozyme activities in plasma. Similar results were obtained in jian carp (C. carpio var. Jian) and large yellow croaker (Pseudosciaena crocea) fed with combination of Astragalus root and Chinese Angelica root (Radix Angelicae sinensis) [18,19]. Extracellular respiratory burst activities in carp fed with herbs alone or in combination with vaccine were elevated. However, there were no significant differences among groups fed with different herbs and fish treated with combination of vaccine and herbs. Injection with glucan increased head kidney macrophages’ extracellular respiratory burst activity [20]. Extracellular activity was very high in rainbow trout fed with dietary glucan [21]. Rainbow trout fed with ginger (Zingiber officinale) extract had
significantly higher extracellular activity of phagocytic cells in blood, while in trout fed with nettle and mistletoe extracts the production of extracellular superoxide anion was on the same level as in the control fish [22]. In our previous study we could not detect such differences in respiratory burst activity in tilapia fed with Astragalus extract and in the case of fish fed with Scutellaria there was significant inhibition of extracellular superoxide anion production [10]. Other dietary components, namely vitamins E, C and A, have been shown to have little effect upon macrophage or phagocytic cells respiratory burst activity [23–25]. Changes in superoxide anion production due to the multiple injections of b-glucan were independent of different dosages [26]. Fishes treated with immunostimulants usually show enhanced phagocytosis. Several studies have reported that oral administration of yeast products (MacroGard; Vitastim; Saccharomyces cerevisiae) [21,27,28], chitin [29], plant extracts of four Chinese herbs (R. officinale, A. paniculata, I. indigotica, L. japonica) increased phagocytosis of white blood cells of crucian carp [6], naturally occurring tetrapeptide tuftsin enhanced phagocytosis in Labeo rohita [30]. In this study, phagocytic activity of blood leucocytes was increased in carp fed with herbs only on weeks 3 and 4, while in vaccinated fish, elevated phagocytic activities were measured on week 5 compared to control. Astragalus has been reported to increase the phagocytosis of the blood cells of soft-shelled turtles
Fig. 3. Plasma lysozyme activity in carp fed diets containing Astragalus radix, Ganoderma lucidum and combination of them (a) and vaccinated carp fed the same diets (b). Legends are the same as on Fig. 1.
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Fig. 4. Circulatory antibody titres against Aeromonas hydrophila in vaccinated carp fed diets containing Astragalus radix, Ganoderma lucidum and combination of them (a) and vaccinated carp fed the same diets (b). Legends are the same as on Fig. 1.
(Pelodiscus sinensis) [31] and alveolus macrophage of pneumoconiosis rats [32]. Astragalus polysaccharide (APS) is the major active component of A. radix. The role of APS on the specific and nonspecific immune responses has been reviewed [33]. APS modulates the functions of the immune cells including T cells, B cells, NK cells and macrophage [34]. Polysaccharides of Ganoderma have been reported to be effective in modulating immune functions, inhibiting tumour growth [35]. However, there is no data available on use of Ganoderma in fish. On the second and the third weeks vaccinated fish fed with herbs showed significantly lower phagocytosis when compared to both controls. In our previous study it was found that feeding tilapia with Scutellaria extract with higher doses (0.5 and 1.0%) caused reduction of function in phagocytic cells, while, when fish were fed with low dose of Scutellaria (0.1%) there was no stimulation on phagocytic activities [10]. Other studies, where higher doses of immunostimulants were used together with vaccine have been found to be even suppressive [36,37].
As a first line of defence, various peptides, such as lysozymes are present in serum where they prevent adherence and colonisation by microorganisms [38]. The supplemented feeds also enhanced the lysozyme activity in all the treatments. Similarly many authors have reported that administration of b-glucan enhances lysozyme activity in Atlantic salmon and turbot [26,39–42]. Other immunostimulants, such as chitosan, levamisole and chitin also elevated lysozyme levels in plasma of carp [43]. In our previous study with tilapia elevated lysozyme activity was measured when fish were fed with Astragalus, while in groups fed with Scutellaria there were no significant changes in plasma lysozyme activities [10]. Lysozyme is a cationic enzyme that breaks b-1,4 glycosidic acids and N-acetyl glucosamine in the peptidoglucan of bacterial cell walls. This action is known to attack mainly Gram positive bacteria as well as some Gram negative bacteria in conjunction with complement [38]. Robertson et al. [20] showed an increased protection against fish bacterial infection, which correlated with an increment in serum lysozyme levels, phagocytic activity and bactericidal activity of head kidney leucocytes. Such enhancement in lysozyme levels could also be correlated with enhanced phagocytic activity. When using herb extracts with vaccine the specific immune response was also elevated, although there were no significant differences among vaccinated only group and vaccinated fish fed with herb extracts. The antibody titres against A. hydrophila were not significantly different to the control in L. rohita [30]. While in tilapia it was found that medicinal plants Azadirachta indica enhanced the primary and secondary antibody responses in inverse dose dependant mode [44]. In this study after challenge with A. hydrophila, mortalities were significantly reduced in all groups compared to controls, with the lowest mortality in vaccinated fish fed with both herbs. It was considered that we would be able to use the herbs without the addition of a specific vaccine for inducing protection in carp against A. hydrophila. The dose of bacteria we used for challenge was very high, resulting in 90% mortality of control fish. Sharifpour [45] observed that intramuscular injection of A. hydrophila at a concentration of 5.3 106 cells ml1 caused infection in carp, and fish started to die within 12 h with one quarter of the injected fish dying between 12 and 24 h post-infection. Sarder et al. [46] injected tilapia intraperitoneally with A. hydrophila suspension (5 106 cells ml1) and fish started to die within 12 h. Mortality due to infections with A. hydrophila was reduced significantly by
Fig. 5. Cumulative mortalities after artificial challenging with Aeromonas hydrophila in carp fed diets containing Astragalus radix, Ganoderma lucidum and combination of them (a) and vaccinated carp fed the same diets (b). Significant differences from the untreated control are indicated by asterisks (*P < 0.05; **P < 0.001).
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injecting different doses of glucan, the least mortality was observed in the group of fish injected with the medium dose (10 mg kg1 body weight) [26]. The use of immunostimulants and herbs with vaccine to increase efficacy of vaccine has been considered by others [47,48]. Anderson and Jeney [49] and Jeney and Anderson [3] showed that use of QAC, ISK and levamisole given by injection or bath enhanced protection in rainbow trout vaccinated with A. salmonicida bacterin to A. salmonicida infection. The carp used in this experiment is regarded as more resistant, however, even the challenge doses given, quickly killed negative control fish. Results obtained in this study showed that there is a possibility of using Astragalus and Ganoderma extracts as an immunostimulants in immunocompromised fish raised at high density in intensive aquaculture farms. Astragalus may be used as an immunostimulant in aquaculture as it can be easily obtained and is not expensive. Ganoderma is more expensive; however, the by-products are cheap and can be used in aquaculture, because the use of highly purified products is not needed. Furthermore, the use of such plants products as immunostimulants in aquaculture systems may also have environmental value because of their biodegradability. References [1] Karunasagar GM, Rosalind GM, Karunasagar I. Immunological response of the Indian major carps to Aeromonas hydrophila vaccine. Fish Shellfish Immunol 1993;3:413–7. [2] Jeney Z, Jeney G. Recent achievements in studies of diseases of common carp (Cyprinus carpio L.). Aquaculture 1995;129:397–420. [3] Jeney G, Anderson DP. Enhanced immune response and protection in rainbow trout to Aeromonas salmonicida bacterin following prior immersion in immunostimulants. Fish Shellfish Immunol 1993;3:51–8. [4] Luo R. Induction of immunity substance in Penaeus chinensis by Chinese herbal medicine. Oceanol Limnol Sin 1997;28:573–7 [China]. [5] Chansue N, Ponpornpisit A, Endo M, Sakai M, Satoshi Y. Improved immunity of tilapia, Oreochromis niloticus by C-UP III, a herb medicine. Fish Pathol 2000;35:89–90. [6] Chen X, Wu Z, Yin J, Li L. Effects of four species of herbs on immune function of Carassius auratus gibelio. J Fish Sci China 2003;10:36–40 [China]. [7] Jian J, Wu Z. Effect of Chinese herbal medicine on non-specific immunity of Jian common carp (Cyprinus carpio var. Jian). J Dalian Fish Univ 2002;17:114–9 [China]. [8] Wang R, Li D, Bourne S. Can 2000 years of herbal medicine history help us to solve problems in the year 2000? In: Biotechnology in the feed industry, Proceedings of Alltech’s 14th annual symposium; 1999. p. 273–91. [9] Liu HB. Research status of Chinese herbal immunostimulants and their application in aquaculture. J Fish 2002;15:91–4 [China]. [10] Yin G, Jeney G, Racz T, Xu P, Jun X, Jeney Z. Effect of two Chinese herbs (Astragalus radix and Scutellaria radix) on non-specific immune response of tilapia, Oreochromis niloticus. Aquaculture 2006;253:39–47. [11] Lin ZB, Zhang HN. Anti-tumor and immunoregulatory activities of Ganoderma lucidum and its possible mechanisms. Acta Pharmacol Sin 2004;25:1387–95 [China]. [12] You YH, Lin ZB. Protective effects of Ganoderma lucidum polysaccharides peptide on injury of macrophages induced by reactive oxygen species. Acta Pharmacol Sin 2002;23:787–91. [13] Zhang J, Tang Q, Zimmerman-Kordman M, Reutter W, Fan H. Activation of B lymphocytes by GLIS, a bioactive proteoglucan from Ganoderma lucidum. Life Sci 2002;71:623–38. [14] Secombes CJ. Isolation of salmonid macrophages and analysis of their killing activity. In: Stolen JS, Anderson DP, Robertson BS, van Muiswinkel WB, editors. Techniques in fish immunology. Fair Haven: SOS Publications; 1990. p. 137–54. [15] Seeley KR, Gillespie PD, Weeks BA. A simple technique for the rapid spectrophotometric determination of phagocytosis by fish macrophages. Mar Environ Res 1990;30:123–8. [16] Sankaran K, Gurnani S. On the variation in the catalytic activity of lysozyme in fishes. Indian J Biochem Biophys 1972;9:162–5. [17] Adams A. Sandwich enzyme linked immunosorbent assay (ELISA) to detect and quantify bacterial pathogens in fish tissue. In: Stolen JS, Fletcher TC, Katari SL, Rowley AF, editors. Techniques in fish immunology, vol. 2. Fair Haven, NJ, USA: SOS Publications; 1992. p. 177–84. [18] Jian J, Wu Z. Effects of traditional Chinese medicine on nonspecific immunity and disease resistance of large yellow croaker, Pseudosciaena crocea (Richardson). Aquaculture 2003;218:1–9. [19] Jian J, Wu Z. Influences of traditional Chinese medicine on non-specific immunity of Jian carp (Cyprinus carpio var. Jian). Fish Shellfish Immunol 2004;16:185–91. [20] Roberson B, Rostadt G, Engstadt R, Raa J. Enhancement of non-specific disease resistance in Atlantic salmon, Salmo salar L., by a glucan from Saccharomyces cerevisiae cell walls. J Fish Dis 1990;13:391–400.
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Acta Pharmacologica Sinica 2004,25: 1387–95 [China]. [36] Mulero I, Pilar Sepulcre M, Fuentes I, Garcı´a-Alca´zar A, Meseguer A, Garcı´aAyala A, Mulero V. Vaccination of larvae of the bony fish gilthead seabream reveals a lack of correlation between lymphocyte development and adaptive immunocompetence. Mol Immunol 2008;45:2981–9. [37] Logambal SM, Michael RD. Azadirachtin – an immunostimulant for Oreochromis mossambicus. J Aqua Trop 2001;16:339–47. [38] Alexander JB, Ingram GA. Non-cellular and non-specific defense mechanisms of fish. Annu Rev Fish Dis 1992;2:249–80. [39] Engstadt RJ, Robertsen B, Frivold B. Yeast glucan induces increase in lysozyme and complement-mediated haemolytic activity in Atlantic salmon blood. Fish Shellfish Immunol 1992;2:287–97. [40] Matsuyama H, Mangindaan REP, Yano T. Protective effect of schizophyllan and scleroglucan against Streptococcus sp. infection in yellowtail (Seriola quinqueradiata). Aquaculture 1992;101:197–203. [41] Santarem M, Novoa B, Figueras A. Effects of b-glucan on the non-specific immune responses in turbot (Scophthalmus maximus L.). Fish Shellfish Immunol 1997;7:429–37. [42] Paulsen SM, Engstadt RE, Robertsen B. Enhanced lysozyme production in Atlantic salmon (Salmo salar L.) macrophages treated with yeast b-glucan and bacterial lipopolysaccharide. Fish Shellfish Immunol 2001;11:23–37. [43] Gopalakannan A, Arul V. 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 2006;255:179–87. [44] Venkatalakshmi S, Michael RD. Immunostimulation by leaf extract of Ocimum sanctum L. in Oreochromis mossambicus (Peters). J Aqua Trop 2001;16:1–10. [45] Sharifpour I. Histology of inflammatory response of carp (Cyprinus carpio L.) to various stimuli. Ph.D. thesis, Institute of Aquaculture, University of Stirling, Scotland; 1997. 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Chinese herbs (Astragalus radix and Ganoderma lucidum) enhance immune response of carp, Cyprinus carpio, and protection against Aeromonas hydrophila Guojun Yin a, L. Ardo´ b, K.D. Thompson c, A. Adams c, Z. Jeney b, G. Jeney b, * a
Key Open Laboratory for Genetic Breeding of Aquatic Animals and Aquaculture Biology, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Quitang 1, Wuxi 214081, China b Research Institute for Fisheries, Aquaculture and Irrigation, Anna liget 8, Szarvas H-4440, Hungary c Institute of Aquaculture, University of Stirling, Stirling, Scotland FK9 4LA, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 June 2008 Received in revised form 19 August 2008 Accepted 22 August 2008 Available online 6 September 2008
The effect of Chinese herbs (Astragalus radix and Ganoderma lucidum) on immune response of carp was investigated. Fish were fed diets containing Astragalus (0.5%), Ganoderma (0.5%) and combination of two herbs (Astragalus 0.5% and Ganoderma 0.5%) for 5 weeks. Other groups of fish were vaccinated (i.p.) against Aeromonas hydrophila/Aeromonas salmonicida (Shering Plough, Essex, U.K.) at the beginning of the experiment and fed the same diets as described above. Control fish (negative control) and fish vaccinated only (positive control) were fed basal diets without supplements of herbs. The respiratory burst activity, phagocytosis, lysozyme activity and circulatory antibody titres in plasma were monitored. Following 5 weeks after feeding, fish were infected with A. hydrophila and mortalities were recorded. The results of this study showed that feeding non-vaccinated and vaccinated carp with combination of Astragalus and Ganoderma stimulated respiratory burst activity, phagocytosis of phagocytic cells in blood and lysozyme and circulatory antibody titres in plasma in vaccinated carp. Fish challenged with A. hydrophila had variable survival. The best survival (60%) was in vaccinated group fed with both herbs, while almost 90% of control fish (negative control) and 60% of fish vaccinated only (positive control) died. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Immune response Astragalus radix Ganoderma lucidum Aeromonas hydrophila Common carp Cyprinus carpio Vaccine Immunostimulant
1. Introduction Several Aeromonads are noted as causing major problems for carp aquaculture. Although motile Aeromonads are fish pathogens it is important to note that these bacteria also compose part of the normal intestinal microflora of healthy fish [1] and consequently stress is often considered to be a contributing factor in disease outbreaks caused by these bacteria. Aeromonas hydrophila is more abundant in waters with a high organic load than in relatively unpolluted water [2]. Vaccines are being developed against A. hydrophila but these are not yet commercially available. A. hydrophila is such a heterogeneous species, having variable antigens, that vaccine development is extremely complex. Using immunostimulants in combination with fish vaccine is an attractive method for increasing the protective capabilities of fish, and by boosting the potency of the vaccine smaller doses can be given [3]. In our research we used herbs to give early activation to
* Corresponding author. Tel.: þ86 66515317; fax: þ86 66312142. E-mail address: [email protected] (G. Jeney). 1050-4648/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2008.08.015
the non-specific defence mechanisms and to elevate the specific immune response. Chinese herbs have been used as traditional medicine and immune booster for human beings for thousands of years in China. Recently, growing interest has been paid to the immune stimulating function of some herbs in aquaculture, non-specific immunity such as bacteriolytic activity and leucocyte function was improved by mixtures of Chinese herbs in shrimp (Penaeus chinensis) and tilapia [4,5]. Phagocytosis by white blood cells and lysozyme activity in the serum of crucian carp were both increased by feeding four different herbs: Rheum officinale, Andrographis paniculata, Isatis indigotica, Lonicera japonica [6], NBT positive cells and lysozyme activities were increased in jian carp (Cyprinus carpio var. Jian) by feeding herbal mixture of traditional Chinese medicine [7]. In this study extracts of two Chinese herbs from Astragalus root (Astragalus radix extracted from Astragalus membranaceus) and from Ganoderma mushroom (Ganoderma lucidum) were chosen because of their recorded ability to enhance the immune system. Analysis shows that A. radix contains polysaccharides, monosaccharides, flavonoid and alkaloid, together with choline, betaine, folic acid, various amino acids, mucoitin, gum, cellulose, and 14 trace minerals, including selenium, zinc, and iron, which are
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essential micronutrients for man and animals. Recently published research has found that some components such as: polysaccharides, organic acids, alkaloids, glucosides and volatile oil can enhance immune function [8,9]. In previously reported experiments with tilapia we showed that A. radix had a positive influence on the immune system by acting as a booster [10]. G. lucidum is an important medicinal herb containing polysaccharides. Ganoderma polysaccharides have been reported to be effective in modulating immune functions, inhibiting tumour growth [11], preventing oxidative damage [12], protecting liver, reducing serum glucose levels, while producing no toxic effects [13]. In the study reported here, carp (C. carpio) were fed with different doses of two extracts of Chinese herbs: A. radix and G. lucidum to investigate the effect of these substances on the nonspecific and specific immune response of non-vaccinated and vaccinated carp and to determine resistance against A. hydrophila. After feeding the herbs, increased activity of the non-specific defence mechanism was monitored. Parameters measured included, neutrophil oxidative activity by reduction of ferricytochrome c and phagocytic activity. These non-specific parameters and the specific immune response were also observed when fish were injected with vaccine. Specific immune response activity was demonstrated by increased circulating antibody titres. Furthermore we determined resistance to A. hydrophila by challenge the fish with virulent pathogen. 2. Materials and methods 2.1. Fish Carp (C. carpio) (62.8 5.40 g) were held in a recirculation system of the Research Institute for Fisheries, Aquaculture and Irrigation (HAKI, Szarvas, Hungary). Fish were fed with a dry feed, produced in the experimental milling facility of the Institute and kept in 2000 l fibreglass tanks at water temperature of 22–23 C.
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2.4. Separation of leucocytes from the blood Leucocytes for assay were separated from each blood sample by density-gradient centrifugation. One millilitre of histopaque 1.119 (Sigma) containing 100 ml of bacto hemagglutination buffer, pH 7.3 (Difco, USA) was dispensed into siliconised tubes. One millilitre of a mixture of 1.077 density histopaque and hemagglutination buffer and 1 ml of blood was carefully layered on the top. The sample preparations were centrifuged at 700 g for 15 min at 4 C. After centrifugation, plasma was collected and stored at 80 C for future analysis. Separated leucocytes were gently removed and dispensed into siliconised tubes, containing phenol red free Hanks Balanced Salt Solution (HBSS, Sigma). Cells were then washed twice in HBSS and adjusted to 1 107 viable cells ml1.
2.5. Respiratory burst activity Respiratory burst activity of isolated leucocytes was quantified by reduction of ferricytochrome c [14]. Briefly, 100 ml of leucocyte suspension and an equal volume of cytochrome c (2 mg l1 in phenol red free HBSS) containing phorbol 12-myristate 13-acetate (PMA, Sigma) at 1 mg ml1 were placed in triplicate in microtitre plates. In order to test specificity, another 100 ml of leucocyte suspensions and solutions of cytochrome c containing PMA and superoxide dismutase (SOD, Sigma) at 300 U ml1 were prepared in duplicate in microtitre plates. Samples were then mixed and incubated at room temperature for 15 min. Extinctions were measured at 550 nm against a cytochrome c blank in a multiscan spectrophotometer. Readings were converted to nmol O 2 by subtracting the O.D. of the PMA/SOD treated supernatant from that treated with PMA given alone for each fish, and converting O.D. to nmol O 2 by multiplying 15.87. Final results were expressed as 5 nmol O 2 produced per 10 blood leucocytes.
2.2. Herbal extracts
2.6. Phagocytosis assay
Astragalus extract containing 40% Astragalus polysaccharide and Ganoderma extract containing 30% polysaccharides were commercial products from Xuancheng Baicao Plants Industry and Trade Ltd., China.
Phagocytic activity of blood leucocytes was determined spectrophotometrically by the method as described in Ref. [15]. This assay involves the measurement of congo red-stained yeast cells that have been phagocytised by cells. To perform the assay, 1000 ml of the leucocyte solution was mixed with 2000 ml of the congo redstained and autoclaved yeast cell suspension (providing a yeast cell: leucocyte ratio of 20:1). The mixtures were incubated at room temperature for 60 min. Following incubation, 1 ml ice-cold HBSS was added and 1 ml of histopaque (1.077) was injected into the bottom of each sample tube. The samples were centrifuged at 850 g for 5 min to separate leucocytes from free yeast cells. Leucocytes were harvested and washed twice in HBSS. The cells were then resuspended in 1 ml trypsin–EDTA solution (5.0 g l1 trypsin and 2.0 g l1 EDTA, Sigma) and incubated at 37 C overnight. The absorbance of the samples was measured at 510 nm using trypsin– EDTA as a blank.
2.3. Experimental design and sampling procedure All experiments were carried out in the recirculation system of the HAKI Institute, Szarvas, Hungary. Batches (eight groups) of 60 five-month old carp with an average initial weight of 62.8 5.40 g were held in 100 l fibreglass tanks. Water temperature and pH were constant (22–23 C; pH 8.5) during the experimental period, and dissolved oxygen was maintained at 80–90% of saturation. Water flow was maintained at 7 l/ min. Fish were fed ad libitum 6 times a day with a pelleted feed containing either A. radix (0.5%), or Ganoderma (0.5%), or combination of Astragalus (0.5%) and Ganoderma (0.5%) for 5 weeks. At the beginning of experiment four groups of fish were vaccinated (i.p. 0.1 ml/fish) using vaccine developed against A. hydrophila/A. salmonicida (Shering Plough, Essex, U.K.) and fed the same diets as described above. Control fish (negative control) and fish vaccinated only (positive control) were fed basal diets without supplements of herbs. Blood samples (five fish/group) were collected from caudal vein 1, 2, 3, 4 and 5 weeks after the 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.
2.7. Lysozyme assay Plasma lysozyme activity was measured spectrophotometrically according to the method as described in Ref. [16]. The lysozyme substrate was a 0.02% (w/v) suspension of Micrococcus lysodeikticus made up in phosphate buffer (0.05 M, pH 6.2). Lyophilised hen egg white lysozyme was used as a standard. A new standard curve was prepared for each assay. Standard solutions as well as samples were added to the substrate at 25 C. The results were expressed as mg ml1 equivalent of hen egg white enzyme activity.
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2.8. ELISA assay for determining antibody titres Antibody titres were measured against A. hydrophila. Serum antibody titres were determined using an indirect ELISA [17]. Plates were coated with suspensions of A. hydrophila adjusted to an OD610 of 1.0. Serum samples from vaccinated fish were serially diluted (twofold dilutions) in PBS, and a monoclonal antibody (anti-carp IgM monoclonal antibody, Aquatic diagnostic Ltd, Stirling, Scotland) was used as the second antibody. 2.9. Challenges with virulent pathogen The susceptibility of the fish fed herbs to a bacterial challenge was examined in vivo A. hydrophila (strain OB 212 Bacteriology Unit, Institute of Aquaculture University of Stirling, Scotland) was used as the challenge strain. Groups of 30 fish fed with herbs alone or in combination of herbs and vaccine were challenged 5 weeks after the start of treatments. Carp were injected intraperitoneally with a 1-day growth of virulent pathogen adjusted to 1 106 cells per fish. The fish were observed regularly at 4 h intervals for behavioural changes and mortalities. All the dead fish were removed and bacterial swabs were taken from the kidneys, and cultured on tryptone soya agar (TSA) plates. Bacteria isolated from the fish were confirmed as A. hydrophila using conventional methods. 2.10. Statistics Results are presented as the average (standard error) for five fish, and were compared at each time point using one-way ANOVA and Dunn’s multiple range tests (SigmaStat 3.2). Significant differences between experimental groups were expressed at a significant level of P < 0.05. Mortality (obtained at the end of experiment) in each treated group was compared statistically with control group using Chi-square test (SigmaStat 3.2). Significant differences between control and treated groups were expressed at a significant level of P < 0.001 and P < 0.05. 3. Results The effect of herbs and combination of herbs and vaccination on respiratory burst activities of isolated phagocytic cells is shown in Fig. 1. Respiratory burst activities were elevated in the group fed with Ganoderma on week 1 and in fish fed with Astragalus on week 3 compared to the negative control (Fig. 1a). Otherwise there was
no effect on respiratory burst activities in fish fed with herbs only during the whole experiment, although in some cases fish fed with Astragalus had lower values of respiratory burst activities, the differences were not significant comparing to control. When using the combination of herbs and vaccine in fish, elevation of respiratory burst activities was noticed on weeks 3 and 4 compared to control (Fig. 1b), whereas significant inhibition of respiratory burst activities was observed on week 3 in vaccinated groups fed with herbs compared to positive control (vaccinated only). At the end of experiment, vaccinated fish fed with Astragalus and combination of Astragalus and Ganoderma showed significantly higher values of respiratory burst activities compared to both controls – negative and positive (Fig. 1b). Elevated phagocytic activity was noted after 3 weeks in fish fed with herbs (Fig. 2a), while in vaccinated groups fed with Ganoderma and the combination of two herbs, elevated levels were noted on week 1 and at the end of experiment compared to both controls (Fig. 2b). On the second and the third weeks vaccinated fish fed with herbs phagocytosis was significantly lower when compared to both controls. There were no significant differences among groups fed with different herbs and fish treated with the combination of vaccine and herbs. Plasma lysozyme activities were significantly higher after 1 week in groups fed with herbs compared to control (Fig. 3a), whereas in vaccinated fish significant differences were measured only in groups fed with Ganoderma (second week), Astragalus (third week) and in fish fed with the combination of herbs a significant difference was observed at the end of experiment (Fig. 3b). Following vaccination all the vaccinated fish were found to have circulatory antibody 1 week post-vaccination (Fig. 4). Antibodies were not detected in control fish and fish fed with herbs only, whereas all fish immunised with the vaccine were responding well. Significant differences (P < 0.05) were noted between immunised fish and control throughout the whole experiment, the highest antibody titres were measured at the end of experiment. However, there were no significant differences among fish vaccinated only and fish treated with the combination of vaccine and herbs. Fish infected with A. hydrophila started to die after 36 h after injection. Mortalities due to A. hydrophila first occurred in control fish reaching almost 50% after 2 days post-infection and a cumulative mortality over the 6-day experimental period was 87% (Fig. 5a). Cumulative mortalities in fish fed with herbs were at significantly lower level comparing to control, reaching 58% and 60%. Fish treated with vaccine only started to die after 2 days post-infection,
Fig. 1. Respiratory burst activity of isolated blood cells in carp fed diets containing Astragalus radix, Ganoderma lucidum and combination of them (a) and vaccinated carp fed the same diets (b). Data is expressed as the mean of five fish SEM. Significant differences (P < 0.05) from the untreated control are indicated by asterisks. Significant differences (P < 0.05) from the vaccinated group only (positive control) are indicated by letter a.
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Fig. 2. Phagocytic activity of isolated blood cells in carp fed diets containing Astragalus radix, Ganoderma lucidum and combination of them (a) and vaccinated carp fed the same diets (b). Legends are the same as on Fig. 1.
cumulative mortality reached 50% at 4-day experimental period. The same tendency was observed in vaccinated fish fed with Astragalus. The significantly lowest mortality comparing to control was noted in vaccinated fish fed with combination of two herbs with a cumulative mortality 38%. 4. Discussion Both herb extract used alone or in combination with vaccine can modulate the non-specific defence mechanism. The present results showed that fish fed with Chinese herbs alone or in combination with vaccine significantly enhanced respiratory burst activity of phagocytic cells, phagocytosis and lysozyme activities in plasma. Similar results were obtained in jian carp (C. carpio var. Jian) and large yellow croaker (Pseudosciaena crocea) fed with combination of Astragalus root and Chinese Angelica root (Radix Angelicae sinensis) [18,19]. Extracellular respiratory burst activities in carp fed with herbs alone or in combination with vaccine were elevated. However, there were no significant differences among groups fed with different herbs and fish treated with combination of vaccine and herbs. Injection with glucan increased head kidney macrophages’ extracellular respiratory burst activity [20]. Extracellular activity was very high in rainbow trout fed with dietary glucan [21]. Rainbow trout fed with ginger (Zingiber officinale) extract had
significantly higher extracellular activity of phagocytic cells in blood, while in trout fed with nettle and mistletoe extracts the production of extracellular superoxide anion was on the same level as in the control fish [22]. In our previous study we could not detect such differences in respiratory burst activity in tilapia fed with Astragalus extract and in the case of fish fed with Scutellaria there was significant inhibition of extracellular superoxide anion production [10]. Other dietary components, namely vitamins E, C and A, have been shown to have little effect upon macrophage or phagocytic cells respiratory burst activity [23–25]. Changes in superoxide anion production due to the multiple injections of b-glucan were independent of different dosages [26]. Fishes treated with immunostimulants usually show enhanced phagocytosis. Several studies have reported that oral administration of yeast products (MacroGard; Vitastim; Saccharomyces cerevisiae) [21,27,28], chitin [29], plant extracts of four Chinese herbs (R. officinale, A. paniculata, I. indigotica, L. japonica) increased phagocytosis of white blood cells of crucian carp [6], naturally occurring tetrapeptide tuftsin enhanced phagocytosis in Labeo rohita [30]. In this study, phagocytic activity of blood leucocytes was increased in carp fed with herbs only on weeks 3 and 4, while in vaccinated fish, elevated phagocytic activities were measured on week 5 compared to control. Astragalus has been reported to increase the phagocytosis of the blood cells of soft-shelled turtles
Fig. 3. Plasma lysozyme activity in carp fed diets containing Astragalus radix, Ganoderma lucidum and combination of them (a) and vaccinated carp fed the same diets (b). Legends are the same as on Fig. 1.
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Fig. 4. Circulatory antibody titres against Aeromonas hydrophila in vaccinated carp fed diets containing Astragalus radix, Ganoderma lucidum and combination of them (a) and vaccinated carp fed the same diets (b). Legends are the same as on Fig. 1.
(Pelodiscus sinensis) [31] and alveolus macrophage of pneumoconiosis rats [32]. Astragalus polysaccharide (APS) is the major active component of A. radix. The role of APS on the specific and nonspecific immune responses has been reviewed [33]. APS modulates the functions of the immune cells including T cells, B cells, NK cells and macrophage [34]. Polysaccharides of Ganoderma have been reported to be effective in modulating immune functions, inhibiting tumour growth [35]. However, there is no data available on use of Ganoderma in fish. On the second and the third weeks vaccinated fish fed with herbs showed significantly lower phagocytosis when compared to both controls. In our previous study it was found that feeding tilapia with Scutellaria extract with higher doses (0.5 and 1.0%) caused reduction of function in phagocytic cells, while, when fish were fed with low dose of Scutellaria (0.1%) there was no stimulation on phagocytic activities [10]. Other studies, where higher doses of immunostimulants were used together with vaccine have been found to be even suppressive [36,37].
As a first line of defence, various peptides, such as lysozymes are present in serum where they prevent adherence and colonisation by microorganisms [38]. The supplemented feeds also enhanced the lysozyme activity in all the treatments. Similarly many authors have reported that administration of b-glucan enhances lysozyme activity in Atlantic salmon and turbot [26,39–42]. Other immunostimulants, such as chitosan, levamisole and chitin also elevated lysozyme levels in plasma of carp [43]. In our previous study with tilapia elevated lysozyme activity was measured when fish were fed with Astragalus, while in groups fed with Scutellaria there were no significant changes in plasma lysozyme activities [10]. Lysozyme is a cationic enzyme that breaks b-1,4 glycosidic acids and N-acetyl glucosamine in the peptidoglucan of bacterial cell walls. This action is known to attack mainly Gram positive bacteria as well as some Gram negative bacteria in conjunction with complement [38]. Robertson et al. [20] showed an increased protection against fish bacterial infection, which correlated with an increment in serum lysozyme levels, phagocytic activity and bactericidal activity of head kidney leucocytes. Such enhancement in lysozyme levels could also be correlated with enhanced phagocytic activity. When using herb extracts with vaccine the specific immune response was also elevated, although there were no significant differences among vaccinated only group and vaccinated fish fed with herb extracts. The antibody titres against A. hydrophila were not significantly different to the control in L. rohita [30]. While in tilapia it was found that medicinal plants Azadirachta indica enhanced the primary and secondary antibody responses in inverse dose dependant mode [44]. In this study after challenge with A. hydrophila, mortalities were significantly reduced in all groups compared to controls, with the lowest mortality in vaccinated fish fed with both herbs. It was considered that we would be able to use the herbs without the addition of a specific vaccine for inducing protection in carp against A. hydrophila. The dose of bacteria we used for challenge was very high, resulting in 90% mortality of control fish. Sharifpour [45] observed that intramuscular injection of A. hydrophila at a concentration of 5.3 106 cells ml1 caused infection in carp, and fish started to die within 12 h with one quarter of the injected fish dying between 12 and 24 h post-infection. Sarder et al. [46] injected tilapia intraperitoneally with A. hydrophila suspension (5 106 cells ml1) and fish started to die within 12 h. Mortality due to infections with A. hydrophila was reduced significantly by
Fig. 5. Cumulative mortalities after artificial challenging with Aeromonas hydrophila in carp fed diets containing Astragalus radix, Ganoderma lucidum and combination of them (a) and vaccinated carp fed the same diets (b). Significant differences from the untreated control are indicated by asterisks (*P < 0.05; **P < 0.001).
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