Aquacult Int (2011) 19:945–956 DOI 10.1007/s10499-010-9412-x
Dietary polysaccharide from Angelica sinensis enhanced cellular defence responses and disease resistance of grouper Epinephelus malabaricus Qing-Kui Wang • Cheng-Xun Chen • Yong-Jun Guo • Hai-Yun Zhao Jing-Feng Sun • Shen Ma • Ke-Zhi Xing
•
Received: 23 August 2010 / Accepted: 28 December 2010 / Published online: 19 January 2011 Ó Springer Science+Business Media B.V. 2011
Abstract The purpose of this study was to determine the effect of Angelica sinensis polysaccharide (ASP) supplemented in diet on the innate cellular immune response and disease resistance in grouper, Epinephelus malabaricus. Fish were fed diets containing different doses of ASP (0, 500 and 3,000 mg kg-1 diet) for 12 weeks. After 12 week feeding, the respiratory burst activity, phagocytic activity, and leukocytes proliferation in head kidney were assayed. The functional immunity in terms of cumulative mortality was also assessed by a challenge with live Edwardsiella tarda. Results showed that the respiratory burst activities in ASP-supplemented groups were increased significantly. The respiratory burst index was the highest in fish-fed 3000 mg kg-1 diet and the lowest in control. The phagocytic activities in ASP-supplemented groups were significantly higher than that of control. No significant difference in phagocytic activity was observed between ASP-supplemented groups. ASP stimulated the head kidney leukocytes proliferation significantly, despite the absence of lipopolysaccharide (LPS) or not. The cumulative mortalities of fish fed with 3000 mg ASP kg-1 diet were significantly lower than those fed with 500 mg ASP kg-1 diet and control diet after 96 h of challenge. In conclusion, dietary ASP enhanced some cellular immune parameters and disease resistance against E. tarda in grouper. Keywords Angelica sinensis Epinephelus malabaricus Leukocytes proliferation Phagocytic activity Protection Polysaccharide Respiratory burst Abbreviations ASP Angelica sinensis polysaccharide. LPS Lipopolysaccharide Q.-K. Wang S. Ma The Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, 5 Yu Shan Road, 266003 Qingdao, People’s Republic of China Q.-K. Wang C.-X. Chen Y.-J. Guo H.-Y. Zhao J.-F. Sun K.-Z. Xing (&) Tianjin Key Laboratory of Aqua-Ecology and Aquaculture, Department of Fishery Science, Tianjin Agricultural University, 22 Jin Jing Road, 300384 Tianjin, People’s Republic of China e-mail:
[email protected]
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RAS PBS GSRBCs FBS HBSS NBT DMSO RBI MTT ANOVA ROS
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Roots of Angelica sinensis (Oliv.) Diels Phosphate buffer saline Glutaraldehyde-fixed sheep red blood cells Fetal bovine serum Hanks’ balanced salt solution Nitroblue tetrazolium Dimethyl sulphoxide Respiratory burst index 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide One-way analysis of variance Reactive oxygen species
Introduction Intensive fish farming tends to adversely affect fish health status and increase their susceptibility to infections. As traditional therapeutic agents, antibiotics and chemotherapeutics used to control fish diseases can result in the development of drug-resistant bacteria, environmental pollution, and unwanted residues in aquaculture (Reilly and Ka¨ferstein 1997). Studies showed that immunostimulants could be an alternative to antibiotics and chemotherapeutics in aquaculture (Sakai 1999). Among the immunostimulants, botanical polysaccharides, with biocompatible, biodegradable, effective, and safe for the environment, have attracted a great deal of public attention for their profound and extensive effects on animals (Tzianabos 2000; Han et al. 2003; Lee and Jeon 2003; Schepetkin and Quinn 2006; Sun et al. 2009). The roots of Angelica sinensis (Oliv.) Diels (RAS), a well-known oriental herb belonging to the Umbelliferae family, have been used for thousands of years in traditional Chinese medicinal prescriptions (Hsu and Peacher 1976; Lao et al. 2004; Kim et al. 2005a, b). A. sinensis polysaccharide (ASP) is one of the major active ingredients in RAS, and it has extensive effects on animals, such as radioprotective effect (Mei et al. 1988; Hong et al. 2000, 2002), gastrointestinal protection (Ye et al. 2001), anti-ulcer (Ye et al. 2003), hematopoietic effect (Liu et al. 2010), antitumor activity (Zheng and Wang 2002; Yang et al. 2004; Cao et al. 2006; Song et al. 2008), antioxidative activity (Ling et al. 2002; Liu et al. 2003, 2004), antivirus activity (Hu et al. 2003; Jia et al. 2005), and immunomodulative activity (Yang et al. 2006; Chen et al. 2010). Although ASP can modulate the immune system of mammals and poultries, its immunostimulatory effects on aquatic animals have currently not been investigated. Groupers, Epinephelus spp., are economically important fish in tropical and subtropical waters around the world. E. malabaricus is one of the grouper species that is widely cultured in Asia. The intensive culture of E. malabaricus has dramatically developed in south China during the past decade. Various diseases result from intensive grouper culture occurred in China in recent years (Qin et al. 2004; Chen et al. 2004; Hu et al. 2005). Seeking effective botanical polysaccharides to enhance innate immunity and disease resistance of groupers is urgent in mariculture. This investigation aimed to study the effects of dietary ASP on the innate cellular immune responses. The respiratory burst, phagocytosis and proliferation in head kidney leukocytes, and disease resistance in E. malabaricus were performed.
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Materials and methods Extraction of ASP RAS was purchased from Da Ren Tang pharmacy in Tianjin, China. ASP was extracted as the following procedure: RAS was ground, boiled in distilled water, and centrifuged to remove undissolved matter. The polysaccharide in supernatant was precipitated with 80% ethanol and washed several times with ethanol to eliminate coloring matter. Protein in the sediment was removed using Sevag method (Staub 1965; Zhao et al. 2009). Briefly, the sediment was dissolved with distilled water, mixed with Sevag reagent, and stirred for 30 min to remove protein. Then, the mixture was centrifuged and upper polysaccharide solution was collected. The polysaccharide solution was deproteinized with Sevag reagent for six or seven times until there was no white layer between polysaccharide solution and Sevag reagent and no specific absorbance at 260 nm (nucleic acid) and 280 nm (protein). The resulting polysaccharide solution was dialyzed with semipermeable membrane (MW: 8,000–14,400, BioSharp, USA), deposited with ethanol, and lyophilized for later use. Total sugar, reducing sugar, uronic acid, and protein in the resulting ASP were assayed. Total sugar was measured by phenol-sulfuric acid method (Dubois et al. 1956) with glucose as standard. Reducing sugar was measured as described by Wen et al. (2005, pp. 122–123). Uronic acid was measured as described by Meseguer (1988, pp. 285–291) and Yu et al. (2009, pp. 533–535). Protein was measured using Bradford method (1976, pp. 242–254), using bovine serum albumin as standard. Preparation of diets Three diets containing different levels of ASP were prepared as described in Table 1. The basal diet containing 3% cellulose served as the control. ASP was added to the test diets at levels of 500 and 3,000 mg kg-1 diet at the expense of cellulose. The ingredients were ground to pass through a 60-mesh screen. Diets were prepared by mixing the dry ingredients with oil and then adding water until a stiff dough resulted. Each diet was then extruded through a mincer with a die. The resulting strands were shadow-dried, broken up, sieved into pellets (3 mm 9 5 mm), and stored in plastic bags at 4°C until use. Fish and rearing conditions E. malabaricus were obtained from the fishfarm of Tianjin Haifa Seafood Co. Ltd. (Tianjin China). Before commencing the feeding trial, fish were acclimated for 3 weeks. At the beginning of the experiment, a total of 225 apparently healthy fish were individually weighed (average weight 158.64 ± 2.14 g) and randomly assigned to nine flow-through tanks of 139 l capacity. Seawater flow rate was 2 000 ml min-1 and was continuously aerated. The light intensity was less than 500 lx and the photoperiod maintained at 12 h light/12 h dark. During the 12-week feeding trial, the water temperature ranged between 22 and 25°C and the salinity ranged between 32 and 34%. The fish were hand fed twice daily at 08:30 h and 16:30 h. Uneaten food and fecal matter, if any, were removed before each feeding. Initial feeding was in excess (approximately 3% of body weight), and feeding rates were thereafter adjusted according to prior feeding responses.
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Table 1 Composition of the basal diet (g kg-1) for Epinephelus malabaricus Ingredients
ASP in diet (mg kg-1) 0 (Control)
500
3,000
Fish meal
580
580
580
Soybean meal
170
170
170
Shrimp meal
50
50
50
Yeast meal
50
50
50
Wheat flour
67
67
67
Fish oil
10
10
10
Soybean oil
10
10
10
0
500
3,000
ASP Cellulose
3
2.5
0
Vitamin mixturea
20
20
20
Mineral mixtureb
40
40
40
Proximate analysis of basal diet (mean of a triplicate measurement): moisture, 5.6%; ash,13.5%; crude protein, 50.3%; crude lipid, 10.4% a Vitamin mixture (mg g-1 mixture): thiamin hydrochloride, 2.5; riboflavin, 10; calcium pantothenate, 25; nicotinic acid, 37.5; pyridoxine hydrochloride, 2.5; folic acid, 0.75; inositol, 100; L-ascorbyl-2-monophosphate Mg, 5; choline chloride, 250; menadione, 2; alpha-tocopheryl acetate, 5; retinyl acetate, 1; cholecalciferol, 0.0025; biotin, 0.25; vitamin B12, 0.05. All ingredients were diluted with wheat flour b Mineral mixture (mg g mixture): calcium lactate, 327; K2PO4, 239.8; CaHPO42H2O, 135.8; MgSO47H2O, 132; Na2HPO42H2O, 87.2; ferric citrate, 29.7; ZnSO47H2O, 3; CoCl26H2O, 1; MnSO4H2O, 0.8; KI, 0.15; CuSO45H2O, 80; Na2O3Se, 0.04. All ingredients were diluted with wheat flour
Experimental design Fish were fed with the three diets for 12 weeks. Each dietary treatment was carried out in triplicate, and 25 fish were placed in each tank. At the end of the feeding trial, 12 fish in each treatment (four fish from each tank) were sampled randomly to assay the respiratory burst, phagocytic activity, and proliferation in head kidney leukocytes. Another 27 fish in each treatment (nine fish from each tank, with even weight distribution) were sampled, distributed to nine tanks (nine fish per tank), and challenged with Edwardsiella tarda. Each fish was intraperitoneally injected with 200 ll E. tarda stock suspension (9 9 108 cfu ml-1). Challenged fish were observed for seven days. During the challenge period, the fish were fed twice a day with their respective diets. Dead fish was removed each day, and cumulative mortalities were calculated. The tanks used for the challenge test were the same as those used for feeding test. In a preliminary challenge test, fish were reared in the same tanks and intraperitoneally injected with different dosages of E. tarda. Control fish were injected with sterile phosphate buffer saline (PBS, 0.15 M, pH 7.2). Fish injected with E. tarda died, and mortalities varied with the dosages of the bacteria. No mortality occurred in control fish during the preliminary test. Culture of E. tarda E. tarda, which was separated and identified from diseased E. malabaricus cultured in the local fish farm, was cultured on nutrient agar for 24 h at 28°C before being washed with
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sterile PBS (0.15 M, pH 7.2). The bacterial pellets were resuspended in PBS at 9 9 108 cfu ml-1 as the stock bacterial suspension for the susceptibility study in grouper. Preparation of glutaraldehyde-fixed sheep red blood cells (GSRBCs) GSRBCs were prepared according to the method described by Wu et al. (2008, p. 8885) with some modifications. Sheep red blood cells mixed with Alsever’s solution (sodium citrate 0.8 g, citric acid 0.055 g, sodium chloride 0.42 g, and glucose 2.05 g were dissolved in 100 ml distilled water and sterilized at 120°C for 20 min.) were washed three times with PBS (0.15 M, pH 7.2). Two microliters of 10% sheep red blood cells (v/v) was mixed with 2 ml PBS and 1 ml 2.5% glutaraldehyde. The mixture was incubated at room temperature for 2 h with regular gentle agitation. Then, the sheep red blood cells were washed three times, resuspended with PBS, and stored at 4°C for later use. Prier to use, GSRBCs were resuspended in RPMI maintenance medium (mRPMI, RPMI-1640 (Gibco) with 5% fetal bovine serum (FBS), 100 IU ml-1 ampicillin (Genview), 0.1 mg ml-1 streptomycin sulfate (Genview)), counted in a Neubauer hemocytometer, and adjusted to 1.8 9 107 cells ml-1. Isolation of head kidney leukocytes Head kidney of each sampled fish was processed individually. Thus, there were twelve replicates in each treatment for the leukocytes preparation. Head kidney leukocytes were isolated according to the method described by Li et al. (2007, p. 982) with some modifications. Briefly, head kidney was excised, cut into fragments, and transferred to 5 ml of RPMI-1640 contained 20 IU ml-1 heparin. Cell suspensions were obtained by forcing fragments of the organ through a 150 lm stainless steel mesh with a syringe rod. After two washes, the cells were resuspended in mRPMI, placed on a 1.020/1.059 Percoll (Sigma) density gradient, and centrifuged at 840g for 10 min at 4°C. Subsequently, the isolated cells were harvested at the Percoll interface and washed twice with mRPMI. Cell viability was examined by trypan blue exclusion in a Neubauer hemocytometer. In all samples, this was greater than 95%. The leukocytes including neutrophils and macrophages were adjusted to 2 9 107 cells ml-1 of mRPMI for later use. A turbidity comparator (vitek densichek M004120, Italy) was used to adjust the leukocytes concentration. Respiratory burst Respiratory burst activity produced by phagocytes in the head kidney was measured based on the method described by Cheng et al. (2007, p. 197) with minor modifications. Briefly, a 100 ll aliquot of leukocytes suspension (5 9 106 cells ml-1) was placed in flat-bottomed 96-well cell culture plate (Greiner Bio-one, Germany) and incubated for 2 h at 27°C. Then, the non-adherent cells were removed by washing the wells with Hanks’ balanced salt solution (HBSS, Beijing Dingguo Changsheng Biotech. Co. Ltd., China). A 100 ll aliquot of lipopolysaccharide (LPS, Sigma) at 100 lg ml-1 in HBSS was added to four wells to assay the stimulated respiratory burst activity, and 100 ll HBSS was added to another four wells to assay the basal respiratory burst. One hundred microliters of nitroblue tetrazolium (NBT, 2 mg ml-1, Sigma) was added and incubated at 27°C for 30 min. Then, the HBSS was discarded and the reaction was stopped by adding methanol. After washing twice with 70% methanol, the formazan formed in each well was dissolved by adding 120 ll of 2 M
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KOH and 140 ll of dimethyl sulphoxide (DMSO). The NBT reduction was measured using a microplate reader (VarioskanFlash) at 690 nm. Cells from each fish were in eightfold wells. Respiratory burst activity was expressed as NBT reduction. Respiratory burst index (RBI) was calculated by subtracting O.D. values of the basal wells from the values of the stimulated wells, expressed as O.D690 nm per 5 9 106 cells. Phagocytic activity Phagocytic activity was measured according to the method described by Gebran et al. (1992, p. 255) and Wu et al. (2003, p. 223). A 100-ll aliquot of leukocytes suspension was placed in wells (eight replicates for each sample) of a 96-well cell culture plate and incubated for 3 h in a humidified atmosphere containing 5% CO2. Non-adherent cells were removed by washing the cultures twice with mRPMI. The remaining monolayer was incubated for 24 h in mRPMI in a humidified atmosphere containing 5% CO2. After incubation, half of the cells from the same sample (four wells) were fixed with 0.5% (v/v) paraformaldehyde (Beijing Dingguo Changsheng Biotech. Co. Ltd., China) in PBS for 60 min and treated as the control. All wells were then washed once with mRPMI, and 100 ll GSRBCs (1.8 9 107 cells ml-1 in mRPMI) was added to each well. Phagocytosis was allowed to proceed for 30 min, and the wells were washed three times to remove noningested and non-attached GSRBCs. Each well was incubated with 100 ll 0.2 M Tris–HCl (pH 7.4 in 6 M urea) and left for 5 min before the addition of 100 ll 2,4-diaminofluorene substrate solution (Sigma) (containing 0.27% H2O2). The plates were incubated for 5 min, and the optical density (O.D.) at 608 nm was determined using the microplate reader mentioned above. Phagocytosis activity was calculated by subtracting O.D. values of the control wells from the values of the experimental wells. The mean cell numbers per well were determined by counting the numbers of nuclei released after replacing the medium with 100 ll lysis buffer containing 0.1 M citric acid, 1% Tween 20 (Genview), and 0.05% crystal violet (Secombes 1990). The cell count in each well was approximately 2 9 105. Leukocytes proliferation Leukocytes proliferation was measured according to the method described by Wu et al. (2003, p. 223) with minor modifications. The head kidney leukocytes were resuspended in proliferation medium (RPMI-1640, containing 10% FBS, 2% heat-inactivated grouper sera, 100 IU ml-1 ampicillin, and 0.1 mg ml-1 streptomycin sulfate). Sera collected from non-experiment grouper were pooled and inactivated at 45°C for 30 min. A 90 ll aliquot of proliferation medium containing 5 9 105 cells and 10 ll LPS (100 lg ml-1 mRPMI) or HBSS was added to wells of a 96-well cell culture plate and incubated for 24 h or 48 h at 27°C in a humidified atmosphere with 5% CO2. A 20 ll aliquot of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT, 1 mg ml-1 HBSS) was added to each well and incubated for 4 h at 27°C. The plate was then centrifuged at 500g for 10 min at 4°C. The supernatant was discarded, and the formazan crystals in each well were dissolved by adding 200 ll DMSO and 25 ll of glycine buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5). The contents of the wells were then thoroughly mixed, and the O.D. at 550 nm of the resulting suspension was measured in the microplate reader mentioned above 10 min later.
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Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA), and any significant difference was determined at the 0.05 level by Duncan’s multiple range tests. The analyses were carried out with the SPSS software (version 16.0).
Results Total sugar, reducing sugar, uronic acid, and protein in ASP Total sugar, reducing sugar, uronic acid, and protein in resulting ASP were 76.72 ± 1.70%, 4.02 ± 0.12%, 13.82 ± 0.41%, and 4.87 ± 0.05%, respectively. Respiratory burst and phagocytic activity The basal respiratory burst in fish-fed 500 and 3,000 mg kg-1 diet increased significantly by 107% and 68%, when compared to that of control. The LPS-stimulated respiratory burst in fish-fed 500 and 3,000 mg kg-1 diet increased significantly by 140% and 160%, when compared to that of control (Fig. 1). RBIs among treatments were significantly different, of which the highest was found in fish-fed 3000 mg ASP kg-1 and the lowest in control. The phagocytic activity of fish received ASP at 500 and 3,000 mg kg-1 diet increased significantly by 173% and 171%, when compared to that of control after 12-week feeding (Fig. 2). No significant difference in phagocytic activity was observed between the ASPsupplemented groups. Leukocytes proliferation
Respiratory burst activity (O.D. 690 nm)
ASP stimulated the head kidney leukocytes proliferation significantly, despite the absence of LPS or not (Fig. 3). The difference between LPS-stimulated and unstimulated leukocytes proliferation in the same treatment after 48 h incubation was greater than that after
basal LPS-stimulated RBI
0.1 0.09 0.08 0.07
b b b
0.06
b
0.05 0.04
a
c
a
0.03
b
0.02
a
0.01 0
0
500
3000 -1
ASP (mg kg )
Fig. 1 Respiratory burst activity in head kidney leukocytes of Epinephelus malabaricus. basal: Leukocytes were not stimulated by lipopolysaccharide during incubation. LPS-stimulated: Leukocytes were stimulated by lipopolysaccharide during incubation. RBI: respiratory burst index, calculated by subtracting O.D. values of the basal wells from the values of the stimulated wells. Each bar represents mean value from twelve fish with standard error. Data at the same type of bar with different letters are significantly different (P \ 0.05) among treatments
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Phagocytic activity
0.6
b
b
500
3000
0.5 0.4 0.3
a
0.2 0.1 0
0
ASP (mg kg -1) Fig. 2 Phagocytic activity in head kidney leukocytes of Epinephelus malabaricus. Each bar represents mean value from twelve fish with standard error. Data with different letters are significantly different (P \ 0.05) among treatments
Leucocyte proliferation (O.D. 550 nm)
(a)
unstimulated
0.35
LPS-stimulated
0.3
b
b
b
b
0.25 0.2
a
a
0.15 0.1 0.05 0
0
500
3000
ASP (mg kg -1 )
Leucocytes proliferation (O.D. 550 nm)
(b)
0.7
unstimulated
0.6
LPS-stimulated
b b c
a 0.5 0.4
b a
0.3 0.2 0.1 0
0
500
3000
ASP (mg kg -1 )
Fig. 3 Leukocytes proliferation of Epinephelus malabaricus, incubated for 24 h (a) and 48 h (b). unstimulated: leukocytes were not stimulated by lipopolysaccharide during incubation. LPS-stimulated: leukocytes were stimulated by lipopolysaccharide during incubation. Each bar represents mean value from twelve fish with standard error. Data at the same type of bar with different letters are significantly different (P \ 0.05) among treatments
24 h incubation. After 48 h incubation, the unstimulated leukocytes proliferation in fish-fed ASP at 500 and 3,000 mg kg-1 diet was enhanced by 35 and 72%, when compared to that of control. The LPS-stimulated leukocytes proliferation in fish-fed ASP at 500 and 3,000 mg kg-1 diet was enhanced by 20 and 29%, when compared to that of control after 48 h incubation.
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Cumulative mortality In the challenge test, death occurred 48 h after bacterial injection (Fig. 4). The cumulative mortalities in fish-fed 500 and 3,000 mg ASP kg-1 diet were significantly lower than that of control after 72 h. The cumulative mortality in 3,000 mg ASP kg-1 diet group was significantly lower than that in 500 mg ASP kg-1 diet group and control after 96 h of challenge. After 96 h of challenge, the cumulative mortalities in 500 and 3000 mg ASP kg-1 diet groups decreased by 18.5% and 40.7%, respectively, compared to that of control, and maintained stable afterward.
Discussion
Cumulative mortality
Studies showed that botanical polysaccharides can enhance the innate immunity and resistance to pathogens of aquatic animals (Huang et al. 2006; Cheng et al. 2007; Yeh et al. 2008; Wang et al. 2009). The present results clearly indicate that dietary ASP boosts the cellular immunity of E. malabaricus, when assayed by respiratory burst activity, phagocytic activity, and leukocytes proliferation. Dietary ASP can also enhance disease resistance against E. tarda in grouper. High dietary ASP supplement level (3,000 mg kg-1 diet) is more effective than low level (500 mg kg-1 diet). Respiratory burst, phagocytosis, and leukocytes proliferation are important indicators of non-specific defence in aquatic animals. As a primitive defence mechanism in fish, phagocytosis has been recognized as an important cellular component of the innate immune system against invading microorganisms of fish (MacArthur and Fletcher 1985). Phagocytes can engulf microorganisms and kill them principally by the production of reactive oxygen species (ROS) during the so-called respiratory burst. Fish treated with polysaccharide like chitin (Sakai et al. 1992), chitosan (Siwicki et al. 1994), sodium alginate (Yeh et al. 2008), and PS-K isolated from Coriolus versicolor (Park and Jeong 1996) showed increased phagocytosis as well as respiratory burst activity (Sakai 1999). Figure 1 showed that dietary ASP enhanced both basal and LPS-stimulated respiratory burst in head kidney leukocytes significantly. This suggested that ASP has a priming effect and the cells could still respond to further stimulation. Similar results were found in turbot (Psetta maxima L.) phagocytes, which were pre-incubated with polysaccharides extracted
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
a a
a
a
a
a
a
a
a
a
b
b
b
b
144
168
b a
c
a 0 500 3000
24
48
72
96
120
Hours post challenge Fig. 4 Cumulative mortality of Epinephelus malabaricus after i.p. injection of Edwardsiella tarda. 0: fish fed with 0 mg ASP kg-1 diet. 500: fish fed with 500 mg ASP kg-1 diet. 3000: fish fed with 3000 mg ASP kg-1 diet. Each bar represents mean value from 27 fish with standard error. Data at the same time with different letters are significantly different (P \ 0.05) among treatments
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from seaweeds Ulva rigida and Chondrus crispus (Castro et al. 2004). However, the basal respiratory burst is not the higher the better. Over enhanced basal respiratory burst could result in cells being exhausted and unable to respond to further stimulation by pathogens (Castro et al. 2004). In this paper, LPS was used in leukocytes respiratory burst and proliferation assays to examine whether it has synergy or antagonism effect with ASP on head kidney leukocytes and whether ASP contributes to the humoral immunity of E. malabaricus. Results showed that LPS has synergy effect with ASP on respiratory burst and proliferation of head kidney leukocytes (Figs. 1, 3). The LPS-responsive lymphocytes belong to the B-cell subpopulation, which involves humoral immunity (Sizemore et al. 1984). Data in Fig. 3 implied that oral administration of ASP might contribute to the humoral immunity of grouper. In the same treatment, the difference between LPS-stimulated and unstimulated leukocytes proliferation after 48 h incubation was greater than that after 24 h incubation. Maybe the stimulating potential of LPS exerted more after 48 h incubation than after 24 h incubation. In conclusion, dietary ASP enhanced some cellular immune parameters and disease resistance against E. tarda in grouper. Further research of ASP on grouper should be focused on humoral immunity and immune-related gene expression to reveal its innate immunomodulation mechanism. Acknowledgments This work was financially supported by National High-Tech Research and Development Program of China (2007AA10Z232361; 2007AA10Z23236103), National Spark Program of China (2007EA610021), scientific program of Tianjin city (2004zd10; 06TXTJJC14201; 06YFGZNC08100; 07ZHXHNC05400; 033121411; 08JCYBJC27800; 2009D05), and Shandong province (200802040). Many thanks to the staff, especially Shu-Sen Zhang, Yong-Hai Yang, Zhi-Min Lv, Jin-Cheng Hu, Ya-Guang Zhu, and Zhen-Hui Wang, in Tianjin Haifa seafood Co. Ltd. for their assistance during the rearing of grouper. The authors would like to thank professor Dong-Qing Bai, Department of Fishery Science, Tianjin Agricultural University for her donation of virulent pathogen Edwardsiella tarda.
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