Fish & Shellfish Immunology 42 (2015) 353e362
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Dietary tryptophan and methionine as modulators of European seabass (Dicentrarchus labrax) immune status and inflammatory response nio Afonso a, b, Marina Machado a, b, *, Rita Azeredo a, c, Patricia Díaz-Rosales a, Anto a a, c a, * Helena Peres , Aires Oliva-Teles , Benjamín Costas a b c
~o Marinha e Ambiental (CIIMAR/CIMAR), Universidade do Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal Centro Interdisciplinar de Investigaça Instituto de Ci^ encias Biom edicas Abel Salazar (ICBAS-UP), Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal Departamento de Biologia, Faculdade de Ci^ encias da Universidade do Porto (FCUP), 4169-007 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 August 2014 Received in revised form 14 November 2014 Accepted 15 November 2014 Available online 25 November 2014
Amino acids regulate key metabolic pathways important to immune responses and their nutritional supply may increase synthesis of immune-related proteins. The present study aimed to evaluate the effects of dietary supplementation of tryptophan and methionine on European seabass (Dicentrarchus labrax) cellular and humoral status. The immunomodulatory effects of tryptophan and methionine during an inflammatory insult was also evaluated after intraperitoneal injection with inactivated Photobacterium damselae subsp. piscicida (Phdp). A practical isonitrogenous (45% crude protein) and isolipidic (16% crude fat) diets was formulated to include fish meal and a blend of plant feedstuffs as protein sources and fish oil as the main lipid source (CRL diet). Two other diets were formulated similar to the control but including L-tryptophan or Lmethionine at 2 the requirement level (diets TRP and MET, respectively). European seabass weighing 275 g were fed the experimental diets for a period of 15 days before being sampled (trial 1). Then, fish were subjected to a peritoneal inflammation by intraperitoneally injecting UV killed Phdp (106 colony forming units ml1) and sampled following 4 and 24 h post-injection (trial 2). Fish injected with a saline solution served as control. The haematological profile, peripheral cell dynamics and several plasma immune parameters were determined in trials 1 and 2, whereas cell migration to the inflammatory focus was also determined in trial 2. MET positively affected European seabass immune status by improving the peripheral leucocyte response, complement activity and bactericidal capacity, a stronger cellular recruitment to the inflammatory focus, and higher plasma peroxidase and bactericidal activities. TRP also seemed to improve immunostimulation, as there was a trend to augment both cell-mediated immunity and humoral capacity. However, TRP failed to improve an inflammatory response, verified by a decrease in blood phagocyte numbers and lack of immune cells recruitment. In summary, it is confirmed that MET has a pronounced influence on the innate immune response to inflammation, which is more evident than TRP, and raises its potential to incorporate in functional feeds to be used in prophylactic strategies against predictable unfavourable events. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Amino acids Immunomodulation Inflammation Immune response Photobacterium damselae subsp. piscicida
1. Introduction
~o Marinha * Corresponding authors. Centro Interdisciplinar de Investigaça e Ambiental (CIIMAR/CIMAR), Rua dos Bragas 289, 4050-123 Porto, Portugal. Tel.: þ351 223401850; fax: þ351 223401838. E-mail addresses: [email protected] (M. Machado), [email protected] (B. Costas). http://dx.doi.org/10.1016/j.fsi.2014.11.024 1050-4648/© 2014 Elsevier Ltd. All rights reserved.
European seabass, Dicentrarchus labrax (Linnaeus 1758), has a long history in Mediterranean aquaculture where it is produced under intensive, semi-intensive or extensive systems [1]. Farming practices alter the natural equilibrium between host and pathogens found in the wild, favouring the emergence of diseases and posing a major problem for the aquaculture industry [2]. Bacteria are the
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most common group of pathogens in cultured fish, some of them acting as primary pathogens, but others are opportunistic and cause disease in damaged hosts [3]. Photobacterium damselae subsp. piscicida (Phdp), the etiological agent of photobacteriosis (formerly pasteurellosis), has been a major cause of disease in species such as gilthead seabream (Sparus aurata) and European seabass [4e6]. Several types of commercial vaccines against photobacteriosis are available in the market, but their efficacy is dependent on the fish species, fish size, vaccine formulation and combined use with immunostimulants [7]. Fish have a well-developed immune system, composed by innate and acquired humoral and cell-mediated mechanisms which are responsible for resistance to diseases [8]. The innate immune system is the more primitive system of protection, as it is present in some way in all organisms, and it is well developed in teleost fish [9]. When external barriers are breached an inflammatory response is initiated in order to resolve the infection or wound, repairing the damage and re-establishing homeostasis [10]. This response to infection is characterized by an influx of phagocytic cells to the infection focus, which produce a number of bactericidal instruments capable of degrading and kill invading organisms [11,12]. Both macrophages and neutrophils efficiently attack pathogens by generating lysozyme, complement factors and anti-proteases compounds [10,13]. Furthermore, these cells produce high amounts of reactive oxygen species (ROS) such as the superoxide anion when in contact with bacteria [14]. The ideal inflammatory response is rapid and destructive, yet specific and self-limiting [9] and it is likely that nutrients influence several aspects of the immune system [15]. Therefore, modulation of the immune system of aquaculture fish may be achieved through nutritional strategies [16,17]. Li et al. [18] suggested that diet supplementation with amino acids (AA) may improve fish performance. AA are primarily the building blocks for protein synthesis but are also important regulators of key metabolic pathways with important regulatory roles in growth and immune responses [19,20]. Indispensable AA such as methionine and tryptophan have important roles in inflammation and both are currently available as feed grade, thus their potential use in functional feeds can be done at acceptable costs [21]. Methionine was shown to modulate the mammalian immune system by improving both cellular and humoral immune responses [22]. Methionine may also affect cell proliferation and differentiation of lymphocytes since it is involved in polyamine biosynthesis [21]. It is also precursor of glutathione (GSH), a molecule involved in reducing ROS and thus protecting cells from oxidative stress during the inflammatory process [22]. On the other hand, tryptophan is the precursor of both serotonin (5-hydroxytryptamine, 5-HT) and melatonin (N-acetyl-5methoxytryptamine), which are potent scavengers of damaging free radicals and play key roles on cell redox balance by promoting the activity of antioxidative enzymes [23]. Additionally, tryptophan catabolism by indoleamine 2,3-dioxygenase (IDO) seems to have a major immunomodulatory effect during inflammation in mammals [24]. In fact, a progressive decline in plasma tryptophan concentration is observed during inflammation, thereby affecting both macrophages and lymphocyte functions [25]. Recent studies have also reported that a deficiency of dietary AA reduces their concentration in plasma compromising the immune system [15,20]. Hence, dietary supplementation of key AA may represent a strategy to improve the fish immune system and welfare, being a less costly and dramatic solution than the use of antibiotics [19]. Few studies were performed to elucidate the effect of methionine and tryptophan on the innate immune response, and most of them were performed in mammals. Therefore, the present study aimed to assess the effects of dietary supplementation with methionine or tryptophan on the European seabass cellular and
humoral innate immune status, and to investigate their immunomodulation potential during the inflammatory response. 2. Material and methods 2.1. Diets A practical isonitrogenous (45% crude protein) and isolipidic (16% crude fat) diets was formulated to include fish meal and a blend of plant feedstuffs as protein sources and fish oil as the main lipid source (CRL diet). The CRL diet was formulated to include an indispensable AA level meeting the ideal protein indispensable AA pattern estimated for European seabass [26]. Two other diets were formulated similar to the control but including L-tryptophan or Lmethionine at 2 the tryptophan or methionine levels of control diet (diets TRP and MET, respectively). All dietary ingredients were finely ground, well mixed and dry pelleted in a laboratory pellet mill (California Pellet Mill, Crawfordsville, IN, USA). Ingredient composition and proximate analysis of the experimental diets are presented in Table 1 and the AA profile of the diets is presented in Table 2. Proximate analysis of the diets was performed according to Association of Official Analytical Chemists methods [27] and AA analysis according to Peres and Oliva-Teles [28]. Briefly, diet samples were hydrolyzed for 23 h with 6 N hydrochloric acid at 110 C under nitrogen atmosphere and derivatized with phenylisothiocyanate (PITC; Pierce) reagent before separation by high performance liquid chromatography (HPLC) in a Waters Reversed-
Table 1 Composition and proximate analysis of the experimental diets. Experimental diets
Ingredients (% DM) Fish meala Soybean mealb Corn glutenc Wheat glutend Wheat meale Fish oil Vitamin premixf Choline chloride (50%) Mineral premixg Binderh Agar Dibasic calcium phosphate L-Methionine L-Tryptophan Proximate analyses (% dry weight) Dry matter (%) Crude protein Crude lipid Ash
CRL
TRP
MET
34.1 15.0 10.0 5.0 16.7 13.9 1.0 0.5 1.0 1.0 1.0 0.8 e e
33.5 15.0 10.0 5.0 16.6 13.4 1.0 0.5 1.0 1.0 1.0 0.9 e 0.5
33.2 15.0 10.0 5.0 16.2 14.0 1.0 0.5 1.0 1.0 1.0 0.9 1.2 e
95.2 44.9 15.5 10.5
94.3 45.0 16.9 10.5
94.9 45.2 16.5 10.4
a Pesquera Centinela, Steam Dried LT, Chile (CP: 71.4%; CL:9.3%). Sorgal, S.A. Ovar, Portugal. b Soybean meal (CP: 54.9%; CL:2.1%), Sorgal, S.A. Ovar, Portugal. c Corn gluten (CP: 72.2%; CL: 2.0%), Sorgal, S.A. Ovar, Portugal. d Wheat gluten (CP: 84.4%; CL: 2.1%), Sorgal, S.A. Ovar, Portugal. e Wheat meal (CP: 13.9%; CL: 1.8%), Sorgal, S.A. Ovar, Portugal. f Vitamins (mg kg1 diet): retinol, 18,000 (IU kg1 diet); calciferol, 2000 (IU kg1 diet); alpha tocopherol, 35; menadion sodium bis., 10; thiamin, 15; riboflavin, 25; Ca pantothenate, 50; nicotinic acid, 200; pyridoxine, 5; folic acid, 10; cyanocobalamin, 0.02; biotin, 1.5; ascorbyl monophosphate, 50; inositol, 400. g Minerals (mg kg1 diet): cobalt sulphate, 1.91; copper sulphate, 19.6; iron sulphate, 200; sodium fluoride, 2.21; potassium iodide, 0.78; magnesium oxide, 830; manganese oxide, 26; sodium selenite, 0.66; zinc oxide, 37.5; dicalcium phosphate, 8.02 (g kg1 diet); potassium chloride, 1.15 (g kg1 diet); sodium chloride, 0.4 (g kg1 diet). h Aquacube. Agil, UK.
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to duplicate tanks and fish were fed by hand twice a day, to apparent visual satiety.
Table 2 Amino acid composition (g 16 g1 N) of the experimental diets. Experimental diets
Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Tyrosine Threonine Tryptophan Valine Aspartic Acid Glutamic Acid Serine Glycine Alanine Proline
CRL
TRP
MET
7.74 3.78 5.05 9.73 6.66 2.57 5.39 4.04 4.68 1.12 5.38 8.20 16.39 4.15 3.98 4.89 4.99
7.11 3.63 4.64 9.71 6.96 2.42 5.16 3.96 4.38 2.24 5.10 7.54 16.14 4.42 4.18 4.92 5.50
7.02 4.12 4.71 9.49 6.68 4.95 5.16 3.98 4.51 1.10 5.12 7.60 16.09 4.29 4.20 4.97 4.84
Phase Amino Acid Analysis System equipped with a PicoTag column, using the conditions described by Cohen et al. [29]. External standards were prepared along with the samples and norleucine was used as an internal standard. Chromatographic peaks were analysed with the Breeze software (Waters). Tryptophan was measured by a spectrophotometric method as described by De Vries et al. [30]. 2.2. Bacterial growth and inoculum preparation Photobacterium damselae subsp. piscicida (Phdp), strain PP3, was kindly provided by Dr. Ana do Vale (Institute for Molecular and Cell Biology, University of Porto, Portugal) and isolated from yellowtail (Seriola quinqueradiata; Japan) by Dr Andrew C. Barnes (Marine Laboratory, Aberdeen, UK). Bacteria were routinely cultured at 22 C in tryptic soy broth (TSB) or tryptic soy agar (TSA) (both from Difco Laboratories) supplemented with NaCl to a final concentration of 1% (w/v) (TSB-1 and TSA-1, respectively) and stored at 70 C in TSB-1 supplemented with 15% (v/v) glycerol. To prepare the inoculum for injection into the fish peritoneal cavities, stocked bacteria were cultured for 48 h at 22 C on TSA-1 and then inoculated into TSB-1 and cultured overnight at the same temperature, with continuous shaking (100 rpm). Exponentially growing bacteria were collected by centrifugation at 3500 g for 30 min, resuspended in sterile HBSS and adjusted to 1 106 colony forming units (cfu) ml1 according to Costas et al. [31]. Bacteria were then killed by UV exposure for 2 h. Loss of bacterial viability following UV was confirmed by plating resulting cultures on TSA-1 plates and failing to see any bacterial growth. 2.3. Experimental design This study was directed by trained scientists (following FELASA category C recommendations) and conducted according to the guidelines on the protection of animals used for scientific purposes from the European directive 2010/63/UE. The trials were performed at the Marine Zoological Station, Porto, Portugal with European seabass weighing 274.7 ± 20.4 g that were obtained in a commercial fish farm (Maresa, Huelva, Spain). Fifteen fish were randomly distributed into each of 6 fibreglass tanks of 300 L water capacity in a recirculating aerated seawater system supplied with a continuous flow of filtered seawater. A 12 h light/12 h dark photoperiod was adopted; dissolved oxygen was maintained at 90%, water temperature was 25 ± 0.5 C, and salinity averaged 35 ± 1%0. The experimental diets were randomly assigned
2.3.1. Trial 1 The feeding trial lasted 15 days, to assess the effects of short term dietary supplementation with methionine or tryptophan on European seabass cellular and humoral immune status. It has been previously reported that 14 days of feeding diets supplemented with individual AA are enough to modulate physiological and immune responses in fish [17]. At the end of the trial, after a fasting period of 18 h, three fish per tank were randomly selected, anesthetized by immersion in 2-phenoxyethanol (1500 ppm; Sigma) and sampled for blood collection. 2.3.2. Trial 2 This experiment was designed to investigate an eventual immunomodulation during the inflammatory response in fish previously fed the experimental diets. For that purpose, at the end of Trial 1, 12 fish per tank were anesthetized as described above and intraperitoneally (i.p.) injected with either Hank's Balanced Salt Solution (HBSS) or UV killed Phdp (1 106 cfu ml1). Afterwards, stimulated specimens were reallocated in duplicate tanks in the same recirculated seawater system according to dietary treatments and stimuli. Fish were then sampled for blood and peritoneal exudates collection at 4 and 24 h after i.p. injection. 2.4. Haematological procedures Blood was collected from the caudal vein using heparinized syringes. The haematological profile consisted of total white (WBC) and red (RBC) blood cells counts, haematocrit (Ht) and haemoglobin (Hb; SPINREACT kit, ref. 1001230, Spain). The mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were also calculated as follows: - MCV (mm3) ¼ (Ht/RBC) 10 - MCH (pg cell1) ¼ (Hb/RBC) 10 - MCHC (g 100 ml1) ¼ (Hb/Ht) 100 The remaining blood was centrifuged at 10000 g during 10 min at 4 C and plasma stored at e80 C until assayed. Immediately after blood collection, blood smears were performed from homogenized blood, air dried, and stained with Wright's stain (Haemacolor; Merck) after fixation with formoleethanol (10% of 37% formaldehyde in absolute ethanol). Detection of peroxidase activity was carried out as described by Afonso et al. [11] in order to facilitate detection of neutrophils. The slides were examined (1000), and at least 200 leucocytes were counted and classified as thrombocytes, lymphocytes, monocytes and neutrophils. The relative percentage and absolute value (104 ml1) of each cell type was calculated. 2.5. Analytical procedures with peritoneal leucocytes The peritoneal cells were only collected in fish from trial 2, according to the procedure initially described for mice by Silva et al. [32] and posteriorly adapted for fish by Afonso et al. [33]. Briefly, following fish anaesthesia and bleeding by the caudal vessel, 5 ml of cold HBSS supplemented with 30 units heparin ml1 was injected into the peritoneal cavity. Then, the peritoneal area was slightly massaged in order to disperse the peritoneal cells in the injected HBSS. The i.p. injected HBSS containing suspended cells was finally collected. Total peritoneal cell counts were performed with a haemocytometer. Cytospin preparations were then made with a
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THARMAC Cellspin apparatus and stained as indicated above for blood smears. The lymphocytes, macrophages and neutrophils in the peritoneal exudates were differentially counted, and the percentage of each cell type was established after counting a minimum of 300 cells per slide. The concentration (104 ml1) of each leucocyte type was also calculated. 2.6. Innate humoral parameters Alternative complement pathway (ACP) activity was estimated as described by Sunyer and Tort [34]. The following buffers were used: GVB (Isotonic veronal buffered saline), pH 7.3, containing 0.1% gelatin; EDTA-GVB, as previous one but containing 20 mM EDTA; and Mg-EGTA-GVB, which is GVB with 10 mM Mgþ2 and 10 mM EGTA. Rabbit red blood cells (RaRBC; Probiologica Lda, Portugal) were used for ACP determination. RaRBC were washed four times in GVB and resuspended in GVB to a concentration of 2.5 108 cells ml1. Ten ml of RaRBC suspension were then added to 40 ml of serially diluted plasma in Mg-EGTA-GVB buffer. Samples were incubated at room temperature for 100 min with regular shaking. The reaction was stopped by adding 150 ml of cold EDTAGVB. Samples were then centrifuged and the extent of haemolysis was estimated by measuring the optical density of the supernatant at 414 nm in a Synergy HT microplate reader (Biotek). The ACH50 units were defined as the concentration of plasma giving 50% haemolysis of RaRBC. All analyses were conducted by triplicates. Lysozyme activity was measured using a turbidimetric assay as described by Costas et al. [35]. Briefly, a solution of Micrococcus lysodeikticus (0.5 mg ml1, 0.05 M sodium phosphate buffer, pH 6.2) was prepared. To a microplate, 15 ml of plasma in triplicates and 250 ml of the above suspension were added to give a final volume of 265 ml. The reaction was carried out at 25 C and the absorbance (450 nm) was measured after 0.5 and 4.5 min in a Synergy HT microplate reader. Lyophilized hen egg white lysozyme (Sigma) was serially diluted in sodium phosphate buffer (0.05 M, pH 6.2) and used to develop a standard curve. The amount of lysozyme in the sample was calculated using the formula of the standard curve. Total peroxidase activity in plasma was measured following the procedure described by Quade and J.A [36]. Briefly, 15 ml of plasma in triplicates were diluted with 135 ml of HBSS without Caþ2 and Mgþ2 in flat-bottomed 96-well plates. Then, 50 ml of 20 mM 3,30 ,5,50 -tetramethylbenzidine hydrochloride (TMB; Sigma) and 50 ml of 5 mM H2O2 were added. The color-change reaction was stopped after 2 min by adding 50 ml of 2 M sulphuric acid and the optical density was read at 450 nm in a Synergy HT microplate reader. Wells without plasma were used as blanks. The peroxidase activity (units ml1 plasma) was determined by defining one unit of peroxidase as that which produces an absorbance change of 1 OD. Total nitrite plus nitrate in plasma was analysed in duplicates using a nitrite/nitrate colorimetric method kit (Roche Diagnostics GmbH, Mannheim, Germany), adapted for 96-well microplates. Briefly, 100 ml of diluted plasma samples in duplicates were added to a microplate. Then, 50 ml of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and 4 ml of the enzyme nitrate reductase were added to each well in order to reduce nitrate to nitrite. The plates were then incubated for 30 min at 25 C. Afterwards, 50 ml of sulfanilamide and 50 ml of N-(1-naphthyl)-ethylenediamine dihydrochloride were added and incubated for 10 min at 25 C. The absorbance was then read at 540 nm a Synergy HT microplate reader after the two incubation periods. Water instead of plasma was used as blank. Total nitrite was determined by comparison with a sodium nitrite standard curve. Since nitrite and nitrate are endogenously produced as oxidative metabolites of the messenger molecule NO, these compounds are considered as indicative of NO production [37].
The anti-protease activity was determined as described by Ellis [38] with some modifications. Briefly, 10 ml of plasma were incubated with the same volume of a trypsin solution (5 mg ml1 in NaHCO3, 5 mg ml1, pH 8.3) for 10 min at 22 C in polystyrene microtubes. To the incubation mixture, 100 ml of phosphate buffer (NaH2PO4, 13.9 mg ml1, pH 7.0) and 125 ml of azocasein (20 mg ml1 in NaHCO3, 5 mg ml1, pH 8.3) were added and incubated for 1 h at 22 C. Finally, 250 ml of trichloroacetic acid were added to each microtube and incubated for 30 min at 22 C. The mixture was centrifuged at 10,000 g for 5 min at room temperature. Afterwards, 100 ml of the supernatant was transferred to a 96 well-plate that previously contained 100 ml of NaOH (40 mg ml1) per well. The OD was read at 450 nm in a Synergy HT microplate reader. Phosphate buffer in place of plasma and trypsin served as blank whereas the reference sample was phosphate buffer in place of plasma. The percentage inhibition of trypsin activity compared to the reference sample was calculated. All analyses were conducted in duplicates. Phdp strain PP3 was used in the bactericidal activity assay. Bacteria were cultured as described above and exponentially growing bacteria were resuspended in sterile HBSS and adjusted to 1 106 cfu ml1. Plating serial dilutions of the suspensions onto TSA1 plates and counting the number of cfu following incubation at 22 C confirmed bacterial concentration of the inoculum. Plasma bactericidal activity was then determined following the method of Graham et al. [39] with modifications. Briefly, 20 ml of plasma were added to duplicate wells of a U-shaped 96-well plate. HBSS was added to some wells instead of plasma and served as positive control. To each well, 20 ml of Phdp (1 106 cfu ml1) were added and the plate was incubated for 2.5 h at 25 C. To each well, 25 ml of 3-(4,5 dimethyl-2-yl)-2,5-diphenyl tetrazolium bromide (1 mg ml1; Sigma) were added and incubated for 10 min at 25 C to allow the formation of formazan. Plates were then centrifuged at 2000 g for 10 min and the precipitate was dissolved in 200 ml of dimethyl sulfoxide (Sigma). The absorbance of the dissolved formazan was measured at 560 nm. Bactericidal activity is expressed as percentage, calculated from the difference between bacteria surviving compared to the number of bacteria from positive controls (100%). 2.7. Data analysis All results are expressed as means ± standard deviation (SD). Data from trial 2 are presented as fold change levels (means ± SD), calculated by dividing each parameter value from fish i.p. injected with UV Killed Phdp by the mean value from control fish, i.p. injected with HBBS, minus one. Fold values higher than 0 express an increase and lower than 0 a decrease in the parameters assessed relative to fish i.p. injected with HBSS (sham solution). Data were analysed for normality and homogeneity of variance and, when necessary, transformed before being treated statistically. All data expressed as percentage were arcsine transformed [40]. Data from trial 1 were analysed by one-way ANOVA whereas foldchange values from trial 2 were analysed by two-way ANOVA, with time and diet as factors. Both procedures were followed by Tukey post hoc test to identify differences in the experimental treatments. All statistical analyses were performed using the computer package STATISTICA 12 for WINDOWS. The level of significance used was P 0.05 for all statistical tests. 3. Results 3.1. Haematology In trial 1, Ht, Hb, MCV, MCH, MCHC and RBC remained unchanged in fish fed the different diets whereas fish fed MET diet increased total WBC numbers compared to the control group
M. Machado et al. / Fish & Shellfish Immunology 42 (2015) 353e362
(Table 3). The percentage and total concentration of peripheral thrombocytes, lymphocytes and monocytes also did not change among fish fed the dietary treatments (Table 4) while both the relative proportion (p ¼ 0.047) and absolute values (p ¼ 0.006) of peripheral neutrophils were increased comparatively to the control in fish fed the MET diet. In trial 2, Ht increased comparatively to the control group in fish fed the MET following inoculation with the inactivated bacteria at both times (p ¼ 0.025), while MCHC decreased in fish fed both the TRP and MET diets (p < 0.001) (Table 5). An increase of RBC following injection in the peritoneal cavity was observed in all dietary treatments (p ¼ 0.026). Similarly, an augmentation of total WBC with time (p ¼ 0.002) in all groups and in fish fed the MET diet (p < 0.001) was also observed (Table 5). Fold change values of peripheral blood leucocytes are presented in Table 6. Circulating thrombocyte numbers in fish fed MET decreased compared to fish fed TRP at 4 h post-injection (p ¼ 0.05) but no differences relatively to fish fed the control diet were observed in both experimental groups. Fish fed TRP, but not the other diets, showed decreasing peripheral thrombocyte numbers from 4 h to 24 h following inflammation (p ¼ 0.017). Lymphocytosis was observed at 4 h but not at 24 h post-injection in fish fed MET compared to fish fed CRL and TRP (p < 0.001). At 24 h following inflammation, fish fed MET presented monocytosis compared to fish fed CRL and TRP (p ¼ 0.003). Neutrophil numbers increased from 4 h to 24 h in all experimental groups (p < 0.001), and were lower in fish fed TRP than in the other groups (p < 0.001). 3.2. Peritoneal leucocytes Total peritoneal leucocytes found in the inflamed peritoneal cavity of European seabass presented similar values among fish fed the experimental diets at 4 h after bacterial injection, but increased in fish fed MET compared to fish fed CRL and TRP at 24 h post-injection (p ¼ 0.036) (Table 7). Moreover, an increase in circulating leucocytes from 4 h to 24 h was also observed in fish fed MET (p ¼ 0.016). Fish fed METalso presented increased macrophage (p ¼ 0.014) and neutrophil (p ¼ 0.035) numbers compared to fish fed TRP, whereas an increase of neutrophil counts with time was observed in all dietary treatments (p ¼ 0.05) (Table 7). Fish fed TRP showed a decrease in lymphocyte numbers in the inflamed peritoneal cavity compared to fish fed CRL, after 24 h following Phdp injection (p ¼ 0.004) (Table 7). 3.3. Innate immune parameters in plasma In trial 1, fish fed the MET diet showed an increase in plasma ACP (p ¼ 0.008) and bactericidal activities (p ¼ 0.050) compared to fish Table 3 Haematocrit, haemoglobin, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), red blood cells (RBC) and white blood cells (WBC) in European seabass fed dietary treatments during 15 days. Parameters
Dietary treatments Control
Haematocrit Haemoglobin MCVa MCHb MCHCc RBC WBC
(%) (g dl) (mm3) (pg cell1) (g 100 ml1) (106 ml) (104 ml)
31.7 4.5 113.5 16.0 14.2 2.8 2.9
± ± ± ± ± ± ±
Tryptophan 6.2 1.3 15.2 2.8 2.0 0.7 0.3a
33.5 4.5 115.6 15.5 13.4 3.0 3.2
± ± ± ± ± ± ±
2.8 0.4 25.9 4.2 1.5 0.5 0.5a,b
Methionine 32.3 4.4 110.7 15.0 13.8 3.0 3.9
± ± ± ± ± ± ±
4.7 0.7 22.7 1.7 2.2 0.4 0.8b
Values are expressed as means ± SD (n ¼ 6). Different letters in the same row mean significant differences among dietary treatments (p < 0.05). a MCV (mm3) ¼ (Ht/RBC) 10. b MCH (pg cell1) ¼ (Hb/RBC) 10. c MCHC (g 100 ml1) ¼ (Hb/Ht) 100.
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Table 4 Relative proportion and absolute values of peripheral blood leucocytes (thrombocytes, lymphocytes, monocytes and neutrophils) of European seabass fed dietary treatments during 15 days. Parameters
Dietary treatments Control
Thrombocytes Lymphocytes Monocytes Neutrophils
(% WBC) (104 ml) (% WBC) (104 ml) (% WBC) (104 ml) (% WBC) (104 ml)
35.6 1.0 58.5 1.6 2.6 0.1 3.3 0.1
± ± ± ± ± ± ± ±
10.1 0.4 10.2 0.3 0.9 0.0 0.7a 0.0a
Tryptophan 40.4 1.3 52.6 1.7 2.8 0.1 4.3 0.1
± ± ± ± ± ± ± ±
6.0 0.1 5.3 0.4 0.9 0.0 0.8ab 0.0ab
Methionine 39.7 1.6 50.9 1.9 3.8 0.1 5.7 0.2
± ± ± ± ± ± ± ±
9.8 0.6 8.8 0.5 1.5 0.1 2.4b 0.1b
Values are expressed as means ± SD (n ¼ 6). Different letters in the same row mean significant differences among dietary treatments (p 0.05).
fed CRL whereas non-significant changes related to diet composition were observed for NO levels and lysozyme, peroxidase and anti-protease activities. Moreover, seabass fed TRP presented a general increase in anti-protease, ACP and bactericidal activities (Table 8). In trial 2, plasma ACP levels increased from 4 h to 24 h following inflammation in fish fed CRL (p ¼ 0.048) whereas no changes were observed among fish fed dietary treatments (Fig. 1A). Lysozyme activity augmented in fish fed TRP and MET compared to fish fed CRL at 4 h following pathogen injection (p < 0.001), but no differences between dietary treatments were observed at 24 h following pathogen injection (Fig. 1B). Comparatively to values at 4 h, lysozyme activity increased several folds in all groups at 24 h following pathogen injection. Plasma peroxidase levels were similar among fish fed dietary treatments at 4 h after inflammation whereas those values increased at 24 h in fish fed both CRL and MET, values being several fold higher in fish fed MET than in the control group (p < 0.001) (Fig. 1C). Plasma NO decreased in fish fed TRP compared to fish fed CRL and MET both at 4 h and 24 h (p < 0.001). Moreover, NO production augmented with time in fish fed CRL and TRP diets but not the MET diet (p < 0.001) (Fig. 1D). The anti-protease activity in fish fed the CRL diet was identical at 4 h and 24 h after bacterial injection while it increased with time in fish fed the MET diet (p < 0.001) (Fig. 1E). In fish fed the TRP diet the anti-protease activity was higher than in the other groups at 4 h but decreased to the control levels at 24 h after bacterial injection (p < 0.001). An enhanced plasma bactericidal capacity was observed in fish fed MET compared to fish fed CRL and TRP at 4 h and 24 h (p < 0.001). Furthermore, the bactericidal capacity augmented in time regardless of dietary treatment (p < 0.001) (Fig. 1F). 4. Discussion Dietary tryptophan and methionine have been proven to have important roles in the mammalian immune response, and the capacity to modulate metabolic pathways involved in the development of an improved immune response [21,24,41]. While the inflammatory response of European seabass after experimental infection with Phdp is well known [4,42e46], this is the first study showing the modulatory effects of tryptophan and methionine supplementation on the innate immune response following stimulation with bacteria, as well as the leucocyte migration dynamics to the inflammatory focus. In the present study, the overall haematological profile from trial 1 was not significantly altered by either TRP or MET, while a significantly higher concentration of WBC was observed in fish fed MET after 15 days of feeding which translated in higher neutrophil
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Table 5 Fold change values of haematocrit, haemoglobin, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), red blood cells (RBC) and white blood cells (WBC) in European seabass fed dietary treatments at 4 and 24 h after peritoneal inflammation. Parameters
Dietary treatments Control
Tryptophan
4h Haematocrit Haemoglobin MCV MCH MCHC RBC WBC
Fold change
24 h
0.11 0.11 0.00 0.06 0.06 0.27 0.13
± ± ± ± ± ± ±
0.07 0.06 0.26 0.06 0.05 0.19 0.17
Methionine
4h
0.02 0.21 0.22 0.21 0.21 0.36 0.49
± ± ± ± ± ± ±
0.12 0.16 0.18 0.12 0.12 0.19 0.37
24 h
0.08 0.02 0.20 0.01 0.01 0.13 0.13
± ± ± ± ± ± ±
0.03 0.11 0.18 0.17 0.18 0.14 0.22
4h
0.10 0.01 0.12 0.12 0.12 0.33 0.31
± ± ± ± ± ± ±
0.09 0.24 0.16 0.08 0.09 0.14 0.24
24 h
0.15 0.69 0.13 0.12 0.12 0.14 0.65
± ± ± ± ± ± ±
0.07 0.18 0.11 0.17 0.17 0.11 0.48
0.12 0.14 0.16 0.03 0.03 0.41 0.81
± ± ± ± ± ± ±
0.09 0.06 0.25 0.05 0.06 0.31 0.45
Parameters
Time
Diet
Time diet
Control
Tryptophan
Methionine
Haematocrit Haemoglobin MCV MCH MCHC RBC WBC
ns ns ns ns ns 0.026 0.002
0.025 ns ns ns
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Full length article
Dietary tryptophan and methionine as modulators of European seabass (Dicentrarchus labrax) immune status and inflammatory response nio Afonso a, b, Marina Machado a, b, *, Rita Azeredo a, c, Patricia Díaz-Rosales a, Anto a a, c a, * Helena Peres , Aires Oliva-Teles , Benjamín Costas a b c
~o Marinha e Ambiental (CIIMAR/CIMAR), Universidade do Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal Centro Interdisciplinar de Investigaça Instituto de Ci^ encias Biom edicas Abel Salazar (ICBAS-UP), Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal Departamento de Biologia, Faculdade de Ci^ encias da Universidade do Porto (FCUP), 4169-007 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 August 2014 Received in revised form 14 November 2014 Accepted 15 November 2014 Available online 25 November 2014
Amino acids regulate key metabolic pathways important to immune responses and their nutritional supply may increase synthesis of immune-related proteins. The present study aimed to evaluate the effects of dietary supplementation of tryptophan and methionine on European seabass (Dicentrarchus labrax) cellular and humoral status. The immunomodulatory effects of tryptophan and methionine during an inflammatory insult was also evaluated after intraperitoneal injection with inactivated Photobacterium damselae subsp. piscicida (Phdp). A practical isonitrogenous (45% crude protein) and isolipidic (16% crude fat) diets was formulated to include fish meal and a blend of plant feedstuffs as protein sources and fish oil as the main lipid source (CRL diet). Two other diets were formulated similar to the control but including L-tryptophan or Lmethionine at 2 the requirement level (diets TRP and MET, respectively). European seabass weighing 275 g were fed the experimental diets for a period of 15 days before being sampled (trial 1). Then, fish were subjected to a peritoneal inflammation by intraperitoneally injecting UV killed Phdp (106 colony forming units ml1) and sampled following 4 and 24 h post-injection (trial 2). Fish injected with a saline solution served as control. The haematological profile, peripheral cell dynamics and several plasma immune parameters were determined in trials 1 and 2, whereas cell migration to the inflammatory focus was also determined in trial 2. MET positively affected European seabass immune status by improving the peripheral leucocyte response, complement activity and bactericidal capacity, a stronger cellular recruitment to the inflammatory focus, and higher plasma peroxidase and bactericidal activities. TRP also seemed to improve immunostimulation, as there was a trend to augment both cell-mediated immunity and humoral capacity. However, TRP failed to improve an inflammatory response, verified by a decrease in blood phagocyte numbers and lack of immune cells recruitment. In summary, it is confirmed that MET has a pronounced influence on the innate immune response to inflammation, which is more evident than TRP, and raises its potential to incorporate in functional feeds to be used in prophylactic strategies against predictable unfavourable events. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Amino acids Immunomodulation Inflammation Immune response Photobacterium damselae subsp. piscicida
1. Introduction
~o Marinha * Corresponding authors. Centro Interdisciplinar de Investigaça e Ambiental (CIIMAR/CIMAR), Rua dos Bragas 289, 4050-123 Porto, Portugal. Tel.: þ351 223401850; fax: þ351 223401838. E-mail addresses: [email protected] (M. Machado), [email protected] (B. Costas). http://dx.doi.org/10.1016/j.fsi.2014.11.024 1050-4648/© 2014 Elsevier Ltd. All rights reserved.
European seabass, Dicentrarchus labrax (Linnaeus 1758), has a long history in Mediterranean aquaculture where it is produced under intensive, semi-intensive or extensive systems [1]. Farming practices alter the natural equilibrium between host and pathogens found in the wild, favouring the emergence of diseases and posing a major problem for the aquaculture industry [2]. Bacteria are the
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most common group of pathogens in cultured fish, some of them acting as primary pathogens, but others are opportunistic and cause disease in damaged hosts [3]. Photobacterium damselae subsp. piscicida (Phdp), the etiological agent of photobacteriosis (formerly pasteurellosis), has been a major cause of disease in species such as gilthead seabream (Sparus aurata) and European seabass [4e6]. Several types of commercial vaccines against photobacteriosis are available in the market, but their efficacy is dependent on the fish species, fish size, vaccine formulation and combined use with immunostimulants [7]. Fish have a well-developed immune system, composed by innate and acquired humoral and cell-mediated mechanisms which are responsible for resistance to diseases [8]. The innate immune system is the more primitive system of protection, as it is present in some way in all organisms, and it is well developed in teleost fish [9]. When external barriers are breached an inflammatory response is initiated in order to resolve the infection or wound, repairing the damage and re-establishing homeostasis [10]. This response to infection is characterized by an influx of phagocytic cells to the infection focus, which produce a number of bactericidal instruments capable of degrading and kill invading organisms [11,12]. Both macrophages and neutrophils efficiently attack pathogens by generating lysozyme, complement factors and anti-proteases compounds [10,13]. Furthermore, these cells produce high amounts of reactive oxygen species (ROS) such as the superoxide anion when in contact with bacteria [14]. The ideal inflammatory response is rapid and destructive, yet specific and self-limiting [9] and it is likely that nutrients influence several aspects of the immune system [15]. Therefore, modulation of the immune system of aquaculture fish may be achieved through nutritional strategies [16,17]. Li et al. [18] suggested that diet supplementation with amino acids (AA) may improve fish performance. AA are primarily the building blocks for protein synthesis but are also important regulators of key metabolic pathways with important regulatory roles in growth and immune responses [19,20]. Indispensable AA such as methionine and tryptophan have important roles in inflammation and both are currently available as feed grade, thus their potential use in functional feeds can be done at acceptable costs [21]. Methionine was shown to modulate the mammalian immune system by improving both cellular and humoral immune responses [22]. Methionine may also affect cell proliferation and differentiation of lymphocytes since it is involved in polyamine biosynthesis [21]. It is also precursor of glutathione (GSH), a molecule involved in reducing ROS and thus protecting cells from oxidative stress during the inflammatory process [22]. On the other hand, tryptophan is the precursor of both serotonin (5-hydroxytryptamine, 5-HT) and melatonin (N-acetyl-5methoxytryptamine), which are potent scavengers of damaging free radicals and play key roles on cell redox balance by promoting the activity of antioxidative enzymes [23]. Additionally, tryptophan catabolism by indoleamine 2,3-dioxygenase (IDO) seems to have a major immunomodulatory effect during inflammation in mammals [24]. In fact, a progressive decline in plasma tryptophan concentration is observed during inflammation, thereby affecting both macrophages and lymphocyte functions [25]. Recent studies have also reported that a deficiency of dietary AA reduces their concentration in plasma compromising the immune system [15,20]. Hence, dietary supplementation of key AA may represent a strategy to improve the fish immune system and welfare, being a less costly and dramatic solution than the use of antibiotics [19]. Few studies were performed to elucidate the effect of methionine and tryptophan on the innate immune response, and most of them were performed in mammals. Therefore, the present study aimed to assess the effects of dietary supplementation with methionine or tryptophan on the European seabass cellular and
humoral innate immune status, and to investigate their immunomodulation potential during the inflammatory response. 2. Material and methods 2.1. Diets A practical isonitrogenous (45% crude protein) and isolipidic (16% crude fat) diets was formulated to include fish meal and a blend of plant feedstuffs as protein sources and fish oil as the main lipid source (CRL diet). The CRL diet was formulated to include an indispensable AA level meeting the ideal protein indispensable AA pattern estimated for European seabass [26]. Two other diets were formulated similar to the control but including L-tryptophan or Lmethionine at 2 the tryptophan or methionine levels of control diet (diets TRP and MET, respectively). All dietary ingredients were finely ground, well mixed and dry pelleted in a laboratory pellet mill (California Pellet Mill, Crawfordsville, IN, USA). Ingredient composition and proximate analysis of the experimental diets are presented in Table 1 and the AA profile of the diets is presented in Table 2. Proximate analysis of the diets was performed according to Association of Official Analytical Chemists methods [27] and AA analysis according to Peres and Oliva-Teles [28]. Briefly, diet samples were hydrolyzed for 23 h with 6 N hydrochloric acid at 110 C under nitrogen atmosphere and derivatized with phenylisothiocyanate (PITC; Pierce) reagent before separation by high performance liquid chromatography (HPLC) in a Waters Reversed-
Table 1 Composition and proximate analysis of the experimental diets. Experimental diets
Ingredients (% DM) Fish meala Soybean mealb Corn glutenc Wheat glutend Wheat meale Fish oil Vitamin premixf Choline chloride (50%) Mineral premixg Binderh Agar Dibasic calcium phosphate L-Methionine L-Tryptophan Proximate analyses (% dry weight) Dry matter (%) Crude protein Crude lipid Ash
CRL
TRP
MET
34.1 15.0 10.0 5.0 16.7 13.9 1.0 0.5 1.0 1.0 1.0 0.8 e e
33.5 15.0 10.0 5.0 16.6 13.4 1.0 0.5 1.0 1.0 1.0 0.9 e 0.5
33.2 15.0 10.0 5.0 16.2 14.0 1.0 0.5 1.0 1.0 1.0 0.9 1.2 e
95.2 44.9 15.5 10.5
94.3 45.0 16.9 10.5
94.9 45.2 16.5 10.4
a Pesquera Centinela, Steam Dried LT, Chile (CP: 71.4%; CL:9.3%). Sorgal, S.A. Ovar, Portugal. b Soybean meal (CP: 54.9%; CL:2.1%), Sorgal, S.A. Ovar, Portugal. c Corn gluten (CP: 72.2%; CL: 2.0%), Sorgal, S.A. Ovar, Portugal. d Wheat gluten (CP: 84.4%; CL: 2.1%), Sorgal, S.A. Ovar, Portugal. e Wheat meal (CP: 13.9%; CL: 1.8%), Sorgal, S.A. Ovar, Portugal. f Vitamins (mg kg1 diet): retinol, 18,000 (IU kg1 diet); calciferol, 2000 (IU kg1 diet); alpha tocopherol, 35; menadion sodium bis., 10; thiamin, 15; riboflavin, 25; Ca pantothenate, 50; nicotinic acid, 200; pyridoxine, 5; folic acid, 10; cyanocobalamin, 0.02; biotin, 1.5; ascorbyl monophosphate, 50; inositol, 400. g Minerals (mg kg1 diet): cobalt sulphate, 1.91; copper sulphate, 19.6; iron sulphate, 200; sodium fluoride, 2.21; potassium iodide, 0.78; magnesium oxide, 830; manganese oxide, 26; sodium selenite, 0.66; zinc oxide, 37.5; dicalcium phosphate, 8.02 (g kg1 diet); potassium chloride, 1.15 (g kg1 diet); sodium chloride, 0.4 (g kg1 diet). h Aquacube. Agil, UK.
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to duplicate tanks and fish were fed by hand twice a day, to apparent visual satiety.
Table 2 Amino acid composition (g 16 g1 N) of the experimental diets. Experimental diets
Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Tyrosine Threonine Tryptophan Valine Aspartic Acid Glutamic Acid Serine Glycine Alanine Proline
CRL
TRP
MET
7.74 3.78 5.05 9.73 6.66 2.57 5.39 4.04 4.68 1.12 5.38 8.20 16.39 4.15 3.98 4.89 4.99
7.11 3.63 4.64 9.71 6.96 2.42 5.16 3.96 4.38 2.24 5.10 7.54 16.14 4.42 4.18 4.92 5.50
7.02 4.12 4.71 9.49 6.68 4.95 5.16 3.98 4.51 1.10 5.12 7.60 16.09 4.29 4.20 4.97 4.84
Phase Amino Acid Analysis System equipped with a PicoTag column, using the conditions described by Cohen et al. [29]. External standards were prepared along with the samples and norleucine was used as an internal standard. Chromatographic peaks were analysed with the Breeze software (Waters). Tryptophan was measured by a spectrophotometric method as described by De Vries et al. [30]. 2.2. Bacterial growth and inoculum preparation Photobacterium damselae subsp. piscicida (Phdp), strain PP3, was kindly provided by Dr. Ana do Vale (Institute for Molecular and Cell Biology, University of Porto, Portugal) and isolated from yellowtail (Seriola quinqueradiata; Japan) by Dr Andrew C. Barnes (Marine Laboratory, Aberdeen, UK). Bacteria were routinely cultured at 22 C in tryptic soy broth (TSB) or tryptic soy agar (TSA) (both from Difco Laboratories) supplemented with NaCl to a final concentration of 1% (w/v) (TSB-1 and TSA-1, respectively) and stored at 70 C in TSB-1 supplemented with 15% (v/v) glycerol. To prepare the inoculum for injection into the fish peritoneal cavities, stocked bacteria were cultured for 48 h at 22 C on TSA-1 and then inoculated into TSB-1 and cultured overnight at the same temperature, with continuous shaking (100 rpm). Exponentially growing bacteria were collected by centrifugation at 3500 g for 30 min, resuspended in sterile HBSS and adjusted to 1 106 colony forming units (cfu) ml1 according to Costas et al. [31]. Bacteria were then killed by UV exposure for 2 h. Loss of bacterial viability following UV was confirmed by plating resulting cultures on TSA-1 plates and failing to see any bacterial growth. 2.3. Experimental design This study was directed by trained scientists (following FELASA category C recommendations) and conducted according to the guidelines on the protection of animals used for scientific purposes from the European directive 2010/63/UE. The trials were performed at the Marine Zoological Station, Porto, Portugal with European seabass weighing 274.7 ± 20.4 g that were obtained in a commercial fish farm (Maresa, Huelva, Spain). Fifteen fish were randomly distributed into each of 6 fibreglass tanks of 300 L water capacity in a recirculating aerated seawater system supplied with a continuous flow of filtered seawater. A 12 h light/12 h dark photoperiod was adopted; dissolved oxygen was maintained at 90%, water temperature was 25 ± 0.5 C, and salinity averaged 35 ± 1%0. The experimental diets were randomly assigned
2.3.1. Trial 1 The feeding trial lasted 15 days, to assess the effects of short term dietary supplementation with methionine or tryptophan on European seabass cellular and humoral immune status. It has been previously reported that 14 days of feeding diets supplemented with individual AA are enough to modulate physiological and immune responses in fish [17]. At the end of the trial, after a fasting period of 18 h, three fish per tank were randomly selected, anesthetized by immersion in 2-phenoxyethanol (1500 ppm; Sigma) and sampled for blood collection. 2.3.2. Trial 2 This experiment was designed to investigate an eventual immunomodulation during the inflammatory response in fish previously fed the experimental diets. For that purpose, at the end of Trial 1, 12 fish per tank were anesthetized as described above and intraperitoneally (i.p.) injected with either Hank's Balanced Salt Solution (HBSS) or UV killed Phdp (1 106 cfu ml1). Afterwards, stimulated specimens were reallocated in duplicate tanks in the same recirculated seawater system according to dietary treatments and stimuli. Fish were then sampled for blood and peritoneal exudates collection at 4 and 24 h after i.p. injection. 2.4. Haematological procedures Blood was collected from the caudal vein using heparinized syringes. The haematological profile consisted of total white (WBC) and red (RBC) blood cells counts, haematocrit (Ht) and haemoglobin (Hb; SPINREACT kit, ref. 1001230, Spain). The mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were also calculated as follows: - MCV (mm3) ¼ (Ht/RBC) 10 - MCH (pg cell1) ¼ (Hb/RBC) 10 - MCHC (g 100 ml1) ¼ (Hb/Ht) 100 The remaining blood was centrifuged at 10000 g during 10 min at 4 C and plasma stored at e80 C until assayed. Immediately after blood collection, blood smears were performed from homogenized blood, air dried, and stained with Wright's stain (Haemacolor; Merck) after fixation with formoleethanol (10% of 37% formaldehyde in absolute ethanol). Detection of peroxidase activity was carried out as described by Afonso et al. [11] in order to facilitate detection of neutrophils. The slides were examined (1000), and at least 200 leucocytes were counted and classified as thrombocytes, lymphocytes, monocytes and neutrophils. The relative percentage and absolute value (104 ml1) of each cell type was calculated. 2.5. Analytical procedures with peritoneal leucocytes The peritoneal cells were only collected in fish from trial 2, according to the procedure initially described for mice by Silva et al. [32] and posteriorly adapted for fish by Afonso et al. [33]. Briefly, following fish anaesthesia and bleeding by the caudal vessel, 5 ml of cold HBSS supplemented with 30 units heparin ml1 was injected into the peritoneal cavity. Then, the peritoneal area was slightly massaged in order to disperse the peritoneal cells in the injected HBSS. The i.p. injected HBSS containing suspended cells was finally collected. Total peritoneal cell counts were performed with a haemocytometer. Cytospin preparations were then made with a
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THARMAC Cellspin apparatus and stained as indicated above for blood smears. The lymphocytes, macrophages and neutrophils in the peritoneal exudates were differentially counted, and the percentage of each cell type was established after counting a minimum of 300 cells per slide. The concentration (104 ml1) of each leucocyte type was also calculated. 2.6. Innate humoral parameters Alternative complement pathway (ACP) activity was estimated as described by Sunyer and Tort [34]. The following buffers were used: GVB (Isotonic veronal buffered saline), pH 7.3, containing 0.1% gelatin; EDTA-GVB, as previous one but containing 20 mM EDTA; and Mg-EGTA-GVB, which is GVB with 10 mM Mgþ2 and 10 mM EGTA. Rabbit red blood cells (RaRBC; Probiologica Lda, Portugal) were used for ACP determination. RaRBC were washed four times in GVB and resuspended in GVB to a concentration of 2.5 108 cells ml1. Ten ml of RaRBC suspension were then added to 40 ml of serially diluted plasma in Mg-EGTA-GVB buffer. Samples were incubated at room temperature for 100 min with regular shaking. The reaction was stopped by adding 150 ml of cold EDTAGVB. Samples were then centrifuged and the extent of haemolysis was estimated by measuring the optical density of the supernatant at 414 nm in a Synergy HT microplate reader (Biotek). The ACH50 units were defined as the concentration of plasma giving 50% haemolysis of RaRBC. All analyses were conducted by triplicates. Lysozyme activity was measured using a turbidimetric assay as described by Costas et al. [35]. Briefly, a solution of Micrococcus lysodeikticus (0.5 mg ml1, 0.05 M sodium phosphate buffer, pH 6.2) was prepared. To a microplate, 15 ml of plasma in triplicates and 250 ml of the above suspension were added to give a final volume of 265 ml. The reaction was carried out at 25 C and the absorbance (450 nm) was measured after 0.5 and 4.5 min in a Synergy HT microplate reader. Lyophilized hen egg white lysozyme (Sigma) was serially diluted in sodium phosphate buffer (0.05 M, pH 6.2) and used to develop a standard curve. The amount of lysozyme in the sample was calculated using the formula of the standard curve. Total peroxidase activity in plasma was measured following the procedure described by Quade and J.A [36]. Briefly, 15 ml of plasma in triplicates were diluted with 135 ml of HBSS without Caþ2 and Mgþ2 in flat-bottomed 96-well plates. Then, 50 ml of 20 mM 3,30 ,5,50 -tetramethylbenzidine hydrochloride (TMB; Sigma) and 50 ml of 5 mM H2O2 were added. The color-change reaction was stopped after 2 min by adding 50 ml of 2 M sulphuric acid and the optical density was read at 450 nm in a Synergy HT microplate reader. Wells without plasma were used as blanks. The peroxidase activity (units ml1 plasma) was determined by defining one unit of peroxidase as that which produces an absorbance change of 1 OD. Total nitrite plus nitrate in plasma was analysed in duplicates using a nitrite/nitrate colorimetric method kit (Roche Diagnostics GmbH, Mannheim, Germany), adapted for 96-well microplates. Briefly, 100 ml of diluted plasma samples in duplicates were added to a microplate. Then, 50 ml of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and 4 ml of the enzyme nitrate reductase were added to each well in order to reduce nitrate to nitrite. The plates were then incubated for 30 min at 25 C. Afterwards, 50 ml of sulfanilamide and 50 ml of N-(1-naphthyl)-ethylenediamine dihydrochloride were added and incubated for 10 min at 25 C. The absorbance was then read at 540 nm a Synergy HT microplate reader after the two incubation periods. Water instead of plasma was used as blank. Total nitrite was determined by comparison with a sodium nitrite standard curve. Since nitrite and nitrate are endogenously produced as oxidative metabolites of the messenger molecule NO, these compounds are considered as indicative of NO production [37].
The anti-protease activity was determined as described by Ellis [38] with some modifications. Briefly, 10 ml of plasma were incubated with the same volume of a trypsin solution (5 mg ml1 in NaHCO3, 5 mg ml1, pH 8.3) for 10 min at 22 C in polystyrene microtubes. To the incubation mixture, 100 ml of phosphate buffer (NaH2PO4, 13.9 mg ml1, pH 7.0) and 125 ml of azocasein (20 mg ml1 in NaHCO3, 5 mg ml1, pH 8.3) were added and incubated for 1 h at 22 C. Finally, 250 ml of trichloroacetic acid were added to each microtube and incubated for 30 min at 22 C. The mixture was centrifuged at 10,000 g for 5 min at room temperature. Afterwards, 100 ml of the supernatant was transferred to a 96 well-plate that previously contained 100 ml of NaOH (40 mg ml1) per well. The OD was read at 450 nm in a Synergy HT microplate reader. Phosphate buffer in place of plasma and trypsin served as blank whereas the reference sample was phosphate buffer in place of plasma. The percentage inhibition of trypsin activity compared to the reference sample was calculated. All analyses were conducted in duplicates. Phdp strain PP3 was used in the bactericidal activity assay. Bacteria were cultured as described above and exponentially growing bacteria were resuspended in sterile HBSS and adjusted to 1 106 cfu ml1. Plating serial dilutions of the suspensions onto TSA1 plates and counting the number of cfu following incubation at 22 C confirmed bacterial concentration of the inoculum. Plasma bactericidal activity was then determined following the method of Graham et al. [39] with modifications. Briefly, 20 ml of plasma were added to duplicate wells of a U-shaped 96-well plate. HBSS was added to some wells instead of plasma and served as positive control. To each well, 20 ml of Phdp (1 106 cfu ml1) were added and the plate was incubated for 2.5 h at 25 C. To each well, 25 ml of 3-(4,5 dimethyl-2-yl)-2,5-diphenyl tetrazolium bromide (1 mg ml1; Sigma) were added and incubated for 10 min at 25 C to allow the formation of formazan. Plates were then centrifuged at 2000 g for 10 min and the precipitate was dissolved in 200 ml of dimethyl sulfoxide (Sigma). The absorbance of the dissolved formazan was measured at 560 nm. Bactericidal activity is expressed as percentage, calculated from the difference between bacteria surviving compared to the number of bacteria from positive controls (100%). 2.7. Data analysis All results are expressed as means ± standard deviation (SD). Data from trial 2 are presented as fold change levels (means ± SD), calculated by dividing each parameter value from fish i.p. injected with UV Killed Phdp by the mean value from control fish, i.p. injected with HBBS, minus one. Fold values higher than 0 express an increase and lower than 0 a decrease in the parameters assessed relative to fish i.p. injected with HBSS (sham solution). Data were analysed for normality and homogeneity of variance and, when necessary, transformed before being treated statistically. All data expressed as percentage were arcsine transformed [40]. Data from trial 1 were analysed by one-way ANOVA whereas foldchange values from trial 2 were analysed by two-way ANOVA, with time and diet as factors. Both procedures were followed by Tukey post hoc test to identify differences in the experimental treatments. All statistical analyses were performed using the computer package STATISTICA 12 for WINDOWS. The level of significance used was P 0.05 for all statistical tests. 3. Results 3.1. Haematology In trial 1, Ht, Hb, MCV, MCH, MCHC and RBC remained unchanged in fish fed the different diets whereas fish fed MET diet increased total WBC numbers compared to the control group
M. Machado et al. / Fish & Shellfish Immunology 42 (2015) 353e362
(Table 3). The percentage and total concentration of peripheral thrombocytes, lymphocytes and monocytes also did not change among fish fed the dietary treatments (Table 4) while both the relative proportion (p ¼ 0.047) and absolute values (p ¼ 0.006) of peripheral neutrophils were increased comparatively to the control in fish fed the MET diet. In trial 2, Ht increased comparatively to the control group in fish fed the MET following inoculation with the inactivated bacteria at both times (p ¼ 0.025), while MCHC decreased in fish fed both the TRP and MET diets (p < 0.001) (Table 5). An increase of RBC following injection in the peritoneal cavity was observed in all dietary treatments (p ¼ 0.026). Similarly, an augmentation of total WBC with time (p ¼ 0.002) in all groups and in fish fed the MET diet (p < 0.001) was also observed (Table 5). Fold change values of peripheral blood leucocytes are presented in Table 6. Circulating thrombocyte numbers in fish fed MET decreased compared to fish fed TRP at 4 h post-injection (p ¼ 0.05) but no differences relatively to fish fed the control diet were observed in both experimental groups. Fish fed TRP, but not the other diets, showed decreasing peripheral thrombocyte numbers from 4 h to 24 h following inflammation (p ¼ 0.017). Lymphocytosis was observed at 4 h but not at 24 h post-injection in fish fed MET compared to fish fed CRL and TRP (p < 0.001). At 24 h following inflammation, fish fed MET presented monocytosis compared to fish fed CRL and TRP (p ¼ 0.003). Neutrophil numbers increased from 4 h to 24 h in all experimental groups (p < 0.001), and were lower in fish fed TRP than in the other groups (p < 0.001). 3.2. Peritoneal leucocytes Total peritoneal leucocytes found in the inflamed peritoneal cavity of European seabass presented similar values among fish fed the experimental diets at 4 h after bacterial injection, but increased in fish fed MET compared to fish fed CRL and TRP at 24 h post-injection (p ¼ 0.036) (Table 7). Moreover, an increase in circulating leucocytes from 4 h to 24 h was also observed in fish fed MET (p ¼ 0.016). Fish fed METalso presented increased macrophage (p ¼ 0.014) and neutrophil (p ¼ 0.035) numbers compared to fish fed TRP, whereas an increase of neutrophil counts with time was observed in all dietary treatments (p ¼ 0.05) (Table 7). Fish fed TRP showed a decrease in lymphocyte numbers in the inflamed peritoneal cavity compared to fish fed CRL, after 24 h following Phdp injection (p ¼ 0.004) (Table 7). 3.3. Innate immune parameters in plasma In trial 1, fish fed the MET diet showed an increase in plasma ACP (p ¼ 0.008) and bactericidal activities (p ¼ 0.050) compared to fish Table 3 Haematocrit, haemoglobin, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), red blood cells (RBC) and white blood cells (WBC) in European seabass fed dietary treatments during 15 days. Parameters
Dietary treatments Control
Haematocrit Haemoglobin MCVa MCHb MCHCc RBC WBC
(%) (g dl) (mm3) (pg cell1) (g 100 ml1) (106 ml) (104 ml)
31.7 4.5 113.5 16.0 14.2 2.8 2.9
± ± ± ± ± ± ±
Tryptophan 6.2 1.3 15.2 2.8 2.0 0.7 0.3a
33.5 4.5 115.6 15.5 13.4 3.0 3.2
± ± ± ± ± ± ±
2.8 0.4 25.9 4.2 1.5 0.5 0.5a,b
Methionine 32.3 4.4 110.7 15.0 13.8 3.0 3.9
± ± ± ± ± ± ±
4.7 0.7 22.7 1.7 2.2 0.4 0.8b
Values are expressed as means ± SD (n ¼ 6). Different letters in the same row mean significant differences among dietary treatments (p < 0.05). a MCV (mm3) ¼ (Ht/RBC) 10. b MCH (pg cell1) ¼ (Hb/RBC) 10. c MCHC (g 100 ml1) ¼ (Hb/Ht) 100.
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Table 4 Relative proportion and absolute values of peripheral blood leucocytes (thrombocytes, lymphocytes, monocytes and neutrophils) of European seabass fed dietary treatments during 15 days. Parameters
Dietary treatments Control
Thrombocytes Lymphocytes Monocytes Neutrophils
(% WBC) (104 ml) (% WBC) (104 ml) (% WBC) (104 ml) (% WBC) (104 ml)
35.6 1.0 58.5 1.6 2.6 0.1 3.3 0.1
± ± ± ± ± ± ± ±
10.1 0.4 10.2 0.3 0.9 0.0 0.7a 0.0a
Tryptophan 40.4 1.3 52.6 1.7 2.8 0.1 4.3 0.1
± ± ± ± ± ± ± ±
6.0 0.1 5.3 0.4 0.9 0.0 0.8ab 0.0ab
Methionine 39.7 1.6 50.9 1.9 3.8 0.1 5.7 0.2
± ± ± ± ± ± ± ±
9.8 0.6 8.8 0.5 1.5 0.1 2.4b 0.1b
Values are expressed as means ± SD (n ¼ 6). Different letters in the same row mean significant differences among dietary treatments (p 0.05).
fed CRL whereas non-significant changes related to diet composition were observed for NO levels and lysozyme, peroxidase and anti-protease activities. Moreover, seabass fed TRP presented a general increase in anti-protease, ACP and bactericidal activities (Table 8). In trial 2, plasma ACP levels increased from 4 h to 24 h following inflammation in fish fed CRL (p ¼ 0.048) whereas no changes were observed among fish fed dietary treatments (Fig. 1A). Lysozyme activity augmented in fish fed TRP and MET compared to fish fed CRL at 4 h following pathogen injection (p < 0.001), but no differences between dietary treatments were observed at 24 h following pathogen injection (Fig. 1B). Comparatively to values at 4 h, lysozyme activity increased several folds in all groups at 24 h following pathogen injection. Plasma peroxidase levels were similar among fish fed dietary treatments at 4 h after inflammation whereas those values increased at 24 h in fish fed both CRL and MET, values being several fold higher in fish fed MET than in the control group (p < 0.001) (Fig. 1C). Plasma NO decreased in fish fed TRP compared to fish fed CRL and MET both at 4 h and 24 h (p < 0.001). Moreover, NO production augmented with time in fish fed CRL and TRP diets but not the MET diet (p < 0.001) (Fig. 1D). The anti-protease activity in fish fed the CRL diet was identical at 4 h and 24 h after bacterial injection while it increased with time in fish fed the MET diet (p < 0.001) (Fig. 1E). In fish fed the TRP diet the anti-protease activity was higher than in the other groups at 4 h but decreased to the control levels at 24 h after bacterial injection (p < 0.001). An enhanced plasma bactericidal capacity was observed in fish fed MET compared to fish fed CRL and TRP at 4 h and 24 h (p < 0.001). Furthermore, the bactericidal capacity augmented in time regardless of dietary treatment (p < 0.001) (Fig. 1F). 4. Discussion Dietary tryptophan and methionine have been proven to have important roles in the mammalian immune response, and the capacity to modulate metabolic pathways involved in the development of an improved immune response [21,24,41]. While the inflammatory response of European seabass after experimental infection with Phdp is well known [4,42e46], this is the first study showing the modulatory effects of tryptophan and methionine supplementation on the innate immune response following stimulation with bacteria, as well as the leucocyte migration dynamics to the inflammatory focus. In the present study, the overall haematological profile from trial 1 was not significantly altered by either TRP or MET, while a significantly higher concentration of WBC was observed in fish fed MET after 15 days of feeding which translated in higher neutrophil
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Table 5 Fold change values of haematocrit, haemoglobin, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), red blood cells (RBC) and white blood cells (WBC) in European seabass fed dietary treatments at 4 and 24 h after peritoneal inflammation. Parameters
Dietary treatments Control
Tryptophan
4h Haematocrit Haemoglobin MCV MCH MCHC RBC WBC
Fold change
24 h
0.11 0.11 0.00 0.06 0.06 0.27 0.13
± ± ± ± ± ± ±
0.07 0.06 0.26 0.06 0.05 0.19 0.17
Methionine
4h
0.02 0.21 0.22 0.21 0.21 0.36 0.49
± ± ± ± ± ± ±
0.12 0.16 0.18 0.12 0.12 0.19 0.37
24 h
0.08 0.02 0.20 0.01 0.01 0.13 0.13
± ± ± ± ± ± ±
0.03 0.11 0.18 0.17 0.18 0.14 0.22
4h
0.10 0.01 0.12 0.12 0.12 0.33 0.31
± ± ± ± ± ± ±
0.09 0.24 0.16 0.08 0.09 0.14 0.24
24 h
0.15 0.69 0.13 0.12 0.12 0.14 0.65
± ± ± ± ± ± ±
0.07 0.18 0.11 0.17 0.17 0.11 0.48
0.12 0.14 0.16 0.03 0.03 0.41 0.81
± ± ± ± ± ± ±
0.09 0.06 0.25 0.05 0.06 0.31 0.45
Parameters
Time
Diet
Time diet
Control
Tryptophan
Methionine
Haematocrit Haemoglobin MCV MCH MCHC RBC WBC
ns ns ns ns ns 0.026 0.002
0.025 ns ns ns
