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Effect of dietary β-glucan on growth, survival and regulation of immune processes inrainbow trout (Oncorhynchus mykiss) infected by Aeromonas salmonicida
Liqin Ji, Guoxiang Sun, Jun Li, Yi Wang, Yishuai Du, Xian Li, Ying Liu
PII: S1050-4648(17)30134-1
DOI: 10.1016/j.fsi.2017.03.015
Reference: YFSIM 4484
To appear in: Fish and Shellfish Immunology
Received Date: 17 December 2016
Revised Date: 3 March 2017
Accepted Date: 4 March 2017
Please cite this article as: Ji L, Sun G, Li J, Wang Y, Du Y, Li X, Liu Y, Effect of dietary β-glucan ongrowth, survival and regulation of immune processes in rainbow trout (Oncorhynchus mykiss) infectedby Aeromonas salmonicida, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.03.015.
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Effect of dietary β-glucan on growth, survival and regulation of 1
immune processes in rainbow trout (Oncorhynchus mykiss) infected 2
by Aeromonas salmonicida 3
Liqin Jia, b, Guoxiang Suna, Jun Lic, Yi Wangd, Yishuai Dua, Xian Lia, Ying Liud, * 4
a Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China 5
b University of Chinese Academy of Sciences, Beijing, 100039, China 6
c School of Biological Sciences, Lake Superior State University, Sault Ste. Marie, MI, 49783 7
d School of Marine Science and Environment Engineering, Dalian Ocean University, Dalian, 116023, China 8
ABSTRACT 9
The present study evaluated the effects of dietary β-glucan (0, 0.05%, 0.1%, and 0.2%) on 10
growth performance after 42 days of feeding. Thereafter, rainbow trout (Oncorhynchus 11
mykiss) were infected with Aeromonas salmonicida, and survival rates as well as the 12
regulating processes of stress- and immune-related factors were analyzed. In general, 13
higher dietary β-glucan levels obviously improved specific growth rate (SGR), weight gain 14
(WG) and feed efficiency (FE) (P ≤ 0.05). Survival rates in β-glucan groups increased 15
significantly compared with the control group after A. salmonicida infection (P ≤ 0.05). 16
Serum total superoxide dimutase (T-SOD), peroxidase (POD) as well as catalase (CAT) 17
activities, and their mRNA expressions in the head kidney of fish in the β-glucan groups 18
generally increased to higher levels after infection, and more quickly, compared with in the 19
control group. Serum lysozyme (LSZ) and its expression in the head kidney in β-glucan 20
groups reached a higher peak earlier than in the control group. 21
22
*Corresponding author. Tel.: +86-411-84762010; Fax. : +86-411-84763520 23
E-mail address: [email protected] (Y. Liu) 24
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Serum glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) 25
levels in the β-glucan groups were significantly lower than in the control group (P ≤ 0.05). 26
The peak of heat shock protein 70 (HSP70) expression in the 0.2% β-glucan group was 27
higher and occurred earlier than in other groups (P ≤ 0.05). These results confirm that 0.1% 28
and 0.2% dietary β-glucan are beneficial for promoting growth in rainbow trout and 29
enhancing resistance against A. salmonicida. Furthermore, β-glucan could play an 30
important role in regulating stress- and immune-related factors in rainbow trout to more 31
quickly fight against bacterial infection. 32
Keywords: β-glucan; Oncorhynchus mykiss; growth; survival; immune-related factors; 33
Aeromonas salmonicida 34
1. Introduction 35
Rainbow trout (Oncorhynchus mykiss) is one of the most widely cultured fish 36
species worldwide for its fast growth and adaptation to low temperature [1]. Despite 37
the large market demand, natural resources are limited. These factors have led to the 38
intensification of aquaculture production systems. Intensive farming, along with 39
overcrowding and poor water quality, is likely to alter fish physiological status and 40
therefore increase the susceptibility of pathogen infection [2]. In the last few years, 41
abuse of antibiotics to prevent the uncontrolled spread of pathogens has resulted in 42
the emergence of several resistant pathogens in aquaculture. Therefore, it is urgent 43
to find suitable ways to control disease outbreaks [3]. Immunostimulants are the 44
current primary approach for enhancing resistance against pathogens in aquaculture. 45
Although numerous substances have been investigated as immunostimulants, only a 46
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few of these are appropriate for use in aquaculture [4, 5]. 47
In recent years, the effective immunomodulatory properties of β-1,3/1,6-glucan 48
derived from yeast have been extensively proved, not only in mammals but also in 49
fish [6, 7]. β-glucan naturally form polysaccharides with glucose linked by 50
β-glycosidic bonds [8] and can stimulate macrophages to actively fight against fish 51
pathogens [9]. They can also enhance the activity of non-specific immune factors 52
such as lysozyme and the complement system [10, 11]. Altering immune 53
cytokine-like gene expression, such as tumor necrosis factor-ɑ (TNF-ɑ) and 54
interleukin-1β (IL-1β), is a primary channel through which β-glucan improves 55
innate immunity in various fish [12-14]. 56
Immune efficacy can vary according to several factors such as the dietary dose of 57
glucan, feeding regime, and glucan type [15]. Although the immunostimulatory 58
effects of various derivations of β-glucan have been studied against a diverse range 59
of pathogens, such as Yersinia ruckeri [16], A. hydrophila [17], and bacterial 60
lipopolysaccharides [18], few studies have investigated its disease resistance against 61
A. salmonicida. 62
Therefore, the aim of the present study was to evaluate the effects of dietary 63
β-glucan derived from yeast cells on growth promotion, disease resistance and 64
immune response to A. salmonicida in rainbow trout. Furthermore, the manner in 65
which β-glucan regulates the continuous process of immunity after A. salmonicida 66
infection was investigated. 67
2. Materials and Methods 68
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2.1. Fish husbandry and experimental facilities 69
This trial was conducted at Shandong Oriental Ocean Sci-Tech Co., Ltd 70
(Shandong, China). The average initial body weight of rainbow trout was 293.0 ± 71
18.3 g. At the farming trial, 1200 farmed individuals were randomly placed into a 72
100-m3 tank containing 80-m3 seawater (average parameters: water flow 7 L/min, 73
water temperature 12 ℃, oxygen saturation 7.2 mg/L, pH 7.8, and salinity 30‰). 74
Fish were fed with commercial dry pellets without β-glucan inclusion (Beijing 75
HanYe Science & Technology Co., Ltd., Beijing, China) prior to the start of the trial. 76
They were fed 2% of their body weight every day. The feed was divided into five 77
portions and distributed automatically per day using automatic feeders 78
(Hangzhou Ecological Environmental Engineering Co., Ltd). 79
2.2. Experimental diets 80
β-1, 3-glucan produced by Saccharomyces cerevisiae was purchased from 81
Angel Yeast Co., Ltd (Hubei, China). The basal diet (commercial diet) was used as 82
the control diet. For the experimental diets, the basal diet was supplemented with 83
different levels of β-1, 3-glucan (0.05%, 0.1% and 0.2%). The formulation of the 84
basal diet is listed in Table 1. 85
Table 1 86
Composition of the experimental basal diet. 87
Ingredients Percentage (%)
Fish meala 42.8
Soybean meala 21.2
DL-methionineb 2.65
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Fish oilc 15.7
Wheat flourd 10.8
Wheat starchd 2.5
ɑ-Cellulosee 3.5
Vitamin and mineralf
0.85
Proximate composition
Crude protein 42.18
Crude lipid
Ash
11.23
11.51
a Fish meal: crude protein 75.3% dry matter, crude lipid 2.5% dry matter; soybean meal: 51.7% crude 88
protein dry matter, 2.0% crude lipid of dry matter. Both meals were from Qingdao Fusen Co., Ltd., 89
Qingdao, China. 90
b DL-methionine: Guangzhou Shuomu biological technology Co., Ltd., Guangzhou, China. 91
c Fish oil: Qingdao Fusen Co., Ltd., Qingdao, China. 92
d Wheat flour and wheat starch: Haixing Huicheng feed marketing Co., Ltd., Cangzhou, China.
93
e ɑ-Cellulose: Sahn chemical technology Co., Ltd., Shanghai, China.f Vitamin (mg or g/kg diet): thiamin, 94
25 mg; riboflavin, 45 mg; pyridoxine-HCl, 20 mg; vitamin B12, 0.1mg; vitamin K3, 10 mg; inositol, 800 95
mg; pantothenic acid, 60 mg; niacin acid, 200 mg; folic acid, 20 mg; biotin, 1.20 mg; retinol acetate, 32 96
mg; cholecalciferol, 5 mg; alpha-tocopherol, 120 mg; ascorbic acid, 2000 mg; choline chloride, 2000 mg; 97
ethoxyquin, 150 mg; microcrystalline cellulose, 14.52 g. 98
Mineral (mg or g/kg diet): NaF, 2 mg; KI, 0.8 mg; CoCl2·6H2O(1%), 50 mg; CuSO4·5H2O, 10 mg; 99
FeSO4·H2O, 80 mg; ZnSO4·H2O, 50 mg; MnSO4·H2O, 60 mg; MgSO4·7H2O, 1200 mg; Ca 100
(H2PO4)2·H2O, 3000 mg; NaCl, 100 mg; Zoelite, 15.448 g. 101
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2.3. Trial design 102
The trial consisted of two stages: the first stage comprised fish culturing, which 103
was performed in 12 100-m3 concrete tanks of a commercial land-based 104
recirculating aquaculture system. The 12 tanks were randomly divided into four 105
groups with triplicate tanks. The four groups consisted of a control group and three 106
experimental groups corresponding to the three levels of β-glucan (0.05%, 0.1%, 107
and 0.2%). Fish were fed with the corresponding pellets at a feeding rate of 2% body 108
weight with a feeding frequency of five times daily for 42 days. A total of 400 109
healthy individuals were randomly selected in each tank, and weighed at the 110
beginning and the end of the first stage. 111
The second trial stage was performed to study the role of β-glucan in regulating 112
immune-related functions in individuals infected with A. salmonicida. When the 113
first stage was completed, 100 individuals from each 100-m3 tank were injected with 114
A. salmonicida, and then transferred to each corresponding small tank. One hundred 115
individuals injected with 100 µl 0.9% NaCl from each control tank were transferred 116
to each of three small tanks as the non-infection control. The number of fish dying 117
from A. salmonicida was recorded daily. Every other day post infection (dpi) at 2, 4, 118
and 6 dpi, five live individuals were sampled from each tank to study the process of 119
metabolic change after infection. At 7 dpi, the survival rate was analyzed and the 120
results were used as an indication of the ability of rainbow trout fed with different 121
dosage β-glucan diets to resist bacterial challenge. 122
2.4. Challenge test and survival rate 123
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The second trial stage was carried out in 15 small round tanks (radius 1.2 m, 124
height 1.8 m, and volume 8.14 m3) of a commercial land-based recirculating 125
aquaculture system containing 7-m3 seawater (average parameters: water flow 3 126
L/min, water temperature 12 ℃, oxygen saturation 72%, pH 7.8, and salinity 127
30‰). The 15 round tanks included five groups with triplicate tanks: non-infected 128
control group (NCG), infected control group (ICG), and three infected experiment 129
groups (0.05%, 0.1% and 0.2% β-glucan). According to the preliminary 130
experiment, 100 µl 3×105 A. salmonicida resulted in fish mortality in 7 days. 131
Before transferring to small tanks, with the exception of the non-infected control, 132
fish were injected with 100 µl 3×105 A. salmonicida. In this experiment, 100 133
individuals from the previous experiment were initially added to each small tank. 134
The survival rate of fish in each group was calculated at 7 dpi as follows: 135
Survival rate = the number of live fish total fish-sampled fish ×100 136
2.5. Sampling 137
Sampling (n=5) occurred at the beginning and the end of the first experiment. For 138
the second experiment, sampling (n=5) took place at every other day after infection 139
(day 2, 4, and 6 dpi). Fish for dissection were anesthetized with tricaine 140
methanesulfonate before sampling. Blood samples for subsequent biochemical 141
analysis were drawn using non-heparinized 5 ml syringes through the caudal vein 142
and kept at 4 °C for 5 h. After that, the blood samples were centrifuged at 4000 g for 143
20 min to obtain serum, which was stored at -80 °C until use. Furthermore, head 144
kidney tissue was also sampled, frozen quickly in liquid nitrogen and stored at 145
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-80 °C for subsequent gene expression tests. 146
2.6. Growth measurements 147
Four hundred individuals randomly selected from each tank were weighed 148
individually at the beginning and the end of the feeding trial. After the first stage, 149
the specific growth rate (SGR), weight gain (WG), feed efficiency (FE) in all groups 150
were calculated according to previously published methods [19] as follows: 151
SGR= ln final weight-ln initial weigh
Total duration of the experiment ×100 152
WG= final weight- initial weight
initial weight ×100 153
Feed efficiency= final weight-initial weight
feed consumed (g, dry weight) 154
2.7. Analysis of serum enzyme activities 155
The activities of total superoxide dismutase (T-SOD), catalase (CAT), peroxidase 156
(POD), lysozyme (LSZ), glutamate pyruvate transaminase (GPT), glutamic 157
oxalacetic and transaminase (GOT) in serum were detected by reagent kit (NanJing 158
JianCheng Bio Inst, Nanjing, China) and guided by the manufacturer's instruction. 159
2.8. RNA extraction, cDNA synthesis and qRT-PCR analysis 160
Total RNA from head kidney tissue was extracted with RNAfast kit 161
(FASTAGEN, Shanghai, China) following the manufacturer's instruction. A 162
Biodropsis BD-1000 spectrophotometer (OSTD, Beijing Co., Ltd., China) was used 163
to assess the purity of RNA by determining the ratio of the absorbance at 260/280 164
nm and quantify the concentration of RNA. All samples had 260/280 nm ratios 165
between 1.8 and 2.0. Additionally, the integrity of the RNA samples was verified by 166
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1.0% agarose gel electrophoresis. A sample of 1 µg of RNA was used as the 167
template for synthesis of the first strand of cDNA in a final reaction volume of 20 168
µL using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, 169
China). The cDNA was diluted 10-fold for qRT-PCR. 170
2.9. RNA extraction, cDNA synthesis and qRT-PCR analysis 171
The primers designed for β-actin, LSZ, Cu/Zn-SOD, CAT and POD were listed in 172
Table 2. β-actin was chosen as a reference housekeeping gene. Real-time PCR was 173
performed using an ABI 7500 Fast instrument and 7500 software v2.0.1 (Applied 174
Biosystems, USA). The PCR mixture contained 2 µL diluted cDNA, 10 µL 2 × 175
SYBR Green PCR Mix (Takara, Dalian, China), 4 µM of each gene-specific primer, 176
0.4 µL ROX Reference Dye and 6.8 µL distilled water in a final volume of 20 µL. 177
Cycling parameters were: 95 ℃ for 30 s, followed by 40 cycles of 95 ℃ for 5 s and 178
specific annealing temperature for 30 s and then a melt curve stage after the cycling 179
stage. Specificity of qRT-PCR was analyzed by agarose gel and melting curve 180
analysis. The expression levels of target gene were calculated by (target gene/β-actin) 181
using the 2 ( -∆∆Ct ) method [20]. Finally, data were expressed as fold change to 182
control group. 183
Table 2 184
Primers designed in the qRT-PCR 185
Gene name Primer name Accession no. Primer sequence Amplification
β-actin ACTINF1 NM_001124235.1 ATGGGCCAGAAAGACAGCTACGTG 140 bp
β-actin ACTINR1 NM_001124235.1 CTTCTCCATGTCGTCCCAGTTGGT 140 bp
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LSZ LSZF1 BT073234 GGTTATAGCGGGGTGACTGC 228 bp
LSZ LSZR1 BT073234 TAGCCCAAAGCCTCTTCCAT 228 bp
Cu/Zn-SOD SODF1 NM_001160614 GAGATGGTGCTGAAGGCTGTT 226 bp
Cu/Zn-SOD SODR1 NM_001160614 GTCCTCCGTGGGTCTTGTTG 226 bp
POD PODF2 NM_001165188 GTCCCAAACGTCCAGAAGAG 128 bp
POD PODR2 NM_001165188 GTCAGCGATCCAGCTAACAA 128 bp
HSP70
HSP70
HSP70F2
HSP70R2
AB062281.1
AB062281.1
GACGCTGACAAATACAAAGCT
TGTTCTCCAACCAGGAAATG
190 bp
190 bp
2.10. Statistical analysis 186
The results were analyzed by one-way analysis of variance using SPSS 19.0 187
(SPSS, Chicago, IL, USA). Significant differences were indicated by Duncan's 188
multiple range test. Data were presented as mean ± SD (standard deviation of the 189
mean) and considered to be significantly different at P ≤ 0.05 level. 190
3. Results 191
3.1. Growth performance 192
In general, the SGR, WG and FE were significantly higher (P ≤ 0.05) when the 193
dietary β-glucan level increased. After administration of dietary β-glucan for 42 194
days, the SGR, WG and FE of the 0.1% and 0.2% β-glucan groups were 195
significantly higher than the control group (P ≤ 0.05). These three indicators in the 196
0.05 group have no significant difference with the control group (P > 0.05) (Tab. 3). 197
Table 3 Growth performance indices of rainbow trout fed different dietary β-glucan for 6 198
weeks. 199
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CG 0.05 0.1 0.2 WG (%)
61.57±1.62a 63.43±1.13a 73.29±1.60b 81.43±3.78c
SGR (%) 1.24±0.03a 1.3±0.02a 1.47±0.03b 1.62±0.05c
FE (%)
0.61±0.02a
0.63±0.01a
0.73±0.02b
0.81±0.04c
Tab. 3. CG denotes control group, 0.05 denotes experiment group fed 0.05% dietary β-glucan, 200
0.10 denotes experiment group fed 0.1% dietary β-glucan and 0.20 denotes experiment group 201
fed 0.2% dietary β-glucan. Data are expressed as mean ± standard deviation. Different 202
superscripts indicate significant (P ≤ 0.05) difference between groups. 203
3.2. Survival rate 204
Overall, the survival rate was significantly affected by different dosages of dietary 205
β-glucan. In the NCG, survival rate was 92%, which was significantly higher (P ≤ 206
0.05) than in the other groups. The ICG had the lowest survival rate (32%) that was 207
significantly lower than other groups (P ≤ 0.05). As shown in Fig. 1, the survival 208
rate was significantly (P ≤ 0.05) improved by increasing the dosage of dietary 209
β-glucan. Among the experimental groups, the survival rate of 0.2% the β-glucan 210
group was significantly higher than the other groups (P ≤ 0.05). However, there was 211
no significant difference between the 0.05% and 0.1% β-glucan groups (P ≤ 0.05) 212
(Fig.1). 213
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214
Fig. 1. Survival rate (%) of rainbow trout infected by A. salmonicida after feeding for 42 days. 215
NCG denotes control group, ICG denotes infected control group, 0.05 denotes experiment 216
group fed 0.05% dietary β-glucan, 0.10 denotes experiment group fed 0.1% dietary β-glucan 217
and 0.20 denotes experiment group fed 0.2% dietary β-glucan. Data are expressed as mean ± 218
standard deviation. Different superscripts indicate significant (P ≤ 0.05) difference between 219
groups. 220
3.3. Effect of dietary β-glucan in different dosages on stress- and immune-related 221
index of rainbow trout after infection by A. salmonicida 222
3.3.1. Serum T-SOD activity and relative Cu-Zn/SOD mRNA expression in the 223
head kidney 224
After feeding for 42 days, T-SOD activity in all groups decreased significantly 225
compared with day 0 (P ≤ 0.05); it was higher in the 0.1% and 0.2% groups than in 226
the other groups (P ≤ 0.05). At 2 dpi, the T-SOD activity reached lowest level in the 227
ICG and reached highest point in the 0.2% group (P ≤ 0.05). In all β-glucan groups, 228
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T-SOD activity was significantly higher than in the NCG and there was no 229
difference between 0.05% and 0.1% groups (P > 0.05). At 4 dpi, the T-SOD activity 230
in the 0.1% group was significantly higher than in the other groups (P ≤ 0.05). The 231
T-SOD activity in the 0.2% group was significantly lower than in the ICG (P ≤ 0.05); 232
however, there was no significant difference between the ICG and 0.05% group (P > 233
0.05). At 6 dpi, T-SOD activity in the β-glucan groups was significantly higher than 234
in the ICG. T-SOD activity in the ICG decreased to minimum at 2 dpi (P ≤ 0.05), 235
increased to maximum at 4 dpi (P ≤ 0.05). In the 0.05% and 0.1% groups, T-SOD 236
activity improved to maximum at 4 dpi (P ≤ 0.05). In the 0.2% group, T-SOD 237
activity improved to maximum at 2 dpi (P ≤ 0.05) (Fig. 2A). 238
After feeding for 42 days, Cu-Zn/SOD gene expression in the 0.05% and 0.1% 239
groups was considerably higher than in the other groups; however, in the 0.2% 240
group it was significantly lower than in the other groups (P ≤ 0.05). At 2 dpi, 241
Cu-Zn/SOD expression in the 0.1% and 0.2% groups was higher than in other 242
groups while it reached the lowest point in the 0.05% group (P ≤ 0.05). At 4 dpi, 243
Cu-Zn/SOD expression in dietary β-glucan groups was remarkably higher than in 244
the ICG (P > 0.05). At 6 dpi, Cu-Zn/SOD expression in 0.2% group was 245
considerably higher than in the ICG (P ≤ 0.05). Cu-Zn/SOD expression in the ICG, 246
0.1%, and 0.2% groups reached a maximum at 2 dpi, while in the 0.05% group it 247
increased to the highest at 4 dpi (Fig. 2B). 248
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249
250
Fig. 2. T-SOD activity (A) and relative Cu-Zn/SOD expression (B) in the head kidney of 251
rainbow trout at different times. NCG denotes control group, ICG denotes infected control 252
group, 0.05 denotes experiment group fed 0.05% dietary β-glucan, 0.10 denotes experiment 253
group fed 0.1% dietary β-glucan, 0.20 denotes experiment group fed 0.2% dietary β-glucan, 254
and dpi denotes days post infection. Data are expressed as mean ± standard deviation. Different 255
lowercases indicate significant differences (P ≤ 0.05) among time points in the same group. 256
Different uppercases indicate significant differences (P ≤ 0.05) among the groups at the same 257
time points. 258
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3.3.2. Serum POD activity and relative POD mRNA expression in the head kidney 259
After 42 days of feeding, POD decreased significantly compared with 0 day in all 260
groups (P ≤ 0.05), and no significant difference was found among the different 261
groups (P > 0.05). At 2 dpi, the POD activity in the dietary β-glucan groups was 262
significantly higher than in the NCG (P ≤ 0.05) and it was the highest in the 0.05% 263
group. At 4 dpi, POD activity in the 0.1% and 0.2% groups was significantly higher 264
than others (P ≤ 0.05). At 6 dpi, POD activity in the NCG and 0.05% group was 265
significantly higher than in the other groups (P ≤ 0.05). 266
POD activity in the ICG and 0.05% group increased to its maximum at 6 dpi 267
while POD increased to its maximum at 4 dpi in the 0.1% and 0.2% groups (P ≤ 268
0.05) (Fig. 3A). 269
After feeding for 42 days, POD expression in the 0.1% and 0.2% groups was 270
considerably higher than in the other groups (P ≤ 0.05). At 2 dpi, POD expression in 271
the 0.05% and 0.2% groups was higher than in the other groups (P ≤ 0.05). At 4 dpi, 272
POD expression in the 0.1% and 0.2% groups was markedly higher than in the ICG 273
and 0.05% group (P > 0.05). At 6 dpi, POD expression in the ICG was significantly 274
higher than in the other infected groups (P ≤ 0.05). POD expression in the 0.1% and 275
0.2% groups reached a maximum at 4dpi while in the 0.05% group and ICG it was 276
the highest at 6dpi (Fig. 3B). 277
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278
279
Fig. 3. POD activity (A) and relative POD expression (B) in the head kidney of rainbow trout 280
at different times. NCG denotes control group, ICG denotes infected control group, 0.05 281
denotes experiment group fed 0.05% dietary β-glucan, 0.10 denotes experiment group fed 0.1% 282
dietary β-glucan, 0.20 denotes experiment group fed 0.2% dietary β-glucan, and dpi denotes 283
days post infection. Data are expressed as mean ± standard deviation. Different lowercases 284
indicate significant differences (P ≤ 0.05) among time points in the same group. Different 285
uppercases indicate significant differences (P ≤ 0.05) among the groups at the same time 286
points. 287
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3.3.3. Serum CAT activity and relative CAT mRNA expression in the head kidney 288
After 42 days of feeding, CAT activity decreased significantly compared with 0 289
day in all groups (P ≤ 0.05), and no significant difference was found among groups 290
(P > 0.05). At 2 dpi, CAT activity was the lowest in the ICG and the highest in the 291
0.1% group. It was significantly higher in the 0.05% and 0.2% groups than in the 292
NCG (P ≤ 0.05); At 4 dpi, CAT activity in all three β-glucan groups was higher than 293
in the ICG (P ≤ 0.05) and it was highest in the 0.2% group (P ≤ 0.05). It was 294
significantly higher in the 0.05% and 0.2% groups than in the 0.1% group (P ≤ 0.05). 295
At 6 dpi, CAT activity was the highest in the ICG, significantly higher in the 0.05% 296
group than in 0.1% and 0.2% groups (P ≤ 0.05). 297
CAT activity in the ICG and 0.05% group increased to its maximum at 6 dpi, while 298
in the 0.1% and 0.2% groups, it increased to its maximum at 4 dpi (P ≤ 0.05) (Fig. 299
4A). 300
CAT mRNA expression was not significantly different among groups after 301
feeding for 42 days (P ≤ 0.05). At 2 dpi, CAT expression in the dietary β-glucan 302
groups was higher than in the ICG; it was highest in the 0.1% group (P ≤ 0.05). At 4 303
dpi, CAT expression in the 0.2% group was significantly higher than in the other 304
groups (P ≤ 0.05), but there was no difference among the infected groups (P > 0.05). 305
At 6 dpi, CAT expression in the ICG was considerably higher than in other groups 306
(P ≤ 0.05). 307
CAT expression in the 0.05% and 0.1% groups reached a maximum at 2 dpi and 308
was the highest in the 0.2% group at 4 dpi, while the ICG group reached its highest 309
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level at 6 dpi (Fig. 4B) 310
311
312
Fig. 4. CAT activity (A) and relative CAT expression (B) in the head kidney of rainbow trout 313
at different times. NCG denotes control group, ICG denotes infected control group, 0.05 314
denotes experiment group fed 0.05% dietary β-glucan, 0.10 denotes experiment group fed 0.1% 315
dietary β-glucan, 0.20 denotes experiment group fed 0.2% dietary β-glucan, and dpi denotes 316
days post infection. Data are expressed as mean ± standard deviation. Different lowercases 317
indicate significant differences (P ≤ 0.05) among time points in the same group. Different 318
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uppercases indicate significant differences (P ≤ 0.05) among the groups at the same time 319
points. 320
3.3.4. Serum LSZ activity and relative LSZ mRNA expression in the head kidney 321
Dietary β-glucan had no significant effect on serum LSZ in rainbow trout after 42 322
days of feeding (P > 0.05). At 2 dpi, LSZ activity in groups infected by A. 323
salmonicida was significantly lower than in the NCG (P ≤ 0.05) and was the lowest 324
in the ICG. At 4 dpi, LSZ activity in the dietary β-glucan groups was remarkably 325
higher than control groups (P ≤ 0.05) and it was the highest in the 0.1% group. At 6 326
dpi, LSZ in the NCG was significantly higher than in the other groups (P ≤ 0.05). It 327
was significantly higher in the 0.01% group than the other β-glucan groups (P ≤ 328
0.05). LSZ activity in the ICG group increased to its maximum at 6 dpi, while LSZ 329
in the dietary β-glucan groups reached maximum at 4 dpi (P ≤ 0.05) (Fig. 5A). 330
LSZ expression was not significantly different among groups (P ≤ 0.05) before 331
infection. At 2 dpi, LSZ expression in the 0.1% group was higher than in the 0.05% 332
group (P ≤ 0.05). At 4 dpi, LSZ expression in 0.1% group was the highest (P ≤ 0.05), 333
and significantly higher in the 0.1% and 0.2% groups compared with the other 334
groups (P ≤ 0.05). At 6 dpi, LSZ expression in the ICG was significantly higher than 335
in other groups while the 0.2% group had the lowest value (P ≤ 0.05). 336
LSZ in the dietary β-glucan groups reached its maximum at 4 dpi and the ICG group 337
reached its highest level at 6dpi (Fig. 5B). 338
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339
340
Fig. 5. LSZ activity (A) and relative LSZ expression (B) in head kidney of rainbow trout at 341
different times. NCG denotes control group, ICG denotes infected control group, 0.05 denotes 342
experiment group fed 0.05% dietary β-glucan, 0.10 denotes experiment group fed 0.1% dietary 343
β-glucan, 0.20 denotes experiment group fed 0.2% dietary β-glucan, and dpi denotes days post 344
infection. Data are expressed as mean ± standard deviation. Different lowercases indicate 345
significant differences (P ≤ 0.05) among time points in the same group. Different uppercases 346
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indicate significant differences (P ≤ 0.05) among the groups at the same time points. 347
3.3.5. GPT activity in serum 348
GPT in dietary β-glucan groups was significantly higher than in the control group 349
after 42 days of feeding (P > 0.05). At 2 dpi, GPT activity in infected groups was 350
significantly lower than in the NCG and was significantly higher in the ICG than in 351
other groups (P ≤ 0.05). At 4 dpi, GPT activity in the 0.05% group was remarkably 352
higher than in other groups (P ≤ 0.05). With the exception of the NCG, GPT activity 353
was lower in the 0.2% group compared with the other groups (P ≤ 0.05). At 4 dpi, 354
GPT activity in the ICG was significantly higher than in the other groups (P ≤ 0.05), 355
but had returned to the initial levels in the other groups (P ≤ 0.05). GPT activity in 356
the ICG group showed an increasing trend until 6 dpi. In dietary β-glucan groups, it 357
increased up to 4 dpi and then decreased to initial level at 6dpi (P ≤ 0.05) (Fig. 6). 358
359
Fig. 6. GPT activity in serum of rainbow trout at different times. NCG denotes control group, 360
ICG denotes infected control group, 0.05 denotes experiment group fed 0.05% dietary β-glucan, 361
0.10 denotes experiment group fed 0.1% dietary β-glucan, 0.20 denotes experiment group fed 362
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0.2% dietary β-glucan, and dpi denotes days post infection. Data are expressed as mean ± 363
standard deviation. Different lowercases indicate significant differences (P ≤ 0.05) among time 364
points in the same group. Different uppercases indicate significant differences (P ≤ 0.05) 365
among the groups at the same time points. 366
3.3.6. GOT activity in serum 367
GOT activity improved significantly in all groups (P ≤ 0.05) after feeding for 42 368
days, but no significant difference was recorded among them (P > 0.05). At 2 dpi, 369
with the exception of the 0.2% group, GOT activity in the infected groups was 370
significantly higher than in the NCG, and was significantly higher in the ICG than in 371
the other groups (P ≤ 0.05). At 4 dpi, GOT activity in the ICG and 0.05% group was 372
markedly higher than others and that in 0.2% group was significantly lower than 373
other groups (P ≤ 0.05). 6 days post-infection, GOT in 0.05% group was 374
significantly higher than in the other groups (P ≤ 0.05). 375
GOT activity in the ICG group reached to its maximum at 2 dpi and was at a 376
higher level than the dietary β-glucan groups. In the 0.2% β-glucan group, the GOT 377
level remained at a low level comparable with the control group (P ≤ 0.05) (Fig. 7). 378
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379
Fig. 7. GOT activity in serum of rainbow trout at different times. NCG denotes control group, 380
ICG denotes infected control group, 0.05 denotes experiment group fed 0.05% dietary β-glucan, 381
0.10 denotes experiment group fed 0.1% dietary β-glucan, 0.20 denotes experiment group fed 382
0.2% dietary β-glucan, and dpi denotes days post infection. Data are expressed as mean ± 383
standard deviation. Different lowercases indicate significant differences (P ≤ 0.05) among time 384
points in the same group. Different uppercases indicate significant differences (P ≤ 0.05) 385
among the groups at the same time points. 386
3.3.7. HSP70 mRNA expression in the head kidney 387
HSP70 expression was not significantly different among groups (P ≤ 0.05) before 388
infection. At 2 dpi, HSP70 expression in the 0.1% group was significantly higher 389
than in the other groups (P ≤ 0.05). At 4 dpi, HSP70 expression in the 0.2% group 390
reached the highest level (P ≤ 0.05) and there was no significant difference between 391
the other groups (P > 0.05). At 6 dpi, HSP70 expression in the ICG was 392
considerably higher than in the other groups, while it was lower in the 0.05% group 393
compared with other infected groups (P ≤ 0.05). 394
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HSP70 expression in the 0.2% group reached its maximum at 4dpi, while in all 395
other infected groups it reached the highest level at 6dpi (Fig. 8). 396
397
Fig. 8. Relative HSP70 expression in head kidney of rainbow trout at different times. NCG 398
denotes control group, ICG denotes infected control group, 0.05 denotes experiment group fed 399
0.05% dietary β-glucan, 0.10 denotes experiment group fed 0.1% dietary β-glucan, 0.20 400
denotes experiment group fed 0.2% dietary β-glucan, and dpi denotes days post infection. Data 401
are expressed as mean ± standard deviation. Different lowercases indicate significant 402
differences (P ≤ 0.05) among time points in the same group. Different uppercases indicate 403
significant differences (P ≤ 0.05) among the groups at the same time points. 404
4. Discussion 405
Fish are more prone to infection by various pathogens in industrial aquaculture 406
due to high stocking density and management practices leading to an adverse 407
farming environment [21]. β-glucan, as one of the commonly known 408
immunostimulants, has been demonstrated to enhance the innate response with little 409
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impairment to the development of animal [22, 23]. However, to the best of the 410
experts’ knowledge, little has been published on the process of how β-glucan 411
regulates the innate response during pathogen infection. In the present study, 412
changes in serum enzyme activities and a series of stress- and immune-related genes 413
expression in the head kidney of rainbow trout were assessed during the challenge 414
period. 415
The present findings revealed that dietary β-glucan can promote the growth of 416
rainbow trout. Within the range of 0.2% β-glucan concentration, growth 417
performance improved with increased dosage. The survival rate of rainbow trout 418
infected by A. salmonicida was significantly higher in the dietary β-glucan groups, 419
and the 0.2% group had a stronger level of protection than both the 0.5% and 0.1% 420
groups. This result is consistent with many other studies in which β-glucan was 421
shown to increase the growth performance and the survival rate in numerous aquatic 422
species, such as koi carp (Cyprinus carpio koi) [24], large yellow croaker 423
(Pseudosciaena crocea) [15], and juvenile pompano (Trachinotus ovatus) [25]. 424
Besides dosage, feeding duration and the mode of administration among several 425
other factors can influence the effectiveness of β-glucan [26]. Therefore, further 426
studies are required to determine the contributing factors and mechanism involved in 427
the enhancement of growth performance. 428
In an adverse setting such as pathogens infection or cold stress, respiratory 429
burst can occur in the immune cells and NADPH oxidase is activated. After 430
activation, NADPH oxidase generates the production of a series of reactive 431
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oxygen species (ROS) such as superoxide anion (O2-) and hydrogen peroxide 432
(H2O2) which are effective antimicrobial substances. However, excessive ROS 433
can the membranes and DNA [27, 28]. The antioxidant system consisting of SOD, 434
CAT, and POD can effectively eliminate ROS in order to maintain a stable 435
internal environment [29]. Therefore SOD, CAT, and POD can indirectly reflect 436
the ability of the host to remove ROS, and are generally recognized as indicators 437
of the immune potential [30]. In Atlantic salmon, serum SOD, POD, and CAT 438
activities, along with their mRNA expression, have been shown to significantly 439
decrease after bacterial challenge [31]. However, in the present study, serum T- 440
SOD, POD, and CAT activities, and their mRNA expression in the head kidney 441
generally increased to a higher level after infection. This indicates that during 442
infection in rainbow trout, β-glucan can enhance the host’s antioxidant ability to 443
clear more excessive ROS triggered by bacteria. 444
Lysozyme, in the serum, mucus, and ova [32], can attack the peptidoglycan layer 445
of bacterial cell walls causing lysis either directly or together with the complement 446
system [11]. Lysozyme in rainbow trout have a strong bactericidal effect on a few 447
Gram-negative fish pathogenic bacteria, such as Y. ruckeri [33]. In the present study, 448
serum LSZ and LSZ mRNA expression in the head kidney after infection were 449
higher than baseline levels (P ≤ 0.05). In the dietary β-glucan groups, serum LSZ 450
and its expression in the head kidney reached a higher peak earlier than in the 451
control group which was in accordance with the findings of other studies [11, 34]. 452
Such enhancement might be related to improved phagocytic activity of phagocytic 453
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cells [35]. After activation by β-glucan, the in vivo phagocytic cells might trigger 454
other antimicrobial mechanisms such as the release of lysosomal enzymes, the 455
complement system and production of ROS [36]. In a murine model, orally 456
administered β-glucan could be transferred to the bone marrow by macrophages 457
where they appeared 4 days post β-glucan administration [37]. As the head kidney 458
of fish is equivalent to mammalian bone marrow [38, 39], it is deduced that the 459
lysozyme gene regulations in the rainbow trout head kidney could be the 460
consequence of the same type of mechanism [37]. However, this needs to be 461
investigated further. 462
GPT and GOT are serum non-functional enzymes. They are normally 463
distributed in the cells of the liver, kidneys, heart, and other organs [40]; however, 464
the activities of these two enzymes in serum increase significantly when tissue 465
cells, especially those in the liver and heart, are impaired. Therefore, these 466
enzymes are considered to be important for assessing the state of liver [41]. In 467
the present study, serum GPT and GOT activity was considerably increased after 468
infection; however, in the dietary β-glucan groups, they were lower than in the 469
control group. This indicates that β-glucan can protect the liver of rainbow trout 470
from damage by bacteria especially at higher dosages. 471
In aquatic animals, the HSP family has been shown to play an important part 472
in the host response to environmental stressors, along with specific and 473
non-specific immune responses to bacterial and viral infections in both fish and 474
shrimp. Regulation of HSP genes relating to infection has also been reported in 475
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many aquatic species such as Penaeus monodon [42] and sea bass [43]. In the 476
present study, HSP70 expression in all groups increased considerably after 477
infection; the most dramatical and earlier increase was found in the 0.2% 478
β-glucan group than others. The HSP response has been shown to regulate the 479
mammalian immune response and, in particular, the T-cell-mediated response 480
[44]. This produces inflammatory products such as cytokines and adhesins. 481
Despite insufficient evidence, it has been deduced that HSPs also play a similar 482
role in regulating the fish immune response and correlate with the T-cell function 483
in immune response. Therefore HSPs may be a vital factor in immunity [45]. In 484
the present study, earliest and highest levels of expression of HSP70 in the high 485
dosage β-glucan group indicate that β-glucan is likely to alter the immune 486
response of rainbow trout via regulation of HSP70 expression. 487
This study confirms that dietary β-glucan is beneficial for promoting growth 488
and enhancing resistance to A. salmonicida in rainbow trout. According to the 489
analysis of stress- and immune-related factors, it can be assumed that β-glucan 490
helps the organism to resist bacterial infection in two ways. It can stimulate 491
stress- and immune-related factors at higher levels and quickly it can trigger the 492
immune reaction to fight against bacterial infection. The present findings indicate 493
that the optimal level of dietary β-glucan concentration for rainbow trout is 0.2%. 494
However, the regulating mechanism of β-glucan in the immune response requires 495
further research. 496
Ethics statement 497
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The use of non-human primates in research. All fish in this study were handled 498
in strict accordance with China legislation on scientific procedures on living 499
animals. The protocol was approved by the ethics committee at University of 500
Chinese Academy of Science (permit number: 20021101). 501
Conflict of interest 502
No potential conflict of interest. 503
Acknowledgment 504
This work was financially supported by the National Natural Science 505
Founds (grant nos. 41306152, 31472312, 31402283), the Jiangsu Province key 506
R&D Project (BE2015325), the National Key Technologies R&D 507
Program (2014BAD08B09), the 56th China Postdoctoral Science Foundation 508
(2014M560580), Postdoctoral Innovation Project Special Funds of Shandong 509
Province (201402005), and Independent Innovation and Achievements 510
Transformation Special Foundation of Shandong Province (2014ZZCX06204). 511
We particularly thank Dr Zaki Zaki Sharawy for carefully revising the paper. 512
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1
Highlight: 2
1、 Dietary β-glucan in dosage of 0.1% and 0.2% can enhance growth 3
performance of rainbow trout. 4
2、 Dietary β-glucan (from 0.05% to 0.2%) is able to significantly 5
enhance resistance to A. salmonicida in rainbow trout. 6
3、 It can stimulate stress- and immune-related factors at higher levels 7
and it can trigger the immune reaction more quickly to fight against 8
bacterial infection. 9
4、 In this paper, the optimal level of dietary β-glucan concentration for 10
rainbow trout is 0.2%. 11
12