combined effects of dietary fructooligosaccharide and bacillus licheniformis on innate immunity,...

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Fish & Shellfish Immunology xxx (2013) 1e7

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Fish & Shellfish Immunology

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Combined effects of dietary fructooligosaccharide and Bacilluslicheniformis on innate immunity, antioxidant capability and diseaseresistance of triangular bream (Megalobrama terminalis)

Chun-Nuan Zhang, Xiang-Fei Li, Wei-Na Xu, Guang-Zhen Jiang, Kang-Le Lu, Li-Na Wang,Wen-Bin Liu*

Key Laboratory of Aquatic Nutrition and Feed Science of Jiangsu Province, College of Animal Science and Technology, Nanjing Agricultural University,No. 1 Weigang Road, Nanjing 210095, PR China

6768
6970717273747576777879808182
a r t i c l e i n f o

Article history:Received 15 April 2013Received in revised form30 July 2013Accepted 31 July 2013Available online xxx

Keywords:Megalobrama terminalisFructooligosaccharideBacillus licheniformisInnate immunityAntioxidant capabilityDisease resistance

* Corresponding author. Tel./fax: þ86 025 8439538E-mail address: [email protected] (W.-B. Liu).

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Please cite this article in press as: Zhang Cimmunity, antioxidant capability and diseahttp://dx.doi.org/10.1016/j.fsi.2013.07.047

a b s t r a c t

This study was conducted to investigate the effects of fructooligosaccharide (FOS) and Bacillus lichen-iformis (B. licheniformis) and their interaction on innate immunity, antioxidant capability and diseaseresistance of triangular bream Megalobrama terminalis (average initial weight 30.5 � 0.5 g). Nineexperimental diets were formulated to contain three FOS levels (0, 0.3% and 0.6%) and threeB. licheniformis levels (0, 1 � 107, 5 � 107 CFU g�1) according to a 3 � 3 factorial design. At the end of the8-week feeding trial, fish were challenged by Aeromonas hydrophila (A. hydrophila) and survival rate wasrecorded for the next 7 days. The results showed that leucocyte counts, alternative complement activityas well as total serum protein and globulin contents all increased significantly (P < 0.05) as dietaryB. licheniformis levels increased from 0 to 1 � 107 CFU g�1, while little difference (P > 0.05) was observedin these parameters in terms of dietary FOS levels. Both plasma alkaline phosphatase and phenoloxidaseactivities were significantly (P < 0.05) affected only by dietary FOS levels with the highest valuesobserved in fish fed 0.6 and 0.3% FOS, respectively. Both immunoglobulin M content and liver superoxidedismutase (SOD) activity were significantly affected (P > 0.05) by both FOS and B. licheniformis. Livercatalase, glutathione peroxidase as well as plasma SOD activities of fish fed 1 � 107 CFU g�1

B. licheniformis were all significantly (P < 0.05) higher than that of the other groups, whereas theopposite was true for malondialdehyde content. After A. hydrophila challenge, survival rate was notaffected (P > 0.05) by either FOS levels or B. licheniformis contents, whereas a significant (P < 0.05)interaction between these two substances was observed with the highest value observed in fish fed 0.3%FOS and 1 � 107 CFU g�1 B. licheniformis. The results of this study indicated that dietary FOS andB. licheniformis could significantly enhance the innate immunity and antioxidant capability of triangularbream, as well as improve its disease resistance. The best combination of these two prebiotics and/orprobiotics was 0.3% FOS and 1 � 107 CFU g�1 B. licheniformis.

� 2013 Published by Elsevier Ltd.

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1. Introduction

As the global aquaculture rapidly develops, the environmentchanges which subsequently adversely affects the aquatic animals.Accordingly, the aquaculture industry has been limited by outbreaksof infectious, particularly those caused by viruses and bacteria [1].One of the most common ways to prevent aquatic diseases is toadminister antibiotics. However, recently, there are strict regulationson the use of antibiotics in aquaculture because of its seriously

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negative effects [2]. Therefore, it is becoming increasingly importantto use preventive approaches to control diseases through improvedimmunity, pathogen inhibition and so on. An alternative method toantibiotic treatment is the use of immunostimulants. Among theimmunostimulants commonly used in aquaculture, probiotics havebeen reported to positively affect the innate immune response anddisease resistance of aquatic animals [3]. Furthermore, prebiotics arealso an effective addition to disease control strategies in aquaculture,including mannanoligosaccharides (MOS), fructooligosaccharides(FOS), inulin and vitamin C [1,4,5]. Thus, during the last decade, theapplication of both prebiotics and probiotics taking advantage oftheir pathogen control potentials has been increasing in aquaculture.

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of dietary fructooligosaccharide and Bacillus licheniformis on innateeam (Megalobrama terminalis), Fish & Shellfish Immunology (2013),

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Table 1Ingredients and proximate composition of the basal diets.

Ingredients (%) Proximate composition (% air-dry basis)

Fish meal 8 Moisture 12.32Soybean meal 30 Crude protein 33.42Cottonseed meal 16 Crude lipid 6.28Rapeseed meal 16 Energy (MJ kg�1) 14.26Soybean oil 2Fish oil 2Wheat bran 5Wheat flour 18Ca(H2PO4)2 1.8Premixa 1Salt 0.2

a Premix supplied the following minerals (g kg�1) and vitamins (IU or mg kg�1):CuSO4$5H2O, 2.0 g; FeSO4$7H2O, 25 g; ZnSO4$7H2O, 22 g; MnSO4$4H2O, 7 g;Na2SeO3, 0.04 g; KI, 0.026 g; CoCl2$6H2O, 0.1 g; Vitamin A, 900,000 IU; Vitamin D,200,000 IU; Vitamin E, 4500 mg; Vitamin K3, 220 mg; Vitamin B1, 320 mg; VitaminB2, 1090mg; Vitamin B5, 2000mg; Vitamin B6, 500mg; Vitamin B12, 1.6mg; VitaminC, 5000 mg; pantothenate, 1000 mg; folic acid, 165 mg; choline, 60,000 mg.

C.-N. Zhang et al. / Fish & Shellfish Immunology xxx (2013) 1e72

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Among the prebiotics and probiotics commonly adopted, bothFOS and Bacillus have been shown as environmentally alternativesto antibiotics in aquaculture, especially for fish [6e10]. FOS, one ofthe most studied prebiotics in fish, offers innumerable advantagesto overcome the limitations and side effects of antibiotics and otherdrugs, and also leads to high production through enhanced growth,stimulated immunity and increased resistance to pathogens of fish.Several studies have demonstrated the efficacy of FOS to increasethe non-specific immune responses of both fish and shellfish [6,7].Bacillus licheniformis (B. licheniformis) is a gram-positive, oxidase-positive and catalase-positive endospore forming non-pathogenicbacterium, belonging to the genus Bacillus [11,12]. So far,B. licheniformis has been shown to act as an antiviral and immu-noregulatory agent [13]. However, to the best of our knowledge,there is limited information concerning the application ofB. licheniformis in freshwater fish. In addition, little research isavailable regarding the combined effects of FOS and B. licheniformison innate immunity and disease resistance of fish. Furthermore, itwas reported that the administration of synbiotics yielded signifi-cantly better results than individual application of probiotics orprebiotics [6,14,15]. Thus, the investigation of the potentiallyinteractive effects of FOS and B. licheniformis in fish is of greatsignificance. According to previous studies, large variations some-times even contradictions have been reported in the application ofboth prebiotics and probiotics in aquaculture, thus warrantingextensive studies [6,16].

Triangular bream (Megalobrama terminalis) is a typical herbiv-orous freshwater fish which belongs to the Cyprinidae family. It hasa high potential for intensive aquaculture since it shares manyadmirable characteristics such as rapid growth, high diseaseresistance, good taste and high market value. Until now, it hasbecome one of the commercially important species in China.However, the rapid expansion of triangular bream aquacultureresulted in several serious diseases, mainly skin ulceration syn-drome, which is highly infectious and lethal to this species, andleads to significant economic loss. Therefore, it is quite urgent tofind a safe and effective method to prevent and/or control thediseases of this species. Bearing this in mind, the present study wasconducted to investigate the effects of FOS and B. licheniformis andtheir interaction upon non-specific immunity, antioxidant capa-bilities and disease resistance of juvenile triangular bream. The dataobtained here may give some instructions for the application ofboth probiotics and prebiotics in this fish as well as the otherspecies sharing the same feeding habit.

2. Materials and methods

2.1. FOS and B. licheniformis

The FOS used in this study was produced by Meiji Holdings Co.,Ltd, Japan. The minimum level of sucrose combined with 1e3fructoses in the product was 95% and the level of other componentswas no more than 5%, mainly including glucose, fructose andsucroser.

The commercially available probiotic product (Alpharma Inc.America.) contains Bacillus licheniformis 5 � 109 CFU g�1.

2.2. Experimental diets

Prior to use, all feed ingredients were analyzed for proximatecomposition and the data obtained was used as a basis for the feedformulation. Fish meal, soybean meal, cottonseed meal and rape-seed meal were used as protein sources. Equal portions of fish oiland soybean oil were used as lipid sources. Wheat flour was used ascarbohydrate source. Nine experimental diets were formulated to

Please cite this article in press as: Zhang C-N, et al., Combined effectsimmunity, antioxidant capability and disease resistance of triangular brhttp://dx.doi.org/10.1016/j.fsi.2013.07.047

contain three FOS (0, 0.3 and 0.6%) levels and three B. licheniformis(0, 1 and 5 � 107 CFU g�1) levels following a 3 � 3 factorial design.Graded doses of FOS and B. licheniformiswere supplemented to thebasal diet at the expense of wheat flour to obtain the levelsrequired. Feed ingredients were ground into fine powder thenthoroughly mixed and blended oil and sufficient water to form softdough. The doughwas then pelleted (without injected steam) usinga pellet mill with a 2 mm diameter die. The experimental feed wasair-dried at 33 �C overnight and stored in sealed plastic bagsat�4 �C until use. The ingredients and composition of the basal dietare given in Table 1.

2.3. Fish and experimental design

Triangular bream were obtained from a local fish hatchery(Hangzhou, China). Prior to the feeding trial, fishwere acclimated atlaboratory conditions for 4 weeks. During the acclimation period,fish were fed a commercial feed thrice a day. After starvation for48 h, 720 healthy fish with an initial body weight of 30.5 � 0.5 gwere randomly distributed into 36 cages (1 �1 �1 m, L:W:H). Eachcage has 20 fish. During the experimental period, fish were fedthree times daily at 6:30, 12:00 and 17:30 h, respectively, for 8weeks. Fish were hand-fed to apparent satiation with utmost careto minimize feed waste. Fish were held under natural photoperiodcondition throughout the feeding trail. Water temperature, pH anddissolved oxygenwere monitored using a YSI 556 MPS multi-probefield meter (Geotech, USA). Water temperature ranged from 23 to28 �C, pH fluctuated between 6.5 and 7.6, and dissolved oxygenwasmaintained approximately at 5.0 mg L�1 during the feeding trial.

2.4. Sampling and analysis

2.4.1. SamplingAt the end of the feeding trial, fish were starved for 24 h before

sampling. Three fish from each replicate were anesthetized indiluted MS-222 (tricaine methanesulfonate, Sigma, USA) at theconcentration of 100 mg L�1. Blood samples were taken from thecaudal vein using heparinized plastic syringes. One part of theblood sample was placed into tubes containing heparin as ananticoagulant for the determination of total erythrocyte and leu-cocyte count. The rest blood was separated by centrifugation, andthe supernatant was pooled and stored at �70 �C for subsequentanalysis. Individual livers were washed thoroughly with chilledsaline (0.9 g NaCl L�1), dried quickly over a piece of filter paper andstored at �70 �C for subsequent analysis.


C.-N. Zhang et al. / Fish & Shellfish Immunology xxx (2013) 1e7 3

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2.4.2. Proximate analysis of the experimental dietsThe proximate composition of the experimental diets was

determined according to the standard AOAC method [17]. Mois-ture was estimated by oven drying at 105 �C till constant weight.Crude protein was analyzed by micro-Kjeldahl method andmultiplying with a factor 6.25 after acid digestion using an AutoKjeldahl System (1030-Auto-analyzer, Tecator, Hoganas, Sweden).Crude lipid was determined by solvent extraction with a SoxtecSystem HT (Soxtec System HT6, Tecator, Hoganas, Sweden); ash bycombustion at 550 �Cfor 4 h. Gross energy in diets was measuredby a Bomb Calorimeter (Parr 1281, Parr Instrument Company,Moline, IL, USA).

2.4.3. Immune parametersPlasma acid phosphatase (ACP) activity was measured using a

disodium phenyl phosphate method [18]. Alkaline phosphatase(AKP) activity was carried out based on the procedures given byRausch et al. [19]. Plasma total protein and globulin contents wereestimated by the Biuret and BCG dye binding method and thebromocresol green binding method, respectively [20]. Plasmaalternative complement (ACH50) pathway activity was assayedfollowing the method described by Montero et al. [21]. Lysozymeactivity was measured based on the turbidimetric method [22].Phenoloxidase (PO) activity was estimated following a spectro-photometric method as described by Hernández-López et al [23].Total serum immunoglobulin M (IgM) content was determined bythe enzyme-linked immunosorbent assay [24].

Total erythrocyte and leucocyte number were counted based onthe procedures given by Johnson et al. [25]. The number of eryth-rocyte and leucocyte per mL of the blood sample were calculatedusing the following formula.

No. of cells/mL ¼ (No. of cells counted � dilution)/(Areacounted � depth of fluid)

2.4.4. Analysis of antioxidant capabilitiesLiver sample was homogenized in ten volumes (v/w) of in a

tissue homogenizer and centrifuged at 3000 g at 4 �C for 10 min.The supernatant was then stored at�70 �C for subsequent analysis.All enzyme preparations were carried out on ice. Dilution of thesample was done when required.

Total superoxide dismutase (t-SOD) activity was measuredfollowing the method described by Wang and Chen [26]. Bothcatalase (CAT) and glutathione peroxidase (GPX) activities weredetermined following the methods described by Lygren et al. [27].Malondialdehyde (MDA) content was determined by the thio-barbituric acid method [28].

2.5. Challenge test

Aeromonas hydrophila (A. hydrophila) (Ah, BSK-10) was providedby Freshwater Fisheries Research Center, Chinese Academy ofFishery Sciences (Wuxi, Jiangsu Province, China) and was activatedfollowing themethods described by Alexander et al. [29]. The 7-dayLD50 (Ah, BSK-10 does that killed 50% of the test fish) was deter-mined by an intraperitoneal injection of 50 fish with graded dosesof V (106, 107, 5 � 107, 108) and the result showed that the LD50 onday 7 was 5 � 107 cfu/mL.

At the termination of the feeding experiment, 10 fish from eachtreatment were injected intraperitoneally with 1 mL/kg bodyweightof A. hydrophila using medical syringes. Fish continued to receivetheir assigned diets after the injection. Fishwere carefully monitoredand mortality was recorded twice daily for the next 7 days. Survivalrate was determined at the 8th day using the following formula.


Survival rate (%) ¼ Nt � 100/N0

where Nt and N0 were the final and initial number of fish.

2.6. Statistical analysis

Datawere analyzed using the SPSS General LinearModels (GLM)procedure (SPSS 7.5, Michigan Avenue, Chicago, IL, USA) for sig-nificant differences among treatment means based on dietary FOS,B. licheniformis and their interaction. If significant (P < 0.05) dif-ferences were found in factors, Duncan’s multiple range test(Duncan, 1955) was used to rank the means. All data were pre-sented as means � S.E.M (standard error of the mean) of fourreplications.

3. Results

3.1. Immune responses

Parameters indicating the immune responses of triangularbream were presented in Table 2. Erythrocyte counts showed nosignificant (P > 0.05) difference among all the treatments. PlasmaACP and lysozyme activities were not affected (P > 0.05) by eitherFOS levels or B. licheniformis contents, whereas the opposite wastrue for plasma IgM content. IgM content of fish fed 0.3% FOS wassignificantly (P< 0.05) higher than that of the other groups in termsof dietary FOS levels, as also held true for that of fish fed1 � 107 CFU g�1 B. licheniformis. Leucocyte counts, ACH50 activity,total serum protein and globulin content all increased significantly(P < 0.05) as dietary B. licheniformis levels increased from 0 to1 � 107 CFU g�1, while little difference (P > 0.05) was observed inthese parameters in terms of dietary FOS levels. Contrary to the casementioned above, both plasma AKP and PO activities were signifi-cantly (P < 0.05) affected only by dietary FOS levels with thehighest values observed in fish fed 0.6 and 0.3 FOS, respectively. Inaddition, a significant (P < 0.05) interaction between dietary FOSand B. licheniformis was observed in leucocyte counts and ACH50,ACP, PO and lysozyme activities as well as globulin and IgM contentwith the highest values mostly observed in fish fed diet 1/0.3.

3.2. Liver and plasma antioxidant capabilities

Liver and plasma antioxidant capabilities of triangular breamwere both presented in Table 3. Both plasma CAT and GPX activitieswere not affected (P > 0.05) by either FOS levels or B. licheniformiscontents, whereas the opposite was true for liver SOD activity. LiverSOD activity of fish fed 0.3% FOS was significantly (P < 0.05) higherthan that of the other groups in terms of dietary FOS levels, as alsoheld true for that of fish fed 1 � 107 CFU g�1 B. licheniformis. Con-trary to MDA content (both in liver and plasma), liver CAT and GPXaswell as plasma SOD activities all increased significantly (P< 0.05)as dietary B. licheniformis levels increased from 0 to 1�107 CFU g�1,while little difference (P > 0.05) was observed in these parametersin terms of dietary FOS levels. In addition, a significant (P < 0.05)interaction between dietary FOS and B. licheniformis was observedin liver SOD and CAT as well as plasma SOD activities with thehighest values all observed in fish fed diet 1/0.3. MDA contentboth in liver and plasmawas also significantly (P< 0.05) affected bythis interaction, but the lowest values were obtained in fish fed diet1/0.3.

3.3. Survival rate after A. hydrophila challenge

After challenge with A. hydrophila, the first mortality wasrecorded after 24 h. Cumulative mortality was recorded up to 7


Table 2Combined effects of dietary FOS and Bacillus in practical diets on haemato-immunological responses of triangular bream.a

Diets Erythrocytecount (106 mL�1)

Leucocytecount (105 mL�1)

AKP (U L�1) ACP (U L�1) Lysozyme(U mL�1)

ACH50(U mL�1)

PO (U L�1) Total serumprotein (g L�1)

Globulin(g L�1)

IgM (g L�1)

0/0 2.07 � 0.13 1.40 � 0.15a 21.6 � 1.9a 12.5 � 0.6a 202 � 10a 198 � 4a 87.1 � 2.6a 26.1 � 0.8a 6.83 � 0.30a 22.0 � 0.5a

0/0.3 2.07 � 0.15 1.64 � 0.07ab 25.1 � 1.5ab 14.0 � 0.6abc 247 � 18ab 224 � 5bc 104 � 3bc 26.9 � 0.4ab 7.60 � 0.42ab 25.7 � 0.4bc

0/0.6 2.02 � 0.12 1.87 � 0.09b 30.2 � 1.2b 14.5 � 0.9abc 269 � 20abc 239 � 7c 106 � 2bc 27.6 � 0.5ab 8.34 � 0.16bc 26.2 � 0.6bc

1/0 2.13 � 0.22 1.53 � 0.13a 26.9 � 3.0ab 14.2 � 0.6abc 270 � 12abc 240 � 8c 93.8 � 2.9ab 27.5 � 0.5ab 8.07 � 0.43bc 25.8 � 1.0bc

1/0.3 2.09 � 0.16 1.87 � 0.11b 29.1 � 2.4ab 16.2 � 0.6c 303 � 29bc 241 � 9c 107 � 2c 27.9 � 1.2ab 9.02 � 0.32c 27.1 � 0.7c

1/0.6 2.02 � 0.06 1.87 � 0.12b 31.7 � 3.0b 14.7 � 0.5abc 282 � 19abc 231 � 6c 101 � 7bc 28.5 � 0.7b 8.23 � 0.34bc 25.6 � 0.6bc

5/0 2.11 � 0.16 1.64 � 0.11ab 26.8 � 1.9ab 15.8 � 0.4c 345 � 56c 239 � 6c 105 � 2bc 28.5 � 0.7b 8.26 � 0.31bc 25.9 � 0.6bc

5/0.3 2.03 � 0.21 1.45 � 0.10a 32.9 � 2.2b 15.4 � 1.1bc 242 � 29ab 224 � 7bc 95.3 � 5.1abc 28.0 � 0.7ab 7.78 � 0.27ab 25.6 � 0.4bc

5/0.6 2.10 � 0.14 1.46 � 0.06a 29.0 � 3.4ab 13.3 � 1.0ab 213 � 11a 205 � 5ab 93.7 � 4.0ab 28.3 � 0.8ab 7.49 � 0.39ab 25.0 � 0.3bc

Two-way ANOVAFOS ns ns * ns ns ns * ns ns **B. licheniformis ns * ns ns ns ** ns * * *Interaction ns * ns * ** *** * ns * ***

a Values are means � S.E.M of four replicates. Means in the same column with different superscripts were significantly different (P < 0.05).


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days after injection. Survival rate was determined at the 8th dayand was presented in Fig. 1. Survival rate of triangular bream wasnot affected (P > 0.05) by either FOS levels or B. licheniformis con-tents. However, it was significantly (P < 0.05) affected by theinteraction with the highest value observed in fish fed diet 1/0.3.

4. Discussion

4.1. Immune response

The roles of both prebiotics and probiotics enhancing the im-mune system have been extensively investigated and reviewed notonly inmammals [30] but also in aquatic animals [1,3e5]. Among theprebiotics and/or probiotics widely adopted, both oligosaccharidesand Bacillus have been successfully used in aquaculture. Previousstudies showed that both FOS and Bacillus could enhance the diseaseresistance of fish and shrimp mainly by suppressing the pathogens,enhancing immunity or improving water quality [6e10]. However,most studies mentioned above tested only one kind of prebioticsand/or probiotics. It was noted that the administration of synbioticsyielded significantly better results than individual applications. Thus,the investigation of the potentially interactive effects between pre-biotics and probiotics in aquaculture is of great significance.

In the present study, the application of FOS and B. licheniformisboth significantly improved the innate immunity of triangularbream. This was supported by the fact that fish fed experimentaldiets obtained significantly higher leucocyte counts and ACH50,AKP and PO activities as well as total serum protein and IgM

Table 3Combined effects of dietary FOS and Bacillus in practical diets on liver and plasma antio

Diets Liver

SOD (U mg�1

protein)MDA (nmol mg�1

protein)CAT (U mg�1

protein)GPXprot

0/0 145 � 3a 3.73 � 0.36c 12.8 � 0.4a 36.80/0.3 154 � 3b 3.21 � 0.31bc 14.1 � 1.0ab 40.70/0.6 165 � 2c 2.65 � 0.34ab 15.2 � 1.2ab 45.41/0 157 � 3bc 2.66 � 0.29ab 15.0 � 0.5ab 45.31/0.3 165 � 1c 1.86 � 0.13a 17.8 � 0.5c 57.41/0.6 162 � 2bc 2.70 � 0.32ab 14.2 � 0.3ab 56.05/0 158 � 3bc 2.75 � 0.29ab 16.7 � 1.0b 58.45/0.3 159 � 2bc 3.24 � 0.42bc 14.4 � 2.0ab 54.45/0.6 154 � 4b 3.49 � 0.33bc 13.8 � 0.9ab 45.4Two-way ANOVAFOS * ns ns nsB. licheniformis ** ** * **Interaction * * ** ns

a Values are means � S.E.M of four replicates. Means in the same column with differe


contents, compared with fish fed control diet. Similar to our results,the application of dietary oligosaccharides (FOS, MOS and inulin)and probiotics (lactic acid bacteria and Bacillus spp) has also beenreported to improve the innate immunity of some fish and shellfishspecies [1,4e7,31,32]. According to previous reports, the immu-nostimulatory nature of prebiotics may be attributed to its stimu-lation of the growth of beneficial bacteria such as lactic acidbacteria and Bacillus spp. [31,32]. These beneficial bacteria possesscell wall components such as lipopolysaccharides which haveimmunostimulatory properties [33,34]. Besides, these oligosac-charides can be selectively used by bifidobacterium to reproduceprobiotic bacteria and restrain the adherence and colonization ofpathogenic microorganism [35], as modulates the beneficial bac-teria in gut, thus resulting in the enhanced immune function of host[36]. Soleimani et al. reported that Caspian roach (Rutilus rutilus) fryfed 2% and 3% FOS obtained an improved immunity [37]. Unlike thecase mentioned above, Ai et al. reported that FOS had little effect onthe immune response and disease resistance of large yellowcroaker (Larimichthys crocea) [38]. This contradiction is justifiabledue to the fact that the effects of both prebiotic and probiotics couldbe affected by various factors, including dosage, duration of pre-biotic administration, fish species and even the life stage [5]. Unlikethe case of prebiotics, the beneficial effects of probiotics was orig-inally thought to stem from the improvements in intestinal mi-crobial balance and the microbiota bacteria which secrete manyenzymes to compete for nutrients and sites and inhibit otherbacteria if probiotics are presented in high numbers [39]. Corre-spondingly, the administrations of probiotic strains have been

xidant capabilities of triangular bream.a

Plasma

(U mg�1

ein)SOD (U mL�1) MDA

(nmol mL�1)CAT (U mL�1) GPX

(U mL�1)

� 4.4a 65.0 � 0.8a 9.71 � 0.56c 1.21 � 0.07a 16.1 � 0.6� 1.8a 67.8 � 0.5bc 8.28 � 0.59b 1.45 � 0.05ab 16.8 � 0.5� 0.9ab 68.0 � 0.7bc 7.94 � 0.32ab 1.34 � 0.05a 17.2 � 0.6� 3.5ab 67.8 � 1.0bc 7.96 � 0.36ab 1.56 � 0.11b 16.3 � 1.6� 3.8c 68.8 � 2.2c 6.90 � 0.26a 1.52 � 0.11a 17.1 � 0.6� 2.2c 67.9 � 0.4bc 7.99 � 0.37ab 1.45 � 0.07a 16.2 � 0.9� 2.3c 67.6 � 0.7bc 7.80 � 0.31ab 1.36 � 0.11a 16.6 � 0.6� 3.6bc 66.9 � 0.5abc 8.07 � 0.33ab 1.54 � 0.13a 16.1 � 0.7� 4.6ab 66.4 � 0.6ab 8.43 � 0.46b 1.43 � 0.09a 16.2 � 0.9

ns ns ns ns* ** ns ns* * ns ns

nt superscripts were significantly different (P < 0.05).

482483484485486487488489490491492493494495496497498499500


Fig. 1. Survival rate (%) of triangular bream after 7 days of Aeromonas hydrophilachallenge. Each data represents the mean of four replicates. Bars assigned withdifferent superscripts are significantly different (P < 0.05).


501502503504505506507508509510511512513514515516517518519520521522523524525526527528529530531532533534535536537538539540541542543544545546547548549550551552553554555556557558559560561562563564565

566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607608609610611612613614615616617618619620621622623624625626627628629630


demonstrated to be able to augment fish innate and adaptive im-mune responses in the gut-associated lymphoid tissue as well asthe systemic immunity [40,41].

Although fish immunity was enhanced by using FOS orB. licheniformis alone, the interaction between them showed asignificant synergistic effect on the immune function of triangularbream. In this study, fish fed diet containing combination 0.3% FOSand 1 � 107 CFU g�1 B. licheniformis showed significantly higherleucocyte counts and ACP, ACH50 and PO activities as well as IgMcontent, compared with fish administrated single substance. Thesefacts suggested that combination of FOS and B. licheniformis coulddelay the occurrence of immunity fatigue by activating differentpathways of triangular bream immune system. This synergisticaction between prebiotics and probiotics may be explained by thefact that prebiotics could be selectively fermented by specific in-testinal bacteria and modulate the growth and/or the activity ofthese bacteria [42]. It has also reported that FOS can selectivelyenhance growth of certain bacterial species in the gastrointestinaltract of shrimp (Litopenaeus vannamei), and enhance the immunecapacity of host [7]. Similar synergisticactions have also beenobserved in sea cucumber (Apostichopus japonicas), white shrimp(L. vannamei) and European lobster (Homarus gammarus L.) [6,7,15].

In the present study, triangular bream fed diet 1/0.3 obtainedrelatively higher leucocyte counts as well as lysozyme, ACP andACH50 activities, probably suggesting an immune enhancement ofthis species. This was supported by the fact that leucocyte plays animportant role in innate immunity and its counts can commonlyindicate the health status of fish [43]. Most of the immunostimu-lants used in aquaculture are believed to enhance the innate im-munity of fish by stimulating leucocyte counts before an infection[44]. In this study, FOS and B. licheniformis supplemented groupsobtained leucocyte counts. This might be due to the presence of apolypeptideephycocyanin which was thought to be a factor thatenhanced the leucocyte production in mice [45]. However, further


studies in fish are still needed to elucidate this. It is well known infish that lysozyme is one of the important defense elements againstparasitic, bacterial and viral infections, which play an importantrole as an opsonin to the lysis of pathogens, and activate thecomplement and phagocytes [46,47]. The higher lysozyme activityobtained here might be ascribed to the relatively higher leucocytecounts due to the fact that fish serum lysozyme is believed to beleucocyte origin [48]. As a typical lysozymal enzyme, ACP activityexpectedly has the similar trend with that of lysozyme [49]. Thehigh ACH50 activity might be attributed to the enhanced liverfunction (which was not determined in this study), as was sup-ported by the fact that liver is the main source of complementproteins and hepatic enhancementmight therefore affect positivelyon complement [50]. Furthermore, the high plasma PO activityobserved in this group also revealed an enhanced immuneresponse of fish due to the fact that PO is an important system inimmune defense of animals [51,52].

Proteins are the most important compounds in serum and itsconcentration is considered as a basic index for fish health status[29,53]. Increases in serum protein contents are usually thought tobe associated with a stronger innate immune response [46]. Amongthe serum proteins, globulin is the major proteins, which plays asignificant role in the immune response [29]. As a natural antibody,IgM is the main immunoglobulin present in teleosts [54], and couldprovide an instant protection of fish against pathogens of broadspecificity. Therefore, its content could reflect the immune status offish [46,55]. In this study, the contents of serum total protein,globulin and IgM of fish fed FOS and B. licheniformis supplementeddiets were all significantly higher than that of fish fed control diet,indicating an enhanced immune function of triangular bream. Theresults may be explained by the fact that both FOS andB. licheniformis could activate and facilitate the antigen processingand serve to stimulate the initial stages of the immune response[56]. These results are also consistent with the higher leucocytecounts and lysozyme activity observed in fish fed experimentaldiets. In the current study, the IgM levels ranged from 22.54 to28.91 mg/mL, as was higher than that of several other fish species[24]. This discrepancy was justifiable since the IgM levels of fishwere susceptible to immunomodulation by a wide range of prod-ucts [24].

4.2. Antioxidant parameters

Under normal physiological conditions, animal cells producereactive oxygen species (ROS). At the same time, the body hasdesigned several antioxidant defense mechanisms to cope with it.An imbalance between the generation and removal of ROS canproduce oxidative stress [8,57]. As the first line of antioxidantenzymatic defense, SOD, GPX and CAT are the importantbiochemical parameters for antioxidant defense [9]. In general, themeasurement of those activities can provide an indication of theantioxidant status of fish, as can also serve as biomarkers ofoxidative stress [59]. In this study, the B. licheniformis supplemen-tation significantly increased the activities of SOD, GPX and CATboth in liver and plasma of triangular bream, probably suggestingan enhanced antioxidant capability of this species. This was sup-ported by the fact that as an antigen Bacillus can stimulate thesecretion of both antioxidant enzymes and antioxidants, which caneffectively remove excess free radicals and regulate the body’s freeradial balance, thus resulting in the improved antioxidant ability[60]. Similar result was observed in grass carp (Ctenopharyngodonidellus) [60] and shrimp [8]. The enhanced antioxidant capabilitywas further supported by the relatively low MDA content observedboth in liver and plasma of fish fed diets supplementedB. licheniformis. The decreased MDA content usually indicates


2


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increased enzymatic antioxidants of the defense system due to thefact that MDA is a direct evidence of the toxic processes caused byfree radicals [61]. In addition, the supplementation of FOS at 0.3%could also increase the antioxidant capacity of triangular bream.But little information is available on enhanced antioxidant capacityby supplying FOS, so more studies are needed. The antioxidantenzyme activities obtained here was consistent with the resultsobserved in immunity diagnosis. Together, they reflect a conse-quent improvement in the health status of fish fed diets supple-mented FOS and B. licheniformis. It might be possible that the effectsof both FOS and B. licheniformis on SOD, CAT and GSH-Px activitieswas associated with their effects on the translation process and/orpost-translational process of these antioxidant enzymes. However,further investigation is still needed to elucidate this.

4.3. Challenge test

In this study, after the challenge with A. hydrophila, survival ratewas significantly improved by the administration of both FOS andB. licheniformis. The highest survival rate was observed in fish feddiet with 0.3% FOS and 1 � 107 B. licheniformis, This might beascribed to the fact that the innate immune defenses of triangularbream were activated both by FOS and B. licheniformis as conse-quently improve its resistance to pathogenic bacterial infection. Itwas reported that Bacillus bacteria are able to outcompete otherbacteria for nutrients and space and can exclude other bacteriathrough the production of antibiotics, as usually lead to theenhanced immunity of fish [38]. One proposed mechanism is thatthe regulatory signals generated by probiotics stimulate host im-munity to enhance protection against pathogens [62]. In thisaspect, B. licheniformis may act as immune-modulators becausethey enhance IgM levels in fish and is important for pathogenrecognition and activation of the innate immune system via theclassical pathway of complement activation. FOS is a nondigestiblefood ingredient that beneficially affects the host health by stimu-lating the immune capacity [6]. Studies on grass carp (C. idellus)(0.2% FOS) and allogynogenetic silver crucian carp (Carassius aur-atus) (0.1 and 0.2% FOS) both reported the beneficial effects of FOSon fish innate immunity and disease resistance [63,64]. Severalstudies demonstrated that inulin or FOS provide an effective meansof promoting bifidobacterium and lactobacillus growth, whileselectively arresting the growth of pathogenic microorganisms[65,66]. In this study, the non-specific defense system which isimportant in affording protection against diseases [67] wasenhanced by stimulating immune parameters such as plasmalysozyme, ACH50 and PO activities. Similar improved survival rateby dietary supplementation of prebiotics and/or probiotics alsowere observed in other studies [1,4,5,10,68,69]. In addition, a sig-nificant interaction was observed between FOS and B. licheniformison the disease resistance of triangular bream. This result was in linewith the increased innate immunity and antioxidant capabilities. Inaddition, it was reported that sea cucumber fed the diet with 0.25%and 0.50% FOS had significantly lower cumulative mortality at eachB. subtilis level after injecting with Vibrio splendidus [6]. However,unlike the cases mentioned above, dietary B. subtilis and FOS wasreported to have no synergistic effect on disease resistance of seacucumber [16]. This discrepancy may be explained by the fact thatdisease resistance capacity of fish were affected by a wide range offactors such as species, size, physiological status, feeding durationand environmental factors and so on [5,70].

In conclusion, the present study indicated that dietary FOS andB. licheniformis could significantly enhance the innate immunityand antioxidant capability of triangular bream, as well as improveits disease resistance. What’s more, the combination of FOS andB. licheniformis exhibited more advantages than the administration


of individual ones. The best combination of these two prebioticsand/or probiotics for triangular bream was 0.3% FOS and1 � 107 CFU g�1 B. licheniformis.

Uncited reference

[58].

Acknowledgements

The present study was funded by the National TechnologySystem for Conventional Freshwater Fish Industries of China (CARS-46-20) in collaboration with the Science and Technology SupportProgram from Jiangsu Province (China, BE2010394).

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