Short- and long-term effects of a dietary yeast β-glucan (Macrogard) and alginic acid (Ergosan)...

Fish & Shellfish Immunology 18 (2005) 311e325

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Short- and long-term effects of a dietary yeast b-glucan(Macrogard) and alginic acid (Ergosan) preparation onimmune response in sea bass (Dicentrarchus labrax)

M. Bagnia,1, N. Romanob,1, M.G. Finoiaa, L. Abellic, G. Scapigliatib,P.G. Tiscard, M. Sartie, G. Marinoa,*

aICRAM, Institute for the Research Applied to the Sea, Rome, ItalybDepartment of Environmental Sciences, Tuscia University, Viterbo, Italy

cDepartment of Biology, University of Ferrara, ItalydDepartment of Comparative Biomedical Sciences, University of Teramo, Italy

eHendrix, Udine, Italy

Received 5 January 2004; revised 5 July 2004; accepted 6 August 2004

Available online 11 November 2004

Abstract

The present study investigated the immunomodulatory activity of Ergosan, an algal extract containing alginic acid,and Macrogard, a yeast extract containing b-glucans, on innate and specific immunity in sea bass (Dicentrarchus

labrax). Four cycles of experimental feeding using normal fish feed formulation (control group) supplemented withErgosan (0.5%) or Macrogard (0.1%) were performed at 60-day intervals (15 days of treatmentC 45 days ofsuspension). Serum complement, lysozyme, total proteins and heat shock protein (HSP) concentrations were measured

at 15, 30 and 45 days from the end of the first 15-day feeding cycle (short term) and 45 days after the end of each feedingcycle over a 35-week period (long term). The percentage of B- and T-lymphocytes in peripheral blood leucocytes andgut were measured over long-term trial.

Significant elevation (P! 0.05) in serum complement activity occurred in sea bass fed with alginic acid and glucans,

at 15 days from the end of first cycle of treatment. Significant elevation (P! 0.05) in serum lysozyme, gill and liver HSPconcentration were observed in the same experimental groups at 30 days from the end of treatment, whereasa significant increase (P! 0.05) of complement activity was only observed in fish that received an Ergosan diet. At 45

days from the end of treatment, complement, lysozyme and HSP concentration did not differ among groups.Over the long-term period, no significant differences were observed in innate and specific immune parameters,

survival, growth performances and conversion index in treated and control fish. A dramatic decrease of both innate and

* Corresponding author. ICRAM, Aquaculture Department, Via Casalotti 300, 00166, Rome, Italy. Tel.: C39 066157 0495;

fax: C39 066156 1906.

E-mail address: [email protected] (G. Marino).1 These authors contributed equally to this work.

1050-4648/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.fsi.2004.08.003

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312 M. Bagni et al. / Fish & Shellfish Immunology 18 (2005) 311e325

acquired immune parameters was observed during the winter season in all groups, followed by a partial recovery whenwater temperature increased. Reduction in complement and lysozyme activities was significatively correlated (P! 0.01)

to water temperature variation. The results suggested the potential of alginic acid and b-glucans to activate someinnate immune responses in sea bass, and particularly under conditions of immunodepression related to environ-mental stress.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Immunostimulant; Complement; Lysozyme; Lymphocytes; Heat shock proteins

1. Introduction

Immunostimulants are biological extracts and synthetic chemicals which stimulate the immuneresponse by promoting phagocytic cell function, increasing their bactericidal activity and/or non-specificcytotoxic cells and antibody production [1]. Different kinds of substances are known to act asimmunostimulants but only a few are suitable for use in aquaculture [2,3]. Most studies in fish are relatedto the b-glucans, polyglucoses consisting of a linear backbone of b1,3 linked d-glucopyranosyl residues[4] derived from yeast, mushroom and fungi mycelia. Yeast b-glucans enhance immune responses [5,6] inseveral fish species by promoting the production of antimicrobial lytic proteins, such as lysozyme andcomplement [7e9] and stimulating the phagocytic activity of macrophages [10e12]. The immunomod-ulatory effects of glucans are not unequivocal and have been shown to be different in relation to the fishspecies, route of administration, doses and the association with other immunostimulants. The adjuvanteffect of yeast b-glucans has been demonstrated in Atlantic salmon [10], catfish [5] and rainbow trout[13]. Intraperitoneal injections seemed to a more effective administration route than oral [2,14e16]. Theimmunostimulatory activity of yeast b-glucans also varies in relation to the duration of administration.Innate immunity is enhanced after yeast b-glucans immunostimulation, but it is likely to be a short-termresponse [1]. Long-term oral administration of peptido-glucans decreased the immune response inrainbow trout when challenged with Vibrio anguillarum [17], as well as in catfish [14], suggestinga negative feedback effect of b-glucan [1]. Yeast b-glucans seem to modulate the specific immuneresponse by increasing the serum antibodies secreted by plasma cells against Edwarsiella ictaluri in catfish[5] and against Yersinia ruckeri in rainbow trout, when given in combination with vitamin C [18]. Fewstudies have addressed the effects of immunostimulants in sea bass (Dicentrarchus labrax). Elevation inphagocytic activity has been found in sea bass fed on diet supplemented with levamisole and yeastb-glucans [19]. Enhanced lysozyme and complement activity have also been noted in sea bass fed withyeast b-glucans, vitamins C and E [20].

Only few data have been published on the immunostimulatory activity of alginic acid, a polysaccharidederived from several brown macro- and microalgae [21]. In fish, alginic acid enhanced the activity of headkidney phagocytes and their migration in the site of alginate injection by increasing their production ofchemotactic factors and their sensitivity to them [22,23]. Intraperitoneal injection of Ergosan (1% alginicacid) at doses R 2.5 mg kg�1 raised the number of neutrophils, degree of phagocytosis and respiratoryburst activity and expression of interleukins (IL-1b) and chemokines (IL-8), but had no effect on lysozymeand antiprotease activity over a 7 days time period [21].

The aim of the present study is to provide information on the immunomodulatory activity of alginic acid(Ergosan, Schering-Plough Aquaculture, UK), and yeast b1,3 of b1,6 glucans (Macrogard, Biotec-Mackzymal, NO) in 1-year-old sea bass. Fish fed on a commercial diet were supplemented with Ergosan orMacrogard for 15 days after which they were fed only the commercial diet for 45 days. Four cycles of

313M. Bagni et al. / Fish & Shellfish Immunology 18 (2005) 311e325

feeding were performed at 60-day intervals, from November to June. The effect of Ergosan and Macrogardon innate and specific immune parameters was analysed over the short-term period after a single treatmentand in the long-term period after multiple cycles of treatment.

2. Materials and methods

2.1. Animals

The experiment was carried out at Valle Bonello, Rovigo, Italy. Sea bass [initial body weight (BW)80G 12 g] were randomly separated into six experimental groups of 150 fish each and stocked into sixcircular 4000 l indoor tanks. Fish were acclimated to experimental condition for 45 days, under naturalphotoperiod. Seawater was pumped from the adjacent lagoon (30 ppt salinity) in a continuous flow-through during spring/summer (22e26 (C) and recirculated and heated during autumn/winter (13e18 (C).During the trial, fish were kept under veterinary control and any clinical signs of disease were registered.

2.2. Treatments

Two groups of fish were fed with a Macrogard diet containing 0.1% yeast b-glucans derived from thewall of Saccaromyces cerevisiae (Macrogard Biotec-Mackzymal, Norway) included in food beforeextrusion. Two groups received an Ergosan diet prepared according to the manufacturer’s recommenda-tions by suspending algal powder (Laminaria digitata 99% and Ascofillum nodosum 1% Ergosan, Schering-Plough Aquaculture, UK) in PBS sterile solution and mixed in food at 0.5% at 20 (C. As controls, twogroups were fed with normal fish feed formulation (Trouwit, Hendrix). Fish were fed Ergosan orMacrogard diets for 15 days and fed the commercial diet for 45 days. Four cycles of feeding were performedat 60-day intervals, from November to June. All groups were fed twice a day, 7 days a week throughout theexperiment period at a rate ranging between 0.3 and 1.2% BW, according to water temperature.

2.3. Sampling

2.3.1. Short termThe short-term trial was performed at the end of the first 15 days of treatment. Ten fish from each

experimental group (20 per treatment) were sampled before treatment and at 15, 30 and 45 days (15 d, 30 d,45 d) from the end of the first 15 days treatment. Fish were anaesthetised (tricaine methanesulphonate0.1 mgml�1; MS222, Sigma, St. Louis, MO, USA), bled from the caudal vein and sacrificed using anMS222 overdose. Blood samples were centrifuged at 3500 g for 10 min and sera stored at �80 (C for serumcomplement, lysozyme and total protein determinations. Gills and liver were collected and stored at�80 (C for heat shock protein (HSP) determination.

2.3.2. Long termThe long-term trial was performed during the four cycles of treatment (IeIV). Ten fish from each

experimental group (20 per treatment) were sampled 45 days from the end of each cycle of treatment. Fishwere bled as described above and serum complement, lysozyme and total proteins were measured.

Eight fish from each group were also sacrificed using an MS222 overdose and blood and intestine werecollected for lymphocyte determination. Blood was diluted 1:50 with HBSS (Hank’s balanced salt solution,Sigma) containing 10 I.U. heparin and the intestine immersed in the same buffer was stored at 0.5 (Cbefore analysis.


2.4. Aspecific immunity tests

2.4.1. ComplementThe complement activity was measured using the alternative pathway (ACP) in microtiter plates as

described by Bagni et al. [20]. Briefly, 25 ml of rabbit red blood cells (RaRBC) suspension was added toserial dilutions of sea bass serum in 150 ml of Veronal buffer. The plate was incubated at 21 (C for 100 minand centrifuged at 2000 g for 2 min. The extent of haemolysis was estimated by measuring the opticaldensity of the supernatant at 550 nm (OD 550). The total haemolysis value was given by the optical readingof supernatant from 25 ml of RaRBC added to 150 ml of distilled water. The complement titre (ACP50 units ml�1) was the reciprocal of the serum dilution causing 50% lysis of RBC.

2.4.2. LysozymeSerum lysozyme was assessed using the lysoplate assay [24], carried out at 25 (C for 18 h in a humidified

incubator, as modified by Bagni et al. [20]. This method is based on the lysis of Micrococcus lysodeikticus in1% agarose. The diameters of the lysed zones were determined using a measuring viewer and comparedwith the lysed zones of a standard lysozyme preparation (Sigma, M 3770).

2.4.3. Total proteinsSerum protein content was assessed using an automatic reader on dry chemistry strips (Spotchem Dry

Chemistry Analyzer, Menarini, Italy).

2.5. Heat shock proteins

2.5.1. Tissue preparationGills and liver were homogenised on ice by using an Ultraturrax at 30 000 rpm for 30 s in buffer,

containing 100 mM Tris and 5 mM EDTA (pH 7.2). Protease inhibitors were added immediately before tohomogenisation. The samples were then centrifuged at 14 000 g for 10 min and total protein concentrationwas assayed in supernatant fractions according to the Bradford method, using bovine serum albumin asa standard.

2.5.2. Western blottingSome gills and liver samples were tested by Western blotting to confirm specific reactivity of the anti HSP

70 monoclonal antibody BRM-22 (Sigma Chemical) against sea bass HSP. Samples of equal total proteinwere separated by one-dimensional electrophoresis using as positive control a constitutive HSP 70 frombovine brain (Sigma Chemical). After electrophoresis, proteins were blotted onto a PVDF membrane(Biorad) by the semi-dry system; the PVDF membrane was incubated with 5% dry milk in TBS (0.05%Tween-20, pH 7.5) and with the BRM-22. After incubation, horseradish peroxidase-conjugated goat anti-mouse antibodies were used to detect the labelling. The substrate 3,30-diaminobenzidine (DAPI) was usedto visualise the bound antibodies.

2.5.3. Dot-blot analysisThe nitrocellulose membrane (0.45 mm, Biorad) was humidified in TBS buffer and laid on the Bio-Dot

apparatus (Biorad). The samples (200 mg ml�1 TBS) were applied in the 96 wells, filtered through themembrane by gravity flow and washed. The immunoassay was performed using BRM-22. Horseradishperoxidase-conjugated goat anti-mouse antibodies were used to detect the antibody labelling, and DAPIsubstrate was used to visualise the bound antibodies. Membranes were quantified by Gel Doc 2000 GelDocumentation System (Biorad), and HSP levels were reported as relative concentrations with respect to


different dilutions of bovine brain HSP 70. The relationships between concentrations and dots intensitywere linear.

2.6. Specific immunity tests

2.6.1. Leucocyte purificationCell suspensions from intestine and blood were prepared as previously described [25]. Cell suspensions

were layered over a discontinuous gradient of Percoll (Pharmacia AB, Uppsala, Sweden) diluted in HBSSto yield densities of 1.020 and 1.070 g ml�1. After centrifugation (30 min at 840 g) at 4 (C, cells layeredbetween the densities mentioned above were collected and washed twice (10 min at 680 g) at 4 (C. Thepellet was resuspended in HBSS with 1% BSA and 0.1% sodium azide. Peripheral blood leucocytes (PBL)were prepared from heparinised blood (5 ml) diluted 1:1 in HBSS and centrifuged (15 min at 100 g) at 4 (C.The buffy coat was collected, resuspended in HBSS and layered as described above.

2.6.2. AntibodiesThe monoclonal antibody DLT15 (IgG3 isotype class) was obtained by immunising mice with

paraformaldehyde-fixed thymocytes of 1-year-old sea bass and screening hybridoma culture media byimmunocytochemical analysis of both fixed and living cells [26]. The monoclonal antibody DLIg3 (IgG1

isotype class) was obtained by immunising mice with sea bass serum Ig purified by protein A-Sepharoseaffinity chromatography. The hybridoma culture media were screened by dot-blot assay andimmunocytochemistry [27].

2.6.3. Indirect immunofluorescence and flow cytometry (FACS)The cell suspensions (107 cells ml�1) were incubated in 250 ml of undiluted DLT15 or DLIg3 hybridoma

culture supernatants for 45 min at 4 (C [25]. Labelled cells were washed (10 min at 680 g) and incubated at4 (C for 30 min with FITC-conjugated goat anti-mouse Ig serum (GAM, Cappel Europe, Turnhout,Belgium) diluted 1:250 in HBSS. After washing, 20 ml of 106 cells ml�1 were cytocentrifuged at 500 g at4 (C. Ten thousand cells ml�1 were measured with a flow cytometer (FACScalibur, Becton DickinsonImmunocytometry System, Mountain View, CA, USA) by ‘‘cellQuest’’ computer-program (B.D.). Thepercentage of positive cells was calculated by subtracting the results obtained from the negative control.

2.7. Growth performance

Body weight (BW) and standard length (SL) were recorded for growth evaluation at each sampling time.The condition factor (CF) was calculated as BW (g)/SL3 (cm)! 100. The specific growth rate (SGR) wascalculated as the increase of weight (g) in respect to a given time interval (days). The feed conversion ratio(FCR) was calculated for each experimental group as the increase of body weight (g) with respect to feed(g), in the time interval between two successive samplings. The weight of feed dispersed to each tank wasrecorded daily and uneaten feed collected, weighted and not included for FCR calculation.

2.8. Statistical analysis

Data analysis was performed using the SPSS software statistical package. Mean and standard error ofthe mean (S.E.) was calculated for each parameter. Analysis of covariance was performed to verify whetherfish size and sex influenced the parameters analysed. A two-way ANOVA was used to determine thecombined effects of immunostimulation and time. One-way ANOVA was applied to detect any differencedue to immunostimulation at different sampling times.


The Student’s t test was applied to assess any difference in lysozyme, complement, total protein and HSPconcentrations between the two experimental groups subjected to the same treatment. Since no difference inany analysed parameter was recorded, data were pooled together.

Differences among treated and control groups were compared with pre-treatment level by a post hoc test(Bonferroni). When variance was found not homogeneous, the Dunnett’s T3 test was used for multiplecomparisons. The level for accepted statistical significance was P! 0.05.

3. Results

3.1. Short-term trial

Alginic acid and glucans had a significant effect on complement activity, which increased significantly(P! 0.05, by one-way ANOVA, Fig. 1a) in treated fish compared to the controls at 15 days from the end ofthe first cycle of treatment. After 30 days, complement activity was reduced in all groups, althoughDunnett’s T3 pairwise comparison test showed that it was significantly higher (P! 0.05) in fish treatedwith Ergosan compared to the fish that received a Macrogard diet and to the controls. After 45 days fromthe end of the first feeding cycle, complement activity in treated groups returned to control levels.

Serum lysozyme showed high variability during the whole trial (Fig. 1b). A significant effect of time(P! 0.001, two-way ANOVA) on serum lysozyme was observed, not correlated with treatments. Thirtydays from the end of first treatment, serum lysozyme level was significantly higher (P! 0.05, Dunnett’s T3)in fish receiving alginic acid and glucans with respect to the controls.

Western blotting of heat shock protein (HSP) in gills and liver showed a single protein bandimmunoreactive with the monoclonal antibody BRM-22, similar in molecular weight to the constitutive70 kDa HSP purified from bovine brain. Overall, there was a significant effect of time (P! 0.001, by two-way ANOVA), but not of treatment, on both gills and liver HSP. On day 30, after treatment, both gills andliver HSP were significantly increased (P! 0.05) in fish fed with alginic acid and glucans, compared to thecontrols (Fig. 1c, d). No effect of time and treatments were observed on serum total protein level (two-wayANOVA, data not shown).

3.2. Long-term trial

Variation of water temperature (Fig. 2a) influenced feed intake, which ranged between 0.3% at 13 (Cand 1.2% at about 20 (C. SGR showed high variability (from 0.1% to 1.54%) during the experimentalperiod, according to the feeding rate. SGR during the winter months was slightly, but not significantly,higher in fish fed with alginic acid and glucans compared with the controls. FCR ranged from 0.7% to 5%(Fig. 2b), but showed no significant relation with the water temperature or with the immunostimulanttreatments. CF ranged from 0.82 to 1.35, and did not vary among the groups throughout the trial.

Changes of both specific and non-specific immune parameters were not related to fish body weight andsex (ANCOVA, ns), except for serum T-protein which was found significantly higher (P! 0.05) in sea bassmales. Overall, there was a significant effect of time on both specific and aspecific immune parameters,mainly related to water temperature variation. Variations in lysozyme concentration were positivelycorrelated to water temperature variation (rZ 0.299, P! 0.001), being significantly reduced during wintersamplings (Fig. 3b) at water temperatures below 15 (C and recovering when temperature was increased upto 20 (C (two-way ANOVA, P! 0.001). Long-term treatment with Ergosan had a significant effect onlysozyme levels (two-way ANOVA, P! 0.05), but there was no interaction between time and treatment. Asignificant increase of serum lysozyme (P! 0.05) was observed at the end of the fourth and last treatmentin fish fed with Ergosan.


Serum complement level was correlated to water temperature (rZ 0.20, P! 0.001) and changed duringthe experimental period (two-way ANOVA, P! 0.001), even if it did not differ among the treated andcontrol groups (Fig. 3a).

Change in serum T-protein was significantly correlated to water temperature (rZ 0.301, P! 0.001), butnot significantly influenced by treatments with Ergosan and Macrogard (Fig. 3c).

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Fig. 1. Short-term trial. Effect of 15-day treatment with Ergosan and Macrogard on: (a) serum complement, (b) serum lysozyme, heat

shock proteins (HSP) in gills (c) and (d) liver in sea bass. Samplings were performed before treatment (0 d) and at day 15, 30 and 45

(15 d, 30 d, 45 d) after the end of first treatment. Data are expressed as meanC S.E. of 20 fish sera for each treatment. *Differences

(P! 0.05) between treated and control groups at each sampling time.


Profiles of T-lymphocytes (DLT15C cells) and B-lymphocytes (DLIg3C cells) in the gut and PBLanalysed by flow cytometry are shown in Fig. 4. The forward scatter (FSC) and 90( side scatter (SSC)profiles are displayed in Fig. 4a and b, as well as the gate selected for quantitative analysis. Cells with lowside scatter within the gate were considered to be lymphoid cells, whereas cells with high side scatter wereexcluded from the calculations because these were mainly non-lymphoid cells, as proven by electronmicroscopy of sorted cells [25]. During the experimental period T- and B-lymphocyte percentages (Fig. 5)were affected by water temperature variations. The percentage of T-lymphocyte in PBL and B-lymphocyte

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Fig. 2. Effect of four cycles of treatments (IeIV) with Ergosan and Macrogard on body weight (a) and feed conversation rate (b).

Samplings were performed before the first treatment (0 d) and at 15, 30 and 45 days from the end of first treatment (short term) and 45

days from the end of each treatment (45 d/I, 45 d/II, 45 d/III and 45 d/IV, long term). Data are expressed as meanC S.E. of 20 fish at

each sampling point.


in gut was significantly correlated to water temperature (rZ 0.39, P! 0.001; rZ 0.35, P! 0.001), beingreduced in the coldest period in both treated and control fish. The two-way ANOVA analysis also indicateda significant effect of time on T-lymphocytes in blood and gut (P! 0.01, Fig. 5a, b) and on B-lymphocytepercentage in blood (P! 0.001, Fig. 5c) and gut (P! 0.05, Fig. 5d). No significant effect of treatment andno interaction between time and treatment were observed.

4. Discussion

Innate responses mediated by complement and lysozyme have been reported in several fish species[28e30], including sea bass [31,19]. Innate cytotoxic reactions of sea bass macrophages and neutrophilic

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Fig. 3. Long-term trial. Effect of four cycles of treatments (IeIV) with Ergosan and Macrogard on (a) serum complement, (b) serum

lysozyme and (c) serum total protein. Treatments were performed for 15 days at 60-day intervals, from November to June. Samplings

were performed after 45 days from the end of each treatment (45 d/I, 45 d/II, 45 d/III and 45 d/IV). Data are expressed as

meanC S.E. of 20 fish sera for each treatment. *Differences (P ! 0.05) between treated and control groups at each sampling time.


granulocytes have been shown to be mainly responsible for production of complement fractions andlysozyme, respectively [32]. The haemolytic activity of serum, which is mainly based on the alternativecomplement pathway [33], is activated by polysaccharides (including those of bacterial cell wall) aggregatesof immunoglobulins, fungi and viruses [34,35]. The fast response to b-glucan injection has been related tothe presence of a specific receptor on macrophages [36,4]. In this present study, serum complementactivated through the alternative pathway and serum lysozyme are enhanced in the short-term period afterthe administration of b-glucan.

Serum complement and lysozyme levels were significantly increased at 15 days from the end of the firsttreatment withMacrogard and serum lysozyme level at days 30. Such beneficial effect of b-glucan had alreadybeen observed in salmonids on lysozyme [11] and on both complement and lysozyme [7] between 7 and 28days after an intraperitoneal injection of b-glucans. The present results did not show an influence of b-glucanson complement activity at days 30 and 45 from the end of first treatment, in agreement with previous data inrainbow trout [18], that showed a 2-week stimulatory effect of Macrogard and high-dose vitamin C, notobserved 4 weeks from immunostimulation [37]. Moreover, multiple cycles of b-glucans (long-term trial) hadno cumulative effects on serum complement activity and lysozyme. Similarly, long-term dietaryadministration of glucans (35 d) did not influence the complement activity and lysozyme in turbot [38].

This investigation provides the first information on the effect of oral administration of alginic acid oncomplement activity and lysozyme in fish. Dietary administration of Ergosan had a significant effect onserum complement activity at days 15 and 30 from the end of first 15-day treatment and on serum lysozymeat day 30, confirming our preliminary data on the immunomodulatory effect of alginic acid on non-specificimmunity in sea bass under stress [39]. Conversely, intraperitoneal injection of Ergosan did not elicit animmunostimulatory effect on humoral immune parameters (antiprotease, lysozyme and complement),whereas neutrophil migration into the peritoneal cavity, phagocytosis, respiratory burst and cytokineexpression are increased [21]. No impact of sodium alginate on complement activity was found in carp [23].Various factors should be taken into account to explain the apparent discrepancy with our findings: speciesdifferences, different administration route (oral vs. i.p.), much lower oral dosage, different response kinetics,and differential cell recruitment and stimulation in presence/absence of an inflammatory response. Theeffect of alginic acid by oral delivery should be further investigated in relation to these aspects to evaluate itsefficacy in increasing disease resistance.

Heat shock proteins have been recognised among the primary defence cellular mechanisms. Theirexpression has been described in fish cell lines, primary cultures and tissues exposed to stressors, includinginfectious agents [40]. In the present study, HSP were first detected in sea bass by using the mAb BRM-22,that evidenced a heat-inducible protein of approximately 70 kDa, as already reported for other fish species

Fig. 4. The forward scatter (FSC) and 90( side scatter (SSC) profiles of peripheral blood leucocytes (a) and gut leucocytes (b) and the

gate selected for quantitative analysis.


[41]. Increased HSP in gills and liver was observed following both Macrogard and Ergosan dietaryadministration. Western blot analysis showed that BRM-22 did not bindVibrio alginolyticus and Escherichiacoli proteins, providing evidence that increased HSP could not be related to bacterial constituents.Interestingly, HSP enhancement overlapped that of lysozyme 30 days from the end of the first treatment.

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Fig. 5. Long-term trial. Effect of four cycles of treatments (IeIV) with Ergosan and Macrogard on T-lymphocyte percentages in

peripheral blood (a) and in gut (b) and on B-lymphocyte percentages in peripheral blood (c) and in gut (d), analysed by flow cytometry.

Treatments were performed for 15 days at 60-day intervals, from November to June. Samplings were performed after 45 days from the

end of each treatment (45 d/I, 45 d/II, 45 d/III and 45 d/IV). Data are expressed as meanC S.E. of 16 fish for each treatment.


Likewise, HSP and plasma lysozyme levels both increased in trout after Vibrio anguillarum infection,suggesting that fish might share similar responses to killed bacteria, bacterial products or adjuvants [42].These observations could be explained by the origin of HSP and lysozyme due to phagocyte stimulation.

Total serum proteins did not vary significantly in all experimental groups and were not influenced by thefeeding regime during the whole trial, confirming other data [43]. Mean value and range were similar tothose reported for sea bass [44]. Neither lysozyme, complement nor HSP enhancements observed in thisstudy correlated with variation of total proteins, suggesting that their stimulation was not dependent onhigher protein synthesis, as already reported [45,46,20].

The notion that immunostimulants, such as b-glucans and alginic acid, can enhance specific immunity[37] is still debated. b-glucans could modulate the specific response by increasing the serum antibodiessecreted by plasma cells [5], although aspecific immune system stimulation by dietary glucan administrationin turbot [38] and trout [37] could indirectly stimulate lymphocyte activation processes. Overall, b-glucanscould improve resistance to bacterial challenge in Sparidae [47]. Intraperitoneal administration of bothglucans and Ergosan has been shown to activate leucocyte responses that can represent, in turn, a preludeof multifaceted inflammatory-like immune response [11,21]. Since quantitative studies on identifiedlymphocyte subpopulations were lacking, we reanalysed the long-term effects of immunostimulants onT- and B-cells in PBL and gut, in order to assess percentages of circulating and mucosa-associatedlymphocytes [25,48]. The effect of Ergosan and Macrogard treatment did not show a statistical significance,although a positive trend was detected over the trial. Thus, further studies of the functionality oflymphocyte subpopulations under immunostimulation are worth being investigated.

Diets supplemented with immunostimulants did not significantly enhance sea bass growth performance,as previously shown in Dentex dentex [49] and Atlantic salmon, Salmo salar [45,46]. However, watertemperature during the trials was below the optimal temperature for this species (22 (C), and therefore fishdid not express their maximum growth potential. During the coldest season fish reduce feeding and in suchconditions oral administration of glucans and alginic acid induced a positive, although not significant, effecton growth at the lowest temperature (13 (C).

Furthermore, the immune response of temperate teleosts is depressed both in the coldest season andwhen they are kept at low temperature [50e52]. Low temperature can diminish or deplete antibodyproduction [53,54] and inhibit the generation of putative carrier-specific T cells in catfish [55] and primaryantibody response to T-dependent antigens in carp [56]. These effects can be related to decreased number ofcirculating lymphocytes [56,57]. Acute temperature stress provoked in carp a dramatic reduction ofcirculating B-cells that could be due to their redistribution versus head kidney and spleen [56]. In this study,complement significantly reduced during winter season, while lysozyme and lymphocytes percentage wereeven more severely affected, as observed in sea bream [57,58]. Following the coldest period, sea bassrecovered immune parameters, feeding intake and growth, as in sea bream [57].

In conclusion, this study has shown that Ergosan and Macrogard treatment, following themanufacturer’s instructions concerning the protocol of oral immunostimulation of cultured fish, had aninteresting, potential efficacy on innate immunity under temperature stress in sea bass.

Acknowledgements

The authors wish to thank L. Zanella (Veneto Agricoltura, Biotopo, Valle Bonello, Rovigo, Italy) andF. Anfuso for the support at the facilities; L. Archetti and her staff (IZS, Brescia, Italy); M. Fanelli, M.R.Baldassini and M. Forlenza (Tuscia University, Viterbo, Italy); A. Ciarelli and L. Di Gialleonardo(University of Teramo, Italy) for their expert technical assistance. This study was granted by MIPAF,Projects 5C68 and 5C116.


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