Aquaculture 293 (2009) 16–21

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Effects of two closely related probiotics on respiratory burst activity of Senegalesesole (Solea senegalensis, Kaup) phagocytes, and protection against Photobacteriumdamselae subsp. piscicida

P. Díaz-Rosales a, S. Arijo a, M. Chabrillón a, F.J. Alarcón b, S.T. Tapia-Paniagua a, E. Martínez-Manzanares a,M.C. Balebona a, M.A. Moriñigo a,⁎a Department of Microbiology, Group of Prophylaxis and Biocontrol of Fish Diseases, Faculty of Sciences, University of Málaga, 29071 Málaga, Spainb Department of Applied Biology, University of Almería, 04120 Almería, Spain

⁎ Corresponding author.E-mail address: morinigo@uma.es (M.A. Moriñigo).

0044-8486/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.aquaculture.2009.03.050

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 July 2008Received in revised form 26 March 2009Accepted 28 March 2009

Keywords:Solea senegalensisProbioticsP. damselae subsp. piscicidaRespiratory burst activity

The effects of dietary administration of two probiotics, Shewanella putrefaciens Pdp11 and Shewanella balticaPdp13, on growth, respiratory burst activity of phagocytes of Senegalese sole (Solea senegalensis), and survivalof fish challenged with Photobacterium damselae subsp. piscicida have been studied. Fish were fed for 60 dayswith three different diets: one control, and twodiets supplementedwith 109 cfu g−1of probiotics Pdp11 (Pdp11diet) and Pdp13 (Pdp13 diet). Respiratory burst activity of phagocytes from fish fed with diet Pdp11significantly increased after 60 days of feeding, whilst this significant increase was not detected in phagocytesfrom fish fed control and Pdp13 diets. On the other hand, the cumulative percentage of mortality after thechallenges with P. damselae subsp. piscicida, was 100% in the groups fed with control diet, whereas mortalityrates observed in the groups fedwith diets supplementedwith Pdp11 and Pdp13 ranged from75–100% and 65–80%, respectively. These results suggest that an increased respiratory burst activity of the phagocytes is notessential to increase the protection against the P. damselae subsp. piscicida. On the other hand, the use of bothprobiotics improved the growth, and the survival against the pathogen, in comparisonwith those fish receivingthe control diet.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Senegalese sole culture (Solea senegalensis) Kaup 1858 is nowadaysa very promising industry in the Mediterranean countries. However,one of the most serious problems concerning sole production is theexistence of infectious diseases such as pseudotuberculosis causedby Photobacterium damselae subsp. piscicida (Zorrilla et al., 1999;Romalde, 2002; Arijo et al., 2005).

In fish farms, control of this bacterial pathogen is achieved bythe administration of chemotherapeutic agents, which are extensivelyemployed, leading to increasing antibiotic resistance (Miranda andZimelman, 2001; Radu et al., 2003). Therefore, immunoprophylaxis hasbeen suggested as the best way to prevent pseudotuberculosis (Sakai,1999).

Throughout the last 20 years, there has been a variety of studiesanalyzing the effectiveness of immunization in preventing pseudotu-berculosis (Romalde and Magariños, 1997). In the case of Senegalesesole, a divalent vaccine against Vibrio harveyi and P. damselae subsp.piscicida has been reported (Arijo et al., 2005). However, this vaccine

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only provides short-term protection. Taking this into account, it isnecessary to provide aquaculture industrywith alternativemeans thatcould keep a microbiologically healthy environment and enhance itsproduction and economical profits. Searching for new prophylacticmethods, use of probiotics has been suggested as an alternative.

Several modes of probiotic action have been demonstrated, such as(i) competition of adhesion sites (Vine et al., 2004; Chabrillón et al.,2005a), (ii) production of antibiotic substances (Yan et al., 2002),(iii) improvement in feed efficiency (Sáenz de Rodrigáñez et al.,2009), and (iv) competition for energy sources (Gram et al., 1999;Verschuere et al., 2000). Recent studies have been focused on theimmunological stimulation of fish defence mechanisms by probioticbacteria (Irianto and Austin, 2003; Nikoskelainen et al., 2003;Panigrahi et al., 2004, 2005; Salinas et al.,2005; Díaz-Rosales et al.,2006a,b). As immunomodulators, there are evidences pointing toa stimulatory effect of probiotics on the gut immune system, suchas increases of the number of Ig+ cells and acidophilic granulocytes(Picchietti et al., 2007, 2008), and probiotics may enhance phagocyticactivity and increase the production of reactive oxygen species bymacrophages from several fish species (Irianto and Austin, 2003;Díaz-Rosales et al.,2006a,b; Salinas et al., 2006). Themeans of invasionand survival of P. damselae subsp. piscicida inside the host are stillunknown, and while authors have reported the presence of intact

17P. Díaz-Rosales et al. / Aquaculture 293 (2009) 16–21

bacteria inside fish cells, suggesting the ability of the bacterium tosurvive as an intracellular pathogen (Noya et al., 1995; López-Dórigaet al., 2000; Díaz-Rosales et al.,2003; Elkamel et al., 2003), othershave observed that this pathogen is highly susceptible to oxidativeradicals generated during the macrophage respiratory burst (Skarmetaet al., 1995; Barnes et al., 1999). For this reason, the stimulation ofthe respiratory burst activity of Senegalese sole phagocytes couldfacilitate a more effective destruction of P. damselae subsp. piscicida.

In previous studies, we have isolated two bacteria from giltheadseabream (Sparus aurata) skin, named Pdp11 and Pdp13, belongingto Shewanella genus (Salinas et al., 2006). Several Shewanella specieshave shown activity against different fish pathogens (Makridis et al.,2005; Flellheim et al., 2007; Goldschmidt-Clermont et al., 2008).The isolates Pdp11 and Pdp13 showed in vitro characteristics such asbe non virulent for Senegalese sole, interaction with fish pathogenssuch as P. damselae subsp piscicida and V. harveyi (Chabrillón et al.,2005a,b), and ability to modulate in vivo the innate immuneresponses of gilthead seabream (Salinas et al., 2006) and Senegalesesole (Díaz-Rosales et al., 2006a,b). Thus, they were considered aspotential fish probiotics to be tested in fish aquaculture. Hence, theaim of the present study was to evaluate the possible effect of dietaryadministration of two different strains of Shewanella genus on therespiratory burst activity of sole phagocytes, and the protectionagainst P. damselae subsp. piscicida.

2. Materials and methods

2.1. Microorganisms

Shewanella ssp. Pdp11 and Shewanella ssp. Pdp13 were identifiedto species level by the amplification and sequentiation of a fragmentof 16R rDNA. Briefly, this fragment was amplified using the universalprimers SD-Bact-0008-a-S20 (5′ AGA GTT TGA TCC TGG CTC AG 3′)and SD-Bact-1492-a-A-19 (5′ GGT TAC CTT GTT ACG ACT T) (Kim andAustin, 2006). Polymerase chain reactions were carried out in a 50 µlreactionmixture that included 5 pmol of each primer, 200 µM dNTPs,1×PCR buffer, 2 mM MgCl2, 1 U BIOTAQ™ DNA Polymerase (Bioline,London, UK) and 1 µl of a boiled colony suspension. The PCR profilewas as follows: 2 min at 95 °C and 35 cycles of 30 s at 95 °C, 30 s at52 °C and 1.3 min at 72 °C and a final step 5 min at 72 °C. Polymerasechain reaction products were electrophoresed on a 1% agarose geland visualized via ultraviolet transilumination. Following the PCRreaction, unconsumed dNTPs and primers were removed using theHigh Pure PCR Product Purification kit (Roche, Mannheim, Germany).The PCR products were sequenced by cycle sequencing using SD-Bact-0008-a-S20 and SD-Bact-1492-a-A-19 as a sequencing primer.Sequences were obtained on Sequence analyser (Genetic AnalyzerABIPRISM 310 Applied Biosystems). The sequences were aligned tothe closest relative in the GenBank database using BLAST.

Bacterial strains were grown in tubes containing 5 ml of tryptonesoya broth (Oxoid Ltd., Basingstoke, UK) supplemented with 1.5%NaCl (TSBS) for 18 h at 22 °C, with continuous shaking. Appropriatedilutions of the culture were spread onto plates of tryptone soyaagar (Oxoid) supplemented with 1.5% NaCl (TSAS). The number ofculturable bacteria was determined by plate counting on TSAS.Bacteria were recovered from the plates and lyophilized before beingadded to the fish diet. Bacteria were frozen at −80 °C for 12 h, andthen they were freeze-dried at −80 °C for 12 h with a freeze dryerFTS Systems Flexi-dryModel FD-1-84D-O (FTS Systems, Inc. NY, USA)equipped with vacuum pump 2400 A (Alcatel Cit, Annecy Cedex,France). The frozen samples were dried at a pressure of 0.075 mbar.

2.2. Fish experimental design and sampling

Senegalese sole specimens (Solea senegalensis Kaup, 1858) of 10–17 g weight, from a farm located in Carboneras (Almería, Spain), were

distributed in 500 l seawater tanks (200 fish per tank), 36‰ salinity, at20–22 °C (due to natural fluctuations in seawater temperature) andfed with a commercial pellet diet (55% crude protein and 17% etherextract, Gemma 0.5, Skretting, Spain) at PREDOMAR S.L. facilities(Carboneras, Almería, Spain). These tanks were connected to an open-circulating water system. The fish were acclimated to the tanks andexperimental diets for 2 weeks prior to the 60-day experimentalperiod. The tanks were randomly selected and assigned to each of thedifferent dietary treatments. Three replicate tanks of fish were usedfor each dietary treatment.

Experimental diets were prepared in the laboratory by supple-mentation of the before mentioned diet with lyophilized cells ofPdp11 and Pdp13 strains. An amount of the lyophilized bacterial cellpreparation was suspended in sodium alginate solution (Sigma,Sigma-Aldrich, St. Louis, Mo, USA) (2 g kg−1 fed) to obtain a dose of109 cfu g−1 feed. This dose produces health benefits in Sparus aurataand S. senegalensis (Díaz-Rosales et al., 2006a,b). The sodium alginateused in our study is a linear polymer of β-(1-4)-D-mannuronic acidand α(1-4)-L-glucuronic acid residues. Typically, material is com-posed of approximately 61% mannuronic acid and 39% glucuronic acidor M/G ratio 1.56.

The suspension was sprayed into the feed and the mixture waskept under continuous agitation. After 10 min, the obtained uni-form mixture was quickly sprayed with 50 mM calcium chloride(100 ml kg−1 feed) under continuous agitation. The gel was obtainedby spraying the solution of CaCl2 over the pellet impregnated withthe bacterial–alginate solution. The gelation occurred from the surfaceinwards, producing a superficial shell entrapping the bacterial cells intothe surface of pellets. Two different diets were prepared in this manner,each using a different probiotic strain at the same dose (109 cfu g−1)of Pdp11 (diet Pdp11) and Pdp13 (diet Pdp 13). A third diet, preparedin the same manner but using only alginate (alginate diet), and thefourth diet was the commercial fed, which was used as control. Eachdiet was assayed in triplicate. Fish were fed for 60 days at a rate of 15 gdry diet kg−1 biomass (1.5%) twice per day. Satiation was determinedbased on visual observation of acceptance and refusal of feed. Thebiomass of the fish in each tank wasmeasured at the beginning and theend of the experiment, and daily ratio was adjusted accordingly. Nomortality was observed during the experiment.

Samples for determination of the respiratory burst activitywere taken at the middle of the trial and at the end, day 30 and 60,respectively. Specimens were sacrificed by overdose of tricainemethanosulphate (MS222) (Sigma, Sigma-Aldrich, St. Louis, MO,USA).

Fish were weighed at the beginning and at the end of the 60-dayexperiment after being anaesthesized with 0.1 g l−1 MS222 (Sigma).

2.3. Isolation of head kidney phagocytes

The influence of the treatment on respiratory burst activity wastested in phagocytes isolated from sole kidneys following thetechnique described by Secombes (1990). Briefly, the head kidneywas removed aseptically and pushed through a 100 µm nylon meshwith Leibovitz medium (L-15) (Gibco, Gaithersburg, MD, U.S.A.)containing 2% foetal calf serum (FCS, Sigma), 500 Uml−1 penicillin–streptomycin (Sigma), 0.005 mg ml−1 gentamicin (Sigma) (P/S/G)and 10 U heparin ml−1 (Chiesi, Barcelona, Spain). This cell suspen-sion was layered on a 30 to 51% Percoll (Amersham Pharmacia,Piscataway, NJ, U.S.A.) gradient and centrifuged at 600×g for30 min without brake. Then, the band separated at the interface,and corresponding to the phagocytes was collected, centrifuged for15 min at 500×g and resuspended in L-15 medium supplementedwith P/S/G. The viable cell concentration was determined afterstaining with trypan blue and microscope counting. Aliquots of100 µl containing 1×107 cells ml−1 in L-15 medium supplementedwith P/S/G were added to 96-well microtitre plates (Nunc, Roskilde,

Table 1Growth performance of Senegalese sole specimens after 60 day feeding with controldiet and diets supplemented with alginate, probiotics Shewanella putrefaciens Pdp11and Shewanella baltica Pdp13 (mean±standard error).

Parameters Control diet Alginate diet Diet Pdp11 Diet Pdp13

Initial body weight (g) 13.5±2.9 13.4±3.1 13.9±3.9 13.6±3.3Final body weight (g) 20.9±4.2a 21.0±5.5a 27.7±6.5b 26.9±7.2b

Averages in the same rowwith different superscript are significantly different (Pb0.05).

Fig. 1. Respiratory burst activity of kidney phagocytes from sole specimens fed dietscontaining alginate, 109 cfu g−1 Pdp11or 109 cfu g−1 Pdp13 for 30 days (■) or 60 days(□), and incubated with P. damselae subsp. piscicida (108 bacteria ml−1). Results areexpressed as stimulation index obtained by dividing each sample value by the meancontrol value. Symbol ⁎ denotes statistically significant differences (Pb0.05) withrespect to the control group.

18 P. Díaz-Rosales et al. / Aquaculture 293 (2009) 16–21

Denmark). After 3 h incubation at 22 °C, non-adherent cells wereremoved and medium was substituted by L-15 and P/S/G supple-mented with 2% FCS. Monolayers were incubated overnight at 22 °C.

2.4. Respiratory burst activity

Three specimens of each experimental group, fed with control,alginate Pdp11 and Pdp13 diets, were assayed for respiratoryburst activity. The generation of intracellular superoxide radicals bysole phagocytes was determined by the reduction of nitro-bluetetrazolium (NBT) according to the technique described by Secombes(1990) and Boesen et al. (2001). Phagocyte monolayers werewashed with L-15 medium and HBSS (Hank´s Balanced Salt Solution)to remove any trace of antibiotics. Then, 100 µl NBT, dissolved at1 mg ml−1 in HBSS, were added to the wells and the phagocytesincubated at 22 °C for 30 min. Wells containing phagocytes wereinfected with P. damselae subsp. piscicida(108 cells ml−1) and usedto determine the response of the phagocytes to the fish patho-gen. As a positive control, phorbol myristate acetate (PMA, Sigma)(1 µg ml−1), an activating agent of the respiratory burst, wasused to stimulate the respiratory burst of non-infected phagocytes.The specificity of the reaction was tested by adding superoxidedismutase (SOD) (300 IU per well) to some wells containing PMA-stimulated phagocytes.

After incubation, cells were fixed in 70% methanol and reducedformazan within phagocytes was solubilised by adding 120 µl 2 MKOH and 140 µl dimethyl sulfoxide (DMSO, Sigma). Finally, absor-bance was read at 630 nm in a multiscan spectrophotometer (UV-1601 Spectrophotometer, Whitakker Bioproducts). All experimentswere carried out by triplicate.

Respiratory burst activity of isolated phagocytes from assayedsoles was expressed as stimulation index, which was calculated asthe ratio between the absorbance obtained with phagocytes fromfish fed with the different experimental diets and absorbance fromfish fed with the control diet, without any supplementation.

2.5. Challenge with P. damselae subsp. piscicida

Triplicate groups of 20 fish fed with control, alginate, Pdp11and Pdp13 diets per tank assayed were challenged at the end ofthe feeding trial with P. damselae subsp. piscicida (Lgh41/01). Thisstrain was isolated in a previous study from diseased farmedSenegalese sole, showing a lethal dose 50 (LD50) for this fish speciesof 2.2×104 CFU g−1 (Díaz-Rosales et al., 2003). This microorganismwas grown in TSBS and incubated at 22 °C for 48 h to achieve anoptical density at 600 nmof 1 (5×108 cfuml−1 approximately). Soleswere intraperitoneally injected with a dose of P. damselae subsp.piscicida (Lgh41/01)of 5×104 cfu g−1 in phosphate buffer saline (PBS).As control, the same number of fish was inoculated with PBS.Inoculated fish were followed daily for 10 days, and all mortalitieswere recorded, considering only those that could be attributed to thebacterial challenge if re-isolation in pure culture on TSAS frominternal organs of dead fish, and confirmation by Mono-Pp (BIONOR,Skien, Norway) as P. damselae subsp. piscicida was performed.

The relative percentage of survival (RPS) (Amend, 1981) wascalculated to evaluate the potential protection exerted by the pro-

biotics as: [1−(% mortality in fish fed with probiotic diet/% mortalityin control)]×100.

2.6. Statistical analysis

Statistical analyses were conducted using the computerized soft-ware Statistical Package for Social Sciences (SPSS). The data wereanalysed by analysis of variance (ANOVA) two-way in the case ofgrowth and respiratory burst activity data, and one-way for the resultsobtained for challenges with the pathogenic microorganism, andTukey's comparison of means was performed when necessary.Treatment effect was considered statistically significant whenPb0.05. The data were plotted using the program Microsoft Excel(Microsoft Co., Seattle WA).

3. Results

Shewanella Pdp11 and Shewanella Pdp13 were identified as She-wanella putrefaciens and Shewanella baltica, respectively. Data on fishgrowth are resumed in Table 1. Significantly better results were onlyobtained in fish fed any of the diets supplemented with probioticsPdp11 or Pdp13 in comparison with the alginate diet only supple-mented with sodium alginate and control group (Pb0.05). Therespiratory burst activity of phagocytes was measured at days 30and 60. Only the phagocytes from sole specimens fed during 60 dayswith the diet supplementedwith probiotic Pdp11 showed a significantincrease (Pb0.05) of respiratory burst activity (Fig. 1). Fish fed withPdp13 diet showed an enhancement of respiratory burst activity,although after 60 days this increase was not statistically significantcompared to the results obtained from specimens fed with alginatediet and control diet.

Table 2 shows percentages of mortality of Senegalese sole speci-mens inoculated with P. damselae subsp. piscicida after 60 day feedingwith a diet supplemented with probiotics Pdp11 and Pdp13. Mortalityin the groups of Senegalese sole challenged with P. damselae subsp.piscicida Lgh41/01 was observed from the second day post-challenge.The cumulative percentage of mortality observed in the groupsfed with diets Pdp11 and Pdp13 ranged from 75 to 100% and 65 to80%, respectively. In all cases, the mortality in fish receiving the dietsupplemented with Pdp13 was significantly lower (Pb0.05)than that in fish receiving the diet supplemented with Pdp11. Inall groups of fish receiving the alginate diet and control diet thecumulative percentage of mortality was 100%. All the mortalities inthe experimental groups were caused by P. damselae subsp. piscicida

Table 2Percentage of mortality and relative protection survival (RPS) of Senegalese solespecimens after 60 days of feeding with diets supplemented with probiotics Pdp11 andPdp13, and challenge with P. damselae subsp. piscicida.

% Mortality RPS

Pdp11 dietTank 1 100 0Tank 2 80 20Tank 3 75 25Pdp13 dietTank 1 80 20Tank 2 70 30Tank 3 65 35

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Lgh41/01, as demonstrated by culture from the kidney and liver (datanot shown).

4. Discussion

Shewanella Pdp11 and Shewanella Pdp13 were identified as Shewa-nella putrefaciens and Shewanella baltica, respectively. Shewanellaspecies have been isolated from marine fish such as cod and flounder(Satomi et al., 2006, 2007), and Senegalese sole (Martín-Antonio et al.,2007). Members of this genus are part of the intestinal microbiota ofSenegalese sole cultured under extensive, semi-intensive and intensiveproduction systems, although the highest occurrence of these micro-organisms has beendetected in specimens fedwithpolychaeta (Martín-Antonio et al., 2007). On the other hand, Shewanella putrefaciens andShewanella baltica have been considered as important spoilage micro-organisms of low-temperature stored marine seafood (Vogel et al.,2005; Hovda et al., 2007).

Results obtained showed that diets supplemented with theprobiotics Pdp11 or Pdp13 produced significantly higher growth(Pb0.05) than the alginate diet and control diet, suggesting thataddition of these probiotics enhances growth performance of Sene-galese sole specimens. This beneficial effect on the growth ofSenegalese sole by the use of probiotics has also been reported inthe case of other farmed fish, such as European sea bass (Dicentrarchuslabrax) (Carnevali et al., 2006) and Nile tilapia (Oreochromis niloticus)(El-Haroun et al., 2006), when lactic acid bacteria such as Lactobacillusdelbrueckii delbrueckii and dietary probiotic Biogen® were admin-istered. In this study, sodium alginate containing high levels ofmannuronic acid polymers was used to facilitate the incorporationof bacterial cells to the pellet. The results obtained from specimens fedwith alginate diet are not in agreement with the results obtainedby authors, such as Vollstad et al. (2006) and Yeh et al. (2008) whoreported a higher growth in fish of other species fed with high-Malginate compared to control.

In our study, despite their affiliation within the same genus,Shewanella, and same origin of both microorganisms assayed in thisstudy, their effect on the respiratory burst activity was different, andonly Pdp11 significantly (Pb0.05) enhanced superoxide anionproduction by sole kidney phagocytes after 60-day feeding. In thecase of specimens fedwith Pdp13 diet also showed an increase of thisactivity after 60 days was also observed, although it was notsignificant in comparison to the values obtained from the fish fedwith the control diet containing sodium alginate. This could suggestthat the immunostimulation of the respiratory burst of the solephagocytes is strain-dependent rather than a general characteristicof a bacterial genus. This dependence has been described for othercharacteristics such as themucus adhesion of certain isolates of lacticacid bacteria (Rinkinen et al., 2003) and fish isolates (Chabrillónet al., 2005a,b). In a previous study, Díaz-Rosales et al. (2006a)feeding specimens of gilthead seabream with a diet supplementedwith heat-inactivated Pdp11 reported that this bacterial strain didnot induce a significant increase on the respiratory burst activity of

phagocytes from gilthead seabream, although they observed anincrease, but not significant, of the respiratory burst activity ofphagocytes after 4 week feeding. It could suggest that long-termprobiotic treatment could enhance the respiratory burst activity ofthe phagocytes. The high-M alginate is known to be an immunos-timulant with potential in marine aquaculture (Skjermo et al., 1995;2001; Fujiki and Yano, 1997; Peddie et al., 2002; Castro et al., 2004;Skjermo and Bergh, 2004; Bagni et al., 2005; Cheng et al., 2007). Inour study, this enhanced the respiratory burst activity has not beendetected in the case of phagocytes from specimens fed with alginatediet. Likely, Huttenhuis et al. (2006) reported some immunosuppre-sive effects on juvenile carps.

Several studies have reported that sodium alginate extracted frombrown algae enhance the resistance against pathogens in fish such ascommon carp (Fujiki and Yano, 1997), turbot (Skjermo et al., 1995)and grouper (Cheng et al., 2007). Yeh et al. (2008) carried out oneexperiment to determine the effects of the dietary administration ofsodium alginate on the growth of fingerling grouper, and theirresistance to Streptococcus sp. and iridovirus infections. They reportedthat after 12 days of the challenge with the pathogens a significantlyhigher survival rate of fish fed with a diet containing 2 g kg−1 sodiumalginate was observed compared to those fish with a control diet.However, in this study, the use of this same level of sodium alginatedid not produce any protection against P. damselae subsp. piscicida,because in all challenges the fish mortalities were 100%. On thecontrary, when fish were fed with Pdp11 and Pdp13 diets, survivalpercentages ranging from 0 to 25% and 20–35% were achieved,respectively. This positive effect of Shewanella species is in agreementwith the data reported by Makridis et al. (2008), who obtainedimproved survival of larvae of gilthead seabream and Senegalese solewhen different bacteria, among them Shewanella sp. strains wereadded, to the larval culture. This improvement of disease resistancealso has been observed by Carnevali et al. (2004) and Gildberg et al.(1997), who reported that the probiotic treatment significantlydecreased larvae and fry mortality of atlantic cod (Gadusmorrhua)and gilt head seabream, respectively.

Although several authors reported increases in respiratory burstactivity in fish by probiotics (Nikoskelainen et al., 2003; Gullian et al.,2004; Brunt and Austin, 2005; Aubin et al., 2005), it is not a generalcharacteristic of the probiotics. The fact that the probiotic Pdp13did not significantly increase the superoxide anion production, butsurvival rates of the fish fed with a diet supplemented with thismicroorganism were higher after the challenges with the pathogen,demonstrate that an increase of the respiratory burst is not essen-tial to enhance survival after the challenge with P. damselae subsp.piscicida. Several authors have reported a stimulatory effect of someprobiotics on different immunological parameters (Irianto andAustin, 2003; Nikoskelainen et al., 2003; Panigrahi et al., 2004;Aubin et al., 2005; Brunt and Austin, 2005; Salinas et al., 2005; Díaz-Rosales et al., 2006a,b). Thus, Pdp13 could exert its effects on immu-nological abilities different from respiratory burst. However, addi-tional experiments are necessary to test this hypothesis. Althoughthe immunostimulant effects of probiotics and sodium alginate aredemonstrated, Vollstad et al. (2006) warned about the negativeeffects that could result from inappropriate dose or treatmentduration. For this reason, additional information is required ondose–response relationships, kinetics, administration route andsurvival percentages at different ages. However, the use of theseprobiotics assayed in this study had a beneficial effect on theresistance of the fish against P. damselae subsp. piscicida.

5. Conclusion

The results obtained in this study showed a significantly bettergrowth of Senegalese sole specimens fed with the diets supplementedwith probiotics Shewanella putrefaciens Pdp11, and Shewanella baltica

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Pdp13, than of fish fed with a control diet including sodium alginate.On the other hand, the results derived from the evaluation of therespiratory burst activity of the phagocytes of specimens receiving thedifferent diets, and the challenges with P. damselae subsp. piscicidademonstrated that an increase of the respiratory burst of the phagocytesis not essential to obtain enhanced survival against the pathogen.

Acknowledgements

The authors thank PREDOMAR (Carboneras, Almería, Spain)fishery for help and participation in this study and the financialsupport by the Ministerio Educación y Ciencia (PTR1995-0943-OP andAGL2005-07454-C02-02), and the University of Almería and PRE-DOMAR (ACU03-022-C2-1 and 2). P. Díaz-Rosales thanks MinisterioEspañol de Educación y Ciencia for a F.P.U. scholarship.

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