Fish & Shellfish Immunology

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1 Fish & Shellfish Immunology 33 (212) 984e992 Contents lists ville t SciVerse ScienceDirect Fish & Shellfish Immunology journl homepge: Effects of mrine silges enriched with Lctocillus skei 5-4 on hemtoimmunologicl nd growth response in Pcific red snpper (Lutjnus peru) exposed to Aeromons veronii Mrth Reyes-Becerril,, Felipe Ascencio-Vlle, Mrí Esther Mcis,d, Minerv Mldondo, Murili Rojs c, Mrí Ángeles Esten, * Centro de Investigciones Biológics del Noroeste (CIBNOR), Mr Bermejo 195, Col. Ply Plo de Snt Rit, L Pz, B.C.S. 239, Mexico Fish Innte Immune System Group, Deprtment of Cell Biology nd Histology, Fculty of Biology, Regionl Cmpus of Interntionl Excellence Cmpus Mre Nostrum, University of Murci, 31 Murci, Spin c Lortorio de Cienci y Tecnologí de Alimentos, Áre Interdisciplinri de Ciencis Agropecuris, Universidd Autónom de Bj Cliforni Sur. Km 5.5, Crreter l Sur, 238 L Pz, Bj Cliforni Sur, Mexico d Centro Universitrio de Ciencis Excts e Ingenierís, Universidd de Gudljr, 4443 Gudljr, Jlisco, Mexico rticle info strct Article history: Received 5 June 212 Received in revised form 2 July 212 Accepted 13 August 212 Aville online 24 August 212 Keywords: Mrine silges Lctocillus skei 5-4 Immune system Histopthology Pcific red snpper Comined effects of mrine silges enriched with Lctocillus skei 5-4 were evluted on growth performnce, immune ctivity nd disese resistnce of Pcific red snpper (Lutjnus peru) ginst Aeromons veronii infection. The experimentl fish were divided into three groups which were fed with ech one of the following diets: silge-proiotic-free diet (control, C group), Pcific creole-fish silge diet supplemented with live L. skei (1 6 CFU g 1 )(FSLct group) nd Humoldt squid silge diet supplemented with live L. skei (1 6 CFU g 1 )(SSLct group) for 6 weeks. After 6 weeks, fish were immunocompromised with pthogenic A. veronii nd spleen nd liver smples were processed for histopthologicl studies. Generlly, the results showed enhnced growth performnce in fish fed the diet contining SSLct t 6 nd 7 weeks compred with fish fed control diet. Addition of SSLct hd n increse in plsmtic protein t week 6 nd post-chllenge. Hemogloin concentrtion incresed fter chllenge in fish fed with SSLct compred to control group. At week 6 nd post-chllenge the results indicted tht, the fish groups which received diet supplemented with SSLct reveled significnt increse in humorl immune prmeters. Histologiclly, fish fed C diets showed mrked ftty degenertion nd gret ctivtion of melnomcrophge centers compre with SSLct nd FSLct groups. These results support the ide tht the mrine silges with squid s protein source enriched or comined with L. skei 5-4 increses the ody weight nd stimultes the physiologicl nd humorl immune prmeters in Pcific red snpper infected with A. veronii. Ó 212 Elsevier Ltd. All rights reserved. 1. Introduction The Pcific red snpper Lutjnus is distriuted throughout tropicl nd sutropicl regions, from the Gulf of Cliforni to Peru [1]. Lutjnus peru is n importnt species of the locl, smll-scle fisheries of Mexico. It hs high mrket vlue, sufficient to e considered for quculture [2]. Despite its importnce, ssessment studies for the Pcific red snpper re so scrce. Only few studies hve een devoted to its iologicl spect [3,4] nd recently spect of culture conditions on feeding response of lrve hs een evluted [5]; however, immunology studies remin unknown. * Corresponding uthor. Tel.: þ ; fx: þ E-mil ddress: esten@um.es (M.Á. Esten). The growth of quculture, ssocited to the intensifiction of production systems, hs incresed the demnd for high-qulity feedstuff, economiclly vile, nd environmentlly-friend diets [6]. The dministrtion of immunostimulnts hs ppered s very promising iologicl control for quculture. The immunostimulnts re iologicl nd synthetic compounds tht enhnce innte defense mechnism in fish [7] nd they confer protection under dverse conditions. To dte, mny different immunostimulnts hve een found to e effective in different fish species [8e11]. Dietry silges in quculture hve een widely studied over the lst few yers since the point of view of some nutritionl spect s digestiility [12,13] or fish qulity [14]. Fish silge is defined s liquid product produced from the whole fish or prts of it, to which cids, enzymes or lctic cid-producing cteri re dded, /$ e see front mtter Ó 212 Elsevier Ltd. All rights reserved.

2 M. Reyes-Becerril et l. / Fish & Shellfish Immunology 33 (212) 984e with the liquefction of the mss provoked y the ction of enzymes from the fish [15,16]. Studies of silges (s lterntive using high-qulity nd economicl protein source) comined with cid lctic cterium s immunostimulnt source re still very scrce. Concerning the immune point of view, while mny reserches showed improvement in the immune response of fishes treted with Lctocillus sp. [17,18] owing to their ility to colonize the digestive trct. This lters the nturl lnce of the intestinl microiot nd could enhnce the immune system [19,2] or confer protection ginst severl mjor fish pthogens such s Aeromons in different species, including rinow trout (Oncorhynchus mykiss) nd Africn ctfish (Ictlurus puncttus) [1,21]. One of the mjor cteril diseses is cused y the Grmnegtive cterium Aeromons veronii, which produces diseses known s motile eromond septicemi of cultured wrm-wter fish nd Aeromons hydrophil is considered mjor economic prolem for quculture industry [22]. This cterium cn lso ehve s secondry opportunistic pthogen, y ssiling lredy compromised or stressed hosts [23]. Tken into ccount ll the ove considertions nd in order to use efficiently the locl protein sources from low-vlue species, prticulrly muscle Pcific creole-fish nd Humoldt squid, the im of the present study ws to determine the effect of two mrine silge diets, enriched with the proiotic Lctocillus skei on growth performnce nd immunologicl prmeters fter n experimentl infection with A. veronii in juvenile Pcific red snpper (L. peru). 2. Mterils nd methods 2.1. Silges Sterile 1 l volume glss ottles with screw cp were used for the replicte preprtion of iologicl silges ccording with the method previously reported y Ottti et l. [24], with some modifictions. Briefly, 85 g of commercil muscle of Pcific creole-fish (Prnthis colonus) nd Humoldt squid (Dosidicus gigs) were grounded nd pssed through 1.5 mm sieve. Grounded muscle ws perfectly mixed with 1 g of crohydrte source (food-grde sucrose), 5 g of n overnight-wet pellet of L. skei strin 5-4 s strter culture nd 2.5 g of soric cid nd then dispensed into the ottles. Cultures were incuted t 3 C for 72 h nd then stored t 4 C until used Proiotic cteri nd culture condition L. skei strin 5-4 ws isolted from intestinl mucus from notconfined juvenile spotted snd ss Prlrx mcultofcitus. Their moleculr identifiction ws performed y direct nlysis of the 16S rdna gene s previously descried y Mcís-Rodríguez et l. [25]. For routine use, primry culture from stocks stored t 8 C ws grown overnight t 3 C in pltes of Mnn, Rogos nd Shrpe Agr (MRS, Difco) under neroic conditions. From single isolted colony, 1 ml of MRS roth ws inoculted nd grown overnight under sttic conditions for 12e14 h. For preprtion of the experimentl silges, the cteril iomss ws hrvested nd wshed in Phosphte-Sline Buffer (PBS; 145 mm NCl, 2.87 mm KH 2 PO 4, nd 6.95 mm K 2 HPO 4, ph 7.2) nd centrifuged t 35 g for 1 min t room temperture. Wet pellets were used to inoculte the silge components s descried ove Experimentl diets Ingredients used for experimentl diets re given in Tle 1. The proximte chemicl compositions of the min ingredients in the Tle 1 Ingredient composition (g/1 g diet) of experimentl diets contining different source of protein used in the present study. Diets Ingredients Control Squid silge Fish silge Fish mel Fish silge Squid silge Whet flour Soyen mel c Yest g Cod liver oil d Soyen lecithin e Minerl premix h Vitmin premix i Alginic cid f Soyen oil e Glycine Choline chloride j Vitmin C k Fungus inhiitor BHT ntioxidnt l Pprik Srdine mel (66.2 crude protein, 9.2 lipids g 1 g 1 ). Conserver Sn Crlos, Puerto Sn Crlos, B.C.S., Mexico. Whole whet flour (12.7 crude protein,.7 lipids g 1 g 1 ) nd squid mel (71.2 crude protein, 3.3 lipids g 1 g 1 ). Proteíns Mrins y Agropecuris (Gudljr, Jlisco, Mexico). c Soyen mel (83.2 crude protein, 1.5 lipids g 1 g 1 ). Prost. (Toluc, Edo de Mexico, Mexico). d Frmci Pris (Mexico, D.F, Mexico). e Soyen lecithin nd oil. Distriuidor de Alimentos Nturles y Nutricionles, S.A. de C.V. (Mexico, D.F., Mexico). f Sigm A-7128 (St. Louis, MO, U.S.A.). g Otined in our lortory. h Minerl premix (g kg 1 ). KCl,.5; MgSO 4 $7H 2 O,.5; ZnSO 4 $7H 2 O,.9; MnCl 2 $4H 2 O,.234; CuSO 4 $5H 2 O,.5; KI,.5; CoCl 2 $2H 2 O,.25; N 2 HPO 4, i Vitmin premix (All vlues re in mg kg 1, except where indicted). Vitmin A Retinol, 5 IU; Vitmin D3 4 IU; Vitmin E Tocopherol, 1; Vitmin K Mendione, 5; Thimine, 6; Rioflvin, 25; Pyridoxine, 5; DL-Pntothenic cid, 75; Nicin, 4; Biotin, 1; Inositol, 4; Cynocolmin,.2; Folic cid, 1. j ICN Biomedicls Inc. (Auror, OH, U.S.A.). k Sty C (35% ctive gent) Roche, D.F., Mexico. l Butylted Hydroxytoluene. ICN Biomedicls Inc. (Auror, OH, U.S.A.). diets were nlyzed follow the Officil Methods of Anlysis [26] (Tle 2). Experimentl-diet preprtion ws performed ccording to the method descried y Goytortú-Bores [27]. Three diets were used nd formulted: control or silge-l. skei free (C) used fish mel s protein source, Pcific creole-fish silge þ L. skei (1 6 cfu g 1 ) (FSLc) nd Humoldt squid silge þ L. skei (1 6 cfu g 1 ) (SSLc). Briefly, dry ingredients were pulverized, sieved (.5-mm mesh) nd thoroughly mixed in food mixer prior to the ddition of fish oil nd soyen lecithin. After dispersion of the oil, wter ws dded to pproximtely 4% of the totl ingredient weight (where silges diets were dded t the desired volume or concentrtion). The finl product ws extruded t room temperture with met grinder nd 2-mm die, nd the resulting Tle 2 Chemicl nlysis of the experimentl diets used in the present study. Component % Control Fish silge Squid silge Crude protein Lipids Crude fier Ash Nitrogen-free extrct Methods of nlyses used the following AOAC methods (199).

3 986 M. Reyes-Becerril et l. / Fish & Shellfish Immunology 33 (212) 984e992 pellets were dried in forced-ir oven t 37 C for 24 h. Diets were pckged in polypropylene gs nd stored t 4 C until used Fish nd experimentl design Ninety specimens (1 5 g men ody weight) of juveniles Pcific red snpper were rndomly plced in nine running sewter tnks (1 fish per tnk) (flow rte 15 l h 1 ). Wter qulity ws monitored weekly nd wter temperture ws mintined t 26 C (with 12 h drk/12 h light photoperiod), dissolved oxygen t 4.3e 6.9 mg L 1 nd ph t 7.7e8.1. Totl mmoni nd nitrite concentrtion remined elow.2 mg L 1. Juveniles were fed three different diets (C, FSLct nd SSLct) for triplicte for 6 weeks. The control diet ws dministered to ll fish for 2 week conditioning period previous to the dministrtion of experimentl diets. The iomss in ech qurium ws mesured efore the experiment nd the dily rtion ws djusted ccordingly fter ech smpling. The fish were fed twice dily t 2% of their iomss. On dy nil, efore morning feeding, ech fish from every tnk ws individully weighed, nd this mesurement ws repeted weekly (initil, 6 nd 7 weeks) to otin the verge ody weight A. veronii The A. veronii iotype veronii strin A186 used in the present study ws otined from the Microil Culture Collection of the Hospitl of the University of Lund (Sweden) nd ws kindly provided y Prof. T. Wdström. A. veronii ws identified ccording to Vázquez-Juárez et l. [28] nd re-ctivted ccording to Reyes et l. [29]. Finlly, the cteril suspensions were diluted with phosphte-uffered sline (PBS, ph 7.4) to finl concentrtion of cells ml 1 for the chllenge experiment. The stock cultures were kept in suspension in 25% glycerol TSA nd stored t 8 C Infection test The LD 5 of the A. veronii ws 1 8 CFU, nd infected fish died within 48 h. No mortlity ws reported t concentrtions of e1 7 CFU in period of 2 weeks. In the present study, only immunocompromised fish were required eing used concentrtion of 1 6 CFU for the infection test. After 6 weeks of the experimentl feeding tril, the orgnisms were infected y intrperitonel injection of pthogenic A. veronii (1 6 CFU,.1 ml dose) or PBS. All fish were kept under oservtion for seven dys to record clinicl signs nd dily mortlity. At the end of the experiment (1 week post-infection), liver, lood nd intestine smples of oth chllenged nd non-infected fish were exmined to determine the presence or sence of A. veronii nd their identities were confirmed ccording to Popoff criteri [3] nd polymerse chin rection (PCR) detection Fish smple collection Five fish from ech qurium were rndomly smpled t week 6 of the feeding tril or 7 (1 week post-infection). Before smpling, the fish were strved for 24 h nd lood ws collected from the cudl vein with 27-guge needle nd 1-ml syringe nd llowed to clot t 4 C for 4 h. Serum ws otined y centrifugtion (1 min, 2 g,4 C) nd then stored t 8 C until used for physiologicl nd humorl immune prmeters determintion. Liver nd spleen were cut nd fixed in 1% neutrl formlin for histologicl exmintion Hemtologicl nlysis The totl protein content in plsm ws mesured y the method descried y Brdford [31]. Totl solule protein concentrtion ws ssessed in microplte reder t 595 nm y protein ssy (Bio-Rd Lortories). Bovine serum lum (BSA) ws used to uild stndrd curve to determine the protein concentrtion of ech smple. Fresh lood ws used to nlyze totl hemogloin levels y spectrophotometry t 54 nm, monitoring the rection with Drkin s regent [32] Humorl immune prmeters Myeloperoxidse content Totl myeloperoxidse (MPO) content in serum smples ws mesured ccording to Qude nd Roth [33] with slight modifictions. Ten microliters of serum ws diluted with 9 ml of Hnk s lnced slt solution (HBSS) without C 2þ or Mg 2þ in 96-well pltes. Then, 35 ml of 2 mm 3,3,5,5 -tetrmethylenzidine hydrochloride (TMB) (Genei, Indi) nd 5 mm H 2 O 2 were dded (oth sustrtes of MPO nd prepred on the sme dy). The colorimetric rection ws stopped fter 2 min y dding 35 ml of 4 M sulphuric cid (H 2 SO 4 ). The opticl density (OD) ws red t 45 nm in microplte reder. Stndrd smples without serum were used s lnks Enzyme-linked immunosorent ssy (immunogloulin M) Plsm totl IgM levels were mesured y n indirect enzymelinked immunosorent ssy (ELISA) ccording to Cuest et l. [34]. Briefly, flt-ottomed 96-well pltes were coted overnight with serem plsm (plsm diluted 1/5 in 5 mm cronte icronte uffer, ph 9.6). Smples were locked with ovine serum lumin nd incuted for 1 h with the primry ntiody (mouse nti-pcific red snpper IgM monoclonl ntiody; Aqutic Dignostics Ltd., 1/1 in locking uffer). After incution with the secondry ntiody nti-mouse IgG-HRP (1/1 in locking uffer), smples were developed with 3,3,5,5 -tetrmethylenzidine hydrochloride (TMB, Sigm) nd H 2 O 2. The pltes were red t 45 nm in plte reder (BMG, Fluoro Str Glxy). Negtive controls consisted of smples with or without plsm or primry ntiody, nd these OD vlues were sutrcted for ech smple vlue Lysozyme ctivity Bsed on the method y Lnge et l. [35], lysozyme ctivity ws tested in fish serum. On flt-ottomed 96-well microtitre plte, 1 ml of.4 mg ml 1 suspension of Micrococcus lysodeikticus (Sigm) in.5 mol l 1 sodium phosphte uffer (PBS, ph 5.2) ws dded to 1 ml of serum in seril dilutions of 1/5 to 1/4. The OD ws tken t 57 nm using n ELISA plte reder t, 15, 3, 45 nd 6 min. A unit of lysozyme ctivity ws defined s the mount of serum cusing decrese in sornce of.1 units per min Serum ntiproteses Totl ntiprotese ctivity ws determined s indicted y the cpcity of serum to inhiit trypsin ctivity [36,37]. Briefly, 2 ml of serum ws incuted with 2 ml of stndrd trypsin solution (1e2 BAEE, 5 mg ml 1 ; SigmeAldrich T-749) for 1 min t 22 C in Eppendorf tues. Then, 2 ml of.1 M PBS (ph 7.) nd 25 ml of 2% (w/v) zocsein (SigmeAldrich) in PBS were dded, nd incuted for 1 h t 22 C. The rection ws stopped y the ddition of 5 ml of 1% (v/v) trichlorocetic cid (TCA, Sigme Aldrich), incuted for 3 min t 22 C, nd then centrifuged t 6 g for 5 min. The superntnts (1 ml) were trnsferred to 96-well microtitre plte (Nlge Nunc) contining 1 ml well 1 of

4 M. Reyes-Becerril et l. / Fish & Shellfish Immunology 33 (212) 984e N sodium hydroxide (NOH, BDH). The OD ws red t 45 nm using plte reder. For positive (1%) control, uffer replced the serum, nd for negtive control, uffer replced oth serum nd trypsin. The inhiitory ility of ntiprotese ws expressed in terms of percentge trypsin inhiition ccording to the formul: %Trypsin inhiition ¼ Trypsin OD Smple OD=Trypsin OD Histologicl exmintion For histopthologicl studies, tissue smples were otined from liver nd spleen, fixed in 1% neutrl formlin, dehydrted in ethnol nd emedded in prffin ccording to stndrd histologicl techniques. Sections of pproximtely 5 mm were otined nd stined with hemtoxylin nd eosin ccording to the method descried y Drury et l. [38]. Spleen nd liver segments were exmined under Leic DMLB light microscope nd imges were cquired y mens of Leic DC 3 digitl cmer Sttisticl nlysis All iossys nd mesurements were performed in triplicte nd the men SE for ech dietry group ws clculted. A onewy nlysis of vrince (ANOVA) ws performed to determine the effects of experimentl diets on immunologicl prmeters using SPSS v.12. softwre (SPSS, Richmond, VA, USA). Mens were seprted y Tukey multiple rnge test. Student s t test ws used to determine the significnt difference in growth performnce. Differences were considered significnt t P Results 3.1. Infection test In the present study, for the infection test, we required only immunocompromised fish using concentrtion of CFU per fish for the experimentl infection nd no mortlity ws oserved in the experimentl or control groups. In prticulr, fish fed with FSLc showed typicl externl signs of cute septicemi with swollen round the cudl fins. Internl clinicl signs were oserved during the 7 dys following intrperitonel inocultion (viscerl inflmmtion, principlly spleen, nd ple liver) in fish fed with FSLc nd control groups. On the other hnd, externl or internl symptoms were not oserved in C or SSlct group. Externl nd internl exmintion nd cteriologicl nlysis of fish during the study reveled the presence of A. veronii in ll cses. A. veronii ws recovered in infected fish, minly in intestine smples Growth performnce During the whole experimentl period, weight gin ws generlly higher in fish fed with the SSLc diet t weeks 6 nd 7 (135.8 nd g, respectively) compred with thn tht oserved in fish fed control diet (16 nd g) (P <.5) (Fig. 1) Serologicl nlysis Fish fed with SSLc diets exhiited higher levels of plsmtic protein t week 6 (38.5 mg/ml) compred to C or FSlct groups (28 nd 29 mg/ml, respectively). After chllenge, incresed plsmtic Averge ody weight (g/fish) Initil protein ws oserved in SSLc diet (49.6 mg/ml) compred to those C or FSlct groups (33 nd 35 mg/ml, respectively) (Fig. 2). No significnt difference were found in hemogloin levels of Pcific red snpper fed with different experimentl diets t week 6. On the contrry, fter chllenge, only fish fed SSLc (1.36 g/dl) cused significnt increse in this prmeter compred with C or FSlct diets (8.2 nd 8.3 g/dl, respectively) (Fig. 3) Humorl immune prmeters There were not significnt chnges in mieloperoxidse ctivity t week 6 with ny of tretments used in this experiment. After these prmeters ws significntly increse in SSLct group (15 U/ml) compred with the C or FSlct diets (86 nd 18 U/ml). On the other hnd, dietry dministrtion for 6 weeks nd fter chllenge in fish fed SSLct showed significnt increse on IgM levels (.194 nd.27 OD 45 nm, respectively) in Pcific red snpper compred with C or FSlct diets. Lysozyme ctivity ws significntly higher (32.7 nd 36 U/ml) (P <.5) in fish fed SSLc group t week 6 compred with C or FSlct groups nd fter chllenge. Finlly, there ws significnt vrition in totl ntiproteses in fish fed SSLct for 6 nd fter chllenge (75 nd 78% of trypsin inhiition) compred to C nd FSlct fish (Fig. 4) Histopthologicl oservtion 6 Weeks The histopthologicl oservtion included vrying degrees of heptocyte lipid infiltrtion (ftty degenertion from slight to excessive), congestion of lood vessels in liver nd different 7 Control FSLct SSLct Fig. 1. Averge ody weight (fish g 1 ) in Pcific red snpper fed non-supplemented (control) nd silges enriched with Lctocillus skei 5-4 diets. Dt represent the men SE (n ¼ 15). Vlues with different letters re significntly different (P.5). FSlct: Fish silge in conjunction with L. skei 5-4; SSLct: Squid silge in conjunction with L. skei 5-4. Plsmtic protein (mg/ml) Postchllenge Control FSLct SSLct Fig. 2. Plsmtic protein levels from Pcific red snpper fed with three different diets t 6 weeks nd post-chllenge with Aeromons veronii infection. Dt represent the mens SE (n ¼ 5). Columns showing different letters re significntly different t P.5. FSlct: Fish silge in conjunction with L. skei 5-4; SSLct: Squid silge in conjunction with L. skei 5-4.

5 988 M. Reyes-Becerril et l. / Fish & Shellfish Immunology 33 (212) 984e992 Hemogloin (g/dl) Control FSLct SSLct contining high mounts of hemosiderin. High frequency of dispersed MMO ws oserved in fish fed C nd SSlct diets. On the other hnd, low frequency of MMC nd MMC with hemosiderosis ws oserved in fish fed C nd SSlct (Fig. 6) diets. Finlly, the exmintion of spleen in fish fed FSlct reveled no or miniml numers of these cells (MMC or MMO) on this group. 4. Discussion Postchllenge Fig. 3. Hemogloin concentrtion from Pcific red snpper fed with three different diets t 6 weeks nd post-chllenge with Aeromons veronii infection. Dt represent the mens SE (n ¼ 5). Columns showing different letters re significntly different t P.5. FSlct: Fish silge in conjunction with L. skei 5-4; SSLct: Squid silge in conjunction with L. skei 5-4. ltertions in splenic melnomcrophge centers (Tle 3). Liver Pcific red snpper fed C diet nd infected with A. veronii showed severe ftty degenertion. Fish supplemented with FSLct diet showed moderte liver ftty degenertion while mild ftty degenertion ws oserved in fish fed with SSlct (Fig. 5). Severe congestion ws oserved in liver from C group fter infection with A. veronii compred with mild congestion present in liver from fish fed FSLct nd SSLct diets. The exmintion of spleen in fish fed-supplemented diets fter chllenge reveled principlly the presence of dispersed melnomcrophges (MMO) nd melnomcrophges centers (MMC) In the qutic industry, rnge of feed ingredients nd dditives is used to formulte nutritionlly dequte fish feeds. Feed cost is mjor operting cost in fish frming minly due to lck of chep sources of protein [16]. Thus, it is importnt to find lterntive protein sources tht not only meet the nutritionl requirements of the trget species ut re economiclly vile y eing competitively priced. Moreover to ensure high fish qulity for trde nd to control disese outrek it is importnt for potentil protein sources to fulfill the nutritionl requirements of the immune system. The mrine silge production process is simple, prcticl nd economicl, not requiring expensive equipment nd procedures, such s those used in the production of fish mel [39]. Therefore, control of the microiot in mrine silge cultures represents significnt interest to relize stle fermenttion (lctic cid cterium, Lctocillus) nd to gurntee the microil sfety of the products [4]. This investigtion ws plnned to evlute the effect of mrine silge comined with the proiotic L. skei 5-4 on growth performnce nd immune prmeters in Pcific red snpper under the infection with the cterium A. veronii. In this study, supplementtion with SSLc improved Myeloperoxidse content (U/ml) c Postchllenge IgM (OD 45 nm) c Postchllenge Lysozyme ctivity (U/ml) % tryps in inhiition Postchllenge Postchllenge Control FSlct SSlct Fig. 4. Humorl immune prmeters from Pcific red snpper fed with three different diets t 6 weeks nd post-chllenge with Aeromons veronii infection. Dt represent the mens SE (n ¼ 5). Columns showing different letters re significntly different t P.5. FSlct: Fish silge in conjunction with L. skei 5-4; SSLct: Squid silge in conjunction with L. skei 5-4.

6 M. Reyes-Becerril et l. / Fish & Shellfish Immunology 33 (212) 984e Tle 3 Histopthologicl chnges undergone y Pcific red snpper (Lutjnus peru) spleen nd liver following the exposure to Aeromons veronii. Histopthologicl oservtions Control FSlct d SSlct e Liver Ftty degenertion Congestion Spleen c Dispersed MMO MMC 1 MMC with hemosiderosis 1 1 Pthologicl chnges grded: 1 (imperceptile); 2 (mild); 3 (moderte); 4 (severe); 5 (mrked). Pthologicl chnges grded: (norml); 1 (mild); 2 (moderte); 3 (severe). c Tissue chnges were ssessed s follows: () not oserved; (1) low frequency; (2) moderte frequency; (3) high frequency. MMO: melnomcrophges. MMC: melnomcrophge centers. d FSlct: Fish silge in conjunction with L. skei 5-4. e SSLct: Squid silge in conjunction with L. skei 5-4. significntly growth in juveniles of Pcific red snpper fter 6 nd 7 weeks of feeding compred to control diet. A numer of orgnic compounds, including L-mino cids, etine, nucleosides, nucleotides nd extrcts from mrine nimls, hve een proposed s feeding stimulnts for fish [41]. On the other hnd, enhnced fish growth ssocited to proiotics such s Scchromyces cerevisie [42], Leuconostoc mesenteroides CLFP 196 nd Lctocillus plntrum CLFP 238 [43] my e due to improve the nutrient digestiility nd/or to lter the intestinl microiot, s well s immunostimultion. The present investigtion demonstrted tht replcing herring fish mel silge with fish or squid silge comined with the proiotic L. skei 5-4 showed positive effects on growth nd immune system. Squid silge tretment is high protein nd economicl source tht could replce fish mel protein. According to the results is giel crp (Crssius urtus), the squid extrct ws effective s ttrctnts y stimulting the fish immune response [44]. The use of Aeromons s model pthogen to elucidte immune mechnisms in Pcific red snpper is of significnce, ecuse cute septicemi cused y this pthogen is serious thret to fish production [45]. To dte, the eneficil effects of proiotics dministrtion ginst Aeromons infection in fish hve een demonstrted with dietry dministrtion of S. cerevisie [42], Deryomyces hnsenii CBS 8339 [29] nd Lctocillus cidophilus [1]. The present study confirmed the enefits of mrine silges dded with L. skei ginst A. veronii infection. The min histologicl ltertions were oserved in fish fed control diet in which severe congestion in the liver nd splenic melnomcrophge centers nd high intensity of hemosiderin pigment were oserved. Furthermore, mrked ccumultion of ft in the liver in control group ws oserved compred to fish fed mrine silges enriched with L. skei. The effects of proiotics hve een widely studied in cultured qutic species, prticulrly to enhncement of the innte immune system [9,11,18] which is fvorle to pthogen control [17]. Hemtologicl prmeters re typiclly used s indictor of helth sttus nd to detect physiologicl chnges in severl fish species [46]. In this work, protein nd hemogloin prmeters of Pcific red snpper were nlyzed nd were within the verge rnge of the other mrine nd freshwter fish, such s tilpi (Oreochromis niloticus) [47], rinow trout (O. mykiss) [48] or leoprd grouper (Mycteroperc rosce) [49]. Dt for the totl serum protein is reflection of innte immunity [5]. In this study, physiologicl prmeters such s plsmtic protein nd hemogloin were incresed in fish fed with SSLc diet t week 6 nd fter infection with A. veronii. Newj-Fyzul et l. [21] found significnt increse in totl protein levels fter dministrtion of proiotics feed dditives in rinow trout for 14 dys, s well s enhnced in myeloperoxidse ctivity. In this work cler incresing in myeloperoxidse ctivity ws oserved fter chllenge with A. veronii in fish fed with SSLct diet. The corresponding increse in peroxidse ctivity ws not surprising ecuse these enzymes will e required to remove rective-free rdicls Fig. 5. Liver ftty degenertion. ) Control diet; ) Fish silge in conjunction with L. skei 5-4; c) Squid silge in conjunction with L. skei 5-4.

7 99 M. Reyes-Becerril et l. / Fish & Shellfish Immunology 33 (212) 984e992 Fig. 6. The spleenic tissue with melnomcrophge centers nd hemosiderosis. ) Control diet; ) Fish silge in conjunction with L. skei 5-4; c) Squid silge in conjunction with L. skei 5-4. tht my e hrmful to the fish [21]. A similr peroxidse ctivity hs een reported in Kelp grouper (Epinephelus runeus) fed with L. skei BK19 nd chllenged with Scchromyces inie t weeks 2 nd 4 [17]. On the other hnd, IgM levels nd lysozyme ctivity were significntly incresed t week 6 nd fter chllenge in fish fed with SSLc in fish exposed to infection. Serum immunogloulins re mjor components of the humorl immune system nd IgM is the min immunogloulin present in fish [51]. In greement with these results, Nikoskelinen et l. [19] oserved tht serum immunogloulin levels were significntly rised in fish fed-supplemented diet with Lctocillus rhmnosus t CFU/g feed (LAB8) on week 1 nd in LAB4 ( CFU/g feed) nd LAB8 t the end of the tril (week 4). An increse in the lysozyme concentrtion in fish lood cn e cused y infections or invsion y foreign mteril [52]. The stimultion of lysozyme ctivity hs een recognized fter 2, 4 nd 6 weeks of feeding with Kocuri SM1 in rinow trout O. mykiss [48], S. cerevisie in serem Sprus urt [53], nd L. plntrum in grouper Epinephelus coioides [54], respectively. Fish plsm contins numer of protese inhiitors, principlly 1-ntiprotese, 2-ntiplsmin nd 2-mcrogloulin, which hve een demonstrted to hve role in restricting the ility of cteri to survive in vivo [55]. These ctivities ppered to e hrdly modulted in fish were immuniztion or infection [56]. In the present study, serum ntiproteses ctivity ws significntly higher in fish fed with SSlct diet t week 6 nd post-infection compred with control nd FSlct group. Similr result ws reported in kelp grouper, E. runeus fed with L. skei nd chllenge with or without S. inie t weeks 2 nd 4 [17]. In this work, growth performnce nd hemtologicl prmeters increse in fish fed with squid silge comined with L. skei 5-4 (SSLct diet), principlly fter infection. The fcts suggest tht n immunostimulnt effect fter 6 weeks of proiotic feeding ws the key determinnt in mximizing host resistnce to disese. Mny qutic niml extrcts hve een proven to e feeding stimulnts for fish, such s squid, shrimp, mullet, nd cr [57,58], nd squid extrct hs een used s feeding stimulnt for slmonids [59], turot [6] nd loster Homrus gmmrus [61]. Active components in these niml extrcts included free mino cids, nucleotides, nucleosides nd quternry mmonium ses [44]. More studies re needed to determine which of these components re responsile of the immunostimulnt effect oserved. To conclude, the results of the present study clerly demonstrte tht the infected groups with the cterium A. veronii mintined with mrine silge diets, principlly using squid s protein source nd enriched with L. skei, yielded significntly etter growth performnce, hemtologicl prmeters nd we oserved decresed the histopthologicl dmge compred with fish fed control diet. This supports the eneficil effects of proiotic cteri (L. skei) s io-control gent in ssisting fish (Aeromons) to etter withstnd cteril infections. Furthermore, use of silges s lterntive to replce mel fish y proteins otined mrine y-products comined with proiotics give us economicl nd qulity diet which could provide helthy nd sfe fish production from quculture replcing the commercil diets. Acknowledgements We thnk Gspr Pettit for their technicl support nd Dr. Mrcos Cden for silges preprtion. The first uthor received postdoctorl fellowships from Consejo Ncionl de Cienci y Tecnologí of Mexico (CONACYT grnt ) nd the project ws funded under SAGARPA-CONACYT (grnt 24-44) nd Ministerio de Cienci e Innovción of Spin (AGL C3-1). Dr. MA Esten is memer of Grupo de Excelenci de l Región de Murci (4538/GERM/6).

8 M. Reyes-Becerril et l. / Fish & Shellfish Immunology 33 (212) 984e References [1] Allen GR. FAO species ctlogue, vol. 6. Snppers of the Word. An nnotted nd illustrted ctlogue of Lutjnid species know to dte. FAO Fisheries Synop 1985;125:1e28. [2] Avilés-Quevedo A, Reyes L, Hirles O, Rodríguez R, McGregor U. Resultdos preliminres en el cultivo del huchinngo del Pcífico Lutjnus peru (Nichols nd Murphy, 1922) en juls flotntes en hí Fls, BCS, México. In: Silv A, Merino G, editors. Acuicultur en Ltinomeric. IX Congreso ltinomericno de cuicultur. 2 Simposio vnces y perspectivs de l cuicultur en Chile; p. 248e5 [Coquimo, Chile]. [3] Roch OA, Sndovl CJ. Mitocondril diversity nd genetic structure in lloptric popultion of the Pcific red snpper Lutjnus peru. Ciencis Mrins 23;29:197e29. [4] Gllrdo CM, Sri MM, Espino BE, Anisldo TV. Biologicl spects of Lutjnus peru in Bufdero By, Michocán, México: growth, reproduction nd condition fctors. Rev Biol Mr Ocenogr 21;45:25e15. [5] Zvl LI, Dums S, Peñ R, Contrers OM, Hernández CD. Effects of culture conditions on feeding response of lrvl Pcific red snpper (Lutjnus peru, Nichols & Murphy) t first feeding. Aqucult Res 212:1e /j x. [6] Espe M, Sveier H, Høgøy I, Lied E. Nutrient sorption nd growth of Atlntic slmon (Slmo slr) fed fish protein concentrte. Aquculture 1999;174: 119e37. [7] Anderson DP. Immunostimulnts, djuvnts, nd vccine crriers in fish: pplictions to quculture. Ann Rev Fish Dis 1992;2:281e37. [8] Goplknnn A, Arul V. Enhncement of the innte immune system nd disese-resistnt ctivity in Cyprinus crpio y orl dministrtion of -glucn nd whole cell yest. Aqucult Res 21;41:884e92. [9] Reyes BM, Ascencio VF, Meseguer J, Tpi PST, Moriñigo MA, Esten AMA. Deryomyces hnsenii L2-enriched diet enhnces the immunity sttus, gene expression nd intestine functionlity in gilthed serem (Sprus urt L.). Aqucult Res 211:1e12. [1] Al-Dohil MA, Hshim R, Aliyu-Piko M. Evluting the use of Lctocillus cidophilus s iocontrol gent ginst common pthogenic cteri nd the effects on the hemtology prmeters nd histopthology in Africn ctfish Clris griepinus juveniles. Aqucult Res 211;42:196e29. [11] Mouriño JLP, Nscimento VFD, Jto AB, D Silv BC, Jesus GFA, Seiffert WQ, et l. Effects of dietry supplementtion of inulin nd W. ciri on hemtoimmunologicl prmeters of hyrid suruim (Pseudopltystom sp). Aqucult Nutr 212;18:73e8. [12] Borghesi R, Portz L, Oetterer M, Cyrino JEP. Apprent digestiility coefficient of protein nd mino cids of cid, iologicl nd enzymtic silge for Nile tilpi (Oreochromis niloticus). Aqucult Nutr 28;14:242e8. [13] Mch DTN, Nguyen MD, Nortvedt R. Effects on digestiility nd growth of juvenile coi (Rchycentron cndum) fed fish or cr silge protein. Aqucult Nutr 21;16:35e12. [14] Mch DTN, Nortvedt R. Chemicl nd nutritionl qulity of silge mde from rw or cooked lizrd fish (Surid undosqumis) nd lue cr (Portunus pelgicus). J Sci Food Agric 29;89:2519e26. [15] Oetterer M. Proteíns do pescdo-processmento com intervenção protéic. In: Oetterer M, Regitno d Arce MA, Spoto MHF, editors. Fundmentos de Ciênci e Tecnologi de Alimentos. Brueri: Mnole; 26. p. 99e134. [16] FAO. Animl feed resources informtion system, 2 Mr. 27. [17] Hrikrishnn R, Blsundrm C, Heo M-S. Lctocillus skei BK19 enriched diet enhnces the immunity sttus nd disese resistnce to streptococcosis infection in kelp grouper, Epinephelus runeus. Fish Shellfish Immunol 21; 29:137e43. [18] Pnigrhi A, Viswnth K, Stoh S. Rel-time quntifiction of the immune gene expression in rinow trout fed different forms of proiotic cteri Lctocillus rhmnosus. Aqucult Res 211;42:96e17. [19] Nikoskelinen S, Ouwehnd A, Bylund G, Slminen S, Lilius EM. Immune enhncement in rinow trout (Oncorhynchus mykiss) y potentil proiotic cteri (Lctocillus rhmnosus). Fish Shellfish Immunol 23;5:443e52. [2] Blcázr JL, de Bls I, Ruiz-Zrzuel I, Vendrell D, Clvo AC, Márquez I, et l. Chnges in intestinl microiot nd humorl immune response following proiotic dministrtion in rown trout (Slmo trutt). Br J Nutr 27;97: 522e7. [21] Newj-Fyzul A, Adesiyun AA, Mutni A, Rmsuhg A, Brunt J, Austin B. Bcillus sutilis AB1 controls Aeromons infection in rinow trout (Oncorhynchus mykiss, Wlum). J Appl Microiol 27;5:1699e76. [22] Austin B, Austin DA. Bcteril fish pthogens: disese in frmed nd wild fish. New York: Springer-Verlg; [23] Tellez BMC, Snterre A, Css-Solis J, Zirsev G. Endosulfn increses seric interleukin-2 like (IL-2L) fctor nd immunogloulin M (IgM) of Nile tilpi (Oreochromis niloticus) chllenged with Aeromons hydrophil. Fish Shellfish Immunol 21;28:41e5. [24] Ottti M, Gutiérrez M, Bello R. Preprtion of microil silge from fish of underutilized species. Arch Ltinom Nutr 199;4:48e25. [25] Mcís-Rodríguez ME, Zgorec M, Ascencio F, Rojs M. Potentil proiotic Lctocillus strins for piglets from n rid cost. Ann Microiol 28;58:1e8. [26] AOAC e Off methods nl, vol. 985; 199. p. 115e6. [27] Goytortú-Bores E. Evlución de l digestiilidd de diets compuests se de hrin de lngostill (Pleuroncodes plnipes) y su efecto en el crecimiento del cmrón lnco (Peneus vnnmei). Tesis de Licencitur. Universidd Autónom de Sn Luis Potosí. S.L.P.; pp. [28] Vázquez-Juárez RC, Brrer-Sldñ HA, Hernández-Svedr NY, Gómez- Chirri M, Ascencio F. Moleculr cloning, sequencing nd chrcteriztion of omp48, the gene encoding for n ntigenic outer memrne protein from Aeromons veronii. J Appl Microiol 23;94:98e18. [29] Reyes BM, Tovr RD, Ascencio VF, Civer CR, Grci LV, Bros SV. Effects of dietry supplementtion with proiotic live yest Deryomyces hnsenii on the immune nd ntioxidnt systems of leoprd grouper Mycteroperc rosce infected with Aeromons hydrophil. Aqucult Res 21:1e11. dx.doi.org/1.1111/j x. [3] Popoff M. Aeromons. In: Krieg NR, editor. Bergey s mnul of systemtic cteriology. Bltimore, MD: Willims & Wilkins; p. 545e8. [31] Brdford MM. A rpid nd sensitive method for the quntittion of microgrm quntities of protein utilizing the principle of proteinedye inding. Anl Biochem 1976;72:248e54. [32] Drkin DL, Austin JH. Spectrophotometric studies. II. Preprtions from wshed lood cells; nitric oxide hemogloin nd sulphemogloin. J Biol Chem 1935;112:51e64. [33] Qude MJ, Roth JA. A rpid, direct ssy to mesure degrnultion of ovine neutrophil primry grnules. Vet Immunol Immunopthol 1997;58:239e48. [34] Cuest A, Esten MA, Meseguer J. Totl serum immunogloulin M levels re ffected y immunomodultors in serem (Sprus urt L.) specimens. Vet Immunol Immunopthol 24;11:23e1. [35] Lnge S, Gudmundsdóttir B, Mgndottir B. Humorl immune prmeters of cultured Atlntic hliut (Hippoglossus hippoglossus L.). Fish Shellfish Immunol 21;11:523e35. [36] Ellis AE. Serum ntiproteses in fish nd lysozyme ssys. In: Stolen JS, Fletcher TC, Anderson DP, Roerson BS, vn Muiswinkel WB, editors. Techniques in fish immunology. Fir Hven NJ: SOS Pulictions; 199. p. 95e13. [37] Mgndottir B, Jonsdottir H, Helgson S, Bjornsson B, Jørgensen TØ, Pilstrom L. Humorl immune prmeters in Atlntic cod (Gdus morhu L.): the effects of environmentl temperture. Comp Biochem Physiol 1999;B;122:173e8. [38] Drury RA, Wllington EA, Cncerson R. Crlton s histopthologicl techniques. 4th ed. Oxford, London, New York: Oxford University Press; [39] de Arrud LF, Borghesi R, Portz L, Cyrino JEP, Oetterer M. Fish silge in Blck ss (Micropterus Slmoides) feed s n lterntive to fish mel. Brz Arch Biol Technol 29;52:1261e6. [4] Uchid M, Amksu H, Stoh Y, Murt M. Comintions of lctic cid cteri nd yest suitle for preprtion of mrine silge. Fish Sci 24;7:57e17. [41] Pptryphon E, Sores JH. Optimizing the levels of feeding stimulnts for use in high-fish mel nd plnt feedstuff-sed diets for striped ss, Moronesxtilis. Aquculture 21;22:279e88. [42] Adel-Tww M, Adel-Rhmn A, Ismel M. Evlution of commercil live kers yest, Scchromyces cerevisie s growth nd immunity promoter for Fry Nile tilpi, Oreochromis niloticus (L.) chllenged in situ with Aeromons hydrophil. Aquculture 28;28:185e9. [43] Vendrell D, Blczr JL, De Bls I, Ruiz-Zrzuel I, Girone SO, Murquiz JL. Protection of rinow trout (Oncorhynchus mykiss) from lctococcosis y proiotic cteri. Comp Immunol Microiol Infect Dis 28;31:337e45. [44] Xue M, Cui Y. Effect of severl feeding stimulnts on diet preference y juvenile giel crp Crssius urtus gielio, fed diets with or without prtil replcement of fish mel y met nd one mel. Aquculture 21;198:281e92. [45] Fng HM, Ling KC, Ge R, Sin YM. Enhncement of protective immunity in lue gourmi, Trichogster trichopterus (Plls), ginst Aeromons hydrophil nd Virio nguillrum ya. hydrophil mjor dhesion. J Fish Dis 2;23:137e45. [46] Atmnlp M, Ynik T. Altertions in hemtologicl prmeters of rinow trout (Oncorhynchus mykiss) exposed to mncoze. Turk J Vet Anim Sci 23;27:1213e7. [47] Aly SM, Ahmed YAG, Ghree AAA, Mohmed MF. Studies on Bcillus sutilis nd Lctocillus cidophilus, s potentil proiotics, on the immune response nd resistnce of Tilpi nilotic (Oreochromis niloticus) to chllenge infections. Fish Shellfish Immunol 28;25:128e36. [48] Shrifuzzmn SM, Austin B. Influence of proiotic feeding durtion on disese resistnce nd immune prmeters in rinow trout. Fish Shellfish Immunol 29;27:44e5. [49] Reyes BM, Tovr RD, Ascencio VF, Civer CR, Grci LV, Bros SV. Effects of dietry live yest Deryomyces hnsenii on the immune nd ntioxidnt system in juvenile leoprd grouper Mycteroperc rosce exposed to stress. Aquculture 28;28:39e44. [5] Wiegertjes GF, Stet RJM, Prmentier HK, vn Muiswinkel WB. Immunogenetics of disese resistnce in fish: comprle pproch. Dev Comp Immunol 1996;2:365e81. [51] Wtts M, Mundy BL, Burke CM. cdna sequences nd orgniztion of IgM hevy chin genes in two holosten fish. Dev Comp Immunol 21;9:153e64. [52] Siwicki AK, Studnick M, Mornd M, Pozet F, Mjewsk ET. Comprtive immunotoxicology new direction. Act Vet (Brno) 1998;67:295e31. [53] Rodríguez A, Cuest A, Ortuño J, Esten MA, Meseguer J. Immunostimulnt properties of cell wll-modified whole Scchromyces cerevisie strin dministered y diet to serem (Sprus urt L.). Vet Immunol Immunopthol 23;96:183e92.

9 992 M. Reyes-Becerril et l. / Fish & Shellfish Immunology 33 (212) 984e992 [54] Son VM, Chng CC, Wu MC, Guu YK, Chiu CH, Cheng W. Dietry dministrtion of the proiotic, Lctocillus plntrum, enhnced the growth, innte immune responses, nd disese resistnce of the grouper Epinephelus coioides. Fish Shellfish Immunol 29;26:691e8. [55] Ellis AE. Innte host defense mechnisms of fish ginst viruses nd cteri. Dev Comp Immunol 21;25:827e39. [56] Slins I, Aelli L, Bertoni F, Picchietti S, Roque A, Furones D, et l. Monospecies nd multispecies proiotic formultions produce different systemic nd locl immunostimultory effects in the gilthed serem (Sprus urt L.). Fish Shellfish Immunol 28;25:114e23. [57] Crr WES, Kenneth M, Blumenthl J, Netherton III C. Chemoreception in the pigfish, Orthopristis chrysopterus, the contriution of mino cids nd etine to stimultion of feeding ehvior y vrious extrcts. Comp Biochem Physiol 1977;58A:69e73. [58] Mckie AM, Mitchell AI. Further studies on the chemicl control of feeding ehviour in the Dover sole Sole sole. Comp Biochem Physiol 1982;73A:89e93. [59] Toften H, Jørgensen EH, Joling M. The study of feeding preference using rdiogrphy: oxytetrcycline s feeding deterrent nd squid extrct s feeding stimulnt in diets for slmon. Aqucult Nutr 1995;1:145e9. [6] Mckie AM, Adron JW. Identifiction of inosine nd inosine 5 -monophosphte s the gusttory feeding stimulnts for the turot, Scophthlmus mximus. Comp Biochem Physiol 1978;6A:79e83. [61] Mckie AM. The chemicl sis of food detection in the loster Homrus gmmrus. Mr Biol 1973;21:13e8.