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Fish & Shellfish Immunology 29 (2010) 2e14

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Fish & Shellfish Immunology

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Review

Probiotics and immunity: A fish perspective

S.K. NayakLaboratory of Fish Pathology, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Japan

a r t i c l e i n f o

Article history:Received 6 November 2009Received in revised form12 February 2010Accepted 19 February 2010Available online 26 February 2010

Keywords:FishGut immunityInnate immunityProbiotics

E-mail address: sukantanayak@rediffmail.com

1050-4648/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.fsi.2010.02.017

a b s t r a c t

Probiotics are usually live microorganisms which when administered in adequate amounts confera health benefits on host. Nowadays, probiotics are also becoming an integral part of the aquaculturepractices to obtain high production. The common probiotics that are used for aquaculture practicesinclude Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Carnobacterium, Shewanella, Bacillus, Aero-monas, Vibrio, Enterobacter, Pseudomonas, Clostridium, and Saccharomyces species. The involvement ofprobiotics in nutrition, disease resistance and other beneficial activities in fish has proven beyond anydoubt. Among the numerous health benefits attributed to probiotics, modulation of immune system isone of the most commonly purported benefits of the probiotics and their potency to stimulate thesystemic and local immunity under in vitro and in vivo conditions is noteworthy. Different probioticseither monospecies or multispecies supplementation can eventually elevate phagocytic, lysozyme,complement, respiratory burst activity as well as expression of various cytokines in fish. Similarly,probiotics can stimulate the gut immune system of fish with marked increase in the number of Igþ cellsand acidophilic granulocytes. Furthermore, mono-bacterial association studies (with non-probioticbacterial strains) in gnotobiotic fish also indicate the up-regulation of various immune related genes.Though the exact mode of action of probiotics is yet to be established in any animal including fish,probiotics often exert host specific and strain specific differences in their activities. Various factors likesource, type, dose and duration of supplementation of probiotics can significantly affect the immuno-modulatory activity of probiotics. The review is therefore, aiming to highlight the immunomodulatoryactivity of probiotics and also to evaluate the factors that regulate for the optimum induction of immuneresponses in fish.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Over the years various strategies tomodulate the composition ofthe gut microbiota for better growth, digestion, immunity, anddisease resistance of the host have been investigated in variouslivestock as well as in human beings [1]. The manipulation of thegut microbiota through dietary supplementation of beneficialmicrobe(s) is a novel approach not only from nutritional point ofview but also as an alternate viable therapeutic modality to over-come the adverse effects of antibiotics and drugs. Those beneficialmicroorganisms are usually referred as “probiotics” which afteradministration can able to colonize and multiply in the gut of hostand execute numerous beneficial effects by modulating variousbiological systems in host [2]. Probiotics are originally defined asthe organisms and substances which contribute to the intestinalmicrobial balance [3]. The term probiotic was originated from the

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Greek words “pro” and “bios” which mean “for life” [4] and areoften called as promoter of life that help in a natural way toimprove the overall health status of the host organism. According tothe currently adopted definition by Food and Agricultural Organi-zation/World Health Organization, probiotics are live microorgan-isms which when administered in adequate amounts confera health benefit on the host [5].

Probiotics, thus, open a new era in health management strategyfrom human to fish/shellfish. Probiotics are gaining increasingscientific and commercial interest and are now quite commonplacein health promoting functional foods to therapeutic, prophylacticand growth supplements [6,7]. The success of probiotics, has laidthe foundation for other concepts like “prebiotics” which are thenon-digestible food ingredients that selectively stimulate thegrowth and/or activity of one or limited microbes and “synbiotics”,the nutritional supplements combining probiotics and prebiotics[8,9]. The obvious potential advantages of such approaches are thatthey promote specific microbe(s) in the intestine for restoring theintestinal microbial balance and exerting numerous beneficialeffects in host [2,8,9].

S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e14 3

2. Probiotics in fish culture

Fish is one of the richest sources of animal protein and is thefastest food producing sector in the world. Worldwide, peopleobtain about 25% of their animal protein from fish and shellfish andconsumer's demand for fish continues to climb [10]. Over the years,aquaculture sector has undergone a sea change in order tomeet theincreasing demand. The production is maximized through inten-sification with addition of commercial diets, growth promoters,antibiotics, and several other additives. Application of thesemeasures leads to high production beyond any doubt, but the mostworrisome factor is that the routine use of these products causessevere complications and even a stage has come where itssustainability is in stake [11].

In aquaculture practices, probiotics are used for a quite longtime but in last few years probiotics became an integral part of theculture practices for improving growth and disease resistance. Thisstrategy offers innumerable advantages to overcome the limita-tions and side effects of antibiotics and other drugs and also leads tohigh production through enhanced growth and disease prevention[12e15]. In aquaculture, the range of probiotics evaluated for use isconsiderably wider than in terrestrial agriculture. Several pro-biotics either as monospecies or multispecies supplements arecommercially available for aquaculture practices [16e20]. Apartfrom the nutritional and other health benefits [21e25], certainprobiotics as water additives can also play a significant role indecomposition of organic matter, reduction of nitrogen and phos-phorus level as well as control of ammonia, nitrite, and hydrogensulfide [26].

Numerous microbes have been identified as probiotics foraquaculture practices, many of which differ markedly in their modeof action. There are, however, some commonmechanisms of actionthat have been reported for the majority of probiotic strains.Probiotics help in feed conversion efficiency and live weight gain[27,28] and confer protection against pathogens by competitiveexclusion for adhesion sites [29,30], production of organic acids(formic acid, acetic acid, lactic acid), hydrogen peroxide and severalother compounds such as antibiotics, bacteriocins, siderophores,lysozyme [31e35] and also modulate physiological and immuno-logical responses in fish [36,37].

3. Probiotics and fish immunity

Among the numerous beneficial effects of probiotics, modula-tion of immune system is one of the most commonly purportedbenefits of the probiotics. The role of probiotics in modulating theimmune system has been extensively investigated and reviewed inhumans and animals [38e41]. Most of the earlier studies in fish,dealt with growth promoting and disease protective ability ofprobiotics. However, in recent times much attention has beenhitherto towards the immunmodulating effects of probiotics inpiscine system. A lot of immunological studies have beenperformedin several fish using different probiotics and their potency tostimulate the teleost immunity both under in vivo and in vitroconditions is noteworthy [42e79]. Perusal of available literaturesindicates that several probiotics either individually or in combina-tion can enhance both systemic as well as local immunity in fish.The review is therefore, aiming to highlight the immunomodulatoryactivity of probiotics and also to evaluate the factors that regulatefor the optimum induction of immune responses in piscine system.

4. Effect of probiotics on systemic immunity

Studies on human and animal models provide a baselineunderstanding of the degree and type of immune responses

induced by different probiotics [80]. Unlike other animals, pro-biotics also modulate various immunohaematological parametersin teleosts and is presented in Table 1. Probiotics interact with theimmune cells such as mononuclear phagocytic cells (monocytes,macrophages) and polymorphonuclear leucocytes (neutrophils)and NK cells to enhance innate immune responses. Like highervertebrates, certain probiotics can enhance the number of eryth-rocytes, granulocytes, macrophages and lymphocytes in differentfish [53,57]. Similarly, probiotics, in both in vitro and in vivoconditions, actively stimulate the proliferation of B lymphocytes infish. Elevation of immunoglobulin level by probiotics supplemen-tation is reported in many animals including fish [27,58,63].Furthermore, Song et al. [75] recorded high immunoglobulin levelin skin mucusa of Miichthys miiuy by Clostridium butyricum.Different Lactic acid bacteria (LAB) group of probiotics either inviable or non-viable form can elevate immunoglobulin level in fish[64] and even one week supplementation of probiotioc like Lacto-bacillus rhamnosus@2.8� 108 CFU/g feed was found to significantlyincrease the immunoglobulin level in rainbow trout (Oncorhynchusmykiss) [60]. However, Balczar et al. [44] only found rise inimmunoglobulin level in Salmo trutta but not at significant level byfeeding LAB groups of probiotics namely Lactococcus lactis ssp.lactis, Lactobacillus sakei and Leuconostoc mesenteroides supple-mented @106 CFU/g feed for a period of 2 weeks.

4.1. Phagocytic activity

Phagocytic activity is responsible for early activation of theinflammatory response before antibody production and plays animportant role in antibacterial defenses. Probiotics can effectivelytrigger the pahgocytic cells in host and enhancement of phago-cytic activity by LAB group of probiotics such as L. rhamnosus,L. lactis and Lactobacillus acidophilus has already been observed inseveral animals [81]. These probiotics are often used in aqua-culture practices and supplementation of these probiotics eitherin viable or inactivated form is found to stimulate phagocyticactivity in several fish species [47,48,54,63,67]. In tilapia (Oreo-chromis niloticus) a 2 weeks feeding of L. rhamnosus significantlystimulated the phagocytic activity [68]. Likewise, oral adminis-tration of C. butyricum bacteria to O. mykiss has also beenreported to enhance the phagocytic activity of O. mykiss [69].However, probiotic like L. lactis failed to enhance the phagocyticactivity of head kidney macrophages of turbot (Scophthalmusmaximus) [77].

4.2. Respiratory burst activity

Respiratory burst activity is an important innate defensemechanism of fish. The findings of respiratory burst activityfollowing probiotics treatment in fish are often contradictory.While some studies indicate probiotics do not have significantimpact on this non-specific defense mechanism of fish [52,58,73],several in vitro and in vivo studies showed significant increase inrespiratory burst activity by various probiotics in many aquaticanimals including fish. Probiotics like Bacillus subtilis and certainmembers of LAB group can stimulate respiratory burst activity infish [60,70,71,79]. Nevertheless 5 � 107 CFU/ml heat inactivatedLactobacillus delbrueckii subsp. lactis and B. subtilis under in vitrocondition also found to enhance this activity of head kidney leu-cocytes of gilthead sea bream (Sparus aurata) [71].

4.3. Lysozyme

Lysozyme, one of the important bactericidal enzymes of innateimmunity is an indispensable tool of fish to fight against

Table 1Effect of different probiotics supplementation on various immune responses of fish.

Sl.no

Probiotics Form ofprobiotics

Mode ofProbioticssupplementation

Assayconditions

Immunological effects Reference

1 Bacillus subtilis,Lactobacillus acidophilus

Viable Individual andCombination

In vivo Respiratory burst activity ([), Serum bactericidal activity ([C),Neutrophil adherence ([), Lysozyme ([), Heamtocrit percentage ([Y)

[42]

2 Lactobacillus sakei,Lactococcus lactis,Leuconostoc mesenteroides

Viable Individual In vivo Respiratory burst activity against live A. salmonicida ([ only inL. lactis), Respiratory burst activity against dead A. salmonicida ([)

[43]

3 Lactobacillus sakei,Lactococcus lactis,Leuconostoc mesenteroides

Viable Individual In vivo Immunoglobulin ([Y), Lysozyme ([ L. lactis, L. mesenteroidesbut [Y for L. sakei), Complement activity ([)

[44]

4 Lactobacillus sakei,Lactococcus lactis,Leuconostoc mesenteroides

Viable Individual In vivo Lysozyme ([Y), Complement activity ([), Phagocytic activity ([),Respiratory burst activity ([ for all except L. sakei [Y)

[45]

5 Lactococcus lactis,Leuconostoc mesenteroides

Viable Individual In vivo Phagocytic activity ([) [46]

6 Aeromonas sobria Viable Individual In vivo Leucocytes ([), Phagocytic activity ([), Respiratory burst activity ([) [47]7 Bacillus species,

Aeromonas sobriaViable Individual In vivo Serum lysozyme ([ Bacillus species [Y A. sobria), Mucus lysozyme

([ Bacillus species, [Y A. sobria), Respiratory burst activity ([),Phagocytic activity ([), Anti-peroxidase ([Y),Leucocytes ([Y), Erythrocytes ([Y)

[48]

8 Pdp11, 51M6 Inactivated(Heat -Killed)

Individual andCombination

In vivo Cytotoxic ([ for 51M6 and C, [Y Pdp11), Respiratory burst activity ([Y),Phagocytic activity ([), Peroxidase activity of serum and head kidneyleucocytes ([Y), Complement activity ([Y)

[49]

9 Shewanella putrefaciens,Shewanella baltica

Inactivated Individual andCombination

In vivo Phagocytic activity ([), Respiratory burst activity ([Y), Complementactivity ([), Peroxidase activity ([), Cyotoxic activity ([ only in S. baltica)

[50]

10 Shewanella putrefaciens,Shewanella baltica

Viable Individual In vivo Respiratory burst activity ([) [51]

11 Shewanella putrefaciens,Shewanella baltica

Viable Individual In vivo Respiratory burst activity ([ S. putrefaciencs, [Y S. baltica) [52]

12 Vibrio fluvialis,Micrococcus luteus,Aeromonas hydrophila,Carnobacterium species

Viable Individual andCombination

In vivo Erythrocytes ([), Macrophages ([), lymphocytes ([), leucocytes ([),lysozyme activity ([)

[53]

13 Gram þve coccus, V. fluvialis,Aeromonas hydrophila,Carnobacterium species

Inactivated Individual In vivo Erythrocytes ([Y), Macrophages ([), leucocytes ([), Lysozyme ([Y),Phagoctic activity ([)

[54]

14 Carnobacterium maltaromaticum,Carnobacterium divergens

Viable Individual In vivo Respiratory burst activity ([Y), Lysozyme ([ Serum and Mucus),Phagocytic activity ([)

[55]

15 Carnobacterium maltaromaticum,Carnobacterium divergens

Viable Individual In vitro IL-8 ([Y) and TGF b ([Y) of gut cells, In head kidney leucocytes:TNF a ([), TCR b ([), IL1b ([), CD8 ([Y), CD4 ([Y),IL8 (Y), TGF b (Y)

[56]

16 Bacillus subtilis Viable Individual In vivo Respiratory burst activity ([), Serum bactericidal activity ([) [57]17 Bacillus subtilis Viable Individual In vivo Immunoglobulin ([), Lysozyme ([), Respiratory burst activity ([Y),

Specific antibody titre against E. tarda ([), Leucocytes ([)[58]

18 Bacillus subtilis Viable Individual In vivo Gut mucus and serum lysozyme ([), Respiratory burst activity ([),Phagocytic activity ([), Anti-Peroxidase ([), Leucocytes ([),Erythrocytes ([Y), Bactericidal activity ([), a1-anti-protease level ([),Peroxidase assay ([), Complement activity ([Y)

[59]

19 Lactobacillus rhamnosus Viable Individual In vivo Immunoglobulin ([), Respiratory burst activity ([),Complement activity ([)

[60]

20 Saccharomyces cerevisiae Viable Individual In vivo Phagocytic activity ([), Respiratory burst activity ([),Complement activity ([Y), Myeloperoxidase ([),

[61]

21 Clostridium butyricum Viable,Inactivated

Individual In vivo Lysozyme ([ gut mucusa and serum), Phagocytic activity ([),Immunoglobulin ([ gut mucusa and serum)

[62]

22 Lactobacillus rhamnosus Viable Individual In vivo Phagocytic activity ([), Respiratory burst activity ([Y),Complement activity ([)

[63]

23 Lactobacillus rhamnosus Viable Individual In vivo Immunoglobulin ([), Respiratory burst activity ([Y),Lysozyme ([Y), Complement activity ([),

[64]

Inactivated(Heat-killed)

Immunoglobulin ([), Respiratory burst activity ([Y),Lysozyme ([Y), Complement activity (Y)

24 Lactobacillus rhamnosus,Bacillus subtilis,Enterococcus faecium

Viable(Freeze dried)

Individual In vivo IL-1b1 ([ spleen and [Y head kidney by L. rhamnosus, [Y for spleen andhead kidney by E. faecium, [Y for spleen and head kidney by B. subtilis,),TNF 1 and 2 ([ head kidney and spleen by L. rhamnosus and E. faecium,[Y for B. subtilis), TGF-b ([Yspleen and head kidney by L. rhamnosus,[ for spleen and [Y head kidney by B. subtilis, [ for spleen and headkidney by E. faecium), Complement activity ([Y L. rhamnosus, [for B. subtilis, E. faecium), Respiratory burst activity([Y L. rhamnosus, B. subtilis and [ for E. faecium)

[65]

25 Lactobacillus delbrueckii Viable Individual In vivo(Throughartemia)

TCR b ([), Immunoglobulin ([Y),CD8 ([Y), CD4 ([Y), IL-1b (Y), IL-10 (Y), COX-2 (Y), TGF b (Y)

[66]

26 Aeromonas sobria,Brochothrix thermosphacta

Viable Individual In vivo Respiratory burst activity ([ B. thermosphacta, [Y for A. sobria),Complement activity ([Y), Phagocytic activity ([), Serum and epidermalmucus lysozyme ([Y), lecucocytes ([Y), lymphocytes ([Y),Pinocytic activity ([Y)

[67]

S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e144

Table 1 (continued )

Sl.no

Probiotics Form ofprobiotics

Mode ofProbioticssupplementation

Assayconditions

Immunological effects Reference

27 Lactobacillus rhamnosus Viable Individual In vivo Complement activity ([), Phagocytic activity ([) [68]28 Clostridium butyricum Viable Individual In vivo Leucocytes ([), Phagocytic activity ([), Respiratory burst activity ([) [69]29 Lactobacillus delbrueckii

ssp. lactis,Bacillus subtilis

Viable Individual andCombination

In vivo Phagocytic activity ([ I), Respiratory burst activity ([Y),Cyotoxic activity ([ C, [Y I), Peroxidase activity ofhead kidney leucocytes ([Y)

[70]

30 Lactobacillus delbrueckii,Bacillus subtilis, Pdp11, 51M6

Inactivated Individual In vitro Peroxidase activity of head kidney leucocytes ([Y), Respiratoryburst activity ([), Cyotoxic activity ([ but [Y Pdp11)

[71]

31 Lactobacillus delbrueckii,Bacillus subtilis

Inactivated(Heat-Killed)

Individual andCombination

In vivo Respiratory burst activity ([Y), Serum peroxidase ([YC, B. subtilis),Peroxidase activity of head kidney leucocytes ([Y), Complement ([C),Phagocytic activity ([C), Immunoglobulin ([C), Cytotoxic activity ([Y)

[72]

32 Kocuria species Viable Individual In vivo Lysozyme ([), Peroxidase ativity of head kidney macrophage ([),Respiratory burst activity ([Y), Phagocytic activity ([), Anti-proteaseactivity ([)

[73]

33 Lactobacillus plantarum Viable Individual In vivo Lysozyme ([), Phagocytic activity ([), Peroxidase activity ([),Complement activity ([), Superoxide dismutase (Y), Glutathioneperoxidase ([)

[74]

34 Clostridium butyricum Viable Individual In vivo Phenoloxidase activity ([), Acid phosphatases activity ([ whentreated @ 109 CFU/g), Lysozyme ([ for serum at 107 CFU/gand skin at 109 CFU/g), Immunoglobulin M ([ serum and skin mucusboth at 107 CFU/g)

[75]

35 Bacillus subtilis,Lactobacillus acidophilus,Clostridium butyricum,Saccharomyces cerevisiae(Commercial probioticspreparation)

Viable,Inactivated

Combination In vivo Neutrophils migration ([), Bactericidal activity ([), Lysozyme([ mucus and serum), Respiratory burst activity ([Y), Skinprotease activity ([)

[76]

36 Lactococcus lactis,Leuconostoc mesenteroides

Viable,Inactivated

Individual In vitro Nitric oxide ([), Phagocytic activity ([) [77]

37 Enterococcus faecium Viable Individual In vivo(Throughwater)

Lysozyme ([Y), Complement activity ([), Respiratory burstactivity ([), Myeloperoxidase activity ([)

[78]

38 Bacillus coagulans,Bacillus subtilis,Rhodopseudomonas palustris

Viable Individual In vivo(Throughwater)

Respiratory burst activity ([), Superoxide dismutase activity ([),Catalase ([), lysozyme ([Y), Total antioxidation competence ([Y),Myeloperoxidase activity ([ in B. coagulans, [Y in B. subtilis, R. palustris)

[79]

*[: Significantly high/up regulated; [Y: No change/high but not at significant level; C: Combination; I: Individual.

S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e14 5

infectious agents [82]. Probiotics either single or in combinationare found to trigger the lysozyme level in teleosts. Theenhancement of lysozyme level by probiotics like L. rhamnosus,Carnobacterium maltaromaticum, Carnobacterium divergens in O.mykiss [56,63], L. lactis ssp. lactis, L. mesenteroides and L. sakei inbrown trout (Salmo trutta) [44] is reported. Apart from serumlysozyme content, probiotics can also enhance the lysozyme levelin skin mucosa of fish [75,76]. Taoka et al. [76] reported signifi-cantly high lysozyme level in skin mucosa by supplementingcommercial probiotics through water in comparison to oralsupplementation in O. niloticus.

On contrary, dietary supplementation of probiotics like L. sakeiin S. trutta [44], L. sakei, L. lactis ssp. lactis, L. mesenteroides, andL. rhamnosus in O. mykiss [45,64], Aeromonas sobria in O. mykiss [48]as well as water supplementation of Bacillus coagulans, B. subtilisand Rhodopseudomonas palustris and Enterococcus faecium inO. niloticus [78,79] failed to elevate lysozyme level. Similarly, Peterset al. [67] failed to detect any specific change in serum and skinmucosa lysozyme level by feeding A. sobria @108 cells/g feedand B. thermosphacta @1010 cells/g feed in O. mykiss for a period of14 days.

4.4. Peroxidase and anti-protease activity

The peroxidase is an important enzyme that utilizes oxidativeradicals to produce hypochlorous acid to kill pathogens. Duringoxidative respiratory burst, it is mostly released by the azurophilicgranules of neutrophils. Dietary supplement of probiotic likeB. subtilis alone or in combinationwith L. delbrueckii ssp. lactis for 3weeks lead to high serum protease activity but failed to enhance

the peroxidase activity of head kidney leucocytes of S. aurata [72].Similarly, probiotics like E. faecium also elevated the serum perox-idase level in O. niloticus when supplemented through water@1 � 107 CFU/ml in every 4 days for 40 days [78]. On contrary tothese findings, probiotics like L. delbrueckii, B. subtilis, Bacillus JB-1,A. sobria, Shewanella putrefaciens (Pdp11) and 51M6 did not affectthe protease activity in fish like O. mykiss and S. aurata [48,49,71].

Similarly, anti-protease activities of serum and other body fluidsare mainly due to a1 and a2-antiprotease, and a2-macroglobulinand also responsible for preventing proteolytic pathogens [83,84].Though, these activities are normally high in fish and hardlymodulated even after immunization or infection [84], certain pro-biotics can successfully elevate this activity in fish [48,59,73].Sharifuzzaman and Austin [73] reported significantly high anti-protease activity in O. mykiss within 2 weeks of supplementationof probiotic belong to Kocuria species (z108 cells/g feed).

4.5. Complement activity

In teleosts, complement system plays a key role in adaptiveimmune responses and involved in chemotaxis, opsonization,phagocytosis and degradation of pathogens. Complement,a component of the non-specific immune response, may haveeffector mechanisms like direct killing of microorganisms by lysis[85]. Probiotics can enhance natural complement activity of fish[65,72] and dietary as well as water treatment of many probioticsare often reported to stimulate the piscine complement compo-nents [64,78]. It is also worth noting that non-viable probioticscan stimulate complement components in fish. Choi and Yoon[49] recorded an increased complement activity in O. mykiss

S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e146

from at 4th week of feeding the heat inactivated probiotics(Pdp11 or 51M6).

4.6. Cytokines

Cytokines are protein mediators produced by immune cells andcontribute to cell growth, differentiation and defense mechanismsof the host [86]. Perusal of available literatures indicate thata number of probiotics can effectively modulate the production ofpro-inflammatory cytokines such as interleukin-1 (IL-1), IL-6, IL-12, tumor necrosis factor a (TNF-a), and gamma interferon (IFN-g)and anti-inflammatory cytokines such as IL-10 and transforminggrowth factor b (TGF-b) in many animals [87e89]. Probiotics likeBifidobacterium longum, L. acidophilus, L. lactis, Lactobacillus para-cascei and Lactobacillus plantarum can up regulate the expressionof various types of cytokines in various hosts [81,90]. Differentstrains of LAB can induce regulatory and pro-inflamatory cyto-kines while others probiotics can increase intestinal inflammatoryresponses [91].

Probiotics like L. rhamnosus, E. faecium and B. subtilis are foundto up regulate the pro-inflammatory cytokines like IL-1b1 and TGF-b in the spleen and head kidney of O. mykiss [65]. Similarly, theexpression of IL-1b, IL-8, TNF-a, and TGF-b in head kidney of O.mykiss by C. maltaromaticum and C. divergens indicates theirpossible involvement in anti-inflammatory responses as well [56].On the other hand, Picchietti et al. [66] recorded down-regulationof Cyclooxygenase 2 (Cox-2) transcripts along with TGF-b and IL-10 genes by L. delbrueckii supplemented through live carrier inDicentrarchus labrax. COX-2 promotes intestinal wound healing butits chronic over expression can lead to inflammatory diseases [92].Therefore, a balanced expression of COX-2 is essential for main-taining the intestinal homeostasis and modulation of the inflam-matory gene Cox-2 could be a keymechanism of anti-inflammatoryaction of certain probiotics [66].

5. Effect of probiotics on gut immunity

The gut is the organwhere probiotics not only establish but alsoexecute their functions including immunostimulaory activity.Therefore, the cross talk between probiotics, epithelial cells and gutimmune system warrants high consideration. The immune systemof the gut is referred to as gut associated lymphoid tissue (GALT)and the piscine gut immune system is quite different frommammals. Unlikemammals, fish lack Peyer's patches, secretory Ig Aand antigen-transporting M cells in the gut [93]. However, manydiffusely organized lymphoid cells, macrophages, granulocytes andmucus IgM found in the intestine of fish constitutes the immunefunction [94e99].

The interaction of non-commensal and probiotics with gutimmune system of host is well documented in higher animals[100]. It is believed that probiotics and/or their components/products interact with GALT to induce immune response. Thewhole bacteria can't be introduced through the epithelial cells andthat only the antigenic particles or degraded products of thebacteria are able to make contact with immune cells [101]. Theaugmentation of the immune response by probiotic bacteria,a phenomenon similar to that of cholera toxin, may also occur inadherence with GALT and may therefore directly affect immunecells like leukocytes [102]. Fish possess strong antigen uptakecapacity in the second gut segment and the uptake and transport ofantigens followed by their processing by intraepithelial macro-phages is also reported in carp [103,104].

The effect of probiotics in stimulating the systemic immuneresponses are nowwell documented in several fish species but thatof local gut immunity is lacking. Limited attempts due to lack of

suitable tools, are made to access the gut immune responsefollowing probiotics treatment. Few studies that were conducted inrecent times indicate that probiotics can stimulate the piscine gutimmune system with marked increase in the number of Igþ cellsand acidophilic granulocytes (AGs) [66,72,105,106]. Probioticssupplementation at early developmental stages can be helpful inincreasing specific AGs subpopulations [105]. The presence ofT-cells in the GALT has been documented in many fish [97,107,108]and probiotics can lead to a significant increase in T-cells in fish. Ina study, Picchietti et al. [66] recorded increased T lymphocytes ingut without any change in CD4 and CD8a transcript in sea bass(D. labrax) by L. delbrueckii ssp. delbrueckii supplemented throughlive carriers like artemia and rotifers.

Apart from this, enhancement of gut mucosal lysozyme byC. maltaromaticum and C. divergens [55] and phagocytic activity ofmucosal leucocytes by LAB group of probiotics such as L. lactis ssp.lactis, L. mesenteroides and L. sakei [43] are also reported in fish likeO. mykiss.

6. Probiotics and gnotobiotic approaches

The detailed mode of action of probiotics has not yet beenestablished in any animals. It is often difficult to derive consensuson a particular pattern of stimulation. Hence gnotobiotic approchcan be instrumental in understanding the basic mechanism ofprobiotic action [109]. Gnotobiotic studies in different animalmodels indicate the effect of different probiotics on the composi-tion and functioning of reconstituted gut microbiota, difference intheir modes of action as well as involvement in both local andsystemic immune responses of host [110e112]. Involvement ofprobiotics in up-regulating the gene expression of cryptdins andmatrilysin, the first line of defense mechanism and in lipidabsorption and metabolism, such as intestinal fatty acid-bindingprotein in higher vertebrates are already recorded [111,113e115].

The mono-bacterial association studies in gnotobiotic fish alsoindicate the microbial up-regulation of serum amyloid A1, C-reac-tive protein, complement component 3, angiogenin 4, glutathioneperoxidase, myeloperoxidase as well as glycoprotein production[116e119]. However, these studies are not exclusively conductedwith probiotic strains but studies involving probiotics in gnotobi-otic fish will certainly able to address the inter- and intra-speciesdifference among different probiotics as well as well can unlockseveral aspects of their role in piscine immune system at molecularlevel.

7. Factors affecting the immunomodulatingpotency of probiotics

Modulation of host immunity is one of the most purportedbenefits of probiotics consumption [120] and fish is no exception.However, the mechanisms by which probiotics affect the immunesystem of host are unknown [39,121]. While factors such as adhe-sion properties, attachment site, stress factors, diet and environ-mental conditions determine the colonization of probiotics in thegut of host [122], probiotics often exert host specific [123] andstrain specific differences in their modes of action [124]. Never-theless the origin and source of probiotics [73], viability [125], dose[126] and duration of supplementation [127] can regulate theiractivities. There is no doubt that probiotics can stimulate piscineimmune system like other animals but inappropriate dose and/orduration of probiotics supplementation can cause undesirableresults [127]. Therefore, the type of probiotics, dose kinetics, andmethod of administration with respect to fish are critical factorsthat can regulate immune responses in fish.

S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e14 7

7.1. Types of strain

The dominant group of probiotics that are used in fish culturebelong to Gram þve especially LAB, Bacillus (B. subtilis, B. lichen-iformis, B. circulans) and bifidobacteria groups. On the other handcertain strains of Aeromonas (Aeromonas hydrophila, A. sobria),Vibrio (Vibrio fluvialis), Pseudomonas and Enterobacteria species arethe Gram �ve probiotics [128]. All these bacteria differ greatly intheir mode of action including the ability to trigger immune systemand therefore every probiotics differ from each other by theirfunctional role. It is recognized that each strain has unique prop-erties and the probiotic effects of a specific strain must not beextrapolated to other strains [129,130]. Gnotobiotic studies alsoindicate the strain specific differences among probiotics in stimu-lating immune system in animals [111,115].

Previous studies in piscine system also documented inter- andintra-species differences in immunostimulating ability of differentprobiotics [52,65,67,70]. Such type of difference is also evident bythe difference in triggering respiratory burst activity, by closelyrelated species like S. putrefaciens and Shewanella baltica in Sene-galese sole (Solea senegalensis) [52] even at a proven beneficiaryeffective dose of the same probiotics in S. aurata and S. senegalensis[50,51]. Similarly, variation in stimulating cellular innate immuneresponses under in vitro and in vivo conditions among probioticsbelong to 51M6, L. delbrueckii subsp. lactis and B. subtilis group arealso recorded [50,54,70,71]. Recently, Peters et al. [67] not onlyrecorded difference in immunostimulating activity but also indisease protecting ability among two established probiotics namelyA. sobria and B. thermosphacta in O. mykiss.

7.1.1. Indigenous vs exogenousSelection of probiotics is very critical because inappropriate

microorganisms can lead to undesirable effects in host [131]. Anideal probiotic, irrespective of its source should able to colonize,establish and multiply in the host gut. Most of the commercialprobiotics used for terrestrial animals are now being used inaquaculture practices. Although, these probiotics are exogenous,their success in aquaculture practices can't be overlooked.However, sometimes commercially available probiotics are rela-tively ineffective because of non-fish origin they are unable tosurvive and/or remain viable at optimum concentration in gut[18,132,133].

On the other hand probiotics from the same species and/or itsnatural environment could be best approach for better efficacy inhost [134]. The strategy of isolating probiotics from the gut ofmature animals and then use in immature animals of the samespecies has been successfully applied in fish [17,23,24,135]. There isa general consensus that probiotics from autochthonous sourcehave a greater chance of competing with resident microbes and ofbecoming predominant within a short period of intake and topersist in the colonic environment for some time after the with-drawal of probiotics [136,137]. For instance, Carnevali et al. [137],recorded a significantly decreased larvae and fry mortality by usingLactobacillus fructivorans, isolated from gut of S. aurata. Further-more, it is assumed that host immune cells do not react withbacteria that are naturally occurring on their surfaces and autoch-thonous in nature [71].

7.1.2. Monospecies vs multispeciesA wide range of probiotics, containing either monospecies or

multispecies of microorganisms are commercially available. Inrecent times a number of studies have confirmed the beneficialeffects of both forms of probiotics under in vitro and in vivoconditions. However, it is postulated that multispecies/multistrainprobiotics aremore effective and consistent than theirmonospecific

counter parts since mixed cultures may exert synergistic probioticproperties [138]. Many times induction of greater systemic innateimmunity has been recorded by using multispecies probiotics infish [42,53,71]. Aly et al. [42] reported significantly high respiratoryburst activity and lysozyme level inO. niloticus fedwith amixture ofB. subtilis and L. acidophilus. Besides systemic effects, multispeciesformulation of probiotics was the most effective in triggering thelocal gut immunity [72,105]. Salinas et al. [72] recorded thatB. subtilis and L. delbrueckii subsp. lactis in combination can increasethe numbers of IgMþ cells and AGs in the intestinal mucosa ofS. aurata juvenile within 3 weeks of supplementation whilst indi-vidually both the probiotic strains failed to induce any change.

However, different probiotics when supplemented in combinedform should complement each other and acquire different nicheswithin the gut microflora environment for executing desirableimmune stimulatory and other beneficial effects in host [70].Nevertheless, the probiotic sources and their relatedness can alsoaffect the synergistic effects in combined form i.e., multispeciescontaining different species may be more effective as compared tomultistrain probiotics [49]. For example, probiotics like Pdp11 and51M6 which belong to Vibrionaceae family showed no synergisticimmunostimulatory activity in combined form as compared toindividual treatment in O. mykiss [49] but other probiotics belong todifferent families such as Lactobacillus and Bacillus species arefound to complement each other by exerting synergistic immu-nomodulating responses in fish [70].

7.1.3. Spore former vs non-spore formerBacteria belong to both spore former and non-spore formers are

used as probiotics. Several spore forming bacteria which producea wide range of antagonistic compounds can be valuable as pro-biotics [139]. Among spore formers, Bacillus spores are routinelybeing used as probiotics in human and animal practices due to theirimmunostimulatory properties [140,141]. In aquaculture, B. subtilisand B. licheniformis are most commonly used probiotics [139]. InO. mykiss, Raida et al. [143] reported immunity enhancement andsignificant protection against yersioniosis by using commercialprobiotics containing B. subtilis and B. licheniformis spores. Bacillusspores have been shown to increase the survival and production ofchannel catfish [144]. Similarly, B. subtilis spores when introducedinto rearing water eliminated Vibrio species from the larvae ofsnook [145]. The spores of Bacillus toyoi and other Bacillus specieswhen used as feed additive increased the growth of S. maximus[146,147] and common snook (Centropomus undecimalis) [53].

Spore formers possess additional advantage that they can resistadverse environmental conditions. The long term advantages ofusing spores as probiotics is that they are heat-stable and cansurvive transit across the stomach barrier, properties that cannot beassured with other probiotics that are given in the vegetative form[142]. However, the majority of probiotics currently available arebacteria which are non-spore formers i.e., they are given as vege-tative cells (usually as lyophilized preparations) and several non-spore probiotics like LAB group of bacteria exhibit very promisingimmunostimulatory results. Nevertheless, the combination of bothspore former and non- spore former are also found to increaseimmunity in fish [70,72,76].

7.1.4. Viable vs non-viableProbiotics, as per definition, are viable microorganisms with

documented beneficial effects on the overall health status of host.Viability is an important property of any probiotics which helpthem to adhere and subsequent colonization in the intestinal tractof host [148]. However, certain probiotics in inactivated form canpotentially elicit similar effects in host compared to viableprobiotics. Furthermore, several bacteria in non-viable form are

S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e148

found not only to adhere to tissue culture cells of animals [149,150]but also to augment the systemic and mucosal immune responsesin host [151]. Therefore, the concept of incorporating inactivatedprobiotics has surfaced for aquaculture practices especially due tothe fact that probiotics are usually found in a transient state andoften expelled out immediately after the withdrawal of the feed inhydrobionts [70].

Different probiotics in inactivated form also exhibited promisingimmunomodulatory and protection in various fish species.Although, immunostimulating potency of inactivated probioticsunder in vitro [71] and in vivo conditions [64] as well as ability tocontrol diseases [54] has been documented, viable probiotics areproved to be better stimulator of immune system in any animalsincluding fish [76,101,152,153]. In a comparative study, Panigrahiet al. [64] reported thatprobiotic strainof L. rhamnosus inviable formis a better immune inducer compared to its heat inactivated form.

The immunomodulating activity of non-viable probionts couldpossibly be attributed to the presence of certain conserved micro-bial components such as capsular polysaccharides, peptidoglycansand lipoteichoic acids which are the potent stimulator of piscineimmune system [153,154]. Therefore, the whole and/or certaincomponents of the inactivated probiotics are believed to interactwith the epithelial cells of the gut and ultimately leading to anenhanced immune response in host.

7.2. Dose of probiotics

Dose of probiotics could be limiting factor for achievingoptimum beneficial effects in any host [155,156]. The optimumconcentration of probiotics is not only required for establishmentand subsequent proliferation in gut but also need to exert variousbeneficial effects including immunostimulatory activity. Differentin vitro and in vivo studies indicate that immune response of fishvaries with the concentration of probiotics. The dose of probiotics isusually selected based on their ability to enhance the growth andprotection in host. For instance, Brunt et al. [48] determined theeffective dose of the probiotic strain belong to Bacillus species to be2 � 108 cells at which they have recorded least percentagemortality in O. mykiss during challenge study. The in vitro stimu-latory activities of probiotics like Pdp11, 51M6, L. delbrueckii subsp.lactis and B. subtilis are found to be dose dependent [71]. Similarly,immunostimulatory activities of LAB and B. subtilis in fish under invivo condition also vary in a dose dependant manner [57,64].

In aquaculture the dose of probiotics usually varies from106e10 CFU/g feed. The optimum dose of a probiotics can vary withrespect to host and also type of immune parameters. Panigrahi et al.[63] recorded high serum lysozyme, phagocytic activity of headkidney leucocyte and complement activities in O. mykiss fed for 30days with L. rhamnosus strain at 1011 CFU/g feed but not at a dose of109 CFU/g feed. Furthermore, stimulation of a particular immuneresponse with respect to different tissue/organ also varies withdose. For instance, elevation of lysozyme activity in serum and skininM. miiuy is reported at two different doses i.e., 107 and 109 CFU ofC. butyricum/g feed, respectively [75]. On the other hand Son et al.[74], found best dose of probiotic for grouper (Epinephelus coioides)to be 108 CFU/kg of feed compared to 106 and 1010 CFU/kg ofL. plantarum in terms of growth, immune enhancement andprotection. Therefore, lower dose can fail to stimulate the piscineimmune systemwhile high dose can exert deleterious effects [157].In another study, Son et al. [75] found higher dose (i. e. 1010 CFU/kg feed) of L. platarum failed to protect fish on challenge studydespite enhancement of certain immune parameters at theparticular dose. Earlier, Nikoskelainen et al. [157] also recordedhigher percentage of mortality in O. mykiss fed at high dose ofL. rhamnosus (1012 CFU/g feed) compared to lower dose (109 CFU/g

feed). Therefore, the dose of the individual probiotics needs to bedetermined for a particular host.

7.3. Duration of feeding

Duration of the probiotics feeding is another important factorthat can affect the establishment, persistence and subsequentinduction of immune responses in a host. In fish most of thebeneficial effects like live weight gain, improved immunity anddisease resistance have been recorded within a dietary probioticsfeeding regime of 1e10 weeks. The time course for optimuminduction of immune response differs with respect to probioticstrain and also type of immune parameter.

The time course of probiotics feeding for stimulating innateimmunity can also vary among different strains of probiotics in thesame family [49]. Similarly difference in stimulating specificimmune parameter is also dependent on feeding duration. Forexample Diaz-Rosales et al.[52] observed significant enhancementof respiratory burst activity by feeding probiotics for 60 days butearlier Dıaz-Rosales et al. [50], failed to detect significantenhancement of this activity when fed the heat inactivated form ofthe same probiotics for 4 weeks.

However, several probiotics are often found to stimulate thepiscine immune system within 2 weeks of supplementation. Shar-ifuzzaman and Austin [73] recorded highest cellular and humoralimmunity at 2 weeks of feeding regime and further supplementa-tion lead to lowering at 3rd and 4th weeks of feeding. While someresearchers believe a long feeding regime is not necessary for pro-biotics [49], the shorter feeding regime can cause sharp decline inimmune response in fish [64]. Such type of decline may be due thefailure of the probiotic strains to establish and multiply in the fishgut. Though, a long dietary feeding regime is advantageous to hostin many aspects, more studies are required to establish the benefi-cial effect of short term regime is not just adjuvant effects.

7.4. Mode of supplementation

Although probiotics are used as dietary supplements, Moriarty[19] proposed to extend the definition of probiotics in aquacultureto microbial ‘‘water additives’’ and several probiotics are alsodirectly used as water additives with documented health andenvironmental benefits [79]. In fish, probiotics are applied indifferent methods like bath, suspension and feed. Howeversupplementation of probiotics as feed additive is best method forsuccessful colonization and establishment in gut [19,23,158,159].Oral administration of probiotics is more effective in enhancingimmunity as well as subsequent protection as compared to watersupplementation [76]. Likewise suspension or bioencapsulation ofprobiotics is usually adopted for fish larvae [146,159e163]. Pro-biotics like L. delbrueckii spp. delbrueckii when supplementedthrough live carriers like rotifers and artemia succeeded in stimu-lating local immunity in larvae [66,105].

Apart from dietary supplementation, water borne uptake ofprobiotics can also modulate the piscine immune system withelevation of several immune parameters [76,78,79]. In a study Zhouet al. [79] found that among three probiotics (B. subtilis, B. coagulans,R. palustris) supplemented into water @1 � 107 CFU/ml in every2 days for 40 days, B. coagulans and R. palustris, showed promisingresult with improved growth, immunity and health status ofO. niloticus.

7.5. Environmental conditions

The effectiveness of probiotics is dependent on the successfulestablishment of the probiotics in the gut. Several factors that

Table 2Effect of different probiotics supplementation on disease resistance of fishes.

Sl. no Probiotics Fish Dose (CFU/g) Duration (Weeks) Pathogen (s) challenged Reference

1 V. alginolyticus S. salar e e Aeromonas salmonicida,Vibrio anguillarum, Vibrio ordalii

[21]

2 C. divergens G. morhua 2 � 109 3 V. anguillarum [23]3 P. fluorescens S. salar 105e6 cells/ml 3e5 days A. salmonicida (No specific protection

on co-habitant challenge)[24]

4 Pseudomonas,Micrococcus, Vibrio species

S. aurata 108 15 days Vibrio harveyi [29]

5 B. subtilis,L. acidophilus

O. niloticus 107 2 Aeromonas hydrophila, Pseudomonas fluorescens,Streptococcus iniae

[42]

6 L. sakei,L. lactis,L. mesenteroides

O. mykiss 106 2 A. salmonicida ssp. salmonicida [45]

7 L. lactis,L. mesenteroides

S. trutta 106 30 days A. salmonicida [46]

8 A. sobria O. mykiss 5 � 107 2 Lactococcus garvieae,S. iniae

[47]

9 Bacillus species,A. sobria

O. mykiss 2 � 108 2 A. salmonicida, S. iniae, V. ordalii, L. garvieae,V. anguillarum,Yersinia ruckeri

[48]

10 L. rhamnosus O. niloticus 108 and 1010 2 Edwardsiella tarda [49]11 S. putrefaciens,

S. balticaS. senegalensis 109 60 days Photobacterium damselae subsp. piscicida [52]

12 Vibrio fluviales,A. hydrophila,M. luteusCarnobacterium species

O. mykiss 106e108 7 and 14 days A. salmonicida [53]

13 Vibrio fluviales,A. hydrophila,Gram þve coccusCarnobacterium species

O. mykiss 107 14 days A. salmonicida [54]

14 C. maltaromaticum,C. divergens

O. mykiss 107 2 A. salmonicida,Y. ruckeri

[55]

15 B. subtilis L. rohita 1 � 107a 15 days A. hydrophila [57]16 B. subtilis L. rohita 108 60 days E. tarda [58]17 C. butyricum M. miiuy 108 30 days V. anguillarum [62]18 A. sobria O. mykiss 108 cells for A. sobria 2 A. bestiarum, Ichthyophthirius multifiliis (Ich)

A. sobria (More effective against Ich)[67]

B. thermosphacta 1010 for B. thermosphacta19 C. butyricum O. mykiss 300 mg/kg fish 3 days V. anguillarum [69]20 Kocuria species O. mykiss 108 4 V. anguillarum [73]21 L. plantarum Epinephelus coioides 105a 4 Streptococcus species, Iridovirus of E. coioides [74]22 Commercial probiotics O. niloticus 1% 30a E. tarda [75]23 Commercial probiotics Carassius auratus,

Xiphophorus helleri5 g/kg 30 days P. fluorescens

(No significant protection)[132]

24 B. subtilis,B. licheniformis

O. mykiss 4 � 104 spores 42 days Y. ruckeri [143]

25 L. rhamnosus O. mykiss 109a 51 days A. salmonicida [157]26 Carnobacterium species S. salar,

O. mykiss5 � 107 2a A. salmonicida,

V. ordalii,Y. ruckeri,V. anguillarum,(No specific protection againstV. anguillarum)

[159]

27 Commercial probiotics(E. faecium, B. toyoi)

Anguilla anguilla 1 g/kg 2 E. tarda [167]

28 L. plantarumL. mesenteroides

O. mykiss 107a 4 L. garvieae [168]

29 S. putrefaciens S. aurata 108 15 days L. anguillarum [169]30 Roseobacter species S. maximus 107 CFU/ml e V. anguillarum,

Vibrio splendidus,Pseudoalteromonas sp.

[170]

31 Lactic Acid Bacteria O. mossambicus 106 25 days A. hydrophila [173]32 L. plantarum S. salar 2.5 � 109 5 A. slmonicida (No specific protection) [174]33 C. divergens G. morhua 108 3 V. anguillarum (No specific protection) [175]

a Experiment was conducted at different concentration/days but optimum result was recorded in that particular concentration/days of sampling.

S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e14 9

influence the establishment and stability of probiotics and subse-quent action include water quality, hardness, dissolved oxygen,temperature, pH, osmotic pressure and mechanical friction [12].Apart from these, stress due to high stocking density can affect theperformance of the probiotics. Mehrim [13] conducted the effect ofprobiotics on the O. niloticus at different stocking density rangingfrom 10 to 60 fish/m3 and found best growth, haematolgical

parameters and economic efficacy of probiotics within a stockingdensity of 30 fish/m3. Similarly, in laying hens improved immuneresponse and other beneficial effects by using probiotics duringstress condition due to high temperature was also recorded [164].Likewise probiotics can help to overcome stress due to salinity asreported by slightly enhanced salinity tolerance of O. niloticus bycommercial probiotics [76].

S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e1410

However, in aquaculture it is a neglected aspect and nosystematic attempt has been made to correlate effect of probioticson the immunity of fish at various environmental conditions.Temperature could be crucial since a probiotic would be mosteffective when used in its optimum temperature range matchesthat of fish which is identical with surrounding environment [65].Panighagi et al. [65] found better immunoefficacy of E. faecium incomparison to L. rhamnosus and B. subtilis due to its mesophilic andmore psychrotolerant nature.

8. Probiotics and disease protection

Probiotic therapy offers a suitable alternative for controllingpathogens thereby overcoming the adverse consequences of anti-biotics and chemotherapeutic agents. In fish culture, probioticseither in diet or bioencapsulation help in achieving natural resis-tance and high survivability of larvae and post larvae of fishes[159,165]. Significant increase in the mean weight and naturalsurvival rate of larvae of S. maximus fed rotifers enriched in LAB aswell as high protection against a pathogenic Vibrio species wasrecorded [147]. Probiotic like Pediococcus acidilactici is also found tobe effective against vertebral column compression syndrome inO. mykiss [166].

Furthermore, the effectiveness of probiotics in terms ofprotection against infectious pathogens is often attributed to theelevate immunity. Protection against edwadsiellosis [58,76,167],enteric red mouth disease [55,143], furunculosis [54,60,157], lac-tococossi [47,168], streptococcosis [47], and several other diseases[73,169e173] are successfully accomplished through probioticsfeeding (Table 2). Furthermore, probiotics treatment leads tobetter protection of fish from multiple diseases [42,48,159]. Apartfrom protection against bacterial pathogens, probiotics can protectagainst viral and protozoan infections as well. Recently, successfulcontrol of Ichthyophthiriasis (Ichthyophthirius multifiliis, Ich) byA. sobria in O. mykiss [67] and iridovirus of grouper E. coioides byL. plantarum [74] is achieved.

Despite, several reports also indicate deleterious effects likereduced growth, non stimulation of immune responses and nosignificant protection by various probiotics in fish [24,165,174e177].Therefore, detailed information of the probiotics and their mode ofaction can enable the selection of effective strain with a morecredible scientific rationale [8].

9. Conclusions

The beneficial effects of dietary supplements like probiotics,prebiotics and synbiotics have been recorded in a wide range ofanimal models including fish. Amongst probiotics concept hasalready been established in aquaculture practices especially asa promising alternative to chemicals and antibiotics [178,179]. Overthe years several candidate probiotics strains belonging to Gramþve and Gram eve groups of bacteria are introduced into culturepractices. However, certain autochthonous probiotic strains belongto Aeromonas, Pseudomonas and Vibrio species could be of note-worthy interest for aquaculture practices as the chance of re-establishment of such probiotics in the gut of host is high.Although, careful selection of the probiotic strains can lead tospecies specific advantages [18,180], many times lack scientificcredibility. Undoubtly, most of the probiotics are usually safe buttheir safety is an important issue, especially in case of newlyintroduced candidate species [181] as well as the possibility ofacquisition of genes encoding the virulence and antimicrobial drugresistance traits from pathogens to probiotics through horizontaltransfer of genes [182,183]. Although, no such report has beendocumented in the aquaculture practices till date, the possibility of

such horizontal virulence gene transfer phenomenon from path-ogenic strains to probiotics can't be ruled out as all forms ofmicroorganisms viz., non-pathogens, pathogens and probioticsare co-existing in the intestinal tract of fish. Therefore, morefundamental research is needed not only to address inter- and intra-species differences among various probiotics but also to evaluatetheir safety aspects. Nevertheless, looking into the fact thatmost of the probiotics can exert immunomodulatory effect infish, a complete understanding of the interactions between gutmicrobes, the intestinal epithelium, and the gut immune system isalso necessary so that proper strategy can be developed for stimu-lating the local as well as systemic immunity throughmanipulationof gut microbiota with suitable probiotics/prebiotics/synbioticswithout altering the intestinal homeostasis.

Acknowledgement

The author is thankful to Professor T. Nakanishi, Laboratory ofFish Pathology, Department of Veterinary Medicine, NihonUniversity, Japan for his guidance in writing and modifying thearticle. The author is also thankful to Dr. S. C. Mukherjee, CentralInstitute of Fisheries Education, Mumbai, India for his help inpreparing the review article.

References

[1] Burr G, Gatlin III D, Ricke S. Microbial ecology of the gastrointestinal tract offish and the potential application of prebiotics and probiotics in finfishaquaculture. J World Aquacul Soc 2007;36(4):425e36.

[2] Cross ML. Microbes versus microbes: immune signals generated by probioticlactobacilli and their role in protection against microbial pathogens. FEMSImmunol Med Microbiol 2002;34:245e53.

[3] Parker RB. Probiotics, the other half of the antibiotic story. Anim Nutr Health1974;29:4e8.

[4] Gismondo MR, Drago L, Lombardi A. Review of probiotics available to modifygastrointestinal flora. Int J Antimicrob Agents 1999;12:287e92.

[5] Food and Agriculture Organization of the United Nations (FAO). 2001. Healthand Nutritional Properties of Probiotics in Food including Powder Milk withLive Lactic Acid Bacteria. In the Joint FAO/WHO Expert Consultation report onEvaluation of Health and Nutritional Properties of Probiotics in FoodIncluding Powder Milk with Live Lactic Acid Bacteria (October 2001).

[6] Ouwehand AC, Salminen S, Isolauri E. Probiotics: an overview of beneficialeffects. Antonie Van Leewenhoek 2002;82:279e89.

[7] O'Sullivan DJ. Screening of intestinal microflora for effective probioticbacteria. 1. Agric Food Chem 2001;49(4):751e60.

[8] Andersson H, Asp NG, Bruce A, Roos S, Wadstrom T, Wold A. Health effects ofprobiotics and prebiotics: a literature review on human studies. Scand J Nutr2001;45:58e75.

[9] Morelli L, Zonenschain D, Callegari ML, Grossi E, Maisano F, Fusillo M.Assessment of a new synbiotic preparation in healthy volunteers: survival,persistence of probiotic strains and its effect on the indigenous flora. Nutr J2003;2:11e6.

[10] Naylor RL, Goldburg RJ, Primavera J, Kautsky N, Beveridge MCM, Clay J, et al.Effects of aquaculture on world fish supplies. Issues Ecol 2001;8:1e12.

[11] Panigrahi A, Azad IS. Microbial intervention for better fish health in aqua-culture: the Indian scenario. Fish Physiol Biochem 2007;33:429e40.

[12] Das S, Ward LR, Burke C. Prospects of using marine Actinobacteria as pro-biotics in aquaculture. Appl Microbiol Biotechnol 2008;81:419e29.

[13] Mehrim AI. Effect of dietary supplementation of Biogen� (Commercial pro-biotic) on mono-sex Nile tilapia Oreochromis niloticus under differentstocking densities. J Fisher Aquat Sci 2009;4(6):261e73.

[14] Mishra S, Mohanty S, Pattnaik P, Ayyappan S. Probiotics: possible applicationin aquaculture. Fish Chimes 2001;21(1):31e7.

[15] Sahu MK, Swarnakumar NS, Sivakumar K, Thangaradjou T, Kannan L. Pro-biotics in aquaculture: importance and future perspectives. Ind J Microbiol;2008:1e10.

[16] Decamp O, Moriarty DJW. Probiotics as alternative to antimicrobials: limi-tations and potential. World Aquac 2006;37(4):60e2.

[17] Gatesoupe FJ. The use of probiotics in aquaculture. Aquaculture1999;180:147e65.

[18] Ghosh S, Sinha A, Sahu C. Isolation of putative probionts from the intestinesof Indian major carps. Israeli J Aquac e Bam 2007;59(3):127e32.

[19] Moriarty DJW. Control of luminous Vibrio sp in penaeid aquaculture ponds.Aquaculture 1998;164:357e8.

[20] Moriarty DJW. Disease control in shrimp aquaculture with probiotic bacteria.Proceeding of the 8th International Symposium on Microbial Ecology. In:Bell CR, Brylinsky M, Johnson-Green P, editors. Microbial biosynthesis: new

S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e14 11

frontiers. Halifax, Canada: Atlantic Canada Society for Microbial Ecology;1999. p. 7 [p].

[21] Austin B, Struckey LF, Robertson PAW, Effendi I, Griffith DRW. A probioticstrain of Vibrio alginolyticus effective in reducing disease caused by Aero-monas salmonicida, Vibrio anguillarum and Vibrio ordalii. J Fish Dis1995;18:93e6.

[22] Carnevali O, De Vivo L, Sulpizio R, Gioacchin G, Olivotto I, Silvi S, et al. Growthimprovement by probiotic European sea bass juveniles (Dicentrarchus labrax,L.), with particular attention to IGF-1, myostatin and cortisol gene expression.Aquaculture 2006;258:430e8.

[23] Gildberg A, Mikkelsen H, Sandaker E, Ringo E. Probiotic effect of lactic acidbacteria in the feed on growth and survival of fry of Atlantic cod (Gadusmorhua). Hydrobiologia 1997;352:279e85.

[24] Gram L, Melchiorsen J, Spangaard B, Huber I, Nielsen TF. Inhibition of Vibrioanguillarum by Pseudomonas fluorescens AH2, a possible probiotic treatmentof fish. Appl Environ Microbiol 1999;65:969e73.

[25] Smith P, Davey S. Evidence for the competitive exclusion of Aeromonas sal-monicida from fish with stress-inducible furunculosis by a fluorescentpseudomonad. J Fish Dis 1993;16:521e4.

[26] Boyd CE, Massaut L. Risks associated with the use of chemicals in pondaquaculture. Aquac Eng 1999;20:113e32.

[27] Al-Dohail MA, Hashim R, Aliyu-Paiko M. Effects of the probiotic, Lactobacillusacidophilus, on the growth performance, haematology parameters andimmunoglobulin concentration in African Catfish (Clarias gariepinus, Burchell1822) fingerling. Aquac Res 2009;40:1642e52.

[28] Saenz de Rodriguez MA, Diaz-Rosales P, Chabrillon M, Smidt H, Arijo S, Leon-Rubio JM, et al. Effect of dietary administration of probiotics on growth andintestine functionally of juvenile Senegalese sole (Solea senegalensis, Kaup1858). Aquac Nutr 2009;15:177e85.

[29] Chabrillon M, Rico RM, Balebona MC, Morinigo M. Adhesion to sole Soleasenegalensis Kaup, mucus of microorganisms isolated from farmed fish, andtheir interaction with Photobacterium damselae subsp. Piscicida. J Fish Dis2005;28:229e37.

[30] Vine NG, Leukes WD, Kaiser H, Daya S, Baxter J, Hecht T. Competition forattachment of aquaculture candidate probiotic and pathogenic bacteria onfish intestinal mucus. J Fish Dis 2004;27:319e26.

[31] El-Dakar AY, Shalaby SM, Saoud IP. Assessing the use of dietary probiotic/prebiotic as an enhancer of spinefoot rabbitfish Singanus rivulatus survivaland growth. Aquac Nutr 2007;13:407e12.

[32] Gibson LF. Bacteriocin activity and probiotic activity of Aeromonas media.J Appl Microbiol Symp Suppl 1999;85:243Se8S.

[33] Silva M, Jacobus NV, Deneke C, Gorbach SL. Antimicrobial substance froma human Lactobacillus strain. Antimicrob Agents Chemother 1987;8:1231e3.

[34] Sugita H, Hirose Y, Matsuo N, Deguchi Y. Production of the antibacterialsubstance by Bacillus sp. strain NM 12, an intestinal bacterium of Japanesecoastal fish. Aquaculture 1998;165:269e80.

[35] Yan I, Boyd KG, Burgess JG. Surface attachment induced production of anti-microbial compounds by marine epiphytic bacteria using modified rollerbottle cultivation. Mar Biotechnol 2002;4:356e66.

[36] Balcazar JL, de Blas I, Ruiz-Zarzuela I, Cunningham D, Vendrell D, Muzquiz JL.The role of probiotics in aquaculture. Vet Microbiol 2006;114:173e86.

[37] Khattab YAE, Shalaby AME, Sharaf SM, El-Marakby H, RizlAlla EH. Thephysiological changes and growth performance of the Nile tilapiaOreochromis niloticus after feeding with Biogen� as growth promoter. EgyptJ Aquat Biol Fish 2004;8(2):145e58.

[38] Fooks LJ, Fuller R, Gibson GR. Prebiotics, probiotics and human gut micro-biology. Int Dairy J 1999;9:53e61.

[39] Galdeano CM, Perdigon G. The probiotic bacterium Lactobacillus casei inducesactivation of the gut mucosal immune system through innate immunity. ClinVacc Immunol 2006;13:219e26.

[40] Herich R, Levkut M. Lactic acid bacteria, probiotics and immune system. VetMed e Czech 2002;47(6):169e80.

[41] Sartor RB. Therapeutic manipulation of the enteric microflora in inflamma-tory bowel diseases: antibiotics, probiotics and prebiotics. Gastroenterology2004;126:1620e33.

[42] Aly SM, Ahmed YAG, Ghareeb AAA, Mohamed MF. Studies on Bacillus subtilisand Lactobacillus acidophilus, as potential probiotics, on the immuneresponse and resistance of Tilapia nilotica (Oreochromis niloticus) to challengeinfections. Fish Shellfish Immunol 2008;25:128e36.

[43] Balcazar JL, Vendrell D, de Blas I, Ruiz-Zarzuela I, Girones O, Muzquiz JL.Immune modulation by probiotic strains: quantification of phagocytosis ofAeromonas salmonicida by leukocytes isolated from gut of rainbow trout(Oncorhynchus mykiss) using a radiolabelling assay. Comp Immunol Micro-biol Infect Dis 2006;29(5e6):335e43.

[44] Balcazar JL, de Blas I, Ruiz-Zarzuela I, Vendrell D, Calvo AC, Marquez I, et al.Changes in intestinal microbiota and humoral immune response followingprobiotic administration in brown trout (Salmo trutta). Br J Nutr 2007;97:522e7.

[45] Balcazar JL, de Blas I, Ruiz-Zazuela I, Vandrell D, Girones O, Muzquiz JL.Enhancement of the immune response and protection induced by probioticlactic acid bacteria against furunculosis in rainbow trout (Oncorhynchusmykiss). FEMS Immunol Med Microbiol 2007;51:185e93.

[46] Balcazar JL, Vendrell Dl, de Blas I, Ruiz-Zarzuela I, Muzquiz JL. Effect ofLactococcus lactis CLFP 100 and Leuconostoc mesenteroides CLFP 196 on

Aeromonas salmonicida infection in brown trout (Salmo trutta. J Mol Micro-biol Biotechnol 2009;17:153e7.

[47] Brunt J, Austin B. Use of a probiotic to control lactococcosis and strepto-coccosis in rainbow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis2005;28:693e701.

[48] Brunt J, Newaj-Fyzul A, Austin B. The development of probiotics for thecontrol of multiple bacterial diseases of rainbow trout, Oncorhynchus mykiss(Walbaum). J Fish Dis 2007;30:573e9.

[49] Choi SH, Yoon TJ. Non-specific immune response of rainbow trout (Onco-rhynchus Mykiss) by dietary heat-inactivated potential probiotics. ImmuneNetw 2008;8(3):67e74.

[50] Diaz-Rosales P, Salinas I, Rodriguez A, Cuesta A, Chabrillon M, Balebona MC,et al. Gilthead seabream (Sparus aurata L.) innate immune response afterdietary administration of heat-inactivated potential probiotics. Fish ShellfishImmunol 2006;20:482e92.

[51] Diaz-Rosales P, Rico RM, Arijo S, Chabrillon M, Balebona MC, deRodriguez MAS, et al. Effect of two probiotics on respiratory burst ofphagocytes from sole (Solea senegalensis, Kaup 1858). Aquaculture Europe2006. Florence, Italy: Linking Tradition and Technology. Highest Quality forthe Consumer; 2006.

[52] Diaz-Rosales P, Arijo S, Chabrillon M, Alarcon FJ, Tapia-Paniagua ST, Martinez-Manzanares E, et al. Effects of two closely related probiotics on respiratoryburst activity of Senegalese sole (Solea senegalensis, Kaup) phagocytes, andprotection against Photobacterium damselae subsp. piscicida. Aquaculture2009;293:16e21.

[53] Irianto A, Austin B. Use of probiotics to control furunculosis in rainbow trout,Oncorhynchus mykiss (Walbaum). J Fish Dis 2002;25:333e42.

[54] Irianto A, Austin B. Use of dead probiotic cells to control furunculosis inrainbow trout, Onchorhynchus mykiss (Walbaum). J Fish Dis 2003;26:59e62.

[55] Kim DH, Austin B. Cytokine expression in leucocytes and gut cells of rainbowtrout, Oncorhynchus mykiss Walbaum, induced by probiotics. Vet ImmunolImmunopathol 2006;114:297e304.

[56] Kim DH, Austin B. Innate immune responses in rainbow trout (Oncorhynchusmykiss, Walbaum) induced by probiotics. Fish Shellfish Immunol 2006;21:513e24.

[57] Kumar R, Mukherjee SC, Ranjan R, Nayak SK. Enhanced innate immuneparameters in Labeo rohita (Ham.) following oral administration of Bacillussubtilis. Fish Shellfish Immunol 2008;24:168e72.

[58] Nayak SK, Swain P, Mukherjee SC. Effect of dietary supplementation ofprobiotic and vitamin C on the immune response of Indian major carp, Labeorohita (Ham). Fish Shellfish Immunol 2007;23:892e6.

[59] Newaj-Fyzul A, Adesiyun AA, Mutani A, Ramsubhag A, Brunt J, Austin B.Bacillus subtilis AB1 controls Aeromonas infection in rainbow trout (Onco-rhynchus mykiss, Walbaum). J Appl Microbiol 2007;103:1699e706.

[60] Nikoskelainen S, Ouwehand AC, Bylund G, Salminen S, Lilius E. Immuneenhancement in rainbow trout (Oncorhynchus mykiss) by potential pro-biotic bacteria (Lactobacillus rhamnosus). Fish Shellfish Immunol 2003;15:443e52.

[61] Ortuno J, Cuesta A, Rodriguez A, Esteban MA, Meseguer J. Oral administrationof yeast Saccharomyces cereviceae enhances the cellular innate immuneresponse of gilthead seabream (Sparus aurata L.). Vet Immunol Immunopa-thol 2002;85:41e50.

[62] Pan X, Wu T, Song Z, Tang H, Zhao Z. Immune responses and enhanceddisease resistance in Chinese drum, Miichthys miiuy (Basilewsky), after oraladministration of live or dead cells of Clostridium butyricum CB2. J Fish Dis2008;31:679e86.

[63] Panigrahi A, Kiron V, Kobayashi T, Puangkaew J, Satoh S, Sugita H. Immuneresponses in rainbow trout Oncorhynchus mykiss induced by a potentialprobiotic bacteria Lactobacillus rhamnosus JCM 1136. Vet Immunol Immu-nopathol 2004;102:379e88.

[64] Panigrahi A, Kiron V, Puangkaew J, Kobayashi T, Satoh S, Sugita H. Theviability of probiotic bacteria as a factor influencing the immune response inrainbow trout Oncorhynchus mykiss. Aquaculture 2005;243:241e54.

[65] Panigrahi A, Kiron V, Satoh S, Hirono I, Kobayashi T, Sugita H, et al. Immunemodulation and expression of cytokine genes in rainbow trout Onco-rhynchus mykiss upon probiotic feeding. Dev Comp Immunol 2007;31:372e82.

[66] Picchietti S, Fausto AM, Randelli E, Carnevali O, Taddei AR, Buonocore F,et al. Early treatment with Lactobacillus delbrueckii strain induces anincrease in intestinal T-cells and granulocytes and modulates immune-related genes of larval Dicentrarchus labrax (L.). Fish Shellfish Immunol2009;26:368e76.

[67] Pieters N, Brunt J, Austin B, Lyndon AR. Efficacy of in-feed probiotics againstAeromonas bestiarum and Ichthyophthirius multifiliis skin infections inrainbow trout (Oncorhynchus mykiss, Walbaum). J Appl Microbiol 2008;105:723e32.

[68] Pirarat N, Kobayashi T, Katagiri T, Maita M, Endo M. Protective effects andmechanisms of a probiotic bacterium Lactobacillus rhamnosus againstexperimental Edwardsiella tarda infection in tilapia (Oreochromis niloticus).Vet Immunol Immunopathol 2006;113:339e47.

[69] Sakai M, Yoshida T, Astuta S, Kobayashi M. Enhancement of resistance tovibriosis in rainbow trout, Oncorhynchus mykiss (Walbaum) by oraladministration of Clostridium butyricum bacteria. J Fish Dis 1995;18:187e90.

S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e1412

[70] Salinas I, Cuesta A, Esteban MA, Meseguer J. Dietary administration ofLactobacillus delbrueckii and Bacillus subtilis, single or combined, on giltheadseabream cellular innate immune responses. Fish Shellfish Immunol2005;19:67e77.

[71] Salinas I, Diaz-Rosales P, Cuesta A, Meseguer J, Chabrillon M, Morinigo MA,et al. Effect of heat-inactivated fish and non-fish derived probiotics on theinnate immune parameters of a teleost fish (Sparus aurata L.). Vet ImmunolImmunopathol 2006;111:279e86.

[72] Salinas I, Abelli L, Bertoni F, Picchietti S, Roque A, Furones D, et al. Mono-species and multispecies probiotic formulations produce different systemicand local immunostimulatory effects in the gilthead seabream (Sparus aurataL.). Fish Shellfish Immunol 2008;25:114e23.

[73] Sharifuzzaman SM, Austin B. Influence of probiotic feeding duration ondisease resistance and immune parameters in rainbow trout. Fish ShellfishImmunol 2009;27:440e5.

[74] Son VM, Changa CC, Wu MC, Guu YK, Chiu CH, Cheng W. Dietary adminis-tration of the probiotic, Lactobacillus plantarum, enhanced the growth, innateimmune responses, and disease resistance of the grouper Epinepheluscoioides. Fish Shellfish Immunol 2009;26:691e8.

[75] Song Z, Wu T, Cai L, Zhang L, Zheng X. Effects of dietary supplementationwith Clostridium butyricumon the growth performance and humoralimmune response in Miichthys miiuy. J Zhejiang Univ Sci B 2006;7(7):596e602.

[76] Taoka Y, Maeda H, Jo JY, Kim SM, Park S, Yoshikawa T, et al. Use of live anddead probiotic cells in tilapia Oreochromis niloticus. Fisher Sci 2006;72:755e66.

[77] Villamil L, Tafalla C, Figueras A, Novoa B. Evaluation of immunomodulatoryeffects of lactic acid bacteria in turbot (Scophthalmus maximus). Clin DiagnLab Immunol 2002;9:1318e23.

[78] Wang YB, Tian ZQ, Yao JT, Li WF. Effect of probiotics, Enteroccus faecium, ontilapia (Oreochromis niloticus) growth performance and immune response.Aquaculture 2008;277:203e7.

[79] Zhou X, Tian Z, Wang Y, Li W. Effect of treatment with probiotics as wateradditives on tilapia (Oreochromis niloticus) growth performance and immuneresponse. Fish Physiol Biochem 2009;0920e1742:1573e5168 [OnlineAvailable].

[80] Mishra VK, Mohammad G, Jha A. Immunomodulation and anticancerpotentials of yogurt probiotic. EXCLI J 2008;7:177e84.

[81] Rutherfurd-Markwick KJ, Gill HS. Probiotics and immunomodulation. In:Hughes DA, Darlington LG, Bendich A, editors. Diet and human immunefunction. Totowa, NJ: Humana Press; 2004. p. 327e44.

[82] Lindsay GJH. The significance of chitinolytic enzymes and lysozyme inrainbow trout (Salmo gairdneri) defence. Aquaculture 1986;51:169e73.

[83] Ellis AE. Innate host defense mechanisms of fish against viruses and bacteria.Dev Comp Immunol 2001;25:827e39.

[84] Magnadottir B, Gudmundsdottir BK, Lange S, Steinarsson A, Oddgeirsson M,Bowden T, et al. Immunostimulation of larvae and juveniles of cod, Gadusmorhua L. J Fish Dis 2006;29(3):147e55.

[85] Ellis AE. Immunity to bacteria in fish. Fish Shellfish Immunol1999;9:291e308.

[86] Peddie S, Zou J, Secombes CJ. Immunostimulation in the rainbow trout(Oncorhynchus mykiss) following intraperitoneal administration of ergosan.Vet Immunopathol 2002;86:101e13.

[87] Christensen HR, Frokiaer H, Pestka JJ. Lactobacilli differentially modulateexpression of cytokines and maturation surface markers in murine dendriticcells. J Immunol 2002;168:171e8.

[88] Niers LEM, Timmerman HM, Rijkers GT, van Bleek GM, van Uden NOP,Knol EF, et al. Identification of strong interleukin-10 inducing lactic acidbacteria which down regulate T helper type 2 cytokines. Clin Exp Allergy2005;35:1481e9.

[89] von der Weid T, Bulliard C, Schiffrin EJ. Induction by a lactic acid bacterium ofa population of CD4þ T cells with low proliferative capacity that producetransforming growth factor b and interleukin-10. Clin Diagn Lab Immunol2001;8:695e701.

[90] Kato I, Tanaka K, Yokokura T. Lactic acid bacterium potently induces theproduction of interleukin-12 and interferon-gamma by mouse splenocytes.Int J Immunopharmacol 1999;21:121e31.

[91] Perdigon G, Maldonado Galdeano C, Valdez JC, Medici M. Interaction oflactic acid bacteria with the gut immune system. Eur J Clin Nutr 2002;56(S4):21e6.

[92] Nurmi JT, Puolakkainen PA, Rautonen NE. Bifidobacterium lactis sp. 420 up-regulates cyclooxygenase (Cox)-1 and down-regulates Cox-2 gene expres-sion in a Caco-2 cell culture model. Nutr Cancer 2005;51:83e92.

[93] Buddington RK, Krogdahl A, Bakke-Mckellep AM. The intestines of carnivo-rous fish: structure and functions and the relations with diet. Acta PhysiolScand Suppl 1997;638:67e80.

[94] Bakke-McKellep AM, Froystad MK, Lilleeng E, Dapra F, Refstie S, Krogdahl A,et al. Response to soy: T-cell-like reactivity in the intestine of Atlanticsalmon, Salmo salar L. J Fish Dis 2007;30:13e25.

[95] Inami M, Taverne-Thiele AJ, Schroder MB, Kiron V, Rombout JHWM. Immu-nological differences in intestine and rectum of Atlantic cod (Gadus morhuaL.). Fish Shellfish Immunol 2009;26:751e9.

[96] Joosten PHM, Kruijer WJ, Rombout JHWM. Anal immunisation of carp andrainbow trout with different fractions of a Vibrio anguillarum bacterin. FishShellfish Immunol 1996;6:541e51.

[97] Picchietti S, Terribili FR, Mastrolia L, Scapigliati G, Abelli L. Expression oflymphocyte antigenic determinants in developing GALT of the sea bassDicentrarchus labrax (L.). Anat Embryol 1997;196:457e63.

[98] Rombout JHWM, Taverne-Thiele AJ, Villena MI. The gut-associated lymphoidtissue (GALT) of carp (Cyprinus carpio L.): an immunocytochemical analysis.Dev Comp Immunol 1993;17:55e66.

[99] Uran PA, Aydin R, Schrama JW, Verreth JAJ, Rombout JHWM. Soybean mealinduced uptake block in the distal enterocytes of Atlantic salmon (SalmosalarL.). J Fish Biol 2008;73:2571e9.

[100] Perdigon G, Fuller R, Raya R. Lactic acid bacteria and their effect on theimmune system. Curr Issues Intest Microbiol 2001;2:27e42.

[101] Galdeano CM, Perdigon G. Role of viability of probiotic strains in theirpersistence in the gut and in mucosal immune stimulation. J Appl Microbiol2004;97:673e81.

[102] De Simone C, Salvadori BB, Negri R, Ferrazzi M, Baldinelli L, Vesely R. Theadjuvant effect of yogurt on production of g-interferon by Con A-stimulatedhuman peripheral blood lymphocytes. Nutr Rep Int 1986;33:419e33.

[103] Rombout JHWM, Van den Berg AA. Uptake and transport of ferritin in theepithelium of carp (Cyprinus carpio L.) and the possible immunologicalimplications. Cell Biol Int Rep 1985;9:516.

[104] Rombout JHWM, van der Berg AA. Immunological importance of the secondgut segment of carp. I. Uptake and processing of antigens by epithelial cellsand macrophages. J Fish Biol 1989;35:13e22.

[105] Picchietti S, Mazzini M, Taddei AR, Renna R, Fausto AM, Mulero V, et al.Effects of administration of probiotic strains on GALT of larval giltheadseabream: immunohistochemical and ultrastructural studies. Fish ShellfishImmunol 2007;22:57e67.

[106] Picchietti S, Guerra L, Selleri L, Buonocore F, Abelli L, Scapigliati G, et al.Compartmentalisation of T cells expressing CD8a and TCR b in developingthymus of sea bass Dicentrarchus labrax (L.). Dev Comp Immunol2008;32:92e9.

[107] Romano N, Rossi F, Abelli L, Caccia E, Piergentili R, Mastrolia L, et al. Majorityof TcRbþ T-lymphocytes located in thymus and midgut of the bony fish,Dicentrarchus labrax (L). Cell Tissue Res 2007;329:479e89.

[108] Scapigliati G, Romano N, Abelli L, Meloni S, Ficca AG, Buonocore F, et al.Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.). FishShellfish Immunol 2000;10:329e41.

[109] Tinh NTN, Dierckens K, Sorgeloos P, Bossier P. A review of the functionality ofprobiotics in the larviculture food chain. Mar Biotechnol 2008;10:1e12.

[110] Bjursell MK, Martens EC, Gordon JI. Functional genomic and metabolicstudies of the adaptations of a prominent adult human gut symbiont,Bacteroides thetaiotaomicron to the suckling period. J Biol Chem 2006;281:36269e79.

[111] Prioult G, Fliss I, Pecquet S. Effect of probiotic bacteria on induction andmaintenance of oral tolerance to beta-lactoglobulin in gnotobiotic mice. ClinDiagn Lab Immunol 2003;10:787e92.

[112] Samuel BS, Gordon JI. A humanized gnotobiotic mouse model ofhost-archaeal-bacterial mutualism. Proc Natl Acad Sci U S A 2006;103:10011e6.

[113] Menard O, Butel MJ, Gaboriau-Routhiau V, Waligora-Dupriet AJ. Gnotobioticmouse immune response induced by Bifidobacterium sp. strains isolated frominfants. Appl Environ Microbiol 2008;74(3):660e6.

[114] Rodrigues ACP, Cara DC, Fretez SHGG, Cunha FQ, Vieira EC, Nicoli JR, et al.Saccharomyces boulardii stimulates sIgA production and the phagocyticsystem of gnotobiotic mice. J Appl Microbiol 2000;89:404e14.

[115] Shima T, Fukushima K, Setoyama H, Maoka A, Matsumoto S, Hara T, et al.Differential effects of two probiotic strains with different bacteriologicalproperties on intestinal gene expression, with special reference to indigenousbacteria. FEMS Immunol Med Microbiol 2008;52:69e77.

[116] Bates JM, Mittge E, Kuhlman J, Baden KN, Cheesman SE, Guillemin K. Distinctsignals from the microbiota promote different aspects of zebrafish gutdifferentiation. Dev Biol 2006;297:374e86.

[117] Rawls JF, Samuel BS, Gordon JI. Gnotobiotic zebra fish reveal evolutionarilyconserved responses to the gut microbiota. Proc Natl Acad Sci U S A2004;101:4596e601.

[118] Rawls JF, Mahowald MA, Goodman AL, Trent CM, Gordon JI. In vivo imagingand genetic analysis link bacterial motility and symbiosis in the zebra fishgut. Proc Natl Acad Sci U S A 2007;104:7622e7.

[119] Rekecki A, Dierckens K, Laureau S, Boon N, Bossier P, Van den Broeck W. Effectof germ-free rearing environment on gut development of larval sea bass(Dicentrarchus labrax L.). Aquaculture 2009;293:8e15.

[120] Medina M, Izquierdo E, Ennahar S, Sanz Y. Differential immunomodulatoryproperties of Bifidobacterium logum strains: relevance to probiotic selectionand clinical applications. Clin Exp Immunol 2007;150:531e8.

[121] Corr SC, Hill C, Gahan CGM. Chapter 1 Understanding the mechanisms bywhich probiotics inhibit gastrointestinal pathogens. Adv Food Nutr Res2009;56:1e15.

[122] Skjermo J, Vadstein O. Techniques for microbial control in the intensiverearing of marine larvae. Aquaculture 1999;177:333e43.

[123] Madsen K. Probiotics and the immune response. J Clin Gastroenterol 2006;40(3):232e4.

[124] Ibnou-Zekri N, Blum S, Schiffrin EJ, von der Weid T. Divergent patterns ofcolonization and immune response elicited from two intestinal Lactobacillusstrains that display similar properties in vitro. Infect Immun 2003;71:428e36.

S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e14 13

[125] Gill H, Rutherfurd K, Cross M. Dietary probiotic supplementation enhancesnatural killer cell activity in the elderly: an investigation of age-relatedimmunological changes. J Clin Immunol 2001;21(4):264e71.

[126] Donnet-Hughes A, Rochat F, Serrant P, Aeschlimann JM, Schiffrin EJ. Modu-lation of nonspecific mechanisms of defense by lactic acid bacteria: effectivedose. J Dairy Sci 1999;82:863e9.

[127] Vollstad D, Bogwald J, Gaserod O, Dalmo RA. Influence of high-M alginate onthe growth and survival of Atlantic cod (Gadus morhua L.) and spottedwolffish (Anarhichas minor Olafsen) fry. Fish Shellfish Immunol 2006;20:548e61.

[128] Kesarcodi-Watson A, Kaspar H, Lategan MJ, Gibson L. Probiotics in aquacul-ture: the need, principles and mechanisms of action and screening processes.Aquaculture 2008;274:1e14.

[129] Boyle RJ, Robins-Browne RM, Tang ML. Probiotic use in clinical practice: whatare the risks? Am J Clin Nutr 2006;83:1256e64.

[130] Pineiro M, Stanton C. Probiotic bacteria: legislative framework e require-ments to evidence basis. J Nutr 2007;137:850e3.

[131] Gomez-Gil B, Roque A. Selection of probiotic bacteria for use in aquaculture.In: Flegel TW, editor. Advances in shrimp biotechnology. Bangkok: NationalCenter for Genetic Engineering and Biotechnology; 1998.

[132] Abraham TJ, Mondal S, Babu CS. Effect of commercial aquaculture probioticand fish gut antagonistic bacterial flora on the growth and disease resistanceof ornamental fishes Carassius auratus and Xiphophorus helleri. J Fish AquatSci 2008;25(1):27e30.

[133] Moriarty DJW. Microbial biotechnology: a key ingredient for sustainableaquaculture. Info Fish Int 1996;4/96:29e33.

[134] Verschuere L, Rombaut G, Sorgeloos P, Verstraete W. Probiotic bacteria asbiological control agents in aquaculture. Microbiol Mol Biol Rev 2000;64:655e71.

[135] Gomez-Gil B, Roque A, Turnbull JF. The use and selection of probiotic bacteriafor use in the culture of larval aquatic organisms. Aquaculture 2000;191:259e70.

[136] Goldin BR, Gorbach SL. Probiotics for humans. In: Fuller R, editor. Probiotics.London: Chapman and Hall; 1992. p. 355e76.

[137] Carnevali O, Zamponi MC, Sulpizio R, Rollo A, Nardi M, Orpianesi C, et al.Administration of probiotic strain to improve seabream wellness duringdevelopment. Aquac Int 2004;12:377e86.

[138] Timmerman HM, Koning CJM, Mulder L, Rombout FM, Beynen AC. Mono-strain, multistrain and multispecies probiotics e a comparison of function-ality and efficacy. Int J Food Microbiol 2004;96:219e33.

[139] Moriarty DJW. Probiotics in aquaculture. Microbiol Aust 2003;24:15e7.[140] Casula G, Cutting SM. Bacillus probiotics: spore germination in the gastro-

intestinal tract. Appl Environ Microbiol 2002;68(5):2344e52.[141] Hong HA, Duc LH, Cutting SM. The use of bacterial spore formers as pro-

biotics. FEMS Microbiol Rev 2005;29:813e35.[142] Huang JM, La Ragione RM, Nunez A, Cutting SM. Immunostimulatory activity

of Bacillus spores. FEMS Immunol Med Microbiol 2008;53:195e203.[143] Raida MK, Larsen JL, Nielsen ME, Buchmann K. Enhanced resistance of

rainbow trout, Oncorhynchus mykiss (Walbaum), against Yersinia ruckerichallenge following oral administration of Bacillus subtilis and B. licheniformis(BioPlus2B). J Fish Dis 2003;26:495e8.

[144] Queiroz JF, Boyd CE. Effects of a bacterial inoculum in channel catfish ponds.J World Aquac Soc 1998;29:67e73.

[145] Kennedy SB, Tucker JW, Neidig CL, Vermer GK, Cooper VR, Jarrell JL, et al.Bacterial Management Strategies for stock enhancement of warm watermarine fish: a case study with common snook (Centropomus underci malis).Bull Mar Sci 1998;62:573e88.

[146] Gatesoupe FJ. The effect of three strains of lactic bacteria on the productionrate of rotifers, Brachionus plicatilis, and their dietary value for larval turbot,Scophthalmus maximus. Aquaculture 1991;96:335e42.

[147] Gatesoupe FJ. Lactic acid bacteria increase the resistance of turbot larvae,Scophthalmus maximus, against pathogenic Vibrio. Aquat Living Resour1994;7:277e82.

[148] Beachey EH. Bacterial adherence: adhesion-receptor interactions mediatingthe attachment of bacteria to mucosal surfaces. J Infect Dis 1981;143:325e45.

[149] Coconier MH, Bernet MF, Chauviere G, Servin AL. Adhering heat-killedhuman lactobacillus acidophilus, strain LB, inhibits the process of pathoge-nicity of diarrhoeagenic bacteria in cultured human intestinal cells. J Diar-rhoeal Dis Res 1993;11:235e42.

[150] Hood SK, Zottola EA. Effect of low pH on the ability of Lactobacillus acid-ophilus to survive and adhere to human intestinal cells. J Food Sci 1988;53:1514e6.

[151] He F, Hirotsugu M, Kubota A, Ouwehand AC, Hosoda M, Hiramatsu M,et al. Effect of orally administered non-viable Lactobacillus cells onmurine humoral immune responses. Microbiol Immunol 2005;49(11):993e7.

[152] Haller D, Bode C, Hammes WP, Pfeifer AM, Schiffrin EJ, Blum S. Non-patho-genic bacteria elicit a differential cytokine response by intestinal epithelialcell/leucocyte co-cultures. Gut 2000;47:79e87.

[153] Miettinen M, Vuopio-Varkila J, Varkila K. Production of human tumornecrosis factor alpha, interleukin-6, and interleukin-10 is induced by lacticacid bacteria. Infect Immun 1996;64:5403e5.

[154] Secombes CJ, Wang T, Hong S, Peddie S, Crampe M, Laing KJ, et al. Cytokinesand innate immunity of fish. Dev Comp Immunol 2001;25:713e23.

[155] Minelli EB, Benini A. Relationship between number of bacteria and theirprobiotic effects. Microb Ecol Health Dis 2008;20:180e3.

[156] Kishi A, Uno K, Matsubara Y, Okuda C, Kishida T. Effect of the oral adminis-tration of Lactobacillus brevis subsp coagulans on interferon alpha producingcapacity in humans. J Am Coll Nutr 1996;15:408e12.

[157] Nikoskelainen S, Ouwehand A, Bylund G, Salminen S. Protection of rainbowtrout (Oncorhynchus mykiss) from furunculosis by Lactobacillus rhamnosus.Aquaculture 2001;198:229e36.

[158] Rengpipat S, Phianphak W, Piyatiratitivorakul S, Menasaveta P. Effects ofa probiotic bacterium in black tiger shrimp Penaeus monodon survival andgrowth. J Aquacult 1998;167:301e13.

[159] Robertson PAW, O'Dowd C, Burrells C, Williams P, Austin B. Use of Carno-bacterium sp. as a probiotic for Atlantic salmon (Salmo salar L.) andrainbow trout (Oncorhynchus mykiss Walbaum. Aquaculture 2000;185:235e43.

[160] Gatesoupe FJ. Bacillus sp. spores as food additive for the rotifer Brachionusplicatilis: improvement of their bacterial environment and their dietary valuefor larval turbot, Scophthalmus maximus L. 4th Int. Symp. Fish Nutr. Feeding24e27 June 1991. In: Kaushik SJ, Luquet P, editors. Fish nutrition in practice.Biarritz, France: Les Colloques, no. 61, Institut National de la RechercheAgronomique, Paris; 1993. p. 561e8.

[161] Keskin M, Keskin M, Rosenthal H. Pathways of bacterial contaminationduring egg incubation and larval rearing of turbot, Scophthalmus maximus. JAppl Ichthyol 1994;10:1e9.

[162] Munro PD, Henderson RJ, Barbour A, Birkbeck TH. Partial decontamination ofrotifers with ultraviolet radiation: the effect of changes in the bacterial loadand flora of rotifers on mortalities in start-feeding larval turbot. Aquaculture1999;170:229e44.

[163] Nicolas JL, Robic E, Ansquer D. Bacterial flora associated with a trophic chainconsisting of microalgae, rotifers and turbot larvae: influence of bacteria onlarval survival. Aquaculture 1989;83:237e48.

[164] Asli MM, Hosseini SA, Lotfollahian H, Shariatmadari F. Effect of probiotics,yeast, vitamin e and vitamin c supplements on performance and immuneresponse of laying hen during high environmental temperature. Int J Poul Sci2007;6(12):895e900.

[165] Abraham TJ, Babu CHS, Mondal S, Banerjee T. Effects of dietary supplemen-tation of commercial human probiotic and antibiotic on the growth rate andcontent of intestinal microflora in ornamental fishes. Bangladesh J Fish Res2007;11(1):57e63.

[166] Aubin J, Gatesoupe FJ, Labbe L, Lebrun L. Trial of probiotics to prevent thevertebral column compression syndrome in rainbow trout (Oncorhynchusmykiss Walbaum). Aquac Res 2005;36:758e67.

[167] Chang CI, Liu WY. An evaluation of two probiotic bacterial strains, Entero-coccus faecium SF68 and Bacillus toyoi, for reducing edwardsiellosis incultured European eel, Anguilla anguilla L. J Fish Dis 2002;25:311e5.

[168] Vendrell D, Balcazar JL, de Blas I, Ruiz-Zarzuela I, Girones O, Muzquiz JL.Protection of rainbow trout (Oncorhynchus mykiss) from lactococcosisby probiotic bacteria. Comp Immunol Microbiol Infect Dis 2008;31:337e45.

[169] Chabrillon M, Arijo S, Diaz-Rosale P, Balebonz MC, Morinigo MA. Interferenceof Listonella anguillarum with potential probiotic microorganisms isolatedfrom farmed gilthead seabream (Sparus aurata, L.). Aquac Res 2006;37:78e86.

[170] Hjelm M, Bergh O, Riaza A, Nielsen J, Melchiorsen J, Jensen S, et al. Selectionand identification of autochthonous potential probiotic bacteria from turbotlarvae (Scophthalmus maximus) rearing units. Syst Appl Microbiol 2004;27:360e71.

[171] Kodama H, Moustofa M, Mikami T, Izawa H. Partial purification of extracel-lular substance of Vibrio anguillarum toxigenic for rainbow trout and mouse.Fish Pathol 1985;20:173e9.

[172] Kodama H, Nakamura H, Kashima M, Togase H. Protection against atypicalAeromonas salmonicida infection in carp (Cyprinus carpio L.) by oraladministration of probiotic bacterial culture. Proceedings, The 15th Congressof FAVA 27e30 October, FAVA -OIE Joint Symposium on Emerging Diseases69e71.

[173] Vijayabaskar P, Somasundaram ST. Isolation of bacteriocin producing Lacticacid bacteria from fish gut and probiotic activity against common fresh-water fish pathogen Aeromonas hydrophila. Biotechnology 2008;7(1):124e8.

[174] Gildberg A, Johansen A, Bogwald J. Growth and survival of Atlantic salmon(Salmo salar) fry given diets supplemented with fish protein hydrolysate andlactic acid bacteria during a challenge trial with Aeromonas salmonicida.Aquaculture 1995;138:23e34.

[175] Gildberg A, Mikkelsen H. Effect of supplementing the diet to Atlantic cod(Gadus morhua) fry with lactic acid bacteria and immuno-stimulatingpeptides during a challenge trial with Vibrio anguillarum. Aquaculture1998;167:103e13.

[176] Murthy HS, Naik ATR. Growth performance of Cirrhinus mrigala in relationto a natural probiotic feed supplement, Biovet-YC. Fish Chimes 2002;22:54e6.

[177] Shelby RA, Lim CE, Aksoy M, Klesius PH. Effects of probiotic bacteria asdietary supplements on growth and disease resistance in young channelcatfish, Ictalurus punctatus (Rafinesque). J Appl Aquac 2007;19(1):81e91.

[178] Li J, Tan B, Mai K, Ai Q, Zhang W, Xu W, et al. Comparative study betweenprobiotic bacterium Arthrobacter XE-7 and chloramphenicol on protection of

S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e1414

Penaeus chinensis post-larvae from pathogenic vibrios. Aquaculture 2006;253:140e7.

[179] Vaseeharan B, Ramasamy P. Control of pathogenic Vibrio spp.by Bacillussubtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeusmonodon. Lett Appl Microbiol 2003;36:83e7.

[180] Hai NV, Fotedar R, Buller N. Selection of probiotics by various inhibition testmethods for use in the culture of western king prawns, Penaeus latisulcatus(Kishinouye). Aquaculture 2007;272:231e9.

[181] Wang YB, Li JR, Lin J. Probiotics in aquaculture: challenges and outlook.Aquaculture 2008;281(1e4):1e4.

[182] Mater DDG, Langella P, Corthier G, Flores MJ. A probiotic Lactobacillus straincan acquire vancomycin resistance during digestive transit in mice. J MolMicrobiol Biotechnol 2008;14:123e7.

[183] Capozzi V, Spano G. Horizontal gene transfer in the gut: is it a risk? Food ResInt 2009;42:1501e2.