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Review

Probiotics and immunity: A sh perspective

S.K. Nayak

Laboratory 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 2009

Received in revised form

12 February 2010

Accepted 19 February 2010

Available online 26 February 2010

Keywords:

Fish

Gut immunity

Innate immunity

Probiotics

a b s t r a c t

Probiotics are usually live microorganisms which when administered in adequate amounts confer

a health benets on host. Nowadays, probiotics are also becoming an integral part of the aquaculture

practices 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 of

probiotics in nutrition, disease resistance and other benecial activities in sh has proven beyond any

doubt. Among the numerous health benets attributed to probiotics, modulation of immune system is

one of the most commonly purported benets of the probiotics and their potency to stimulate the

systemic and local immunity under in vitro and in vivo conditions is noteworthy. Different probiotics

either monospecies or multispecies supplementation can eventually elevate phagocytic, lysozyme,

complement, respiratory burst activity as well as expression of various cytokines in sh. Similarly,

probiotics can stimulate the gut immune system ofsh with marked increase in the number of Ig cells

and acidophilic granulocytes. Furthermore, mono-bacterial association studies (with non-probiotic

bacterial strains) in gnotobiotic sh 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 sh,

probiotics often exert host specic and strain specic differences in their activities. Various factors like

source, type, dose and duration of supplementation of probiotics can signi cantly affect the immuno-

modulatory activity of probiotics. The review is therefore, aiming to highlight the immunomodulatory

activity of probiotics and also to evaluate the factors that regulate for the optimum induction of immuneresponses in sh.

2010 Elsevier Ltd. All rights reserved.

1. Introduction

Over the years various strategies to modulate the composition of

the gut microbiota for better growth, digestion, immunity, and

disease resistance of the host have been investigated in various

livestock as well as in human beings [1]. The manipulation of the

gut microbiota through dietary supplementation of benecial

microbe(s) is a novel approach not only from nutritional point of

view but also as an alternate viable therapeutic modality to over-come the adverse effects of antibiotics and drugs. Those benecial

microorganisms are usually referred as probiotics which after

administration can able to colonize and multiply in the gut of host

and execute numerous benecial effects by modulating various

biological systems in host[2]. Probiotics are originally dened as

the organisms and substances which contribute to the intestinal

microbial balance[3]. The term probiotic was originated from the

Greek words pro and bios which mean for life [4] and are

often called as promoter of life that help in a natural way to

improve the overall health statusof the host organism. According to

the currently adopted denition by Food and Agricultural Organi-

zation/World Health Organization, probiotics are live microorgan-

isms which when administered in adequate amounts confer

a health benet on the host[5].

Probiotics, thus, open a new era in health management strategy

from human to sh/shellsh. Probiotics are gaining increasingscientic and commercial interest and are now quite commonplace

in health promoting functional foods to therapeutic, prophylactic

and growth supplements[6,7]. The success of probiotics, has laid

the foundation for other concepts like prebiotics which are the

non-digestible food ingredients that selectively stimulate the

growth 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 that

they promote specic microbe(s) in the intestine for restoring the

intestinal microbial balance and exerting numerous benecial

effects in host[2,8,9].E-mail address: [email protected]

Contents lists available at ScienceDirect

Fish & Shellsh Immunology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m/ l o c a t e / f s i

1050-4648/$e see front matter 2010 Elsevier Ltd. All rights reserved.

doi:10.1016/j.fsi.2010.02.017

Fish & Shellsh Immunology 29 (2010) 2e14
mailto:[email protected]://www.sciencedirect.com/science/journal/10504648http://www.elsevier.com/locate/fsihttp://www.elsevier.com/locate/fsihttp://www.sciencedirect.com/science/journal/10504648mailto:[email protected]

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2. Probiotics in sh culture

Fish is one of the richest sources of animal protein and is the

fastest food producing sector in the world. Worldwide, people

obtain about 25% of their animal protein from sh and shellsh and

consumer's demand for sh continues to climb[10]. Over the years,

aquaculture sector has undergone a sea change in order to meet the

increasing demand. The production is maximized through inten-

sication with addition of commercial diets, growth promoters,

antibiotics, and several other additives. Application of these

measures leads to high production beyond any doubt, but the most

worrisome factor is that the routine use of these products causes

severe complications and even a stage has come where its

sustainability is in stake[11].

In aquaculture practices, probiotics are used for a quite long

time but in last few years probiotics became an integral part of the

culture practices for improving growth and disease resistance. This

strategy offers innumerable advantages to overcome the limita-

tions and side effects of antibiotics and other drugs andalso leads to

high production through enhanced growth and disease prevention

[12e15]. In aquaculture, the range of probiotics evaluated for use is

considerably wider than in terrestrial agriculture. Several pro-

biotics either as monospecies or multispecies supplements arecommercially available for aquaculture practices [16e20]. Apart

from the nutritional and other health benets [21e25], certain

probiotics as water additives can also play a signicant role in

decomposition of organic matter, reduction of nitrogen and phos-

phorus level as well as control of ammonia, nitrite, and hydrogen

sulde[26].

Numerous microbes have been identied as probiotics for

aquaculture practices, many of which differ markedly in their mode

of action. There are, however, some common mechanisms of action

that have been reported for the majority of probiotic strains.

Probiotics help in feed conversion efciency and live weight gain

[27,28] and confer protection against pathogens by competitive

exclusion 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 sh[36,37].

3. Probiotics and sh immunity

Among the numerous benecial effects of probiotics, modula-

tion of immune system is one of the most commonly purported

benets of the probiotics. The role of probiotics in modulating the

immune system has been extensively investigated and reviewed in

humans and animals[38e41]. Most of the earlier studies in sh,

dealt with growth promoting and disease protective ability of

probiotics. However, in recent times much attention has been

hitherto towards the immunmodulating effects of probiotics inpiscine system. A lot of immunological studies havebeen performed

in several sh using different probiotics and their potency to

stimulate the teleost immunity both under in vivo and in vitro

conditions is noteworthy[42e79]. Perusal of available literatures

indicates that several probiotics either individually or in combina-

tion can enhance both systemic as well as local immunity in sh.

The review is therefore, aimingto highlight the immunomodulatory

activity of probiotics and also to evaluate the factors that regulate

for the optimum induction of immune responses in piscine system.

4. Effect of probiotics on systemic immunity

Studies on human and animal models provide a baseline

understanding of the degree and type of immune responses

induced by different probiotics [80]. Unlike other animals, pro-

biotics also modulate various immunohaematological parameters

in teleosts and is presented inTable 1.Probiotics interact with the

immune cells such as mononuclear phagocytic cells (monocytes,

macrophages) and polymorphonuclear leucocytes (neutrophils)

and NK cells to enhance innate immune responses. Like higher

vertebrates, certain probiotics can enhance the number of eryth-

rocytes, granulocytes, macrophages and lymphocytes in different

sh [53,57]. Similarly, probiotics, in both in vitro and in vivo

conditions, actively stimulate the proliferation of B lymphocytes in

sh. Elevation of immunoglobulin level by probiotics supplemen-

tation is reported in many animals including sh [27,58,63].

Furthermore, Song et al.[75]recorded high immunoglobulin level

in skin mucusa of Miichthys miiuy by Clostridium butyricum.

Different Lactic acid bacteria (LAB) group of probiotics either in

viable or non-viable form can elevate immunoglobulin level insh

[64]and even one week supplementation of probiotioc like Lacto-

bacillus rhamnosus @2.8 108 CFU/g feed was found to signicantly

increase the immunoglobulin level in rainbow trout (Oncorhynchus

mykiss) [60]. However, Balczar et al. [44] only found rise in

immunoglobulin level inSalmo truttabut not at signicant level by

feeding 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 the

inammatory response before antibody production and plays an

important role in antibacterial defenses. Probiotics can effectively

trigger the pahgocytic cells in host and enhancement of phago-

cytic activity by LAB group of probiotics such as L. rhamnosus,

L. lactisand Lactobacillus acidophilus has already been observed in

several animals [81]. These probiotics are often used in aqua-

culture practices and supplementation of these probiotics either

in viable or inactivated form is found to stimulate phagocytic

activity in several sh species [47,48,54,63,67]. In tilapia (Oreo-chromis niloticus) a 2 weeks feeding of L. rhamnosus signicantly

stimulated the phagocytic activity [68]. Likewise, oral adminis-

tration of C. butyricum bacteria to O. mykiss has also been

reported to enhance the phagocytic activity of O. mykiss [69].

However, probiotic like L. lactis failed to enhance the phagocytic

activity of head kidney macrophages of turbot (Scophthalmus

maximus) [77].

4.2. Respiratory burst activity

Respiratory burst activity is an important innate defense

mechanism of sh. The ndings of respiratory burst activity

following probiotics treatment in sh are often contradictory.

While some studies indicate probiotics do not have signicantimpact on this non-specic defense mechanism ofsh[52,58,73],

severalin vitro and in vivo studies showed signicant increase in

respiratory burst activity by various probiotics in many aquatic

animals including sh. Probiotics like Bacillus subtilis and certain

members of LAB group can stimulate respiratory burst activity in

sh [60,70,71,79]. Nevertheless 5 107 CFU/ml heat inactivated

Lactobacillus delbrueckii subsp. lactis and B. subtilis under in vitro

condition 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 innate

immunity is an indispensable tool of

sh to

ght against

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Table 1

Effect of different probiotics supplementation on various immune responses ofsh.

Sl.

no

Probiotics Form of

probiotics

Mode of

Probiotics

supplementation

Assay

conditions

Immunological effects Reference

1 Bacillus subtilis,

Lactobacillus acidophilus

Viable Indiv idual and

Combination

In vivo Respiratory burst activity ([), Serum bactericidal activity ([C),

Neutrophil adherence ([), Lysozyme ([), Heamtocrit percentage ([Y)

[42]

2 Lactobacillus sakei,

Lactococcus lactis,Leuconostoc mesenteroides

Viable I ndividual In vivo Respiratory burst activity against liveA. salmonicida([only in

L. lactis), Respiratory burst activity against dead A. salmonicida([)

[43]


Lactococcus lactis,

Leuconostoc mesenteroides

Viable I ndividual In vivo Immunoglobulin ([Y), Lysozyme ([L. lactis, L. mesenteroides

but[Y for L. sakei), Complement activity ([)

[44]


Lactococcus lactis,


Viable I ndividual In vivo Lysozyme ([Y), Complement activity ([), Phagocytic activity ([),

Respiratory burst activity ([for all except L. sakei [Y)

[45]

5 Lactococcus lactis,


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

6 Aeromonassobria Viable I ndividual In vivo Leucocytes ([), Phagocytic activity ([), Respiratory burst activity ([) [47]

7 Bacillusspecies,

Aeromonas sobria

Viable I ndividual 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 and

Combination

In vivo Cytotoxic ([for 51M6 and C, [Y Pdp11), Respiratory burst activity ([Y),

Phagocytic activity ([), Peroxidase activity of serum and head kidney

leucocytes ([Y), Complement activity ([Y)

[49]

9 Shewanella putrefaciens,

Shewanella baltica

Inactivated Individual and

Combination

In vivo Phagocytic activity ([), Respiratory burst activity ([Y), Complement

activity ([), Peroxidase activity ([), Cyotoxic activity ([only inS. baltica)

[50]


Shewanella baltica

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


Shewanella baltica

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

12 Vibrio uvialis,

Micrococcus luteus,

Aeromonas hydrophila,

Carnobacteriumspecies

Vi able Indiv idual and

Combination

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

lysozyme activity ([)

[53]

13 Gram ve coccus, V. uvialis,

Aeromonas hydrophila,


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:

TNFa ([), TCRb ([), IL1b([), CD8 ([Y), CD4 ([Y),IL8 (Y), TGFb (Y)

[56]

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

Specic 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 I ndividual In vivo Immunoglobulin ([), Respiratory burst activity ([),

Complement activity ([)

[60]

20 Saccharomyces cerevisiae Viable I ndividual 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 and

head kidney by E. faecium, [Y for spleen and head kidney by B. subtilis,),

TNF 1 and 2 ([head kidney and spleen by L. rhamnosusand E. faecium,

[Yfor B. subtilis), TGF-b([Yspleen and head kidney by L. rhamnosus,

[for spleen and [Y head kidney by B. subtilis, [ for spleen and head

kidney byE. faecium), Complement activity ([YL. rhamnosus, [

forB. subtilis, E. faecium), Respiratory burst activity

([YL. rhamnosus, B. subtilisand [ for E. faecium)

[65]

25 Lactobacillus delbrueckii Viable I ndividual In vivo

(Through

artemia)

TCRb ([), Immunoglobulin ([Y),

CD8 ([Y), CD4 ([Y), IL-1b(Y), IL-10 (Y), COX-2 (Y), TGFb (Y)

[66]

26 Aeromonas sobria,

Brochothrix thermosphacta

Viable I ndividual In vivo Respiratory burst activity ([B. thermosphacta, [Y for A. sobria),

Complement activity ([Y), Phagocytic activity ([), Serum and epidermal

mucus lysozyme ([Y), lecucocytes ([Y), lymphocytes ([Y),

Pinocytic activity ([Y)

[67]

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infectious agents [82]. Probiotics either single or in combination

are 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 in

brown trout (Salmo trutta) [44] is reported. Apart from serum

lysozyme content, probiotics can also enhance the lysozyme level

in skin mucosa of sh [75,76]. Taoka et al. [76] reported signi-

cantly high lysozyme level in skin mucosa by supplementing

commercial probiotics through water in comparison to oral

supplementation in O. niloticus.

On contrary, dietary supplementation of probiotics like L. sakei

in S. trutta [44], L. sakei, L. lactis ssp. lactis, L. mesenteroides, and

L. rhamnosus in O. mykiss [45,64],Aeromonas sobria in O. mykiss [48]

as well as water supplementation of Bacillus coagulans, B. subtilis

and Rhodopseudomonas palustris and Enterococcus faecium inO. niloticus[78,79]failed to elevate lysozyme level. Similarly, Peters

et al. [67] failed to detect any specic change in serum and skin

mucosa lysozyme level by feeding A. sobria @108 cells/g feed

and B. thermosphacta @1010 cells/g feed inO. mykissfor a period of

14 days.

4.4. Peroxidase and anti-protease activity

The peroxidase is an important enzyme that utilizes oxidative

radicals to produce hypochlorous acid to kill pathogens. During

oxidative respiratory burst, it is mostly released by the azurophilic

granules of neutrophils. Dietary supplement of probiotic like

B. subtilisalone or in combination withL. delbrueckiissp.lactisfor 3

weeks lead to high serum protease activity but failed to enhance

the peroxidase activity of head kidney leucocytes ofS. aurata [72].

Similarly, probiotics likeE. faeciumalso 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 to

thesendings, probiotics like L. delbrueckii,B. subtilis,Bacillus JB-1,

A. sobria, Shewanella putrefaciens (Pdp11) and 51M6 did not affect

the protease activity in sh likeO. mykiss and S. aurata[48,49,71].

Similarly, anti-protease activities of serum and other bodyuids

are mainly due to a1 and a2-antiprotease, and a2-macroglobulin

and also responsible for preventing proteolytic pathogens [83,84].

Though, these activities are normally high in sh and hardly

modulated even after immunization or infection[84], certain pro-

biotics can successfully elevate this activity in sh [48,59,73].

Sharifuzzaman and Austin [73] reported signicantly high anti-

protease activity in O. mykiss within 2 weeks of supplementation

of probiotic belong to Kocuria species (z

10

8

cells/g feed).

4.5. Complement activity

In teleosts, complement system plays a key role in adaptive

immune responses and involved in chemotaxis, opsonization,

phagocytosis and degradation of pathogens. Complement,

a component of the non-specic immune response, may have

effector mechanisms like direct killing of microorganisms by lysis

[85]. Probiotics can enhance natural complement activity ofsh

[65,72]and dietary as well as water treatment of many probiotics

are often reported to stimulate the piscine complement compo-

nents [64,78]. It is also worth noting that non-viable probiotics

can stimulate complement components in sh. Choi and Yoon

[49] recorded an increased complement activity in O. mykiss

Table 1 (continued )

Sl.

no

Probiotics Form of

probiotics

Mode of

Probiotics

supplementation

Assay

conditions

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

Vi able Indivi dual an d

Combination

In vivo Phagocytic activity ([I), Respiratory burst activity ([Y),

Cyotoxic activity ([C, [Y I), Peroxidase activity of

head kidney leucocytes ([Y)

[70]

30 Lactobacillus delbrueckii,

Bacillus subtilis, Pdp11, 51M6

Inactivated Individual In vitro Peroxidase activity of head kidney leucocytes ([Y), Respiratory

burst activity ([), Cyotoxic activity ([but [Y Pdp11)

[71]

31 Lactobacillus delbrueckii,

Bacillus subtilis

Inactivated

(Heat-Killed)

Individual and

Combination

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 Kocuriaspecies Viable Individual In vivo Lysozyme ([), Peroxidase ativity of head kidney macrophage ([),

Respiratory burst activity ([Y), Phagocytic activity ([), Anti-protease

activity ([)

[73]

33 Lactobacillus plantarum Viable Individual In vivo Lysozyme ([), Phagocytic activity ([), Peroxidase activity ([),

Complement activity ([), Superoxide dismutase (Y), Glutathione

peroxidase ([)

[74]

34 Clostridium butyricum Viable Individual In vivo Phenoloxidase activity ([), Acid phosphatases activity ([when

treated @ 109 CFU/g), Lysozyme ([for serum at 107 CFU/g

and skin at 109 CFU/g), Immunoglobulin M ([serum and skin mucus

both at 107 CFU/g)

[75]

35 Bacillus subtilis,

Lactobacillus acidophilus,

Clostridium butyricum,

Saccharomyces cerevisiae

(Commercial probiotics

preparation)

Viable,

Inactivated

Combination In vivo Neutrophils migration ([), Bactericidal activity ([), Lysozyme

([mucus and serum), Respiratory burst activity ([Y), Skin

protease activity ([)

[76]

36 Lactococcus lactis,


Viable,

Inactivated

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

37 Enterococcus faecium Viable Individual In vivo

(Through

water)

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

activity ([), Myeloperoxidase activity ([)

[78]

38 Bacillus coagulans,

Bacillus subtilis,

Rhodopseudomonas palustris

Viable Individual In vivo

(Through

water)

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]

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


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from at 4th week of feeding the heat inactivated probiotics

(Pdp11 or 51M6).

4.6. Cytokines

Cytokines are protein mediators produced by immune cells and

contribute to cell growth, differentiation and defense mechanisms

of the host [86]. Perusal of available literatures indicate that

a number of probiotics can effectively modulate the production of

pro-inammatory cytokines such as interleukin-1 (IL-1), IL-6, IL-

12, tumor necrosis factora (TNF-a), and gamma interferon (IFN-g)

and anti-inammatory cytokines such as IL-10 and transforming

growth factor b (TGF-b) in many animals[87e89]. Probiotics like

Bidobacterium longum, L. acidophilus, L. lactis, Lactobacillus para-

cascei and Lactobacillus plantarum can up regulate the expression

of various types of cytokines in various hosts [81,90]. Different

strains of LAB can induce regulatory and pro-inamatory cyto-

kines while others probiotics can increase intestinal inammatory

responses [91].

Probiotics likeL. rhamnosus, E. faecium and B. subtilis are found

to up regulate the pro-inammatory cytokines like IL-1b1 and TGF-

b in the spleen and head kidney of O. mykiss [65]. Similarly, the

expression of IL-1b, IL-8, TNF-a, and TGF-b in head kidney of O.mykiss by C. maltaromaticum and C. divergens indicates their

possible involvement in anti-inammatory responses as well[56].

On the other hand, Picchietti et al.[66]recorded down-regulation

of Cyclooxygenase 2 (Cox-2) transcripts along with TGF-b and IL-

10 genes by L. delbrueckii supplemented through live carrier in

Dicentrarchus labrax. COX-2 promotes intestinal wound healing but

its chronic over expression can lead to inammatory diseases[92].

Therefore, a balanced expression of COX-2 is essential for main-

taining the intestinal homeostasis and modulation of the inam-

matory gene Cox-2 could be a key mechanism of anti-inammatory

action of certain probiotics[66].

5. Effect of probiotics on gut immunity

The gut is the organ where probiotics not only establish but also

execute their functions including immunostimulaory activity.

Therefore, the cross talk between probiotics, epithelial cells and gut

immune system warrants high consideration. The immune system

of the gut is referred to as gut associated lymphoid tissue (GALT)

and the piscine gut immune system is quite different from

mammals. Unlike mammals,sh lack Peyer's patches, secretory Ig A

and antigen-transporting M cells in the gut[93]. However, many

diffusely organized lymphoid cells, macrophages, granulocytes and

mucus IgM found in the intestine ofsh constitutes the immune

function[94e99].

The interaction of non-commensal and probiotics with gut

immune 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. The

whole bacteria can't be introduced through the epithelial cells and

that only the antigenic particles or degraded products of the

bacteria are able to make contact with immune cells [101]. The

augmentation of the immune response by probiotic bacteria,

a phenomenon similar to that of cholera toxin, may also occur in

adherence with GALT and may therefore directly affect immune

cells like leukocytes [102]. Fish possess strong antigen uptake

capacity in the second gut segment and the uptake and transport of

antigens followed by their processing by intraepithelial macro-

phages is also reported in carp [103,104].

The effect of probiotics in stimulating the systemic immune

responses are now well documented in severalsh species but that

of local gut immunity is lacking. Limited attempts due to lack of

suitable tools, are made to access the gut immune response

following probiotics treatment. Few studies that were conducted in

recent times indicate that probiotics can stimulate the piscine gut

immune system with marked increase in the number of Ig cells

and acidophilic granulocytes (AGs) [66,72,105,106]. Probiotics

supplementation at early developmental stages can be helpful in

increasing specic AGs subpopulations [105]. The presence of

T-cells in the GALT has been documented in many sh[97,107,108]

and probiotics can lead to a signicant increase in T-cells in sh. In

a study, Picchietti et al. [66]recorded increased T lymphocytes in

gut without any change in CD4 and CD8a transcript in sea bass

(D. labrax) by L. delbrueckii ssp. delbrueckii supplemented through

live carriers like artemia and rotifers.

Apart from this, enhancement of gut mucosal lysozyme by

C. maltaromaticum and C. divergens [55]and phagocytic activity of

mucosal leucocytes by LAB group of probiotics such as L. lactisssp.

lactis,L. mesenteroidesandL. sakei[43]are also reported in sh like

O. mykiss.

6. Probiotics and gnotobiotic approaches

The detailed mode of action of probiotics has not yet beenestablished in any animals. It is often difcult to derive consensus

on a particular pattern of stimulation. Hence gnotobiotic approch

can be instrumental in understanding the basic mechanism of

probiotic action [109]. Gnotobiotic studies in different animal

models indicate the effect of different probiotics on the composi-

tion and functioning of reconstituted gut microbiota, difference in

their modes of action as well as involvement in both local and

systemic immune responses of host [110e112]. Involvement of

probiotics in up-regulating the gene expression of cryptdins and

matrilysin, the rst line of defense mechanism and in lipid

absorption and metabolism, such as intestinal fatty acid-binding

protein in higher vertebrates are already recorded [111,113e115].

The mono-bacterial association studies in gnotobiotic sh also

indicate the microbial up-regulation of serum amyloid A1, C-reac-

tive protein, complement component 3, angiogenin 4, glutathione

peroxidase, myeloperoxidase as well as glycoprotein production

[116e119]. However, these studies are not exclusively conducted

with probiotic strains but studies involving probiotics in gnotobi-

otic sh will certainly able to address the inter- and intra-species

difference among different probiotics as well as well can unlock

several aspects of their role in piscine immune system at molecular

level.

7. Factors affecting the immunomodulating

potency of probiotics

Modulation of host immunity is one of the most purported

benets of probiotics consumption[120]and sh is no exception.However, the mechanisms by which probiotics affect the immune

system 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 the

gut of host [122], probiotics often exert host specic [123] and

strain specic 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 their

activities. There is no doubt that probiotics can stimulate piscine

immune system like other animals but inappropriate dose and/or

duration of probiotics supplementation can cause undesirable

results[127]. Therefore, the type of probiotics, dose kinetics, and

method of administration with respect to sh are critical factors

that can regulate immune responses in

sh.


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7.1. Types of strain

The dominant group of probiotics that are used in sh culture

belong to Gram ve especially LAB, Bacillus (B. subtilis, B. lichen-

iformis, B. circulans) and bidobacteria groups. On the other hand

certain strains of Aeromonas (Aeromonas hydrophila, A. sobria),

Vibrio (Vibriouvialis), Pseudomonas and Enterobacteria species are

the Gram ve probiotics [128]. All these bacteria differ greatly in

their mode of action including the ability to trigger immune system

and therefore every probiotics differ from each other by their

functional role. It is recognized that each strain has unique prop-

erties and the probiotic effects of a specic strain must not be

extrapolated to other strains [129,130]. Gnotobiotic studies also

indicate the strain specic differences among probiotics in stimu-

lating immune system in animals[111,115].

Previous studies in piscine system also documented inter- and

intra-species differences in immunostimulating ability of different

probiotics [52,65,67,70]. Such type of difference is also evident by

the difference in triggering respiratory burst activity, by closely

related species like S. putrefaciens and Shewanella baltica in Sene-

galese sole (Solea senegalensis) [52] even at a proven beneciary

effective dose of the same probiotics in S. aurataandS. senegalensis

[50,51]. Similarly, variation in stimulating cellular innate immuneresponses under in vitro and in vivo conditions among probiotics

belong to 51M6,L. delbrueckii subsp.lactisand B. subtilisgroup are

also recorded [50,54,70,71]. Recently, Peters et al. [67] not only

recorded difference in immunostimulating activity but also in

disease protecting ability among two established probiotics namely

A. sobriaand B. thermosphacta in O. mykiss.

7.1.1. Indigenous vs exogenous

Selection of probiotics is very critical because inappropriate

microorganisms can lead to undesirable effects in host [131]. An

ideal probiotic, irrespective of its source should able to colonize,

establish and multiply in the host gut. Most of the commercial

probiotics used for terrestrial animals are now being used in

aquaculture 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-sh origin they are unable to

survive and/or remain viable at optimum concentration in gut

[18,132,133].

On the other hand probiotics from the same species and/or its

natural environment could be best approach for better efcacy in

host [134]. The strategy of isolating probiotics from the gut of

mature animals and then use in immature animals of the same

species has been successfully applied in sh[17,23,24,135]. There is

a general consensus that probiotics from autochthonous source

have a greater chance of competing with resident microbes and of

becoming predominant within a short period of intake and to

persist in the colonic environment for some time after the with-drawal of probiotics [136,137]. For instance, Carnevali et al. [137],

recorded a signicantly decreased larvae and fry mortality by using

Lactobacillus fructivorans, isolated from gut ofS. aurata. Further-

more, it is assumed that host immune cells do not react with

bacteria that are naturally occurring on their surfaces and autoch-

thonous in nature[71].

7.1.2. Monospecies vs multispecies

A wide range of probiotics, containing either monospecies or

multispecies of microorganisms are commercially available. In

recent times a number of studies have conrmed the benecial

effects of both forms of probiotics under in vitro and in vivo

conditions. However, it is postulated that multispecies/multistrain

probiotics are moreeffectiveand consistent than their monospeci

c

counter parts since mixed cultures may exert synergistic probiotic

properties [138]. Many times induction of greater systemic innate

immunity has been recorded by using multispecies probiotics in

sh[42,53,71]. Aly et al.[42]reported signicantly high respiratory

burst activity andlysozyme level in O. niloticus fed with a mixture of

B. subtilisand L. acidophilus. Besides systemic effects, multispecies

formulation of probiotics was the most effective in triggering the

local gut immunity [72,105]. Salinas et al. [72] recorded that

B. subtilis and L. delbrueckii subsp. lactis in combination can increase

the numbers of IgM cells and AGs in the intestinal mucosa of

S. 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 combined

form should complement each other and acquire different niches

within the gut microora environment for executing desirable

immune stimulatory and other benecial effects in host [70].

Nevertheless, the probiotic sources and their relatedness can also

affect the synergistic effects in combined form i.e., multispecies

containing different species may be more effective as compared to

multistrain probiotics[49]. For example, probiotics like Pdp11 and

51M6 which belong to Vibrionaceae family showed no synergistic

immunostimulatory activity in combined form as compared to

individual treatment in O. mykiss [49] butotherprobiotics belong todifferent families such as Lactobacillus and Bacillus species are

found to complement each other by exerting synergistic immu-

nomodulating responses in sh[70].

7.1.3. Spore former vs non-spore former

Bacteria belong to both spore former and non-spore formers are

used as probiotics. Several spore forming bacteria which produce

a wide range of antagonistic compounds can be valuable as pro-

biotics[139]. Among spore formers, Bacillus spores are routinely

being used as probiotics in human and animal practices due to their

immunostimulatory properties[140,141]. In aquaculture,B. subtilis

and B. licheniformis are most commonly used probiotics [139]. In

O. mykiss, Raida et al. [143]reported immunity enhancement and

signicant protection against yersioniosis by using commercialprobiotics containingB. subtilisand B. licheniformisspores.Bacillus

spores have been shown to increase the survival and production of

channel catsh[144].Similarly,B. subtilis spores when introduced

into rearing water eliminated Vibrio species from the larvae of

snook[145]. The spores ofBacillus toyoiand other Bacillus species

when used as feed additive increased the growth ofS. maximus

[146,147]and common snook (Centropomus undecimalis)[53].

Spore formers possess additional advantage that they can resist

adverse environmental conditions. The long term advantages of

using spores as probiotics is that they are heat-stable and can

survive transit across the stomach barrier, properties that cannot be

assured with other probiotics that are given in the vegetative form

[142]. However, the majority of probiotics currently available are

bacteria 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 promising

immunostimulatory results. Nevertheless, the combination of both

spore former and non- spore former are also found to increase

immunity in sh [70,72,76].

7.1.4. Viable vs non-viable

Probiotics, as per denition, are viable microorganisms with

documented benecial effects on the overall health status of host.

Viability is an important property of any probiotics which help

them to adhere and subsequent colonization in the intestinal tract

of host[148]. However, certain probiotics in inactivated form can

potentially elicit similar effects in host compared to viable

probiotics. Furthermore, several bacteria in non-viable form are


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found not only to adhere to tissue culture cells of animals[149,150]

but also to augment the systemic and mucosal immune responses

in host [151]. Therefore, the concept of incorporating inactivated

probiotics has surfaced for aquaculture practices especially due to

the fact that probiotics are usually found in a transient state and

often expelled out immediately after the withdrawal of the feed in

hydrobionts[70].

Different probiotics in inactivated form also exhibited promising

immunomodulatory and protection in various sh species.

Although, immunostimulating potency of inactivated probiotics

under in vitro[71]and in vivoconditions[64]as well as ability to

control diseases[54] has been documented, viable probiotics are

proved to be better stimulator of immune system in any animals

including sh [76,101,152,153]. In a comparative study, Panigrahi

etal. [64] reportedthat probiotic strain ofL. rhamnosus inviableform

is a better immune inducer compared to its heat inactivated form.

The immunomodulating activity of non-viable probionts could

possibly be attributed to the presence of certain conserved micro-

bial components such as capsular polysaccharides, peptidoglycans

and lipoteichoic acids which are the potent stimulator of piscine

immune system [153,154]. Therefore, the whole and/or certain

components of the inactivated probiotics are believed to interact

with 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 achieving

optimum benecial effects in any host [155,156]. The optimum

concentration of probiotics is not only required for establishment

and subsequent proliferation in gut but also need to exert various

benecial effects including immunostimulatory activity. Different

in vitro and in vivo studies indicate that immune response ofsh

varies with the concentration of probiotics. The dose of probiotics is

usually selected based on their ability to enhance the growth and

protection in host. For instance, Brunt et al. [48] determined the

effective dose of the probiotic strain belong to Bacillus species to be2 108 cells at which they have recorded least percentage

mortality in O. mykiss during challenge study. The in vitro stimu-

latory activities of probiotics like Pdp11, 51M6,L. delbrueckiisubsp.

lactisand B. subtilisare found to be dose dependent [71]. Similarly,

immunostimulatory activities of LAB and B. subtilisin sh underin

vivocondition also vary in a dose dependant manner [57,64].

In aquaculture the dose of probiotics usually varies from

106e10 CFU/g feed. The optimum dose of a probiotics can vary with

respect to host and also type of immune parameters.Panigrahi et al.

[63] recorded high serum lysozyme, phagocytic activity of head

kidney leucocyte and complement activities in O. mykissfed for 30

days withL. rhamnosusstrain at 1011 CFU/g feed but not at a dose of

109 CFU/g feed. Furthermore, stimulation of a particular immune

response with respect to different tissue/organ also varies withdose. For instance, elevation of lysozyme activity in serum and skin

inM. miiuyis reported at two different dosesi.e., 107 and 109 CFU of

C. 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 of

L. plantarum in terms of growth, immune enhancement and

protection. Therefore, lower dose can fail to stimulate the piscine

immune system while 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 sh on challenge study

despite enhancement of certain immune parameters at the

particular dose. Earlier, Nikoskelainen et al. [157] also recorded

higher percentage of mortality in O. mykiss fed at high dose of

L. rhamnosus(10

12

CFU/g feed) compared to lower dose (10

9

CFU/g

feed). Therefore, the dose of the individual probiotics needs to be

determined for a particular host.

7.3. Duration of feeding

Duration of the probiotics feeding is another important factor

that can affect the establishment, persistence and subsequent

induction of immune responses in a host. In sh most of the

benecial effects like live weight gain, improved immunity and

disease resistance have been recorded within a dietary probiotics

feeding regime of 1e10 weeks. The time course for optimum

induction of immune response differs with respect to probiotic

strain and also type of immune parameter.

The time course of probiotics feeding for stimulating innate

immunity can also vary among different strains of probiotics in the

same family [49]. Similarly difference in stimulating specic

immune parameter is also dependent on feeding duration. For

example Diaz-Rosales et al.[52]observed signicant enhancement

of respiratory burst activity by feeding probiotics for 60 days but

earlier Daz-Rosales et al. [50], failed to detect signicant

enhancement of this activity when fed the heat inactivated form of

the 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 humoral

immunity at 2 weeks of feeding regime and further supplementa-

tion lead to lowering at 3rd and 4th weeks of feeding. While some

researchers believe a long feeding regime is not necessary for pro-

biotics[49], the shorter feeding regime can cause sharp decline in

immune response in sh[64]. Such type of decline may be due the

failure of the probiotic strains to establish and multiply in the sh

gut. Though, a long dietary feeding regime is advantageous to host

in many aspects, more studies are required to establish the bene-

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 denition of probiotics in aquaculture

to microbial water additives and several probiotics are also

directly used as water additives with documented health and

environmental benets [79]. In sh, probiotics are applied in

different methods like bath, suspension and feed. However

supplementation of probiotics as feed additive is best method for

successful colonization and establishment in gut [19,23,158,159].

Oral administration of probiotics is more effective in enhancing

immunity as well as subsequent protection as compared to water

supplementation[76]. Likewise suspension or bioencapsulation of

probiotics is usually adopted for sh larvae [146,159e163]. Pro-

biotics like L. delbrueckii spp. delbrueckii when supplemented

through live carriers like rotifers and artemia succeeded in stimu-lating local immunity in larvae[66,105].

Apart from dietary supplementation, water borne uptake of

probiotics can also modulate the piscine immune system with

elevation of several immune parameters[76,78,79]. In a study Zhou

et al.[79] found that among three probiotics (B. subtilis,B. coagulans,

R. palustris) supplemented into water @1 107 CFU/ml in every

2 days for 40 days, B. coagulansand R. palustris, showed promising

result with improved growth, immunity and health status of

O. niloticus.

7.5. Environmental conditions

The effectiveness of probiotics is dependent on the successful

establishment of the probiotics in the gut. Several factors that


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inuence 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 the

performance of the probiotics. Mehrim[13]conducted the effect of

probiotics on the O. niloticus at different stocking density ranging

from 10 to 60

sh/m

3

and found best growth, haematolgical

parameters and economic efcacy of probiotics within a stocking

density of 30 sh/m3. Similarly, in laying hens improved immune

response and other benecial effects by using probiotics during

stress condition due to high temperature was also recorded [164].

Likewise probiotics can help to overcome stress due to salinity as

reported by slightly enhanced salinity tolerance of O. niloticus by

commercial probiotics[76].

Table 2

Effect of different probiotics supplementation on disease resistance ofshes.

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.uorescens S. salar 105e6 cells/ml 3e5 days A. salmonicida(No specic protection

on co-habitant challenge)

[24]

4 Pseudomonas,Micrococcus,VibriospeciesS. aurata 10

8

15 days Vibrio harveyi [29]

5 B. subtilis,

L. acidophilus

O. niloticus 107 2 Aeromonas hydrophila,Pseudomonasuorescens,

Streptococcus iniae

[42]

6 L. sakei,

L. lactis,

L. mesenteroides

O. mykiss 106 2 A. salmonicidassp.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 Bacillusspecies,

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. baltica

S. senegalensis 109 60 days Photobacterium damselaesubsp.piscicida [52]

12 Vibriouviales,

A. hydrophila,

M. luteus


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

13 Vibriouviales,

A. hydrophila,

Gram ve coccus


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 forA. sobria 2 A. bestiarum,Ichthyophthirius multiliis(Ich)

A. sobria (More effective against Ich)

[67]

B. thermosphacta 1010 forB. thermosphacta

19 C. butyricum O. mykiss 300mg/kg sh 3 days V. anguillarum [69]

20 K ocuria species O. mykiss 108 4 V. anguillarum [73]

21 L. plantarum Epi nephelus coioides 105a 4 Streptococcusspecies, IridovirusofE. coioides [74]

22 Commercial probiotics O. niloticus 1% 30a E. tarda [75]23 Commercial probiotics Carassius auratus,

Xiphophorus helleri

5 g/kg 30 days P.uorescens

(No signicant 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 Carnobacteriumspecies S. salar,

O. mykiss

5 107 2a A. salmonicida,

V. ordalii,

Y. ruckeri,

V. anguillarum,

(No specic protection against

V. anguillarum)

[159]

27 Commercial probiotics

(E. faecium, B. toyoi)

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

28 L. plantarum

L. mesenteroides

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

29 S. putrefaciens S. aurata 108 15 days L. anguillarum [169]

30 Roseobacterspecies S. maximus 10

7

CFU/ml e

V. anguillarum,Vibrio splendidus,

Pseudoalteromonassp.

[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 specic protection) [174]

33 C. divergens G. morhua 108 3 V. anguillarum(No specic protection) [175]

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


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However, in aquaculture it is a neglected aspect and no

systematic attempt has been made to correlate effect of probiotics

on the immunity of sh at various environmental conditions.

Temperature could be crucial since a probiotic would be most

effective when used in its optimum temperature range matches

that ofsh which is identical with surrounding environment[65].

Panighagi et al. [65]found better immunoefcacy ofE. faecium in

comparison to L. rhamnosusandB. subtilisdue to its mesophilic and

more psychrotolerant nature.

8. Probiotics and disease protection

Probiotic therapy offers a suitable alternative for controlling

pathogens thereby overcoming the adverse consequences of anti-

biotics and chemotherapeutic agents. In sh culture, probiotics

either in diet or bioencapsulation help in achieving natural resis-

tance and high survivability of larvae and post larvae of shes

[159,165]. Signicant increase in the mean weight and natural

survival rate of larvae ofS. maximusfed rotifers enriched in LAB as

well as high protection against a pathogenic Vibrio species was

recorded [147]. Probiotic like Pediococcus acidilactici is also found to

be effective against vertebral column compression syndrome in

O. mykiss[166].Furthermore, the effectiveness of probiotics in terms of

protection against infectious pathogens is often attributed to the

elevate 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 probiotics

feeding (Table 2). Furthermore, probiotics treatment leads to

better protection ofsh from multiple diseases[42,48,159]. Apart

from protection against bacterial pathogens, probiotics can protect

against viral and protozoan infections as well. Recently, successful

control of Ichthyophthiriasis (Ichthyophthirius multiliis, Ich) by

A. sobriain O. mykiss [67]and iridovirus of grouper E. coioides by

L. plantarum [74] is achieved.

Despite, several reports also indicate deleterious effects likereduced growth, non stimulation of immune responses and no

signicant protection by various probiotics in sh [24,165,174e177].

Therefore, detailed information of the probiotics and their mode of

action can enable the selection of effective strain with a more

credible scientic rationale[8].

9. Conclusions

The benecial effects of dietary supplements like probiotics,

prebiotics and synbiotics have been recorded in a wide range of

animal models including sh. Amongst probiotics concept has

already been established in aquaculture practices especially as

a promising alternative to chemicals and antibiotics[178,179]. Over

the years several candidate probiotics strains belonging to Gramve and Gram eve groups of bacteria are introduced into culture

practices. However, certain autochthonous probiotic strains belong

to 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 to

species specic advantages [18,180], many times lack scientic

credibility. Undoubtly, most of the probiotics are usually safe but

their safety is an important issue, especially in case of newly

introduced candidate species [181] as well as the possibility of

acquisition of genes encoding the virulence and antimicrobial drug

resistance traits from pathogens to probiotics through horizontal

transfer of genes [182,183]. Although, no such report has been

documented 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 of

microorganisms viz., non-pathogens, pathogens and probiotics

are co-existing in the intestinal tract of sh. Therefore, more

fundamental research is needednot only to address inter- andintra-

species differences among various probiotics but also to evaluate

their safety aspects. Nevertheless, looking into the fact that

most of the probiotics can exert immunomodulatory effect in

sh, a complete understanding of the interactions between gut

microbes, the intestinal epithelium, and the gut immune system is

also necessary so that proper strategy can be developed for stimu-

lating the local as well as systemic immunity through manipulation

of gut microbiota with suitable probiotics/prebiotics/synbiotics

without altering the intestinal homeostasis.

Acknowledgement

The author is thankful to Professor T. Nakanishi, Laboratory of

Fish Pathology, Department of Veterinary Medicine, Nihon

University, Japan for his guidance in writing and modifying the

article. The author is also thankful to Dr. S. C. Mukherjee, Central

Institute of Fisheries Education, Mumbai, India for his help in

preparing the review article.

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