A Review on the Interactions Between Gutmicrobiota and Innate Inmunity of Fish

8/12/2019 A Review on the Interactions Between Gutmicrobiota and Innate Inmunity of Fish

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M I N I R E V I E W

A review on the interactions between gut microbiota and innate

immunity of sh

Geovanny D. Gomez1 & Jose Luis Balcazar2

1Mariculture Research Laboratory, Ocean University of China, Qingdao, China; and 2 Instituto de Investigaciones Marinas, Consejo Superior de

Investigaciones Cientficas (CSIC), Eduardo Cabello, Vigo, Spain

Correspondence:Jos e Luis Balc azar,

Instituto de Investigaciones Marinas, Consejo

Superior de Investigaciones Cientficas (CSIC),

Eduardo Cabello 6, 36208 Vigo, Spain.

Tel.: 134 986 214 457; fax:134 986 292

762; e-mail: [email protected]

Received 20 April 2007; revised 12 September2007; accepted 12 September 2007.

First published online 17 December 2007.

DOI:10.1111/j.1574-695X.2007.00343.x

Editor: Willem van Leeuwen

Keywords

innate immunity; gut microbiota; probiotics;

fish.

Abstract

Although fish immunology has progressed in the last few years, the contribution

of the normal endogenous microbiota to the overall health status has been so

far underestimated. In this context, the establishment of a normal or protective

microbiota constitutes a key component to maintain good health, through

competitive exclusion mechanisms, and has implications for the development

and maturation of the immune system. The normal microbiota influences theinnate immune system, which is of vital importance for the disease resistance of

fish and is divided into physical barriers, humoral and cellular components. Innate

humoral parameters include antimicrobial peptides, lysozyme, complement

components, transferrin, pentraxins, lectins, antiproteases and natural antibodies,

whereas nonspecific cytotoxic cells and phagocytes (monocytes/macrophages and

neutrophils) constitute innate cellular immune effectors. Cytokines are an integral

component of the adaptive and innate immune response, particularly IL-1b,

interferon, tumor necrosis factor-a, transforming growth factor-b and several

chemokines regulate innate immunity. This review covers the innate immune

mechanisms of protection against pathogens, in relation with the installation and

composition of the normal endogenous microbiota in fish and its role on health.

Knowledge of such interaction may offer novel and useful means designingadequate therapeutic strategies for disease prevention and treatment.

Introduction

The health status of aquatic organisms is uniquely related to

their immediate environments, which can contain very high

concentrations of microorganisms. Many of these micro-

organisms are saprophytic, some are pathogenic and both

types are capable of infecting fish when conditions become

favorable for multiplication. However, under normal condi-

tions fish maintain a healthy status by defending themselves

against these potential invaders using a repertoire of innate

and specific defense mechanisms (Ellis, 2001).

The immune systems of fish and higher vertebrates are

similar and both have two integral components: (1) the

innate, natural or nonspecific defense system formed by a

series of cellular and humoral components, and (2) the

adaptive, acquired or specific immune system characterized

by the humoral immune response through the production

of antibodies and by the cellular immune response, which is

mediated by T-lymphocytes, capable of reacting specifically

with antigens.

The innate immune system, unlike the specific immune

system, lacks the ability to acquire memory and specific

recognition after an encounter with foreign agents. How-

ever, this system is quite important in fish since the synthesis

of antibodies is relatively slow in comparison with antibody

production in the higher vertebrates. An adaptive immune

response in ectothermic vertebrates takes considerable time

(e.g., antibody production in salmonids takes at least 46

weeks) to respond and is very temperature-dependent (Ellis,

2001).

The main function of the innate immune system, i.e., the

innate immune reactions mediated by monocytes/macro-

phages, comprises antigen presentation and regulation

of the functional balance of immune response related to

cytokine and chemokine receptor profiles. Although the

host has evolved various tolerogenic mechanisms allowing

FEMS Immunol Med Microbiol 52(2008) 145154 c 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved


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a peaceful and productive coexistence with its normal

endogenous microbiota, it remains highly responsive to

enteropathogenic bacteria. This discriminatory ability re-

presents a pivotal feature of efficient tolerance and homeo-

static mechanisms.

Recently, the use of gnotobiotic animals has shown that

bacteria have a profound impact on the anatomical, physio-logical and immunological development of the host (Rawls

et al., 2004). Thus, establishing a healthy microbiota plays an

important role in the generation of immunophysiologic

regulation in the host by providing crucial signals for the

development and maintenance of the immune system

(Salminenet al., 2005).

Therefore, the focus of this review will be primarily on

innate immune mechanisms of protection against pathogens

as well as on the composition of the gut microbiota in fish,

and particularly its role in maintaining health of the host.

Innate immune systemThe primary line of defense in fish is the skin and mucus

membranes. However, when pathogenic microorganisms

enter the host, cellular and humoral innate defense mechan-

isms are activated (Magnadottir, 2006). The most important

mechanism involved in this defense is phagocytic activity,

which will be described in detail later.

Epithelial barriers

Physical and chemical barriers, such as the dermis, epider-

mis, scales and mucus, constitute the first line of defense

against disease-causing microorganisms in fish. The epider-mal cells are capable of reacting against different aggressors

and the integrity of these cells is fundamental to maintaining

osmotic equilibrium, as well as impeding the entrance of

foreign agents (Shephard, 1994).

Mucus, composed mainly of glycoprotein, prevents the

colonization of foreign agents. The continuously main-

tained mucus layer provides a substrate in which the anti-

bacterial mechanisms can occur by virtue of biologically

active components including antibodies, antibacterial pep-

tides, lysozymes, complement proteins, lectins and pentraxins

(Nowak, 1999; Nagashimaet al., 2001; Hellioet al., 2002).

Innate humoral immunity

The body fluids of the fish contain proteins and peptides

that react against a great variety of microorganisms and

microbial products. These nitrogenous compounds form

part of the defense of the innate humoral immunity, and

consist of antimicrobial peptides, lysozyme, complement,

transferrin, pentraxins, lectins and antiproteases (Ellis,

1989).

Antimicrobial peptides (AMPs)

AMPs are present in tissues exposed to microorganisms

such as mucosal surfaces and skin (Cole et al., 1997) and

immune cells such as mast cells (Silphaduang & Noga, 2001;

Murrayet al., 2003). One type of AMPs expressed by fish

mast cells (also known as eosinophilic granule cells) is

piscidin, which has potent, broad-spectrum antibacterialactivity against fish pathogens (Silphaduang & Noga, 2001).

Recently, other AMPs present in gill mast cells have been

identified such as chrysophsin and pleurocidin, which have

been isolated from red sea bream (Chrysophrys major) and

winter flounder (Pleuronectes americanus), respectively (Iiji-

maet al., 2003; Murrayet al., 2003).

Lysozyme

Lysozyme is a cationic enzyme widely distributed in the

serum, mucus, kidney, spleen and intestine of the fish (Lie

et al., 1989). This enzyme is primarily associated with andsynthesized by monocytesmacrophages and neutrophils

(Murray & Fletcher, 1976; Nathan, 1987).

Lysozyme has the capacity to hydrolyze the chemical

bond between the N-acetylmuramic acid and N-acetylglu-

cosamine present in the peptidoglycan of bacterial cell walls.

Lysozyme is able to lyse certain Gram-positive bacteria and,

in conjunction with complement, even some Gram-negative

bacteria (Paulsenet al., 2001).

Complement

The complement system comprises more than 35 solubleplasma proteins that are key to innate and adaptive im-

munity. Activation of the complement system initiates a

cascade of biochemical reactions accompanied by the gen-

eration of biologically active mediators that result in antigen

elimination via cell membrane lysis and activation of non-

specific mediators of inflammation (Holland & Lambris,

2002). There are three pathways that can activate the

complement system: the classical pathway, which requires

the presence of the antigenantibody complex; the lectin

pathway, which depends on the interaction of lectins such as

mannose-binding lectin and ficolins with sugar moieties

found on the surface of microorganisms, and finally the

alternative pathway, which is activated directly by viruses,

bacteria, fungi or even tumor cells and is independent of

antibody (Boshraet al., 2006).

Transferrin

Transferrin, a bi-lobed monomeric glycoprotein, is respon-

sible for the transport and delivery of iron to cells. Binding

of iron to transferrin creates a bacteriostatic environment by

FEMS Immunol Med Microbiol52(2008) 145154c 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

146 G.D. G omez & J.L. Balc azar


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limiting the availability of iron to replicating pathogens.

Transferrin is also an acute phase protein invoked during an

inflammatory response to remove iron from damaged tissue

(Bayne & Gerwick, 2001) and also functions as an activator

of fish macrophages (Stafford & Belosevic, 2003).

Interferon

Interferons (IFNs) are secreted by host cells, including

macrophages, lymphocytes, natural killer cells and fibro-

blasts, in response to recognition of viral double-stranded

RNA intermediates (Halleret al., 2006).

Two families of interferons can be distinguished on the

basis of gene sequences, protein structure and functional

properties. Type I IFNs, represented by the IFN-a and the

IFN-b, which have a very similar biological activity. The

IFN-ais synthesized mainly by the leukocytes and IFN-bby

fibroblasts. Both types of interferons are produced in

response to viral infections.

Type II IFN, as represented by IFN-g, is produced by

natural killer cells and T-lymphocytes in response to IL-12,

IL-18, mitogens or antigens (Robertsen, 2006). In contrast

to type I IFNs, IFN-g is a key activator of macrophages for

increased killing of bacterial, protozoal and viral pathogens.

Pentraxins: C-reactive protein (CRP) and serum

amyloid protein (SAP)

Both CRP and SAP belong to a family of pentameric

proteins called the pentraxins that bind their ligands in a

calcium-dependent manner. They are commonly associated

with the acute phase response.CRP was discovered and named because of its reactivity

with the phosphorylcholine residues of C-polysaccharide,

the teichoic acid of Streptococcus pneumoniae (Tillett &

Francis, 1930). The main biologic function of CRP is the

ability to recognize pathogens and damaged cells of the host

and to mediate their elimination by recruiting the comple-

ment system and phagocytic cells (Volanakis, 2001). In

rainbow trout, CRP has showed opsonic activity for head

kidney cells, resulting in enhanced phagocytic and chemo-

kinetic activities (Kodamaet al., 1999).

CRP is distinguished from SAP by its binding affinity for

phosphorylcholine and phosphorylethanolamine. SAP only

binds to phosphory-ethanolamine and can be purified as a

result of its affinity for agarose.

Lectins

Lectins are usually constitutive proteins or glycoproteins,

which possess binding activity towards carbohydrate resi-

dues. They have been grouped into classes based on the

nature of their carbohydrate ligands, the biological processes

in which they participate, their subcellular localization and

their dependence on divalent cations (Drickamer & Taylor,

1993). A mannose-binding lectin, isolated from the serum

of Atlantic salmon, has been shown to have opsonizing

activity for a virulent strain of Aeromonas salmonicida

(Ottingeret al., 1999).

Antiproteases

These antienzymes are characterized by their capacity to

inhibit the action of proteases that some microorganisms

utilize to penetrate the host. In teleost fish, an analogous

protein to a1-antitrypsin was demonstrated (Hjelmeland,

1983). Another protein, which was demonstrated as homo-

logous to a2-macroglobulin (Starkey et al., 1982), was

reportedly capable of inhibiting several types of proteinases,

including serine-, cysteine-, aspartic- and metallo-protei-

nases (Alexander & Ingram, 1992).

In addition, it has been observed that a2-macroglobulin

present in the serum of rainbow trout is capable of inhibit-

ing A. salmonicida protease (Ellis, 1987). The combined

action of antithrombin anda2-macroglobulin in the plasma

of Atlantic salmon was reported to inhibit the action of

a serine protease ofA. salmonicida (Salteet al., 1992). The

differences in the a2-macroglobulin activity between the

species of rainbow trout and brook trout have been directly

correlated with their differing resistance to the infection

caused byA. salmonicida(Freedman, 1991).

Natural antibodies (NA)

NA are secreted by B-cells without prior antigen-specific

activation or antigen-driven selection. A large proportion of

NA is polyreactive to phylogenetically conserved structures,

such as nucleic acids, heat shock proteins, carbohydrates

and phospholipids (Boes, 2000). The importance of NA

functions in fish may be even greater than for higher

vertebrates given that fish have neither appreciable affinity

maturation responses nor class switch capabilities (Magor &

Magor, 2001).

Recently, Sinyakovet al. (2002) observed that NA in the

serum of goldfish (Carassius auratus) can be directly in-

volved in the first line of resistance against A. salmonicida

infection. In addition, these authors indicated that NA alsomay influence the level of antibody response since only the

low NA carriers were capable of developing effective anti-

body response, and vice versa, the high NA carriers did not

possess potential for active immunization.

Innate cellular immunity

The adaptive immunity effector function is mediated by

T-lymphocytes, whereas nonspecific cytotoxic cells and


147The role of gut microbiota in fish innate immunity


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phagocytes (monocytes/macrophages and neutrophils) con-

stitute innate cellular immune effectors.

Nonspecific cytotoxic cells (NCC)

The NCC perform functions very similar to those of the

higher vertebrates, acting on a wide variety of target cells,including allogeneic and xenogeneic tumor cells, virus-

infected cells and protozoan parasites. NCC may also

participate in antibacterial immunity by eliciting cytokine

production and secretion (Jaso-Friedmannet al., 2001).

Phagocytosis

The innate cellular immune system is formed by a series of

cells with essential functions to the host survival. Among

these cells are the phagocytic cells, monocytes/macrophages

and neutrophils, which play a fundamental role in protec-

tion and survival during adverse conditions. For example,antibody production is slow when there is a drop in

temperature, therefore the host defense will depend almost

exclusively on the phagocytic capacity.

Phagocytosis occurs when foreign objects such as bacteria

adhere to the surface of the phagocyte, mediated by hydro-

phobic interactions or sugar/lectin interactions (Secombes,

1996). However, the most active promoter of phagocytosis is

the C3 component of complement, which is bound to the

bacterial surface lipopolysaccharide directly via the alterna-

tive pathway or indirectly via lectin or CRP (Ellis, 2001).

Antimicrobial response of fish phagocytes

Fish macrophages and neutrophils produce bactericidal

reactive oxygen species (ROS) during the respiratory burst

on contact with the particles or during phagocytosis or upon

stimulation with a variety of agents. This process involves

reduction of oxygen (O2) to the anionic radical superoxide

(O2), which is catalyzed by an NADPH oxidase localized in

the plasma and phagosomal membranes. Production of

superoxide anion (O2) results in the spontaneous or en-

zyme-catalyzed production of an array of reactive oxygen

products including hydrogen peroxide (H2O2), hydroxyl

radical (OH ), hypochlorous acid (OCl) and peroxynitrite

(ONOO), which have potent antimicrobial effects.

Production of nitric oxide (NO) constitutes another

bactericidal mechanism, which is catalyzed by a NO

synthase. Schoor & Plumb (1994) demonstrated inducible

NO production, using enzyme histochemical techniques,

from the anterior kidney of channel catfish (Ictalurus

punctatus) infected with Edwardsiella ictaluri. Recently,

Stafford et al. (2001) have characterized the molecules

present in crude leukocyte supernatants that induce NO

production in goldfish macrophages, suggesting that trans-

ferrin appears to be an important mediator for the activa-

tion of both fish macrophages and granulocytes.

Integration of the immune response -- cytokines

Communication within the acquired immune system and

between the innate and acquired systems is brought about bydirect cell-to-cell contact involving adhesion molecules and

by the production of chemical messengers. Chief among these

chemical messengers are proteins called cytokines, which can

induce a broad range of activities via multiple target cell types

and their redundancy, indicated by the overlap in activities

among different cytokines (Engelsmaet al., 2002).

There are three functional categories of cytokines: (1)

cytokines that regulate innate immune response; (2) cyto-

kines that regulate adaptive immune response; and (3)

cytokines that stimulate hematopoiesis.

Cytokines that regulate innate immunity are produced

primarily by macrophages although they can also be pro-

duced by lymphocytes, NCC and other cells. They are

produced in response to microbial antigens or compounds

released from damaged cells. Among the mediators of

inflammation released by activated phagocytes are the cyto-

kines, particularly IL-1b, an important pro-inflammatory

cytokine, interferon, tumor necrosis factor-a (TNF-a), trans-

forming growth factor-b(TGF-b) and several chemokines.

TNF-ais one of the principal mediators of the inflamma-

tory response in mammals, transducing differential signals

that regulate cellular activation and proliferation, cytotoxicity

and apoptosis. When an inflammatory response is induced,

the cascade of cytokine secretion begins with the release of

TNF-a. This stimulates the release of IL-1b, which is thenfollowed by the release of IL-6. The initiation of inflammation

leads to the release of a myriad of other cytokines, which

include chemoattractants that signal neutrophils and macro-

phages to migrate to the site of infection (e.g. chemokines).

Influence of gut microbiota on thehealth of fish

As has been indicated previously, fish health status is depen-

dent on or conditioned to the immediate environment, since

they are intimately in contact with a wide variety of micro-

organisms, including pathogenic and opportunistic bacteria

that may colonize the external and internal body surfaces

(Ellis, 2001). Thus, the establishment of a normal or protec-

tive microbiota is a key component in excluding potential

invaders and maintaining health (Balcazaret al., 2006a). This

is accomplished through competitive exclusion mechanisms

and facilitates immune system development and maturation.

Colonization of the gastrointestinal tract of fish larvae

starts immediately after hatching and is completed within a

few hours. Colonizing bacteria can modulate expression of




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genes in the digestive tract, thus creating a favorable habitat

for themselves and preventing invasion by other bacteria

introduced later into the ecosystem (Balcazaret al., 2006b).

Traditionally, the influences of microbiota on the fish

host have been obtained from comparisons of the physiolo-

gical characteristics of germfree and conventional fish, but

comparative research of this type can now be performed atthe genomic level. The potential for obtaining exciting

knowledge of mechanistic influences of the microbiota on

the host by this approach has been demonstrated by the

pioneering work of Rawls & colleagues (2004), who studied

the effect of colonization by components of the microbiota

in zebrafish (Danio rerio). Some genes were always ex-

pressed, independent of the type of bacteria used, while

the expression of other genes was bacteria-specific, suggest-

ing that at least a subset of zebrafish genes is sensitive to

unknown factors induced by specific bacteria present in the

gut microbiota.

Composition of gut microbiota

The relatively recent introduction of molecular techniques

for the detection and quantification of microorganisms has

led to a greater understanding of microbial diversity and its

role in nature. Several studies involving molecular techni-

ques have demonstrated that bacteria are the main consti-

tuent of the gut microbiota in fish (Spanggaardet al., 2000;

Pond et al., 2006). However, some authors have also

reported the presence of yeast (Andlid et al., 1998; Gate-

soupe, 2007).

Although the composition of endogenous microbiota

depends on genetic, nutritional and environmental factors,

it is generally accepted that Gram-negative facultative

anaerobic bacteria such asAcinetobacter,Alteromonas,Aero-

monas, Flavobacterium/Cytophaga, Micrococcus, Moraxella,

Pseudomonasand Vibrioconstitute the predominant endo-

genous microbiota of a variety of species of marine fish

(Cahill, 1990; Onarheimet al., 1994; Blanchet al., 1997). In

contrast to saltwater fish, the endogenous microbiota of

freshwater fish species tends to be dominated by members

of the genera Aeromonas, Acinetobacter, Pseudomonas,

Flavobacterium, representatives of the familyEnterobacter-

iaceae, and obligate anaerobic bacteria of the generaBacter-

oides, Clostridium and Fusobacterium (Sakata, 1990; Huber

et al., 2004; Kimet al., 2007). In addition, various species oflactic acid bacteria have also been demonstrated to comprise

part of this microbiota (Ring & Gatesoupe, 1998; Balcazar

et al., 2007a).

Immunity to bacterial pathogens

The external surface of fish is covered by a mucus layer,

which acts as a medium for biologically active molecules

(e.g. antibacterial peptides, lysozyme, lectins and proteases),

and functions as the primary barrier to the adhesion and

penetration of bacterial pathogens. Moreover, the gastro-

intestinal tract contains a diverse and complex endogenous

microbiota, acids, bile salts and enzymes that can create a

hostile environment for many pathogens. In most cases

these properties are sufficient to protect against bacterial

pathogens, which often only produce disease when condi-tions become favorable for their multiplication. If bacterial

pathogens can breach these early lines of defense, cellular

and humoral mechanisms are activated for preventing

further spread of the infection. The complement system

plays an essential role in alerting the host of the presence of

microbial pathogens, as well as in their clearing. Comple-

ment can be activated directly by foreign surfaces and

also indirectly by other factors, principally CRP and lectin.

Plasma also contains a number of soluble factors like

antibacterial peptides, proteases and acute-phase proteins

(pentraxins, transferrin, a2-macroglobulin, complement

component C3, lysozyme and lectins). At the same time

the cellular component of innate immunity is activated

upon the recognition of pathogen-derived pathogen-

associated molecular patterns, including lipopolysaccharide

and double-stranded RNA as well as by host-derived cyto-

kines. The latter group includes typical proinflammatory

cytokines such as IL-1b, TNF-aand chemokines, which are

of pivotal importance in recruiting monocytes/macrophages

and neutrophils to the site of inflammation (Huising et al.,

2003).

Probiotics as a strategy for improving health

The demonstration that the gut microbiota is an importantcomponent of mucosal barrier has resulted in the promo-

tion of the use of beneficial probiotics. Probiotics have been

defined by the World Health Organization-Food and Agri-

culture Organization as live microorganisms which when

administered in adequate amounts, confer a health benefit

on the host (FAO/WHO, 2001).

Probiotic microorganisms consist mostly of strains of

Bacillus(Salinaset al., 2005; Panigrahiet al., 2007),Carno-

bacterium (Robertson et al., 2000; Irianto & Austin, 2002;

Kim & Austin, 2006a) and Lactobacillus (Nikoskelainen

et al., 2001b; Panigrahi et al., 2004; Vendrell et al., 2007;

Balcazaret al., 2007c), although the use of other species such

as Aeromonas and Vibrio has also been explored (Austin

et al., 1995; Irianto & Austin, 2002; Brunt & Austin, 2005).

Intake of probiotics has been demonstrated to modify the

composition of the microbiota, and therefore assist in

returning a disturbed microbiota (by antibiotics or other

risk factors) to its normal beneficial composition. Mechan-

isms that may be implicated include the production of

antimicrobial substances such as organic acids or bacterio-

cins (Balcazar et al ., 2006c, 2007b), competition for




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nutrients or adhesion receptors (Nikoskelainenet al., 2001a;

Vine et al., 2004; Balcazar et al., 2007b), inhibition of

virulence gene expression (Defoirdt, 2007), and enhance-

ment of the immune response (Nikoskelainen et al., 2003;

Kim & Austin, 2006a; Balcazaret al., 2007c, d).

There is increasing evidence that probiotics enhance

innate host resistance to microbial pathogens (Table 1).The findings of Irianto & Austin (2002) demonstrated that

after feeding rainbow trout with probiotics containing

Aeromonas hydrophila, Vibrio fluvialis, Carnobacterium sp.

andMicrococcus luteusfor 2 weeks, stimulation of humoral

and cellular immunity was detected as demonstrated by an

increase in lysozyme activity and in the number of erythro-

cytes, macrophages and lymphocytes. This finding offers an

important example of the ability of nonpathogenic, endo-

genous microbial species to enhance the immunological

functions of the host.

Probiotic strains have been shown to modulate the innate

humoral responses and thereby facilitate the exclusion of

potential pathogens. Panigrahi et al. (2004) fed rainbow

trout a diet containing the probioticLactobacillus rhamnosus

JCM1136. Evidence of an enhanced innate immune re-

sponse was observed, including increased levels of serum

lysozyme and complement activity. Similar observations

have been described by Balcazaret al. (2007d), who demon-

strated a positive effect on humoral immune response

following probiotic administration (Lactococcus lactis ssp.

lactis, Leuconostoc mesenteroides and Lactobacillus sakei) inbrown trout (Salmo trutta).

An enhancement of phagocytic activity, which is respon-

sible for early activation of the inflammatory response

before antibody production, has also been reported in fish.

Pirarat et al. (2006) demonstrated that after feeding tilapia

(Oreochromis niloticus) withLactobacillus rhamnosusATCC

53103 for 2 weeks, stimulation of cellular immunity was

detected as demonstrated by an increase in phagocytic

activity. Similarly, Balcazar et al. (2006d) observed after

feeding rainbow trout with probiotics containingLactococ-

cus lactis ssp. lactis, Leuconostoc mesenteroides and Lactoba-

cillus sakei for 2 weeks, an enhanced phagocytosis of

Aeromonas salmonicidaby leukocytes isolated from muco-

sa-associated lymphoid tissues.

Table 1. Probiotics used in fish and the effect on their host

Probiotic strain Host species Effect Reference

Vibrio fluvialesA3-47S,

Aeromonas hydroph ilaA3-51,

Carnobacterium sp. BA211,

Micrococcus luteusA1-6

Oncorhynchus

mykiss

Immune stimulation and improved survival after

challenge withAeromonas salmonicida

Irianto & Austin

(2002)

Lactobacillus rhamnosusATCC 53103 Oncorhynchus

mykiss


challenge withAeromonas salmonicida

Nikoskelainenet al.

(2001b, 2003)

Lactococcus lactisCECT 539 Scophthalmus

maximus

Immune stimulation Villamilet al. (2002)

Lactobacillus rhamnosusJCM 1136 Oncorhynchus

mykiss

Immune stimulation Panigrahiet al. (2004)

Lactobacillus delbriieckiiCECT 287,

Bacillus subtilisCECT 35

Sparus aurata Immune stimulation Salinaset al. (2005)

Aeromonas sobria GC2 Oncorhynchus

mykiss


challenge withLactococcus garvieaeand

Streptococcus iniae

Brunt & Austin (2005)

Bacillus subtilis, Lactobacillus acidophilus,

Clostridium butyricum, Saccharomyces

cerevisiae

Paralichthys

olivaceus


challenge withVibrio anguillarum

Taokaet al. (2006)

Carnobacterium maltaromaticumB26

Carnobacterium divergensB33

Oncorhynchus

mykiss


challenge withAeromonas salmonicidaandYersinia

ruckeri. Expression of cytokine genes

Kim & Austin

(2006a, b)

Lactobacillus rhamnosusATCC 53103 Oreochromis

niloticus


challenge withEdwardsiella tarda

Piraratet al. (2006)

Lactobacillus rhamnosusATCC 53103

Bacillus subtilis

Enterococcus faecium

Oncorhynchus

mykiss

Immune stimulation and expression of cytokine

genes

Panigrahiet al. (2007)

Lactobacillus sakeiCLFP 202,

Lactococcus lactisCLFP 100

Leuconostoc mesenteroidesCLFP 196

Oncorhynchus

mykiss,Salmo trutta

Immune stimulation and improved survival

after challenge withAeromonas salmonicida

Balc azaret al.

(2006d, 2007c,

2007d)

Lactobacillus plantarumCLFP 238

Leuconostoc mesenteroidesCLFP 196

Oncorhynchus

mykiss

Competitive exclusion and improved survival

after challenge withLactococcus garvieae

Vendrellet al. (2007)




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Probiotics can also modify the immune response of the

host by interacting with epithelial cells and by modulating

the secretion of anti-inflammatory cytokines, which could

result in a reduction of inflammation. Recently, studies

showed that IL-1b, IL-8, TNF-a, and TGF-bexpression was

not induced in rainbow trout gut cells following adminis-

tration of the probiotic bacteria Carnobacterium maltaro-maticumB26 and Carnobacterium divergensB33. However,

detection of significantly higher IL-1b and TNF-a expres-

sion in head kidney cells indicates induction of an anti-

inflammatory effect (Kim & Austin, 2006b).

Selecting probiotic strains

To be a probiotic, a bacterial strain has to fulfil several

criteria. Potential probiotics must be safe and free of

plasmid-encoded antibiotic resistance genes, that could be

passed to pathogenic organisms in the host. They must

persist in the gastrointestinal tract long enough to elicit an

effect. Ability to adhere and persist are also closely relatedto potential immune effects. They must have the ability

to improve host health, they must be amenable

to industrial processes necessary for commercial production

and finally they must remain viable in the food product and

during storage (Verschuere et al., 2000; Vine et al., 2006;

Balcazaret al., 2006b).

Concluding remarks

The maintenance of a healthy status is complex and relies on

a delicate balance between the immune system and the

normal endogenous microbiota. The normal microbiota

confers many benefits to the intestinal physiology of thehost. Some of these benefits include the metabolism of

nutrients and organic substrates, and the contribution of

the phenomenon of colonization resistance. However, when

this balance is upset, pathogens that arrive or that have

already been present but in numbers too small to cause

disease take the opportunity to multiply. The chemother-

apeutic agents may also have a greater effect on the host

normal microbiota than on the pathogens, thus upsetting

the balance.

Therefore, probiotic supplementation can assist in re-

turning a disturbed microbiota to its normal beneficial

composition, and influence the fish immune response in

different ways. They can induce the proportion of phagocy-

tically active cells and the activation of complement receptor

expression. They also can modulate the secretion of anti-

inflammatory cytokines.

Understanding how the fish immune system generally

responds to gut microbiota may be an important basis for

targeting manipulation of the microbial composition. This

might be of special interest to design adequate therapeutic

strategies for disease prevention and treatment.

Acknowledgements

J.L.B. was supported by a postdoctoral I3P contract from

Consejo Superior de Investigaciones Cientficas (CSIC). The

authors thank J. Rhodes, C. Peter and L. Rivera for critical

reading of the manuscript.

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