MI NI R E V I E W

Areviewontheinteractions betweengut microbiotaandinnate

immunityof sh
Geovanny D. G omez
1
& Jos e Luis Balc azar
2
1
Mariculture Research Laboratory, Ocean University of China, Qingdao, China; and
2
Instituto de Investigaciones Marinas, Consejo Superior de
Investigaciones Cientcas (CSIC), Eduardo Cabello, Vigo, Spain
Correspondence: Jos e Luis Balc azar,
Instituto de Investigaciones Marinas, Consejo
Superior de Investigaciones Cientcas (CSIC),
Eduardo Cabello 6, 36208 Vigo, Spain.
Tel.: 134 986 214 457; fax: 134 986 292
762; e-mail: balcazar@iim.csic.es
Received 20 April 2007; revised 12 September
2007; 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;
sh.
Abstract
Although sh 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 inuences the
innate immune system, which is of vital importance for the disease resistance of
sh 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 nonspecic 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 sh and its role on health.
Knowledge of such interaction may offer novel and useful means designing
adequate 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 sh when conditions become
favorable for multiplication. However, under normal condi-
tions sh maintain a healthy status by defending themselves
against these potential invaders using a repertoire of innate
and specic defense mechanisms (Ellis, 2001).
The immune systems of sh and higher vertebrates are
similar and both have two integral components: (1) the
innate, natural or nonspecic defense system formed by a
series of cellular and humoral components, and (2) the
adaptive, acquired or specic 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 specically
with antigens.
The innate immune system, unlike the specic immune
system, lacks the ability to acquire memory and specic
recognition after an encounter with foreign agents. How-
ever, this system is quite important in sh 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 proles. Although the
host has evolved various tolerogenic mechanisms allowing
FEMS Immunol Med Microbiol 52 (2008) 145154 c 2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
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 efcient 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
(Salminen et 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 sh,
and particularly its role in maintaining health of the host.
Innate immune system
The primary line of defense in sh is the skin and mucus
membranes. However, when pathogenic microorganisms
enter the host, cellular and humoral innate defense mechan-
isms are activated (Magnad ottir, 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 rst line of defense
against disease-causing microorganisms in sh. 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; Nagashima et al., 2001; Hellio et al., 2002).
Innate humoral immunity
The body uids of the sh 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;
Murray et al., 2003). One type of AMPs expressed by sh
mast cells (also known as eosinophilic granule cells) is
piscidin, which has potent, broad-spectrum antibacterial
activity against sh pathogens (Silphaduang & Noga, 2001).
Recently, other AMPs present in gill mast cells have been
identied such as chrysophsin and pleurocidin, which have
been isolated from red sea bream (Chrysophrys major) and
winter ounder (Pleuronectes americanus), respectively (Iiji-
ma et al., 2003; Murray et al., 2003).
Lysozyme
Lysozyme is a cationic enzyme widely distributed in the
serum, mucus, kidney, spleen and intestine of the sh (Lie
et al., 1989). This enzyme is primarily associated with and
synthesized 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 (Paulsen et al., 2001).
Complement
The complement system comprises more than 35 soluble
plasma 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-
specic mediators of inammation (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 colins with sugar moieties
found on the surface of microorganisms, and nally the
alternative pathway, which is activated directly by viruses,
bacteria, fungi or even tumor cells and is independent of
antibody (Boshra et 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 Microbiol 52 (2008) 145154 c 2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
146 G.D. G omez & J.L. Balc azar
limiting the availability of iron to replicating pathogens.
Transferrin is also an acute phase protein invoked during an
inammatory response to remove iron from damaged tissue
(Bayne & Gerwick, 2001) and also functions as an activator
of sh macrophages (Stafford & Belosevic, 2003).
Interferon
Interferons (IFNs) are secreted by host cells, including
macrophages, lymphocytes, natural killer cells and bro-
blasts, in response to recognition of viral double-stranded
RNA intermediates (Haller et 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-a is synthesized mainly by the leukocytes and IFN-b by
broblasts. 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 (Kodama et al., 1999).
CRP is distinguished from SAP by its binding afnity for
phosphorylcholine and phosphorylethanolamine. SAP only
binds to phosphory-ethanolamine and can be puried as a
result of its afnity 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
(Ottinger et 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 sh, an analogous
protein to a
1
-antitrypsin was demonstrated (Hjelmeland,
1983). Another protein, which was demonstrated as homo-
logous to a
2
-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 a
2
-macroglobulin
present in the serum of rainbow trout is capable of inhibit-
ing A. salmonicida protease (Ellis, 1987). The combined
action of antithrombin and a
2
-macroglobulin in the plasma
of Atlantic salmon was reported to inhibit the action of
a serine protease of A. salmonicida (Salte et al., 1992). The
differences in the a
2
-macroglobulin activity between the
species of rainbow trout and brook trout have been directly
correlated with their differing resistance to the infection
caused by A. salmonicida (Freedman, 1991).
Natural antibodies (NA)
NA are secreted by B-cells without prior antigen-specic
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 sh may be even greater than for higher
vertebrates given that sh have neither appreciable afnity
maturation responses nor class switch capabilities (Magor &
Magor, 2001).
Recently, Sinyakov et al. (2002) observed that NA in the
serum of goldsh (Carassius auratus) can be directly in-
volved in the rst line of resistance against A. salmonicida
infection. In addition, these authors indicated that NA also
may inuence 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 nonspecic cytotoxic cells and
FEMS Immunol Med Microbiol 52 (2008) 145154 c 2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
147 The role of gut microbiota in sh innate immunity
phagocytes (monocytes/macrophages and neutrophils) con-
stitute innate cellular immune effectors.
Nonspecic 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-Friedmann et 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 sh 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 (O
2
) to the anionic radical superoxide
(O
2

), which is catalyzed by an NADPH oxidase localized in

the plasma and phagosomal membranes. Production of
superoxide anion (O
2

) results in the spontaneous or en-

zyme-catalyzed production of an array of reactive oxygen
products including hydrogen peroxide (H
2
O
2
), 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 catsh (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 goldsh macrophages, suggesting that trans-
ferrin appears to be an important mediator for the activa-
tion of both sh 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 by
direct 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 (Engelsma et 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
inammation released by activated phagocytes are the cyto-
kines, particularly IL-1b, an important pro-inammatory
cytokine, interferon, tumor necrosis factor-a (TNF-a), trans-
forming growth factor-b (TGF-b) and several chemokines.
TNF-a is one of the principal mediators of the inamma-
tory response in mammals, transducing differential signals
that regulate cellular activation and proliferation, cytotoxicity
and apoptosis. When an inammatory response is induced,
the cascade of cytokine secretion begins with the release of
TNF-a. This stimulates the release of IL-1b, which is then
followed by the release of IL-6. The initiation of inammation
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 the
health of fish
As has been indicated previously, sh 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 (Balc azar et al., 2006a). This
is accomplished through competitive exclusion mechanisms
and facilitates immune system development and maturation.
Colonization of the gastrointestinal tract of sh larvae
starts immediately after hatching and is completed within a
few hours. Colonizing bacteria can modulate expression of
FEMS Immunol Med Microbiol 52 (2008) 145154 c 2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
148 G.D. G omez & J.L. Balc azar
genes in the digestive tract, thus creating a favorable habitat
for themselves and preventing invasion by other bacteria
introduced later into the ecosystem (Balc azar et al., 2006b).
Traditionally, the inuences of microbiota on the sh
host have been obtained from comparisons of the physiolo-
gical characteristics of germfree and conventional sh, but
comparative research of this type can now be performed at
the genomic level. The potential for obtaining exciting
knowledge of mechanistic inuences 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 zebrash (Danio rerio). Some genes were always ex-
pressed, independent of the type of bacteria used, while
the expression of other genes was bacteria-specic, suggest-
ing that at least a subset of zebrash genes is sensitive to
unknown factors induced by specic bacteria present in the
gut microbiota.
Composition of gut microbiota
The relatively recent introduction of molecular techniques
for the detection and quantication 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 sh (Spanggaard et 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 as Acinetobacter, Alteromonas, Aero-
monas, Flavobacterium/Cytophaga, Micrococcus, Moraxella,
Pseudomonas and Vibrio constitute the predominant endo-
genous microbiota of a variety of species of marine sh
(Cahill, 1990; Onarheim et al., 1994; Blanch et al., 1997). In
contrast to saltwater sh, the endogenous microbiota of
freshwater sh species tends to be dominated by members
of the genera Aeromonas, Acinetobacter, Pseudomonas,
Flavobacterium, representatives of the family Enterobacter-
iaceae, and obligate anaerobic bacteria of the genera Bacter-
oides, Clostridium and Fusobacterium (Sakata, 1990; Huber
et al., 2004; Kim et al., 2007). In addition, various species of
lactic acid bacteria have also been demonstrated to comprise
part of this microbiota (Ring & Gatesoupe, 1998; Balc azar
et al., 2007a).
Immunity to bacterial pathogens
The external surface of sh 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 sufcient 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, a
2
-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 proinammatory
cytokines such as IL-1b, TNF-a and chemokines, which are
of pivotal importance in recruiting monocytes/macrophages
and neutrophils to the site of inammation (Huising et al.,
2003).
Probiotics as a strategy for improving health
The demonstration that the gut microbiota is an important
component of mucosal barrier has resulted in the promo-
tion of the use of benecial probiotics. Probiotics have been
dened by the World Health Organization-Food and Agri-
culture Organization as live microorganisms which when
administered in adequate amounts, confer a health benet
on the host (FAO/WHO, 2001).
Probiotic microorganisms consist mostly of strains of
Bacillus (Salinas et al., 2005; Panigrahi et 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;
Balc azar et 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 benecial composition. Mechan-
isms that may be implicated include the production of
antimicrobial substances such as organic acids or bacterio-
cins (Balc azar et al., 2006c, 2007b), competition for
FEMS Immunol Med Microbiol 52 (2008) 145154 c 2007 Federation of European Microbiological Societies
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149 The role of gut microbiota in sh innate immunity
nutrients or adhesion receptors (Nikoskelainen et al., 2001a;
Vine et al., 2004; Balc azar et al., 2007b), inhibition of
virulence gene expression (Defoirdt, 2007), and enhance-
ment of the immune response (Nikoskelainen et al., 2003;
Kim & Austin, 2006a; Balc azar et al., 2007c, d).
There is increasing evidence that probiotics enhance
innate host resistance to microbial pathogens (Table 1).
The ndings of Irianto & Austin (2002) demonstrated that
after feeding rainbow trout with probiotics containing
Aeromonas hydrophila, Vibrio uvialis, Carnobacterium sp.
and Micrococcus luteus for 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 nding 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 probiotic Lactobacillus 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 Balc azar et al. (2007d), who demon-
strated a positive effect on humoral immune response
following probiotic administration (Lactococcus lactis ssp.
lactis, Leuconostoc mesenteroides and Lactobacillus sakei) in
brown trout (Salmo trutta).
An enhancement of phagocytic activity, which is respon-
sible for early activation of the inammatory response
before antibody production, has also been reported in sh.
Pirarat et al. (2006) demonstrated that after feeding tilapia
(Oreochromis niloticus) with Lactobacillus rhamnosus ATCC
53103 for 2 weeks, stimulation of cellular immunity was
detected as demonstrated by an increase in phagocytic
activity. Similarly, Balc azar et al. (2006d) observed after
feeding rainbow trout with probiotics containing Lactococ-
cus lactis ssp. lactis, Leuconostoc mesenteroides and Lactoba-
cillus sakei for 2 weeks, an enhanced phagocytosis of
Aeromonas salmonicida by leukocytes isolated from muco-
sa-associated lymphoid tissues.
Table 1. Probiotics used in sh and the effect on their host
Probiotic strain Host species Effect Reference
Vibrio uviales A3-47S,
Aeromonas hydrophila A3-51,
Carnobacterium sp. BA211,
Micrococcus luteus A1-6
Oncorhynchus
mykiss
Immune stimulation and improved survival after
challenge with Aeromonas salmonicida
Irianto & Austin
(2002)
Lactobacillus rhamnosus ATCC 53103 Oncorhynchus
mykiss
Immune stimulation and improved survival after
challenge with Aeromonas salmonicida
Nikoskelainen et al.
(2001b, 2003)
Lactococcus lactis CECT 539 Scophthalmus
maximus
Immune stimulation Villamil et al. (2002)
Lactobacillus rhamnosus JCM 1136 Oncorhynchus
mykiss
Immune stimulation Panigrahi et al. (2004)
Lactobacillus delbriieckii CECT 287,
Bacillus subtilis CECT 35
Sparus aurata Immune stimulation Salinas et al. (2005)
Aeromonas sobria GC2 Oncorhynchus
mykiss
Immune stimulation and improved survival after
challenge with Lactococcus garvieae and
Streptococcus iniae
Brunt & Austin (2005)
Bacillus subtilis, Lactobacillus acidophilus,
Clostridium butyricum, Saccharomyces
cerevisiae
Paralichthys
olivaceus
Immune stimulation and improved survival after
challenge with Vibrio anguillarum
Taoka et al. (2006)
Carnobacterium maltaromaticum B26
Carnobacterium divergens B33
Oncorhynchus
mykiss
Immune stimulation and improved survival after
challenge with Aeromonas salmonicida and Yersinia
ruckeri. Expression of cytokine genes
Kim & Austin
(2006a, b)
Lactobacillus rhamnosus ATCC 53103 Oreochromis
niloticus
Immune stimulation and improved survival after
challenge with Edwardsiella tarda
Pirarat et al. (2006)
Lactobacillus rhamnosus ATCC 53103
Bacillus subtilis
Enterococcus faecium
Oncorhynchus
mykiss
Immune stimulation and expression of cytokine
genes
Panigrahi et al. (2007)
Lactobacillus sakei CLFP 202,
Lactococcus lactis CLFP 100
Leuconostoc mesenteroides CLFP 196
Oncorhynchus
mykiss, Salmo trutta
Immune stimulation and improved survival
after challenge with Aeromonas salmonicida
Balc azar et al.
(2006d, 2007c,
2007d)
Lactobacillus plantarum CLFP 238
Leuconostoc mesenteroides CLFP 196
Oncorhynchus
mykiss
Competitive exclusion and improved survival
after challenge with Lactococcus garvieae
Vendrell et al. (2007)
FEMS Immunol Med Microbiol 52 (2008) 145154 c 2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
150 G.D. G omez & J.L. Balc azar
Probiotics can also modify the immune response of the
host by interacting with epithelial cells and by modulating
the secretion of anti-inammatory cytokines, which could
result in a reduction of inammation. Recently, studies
showed that IL-1b, IL-8, TNF-a, and TGF-b expression was
not induced in rainbow trout gut cells following adminis-
tration of the probiotic bacteria Carnobacterium maltaro-
maticum B26 and Carnobacterium divergens B33. However,
detection of signicantly higher IL-1b and TNF-a expres-
sion in head kidney cells indicates induction of an anti-
inammatory effect (Kim & Austin, 2006b).
Selecting probiotic strains
To be a probiotic, a bacterial strain has to full 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 related
to potential immune effects. They must have the ability
to improve host health, they must be amenable
to industrial processes necessary for commercial production
and nally they must remain viable in the food product and
during storage (Verschuere et al., 2000; Vine et al., 2006;
Balc azar et 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 benets to the intestinal physiology of the
host. Some of these benets 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 benecial
composition, and inuence the sh 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-
inammatory cytokines.
Understanding how the sh 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 Cientcas (CSIC). The
authors thank J. Rhodes, C. Peter and L. Rivera for critical
reading of the manuscript.
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