Fish & Shellfish Immunology 29 (2010) 2e14

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Fish & Shellfish Immunology

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Review

Probiotics and immunity: A fish perspective

S.K. 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 a b s t r a c t

Article history: Probiotics are usually live microorganisms which when administered in adequate amounts confer
Received 6 November 2009 a health benefits on host. Nowadays, probiotics are also becoming an integral part of the aquaculture
Received in revised form practices to obtain high production. The common probiotics that are used for aquaculture practices
12 February 2010
include Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Carnobacterium, Shewanella, Bacillus, Aero-
Accepted 19 February 2010
Available online 26 February 2010
monas, Vibrio, Enterobacter, Pseudomonas, Clostridium, and Saccharomyces species. The involvement of
probiotics in nutrition, disease resistance and other beneficial activities in fish has proven beyond any
doubt. Among the numerous health benefits attributed to probiotics, modulation of immune system is
Keywords:
Fish
one of the most commonly purported benefits of the probiotics and their potency to stimulate the
Gut immunity systemic and local immunity under in vitro and in vivo conditions is noteworthy. Different probiotics
Innate immunity either monospecies or multispecies supplementation can eventually elevate phagocytic, lysozyme,
Probiotics complement, respiratory burst activity as well as expression of various cytokines in fish. Similarly,
probiotics can stimulate the gut immune system of fish with marked increase in the number of Igþ cells
and acidophilic granulocytes. Furthermore, mono-bacterial association studies (with non-probiotic
bacterial strains) in gnotobiotic fish also indicate the up-regulation of various immune related genes.
Though the exact mode of action of probiotics is yet to be established in any animal including fish,
probiotics often exert host specific and strain specific differences in their activities. Various factors like
source, type, dose and duration of supplementation of probiotics can significantly affect the immuno-
modulatory activity of probiotics. The review is therefore, aiming to highlight the immunomodulatory
activity of probiotics and also to evaluate the factors that regulate for the optimum induction of immune
responses in fish.
Ó 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 beneficial
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 beneficial
microorganisms are usually referred as “probiotics” which after
administration can able to colonize and multiply in the gut of host
and execute numerous beneficial effects by modulating various
biological systems in host [2]. Probiotics are originally defined as
the organisms and substances which contribute to the intestinal
microbial balance [3]. The term probiotic was originated from the
E-mail address: sukantanayak@rediffmail.com
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 status of the host organism. According to
the currently adopted definition by Food and Agricultural Organi-
zation/World Health Organization, probiotics are live microorgan-
isms which when administered in adequate amounts confer
a health benefit on the host [5].
Probiotics, thus, open a new era in health management strategy
from human to fish/shellfish. Probiotics are gaining increasing
scientific 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 specific microbe(s) in the intestine for restoring the
intestinal microbial balance and exerting numerous beneficial
effects in host [2,8,9].

1050-4648/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fsi.2010.02.017
 S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e14
2. Probiotics in fish 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 fish and shellfish and
consumer's demand for fish 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-
sification 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 and also 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 are
commercially available for aquaculture practices [16e20]. Apart
from the nutritional and other health benefits [21e25], certain
probiotics as water additives can also play a significant role in
decomposition of organic matter, reduction of nitrogen and phos-
phorus level as well as control of ammonia, nitrite, and hydrogen
sulfide [26].
Numerous microbes have been identified 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 efficiency 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 several
other compounds such as antibiotics, bacteriocins, siderophores,
lysozyme [31e35] and also modulate physiological and immuno-
logical responses in fish [36,37].
3. Probiotics and fish immunity
Among the numerous beneficial effects of probiotics, modula-
tion of immune system is one of the most commonly purported
benefits 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 fish,
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 in
piscine system. A lot of immunological studies have been performed
in several fish 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 fish.
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 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
3
induced by different probiotics [80]. Unlike other animals, pro-
biotics also modulate various immunohaematological parameters
in teleosts and is presented in Table 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
fish [53,57]. Similarly, probiotics, in both in vitro and in vivo
conditions, actively stimulate the proliferation of B lymphocytes in
fish. Elevation of immunoglobulin level by probiotics supplemen-
tation is reported in many animals including fish [27,58,63].
Furthermore, Song et al. [75] recorded high immunoglobulin 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 in fish
[64] and even one week supplementation of probiotioc like Lacto-
bacillus rhamnosus @2.8  108 CFU/g feed was found to significantly
increase the immunoglobulin level in rainbow trout (Oncorhynchus
mykiss) [60]. However, Balczar et al. [44] only found rise in
immunoglobulin level in Salmo trutta but not at significant 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
inflammatory 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. lactis and 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 fish species [47,48,54,63,67]. In tilapia (Oreo-
chromis niloticus) a 2 weeks feeding of L. rhamnosus significantly
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 fish. The findings of respiratory burst activity
following probiotics treatment in fish are often contradictory.
While some studies indicate probiotics do not have significant
impact on this non-specific defense mechanism of fish [52,58,73],
several in vitro and in vivo studies showed significant increase in
respiratory burst activity by various probiotics in many aquatic
animals including fish. Probiotics like Bacillus subtilis and certain
members of LAB group can stimulate respiratory burst activity in
fish [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 fish to fight against
4 S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e14

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

Sl. Probiotics Form of Mode of Assay Immunological effects Reference

no probiotics Probiotics conditions
supplementation
1 Bacillus subtilis, Viable Individual and In vivo Respiratory burst activity ([), Serum bactericidal activity ([C), [42]
Lactobacillus acidophilus Combination Neutrophil adherence ([), Lysozyme ([), Heamtocrit percentage ([Y)
2 Lactobacillus sakei, Viable Individual In vivo Respiratory burst activity against live A. salmonicida ([ only in [43]
Lactococcus lactis, L. lactis), Respiratory burst activity against dead A. salmonicida ([)
Leuconostoc mesenteroides
3 Lactobacillus sakei, Viable Individual In vivo Immunoglobulin ([Y), Lysozyme ([ L. lactis, L. mesenteroides [44]
Lactococcus lactis, but [Y for L. sakei), Complement activity ([)
Leuconostoc mesenteroides
4 Lactobacillus sakei, Viable Individual In vivo Lysozyme ([Y), Complement activity ([), Phagocytic activity ([), [45]
Lactococcus lactis, Respiratory burst activity ([ for all except L. sakei [Y)
Leuconostoc mesenteroides
5 Lactococcus lactis, Viable Individual In vivo Phagocytic activity ([) [46]
Leuconostoc mesenteroides
6 Aeromonas sobria Viable Individual In vivo Leucocytes ([), Phagocytic activity ([), Respiratory burst activity ([) [47]
7 Bacillus species, Viable Individual In vivo Serum lysozyme ([ Bacillus species [Y A. sobria), Mucus lysozyme [48]
Aeromonas sobria ([ Bacillus species, [Y A. sobria), Respiratory burst activity ([),
Phagocytic activity ([), Anti-peroxidase ([Y),
Leucocytes ([Y), Erythrocytes ([Y)
8 Pdp11, 51M6 Inactivated Individual and In vivo Cytotoxic ([ for 51M6 and C, [Y Pdp11), Respiratory burst activity ([Y), [49]
(Heat -Killed) Combination Phagocytic activity ([), Peroxidase activity of serum and head kidney
leucocytes ([Y), Complement activity ([Y)
9 Shewanella putrefaciens, Inactivated Individual and In vivo Phagocytic activity ([), Respiratory burst activity ([Y), Complement [50]
Shewanella baltica Combination activity ([), Peroxidase activity ([), Cyotoxic activity ([ only in S. baltica)
10 Shewanella putrefaciens, Viable Individual In vivo Respiratory burst activity ([) [51]
Shewanella baltica
11 Shewanella putrefaciens, Viable Individual In vivo Respiratory burst activity ([ S. putrefaciencs, [Y S. baltica) [52]
Shewanella baltica
12 Vibrio fluvialis, Viable Individual and In vivo Erythrocytes ([), Macrophages ([), lymphocytes ([), leucocytes ([), [53]
Micrococcus luteus, Combination lysozyme activity ([)
Aeromonas hydrophila,
Carnobacterium species
13 Gram þve coccus, V. fluvialis, Inactivated Individual In vivo Erythrocytes ([Y), Macrophages ([), leucocytes ([), Lysozyme ([Y), [54]
Aeromonas hydrophila, Phagoctic activity ([)
Carnobacterium species
14 Carnobacterium maltaromaticum, Viable Individual In vivo Respiratory burst activity ([Y), Lysozyme ([ Serum and Mucus), [55]
Carnobacterium divergens Phagocytic activity ([)
15 Carnobacterium maltaromaticum, Viable Individual In vitro IL-8 ([Y) and TGF b ([Y) of gut cells, In head kidney leucocytes: [56]
Carnobacterium divergens TNF a ([), TCR b ([), IL1b ([), CD8 ([Y), CD4 ([Y),IL8 (Y), TGF b (Y)
16 Bacillus subtilis Viable Individual In vivo Respiratory burst activity ([), Serum bactericidal activity ([) [57]
17 Bacillus subtilis Viable Individual In vivo Immunoglobulin ([), Lysozyme ([), Respiratory burst activity ([Y), [58]
Specific antibody titre against E. tarda ([), Leucocytes ([)
18 Bacillus subtilis Viable Individual In vivo Gut mucus and serum lysozyme ([), Respiratory burst activity ([), [59]
Phagocytic activity ([), Anti-Peroxidase ([), Leucocytes ([),
Erythrocytes ([Y), Bactericidal activity ([), a1-anti-protease level ([),
Peroxidase assay ([), Complement activity ([Y)
19 Lactobacillus rhamnosus Viable Individual In vivo Immunoglobulin ([), Respiratory burst activity ([), [60]
Complement activity ([)
20 Saccharomyces cerevisiae Viable Individual In vivo Phagocytic activity ([), Respiratory burst activity ([), [61]
Complement activity ([Y), Myeloperoxidase ([),
21 Clostridium butyricum Viable, Individual In vivo Lysozyme ([ gut mucusa and serum), Phagocytic activity ([), [62]
Inactivated Immunoglobulin ([ gut mucusa and serum)
22 Lactobacillus rhamnosus Viable Individual In vivo Phagocytic activity ([), Respiratory burst activity ([Y), [63]
Complement activity ([)
23 Lactobacillus rhamnosus Viable Individual In vivo Immunoglobulin ([), Respiratory burst activity ([Y), [64]
Lysozyme ([Y), Complement activity ([),
Inactivated Immunoglobulin ([), Respiratory burst activity ([Y),
(Heat-killed) Lysozyme ([Y), Complement activity (Y)
24 Lactobacillus rhamnosus, Viable Individual In vivo IL-1b1 ([ spleen and [Y head kidney by L. rhamnosus, [Y for spleen and [65]
Bacillus subtilis, (Freeze dried) head kidney by E. faecium, [Y for spleen and head kidney by B. subtilis,),
Enterococcus faecium TNF 1 and 2 ([ head kidney and spleen by L. rhamnosus and E. faecium,
[Y for B. subtilis), TGF-b ([Yspleen and head kidney by L. rhamnosus,
[ for spleen and [Y head kidney by B. subtilis, [ for spleen and head
kidney by E. faecium), Complement activity ([Y L. rhamnosus, [
for B. subtilis, E. faecium), Respiratory burst activity
([Y L. rhamnosus, B. subtilis and [ for E. faecium)
25 Lactobacillus delbrueckii Viable Individual In vivo TCR b ([), Immunoglobulin ([Y), [66]
(Through CD8 ([Y), CD4 ([Y), IL-1b (Y), IL-10 (Y), COX-2 (Y), TGF b (Y)
artemia)
26 Aeromonas sobria, Viable Individual In vivo Respiratory burst activity ([ B. thermosphacta, [Y for A. sobria), [67]
Brochothrix thermosphacta Complement activity ([Y), Phagocytic activity ([), Serum and epidermal
mucus lysozyme ([Y), lecucocytes ([Y), lymphocytes ([Y),
Pinocytic activity ([Y)
 S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e14 5

Table 1 (continued )

Sl. Probiotics Form of Mode of Assay Immunological effects Reference

no probiotics Probiotics conditions
supplementation
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 Viable Individual and In vivo Phagocytic activity ([ I), Respiratory burst activity ([Y), [70]
ssp. lactis, Combination Cyotoxic activity ([ C, [Y I), Peroxidase activity of
Bacillus subtilis head kidney leucocytes ([Y)
30 Lactobacillus delbrueckii, Inactivated Individual In vitro Peroxidase activity of head kidney leucocytes ([Y), Respiratory [71]
Bacillus subtilis, Pdp11, 51M6 burst activity ([), Cyotoxic activity ([ but [Y Pdp11)
31 Lactobacillus delbrueckii, Inactivated Individual and In vivo Respiratory burst activity ([Y), Serum peroxidase ([YC, B. subtilis), [72]
Bacillus subtilis (Heat-Killed) Combination Peroxidase activity of head kidney leucocytes ([Y), Complement ([C),
Phagocytic activity ([C), Immunoglobulin ([C), Cytotoxic activity ([Y)
32 Kocuria species Viable Individual In vivo Lysozyme ([), Peroxidase ativity of head kidney macrophage ([), [73]
Respiratory burst activity ([Y), Phagocytic activity ([), Anti-protease
activity ([)
33 Lactobacillus plantarum Viable Individual In vivo Lysozyme ([), Phagocytic activity ([), Peroxidase activity ([), [74]
Complement activity ([), Superoxide dismutase (Y), Glutathione
peroxidase ([)
34 Clostridium butyricum Viable Individual In vivo Phenoloxidase activity ([), Acid phosphatases activity ([ when [75]
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)
35 Bacillus subtilis, Viable, Combination In vivo Neutrophils migration ([), Bactericidal activity ([), Lysozyme [76]
Lactobacillus acidophilus, Inactivated ([ mucus and serum), Respiratory burst activity ([Y), Skin
Clostridium butyricum, protease activity ([)
Saccharomyces cerevisiae
(Commercial probiotics
preparation)
36 Lactococcus lactis, Viable, Individual In vitro Nitric oxide ([), Phagocytic activity ([) [77]
Leuconostoc mesenteroides Inactivated
37 Enterococcus faecium Viable Individual In vivo Lysozyme ([Y), Complement activity ([), Respiratory burst [78]
(Through activity ([), Myeloperoxidase activity ([)
water)
38 Bacillus coagulans, Viable Individual In vivo Respiratory burst activity ([), Superoxide dismutase activity ([), [79]
Bacillus subtilis, (Through Catalase ([), lysozyme ([Y), Total antioxidation competence ([Y),
Rhodopseudomonas palustris water) Myeloperoxidase activity ([ in B. coagulans, [Y in B. subtilis, R. palustris)

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

infectious agents [82]. Probiotics either single or in combination
are found to trigger the lysozyme level in teleosts. The
enhancement 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 fish [75,76]. Taoka et al. [76] reported signifi-
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 in
O. niloticus [78,79] failed to elevate lysozyme level. Similarly, Peters
et al. [67] failed to detect any specific change in serum and skin
mucosa lysozyme level by feeding A. sobria @108 cells/g feed
and B. thermosphacta @1010 cells/g feed in O. mykiss for 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. subtilis alone or in combination with L. delbrueckii ssp. lactis for 3
weeks lead to high serum protease activity but failed to enhance
the peroxidase activity of head kidney leucocytes of S. aurata [72].
Similarly, probiotics like E. faecium also elevated the serum perox-
idase level in O. niloticus when supplemented through water
@1  107 CFU/ml in every 4 days for 40 days [78]. On contrary to
these findings, probiotics like L. delbrueckii, B. subtilis, Bacillus JB-1,
A. sobria, Shewanella putrefaciens (Pdp11) and 51M6 did not affect
the protease activity in fish like O. mykiss and S. aurata [48,49,71].
Similarly, anti-protease activities of serum and other body fluids
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 fish and hardly
modulated even after immunization or infection [84], certain pro-
biotics can successfully elevate this activity in fish [48,59,73].
Sharifuzzaman and Austin [73] reported significantly high anti-
protease activity in O. mykiss within 2 weeks of supplementation
of probiotic belong to Kocuria species (z108 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-specific immune response, may have
effector mechanisms like direct killing of microorganisms by lysis
[85]. Probiotics can enhance natural complement activity of fish
[65,72] and dietary as well as water treatment of many 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 fish. Choi and Yoon
[49] recorded an increased complement activity in O. mykiss
6 S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e14
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-inflammatory cytokines such as interleukin-1 (IL-1), IL-6, IL-
12, tumor necrosis factor a (TNF-a), and gamma interferon (IFN-g)
and anti-inflammatory cytokines such as IL-10 and transforming
growth factor b (TGF-b) in many animals [87e89]. Probiotics like
Bifidobacterium 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-inflamatory cyto-
kines while others probiotics can increase intestinal inflammatory
responses [91].
Probiotics like L. rhamnosus, E. faecium and B. subtilis are found
to up regulate the pro-inflammatory 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-inflammatory 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 inflammatory diseases [92].
Therefore, a balanced expression of COX-2 is essential for main-
taining the intestinal homeostasis and modulation of the inflam-
matory gene Cox-2 could be a key mechanism of anti-inflammatory
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, fish 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 of fish 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 several fish 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 Igþ cells
and acidophilic granulocytes (AGs) [66,72,105,106]. Probiotics
supplementation at early developmental stages can be helpful in
increasing specific AGs subpopulations [105]. The presence of
T-cells in the GALT has been documented in many fish [97,107,108]
and probiotics can lead to a significant increase in T-cells in fish. 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. lactis ssp.
lactis, L. mesenteroides and L. sakei [43] are also reported in fish like
O. mykiss.
6. Probiotics and gnotobiotic approaches
The detailed mode of action of probiotics has not yet been
established in any animals. It is often difficult 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 first 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 fish 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 fish 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
benefits of probiotics consumption [120] and fish 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 specific [123] and
strain specific differences in their modes of action [124]. Never-
theless the origin and source of probiotics [73], viability [125], dose
[126] and duration of supplementation [127] can regulate 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 fish are critical factors
that can regulate immune responses in fish.
 S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e14
7.1. Types of strain
The dominant group of probiotics that are used in fish culture
belong to Gram þve especially LAB, Bacillus (B. subtilis, B. lichen-
iformis, B. circulans) and bifidobacteria groups. On the other hand
certain strains of Aeromonas (Aeromonas hydrophila, A. sobria),
Vibrio (Vibrio fluvialis), 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 specific strain must not be
extrapolated to other strains [129,130]. Gnotobiotic studies also
indicate the strain specific 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 beneficiary
effective dose of the same probiotics in S. aurata and S. senegalensis
[50,51]. Similarly, variation in stimulating cellular innate immune
responses under in vitro and in vivo conditions among probiotics
belong to 51M6, L. delbrueckii subsp. lactis and B. subtilis group 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. sobria and 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-fish 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 efficacy 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 fish [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 significantly decreased larvae and fry mortality by using
Lactobacillus fructivorans, isolated from gut of S. 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 confirmed the beneficial
effects of both forms of probiotics under in vitro and in vivo
conditions. However, it is postulated that multispecies/multistrain
probiotics are more effective and consistent than their monospecific
7
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
fish [42,53,71]. Aly et al. [42] reported significantly high respiratory
burst activity and lysozyme level in O. niloticus fed with a mixture of
B. subtilis and 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 IgMþ 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 microflora environment for executing desirable
immune stimulatory and other beneficial 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] but other probiotics belong to
different families such as Lactobacillus and Bacillus species are
found to complement each other by exerting synergistic immu-
nomodulating responses in fish [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
significant protection against yersioniosis by using commercial
probiotics containing B. subtilis and B. licheniformis spores. Bacillus
spores have been shown to increase the survival and production of
channel catfish [144]. Similarly, B. subtilis spores when introduced
into rearing water eliminated Vibrio species from the larvae of
snook [145]. The spores of Bacillus toyoi and other Bacillus species
when used as feed additive increased the growth of S. maximus
[146,147] and common snook (Centropomus undecimalis) [53].
Spore formers possess additional advantage that they can 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 fish [70,72,76].
7.1.4. Viable vs non-viable
Probiotics, as per definition, are viable microorganisms with
documented beneficial effects on the overall health status of host.
Viability is an important property of any probiotics which 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
8 S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e14
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 fish species.
Although, immunostimulating potency of inactivated probiotics
under in vitro [71] and in vivo conditions [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 fish [76,101,152,153]. In a comparative study, Panigrahi
et al. [64] reported that probiotic strain of L. rhamnosus in viable form
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 an
enhanced immune response in host.
7.2. Dose of probiotics
Dose of probiotics could be limiting factor for achieving
optimum beneficial 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
beneficial effects including immunostimulatory activity. Different
in vitro and in vivo studies indicate that immune response of fish
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 be
2  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. delbrueckii subsp.
lactis and B. subtilis are found to be dose dependent [71]. Similarly,
immunostimulatory activities of LAB and B. subtilis in fish under in
vivo condition 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. mykiss fed for 30
days with L. rhamnosus strain 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 with
dose. For instance, elevation of lysozyme activity in serum and skin
in M. miiuy is reported at two different doses i.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 fish 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 (1012 CFU/g feed) compared to lower dose (109 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 fish most of the
beneficial 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 specific
immune parameter is also dependent on feeding duration. For
example Diaz-Rosales et al.[52] observed significant enhancement
of respiratory burst activity by feeding probiotics for 60 days but
earlier Dıaz-Rosales et al. [50], failed to detect significant
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 the
piscine 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 fish [64]. Such type of decline may be due the
failure of the probiotic strains to establish and multiply in the fish
gut. Though, a long dietary feeding regime is advantageous to host
in many aspects, more studies are required to establish the benefi-
cial effect of short term regime is not just adjuvant effects.
7.4. Mode of supplementation
Although probiotics are used as dietary supplements, Moriarty
[19] proposed to extend the definition of probiotics in aquaculture
to microbial ‘‘water additives’’ and several probiotics are also
directly used as water additives with documented health and
environmental benefits [79]. In fish, 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 fish 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. coagulans and 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
 S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e14 9

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

Sl. no Probiotics Fish Dose (CFU/g) Duration (Weeks) Pathogen (s) challenged Reference
1 V. alginolyticus S. salar e e Aeromonas salmonicida, [21]
Vibrio anguillarum, Vibrio ordalii
9
2 C. divergens G. morhua 2  10 3 V. anguillarum [23]
3 P. fluorescens S. salar 105e6 cells/ml 3e5 days A. salmonicida (No specific protection [24]
on co-habitant challenge)
4 Pseudomonas, S. aurata 108 15 days Vibrio harveyi [29]
Micrococcus, Vibrio species
7
5 B. subtilis, O. niloticus 10 2 Aeromonas hydrophila, Pseudomonas fluorescens, [42]
L. acidophilus Streptococcus iniae
6
6 L. sakei, O. mykiss 10 2 A. salmonicida ssp. salmonicida [45]
L. lactis,
L. mesenteroides
7 L. lactis, S. trutta 106 30 days A. salmonicida [46]
L. mesenteroides
7
8 A. sobria O. mykiss 5  10 2 Lactococcus garvieae, [47]
S. iniae
8
9 Bacillus species, O. mykiss 2  10 2 A. salmonicida, S. iniae, V. ordalii, L. garvieae, [48]
A. sobria V. anguillarum,
Yersinia ruckeri
10 L. rhamnosus O. niloticus 108 and 1010 2 Edwardsiella tarda [49]
11 S. putrefaciens, S. senegalensis 109 60 days Photobacterium damselae subsp. piscicida [52]
S. baltica
12 Vibrio fluviales, O. mykiss 106e108 7 and 14 days A. salmonicida [53]
A. hydrophila,
M. luteus
Carnobacterium species
13 Vibrio fluviales, O. mykiss 107 14 days A. salmonicida [54]
A. hydrophila,
Gram þve coccus
Carnobacterium species
14 C. maltaromaticum, O. mykiss 107 2 A. salmonicida, [55]
C. divergens Y. ruckeri
15 B. subtilis L. rohita 1  107a 15 days A. hydrophila [57]
16 B. subtilis L. rohita 108 60 days E. tarda [58]
17 C. butyricum M. miiuy 108 30 days V. anguillarum [62]
18 A. sobria O. mykiss 108 cells for A. sobria 2 A. bestiarum, Ichthyophthirius multifiliis (Ich) [67]
B. thermosphacta 1010 for B. thermosphacta A. sobria (More effective against Ich)
19 C. butyricum O. mykiss 300 mg/kg fish 3 days V. anguillarum [69]
20 Kocuria species O. mykiss 108 4 V. anguillarum [73]
21 L. plantarum Epinephelus coioides 105a 4 Streptococcus species, Iridovirus of E. coioides [74]
22 Commercial probiotics O. niloticus 1% 30a E. tarda [75]
23 Commercial probiotics Carassius auratus, 5 g/kg 30 days P. fluorescens [132]
Xiphophorus helleri (No significant protection)
24 B. subtilis, O. mykiss 4  104 spores 42 days Y. ruckeri [143]
B. licheniformis
25 L. rhamnosus O. mykiss 109a 51 days A. salmonicida [157]
26 Carnobacterium species S. salar, 5  107 2a A. salmonicida, [159]
O. mykiss V. ordalii,
Y. ruckeri,
V. anguillarum,
(No specific protection against
V. anguillarum)
27 Commercial probiotics Anguilla anguilla 1 g/kg 2 E. tarda [167]
(E. faecium, B. toyoi)
28 L. plantarum O. mykiss 107a 4 L. garvieae [168]
L. mesenteroides
8
29 S. putrefaciens S. aurata 10 15 days L. anguillarum [169]
30 Roseobacter species S. maximus 107 CFU/ml e V. anguillarum, [170]
Vibrio splendidus,
Pseudoalteromonas sp.
31 Lactic Acid Bacteria O. mossambicus 106 25 days A. hydrophila [173]
32 L. plantarum S. salar 2.5  109 5 A. slmonicida (No specific protection) [174]
33 C. divergens G. morhua 108 3 V. anguillarum (No specific protection) [175]
a
Experiment was conducted at different concentration/days but optimum result was recorded in that particular concentration/days of sampling.

influence the establishment and stability of probiotics and subse-
quent action include water quality, hardness, dissolved oxygen,
temperature, pH, osmotic pressure and mechanical friction [12].
Apart from these, stress due to high stocking density can affect 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 fish/m3 and found best growth, haematolgical
parameters and economic efficacy of probiotics within a stocking
density of 30 fish/m3. Similarly, in laying hens improved immune
response and other beneficial 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].
10 S.K. Nayak / Fish & Shellfish Immunology 29 (2010) 2e14
However, in aquaculture it is a neglected aspect and no
systematic attempt has been made to correlate effect of probiotics
on the immunity of fish at various environmental conditions.
Temperature could be crucial since a probiotic would be most
effective when used in its optimum temperature range matches
that of fish which is identical with surrounding environment [65].
Panighagi et al. [65] found better immunoefficacy of E. faecium in
comparison to L. rhamnosus and B. subtilis due 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 fish culture, probiotics
either in diet or bioencapsulation help in achieving natural resis-
tance and high survivability of larvae and post larvae of fishes
[159,165]. Significant increase in the mean weight and natural
survival rate of larvae of S. maximus fed 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 of fish 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 multifiliis, Ich) by
A. sobria in O. mykiss [67] and iridovirus of grouper E. coioides by
L. plantarum [74] is achieved.
Despite, several reports also indicate deleterious effects like
reduced growth, non stimulation of immune responses and no
significant protection by various probiotics in fish [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 scientific rationale [8].
9. Conclusions
The beneficial effects of dietary supplements like probiotics,
prebiotics and synbiotics have been recorded in a wide range of
animal models including fish. 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 Gram
þve 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 specific advantages [18,180], many times lack scientific
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 fish. Therefore, more
fundamental research is needed not only to address inter- and intra-
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
fish, 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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