Probiotics in aquaculture: a current assessmentTania P�erez-S�anchez1, Imanol Ruiz-Zarzuela1, Ignacio de Blas1 and Jos�e L. Balc�azar2

1 Laboratory of Fish Pathology, Faculty of Veterinary Medicine, Universidad de Zaragoza, Zaragoza, Spain

2 Catalan Institute for Water Research (ICRA), Scientific and Technological Park of the University of Girona, Girona, Spain

Correspondence

Tania P�erez-S�anchez, Laboratory of Fish

Pathology, Universidad de Zaragoza, 50013

Zaragoza, Spain. Email: taniaper@unizar.es

Received 15 October 2012; accepted 11 March

2013.

Abstract

Because of health and environmental concerns, the use of chemotherapeutic

agents has been restricted in many countries over recent years. This restriction

has resulted in a demand for alternative strategies to improve aquaculture pro-

duction and enhance disease resistance. Among these options, probiotics, live

micro-organisms that confer a health benefit to the host by providing both a

nutritional benefit and protection against pathogens, represent an important

option for the management of diseases and their use may replace some of the

therapeutic chemicals commonly used in aquaculture. Our review explores the

current state of knowledge on the impacts of probiotics on aquaculture, with

particular emphasis on the criteria used for selection and evidence of their

beneficial effects.

Key words: aquaculture, control, diseases, probiotic, resistance.

Introduction

Aquaculture has become an important economic activity in

many countries. Although this activity has expanded, diver-

sified and intensified, the emergence of a large variety of

pathogens is considered to be the major limiting factor.

Antibiotics have been used not only for the treatment of

bacterial infections, but also for preventing them (Cabello

2006; Taylor et al. 2011). However, antibiotic use in aqua-

culture may be detrimental to the environment and human

health, because it promotes the development and transfer

of resistance to other bacteria, including human and fish

pathogens (Sharifuzzaman & Austin 2009a). Moreover,

application of antibiotics and chemicals is a partially effec-

tive strategy for disease management (Harikrishnan et al.

2011). Although their application in hatcheries may be

cost-effective due to the limited water volume, antibiotic

treatment in grow-out systems with large-volume water

exchange is costly and avoided by most producers (Hari-

krishnan et al. 2010). In this sense, several alternative strat-

egies for the prevention and control of diseases have been

proposed, such as the use of vaccines, immunostimulants

and probiotics (Gatesoupe 1999; Balc�azar et al. 2006).

Several studies have demonstrated that the endogenous

microbiota is an important component of the mucosal bar-

rier, which represents the first line of defence against patho-

gens (G�omez & Balc�azar 2008). Although there is a huge

range of variation in the taxa present in the endogenous

microbiota of marine and freshwater species, which can be

influenced by genetic, nutritional and environmental fac-

tors, it has been suggested that members of the genera

Aeromonas, Alcaligenes, Alteromonas, Carnobacterium,

Flavobacterium, Micrococcus, Moraxella, Photobacterium,

Pseudomonas and Vibrio constitute the dominant microbi-

ota of a variety of marine species (Balc�azar et al. 2010;

Dhanasiri et al. 2011; Askarian et al. 2012), while in fresh-

water species has been reported the presence of Acineto-

bacter, Aeromonas, Flavobacterium, Lactococcus and

Pseudomonas, representatives of the family Enterobacteria-

ceae, and obligate anaerobic bacteria of the genera Bactero-

ides, Clostridium and Fusobacterium (Li et al. 2012; Wu

et al. 2012). Probiotics are usually members of the healthy

microbiota associated with the host; therefore, they may

provide an alternative way to reduce the use of antibiotics

in aquaculture (P�erez et al. 2010). Probiotics may prevent

bacterial diseases through a variety of mechanisms, such as

the creation of a hostile environment for pathogens by the

production of inhibitory compounds, by competing for

essential nutrients and adhesion sites or by modulating the

immune responses (Balc�azar et al. 2006; Merrifield et al.

2010).

The term probiotic, meaning ‘for life’, is derived from

the Greek words ‘pro’ and ‘bios’. It was first used by Lilly

and Stillwell in 1965 to describe ‘substances secreted by one

micro-organism which stimulates the growth of another’

(Lilly & Stillwell 1965) and was thus contrasted with the

© 2013 Wiley Publishing Asia Pty Ltd 1

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term antibiotic. The Joint Food and Agriculture Organiza-

tion of the United Nations and World Health Organization

(FAO/WHO) have stated that probiotics are ‘live microor-

ganisms, which when consumed in adequate amounts con-

fer a health benefit on the host’ (FAO/WHO 2001). It is

important to point out that probiotic effects in aquaculture

are not limited to the intestinal tract, but also can improve

the health of the host by controlling pathogens and

improving water quality by modifying the microbial com-

munity composition of the water and sediment (Verschu-

ere et al. 2000; Zheng et al. 2012).

Concerns have been voiced against the development of

virulence traits through horizontal gene transfer, the patho-

genicity to humans and antagonism with other beneficial

bacteria. However, it should be emphasized that, to date,

this possibility has never been documented, and the use of

purified cell components from bacteria with beneficial

health properties may eliminate any problem associated

with virulence (Sharifuzzaman et al. 2010).

Selection of probiotics

In aquaculture, there are several criteria to be considered

when choosing the appropriate probiotic strain. The char-

acteristics to consider include the following: host origin,

safety of the strain, production of antimicrobial substances,

ability to modulate host immune response or efficient com-

petition with pathogens for intestinal mucosa adhesion

sites. One of the most common ways to obtain a source of

these bacteria is to perform in vitro antagonism tests, in

which pathogens are exposed to the candidate probiotics or

their extracellular products in liquid and/or solid medium.

However, previous studies have demonstrated that the

in vitro tests may not always predict a possible in vivo effect

and thus these observations should be interpreted with

great caution (Gram et al. 2001). In fact, three potential

probiotic strains, Lactococcus lactis, Leuconostoc mesentero-

ides and Lactobacillus plantarum, showed the ability to sup-

press Lactococcus garvieae under in vitro conditions (P�erez-

S�anchez et al. 2011a). Despite showing in vitro antagonism,

only L. plantarum conferred protection in rainbow trout

(Oncorhynchus mykiss) against lactococcosis (P�erez-

S�anchez et al. 2011b).

To determine the ability of a probiotic to control or pre-

vent disease outbreaks, in vivo testing must be performed

(Burbank et al. 2011). Based on the results of in vitro tests,

we could consider whether the candidate strains could be

analysed in vivo (Sorroza et al. 2012).

It is essential to know the origin; thus, the use of strains

isolated from the target host should be recommended

(Fjellheim et al. 2010) as they may be more efficient or

effective than other strains. It must be safe and have the

ability to survive the transit through the gastrointestinal

tract, such as resistance to bile salts, low pH and enzymes.

In the case of shrimp, application of probiotics has been

either to larval-rearing tank water, pond water or added to

the feed. Feed supplementation has been found to be more

effective than direct addition to rearing water (Hai et al.

2009), but this may depend on the intended purpose in

using the probiotic (Karunasagar et al. 2010).

Selection of probiotics is very critical because inappro-

priate micro-organism can lead to undesirable effects on

the host. There is a general consensus that probiotics from

autochthonous source have a greater chance of competing

with resident micro-organisms (Sun et al. 2013); therefore,

isolation from the appropriate fish species can be an effi-

cient method of selecting effective probiotics (Burbank

et al. 2012). For instance, Carnevali et al. (2004) recorded

significantly decreased larvae and fry mortality using Lacto-

bacillus fructivorans, isolated from gut of adult seabream

(Sparus aurata). Vine et al. (2006) proposed in the proto-

col for the selection of endogenous probiotics that candi-

date probiotics should be derived from healthy individuals,

preferably of the target species. Studies by our research

group have also demonstrated that feeding rainbow trout

with members of its intestinal microbiota resulted in a

higher survival rate after challenge with Aeromonas salmon-

icida and L. garvieae (Balc�azar et al. 2007a; Vendrell et al.

2008; P�erez-S�anchez et al. 2011b).

According to Fuller (1989), a probiotic not only should

be harmless but must also provide a beneficial effect to the

host. Potential probiotics must therefore be safe, meaning

neither invasive nor pathogenic. They must be amenable to

industrial processes necessary for commercial production,

must remain viable in the food product and during storage

and must be metabolically active to elicit an effect.

The ability to colonize the intestinal tract or other epi-

thelial surfaces is clearly another important property of a

probiotic. In fact, Lc. lactis ssp. lactis, Lactobacillus sakei

and Leuc. mesenteroides, isolated from the endogenous

microbiota of salmonids, were administered to brown

trout (Salmo trutta) and it was observed that these strains

have a strong ability to adhere to and survive in the

intestinal mucus, because after a week of feeding, these

bacteria were detected in high amounts in the intestine

(Balc�azar et al. 2007b). Four probiotic strains, Vibrio

alginolyticus, Phaeobacter gallaeciensis (formely Roseobacter

gallaeciensis) and Pseudomonas aestumarina, isolated from

the gastrointestinal tract of white shrimp (Litopena-

eus vannamei), were administered to the same shrimp

species and it was observed that these strains were able

to survive in the digestive tract and confer protection

against vibriosis (Balc�azar et al. 2007d). Other studies

have showed that inoculation with a probiotic strain dur-

ing cultivation of larval L. vannamei (nauplii stage V)

prevented colonization by a pathogenic strain, because

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T. P�erez-S�anchez et al.

the probiotic succeeds in colonizing the gut of the larvae

(G�omez-Gil et al. 2000).

However, colonization experiments of fingerling perch

(Perca fluviatilis) with xylB-labelled Pseudomonas chlorora-

phis revealed that the bacterium only colonizes the fish

transiently or perhaps remains in the intestine at levels that

were undetected by in vitro bacterial culture. If Ps. chlorora-

phis were to be used as a probiotic, it would probably have

to be administered to the host at regular intervals (Gobeli

et al. 2009).

The selection of probiotics requires several in vitro

screening experiments, including assays for the production

of antagonistic compounds, attachment to fish intestinal

mucus and the production of other beneficial compounds

such as vitamins, fatty acids and digestive enzymes (Vine

et al. 2006). In vitro studies are necessary to optimize the

feeding doses or the viability of the bacteria, in order to

perform the in vivo challenge (Rom�an et al. 2012).

The study of probiotic bacteria has traditionally

depended on culture-based methods, which rely on isola-

tion, growth, and laboratory identification using morphol-

ogy and biochemical tests. However, culture-based and

other traditional identification methods have several limita-

tions, as they tend to give false-positive or false-negative

results. The relatively recent development of molecular

techniques to detect and quantify micro-organisms has fos-

tered a better understanding of microbial diversity and its

role in nature. Modern molecular methods, such as broad-

range sequencing of 16S ribosomal RNA from amplified

nucleic acid, allow evolutionary divergence to be used to

identify and classify micro-organisms. Such tools should be

utilized to identify probiotic species, helping to guarantee

the bacteria harmless to the host are chosen.

Studies with probiotics to date have involved the use of

different feeding durations, for example from 1 to 8 weeks

feeding regimes, but the basis of choosing these periods is

often unclear (Sharifuzzaman & Austin 2009a). In this

sense, dietary supplementation of Kocuria sp. SM1, between

1 and 4 weeks, reduced the mortality rate of rainbow trout

after challenge with Vibrio anguillarum. In particular, a

2-week feeding regime led to the maximum reduction in

mortalities (Sharifuzzaman & Austin 2009a). Two potential

beneficial Bacillus strains isolated from the gut of groupers

(Epinephleus coioides) were evaluated in a 60-day feeding

trial and significant decrease in feed conversion ratio was

observed in fish fed supplemented diets (Sun et al. 2010).

Paralichthys olivaceus fed with three enriched diets with

Zooshikella sp. JE-34 did not significantly increase the per-

cent weight gain (PGW) on first week, but after fourth

week, all the enriched diets significantly increased the PGW

(Kim et al. 2010).

For a bacterial strain to be accepted as a commercial pro-

biotic by the feed industry, extensive in vitro and in vivo

evidences on their safety, adaptability and suitability to the

target microbial environment, functionality, including their

physiological interactions with the host, and resistance to

industrial processes are necessary (Kiron 2012). Finally, an

economical analysis must be carried out to assure its com-

mercial distribution.

The commercialization of probiotics is regulated by gov-

ernment agencies and organizations, such as the US Food

and Drug Administration, the Japanese Foods for Specified

Health Use, the European Food Safety Authority among

others, which are responsible for establishing the policy of

food safety. In the European Union, some micro-organisms

have been authorized for use as probiotics in animal feed,

mainly bacterial strains belonging to the genera Bacillus,

Lactobacillus, Pediococcus and Streptococcus; however, only

Pediococcus acidilactici strain CNCM MA 18/5M has been

the unique probiotic strain authorized for use in aquacul-

ture (European Commission 2013).

Given this, the selection and design of probiotics remains

an important challenge and requires a solid foundation of

basic information regarding the physiology and genetics of

candidate strains, especially as related to their intestinal

roles, functional activities and interaction with other resi-

dent microbiota (Klaenhammer & Kullen 1999). In aqua-

culture enterprises, the selection is usually based on results

of tests showing antagonism towards the pathogens, an

ability to survive and colonize the intestine, and a capacity

to increase an immune response in the host (Luis-Vil-

lase~nor et al. 2011).

Mechanisms of action

Most studies in aquaculture suggest that most common

mechanisms by which probiotics may offer a beneficial

effect include (i) competitive exclusion of pathogenic bac-

teria, (ii) enhancement of host nutrition and enzymatic

contribution to digestion and (iii) stimulation of host

immune response (Irianto & Austin 2002a; G�omez &

Balc�azar 2008; Merrifield et al. 2010).

Competitive exclusion: antagonistic compounds and

adhesion

Competitive exclusion is a phenomenon whereby an estab-

lished microbiota prevents or reduces the colonization of

competing organisms for the same intestinal sites (Lara-

Flores & Aguirre-Guzm�an 2009). Micro-organisms use a

variety of mechanisms to compete for numerous resources,

including nutrients, space (adhesion sites on epithelial

surfaces) and oxygen.

One important mechanism of competition that also has

important implications for pathogen control is the produc-

tion of inhibitory compounds. This phenomenon was first

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Probiotics in aquaculture

noted in 1889 by De Giaxa, when marine bacteria were

observed to inhibit Vibrio species (De Giaxa 1889). In 1947,

Rosenfeld and ZoBell investigated the bacteriostatic or bac-

tericidal properties of seawater on nonmarine bacteria in

culture and reported the presence of antibiotic activity in

marine micro-organisms (Rosenfeld & ZoBell 1947). These

studies stimulated a great interest in finding micro-organ-

isms that can synthesize inhibitory compounds. For

instance, P. gallaeciensis has been used as probiotics in fish

aquaculture, as this bacterium produces tropodithietic acid

that is an efficient inhibitor of fish-pathogenic bacteria

(Berger et al. 2011; D’Alvise et al. 2012).

However, antibiotic substances are not alone in produc-

ing inhibition. Antagonism may be mediated by other sub-

stances besides antibiotics, such as organic acids, hydrogen

peroxide, lytic enzymes, iron-chelating compounds and

bacteriocins (Verschuere et al. 2000; Balc�azar et al. 2006).

Bacteriocins are small peptides that disrupt integrity of bac-

terial cell membranes (Teplitski et al. 2009). Previous stud-

ies have shown that some endogenous lactic acid bacteria

(LAB), such as those belonging to the genus Carnobacteri-

um, Lactobacillus, Lactococcus and Enterococcus, are able to

produce bacteriocins that can inhibit the growth of some

pathogenic bacteria (Ringø & Gatesoupe 1998; Campos

et al. 2006; Gatesoupe 2008). Among them, divercins and

piscicocins, two bacteriocins belonging to class II, have

been characterized from Carnobacterium species isolated

from fish intestine (Bhugaloo-Vial et al. 1996; M�etivier

et al. 1998). Therefore, the establishment of a normal or

protective microbiota is a key component in excluding

potential invaders and maintaining health.

Colonization of intestinal mucosal surfaces with a nor-

mal microbiota has also a positive effect on immune regu-

latory functions, and disturbance of these functions by an

imbalanced microbiota may contribute to the development

of diseases. It has also been suggested that an imbalance of

the microbiota could potentially lead to immune-related

disadvantages (Rombout et al. 2011). Significant attention

has therefore been focused on the role of probiotics in the

induction or restoration of a disturbed microbiota to its

normal beneficial composition (P�erez et al. 2010).

The ability of a probiotic strain to colonize the gut and

adhere to the mucus layer is considered a good criterion

when selecting probiotic candidates (Balc�azar et al. 2006;

Vine et al. 2006). Such a feature is important because a

longer residence time in the intestinal tract could extend

potential beneficial effects (Balc�azar et al. 2007b). However,

in aquaculture systems, the interaction between the micro-

biota and the host is not limited to the intestinal tract. Pro-

biotic bacteria can also be active on the gills or the skin of

the host or in its surrounding environment (Tinh et al.

2008). It has been reported that bacterial strains associated

with intestinal and skin mucus of adult marine turbot

(Scophtalmus maximus) and dab (Limanda limanda) sup-

pressed the growth of the fish pathogen V. anguillarum

(Olsson et al. 1992). Burbank et al. (2011) suggested that

probiotic bacteria may be able to colonize the fish skin and

competitively exclude Flavobacterium psychrophilum at the

site of infection.

Balc�azar et al. (2007c) demonstrated that probiotic

strains (Lc. lactis subsp. lactis, Lc. lactis subsp. cremoris

and L. sakei) reduced the adhesion of fish pathogens,

such as A. salmonicida, Carnobacterium piscicola and

Yersinia ruckeri, to intestinal mucus from rainbow trout.

This sort of evidence suggests that it is desirable to use

adhering strains when designing probiotic supplements.

The administration of dead/inactivated cells or the

supernatant of probiotics does not necessarily reduce bacte-

rial infections, indicating that the maximum benefits of

probiotics are mediated in some cases by live bacterial cells

(Sharifuzzaman & Austin 2009b). Abbass et al. (2010)

demonstrated the potential of using subcellular compo-

nents of the probiotics Aeromonas sobria GC2 and Bacil-

lus subtilis JB-1. When administered to rainbow trout, they

conferred protection against a new biogroup of Y. ruckeri.

Different growth conditions, such as the growth phase,

nutrient level, pH, temperature and the quantity of inocula,

can vary the protection rate by extracellular components of

bacteria (Sharifuzzaman et al. 2010).

Source of nutrients and enzymatic contribution to

digestion

Probiotics are also expected to have a direct growth-pro-

moting effect on the host either by a direct involvement in

nutrient uptake or by providing nutrients or vitamins

(Ringø & Gatesoupe 1998), with a consequent improve-

ment of the digestibility and weight gain (Lin et al. 2012).

Some members of the genera Bacteroides and Clostridium

have been reported to contribute to host nutrition by sup-

plying fatty acids and vitamins (Sakata 1990). In some fish

species, such as tilapia (Oreochromis niloticus) or Japanese

eel (Anguilla japonica), members of the genera Aeromonas

and Pseudomonas have been implicated in the production

of amylase (Sugita et al. 1996). Moreover, members of the

genera Agrobacterium, Pseudomonas, Brevibacterium,Micro-

bacterium, and Staphylococcus have been also shown to

contribute to nutritional processes in Arctic charr (Salveli-

nus alpinus) (Ringø et al. 1995). A previous study has dem-

onstrated that European sea bass (Dicentrarchus labrax)

larvae fed live yeast (Debaryomyces hansenii) showed

increased activity and concentrations of mRNA trypsin and

lipase, suggesting that the dose-dependent effect of yeast on

larval performance could be attributed to the amount of

polyamines secreted by the yeast in the gut lumen of larvae

(Tovar-Ram�ırez et al. 2004).

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T. P�erez-S�anchez et al.

Probiotics can also influence the digestive processes by

improving the microbial population and by enhancing the

enzyme activity that contributes to more efficient digestion

and food utilization. For instance, the supplementation of

whole cell yeast (Saccharomyces cerevisiae), n-3 HUFA-

enriched yeast and treated yeast cells with beta-mercapto-

ethanol improved rainbow trout growth and may be possi-

bly be due to improved nutrient digestibility (Tukmechi

et al. 2011). However, the significance of bacterially-pro-

duced enzymes to host health in vivo remains unclear and

thus additional studies are needed to characterize enzyme

contributions to the host.

Enhancement of the immune response

Probiotics can stimulate the host immune response because

bacterial walls have components (lipopolysaccharides, pep-

tidoglycan and b-glucans) that may cause a range of innate

and adaptive host immune responses. Many immunostim-

ulants have been tested on fish and shellfish and some of

them originated from microbial cell walls, for example

muramyl dipeptide, glucans and lipopolysaccharides

(Anderson 1992).

Modulating immune responses with probiotic bacteria

has been shown to have several effects in a variety of fish,

such as induction of pro-inflammatory cytokines, stimulat-

ing the activity of natural killer cells, increasing mucosal

and systemic antibody production, activating phagocytic

activity and increasing lysozyme and complement activity

(Harikrishnan et al. 2010). Certainly, the immune-stimu-

lating properties of probiotics have been recognized as the

key mode of action protecting fish against bacterial infec-

tions (Sharifuzzaman & Austin 2010) and some factors

such as the source, type, dose and duration of supplemen-

tation can significantly affect the immunomodulatory

activities of probiotics (Liu et al. 2012).

Probiotic strains have been shown to modulate innate

immune responses in their hosts and thereby facilitate the

exclusion of potential pathogens. In rainbow trout, the oral

administration of Clostridium butyricum has been shown to

enhance the trout resistance to vibriosis by increasing the

phagocytic activity of leucocytes (Sakai et al. 1995). Signifi-

cant increases in phagocytic activity and phagocytic index

were also observed in Epinephelus coioides fed Bacil-

lus pumilus or Bacillus clausii containing diets for 60 days

compared with those fed the control diet (Sun et al. 2010).

Phagocytosis is a form of endocytosis where large parti-

cles (i.e. cellular debris or micro-organisms) are ingested

into endocytic vesicles called phagosomes. The fundamen-

tal role of phagocytic cells (monocytes/macrophages and

neutrophils) in host defence is to limit the initial dissemi-

nation and/or growth of infectious organisms (Neumann

et al. 2001).

The augmentation of lysozyme activity is another

mechanism of probiotic protection, and as it is influ-

enced by several external factors, it has been frequently

employed in fish nutrition research (Kiron 2012). Lyso-

zyme is widely distributed in several fish tissues, includ-

ing the serum, kidney, spleen and intestine. This enzyme

breaks the b-1,4 glycosidic bond between N-acetylmu-

ramic acid and N-acetylglucosamine in the cell wall of

Gram-positive bacteria, and in association with comple-

ment components, some Gram-negative bacteria may be

affected as well (G�omez & Balc�azar 2008; Marsh & Rice

2010). Irianto and Austin (2002b) demonstrated that

administration of Aeromonas hydrophila A3-51, Vibrio

fluvialis A3-47S, Carnobacterium sp. BA211 and Micrococ-

cus luteus A1-6 was able to increase lysozyme activity. In

contrast, Merrifield et al. (2009) demonstrated that the

effect of B. subtilis on serum lysozyme activity of rainbow

trout was negligible in a 10-week feeding trail.

Sharifuzzaman and Austin (2009a) observed that rainbow

trout fed Kocuria sp. SM1 at concentrations of 108 cells g�1

feed and then challenged intraperitoneally with V. anguilla-

rum at weekly intervals, enhanced cellular and humoral

immune response, notably greater head kidney macrophage

phagocytic and peroxidise activities, and higher serum lyso-

zyme and total protein levels were recorded.

Low mortality rate after lymphocystis disease virus infec-

tion was seen in olive flounder fed with Lactobacil (com-

mercial product) individually or mixed with Sporolac

(commercial product) supplemented diets and significantly

enhanced the immune parameters such as phagocytic activ-

ity superoxide anion production, complement activity and

plasma lysozyme (Harikrishnan et al. 2010).

Nikoskelainen et al. (2003) showed that the administra-

tion of a lactic acid bacterium Lactobacillus rhamnosus

stimulated respiratory burst in rainbow trout. The adminis-

tration of L. rhamnosus has also been involved in the stim-

ulation of superoxide anion production from head kidney

leukocytes in the same species (Panigrahi et al. 2005). In

addition, Balc�azar et al. (2007b) observed a correlation

between colonization with probiotic bacteria (Lc. lactis

subsp. lactis, Leuc. mesenteroides and L. sakei) and host

innate humoral responses, such as alternative complement

pathway activity and lysozyme activity in brown trout. Pro-

biotics have also been found able to modulate the produc-

tion of pro- and anti-inflammatory cytokines, which are

protein mediators produced by immune cells that contrib-

ute to cell growth, differentiation and defence mechanism

of the host (Nayak 2010). Probiotics such as L. rhamnosus,

Enterococcus faecium and B. subtilis were found to up regu-

late the pro-inflammatory cytokines like interleukin-1b1and transforming growth factor b (TGF-b) in rainbow

trout (Panigrahi et al. 2007). P�erez-S�anchez et al. (2011b)

also observed that IL-1b, IL-10 and TNF-a gene

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Probiotics in aquaculture

expressions were significantly up-regulated by L. plantarum

in rainbow trout.

Stimulation of the immune system by probiotic bacteria

has also been reported in crustaceans, particularly shrimp.

It is important to state that there are three types of circulat-

ing haemocytes in crustaceans: hyaline, semigranular and

large granular cells. Haemocytes are involved not only in

phagocytosis but also in the production of melanin via the

prophenoloxidase system, which is an important compo-

nent of the cellular defence reaction (Johansson &

S€oderh€all 1989). In this sense, the administration of Bacillus

S11 stimulated phagocytic activity in tiger shrimp Pena-

eus monodon (Rengpipat et al. 2000). Moreover, the

administration of L. plantarum stimulated phenoloxidase

and superoxide dismutase activities, enhanced the

efficiency of Vibrio alginolyticus clearance and augmented

peroxinectin mRNA transcription in white shrimp L. van-

namei (Chiu et al. 2007).

Probiotic strains studied in aquaculture

One of the first studies on this subject was conducted by

Yasuda and Taga (1980), who suggested that bacteria could

control fish disease and activate nutrient regeneration in

addition to serving as food. Since then, research effort has

been dedicated to identifying beneficial micro-organisms,

including Gram-positive and Gram-negative bacteria and

yeasts (Table 1).

Gram-positive bacteria

Most probiotics under consideration in aquaculture belong

to the LAB (Lactobacillus and Carnobacterium species) and

Bacillus (Balc�azar et al. 2006; Wang et al. 2008). Lactic acid

bacteria are a group of Gram-positive rod- and coccus-

shaped organisms that do not form spores, are nonmotile

and produce lactic acid as their major end product during

the fermentation of carbohydrates. They are generally

regarded as safe for use in food production and are widely

used as probiotics in aquaculture because of their proper-

ties, such as low or no virulence, outcompeting harmful

bacteria or the capacity to colonize the digestive tract (Ley-

va-Madrigal et al. 2011). Over the last decades, LAB have

received much attention as we begin to understand their

importance to the host with regards to digestive function,

development, immunity and disease resistance (Hoseinifar

et al. 2011).

The LAB probiotics have been shown to be effective

against edwardisellosis, furunculosis and vibriosis (Hari-

krishnan et al. 2010). In fact, a study demonstrated a signif-

icant increase in the mean weight and survival rate of

turbot larvae that were fed rotifers enriched with LAB and

these strains provided a significant protection against a

pathogenic Vibrio (Gatesoupe 1994). Subsequently, Carno-

bacterium inhibens, which was isolated from the gastroin-

testinal tract of Atlantic salmon (Salmo salar), produced

inhibitory substances active against several fish pathogens

in vitro. Furthermore, in vivo results demonstrated that this

strain is metabolically active in both the intestinal mucus

and the faeces of salmonids and does not have a detrimen-

tal effect on the host (J€oborn et al. 1997).

Ferguson et al. (2010) observed that fish fed the diet sup-

plemented with Pediococcus acidilactici showed a survival

rate of 100%, whereas survival was 88.33% in those fed the

control diet. The reason for this has not been elucidated as

no pathogenic challenge was conducted, and no signs of dis-

ease were detected during the trial, but perhaps P. acidilactici

modulates intestinal bacterial communities and stimulates

some aspects of the nonspecific immune response.

Some lactic acid strains that are generally used as human

probiotics (e.g. lactobacilli and enterococci) have been con-

sidered for use in fish (Nikoskelainen et al. 2001; Panigrahi

et al. 2010). Administration of L. rhamnosus to rainbow

trout for 51 days reduced host mortality caused by A. sal-

monicida from 52.6% in the control to 18.9% and 46.3% in

the 109 cells g�1 feed and the 1012 cells g�1 feed groups,

respectively (Nikoskelainen et al. 2001). It is apparent that

a high dose does not necessarily result in a greater amount

of protection.

Panigrahi et al. (2010) assessed the effect of feeding via-

ble and nonviable forms of L. rhamnosus on blood profiles

in rainbow trout. A significant elevation of plasma choles-

terol and triglyceride and alkaline phosphatise activity level

was found in the freeze-dried (FD) probiotic fed groups at

20 and 30 days post-feeding. This was concomitant with

the increased plasma protein and haematocrit values in FD

group at 20 and 30 days. Likewise, the heat-killed probiotic

fed group registered significantly high values of triglyce-

rides, alkaline phosphatase activity and plasma protein after

20 days of feeding. Alterations in the blood profiles could

serve as supplementary information when examining the

benefits of probiotics for fish.

In addition, survival rates of European eels

(Anguilla anguilla) fed with E. faecium were significantly

higher compared to controls following challenge with

Edwardsiella tarda (Chang & Liu 2002). This strain has also

been useful in improving the growth of sheat fish (Silu-

rus glanis). After feeding for 58 days on a dose equivalent to

2 9 108 bacteria g�1 of food, fish achieved better growth and

also showed reduced incidence of Escherichia coli, Staphylo-

coccus aureus andClostridium spp. (Bogut et al. 2000).

The administration of Leuc. mesenteroides also conferred

protection against A. salmonicida and L. garvieae in rain-

bow trout. Competition for nutrients and adhesion recep-

tors could be the basis for this protection because

microbiological and molecular analyses have revealed the

Reviews in Aquaculture (2013) 5, 1–14

© 2013 Wiley Publishing Asia Pty Ltd6

T. P�erez-S�anchez et al.

Table 1 Probiotics used in aquaculture and the effect on their hosts

Host species Potential probiotic Effect on host References

Anguilla anguilla Enterococcus faecium SF68 and

Bacillus toyoi

Protection against edwardsiellosis Chang and Liu (2002)

Centropomus

undecimalis

Bacillus subtilis sp. 48 Reduction of Vibrio levels Kennedy et al. (1998)

Crassostrea gigas Aeromonas media Reduced proliferation of Vibrio tubiashii Gibson et al. (1998)

Dicentrarchus labrax Saccharomyces cerevisiae,

Debaryomyces hansenii

Improved larval survival Tovar-Ram�ırez et al. (2002)

Dicentrarchus labrax Debaryomyces hansenii Improved survival and reduced malformations Tovar-Ram�ırez et al. (2004)

Epinephelus coioides Bacillus pumilus SE5, Bacillus

clausii DE5

Improved growth and reduction of Vibrio levels Sun et al. (2010)

Epinephelus coioides Bacillus subtilis E20 Immune stimulation and enhanced resistance

to Streptococcus sp. and an iridovirus

Liu et al. (2012)

Labeo rohita Pseudomonas aeruginosa

VSG-2

Immune stimulation and enhanced resistance

to Aeromonas hydrophila

Giri et al. (2012)

Labeo rohita Lactobacillus plantarum

VSG3

Improved growth, immune stimulation and

enhanced resistance to Aeromonas hydrophila

Giri et al. (2013)

Litopenaeus vannamei Vibrio alginolyticus Improved survival and growth Garriques and Arevalo (1995)

Litopenaeus vannamei Phaffia rhodozyma Protection against vibriosis Scholz et al. (1999)

Litopenaeus vannamei Vibrio alginolyticus UTM 102,

Phaeobacter gallaeciensis

SLV03, Pseudomonas

aestumarina SLV22

Protection against Vibrio parahaemolyticus Balc�azar et al. (2007d)

Litopenaeus vannamei Lactobacillus plantarum 7-40

(NTU 102)

Immune modulation and enhanced resistance

to Vibrio alginolyticus

Chiu et al. (2007)

Litopenaeus vannamei Bacillus subtilis L10 and G1 Improved growth, expression of immune-related

genes and enhanced resistance to Vibrio harveyi

Zokaeifar et al. (2012)

Huso huso Saccharomyces cerevisiae var.

ellipsoideus

Improved growth and modulation of intestinal

microbiota

Hoseinifar et al. (2011)

Mycteroperca rosacea Debaryomyces hansenii CBS

8339

Immune stimulation and enhanced resistance

to Amyloodinium ocellatum

Reyes-Becerril et al. (2008)

Oncorhynchus mykiss Carnobacterium inhibens K1 Growth inhibition of Vibrio anguillarum and

Aeromonas salmonicida in fish intestinal mucus

J€oborn et al. (1997)

Oncorhynchus mykiss Pseudomonas fluorescens Improved survival after challenge with Vibrio

anguillarum

Gram et al. (1999)

Oncorhynchus mykiss Lactobacillus rhamnosus ATCC

53103

Immune stimulation and enhanced resistance to

Aeromonas salmonicida

Nikoskelainen et al. (2001)

Oncorhynchus mykiss Vibrio fluvialis A3-47S,

Aeromonas hydrophila A3-51,

Carnobacterium sp. BA211,

Micrococcus luteus A1-6

Immune stimulation and enhanced resistance to

Aeromonas salmonicida.

Irianto and Austin (2002b)

Oncorhynchus mykiss Lactobacillus sakei CLFP 202,

Lactococcus lactis CLFP 100,

Leuconostoc mesenteroides

CLFP 196

Immune stimulation and enhanced resistance to

Aeromonas salmonicida

Balc�azar et al. (2007a)

Oncorhynchus mykiss Lactobacillus rhamnosus ATCC

53103, Bacillus subtilis,

Enterococcus faecium

Immune stimulation and expression of

cytokine genes

Panigrahi et al. (2007)

Oncorhynchus mykiss Lactobacillus plantarum CLFP

238, Leuconostoc

mesenteroides CLFP 196

Competitive exclusion and enhanced resistance to

Lactococcus garvieae

Vendrell et al. (2008)

Oncorhynchus mykiss Bacillus subtilis, Bacillus

licheniformis, Enterococcus

faecium

Improved feed conversion and modulation of

intestinal microbiota

Merrifield et al. (2009)

Oncorhynchus mykiss Kocuria sp. SM1 Enhanced resistance to Vibrio anguillarum Sharifuzzaman and Austin

(2009a)

(continued)

Reviews in Aquaculture (2013) 5, 1–14

© 2013 Wiley Publishing Asia Pty Ltd 7

Probiotics in aquaculture

presence of this probiotic strain in the fish intestine

(Balc�azar et al. 2007a; Vendrell et al. 2008).

The genus Bacillus constitutes a diverse group of rod-

shaped, Gram-positive bacteria that are characterized by

their ability to produce endospores in response to adverse

environmental conditions. Most Bacillus species are not

harmful to humans or animals and are commercially

important as producers of high and diverse amount of sec-

ondary metabolites, including antibiotics and enzymes.

Diverse species of Bacillus have been examined as probiot-

ics, such as B. subtilis, B. claussi, B. cereus, B. licheniformis

and B. coagulans (Cutting 2011). When a B. subtilis strain,

isolated from the common snook (Centropomus undecimal-

is), was inoculated into the rearing water, there was an

apparent elimination of Vibrio species from whole snook

larvae (Kennedy et al. 1998). The use of several Bacillus

Table 1 (Continued)

Host species Potential probiotic Effect on host References

Oncorhynchus mykiss Lactobacillus rhamnosus JCM

1136

Alterations in the blood profiles Panigrahi et al. (2010)

Oncorhynchus mykiss Aeromonas sobria GC2,

Bacillus subtilis JB-1

Enhanced resistance to Yersinia ruckeri Abbass et al. (2010)

Oncorhynchus mykiss Enterobacter sp. C6-6 and C6-8 Enhanced resistance to Flavobacterium psychrophilum Burbank et al. (2011)

Oncorhynchus mykiss Lactobacillus plantarum CLFP3,

Lactococcus lactis CLFP 25,

Leuconostoc mesenteroides

CLFP 68

Expression of cytokine genes and enhanced resistance

to Lactococcus garvieae

P�erez-S�anchez et al. (2011b)

Oncorhynchus mykiss Pseudomonas sp. MSB1 Inhibition of Flavobacterium psychrophilum in vitro Str€om-Bestor and Wiklund

(2011)

Oncorhynchus mykiss Saccharomyces cerevisiae Immune stimulation, improved growth and enhanced

resistance to Yersinia ruckeri

Tukmechi et al. (2011)

Oncorhynchus mykiss Saccharomyces cerevisiae Increased growth performance and immune stimulation Sheikhzadeh et al. (2012)

Oreochromis niloticus Saccharomyces cerevisiae Protection against vibriosis Lara-Flores et al. (2003)

Oreochromis niloticus Pediococcus acidilactici

CNCM MA 18/5 M

Modulation of intestinal bacterial communities and

stimulation of the nonspecific immune response

Ferguson et al. (2010)

Oreochromis niloticus Bacillus sp. C5I18 Growth inhibition of Aeromonas and Pseudomonas

species in fish intestine

Del’Duca et al. (2013)

Oreochromis niloticus

Anguilla japonica

Aeromonas, Pseudomonas High amylase production Sugita et al. (1996)

Paralichthys olivaceus Lactobacil and Sporolac

(commercial products)

Immune stimulation and enhanced resistance to

lymphocystis disease virus infection

Harikrishnan et al. (2010)

Paralichthys olivaceus Zooshikella sp. JE-34 Immune stimulation and enhanced resistance to

Streptococcus iniae

Kim et al. (2010)

Pecten maximus Alteromonas haloplanktis Enhanced resistance to Vibrio anguillarum Riquelme et al. (1996)

Pecten maximus Roseobacter BS107 Improved larval survival Ruiz-Ponte et al. (1999)

Penaeus latisulcatus Pseudomonas synxantha,

Pseudomonas aeruginosa

Enhanced resistance to Vibrio harveyi Hai et al. (2009)

Penaeus monodon Bacillus Enhanced resistance to luminescent Vibrio spp Moriarty (1998)

Penaeus monodon Bacillus S11 Immune stimulation and enhanced resistance to

Vibrio harveyi

Rengpipat et al. (2000)

Perca fluiviatilis Pseudomonas chlororaphis

JF3835

Intestinal colonization and enhanced resistance to

Aeromonas sobria

Gobeli et al. (2009)

Salmo trutta Lactococcus lactis CLFP 100,

Leuconostoc mesenteroides

CLFP 196

Immune stimulation and enhanced resistance to

Aeromonas salmonicida

Balc�azar et al. (2007b)

Salvelinus alpinus Agrobacterium, Pseudomonas,

Brevibacterium,

Microbacterium, Staphylococcus

Contribution to nutritional process Ringø et al. (1995)

Scophtalmus maximus Lactobacillus plantarum or

Carnobacterium sp.

Protection against vibriosis Gatesoupe (1994)

Silurus glanis Enterococcus faecium Improved growth and reduced incidence of Escherichia

coli, Staphylococcus aureus and Clostridium spp.

Bogut et al. (2000)

Sparus aurata Lactobacillus fructivorans Decreased larvae and fry mortality Carnevali et al. (2004)

Sparus aurata Bacillus subtilis CECT 35 Expression of different genes involved in inflammation,

development and digestion processes

Cerezuela et al. (2013)

Reviews in Aquaculture (2013) 5, 1–14

© 2013 Wiley Publishing Asia Pty Ltd8

T. P�erez-S�anchez et al.

spp. cultures to shrimp culture ponds also permitted the

continuous culture of the shrimp for over 160 days. Farms

that did not use Bacillus spp. cultures experienced almost

complete failure in all ponds in <80 days as a result of mor-

tality caused by luminescent Vibrio spp. pathogens (Mori-

arty 1998). Similarly, the use of B. subtilis reduced the

mortality of white shrimp infected with a pathogenic Vib-

rio parahaemolyticus strain. Controls inoculated with the

pathogenic bacterium had a cumulative mortality of 33%

at 14 days post-infection, whereas the mortality was 19%

in the treated shrimp (Balc�azar et al. 2007d). In addition,

the use of B. subtilis improved feed efficiency and growth

rate of grouper and induced up-regulation of innate

cellular and humoral immune responses (Liu et al. 2012).

This bacterium had also a beneficial effect in juvenile sea

cucumbers (Apostichopus japonicus) after challenge with

Vibrio splendidus (Zhao et al. 2012).

Bacillus species have also been used to improve water

quality in aquaculture (Song et al. 2011). Recently, the

denitrification characteristics of Bacillus sp. strain YX-6

were evaluated, and results indicated that this strain could

degrade the nitrite nitrogen (nitrite-N) concentrations

under aerobic conditions. Accumulation of nitrite and

nitrate is mainly the result of biological oxidation of

ammonia, and these substances are toxic to aquatic species,

thus the application of probiotics, that is, Bacillus species,

may improve the quality of the surrounding environment

of aquatic organisms (Song et al. 2011).

Gram-negative bacteria

The common Gram-negative probiotics that are used for

aquaculture practices include Aeromonas, Enterobacter,

Pseudomonas, Shewanella and Vibrio species (Nayak 2010).

However, the use of Gram-negative bacteria is accompanied

by the risk of transfer of genetic material encoding resistance

to antibiotics or virulence (Kesacordi-Watson et al. 2012).

The increased survival and growth of white shrimp post-

larvae in Ecuadorian hatcheries has been attributed to the

probiotic properties of V. alginolyticus. The authors suggest

that beneficial effect observed derives from the competitive

exclusion of potential pathogenic bacteria in intensive larvi-

culture systems (Garriques & Arevalo 1995). Similar obser-

vations have been described by Balc�azar et al. (2007d), who

noted that administration of V. alginolyticus offer protec-

tion against V. parahaemolyticus in white shrimp.

Riquelme et al. (1996) showed a significantly improved

rate of survival in scallop larvae (Pecten maximus) treated

with Alteromonas haloplanktis following challenge with

V. anguillarum. Gibson et al. (1998) also found that Aero-

monas media reduced the proliferation of Vibrio tubiashii

in Pacific oyster larvae (Crassostrea gigas). Similarly, Ruiz-

Ponte et al. (1999) found that Roseobacter sp. in co-culture

with V. anguillarum displayed an inhibitory effect on Vib-

rio, enhancing the survival of larval scallop.

Gram et al. (1999) reported that Pseudomonas fluores-

cens reduced the mortality of rainbow trout infected with a

pathogenic V. anguillarum strain. Controls inoculated with

the pathogenic bacterium had a cumulative mortality of

47% after 7 days, whereas in the treated fish, mortality was

only 32%. Moreover, Irianto and Austin (2002b) reported

that cultures of A. hydrophila and V. fluvialis were effective

at controlling A. salmonicida infections in rainbow trout.

Pseudomonas sp. isolate MSB1 efficiently inhibited, under

in vitro conditions, the growth of F. psychrophilum, which

suggested a potential use of MSB1 as a probiotic in rainbow

trout aquaculture, especially in early life stages of the fish,

but further in vivo experiments need to be carried out

(Str€om-Bestor & Wiklund 2011). The protection of Pseudo-

monas aeruginosa VSG-2 against A. hydrophila was evalu-

ated in Labeo rohita and the challenge test revealed that

long-term oral administration of probiotic-supplemented

feed enhanced the resistance of L. rohita to bacterial infec-

tions (Giri et al. 2012).

The effect of probiotic bacteria belonging to genera with

pathogenic species, such as Pseudomonas, Vibrio or Aero-

monas, has been observed in research experiments (Leyva-

Madrigal et al. 2011), although regulatory agencies are

responsible to authorize their use in aquaculture.

Yeasts

Yeasts have the advantage that they are not affected by anti-

biotics, which may help to re-establish the normal microbi-

ota after antibiotic treatment. They could also be an

appropriate organism because some strains synthesize and

secrete different polyamine molecules (Tovar-Ram�ırez et al.

2004), and they have strong adhesion to fish intestinal

mucus (Andlid et al. 1995). In fact, Tovar-Ram�ırez et al.

(2002) investigated the secretion of digestive enzymes in sea

bass larvae fed a compound diet supplemented with differ-

ent strains of Saccharomyces cerevisiae and D. hansenii. The

growth and survival of the larvae fed yeast-incorporated diet

were higher than those of the larvae fed control diet.

The probiotic properties of both bacteria and yeasts have

been evaluated in catla (Catla catla) and both have been

found to increase fish survival and body weight (Mohanty

et al. 1996). It has also been demonstrated that the addition

of Phaffia rhodozyma to the diet confers protection against

vibriosis in juvenile white shrimp (Scholz et al. 1999). Sim-

ilar results have been observed with S. cerevisiae in tilapia

(Lara-Flores et al. 2003). Reyes-Becerril et al. (2008) dem-

onstrated that diet supplemented with live yeast D. hansenii

stimulated the immune system of juvenile leopard grouper

(Mycteroperca rosacea). Growth parameters were improved

by the inclusion of 2% dietary S. cerevisiae in juvenile

Reviews in Aquaculture (2013) 5, 1–14

© 2013 Wiley Publishing Asia Pty Ltd 9

Probiotics in aquaculture

beluga (Huso huso) (Hoseinifar et al. 2011). In rainbow

trout, the supplementation of fully fermented S. cerevisiae

improved the growth, which may be due to essential and

nonessential amino acids frequently responsible for increas-

ing palatability, the better digestibility and immunomodu-

latory effects (Sheikhzadeh et al. 2012).

Concluding remarks

While antibiotics have traditionally been used to treat

disease in aquaculture, indiscriminate use can lead to

increased antibiotic resistance (Cabello 2006). Chemother-

apeutic agents can also alter the community composition

of the endogenous intestinal or environmental microbiota,

and such assemblages play an important role in aquatic ani-

mal health. Probiotics are usually members of this microbi-

ota, and their addition can assist a disturbed microbiota in

returning to normal (Nayak 2010; P�erez et al. 2010).

Nowadays, probiotics are becoming an integral part of the

aquaculture practices for improving growth and disease

resistance and obtaining high production (Nayak 2010)

and the concept of maintaining the health of fish through

the best possible nutrition is well-accepted in modern fish

farming (Kiron 2012).

The use of probiotic bacteria as biological control agents

should be considered as an alternative to the chemothera-

peutic agents commonly used in aquaculture for disease

prevention, as probiotics may provide protection through

the creation of a hostile environment for pathogenic bacte-

ria. Furthermore, they may contribute to the host’s nutri-

tion, especially by supplying nutrients, vitamins and

enzymes (P�erez et al. 2010).

Therefore, the mechanisms by which probiotics act in

vivo merit further research. It is also crucial to investigate

the interaction between probiotic bacteria and the host

gastrointestinal microbiota. Such research will allow us to

establish efficient criteria for probiotic strain selection,

which will lead to the use of effective and safe (harmless to

the host) micro-organisms in aquaculture. The selection

and production of probiotics need to be adapted to a range

of environmental conditions, and cost efficiency needs to

be evaluated before large-scale application (Karunasagar

et al. 2010).

Acknowledgement

This work has been supported by the Spanish Ministry of

Science and Innovation (project AGL2011-24889).

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