MINIREVIEW

The use of probiotics in shrimp aquaculture

Ali Farzanfar
Iranian Fisheries Research Organization, Tehran, Iran

Correspondence: Ali Farzanfar, Iranian Abstract

Fisheries Research Organization (IFRO), No.
297, West Fatemi Ave., Tehran, Iran. Tel.:198
912 3153788; fax:198 192 4562534; e-mail:
afarzanfar@yahoo.com
Received 13 February 2006;
accepted 7 April 2006.
First published online 20 June 2006.
DOI:10.1111/j.1574-695X.2006.00116.x
Editor: Willem van Leeuwen
Keywords
shrimp; aquaculture; probiotic; lactic acid
bacteria; Streptococcus spp.; Lactobacillus
spp.; Bacillus spp.
Shrimp aquaculture, as well as other industries, constantly requires new techniques
in order to increase production yield. Modern technologies and other sciences
such as biotechnology and microbiology are important tools that could lead to a
higher quality and greater quantity of products. Feeding and new practices in
farming usually play an important role in aquaculture, and the addition of various
additives to a balanced feed formula to achieve better growth is a common practice
of many fish and shrimp feed manufacturers and farmers. Probiotics, as ‘bio-
friendly agents’ such as lactic acid bacteria and Bacillus spp., can be introduced into
the culture environment to control and compete with pathogenic bacteria as well
as to promote the growth of the cultured organisms. In addition, probiotics are
nonpathogenic and nontoxic microorganisms without undesirable side-effects
when administered to aquatic organisms. These strains of bacteria have many
other positive effects, which are described in this article.

Introduction Background of study

The use of probiotics as farm animal feed supplements dates
back to the 1970s. They were originally incorporated into
feed to increase the animal’s growth and improve its health
by increasing its resistance to disease. The results obtained in
many countries have indicated that some of the bacteria
used in probiotics (Lactobacilli) are capable of stimulating
the immune system (Fuller, 1992).
The beneficial effect of the application of certain bene-
ficial bacteria in human, pig, cattle and poultry nutrition
has been well documented. However, the use of such
probiotics in aquaculture is a relatively new concept.
With interest in treatments with friendly bacterial candi-
dates increasing rapidly in aquaculture, several research
projects that deal with the growth and survival of fish
larvae, crustaceans and oysters have been undertaken (Ali,
2000).
Yasudo and Taga (1980) predicted that some bacteria
would be found to be useful not only as food but also as
biological controllers of fish disease and activators of
nutrient regeneration. It was only in the late 1980s that the
first publication on biological control in aquaculture
emerged, and since then the research effort has continually
increased (Verschuere et al., 2000).
On fishes
Bacteria live in every corner of the aquatic environment. The
fish egg is the first stage of a fish life-cycle that could be
exposed to bacteria. Therefore, a relatively dense, nonpatho-
genic, and diverse adherent microbiota present on the eggs
would probably be an effective barrier against the formation
of a colony by pathogens on fish eggs. In addition, the
establishment of a normal gut microbiota may be regarded
as complementary to the establishment of the digestive
system, and under normal conditions it serves as a barrier
against invading pathogens. Larvae may ingest substantial
amounts of bacteria. It is obvious that the egg microbiota
will affect the primary colonization of the fish larvae
(Verschuere et al., 2000).
Kennedy et al. (1998) used probiotic bacteria in the
culture of marine fish larvae. They identified and used
probionts for the culture of common snook, red drum,
spotted sea trout and striped mullet. They then observed
that the application of probiotic bacteria to larval fish tanks
(from egg through transformation) increased survival, size
uniformity, and growth rate. The periodic addition of
bacteria to the tanks altered the microbial communities of

FEMS Immunol Med Microbiol 48 (2006) 149–158

c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
150
both tanks and fish. In addition, they noticed that the fish
eggs incubated with probiotic bacteria were less likely to
develop bacterial overgrowth and die than those incubated
without probiotic bacteria.
Carnevali et al. (2004) isolated Lactobacillus fructivorans
(AS17B) from sea bream (Sparus aurata) gut, and then
administered it during sea bream development using Bra-
chinons plicatilis and/or Artemia salina and dry feed as
vectors. At the end of the experiments, they found a
significantly decreased larvae and fry mortality in their
treated groups.
Previously, Gildberg et al. (1997) had analysed the effect
of a probiotic of lactic acid bacteria in the feed of Atlantic
cod fry (Gadus morha) on growth and survival rates. In their
study, a dry feed containing lactic acid bacteria (Carnobac-
terium divergens) that had been isolated from adult intes-
tines was given to cod fry. After 3 weeks of feeding the fry
were exposed to a virulent strain of Vibrio anguillarum. The
number of deaths was recorded during a further 3 weeks of
feeding with feed supplemented with lactic acid bacteria. A
certain improvement in disease resistance was obtained, and
at the end of the experiment lactic acid bacteria dominated
the intestinal flora in surviving fish given feed supplemented
with lactic acid bacteria.
Lara-Flores et al. (2003) used two probiotic bacteria and
the yeast, Saccharomyces cerevisiae as growth promoters in
Nile tilapia (Oreochromis niloticus) fry. The results of this
study indicated that the fry subjected to diets with a
probiotic supplement exhibited greater growth than those
fed with the control diet. In addition, they suggested that the
yeast is an appropriate growth-stimulating additive in tilapia
cultivation.
On crustaceans
During the last few decades, aquaculture has become the
world’s fastest growing food production sector, with cul-
tured shrimp growing at an annual rate of 16.8%. Mean-
while, according to a World Bank report, global losses
resulting from shrimp diseases are around 3 billion US
dollars. The potential negative consequences of using anti-
biotics in aquaculture, such as the development of drug-
resistant bacteria and the reduced efficiency of antibiotic
resistant for human and animal diseases, have led to sugges-
tions of the use of nonpathogenic bacteria as probiotic
control agents (Vaseeharan & Ramasamy, 2003).
Moriarty (1999) reported on his successful experiences of
using probiotic bacteria instead of antibiotics to control
Luminus vibrios in shrimp farms in Negros, Philipine. The
effects of ozone and probiotics on the survival of black tiger
shrimp (Penaeus monodon) were recorded by Meunpol et al.
(2003). They investigated the effects of ozone with and
without feeds supplemented with the probiotic Bacillus S11
c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
A. Farzanfar
on bacterial (Vibrio harveyi) growth and shrimp (P. mono-
don) survival. According to the results of their study,
shrimp survival after probiotic treatment, coupled with
ozonation, increased significantly compared with controls.
The antagonistic effect of Bacillus against the pathogenic
Vibrios was evaluated in black tiger shrimp (P. monodon),
and it was suggested as an alternative treatment factor
instead of antibiotics in shrimp aquaculture (Vaseeharan &
Ramasamy, 2003).
In another experiment that was performed by Rengpipat
et al. (2003), the growth and resistance to Vibrio in black
tiger shrimp (P. monodon) fed with a Bacillus probiotic
(BS11) were studied. It was found that the growth and
survival rates of shrimps fed on the probiotic supplement
were significantly greater than those of the controls. Some
strains of Gram-negative bacteria have been used as probio-
tics in shrimps too. For instance, Alvandi et al. (2004)
isolated Pseudomonas sp. PM11 and Vibrio fluvialis PM17
as candidate probions from the gut of farm-reared subadult
shrimp and tested for their effect on the immunity indica-
tors of black tiger shrimp. The results of the study suggest
that the criteria used for the selection of putative probiotic
strains, such as predominant growth on primary isolation
media, ability to produce extracellular enzymes and side-
ropheros, did not bring about the desired effect in vivo and
improve the immune system in shrimp.
Nogami and Maeda (1992) found that production of crab
(Portunus trituberculatus) larvae increased following the
addition of bacterial strain PM-4 to their culture water. He
isolated PM-4 from a crustacean culturing pond and
cultured it in large quantities to add daily to the water of
crab larvae. When bacteria increased to more than a specific
population, the protozoan population grew rapidly and
reduced the bacterial population.
On bivalve mollusks
The mass culture of scallops and oysters has been introduced
in many countries. However, mass mortalities of larvae have
frequently occurred, limiting the success of the hatcheries.
To prevent these mortalities, most farmers routinely use
antibiotics. As mentioned above, antibiotics have limited
applicability, because of the ability of a large variety of
pathogens to develop multiple antibiotic resistance. An
alternative method for controlling pathogenic bacterial
strains in bivalve farms may be the addition of pure culture
of natural bacteria isolates (probiotics), which have been
shown through experimentation to produce chemical sub-
stances inhibitory to bacterial pathogens (Gildberg et al.,
1997; Riquelme et al., 1997; Vaseeharan & Ramasamy, 2003).
Alteromons haloplanktis was isolated from the gonads of
Chilean scallop (Argopecten purpuratus) brood stock and
displayed in vitro inhibitory activity against the known
FEMS Immunol Med Microbiol 48 (2006) 149–158
Use of probiotics in shrimp aquaculture
pathogens Vibrio ordalii, V. parahaemolyticus, V. anguillar-
um, V. alginolyticus and Aeromonas hydrophila. In an experi-
mental infection, the A. haloplanktis and a Vibrio strain 11
(that showed in vitro inhibition effects on V. anguillarum)
protected the scallop larvae against the V. anguillarum
(Riquelme et al., 1997; Verschuere et al., 2000).
Douillet & Langdon (1994) added a bacteria strain (CA2)
as a food supplement to larval cultures of the oyster
Crassostrea gigas. They found more growth in larvae that
had been treated by CA2 bacteria cells.
On water quality
There are no serious problems for water quality during the
initial stages of farming aquatic organisms, when the
stocked organisms are small and their metabolism rate and
amounts of supplementary feed are low. However, with the
progress of culture the organisms grow, leading to a rapid
increase in biomass, and water quality deteriorates, mainly
as a result of the accumulation of metabolic waste of
cultured organisms, decomposition of unutilized feed, and
decay of biotic materials (Prabhu et al., 1999). At this time,
the application of a group of beneficial microorganisms
(such as Lactobacillus, Bacillus, Nitrosomonas, Cellulomonas,
Nitrobacter, Pseudomonas, Rhodoseudomonas, Nitrosomonas
and Acinetobacter) would be very useful for controlling the
pathogenic microorganisms and water quality (Prabhu
et al., 1999; Shariff et al., 2001; Irianto & Austin, 2002).
By definition, bacteria added directly to pond water are
not probiotics, and should not be compared with living
microorganisms added to feed (Rengpipat et al., 2003).
Many workers have evaluated some specific microorganisms
as biological improvers for water quality: Douilett (1998)
used a probiotic additive consisting of a blend of bacteria in
a liquid suspension in intensive production systems. The
probiotic blend improved water quality in fish and crusta-
cean cultures by reducing the concentration of organic
materials (OM) and ammonia. This procedure was accom-
plished by a series of enzymatic processes carried out in
succession by the various strains present in the probiotic
blend. The addition of this blend to culture systems reduced
the concentration of Vibrio strains and thus controlled
diseases caused by Vibrio strains. In addition, Bacillus spp.
have been evaluated as probiotics, with uses including the
improvement of water quality by influencing the composi-
tion of water-borne microbial populations and reducing the
number of pathogens in the vicinity of the farm species.
Thus, the Bacilli are thought to antagonize potential patho-
gens in the aquatic environment (Irianto & Austin, 2002).
Bacterial species belonging to the genera Bacillus, Pseudo-
monas, Nitrosomonas, Nitrobacter, Acinetobacter and Cellu-
lomonas are known to help in the mineralization of organic
water and in reducing the accumulation of organic loads
FEMS Immunol Med Microbiol 48 (2006) 149–158
151
(Shariff et al., 2001). Furthermore, there are many reports of
the use of microbial products in aquaculture ponds for
increasing the removal rate of ammonia. Prabhu et al.
(1999) used some microorganisms in a shrimp farm to
evaluate them as a factor for controlling the water quality.
According to the results of this study, all factors of water-
quality parameters were at optimum levels in the experi-
mental ponds compared with the control.
On human consumption
The use of live microorganisms to enhance human health is
not new. For thousands of years, long before the discovery of
antibiotics, people have been consuming live microbial food
supplements such as fermented milks. According to Ayurve-
da, one of the oldest medical sciences that dates back to
around 2500 BC, the consumption of yoghurt is recom-
mended for the maintenance of overall good health. A
scientific explanation of the beneficial effects of lactic acid
bacteria present in fermented milk was first provided in
1907 by the Nobel Prize-winning Russian physiologist Eli
Metchnikoff. In his fascinating treatise ‘The Prolongation of
Life’, Metchnikoff states that, ‘The dependence of the
intestinal microbes on the food makes it possible to adopt
measures to modify the flora in our bodies and to replace
the harmful microbes by useful microbes’ (Talwalkar, 2003).
He proposed that the acid-producing organisms in fermen-
ted dairy products could prevent ‘fouling’ in the large
intestine and thus lead to a prolongation of the life span of
the consumer (Heller, 2001). Probiotics have a great variety
of effects on human health. Probiotic therapy could be used
for applications such as: modulation of the intestinal
microbial communities, immune modulation, controlling
allergic diseases, treating diseases related to the gastrointest-
inal tract such as inflammatory bowel disease, and control-
ling colorectal cancer and constipation (Ouwehand et al.,
2002).
Literature review on probiotics
Definitions and history
The word ‘probiotics’ originates from the Greek word ‘for
life’, and is currently used to name bacteria associated with
beneficial effects for humans and animals. The definition of
probiotics has, however, evolved over time. Lily & Stillwell
(1965) had originally proposed to use the term to describe
compounds produced by one protozoan that stimulated the
growth of another. The scope of this definition was further
expanded by Sperti in the early 1970s to include tissue
extracts that stimulated microbial growth (Gomes & Mal-
cata, 1999). Thereafter, other scientists applied the term to
animal feed supplements having a beneficial effect on the
c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
152
host by contributing to its intestinal microbial balance
(Talwalkar, 2003). Consequently, the term probiotics was
applied to describe ‘organisms and substances that contri-
bute to intestinal microbial balance’. This general definition
was made more precise by Fuller (1989), who defined
probiotics as ‘a live microbial feed supplement that benefi-
cially affects the host animal by improving its intestinal
microbial balance’. This was further revised to ‘viable
microorganisms (lactic acid and other bacteria, or yeasts
applied as dried cells or in a fermented product) that exhibit
a beneficial effect on the health of the host upon ingestion by
improving the properties of its indigenous microflora’
(Havenaar & Huis in’t Veld, 1992). In addition, there is
another definition of probiotics by Coeuret et al. (2004) as
‘living microorganisms that, upon ingestion in certain
numbers, exert health benefits beyond inherent basic nutri-
tion’.
Characterization
Lactic acid bacteria
These are classified in the group of Gram-positive bacteria.
They usually have no mobility and are nonsporulating
bacteria that produce lactic acid. Some members of this
group contain both rods (lactobacilli and carnobacteria)
and cocci (streptococci). Different species of lactic acid
bacteria (such as Streptococcus, Leuconostoc, Pediococcus,
Aerococcus, Enterococcus, Vagococcus, Lactobacillus, Carno-
bacterium) have adapted to grow under widely different
environmental conditions. They are found in the gastro-
intestinal tract of various endothermic animals, in milk and
dairy products, seafood products, and on some plant
surfaces (Ring & Gatesoupe, 1998). Although lactic acid
bacteria are not dominant in the normal intestinal micro-
biota of larval or growing fish, several trials have been
undertaken to induce an artificial dominance of lactic acid
bacteria in aquatic animals (Verschuere et al., 2000).
Lactobacilli, as a major group of lactic acid bacteria
(LAB), have the ability to (Reid, 1999; Vázquez et al., 2005):
(1) adhere to cells;
(2) exclude or reduce pathogenic adherence;
(3) compete for essential nutrients;
(4) stimulate the immunity of the host;
(5) persist and multiply;
(6) produce acids, hydrogen peroxide, and bacteriocins
antagonistic to pathogen growth;
(7) resist vaginal microbicides, including spermicides (for
terrestrial animals);
(8) be safe and therefore noninvasive, noncarcinogenic, and
nonpathogenic;
(9) coaggregate and form a normal, balanced flora.
c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
A. Farzanfar
The incubation temperature, incubation time and nutri-
ent medium are the most important factors when isolating
lactic acid bacteria from fish. Lactic acid bacteria are only
rarely isolated from the larvae of aquatic organisms, because
of limiting factors such as incubation water temperature,
incubation time, and the lack of glucose in the medium. The
latter two are the most important factors for some strains of
lactic acid bacteria, because their growth rate is slow and
they require some special nutrients, such as sugar as an
energy and carbon source, nucleotides, fatty acids, amino-
acids and vitamins in their habitat (Brock & Madigan, 1991;
Ring & Gatesoupe, 1998).
Dietary polyunsaturated fatty acids, competition for
nutrients, chromic oxide, salinity and stress are some of the
factors that affect lactic acid bacteria in the gastrointestinal
tract as follows (Maczulak et al., 1981; Ring & Gatesoupe,
1998).
(1) Food and feeds are the main resources for colonizing
some living acid bacteria in the digestive tract. Dietary
long-chain fatty acids, particularly unsaturated fatty
acids, are toxic for many bacterial species, especially
cellulolytic bacteria. However, it is well known that
small amounts of polyunsaturated fatty acids stimulate
the growth of certain microorganisms, as dietary fatty
acids affect attachment sites, possibly by modifying fatty
acid composition of the intestinal wall.
(2) One of the other factors affecting lactic acid bacteria is
competition for nutrients, which is described further in
the next section. Advantages of the use of probiotics and
mode of action.
(3) In addition, chromic oxide, Cr2O3, is one of the most
widely used indicators for determining nutrient digest-
ibility in mammals and fish. When some aquatic
animals were fed with a diet containing chromic oxide,
there was a decline in Gram-negative bacteria genera in
gut and faecal samples, while lactobacilli counts re-
mained stable. There are two hypotheses for this reac-
tion: (a) chromic oxide affects attachment sites to the
intestinal wall, or (b) oxidase-positive microorganisms
may be more sensitive to the surface of chromic oxide
molecules.
(4) The intestinal microbiota of some fishes usually changes
in different aquatic environments with various sali-
nities. Although, for example, in Arctic charr, Salvelinus
alpina, the counts of some strains such as Leuconostoc
spp. and Streptococcus spp. remained stable when the
charr were reared in seawater.
(5) There are many stressful factors, such as starvation or
reduced food intake, that may cause an unbalancing
situation in the intestinal microbiota of aquatic animals.
These factors cause a decrease in lactobacilli coloniza-
tion, but, on the other hand, stress increases coliforms
in the intestinal tract. However, prebiotics and
FEMS Immunol Med Microbiol 48 (2006) 149–158
Use of probiotics in shrimp aquaculture
probiotics are of potential value in these conditions,
where the balance of the gut microbiota is adversely
affected.
Bacillus bacteria
Bacillus subtilis is currently being used for aquaculture,
terrestrial livestock and in human consumption as an oral
bacteriotherapy and bacterioprophylaxis of gastrointestinal
disorders. Bacillus species are saprophytic Gram-positive,
nonpathogenic, spore-forming organisms normally found
in air, water, dust, soil and sediments (Gatesoupe, 1999;
Green et al., 1999; Moriarty, 1999). These bacteria are
considered allochthonous and enter the gut by association
with food. They are also involved in food spoilage (e.g.
spoilage of milk by B. cereus strains; Hong et al., 2005).
Selection of probiotics
The principal purpose of the use of probiotics is to produce
a proper relationship between useful microorganisms and
the pathogenic microflora of digestive organs and their
environment. Hence, a successful probiotic is expected to
have a few specific properties as follows:
(1) Antagonism to pathogens, which is one property of
probiotic bacteria (Fuller, 1992; Austin et al., 1995;
Moriarty, 1999; Ali, 2000; Verschuere et al., 2000; Chang
& Liu, 2002; Irianto & Austin, 2002; Irianto & Austin,
2003). Probiotics should stimulate the immunity of the
host by increasing the number of erythrocytes, macro-
phages and lymphocytes (Irianto & Austin, 2002). One
sign of antagonistic properties against bacteria is the
production of antimicrobial substances such as organic
acids, hydrogen peroxide, sideropheros and lysozyme (Ali,
2000; Verschuere et al., 2000; Irianto & Austin, 2002).
(2) Benefits to the host animal in some ways. In order to
have a beneficial effect in the form of a growth promoter
or to protect fish against bacterial pathogens, the strains
should produce important substances, for example
vitamins such as biotin and vitamin B12 (Fuller, 1992;
Ali, 2000; Irianto & Austin, 2002).
(3) The capability of surviving in or colonizing the gut of an
aquatic organism by adhesion (Fuller, 1992; Ali, 2000;
Verschuere et al., 2000). Similarly, the presence of a
dominant bacterial strain in high densities in culture
water indicates its ability to grow successfully under the
general conditions, and one can expect that this strain
will compete efficiently for nutrients with possibly
harmful strains. Of course, identification of the isolates
at this stage is not essential (Verschuere et al., 2000).
(4) Adhesion is one of the most important selection criteria
for probiotic bacteria because it is considered a pre-
requisite for colonization (Fuller, 1992; Ali, 2000;
Verschuere et al., 2000).
FEMS Immunol Med Microbiol 48 (2006) 149–158
153
(5) Applied microorganisms should be stable for long
periods under storage as well as in field conditions
(Fuller, 1992).
(6) Probiotic microorganisms will, of course, have to be
nonpathogenic and nontoxic in order to avoid undesir-
able side-effects when administered to aquatic organ-
isms (Fuller, 1992).
(7) Probiotics should be of animal-species origin. This
criterion is based on ecological reasons, and takes into
consideration the original habitat of the selected bacter-
ia (in intestinal flora). Many workers believe these
bacteria have a better chance of out-competing resident
bacteria and establishing themselves at a numerically
significant level in their new host (Rengpipat et al.,
2003; Riquelme et al., 1997; Alvandi et al., 2004; Jöborn
et al., 1997). In addition, the existence of a dominant
bacterial strain in high densities in culture water in-
dicates its ability to grow successfully under the prevail-
ing conditions, and one can expect that this strain will
compete efficiently for nutrients with possible harmful
strains (Verschuere et al., 2000).
Gram-positive bacteria such as Bacillus offer an alternative
to antibiotic therapy for shrimp farming. These species of
bacteria are commonly found in marine sediments and there-
fore are naturally ingested by shrimps that feed in or on the
sediments (Moriarty, 1999). Bacillus subtilis is a gram-positive,
nonpathogenic, spore-forming organism, and the robustness
of spores is thought to enable passage across the gastric barrier,
and population, albeit briefly, of the intestinal tract. In
addition, the clinical effects of B. subtilis as an immunostimu-
latory agent in a variety of diseases in human and animals, as
an in vitro and in vivo stimulant of secretor immunoglobulin
A, and as an in vitro mitogenic agent have been documented
(Green et al., 1999). Furthermore, one of the most important
advantages of using Bacillus species is that they are unlikely to
use genes for antibiotic resistance or virulence from the Vibrios
or related Gram-negative bacteria. There are barriers at the
transcriptional and translational levels to the expression of
genes from plasmid, phages and chromosomal DNA of
Escherichia coli in B. subtilis (Moriarty, 1999).
There are many other reports regarding the advantages of
using Gram-positive bacteria in aquaculture. For instance,
Vaseeharan & Ramasamy (2003) reported on the antagonis-
tic effect of B. sublitis BT23 against the pathogenic Vibrios in
P. monodon, and a 90% reduction in accumulated mortality.
The application of Bacillus as a probiotic bacteria in
common snook, Centropomus undecimalis (Bloch), can
improve the survival rate of larvae, increasing food absorp-
tion by enhancing protease levels, and gave better growth.
Moreover, the probiotic decreased the number of suspected
pathogenic bacteria in the gut (Irianto & Austin, 2002).
Some Gram-negative bacteria such as Pseudomonas I-2
have been reported to inhibit V. hervey and V. fluvialis in
c2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
154
shrimp culture (Irianto & Austin, 2002). However, there is
some evidence concerning the transfer of many antibiotic
resistance genes between pathogenic and nonpathogenic
Gram-negative bacteria in several environments, including
seawater. Moreover, genes for virulence can be transferred by
R plasmids and transposes, as the R plasmids can transfer
genes between widely different bacteria in the Gram-nega-
tive group (Moriarty, 1999).
In addition, Alvandi et al. (2004) isolated some Gram-
negative bacteria (such as Pseudomonas sp. PM11 and Vibrio
fluvialis PM17 from the gut of farm-reared shrimp, P.
monodon, and tested for their effect on the immunity
indicators of black tiger shrimp. However, the results of
their study did not indicate the desirable effect of an
improvement in the immune system in shrimp.
Lactic acid bacteria are not dominant in the normal
intestinal microbiota of fish, at variance with homeotherms,
but some strains can colonize the gut. It is, however, possible
to maintain artificially the lactic acid bacterial population at
a high level by regular intake with food (Ring & Gatesoup,
1998).
The microbial species composition in hatchery tanks or
large aquaculture ponds can be changed by adding selected
bacterial species to displace deleterious normal bacteria
(Moriarty, 1999). Aquatic animals are poikilothermic, and
their associated microbiota may vary with changes of
temperature and salinity. In addition, many marine animals
need to drink constantly to prevent water loss from the
body. This continuous water flow increases the influence of
the surrounding medium, in the same way as the water flow
observed in filter-feeders, such as bivalves, shrimp larvae
and live food organisms. This influence is particularly
important in the larval stages (Gatesoupe, 1999), because
larvae may ingest bacteria by grazing on or filtering the
suspended particles. It is suggested that probiotics may be
most effective when applied to penaeid larval rearing tanks
containing naupliar stages, when the larvae have not yet
Table 1. Probiotics applied in aquaculture (after Irianto & Austin, 2002)
Identity of the probiotic Source
Gram-positive bacteria
Bacillus sp. S11 Penaeus monodon
Bacillus sp. Commercial product
Bacillus sp.
Lactobacillus lactis AR21 Rotifer mass culture
Mixed culture, mostly Bacillus spp. Commercial product
Gram-negative bacteria
Vibrio alginolyticus Beach sand
Yeast
Saccharomyces cerevisiae, S. exiguous, Commercial product
Phaffia rhodozoma
Microalgae
Tetraselmis suecica Commercial product
c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
A. Farzanfar
started feeding (Irianto & Austin, 2002; Rengpipat et al.,
2003), and the digestive tract is not yet developed comple-
tely and the immune system is still incomplete. Therefore,
the intestinal microbiota of larvae may change rapidly with
the intrusion of microorganisms coming from water and
food (Vadstein, 1997; Gatesoupe, 1999; Olafsen, 2001).
When microbial control is desired, single strains of
probiotics are less effective than mixed-culture probiotics.
The approach should be systemic, i.e. based on a number of
strains capable of acting and interacting under a variety of
conditions and able to maintain themselves in a dynamic
way. In addition, as has been argued above, the microbial
community in the gut of aquatic organisms may vary with
changes in many factors. It is unlikely that a single bacterial
species will be able to remain dominant in a continuously
changing environment. Furthermore, the probability that a
beneficial bacterium will dominate the associated microbio-
ta is higher when several bacteria are administered than
when only one probiotic strain is involved (Verschuere et al.,
2000).
The range of probiotics examined for use in shrimp
aquaculture has encompassed Gram-negative and Gram-
positive bacteria, yeasts, and unicellular algae (Table 1;
Irianto & Austin, 2002).
Advantages of the use of probiotics and mode
of action
The use of probiotics such as lactic acid bacteria and Bacillus
has had positive results. The advantages of the use of
probiotics might be obtained by some specific modes of
action, which are described below.
Stimulating the immunity of the host
There are many reports that some bacterial compounds act
as an immunostimulant in fish and shrimp. Generally,
Used on Method of application
Penaeus monodon Premix with feed
Penaeids Water
Water Added to water
Brachionus plicatilis Feed additive
Brachionus plicatilis Mixed in water
Penaeids, salmomids Feed, bath for 10 min
Litopenaeus vannamei Premix with feed
Penaeids, Salmo salar Feed
FEMS Immunol Med Microbiol 48 (2006) 149–158
Use of probiotics in shrimp aquaculture 155

immunity may be improved by the probiotic in three ways Competition for nutrients, space and Fe
(Fuller, 1992):
Theoretically, competition for nutrients can play on impor-
(1) Increasing macrophage activity, shown by the enhanced
tant role in the composition of the microbiota of the
ability to phagocytose microorganisms or carbon parti-
intestinal tract or ambient environment of cultured aquatic
cles;
species (Verschuere et al., 2000). Increasing some strains of
(2) Increasing the production of systematic antibodies,
bacteria such as lactobacillus and bacillus by way of a
usually of immunoglobulin and interferon (a nonspe-
probiotic may thereby decrease the substrate available for
cific antiviral agent);
other bacterial populations (Fooks & Gibson, 2002). Com-
(3) Increasing local antibodies at mucus surfaces such as the
petition for space (adhesion sites) in the gut or other tissues
gut wall.
in the digestive tract would be an antagonistic mechanism to
Irianto & Austin (2002) reported that feeding with Gram-
colonization of pathogenic bacteria by probiotics
positive and Gram-negative probiotics at 107 cells per g of
(Verschuere et al., 2000). In view of the reports on the
feed led to the stimulation of cellular rather than humeral
presence of lactic acid bacteria in the intestinal microflora of
(serum of mucus antibodies) immunity. Notably, there was
aquatic organisms, it may be suggested that there exist lactic
an increase in the number of erythrocytes, macrophages and
acid bacteria that constitute nonpathogenic members of the
lymphocytes, and enhanced lysozyme activity within 2
indigenous intestinal microbiota of healthy aquatic organ-
weeks of feeding with probiotics. In this case, the probiotics
ism. In addition, the gastrointestinal tract may serve as an
were behaving almost like oral vaccines. Vázquez et al.
ecological niche for some probiotics such as lactic acid
(2005) found that lactic acid bacteria have inhibitory effects
bacteria strains to fish via the feed. These strains may be
on the growth of vibrios in turbot (Scophthalmus maximus).
metabolically active in the intestinal mucus and feces of an
They proposed some mechanisms in this regard, such as
aquatic organism and grow more than pathogenic bacteria
inhibition or antibiosis of the unwanted microbiota by
in the digestive tract (Jöborn et al., 1997).
metabolites typical of lactic acid bacteria (organic acids,
Successful probiotic bacteria are usually able to colonize
bacteriocins); the competition for sites of adhesion to the
the intestine, at least temporarily, by adhering to the
mucus or the phenomenon of competition for essential
intestinal mucosa. The adhesive probiotic bacteria could
nutrients; inmunostimulation induced by the probiotics or
prevent the attachment of pathogens, such as coliform
associated metabolites.
bacteria and clostridia, and stimulate their removal from
Recently, it has been shown that b-1.3-glucans from the
the infected intestinal tract (Lee et al., 2000; Vine et al.,
yeast cell wall give improved resistance against various
2004).
infectious diseases, when given either as a feed supplement
Iron is necessary for the growth of microorganisms, and
or as an adjuvant in fish vaccine. Apparently, the b-1.3-
successful bacterial strains are able to compete successfully
glucans stimulate the nonspecific immune defence system of
for iron in the highly iron-stressed gut environment
the fish by activating the macrophages (Gildberg et al., 1997).
(Verschuere et al., 2000). In a challenge test, Smith & Davey
(1993) showed that fluorescent strain pseudomonad bacter-
Production of inhibitory compounds ia can competitively inhibit the growth of the fish pathogen
Aeromonas salmonicida. Their results show that the fluores-
The antibacterial effect of bacteria results from factors such
cence is probably due to competition for free iron (Smith &
as the production of antibiotics, bacteriocins, sideropheros,
Davey, 1993; Gram et al., 1999).
lysozyme, protease, hydrogen peroxide, the alteration of pH
Sideropheros are low-molecular-weight, ferric iron-spe-
values, and the production of organic acids and ammonia
cific chelating agents that can dissolve precipitated iron and
(Verschuere et al., 2000).
make it available for microbial growth (Verschuere et al.,
Lactic acid bacteria and Bacillus produce several com-
2000).
pounds that may inhibit the growth of competing bacteria.
Among these compounds, the bacteriocins are the most
important (Gildberg et al., 1997; Ali, 2000). These are
The potential drawbacks of using antibiotics
proteins, or protein complexes, produced by certain strains
of bacteria that can have an antagonistic action against Antibiotics have been in use since the second word war, and
species that are closely related to the producer bacterium. these drugs have played an important role in curing disease
Bacteriocins are divided into four classes: class I anti- in humans and animals. Moreover, because prevention of
biotics; class II small hydrophobic, heat-stable peptides; disease transmission and enhancement of growth and feed
class III large heat-stable peptides; and class IV efficiency are critical in modern animal husbandry, there has
complex bacteriocins: probiotics with lipid and/or carbohy- been widespread incorporation of antibiotics into animal
drate (Fooks & Gibson, 2002). feeds in many countries (Doyle, 2001). During the last few

FEMS Immunol Med Microbiol 48 (2006) 149–158

c2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
156
decades, the public has become increasingly alarmed by new
scientific data that make their way into the popular media
about the connection between the overuse of antibiotics in
both medicine and the agriculture agrifood industry and
the emergence and spread of antibiotic-resistant bacteria.
Microbial resistance to antibiotics is on the rise (Khacha-
tourions, 1998). The increase in the anxiety about antibio-
tic-resistant microorganisms has led to suggestions of
alternative disease-prevention methods, such as probiotic
bacteria (Vaseeharan & Ramasamy, 2003).
Vibrio spp., especially the luminous V. harveyi, have been
implicated as the main bacterial pathogens of shrimps.
Antibiotics such as chloramphenicol, furazolidone, oxyte-
tracycline and streptomycine have been used in attempts to
control these bacteria, but their efficacy is now, in general,
very poor. Chlorine is widely used in hatcheries and ponds
for killing zooplankton before stocking shrimp, but its use
stimulates the development of multiple antibiotic resistance
genes in bacteria. There is a rapid increase in V. harveyi
numbers after the chlorine is removed from ponds, because
chlorine treatment lowers the numbers of competitors for
nutrients and kills algae, thus increasing food resources. If
antibiotics or disinfectants are used to kill bacteria, some
bacteria will survive, because they carry genes for resistance.
These will then grow rapidly because their competitors are
removed (Moriarty, 1999). Two conditions are needed for
antibiotic resistance to develop in bacteria. First, the organ-
ism must come into contact with the antibiotic. Then,
resistance against the agent must develop, along with a
mechanism to transfer the resistance to daughter organisms
or directly to other member of the same species (Khacha-
tourians, 1998).
Stimulating the growth and improving the
nutrients in the host
Aquaculture is one of the most important options in animal
protein production, and requires high-quality feeds with a
high protein content as well as some complementary
additives to keep organisms healthy and favour growth.
Owing to some problems and limitations in using hormones
and antibiotics for animals and the final consumer, probio-
tic bacteria are a good candidate for improving the digestion
of nutrients and growth in aquatic organisms (Irianto &
Austin, 2002; Lara-Flores et al., 2003). The effects of some
bacteria strains have been studied by Lara-Flores et al.
(2003). They found that all the probiotic-supplemented
diets resulted in growth higher than that with the control
diets. In addition, they suggested that the probiotics could
mitigate the effects of the stress factors. The nutrients in
organisms could be improved by the detoxification of
potentially harmful compounds in the diet by hydrolytic
enzymes, including amylases and proteases, and the produc-
c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
A. Farzanfar
tion of vitamins such as biotin and vitamin B12 (Irianto &
Austin, 2002).
Venkat et al. (2004) evaluated the effects of some probio-
tics on the growth of postlarvae of Macrobranchium rosen-
bergii. According to their results, significant growth was
observed for larvae fed diets supplemented with probiotics.
The highest protein gain (more than 55%) and the protein
efficiency ratio were significantly higher in the treatments
that fed on probiotic supplements. Bacteria, by virtue of
their extra cellular enzymes, have been reported to play an
important role in the process of digestion and the assimila-
tion of nutrients in the gut of the host by modifying the gut
flora.
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