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M I N I R E V I E W

The use of probiotics in shrimp aquaculture

Ali Farzanfar

Iranian Fisheries Research Organization, Tehran, Iran

Correspondence: Ali Farzanfar, Iranian

Fisheries Research Organization (IFRO), No.

297, West Fatemi Ave., Tehran, Iran. Tel.:198

912 3153788; fax:198 192 4562534; e-mail:

[email protected]

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.

Abstract

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 intothe 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

The use of probiotics as farm animal feed supplements dates

back to the 1970s. They were originally incorporated intofeed to increase the animals 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).

Background of study

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) 149158 c 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved


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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

worlds 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

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 factorinstead 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 specificpopulation, 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) 149158c 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

150 A. Farzanfar


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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; Shariffet 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 microorganismsas 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

(Shariffet 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 acidbacteria 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 intestinalmicrobial 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


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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 byB. 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 organicacids, 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).

(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; Joborn

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 ofB. 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 immunoglobulinA, 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 ofB. 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




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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 particularlyimportant 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

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 ofprobiotics 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,

Table 1. Probiotics applied in aquaculture (after Irianto & Austin, 2002)

Identity of the probiotic Source Used on Method of application

Gram-positive bacteria

Bacillus sp. S11 Penaeus monodon Penaeus monodon Premix with feed

Bacillus sp. Commercial product Penaeids Water

Bacillus sp. Water Added to water

Lactobacillus lactis AR21 Rotifer mass culture Brachionus plicatilis Feed additive

Mixed culture, mostly Bacillus spp. Commercial product Brachionus plicatilis Mixed in water

Gram-negative bacteria

Vibrio alginolyticus Beach sand Penaeids, salmomids Feed, bath for 10 min

Yeast

Saccharomyces cerevisiae, S. exiguous,

Phaffia rhodozoma

Commercial product Litopenaeus vannamei Premix with feed

Microalgae

Tetraselmis suecica Commercial product Penaeids, Salmo salar Feed


154 A. Farzanfar


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immunity may be improved by the probiotic in three ways

(Fuller, 1992):

(1) Increasing macrophage activity, shown by the enhanced

ability to phagocytose microorganisms or carbon parti-

cles;

(2) Increasing the production of systematic antibodies,

usually of immunoglobulin and interferon (a nonspe-cific antiviral agent);

(3) Increasing local antibodies at mucus surfaces such as the

gut wall.

Irianto & Austin (2002) reported that feeding with Gram-

positive and Gram-negative probiotics at 107 cells per g of

feed led to the stimulation of cellular rather than humeral

(serum of mucus antibodies) immunity. Notably, there was

an increase in the number of erythrocytes, macrophages and

lymphocytes, and enhanced lysozyme activity within 2

weeks of feeding with probiotics. In this case, the probiotics

were behaving almost like oral vaccines. Vazquez et al.

(2005) found that lactic acid bacteria have inhibitory effects

on the growth of vibrios in turbot (Scophthalmus maximus).

They proposed some mechanisms in this regard, such as

inhibition or antibiosis of the unwanted microbiota by

metabolites typical of lactic acid bacteria (organic acids,

bacteriocins); the competition for sites of adhesion to the

mucus or the phenomenon of competition for essential

nutrients; inmunostimulation induced by the probiotics or

associated metabolites.

Recently, it has been shown that b-1.3-glucans from the

yeast cell wall give improved resistance against various

infectious diseases, when given either as a feed supplement

or as an adjuvant in fish vaccine. Apparently, the b-1.3-

glucans stimulate the nonspecific immune defence system ofthe fish by activating the macrophages (Gildberg et al., 1997).

Production of inhibitory compounds

The antibacterial effect of bacteria results from factors such

as the production of antibiotics, bacteriocins, sideropheros,

lysozyme, protease, hydrogen peroxide, the alteration of pH

values, and the production of organic acids and ammonia

(Verschuere et al., 2000).

Lactic acid bacteria and Bacillus produce several com-

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

proteins, or protein complexes, produced by certain strains

of bacteria that can have an antagonistic action against

species that are closely related to the producer bacterium.

Bacteriocins are divided into four classes: class I anti-

biotics; class II small hydrophobic, heat-stable peptides;

class III large heat-stable peptides; and class IV

complex bacteriocins: probiotics with lipid and/or carbohy-

drate (Fooks & Gibson, 2002).

Competition for nutrients, space and Fe

Theoretically, competition for nutrients can play on impor-

tant role in the composition of the microbiota of the

intestinal tract or ambient environment of cultured aquatic

species (Verschuere et al., 2000). Increasing some strains of

bacteria such as lactobacillus and bacillus by way of a

probiotic may thereby decrease the substrate available forother bacterial populations (Fooks & Gibson, 2002). Com-

petition for space (adhesion sites) in the gut or other tissues

in the digestive tract would be an antagonistic mechanism to

colonization of pathogenic bacteria by probiotics

(Verschuere et al., 2000). In view of the reports on the

presence of lactic acid bacteria in the intestinal microflora of

aquatic organisms, it may be suggested that there exist lactic

acid bacteria that constitute nonpathogenic members of the

indigenous intestinal microbiota of healthy aquatic organ-

ism. In addition, the gastrointestinal tract may serve as an

ecological niche for some probiotics such as lactic acid

bacteria strains to fish via the feed. These strains may bemetabolically active in the intestinal mucus and feces of an

aquatic organism and grow more than pathogenic bacteria

in the digestive tract (Joborn et al., 1997).

Successful probiotic bacteria are usually able to colonize

the intestine, at least temporarily, by adhering to the

intestinal mucosa. The adhesive probiotic bacteria could

prevent the attachment of pathogens, such as coliform

bacteria and clostridia, and stimulate their removal from

the infected intestinal tract (Lee et al., 2000; Vine et al.,

2004).

Iron is necessary for the growth of microorganisms, and

successful bacterial strains are able to compete successfully

for iron in the highly iron-stressed gut environment

(Verschuere et al., 2000). In a challenge test, Smith & Davey

(1993) showed that fluorescent strain pseudomonad bacter-

ia can competitively inhibit the growth of the fish pathogen

Aeromonas salmonicida. Their results show that the fluores-

cence is probably due to competition for free iron (Smith &

Davey, 1993; Gram et al., 1999).

Sideropheros are low-molecular-weight, ferric iron-spe-

cific chelating agents that can dissolve precipitated iron and

make it available for microbial growth (Verschuere et al.,

2000).

The potential drawbacks of using antibiotics

Antibiotics have been in use since the second word war, and

these drugs have played an important role in curing disease

in humans and animals. Moreover, because prevention of

disease transmission and enhancement of growth and feed

efficiency are critical in modern animal husbandry, there has

been widespread incorporation of antibiotics into animal

feeds in many countries (Doyle, 2001). During the last few




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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-

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 proteinefficiency 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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