MINIREVIEW

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: 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.

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 sh and shrimp feed manufacturers and farmers. Probiotics, as biofriendly 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
The use of probiotics as farm animal feed supplements dates back to the 1970s. They were originally incorporated into feed 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 benecial effect of the application of certain benecial 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 candidates increasing rapidly in aquaculture, several research projects that deal with the growth and survival of sh 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 sh disease and activators of nutrient regeneration. It was only in the late 1980s that the rst publication on biological control in aquaculture emerged, and since then the research effort has continually increased (Verschuere et al., 2000).
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Background of study
On shes Bacteria live in every corner of the aquatic environment. The sh egg is the rst stage of a sh life-cycle that could be exposed to bacteria. Therefore, a relatively dense, nonpathogenic, and diverse adherent microbiota present on the eggs would probably be an effective barrier against the formation of a colony by pathogens on sh 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 sh larvae (Verschuere et al., 2000). Kennedy et al. (1998) used probiotic bacteria in the culture of marine sh larvae. They identied 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 sh 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
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both tanks and sh. In addition, they noticed that the sh 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 Brachinons plicatilis and/or Artemia salina and dry feed as vectors. At the end of the experiments, they found a signicantly 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 (Carnobacterium divergens) that had been isolated from adult intestines 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 ora in surviving sh 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 cultured shrimp growing at an annual rate of 16.8%. Meanwhile, according to a World Bank report, global losses resulting from shrimp diseases are around 3 billion US dollars. The potential negative consequences of using antibiotics in aquaculture, such as the development of drugresistant bacteria and the reduced efciency of antibiotic resistant for human and animal diseases, have led to suggestions 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
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on bacterial (Vibrio harveyi) growth and shrimp (P. monodon) survival. According to the results of their study, shrimp survival after probiotic treatment, coupled with ozonation, increased signicantly 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 signicantly greater than those of the controls. Some strains of Gram-negative bacteria have been used as probiotics in shrimps too. For instance, Alvandi et al. (2004) isolated Pseudomonas sp. PM11 and Vibrio uvialis PM17 as candidate probions from the gut of farm-reared subadult shrimp and tested for their effect on the immunity indicators 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 sideropheros, 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 specic 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 substances 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
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pathogens Vibrio ordalii, V. parahaemolyticus, V. anguillarum, V. alginolyticus and Aeromonas hydrophila. In an experimental 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 benecial 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 denition, 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 specic 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 sh and crustacean cultures by reducing the concentration of organic materials (OM) and ammonia. This procedure was accomplished 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 inuencing the composition 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 pathogens in the aquatic environment (Irianto & Austin, 2002). Bacterial species belonging to the genera Bacillus, Pseudomonas, Nitrosomonas, Nitrobacter, Acinetobacter and Cellulomonas are known to help in the mineralization of organic water and in reducing the accumulation of organic loads
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(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 waterquality parameters were at optimum levels in the experimental 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 Ayurveda, one of the oldest medical sciences that dates back to around 2500 BC, the consumption of yoghurt is recommended for the maintenance of overall good health. A scientic explanation of the benecial effects of lactic acid bacteria present in fermented milk was rst 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 ora in our bodies and to replace the harmful microbes by useful microbes (Talwalkar, 2003). He proposed that the acid-producing organisms in fermented 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 gastrointestinal tract such as inammatory bowel disease, and controlling 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 benecial effects for humans and animals. The denition 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 denition was further expanded by Sperti in the early 1970s to include tissue extracts that stimulated microbial growth (Gomes & Malcata, 1999). Thereafter, other scientists applied the term to animal feed supplements having a benecial effect on the
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host by contributing to its intestinal microbial balance (Talwalkar, 2003). Consequently, the term probiotics was applied to describe organisms and substances that contribute to intestinal microbial balance. This general denition was made more precise by Fuller (1989), who dened probiotics as a live microbial feed supplement that benecially 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 benecial effect on the health of the host upon ingestion by improving the properties of its indigenous microora (Havenaar & Huis int Veld, 1992). In addition, there is another denition of probiotics by Coeuret et al. (2004) as living microorganisms that, upon ingestion in certain numbers, exert health benets beyond inherent basic nutrition.

Characterization
Lactic acid bacteria These are classied 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, Carnobacterium) have adapted to grow under widely different environmental conditions. They are found in the gastrointestinal 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 microbiota of larval or growing sh, several trials have been undertaken to induce an articial 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; Vazquez 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 ora.
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The incubation temperature, incubation time and nutrient medium are the most important factors when isolating lactic acid bacteria from sh. 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, aminoacids 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 digestibility in mammals and sh. 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 remained stable. There are two hypotheses for this reaction: (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 shes usually changes in different aquatic environments with various salinities. 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 colonization, but, on the other hand, stress increases coliforms in the intestinal tract. However, prebiotics and
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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 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 microora of digestive organs and their environment. Hence, a successful probiotic is expected to have a few specic 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, macrophages 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) Benets to the host animal in some ways. In order to have a benecial effect in the form of a growth promoter or to protect sh 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 efciently for nutrients with possibly harmful strains. Of course, identication 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 prerequisite for colonization (Fuller, 1992; Ali, 2000; Verschuere et al., 2000).
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(5) Applied microorganisms should be stable for long periods under storage as well as in eld conditions (Fuller, 1992). (6) Probiotic microorganisms will, of course, have to be nonpathogenic and nontoxic in order to avoid undesirable side-effects when administered to aquatic organisms (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 bacteria (in intestinal ora). Many workers believe these bacteria have a better chance of out-competing resident bacteria and establishing themselves at a numerically signicant 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 indicates its ability to grow successfully under the prevailing conditions, and one can expect that this strain will compete efciently 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 therefore 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 briey, of the intestinal tract. In addition, the clinical effects of B. subtilis as an immunostimulatory 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 antagonistic 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 absorption 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. uvialis 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-negative group (Moriarty, 1999). In addition, Alvandi et al. (2004) isolated some Gramnegative bacteria (such as Pseudomonas sp. PM11 and Vibrio uvialis 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 sh, at variance with homeotherms, but some strains can colonize the gut. It is, however, possible to maintain articially 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 ow increases the inuence of the surrounding medium, in the same way as the water ow observed in lter-feeders, such as bivalves, shrimp larvae and live food organisms. This inuence is particularly important in the larval stages (Gatesoupe, 1999), because larvae may ingest bacteria by grazing on or ltering 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 Gram-positive bacteria Bacillus sp. S11 Bacillus sp. Bacillus sp. Lactobacillus lactis AR21 Mixed culture, mostly Bacillus spp. Gram-negative bacteria Vibrio alginolyticus Yeast Saccharomyces cerevisiae, S. exiguous, Phafa rhodozoma Microalgae Tetraselmis suecica Source Penaeus monodon Commercial product Rotifer mass culture Commercial product Beach sand Commercial product

started feeding (Irianto & Austin, 2002; Rengpipat et al., 2003), and the digestive tract is not yet developed completely 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 benecial bacterium will dominate the associated microbiota 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 Grampositive 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 specic 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 sh and shrimp. Generally,

Used on Penaeus monodon Penaeids Water Brachionus plicatilis Brachionus plicatilis Penaeids, salmomids Litopenaeus vannamei

Method of application Premix with feed Water Added to water Feed additive Mixed in water Feed, bath for 10 min Premix with feed

Commercial product

Penaeids, Salmo salar

Feed

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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 particles; (2) Increasing the production of systematic antibodies, usually of immunoglobulin and interferon (a nonspecic antiviral agent); (3) Increasing local antibodies at mucus surfaces such as the gut wall. Irianto & Austin (2002) reported that feeding with Grampositive 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 sh vaccine. Apparently, the b-1.3glucans stimulate the nonspecic immune defence system of the sh 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 compounds 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 antibiotics; class II small hydrophobic, heat-stable peptides; class III large heat-stable peptides; and class IV complex bacteriocins: probiotics with lipid and/or carbohydrate (Fooks & Gibson, 2002).
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Competition for nutrients, space and Fe Theoretically, competition for nutrients can play on important 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 for other bacterial populations (Fooks & Gibson, 2002). Competition 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 microora 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 organism. In addition, the gastrointestinal tract may serve as an ecological niche for some probiotics such as lactic acid bacteria strains to sh via the feed. These strains may be metabolically active in the intestinal mucus and feces of an aquatic organism and grow more than pathogenic bacteria in the digestive tract (J born et al., 1997). o 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 uorescent strain pseudomonad bacteria can competitively inhibit the growth of the sh pathogen Aeromonas salmonicida. Their results show that the uorescence is probably due to competition for free iron (Smith & Davey, 1993; Gram et al., 1999). Sideropheros are low-molecular-weight, ferric iron-specic 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 efciency 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 scientic 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 (Khachatourions, 1998). The increase in the anxiety about antibiotic-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, oxytetracycline and streptomycine have been used in attempts to control these bacteria, but their efcacy 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 organism 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 (Khachatourians, 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 nal consumer, probiotic 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 detoxication of potentially harmful compounds in the diet by hydrolytic enzymes, including amylases and proteases, and the produc c

tion of vitamins such as biotin and vitamin B12 (Irianto & Austin, 2002). Venkat et al. (2004) evaluated the effects of some probiotics on the growth of postlarvae of Macrobranchium rosenbergii. According to their results, signicant growth was observed for larvae fed diets supplemented with probiotics. The highest protein gain (more than 55%) and the protein efciency ratio were signicantly 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 assimilation of nutrients in the gut of the host by modifying the gut ora.

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